VMC 311 VETERINARY BACTERIOLOGY & MYCOLOGY

 

 

 

 

 

 

 

 

 

 

 

 

 

 

VOLUME – 3

SYSTEMATIC BACTERIOLOGY

 

 

Also available at

www.geocities.com/john_ivj

For local circulation only.

This course material is not a replacement for any textbook

 

 

 

INSTRUCTORS

J. John Kirubaharan,

N. Daniel Joy Chandran,

Department of Veterinary Microbiology,

Madras Veterinary College,

Chennai – 600 007

E Mail: [email protected]
VMC 311 LECTURE # 1

BACTERIAL CLASSIFICATION

 

LEARNING OBJECTIVES

• Taxonomy and nomenclature
• Taxonomic hierarchy
• Major Phyla

 

 

INTRODUCTION: Taxonomy is defined as the science of classification particularly living forms. The important objective of classification is to establish relationship between organisms. It is the basic and necessary tool in the study of living organisms. The term taxa denote the degree of relatedness among living organisms and provides means for phylogenetic relationship. The taxonomy of bacteria is not very definitively worked out yet, especially the higher levels of classification. It is postulated that the degree of variance between different bacterial groups is sufficient to give them each 'Kingdom (phyla) Status' of their own. The classification scheme laid out in the 2nd edition of "Bergey's Manual of Systematic Biology" will be followed through out this course material.

Brief history of taxonomy:- Taxonomy originated from Aristotle, who classified living organisms into plants and animals. Subsequently in 1859 Charles Darwin published his theory of natural selection that explained similarities among organisms and the similarities were attributed to common ancestors. Initially bacteria were placed under plants by Carl Von Nageli (1857). The term prokaryote was introduced by Edward Chatton (1937) and in 1968 a separate kingdom prokaryote was proposed by RGE Murray.

 

WHAT IS CLASSIFICATION: Classification is the assignment of organisms (species) into an organized scheme of naming and these schemes are based on evolutionary relationships. Thus, classification is concerned with the establishment of criteria for identifying organisms and assignment to groups (what belongs to where). The arrangement of organisms into groups of organisms (e.g., how large or inclusive groups should be; for example, at what level of diversity should a single species be split into two or more species?) is consideration of how evolution resulted in the formation of these groups. Taxon (pleural taxa) refers to a group or category of related organisms (For example, at the lowest level, species is a taxonomic category as is genera and all the way on up to kingdom and domain). These groups become increasingly inclusive as they become larger, going from species to kingdom or domain

 

Two key characteristic of taxa are that

• members of lower level taxa (e.g., species) are more similar to each other than are members of higher level taxa (e.g., kingdoms or domain)
• members of specific taxa are more similar to each other than any are to members of different specific taxa found at the same hierarchical level (e.g., humans are more similar to apes, i.e., comparison between species, than either is similar to, for example, Escherichia coli)

 

TAXONOMIC HIERARCHY: The following is the hierarchy followed for classification of bacteria

–Kingdom

–Phylum of Division

–Class

–Order

–Family

–Genus

–Species

 

Hierarchy	Suffix	Description
Class	-al	A class consists of related orders
Order	-ales	An order contains a group of related families
Family	-aceae	In this category are placed closely related genera or tribes
Tribe	-ieae	A tribe contains closely related genera
Genus		This most important category contains closely related species
Species		A bacterial species is defined by the similarities found among its members. Properties such as biochemical reactions, chemical composition, cellular structures, genetic characteristics, and immunological features are used in defining a bacterial species. Identifying a species and determining its limits presents the most challenging aspects of biological classification—for any type of organism. A formal means of distinguishing bacterial species is by employing a dichotomous key to guide the selection of tests used to efficiently determine those bacterial properties most relevant to bacterial identification
Subspecies		Some species may be further subdivided into subspecies on the basis of small but consistent differences, e.g., Campylobacter fetus subsp. Intestinalis, subsp. Jejuni
Strain		A strain consists of the descendants (clone) of a single isolate in pure culture
Serovar		A strain with distinctive antigenic properties
Biovar		A strain with special biochemical or physiological properties

 

BINOMIAL NOMENCLATURE: Bacteria are named using binomial nomenclature Binomial nomenclature employs the names of the two lower level taxa, genus and species, to name a species. Some of the important points to be borne in mind while writing the name of species of bacteria are provide below;    

 

• Genus comes before species (e.g., Bacillus anthracis)
• Genus name is always capitalized (e.g., Bacillus)
• Species name is never capitalized (e.g., anthracisi)
• Both names are always either italicized or underlined (e.g., I Bacillus anthracis)
• The genus name may be used alone, but not the species name (i.e., saying or writing "Bacillus," alone is legitimate while saying or writing "anthracis" is not)
• The genus name may be abbreviated but it must be used first without abbreviation
• If abbreviated it must be used with the species name (no B. all by itself)
• It must be abbreviated unambiguously
• If abbreviating as the first letter of the genus is unambiguous, then abbreviating as the first letter is what one does (e.g., Bacillus abbreviated as B. but only if no other genera considered also starts with B)
• Genus abbreviations are only used in conjunction with the species name (i.e., B. anthracis)

The names are written in Latin and hence they are written in italics. The names are assigned to bacteria by the International Committee on Systematic Bacteriology, which publishes Bacteriological code and International Journal of Systematic Bacteriology. The rules for assigning names to newly classified bacteria and for assigning bacteria to taxa are published in Bacteriological code and the evidence for their classification are published in International Journal of Systematic Bacteriology. Later it is also published in Bergey’s Manual of (Determinative) Systematic Bacteriology, which is also referred as Bible of Bacteriology.

 

DICHOTOMOUS KEY: A means of assigning an organism to a specific taxonomic category typically involves the use of specific criteria that may be posed as questions (e.g., what does the organism look like? etc.). Relevant criteria may be arranged as a dichotomous key. In a dichotomous key questions are arranged hierarchically (just as taxonomic categories are) with more general questions (i.e., those arranging organisms into large categories) are asked first, with questions becoming more specific (better suited to arranging organisms into more specific taxa) asked subsequently. In addition, questions are dichotomous, meaning that they each have two possible answers, with each answer distinguishing the organisms as well as the path to the next question

 

MAJOR PHYLA: The major phyla or divisions of bacteria as provided in the Bergeys Manual of Systematic Bacteriology (I Edn) are provided below

 

 

Phylum 1 – Aquificae: This is a small group of thermophilic to hyperthermophilic chemolithotrophic bacteria, meaning that they derive their energy from inorganic molecules and they live in hot environments. Members of the genus Aquifex can live at temperatures as high as 95 degrees C and they have an optimum growth temperature of 85 degrees. Bacteria of veterinary importance are not found in this phylum

 

Phylum 2 – Xenobacteria: This group comprises a number of aerobic chemoorganotrophic bacteria. The two best studied genera are Thermus and Deinococcus. Thermus is a thermophilic bacterium. The enzyme Taq DNA Polymerase comes from Thermus aquaticus. This is the major enzyme used in Polymerase Chain Reaction (PCR) techniques for amplyfying DNA. Bacteria of veterinary importance are not found in this phylum.

 

Phylum 3 – Chryosogenates: Not very important. Bacteria of veterinary importance are not found in this phylum

 

Phylum 4 – Thermomicrobium: This is a small phylum of chemotrophic and autotrophic bacteria. Bacteria of veterinary importance are not found in this phylum

 

Phylum 5 – Cyanobacteria: The cyanobacteria are morphologically a heterogeneous mixture of bacteria. Cyanobacteria are photosynthetic organisms; like plants they trap the energy of the sun (autotrophically) to use in their own metabolism and give off oxygen in the process. In order to achieve this they have their own chlorophyll called 'Chlorophyll a'. Cyanobacteria are often called blue-green algae, though they are not all a blue-green colour and they are not algae at all. Bacteria of veterinary importance are not found in this phylum

 

Phylum 6 – Chlorobia: This is a small phylum referred to as Green Sulphur Bacteria. They are all obligately anaerobic phototrophic species. Bacteria of veterinary importance are not found in this phylum

 

Phylum 7 – Proteobacteria: Proteobacteria is the second largest group of bacteria. This phylum contains 1534 species or 32.3% of all known bacteria. Proteobacteria are all gram negative, but otherwise represent a diverse range of organisms. The groups are given below.This phylym contains bacteria of veterinary importance (given in bold letters)

• Purple Phototrophic Bacteria
• Nitrifying bacteria
• Sulphur and Iron Oxidising Bacteria
• Spirilla
• Methanotrophs
• Acetic Acid Bacteria
• Free-living Aerobic Nitrogen Fixers
• Other Gram Negative Bacteria
• Enteric Bacteria
• Bioluminescent and related bacteria
• Rickettsias
• Hydrogen Oxidising Bacteria
• Sheathed Proteobacteria
• Budding and Stalked Bacteria
• Gliding Myxobacteria
• Sulphate and Sulphur Reducing Proteobacteria

 

Phylum 8 – Firmicutes: This is the single largest grouping of bacteria. Approximately 2475 species in 255 genera, 40% of these species are aggregated in just 6 genera; Lactobacillus - 100 sp., Mycoplasma - 110 sp., Bacillus - 114 sp, Clostridium 146 sp and Streptomyces 509. The Firmicutes are all gram-positive bacteria. The Firmicutes are further divided according to their GC ratios. This is the ratio of Guanine and Cytosine to Guanine, Cytosine, Adenine and Thymine in the cell, thus a GC ratio = G+C divided by G+C+A+T times 100. Fermicutes are either High GC or Low GC. . The groups are given below. This phylum contains bacteria of veterinary importance (given in bold letters)

• Spore forming
• Non-spore forming
• Wall-less forms
• Actinomycetes
• Mycobacteria
• Propionic Acid Bacteria
•  

Phylum 9 - Planctomyces and Allies: The group Chlamydiaceae is placed under this phylum. Chlamydiaceae contain only 3 species all in the genus Chlamydia. All three are obligate parasites of warm-blooded animals; C. trachomatis and C. pneumoniae of humans and C. psittaci of birds and occasionally mammals, including humans. All are pathogenic.

 

Phylum 10 – Spirochetes: Spirochetes are a distinct group of bacteria, they are gram negative and most of them are tightly-coiled, long and slender in shape. They have one or more flagella (up to 100) at each end of their cells which when rotated allow them to move. This phylym contains bacteria of veterinary importance

 

Phylum 11 – Fibrobacter: Not very important. Bacteria of veterinary importance are not found in this phylum

 

Phylum 12 – Bacteroides: With 130 species in 20 genera this is the third largest phylum of bacteria. It contains a variety of physiological types including both obligate aerobes and obligate anaerobes. This phylum contains bacteria of veterinary importance

 

Phylum 13 – Flavobacterium: It contains primarily aquatic species though they are also found in food processing plants. This phylum contains bacteria of veterinary importance

 

Phylum 14 – Spingobacteria: They are widespread soil species with the habit of attaching themselves to cellulose strands before digesting them. Bacteria of veterinary importance are not found in this phylum

 

Phylum 15 – Fusobacteria: The Fusobacteria is a small phylum of bacteria most of which occur in the genus Fusobacteria. Fusobacteria are filamentous bacteria which secondary colonists on the dental plaque in teeth. This phylum contains bacteria of veterinary importance

 

Phylum 16 Verrucomicrobia: Not very important. Bacteria of veterinary importance are not found in this phylum

 

CRITERIA FOR CLASSIFICATION: The following are the characters that are normally used to classify bacteria.

• Morphological characteristics
• Differential staining
• Biochemical tests
• Serology
• Phage typing
• Amino acid sequencing
• Protein analysis
• Base composition of nucleic acid G+C content (paradox ?) – DNA fingerprints
• Nucleic acid hybridization (Southern blot)
• Flow cytometry

A SIMPLE KEY FOR CLASSIFICATION OF BACTERIA OF VETERINARY IMPORTANCE

––––– Obligate Intracellular Bacteria –––––
+	–
Rickettsia	––––––––––––––– Cell wall –––––––––––––––
Chlamydia	+		–
––––––––––––––– Spiral-shaped –––––––––––––––			Mycoplasma
+		–
Treponema	––––––––––––––– Gram stain –––––––––––––––
Leptospira	+		–
Borrelia
	Gram-positive		Gram-negative

 

GRAM NEGATIVE BACTERIA

Cocci					Bacilli/Coccobacilli
Neisseria					–––––––––– Aerobic ––––––––––
			+						–
			–––––––– Growth on SBA ––––––––						Bacteriodes
	+						–
	––––––– Oxidase –––––––						Haemophilus
+				–			Legionella
Glucose				Lactose
––– fermented –––				––– fermented –––
+		–		+		–
Vibrio		Brucella		Escherichia		Salmonella
		Bordetella		Klebsiella		Shigella
		Campylobacter				Proteus
		Pseudomonas				Yersinia

 

 

 

GRAM POSITIVE BACTERIA

––––– Obligate Intracellular Bacteria –––––
+	–
Rickettsia	––––––––––––––– Cell wall –––––––––––––––
Chlamydia	+		–
––––––––––––––– Spiral-shaped –––––––––––––––			Mycoplasma
+		–
Treponema	––––––––––––––– Gram stain –––––––––––––––
Leptospira	+		–
Borrelia
	Gram-positive		Gram-negative

 

IMPORTANT BACTERIA OF VETERINARY AND MEDICAL IMPORTANCE

 

 

 

 

 

 

VMC 311 LECTURE # 2

TOOLS IN THE STUDY OF SYSTEMATIC BACTERIOLOGY

LEARNING OBJECTIVES

1. General points in the study of Systematic bacteriology
2. Important study points
1. Morphology
2. Isolation
3. Growth
4. Colonial morphology
5. Biochemical characters
6. Antigenic characters
7. Pathogenesis
8. Diagnosis

 

INTRODUCTION: Systematic veterinary bacteriology deals with the study of bacteria that causes diseases in animals and birds in a particular method so that the infection can be controlled. Another important aspect in the study of systematic veterinary bacteriology deals with differential diagnosis in other words identifying the exact causative agent by differentiating with other causative agents that causes infection whose symptoms and lesions are closely related to the first one. Following are some of the points in which a thorough understanding is essential to study the systematic veterinary bacteriology.

 

IMPORTANT POINTS:

1.      Morphological characters of bacteria with arrangement

2.      General points to be considered for isolation of causative organisms

3.      Cultural characters – Appearance of the colonies in culture media

4.      Biochemical characters – Dependent of metabolic activities of the organisms

5.      Antigenic characters – Different antigens like toxins, capsule antigens, flagellar antigens, fimbrial antigens, somatic antigens and such other antigens

6.      Pathogenesis – Mechanisms by which the infection is produced, host range, spread, symptoms and lesions etc.

7.      Diagnosis – Confirming the etiology, various diagnostic tests like serological, molecular and biological tests etc.

 

1. MORPHOLOGY: The most common bacterial shapes are RODS, COCCI, and SPIRAL. However, within each of these groups are hundreds of unique variations.

Cocci     Rod     Curved rod

1. Rods may be long, short, thick, thin, have rounded or pointed ends, thicker at one end than the other etc.
2. Cocci may be large, small, or oval shaped to various degrees.
3. Spiral shaped bacteria may be fat, thin, loose spirals or very tight spirals.
4. There are square bacteria, star-shaped bacteria, stalked bacteria, comma shaped bacteria, budding bacteria that grow in net-like arrangements and many other morphologies.  
5. Arrangement: Arrangement is also referred as “GROUP ASSOCIATIONS of microbes”. Bacteria may exist as single cell or as common grouping such as chains, uneven clusters, pairs, tetrads, octads and other packets. They may also exist as masses embedded within a capsule.

 

f.       Study on morphology also includes staining characters. Simple staining (Methylene blue) is used to study the morphology and arrangement. Differential staining is used to differentiate organisms into groups based on staining properties (Grams staining, Acid fasti staining). Special staining is used to study presence of absence of certain special structures (Alberts staining is used to stain volutin granules). Of the different staining techniques Gram’s staining technique is very important and most of the bacteria are grouped into two groups of gram positive and negatives based on staining property. The staining property is decided by the cell wall characters. Gram-positive bacteria have a relatively thick wall composed of many layers of the polymer peptidoglycan (sometimes termed murein). The thickness of this wall blocks the escape of the crystal violet-iodine complex when the cells are washed with alcohol or acetone. Gram-negative bacteria have only a thin layer of peptidoglycan, surrounded by a thin outer membrane composed of lipopolysaccharide (LPS). The region between the peptidoglycan and LPS layers is termed the periplasmic space. it is a fluid or gel-like zone containing many enzymes and nutrient-carrier proteins. The crystal violet-iodine complex is easily lost through the LPS and thin peptidoglycan layer when the cells are treated with a solvent.

7.  Study on morphology also includes presence or absence and arrangement of following structures of bacteria

                                                              i.      Capsule or slime layer – present or absent

                                                             ii.      Flagella – Present or absent; number and location of flagella on the surface of bacteria

                                                           iii.      Fimbriae and pili

                                                          iv.      Endospores – formed or not formed

                                                            v.      Special structures like – axial filament (spirochaetes)

                                                          vi.      Cytoplasmic granules and inclusions

8. Study on morphology also includes particulars about the measurement. The standard measure for bacteria is micron ( 1 in 1000 of a mm). The measurement for rod shaped organisms are expressed for length and breadth and for spherical shaped organisms only the diameter is expressed. The size of bacteria varies considerably. Most rods measure between 2 to 5 mm in length by 0.5 to 1 mm in width. Spirochetes are longer – up to 20 mm and narrower 0.1 to 0.2 mm. Cocci approximately have 1mm diameter. Based on size bacteria are grouped into large, medium, small and very small organisms.

 

2. CULTURAL CHARACTERS: In the identification of bacteria much weight is placed on how the organism grows in or on media. The cultural characteristics of bacterium is studied by the colonial morphology it produce on an agar plates. Bacteria grow on solid media as colonies.  A colony is defined as a visible mass of microorganisms all originating from a single mother cell, therefore a colony constitutes a clone of bacteria all genetically alike. The cause of an infection is confirmed mostly by isolating and culturing microorganism either in artificial media or in a living host. Bacteria are cultured in either liquid (broth) or on solid (agar) artificial media. Liquid media provide greater sensitivity for the isolation of small numbers of microorganisms; however, identification of mixed cultures growing in liquid media requires subculture onto solid media so that isolated colonies can be processed separately for identification. Growth in liquid media also cannot ordinarily be quantitated. Solid media, although somewhat less sensitive than liquid media, provide isolated colonies that can be quantified if necessary and identified. Some genera and species can be recognized on the basis of their colony morphologies. Bacteria can also be identified by differential carbohydrate fermentation capabilities of microorganisms by incorporating one or more carbohydrates in the medium along with a suitable pH indicator. Such media are called differential media (e.g., eosin methylene blue or MacConkey agar) and are commonly used to isolate enteric bacilli. Culture media can also be made selective by incorporating compounds such as antimicrobial agents that inhibit the indigenous flora while permitting growth of specific microorganisms resistant to these inhibitors. One such example is Thayer-Martin medium, which is used to isolate Neisseria sp.. This medium contains vancomycin to inhibit Gram-positive bacteria, colistin to inhibit most Gram-negative bacilli, trimethoprim-sulfamethoxazole to inhibit Proteus species and other species that are not inhibited by colistin and anisomycin to inhibit fungi. The pathogenic Neisseria species are ordinarily resistant to the concentrations of these antimicrobial agents in the medium. Certain bacteria like Salmonella require enrichment and in the process of enrichment the growth of Salmonella alone are favoured. Eg. Selenite F broth. The numbers of bacteria in specimens are expressed as Colony forming units (CFU / ml of inoculum).
1. Studies on cultural characters include ability of the organisms to grow on

                                                              i.      Different media – Liquid, Solid and special

                                                             ii.      Incubation conditions – temperature, pH, requirement of gases like oxygen, carbondioixide etc.

                                                        iii.      Days of incubation – overnight, long duration

2. A nutrient material prepared for the growth of microorganisms in a laboratory is called culture medium. The microbes that grow and multiply in or on a culture medium are referred to as a culture. Before introducing organisms into culture medium, it should be sterile and should not contain any contaminating organisms. The culture media are broadly divided into solid, liquid and semisolid depending upon the per centage of agar in it. Agar is complex polysaccharide derived from marine algae. The solid agar media are usually contained in petriplates and in test tubes as slant or as deep. For good microbial growth, a medium must provide energy sources of carbon, sulfur, phosphorus and other growth factor.

                                                              i.      Chemically defined media:  It is one whose exact chemical composition in known.

                                                             ii.      Complex media: They are made up of nutrients such as extracts from yeasts, meat or plants or digests of proteins from these and other sources. The energy, carbon, sulfur and nitrogen requirements are provided by proteins in these media. In these media, the larger proteins are broken into short chain aminoacid substances called peptones, which are easily unitlised by bacteria. Eg. Nutrient agar, Nutrient broth

                                                           iii.      Selective media: In selective media, the growth of unwanted microbes are suppressed and only the growth of desired organisms are supported. Eg. Bismuth sulfite agar, which allows the growth of typhoid bacteria alone.

                                                          iv.      Differential media: In these media, desired organisms are differentiated from other organisms by colony characters. Eg. Blood agar, MacConkey agar

                                                            v.      Enrichment media: These media favour the growth of a particular microbe but not other microbe. It is used for bacteria that are present in small number in environment and likely to be missed by routine media. Eg. Selenite F broth

3. The following are the points one should look for while studying the colonial characters.

                                                              i.      WHOLE SHAPE OF COLONY SIZE OF COLONY (measure with a millimeter rule), less than 1mm = punctiform (pin-point).

                                                             ii.      EDGE/MARGIN OF COLONY:  magnified edge shape

                                                           iii.      CHROMOGENESIS (pigmentation): white, buff, red, purple, etc. Some pigments are water-soluble, others are not.  Water soluble pigments can be studied in liquid medium.

                                                          iv.      OPACITY OF COLONY: transparent (clear), opaque, translucent (almost clear, but distorted vision–like looking through frosted glass), iridescent (changing colors in reflected light)

                                                            v.      ELEVATION OF COLONY: Raised, flat, convex, umbonate etc.

                                                          vi.      (SURFACE OF COLONY: smooth, glistening, rough, dull (opposite of glistening), rugose (wrinkled)

                                                         vii.      CONSISTENCY: butyrous (buttery), viscid (sticks to loop, hard to get off), brittle/friable (dry, breaks apart), mucoid

                                                       viii.      EMULSIFIABILITY OF COLONY:Forming a uniform suspension, a granular suspension, or no emulsification at all.

                                                           ix.      ODOR: Absent or present. If it has an odor, what does it smell like?

                                                            x.      HAEMOLYTIC CHARACTERS: Studied on blood agar. Indicated as zone of haemolysis around colonies. There are four types alpha, beta, gamma and  alpha prime

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                                           xi.       

3. BIOCHEMICAL CHARACTERS: Biochemical reactions are normally performed to identify at species level. Most of the biochemical reactions are based on the metabolic activities of bacteria. In biochemical reaction the end products of a chemical reaction are identified by using specific reagents. Some of the common biochemical reactions are based on
1. Utilization of sugars like glucose, mannose etc.
2. Utilization and breakdown of amino acids
3. Liquefaction of substances like gelatin
4. Utilization of certain substrates like citrate
5. Utilization of milk or egg protein
6. Ability of the organism to produce certain enzymes like oxidase, catalase etc.
7. Growth in the presence of certain specific substances
4. ANTIGENIC CHARACTERS: Bacteria possess different types of antigen mainly based on its morphological features. Some of the common bacterial antigens are somatic antigen (O), Capsular antigen (K), flagella antigen (H) and fimbrial or pili antigen. Phase variations in antigens are also used in grouping of organisms. The major constituent of the cell wall of the gram-positive bacteria is the peptidoglycan (PG), where as it is lipopolysaccharide (LPS) in gram-negative bacteria. In gram-negative bacteria the antigenicity is associated with polysaccharide compound (an oligosaccharide attached to lipid A - LPS). The LPS is also referred as endotoxins. In the same way the capsule of bacteria is also rich in polysaccharide, which are good antigens. Apart from these antigens that are associated with morphology, bacteria also possess other types of antigens. Some of the important antigens include porins, heat shock proteins (HSP) and exotoxins. The porins are proteins that form pores on the cell wall of gram-negative bacteria. HSP are produced in large quantities by bacteria undergoing stress. Exotoxins are highly immunogenic and stimulate the production of antibodies. The antibodies against exotoxins are called antitoxins. When these exotoxins are precipitated by mild protein denaturing agents such as formaldehyde, the exotoxin loses its pathogenicity but retains its immunogenicity. Such precipitated toxins are called as toxoids. The characteristics of exotoxins and endotoxins are summarised in the table below.

 

 

 

CHARACTERISTICS OF BACTERIAL ENDOTOXINS AND EXOTOXINS
PROPERTY	ENDOTOXIN	EXOTOXIN
Chemical Nature	Lipopolysaccharide(mw = 10kDa)	Protein (mw = 50-1000kDa)
Relationship To Cell	Part of outer membrane	Extracellular, diffusible
Denatured By Boiling	No	Usually
Antigenic	Yes	Yes
Form Toxoid	No	Yes
Potency	Relatively low (>100ug)	Relatively high (1 ug)
Specificity	Low degree	High degree
Enzymatic Activity	No	Usually
Pyrogenicity	Yes	Occasionally

 

VMC 311 LECTURE # 3

TOOLS IN THE STUDY OF SYSTEMATIC BACTERIOLOGY - II

LEARNING OBJECTIVES

1. General points in the study of Systematic bacteriology
2. Important study points
1. Morphology
2. Isolation
3. Growth
4. Colonial morphology
5. Biochemical characters
6. Antigenic characters
7. Pathogenesis
8. Diagnosis

 

 

5. PATHOGENESIS

INTRODUCTION: A pathogen is defined as microorganism, which is able to cause disease in a plant, animal or insect. The term pathogenicity is defined as the ability of these microorganisms to produce disease in a host organism. The pathogenicity is expressed by means of their virulence, which refers to the degree of pathogenicity of the microbe. Hence the virulence determinants of a pathogen are may be one or combination of its genetic or biochemical or structural features that enable it to produce disease in a host. The production of an infection depends on the virulence of the pathogen and the relative degree of resistance or susceptibility of the host, mainly due to the effectiveness of the host defense mechanisms.

 

MECHANISMS OF BACTERIAL PATHOGENICITY: Production of infection by pathogenic bacteria is mainly due to its invasiveness or toxigenesis or both. 

1.     Invasiveness – It is defined as the ability of the microorganisms to invade tissues Invasiveness includes mechanisms

a.      Colonization – adherence to a surface and initial multiplication

b.      Ability to bypass or overcome host defense mechanisms

c.      Phe production of extracellular substances which facilitate invasion.

2.     Toxigenesis – It is defined as the ability of microorganisms to produce toxins. Bacteria produce two types of toxins called exotoxins and endotoxins. Exotoxins are released from bacterial cells and may act at tissue sites removed from the site of bacterial growth. Endotoxins are cell-associated substances that are structural components of the cell walls of Gram-negative bacteria. However, endotoxins may be released from growing bacterial cells or from cells, which are lysed as a result of effective host defense (e.g. lysozyme) or the activities of certain antibiotics (e.g. penicillins and cephalosporins). Hence, bacterial toxins, both soluble and cell-associated, may be transported by blood and lymph and cause cytotoxic effects at tissue sites remote from the original point of invasion or growth. Some bacterial toxins may also act at the site of colonization and play a role in invasion.

INVASIVENESS: The first stage of microbial infection is called colonization, which refers to the establishment of the pathogen at the appropriate point of entry. Pathogens usually colonize host tissues that are in contact with the external environment. Sites of entry in animal hosts include the urogenital tract, the digestive tract, the respiratory tract and the conjunctiva. Organisms that enter and infect through these regions should have tissue adherence mechanisms and ability to overcome host defenses on the surface. Bacterial adherence requires a receptor host cell usually specific carbohydrate or peptide residues and an adhesin. The bacterial adhesin is typically a macromolecular component of the bacterial cell surface, which interacts with the host cell receptor. Adhesins and receptors usually interact in a complementary and specific fashion. The specificity of adherence of bacteria to host cells or tissues is contributed by

1.      Tissue tropism: particular bacteria are known to have an apparent preference for certain tissues over others

2.      Species specificity: certain pathogenic bacteria infect only certain species of animals, e.g. nteropathogenic E. coli K-88 infections are limited to pigs; E. coli CFA I and CFA II infect humans; E.coli K-99 strain infects calves. Group A streptococcal infections occur only in humans.

3.      Genetic specificity within a species: certain strains or races within a species are genetically immune to a pathogen , e.g. Certain pigs are not susceptible to E. coli K-88 infections

 

Mechanisms of Adherence to Cell or Tissue Surfaces

The mechanisms for adherence may involve two steps:

1.       Nonspecific adherence: reversible attachment of the bacterium to the eukaryotic surface (sometimes called "docking")

2.       Specific adherence: permanent attachment of the microorganism to the surface (sometimes called "anchoring").

Nonspecific adherence involves nonspecific attractive forces, which allow approach of the bacterium to the eukaryotic cell surface. Possible interactions and forces involved are:

1.      Hydrophobic interactions

2.      Electrostatic attractions

3.      Atomic and molecular vibrations resulting from fluctuating dipoles of similar frequencies

4.      Brownian movement

5.      Recruitment and trapping by biofilm polymers interacting with the bacterial glycocalyx (capsule)

Specific adherence involves permanent formation of many specific lock-and-key bonds between complementary molecules on each cell surface. Complementary receptor and adhesin molecules must be accessible and arranged in such a way that many bonds form over the area of contact between the two cells. Once the bonds are formed, attachment under physiological conditions becomes virtually irreversible. The receptor and/or adhesin molecules mediate specificity of adherence of bacteria to host cells or tissues.

 

INVASION: It is the second stage after colonization. The invasion of a host by a pathogen may be aided by the production of bacterial extracellular substances, which act against the host by breaking down primary or secondary defenses of the body. These substances are referred as invasins. Invasins are proteins (enzymes) that act locally to damage host cells and/or have the immediate effect of facilitating the growth and spread of the pathogen. The damage to the host as a result of this invasive activity may become part of the pathology of an infectious disease. The extracellular proteins produced by bacteria, which promote their invasion are not clearly distinguished from some extracellular protein toxins ("exotoxins") which also damage the host. Invasins usually act at a short range (in the immediate vicinity of bacterial growth) and may not actually kill cells in their range of activity; exotoxins are often cytotoxic and may act at remote sites (removed from the site of bacterial growth). Also, exotoxins typically are more specific and more potent in their activity than invasins.

 

EVASION OF HOST DEFENSES: Some pathogenic bacteria are inherently able to resist the bactericidal components of host tissues. For example, the poly-D-glutamate capsule of Bacillus anthracis protects the organisms against cell lysis by cationic proteins in sera or in phagocytes. The outer membrane of Gram-negative bacteria is a formidable permeability barrier that is not easily penetrated by hydrophobic compounds such as bile salts, which are harmful to the bacteria. Pathogenic mycobacteria have a waxy cell wall that resists attack or digestion by most tissue bactericides. And intact lipopolysaccharides (LPS) of Gram-negative pathogens may protect the cells from complement-mediated lysis or the action of lysozyme. Microbial strategies to avoid phagocytic killing are numerous and diverse, but are usually aimed at blocking one or of more steps in the phagocytic process. They are

1.     Avoiding Contact with Phagocytes - Bacteria can avoid the attention of phagocytes in a number of ways.

a.      Invade or remain confined in regions inaccessible to phagocytes.

b.      Avoid provoking an overwhelming inflammatory response

c.      Inhibit phagocyte chemotaxis. e.g. Streptococcal streptolysin (which also kills phagocytes) suppresses neutrophil chemotaxis

d.      Hide the antigenic surface of the bacterial cell. Some pathogens can cover the surface of the bacterial cell with a component which is seen as "self" by the host phagocytes and immune system. Phagocytes cannot recognize bacteria upon contact and the possibility of opsonization by antibodies to enhance phagocytosis is minimized.

2.     Inhibition of Phagocytic Engulfment - Some bacteria employ strategies to avoid engulfment (ingestion) if phagocytes do make contact with them. Resistance to phagocytic ingestion is usually due to a component of the bacterial cell wall, or fimbriae, or a capsule enclosing the bacterial wall.

3.     Survival Inside of Phagocytes - Some bacteria survive inside of phagocytic cells, in either neutrophils or macrophages. Most intracellular parasites have special (genetically-encoded) mechanisms to get themselves into their host cell as well as special mechanisms to survive once they are inside. Intracellular parasites usually survive by virtue of mechanisms, which interfere with the bactericidal activities of the host cell. Some of these bacterial mechanisms include

a.      Inhibition of phagosome-lysosome fusion

b.      Survival inside the phagolysosome

c.      Escape from the lysosome

4.     Products of Bacteria that Kill or Damage Phagocytes - One of the important strategy in defense against phagocytosis is direct attack by the bacteria upon the phagocytes. The substances that pathogens produce that cause damage to phagocytes are referred to as "aggressins". Most of these are actually extracellular enzymes or toxins that kill phagocytes. Phagocytes may be killed by a pathogen before or after ingestion.

5.     Killing phagocytes before ingestion - Many Gram-positive pathogens, particularly the pyogenic cocci, secrete extracellular enzymes, which kill phagocytes.

6.     Killing phagocytes after ingestion - Some bacteria exert their toxic action on the phagocyte after ingestion has taken place. They may grow in the phagosome and release substances, which can pass through the phagosome membrane and cause discharge of lysosomal granules, or they may grow in the phagolysosome and release toxic substances which pass through the phagolysosome membrane to other target sites in the cell.

7.     Overcoming Host Phagocytic Defenses - On epithelial surfaces the main antibacterial immune defense of the host is the protection afforded by secretory antibody (IgA). Once the epithelial surfaces have been penetrated, however, the major host defenses of inflammation, complement, phagocytosis, Antibody-mediated Immunity (AMI), and Cell-mediated Immunity (CMI) are encountered.

8.     Ability to defeat the immune defenses may play a major role in the virulence of a bacterium and in the pathology of disease. Some bacterial defenses are described below.

a.      Immunological Tolerance to a Bacterial Antigen – It is contributed by

                                                              i.      Fetal exposure to Ag

                                                             ii.      High persistent doses of circulating Ag

                                                           iii.      Molecular mimicry. If a bacterial Ag is very similar to normal host "antigens", the immune responses to this Ag may be weak giving a degree of tolerance. Resemblance between bacterial Ag and host Ag is referred to as molecular mimicry.

b.      Antigenic Disguise - Bacteria may be able to coat themselves with host proteins (fibrin, fibronectin, antibody molecules) or with host polysaccharides (sialic acid, hyaluronic acid) so that they are able to hide their own antigenic surface components from the immunological system.

c.      Immunosuppression Some pathogens (mainly viruses and protozoa, rarely bacteria) cause immunosuppression in the infected host. This means that the host shows depressed immune responses to antigens in general, including those of the infecting pathogen. Suppressed immune responses are occasionally observed during chronic bacterial infections such as leprosy and tuberculosis.

d.      Persistence of a Pathogen at Bodily Sites Inaccessible to the Immune Response - Some pathogens can avoid exposing themselves to immune forces by positioning them in inaccessible locations inside the body.

e.      Induction of Ineffective Antibody - Many types of antibody are formed against a given Ag, and some bacterial components may display various antigenic determinants. Antibodies tend to range in their capacity to react with Ag (the ability of specific Ab to bind to an Ag is called avidity). If Abs formed against a bacterial Ag are of low avidity, or if they are directed against unimportant antigenic determinants, they may have only weak antibacterial action. Such "ineffective" (non-neutralizing) Abs might even aid a pathogen by combining with a surface Ag and blocking the attachment of any functional Abs that might be present.

f.       Antibodies Absorbed by Soluble Bacterial Antigens - Some bacteria can liberate antigenic surface components in a soluble form into the tissue fluids. These soluble antigens are able to combine with and "neutralize" antibodies before they reach the bacterial cells. For example, small amounts of endotoxin (LPS) may be released into surrounding fluids by Gram-negative bacteria.

g.      Antigenic Variation - One way bacteria can avoid forces of the immune response is by periodically changing antigens, i.e., undergoing antigenic variation.

h.      Changing antigens during the course of an infection - Antigenic variation is an important mechanism used by pathogenic microorganisms for escaping the neutralizing activities of antibodies. Antigenic variation usually results from site-specific inversions or gene conversions or gene rearrangements in the DNA of the microorganisms.

i.        Changing antigens between infections - Many pathogenic bacteria exist in nature as multiple antigenic types or serotypes, meaning that they are variant strains of the same pathogenic species.

 

TOXIGENESIS: There are two types of bacterial toxins endotoxins and exotoxins.   

EXOTOXINS: They protein toxins and are soluble proteins secreted by living bacteria during exponential growth. The production of protein toxins is generally specific to a particular bacterial species (e.g. only Clostridium tetani produces tetanus toxin; only Corynebacterium diphtheriae produces the diphtheria toxin). Usually, virulent strains of the bacterium produce the toxin (or range of toxins) while non-virulent strains do not, such that the toxin is the major determinant of virulence. Both Gram-positive and Gram-negative bacteria produce soluble protein toxins. Bacterial protein toxins are the most potent poisons known and may show activity at very high dilutions. The protein toxins resemble enzymes in a number of ways. Like enzymes, bacterial exotoxins: are proteins, are denatured by heat, acid, proteolytic enzymes, have a high biological activity (most act catalytically) and exhibit specificity of action. Usually the site of damage caused by the toxin indicates the location of the substrate for that toxin. Terms such as "enterotoxin", "neurotoxin", "leukocidin" or "hemolysin" are sometimes used to indicate the target site of some well-defined protein toxins. Certain protein toxins have very specific cytotoxic activity (i.e., they attack specific cells, for example, tetanus or botulinum toxins), but some (as produced by staphylococci, streptococci, clostridia, etc.) have fairly broad cytotoxic activity and cause nonspecific death of tissues (necrosis). A few protein toxins obviously bring about the death of the host and are known as "lethal toxins", and even though the tissues affected and the target sites may be known, the precise mechanism by which death occurs is not understood (e.g. anthrax toxin). As "foreign" substances to the host, most of the protein toxins are strongly antigenic. In vivo, specific antibody (antitoxin) neutralizes the toxicity of these bacterial proteins. However, in vitro, specific antitoxin may not fully inhibit their enzymatic activity. Toxoids are detoxified toxins, which retain their antigenicity and their immunizing capacity.

ENDOTOXINS: Endotoxins are part of the outer cell wall of bacteria. Endotoxins are invariably associated with Gram-negative bacteria as constituents of the outer membrane (Lipopolysaccharide) of the cell wall. The biological activity of endotoxin is associated with the lipopolysaccharide (LPS). Toxicity is associated with the lipid component (Lipid A) and immunogenicity (antigenicity) is associated with the polysaccharide components. The cell wall antigens (O antigens) of Gram-negative bacteria are components of LPS. Compared to the classic exotoxins of bacteria, endotoxins are less potent and less specific in their action, since they do not act enzymatically. Endotoxins are heat stable (boiling for 30 minutes does not destabilize endotoxin), but certain powerful oxidizing agents such as , superoxide, peroxide and hypochlorite degrade them. Endotoxins, although strongly antigenic, cannot be converted to toxoids. Endotoxins are toxic to most mammals. They are strong antigens but they seldom elicit immune responses, which give full protection to the animal against secondary challenge with the endotoxin. They cannot be toxoided. Endotoxins released from multiplying or disintegrating bacteria significantly contribute to the symptoms of Gram-negative bacteremia and septicemia, and therefore represent important pathogenic factors in Gram-negative infections. Regardless of the bacterial source, all endotoxins produce the same range of biological effects in the animal host. The biological reactions produced by endotoxins are fever, changes in white blood cell counts, disseminated intravascular coagulation, tumor necrosis, hypotension, shock and lethality. The sequence of events follows a regular pattern: 1. latent period; 2. physiological distress (fever, diarrhea, prostration, shock); 3. death. How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal. Animals vary in their susceptibility to endotoxin. In the LPS the Lipid A is the toxic component of LPS and the polysaccharide side chain (O antigen) of LPS may act as a determinant of virulence in Gram-negative bacteria. The O polysaccharide may supply a bacterium with its specific ligands (adhesins) for colonization, which is essential for expression of virulence. Lastly, the O-polysaccharide is antigenic, and the usual basis for antigenic variation in Gram-negative bacteria rests in differences in their O polysaccharides.

 

6. DIAGNOSIS: The clinical presentation of an infectious disease reflects the interaction between the host and the microorganism. This interaction is affected by the host immune status and microbial virulence factors. Signs and symptoms vary according to the site and severity of infection. Diagnosis requires a composite of information, including history, physical examination, and laboratory data. Infections may be caused by bacteria, viruses, fungi, and parasites. The pathogen may be exogenous (acquired from environmental or animal sources or from other persons) or endogenous (from the normal flora). To establish the microbial etiology of a disease specimen collection is very important. Specimens are selected on the basis of signs and symptoms, should be representative of the disease process, and should be collected before administration of antimicrobial agents. The specimen amount and the rapidity of transport to the laboratory influence the test results. Some of the important aspects in establishing microbial etiology are as follows;

1.     Direct Examination and Techniques: Direct examination of specimens reveals gross pathology. Microscopy may identify microorganisms. Immunofluorescence, immuno-peroxidase staining, and other immunoassays may detect specific microbial antigens. Genetic probes identify genus- or species-specific DNA or RNA sequences.

2.     Culture: Isolation of infectious agents frequently requires specialized media. Nonselective (noninhibitory) media permit the growth of many microorganisms. Selective media contain inhibitory substances that permit the isolation of specific types of microorganisms.

3.     Microbial Identification: Colony and cellular morphology may permit preliminary identification. Growth characteristics under various conditions, utilization of carbohydrates and other substrates, enzymatic activity, immunoassays, and genetic probes are also used.

4.     Serodiagnosis: A high or rising titer of specific IgG antibodies or the presence of specific IgM antibodies may suggest or confirm a diagnosis.

5.     Antimicrobial Susceptibility: Microorganisms, particularly bacteria, are tested in vitro to determine whether they are susceptible to antimicrobial agents.

(Details of diagnostic procedures will be covered in Practical classes)

 

(Annexures provided in the next few pages will give detailed idea about bacterial pathogenicity. These tables are provided for reference purposes only)

ANNEXURE – 1

TERMS USED TO DESCRIBE ADHERENCE FACTORS IN HOST-PARASITE INTERACTIONS
ADHERENCE FACTOR	DESCRIPTION
Adhesin	A surface structure or macromolecule that binds a bacterium to a specific surface
Receptor	A complementary macromolecular binding site on a (eukaryotic) surface that binds specific adhesins or ligands
Lectin	Any protein that binds to a carbohydrate
Ligand	A surface molecule that exhibits specific binding to a receptor molecule on another surface
Mucous	The mucopolysaccharide layer of glucosaminoglycans covering animal cell mucosal surfaces
Fimbriae	Filamentous proteins on the surface of bacterial cells that may behave as adhesins for specific adherence
Common pili	Same as fimbriae
Sex pilus	A specialized pilus that binds mating procaryotes together for the purpose of DNA transfer
Type 1 fimbriae	Fimbriae in Enterobacteriaceae which bind specifically to mannose terminated glycoproteins on eukaryotic cell surfaces
Glycocalyx	A layer of exopolysaccharide fibers on the surface of bacterial cells which may be involved in adherence to a surface
Capsule	A detectable layer of polysaccharide (rarely polypeptide) on the surface of a bacterial cell which may mediate specific or nonspecific attachment
Lipopolysaccharide (LPS)	A distinct cell wall component of the outer membrane of Gram-negative bacteria with the potential structural diversity to mediate specific adherence. Probably functions as an adhesin
Teichoic acids and lipoteichoic acids (LTA)	Cell wall components of Gram-positive bacteria that may be involved in nonspecific or specific adherence

 

ANNEXURE – 2

EXAMPLES OF SPECIFIC ATTACHMENTS OF BACTERIA TO HOST CELL OR TISSUE SURFACES
Bacterium	Adhesin	Receptor	Attachment site	Disease
Streptococcus pyogenes	Protein F	Amino terminus of fibronectin	Pharyngeal epithelium	Sore throat
Streptococcus mutans	Glycosyl transferase	Salivary glycoprotein	Pellicle of tooth	Dental caries
Streptococcus salivarius	Lipoteichoic acid	Unknown	Buccal epithelium of tongue	None
Streptococcus pneumoniae	Cell-bound protein	N-acetylhexosamine-galactose disaccharide	Mucosal epithelium	pneumonia
Staphylococcus aureus	Cell-bound protein	Amino terminus of fibronectin	Mucosal epithelium	Various
Neisseria gonorrhoeae	N-methylphenyl- alanine pili	Glucosamine-galactose carbohydrate	Urethral/cervical epithelium	Gonorrhea
Enterotoxigenic E. coli	Type-1 fimbriae	Species-specific carbohydrate(s)	Intestinal epithelium	Diarrhea
Uropathogenic E. coli	Type 1 fimbriae	Complex carbohydrate	Urethral epithelium	Urethritis
Uropathogenic E. coli	P-pili (pap)	Globobiose linked to ceramide lipid	Upper urinary tract	Pyelonephritis
Bordetella pertussis	Fimbriae ("filamentous hemagglutinin")	Galactose on sulfated glycolipids	Respiratory epithelium	Whooping cough
Vibrio cholerae	N-methylphenylalanine pili	Fucose and mannose carbohydrate	Intestinal epithelium	Cholera
Treponema pallidum	Peptide in outer membrane	Surface protein(fibronectin)	Mucosal epithelium	Syphilis
Mycoplasma	Membrane protein	Sialic acid	Respiratory epithelium	Pneumonia
Chlamydia	Unknown	Sialic acid	Conjunctival or urethral epithelium	Conjunctivitis or urethritis

 

ANNEXURE - 3

SOME EXTRACELLULAR BACTERIAL PROTEINS THAT ARE CONSIDERED INVASINS
Invasin	Bacteria Involved	Activity
Hyaluronidase	Streptococci, staphylococci and clostridia	Degrades hyaluronic of connective tissue
Collagenase	Clostridium species	Dissolves collagen framework of muscles
Neuraminidase	Vibrio cholerae and Shigella dysenteriae	Degrades neuraminic acid of intestinal mucosa
Coagulase	Staphylococcus aureus	Converts fibrinogen to fibrin which causes clotting
Kinases	Staphylococci and streptococci	Converts plasminogen to plasmin which digests fibrin
Leukocidin	Staphylococcus aureus	Disrupts neutrophil membranes and causes discharge of lysosomal granules
Streptolysin	Streptococcus pyogenes	Repels phagocytes and disrupts phagocyte membrane and causes discharge of lysosomal granules
Hemolysins	Streptococci, staphylococci and clostridia	Phospholipases or lecithinases that destroy red blood cells (and other cells) by lysis
Lecithinases	Clostridium perfringens	Destroy lecithin in cell membranes
Phospholipases	Clostridium perfringens	Destroy phospholipids in cell membrane
Anthrax EF	Bacillus anthracis	One component (EF) is an adenylate cyclase which causes increased levels of intracellular cyclic AMP
Pertussis AC	Bordetella pertussis	One toxin component is an adenylate cyclase that acts locally producing an increase in intracellular cyclic AMP

 

ANNEXURE – 4

Table 4. SOURCES AND ACTIVITIES OF BACTERIAL TOXINS
NAME OF TOXIN	BACTERIUM INVOLVED	ACTIVITY
Anthrax toxin (EF)	Bacillus anthracis	Edema Factor (EF) is an adenylate cyclase that causes increased levels in intracellular cyclic AMP in phagocytes and formation of ion-permeable pores in membranes (hemolysis)
Adenylate cyclase toxin	Bordetella pertussis	Acts locally to increase levels of cyclic AMP in phagocytes and formation of ion-permeable pores in membranes (hemolysis)
Cholera enterotoxin	Vibrio cholerae	ADP ribosylation of G proteins stimulates adenlyate cyclase and increases cAMP in cells of the GI tract, causing secretion of water and electrolytes
E. coli LT toxin	Escherichia coli	Similar to cholera toxin
Shiga toxin	Shigella dysenteriae	Enzymatically cleaves rRNA resulting in inhibition of protein synthesis in susceptible cells
Botulinum toxin	Clostridium botulinum	Zn++ dependent protease that inhibits neurotransmission at neuromuscular synapses resulting in flaccid paralysis
Tetanus toxin	Clostridium tetani	Zn++ dependent protease that inhibits neurotransmission at inhibitory synapses resulting in spastic paralysis
Diphtheria toxin	Corynebacterium diphtheriae	ADP ribosylation of elongation factor 2 leads to inhibition of protein synthesis in target cells
Pertussis toxin	Bordetella pertussis	ADP ribosylation of G proteins blocks inhibition of adenylate cyclase in susceptible cells
Staphylococcus enterotoxins*	Staphylococcus aureus	Massive activation of the immune system, including lymphocytes and macrophages, leads to emesis (vomiting)
Toxic shock syndrome toxin (TSST-1)*	Staphylococcus aureus	Acts on the vascular system causing inflammation, fever and shock
Erythrogenic toxin (scarlet fever toxin)*	Streptococcus pyogenes	Causes localized erythematous reactions

 

 

VMC 311 LECTURE # 3 A

KOCH’S POSTULATES

 

Robert Koch, the German physician who has done number of work on different aspects of microbiology has put forth certain conditions, which have to be fulfilled by an etilogical agent. These conditions are called Koch’s postulates. They are as follows.

 

Any organisms to be classified, as an etilogical agent should be

 

1.      isolated from the clinical conditions of the disease.

2.      able to grow in pure culture under laboratory conditions.

3.      able produce the same disease in susceptible animals.

4.      able to isolate from experimentally induced disease.

 

All pathogens do not satisfy all these conditions.

VMC 311 LECTURE # 4

STREPTOCOCCUS

Introduction: The genus Streptococcus is comprised of a wide variety of both pathogenic and commensal gram-positive bacteria, which are found to inhabit a wide range of hosts, including humans, horses, pigs and cows. Within the host, streptococci are often found to colonize the mucosal surfaces of the mouth, nares and pharynx. However, in certain circumstances, they may also inhabit the skin, heart or muscle tissue. This genus contains more than 37 species that include both pathogenic and non-pathogenic species. The pathogenic Streptococci are present in skin and mucous membrane of the genital, upper respiratory and digestive tracts. They are widely distributed in nature. Streptococci are also essential in industrial and dairy processes and as indicators of pollution.

Morphology: Members of the genus Streptococcus are gram-positive, non-motile, non spore forming cocci occurring singly, pairs or in chains. Older cultures may lose their Gram-positive character. Most of them are facultative anaerobes or strict anaerobes. S pyogenes characteristically is a round-to-ovoid coccus 0.6-1.0 µm in diameter. They divide in one plane and thus occur in pairs, or (especially in liquid media or clinical material) in chains of varying lengths. S pneumoniae appears as a 0.5-1.25 µm diplococcus, typically described as lancet-shaped but sometimes difficult to distinguish morphologically from other streptococci

Classification: Different types of classification are followed for Streptococci. Streptococci are classified on the basis of colony morphology, haemolysis, biochemical reactions, and (most definitively) serologic specificity.  

1.      Based on growth characteristics, types of haemolysis and biochemical activities they are classified in to six categories. They are pyogenic streptococci, oral streptococci, enterococci, lactic streptococci, anaerobic streptococci and other streptococci. Most of the pathogenic streptococci are placed in first category.

2.      Based on haemolysis pattern they are grouped into four types. The haemolytic property is the ability of the streptococci to lyse the red blood cells in blood agar medium.

a.      a-Haemolysis: Partial haemolysis surrounded by zone of green coloration around small colonies (haemolysis with an inner zone of partial haemolysis).

b.      b-Haemolysis: Complete haemolysis. Clear zone colourless zone around colonies.

c.      c-Haemolysis: No detectable haemolysis.

d.      a’ Haemolysis: a small zone of partial haemolysis followed by zone of complete haemolysis.

Lancefield classification: In this method streptococci are divided into different groups and each group is further divided into types. This classification of streptococci is based on serologic difference to a carbohydrate substance found on the cell wall of streptococci, which is called as component C. Precipitation test is commonly used in this method to differentiate. Letters A, B, C etc., designates the major groups in this classification and the types under each group are assigned Arabian numbers. The typing under each group is based on serological difference to the M protein. Group A comprises of human pathogens and B, C, G comprise of animal pathogens. Group D streptococci are important etiologic agents of urinary tract infections and infections associated with biliary tract procedures, as well as cases of disseminated infection, bacteremia, and endocarditis. Group F streptococci are associated with abscess formation and purulent disease. Group R streptococci, well-documented causes of meningitis and septicemia in pigs, also pose a serious health hazard to workers in the pork industry. List of important streptococci causing infection in animals are provided below.

Sl.No	Species	Group	Infection produced
1	S.pyogenes	A	Streptococcal disease of human beings, bovine mastitis and lymphangitis in foals
2	S.agalactiae	B	Bovine mastitis. Mastitis in sheep and goat. It is an obligate pathogen. Infection spread by contaminated teat cups and milker’s hands
3	S.dysgalactiae	C	Bovine mastitis and lamb polyarthritis
4	S.equisimilis	C	Associated with Strangles, wound infection, genital infection and mastitis in horses
5	S.equi	C	Strangles and udder infections in mares
6	S.zooepidemicus	C	Genital infections in mares, epididymitis in stallion, naval infection in foals, cervicitis, mastitis and metritis in cattle, arthritis and abortion swines and fatal septicaemia in chickens
7	S.bovis	D	Lactic acidosis and other rumen disorders of cattle
8	S.equinus	D	Alimentary tract infection of horses
9	Enterococcus faecalis S.suis	D, R, S	Meningitis, septicaemia, pyaemia, abscess and encephalitis and abortion in pigs
10	S.uberis	C, D, E, P.U	Bovine mastitis
11	S.porcinus	E, P, U, and V	Mandibular abscess, jowl abscess or cervical lymphadenoma
12	S.avium	Q	Significance not known
13	S.canis	-

Additional groups of streptococci:

1.     Group G: Mastitis in cow and lymphadenitis in cat.

2.     Group M: -

3.     Group N: Syn: Lactococcus lactis or Streptococcus lactis

4.     Viridans streptococci: Commensals

5.     Anaerobic streptococci: Commensals

6.       Nutritionally variant streptococci: Different nutrient requirment

 

Cultural characteristics: The organisms grow well in ordinary laboratory medium enriched with blood or serum. Blood agar and Edwards’s medium are the most preferred media however; growth is luxuriant in nutrient agar also. Streptococci often have a mucoid or smooth colonial morphology. They produce small colonies of 1mm diameter, round, smooth, glistening and look like dewdrops. Haemolytic pattern in blood agar as indicated above may be noticed depending upon the species of streptococci and type of blood used. The colonies produced are mucoid, matt or glossy. Virulent streptococci produce matt colonies and less virulent types produce glossy colonies.

 

Biochemical characters: They are catalase and oxidase negative and fermentative. Ferments sugars like trehalose, sorbitol, mannose, salicin, lactose, raffinose, inulin, esculin etc with variation. Hydrolyse esculin and sodium hippurate. Reduces methylene blue milk and prefers 6.5% sodium chloride for growth.

 

Pathogenesis: The infection may be either endogenous or exogenous. The exogenous infection is through inhalation or ingestion. Aerosols, direct contact and fomites are the important methods of spread. Carrier animals are important source of spread of infection. The streptococcal infection is characterised by production of pus (pyogenic infection). When the pyogenic bacteria invade the system they stimulate inflammatory response that is characterised by vascular dilation and exudation of plasma and neutrophils. Neutrophils engulf the bacteria (phagocytosis). Most of the bacteria are killed in the process of phagocytosis. However, some bacteria can grow in neutrophils resulting in production of toxins, which kill the phagocytic cells. The enzymes liberated by the dead phagocytic cells liquefy dead tissues and phagocytic cells. The liquefied mass is yellow and thick in consistency and called as pus. Variation in consistency of pus is due to deoxyribonucleo protein. Streptococcal is infection is generally localised and rarely it becomes septicaemic or bacterimic. The major virulence factors of streptococci are surface M proteins and hyaluronic acid. The extra cellular products produced by streptococci are important in spread and localisation of the organisms inside the tissue. Some of the extra cellular products are also responsible for damage to the tissue.

 

Extracellular products: The important extra cellular products of streptococci and their role are listed below.

1.      Haemolysin: Two types Streptolysin O and Streptolysin S. Toxic for neutrophils and macrophages.

2.      Streptokinase: Also known as Fibrinolysin. It activates plsminogens to plasmin, which prevents the formation fibrin clots.

3.      DNases A, B, C and D: Also known as Streptodornase. Assist in the production of substances required for growth.

4.      Hyaluronidase A: Major virulence factor. Promotes the spread of organisms in tissues.

5.      Erythrogenic toxins A, B and C: Responsible for rashes in scarlet fever.

6.      NADases: Kills the phagocytic cells.

7.      Proteinase

8.      Lipoproteinase

9.      Amylase

10.  Esterase

 

Diagnosis:

1.     Materials for diagnosis: Pus, joint fluid, milk, organs, blood swab and meningeal swab.

2.     Examination of culture smear by Grams staining method and milk smear by Newman’s staining method.

3.     Cultivation: The media commonly used are nutrient agar, blood agar or Edward’s medium. Most pyogenic bacteria produce beta haemolysis. Edward’s medium contains esculin, crystal violet and thallium acetate.

4.     Biochemical reactions:

a.      Haemolytic patterns

b.      Differentiation in fermentation of sugars trehalose, sorbitol, mannose, salicin, lactose, raffinose, inulin, esculin etc. hydrolysis of esculin and sodium hippurate, reduction of methylene blue milk and preference of 6.5% sodium chloride for growth.

5.     CAMP test (Christie, Atkinson, Muench and Peterson): It is a presumptive test for the diagnosis of S.dysgalactiae. This test is based on the ability of streptococcal organisms to complete partial haemolysis produced by Staphylococcus aureus.

6.     Hydrolysis of esculin: Esculin agar is a selective and differential media that is used primarily to distinguish fecal streptococci (Enterococcus species) from other Streptococcus organisms. Enterococcus is the only organism that can hydrolyze esculin. The hydrolyzed esculin complexes with iron to form a dark, brown-black color in the tube.

7.     The cystine tryptic agar (CTA) sugar fermentation test is used to identify fastidious organisms (especially Streptococcus species), by detecting fermentation reactions. The fermentation of carbohydrates in the media produces acid, which turns the pH indicator to a yellow color. Colonies obtained from a blood agar culture are stabbed into the media in a CTA tube. After incubation, a positive fermentation test will be yellow. A tube of CTA base media is inocculated along with the CTA sugar tube; this base media tube is used to confirm the color change of the CTA sugar tube. If the bacteria grow in the base media tube, that media will remain red.

 

Treatment: Penicillin is the drug of choice. However sulphamethixine, erythromycin, lincomycin and tetracycline are also effective.

 

IMPORTANT STREPTOCOCCAL INFECTIONS:

STRANGLES: Strangles is a highly contagious and serious infection of horses and other equids caused by the bacterium, Streptococcus equi. The disease is characterized by severe inflammation of the mucosa of the head and throat, with extensive swelling and often rupture of the lymph nodes, which produces large amounts of thick, creamy pus. Strangles is caused by Streptococcus equi subspecies equi, better known as Streptococcus equi (S. equi). The organism can be isolated from the nose or lymph nodes of affected animals, and is usually readily identified in the laboratory by simple sugar tests. Horses of all ages are susceptible, though strangles is most common in animals less than 5 years of age and especially in groups of weanling foals or yearlings.

 

Symptoms & lesions: Susceptible horses develop strangles within 3–14 days of exposure. Animals show typical signs of a generalized infectious process (depression, inappetence, and fever of 39°C–39.5°C). More typically of strangles, horses develop a nasal discharge (initially mucoid, rapidly thickening and purulent), a soft cough and slight but painful swelling between the mandibles, with swelling of the submandibular lymph node. Horses are often seen positioning their heads low and extended, so as to relieve the throat and lymph node pain. With the progression of the disease, abscesses develop in the submandibular (between the jaw bones) and/or retropharyngeal (at the back of the throat) lymph nodes. The lymph nodes become hard and very painful, and may obstruct breathing ("strangles"). The lymph node abscesses will burst (or can be lanced) in 7–14 days, releasing thick pus heavily contaminated with S. equi. The horse will usually rapidly recover once abscesses have ruptured. Although the disease process described above is classic, some horses (especially older animals) will develop a mild, short lasting disease without or with minor lymph node abscessation. This is thought to be the result of partial immunity although this may also result from infection by S. equi of relatively low virulence. Classic strangles is a severe infection that can be fatal, usually because of a variety of complications that occur. The main and most important fatal complications of strangles are Bastard strangles and purpura haemorrhagica. Bastard strangles, which describes the dissemination of infection to unusual sites other than the lymph nodes draining the throat. For example, abdominal or lung lymph nodes may develop abscesses and rupture, sometimes weeks or longer after the infection seems to have resolved. A brain abscess may rupture causing sudden death or a retropharyngeal lymph node abscess may burst in the throat and the pus will be inhaled into the lung. Purpura haemorrhagica, which is an immune-mediated acute inflammation of peripheral blood vessels that occurs within 4 weeks of strangles, while the animal is convalescing. It results from the formation of immune complexes between the horse's antibodies and bacterial components. These immune complexes become trapped in capillaries where they cause inflammation, visible in the mucous membranes as pinpoint haemorrhages. These haemorrhages lead to a widespread severe edema of the head, limbs, and other parts of the body. Purpura can also be a complication of routine vaccination.

Diagnosis: Diagnosis can be confirmed by culturing pus from the nose, from abscessated lymph nodes or from the throat of clinically affected horses. Although S. equi isolates are thought to be genetically identical, isolates may vary in virulence and atypical isolates occur, which differ in their sugar tests from typical S. equi.

Treatment: The organism is amenable to Penicillin G.

Prevention and control: A killed and live vaccines are used in Western countries to control the infection. However, isolating the affected animal, cleaning the shed and proper disinfection will help getting rid of the infection.

 

MASTITIS: Mastitis is defined as inflammation of udders and it is the most important disease threatening dairy industry. Clinical mastitis is of greatest concern to producers since the cow is ill and the milk cannot be included in milk for sale. The clinical signs include abnormal milk, sore gland and often sick cow. Mastitis is caused by variety of organisms and S.agalactiae is one of the important organisms causing mastitis. This highly contagious pathogen is found in the mammary gland and spreads from cow to cow. This organism is generally subclinical and causes severe milk loss and a costly disease in a herd. However, S.aglactiae is easily treated with antibiotic therapy in the mammary gland during lactation or during the dry period. There are three methods to control or eliminate S.aglactiae, which are as follows;

1. Use a predipping and post-dipping iodine for teat sanitation.
2. Treat all quarter of all cows at dry off.
3. Treat all quarter of cows identified with Strep ag.

 

California Mastitis Test (CMT). The CMT is a good cow-side test to determine if a quarter has a high milk somatic cell count (SCC). A high SCC in a good indicator of an intramammary infection.

Procedure:

1.      Clean teat and remove a single stripping of milk and discard.

2.      Collect milk (2ml) in appropriate well: A-RF, B-RR, C-LF, D-LR

3.      Add equal amount of CMT solution

4.      Rotate mixture gently for 20 seconds

5.      Determine solution agglutination (gel formation in well)

6.      Record each quarter reaction: quarter with mild to strong should be considered infected

 

VMC 311 LECTURE # 5

STAPHYLOCOCCUS

 

Classification: Family – Staphylococcaceae; Genus – Staphlococcus; Type species – Staphylococcus aureus

 

Introduction: Staphylococcus (der. Greek, "Staphylo" = bunch of grapes"), Size  1 µ dia., Gram positive, non-motile, non-spore forming, facultatively anaerobic (ferments sugars but does not produce gas). Members are pathogenic species associated with suppurative (pus forming) disease (abscesses - hallmark clinical manifestation is a <boil (eg,. pimples)). They can also cause pneumonia, osteomyelitis, meningitis, arthritis in invasion into deeper tissues and organs. They also produce toxic shock syndrome and food poisoning in humans. Organisms of this genus are among the most hardy, most resistant to environmental stress of all of the non-spore forming organisms - resist dehydration, relatively heat resistant and tolerate many common disinfectants. Staphylococci are often called "opportunists" because they wait for suitable conditions before invading the body. Humans carry the bacteria on their hands and arms, in their nose, and in the hairy parts of their body. There are nearly 27 species in this genus and the important animal pathogens are S.aureus, S.intermedius, S.hyicus and S.epidermis. S.aureus is considered as type species for this genus. Generally these organisms are found on the surface of skin as commensal and also on the mucous membrane. Though they are found on the surface of skin, they have habitat preference to head, ears and anterior nares. They are generally non-pathogenic, occasionally causing mastitis, abscesses and wound infections. Some species of staphylococci like S.saprophyticus cause urinary tract infection and wound infection in humans.

 

Morphology: Staphylococci are gram-positive cocci occurring in pairs, short chains or in clusters. The clusters resemble ‘bunches of grapes’. They are aerobic or facultatively anaerobic. They are non motile, non sporulative and fermentative (no gas production). Some strains possess a polysaccharide capsule. They are catalase positive and oxidase negative. These organisms are relatively resistant. They can withstand a temperature of 60^oC for 30 minutes. They are resistant to disinfectants including phenolic compounds and can withstand a high salt concentration.

 

1.      S.aureus: principal pathogenic species and is coagulase-positive. It is so named because of the yellow or gold color of its colonies - due to the production of beta carotene pigment. This pigment is also thought to function as a virulence factor due to its ability to act as an electron-sink (conjugated diene structure) and thus scavenge reactive oxygen species (ROS) produced by inflammatory cells. Pigmented Staph are consistently more resistant to phagocytic oxygen-dependent killing mechanisms However, some strains (particularly pathogenic strains of veterinary significance) lose the ability to produce beta carotene as evidenced by their lack of yellow/gold color.

2.      S.epidermidis - gets its name because of its prevalence on human skin - it is also found on skin, teats, hair and superficial mucosa of many animals but in less numbers than seen in humans - colonies are non-pigmented and non-hemolytic and coagulase negative- it is an opportunistic invader of low virulence.

3.      S.intermedius - most prevalent disease-producing species in dogs and other carnivores. Colonies are non-pigmented, coagulase-positive, vary in hemolytic pattern.

4.      S.hyicus – It was thought to be biotype (biotype 2) of S. epidermidis. It is the etiologic agent of Exudative Epidermitis of Swine (Greasy Pig Disease)

Cultural characteristics: These organisms grow well in ordinary laboratory media. However, the most preferred medium for cultivation is the mannitol salt agar. Blood agar is the preferred medium for primary isolation. Colonies appear as round, smooth, glistening and may or may not have golden yellow pigments. Colonies are roughly 4mm in diameter. They produce double zone haemolysis and b haemolysis. Haemolysis is best demonstrated in bovine red blood cells.  

 

Biochemical tests: The common biochemical tests used to differentiate species of staphylococci are Oxidase test, Coagulase test, beta haemolysis, Pigment production, utilisation of maltose and mannitol, sensitivity to antibiotic novobiocin.

 

Antigenic nature: The antigenic structure is complex and heterogeneous. The cell wall is composed of Teichoic acid, Peptidoglycan and Protein A. Teichoic acid is species specific and Protein A is antiphagocytic. The polysaccharide capsule is also antiphagocytic.

 

Pathogenesis: The infection may be endogenous or exogenous. Endogenous infections are more common. The main mode of spread is by direct contact and by fomites. Some species have the ability to invade the tissues, produce abscesses, pustules, various pyogenic infection, bacteraemia and septicaemia. The inflammatory response following infection results in the infiltration of neutrophils leading to pyogenic response and production of pus. Accumulation of pus results in formation of abscesses. Staphylococci produce number of extra cellular products that are responsible for the establishment of pathogenesis. Not all the extra cellular products produced are toxic. Some of them are enzymes and virulence enhancers. The enzymes are responsible for protecting the organisms from body defence mechanisms. Some of the important extra cellular products and their roles are listed below.

Sl.No	Virulence factors	Important role in pathogenesis
1	Coagulase:	Responsible for clotting of plasma and conversion of prothrombin to thrombin and fibrinogen to fibrin. Most of the staphylococci produce coagulase.
2	Enterotoxin A, B, C1, C2, C3, D and E:	These toxins are highly heat resistant (100oC for 30 minutes). These toxins are produced by S.aureus in custards, raw milk, cream, ice cream, meat gravy, fish, cheese etc. Clinical symptoms in humans include nausea, vomiting and diarrhoea.
3	Haemolysin a, b, n and d (Haemolysins and cytolysins:	The a and b haemolysins are the potent toxins. The a toxin is responsible for the inner clear zone of haemolysis and the b toxin is responsible for outer partial zone of haemolysis. Chemically the b toxin is sphingomylinase C. The role of other two toxins in pathogenesis is not clear.
4	Lipase:	Degrades protective fatty acids on skin and help in abscess formation.
5	Staphylokinase:	Degrades fibrin clots.
6	Leukocidin:	Kills granulocytes and macrophages. It is made up of two heat labile interacting proteins.
7	Exfoliative toxin A and B (Exfoliatin):	Causes cleavage of desmosomes in the stratum granulosum of the epidermis.
8	Toxic shock syndrome toxin
9	Hyaluronidase:	Spreading factor.
10	Penicillinase:
11	Lysostaphin:
12	Protein A:	It is found on the surface of staphylococci and has got a special property of binding with Fc region of antibodies. Used in the co-agglutination test.
13	Slime:	Surface mucoid substance responsible for adhesiveness.
14	Collagenase:
15	Acid and alkaline phosphatase:
16	Pyrogenic exotoxin:
17	Elastase:
18	Catalase:
19	Protease:
20	Nuclease:

Staphylococci of all species are normal inhabitants of the skin and/or mucosal surfaces of most warm-blooded animal and humans. The quantity of an individual Staphylococci species (eg, aureus vs epidermidis) depends on the host - some strains and species are particularly host-adapted.

 

Mechanism of pathogenesis:

1.      Microbial adhesion: not completely understood but initial host cell recognition and attachment may involve binding to glycolipids as well as fibronectin and fibrinogen. S. aureus and especially coagulase-negative strains (CoNS) of S. epidermidis (and presumably intermedius) can also form biofilms. Biofilms are an assemblage of microscopic animals, plants and bacteria attached to a surface. They often produce a slime layer in which they live and which helps them to stick to a surface and provides a protective environment. Following the initial attachment phase, the CoNS adopt a highly characteristic mode of growth in which they begin to produce a mucoid substance or 'slime' which promotes biofilm development. The slime mainly consists of polysaccharides, with about 10-20% proteins that presumably stabilises the biofilms by promoting bacterial cell-to-cell and cell-to-surface associations so that multi-layered cell clusters accumulate on the implant surface.

2.      Penetration, invasion and disease (lesion development and tissue damage) are dependent on a number of factors such as skin trauma, lacerations, macerations, underlying disease, competing flora, immune status, etc. Thus, most often these organisms are opportunistic pathogens - not particularly invasive unless given the opportunity.

3.      Multiplication and spread: Most important factors in Staphylococcal multiplication and spread following invasion are:

a.      resistance to phagocytosis (protein A and polymeric uronic acid capsules

b.      intracellular survival in phagocytes

c.      coagulase & hylauronidase production - toxin production

d.      adhesion to epithelial cells; eg. vet strains of S. aureus are particularly adept at adhesion to intramammary gland epithelia

4.      Damage: (Suppurative (pus producing), abscesses )- local infections lead to the formation of a collection of pus (neutrophils) called an abscess - abscesses in the skin are called boils, pimples, furuncles, etc.

a.      Stages of abscess formation (bacterial and host factors)

                                                              i.      acute inflammation - rapid and extensive infiltration neutrophils

                                                             ii.      chemotactic factors (bacteria products and host-derived complement)

                                                           iii.      outpouring of large amounts of lysosomal enzymes from damaged neutrophils (leucocidin of Staph) causes surrounding tissue damage

                                                          iv.      inflammatory area begins to be "walled off" by a thick-walled fibrin capsule - possible participation by Staph coagulase

b.      Abscess is thus a well-defined area containing pus - from the host's point of view it has contained the invading organism - however, if the abscess happens to be in or near a vital organ or tissue it can often have serious or even life-threatening consequences.

 

Important Staphylococcal Infections

1.     S. aureus

a.      Botyromycosis - The infections caused by staphylococci are referred as botryomycosis. It is a chronic granulomatous lesion involving udder of mare, cow, sow and spermatic cord of stallion invasion into stump of spermatic cord of horses following castration - infected cord becomes greatly enlarged and small pockets of pus are found containing granules of encapsulated organisms.

b.      Mastitis – Characterised by suppurative lesions in cattle mammary gland ductular epithelia. The organisms enter from human hands and opportunistic invasion due to trauma or tissue damage from milking machines. The relative resistance of the organism insures continued environmental contamination and tendency for infection and re-infection. The organisms also have a great adhesive tenacity for epithelium at the tip of the teat. Mastitis varies in severity from subclinical to severe gangrene. Subclinical, chronic form is the most damaging economically because of reduced milk production or milk losses. Staph mastitis cases also occur in sheep, goats, mares, sows, cats and mink.

c.      Purulent synovitis in chickens and turkeys (bumble foot) – Characterised by lameness, swellings in the feet. The organisms enter opportunistically in birds with underlying disease or because of stress-inducing husbandry.

d.      Porcine Necrotizing Staphylococcal Endometritis – Characterised by abortion in swine

e.     Tick Pyemia – Characterised by septicemia in lambs heavily infested with ticks. The ticks inject Staph deep into tissue. If septicemia occurs it is rapidly fatal.

f.       Exfoliative Skin Disease – It comprises a variety of syndromes, generalized exfoliative dermatitis in newborns called as Ritter's disease, toxic epidermal necrolysis in children and occasionally in adults, bullous impetigo and Staphylococcal scarlatina. This group of diseases is collectively called Staphylococcal Scalded Skin Syndrome - as in pigs the disease spreads over the entire skin surface primarily because exfoliatin toxin.

g.     Staphylococcal Food Poisoning: It is due to the powerful enterotoxin. The symptoms are seen within 1-6 hours after ingesting contaminated food and results in severe cramps, nausea, vomiting, and diarrhea.

h.      Toxic Shock Syndrome: It is characterized by fever, skin rash, hypotension, and dysfunction of several systems.

2.     S. intermedius (S. aureus biotype E & F)

a.      Canine Pyoderma - organism enters opportunistically form endogenous normal colonization of nasopharynx and skin. Factors such as dry skin, trauma, ectoparasitism (demodectic mange), matted dirty hair, predispose the animal to invasions of superficial and deeper skin layers. Staphylococcal cellulitis is often a complication of mange or other ectoparasitic disease

3.      S. hyicus (Staphylococcus epidermidis biotype 2)

a.      It is the etiological agent of Exudative Epidermitis of Swine - (Greasy Pig Disease). It is a highly contagious disease and easily spread from pig to pig. Piglets between agegrooup 1-7 weeks are most often affected. Organism often enters through skin lacerations caused by bites from other pigs competing for food. Whole skin can be covered with a moist, greasy exudate similar to "Staphylococcal Scalded Skin Syndrome in humans. The disease starts with lesions as vesicles on hairless skin surfaces (behind the ears), as the disease progresses the skin becomes thickened and layers of epidermis peel off (in milder forms it resembles dandruff-like scaling). The affected pigs usually recover from 15-40 days after symptoms, although it can be fatal.

4.      A type of enterocolitis referred as Staphylococcal enterocolitis is seen in humans after prolonged antibiotic therapy.

 

Diagnosis:

1.     Materials for diagnosis: Pus, affected tissue and milk samples.

2.     Examination of culture and pus smear by Grams staining method will reveal gram-positive organisms with typical arrangement.

3.     Isolation and identification by culture method: These organisms grow well in ordinary laboratory media. However, the most preferred media for cultivation is the mannitol salt agar. Blood agar is the preferred medium for primary isolation. Colonies appear as round, smooth, glistening and may or may not have golden yellow pigments. Colonies are roughly mm in diameter. They produce double zone haemolysis and b haemolysis. Haemolysis is best demonstrated in bovine red blood cells.

4.     Coagulase test: To a loopful of bacterial culture in saline a drop of rabbit plasma is added over a slide. Appearance of clumps within ten minutes is considered as positive. Most of the Staphylococci produce coagulase enzyme. The appearance of the organisms in smear, cultural characteristics and result of this are generally used to confirm staphylococci.

5.     Latex agglutination test:

6.     Rapid miniaturised commercial kits:

7.     Phage typing:

8.     Biochemical tests: The common biochemical tests used to differentiate species of staphylococci are Oxidase test, Coagulase test, beta haemolysis, Pigment production, utilisation of maltose and mannitol, sensitivity to antibiotic novobiocin.

 

Treatment: Penicillin is the drug of choice. However, penicillin resistant strains are also reported. Synthetic penicillins like methicillin, oxacillin and nafcillin, tetracyclines, bacitracin, nitrofurans and erythrocmycin are also used

 

EXTRA READING:

Superantigens: enterotoxins and toxic shock syndrome toxin

S. aureus secretes two types of toxin with superantigen activity, enterotoxins, of which there are six antigenic types (named SE-A, B, C, D, E and G), and toxic shock syndrome toxin (TSST-1). Enterotoxins cause diarrhoea and vomiting when ingested and are responsible for staphylococcal food poisoning. TSST-1 is expressed systemically and is the cause of toxic shock syndrome (TSS). When expressed systemically, enterotoxins can also cause toxic shock syndrome. In fact, enterotoxins B and C cause 50% of non-menstrual cases of TSS. TSST-1 is weakly related to enterotoxins, but it does not have emetic activity. TSST-1 is responsible for 75% of TSS, including all menstrual cases. TSS can occur as a sequel to any staphylococcal infection if an enterotoxin or TSST-1 is released systemically and the host lacks appropriate neutralizing antibodies.

 

Superantigens stimulate T cells non-specifically without normal antigenic recognition. Up to one in five T cells may be activated, whereas only 1 in 10,000 are stimulated during a usual antigen presentation. Cytokines are released in large amounts, causing the symptoms of TSS. Superantigens bind directly to class II major histocompatibility complexes of antigen-presenting cells outside the conventional antigen-binding grove. This complex recognizes only the Vb element of the T cell receptor. Thus any T cell with the appropriate Vb element can be stimulated, whereas normally, antigen specificity is also required in binding.

 

Food poisoning: Symptoms of staphylococcal food poisoning are caused by staphylococcal enterotoxin, not by staphylococcus itself. It is a common cause of food poisoning, and the potential for outbreaks is high when food handlers with skin infections contaminate foods left at room temperature. Custards, cream-filled pastry, milk, processed meat, and fish provide media where coagulase-positive staphylococci grow and produce enterotoxin. Onset is usually abrupt. Symptoms characteristically are severe nausea and vomiting, which begin 2 to 8 h after eating food containing the toxin. Other symptoms may include abdominal cramps, diarrhea, and occasionally, headache and fever. Because the toxin does not cause mucosal ulceration, the diarrhea is usually non-bloody. Acid-base imbalance, prostration, and shock may ensue in severe cases. The attack is brief, often lasting < 12 h, and recovery is usually complete. Rare deaths occur as a result of fluid and metabolic stresses, especially among very young or old or chronically ill patients. Diagnosis relies on recognizing the clinical syndrome. Usually, several persons are similarly affected, constituting a point source outbreak. Diagnostic confirmation, although rarely required, entails isolating coagulase-positive staphylococci from the suspected food. Gram stain of specimens of vomitus may show staphylococci. Careful food preparation is essential for prevention. Persons with furunculosis or impetigo should not prepare food until their lesions have healed. Treatment is described under General Principles of Treatment, above. Rapid IV replacement of electrolytes and fluids often brings dramatic relief

 

VMC 311 LECTURE # 6

BACILLUS

 

Introduction: The anthrax bacillus, Bacillus anthracis, was the first bacterium shown to be the cause of a disease. In 1877, Robert Koch grew the organism in pure culture, demonstrated its ability to form endospores, and produced experimental anthrax by injecting it into animals. This genus contains number of species, which are ubiquitous in nature and are found in air, soil, dust, water etc. They are also common contaminants of the laboratory cultures. This genus consists of four important species B.anthracis, B.cereus, B.thuringiensis and B.subtilis. The only species of pathogenic importance is B.anthracis. Yet another species B.cereus rarely produce disease in animals. It is a normal inhabitant of the soil, but it can also be regularly isolated from foods such as grains and spices. B. thuringiensis is identified by its pathogenicity for lepidopteran insects (moths and caterpillars) and by production of an intracellular parasporal crystal in association with spore formation. The bacteria and protein crystals are sold as "Bt" insecticide, which is used for the biological control of certain garden and crop pests.

 

Morphology: The species under the genus Bacillus are gram-positive; rod shaped organisms measuring 1 - 1.2µm in width x 3 - 5µm in length; arranged singly or in short chains. They are aerobic or facultatively anaerobic. They possess capsule and are sporulative. Most of the species under genus Bacillus are motile, where as the B.anthracis that causes anthrax is non motile. They are catalase positive and fermentative.

B.anthracis has certain special morphological features, which distinguishes it from other anthracoids (anthracoids are normal PM invaders). Morphologically B.anthracis is rod shaped organism with truncated ends and in blood smears they appear either as single organism or chain of one or two organisms. They sporulate with very great frequency in soils with pH of 6 and above and the spores are centrally located. The spores of the organisms are considerably resistant than the vegetative cells to the physical and chemical agents. The spores can remain viable in soil and dried cultures for a long time (many years). Boiling for 10 minutes and by dry heat at 140^oC for 3 hours destroy the spores. However, freezing temperature has no effect on the spores. Only prolong action of chemical disinfectants at high concentration has killing effect on the spores. 5% phenol for 2 days, 10-20% formalin in 10 minutes and autoclaving destroys the spores completely

 

Cultural characteristics: The organisms grow well in all laboratory media. However, serum and blood in the media enhances the growth. In blood agar, the colonies appear with in 24 hours and are flat, rough, grey and non-haemolytic. The colonies are referred as medusa heads or judges wig. On microscopical examination, the edges of the colonies appear as a tangled mass of curly hairs. In nutrient agar the colonies could be smooth and mucoid or rough.

 

The laboratory animals of choice for cultivation of the organisms are guinea pigs and mice. The affected tissues after grinding them in a mortar and pestle or blood as such may be used as an inoculum. The animals will die from 24 hours and anthrax organisms as large capsulated rods can be seen in blood smear and spleen.

 

Antigenic characters: Bacillus anthracis forms a single antigenic type of capsule consisting of a poly-D-glutamate polypeptide. All virulent strains of B. anthracis form this capsule. Production of capsular material is associated with the formation of a characteristic mucoid or "smooth" colony type. "Smooth" (S) to "rough" (R) colonial variants occur, which is correlated with ability to produce the capsule. R variants are relatively avirulent. Capsule production depends on a 60 megadalton plasmid termed as pX02.

 

Pathogenesis: The anthrax bacilli are distributed worldwide. Animals may become infected from contaminated soil, water, bone meal, oil cake, offal etc. Carrion birds and wild animals also have a role in the spread of the infection. The important modes of infection are by ingestion, inhalation and through wounds and scratches. In human beings blood feeding insects are also found to have a role in spread of the infection.

The virulence of Bacillus anthracis is attributable to three bacterial components namely;

1.      Capsular material composed of poly-D-glutamate polypeptide

2.      EF component of exotoxin

3.      LF component of exotoxin

The poly-D-glutamyl capsule is non-toxic, but functions to protect the organism against the bactericidal components of serum and phagocytes, and against phagocytic engulfment. The capsule plays its most important role during the establishment of the infection, and a less significant role in the terminal phases of the disease, which are mediated by the anthrax toxin. The anthrax exotoxin is referred as anthrax toxin, which is protein and comprised of three components I (Oedema factor), II (Protective antigen) and III (lethal factor). Production of the anthrax toxin is mediated by a temperature-sensitive plasmid termed as pX01.

 

Factor I is the edema factor (EF) which is necessary for the edema producing activity of the toxin. EF is known to be an inherent adenylate cyclase, similar to the Bordetella pertussis adenylate cyclase toxin.

Factor II is the protective antigen (PA), because it induces protective antitoxic antibodies in guinea pigs. PA is the binding (B) domain of the anthrax toxin, which has two active (A) domains, EF (above) and LF (below).

Factor III is known as the lethal factor (LF) because it is essential for the lethal effects of the anthrax toxin. Apart from their antigenicity, each of the three factors exhibits no significant biological activity in an animal. However, combinations of two or three of the toxin components yield the following results in experimental animals.

PA+LF combine to produce lethal activity

EF+PA produce edema

EF+LF is inactive

PA+LF+EF produces edema and necrosis and is lethal

Both the capsule and the anthrax toxin play a role in the early stages of infection, through their direct effects on phagocytes. Virulent anthrax bacilli multiply at the site of the lesion. Phagocytes migrate to the area but the encapsulated organisms can resist phagocytic engulfment, or if engulfed, can resist killing and digestion. A short range effect of the toxin is its further impairment of phagocytic activity and its lethal effect on leukocytes, including phagocytes, at the site. After the organisms and their toxin enter the circulation lethality will result. Bacillus anthracis coordinates the expression of its virulence factors in response to a specific environmental signal. Anthrax toxin proteins and the antiphagocytic capsule are produced in response to growth in increased atmospheric CO₂. This CO₂ signal is thought to be of physiological significance for a pathogen, which invades mammalian host tissues.

The spores enter through the skin or mucous membrane and germinate at the site of entry and later spread through out the system. In septicaemic forms the spread is via lymphatics and blood stream. Respiratory failure and anoxia produced by the toxin cause death.

 

In pigs, the infection occur as local form affecting lymph nodes of head and neck. Large number of bacilli is excreted via the natural orifices during the terminal stage. The important virulence factors are the toxin and capsule.

 

Pathogenicity: The infection occur as per-acute, acute, sub-acute, chronic or cutaneous forms. Cattle, horses, sheep, swine and human being are affected by this infection. It is also considered as a zoonotic infection. In per-acute infection, animals suddenly die at the grazing field without exhibiting any symptoms. Mortality and morbidity rates are very high. Generally the symptoms exhibited are very few before death. The important observation during terminal stage is oozing of blood from natural orifice. Since the clotting mechanism is affected the blood will not clot. The important lesion in anthrax infection is the enlargement of spleen (Spleenomegaly). The body cavity will be filled with unclotted blood.

 

In pigs, the infection is usually sub-acute with pharyngitis, swelling and haemorrhage of throat and mouth. An intestinal form with gastroenteritis is also seen. In chronic infection, localisation in the tonsils and lymph nodes of head and cervical region by the organisms are frequent.

 

In human beings, the infection occurs as pulmonary form or as malignant carbuncle or pustule (wool sorter’s disease). Intestinal anthrax is also reported. In humans, anthrax is fairly rare; the risk of infection is about 1/100,000. The most common form of the disease in humans is cutaneous anthrax, which is usually acquired via injured skin or mucous membranes. A minor scratch or abrasion, usually on an exposed area of the face or neck or arms, is inoculated by spores from the soil or a contaminated animal or carcass. The spores germinate, vegetative cells multiply, and a characteristic gelatinous edema develops at the site. This develops into papule within 12-36 hours after infection. The papule changes rapidly to a vesicle, then a pustule (malignant pustule), and finally into a necrotic ulcer from which infection may disseminate, giving rise to septicemia. Lymphatic swelling also occurs within seven days. In severe cases, where the blood stream is eventually invaded, the disease is frequently fatal.

 

Another form of the disease, inhalation anthrax (woolsorters' disease), results most commonly from inhalation of spore-containing dust where animal hair or hides are being handled. The disease begins abruptly with high fever and chest pain. It progresses rapidly to a systemic hemorrhagic pathology and is often fatal if treatment cannot stop the invasive aspect of the infection

 

Diagnosis:

Materials to be sent: As a rule, necropsy should not be conducted on animals suspected to be died of anthrax, since the organisms will sporulate on exposure to atmosphere. Blood smears and blood are ideal material of choice. Isolation under laboratory condition can be attempted with spleen.

 

1.     Direct examination: Smears from tissues or blood stained by Lesihman’s method will reveal anthrax organisms as individual rods with truncated edges. Other staining methods like Giemsa, Grams can also be used. While diagnosing by this method the anthracoids have to be differentiated. The following table lists some important differences between anthrax bacilli and anthracoids.

 

Sl.No	Characters	B.anthracis	Anthracoids
1.	Motility	Non motile	Motile
2.	Edges of bacilli	Truncated	Round
3.	Arrangement	Single	Chain
4.	Salicin utilisation	Slow	Rapid
5.	Methylene blue reduction	Slow	Rapid
6.	Gelatin liquefaction	Slow	Rapid
7.	Haemolysis	Slight	Haemolytic
8.	Susceptibility to Penicillin	Susceptible (some)	Resistant
9.	Capsule	Present(only in virulent organisms)	Absent

2.     McFadyean’s reaction: On staining by methylene blue, the anthrax organism appears as blue colour rods in pink stained amorphous polyglutamic acid capsule. This is used as one of the confirmative tests for anthrax.

3.     Isolation and identification: The organisms grow well in all laboratory media. However, serum and blood in the media enhances the growth. In blood agar, the colonies appear with in 24 hours and are flat, rough, grey and non-haemolytic. The colonies are referred as medusa heads or judges wig. On microscopical examination, the edges of the colonies appear as a tangled mass of curly hairs. In nutrient agar the colonies could be smooth and mucoid or rough.

4.     Isolation in lab animals: The laboratory animals of choice for cultivation of the organisms are guinea pigs and mice. The affected tissues after grinding them in a mortar and pestle or blood as such may be used as an inoculum. The animals will die from 24 hours and anthrax organisms  as large capsulated rods can be seen in blood smear and spleen.

5.     Ascoli’s test: It is an immunological test based on the principles of precipitation reaction. This test is used to detect anthrax in decomposed carcasses. In a narrow tube, extract from the decomposed carcass and antibodies against anthrax bacilli are added. Development of a precipitation ring is an indication of anthrax  infection.

6.     String of pearls test: This test is based on the principle of impairment of cell wall development by penicillin. When anthrax bacilli is grown in a medium containing penicillin, due to cell wall impairment it will produce colonies which will resemble string of pearls.

7.     Phage typing: Gamma phages that specifically lyses B.anthracis are used to identify anthrax bacilli.

8.     Fluorescent antibody test: This test is used to identify anthrax organisms in tissue smears. Either direct or indirect test may be employed.

 

Treatment: Penicillin, tetracycline, gentamycin, streptomycin, enrofloxacin, erythromycin and chlormaphenicol are used to treat the sick animals suspected to have anthrax.

 

Control:

 

1.      Different vaccines like attenuated spore vaccine, avirulent spore vaccine (Sterne’s vaccine) and non-living vaccine containing protective antigen are used to provide protection to risk group. The Sterne’s vaccine is used in Tamil Nadu to control anthrax infection in susceptible population

2.      To prevent the spread of infection, sporulation should be controlled. Hence, necropsy of the animals susceptible to have anthrax should not be performed. The carcass should be buried in a six feet deep pit and covered with lime.

3.      Animal by-products should be screened for the presence of anthrax bacilli and if they are found to contain spores they should be disposed as mentioned in the previous point.

4.      Animals should not be allowed to graze on pastures suspected to have anthrax spores.

VMC 311 LECTURE # 7

CLOSTRIDIUM – PART I

 

Introduction: The clostridia are relatively large, Gram-positive, rod-shaped organisms. All species form endospores and have a strictly fermentative mode of metabolism. They produce end products such as butyric acid, acetic acid, butanol and acetone, and large amounts of gas (CO₂ and H₂) during fermentation of sugars. They also produce foul smelling compounds during the fermentation of amino acids and fatty acids. The clostridia also produce a wide variety of extracellular enzymes to degrade large biological molecules in the environment into fermentable components. Hence, the clostridia play an important role in nature in biodegradation and the carbon cycle. Most clostridia will not grow under aerobic conditions and vegetative cells are killed by exposure to O₂, but their spores are able to survive long periods of exposure to air. The clostridia live in all anaerobic habitats of nature where organic compounds are present, including soils, aquatic sediments and the intestinal tracts of animals. Most of the clostridia are saprophytes but a few are pathogenic for animals. Those that are pathogens have primarily a saprophytic existence in nature and are opportunistic pathogens. Clostridium tetani and Clostridium botulinum produce the most potent biological toxins.

 

Important species:

1.     Clostridium tetani,

2.     Cl.botulinum

3.     Cl.novyi,

4.     Cl.chauvoei,

5.     Cl.septicum,

6.     Cl.perfringes,

7.     C.haemolyticum,

 

Important diseases: Tetanus, Black Quarter, Black disease, Enterotoxaemia, Bacillary haemoglobinuria, botulism (food poisoning)

 

General characters:

1. Morphology: In general, clostridial organisms are gram-positive long rod shaped organisms with rounded ends occurring either singly, short chains or in filaments. They are variable in size measuring 0.4 to 1.2mm in width and 0.3 to 8mm in length. They are motile (except C.perfirngens) by peritrichous flagella and non capsulative. They are known for being anaerobic (some are microaerophilic) and spore forming. The spores are located centrally, terminally or subterminally.
2. Habitat: Clostridial organisms are mainly found on the soil since they are frequently excreted in the faeces of the infected animal. In the soil, they survive as spores. Under favourable conditions, these spores germinate into vegetative cells and cause infection.
3. Classification: Clostridial organisms are classified into three groups according to habitats and natural history. Many species are saprophytes closely associated with soil and are reason for putrefaction of fruits and vegetables. Some are commensals that are seen in the digestive tract. Few species are toxigenic that produce toxins to host tissue. These toxigenic strain are classified into four groups depending upon the tissue they affect viz,
1. Histotoxic clostridia – To a variety of tissue particularly muscle.
2. Hepatotoxc clostridia – To liver
3. Enterotoxic clostridia – To digestive system
4. Neurotxic clostridia – To CNS
4. Resistance: The spores of the organisms are considerably resistant than the vegetative cells to the physical and chemical agents as resistant as the spores of B.anthracis.. The spores can remain viable in soil and dried cultures for a long time (many years). Boiling for 30 minutes, dry heat at 140^oC for 3 hours and autoclaving at 121^oC will destroy spores. However, freezing temperature has no effect on the spores. Only prolong action of chemical disinfectants at high concentration has killing effect on the spores - 5% phenol for 2 days and 10-20% formalin in 10 minutes.
5. Cultural characters: Clostridial organisms are known for their strict anaerobic growth characters. Certain species like C.novyi are very sensitive to oxygen, whereas C.perfirngens grow in microaerophilic conditions. The media commonly used for cultivation are blood agar, cooked meat medium and thioglycolate broth. The colonies are 3 mm in diameter, round or slightly irregular, slightly raised, granular, transparent or translucent with filamentous margins. Special media are used to study the toxin production. When media like blood agar are used for the cultivation of clostridial organisms, the plates have to be incubated in anaerobic conditions. The anaerobic condition can be achieved in laboratory in GasPak system, McIntosh-Fildes jar or by using certain reducing substances. When fluid media are such anaerobic incubation is not necessary since the organisms will grow at the bottom of the media where oxygen tension is low.

 

TETANUS - Clostridium tetani

Morphology and general characters:

1. C.tetani is a long, slender rod shaped organism. They are motile by peritrichous flagella and non-capsulated. The spores of the organisms are round, two times bigger than the diameter of the bacilli and are arranged terminally giving them the characteristic drumstick appearance.
2. These organisms sporulate faster and readily than other clostridia. The spores are destroyed at boiling temperature after 15 minutes, autoclaving and at dry temperature of 150^oC after one hour.

Antigens and toxins: There are 11 strains of C. tetani distinguished primarily on the basis of flagellar antigens. Some strains are toxigenic and some are non toxigenic. Tetanus toxin is one of the three most poisonous substances known, the other two being the toxins of botulism and diphtheria. The toxin is produced by growing cells and released only on cell lysis. Cells lyse naturally during germination the outgrowth of spores, as well as during vegetative growth. After inoculation of a wound with C. tetani spores, only a minimal amount of spore germination and vegetative cell growth are required until the toxin is produced. The toxigenic strains produce two toxic substances termed as tetanolysin and tetanospasmin. Tetanolysin is a haemolysin and cytotoxin. It is responsible for areas of haemolysis in blood agar. It is heat labile and oxygen labile. Tetanospasmin is a potent lethal neurotoxin. It is protein with a molecular weight of 150,000. The toxin is produced inside the cell and not released into the surrounding media. It is released only after the bacterial cell disintegrates. Hence, the toxin is not strictly considered as an exotoxin. Tetanospasmin initially binds to peripheral nerve terminals. It is transported within the axon and across synaptic junctions until it reaches the central nervous system. There it becomes rapidly fixed to gangliosides at the presynaptic inhibitory motor nerve endings, and is taken up into the axon by endocytosis. The effect of the toxin is to block the release of inhibitory neurotransmitters (glycine and gamma-amino butyric acid) across the synaptic cleft, which is required to check the nervous impulse. If nervous impulses cannot be checked by normal inhibitory mechanisms, it produces the generalized muscular spasms characteristic of tetanus. Horses and man are more susceptible to the toxin than other animals. Birds are insusceptible to the toxin. One mg of the toxin contains 100-million mouse lethal dose. Unpurified toxin is denatured at 65^oC for 5 minutes, whereas dried form resists a temperature of 120^oC for one hour.

Cultural characters: They are strict anaerobes and optimum temperature of growth is 37^oC. However, growth is also reported at 14-44^oC. In cooked meat broth good growth along with blackening of meat particles is noticed and addition of egg white to the meat broth yield better growth. In blood agar haemolytic, translucent, grey colonies of 2-4mm diameter with filamentous edges and fuzzy appearance are noticed.

 

Pathogenesis: Many domestic animals are affected by tetanus. However, birds are resistant to the infection. Young animals are more susceptible than older animals. The incubation period is variable (form 6 days to several months). The organism does not have any invasive power and development of tetanus entirely depends on the favourable conditions suitable for germination of spores and production of toxin. The favourable conditions generally occur during traumatic conditions that involve reduced blood supply, necrosis and multiplication of other bacteria which results in reduced oxygen tension in the area. The toxins produced by the bacteria produce two types of infection, local and general. These two forms are more common in man than in animals. In animals general form alone is seen. The toxins move in the interneuronal tissue space of motor nerves and then into interstitial fluid of CNS. The main site of toxin action is the synaptic junction of the motor neurones.

 

Symptoms: Initial symptoms are stiffness and unwillingness to move that is followed by general stiffness of the limbs, head and tail that become stiff. There is mild twitching of muscles that later turn into muscular spasms. Rigidity of the muscles extends from limbs to trunk, with dilated nostrils, erect ears, protruding nictitating membrane and impossible mastication (due to locked jaw) are characteristic symptoms. Respiration is shallow and rapid followed by respiratory failure. The condition of the horses is referred as ‘wooden horse’ since the animals has difficulty in moving.

 

Lesions: No characteristic lesions are noticed.

 

Diagnosis:

1. Based on the symptoms.
2. Micorscopical examination of clinical specimens (wound materials) for ’drumstick bacilli’.
3. Cultural examination on meat broth where the colour of the meat particles turn into black and addition of egg white to the meat broth yield better growth. In blood agar haemolytic, translucent, grey colonies of 2-4mm diameter with filamentous edges and fuzzy appearance are noticed.

 

Control:

1. Tetanus toxoid should be give when wound contamination with C.tetani is suspected. In horses two dosed at an interval of 3-4 weeks produce good response.
2. Hygienic wound management and good animal husbandry practices.

 

BOTULISM – Clostridium botulinum

This organism is found commonly in the soil. The toxin produced by this organism is a very potent one and cause botulism in animals, birds and human beings.

 

Morphology: They are gram-positive rod shaped organism occurring singly, in pairs or in short chains. They are sluggishly motile by peritrichous flagella. They are non capsulative. They produce oval shaped spores that are located centrally, sub-terminally or terminally inside the cell. Seven toxigenic types of the organism exist, each producing an immunologically distinct form of botulinum toxin. The toxins are designated A, B, C1, D, E, F, and G. Not all strains of C. botulinum produce the botulinum toxin. The purified toxins are protein with difference in molecular weight. The toxins are very lethal – 1.0 mg of toxin is lethal for 40 million mice. Rarely the toxins are activated inside the gut by trypsin. This toxin acts on the cholinergic nerve endings of peripheral somatic and autonomic fibres and does not have effect on brain and spinal cord.

 

Cultural characters: They are strict anaerobic organisms and grow better at an incubation temperature of 35^oC.  Growth occurs in ordinary laboratory media and the colonies are transparent and irregular. In blood agar they produce narrow zone of haemolysis. The proteolytic A, B and F cause blackening of the meat particles in the meat medium where as the non proteolytic C, D and E do not blacken the meat. They liquefy gelatin.

 

Pathogenesis: The spores of the organisms are commonly found on the soil. They are ingested by the animals and excreted without any change. Besides the organism have very poor invasive power. The important aspect of botulism is that the disease arises due to consumption of preformed toxin in the putrefied meat or decayed vegetation. C.botulinum does not invade the body and produce toxin inside living body. The ideal environment for the production of toxin is found in putrefied carcases, decomposed feed and decayed vegetation. Another important aspect in production of the disease is that there should be no degeneration of the toxin produced inside the putrefied materials. In cattle the infection is called as lamsiekete and is associated with phosphorus deficiency in cattle. In cattle, phosphorus deficiency is characterised by depraved appetite and the animals will consume pieces of carcass found in the soil and get the infection. In poultry the infection is seen commonly in ducks, chicken and pheasants. In ducks the infection is referred as Duck sickness or Western duck disease. The infection is due to consumption of decayed vegetation containing toxin found on the marshy lands. In chickens and pheasants the infection is termed as limberneck, where the infection is due to consumption of green bottle fly and other insects that feed on dead animals. In brooders the infection is termed as pseudo limberneck and is due to pecking at the litter that may contain botulinum toxin.

 

Symptoms and lesions: The incubation period is 2-10 days and symptoms are characteristic of nervous symptoms. Initially there is excitability followed by in-coordination and paralysis of hind limbs. Paralysis the mouth, pharynx and neck with protrusion of tongue are characteristic of botulism. The animals die of the infection. In poultry, there is paralysis of wings, legs and neck, protrusion of nictitating membrane followed by death.

 

Diagnosis:

1. Based on symptoms and lesion.
2. Detection of toxin in intestinal contents or in feed stuff. The laboratory animals used are mice and guinea pigs
3. Differentiation of C.perfirngens with other clostridia by FAT.

 

Control: A toxoid used in certain countries. However, good animal husbandry practices with prevent the infection.

 

BIG HEAD DISEASE or BLACK DISEASE - Clostridium novyi (C.oedematiens)

Morphology: They are gram-positive rod shaped organism occurring singly, in pairs or in short chains. They are sluggishly motile by peritrichous flagella. They are non capsulative. They produce oval shaped spores that are located centrally, sub-terminally or terminally inside the cell.

 

The organisms are divided into three types based on the antigenic structure and toxins produced.

 

Type A (C.novyi) – Gas gangrene and big head in ram

Type B (C.gigas) – Black disease (chronic hepatitis)

Type C (C.bubalorum) – Osteomyelitis in buffaloes (Kranefeld’s bacillus)

 

Cultural characters: The organisms are strict anaerobes. Primary cultures are difficult to grow and the growth is enhanced by the addition of glucose, fresh blood or brain extract. The growth is characterised by colonial motility (swarming) that is noticed after 3-4 days. Swarming is characterised by movement of daughter colonies away from parent colonies. In plates the swarming looks like arcs or spirals. Later the daughter colonies fuse with parent colonies. In horse blood agar they produce wider zone of haemolysis. In meat medium the meat particles turn into pink. In egg yolk medium, the colonies of type A and B are surrounded by opalescent zone on precipitation that is due to lecithinase. This reaction is called as Nagler’s reaction.

 

Pathogenesis: The spores are commonly found on the soil and the organisms are commensals in the intestine. The big head in ram is a result of contamination of the wounds on the heads of rams. Head wounds are common in ram as result of fighting among themselves. The alpha toxin produced by the organism is responsible for the oedema and death. Black disease occurs in areas where liver fluke infection is common. The organisms multiply the areas where the immature flukes have migrated. The inflammatory response in the tracts of immature fluke migration provide ideal environment for the organism to multiply, produce alpha toxin resulting in death due to toxaemia.

 

Symptoms and lesions: Big head is characterised by gas gangrenous lesion around the face and head. Affected animals will collapse and die. Black disease is an acute toxic infection with sudden death. The symptoms observed are inability to move and unsteady gait. The lesions are more prominent in liver with necrotic areas. There is extensive blood stained oedema that gives the carcass a dark colour. Exudates are seen in heart.

 

Diagnosis:

1. Based on symptoms and lesions.
2. Microscopical observation of liver impression smears
3. Cultural characteristics - The organisms are strict anaerobes. Primary cultures are difficult to grow and the growth is enhanced by the addition of glucose, fresh blood or brain extract. The growth is characterised by colonial motility (swarming) that is noticed after 3-4 days. Swarming is characterised by movement of daughter colonies away from parent colonies. In plates the swarming looks like arcs or spirals. Later the daughter colonies fuse with parent colonies. In horse blood agar they produce wider zone of haemolysis. In meat medium the meat particles turn into pink. In egg yolk medium, the colonies of type A and B are surrounded by opalescent zone on precipitation that is due to lecithinase. This reaction is called as Nagler’s reaction.
4. Detection of toxin in intestinal contents or in feed stuff. The laboratory animals used are mice and guinea pigs.

 

Control: A formalised whole culture vaccine adsorbed onto aluminium hydroxide is used to control the infection.

VMC 311 LECTURE # 8

CLOSTRIDIUM – PART II

BLACK QUARTER - Clostridium Chauvoei

General characters:

1.      Morphology: They are gram-positive rod shaped pleomorphic organisms. They appear as large cigar shaped rods or citron forms. They are sluggishly motile by peritrichous flagella. They are non capsulative. They produce oval shaped spores that are located centrally, sub-terminally or terminally inside the cell. The staining character is highly variable, sometimes only one end of the organisms will take stain and the other side remain unstained.

2.      Habitat: They are commonly found on the pastures where the incidence is endemic. They are also found in the soil.

3.      Resistance: The spores are resistant and are killed by steam in 40-50 minutes and by autoclaving. The spores are also killed by 3 per cent formalin for 15 minutes.

4.      Antigens and toxins: C.chauvoei have one common somatic antigen and the organisms are divided into two groups based on flagellar antigens. They share spore antigen with C.septicum. They produce four different types of toxins alpha (neurotoxin), beta (DNAse), gamma (hyaluronidase) and delta (haemolysin).

5.      Classification: Not very important. They share a spore antigen with C.septicum.

 

Cultural characteristics: They are strict anaerobic organisms. The growth is enhanced by the addition of liver extract of glucose. The optimum temperature for growth is 37C. In blood agar the colonies have irregular edges, 2-4mm in diameter and are surrounded by a zone of haemolysis. In cooked meat medium, the colour of the meat changes into pink and a sour odour is produced. C.chauvoei ferments glucose, lactose, sucrose and maltose with production of acid and gas. However, they do not ferment salicin. This is a differentiating feature with C.septicum.

 

Pathogenesis: The main mode of spread is through wound contamination. The contaminated pasture contains the spores that enter into the animal through wounds. Infection through ingestion is also possible but the spores have to be activated. Activation of spores becomes possible only when the environment becomes anaerobic. Necrotic cells provide ideal environment for the organisms to multiply. Cattle, sheep and goat that are in pasture are more prone to infection than stall fed animals. Young animals are more affected than adult animals.

 

Symptoms: The infection may occur as per acute or acute. In per acute infection, no symptoms are usually seen and animals die suddenly in the pasture. Important symptoms include crepitant swelling on the fore or hindquarter that crackles when rubbed with fingers. It also pits on pressure. This is due to production of large quantities of gas and oedema. The affected animal will have fever, anorexia and becomes lame. Death occurs after 24 hours.

 

Lesions: The muscle becomes dark red in colour, dry necrotic and filled with small gas bubbles. The lesion also emits a rancid odour. The muscle is surrounded by yellow or blood stained fluid.

 

Diagnosis:

1. Based on the symptoms and lesions.
2. Microscopical examination of the impression smears prepared from affected muscles or oedematous fluid  and stained by Gram’s method for C.chauvoei organisms and spores. The spores can also be confirmed by malachite green staining method.
3. Cultural examination: The clinical materials are affected muscle piece either dry of as such. In blood agar the colonies have irregular edges, 2-4mm in diameter and are surrounded by a zone of haemolysis. In cooked meat medium, the colour of the meat changes into pink and a sour odour is produced.
4. Differentiation with C.septicum

                                                              i.      By biochemical reaction – Salicin utilisation

                                                             ii.      Fluorescent antibody test using specific antibodies.

5.      Modern methods like ELISA.

6.      Biological test: Guinea pig is laboratory animal of choice. Suspected material is mixed with Calcium chloride and given as deep intramuscular injection to guinea pigs. Guinea pigs will develop characteristic symptoms of black quarter after 48 hours.

 

Treatment: Penicillin is the drug of choice. Usually it is given coupled with antiserum. Oxytetracycline and chloramphenicol can also be given.

 

Control: Vaccination is the best method to control the infection in endemic areas. Different vaccines are available. The commonly used vaccine is killed alum of aluminium hydroxide adjuvanted vaccine. It protects the animals for 6-24 months.

 

ENTEROTOXAEMIA - Clostridium perfringens

Synonym                 ;        Clostridium welchii

General characters:

1. Morphology: They are gram-positive rod shaped organisms occur, either singly, in pairs or in short chains. Unlike most other clostridia they are non motile, capsulative and sporulate rarely. If sporulate the spores are located centrally, sub-terminally or terminally inside the cell. Young cultures appear as gram-positive where as old cultures appear gram-negative.
2. Habitat: They are present everywhere. They are found in the soil, and intestines commonly.
3. Resistance: They are not as resistant as other clostridia. Boiling for five minutes kills them.
4. Classification: There are five important types.
1. Type A – Causes gas gangrene
2. Type B – Causes lamb dysentery
3. Type C – Causes struck
4. Type D – Causes pulpy kidney or over eating disease
5. Type E – Causes enterotoxaemia in calves.
5. Antigens and toxins: C.perfringens possesses somatic and flagellar antigens but they are of less importance in classification. They produce number of toxins and enzymic substance that are of importance in pathogenesis. Some of important toxins and enzymic substances are listed below.
1. Alpha toxin – (Lecithinase C). Found abundant in type A. Responsible for Nagler reaction.
2. Beta toxin – Found abundant in B and C. It is a lethal necrotising toxin.
3. Gamma toxin – Found in B and C. Minor lethal toxin.
4. Delta toxin – Found in type C and is responsible for struck in sheep. Lethal haemolytic toxin.
5. Epsilon toxin – Found in types B and D. Major lethal necrotising toxin.
6. Eta toxin – Found in type A. Minor lethal toxin.
7. Theta toxin – Haemolysin.
8. Iota toxin – Inert major lethal necrotising toxin that is activated by lambda toxin.
9. Kappa toxin – Collagenase.
10. Lambda toxin – Proteinase and gelatinase
11. Mu toxin – Hyaluronidase like enzyme.
12. Nu toxin – DNAse

 

Cultural characteristics: Unlike other clostridia, C.perfringens are not strict anaerobic and they will grow well in ordinary laboratory media with a temperature range of 37-47C (optimum 43-47C).

1. In blood agar the colonies are small 2-3mm in diameter, smooth, evenly convex and opaque. Occasionally mucoid variants also occur. A narrow zone of complete haemolysis surrounded by wider zone of partial heamolysis is produced in blood agar.
2. The colour of the meat in cooled meat medium changes into pink and it is not digested. The growth in cooled meat medium is characterised by production of large amount of gas. After few hours of incubation gas bubbles can be seen rising from the bottom. This can be achieved when heating at 100C, followed by cooling at 37C, brings down the residual oxygen of the meat medium.
3. In media containing egg yolk or horse sera, C.perfringens produce a marked opalescence. This is caused by lecithinase. This reaction is referred as Nagler reaction or lecithinase reaction.
4. C.perfringens ferments glucose, lactose, sucrose and maltose and produce acid and gas. In litmus milk acid and clot is produced and due to large amount of gas production the clot breaks up in number of areas, this reaction is referred as stormy clot reaction.

 

Pathogenesis: Generally the infections associated with C.perfringens are human food poisoning and gas gangrene.

1.     Gas gangrene: It is caused by Type A. This infection has an incubation period of 12-24 hours. Initially there is pain and oedema in the affected areas followed by accumulation of gas bubbles and extensive haemorrhage. The area, which initially appears pale later, turns into brown, dark green of black. Death follows septicaemia.

2.     Enterotoxaemia in sheep (Lamb dysentery): Lamb dysentery: Type B organisms cause this infection. The organisms are found in the intestinal tract as commensals. Infection occurs as a result of production of large amount of toxins. The toxins are produced when there is heavy consumption of milk  followed by indigestion. The affected animals show signs of abdominal pain, bleat repeatedly, cease to suck, collapse and die. The faeces is blood stained. The intestine consist of intense areas of haemorrhage and ulcers. The stomach is filled with undigested milk.

3.     Enterotoxaemia in sheep (Struck): The disease occurs during early spring and affects adult sheep. The infection is characterised by  fatal toxaemia with ulceration of the intestine. Death occurs suddenly without any symptoms. The affected animals develop symptoms of rigidity, immobility and abdominal pain but no diarrhoea. The important lesions are ulceration of the intestine, degenerative changes in kidneys and fluid accumulation in abdominal and thoracic cavities.

4.     Enterotoxaemia in sheep (Pulpy kidney disease, Over eating disease): It is caused by type D organisms. The infection is due to rapid multiplication of the organisms in the intestine and abomasums and absorption of the toxins produced. Young animals between age groups of 6-12 months are more prone to infection. The infection occurs as result of sheep exposed to lush green pastures after monsoon and resultant over eating. Excessive concentrate feeding is also a predisposing factor. The organisms are found in the soil and they are consumed by the animals resulting in production of small amount of toxin in the intestine. These small quantities are not dangerous. Only when there is large amount of toxin production the infection sets in. The infection is characterised by sudden death of animal in the field. The symptoms exhibited are animals become dullness and weakness. There is spasmodic struggling before the animal collapse. The important lesions are the degeneration of the kidney cortex, soft and pulpy kidneys, degeneration of other organs, accumulation of straw-coloured fluid in the pericardium, endocardial haemorrhages and areas of congestion in abomasums and small intestine.

5.     Enterotoxaemia in cattle: Less significant infection. The infection confuses with lead poisoning.

 

Diagnosis:

1.      Based on symptoms and lesions

2.      Microscopical examination of the smears prepared from intestinal contents reveal gram-positive organisms with characteristic morphology.

3.      Demonstration of toxin activity in mice – Test for lethal action and toxin neutralisation test.

4.      Cultural examination – The intestinal contents from dead animals is the ideal material. The contents are inoculated into cooked meat medium. The colour of the meat in cooled meat medium changes into pink and it is not digested. The growth in cooled meat medium is characterised by production of large amount of gas. After few hours of incubation gas bubbles can be seen rising from the bottom. This can be achieved when the residual oxygen of the meat medium is brought down by heating at 100C, followed by cooling at 37C.

5.      Presence of glucose in the urine is an indication of infection.

 

Treatment: Most of the infections are of acute type. Hence, treatment is of little value.

 

Control: Alum precipitated formalin killed culture is used as vaccine that offers satisfactory immunity. Vaccination of mothers provided passive immunity to the young animals.

VMC 311 LECTURE # 9

CORYNEBACTERIUM

Introduction: Corynebacteria are Gram-positive, aerobic, non-motile, rod-shaped bacteria related to the Actinomycetes. They do not form spores or branch as do the actinomycetes, but they have the characteristic of forming irregular shaped, club-shaped or V-shaped arrangements in normal growth. They undergo snapping movements just after cell division, which brings them into characteristic arrangements resembling Chinese letters. Corynebacterial cell walls contain thin spots, which leads to some Gram variability and "ballooning" that produces a "club-shaped" cell). Old cells store inorganic phosphate, which can appear as metachromatic granules when stained.

Corynebacteria belong in the family Mycobacteriaceae and are part of the CMN group (Corynebacteria, Mycobacteria and Nocardia). As a group, they produce characteristic long chain fatty acids termed mycolic acids. The genus Corynebacterium consists of a diverse group of bacteria including animal and plant pathogens, as well as saprophytes. Some corynebacteria are part of the normal flora of humans, finding a suitable niche in virtually every anatomic site. The best known and most widely studied species is Corynebacterium diphtheriae, the causal agent of the disease diphtheria. The family Mycobacteriaceae are Gram-positive, nonmotile, catalase-positive and have a rodlike to filamentous morphology (Corynebacteria are often pleomorphic.

The genus Corynebacterium consisted of number of important species of veterinary importance, but most of them have been moved to new genus as a result of improved methods of classification. The following table lists important pathogenic species of Corynebacterium and their new names;

Old name	Infection	New name
C. diphtheria	Diphtheria in man
C. equi	Suppurative bronchopenumonia	Rhodococcus equi
C. Pseudotuberculosis	Caseous lymphadenitis
C. pyogenes	Caseous pneumonia in pigs Polyarthritis Chronic suppurative pneumonia, arthritis, umbilical infections in cattle Summer mastitis and pneumonia in sheep and goat	Arcanobacterium pyogenes
C. renale; C.pilosum; C.cystitidis	Pyelonephritis in cattle
C. mastitidis	Mastitis in sheep and goat
C. amycolatum,
C. ulcerans,
C. minutissimum

 

C. pseudotuberculosis

General characters:

1.      Morphology: They are small, Gram-positive, pleomorphic rod bacteria that occur in a characteristic filamentous, angular, palisade arrangement often located intracellularly. They are non-motile, aerobic, fermentative, non-spore forming. C. pseudotuberculosis is a facultative intracellular parasite that has been implicated in the diseases caseous lymphadenitis in sheep and goats, ulcerative lymphangitis in horses and formation of abscesses in many other animal species.

2.      Location: They are facultatively intracellular.

3.      Habitat and resistance: C. pseudotuberculosis is present in the environment following contamination by infected animals. Low temperatures and damp conditions prolong survival time. C. pseudotuberculosis survive for prolonged periods (>6 mths) in contaminated environments and has been isolated from feed, feeders, fences, shears and sheep dips, plus dust and soil samples from around stock handling areas. The main source of infection and potential spread of the organism is via the rupture of affected lymph nodes and abscesses with the discharge of thick caseous pus containing millions of organisms into the environment.

4.      Antigens and toxins:

1.            Cell wall surface lipid. It allows organism to resist digestion by cellular enzymes and to persist as a facultative intracellular parasite within phagocytes. It also cytotoxic and induces caseation giving the organism pathogenic potential.

2.            Heat labile phospholipase exotoxin, phospholipase D (PLD). It is the major virulence factor. The toxin’s major effect of phospholipase relates to the establishment of infection and possible local effects. It helps in the survival and multiplication of the organism in the host. It has effects on phagocytic cells (inhibition of chemotaxis, degranulation, and lethality in neutrophils) or other effects such as compliment depletion. This exotoxin also functions as a permeability factor acting on the local blood vessels and lymphatics. The resulting leakage of plasma increases the extravascular circulation of fluid and therefore the probability of spread of infection both locally and to the regional lymph nodes via the lymphatics. This exotoxin also acts as a partial haemolysin having a limited necrosing action within caseous lesions, and contributing to the degradation of RBC membranes.

Cultural characters: C.pseudotuberculosis is aerobic or facultatively anaerobic. Optimum growth is obtained at 37C at a pH of 7.2 to 7.4. Growth is enhanced by addition of serum to the culture media. On serum agar the organisms produce small, finely granular, translucent colonies with irregular border. After few days the colonies become opaque, concentrically ringed, dry and crumbly. Colonies have cream to orange colored. The organisms grow in Loeffler’s coagulated blood serum containing potassium tellurite salt. The colonies are greyish black in colour. Uniform turbidity is produced in broth.

 

Pathogenesis:

The usual mode of entry of bacteria is via superficial wounds in the skin or mucous membranes followed by extension to the regional lymph nodes and beyond. Organisms also enter via the respiratory tract. Ingestion of bacteria may produce lesions in lymph nodes of head and thorax.

 

Caseous lymphadenitis: It is a chronic purulent disease of sheep and goats characterised by discrete chronic abscesses with palpable enlargement of the superficial lymph nodes, visceral lymph nodes and internal organs. It mainly occurs in sheep. In sheep the, infection is often preempted by wounds obtained during shearing, castration, docking, crutching etc. Other general wounds such as dog bites, scratches etc can also lead to this infection. Contamination of these wounds by bacteria in environments, or via direct contact with infected animals is of importance in transmission. The most common potential source of infection is contaminated sheep dips, with bacteria able to survive in commercial dips up to 24 hours without significant reduction in viable count. Pus from a single discharging node is able to contaminate large volumes. Sheep dipped in contaminated fluid within 2 weeks of being shorn are especially susceptible to infection due to ease of contact between bacteria and the skin. Other predisposing factors are conditions where animals are kept in close contact with infected animals in areas like yards with wooden fences, dirt floors etc., which are difficult to disinfect. In goats skin abrasions, fight wounds or traumatised buccal mucosa act as portals of infection for the organism. Formation of abscess is characteristic lesions in caseous lymphadenitis. The abscesses are most common in superficial nodes, (eg; mandibular, parotid, retropharyngeal, prescapular, subiliac, superficial inguinal) with lung and thoracic lymph node involvement. The lesions are rarely seen in sheep in head and neck lesions and most commonly noticed at prefemoral and prescapular sites, while in the goat mandibular and parotid nodes are frequently involved. Visceral lesions involving lung parenchyma, internal lymph nodes and most other organs develop from haematogenous spread from the regional lesions. Common organs involved are the liver and kidneys plus lungs and thoracic nodes. The Abscess reach a diameter of 5-10 cm and is associated with a firm fibrous capsule. It contains a thick creamy pus which becomes firmer with age leading to a firm, caseous friable dry texture. In sheep the contents are usually light green and often develop a concentric laminated onion-ring appearance due to repeated stages of necrosis and capsule formation. Deposition of calcerous granules frequently occurs at the margin of the expanding lesion. In goats abscess contents are more uniformly pasty than dry, do not have a laminated appearance and rarely calcify. When the disease affects superficial lymph nodes there is palpable enlargement of these nodes. Affected nodes may rupture forming a discharging wound. When internal lesions occur lung and thoracic nodes are frequently involved. Animals with interstitial lung lesions causing pneumonia, pleurisy or other serious internal lesions may show signs of ill thrift. In many cases there are frequently no outward clinical signs, therefore CLA is often referred to as a “hidden disease” and only becomes apparent at slaughter. The major effects of the disease are due to economic losses through trimming and condemnation of carcass, decreases in wool production, value of hides, body weights, milk production and reproductive efficiency.

 

Ulcerative lymphagitis:  It is a contagious disease of equines characterised by inflammation of the subcutaneous lymphatic vessels with formation of nodules, abscesses and ulcers on the ventral abdomen, pectoral region, distal portions of limbs. Lymphatics become corded and infection can spread to form abscesses in internal organs. Superficial skin wounds are a major cause of infection. High incidence is related to poor management, with infection most common in situations of overcrowding when animals are picketed together on muddy standings and subject to kicks, chaffs and other injuries. Contaminated bedding and grooming utensils act as fomites. Flies and other arthropods act as mechanical vectors. The abscesses vary markedly in appearance.
range in diameter from 5-30 cm, often with pitting oedema of adjacent tissues. Most abscesses are localised and tend to come to the surface where they rupture or can be lanced easily. The most common sign is swelling of the pectoral region or ventral abdominal wall. Other common signs include diffuse or localised swellings elsewhere on the body, ventral oedema, ventral midline dermatitis, fever, weight loss plus presence of draining abscesses, wounds or tracts.  Lameness is also noticed and is attributed to stiffness and discomfort from oedema and abscesses adjacent to limbs. Affected animals are often noticeably depressed due to the effects of pyrexia, lameness and pain. The organism has also been cited as a cause of equine abortion, pyelonephritis and equine mastitis.

 

Infections of farm workers and veterinarians exposed to infected animals usually appear as lymphadenitis.

 

Diagnosis

1.           Clinical materials: Pus or affected tissue from lymph nodes.

2.           Based on symptoms and lesions.

3.           Microscopy – Smears prepared from pus smear and culture smear stained by Grams staining method reveal onion ring appearance and Chinese letter shaped organisms

4.           Isolation and identification: Growth is enhanced by addition of serum to the culture media. On serum agar the organisms produce small, finely granular, translucent colonies with irregular border. After few days the colonies become opaque, concentrically ringed, dry and crumbly. Colonies have cream to orange colored. The organisms grow in Loeffler’s coagulated blood serum containing potassium tellurite salt. The colonies are greyish black in colour. Uniform turbidity is produced in broth.

5.           Slide agglutination test

 

Treatment

Treatment is not normally attempted.

 

Control

          A formalised toxin (toxoid) is used to control the infection

 

Contagious bovine pyelonephritis:

Bovine cystitis is an inflammation of the urinary bladder of cattle that may ascend the ureters to cause infection of the kidneys referred as pyelonephritis. A similar condition can occur in sheep. The condition is sporadic and worldwide in distribution. It is most often seen after parturition. The most common causative agents are the Corynebacterium renale group of bacteria, including C renale, C cystitidis, and C pilosum, as well as Escherichia coli . The latter is most often seen in chronic cases and may be an opportunist following the corynebacterial infection. Other organisms that may be involved include, but are not limited to, Staphylococci and Streptococci. The most common causative agents, Corynebacterium spp and E col, are ubiquitous in the environment and are common inhabitants of the vagina and prepuce. The organisms attack or colonize the mucosal lining of the bladder and ureters usually after some traumatic insult (such as parturition or abnormal deformity of the vaginal tract). The stresses of parturition, peak lactation, and a high-protein diet (which increases the pH of the urine and is therefore conducive to colonization of the attacking organisms) are all contributing factors. The symptoms include a slight fever, loss of production, colic with restlessness, tail switching, polyuria, hematuria, or pyuria. In chronic cases, the animal may show colic, diarrhoea, polyuria, polydipsia, stranguria, and anaemia. As the disease progresses, the bladder becomes thickened and inflamed. The ureters become thickened and dilated with a purulent exudate. The involved kidneys develop multiple small abscesses on the surface that may extend into the cortex and medulla. Diagnosis is based on clinical signs; a history of recent parturition; palpation of the left kidney for enlargement, loss of lobulation, and pain; vaginal inspection for detection of inflamed and enlarged ureters; microscopical examination of the urine for WBC and bacteria and urine culture to identify the organism. In early acute cases, enlarged ureters and involvement of the kidney may not be detectable on rectal palpation. Pyelonephritis can be cured by administration of Penicillin.

 

RHODOCOCCUS

Disease caused        :           Suppurative bronchopneumonia in horses

Important species   :         Rhodococcus equi

 

Morphology and general characters:

1. Morphology: This organism was earlier grouped under the genus Corynebacterium. They are gram-positive and weakly acid fast. They are pleomorphic organism occurring as large rods, cocco bacilli or club shaped, arranged in groups or clusters. They are non motile and non sporulative. In contrast to corynebacteria, these organisms do not possess metachromatic granules. Further, they possess large polysaccharide capsule. They are non fermentative and catalase and urease positive.
2. Habitat and resistance: These organisms are soil borne and often found in manure. They are also present in the intestines of horses and persist for long periods in the manure and litter of stables. They are relatively heat resistant and are killed only when exposed to a temperature of 60^oC for 1 hour. Sensitivity of disinfectants is variable.
3. Antigenicity and serotypes: In contrast to corynebacteria, members of the genus Rhodococcus do not produce any toxins. They are serotyped into 10 serotypes based on their capsular (K) antigens. Serotypes 1 and 2 are more predominant.
4. Location: They are facultatively intracellular.

 

Cultural characters: They are aerobic or facultatively anaerobic and grow well on ordinary lab media at 37^oC. In nutrient agar, they produce glistening mucoid colonies that are pale pink in colour. These organisms are known for their ability to produce pale pink to red colour pigments. No haemolysis is produced in blood agar. One of the important characteristics of these organisms is that the primary cultures take a long time to develop. However, the secondary cultures appear with in 24-48 hours. Gelatin and serum are not liquefied.

 

Pathogenesis: The pathogenesis of the organism is not very clear. Infection arise through direct contact, inhalation or from contaminated soil, manure etc. They are pyogenic organisms and produce abscesses. The main route of infection is through respiratory tract. They are facultatively intracellular organisms and have the ability to survive inside macrophages. Though no toxins are produced, two substances phospholipase and cholesterol oxidases are considered as  virulence factors.

 

Pathogenicity: The organism causes suppurative bronchopneumonia in foals, submandibular and cervical lymphadenitis in swine and abscess in lungs and lymphnodes many animals. It is also responsible for abortion in mares. Pneumonia occurs in foals during 2-4 months of age. Affected animals cough, become dull and listless due to pyrexia and respiratory distress. In severe cases, a purulent discharge is noticed from the nostrils. Abscesses are noticed throughout the lungs and associated lymphnodes. Besides these, suppurative lesions are noticed in uterus also.

 

Diagnosis:

1. Clinical materials: Pus or pieces of lung and lymph nodes from dead animals. The clinical material may be mixed with oxalic acid (to a final concentration of 2.5%) for better isolation.
2. Morphological characters – Staining properties:
3. Isolation and identification: They are aerobic or facultatively anaerobic and grow well on ordinary lab media at 37^oC. In nutrient agar, they produce glistening mucoid colonies that are pale pink in colour. These organisms are known for their ability to produce pale pink to red colour pigments. No haemolysis is produced in blood agar. One of the important characteristics of these organisms is that the primary cultures take a long time to develop however, the secondary cultures appear with in 24-48 hours. Gelatin and serum are not liquefied.
4. Based on symptoms and lesions:
5. CAMP reaction: R.equi can produce only partial haemolysis or no haemolysis. When combined with Corynebacterium pseudotuberculosis it causes haemolysis. It also enhances the beta haemolytic action of Stapylococcus aureus.

 

Treatment: Treatment is generally ineffective. It should be started well in advance. Penicillin, lincomycin, rifampicin, neomycin, gentamycin and streptomycin combined with erythromycin are effective.

 

Zoonotic importance: The organisms cause pneumonia and abscess in AIDS and immuno-compromised patients.

VMC 311 LECTURE # 10

ACTINOMYCES, ARCANOBACTERIUM & NOCARDIA

ACTINOMYCES

 

Important species     :        Actinomyces bovis, A.pyogenes

Important diseases    :        Lumpy jaw, Pyaemic infections

 

General characters:

1. Morphology: Members of the genus Actinomyces, Nocardia and Dermatophilus are referred as higher bacteria since they show extensive filamentation, aerial hyphae with asexual spores or conidia. The cultural and morphological characters are similar to fungi than bacteria. Members of the genus Actinomyces are gram-positive, non acid-fast rod shaped organisms that show extensive branching. They are non-motile and non-spore forming. They are catalase negative and fermentative.
2. Habitat: These organisms are commonly found as commensal in the oral cavity of animals. They are obligate parasites in tonsils and carious teeth. These organisms have an affinity to hard tissues.
3. Classification: There are three important species A.bovis affects cattle and produce lumpy jaw. A.israeli produces the same type of infection in human. A.bovis has been moved from the genus Corynebacterium and it causes pyaemic infections.
4. Resistance: They are fragile organisms and are killed at a temperature of 60^oC for 20 minutes.
5. Antigens and toxins: They are antigenically homogenous.

 

Cultural characters: They grow better at anaerobic environment at 5% carbon dioxide tension. The media used for culturing are agar enriched with glucose, serum or blood, Dorset egg medium, Loeffler’s serum, brain heart infusion agar, thioglycollate broth, heart broth or glucose broth. The optimum temperature for growth is 37C. They are slow growing organisms and growth is noticed after 4-6 days of incubation. The colonies are 1 mm in diameter, round, flat and pale yellow. The colonies do not adhere to the medium. In fluid growth occurs as small granules in the bottom of the tube.

 

Pathogenesis: These organisms are commonly found as commensal in the oral cavity of animals. However, they are obligate parasites in crypts of tonsils and carious teeth. These organisms have an affinity to hard tissues. The common areas of infection are cheek muscles, lower jaw, submaxillary region and other areas of throat and head. In pigs, the organisms are found in the udder and suckling piglets get the infection through abrasions found in their mouth. In horses, the organism is associated with fistulous withers and poll evil.

 

 

Lesions: There is suppuration followed by granulation, erosion of bone tissues and formation of new bone tissues. Pockets of pus seen in this area increase in size become fistula discharging the pus. The pus containing small granules referred as sulphur granules. Secondary infection of lung and digestive tract results as a result of swallowing the pus. Microscopically a central area of necrosis surrounded by granulation tissue that is enclosed in fibrous tissue is seen. Lumpy jaw is usually progressive. As the bony swellings continue to enlarge, gross disfiguration of the head can occur, much condition will be lost and death may result

 

Diagnosis:

1. Based on symptoms and lesions and differential diagnosis with Actinobacillosis that affect soft tissue.
2. Microscopical examination of granules found in the pus. The pus granules are collected in a petridish and later transferred to a drop of 10%NaOH on a glass slide, crushed with help of a cover slip and stained by Gram’s method. The organisms are seen as a central mass of filaments that show branching. The periphery of the filamentous mass is covered by a circle of club shaped bodies arranged radially with narrow ends pointing towards centre of filamentous mass (hence, this arrangement is known as ray fungus). The club shaped bodies stain gram-negative.
3. Isolation of the organisms by culturing: The media used for culturing are agar enriched with glucose, serum or blood, Dorset egg medium, Loeffler’s serum, brain heart infusion agar, thioglycollate broth, heart broth or glucose broth. The optimum temperature for growth is 37C. They are slow growing organisms and growth is noticed after 4-6 days of incubation. The colonies are 1 mm in diameter, round, flat and pale yellow. The colonies do not adhere to the medium. In fluid growth occurs as small granules in the bottom of the tube.

 

Actinomyces viscosus: It causes actinomycosis of dogs. The infection in dogs is characterised by granulomatous infection of skin and thoracic tissues.

 

ARCANOBACTERIUM

Important Species : Arcanobacterium pyogenes (Previously C.pyogenes; Actinomyces.pyogenes)

 

Arcanobacterium pyogenes: It is a commensal found on the mucous membrane of nasopharynx of cattle, sheep and pigs. It is also found in the udder. It produces a exotoxin. It is an important pyogenic organism for cattle, sheep and pigs. It causes chronic abscessing mastitis in cattle (summer mastitis), suppurative pneumonia, nephritis, endometritis, pyometra, wound contamination and seminal vesiculitis in bulls.

Summer mastitis: Summer mastitis is produced by Arcanobacterium pyogenes (previously Corynebacterium and Actinomyces pyogenes). It is an acute illness of dry cows and heifers, which causes extensive and painful damage to the udder. The infected quarter is permanently damaged and results in early culling of the cow. Infections are more likely to occur when cows are in an environment where teats can be damaged and fly populations are high. The highest incidence is seen over the summer period (July, August and September), especially if warm and damp where the fly population is high usually close to wooded pastures. Its antibiotic treatment is difficult. In many cases the problem is not rapidly detected because the animals are in the fields and the farmer fails to notice the condition. These bacteria may gain entrance through the wounds caused by the flies, but more often they are commensals in udder.

Symptoms: The exotoxins produced by the organisms are absorbed into the bloodstream making the cow very ill. Affected animals have a high temperature, a swollen udder and stiffness due to joint involvement and friction of the swollen quarter against the side of the leg. When the contents of the teat are drawn they may vary from cheese-like to whitish yellow, to thick reddish and dark clots and a typical rotten smell. The animal may be depressed, the udder may be cold, clammy, secretions are scanty and difficult to drain. Close examination of the teats, reveals a mass of tiny red fly bites. This damage allows the common mastitis germs, usually streptococci, staphylococci and Micrococcus indolicus to multiply and grow in the udder.

 

Treatment & Prevention: In most cases when summer mastitis is detected very late. The first requirement is to remove as much as possible of the toxins in the infected quarter. This is achieved by bathing with hot water, firm massaging of the upper quarter to break pockets of pus and finally stripping or drawing every few hours. This procedure prevents the absorption of toxic materials into the animals system and clears the way for the natural healing process to commence and for intramammary tubes to gain access to the udder tissue. The infection may be so severe that the teat may have to be cut off to allow drainage. Further means to prevention of summer mastitis to the herd are application of teat dips to dry cows every few days, spaying fly repellent on the udders of suspected animals, inspect the udders of dry cows at least once a day, keep all dry cows with the milkers in open fields and bring them in with the milkers twice daily.

 

NOCARDIA

 

Important species               :        Nocardia asteroids, N.caviae, N.farcinica (??)

Important disease               :        Bovine farcy, Nocardiosis

 

These organisms are long filamentous branching with terminal spores. They are gram-positive and some are acid-fast. They are found as saprophytes and few are pathogenic producing farcy characterised by infection of lymphatic vessels. The vessels and glands become enlarged, corded and nodular due to slow formation of caseous material. The exact pathogenesis is not clear. They are also isolated from bovine mastitis.

VMC LECTURE # 11

MYCOBACTERIUM – PART I

 

Introduction: Mycobacterium tuberculosis was the cause of the "White Plague" of the 17th and 18th centuries in Europe. During this period nearly 100 per cent  of the European population was infected with tubeculosis and 25 percent of all adult deaths were caused by tuberculosis.

1. Mycobacterium tuberculosis is the etiologic agent of tuberculosis (TB) in humans. Humans are the only reservoir for the bacterium.
2. Mycobacterium bovis is the etiologic agent of TB in cows and rarely in humans. Both cows and humans can serve as reservoirs. Humans can also be infected by the consumption of unpasteurized milk. This route of transmission can lead to the development of extrapulmonary TB, exemplified in history by bone infections that led to hunched backs.
3. Other human pathogens belonging to the Mycobacterium genus include Mycobacterium avium, which causes a TB-like disease especially prevalent in AIDS patients, and Mycobacterium leprae, the causative agent of leprosy.

 

General characters of M.tuberculosis, M.bovis and M.avium :

1. Morphology: Members of the genus Mycobacteria are gram-positive, nonbranching, acid-fast, slender and small rods. They are non-motile, non-sporulative and do not produce any toxins. They are aerobic organisms. These organisms are known as acid-fast or acid-alcohol-fast organisms since they withstand decolourisation with acid or acid-alcohol.
2. Cell wall structure: Cell wall constituents of mycobacteria: The cell wall constituents of mycobacteria are highly complex. They have a high concentration of lipid 20-40%, which protects the bacteria from destructive actions of disinfectants, strong acids, alkalis and antibodies. Three different types of lipids are commonly found in the cell wall. They are mycolic acid, mycosides and glycolipids. Mycolic acids are b-hydroxy fatty acids that are responsible for acid fastness. Acid fastness is the ability of the organisms to retain the carbol fuchsin after application of decolouriser acid-alcohol. Mycosides are substances that are responsible for the controlling the permeability of the organisms to water, enzymes, disinfectants and antibiotics. These substances are always found in association with Wax-D that in association with proteins induce delayed hypersensitivity. Various virulence factors are found to be mycosides. Glycolipids produce toxicity, granulomatous response and enhance the survival of mycobacteria inside the macrophages. Cord factor, one of the glycolipids is responsible for characteristic cultural characters.
3. Resistance: They are generally killed at a temperature of 60^oC for 15 minutes and comparatively resistant to the lethal action of disinfectants than other organisms. They can remain viable for long periods in the darkness, putrefying material (1-4 years), faeces (150 days), sputum etc. However, they are highly susceptible to ionic detergents and to sunlight. Freezing temperature has little effect on them. Phenol (5%), Lysol (3%), cresols, formaldehyde (3-8%), alkaline gluteraldehyde (2%) and cresylic acid are fairly effective. Sodium hypochlorite at a concentration of 1:2000 or 1:1000, phenol soap mixtures and other phenol derivatives are effective disinfectants.
4. Antigenic structure: A strong relationship exists between different species of mycobacteria. M.tuberculosis and M.bovis are very closely related. The murine type is also very closely related to human and bovine types. Avian type share few antigens with human type. The avian type is very closely related with M.intracellulare (Battey bacilli) and is often indistinguishable. Hence, it is frequently referred as M.avium-intracellulare complex.

 

Classification:

1. This group of organisms are called as classic mycobacteria or classic species since they produce disease in animals and man. They are
1. Mycobacterium tuberculosis
2. M.bovis
3. M.avium
4. M.paratuberculosis
5. M.leprae
2. Anonymous, unclassified or atypical Mycobacteria: The classification of these group of organisms is based on rate of growth, colony morphology and pigment production. This classification was proposed by Runyon (1959). He classified atypical mycobacteria into four groups. These groups are further subdivided into number of subgroups based on biochemical reactions and cultural characteristics.
1. Group I. Photochromogenic: Produce yellow pigmented colonies that appear 7 days or later after exposure to light and slow growing (M.kansasii, M.marinum, M.asiaticum, M.simiae)
2. Group II. Scotochromogenic: Produce yellow or orange pigments in the absence of light and slow growing M.gordonae, M.scrofulaceum, M.szulgai, M.xenopi
3. Group III. Nonphotochromogenic: Produce no or slight pigment after exposure to light and are slow growing M.avium, M.intracellulare, M.terrae, M.ulcerans
4. Group IV: Variable pigmentation and fast growing. Growth appears in less than seven days M.phlei, M.smegmatis, M.fortuitum, and M.chelonei.

 

Cultural characters:

1. M.tuberculosis and M.bovis: These organisms are aerobic and grow at a temperature of 37-38^oC except M.avium that grows at 40^oC and M.piscium that grows at 25^oC. Growth does not occur in ordinary laboratory media but occurs in media that contains egg and serum after 10-14 days. The common media that contain egg and serum – Dorset egg medium is commonly used in cultivation of mycobacteria. The generation time is 18 hours and primary cultures are difficult to grow. The addition of glycerine to egg medium (Lowenstein-Jensen medium) enhances the growth of human strains where as it has no effect or inhibit the growth of bovine strains. The relatively luxuriant growth of human strains in the presence of glycerine is termed as eugoinc and less luxuriant growth of bovine strains are termed as dysgonic. Stonebrink’s medium contains inspissated egg with malachite green to inhibit the growth of contaminants and sodium pyruvate to enhance the growth of bovine strains is commonly used to cultivate bovine strains. In the fluid media these organisms form a tenacious friable bacterial masses instead of homogenous suspension since they strongly adhere to each other. The fluid media that are generally used contain casein hydrolysate, bovine serum albumen, asparagin and various salts.

The growth of M.tuberculosis is dry, crumbly and luxuriant. The colonies are yellow in colour with rough surface. Whereas the growth of M.bovis is dry, sparse, delicate and non-luxuriant. Chains of cells in smears made from in vitro-grown colonies often form distinctive serpentine cords. This observation was first made by Robert Koch who associated cord factor with virulent strains of the bacterium

2. M. avium: The optimal temperature for growth of avian strains is 40^oC. However, growth will also take place at 42-43^oC. Avian strains are fast growing and produce colonies that contain yellow or slightly pink pigments. They grow well in Dorset egg medium with enhanced growth in the presence of glycerine. The growth of M.avium is moist, slimy, glistening and luxuriant.

 

Pathogenesis:

Host affected

1. M.bovis: It mainly affects cattle. Swine are readily and severely infected. Dogs, horses, sheep are also affected. Cats are susceptible and may perpetuate the infection. In cattle pulmonary tuberculosis with involvement of lymph nodes is common. In human beings the infection is mainly due to consumption of milk. Chickens are resistant. Laboratory animals like rabbits, guinea pigs and mice are susceptible to infection.
2. M.avium: It mainly affects chicken. Cattle are sensitised. Infection in horses, dogs and cats is rare. Rabbits and guinea pigs are susceptible.
3. M.tuberculosis: Mainly affects human beings and primates. Cattle are sensitised. Pigs contract the infection by eating squid from infected source. Dogs are infected, where as cats are resistant. Chickens are rarely infected. Guinea pigs and mice are susceptible.

 

The infection is generally exogenous and the most important modes of infection are by ingestion and inhalation. Less frequently the organism also enters through skin and congenital (via placenta). Factors like crowding of animals in stalls, genetic factors like race and breed and resistance to tuberculosis both acquired and natural as a result of previous infection play a major role in the pathogenesis. Other factors like husbandry, hygiene, environment and ability of the organisms to withstand infection also contribute significantly in pathogenesis. M.bovis spread via respiratory discharges, faeces, milk, urine, semen and genital discharge. M.tuberculosis spread via sputum, fomites and respiratory discharges. M.avium spread via faeces. The manifestations of the disease are associated with the local lymphnode (mescentric in case of ingestion and tracheobronchal in case of inhalation). From the lymphnode the organisms reach thoracic duct and disseminated. At the visceral organs they produce tubercle nodules. In acute form, there is general dissemination where tubercle nodules are found through out (miliary tuberculosis). A progressive cellular granulomatous type of lesion with concentration of polymorphs is the first reaction, which is later, replaced by mononuclear cells with number of giant cells. These cells are separated from surrounding cells by macrophages, lymphocytes, plasma cells and fibrous tissue. As the size of the lesion increases, necrosis and caseation develops centrally that discharges caseous material containing bacilli. This caseous material is source of infection for other sites. The lesion later becomes cavity and calcification starts. Alternatively, there is also an exudative form that is characterised by exudate, which contains neutrophils, eosinophils, macrophages and fibrin. The tubercle lesions of M.bovis  are commonly found in lymph nodes of lungs, intestinal wall, mescentric lymph nodes, liver and spleen and lungs. Lesions of M.tuberculosis are found in lungs and associated lymph nodes where as the lesions of M.avium are found in digestive tract, liver, spleen and bone marrow. Lung lesions are rare in M.avium infection.

 

Symptoms: The symptoms depend upon the organs involved and the extent of lesion. In pulmonary TB, dry cough that increases in frequency accompanied by weight loss are the important symptoms. Generally symptoms of tuberculosis in visceral organs are not very specific. Tubercular mastitis is one of the common conditions in M.bovis infection.  In poultry tuberculosis is a disease of digestive tract, the symptoms include severe emaciation, pale mucous membranes and intermittent diarrhoea.

 

Lesions: Tuberculous nodules are seen in lungs in case of pulmonary form and in other visceral organs and associated lymph nodes in generalised infection. The nodules contain thick caseating mass, which tend to calcify. Miliary lesions resembling seeds of millet seeds develop when there s distribution of bacilli via blood stream. In pigs the skeleton especially vertebrae and long bones are common sites for tuberculosis.

 

Diagnosis:

1. Microscopical examination: Sputa, milk, uterine discharges, pleural and peritoneal fluids, urine or faeces are the ideal material for demonstration of organisms under this method. Samples of milk, uterine discharges, and pleural and peritoneal fluids should be centrifuged and the smear should be prepared from the sediment. An initial volume of 20-25 ml is essential.  The smears are stained by Ziehl-Neelsen method. The tubercle bacilli appear in clumps as slender rods stained pink in a blue background. In milk samples, the organisms are commonly seen in epitheloid cells.
2. Based on symptoms and lesions: The symptoms are generally inconclusive, where as the lesions have to differentiated from other bacterial infections where caseation is common.
3. Cultural examination: Demonstration of acid fast tubercle bacilli in the tubercle nodule is one confirmative methods for TB diagnosis. The clinical material have to be processed in the following method before attempting for isolation.
1. The fat has to be trimmed off and the clinical material should be treated with 10ml 4% NaOH containing phenol red,
2. Then the tissue should be crushed in a sterile mortar and pestle with sterile sand.
3. The sediment should be neutralised with 2N HCl for a maximum of 30 minutes.
4. The sediment should be centrifuged at low speed for 20 minutes and decant the supernatant.
5. The sediment should be inoculated into media like Lowenstein-Jensen medium or Stonebrink medium or egg yolk agar. The cultures should be kept for a period of atleast 8 weeks before discarding them as negative.

 

Alternative methods are to treat the samples with 5% oxalic acid,10%trisodium phosphate, 4% caustic soda or 20% antiformin.

 

Colonial morphology:

a.              M.tuberculosis and M.bovis: These organisms are aerobic and grow at a temperature of 37-38^oC except M.avium that grows at 40^oC and M.piscium that grows at 25^oC. Growth does not occur in ordinary laboratory media but occurs in media that contains egg and serum after 10-14 days. The common media that contain egg and serum – Dorset egg medium is commonly used in cultivation of mycobacteria. The generation time is 18 hours and primary cultures are difficult to grow. The addition of glycerine to egg medium (Lowenstein-Jensen medium) enhances the growth of human strains where as it has no effect or inhibit the growth of bovine strains. The relatively luxuriant growth of human strains in the presence of glycerine is termed as eugoinc and less luxuriant growth of bovine strains are termed as dysgonic. Stonebrink’s medium contains inspissated egg with malachite green to inhibit the growth of contaminants and sodium pyruvate to enhance the growth of bovine strains is commonly used to cultivate bovine strains. In the fluid media these organisms form a tenacious friable bacterial masses instead of homogenous suspension since they strongly adhere to each other. The fluid media that are generally used contain casein hydrolysate, bovine serum albumen, asparagin and various salts. The growth of M.tuberculosis is dry, crumbly and luxuriant. The colonies are yellow in colour with rough surface. Whereas the growth of M.bovis is dry, sparse, delicate and non-luxuriant.

b.              M. avium: The optimal temperature for growth of avian strains is 40^oC. However, growth will also take place at 42-43^oC. Avian strains are fast growing and produce colonies that contain yellow or slightly pink pigments. They grow well in Dorset egg medium with enhanced growth in the presence of glycerine. The growth of M.avium is moist, slimy, glistening and luxuriant.

 

4. Animal inoculation: Guinea pigs are the laboratory animals of choice. Rabbits and chicken can also be used. Suspected materials are inoculated into the thighs of guinea pig. Two animals are inoculated for each sample. The animals are killed after 4-6 weeks and examined for typical lesions in the liver, spleen and lymph nodes.

Rabbits are used to differentiate human, bovine and avian types. Rabbits generally die from generalised infection with bovine type 4-5 weeks after intravenous injection, but survive similar infection with human type, Avian type is less virulent for rabbits and produce chronic form of the disease unless the inoculum given in large dose. It does not produce macroscopically visible tubercles. However, the spleen and liver become enlarged containing tubercle bacilli. This type of infection is referred as Yersin type of infection.

5. Polymerase chain reaction
6. DNA probe based diagnosis 

 

Treatment: Treatment is not generally feasible in animals. One of the common drugs is isoniazid. In humans para-aminosalicylic acid, ethambutol combined with streptomycin constitute the triple therapy.

 

Control and prevention:

Tuberculin testing: In the field, tuberculosis is mainly identified by tuberculin testing. The basis of the test is that animals infected with tuberculosis develop a hypersensitivity to M.tuberculosis, so when an extract of the M.tuberculosis  called tuberculin is injected intradermally into the skin, a localised hypersensitive reaction occurs at site of injection. This reaction is not produced in health animals. Two types of tuberculins are commonly used – old tuberculin (OT) and protein purified derivative (PPD). OT was prepared by evaporating a glycerinated brothculture to one-tenth of its volume. This preparation has got only little active priciple besides various non specific proteins. PPD is a pure form, where the active principle is precipitated by trichloracetic acid and some other precipitant. Tuberculin is prepared from human, bovine and avian types. Different types of tests are used to test animals using tuberculin.

1.      Intradermal test: In this method 0.1 ml of tuberculin is injected at caudal fold, vulvar lip or sides of neck. A firm swelling after 72 hours indicates positive reaction. Thickness of swelling 3mm is doubtful and 4mm is positive.

2.      Comparative cervical: Intradermal inoculation of regular and avian tuberculin and various sites on the sides of neck and results are read after 72hours.

3.      Ophthalmic: Used mostly for primates. 0.1 ml of 1:10 dilution of bovine tuberculin is injected intradermally into the upper eyelid.

 

Vaccination:  Though a number of vaccines were tried, the best vaccine is a live attenuated vaccine known BCG (Bacille Calmette Guerin). Animals vaccinated with this vaccine give positive reaction to tuberculin testing and the immunity offered by this vaccine is also questionable. Calfhood vaccination is commonly practiced.

 

Control:

1. The control methods are aimed at identifying the reactors and eliminating them from the herd. For this tuberculin testing is routinely employed in the herd. Comparative tuberculin testing is used when the results of single intradermal test is questionable. Calfhood vaccination is also advisable.
2. The animal sheds containing infected material are sterilised by first washing them with water under pressure and them with disinfectant under pressure. The two common disinfectants used are cresylic compound and sodium orthophenylphenate.
3. Since it is a zoonotic disease and one of the common mode of spread in humans is by ingestion of milk from affected animals, raw milk should never should be consumed and only pasteurised milk should be consumed.
4. The infection may also spread to humans from affected pet animals. Hence such animals should not be maintained in close proximity.
5. Pigs can get human type infection by consuming squid. Hence if pigs are fed with squid the source should be clean.

During meat inspection the affected carcass should be condemned and should not be fed to carnivore animals.

VMC LECTURE # 12

MYCOBACTERIUM – PART II

 

PARATUBERCULOSIS

Organism – Mycobacterium avium sub sp. paratuberculosis (M.johnei)

Johne's disease (pronounced "yo-knees") is a contagious, chronic and usually fatal infection that affects primarily the small intestine of ruminants. All ruminants are susceptible to Johne's disease. Johne's disease is caused by Mycobacterium paratuberculosis, a hardy bacteria related to the agents of leprosy and tuberculosis. The disease is worldwide in distribution. Johne's disease has been diagnosed in cattle, goats, sheep, deer, llamas, camels and other wild ruminants. It is also speculated that M. paratuberculosis is also the causative agent for Crohne's disease in humans. This disease is characterized by ulceration of the intestine causing extreme pain and debilitation. However, to date this research has not been confirmed and the organism has not been cultured from human cases.

 

Morphology and general characters: It is a gram-positive, rod shaped organism with rounded ends. The shape changes in artificial media into club form. It is a non-motile, non-sporulative and non-capsulated organism. It is strongly acid-fast. The cell wall constituents and resistance of M.paratuberculosis resemble other Mycobacteria. The organisms can remain viable for a long time in dried faeces in the pasture. They are resistant to disinfectants and to drying by the sun. Snow cover will help their survival whereas sunlight, drying or exposure to intense cold will reduce their numbers.

 

Strains and antigenic relationship: This organism has got a close antigenic relationship with avian tubercle bacilli. It has been found that animals with paratuberculosis often react positively to avian tuberculin. There are three major strains of this bacteria (which is related but different from Mycobacterium avium subsp. avium, a disease of birds and rarely humans [avium tuberculosis]). There is a cattle strain, a sheep strain (Islandic strain) and an intermediate strain (yellow or orange pigment producing strain). While cattle are susceptible to all three strains, they are usually not infected with the sheep strain. Sheep usually get only the sheep strain but can also succumb to the intermediate strain. Goats usually have the cattle strain.

 

Cultural characters: They are aerobic organisms and are obligatory parasites. They are difficult to cultivate in ordinary laboratory media. However, the organisms will grow slowly in glycerine egg media containing extracts from other killed acid-fast bacteria. The extract of M.phlei (called Mycobactin) is commonly used in the media used for cultivation of M.paratuberculosis. The optimum temperature for growth is 38-39^oC. However, growth is reported from 20-40^oC. Initially the colonies appear as minute, greyish white, friable and irregular. Later, these colonies turn into pale yellow in colour and become large. Subcultures from primary cultures grow better.

 

Pathogenesis: The main mode of infection is by ingestion of the organism from contaminated environment. Young animals are more susceptible. The most common mode of infection for young animals is by ingestion of milk contaminated with faeces from animals having paratuberculosis. The infection is characterised by long incubation period (ranging from 12 months to many years). The pathogenesis of the infection is correlated with high amount of intracellular iron. Huge consumption of iron containing feed is also correlated with the severity of the lesion and shedding of bacteria. Cattle and sheep are mostly affected. Pigs are rarely affected. Mycobacteria are taken up by specialized cells (M cells) in the ileum. M cells present the microbe to macrophages and lymphocytes in Peyer’s patches. The immune system reacts to the invasion by recruiting more macrophages and lymphocytes to the site of infection. Lymphocytes release a variety of cytokines, in an attempt to increase the bacterial killing power of the macrophages. Macrophages fuse forming large cells in an apparent attempt to kill the mycobacteria. Infiltration of infected tissues with millions of lymphocytes and macrophages leads to visible thickening of the intestine. Late in the infection, antibody production by the animal occurs, but this does not affect control of mycobacterium’s multiplication.

 

Sheep become infected through eating feed contaminated with faecal material. Infected sheep on pasture will contaminate that pasture for other grazing animals. Lambs born into these contaminated environments are most susceptible to infection. Dirty udders and wool tags, barn, yards, bedding, etc are all sources of infection to lamb. The bacteria have a very thick cell wall and can survive in the environment for perhaps as long as a year. The bacteria invade the intestine and the intestinal lymph nodes where they become established and interfere with absorption of nutrients, hence weight loss occurs. This progression, however, takes two to seven years to go to completion, depending on the number of bacteria that infect the sheep and the age at which the sheep is infected. When they are first infected, sheep do not shed the bacteria. This period of incubation may be as short as a year or as long as 5 yrs but is often 2 to 3 yrs. Although Johne's disease is primarily a disease of the intestine, during the advanced stages of disease the agent may spread throughout the body. Foetuses are very susceptible to infection while still in the womb. Lambs may be born infected (in cattle, up to 60% of calves born to cows in advanced disease, were infected) and these animals may develop clinical disease at an earlier age.

 

Symptoms:

Cattle: It is a slow progressive infection. Hence, signs of Johne’s disease are not seen until years after initial infection. Cattle may be infected for years before they show any signs of disease.  The signs of Johne’s disease are intermittent bouts with diarrhoea eventually becoming chronic diarrhoea and weight loss (wasting – cachexia) despite a good appetite. Affected cattle do not generally have a fever. Some infected animals appear unthrifty and often weak while others just have chronic diarrhoea. The signs of this disease can easily be confused with several other diseases. In the infected cow or heifer, noticeable signs commonly start within a few weeks after a stressful event like calving. In the sub-clinical stages cattle are more susceptible to other infectious diseases. These animals also may suffer from inefficient utilization of nutrients and drop off in performance especially milk production. In cattle, the bacteria have been isolated from semen from bulls with advanced Johne's disease. Some cows may develop "bottle jaw" due to a low protein edema, or appear unthrifty overall. Clinical signs of the disease occur more commonly during the end stages of the infection, typically at three to six years of age.  

Sheep: Sheep can become infected at any age. When infected as a lamb or foetus, then the disease is manifested at 18 months of age. Stress such as lambing or mastitis will hasten the onset of more severe clinical disease. The ewe is depressed and pale and very thin and about 20% of cases develop diarrhoea. Diarrhoea indicates that the sheep is nearing the end and will die soon. The infection is also mistaken for other wasting type diseases like bad teeth due to incisor loss (broken mouth) or uneven molar wear and gingivitis. Slow virus infections in sheep also cause wasting. Severe parasitism, intestinal abscesses due to caseous lymphadenitis can also cause wasting. Management problems like poor nutrition and competition for feed can also cause wasting. In sheep wasting associated with or without diarrhoea is seen

 

Lesions: Lesions are seen only in the intestine and associated lymph nodes (mesenteric nodes). Lesions are more pronounced in terminal part of the intestine particularly in rectum and ileo-caecal valve. The intestinal lesions are characterised by thickening of the intestinal cell wall. Due to thickening, the intestine looks like a hosepipe. The thickening is due to infiltration of mucosa by plasma cells and endothelial cells.  There is neither giant cell formation nor caseation and calcification of lesions that are characteristic of tuberculosis. In the initial stages, the intestinal wall is oedematous with haemorrhagic streaks. In chronic cases, the intestinal mucosa is wrinkled and corrugated resembling brain. The raised areas are warty where as the depressed areas are smooth. The lymph nodes are oedematous, enlarged and spongy. In sheep infected with pigmented strains, orange discoloration of the mucosal surface is common. Caseation and calcification are sometimes noticed in sheep.

 

Diagnosis:

1.      Based on symptoms and lesions.

2.      Microscopical examination: Rectal pinch smears stained by Ziehl-Neelsen method reveal acid-fast organisms that appear as pink in colour, single or in characteristic clumps. However, to provide a confirmative diagnosis, multiple samples have to be taken from different areas in rectum. However, normally the first diagnosis is often made at necropsy. As mentioned before, the intestinal lymph nodes become enlarged and the intestinal wall may appear thickened. Signs in sheep are more subtle than in cattle, in which the large intestine becomes quite thickened and corrugated like cardboard. Bacteria can be stained and viewed by a microscope. Sometimes the bacteria can be seen in a faecal smear, particularly in animals that have reached the diarrhoea stage. To be diagnostic however, large numbers of bacteria must be seen and follow-up tests need to be done.

3.       Smears from biopsy material of lymph node or terminal ileum stained by Ziehl-Neelsen method reveal acid-fast organisms that appear as pink in colour, single or in characteristic clumps.

4.     Cultural examination: It is not very successful in sheep. In cattle and goats, the bacteria can be cultured about 60% of the time when they are present in the faeces. However, the bacteria are very slow growing and take 4 to 16 weeks to grow. This means that negative results take at least 4 months and then a negative result may only mean failure to grow or that the animal is still in the early stages of disease. The situation in sheep is even worse. Most of the diagnostic laboratories in North America and elsewhere are unable to grow the bacteria with any degree of success. Sensitivity of bacterial culture in sheep even when showing severe clinical disease is only 8% (8 animals culture positive out of 100 shedding animals). Faeces are the material of choice for culturing. Faeces have to be treated with 20 per cent antiformin for twenty minutes or with malachite green and oxalic acid before culturing. Penicillin has to be added to the media. The organisms will grow slowly in glycerine egg media containing extracts from other killed acid-fast bacteria. The extract of M.phlei (called Mycobactin) is commonly used in the media used for cultivation of M.paratuberculosis. The optimum temperature for growth is 38-39^oC. However, growth is reported from 20-40^oC. Initially the colonies appear as minute, greyish white, friable and irregular. Later, the colonies turn into pale yellow in colour and become large. Subcultures from primary cultures grow better.

5.     Serological tests like AGID, ELISA, CFT, RIA, and FAT etc can be performed to diagnose Johne’s disease. These tests have very less sensitivity.

6.     DNA probes are used to rapidly identify M.paratuberculosis in faeces.

7.     Johnin test: PPD prepared from M.paratuberculosis is used to screen animals for paratuberculosis. But this method is not reliable.

8.     Gamma-interferon assay:  It is done in cattle. In cattle, the sensitivity is thought to be between 70 and 94% with excellent specificity (almost 100%). This test proves to be a good test for cattle but evaluation is pending for sheep.

 

Control:

1. Removal of animals with chronic diarrhoea and wasting.
2. Faecal culture of animals (2 years and above) in a herd, once in six months and removal of animals positive for paratuberculosis.
3. Disinfection of the suspected premises with cresylic compounds.
4. Clean animal husbandry practices that include separate rearing of calves.
5. Ploughing infected grazing land will dilute the bacteria and help to kill them.
6. AI from bulls negative in Johnin test.
7. A live vaccine using avirulent M.paratuberculosis strains is used for calves and sheep. This vaccine given to calves at 4 months of age reduces incidence of paratuberculosis. This vaccine given along with an adjuvant that includes mineral oil, liquid paraffin and ground pumice stone.

VMC LECTURE # 13

PASTEURELLA

Taxonomic name: Pasteurella multocida subsp. multocida (Lehmann and Neumann 1899) Rosenbusch and Merchant 1939. [Source: NCBI]

The species Pasteurella multocida includes a heterogeneous group of Gram-negative bacteria that are inhabitants of the upper respiratory tract of many vertebrate hosts including birds, cattle, swine, cats, dogs and rodents. Members of this species are responsible for a number of infections that normally are secondary to colonization of the upper respiratory tract including avian cholera (in waterfowl, chickens and turkeys), respiratory disease and hemorrhagic septicemia in ruminants (cattle, sheep, goats and buffalo), atrophic rhinitis in pigs and snuffles/septicemia in rodents (mice & rabbits). These infections are primarily transmitted by the respiratory route and are associated with crowding and other stressors. P. multocida is also a rare cause of infection in humans that is normally associated with dog or cat bites or scratches.

 

 

General characters:

1. Morphology: Members of the genus Pasteurella gram-negative short ovoid shaped organisms. In laboratory media they also appear as long rods. Generally the shape of the organisms is referred as cocco-bacilli. They are non-motile and non-sporulative organisms. They possess a capsule that is composed of polysaccharide and hyaluronic acid. They are facultatively anaerobic organisms, fermentative and oxidase positive. These organisms are known for their bi-polar staining property in tissue smears stained by Leishman’s stain or methylene blue.
2. Habitat: They are found as commensals in the upper respiratory and digestive tracts of animals. They are also found on mouth, tonsils and nasopharynx.
3. Classification: The latest classification of Pasteurella is based on DNA analysis. Based on this method, the important species of veterinary importance and the diseases produced by them are listed as follows;
1. P.multocida subsp multocida – causes HS in cattle
2. P.multocida subsp septica      - causes wound contamination
3. P.multocida subsp gallicida    - causes fowl cholera
4. P.gallinarum                             - respiratory tract infection
5. P.canis                                       - dog bit wound infections
6. P.avium                                     - Haemophilus avium
4. Resistance: They are not very resistant organisms. They are killed by chemical and agents like 0.5% phenol when exposed for 15 minutes. They are also killed on exposure to sunlight for 3-4 hours and heating at 55^oC for 15 minutes.
5. Antigens and toxins: Based on differences in capsular substances, P.multocida classified into A, B, D, E and F. These types are further subdivided into 16 subtypes based on differences in somatic antigens. These subtypes are assigned numerals. Thus a serotype is designated by an alphabet followed by a number eg. Seroype B2 causes HS. A toxin in addition to endotoxin (LPS) is produced by B serotype.

 

Cultural characters: They are aerobic or facultatively anaerobic organisms and grow at a temperature of 37^oC. In ordinary media, three types of colonies – mucoid, smooth (iridescent) and non-iridescent are produced after overnight incubation. The mucoid colonies are round, flat, mucoid and sticky of 2-3 mm in diameter. Organisms with abundant capsular material produce these types of colonies. Organisms with less capsular material produce the iridescent colonies. Whereas rough colonies are produced by organisms that have no capsular material. Blood agar is preferred medium for growth. There is darkening of the medium with no haemolysis and a specific odour. In broth cultures they produce uniform turbidity.

 

Biochemical characters: HS organisms produce oxidase, catalase and indole, and will reduce nitrates. They do not produce hydrogen sulphide or urease, and fail to use citrate or liquefy gelatin. Glucose and sucrose are always fermented with the production of acid only. Most strains also ferment sorbitol. Some strains ferment arabinose, xylose and maltose, whereas salicin and lactose are almost invariably not fermented

 

A. HAEMORRHAGIC SEPTICAEMIA

Haemorrhagic septicemia (HS) is an acute pasteurellosis, caused by particular serotypes of Pasteurella multocida and manifested by an acute and highly fatal septicemia principally in cattle and water buffaloes; the latter are thought to be more susceptible than cattle. HS occurs infrequently in swine and even less commonly in sheep and goats. It has been reported in bison, camels, elephants, horses, and donkeys, and there is evidence of its occurrence in yak. Presently, two serotypes are recognised – the Asian serotype and the African serotype. The Asian strains belong to capsular type B only while the African strains are types B and E. Epidemic HS is caused by one of two serotypes of P multocida, designated B:2 and E:2.

 

Pathogenesis: Infection is exogenous and animals acquire the infection by contact, inhalation or ingestion. It is hypothesized that animals become susceptible as a result of various stresses, eg, the inanition seen in cattle and water buffalo at the beginning of the rainy season. The heaviest losses occur during the monsoon rains in south east Asia, and it is thought that the organisms, which can survive for hours and probably days in the moist soil and water, are transmitted widely at this time.  Biting arthropods rarely transmits infection. Pasteurella organisms are found in the upper respiratory and digestive tract of animals and birds as commensals. Most of the animals carry these organisms with them with out exhibiting any symptoms. The organisms produce infection when they are exposed to stress conditions like extreme weather conditions, transport, immunosuppression etc. During these conditions the organisms rapidly multiply and excreted via droplets and through digestive tract. The droplets with organisms are the main source of infection for other animals. In Pasteurella infection, the morbidity is always higher. P.multocida is also a secondary invader in respiratory tract infection. The important virulence factor is the endotoxin (LPS). Besides this a thermostable, cell associated toxin that is released by dying cells also plays a role in pathogenesis.   

 

Symptoms: The clinical syndrome of HS consists of an initial phase of temperature elevation (often unnoticed), a phase of respiratory involvement, and a terminal phase of septicaemia and recumbency leading to death. The incubation period is usually 1–3 days, and the course of the disease may range from sudden death, with no observable clinical signs, to a protracted course extending up to 5 days. Buffaloes are generally believed to be more susceptible to HS than cattle, and in this species, the disease course is shorter. In endemic areas, most deaths are confined to older calves and young adults. In nonendemic areas, massive epizootics may occur. Case fatality approaches 100% if treatment is not carried out sufficiently early (in the pyrexic stage). Three forms of HS are noticed among animals – acute, sub-acute (oedematous form) and sub acute (pectoral form). The important symptoms in acute cases are rise in body temperature, drop in milk yield, abdominal pain, severe diarrhoea and dysentery. The respiration becomes rapid before death and the mucous membrane becomes cyanotic. Less acute cases are characterised by rise in body temperature and oedema in the head, neck and brisket region. A purulent, blood stained nasal discharge is also noticed. Occasional cases linger for several days. Recovery is rare. There appears to be no chronic form.

 

Lesions: The most obvious changes in affected animals are the edema, widely distributed hemorrhages, and general hyperemia. In most cases, there is an edematous swelling of the head, neck, and brisket region. Incision of the swellings reveals a clear or straw-colored serous fluid. The edema is also found in the musculature, and the subserous petechial hemorrhages, which are found throughout the animal, are particularly characteristic. Blood-tinged fluid is often found in the pericardial sac and in the thoracic and abdominal cavities. The lesions are also coupled with gastroenteritis, marbled lungs, blood stained stools and enlarged and haemorrhagic mesenteric lymph nodes Petechial hemorrhages are seen in the pharyngeal and cervical lymph nodes. Gastroenteritis is seen only occasionally and, unlike pneumonic pasteurellosis, pneumonia usually is not extensive.

 

Diagnosis:

1. Based on symptoms and lesions.
2. Microscopical examination: The septicaemia in HS occurs at the terminal stage of the disease. Therefore, blood samples taken from sick animals before death may not always contain P. multocida organisms. Also, they are not consistently present in the nasal secretions of sick animals. A blood sample or swab collected from the heart with in few hours after death is most ideal. Blood smears from affected animals are stained with Gram, Leishman’s or methylene blue stains. The organisms appear as Gram-negative, bipolar-staining short bacilli. No conclusive diagnosis can be made on the basis of direct microscopic examinations alone.
3. Isolation and identification: Blood in transport medium is the most ideal material for isolation. If the animal has been dead for a long time, a long bone, free of tissue, can also be taken. The most ideal medium for isolation of Pasteurella is casein/sucrose/yeast (CSY) agar containing 5% blood (calf blood). Freshly isolated P. multocida forms smooth, greyish glistening translucent colonies, approximately 1 mm in diameter, on blood agar after 24 hours’ incubation at 37°C. Colonies grown on CSY agar are larger. Old cultures, particularly those grown on media devoid of blood, may produce smaller colonies.
4. Test to confirm the production of hyaluronidase: HS-causing strains of P. multocida has the ability to produce the enzyme hyaluronidase. A hyaluronic-acid-producing culture is streaked across the centre of a dextrose starch agar plate. The pasteurella culture to be tested for hyaluronidase production is streaked at right angles. The plates are incubated at 37°C for 18 hours. At the point of intersection, the mucoid growth of the hyaluronic acid producer will diminish into a thin line of growth, indicating the production of hyaluronidase by the test culture.
5. Immunological methods: Several immunological tests are used for the identification of the HS-causing serotypes of P. multocida. These consist of a rapid slide agglutination test, an IHA test for capsular typing, the AGID test, and the counter immunoelectrophoresis test (CIEP).
6. Nucleic acid recognition methods: PCR amplification of specific DNA sequences allows rapid detection and presumptive identification of organisms directly from either clinical specimens or from small amounts of mixed or pure bacterial cultures. Some of the common methods performed to identify Pasteurella are as below

                                                              i.      Pasteurella-multocida-specific PCR assay

                                                             ii.      Pasteurella multocida multiplex capsular PCR typing system

                                                           iii.      HS-causing type-B-specific PCR assay

                                                          iv.      Genotypic differentiation of isolates

7. Serological methods: Serological tests for detecting antibodies are not normally used for diagnosis.
8. Animal Inoculation: The mouse usually serves as a biological ‘screen’ for extraneous organisms. A small volume (0.2 ml) of eluted blood swabs or a portion of bone marrow in saline is inoculated subcutaneously or intramuscularly into mice. If viable P. multocida is present, the mice die 24–36 hours following inoculation, and a pure growth of P. multocida can be seen in blood smears.

 

Treatment: Various sulfonamides, tetracyclines, penicillin, and chloramphenicol (are effective if administered early. Because of the rapid course of the disease and the frequent difficulty of access to animals, antimicrobial therapy often is not practicable.

 

Control:

The principal means of prevention is by vaccination. Three kinds of vaccine are widely used: plain bacterin, alum-type precipitated bacterin, and oil-adjuvant bacterin. The most effective bacterin is the oil-adjuvant—one dose provides protection for 9-12 months; it should be administered annually. The alum-precipitated-type bacterin is given at 6-months intervals. A live vaccine prepared from a B:3,4 serotype of deer origin is being used with reported success in southeast Asia.

 

B. FOWL CHOLERA (AVIAN CHOLERA)

Fowl cholera (avian pasteurellosis) is a commonly occurring avian disease that can affect all types of birds and is often fatal. It is contagious and usually occurs as a septicemia of sudden onset with high morbidity and mortality, but chronic and asymptomatic infections also occur.

 

Pathogenesis: Pasteurella multocida, the causal agent, is a small, gram-negative and nonmotile rod. Although P multocida may infect a wide variety of animals, strains isolated from nonavian hosts generally do not produce fowl cholera. Strains that cause fowl cholera represent a number of immunotypes, which complicates widespread prevention by using bacterins. The organism is susceptible to ordinary disinfectants, sunlight, drying, and heat. Turkeys are more susceptible than chickens, older chickens are more susceptible than young ones, and some breeds of chickens are more susceptible than others. Chronically infected birds are considered to be a major source of infection. Dissemination of P multocida within a flock is primarily by excretions from mouth, nose, and conjunctiva of diseased birds that contaminate their environment.

 

Symptoms and lesions: These vary greatly depending on the course of disease. In acute fowl cholera, dead birds are usually the first indication of disease. Fever, depression, anorexia, mucoid discharge from the mouth, ruffled feathers, diarrhea, and increased respiratory rate are usually seen. Many of the lesions are related to vascular disturbances. Hyperemia is especially evident in the vessels of the abdominal viscera. Petechial and ecchymotic hemorrhages are common, particularly in subepicardial and subserosal locations. Increased amounts of peritoneal and pericardial fluids are frequently seen. Livers may be swollen and often develop multiple, small, necrotic foci. Pneumonia is particularly common in turkeys. In chronic fowl cholera, signs and lesions are generally related to localized infections. Sternal bursae, wattles, joints, tendon sheaths, and footpads are often swollen because of accumulated fibrinosuppurative exudate. There may be exudative conjunctivitis and pharyngitis. Torticollis may result when the meninges, middle ear, or cranial bones are infected.

 

Diagnosis:

1.      Based on the symptoms and lesions

2.      Microscopical examination: . The cells are coccobacillary or short rod-shaped, usually 0.2–0.4 by 0.6–2.5 µm in size, stain Gram negative, and generally occur singly or in pairs. Recently isolated organisms or those found in tissue smears show bipolar staining with Wright or Giemsa stains or methylene blue, and are usually encapsulated.

3.      Isolation and identification: Isolation of the organism from visceral organs, such as liver, bone marrow, spleen, or heart blood of birds that succumb to the acute form of the disease, and from exudative lesions of birds with the chronic form of the disease, is generally easily accomplished. Pasteurella multocida is a facultative anaerobic bacterium that grows best at 35–37°C. Primary isolation is usually accomplished using media such as blood agar, trypticase–soy agar or dextrose starch agar, and isolation may be improved by supplementing these media with 5% heat-inactivated serum. Colonies range from 1 to 3 mm in diameter after 18–24 hours of incubation. They usually are discrete, circular, convex, translucent, and butyraceous. Capsulated organisms usually produce larger colonies than those of noncapsulated organisms. Watery mucoid colonies, often observed with mammalian respiratory tract isolates, are very rare with avian isolates.

4.      Nucleic acid identification methods: The most ideal method is the DNA fingerprinting of P. multocida by restriction endonuclease analysis (REA).

5.      Serological tests: Serological tests for the presence of specific antibodies are not used for diagnosis of fowl cholera.

 

Treatment: Sulfonamides and antibiotics are commonly used; early treatment and adequate dosages are important. High levels of tetracycline antibiotics in the feed (0.04%) or administered parenterally may be useful.

 

Control: Good management practices are essential to prevention. Adjuvant bacterins are widely used and generally effective; autogenous bacterins are recommended when polyvalent bacterins are found to be ineffective. Attenuated vaccines are available for administration in drinking water to turkeys and by wing-web inoculation to chickens. These live vaccines can effectively induce immunity against different serotypes of P multocida. They are recommended for use in healthy flocks only.

 

 

C. OTHER IMORTANT PASTEURELLA INFECTIONS

 

 

1. Rabbits: Peracute infection is common in rabbits. In chronic infection coryza like respiratory symptoms are common.
2. Sheep: Mastitis (blue bag) and pneumonia
3. Dogs and cats: Septic contamination of wounds
4. Pigs: Respiratory infection and rhinitis. Atrophic rhinitis is an infectious disease of swine characterised by purulent nasal discharge, shortening or twisting of the snout, atrophy of the turbinate (conchal) bones and reduced productivity. It may occur enzootically or more sporadically, depending on a variety of factors including herd immunity. The most severe progressive form is caused by infection with toxigenic strains of Pasteurella multocida alone or in combination with Bordetella bronchiseptica.

 

OTHER PASTEURELLA INFECTIONS (Organisms renamed)

Mannheimia haemolytica (P. heamolytica): Unlike P.multocida, it is beta haemolytic. Causes transport of shipping fever in cattle. It is a pneumonic condition with rise in temperature, rapid respiration followed by death with 12-48 hours. In less acute cases coughing, debility and death are noticed.

 

Riemeralla anatipestifer (P.anatipestifer): It is a non-fermenting bacterium. It causes infectious serositis (New duck disease) in ducklings. Anatipestifer infection causes high mortality, weight loss and condemnation. In the acute form, listlessness, eye discharge and diarrhea are commonly seen. Ducks show incoordination, shaking of the head and twisted neck. Birds are commonly found on their backs, paddling their legs. Typical lesions found in dead birds are infected air sacs, membranes covering the heart and liver, and meningitis. Preventive management and vaccination are effective means of control. Penicillin, enrofloxacin and sulfadimethoxine-ormetoprim (0.04-0.08% in feed) are effective in reducing mortality.

VMC LECTURE # 14

BRUCELLA

Brucellosis is caused by bacteria of the genus Brucella and is characterized by abortion, retained placenta, and to a lesser extent, orchitis and infection of the accessory sex glands in males. Diagnosis depends on the isolation of Brucella from aborted materials, udder secretions or from tissues removed at post-mortem. Presumptive diagnosis can be made by assessing specific cell-mediated or serological responses to Brucella antigens. The disease is prevalent in most countries of the world. It primarily affects cattle, buffalo, bison, pigs, sheep, goats, dogs, elk, and occasionally horses. The disease in man, sometimes referred to as undulant fever, is a serious public health problem, especially when caused by B melitensis

 

Classification: All members of the Brucella genus are closely related. There are six classical nomenspecies: Brucella abortus, B. melitensis, B. suis, B. neotomae, B. ovis and B. canis. The first three of these are subdivided into biovars based on cultural and serological properties. Two species B. cetaceae and B. pinnipediae have been isolated from marine organisms. Brucella shows close genetic relatedness to some plant pathogens and symbionts of the genera Agrobacterium and Rhizobium, as well as, animal pathogens (Bartonella) and opportunistic or soil bacteria (Ochrobactrum).

Important diseases    -        Brucellosis, Bang’s disease, Malta fever, Undulant

Fever, Fistulous withers, Poll evil

OIE listing: List B disease

 

General characters:

1. Morphology: Brucella are coccobacilli or short rods measuring from 0.6 to 1.5 µm long and from 0.5 to 0.7 µm wide. They are usually arranged singly, and less frequently in pairs or small groups. The organisms appear in pleomorphic forms in old culturs. Brucella are nonmotile. They do not form spores, and do not have flagella, pili, or true capsules. Brucella are Gram negative and usually do not show bipolar staining. Although they are not acid-fast organisms, they withstand decolourisation of 0.5 per cent acetic acid.
2. Resistance: The organisms are killed by heating them to 60^oC for 10 minutes and in milk readily killed by pasteurisation. They are susceptible to acidic pH, disinfectants and sunlight. They can survive for a long time foetus that is not exposed to sunlight. They can remain for a long time in cold temperatures.
3. Antigens and Toxins: Brucella produce two types of colonies smooth and rough that are associated with virulence, auto-agglutination etc. Virulent organisms produce smooth colonies and avirulent organisms produce rough colonies. Certain organisms produce intermediate colonies. B.abortus, B.melitensis and B.suis produce two antigens called A and M. Amount of these antigens in these species varies widely. The A and M antigens occur in ration of 20:1 in B.abortus and still lower in B.suis. Where as it occurs in the ratio of 1:20 B.melitensis. The LPS (endotoxin) is the important virulence factor. The organisms produce no exotoxins.

 

Cultural characters: They are aerobic organisms. However, certain organisms like B.abortus and B.suis require 5-10% CO₂ tension. They are slow growing organisms and growth is visible after 48 hours at a temperature of 37^oC. Numbers of media are used for the cultivation of organisms like nutrient agar, serum agar, liver infusion serum agar, tryptose agar, and dextrose potato and glycerol potato agar. On nutrient agar the colonies are small, round, convex, translucent with a smooth glistening surface. In broth cultures they produce fine granular deposit. Other media used include serum–dextrose agar (SDA) or glycerol dextrose agar. A nonselective, biphasic medium, known as Castañeda’s medium, is used for the isolation of Brucella from blood and other body fluids or milk.

 

Biochemical characters: They utilise carbohydrates but produce very little or no acid and gas. H₂S production is variable and this character is useful to differentiate brucella species. Production of urease by various species is also variable. Inhibition of growth in the presence of different concentration of dyes like thionin, basic fuchsin, methyl violet and pyronin is also useful character in differentiation of various brucella species. In general requirement of CO₂ for growth, hydrogen sulphide production, urease production and sensitivity to various dyes are the characters that are used to differentiate different Brucella species.

 

BRUCELLOSIS IN CATTLE

The disease in cattle, water buffalo, and bison is mostly caused by Brucella abortus. However, B suis or B melitensis occasionally cause infection in cattle.

Pathogenesis: Brucella infection in a herd is through introduction of infected animals. The main sources of infection are aborted foetuses, placentae and genital discharge, milk and semen of infected bulls. Natural transmission occurs by ingestion of organisms, which are present in large numbers in aborted fetuses, fetal membranes, and uterine discharges. Cattle may ingest contaminated feed and water, or lick contaminated genitals of other animals. Venereal transmission by infected bulls to susceptible cows appears to be rare. Transmission may occur by artificial insemination when Brucella -contaminated semen is deposited in the uterus but, reportedly, not when deposited in the midcervix. Brucellae may enter the body through mucous membranes, conjunctivae, wounds, or even intact skin. Calves are relatively insusceptible and excrete the organisms in faeces and serve as source of infection for other animals. B.abortus organisms are excreted in the colostrum and milk after parturition for many months up to 2 years. Mechanical vectors (eg, other animals, including man) may spread infection. The organisms pass from point of entry to the lymphatics to lymph node and multiply in thoracic duct. From there it reaches the paranchymatous organs and other tissues. They have the capacity to survive inside phagocytic cells. In the paranchmatous organs, they develop granulomatous lesions. They have an affinity for gravid uterus and foetal tissues. The affinity of brucella organisms for foetal tissue is due to erythritol – a saccharide alcohol that is present in placenta of cattle, sheep, goat and pigs. In male animals the organisms multiply in testicles, seminal vesicles and epididymis.

Symptoms: Abortion during last stages of pregnancy is the important symptom (seventh or eighth month of pregnancy in cattle, third or fourth month in sheep, 50^th day in dogs). In cattle abortion is associated with retention of placenta. Infections may also cause stillborn or weak calves, retained placenta, and reduced milk yield. Usually, general health is not impaired in uncomplicated abortions.

Seminal vesicles, ampullae, testicles, and epididymides are infected in bulls. Testicular abscesses may occur. Long-standing infections may result in arthritic joints in some cattle.

Lesions: Necrotic placentitis, swollen, hyperaemic cotyledons surrounded by brown exudates, thickened intercotyledonary spaces with leathery appearance are the common lesions. Udder may look normal but cellular infiltration; degeneration and necrosis are also seen. Orchitis and necrosis of the testicular tissues surrounded by fibrous tissue are the common lesions in male animals.

 

 

BRUCELLOSIS IN GOATS

In goats B.melitensis is the causative agent. The disease is prevalent in most countries where goats are a significant part of the animal industry. The signs of brucellosis in goats are similar to those in cattle. B.melitensis organisms have a preference for udder tissue than gravid uterus. Infection occurs primarily through ingestion of the organisms. The disease causes abortion about the fourth month of pregnancy. Arthritis, epididymitis and orchitis also occur.

 

BRUCELLOSIS IN SHEEP

In sheep B ovis is the causative agent, in which epididymitis and orchitis are observed. Occasionally, placentitis and abortion are also seen.

Pathogenesis: The disease can be transmitted among rams by direct contact. Infection in ewes occurs after mating with naturally infected rams. Contaminated pastures do not appear to be important in spread of the disease. Infection frequently persists in rams, and a high percentage shed B ovis intermittently.

Symptoms & Lesions: In sheep apart from abortion, lameness, mastitis, pyrexia and diarrhoea are also seen. Lesions develop rapidly. In rams, the first detectable abnormality is the marked deterioration in semen quality associated with the presence of inflammatory cells and organisms. The epididymal enlargement is unilateral or bilateral. The most prominent lesion is spermatoceles of variable size containing partially inspissated spermatic fluid. The tunics frequently become thickened and fibrous, and extensive adhesions develop between them. The testes show fibrous atrophy. In few cases the organisms are present in semen over long periods without clinically detectable lesions.

 

BRUCELLOSIS IN HORSES

The causative agent is B.abortus. The organisms produce chronic inflammatory conditions referred as fistulous withers, poll evil and joint infections. Fistulous wither and poll evil are clinically same conditions occurring in supraspinous and supra-atlantal bursae respectively. The inflammation of bursae leads to considerable thickening of the wall and the supraspinous bursa distends with a clear, straw-colored, viscid exudates and rupture later. In more chronic, advanced cases, the ligament and the dorsal vertebral spines are also affected become necrosed.

 

BRUCELLOSIS IN PIGS

B.suis is the causative organism for abortion in swine. In pigs’ abortion, birth of weak piglets, lameness, paralysis, affection of spinal column and infertility in male and female animals are the common symptoms.

 

BRUCELLOSIS IN DOGS

B.canis is the causative organism in dogs. In dogs’ abortion, early embryonic deaths, dermatitis of scrotum and testicular atrophy are common symptoms.

 

BRUCELLOSIS IN HUMANS

Infection known as Undulant fever or Malta fever in humans occurs as a result of coming in contact with animals or animal products that are contaminated with these bacteria. Human brucellosis is more common in countries that do not have good standardized and effective public health and domestic animal health programs. Areas currently listed as high risk are the Mediterranean Basin (Portugal, Spain, Southern France, Italy, Greece, Turkey, North Africa), South and Central America, Eastern Europe, Asia, Africa, the Caribbean, and the Middle East. Unpasteurized cheeses, sometimes called "village cheeses," from these areas may represent a particular risk for tourists. Humans are generally infected in one of three ways: eating or drinking something that is contaminated with Brucella, breathing in the organism (inhalation), or having the bacteria enter the body through skin wounds. Direct person-to-person spread of brucellosis is extremely rare. Mothers who are breast-feeding may transmit the infection to their infants. Sexual transmission has also been reported. Veterinarians treating the animals are in risk group along with butchers and animal attendants. The symptoms of brucellosis are similar to the flu and include fever, sweats, headaches, back pains, and physical weakness. Sever infections of the central nervous systems or lining of the heart may occur. Brucellosis can also cause long-lasting or chronic symptoms that include recurrent fevers, joint pain, and fatigue.

Public Health Significance: The World Health Organization (WHO) laboratory biosafety manual classifies Brucella in Risk group III. Brucellosis is readily transmissible to humans, causing acute febrile illness – undulant fever – which may progress to a more chronic form and can also produce serious complications affecting the musculo–skeletal, cardiovascular, and central nervous systems. Infection is often due to occupational exposure and is essentially acquired by the oral, respiratory, or conjunctival routes, but ingestion of dairy products constitutes the main risk to the general public. There is an occupational risk to veterinarians and farmers who handle infected animals and aborted fetuses or placentae. Brucellosis is one of the most easily acquired laboratory infections, and strict safety precautions should be observed when handling cultures and heavily infected samples, such as products of abortion. Specific recommendations have been made for the safety precautions to be observed with Brucella-infected materials.

 

Diagnosis:

1. Based on symptoms and lesion and history of abortion in a herd.
2. Microscopical examination: Smears prepared from chorionic tissues, foetal stomach contents and uterine discharges stained by modification of Macchiavello’s method or Koster’s method will reveal brucella organisms. Brucella are Gram negative and usually do not show bipolar staining. They are not truly acid-fast, but are resistant to decolorisation by weak acids and thus stain red by the Stamp’s modification of the Ziehl–Neelsen method. This is the usual procedure for the examination of smears of organs or biological fluids that have been previously fixed with heat or ethanol, and by this method, Brucella organisms stain red against a blue background. A fluorochrome or peroxidase-labelled antibody conjugate based technique is also used.
3. Bacteriological examination: For the diagnosis of animal brucellosis by cultural examination, the choice of samples usually depends on the clinical signs observed. The important samples are aborted fetuses (stomach contents, spleen and lung), fetal membranes, vaginal secretions (swabs), milk, semen and arthritis or hygroma fluids. From animal carcasses, the preferred tissues for culture are those of the reticulo-endothelial system (i.e. head, mammary and genital lymph nodes and spleen), the late pregnant or early post-parturient uterus, and the udder. When milk products are checked for contamination, they have to be enriched since the milk prouducts contain minimal number of organisms. Albimi agar is the medium of choice. Other media include nutrient agar, serum agar, liver infusion serum agar, tryptose agar, and dextrose potato and glycerol potato agar. The colonies are small, round, convex, and translucent with a smooth glistening surface. In broth cultures they produce fine granular deposit. Growth normally appears after 2–3 days, but cultures should not be discarded as negative until 8–10 days have elapsed.
4. Nucleic acid recognition methods: Polymerase chain reaction is used to differentiate causative organisms from vaccine organisms.

 

5. Animal inoculation:
1. Guinea pigs are the laboratory animals of choice for brucella identification. Clinical materials are inoculated intra muscularly or sub-cutaneously. Two animals are inoculated and one animal is killed after 3 week and another after 6 weeks. Typical lesions include miliary necrotic foci in spleen, liver and lymphatic glands.
2. Strauss reaction: Development of orchitis after intraperitoneal injection of infective material.
6. Serum agglutination tests: These tests are performed to identify antibodies against brucella in animals. Two types of tests a rapid plate test called Rose Bengal plate test  (RBPT), which is a qualitative test and serum agglutination test (SAT), which is a quantitative test are performed to identify animals infected with brucella. In RBPT, the brucella antigen is stained with Rose Bengal dye. 0.3 ml of antigen and 0.3 ml of antibody are mixed and development of agglutination after four minutes is positive reaction. It is a rapid method. In SAT, the serum sample is diluted and brucella antigen without any dye is added. Development of 50% agglutination at dilutions 1:40 and above is considered as positive and 1:20 is considered as suspected. The test is of useful in unvaccinated cattle and in farms where calfhood vaccination is practiced.
7. Milk ring test (MRT) or Abortus Bang’s ring (ABR) test: This test is performed to identify brucella in herds. It is based on detection of antibodies in milk against stained antigen. Milk samples from herd are mixed with stained antigen and incubated. Development of a ring above cream layer is a positive reaction.
8. Whey agglutination test:
9. Coombs test:
10. ELISA: It is a prescribed tests for international trade. 
11. Complement fixation test: It is also a prescribed test for international trade. The CFT is a widely used and accepted confirmatory test although it is complex to perform, requiring good laboratory facilities and adequately trained staff to accurately titrate and maintain the reagents. There are numerous variations of the CFT in use, but this test is most conveniently carried out in a microtitre format.
12. Fluorescence polarisation assay:  It is also a prescribed test for international trade. FPA is a simple technique for measuring antigen/antibody interaction and may be performed in a laboratory setting or in the field. It is a homogeneous assay in which analytes are not separated and it is therefore very rapid. For FPA antigen is labelled with a fluorochrome and added to serum or other fluid to be tested for the presence of antibody. If antibody is present, attachment to the labelled antigen will cause its rotational rate to decrease and this decrease can be measured.
13. Brucellin skin test: It is used for screening unvaccinated herds. A purified (free of sLPS) and standardised antigen preparation (e.g. brucellin) is used.

 

Control and Prevention:

1. Vaccination of calves with strain 19 (avirulent strain) at an age of 3-6 months offer high level of immunity. One dose gives immunity up to fifth pregnancy. Now RB51 strain is used instead of strain 19 vaccine.
2. Periodical testing of herds by SAT and elimination of reactors.
3. Pasteurisation of milk and milk products.
4. Clean animal husbandry procedures.
5. Proper disposal of aborted foetuses.
6. Elimination of male animals from breeding programme

VMC LECTURE # 15

FAMILY – ENTEROBACTERIACEAE – PART I

 

INTRODUCTION:

What is enteric bacterium? Enteric bacterium is one that is found in the intestinal tract of warm-blooded animals in health and disease.  

 

The Enterobacteriaceae are a family of bacteria, including many familiar pathogens, such as Salmonella and E. coli. They are placed among the proteobacteria. The Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter, and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation. The group is defined mainly in terms of ribosomal RNA sequences (recollect Molecular taxonomy – VMC 211), and is named for the Greek god Proteus, who could change his shape, because of the great diversity of forms found in it.

 

Scientific classification: As in Bergey's Manual of Systematic Bacteriology, 2nd Edition, 2001.

Kingdom        :        Bacteria

Phylum             :           Proteobacteria

Class            :        Gamma Proteobacteria

Order            :        Enterobacteriales

Family           :        Enterobacteriaceae

 

          (Bold letter genus are normally pathogenic)

 

General Characters: Members of the family Enterobacteriaceae are Gram-negative, oxidase-negative, rod-shaped bacteria measuring 0.3-1.0 x 1.0-6.0 um. They are motile by peritrichous flagella. They are non-spore forming. Some members possess a prominent capsule and some don’t. Some members also have fimbirae and pili. They are facultative anaerobes. They exhibit both respiratory and fermentative metabolism. Most grow well between 22 and 35°C on media containing peptone or beef extract. They also grow on MacConkey's agar, which may be used for their selective isolation. Most grow on glucose as a sole carbon source, although some require vitamins and/or amino acids for growth.

 

Distribution: They are world wide in distribution and found in water or as commensals. They live as saprophytes, as symbionts, as epiphytes or as parasites.  Their host range includes animals ranging from insects to humans, as well as fruits, vegetables, grains, flowering plants, and trees.  Faecal contamination is one of the important modes of spread. Most of the members produce enteric infection. They are also associated with wound infection, mastitis etc.

 

GENUS – ESCHERICHIA

Type Species – Escherichia coli

This organism was first identified by Theodore Escherich in 1885, who isolated this bateria from the faeces of neonates.

 

General characters:

1. Morphology: They are Gram-negative, oxidase-negative, rod-shaped bacteria measuring 0.3-1.0 x 1.0-6.0 um. They usually occur as single organisms or in short chains. They are motile by peritrichous flagella. They are non-spore forming and capsulative. They also have fimbirae and pili. They are normally present in intestinal tract as commensals.
2. Antigens and toxins: There are over 700 antigenic types (serotypes) recognized based on somatic (O), flagellar (H), and capsular (K) antigens. E.coli organisms are classified based on O, H and K antigens and this system of classification is called as Kauffmann-Knipschidt-Vahlne scheme. The following are the virulence factors of E.coli.
1. Adhesins

                                                              i.      CFAI/CFAII

                                                             ii.      Type 1 fimbriae

                                                           iii.      P fimbriae

                                                          iv.      S fimbriae

                                                            v.      Intimin (non-fimbrial adhesin)

2. Invasins

                                                              i.      hemolysisn

                                                             ii.      siderophores and siderophore uptake systems

                                                           iii.      Shigella-like "invasins" for intracellular invasion and spread

3. Motility/chemotaxis

                                                              i.      Flagella

4. Toxins

                                                              i.      LT toxin

                                                             ii.      ST toxin

                                                           iii.      Shiga-like toxin

                                                          iv.      Cytotoxins

                                                            v.      Endotoxin LPS)

5. Antiphagocytic surface properties

                                                              i.      capsules

                                                             ii.      K antig ens    

                                                           iii.      LPS

6. Defense against serum bactericidal reactions

                                                              i.      LPS

                                                             ii.      K antigens

7. Defense against immune responses

                                                              i.      capsules

                                                             ii.      K antigens

                                                           iii.      LPS antigenic variation

8. Genetic attributes

                                                              i.      genetic exchange by transduction and conjugation

                                                             ii.      transmissible plasmids

                                                           iii.      R factors and drug resistance plasmids

                                                          iv.      toxin and other virulence plasmids

3. Resistance: They are destroyed by 60C at 30 minutes. They are also sensitive to common disinfectants. They are also killed at freezing temperature when exposed to two hours. Variations in the heat sensitivity is observed between strains. E.coli is known for its resitance to antibiotics.

 

Cultural characters: These organisms are aerobic or facultatively anaerobic and fermentative. The optimum temperature for growth is 37.5C. They grow readily on ordinary laboratory media. On nutrient agar, the colonies are white or yellowish white, moist, glistening, opaque and circular with an entire edge. In MacConkey agar the organisms produce pink coloured colonies since they utilize the sugar lactose. In Eosin-Methylene blue agar, the colonies appear as blackish centre with a metallic sheen. In litmus lactose agar the organisms produce red coloured colonies.

 

Biochemical reactions: E.coil produces acid and gas from glucose, lactose, mannitol and number of other sugars. It reduces nitrate and does not produce hydrogen sulphide. The biochemical reactions are commonly referred as IMViC reaction. IMViC is an acronym in which the capital letters stand for Indole, Methyl red, Voges-Proskauer, and Citrate utilization tests). The IMViC tests were developed in order to distinguish strains of E. coli from related species that also produced acid and gas from the fermentation of lactose.

The IMViC set of tests examines: the ability of an organism to

(1) produce Indole;

(2) produce sufficient acid to change the color of Methyl red indicator

(3) produce acetoin, an intermediate in ther butanediol fermentation pathway (a positive result of the Voges-Proskauer test);

(4) the ability to grow on Citrate as the sole source of carbon.

Lactose fermenters are considered E. coli if they are positive in the first two tests and negative in the second two.

 

Pathogenesis

E.coli causes disease in all kinds of domestic and wild animals. As a pathogen, E. coli cause intestinal diseases. Five classes (virotypes) of E. coli that cause diarrheal diseases have been recognized. They are as follows;

1.      Enterotoxigenic E. coli (ETEC)

2.      Enteroinvasive E. coli (EIEC)

3.      Enterohemorrhagic E. coli (EHEC)

4.      Enteropathogenic E. coli (EPEC)

5.      Eeroaggregative E. coli (EaggEC)

1.     Enterotoxigenic E. coli (ETEC): ETEC are an important cause of diarrhoea in infants and travellers in underdeveloped countries or regions of poor sanitation. The diseases vary from minor discomfort to a severe cholera-like syndrome. ETEC also cause infection in piglets and calves. The virulence factors for ETEC adhesins are fimbriae, which are species-specific. For example, the K-88 fimbrial Ag is found on strains from piglets; K-99 Ag is found on strains from calves and lambs. Enterotoxins produced by ETEC include the LT(heat-labile) toxin and/or the ST (heat-stable) toxin. The LT enterotoxin is very similar to cholera toxin in both structure and mode of action. The ST enterotoxin is actually a family of toxins which are peptides of molecular weight about 2,000 daltons. One such toxin STa is responsible for secretion of fluid and electrolytes resulting in diarrhea. Symptoms ETEC infections include diarrhoea without fever.

2.     Enteroinvasive E. coli (EIEC): EIEC resemble Shigella in their pathogenic mechanisms and the kind of clinical illness they produce. EIEC penetrate and multiply within epithelial cells of the colon causing widespread cell destruction. The clinical syndrome is identical to Shigella dysentery and includes a dysentery-like diarrhoea with fever. EIEC apparently lack fimbrial adhesins but do possess a specific adhesin that, as in Shigella, is thought to be an outer membrane protein. Also, like Shigella, EIEC are invasive organisms. They do not produce LT or ST toxin and, unlike Shigella, they do not produce the shiga toxin.

 

3.     Enteropathogenic E. coli (EPEC): EPEC induce watery diarrhoea similar to ETEC, but they do not possess the same colonization factors and do not produce ST or LT toxins.

4.     Enteroaggregative E. coli (EaggEC): These strains are associated with persistent diarrhoea in young children. A heat-labile is associated with this type of infection

5.     Enterohemorrhagic E. coli (EHEC): EHEC is identified with serotype O157:H7, which causes a diarrhoeal syndrome where there is copious bloody discharge and no fever. Usually kidneys are affected in this type of infection

 

There are, three situations where the otherwise harmless E. coli can cause illness:

1. When the bacteria get out of the intestinal tract and into the urinary tract they can cause an infection sometimes referred to as "honeymoon cystitis" in humanbeings.
2. When the bacteria get out of the intestinal tract through a perforation (= a hole or tear, which could be caused by an ulcer, for example) and into the abdomen, they usually cause an infection called "peritonitis" that can be fatal
3. Certain strains of E.coli are toxigenic (some produce a toxin very similar to that seen in dysentery) and can cause food-poisoning usually associated with eating contaminated meat (contaminated during or shortly after slaughter or during storage or display). Severity of the illness varies considerably; it can, rarely, be fatal, but is more often mild.

 

Calves: Calves are susceptible to both systemic colibacillosis and neonatal diarrhoea (calf scours or white scour). The infection is usually fatal if not promptly treated. Specific heat-stable enterotoxigenic E. coli serotypes containing K99 fimbrial adhesin are considered the causative agents. The organisms are also associated with mastitis. ETEC cause diarrhoea in very young calves, less than 3-4 days of age (typically less than 48 hours of age). Calves are depressed, do not drink or suckle, become dehydrated, and die rapidly. Very profuse and watery diarrhoea is typical of ETEC scours. Septicaemic colibacillosis, caused by another serotype of ETEC, is an acute disease with very few diagnostic signs and is the most common cause of acute, fatal illness in neonatal calves. Depressed, weak animals initially have a fever but become hypothermic rapidly. Mortality rates are high and survivors are often affected by post-septicaemic localisation of infection in the form of arthritis, meningitis or pneumonia. Inadequate transfer of passive immunity from the dam is considered the main risk factor for colibacillosis. Multiresistant (to antibiotics) strains of E. coli have been identified and hence antibiotics should not be the main approach to treatment. Control is aimed as two aspects as mentioned below;

Maintenance of immune status

·         Do not separate the dam until 24 hours after calving

·         Provide adequate bedding to allow calf to stand without difficulty

·         Ensure early feeding, assist if needed, monitor the intake as closely as possible and record it ("maximum supervision, minimum interference")

·         Keep a supply of frozen colostrum in case the dam leaks colostrum before calving

·         Feed fresh or fermented colostrum to all calves up to three weeks of age

·         Encourage outdoor calving

·         In cases where ETEC has been identified as a herd problem, immunise pregnant dams before calving as part of a targeted control plan.

Minimisation of exposure

·         Provide adequate numbers of calving pens and clean and disinfect them between batches

·         House calves well away from the calving pens

·         Keep up high hygiene standards throughout the calving period and in calf housing

·         Spread the calving period over a longer period of time (maximum 15 calves per month)

·         Keep the infection out of the herd if not present, by a closed herd policy and the avoidance of visitors from other farms, unless precautions are taken.

Sheep: Infant diarrhea in lambs, is usually caused by strains of E. coli producing a heat-stable enterotoxin. Most of these strains also contain the K-99 fimbrial adhesin.

Pigs: E. coli infections in pigs are as exhibited as diarrhea in piglets, or as edema (edema disease) preceded by mild diarrhoea. Both forms are acute and highly fatal. As in sheep and cows, the causative strains produce a heat-stable enterotoxin, but they may also produce a heat-labile enterotoxin. Swine strains usually possess a K88 fimbrial adhesin, which is antigenically distict from K99.

Chickens: Colisepticaemia is the commonest infectious disease of farmed poultry. It is most commonly seen following upper respiratory disease (such as Infectious Bronchitis) or Mycoplasmosis. It is frequently associated with immunosuppressive diseases such as Infectious Bursal Disease Virus (Gumboro Disease) in chickens or Haemorrhagic Enteritis in turkeys, or in young birds that are immunologically immature. Infection is by the oral or inhalation routes, and via shell membranes/yolk/navel, water, fomites, with an incubation period of 3–5 days. Poor navel healing, mucosal damage due to viral infections and immunosuppression are predisposing factors. The important symptoms are Respiratory signs like coughing, sneezing, dejection, reduced appetite, poor growth and omphalitis. The important lesions are airsacculitis. pericarditis, perihepatitis, swollen liver and spleen, peritonitis, salpingitis, omphalitis, synovitis, arthritis, enteritis, granulomata in liver and spleen (Coligranuloma – Hjarre’s disease) and Cellulitis over the abdomen or in the leg. The infection can be treated with amoxycillin, tetracyclines, neomycin or gentamycin. Good hygiene in handling of hatching eggs, hatchery hygiene and good sanitation of house, feed and water will prevent infection in chickens.

 

Diagnosis

1.      Based on the symptoms and lesions.

2.      Microscopy: Culture smears will reveal gram-negative organisms.

3.      Isolation and identification of E.coli by culture methods and biochemical reactions (IMViC tests)

4.      E. coli as an Indicator of Fecal Pollution: E. coli has been used as the principal indicator of fecal pollution in both tropical and temperate countries. E. coli comprises about 1% of the total fecal bacterial flora of humans and most warm-blooded animals. Tests to identify isolates as E. coli aim to differentiate them from organisms normally associated with uncontaminated water. The term  "coliform" describes E. coli-like organisms. The coliforms are further divided into "fecal coliforms" (E coli) and "nonfecal coliforms" (e.g. Klebsiella and Enterobacter). As per the prevailing Federal standards of US, drinking water should not have any coliforms.

5.      Detection of E. coli in Food: The International Commission on Microbiological Specifications for Foods (ICMSF, 1978) has adopted a set of standard techniques for the enumeration of E. coli in food products, which is also accepted by the International Standards Organization (ISO, 1984). This method employs the use of lauryl sulfate tryptose broth at 35 or 37°C as a mildly selective-enrichment medium. This is followed by growth in EC broth containing 0.15%  bile salts at 45°C as a  second selective step. The ability to produce indole from tryptophan (in tryptone broth) at 45°C defines the strains as E. coli.

6.      Detection of E. coli in Water: There is no method for the detection of E. coli in water that is accepted throughout the world. The presence of E. coli in water is identified by the ability of the organisms to produce gas from lactose and produce indole from tryptophan at 44°C. A method for enumeration employs a standard multiple tube test with a modified glutamate synthetic medium at 37°C as a first selective step, followed by further cultivation in standard media at 44°C.

7.      Detection of E. coli in Clinical Specimens: Direct plating of clinical materials should be attempted when the specimens are supposed to have heavy load of organisms. Media such as MacConkey agar or Eosin Methylene Blue (EMB) agar are commonly used. If the number of E. coli is likely to be very low or the amount of specimen is limited, enrichment in a rich nutrient medium such as brain heart infusion broth may be used.

8.      Rapid Methods for Detecting E. coli : A fluorogenic detection method has been developed based on the cleavage of methylumbelliferyl-D-glucuronide (MUG) to the free methylumbelliferyl moiety, which fluoresces a blue color after irradiation with long-wave ultraviolet radiation. Automated or semi-automated systems are also being used for the detection of E. coli as part of the detection methods for Enterobacteriaceae. Other techniques such as immunoassays and nucleic acid hybridization studies can also be used to enumerate E. coli, and DNA probes directed at a number of genes have also been developed.

 

ISO STANDARDS FOR IDENTIFICATION OF MEMBERS OF ENTEROBACTERIACEAE AND E.COLI

The International Standards Organization has published nearly 35 standards for different bacteria and also for certain microbiological procedures. Some of the standards pertaining to identification of members of the Enterobacteriaceae particularly E.coli are provided below.

 

 

VMC LECTURE # 16

FAMILY – ENTEROBACTERIACEAE – PART II

Genus : Salmonella

Salmonellosis is a bacterial disease caused by strains of Salmonella. It occurs in animals and man. In both cases it is an enteric disease of varying severity, usually involving diarrhoea. In poultry Salmonella causes two important infections namely pullorum disease and fowl typhoid.

 

General characteristics:

1.     Morphology: They are small rod shaped or pleomorphic gram-negative organisms. Most of the species are motile except S.Pullorum and S.gallinarum. They are actively motile by peritrichous flagella. Capsule formation is associated with mucoid strain. Most serotypes also possess pili. They are non spore-forming.

2.     Habitat: They are typical intestinal pathogens and affected animals excrete the organisms in the excreta, contaminate the environment and act as source of infection to other animals. The most important aspect as for as the habitat is concerned is the species specificity of the members. Species affecting one type host does not affect other type of hosts. S.Typhi and S.Paratyphi are essentially pathogens of human beings and do not affect animals. However, S.gallinarum affects both human and animals.

3.     Classification:

a.      Old systems: In this system, classification of the member of the genus Salmonella is based on the antigenic structure, biochemical characters, host specificity, geographic distribution and DNA relatedness. As per this system, there are more than 2000 serotypes of Salmonella (Salmonella – Arizona group) and they are classified into 7 subgroups. Species of Salmonella causing infection in man and warm animals are placed in sub group 1. The other groups contain species that cause infection in cold-blooded animals and species isolated from environment. Further, on the basis of biochemical characteristics the Salmonella organisms are classified into three species namely S.Enteritidis, S.Cholerasuis and S.Typhi.

b.      The other method of classification proposed is placing the members of salmonella under one species. The serovars are further classified into biovars based on variations in biochemical reactions. However, the old method of assigning a name to serotype is still followed considering the simplicity.

c.      According to the latest nomenclature the genus Salmonella consists of only two species: S. enterica and S. bongori. Salmonella enterica is divided into six subspecies, which are distinguishable by certain biochemical characteristics. The subspecies are

 

Strains of Salmonella are classified into serovars on the basis of extensive diversity of lipopolysaccharide (LPS) antigens (O) and flagellar protein antigens (H) in accordance with the Kauffmann–White scheme; currently approximately 2500 serovars are recognised. This number is constantly being increased. The most common serovars that cause infections in humans and food animals belong to subspecies enterica. The serovars of the other subspecies are common in poikilothermic (cold-blooded) animals and in the environment. Names are retained only for subspecies enterica serovars. These names are no longer italicised. The first letter is a capital letter. For example S.Pullorum causing Pullorum disease is written as S.enterica subspecies enterica serovar Pullorum.

4.     Resistance: They are killed by a temperature of 55^oC for 1 hour and 60^oC for 20 minutes. They are destroyed at autoclaving. They are also susceptible to phenol, QAC, cresol and other disinfectants. However, the effect of these disinfectants is reduced in the presence of mucus. Formaldehyde vapour also destroys the organisms (it is used to fumigate egg incubators).

5.     Antigens, toxins and virulence factors: Salmonella possess three types of antigens - somatic (O), flagellar (H) and somatic (Vi). The somatic antigens are designated by numbers (1,2 3 etc.). The flagellar antigens (H) are present in two phases phase 1 and phase 2. Phase 1 antigens are indicated by alphabets and after Z they are indicated by numbers as subscripts to alphabet Z like Z₁₀. Phase 2 antigens are indicated by numbers. Generally Salmonella are grouped based on O antigen and each group is subgrouped based on flagellar antigens. Vi antigen is an important virulence factor for human infection with little significance in animal infections. Salmonella are also classified based on O and H antigens by agglutination reactions.

 

Cultural characteristics: They are aerobic or facultatively anaerobic organisms and grow well at a temperature of 37^oC. The growth is enhanced by the addition of blood, serum, ascitic fluid, glucose etc. The colonies are smooth, round, convex, 2-3 mm in diameter and dewdrop like. During isolation of Salmonella contamination of other enteric organisms are eliminated by enrichment that allows only the growth of Salmonella and destroys the growth of other organisms. Selenite F broth is commonly used as an enrichment media. Numbers of selective media are used for Salmonella isolation. The commonly used selective media are desoxycholate citrate agar (DCA agar), McConkey’s agar and brilliant green agar. Salmonella isolation is generally done first in enrichment media followed by selective media. The suspected materials are first inoculated into enrichment media (Selenite F broth) and incubated for 18-24 hours at 43^oC followed by streaking at selective media like brilliant green agar. The selenite F broth is reincubated again and the contents are streaked again on selective media for better isolation. In McConkey agar they produce pale colour colonies in contrast to pink colonies of E.coli.

 

Salmonella organisms on repeated laboratory cultivation produce rough colonies instead of routine smooth colonies. This is due to loss of O antigen and virulence. This variation is referred as S®R (smooth to rough). Some strains also have transient stage (T) between S and R having intermediate characters. This is referred as S®T®R.

 

Biochemical characters: They ferment glucose, maltose, mannitol, dulcitol and dextrin and produce acid and gas. They do not ferment lactose, sucrose and salicin. They reduce nitrates to nitrites and produce hydrogen sulphide. They produce indole, VP negative, liquefy gelatin and urease negative.

 

Pathogenesis: The infection is generally exogenous in nature. The organisms generally produce enteric infections and affected animals excrete the organisms in the faeces. Faecal contamination of water and fomites are the important source of transmission. Vertical transmission is also common among poultry. Some of the important infections produced by the members of the genus Salmonella are listed below:

 

 

SALMONELLA INFECTIONS IN POULTRY

A. Pullorum disease (S.Pullorum): It is an important poultry disease. The infection spreads both vertically and horizontally. Infected adult birds lay eggs that contain S.Pullorum in the yolk. However, only few such eggs hatch and act as source of infection to other chicks in the incubator. The dried spicules of fluff and faeces of infected source contain S.Pullorum, which spread through the air currents in incubator. The faeces of infected contaminates the feed and water that act as a source of infection. Chick boxes, litter, utensils and chick sexers also spread the infection. Droplet infection is also reported.

OIE Classification: List B disease

Symptoms: The incubation period is usually a few days. Young chicks are more affected with a morality of up to 95%. The chicks appear sleepy, dull and huddle close to the heating source. Inappetance, increased thirst, lour chirping, respiratory distress and nervous symptoms like staggering gait, incoordination of the limbs are other symptoms. The vent of the affected chicks will be covered with adherent mass of faeces. Growth rate is slow among growers with intermittent diarrhoea. In adults decreased egg production is the most important symptoms.

Lesions: In chicks, there is congestion of spleen, yellow colouration of spleen with streaks of haemorrhage. There is also hyperaemia of liver, necrotic foci on kidney, spleen, distended ureters with urates, distended caeca and degeneration of the myocardium. Lung and heart may have white nodules, pericardium may be thickened, with yellow or fibrinous exudates. Gastro-intestinal trac- may have white nodules on the gizzard, caeca and large intestinal wall.  In adult birds the ovaries contain misshaped ovules and detachment of ovules is also noticed. Yolk sac retention, with yolk appearing creamy or caseous are other important lesions. Caseous cores may also be seen in the caeca. Joints may also be swollen with yellow viscous fluid.

Diagnosis:

1. Based on symptoms and lesions.
2. Rapid Whole blood test: It is done with whole blood to identify carrier adult birds. It is based on agglutination principle. The rapid whole blood agglutination test can be used under field conditions for detecting both S. Pullorum and S.Gallinarum, and the reactors can be identified immediately. However, it is not reliable in turkeys as the test results in a significant proportion of false-positive results.
3. Rapid Serum agglutination test: The RST is performed in the same manner, except that serum is substituted for whole blood. For export test purposes an initial screening of sera by RST followed by confirmation of positives by the tube aglutination test is approved.
4. Tube agglutination test: Fresh serum from chickens, turkeys or other birds is used at an initial dilution of 1/25. A titre of 1/50 is usually considered to be positive.
5. Micro agglutination test: This resembles the tube agglutination test, but requires much smaller volumes of reactants. The test is performed in microtest plates.
6. ELISA: The indirect ELISA using lipopolysaccharide antigen is likely to be the most sensitive and specific serological flock test for Salmonella, including S. Gallinarum and S. Pullorum.
7. Isolation of the causative organism by culturing. In clinical cases direct plating on Brilliant Green, McConkey and non-selective agar can be done. Enrichment procedures usually rely on selenite broth followed by plating on selective media.

Control:

1.      Identification of the reactors and removing them from flock.

2.      Periodical disinfection of the incubators using formaldehyde vapour (fumigation).

3.      Fumigation of egg or dipping them in bactericidal agents before incubating them.

4.      Vaccines are not normally used as they interfere with serological testing and elimination of carriers

Treatment

Amoxycillin, poteniated sulponamide, tetracylines, fluoroquinolones.

 

B. Fowl typhoid (S.gallinarum): In contrast to pullorum disease fowl typhoid is a disease of adult birds and growers. Vertical transmission from hen to chicks is also possible. The organism can survive in litter for a long time and the litter acts as source of infection. Broiler parents and brown-shell egg layers are especially susceptible. Chickens are most commonly affected but it also infects turkeys, game birds, guinea fowls, sparrows, parrots and canaries. Infections still occur worldwide in non-commercial poultry but are rare in most commercial systems now. Morbidity is 10–100%; mortality is increased in stressed or immunocompromised flocks and may be up to 100%. The route of infection is oral or via the navel/yolk. Transmission may be transovarian or horizontal by faecal–oral contamination, egg eating etc, even in adults. The bacterium is fairly resistant to normal climate, surviving months, but is susceptible to normal disinfectants.

OIE Classification: List B disease

Symptoms: In chicks the symptoms are as same as pullorum disease. The mortality in adults is up to 50%. The affected birds show are listlessness, green or yellow coloured diarrhoea, purple colouration of comb and wattles, anaemia and intermittent diarrhoea.

Lesions: The most important lesions are enlarged liver that turns into dark red or reddish brown after exposure to atmosphere. Enlargement of spleen, focal necrosis on liver, spleen and myocardium are other lesions. The intestine may show catarrhal inflammation and petechial haemorrhage.

Diagnosis:

1.     Based on symptoms and lesions.

2.     Tests 2-6 mentioned for pullorum can be done for fowl typhoid also but the antigen used should be S.gallinarum.

3.     Isolation of causative organisms by culturing.

Treatment

Amoxycillin, potentiated sulponamide, tetracylines, fluoroquinolones.

Prevention

Biosecurity and clean poultry husbandry practices will reduce the incidence. As with other salmonellae, recovered birds are resistant to the effects of infection but may remain carriers. Vaccines for fowl typhoid have been used in some areas, both live (usually based on the Houghton 9R strain) and bacterins.

 

(C) S. Enteritidis and S. Typhimurium infections

Salmonella Enteritidis and S.Typhimurium are the predominant sero-types associated with human disease in most countries. Infections in chickens, turkeys and ducks cause problems worldwide with morbidity of 0–90% and a low to moderate mortality. The route of infection is oral; many species are intestinal carriers and infection may be carried by faeces, fomites and on eggshells. Vertical transmission may be either by shell contamination or internal transovarian contamination of yolk. Feed and feed raw material contamination is less common than for other sero-types. The bacteria are often persistent in the environment, especially in dry dusty areas, but are susceptible to disinfectants that are suitable for the particular contaminated surfaces and conditions, applied at sufficient concentrations. The infection caused by S.Typhimurium in poultry is also referred as paratyphoid.

Symptoms: The important symptoms in affected birds include dejection. ruffled feathers, closed eyes, diarrhoea, vent pasting, loss of appetite and thirst and  stunting in older birds.

Lesions: The lesions include enteritis, focal necrotic intestinal lesions, foci in liver, unabsorbed yolk, cheesy cores in caecae, pericarditis, perihepatitis and misshapen ovules in the ovaries. 

Diagnosis

1.      Isolation and identification. In clinical cases direct plating on Brilliant Green and McConkey agar will be useful. Enrichment media such as buffered peptone followed by selective broth or semi-solid media (e.g. Rappaport-Vassiliadis) followed by plating on two selective media will greatly increase sensitivity

2.      ELISA to differentiate with S.Pullorum

Treatment

Sulphonamides, neomycin, tetracyclines, amoxycillin and fluoroquinolones

Prevention

1.      Clean poultry husbandry practices.

2.     Routine monitoring of breeding flocks, hatcheries

3.     Vaccines – Inactivated and live vaccines for S. Enteritidis and S. Typhimurium are available.

 

SALMONELLA INFECTION IN CATTLE

The two species that affect cattle are S.Dublin and S.Typhimurium. The infection in cattle spreads by ingestion. Poor health conditions also predispose the animal for infection. The infection in cattle is characterised by high temperature, inappetance, drop in milk yield and diarrhoea. Pregnant animals may abort. Affected animals die in 2-3 days in acute infection. In chronic cased the animal become emaciated, dehydrated and show signs of abdominal pain. In young calves the loss of appetite, weakness and blood stained mucoid are characteristic of salmonellosis. The lesions are found in the intestine and mesenteric lymph nodes. There is extension of the gall bladder and focal necrosis of liver. Diagnosis is based on symptoms and lesions, isolation of the organisms and agglutination tests. The clinical infection can be treated with Chloromycetin, oxytetracycline, neomycin and furazolidone. Both killed and live vaccines are available as protective measure.

 

SALMONELLA INFECTION IN SHEEP AND GOAT

The species affecting sheep and goat are S.Abortus-ovis, S.Typhimurium and S.Dublin. The infection is characterised by abortion during last two months of pregnancy preceded by blood stained purulent vaginal discharge. Affected animals deliver weak lambs that show symptoms of diarrhoea and dysentery from day one. Surviving lambs become debilitated. In adult animals intermittent diarrhoea and fleece loss are the characteristic symptoms. There is congestion and haemorrhage in abomasum, small intestine, myocardium and kidneys.

 

SALMONELLA INFECTION IN PIGS

The pigs act as a reservoir for many serotypes of salmonella. The most common infection in pigs is the pig paratyphoid caused by S.Cholerae-suis. S.Typhimurium can also cause infection in pigs. Pig paratyphoid is an acute, subacute or chronic infection with high temperature, loss of appetite and appearance of bright pin colour areas on the skin. Affected animals will have profuse yellow colour diarrhoea. Survivors act as carriers for other animals.

 

SALMONELLA INFECTION IN DOGS AND CATS

Salmonella infection in dogs and cats are caused by number of serotypes due to consumption of contaminated meat, milk and wild rodents. The infection is characterised by abdominal pain, vomiting, diarrhoea, dysentery and death.

         

PUBLIC HEALTH IMPORTANCE

The main mode of spread to humans is through animal products like meat, milk and egg. Contact with infected pet and domestic animals also act as source of infection.

 

ISO STANDARDS FOR SALMONELLA ENUMERATION

 

ISO 6340:1995         -        Water quality -- Detection and enumeration of

Salmonella

ISO 6579:2002         -        Microbiology of food and animal feeding stuffs –

Horizontal

method for the detection of Salmonella spp.

ISO 6785:2001         -        Milk and milk products -- Detection of Salmonella

spp.

ISO/FDIS 16240        -        Water quality -- Determination of the genotoxicity of

water and waste water -- Salmonella/microsome test (Ames test)

ISO/DIS 19250         -        Water quality -- Determination of Salmonella species

VMC LECTURE # 17

FAMILY – ENTEROBACTERIACEAE – PART III

Genus : Proteus

Important species     :        Proteus vulgaris, P.mirabilis, P.rettgeri

Important infection   :        Enteritis and Otitis

 

General characters:

1. Morphology: They are gram-negative pleomorphic rod shaped organisms. They occur as coccal form or as filaments. They are motile by peritrichous flagella. They are non-spore forming and non-capsulative organisms. Majority of the organisms possess flagella.
2. Habitat: They are found as commonly in the intestine of man and animals, animal manure, sewage, soil and water, meat and eggs. They are also isolated from number of wild animals.
3. Resistance: They are killed by a temperature of 55^oC for 1 hour and 60^oC for 20 minutes. They are destroyed at autoclaving. They are also susceptible to phenol, QAC, cresol and other disinfectants. However, the effect of these disinfectants is reduced in the presence of mucus. Formaldehyde vapour also destroys the organisms (it is used to fumigate egg incubators).
4. Classification: Not very significant.
5. Antigens and toxins: The organisms possess O and H antigens.

 

Cultural characteristics: They are aerobic organisms grow at a temperature range of 20-40^oC. However, the optimal temperature is 37^oC. On the surface of solid media the organisms (colonies) rapidly spread as a thin film and this phenomenon is called swarming. This phenomenon occurs in waves and the culture look like rings. Swarming can be inhibited by sodium azide or media containing 6% agar. A characteristic odour from the plate is also characteristic of proteus.

 

Biochemical characters: They are non-lactose fermenters, indole variable, citrate utilisation negative, and urease positive and produce hydrogen sulphide. Proteus organisms are positive to (phenyl pyruvic acid) PPA reaction. Proteus is the only genus that is positive for this test.

 

Pathogenesis: They are normal inhabitants of the intestine. Under some conditions they produce severe diarrhoea and dysentery. In dogs it produce otitis.

 

Diagnosis: The symptoms are not characteristic. Hence diagnosis is based on isolation of the organisms.

 

GENUS - KLEBSIELLA

Important species     :        Klebsiella pneumoniae (Friedlander’s bacillus); K.mobilis (Enterobacter aerogenes)

Important disease     :        Mastitis, Metritis, Suppurative infection, and atrophic rhinitis

The genus Klebsiella belongs to the tribe Klebsiellae, a member of the family Enterobacteriaceae. The organisms are named after Edwin Klebs, a 19th century German microbiologist. Klebsiellae are nonmotile, rod-shaped, gram-negative bacteria with a prominent polysaccharide capsule. This capsule accounts for their large appearance on samples stained with Gram stain.

General characters:

1.     Morphology: They are gram-negative rod shaped organisms. They are non-motile and non-spore forming organisms. The characteristic feature of the organisms is the presence of a very prominent polysaccharide capsule.

2.     Habitat: These organisms are found as saprophytes in water and soil. They are also found as commensals in the intestinal and respiratory tract of animals.

3.     Classification: There are number of serotypes. The classification is not very important.

4.     Resistance: They are killed at a temperature of 60C for 20 minutes and by number of disinfectants. They can remain for a long time in room temperature.

5.     Antigens and toxins: The O and K antigens are important. Serotypes are based on the structural variability of the capsular polysaccharides (K antigens) and lipopolysaccharides (O antigens). There are 77 K antigens and 8 O antigens. The virulence of all serotypes appears to be similar

 

Cultural characteristics: They grow well in MacConkey’s agar and produce prominent mucoid pink colour colonies. Pink colouration is due to utilisation of lactose and mucoid consistency is due to polysaccharide capsule. No haemolysis is produced in blood agar.

 

Biochemical characters: They are VP positive, MR negative, catalase positive and hydrolyse urea.

 

Pathology: They are generally secondary invaders. In foals they produce pneumonia and metritis. In cattle they produce mastitis and in pigs they produce metritis and atrophic rhinitis. In poultry they cause air sac infection. The symptoms vary according to the system.

 

Diagnosis:

Based on the isolation of the organisms by culturing.

.

 

GENUS– SHIGELLA

Important species       :           Shigella dysenteriae, S.flexneri, S.boydii, S.sonnei

Important disease       :           Human bacillary dysentery.

 

They are pathogens affecting human and cause human bacillary dysentery. Same type of infection has been reported in primates also. The organisms are gram-negative and non motile. S.dysenteriae produce a powerful exotoxin (shiga toxin). The bacillus is also known as shiga bacillus.

 

 

GENUS – YERSINIA

Important Species: Yersinia pestis CO92; Yersinia pestis KIM; Yersinia enterocolitica; Yersinia pseudotuberculosis

Important disease     :        Plague, Pseudotuberculosis

 

Yersinia spp. are responsible for disease syndromes ranging from gastroenteritis to plague. Y. pestis is the cause of the plague and is actually catagorized into three subtypes or biovars; Antiqua, Medievalis, and Orientalis, each associated with a major pandemic. Y. pestis strand KIM belongs to biovar Mediaevalis while strand CO92 is in biovar Orientalis. Biovar Mediaevalis is thought to of descended from the bacteria that caused the second pandemic (the Black Death), while biovar Orientalis bacteria are responsible for the current pandemic (modern plague).

General characters:

1. Morphology: They are gram-negative, short ovoid rod shaped organisms. They have a variable motility character. Y.pseudotuberculosis organisms are non motile at 37C but motile at 22C. Where as Y.pestis is non motile. They appear bipolar stained in fresh isolates. They are non-spore forming. The capsule is more prominent in tissues where as it is not prominent in cultures.
2. Habitat: Y.pseudotuberculosis is world wide in distribution and distributed by wild rodents and pigeons and causes pseudotuberculosis in guinea pigs. Y.pestis is found only in certain pars of Asia, America and Africa and causes bubonic plague or pneumonic plague in man. Rat flea transmits Y.pestis.
3. Resistance: They are not very resistant organisms. They are killed by chemical and agents like 0.5% phenol when exposed for 15 minutes. They are also killed on exposure to sunlight for 3-4 hours and heating at 55^oC for 15 minutes.
4. Classification: Y.pseudotubeculosis is classified based on O antigen and Y.pestis is classified based on O and K antigens.
5. Antigens and Toxins: Y.pseudotuberculosis has got O antigens and Y.pestis has got K antigens. Y.pestis produce a toxin that is lethal to both mice and guinea pigs.

 

Cultural characteristics: They are aerobic organisms that grow in a wide range of temperature. Y.pseudotubeculosis and Y.pestis grow well in MacConkey’s bile salt medium (this character is used to differentiate with Pasteurella multocida). Y.pestis grow between temperature ranges of 14-37C. Their growth is enhanced by blood or serum in the media.

 

Pathogenesis:

1. Y.pseudotuberculosis: The main mode of spread is by ingestion of food contaminated with urine of wild rodents that suffer from clinical infection of Y.pseudotuberculosis. Guinea pigs, rabbits, chickens and turkeys are affected by pseudotuberculosis. The acute infection develops as sudden fatal septicaemia with high mortality. The chronic form is manifested as intermittent diarrhoea and emaciation. The infection remains localised in lymph nodes during chronic infection. The spleen and mesenteric lymph nodes are enlarged and congested. The lesions appear as necrotic caseous foci surrounded by polymorph cells but there is no calcification.
2. Y.pestis: Plague is a disease of rodents and man is considered as an accidental host. The infection is spread through bite of rat flea Xenopsylla cheopis. Bubonic plague that mainly affects lymphatic system is spread through rat flea where as pulmonary plague spreads via infected droplets from affected person.
3. Y.enterocolitica: Associated with contamination of food.

 

Diagnosis:

Based on symptoms, lesions and isolation of organisms by culturing.

 

Treatment and control: Chloramphenicol, tetracyclines and streptomycin are the drugs of choice. Control is mainly by controlling wild rodent population.

 

GENUS - SERRATIA

Serratia marcescens is a Gram negative, facultatively anaerobic, motile bacterium. It is a human pathogen of the family Enterobacteriaceae. Most strains are resistant to several antibiotics because of the presence of R-factors on plasmids. These organisms are characterised by their ability to produce red pigment at room temperature. They occur naturally in soil and water as well as the intestines. They produce nocosomial infection; associated with urinary and respiratory tract infections, endocarditis, osteomyelitis, septicemia, wound infections, eye infections and meningitis. They are transmitted by direct contact, droplets, through catheters, in saline irrigation solutions, and in other supposedly sterile solutions.

 

Serratia marcescant as biological weapon - In 1951 and 1952 the US Army conducted a study called "Operation Sea-Spray" to study wind currents that might carry biological weapons. They filled balloons with S. marcescens and burst them over San Francisco. Shortly thereafter, doctors noted a drastic increase in pneumonia and urinary tract infections

VMC LECTURE # 18

CAMPYLOBACTER

Campylobacteriosis is a bacterial disease caused by Campylobacter jejuni or Campylobacter coli. C.fetus causes abortion and genital infections in animals. Campylobacter jejuni and C. coli are the most frequently isolated Campylobacter species in infected and diseased humans and can be transmitted from animals to humans directly from contact with animals or through consumption and handling of animal food products. Both C. jejuni and C. coli are commonly present in the intestines and being shed. The faecal contamination of meat (especially poultry meat) during processing is considered to be a major source of human food-borne disease. Both C. jejuni and C.coli, like C.fetus, can also be isolated from bovine and ovine aborted foetuses due to translocation across the intestinal mucosa or by ascending infections

 

Important diseases    -        Infertility and abortion, winter dysentery

 

General characters

1. Morphology: Members of the genus are typically Gram-negative, S-shaped or spiral shaped bacteria (0.2–0.8 µm wide and 0.5–5 µm long). The organisms may have two or more curvatures. The shape is also referred as comma shaped, S shaped or flying seagull like appearance. They are pleomorphic rods. They are motile by polar flagella at one or both ends, conferring a characteristic corkscrew-like motility. They are capsulative and non sporulative. They are microaerophilic organisms requiring 3-15% C0₂ tension. They oxidase positive and do not attack carbohydrates.
2. Habitat: These organisms are commonly found on the mucous membrane of reproductive and digestive tracts. Some birds are found to transmit the infection.
3. Classification: The genus Campylobacter has been carved from Vibryo, which is very important genus since one of the members of the genus V.cholera cause cholera in human beings. The species C.fetus is grouped into two sub species C.fetus subsp. fetus and C.fetus subsp. venerealis. The sub species venerealis is further subdivided into two biotypes based on hydrogen sulphide production into venerealis and intermedius. C.fetus subsp fetus causes abortion and C.fetus subsp venerealis causes bovine genital infections.
4. Resistance: They are fragile organisms and cannot withstand drying and sunshine and are readily killed by disinfectants. They are killed when exposed to a temperature of 60^oC for 5 minutes. They can survive in soil, hay and manure for 10-20 days depending upon the humidity and temperature.
5. Antigens and toxins: Somatic (O), capsular (K) and flagellar (H) antigens are found in C.fetus. There are some similarities and dissimilarities between organisms isolated from different species like cattle and sheep.

 

Cultural characters: They are microaerophilic organisms requiring reduced oxygen tension and 3-15 % C0₂ tension for growth. The optimum temperature for growth is 37^oC. They are also slow growing organisms. The media commonly used for isolation are blood agar with or without thioglycollate, serum-dextrose agar, Thiol medium, Clark and Dufty medium and chocolate agar. Colonies appear after 2-6 days as pinpoint, delicate, circular and opaque. The colonies fuse together to produce a large mucoid colony. The surface of the colonies may have a frosted appearance. Organisms without capsule produce rough colonies.

 

Pathogenesis: C.fetus subsp venerealis cause venereal infection in cattle. The main mode of infection is through coitus and fomites. Bulls get the infection from infected cows through coitus and the affected bulls remain as main source of infection to other animals. Bulls can also transmit the infection mechanically for several hours after copulating with an infected cow. In cows, the duration of the carrier state is also variable; some clear the infection rapidly, while others can carry C fetus for ≥2 yr.  The organisms produce both acute and chronic infections. During acute infections the inflammatory changes in the uterus cause death of fertilized ovum leading to either its expulsion (abortion) or absorption. This infection occurs 3-4 week after fertilisation. In cows with immunity abortion occurs in later stage of pregnancy (fifth month) due to inflammation of uterus and placenta. C.fetus subsp venerealis biotype intestinalis causes infection in sheep that is not venereal. The infection spread through ingestion of contaminated water and feed. Birds also spread infection. Rams have little or no role in the pathogenesis. The death of foetus is due to inflammatory changes in the uterus. The important virulence factor is the endotoxin.

Symptoms: In cattle the symptoms are more pronounced in cows than in bulls. The affected bulls remain normal without any symptoms and no abnormalities are found in the semen. In cows endometritis that causes early embryonic death, prolonged luteal phases, irregular oestrous cycles, repeat breeding abortion and infertility are the main symptoms. Abortion occurs during fifth or sixth month of pregnancy. Infertility rate is higher in heifers than in cows. In sheep abortion during end of pregnancy preceded by vaginal discharge for several days are the main symptoms. Large number of animals is generally affected in flock and the condition is referred as abortion storm.

 

Lesions: The carcass of affected foetus is oedematous with pericarditis, peritonitis and petechial haemorrhages. Areas of focal necrosis are seen in liver. The foetal membranes are oedematous with areas of necrosis on cotyledons. Placentitis occurs with hemorrhagic necrotic cotyledons and edematous or leathery intercotyledonary areas. Catarrhal inflammation of vagina and cervix is common in cows. The same lesions are noticed in ewes also. The ewes will appear normal with oedematous uterus and areas of marked hyperaemia. Metritis is also seen in ewes.

 

Diagnosis:

 

1. Based on symptoms, lesion and history of abortion in a herd.
2. Microscopical examination of smears prepared from the foetal stomach contents or visceral organs stained by Gram’s method reveal organisms with characteristic morphology. C.fetus can also be identified in darkfield preparations from abomasal or placental smears or in uterine discharge.
3. Isolation of the bacteria by culturing: Vaginal mucus and preputial washings are the material of choice. Vaginal mucus three weeks after AI or service is the most ideal material. C.fetus will survive only for 6-8 hr after collection, but inoculation into Clark's or similar media will allow survival for >48 hr. Special care must be taken to avoid contamination of the clinical materials with other bacteria. The media commonly used for isolation are blood agar with or without thioglycollate, serum-dextrose agar, Thiol medium, Clark and Dufty medium and chocolate agar. Colonies appear after 2-6 days as pinpoint, delicate, circular and opaque.
4. Vaginal mucus agglutination test: This test is performed in herd basis to detect antibodies against C.fetus, which are found up to 2-12 months after infection. 10% of the herd or at least 10 cows should be sampled. The mucus is collected in sterile tube of tampon. Antibodies in the mucus are separated by mixing it with agar and treating it with phenol-saline. An agglutination test is performed in serial dilution. A positive reaction is atleast 75% agglutination in the first two tubes.
5. Indirect HA test
6. Serum agglutination test
7. ELISA
8. Mating test: Infected bulls are allowed to mate virgin heifers and examination of the vaginal mucosa after 3-4 weeks. But this is seldom practical. Instead the preputial cavity is infused with buffered sterile saline, and the prepuce is massaged vigorously in the area of the fornix. The sheath washing is then examined using a fluorescent antibody test and culture.

 

Treatment: Treatment is usually not feasible. However, penicillin and streptomycin are used as therapeutic agents. Washing the vagina and prepuce of affected animal with streptomycin is of some use.

 

Control: Elimination of the infected bull and treating the semen with streptomycin are the two important aspects in control programme. Killed vaccines have found to reduce the incidence of infertility. Artificial insemination instead of natural coitus will also avoid campylobacteriosis.

 

OTHER SPECIES OF CAMPYLOBACTER

OIE Listing – Covered but not under List A or B

 

The species, particularly C. jejuni, C. coli and C. lari, are thermophilic, growing optimally at 42°C. Hence, in the isolation procedure the media are incubated at 42°C to prevent contamination from other bacteria.

 

C.jujuni: It is found in the intestinal tract of domestic animals and birds as commensals. It causes infectious diarrhoea (winter dysentery) in cattle. Affected animals become listless, show signs of abdominal pain with dark coloured fluid consistency faeces with blood and mucus. The powerful heat labile enterotoxin produced by the organism is responsible for the diarrhoeic symptoms. It causes avian infectious hepatitis (vibryonic hepatitis) in chickens characterised by haemorrhage and necrosis of liver.

 

Pathogenesis: Environmental contamination is the main source of infection for poults, chicks, and ducklings. Litter can remain infective for long periods. Infected chicks and poults become colonized and can continue to excrete C jejuni for up to 2 months. Contaminated water can introduce infection into poultry flocks, and nonchlorinated water derived from a dam, river, or shallow well should be regarded as a possible source. Flocks can be infected by rats, wild birds, and houseflies; equipment and footwear contaminated with faeces from an infected source. Once introduced into the environment, rapid transmission within the flock occurs. C.jejuni is not transmitted vertically, either on the surface of eggs or by transovarial transmission.

 

Symptoms and lesions: Rapid onset of diarrhoea, which may persist for four days is the predominant symptom. Gross lesions include distention of the jejunum, disseminated hemorrhagic enteritis, and in some cases, focal hepatic necrosis. Microscopic lesions include edema of the mucosa of the ileum and cecum with C jejuni in the brush border of enterocytes. Mononuclear infiltration of the submucosa and villous atrophy occur with intraluminal accumulation of mucus, erythrocytes, and both mononuclear and polymorphonuclear cells.

 

Diagnosis:

1.           Isolation and identification by culture method: Fecal specimens should be collected using swabs. Enrichment culture of specimens in semisolid motility medium facilitates isolation when small numbers of C jejuni are present in a sample. The materials should be cultured on selective media containing brucella agar base and bovine blood with up to seven antibiotics that inhibit overgrowth of other Enterobacteriaceae.

2.           Nucleic acid identification method: Bacterial restriction endonuclease DNA analysis can distinguish among various C jejuni isolates.

 

Control:

Control in commercial poultry is based on strict biosecurity, decontamination of housing between successive flocks, exclusion of rodents and wild birds, and insect eradication. Chlorination of drinking water to 2 ppm and operating farms on a strict all-in/all-out basis can reduce the prevalence of infection.

 

Public Health Significance

C.jejuni is a major source of food-borne enteritis in consumers; contaminated, undercooked poultry is responsible for >50% of cases. In addition to contaminated poultry, nonchlorinated ground water, young diarrheic pets, and contaminated beef and pork products may also be responsible for infection of people. C.jejuni 

contamination can be reduced by improved washing of carcasses, the use of counter-flow scalding, elimination of immersion chillers, and reduction in manual handling by installation of advanced automated equipment. Chemical disinfectants, such as glutaraldehyde (0.125%) and succinic acid (3%), and organic compounds, such as lactic and acetic acids, may be used to destroy C jejuni when used as a spray or an immersion treatment.

Gamma irradiation at levels ranging from 1 to 3 kGy effectively eliminates C jejuni from poultry carcasses and products.

The only current measure to reduce the risk of C jejuni infection to consumers is thorough cooking of poultry to achieve a core temperature of 74°C for 1 min. This ensures destruction of C jejuni.

 

C.coli: It causes swine dysentery.

 

ISO STANDARDS IN IDENTIFICATION OF CAMPYLOBACTER

 

ISO 10272:1995   Microbiology of food and animal feeding stuffs -- Horizontal

method for detection of thermotolerant Campylobacter

 

ISO 17995:2002  Isolation of Campylobacter from water (under development)

VMC LECTURE # 19

PSEUDOMONADS - PSEUDOMONAS & BURKHOLDERIA

 

Introduction: Members of the genus are found abundantly as free-living organisms in soils, fresh water and marine environments, and in many other natural habitats. They are also found in associations with plants and animals as normal flora or as agents of disease. The term "pseudomonad" refers to a bacterium with ecophysiological properties similar to members of the genus Pseudomonas. Some of these bacteria were formerly in the genus Pseudomonas but have been moved to other genera, families, or orders. Pseudomonads are globally active in aerobic decomposition and biodegradation, and hence, they play a key role in the carbon cycle. Pseudomonas species are renowned for their abilities to degrade compounds, which are highly refractory to other organisms, including aliphatic and aromatic hydrocarbons, fatty acids, insecticides and other environmental pollutants. Apparently, the only organic compounds that these  pseudomonads can't attack are teflon, styrofoam and one-carbon organic compounds (methane, methanol, formaldehyde, etc.). Pseudomonads are also a regular component of microbial food spoilage in the field, in the market place, and in the home. Pseudomonas and certain other pseudomonads include species pathogenic for humans, domestic animals, and cultivated plants.  Pseudomonas species, as well as species included in the newly-created genera Burkholderia and Ralstonia (ex-Pseudomonas) are among the most important bacteria that are pathogens of plants. They cause economically significant crop disease and crop loss world-wide.  Pseudomonas aeruginosa infects both plants and animals and has evolved into one of the most common and refractory nosocomial pathogens of the post-antibiotic era.

 

Important species     :        Pseudomonas aeruginosa,

P.mallei (Burkholderia mallei)

P.pseudomallei (B. pseudomallei)

Important diseases    :        Pyogenic infection, Melioidosis, Glanders

 

General characters:

1. Morphology: They are gram-negative rod shaped organisms (0.5-0.8 um x 1-3 um). They are actively motile by polar flagella some strains also produce lateral flagella. They are non-capsulative and non-spore forming. They are strictly aerobic. Catalase-positive, Usually oxidase-positive
2. Habitat: They are worldwide in distribution and occur as commensals in the intestine of animals and man. Animal pathogens are less host specific and most are considered to be opportunistic pathogens. One species, B. mallei, is an exception to this rule. This bacterium is the agent of glanders in horses, mules, and donkeys. B. mallei is not found living saprophytically, and it is considered to be the only pseudomonad with exclusive parasitism.
3. Resistance: They are fragile organisms and are lethal to heat and disinfectants. They are killed at a temperature of 55C for 1 hour.
4. Classification: Pseudomonads species are classified into five natural clusters based on ribosomal RNA homology, called “RNA similarity groups”. The Pseudomonas spp. are further classified into subgroups based on O antigen.

Pseudomonas aeruginosa – RNA similarity Group I (Palleroni’s Group I)

Burkholderia mallei and B. pseudomallei – RNA similarity Group II (Palleroni’s Group II)

5. Antigens and toxins: They have somatic (O) antigen. No exotoxins are produced.
6. Metabolism: Pseudomonas species are respiratory and never fermentative. All species respire aerobically, and some respire anaerobically with NO₃ as a final electron acceptor.
7. Pigment production: A common characteristic of the fluorescent pseudomonads is the production of pigments that fluoresce under short wave length (254 nm) ultraviolet light. These pigments and/or their derivatives are known to play a role as siderophores in the iron uptake systems of the  bacteria. P. aeruginosa is capable of producing several pigments, of which the most characteristic is pyocyanin. This blue pigment is an diagnostic character, since no other species has been found to produce it. The other pigments are Pyoverdin (fluorescent pigment), pyorubrin (reddish pigment), and a brown pigment, pyomelanin.  The production of pigments is readily demonstrated by culturing the bacteria in media such as King's Medium B

 

Cultural characteristics: They grow at a wide temperature of 5-43C. Optimum growth is obtained at 37C in ordinary laboratory media. No organic growth factors are required for the growth of these organisms. The colonies are convex, glistening, bluish metallic sheen and have a tendency to spread. The colonies fluoresce due to production of two pigments called pyocyanin and fluorescin. The cultures have a characteristic sticky odour (grape like)

 

Pathogenesis:

P.aeruginosa is found worldwide and is also found in soil. They produce variety of pyogenic infection. The pus is greenish yellow in colour. It also causes bovine mastitis, urogenital infections, liver abscess, calf enteritis, pneumonia etc. In sheep it causes a condition called green wool.

 

GLANDERS – (Syn. Dros, Farcy,    Malleus)

Glanders is an infectious disease caused by the bacterium Burkholderia mallei. Glanders is primarily a disease affecting horses, but it also affects donkeys and mules and can be naturally contracted by goats, dogs, and cats. Human infection occurrS rarely and sporadically among laboratory workers and those in direct and prolonged contact with infected, domestic animals.

 

OIE Listing – List B disease

 

Previous names of the organism: Loefflerella mallei, Pfeifferella mallei, Malleomyces mallei, Actinobacillus mallei, Corynebacterium mallei, Mycobacterium mallei, Bacillus mallei, Pseudomonal mallei

 

Morphology of B.mallei: The organisms are mainly extracellular, fairly straight Gram-negative rods with rounded ends, 2–5 µm long and 0.3–0.8 µm wide with granular inclusions of various size, often stain irregularly. They do not have capsules or form spores. However, the presence of a capsule-like cover has been established by electron microscopy recently. This capsule is composed of neutral carbohydrates and serves to protect the cell from unfavourable environmental factors. Unlike other organisms in the Pseudomonas group, Burkholderia mallei have no flagellae and are therefore nonmotile. The causative organism B.mallei is closely related to B.pseudomallei, the cause of melioidosis, and is serologically indistinguishable. The organism is destroyed by direct sunlight and is sensitive to desiccation. It is readily killed by common disinfectants. It may survive for up to 6 weeks in infected stables

 

Cultural charcters: The organisms are aerobic and facultatively anaerobic only in the presence of nitrate. Optimum temperature for growth is 37°C. It grows well, but slowly, on ordinary culture media, 48-hour incubation of cultures is recommended. Glycerol enrichment enhances the growth. After a few days on glycerol agar confluent, slightly cream-coloured growth that is smooth, moist, and viscid can be observed. With continued incubation, the growth thickens and becomes dark brown and tough. It also grows well on glycerol potato agar and in glycerol broth, on which a slimy pellicle forms. On plain nutrient agar, the growth is much less luxuriant, and growth is poor on gelatin.

 
Pathogenesis: Glanders occurs in horses, mules and donkeys. It also occurs in wild animals eating the meat of carcasses infected with B.mallei. Cattle, pig and poultry are resistant. The disease is introduced into horse populations by diseased or latently infected animals. Ingestion of the pathogen, present in secretions from infected animals, constitutes the major route of infection in glanders. Inhalation and entry through wounds in skin are of minor importance in the natural spread of the disease. Close proximity alone does not usually result in transmission of glanders; transmission is facilitated if the animals share feeding troughs or watering facilities or if they nuzzle each other. The primary infection is seen in lungs. However, digestive tract infection is seen when the organisms are ingested. The susceptibility to infection increases by poor nutrition, over work and other stress factors. The initial distribution of the organisms in the system is via lymphatic system followed by blood stream. The incubation period varies.

Symptoms: The clinical forms of the disease are classified as pulmonary form, nasal form and cutaneous form (farcy). The infection occurs either as any one of the individual form or as combination of forms.

The pulmonary form is characterised by pyrexia and severe cough that produce blood stained mucus. In the nasal form, there is reddening of nasal mucosa and discharge from nostrils that may turn purulent and blood stained later. Ulceration of nasal mucosa and enlargement of the lymph glands are other important symptoms. Farcy is characterised by cutaneous or subcutaneous nodules on the limbs or flanks. Later these nodules turn into hollow ulcers releasing yellow and oily pus. The local lymphatic vessels become corded. These may be referred to as "Farcy pipes."

Lesions: Nodular lesions of glanders are found beneath the pleura of the lung. In some acute cases lobular pneumonia may be present. The nodular lesions, typically about 1 cm in diameter, consist of a gray or white core of necrotic material that may become calcified and are surrounded by a zone of hyperemia and edema. Similar lesions may be found in other viscera. Glanderous orchitis may be seen in intact males.

Nasal lesions consist of submucosal nodules surrounded by a small zone of hyperemia. These nodules may rupture, leaving exudative ulcers. As new lesions develop it is not unusual to find small nodules, ulcers, and scars side by side. Lymphadenitis of associated lymph nodes is a consistent finding. In some cases laryngeal lesions similar to the nasal lesions may be found.

Cutaneous lesions consist of cord-like thickening of subcutaneous lymphatics along which are distributed chains of nodules, some of which are ulcerated. Histologically, there is infiltration of neutrophils, fragmentation of cellular nuclei, giant cell formation and rarely calcification.

 

Diagnosis:

1.      Based on symptoms and lesions.

2.      Mallein test (Test prescribed for International Trade): It is an allergic test. Mallein is the active principle form B.mallei. Various sites are preferred for mallein test like intradermopalpebral, ophthalmic and subcultaneous.

a.      The intradermopalpebral (below the eyelids) test is more reliable. Swelling of the eyelid and congestion of conjunctiva after 36-48 hours indicate a positive reaction.

b.      Ophthalmic: A few drops of mallein are instilled into the eye at the canthus. In an infected animal, the eyelids, and sometimes the side of the face, become swollen and there may be a little discharge from the eye. The reaction may also occur to a lesser extent in the opposite eye.

c.      Subcutaneous: A 10 cm square skin patch in the middle of the neck is clipped and disinfected; 2.5 ml of dilute mallein are injected subcutaneously into the centre of the patch. With a positive test, the horse develops a pyrexia of 104°F (40.0°C) or over during the first 15 hours, and a firm painful swelling with raised edges develops within 24 hours at the injection site. In nonglandered horses, there is no, or minimal, transient local swelling. Doubtful reactors may be retested after 14 days using a double dose of mallein

3.      Strauss reaction: Strauss reaction is observed when infectious material from glanders patients is injected intraperitoneally into male guinea pigs. In positive cases, the guinea pig develops localized peritonitis involving the scrotal sac. Glanderous orchitis follows with painful enlargement of the testes. The testis becomes enlarged and painful and ultimately necrotic and is discharged through the scrotal skin.

4.      Nucleic acid identification methods: Polymerase chain reaction (PCR) is used for the specific detection of B. mallei DNA. This test also helps in the differentiation between B. mallei and B. pseudomallei.

5.      Serological tests: Agglutination, precipitation, ELISA, CFT and conglutination tests (CFT is approved for International Trade) are routinely performed.

6.      Isolation of bacteria from pus in glycerol agar or potato agar or serum agar.

7.      Differential diagnosis:

a.      Strangles is caused by Streptococcus equi. It is characterized by fever, anorexia, and depression with swollen submandibular lymph nodes and mucopurulent nasal discharge. The nasal discharge is usually bilateral, whereas it is most often unilateral in cases of glanders. Skin nodules and typical lung lesions are absent. Animals with strangles will not react to mallein testing or serological tests for glanders. S. equi is readily demonstrable. Strangles does not develop into a chronic, debilitating condition, and most infected horses recover within a few weeks.

b.      Epizootic lymphangitis (caused by Histoplasma farciminosum) is characterized by cutaneous nodules originating from superficial lymph vessels. In epizootic lymphangitis, conjunctivitis is a common lesion. Demonstration of the infectious agent and application of the mallein test and serological testing will help distinguish between these diseases.

c.      Ulcerative lymphangitis (caused by Corynebacterium pseudotuberculosis) is characterized by dermatitis and abscess formation predominantly in the pectoral and ventral abdominal regions. Standard diagnostic tests are again valuable in distinguishing this disease from glanders.

d.      Melioidosis (caused by B.pseudomallei) is characterized by multiple abscesses in a variety of tissues and organs. Unlike glanders, it is not specifically a disease of equids and occurs most often in sheep, goats, and swine. It is characterized by dyspnea and lameness, but a wide array of clinical signs may be elicited. Diagnosis is confirmed by isolation of the causative organism. Serological cross-reactions occur with B.mallei.  

 

Treatment and Control: There is no vaccine. Prevention and control depend on early detection and elimination of affected animals, as well as complete quarantine and rigorous disinfection of the area involved. Treatment is given only in endemic areas. Antibiotics are not very effective. Combinations of sulfazine or sulfamonomethoxine with trimethoprim were found to be efficient in the prevention and treatment of experimental glanders.

 

Zoonotic importance: The human form of the disease is painful and frequently fatal. Laboratory workers and animal attendants are most at risk. Symptoms of glanders in people include nodular eruption on the face, legs, and arms; involvement of the nasal mucosa; and later pyemia and metastatic pneumonia. Human glanders may be confused with a variety of other diseases, including typhoid fever, tuberculosis, syphilis, erysipelas, lymphangitis, pyemia, yaws, and melioidosis. The diagnosis can be confirmed by serology and by isolation of the causative organism.

 

Control of Glanders in India: Glanders was considered as very serious disease and as early as in 1899 and Act was passed to control the infection in horses (includes asses and mules). The extracts are given below;

·         THE GLANDERS AND FARCY ACT 1899 WITH NOTIFICATIONS AND RULES THERE UNDER (ACT    No. XIII OF 1899) PASSED BY THE GOVERNOR GENERAL OF INDIA IN COUNCIL (Received the assent of the Governor General on the 20^th March 1899).

·         It extends to the whole of British India

·         When this Act has been so applied to a local area, the local Government may by notification in the local official Gazette appoint such persons as it thinks fit to be Inspectors under this Act and to exercise and perform within the whole of the local area, of such portions thereof as it may prescribe the powers conferred and the duties imposed by this Act on such officers

·         Within such limits as aforesaid, the Inspector may seize Power of any horse, which he has reason, to believe to be diseased.

·         On any such seizure as aforesaid, the Inspector shall cause the horse seized to be examined as soon as possible by such Veterinary Practitioner as the Local Government may appoint in this behalf.

·         If the Veterinary Practitioner certifies in writing that the horse is diseased, the Inspector shall cause the same to be immediately destroyed.

·         When any diseased horse has been in any building, shed or other enclosed place, or in any open lines, the Inspector may issue a notice to the owner of the building, shed place or lines or to the person in charge thereof, directing him to have the same disinfected and the internal fittings thereof or such other things found therein or near thereto as the Local Government may by rule prescribe, destroyed.

·         On the failure or neglect of such owner or other person as aforesaid to comply with the notice within a reasonable time, the Inspector shall cause the billing, shed place or lines to be disinfected and the fittings or other things to be destroyed, and the expense (if any) thereby incurred may be recovered from the owner or other person as if it were a fine.

·         Whoever refuses or neglects to comply with any notice issued by the Inspector under section 9, or removes any horse in contravention of section 11, shall be punishable with imprisonment for a term which may extend to one month, or with fine which may extend to fifty rupees, or with both.

 

 

VMC LECTURE # 19 (A)

MORAXELLA

 

Important species:    Moraxella bovis

Important disease:    Bovine keratitis, Pink eye, and Bovine kerato-conjunctivitis

 

General characters:

1. Morphology: They are gram-negative rod shaped organisms arranged in pairs and hence are referred as diplobacilli. They are non-motile, non-spore forming organisms with prominent capsules.
2. Habitat: They are found generally found in the eye. They can even remain in the eye without causing any infection.
3. Classification: M.bovis earlier placed in the genus Haemophius. It has been moved out of Haemophius since it does not require X and V factors for growth.
4. Resistance: They are relatively fragile organisms and are sensitive to heat at a temperature of 60^oC for 15 minutes and to number of disinfectants.

 

Cultural characters: It is an aerobic organism and grows at 37^oC on blood agar. The colonies are small, circular, translucent and grey in colour surrounded by beta haemolysis. A granular deposit is noticed in broth culture and gelatin is liquefied.

 

Pathology: The organisms are found in the conjunctiva casing highly contagious kerato-conjunctivitis. The disease is transmitted from one animal to another though infective material. The infection also spreads via conjunctival exudates. There is conjunctivitis followed by keratitis and ulceration of the cornea.

 

Diagnosis is based on isolation of the organisms in blood agar from conjuncival swabs.

 

Treatment: Chloramphenicol, tetracycline and sulphonamides.

.

VMC LECTURE # 20

LEPTOSPIRA

 

The spirochetes comprise of three important genus that caused infection in man and animals. The three genus are

1.      Leptospira

a.      Leptospirosis

2.      Borrelia

a.      Lyme disease or relapsing fever

b.      Fowl spirochetosis

3.      Spirillum

a.      Rat bite fever

 

Leptospirosis is an infectious disease of animals and man caused by a spiral-shaped organism (spirochete) of the genus Leptospira. The important serotypes recognized in livestock are Leptospira pomona, L. canicola, L. icterohaemorrhagiae, L. grippotyphosa and L. hardjo. These organisms have a wide host range, including man. However, humans are accidental hosts in whom this disseminated disease varies in severity from subclinical to fatal. In humans this infection is referred as Weil’s disease.  Among domestic animals, swine, cattle, dogs, and horses are most frequently affected. Known wildlife hosts include many of the small rodents, raccoons, foxes, opossums, skunks, deer, and moose. Because of the nature of the disease, it should not be considered a problem of the individual animal but a problem of a population, either a herd or a species within the area. The disease can be controlled by proper management and the correct use of available vaccines.

 

General Characters

1.     Morphology: Leptospira are thin, tightly coiled obligate aerobes that are highly motile (flexuous type of motility). Leptospira has the general structural characteristics that distinguish spirochetes from other bacteria. The cell is encased in a three- to five-layer outer membrane or envelope. Beneath this outer membrane are the flexible, helical peptidoglycan layer and the cytoplasmic membrane; these encompass the cytoplasmic contents of the cell. The structures surrounded by the outer membrane are collectively called the protoplasmic cylinder. An unusual feature of the spirochetes is the location of the flagella, which lie between the outer membrane and the peptidoglycan layer. They are referred to as periplasmic flagella. The periplasmic flagella are attached to the protoplasmic cylinder subterminally at each end and extend toward the center of the cell. The number of periplasmic flagella per cell varies among the spirochetes. The leptospires have two periplasmic flagella, one originating at each end of the cell.  The motility of bacteria with external flagella is impeded in viscous environments, but that of spirochetes is enhanced. The slender (0. 1 µm by 8 to 20 µm) leptospires are tightly coiled, flexible cells. In liquid media, one or both ends are usually hooked. Leptospires are too slender to be visualized with the bright-field microscope but are clearly seen by dark-field or phase microscopy. They do not stain well with aniline dyes.

2. Habitat: The primary reservoir hosts are wild animals such as rodents, which can shed leptospires throughout their lifetimes. Leptospires have been isolated from approximately 160 mammalian species. Most leptopsires are pathogenic whereas serotypes of L biflexa are not pathogenic and exist in water and soil as free-living organisms. .
3. Classification: All the pathogenic leptospires were formerly classified as members of the species Leptospira interrogans, however the genus has recently been reorganised and pathogenic leptospires are now identified in several species of Leptospira. The recent classification is based on nucleotide sequences. There are more than 200 distinct leptospiral serovars recognised and these are arranged in 23 serogroups. Some of the important pathogenic species are listed below; Leptospira pomona, L. canicola, L. icterohaemorrhagiae, L. grippotyphosa and L. hardjo
4. Resistance: They are fragile and do not withstand dryness or heat. They can survive in alkaline waters for upto six months.
5. Antigens and Toxins: They do not produce exotoxin and do not have endotoxin

 

Cultural characters: The leptospires are the most readily cultivated of the pathogenic spirochetes. They have relatively simple nutritional requirements; long-chain fatty acids and vitamins B1 and B12 are the only organic compounds known to be necessary for growth. When cultivated in media of pH 7.4 at 30°C, their average generation time is about 12 hours. Aeration is required for maximal growth. They can be cultivated in plates containing soft (1 percent) agar medium, in which they form primarily subsurface colonies. They are also grown in semisolid (0.1–0.2% agar) medium containing BSA and either Tween 80. Fastidious leptospires prefer a medium containing o.4–1% rabbit serum (Stuart’s media; Fletchers media). Cultures are incubated at 29 +/- 1°C for at least 16 weeks, and preferably for 26 weeks. 

 

Pathogenesis

OIE Listing – List B (Multi species)

Leptospirosis is generally contracted by the direct splashing of urine from infected or carrier animals into the eyes of susceptible animals. It can also be spread through the skin and mucous membranes from contact with water contaminated with leptospires. Transmission may also occur during breeding through residual urine in the genital tract or through infectious semen. The major sources of contamination are swine, cattle, dogs, and wildlife that have recovered from the disease and have become carriers. Cattle and swine are major sources because of the volume of urine and the extent and duration of leptospires in the urine. Human cases are acquired through direct contact with the urine of infected livestock or with contaminated soil or water. Swimming in water contaminated with the urine of infected livestock also serves as a source for human infection. The organism may survive up to six months in alkaline water.

The mucosa and broken skin are the most likely sites of entry for the pathogenic leptospires. A generalized infection ensues, but no lesion develops at the site of entry. Bacteremia occurs during the acute, leptospiremic phase of the disease. The host responds by producing antibodies that, in combination with complement, are leptospiricidal. The leptospires are rapidly eliminated from all host tissues except the brain, eyes, and kidneys. Leptospires surviving in the brain and eyes multiply slowly if at all; however, in the kidneys they multiply in the convoluted tubules and are shed in the urine (the leptospiruric phase). The leptospires may persist in the host for weeks to months; in rodents they may be shed in the urine for the lifetime of the animal. Leptospiruric urine is the vehicle of transmission of this disease. The mechanism by which leptospires cause disease remains unresolved, as neither endotoxin nor exotoxins have been associated with them. The marked contrast between the extent of functional impairment in leptospirosis and the scarcity of histologic lesions suggests that most damage occurs at the subcellular level. Damage to the endothelial lining of the capillaries and subsequent interference with blood flow appear responsible for the lesions associated with leptospirosis. The most notable feature of severe leptospirosis is the progressive impairment of hepatic and renal function. Renal failure is the most common cause of death.

 

Symptoms:

Cattle: Leptospirosis in cattle may vary in severity from a mild, inapparent infection to an acute infection that may cause death. Acute clinical disease occurs more often in young calves, but all ages may be affected. Cattle may develop a high fever of 104° to 107°F, depression, loss of appetite, decreased milk production, and weakness. Hemoglobinuria (coffee-colored urine), anemia, icterus (jaundice), and bloody milk are also seen. In lactating cows, the udder becomes flaccid and milk flow nearly ceases. The milk is usually abnormal and may contain thick yellow or reddish clots. Reddish milk may develop with the onset of hemoglobinuria. Abortion occurs two to five weeks after initial infection with leptospires in the last three months of pregnancy.

Swine. The disease in swine is largely subclinical (no apparent signs of illness) except for abortions, which usually occur during the last two to three weeks of pregnancy. The sow aborts pigs rapidly with no apparent signs of illness. Some aborted fetuses may have been dead a short time. Piglets may be born weak and die shortly after birth.

Horses. The infected animal may have a slight temperature rise and mild loss of appetite. Leptospirosis caused by L. pomona is widespread among susceptible horse populations. Within 12 to 14 months after the initial infection, the eyes of many horses show evidence of uveitis (inflammation of the internal structures of the eye), a disease commonly known as periodic ophthalmia or "moon blindness." Leptospirosis is not a common cause of abortion in horses.

Sheep and goats. The signs reported are similar to those described for cattle.

Dogs. Leptospira canicola is the most common serotype; L. icterohaemorrhagiae, L. pomona, and L. grippotyphosa are also responsible for some infections. The acute infection is characterized by elevated body temperature, vomiting, muscular stiffness, weakness, and nephritis (inflammation of the kidney). In severe cases, jaundice and death may occur. Central nervous system signs may occur with or without other clinical signs, and organisms may be present in the brain tissue for extended periods. Chronic leptospirosis is primarily associated with chronic kidney degeneration. Shedding of leptospires in the urine may continue for over a year.

Cats: Leptospira infection and disease are very rare in cats.

Rodents. Wild rat (Rattus norvegicus) populations in urban and rural areas are frequently infected with L. icterohaemorrhagiae. Leptospira ballum is found primarily in wild mice (Mus musculus), but is also present in some rat populations and has been present in commercially raised laboratory mice. Other serotypes occur with less frequency in rodent populations.

Diagnosis

1.      Based on symptoms and lesions: Leptospirosis diagnosis in all animals, including livestock, companion, and wild animals, must be confirmed by laboratory tests. There are no clinical signs distinctive to this disease but certain signs are suggestive.

2.      Serological tests: The ideal method for diagnosis of leptospirosis is the demonstration of significant levels of antibodies to leptospires in the serums of recovered animals. Plate agglutination tests or microscopic agglutination tests are normally performed.

a.      Plate agglutination test with killed antigens or the microscopic agglutination (MA) test, in which living organisms are used as antigens. Titers greater than 1:100 by MA test or 1:40 or greater by plate test against one or more serotypes are generally considered significant.

b.      ELISA:

3.      Microscopic demonstration:

a.      Leptospires are too slender to be visualized with the bright-field microscope but are clearly seen by dark-field or phase microscopy.

b.      Leptospires do not stain satisfactorily with aniline dyes, and silver-staining techniques lack sensitivity and specificity.

c.      Immunohistochemical staining: Leptospires can also be demonstrated by a variety of immunochemical staining techniques, e.g. immunofluorescence. This technique is in diagnosing infection in pathological material that is unsuitable for culture or where a rapid diagnosis is required. The technique is less suitable for diagnosing the chronic carrier state, where the numbers of organisms may be very low or localised.

4.      Isolation and identification of leptospires: Leptospires are most readily isolated from blood or milk taken from animals during the acute phase, or from the urine, kidneys, spinal fluid, and brains of recovered animals.

5.      Nucleic acid identification methods: Polymerase chain reaction (PCR)-based assays are now used in the detection of leptospires in tissues and body fluids.

 

Control

1.      Livestock herds can be protected against leptospirosis by a combination of proper management and vaccination procedures.

2.      Cattle used for breeding purposes should be vaccinated during early pregnancy to provide the greatest degree of protection during the last two-thirds of gestation.

3.      The vaccine used in infected herds should be identical with the serotype causing the diseases, as there is little or no cross-protection between vaccine serotypes. The vaccine should be given to all susceptible livestock on the premises where infection has been identified.

4.      Annual vaccination is recommended for dogs with a booster given if an outbreak occurs in the area.

5.      Control of rodents in animal houses.

 

Treatment

Streptomycin, chlortetracycline, or oxytetracycline are the antibiotics of choice for treatment.

VMC LECTURE # 20 (A)

BORRELIA - Fowl spirochetosis

General characters: The causal organism, Borrelia anserina, is an actively motile spirochete, ~0.2-0.3 µm × 8-20 µm and consists of 5-8 loosely arranged coils.

Cultural characters: in vitro cultivation is not possible. However, they can be propagated in embryonating duck or chick embryos or in young ducks or chicks.

 

Pathogenesis: Argas spp transmit the disease in different geographic areas. Generally, an infected Argas tick can transmit the disease at every feeding and maintains the infection throughout larval, nymphal, and adult stages. The ticks also transmit the infection transovarially, ie, the F₁ larvae are infective. Other vectors (lice, mosquitoes, some species of ticks, inanimate objects) can transmit the spirochete mechanically to a susceptible host. Ingestion of bile-stained fecal droppings containing the spirochete, as well as acts of cannibalism during spirochetemia can result in infection. After the bite of an infected tick, the incubation period is ~4-7 days.

 

Symptoms

Signs are highly variable, depending on the virulence of the spirochete, and are not pathognomonic. They include listlessness, depression, somnolence, moderate to marked shivering, and increased thirst. Young birds are affected more severely than older ones. During the initial stages of the disease, there is usually a greenish yellow diarrhoea with increased urates. The course of the disease is 1-2 wk. In tick-infested geographical areas, morbidity can approach 100% and mortality >90% has been recorded.

 

Lesions: An enlarged spleen with petechial or ecchymotic hemorrhages is the most notable gross lesion. Occasionally, the liver may be swollen and contain focal areas of necrosis. Kidneys may be enlarged and pale. A green, catarrhal enteritis is common

 

Diagnosis:

1. Diagnosis rests on demonstration of Borrelia in the blood, either as actively motile Borrelia during darkfield microscopy, or as stained spirochetes in Giemsa-stained blood smears.
2. Agar-gel diffusion and various serological tests have been described but are of questionable value

Treatment

The most widely used antibiotics are penicillin derivatives, but the streptomycins and tetracyclines are also effective.

 

Control

1.

Control of biological vector Argas ticks

2. Bacterins prepared from infective blood are used as vaccines.

 

 

VMC LECTURE # 21

MYCOPLASMA

 

Members of the genus Mycoplasma are the smallest organisms lacking cell walls that are capable of self-replication and cause various diseases in humans, animals, and plants. Mycoplasmas are unique because they are neither virus nor bacteria. Mycoplasmas do not have a protective cell wall and are minimal in the area of genetic material.  Due to this fact it is very hard for the hosts immunity to fight off the disease. The animal's immune system targets disease-causing bacteria for destruction by locking on the shape and size of the bacteria's surface structures.  In the case of mycoplasma, these structures keep changing making their removal difficult for immune system. Mycoplasma often colonize in the respiratory tract, the bloodstream, and other mucosal surfaces.  For this reason it is very hard to control due to animal's contact.  Even in confinement houses animals sneeze and can spread the disease. Over 90 organisms belonging to this group have now been recognized

 

Type species : Mycoplasma mycoides

 

General characteristics:

1. Morphology: Mycoplasma are the smallest and simplest prokaryotic cells capable of self-replication. They do not have cell wall hence they are highly pleomorphic (filamentous, pear shaped, spherical). They are surrounded by a lipoprotein plasma membrane. Some multiply by binary fission where as some have a reproductive cycle. They are poorly gram-negative and are stained by Giemsa method. The genome of mycoplasma is dsDNA and is smallest of any replicating prokaryotic cell. They have the smallest genome size and, as a result, lack many metabolic pathways and require complex media for their isolation.
2. Habitat: They are generally found as commensals in mucous membrane of upper respiratory, digestive, genital tracts and udder.
3. Classification: All mycoplasma belong to the class Mollicutes having two families Mycoplasmataceae and Acholeplasmataceae and three genuses Mycoplasma, Acholeplasma and Ureaplasma.
4. Resistance: They are killed at moist heat and by dry temperatures of 60C for 5 minutes. They can remain viable for long time in freeze dried cultures and frozen lung. They are sensitive to disinfectants.
5. Antigens and toxins: Multiple antigenic components are reported in mycoplasmas. Certain organisms produce exotoxins. M.neurolyticum, which causes rolling disease in mice, produce a neurotoxin.

 

Cultural characteristics: They are fastidious organisms and require cholesterol for their growth. They grow aerobically at a carbon dioxide tension of 10%. The media used for isolation of mycoplasma contain 20% unheated horse serum, 10% fresh yeast agar, and very minute quantity of DNA, penicillin and thallous acetate. The pH should be between 7 and 8. The optimum temperature is 36-38C. The colonies are microscopic and appear after 3-4 days. They have yellowish, opaque centre surrounded by a thin translucent rim resembling poached egg or fried egg appearance. Growth is difficult in fluid medium and a granular type growth is noticed. Seven-day-old embryonated eggs are used for cultivation of mycoplasmas. After two days the embryo will die showing extensive oedema and swelling. Cell culture systems like HeLa cells, chicken heart fibroblasts and human conjunctival cells are used for cultivation. The biochemical reactions, growth inhibition test and metabolic inhibition test are of use in diagnosis.

 

Pathogenesis:

Many Mycoplasmas live as commensals in the respiratory, digestive, urinary tracts and udder. Their plastic nature besides an adhesion protein help the organisms to attach themselves to the mucous membrane. The destruction of cilia in mucous membrane predispose the cells for mycoplasma infection. The fibrinous exudates protect them from phagocytic cells and antimicrobial drugs. The accumulation of mycoplasmal metabolites are also responsible for the pathogenesis. The important virulence factors are adherence factors and toxic metabolic products. The immune response also aids in spread of the infection.

Adherence factors – The adherence proteins are one of the major virulence factors. The adherence protein called P1. The P1 Adhesin localizes at tips of the bacterial cells and binds to sialic acid residues on host epithelial cells. This protein has been identified only in M.pneumoniae and not identified in other Mycoplasmas. Colonization of the respiratory tract results in the cessation of ciliary movement. The normal clearance mechanisms of the respiratory tract do not function, resulting in contamination of the respiratory tract and the development of a dry cough.

Toxic Metabolic Products - The toxic metabolic products of Mycoplasmas accumulate and damage host tissues. Both hydrogen peroxide and superoxide, which are products of mycoplasma metabolism and are responsible for pathogenesis. The mycoplasmas also inhibit host cell catalase and increase the peroxide concentrations further facilitating the infection.

 

Both acute and chronic infections are produced. The important mycoplasma species and the infection produced by them are listed below

 

1. Mycoplasma of birds

Avian mycoplasma are generally transmitted by eggs. Affected birds will have respiratory distress, drop in egg production and poor growth rate.

1.      M.gallisepticum – Chronic respiratory disease (CRD), air sac disease of chicken, turkeys and other fowl.

2.      M.synoviae: Infectious synovitis of chickens and turkeys. Infection are seen in hock and wing joints.

3.      M.meleagridis: Airsacculitis in turkeys.

4.      M.iowae: Airsacculitis

 

2. Mycoplasma of cattle:

1.      M.mycoides ss. mycoides: Causes contagious bovine pleuropnemonia (CBPP). The infection is characterised by septicaemia and suppurative lesions in lungs, pleura and pericardium. It also causes abortion and results in infertility.

 

3. Mycoplasma of sheep and goat:

1.      M.mycoides ss. Capri: Causes contagious caprine pleuropneumonia (CCCP).

2.      M.agalactiae: Contagious agalactia in sheep and goat.

 

4. Mycoplasma of pigs:

1.      M.hyorhinis: Polyserositis and arthritis.

2.      M.hyosynoviae: Arthritis.

 

A. MYCOPLASMA INFECTION IN POULTRY

1. MYCOPLASMA GALLISEPTICUM

Mycoplasma gallisepticum infection is commonly designated as chronic respiratory disease in chickens and infectious sinusitis in turkeys. Infection may also occur in pheasants, chukar partridges, and peafowl. The disease is worldwide. Its effects are most severe in large commercial operations during winter. MG (Mycoplasma gallisepticum) is one of the major health problems facing the poultry industry. The severity and appearance of disease may be influenced by concomitant infection and other pathogens and/or by predisposing factors. Predisposing factors include nutritional deficiencies and intensive management with excessive ammonia and dust. Other pathogens include the viruses of Newcastle disease and infectious bronchitis, including vaccine strains and pathogenic strains of E. coli and Hemophilus gallinarium. Under experimental conditions the incubation period between exposure and infection is from 6 to 21 days. Under field conditions, the incubation period is not established due to many variables, which influence the onset and the extent of disease. Field outbreaks of MG near the onset of production (between the 26th and 38th weeks of age) may suggest a long incubation period. Mycoplasma spread rapidly within a population of birds (7-14 days). The disease spread in the flock by direct contact (bird to bird) with infected carriers, airborne dust and droplets, contact with contaminated equipment, crates, feed, water, and litter, and other species of birds, wild birds and rodents. MG can also spread through the hatching eggs (transovarian) and/or hatchery. The severity and intensity of MG infection is variable. It is more severe and of longer duration in the cold months and it affects younger birds more severely than mature birds. Recovered birds may become carriers of MG.

Symptoms and lesions: Morbidity and mortality in the field outbreaks vary widely depending on the environmental and climatic conditions and type of secondary infection. The infection may vary from mild with low mortality to severe with high mortality In broilers, mortality may be high if management is poor and birds are exposed to stress and other secondary infection. Laying hens usually experience low mortality, but many of the birds have an unthrifty appearance. Very high mortality may be seen in turkeys. Signs of MG may develop slowly in the flock. Respiratory signs usually persist for weeks. The first sign is a nasal discharge followed by a foamy or bubbly condition of the eyes. Birds may show a persistent hacking cough, sneezing and sniffing, and tracheal rales. Poor physical condition and loss of weight are usually apparent. In broilers, the onset of signs are severe and include severe reduced growth rate and feed intake. Layers will show a drop in egg production and feed consumption. Production may continue at a lower level. The primary lesions in turkeys are air sacs and lung lesions. There is also swelling of one or both infraorbital sinuses. Important lesions include swelling of nasal passages, sinuses, trachea, and bronchi. Air sacs are often thick and opaque and may contain slimy or cheesy exudate.

Control strategy for MG infection in poultry

1.      Completely depopulate the infected premises (removing all birds as well as all used feed supplies, removable equipment, and supply items that may be contaminated) to establish a "MG-free" flock.

2.      Remove all of the litter and manure completely.

3.      Wash down all inside surfaces (walls, ceilings, rafters, ventilation openings, air intake ducts, etc.) with a high pressure washer using a large volume of water. The addition of a cleaning agent speeds up and improves the process.

4.      Disinfect all of the interior surfaces of the building with recommended concentration of phenolic or cresylic acid disinfectant solution. Chlorine bleach solution (.025%) may be substituted for phenol or cresylic acid solutions.

5.      Spray the entire floor area, the lower three to four feet of the support poles, the perimeter walls, and the area outside the entrance with 0.1% glutaraldehyde solution at one gallon per 10 square feet. Also, spray a five- to ten- foot band around the entire outside perimeter to prevent contamination from outside the building. Close up the building and make it tight without any ventilation until the next day, and then open it up and air out the formaldehyde fumes.

6.      Spray the entire floor area, support poles, and side walls with carbaryl insecticide at recommended levels to control insect pests. Malathion may be added to, and applied with, the cresylic acid spray in place of the separated carbaryl applications.

7.      Bring back the necessary removable equipment that has been cleaned and disinfected and bring in feed and supply items that are also clean and not contaminated with pathogens.

8.      Allow the buildings to remain empty for at least two weeks after cleanout.

9.      Always consider the area outside the building contaminated, particularly if the previous flock had MG. Set up your traffic pattern to prevent recontaminating the building from the area outside.

10.  Obtain chicks hatched from eggs from MG-free breeder flocks, hatched in a hatchery where no infected eggs are set, and delivered in "MG-free" trucks by people who have no contact with infected birds.

11.  Farm workers go from younger to older birds (not vice-versa) if mutliple ages are present on the farm and cared for by the same person.

12.  Have only one age group on the farm (all-in, all-out) or have barns adequately separated (about 650 feet or more) and treat each age group as a separate unit. Clean up and disinfect barns between flocks.

13.  Enforce regulations on clothing and footwear worn by flock caretakers, servicemen, and equipment repairmen that move between flocks and farms. You can significantly influence the security and isolation of a healthy flock from the rest. Use "on-farm" clothing (including coveralls, boots, hats, etc.) in poultry barns.

14.  Feed truck, egg pick-up, and other service vehicle drivers must stay in the truck if servicing both infected and free farms (or if that is not possible, avoid the barn entrance area).

 

Treatment: Most strains of M gallisepticum are sensitive to a number of antibiotics, such as chlortetracycline, erythromycin, oxytetracycline, spectinomycin, tiamulin, tylosin, or a fluoroquinolone such as enrofloxacin.

 

2. MYCOPLASMA IOWAE: Infection IS common in turkey flocks. It is a relatively uncommon infection of chickens. Mycoplasma iowae is egg transmitted. Many strains of M iowae are lethal to turkey embryos. Affected turkey breeder flocks show no clinical signs other than reduced hatchability (usually 2-5%). In many flocks, the hatchability returns to normal after 1-2 months. Most embryos die during the later stages of incubation. Dead turkey embryos are edematous, congested, and stunted; they may have clubbed down. Poults infected in ovo or at 1 day of age may develop various skeletal deformities such as rotated tibia, deviated toes, chondrodystrophy, or erosion of the articular cartilage of the hock joint. Feathers may also be poorly developed.

 

3. MYCOPLASMA MELEAGRIDIS: It is a widespread, egg-transmitted disease of turkeys found worldwide. The primary lesion is airsacculitis. Mycoplasma meleagridis is thought to be a specific pathogen for turkeys, and the organism is commonly found in the respiratory and reproductive tract.

 

4. MYCOPLASMA SYNOVEAE: The organisms cause airsacculitis and synovitis. It is also a egg transmitted infection.

 

B. MYCOPLASMA INFECTIONS IN CATTLE:

1. CONTAGIOUS BOVINE PLEUROPNEUMONIA (CBPP)

CBPP is a disease of bovines. The causative agent of contagious bovine pleuropneumonia (CBPP) is Mycoplasma mycoides subspecies mycoides SC (small colony). CBPP is a respiratory illness characterized by the presence of sero-fibrinous, interstitial pneumonia, interlobular oedema and hepatization giving a marbled appearance of the lung and capsulated lesions termed sequestra in the lungs of affected cattle. The occurrence of subacute, symptomless infections and chronic carriers after the clinical phase of the disease create major problems in the control of this disease. Infection is spread to a susceptible animal by the inhalation of expired breath laden with M. m. mycoides SC from an infected animal.

Symptoms: There is considerable variation in the degree of symptoms seen in cattle affected with CBPP ranging from the hyperacute through acute to chronic and sub-clinical forms. Respiratory distress and coughing, evident on stimulation of resting animals, are the main signs of CBPP.

Acute Form: Animals show dullness, anorexia, and irregular rumination with moderate fever, and may show signs of respiratory distress. Coughing is usually persistent and is slight or dry. Sometimes body temperature rises from 40 to 42C and the animal prostrates with difficulty of movement.

Hyperacute Form: The clinical signs observed in the hyperacute form are greatly accelerated. The pathological signs are usually characteristic with marked pleural adhesion accompanied by exudative pericarditis. Affected animals may die within a week of exhibiting classical respiratory signs.

Subacute/Chronic FormsL In the subacute form, symptoms may be limited to a slight cough, only noticeable when the animal is exercised.

Lesions: The pathological lesions of this disease are confined to the thoracic cavity and lungs, and lesions are usually unilateral. The thoracic cavity of affected animals may contain many litres of clear yellowish brown fluid containing some pieces of fibrin.  Caseous fibrinous deposits are observed on the parietal and visceral surfaces of the lungs. The interlobular septa of the affected lung show distension with amber-coloured fluid surrounding the distended lymphatics. This fluid separates the lung lobules, which vary in colour with red, grey and yellow hepatization being evident indicating different stages of inflammatory lesions. Consolidation of the lungs with typical marbled appearance, sometimes accompanied by adhesion of the parietal and visceral surfaces is also characteristic. In addition to respiratory disease, affected calves may present exudative peritonitis, arthritis, bursitis and fibrinous arthritis of the carpal and tarsal joints.

 

C. MYCOPLASMA INFECTIONS IN SHEEP AND GOAT

1. CONTAGIOUS AGALACTIA

OIE LISTING – LIST B DISEASE

Contagious agalactia is a disease of sheep and goats that is characterised by mastitis, arthritis and keratoconjunctivitis. It is mainly caused by Mycoplasma agalactiae. Of late M. capricolum subsp. capricolum (Mcc) and M. mycoides subsp. mycoides LC (MmmLC; LC = large colonies) have also been isolated in many countries from sheep and goats with mastitis and arthritis.

Symptoms: Following an incubation period of 60 days from natural exposure, body temperature rises to a range from 41 to 42C. The period of fever corresponds to the time of septicaemia. During early stages, infected animals become depressed and anorectic, and some may die. As it progresses, the disease usually develops signs of keratitis and arthritis and, in female sheep, mastitis and abortion. The acidity of the milk from infected udders changes from a normal pH 6.8 to pH 7.8, becomes yellow in colour and, on standing, separates into a light green supernatant layer and a grumous sediment. Gradually, the udder atrophies and the milk yield diminishes. Joints, especially carpal and tarsal, become swollen, painful, and lame. In some animals, a joint may rupture and discharge exudate. One or both eyes may become infected, leading to impaired vision and blindness. Depending on the stage of disease development, the infected eye may show congestion and swelling, punctiform keratitis with. each opaque focus measuring up to 1mm in diameter, keratitis with yellow opacity of the cornea, or rupture of the cornea. Ewes in the third trimester of gestation may abort either dead or living infected lambs and later develop vaginal discharges.

Lesions: The infected udder is grossly atrophic in either one or both halves of the organ. Microscopically, the chronic inflammatory reaction in the stroma shows increased fibrosis and a reduced number of glandular acini. Infected joint capsules are oedematous, and the synovium may contain clumps of fibrin. Articular surfaces maybe eroded and occasionally ankylosed. In early stages of keratitis, the cornea is oedematous and infiltrated with leukocytes. In advanced stages, abundant purulent exudate infiltrates both cornea and the ciliary body.

 

2. CONTAGIOUS CAPRINE PLEUROPNEUMONIA (CCPP): CCPP is an infectious disease which affects only goats. The causative agent of contagious caprine pleuropneumonia (CCPP) is Mycoplasma capricolum subsp. capripneumoniae (Mccp). In natural infections, the organisms are acquired by susceptible goats by inhalation of contaminated droplets from infected goats. Due to the high sensitivity of mycoplasmas to the external environment, close contact is essential between infected and naive animals for transmission to take place, and, overcrowding and confinement favours close contact and circulation of mycoplasmas. Stress factors due to malnutrition and movement over long distances can predispose the animal to disease. 

Symptoms: The primary clinical signs are cough with animals tending to lie down or lag behind the flock. Affected animals continue to graze for some time but eventually become anorexic, breathing becomes laboured with painful grunting and a rise in temperature up to 41C. Gradually, the respiratory symptoms become prominent, respiration is accelerated and painful, and is followed by violent coughing. In the terminal stages, the animals are unable to move. They stand with their legs abducted, the neck is stiff and extended downward, saliva continuously drips from their mouth and their nose is obstructed by mucopurulent discharge. The tongue protrudes and the animals bleat distressingly. The organism is not reported to affect organ systems other than the respiratory tract of goats. In endemic areas subacute and chronic cases are common and the symptoms are milder, dominated by intermittent coughing.

Lesions: The gross pathological lesions are localized exclusively to lung and pleura and are often unilateral. Affected lungs can be totally hepatized, and have a port wine colour. A lung section shows a fine granular texture with various colours, but usually without any thickening of the interlobular septa. There is often an abundant pleural exudate and conspicuous pleuritis. The pleural exudates can solidify and form a gelatinous covering sometimes over the whole lung. In acute cases, the pleural cavity contains an excess of straw-coloured fluid with fibrin flocculations. In chronic cases there is a black discolouration of the lung tissue and sequestration of the necrotic lung areas. Adhesions between the lung and the pleura are very common and often very thick.

 

Diagnosis:

1. Symptoms and lesions are very conclusive.
2. Isolation and identification by culturing: The medial used for isolation of mycoplasma contain 20% unheated horse serum, 10% fresh yeast agar, and very minute quantity of DNA, penicillin and thallous acetate. The pH should be between 7 and 8. The optimum temperature is 36-38C. The colonies are microscopic and appear after 3-4 days. They have yellowish, opaque centre surrounded by a thin translucent rim resembling poached egg appearance. Growth is difficult in fluid medium and a granular type growth is noticed. Seven-day-old embryonated eggs are used for cultivation of mycoplasmas. After two days the embryo will die showing extensive oedema and swelling. Cell culture systems like HeLa cells, chicken heart fibroblasts and human conjunctival cells are used for cultivation.
3. Nucleic acid identification method
4. Agglutination reaction using specific antisera.
5. Complement fixation test (OIE accepted test).
6. ELISA.
7. Direct and indirect FAT.
8. Growth inhibition test using specific antisera.
9. Biochemical reaction.

 

Treatment and control: screening the flock supplying egg and eliminating the flock can control avian mycoplasma. Treatment of eggs with antiseptics prevents the occurrence of Mycoplasma in poultry. Live and killed vaccines are available for cattle infection.

 

VMC LECTURE # 22

RICKETTSIA

The study about Rickettsia and Chlamydia organisms is called as rickettsiology. Rickettsia and Chlamydia are group of organisms that are placed in between the bacteria and viruses. They have some properties that are found in both bacteria and viruses. However, the majority of the properties of rickettsia are found in bacteria and they are closely related to bacteria. Rickettsias are so named to perpetuate the memory of Dr.H.D.Ricketts, who worked on typhus (important disease caused by Rickettsia organisms) and died of the same disease after accidentally contracting it. The rickettsiae are a diverse collection of obligately intracellular Gram-negative bacteria found in ticks, lice, fleas, mites, chiggers, and mammals. They include the genera Rickettsiae, Ehrlichia, Orientia, and Coxiella. This genus consists of two antigenically defined groups: spotted fever group and typhus group, which are related; scrub typhus rickettsiae differ in lacking lipopolysaccharide, peptidoglycan, and a slime layer, and belong in the separate, although related, genus Orientia. Rickettsias are coccoid or rod shaped organisms in the size range of 0.3-0.7 m wide and 1-2 m long. Except one organism all the remaining rickettsia are obligate parasites (Rochalimaea Quintana – trench fever). They cannot be cultivated in the artificial laboratory media. The following table summarises some of the important properties of rickettsia, Chlamydia, viruses and bacteria

 

Properties	Rickettsia	Chlamydia	Viruses	Bacteria
Structural
1)      Nucleic acid	RNA and DNA	RNA and DNA	RNA or DNA	RNA and DNA
2)      Ribosomes	Present	Present	Absent	Present
3)      Cell wall	Muramic acid, Diaminopimelic acid (DPA) preset	Muramic acid, DPA absent	No cell wall	Muramic acid
4)      Structural integrity during multiplication	Maintained	Maintained	Lost	Maintained
5)      Visibility under light microscopy	Visible	Visible	Not visible	Visible
Metabolic capabilities
1)      Macromolecular synthesis	Carried out	Carried out	Only with use of host machinery	Carried out
2)      ATP generating system	Present	Absent	Absent	Present
3)      Sensitivity to antibiotics	Sensitive	Sensitive (except penicillin)	Not sensitive	Sensitive
4)      Sensitivity to interferon	Not sensitive	Sensitive	Sensitive	--
Replication
1)      Mode of replication	Binary fission	Binary fission and budding	Dependence on host cell machinery	Binary fission
Cultivation
1)      Growth in artificial media	Not possible	Not possible	Not possible	Possible
2)      Intracellular replication	Possible	Possible	Possible	Variable

 

GENERAL PROPERTIES OF RICKETTSIA

DEFINITION: The rickettsias are bacteria that are obligate intracellular parasites. They are considered a separate group of bacteria because they have the common feature of being spread by arthropod vectors (lice, fleas, mites and ticks). They are true bacteria as they have metabolic enzymes and cell walls, use O₂, and are susceptible to antibiotics, although they require living cells for growth. Most rickettsia are maintained in nature by a cycle involving an animal reservoir and an insect vector (usually an arthropod) that infects animals. Most species are found only in the cytoplasm of host cells, but those, which cause spotted fevers multiply in nuclei as well as in cytoplasm. In the laboratory, they may be cultivated in living tissues such as embryonated chicken eggs or vertebrate cell cultures.

 

GENERAL CHARACTERS:

1.     Morphology: The cells are extremely small (0.25 u in diameter) rod-shaped, coccoid and often pleomorphic microorganisms, which have typical bacterial cell walls, no flagella (except for Rickettsia prowazekii), are gram-negative and multiply via binary fission only inside host cells. They occur singly, in pairs, or in strands. The structure of the typical rickettsia is very similar to that of Gram-negative bacteria. The typical envelope consists of three major layers: an innermost cytoplasmic membrane, a thin electron dense rigid cell wall and an outer layer. The outer layer resembles typical membranes in its chemical composition and its tri-laminar appearance. The cell wall is chemically similar to that of Gram-negative bacteria in that it contains diamino pimelic acid and lacks teichoic acid. Intracytoplasmic invaginations of the plasma membrane (mesosomes) and ribosomes are also seen. There are no discrete nuclear structures. The flagellum of R. prowazekii is similar to that of other bacteria. The rickettsia have many of the metabolic capabilities of bacteria, but require an exogenous supply of cofactors to express these capabilities. The response to exogenous cofactors implies an unusually permeable cytoplasmic membrane.

Electron microscopic studies of the rickettsial cell show its close resemblance with bacterial cell. The cell wall contains muramic acid and diaminopimelic acid. The nucleolar material contains both RNA and DNA. The DNA is of double stranded form with GC content varying from 29-33% in different species of rickettsia.

2.     Replication: Rickettsia normally multiply by transverse binary fission with a doubling time of about 8 hours. For binary fission to take place both rickettsial cell and host cell should be in active state. First the rickettsial cell enters into the host cell, multiply inside it and when sufficient numbers or daughter cells are produced they come out of the host cell by rupturing it. Under poor nutritional conditions, the rickettsia cease dividing and grow into long filamentous forms, which subsequently undergo rapid and multiple division into the typical short rod forms when fresh nutrient is added. Immediately after division, the rickettsia engage in extensive movements through the cytoplasm of the cell.

C. burnetii differs from other rickettsia in that it is enclosed in a persistent vacuole during growth and division. Six to ten daughter cells will form within a host cell before the cell ruptures and releases them.

3.     Classification: The family Rickettsiaceae is taxonomically divided as per the following flow chart. The important differentiating features of three important genus are given belo

a.      Rickettsia - obligate intracellular parasites, which do not  multiply within vacuoles and do not parasitise white blood cells.

b.      Ehrlichia - obligate intracellular parasites, which do not multiply within vacuoles but do parasitize white blood cells.

c.      Coxiella -obligate intracellular parasite, which grows preferentially in vacuoles of host, cells.

 

THE RICKETTSIAS

 

 

ORDER                                                  Rickettsiales                                 Chlamydiales

 

 

 

FAMILY              Rickettsiaceae                Bartonellaceae   Anaplamataceae     Chlamydiaceae

 

 

 

 

TRIBE               Ricettsieae                                             Ehrlichiaea

 

 

 

GENUS  Ricettsia            Orientia             Coxiella                                                    Chlamydia

 

                                                                    Ehrlichia    Cytocetes    Cowdria   Neorickettsia

 

 

SPECIES

 

            R.prowazeki       O.tsutsugamushi C.burnetii

            R.mooser                                              E.bovis  C.phagocytophelia     N.helminthoeca

           R.rickettsii                                              E.canis  C.microti      C.ruminantium                                                        E.ovina  C.bovis                                

          C.trachomatis

                                                                                                                                                                C.psittacI

Genus Rochalimaea: Bartonella (Rochalimaea) quintana, the agent of trench fever, was formerly considered as a rickettsial agent and now moved to the family Bartonellaceae. Earlier it was considered as the only rickettsia that can be cultivated extracellularly. Bartonella henselae – cat scratch fever

4.     Resistance: The resistance of different members of the family Rickettsiaceae vary widely. Generally they are fragile organisms and cannot survive for a long time outside a cell. Certain rickettsial organisms like Coxiella burnetti that causes Q fever are very resistant to physical damages since they produce an endospore form. They can withstand even pasteurisation.

5.     Transmission: Rickettsias do not survive outside the host cell for a long time and hence they must be transported between animals by arthropod vectors. When and arthropod bites a rickettsia infected patients, the organisms enters into the arthropod where they penetrate into epithelial cells of gastrointestinal tract, multiply there and are released in through the faeces. Other methods of transmission are also possible.

 

CULTIVATION: They are difficult to cultivate and require a living system for the growth. They are cultivated in embryonated eggs of 6-8 days old by yolk sac route. The antigen is separated from yolk membrane. Lab animals like guinea pigs, rats and feral animals are also used. Most of the organisms are potential pathogens to human beings. Hence, attempts to isolate the organism from clinical cases or routine laboratory cultivation are done in laboratories with containment facilities.

PATHOGENESIS: Cattle, sheep, goat, and dog cats are the important hosts affected by rickettsial organisms. The main mode of spread is through ticks. Ticks act as biological vectors. Other modes of spread are ingestion and droplet infection. Rickettsia generally multiply in the gut epithelium of ticks and in vascular endothelial cells of animals. In their arthropod vectors, the rickettsia multiply in the epithelium of the intestinal tract; they are excreted in the faeces, but occasionally gain access to the arthropods salivary glands. They are transmitted to man, via the arthropod saliva, through a bite. In their mammalian host, they are found principally in the endothelium of the small blood vessels, particularly in those of the brain, skin and heart. Hyperplasia of endothelial cells and localized thrombus formation lead to obstruction of blood flow, with escape of RBC's into the surrounding tissue. Inflammatory cells also accumulate about affected segments of blood vessels. This angiitis appears to account for some of the more prominent clinical manifestations, such as petechial rash, stupor and terminal shock. Death is ascribed to damage of endothelial cells, resulting in leakage of plasma, decrease in blood volume, and shock. It is also assumed that the observed clinical manifestations of a rickettsial infection are due to production of an endotoxin, although this endotoxin is quite different in physiological effects from that produced by members of the Enterobacteriaceae. No evidence for immunopathological damage has been obtained. Both humoral and cell mediated immunity are important in recovery from infection. Antibody-opsonized Rickettsia are phagocytosed and killed by macrophages and delayed type hypersensitivity develops following rickettsial infections.

 

DIAGNOSIS:

1.      Based on the symptoms and lesions.

2.      Microscopical identification of the organisms in tissue or blood. Although the organisms are gram-negative, they only weakly take the counter stain, safranin. Hence, special staining procedures are commonly used.  The special staining procedures used are

a.      Macchiavello stain:-organisms are bright red against the blue       background of the tissue.

b.      Castaneda stain:-blue organisms against a red background.

c.      Giemsa stain:-bluish purple organisms.

3.      Weil-Felix reaction: It is an agglutination test. It is normally done in human beings using Proteus organisms.  In this test, the titre of the agglutinins in the patient's serum against the Proteus strains OX-19, OX-2 and OX-K are determined. These Proteus strains have no etiological role in rickettsial infections, but appear to share antigens in common with certain rickettsia. These antigens are alkali stable polysaccharide haptens, which are distinct from the group-specific and type-specific antigens. In interpreting the results, it must be kept in mind that Proteus infections are fairly common (especially in the urinary tract) and that they, too, may evoke antibodies to the Proteus-OX strains. This test is usually positive seven days after the initial infection.

4.      Complement fixation test: It is a better test than Weil-Felix reaction.

5.      Immuno-fluorescense assay: It is a better test than Weil-Felix reaction and complement fixation test.

6.      Nucleic acid identification methods

 

TREATMENT: The drugs of choice for the treatment of rickettsial diseases are chloramphenicol and tetracycline. The sulfonamides stimulate rickettsial growth and thus are contraindicated in the treatment of these diseases.

 

CONTROL:

1.     Vector control is the best method.

2.     Proper disposal of the dead animals, and foetal membranes also prevent the zoonotic rickettsial infections.

3.     Raw milk should never be consumed.

4.     Vaccination is not normally practiced in animals.

5.     Most of the rickettsia infection are found in few geographical location. Hence, animal movement from endemic areas should be restricted.

 

VMC LECTURE # 23

RICKETTSIA PART II - Q FEVER

 

Causative organism   :        Coxiella burnetii

Synonyms               :        Abattoir fever, Query fever, Burnet’s rickettsiosis

 

INTRODUCTION: Query (Q) fever is a zoonosis that occurs in most countries. Humans acquire infection from animal reservoirs, especially from domestic ruminants. Q fever is a highly infectious disease, which is due to the proliferation of Coxiella burnetii, a small and pleomorphic bacterium. Q fever was first described in Australia in 1937. Cattle, goats, sheep and other animals are affected by the infection. Infection in man usually occurs as a result of inhalation of aerosols containing the organism.

 

GENERAL CHARACTERS:

1.      Morphology: Coxiella burnetii is a small, obligate and pleomorphic bacterium measuring 0.3–1.5 µm long x 0.2–0.4 µm wide. It is acid–alcohol resistant and can be stained by several methods, which include Stamp, modified Ziehl–Neelsen, Gimenez, Giemsa and modified Koster techniques.  They are also weakly gram-negative. However, they stain gram-positive with alcoholic iodine as mordent. Unlike rickettsiae, C. burnetii produces a small, dense, highly resistant spore-like form that is highly stable in the environment. This particular character is important for transmission of the infection. This ability has been attributed to the existence of C. burnetii developmental cycle variants: large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC). The SDC and SCV represent the forms of the bacteria likely to survive extracellularly as infectious particles. The organism has 11 chromosomal genes and 1 plasmid.

2.      Classification: C.burnetii was earlier under the family Rickettsiae, but now as per the molecular taxonomy (based on 16S ribosomes), they are now under a separate family – Coxiellaceae

3.      Habitat: C.burnetii is found in all most all animals. The reservoirs for this organisms include wild and domestic mammals, birds, and arthropods such as ticks. Cattle, goats, and sheep are considered the primary reservoirs from which human contamination occurs.

4.      Antigens: C. burnetii displays antigenic variations similar to the smooth-rough variation in the family Enterobacteriaceae. The organisms exhibit two phases. Phase variation is related mainly to mutational variation in the lipopolysaccharide (LPS).  Phase I is the natural phase found in infected animals, arthropods, or humans. Organisms as Phase I is highly infectious and corresponds to smooth LPS. In contrast, phase II is not very infectious and is obtained only in laboratories after serial passages in cell cultures or embryonated egg cultures. It corresponds to rough LPS.

5.      Resistance: The organism is very stable in environment. It is resistant to drying, chemicals, and many disinfectants. C. burnetii is very resistant to killing in nature and may survive for several weeks in areas where animals have been present; the organism may also be spread by the wind

6.      OIE Classification: List B infection (Multi species)

7.      Human risk: C. burnetii is extremely hazardous to humans, and laboratory infections are common. Because of its low infectious dose (even a single bacterium can cause infection), resistance in the environment, and aerosol route of transmission, C. burnetii is considered a potential agent of bioterrorism. The OIE has classified C.burnetii as risk group 3 agent. Live culture or contaminated material from infected animals must only be handled in facilities that meet the requirements for Containment Group 3 pathogens.

 

CULTURAL CHARACTERS: It cannot be grown in non-living media. Lab animals like guinea pigs are better source for cultivation. In guinea pigs the organisms are cultivated through intraperitoneal route. In male guinea pigs development of orchitis (called as Strauss reaction) is rare. They are also cultivated in embryonated eggs by yolk sac route. C. burnetii infection leads to death of the embryo within 14 days. However, only Phase II bacteria are recovered from embryonated eggs. C. burnetii can also be grown in vitro in a number of cell types, including mouse macrophage-like cells, fibroblast cells and Vero cells

 

PATHOGENESIS:  Cattle, sheep, and goats are the primary reservoirs of C. burnetii.  Infection has been noted in a wide variety of other animals, including other species of livestock and in domesticated pets. Cats and dogs may represent reservoirs of C. burnetii.  Coxiella burnetii does not usually cause clinical disease in these animals, although abortion in goats and sheep has been linked to C. burnetii infection.  Organisms are excreted in milk, urine, and feces of infected animals. Most importantly, during birth the organisms are shed in high numbers within the amniotic fluids and the placenta.  The organisms are resistant to heat, drying, and many common disinfectants.  These features enable the bacteria to survive for long periods in the environment.  Infection of humans usually occurs by inhalation of these organisms from air that contains airborne barnyard dust contaminated by dried placental material, birth fluids, and excreta of infected herd animals.  Humans are often very susceptible to the disease, and very few organisms may be required to cause infection. Ingestion of contaminated milk is also a less common mode of transmission.  Other modes of transmission to humans, including tick bites and human to human transmission, are rare. Ticks are not considered essential in the natural cycle of C. burnetii infection in livestock and domestic animals. However, ticks play a significant role in the transmission of coxiellosis among the wild vertebrates, especially in rodents, and wild birds. Over 40 tick species are naturally infected with C. burnetii, including Rhipicephalus sanguineus found in dogs. C. burnetii infection has been reported less frequently in a number of other domestic or wild mammals, including horses, rabbits, swine, camels, water buffalo, rats, and mice. Birds may also be infected, and C. burnetii has been isolated from pigeons, chickens, ducks, geese, and turkeys. Humans may acquire Q fever from infected domestic poultry by consumption of raw eggs or inhalation of infected fomites.

 

SYMPTOMS: Animals do not usually experience symptomatic C. burnetii infections. In the acute phase, the presence of C. burnetii may be demonstrated in the blood, lungs, spleen, and liver. Most animals remain totally asymptomatic, including a lack of fever. C. burnetii infection often becomes chronic, with persistent shedding of C. burnetii in feces and urine. Animals do not develop chronic endocarditis as is observed in humans. The female uterus and mammary glands are primary sites of chronic C. burnetii infection. Thus, the shedding of C.burnetii into the environment occurs mainly during parturition. Birth products, mainly the placenta, are heavily contaminated with C. burnetii. C.burnetii can also be recovered from milk. The only pathological manifestations that have been associated with chronic C.burnetii infection in animals are abortion, mainly in sheep and goats and lower birth weight and infertility in cattle.

 

In human beings, variety of symptoms ranging from inapparent or mild to severe occur. Acute infection in humans’ presents as a febrile, self-limiting disease marked by symptoms including fever, headache and myalgia. Some of the common symptoms are a sudden onset, chills, headache, muscle cramps, weakness and malaise, severe sweats, hepatitis, meningitis pneumonitis and other respiratory symptoms. The infection may lead to complications in pregnant women. A serious complication of chronic Q fever is endocarditis, generally involving the aortic heart valves, less commonly the mitral valve.

 

DIAGNOSIS:

1. Q fever may be confused with Brucellosis and Leptospirosis. Hence, it should be differentiated from these two infections.
2. Most common method of diagnosis is detection of specific antibodies in serum

3.      Isolation of organisms by culture method is hazardous, therefore not routinely carried out. It also requires specially equipped laboratory with bio-containment facilities. As isolation of the organism is hazardous, the most common method of diagnosis is through the detection of specific antibody in serum, with ELISA considered the most sensitive method.

4. ELISA: It is considered most sensitive method of diagnosis (IgG studies). It correlates with conventional CFT. Diagnosis can be made with single serum specimen. It can measure response to different classes of antibody.
5. Complement fixation test (CFT)- Involves a >2 fold increase in paired sera. Usually phase II antigens are used. Slow production of antibodies and static titres are problems of this test.
6. Immunofluorescense assay (IFA): It is an accepted method of diagnosis. It is more sensitive than CFT with antibodies appearing as early as 5 days. It can measure individual antibody classes to different phases
7. Nucleic acid identification methods like PCR.

 

PREVENTION:

1. Vaccination of abattoir workers and others in hazardous occupations
2. Pasteurisation of milk from cows, goats and sheep to inactivate organism

 

TREATMENT

Tetracyclines administered after patient is afebrile.

Significance for Bioterrorism: Coxiella burnetii is a highly infectious agent that is rather resistant to heat and drying. It can become airborne and inhaled by humans. A single C. burnetii organism may cause disease in a susceptible person.  This agent could be developed for use in biological warfare and is considered a potential terrorist threat.

 

 

 

VMC LECTURE # 23 (A)

RICKETTSIA PART II – HEART WATER or COWDRIOSIS

 

Causative organism            :           Ehrlichia ruminantium

(formerly Cowdria ruminantium)

Synonym                               :           Veld poisoning

 

Definition: Heartwater (also known as cowdriosis) is an infectious, non-contagious rickettsial disease of ruminants caused by Ehrlichia ruminantium (formerly Cowdria ruminantium) and transmitted by Amblyomma ticks. The disease affects domestic and wild ruminants, including cattle, sheep, goats, antelope, and buffalo. Nonruminant hosts of E. ruminantium, such as guinea-fowl, leopard tortoises, and scrub hare, may also be important in the maintenance of the organism in nature because they are all known carriers of the agent. Heartwater is usually an acute disease and is commonly fatal within a week of onset of clinical signs. The name "heartwater" is derived from the hydropericardium, which is commonly seen with this disease. The disease is widespread in most of Africa and on several islands in the West Indies. With increased trade and movement of animals in today's global market, heartwater may present a significant threat to the domestic livestock industry in any country

 

General characters of E.ruminantium:

1.     Morphology: It is a very small (0.4 to 1.0m), pleomorphic, obligate intra cellular parasite. They stain gram-negative and stain blue or purplish blue in Giemsa in smears prepared from affected organs. They have an affinity for multiplication in the vascular endothelial cells. They appear as colonies inside the endothelial cells. In such colonies, the organisms appear as pleomorphic rods to horseshoe shaped structures. It usually occurs in clumps of from less than five to several thousand organisms within the cytoplasm of infected capillary endothelial cells, especially in the brain. They remain firmly attached to RBC and it is difficult to remove the organisms by washing in buffered saline.

2.     Resistance: These organisms are very fragile organisms and cannot survive for longer time at room temperature in blood. Because of its fragility, the organism must be stored in dry ice or liquid nitrogen to preserve its infectivity The organisms remain viable for two years at –80C in spleen and blood.

3.     Habitat: They are obligate intra cellular parasites and are found either in the affected animals or the Ambylomma ticks that are biological vectors.

4.     Antigens: There are many strains of E.ruminantium, which vary in virulence. All strains are apparently pathogenic for sheep and goats and at least one strain is nonpathogenic for cattle.

5.     OIE listing: List B disease (Multi species)

6.     Human risk: Humans are not susceptible to E.ruminantium.

 

Cultivation: The organisms are very difficult to grow and cannot be cultivated in embryonated eggs or in tissue cultures. Of late endothelial cells are used in the isolation of E.ruminantium. Ferrets are the only laboratory animals of choice. The animals used for routine cultivation under laboratory cultivation are the sheep. In mice, the organisms can remain upto 90 days but they do not multiply.

 

Pathogenesis: The important mode of spread is by tick bites. Heartwater is transmitted only by ticks of the genus Amblyomma, with the tropical bont tick as one of the most important vectors. This tick is widely distributed throughout Africa and number of other countries. The life cycle of Amblyomma ticks may take from 5 months to 4 years to complete. Thus, the infection may persist in the environment, inside the tick, for a long time. The immature stages of the tick will feed on a wide variety of livestock, wild ungulates, ground birds, small mammals, reptiles, and amphibians. Cattle egrets have also been implicated in much of the recent spread of heartwater.

 

The incubation period is usually 12 days in sheep and goats and 12-18 days in cattle. After infection of the host with E. ruminantium, from the sites of tick bite the organisms are taken to various organs through blood circulation. Initial replication of the organism takes place in reticuloendothelial cells and macrophages in the regional lymph nodes. From here the organisms are disseminated via the blood stream to invade endothelial cells of blood vessels of various organs where further multiplication occurs. The organisms multiply in vascular endothelial cells and produce a toxin. This toxin increases the permeability of the capillary walls that causes oedema of the lungs and accumulation of fluid in pericardium (this gives the name heartwater to the infection). Endothelial cell parasitization coincides with the onset of fever. There is an increased vascular permeability allowing the seepage of plasma proteins, which result in transudation through the serous membranes with resultant tissue oedema and effusion into body cavities. This causes the drastic fall in blood volume before death. Oedema of the brain is responsible for the nervous signs, hydropericardium contributes to cardiac dysfunction during the terminal stages of the disease and progressive pulmonary oedema and hydrothorax result in asphyxiation.

Symptoms: Heartwater occurs in four different clinical forms  - peracute, acute, subacute and subclinical as determined by variations in susceptibility of the hosts and the virulence of various strains of the HW agent. The peracute form is rare and is seen in exotic breeds of cattle, sheep, and goats introduced to an HW enzootic area. Heavily pregnant cows are especially prone to develop the peracute disease. In this form sudden death occurs, usually preceded only by a fever, severe respiratory distress, and terminal convulsions. Severe diarrhea may be seen in some breeds of cattle. The acute form of heartwater is the most commonly observed form of the disease. A sudden high fever (107 degrees F) is followed by loss of appetite, depression, and respiratory problems. Animals may initially have an increased respiratory rate, followed within a few days by severe respiratory distress. Nervous disorders often follow the respiratory signs and can include a variety of abnormal behaviours such as protrusion of tongue, excessive chewing movements, twitching of eyelids, incoordination, circling, star-gazing, head tilt, overly rigid posture, and walking with a high-stepping gait. These nervous signs usually last upto 48 hours, followed by the animal's death. In some cases, the nervous signs may not be noticed prior to death. The nervous signs increase in severity, and the animal goes down in convulsions. Galloping movements and opisthotonos are commonly seen before death. Hyperesthesia is often observed in the terminal stages of the disease, as is nystagmus and frothing at the mouth. Diarrhea is occasionally seen, especially in younger animals. The acute disease is usually fatal within a week of the onset of signs. The subacute infection is characterized by prolonged fever, coughing (a result of lung edema), and mild incoordination; recovery or death occurs in 1 to 2 weeks. A mild or subclinical form of the disease, known as "heartwater fever," is seen in partially immune cattle or sheep, in calves less than 3 weeks old, in antelope, and in some indigenous breeds of sheep and cattle with high natural resistance to the disease. The only clinical sign in this form of the disease is a transient febrile response.

Lesions: The gross lesions in cattle, sheep, and goats are similar. Hydropericardium is the most important lesions of this infection. The accumulation of straw-colored to reddish fluid in the pericardium is more consistently observed in sheep and goats than in cattle. Ascites, hydrothorax, mediastinal edema, and edema of the lungs, all resulting from increased vascular permeability with consequent transudation, are frequently encountered. Subendocardial petechial hemorrhages are also seen, and submucosal and subserosal hemorrhages may occur elsewhere in the body. Degeneration of the myocardium and liver parenchyma, splenomegaly, edema of lymph nodes, nephrosis, and catarrhal and hemorrhagic abomasitis and enteritis are all commonly encountered. Meningeal congestion and edema are often present. Brain congestion may occur, but brain lesions can be remarkably few when one considers the severity of the nervous signs observed in this disease.

 

Diagnosis:   

1.      Based on the symptoms and lesions. The symptoms should be correlated with the presence of Ambylomma ticks in the region.

2.      Microscopy:

a.      Demonstration of the colonies or the organisms inside the cytoplasm of the vascular endothelial cells. In the colonies the organisms appear as pleomorphic rods to  horseshoe shaped structures.

b.      Demonstration of the organisms: The HW organism stains purplish-blue with Giemsa stain and can be seen by microscopic examination of brain smears prepared from cerebrum, cerebellum, hippocampus, or other well-vascularized portion of the brain. These portions are macerated between two microscope slides. The resultant pulp is then drawn across a slide with varying pressure, which results in "ridges and valleys" on the slide. The slide is then air-dried, fixed with methanol, and stained with Giemsa. Under low magnification, the capillaries will be found extending from the "thick" areas of the slide. Examination of the capillary endothelial cells under oil immersion will reveal the blue to reddish-purple clumps of organisms. A rapid method for obtaining brain tissue for examination is to drive a large nail through the unopened skull and make a smear from the tissue adhering to the nail. The HW organisms can also be observed in smears prepared from the intima of large blood vessels or in stained sections of kidney glomeruli and lymph nodes.

c.      Transmission electron microscopy is also used to demonstrate Ehrlichia organisms in the endothelial cell’s cytoplasm

3.      Isolation of the organisms by culturing: Ehrlichia can be isolated from the blood of reacting animals by cultivation on ruminant endothelial cells. Endothelial cells from umbilical cord, aorta, or the pulmonary artery of different ruminant species (cattle, goat, sheep) are used most often for isolation. No standard cell line has yet been designated for isolation. However, the best method to isolate the organism in susceptible ruminants.

4.      Nucleic acid identification methods: Of late PCR and DNA probes are used to identify infected animals.

5.      Serological tests: Indirect fluorescent assay and cELISA are performed to identify antibodies against these organisms in serum.

6.      Differential diagnosis: The observed nervous system abnormalities suggest other diseases (such as rabies, tetanus, meningitis, or encephalitis) or toxic poisoning. Other diseases to be eliminated are anthrax, piroplasmosis, east coast fever, listeriosis, louping ill etc. A definitive diagnosis of heartwater is made by microscopic examination and observation of the causative rickettsia in a brain tissue smear.

 

Treatment: Administration of chlortetracycline and oxytetracycline intravenously is very effective.

 

Control: Eradiation of Ambylomma sp. ticks is the best method of control. Since the young animals are not normally affected inoculation of young animals with infected blood protects them from infection during adult stage.

 

 

 

VMC LECTURE # 23 (B)

RICKETTSIA PART II – SALMON POISONING

 

Causative organism            :           Neorickettsia heminthoeca

Synonym                               :           Salmon disease of dogs

 

Definition: It is a non-contagious, infectious, fatal acute infection of dogs characterised by extreme thirst, severe depression and frequent vomiting. Salmon poisoning is characterized by fever, lymph node enlargement and severe hemorrhagic enteritis, which may lead to the animal's death if left untreated. The infection is spread by the metacercariae of the fluke Nanophyetus salmincola. The fluke is a parasite of fish and the infection is more commonly seen in areas where the dogs eat raw fishes. The infection is more commonly seen in the northwestern region of American continent. The infection was first reported in 1924. However, the aetiology was confirmed in 1947. It is one of very few rickettsial infections that are spread by trematode vector.

 

General characters of N.helminthoeca:

1.     Morphology: They are very small pleomorphic organisms (coccoid, rod or crescent shaped) often arranged in clusters. They stain gram-negative but stain blue or purple by Giemsa and red with Macchiavello’s stain. In cells the organisms are appear as LCL bodies of Chlamydia. In dogs, they are found inside the reticuloendothelial cells of the lymph nodes, intestinal lymphoid follicles, tonsils, thymus and spleen. Rarely they are also seen inside macrophages.

2.     Habitat: The organisms are normally found in the metacercariae of the fluke Nanophyetus salmincola that affects fish. Dogs get the infection when they consume raw fish.

 

Cultivation:  Very difficult and very scarce information available on this.

Pathogenesis: This microorganism is found in salmon, steelhead, trout, Pacific giant salamanders and fresh water fish found in and around the Pacific Ocean from Northern California to Seattle.  The geographical limitations are likely caused by the limited habitats of infected snails. The infection is commonly seen in dogs, foxes and other carnivores that feed on raw fishes.  

The fluke Nanophyetus salmincola is host to Neorickettsia helminthoeca. The organism develops in snails (Oxytrema plicifer), infects and develops into cysts in fish, is ingested by dogs where they infect the intestinal tract.  The dogs excrete eggs in their stool, the organism re-enters the water, infects snails and the cycle begins again. 

SNAIL ==>  FISH  ===>   DOG ===> SNAIL

The egg matures (3 months) and hatches in water. The miracidium finds and penetrates a snail. Redia develops in the snail. Cercariae develop in the redia, and when mature emerge from the snail. The cercariae swim around until they find a trout or salmon whose skin they penetrate and enscyst as metacercaria in various tissues. When the definitive host eats the fish the metacercaria excyst and develops to the adult stage in the host's small intestine. Eggs are laid in the small intestine and pass out with the faeces.

Symptoms: The onset of symptoms is usually sudden, usually 5-7 days after ingestion but can be delayed up to a month.  Symptoms last for 7-10 days and can be fatal in a majority (up to 90%)of untreated dogs. The dog's temperature can peak (104-107F) suddenly and then return to normal or even below normal. The fever may persist for 37 days. There can be severe and bloody diarrhoea, dehydration, vomition, extreme thirst, severe weight loss and complete loss of appetite.   Some animals show serous nasal discharge and conjunctivitis. Symptoms can look like parvo or distemper. Mortality is as high as upto 90%. Death is generally caused by the toll the symptoms take on the dog's body.  These include electrolyte imbalance, anaemia and dehydration.  

Lesions: There is hyperplasia of carcase, visceral lymph nodes with necrosis. The tonsils, thymus and spleen may be enlarged. Haemorrhagic enteritis is the pathognomonic lesion.

Diagnosis:

1.      Diagnosis is made by finding fluke eggs in the stool.

2.      Demonstration of the organisms in the buffy coat of infected dogs.

3.      Based on symptoms and lesions.

4.      Microscopical examination of smear prepared from affected animals and stained by Romanowsky stains. 

Treatment: Treatments include hydration and nutrition, blood transfusions as well as antibiotics (sulphonamides or any other broad spectrum antibiotics) and related medications. 

Control: No vaccines are available. Dogs that have been infected and recovered can develop immunity. 

 

VMC LECTURE # 24 (A)

RICKETTSIA PART III –

CANINE ROCKY MOUNTAIN SPOTTED FEVER

 

Causative organism            :           Rickettsia rickettsii

 

Rocky Mountain Spotted Fever (RMSF) is an important zoonotic disease that may cause clinical signs in both dogs and humans. It is caused by the organism Rickettsia rickettsii, a small gram-negative obligate intracellular parasite from the family Rickettsiaceae. Although it has been long identified as a human pathogen, RMSF was not recognized in dogs until the 1970’s.

 

General characters:

1.         Morphology: R.rickettsii is a very small bacterium that live inside the cells of its hosts. These bacteria range in size from 0.2 x 0.5 micrometers to 0.3 x 2.0 micrometers. They are difficult to see in tissues by using routine histologic stains and generally require the use of special staining methods. R.rickettsii is a weakly Gram negative. R. rickettsii are observed using Giemsa, Machiavello, and Gimenez staining and by the use of direct fluorescent antibody staining techniques. R rickettsii has ribosomes and indistinct strands of DNA in an amorphous cytosol surrounded by a plasma membrane. In addition, an indistinct microcapsular layer is present on the outer surface of the cell wall. An electron-lucent zone separates this layer from the host cytosol. This zone is thought to represent a slime layer which may be important in pathogenicity

Pathogenesis: R. rickettsii is transmitted to the dog through the bite of an infected tick. Hard-bodied ticks are the vectors of RMSF. The most common vectors are Dermacentor variabili and D.andersoni. Amblyomma americanum and Rhipicephalus sanguineous also have been reported to carry the organisms. The ticks acquire the organism by feeding on parasitemic small mammals such as chipmunks and squirrels. Larger mammals rarely achieve the degree of parasitemia necessary to transmit the disease to a feeding tick. These small mammals function as reservoirs for the organism. Dogs and humans are incidental hosts and are the only reservoirs that display clinical signs of disease. The larvae and nymphs of the Dermacentor species typically feed on small mammals, while the adult ticks prefer larger mammalian hosts. Therefore, it is the larval and nymphal stages that are most often infected with R. rickettsii during feeding. Once infected, ticks spread RMSF among their own population through transfer of bodily fluids during mating, or transovarially spread the organism from the pregnant female to her offspring. Transovarial infection is the primary means by which R. rickettsii propagates in nature. Once infected, ticks then transmit RMSF to vertebrates (including dogs and humans) through their saliva when taking blood meals. The incubation period is 2-14 days. Then the organism enters into the circulation and then invades endothelial cells of the venules and capillaries and begins replicating, causing a vasculitis. The organism prevents apoptosis (programmed death) of the vascular endothelial cell, allowing adequate time for the organism to effectively replicate and spread. Vasculitis lead to edema, hemorrhage, shock, and vascular collapse. Organs like brain, skin, heart and kidneys are affected most often. Vascular leakage also triggers activation of the platelets and coagulation system.

Symptoms & Lesions: The first symptom is development of a red rash approximately 12 days after the initial tick bite. However, it is not very common among dogs. High fever (102.6-104.9º F) approximately 5 days after the tick bite, is the more common clinical finding. Petechiae and ecchymotic hemorrhages associated with destruction of platelets in response to vasculitis are also seen on exposed mucosal surfaces in the dog. Vasculitis also may cause edema in the extremities including the scrotum, prepuce, and ears of affected dogs. Other signs are joint swelling, myalgia, dyspnea, and neurological abnormalities due to meningoencephalitis. Vestibular ataxia is the most common neurologic affliction. Ocular lesions include conjunctival hyperemia, hyphema, retinal hemorrhage, and anterior uveitis. These ophthalmic lesions tend to be mild and usually occur bilaterally.

Diagnosis:

1.      Based on symptoms and lesions in correlation with tick infestation.

2.      Blood picture –

                                                  i.      Thrombocytopenia, with platelet counts ranging from 23,000 to 220,000 /µl.

                                                 ii.      Moderate leukocytosis

                                               iii.      Normocytic, normochromic anemia

3.      Biochemical abnormalities may include elevated glucose and cholesterol concentrations as well as increased activity of alkaline phosphatase (ALP) and alanine aminotransferase (ALT).

4.      Serologic tests: ELISA, IFA and CFT are routinely performed to identify RMSF. A four-fold increase or decrease in IgG antibody titers to R. rickettsii along with appropriate clinical signs of RMSF is considered diagnostic for the disease

5.      Biopsy of skin

6.      FAT on frozen tissue samples

7.      Nucleic acid identification methods – PCR

Treatment: Response to treatment in most dogs with RMSF, treated with tetracycline (22 mg/kg, TID), is rapid. Most dogs without neurologic deficits improve within 24 to 48 hours. Chloramphenicol (15 mg/kg, TID) has been used in pups less than 12 weeks old because of the risk of staining their dental enamel with tetracycline. Dogs with neurologic deficits may have more prolonged recovery periods.

Prevention: Since no vaccine is available for RMSF, the control is aimed at control of ticks.

VMC LECTURE – 24 (B)

CANINE EHRLICHIOSIS

 

Causative organism            :           Ehrlichia canis

Synonyms                             :           canine rickettsiosis, canine hemorrhagic fever,

tracker dog disease, canine tick typhus,

Nairobi bleeding disorder, canine agranulocytic ehrlichiosis and tropical canine pancytopenia

 

Definition: A number of Ehrlichia species can infect dogs and their affinity for hematopoietic cells may result in leukopenia and thrombocytopenia. Monocytic ehrlichioses in dogs (CME – canine monocytic ehrlichiosis) is caused primarily by E. canis. E. chaffeensis, E. ewingii, E. phagocytophila, E. equi and E. platys cause Granulocytic ehrlichiosis (CGE – Canine granulocytic ehrlichiosis). Ehrlichia canis was the first ehrlichial organism to be discovered and is the type species of the genus Ehrlichia. E. canis has worldwide distribution and is the cause of canine agranulocytic ehrlichiosis.. Canine ehrlichiosis is a disease of dogs and wild canids (e.g., wolves) and the infection is found worldwide. This organism is transmitted by the brown dog tick, Rhipicephalus sanguineus under natural conditions. E.canis primarily infect the host’s white blood cells, especially the mononuclear phagocytic cells. The disease is then disseminated to other organ systems including the bone marrow and results in a pancytopaenia (loss of a number of blood cell lines).

 

General characters:

1.      Morphology: They are very small pleomorphic organisms (coccoid or rod or cocco-bacillary) often arranged in clusters or colonies. They are generally referred as morulae. These colonies are small (initial bodies) and contain varying numbers of granules. They stain gram-negative. The initial bodies stain as slate-grey or light blue and the granules stain deep purple in Giemsa method. In dogs, they are found inside the reticuloendothelial cells of the lymph nodes and spleen. They are also seen inside macrophages and other granulocytes.

2.      Habitat: They are worldwide in distribution and the infection is mostly found in Asian countries. The organisms appear in clusters known as morulae in the cytoplasm of infected cells. The cells that are most commonly affected are the white blood cells. Ehrlichial organisms are classified as agranulocytic or granulocytic based on the cells they infect.

3.      Resistance: They are very fragile organisms.

4.      Classification: has been divided into three genogroups based on 16S rRNA. Genogroup I incorporates three species i.e. E. canis, E.chaffeensis, and E. ewingii. Genogroup II includes E.phagocytophila and E. equi. Genogroup III covers two species E. sennetsu and E. risticii.

Cultivation: These organisms are very difficult to cultivate in embryonated eggs or in cell cultures. However, they can be cultured in vitro in mammalian-derived cell line (DH82) and in an invertebrate cell line (IDE8).

Pathogenesis: The brown dog tick, Rhipicephalus sanguineus, transmits Ehrlichia. The tick Dermacenter variabilis also transmits it. The immature form of the tick feeds on an animal infected with Ehrlichia. When these immature forms or a mature form of the tick feeds on another animal, the Ehrlichia is passed on to that animal. The Ehrlichia can remain alive in the developing tick for up to 5 months. Because the brown dog tick transmits the disease, it can occur wherever brown dog ticks are found.

Symptoms and Lesions:

Canine Monocytic Ehrlichiosis: CME is characterized by three stages. The first, acute stage, beginning after 8–20 days following transmission by infected tick, lasts 2–4 weeks. The acute phase may be manifested by fever (103-106⁰F), depression, dyspnoea, anorexia, and slight weight loss. The laboratory findings are thrombocytopenia, leucopoenia, mild anaemia, and hypergammaglobulinemia are observed. Dogs may have bleeding tendencies, mainly petechiae and echymoses of the skin and mucous membranes, and occasionally, epistaxis may also occur. Ocular signs include corneal opacity, anterior uveitis, hyphema, tortuous retinal vessels and focal chorioretinal lesions consisting of central pigmented spots with surrounding areas of hyper-reflectivity. Subretinal haemorrhages, resulting in retinal detachment may occur and lead to blindness. Other clinical signs may include vomiting, serous to purulent oculonasal discharge, lameness and ataxia.

 

The second phase is subclinical and follows the acute phase and may last 40–120 days or even years, in which dogs can remain persistently infected for years without clinical signs but with mild thrombocytopenia. In the sub clinical phase the animal may show only slight anaemia. During this phase the dog either eexcretes the Ehrlichia from the body or the infection may progress to the chronic phase.

 

The ultimately stage is chronic,characterized by haemorrhages, epitasis and edema, with clinical signs. The results of laboratory study resemble the first phase of the disease. The course of this phase may often be complicated by superinfections by other micro organisms. The chronic phase generally develops 1-4 months after the tick bite and can be either mild or severe. The common clinical signs of the chronic disease are weakness, depression, anorexia, chronic weight loss and emaciation, pale mucous membranes, fever and peripheral oedema, especially of the hind limbs and the scrotum. Platelet-related bleeding, such as petechiae and echymoses of the skin and mucous membranes and epistaxis are common findings.   Secondary bacterial and protozoal infections, interstitial pneumonia, renal failure, and arthritis may occur during chronic severe disease.  Some reproductive disorders have also been associated with chronic CME including; prolonged bleeding during oestrus, inability to conceive, abortion and neonatal death.  Polymyositis has also been associated with CME.

 

Neurological signs may occur during the acute and chronic disease. These include signs of meningoencephalitis, e.g. arched back, severe neck or back pain, paraparesis or tetraparesis, ataxia, cranial nerve deficits and convulsions. Neurological signs may be attributed to haemorrhages, vasculitis and extensive plasma cell infiltration and perivascular cuffing of the meninges. Dogs infected with E. canis remain infected for their entire lives, even if they received antibiotic treatment with doxycycline.

 

Blood tests show that one or all of the different blood cell types are decreased. One cell type, the lymphocyte may increase and be abnormal in appearance. This can sometimes be confused with certain types of leukaemia. If a dog becomes chronically infected, the disease can keep coming back, especially during periods of stress. Decrease in the number of platelets (platelets help the blood clot) in the blood is the most common laboratory finding in all phases of the disease. Changes in the protein levels in the blood are common. The most common protein, albumin, is decreased and other types of protein called "globulins" are increased. Since one tick could be infected with and transmit more than one disease (e.g., haemobortenellosis or babesiosis), it is not all that uncommon to see a dog infected with more than one of these diseases at a time which generally causes more severe symptoms.

 

Canine granulocytic ehrlichiosis: Two clinically distinct disease syndromes, including chronic, moderate to sever anaemia and polyarthritis, are associated with CGE. Clinical signs are nonspecific and include fever, lethargy, anorexia, vomiting, and diarrhoea. The most frequent laboratory abnormalities are normocytic, normochromic nonregenerative anaemia, moderate thrombocytopenia with large platelets, lymphopenia, and eosinopenia. In beagles, inoculated experimentally with granulocytic Ehrlichia organism, after an incubation period of 4–11 days, the most prominent clinical signs were high fever for 2–5 days, and depression

 

Diagnosis:

1.      Based on the symptoms and lesions.

2.      Examination of blood smear: A small drop of blood is spread over a microscope slide, stained and examined under the microscope. The organism can be seen as morulae and only be found in the blood stream for about 3 days during the acute phase of the disease. So this method of diagnosis could miss some cases of the disease.

3.      Immunofluorescet antibody test (IFA): IFA is a highly accurate blood test that detects antibodies against E.canis in dogs. As the disease progresses, the antibody level will rise significantly. Often two tests will be done 2 weeks apart and the results compared. Dogs with an active infection will show a significant rise in the amount of antibody present.

4.      ELISA:  This test also determines the amount of antibodies present.

5.      Nucleic acid identification methods: Polymerase chain reaction.

6.      Western blotting.

 

Treatment: The antibiotics tetracycline or doxycycline is used. Treatment is for 2-3 weeks. Some dogs will need blood transfusions or intravenous fluids depending on the severity of the disease. The drug imidocarb dipropionate is sometimes used in conjunction with the antibiotics. Some of the damage caused by Ehrlichia may be due to the dog's own immune response to the organism. For this reason, high doses of corticosteroids (e.g., prednisolone) are often given during the early phase of the disease.

 

Control: Tick control is the main way to prevent ehrlichiosis. Products that repel and kill ticks, tick collars containing the active ingredient amitraz (Preventic collars) are also used. There is no vaccine for ehrlichiosis.

 

Zoonotic importance:

The common symptoms in man include fever, chills, headache, and muscle aches. Other less common symptoms include nausea, loss of appetite, weight loss, abdominal pain, cough, diarrhoea and change in mental status. However, man does not get infected directly from a dog, but through a tick bite. A different tick than the brown dog tick may spread human ehrlichiosis. Research suggests the Lone Star tick may be involved. Also, the Ehrlichia species most often implicated in human infections is E. chaffeensis.

VMC LECTURE – 25 (A)

 

RICKETTSIOSIS PART IV - ANAPLASMOSIS

 

1. Bovine Anaplasmosis

Causative organism   :        Anaplasma marginale; A.marginale subsp.centrale

 

Bovine anaplasmosis is an arthropod-borne hemolytic disease of cattle that is caused by the rickettsia Anaplasma marginale (Rickettsiales: Anaplasmataceae). It is a tick-borne disease and is endemic in tropical and subtropical areas of the world. The disease causes considerable economic loss to the dairy industry. The organism parasitises red blood cells, causing their destruction and producing emaciation, anaemia, jaundice, and, occasionally death.  Analyses of 16S rRNA, groESL, and surface proteins have resulted in the recent reclassification of the order Rickettsiales. The genus Anaplasma now also includes A. bovis, A. platys, and A. phagocytophilum, which were previously known as Ehrlichia bovis, E. platys, and the E. phagocytophila respectively. Anaplasma marginale are called so, since they are found in the periphery of the erythrocytes. One more organism of importance in cattle A.centrale is less pathogenic and found slightly interior of the erythrocytes.

 

GENERAL CHARACTERS:

1.      Morphology: A. marginale appear as dense, rounded, intraerythrocytic bodies approximately 0.3–1.0 µm in diameter with most situated on or near the margin of the erythrocyte. Anaplasma centrale is similar in appearance, but most of the organisms are situated away from the margin of the erythrocyte. The organisms appear as very minute roundish body at the periphery of erythrocytes (A). Within these cells the membrane-bound inclusions (also called initial bodies) contain four to eight organisms (B). The small genome of A. marginale is circular, and the size is estimated at 1.2 to 1.6 Mb. The organism also has six major surface proteins.

2.      Classification: The organisms in the order Rickettsiales were recently reclassified based on biological characteristics and genetic analyses of 16S rRNA genes and surface protein genes. There are four distinct genus under the family Anaplasmataceae:  -  Anaplasma, Ehrlichia, Wolbachia and Neorickettsia Organisms classified within the family Rickettsiaceae (genera Rickettsia and Orientia) are all obligate intracellular bacteria that grow freely within the cytoplasm of eukaryotic cells. While organisms placed in the family Anaplasmataceae are also obligate intracellular organisms, they are found exclusively within membrane-bound vacuoles in the host cell cytoplasm. All organisms in the family Anaplasmataceae multiply in both vertebrates and invertebrates (primarily ticks and trematodes).The genus Anaplasma, includes three species that infect ruminants: A. marginale (the type species). A. marginale subsp. centrale (referred A. centrale), and A. ovis. The genus Anaplasma also includes A. phagocytophilum (formerly Ehrlichia equi, E. phagocytophila), A. bovis (formerly E. bovis), and A. platys (formerly E. platys).

3.     Habitat: The organisms are found in cattle. The cattle tick Boophilus microplus act as biological vector.

4.     Resistance: As other rickettsial organisms

5.     OIE Listing: List B disease (Bovine)

6.     Human risk: No human risk

 

CULTURAL CHARACTERS: Very difficult to cultivate and susceptible animals are normally used.

 

PATHOGENESIS: Cattle are the most important host affected. Sheep, goats and certain wild animals are also affected.. Ticks are the natural vectors and a range of tick species has been shown to be capable of transmitting infection. In the absence of ticks, biting flies can maintain the disease though these are less efficient vectors. Fourteen different ticks transmit A. marginale experimentally. The important tick species are Argas persicus, Ornithodoros lahorensis, Boophilus annulatus, B. decoloratus, B. microplus, Dermacentor albipictus, D. andersoni, D. occidentalis, D. variabilis, Hyalomma excavatum, Ixodes ricinus, Rhipicephalus bursa, R. sanguineus and R. simus, However, the cattle tick, Boophilus microplus and Rhipicephalus sanguineus are the most important vectors. Various other biting arthropods have also been found to act as mechanical vectors in the transmission of Anaplasma. Mechanical transmission frequently occurs via blood-contaminated fomites, including needles, dehorning saws, nose tongs, tattooing instruments, ear-tagging devices, and castration instruments. In addition to mechanical and biological transmission, A. marginale can be transmitted from cow to calf transplacentally during gestation

Tick transmission can occur from stage to stage (transstadial) or within a stage (intrastadial). Transovarial transmission from one tick generation to the next does not occur. After the tick feeds on an infected animal, infected erythrocytes are ingested by ticks. The first site of infection of A. marginale in ticks is the gut cells. When the ticks feed a second time, many tick tissues become infected, including salivary gland cells, from where the rickettsia is transmitted back to cattle. Two forms of A. marginale, reticulated and dense forms, are found in infected tick cells. Reticulated forms appear first and are the vegetative stage that divides by binary fission. The reticulated form changes into the dense form, which is the infective form and can survive extracellularly. The organism can then be transmitted to succeeding generations of ticks. By this method, the infectious organism can survive outside of infected cattle for long periods of time. When future generations of infected ticks feed on susceptible cattle, the disease is transmitted.

SYMPTOMS: There is considerable variability in the severity of anaplasmosis with severity generally increasing with the age of the animal. Affected cattle have steadily increasing temperatures (up to 105F), anaemia, weakness and respiratory distress particularly after exercise, depression and anorexia become more obvious as the disease progresses, jaundice and frequently a marked loss of condition and urine is often brown due to the presence of bile pigments. Severely affected animals may die

 

LESIONS: Gross pathology findings are due to anaemia and resulting anoxia. These include pale and often jaundiced tissues, watery blood and an enlarged spleen with a soft reddish-brown pulp. The liver is also enlarged, yellow-brown and often mottled.

 

DIAGNOSIS:

1.     Based on symptoms and lesion

2.      Direct Microscopy: The clinical sample from live animal includes thin blood smears and blood collected into an anticoagulant. From dead animals air-dried thin smears from the liver, kidney, heart and lungs should be collected. Both blood and organ smears are fixed in absolute methanol for 1 minute and stained in 10% Giemsa stain for 30 minutes. In Giemsa-stained smears, A. marginale appear as dense, rounded and deeply stained intraerythrocytic bodies, approximately 0.3–1.0 µm in diameter. Most of these bodies are located on or near the margin of the erythrocyte. This feature distinguishes A. marginale from A. centrale, as in the latter most of the organisms have a more central location in the erythrocyte. Fluorescent antibody staining may be used as an alternative staining technique for detecting Anaplasma in smears taken at post-mortem.

3.      Isolation of the organism: The organisms are identified by inoculating blood from suspected cattle into spleenectomized calves, in which the infection is confirmed by examination of blood smear.

4.      Nucleic acid identification methods: PCR technique and a RNA probe is used to confirm the infection. Restriction enzyme analysis, Southern blotting and DNA sequencing are the other methods of diagnosis.

5.      Serological tests: Serological tests include competitive ELISA, card agglutination test (CAT), CFT, indirect ELISA, dot ELISA and IFA.

 

TREATMENT: The anaplasmosis organism is susceptible to oxytetracycline or chlortetracycline (antibiotics. Cattle showing clinical signs usually respond to high dosages of oxytetracycline given by injection. When diagnosed in an individual in the herd, it is advisable to immediately treat the entire herd with tetracylines to prevent spread of the infection and possible development of other cases of disease

 

CONTROL: Tick control by acaracide dipping is widely used in endemic areas. Acaracides used for this purpose include various synthetic pyrethroids, amitraz, and some organophosphates. Dipping may be done as frequently as every 4-6 weeks in heavily infested areas. Anaplasmosis vaccines are readily available and are highly effective. The most commonly used vaccine is a live Anaplasma centrale vaccine used either singly or in combination with Babesia bovis vaccine. Vaccination is particularly important for susceptible cattle entering endemic areas. An anti-tick vaccine is also commercially available in Australia. This vaccine is of limited use, but can be used as part of an integrated program for the control of ticks.

 

 

2. BOVINE PETECHIAL FEVER

 

Synonyms      :        Bovine infectious petechial fever, transmissible petechial

fever, Ondiri disease, Nairobi quarantine disease

Etiology         :        Anaplasma phagocytophilum (E.phagocytophilia; E.ondiri)

 

It is an infectious non-contagious disease of cattle that is characterised by fever, oedema of the conjunctiva, petechiae of the mucous membrane and in very severe cases the affected animal collapse and dies.

 

General properties:

1.     Morphology: It is caused by  E.ondiri. E.ondiri is an obligate intracellular organism, which cannot survive outside the cell. The organisms occur in polymorphonucelar phagocytic cells like neutrophils and eosinophils and monocytes. They are arranged either singly or as colonies. They are very small pleomorphic organisms with small variations in size. The initial bodies stain as  slate-grey or light blue and the granules stain deep purple in Giemsa method. The particles are arranged as discrete units in the periphery of the colonies and the appearance is attributed to ‘broken dinner plate’.

2.     Habitat: The organisms are normally seen in phagocytic cells.

3.     Resistance: They are fragile organisms and can withstand 37C for only two days and retain its infectivity at refrigeration temperature for only four hours.

 

Cultivation: The organisms are difficult to cultivate in embryonated eggs, tissue cultures or laboratory animals like mice and guinea pig. Cattle and very few wild animals are the only host susceptible for this organism. Heparinised blood is the main source for infecting cattle in which the organisms are found in the leucocyte fraction.

 

Pathogenesis: Cattle are the main host and certain wild ruminants, sheep and goat are less severely affected by this infection. Blood sucking arthropods and ticks spread the infection between animals. The ticks Rhipicephalus hurtis, R.kochi and Ixodus are involved in transmission of the infection. The wild ruminants also act as reservoirs of infection. The incubation period is 7-14 days.

 

Symptoms: Infected animals have high fever with sharp decline in milk yield in lactating cows. On the second day of infection petechial haemorrhages appear on the gums, under the tongue, conjunctiva and mucosa of vulva and vagina. Persistent and foetid smelling diarrhoea may remain for 1-10 days. As a result of swelling and eversion of the conjunctival sac and accumulation of blood in the lower part of aqueous humor, the appearance of the eye is referred as ‘poached-egg eye’ appearance. Affected pregnant animals may abort. The mortality rate is very high.

 

Lesions: There is persistent haemorrhage, oedema and hyperplasia through out the body. Petechial haemorrhages are seen on the gums, under the tongue, conjunctiva and mucosa of vulva and vagina. Different types of haemorrhages on the inner and outer surface of heart, epicardial and endocardial surfaces are characteristic of this infection. Haemorrhages may also be present in various other organs and glands. Oedema of the intermuscular tissue is the frequent cause of death.

 

Diagnosis:

1.     Based on symptoms and lesions.

2.     Microscopical examination of blood film and identification of causative organism.

3.     The infection should be differentiated from other acute infection of cattle like anthrax, Rift valley fever etc.

 

Treatment: No satisfactory treatment is available. Tetracyclines may be effective.

 

Control: Control is aimed at elimination of arthropod vectors and prevention of wild ruminants in grazing areas. No vaccines are available.

 

3. TICK BORNE FEVER

 

Etiology         :        Cytocetes phagocytophilia (E.phagocytophilia

A,phagocytophilum)(for sheep); C.bovis (cattle)

 

Definition: It is an infectious, non-contagious febrile disease of sheep and occasionally cattle. This infection is spread by ticks.

 

General characters:

1.     Morphology: They are very small pleomorphic obligate intracellular organisms that are seen in phagocytic cells. They appear as pleomorphic intracytoplasmic bodies. They are usually stained by Macchiavello’s or modified Ziehl-Neelsen method. In contrast to other organisms they don’t retain the basic fuchsin and take the counter stain methylene blue or malachite green. Various developmental forms like small spherical granules, large round or oval bodies and large morulae are seen.

2.     Habitat: The organisms are commonly seen in neutrophils, eosinophils, basophils and monocytes.

3.     Resistance: They are less fragile organisms than heartwater and Ondiri infection agents.

 

Cultivation: The organisms are difficult to cultivate in embryonated eggs and tissue culture. The organisms can be cultivated only in sheep or in cattle.

 

Pathogenesis:  Sheep and cattle are the main hosts affected by the infection. The infection is spread by Ixodes ricinus ticks. The incubation period is 4-8 days.

 

Symptoms: In sheep the infection is characterised by high fever (40-42C) and is associated with presence of parasites in phagocytic cells. The fever curve follows a plateau type with fever persisting for 6-22 days. The infection makes the sheep more prone for other infectious diseases. In cattle the disease relatively insignificant and seen in cattle that have come to a tick infested area from tick free area. In sever cases abortion may result.

Lesions: There are no characteristic lesions.

 

Diagnosis:

1.     Microscopical examination of blood film and demonstration of the organisms in phagocytic cells.

2.     Production of the infection in sheep by infecting them with blood from susceptible animals. Instead of cattle splenectomised guinea-pigs may also be used.

 

Treatment & Control: Tetracycline is the best drug. Eradication of ticks will prevent the occurrence of infection.

 

VMC LECTURE – 25 (B)

HAEMOBARTONELLOSIS

 

Causative organism:            Haemobartonella felis

 

Disease caused:                 Feline infectious anaemia

 

Feline Infectious Anaemia (FIA) is caused Haemobartonella felis, which lives on the surface of red blood cells. The resulting structural damage can cause anaemia if the red blood cells are destroyed. The cat's own immune system may also cause death of red blood cells as it tries to kill the parasite attached to them. Clinical signs usually reflect the underlying anaemia. Cats, which have been infected with FIA, may remain carriers of the parasite for life. FIA causes anaemia, which may be accompanied by a fever in the early stages of infection. Methods of spread of H.felis infection are not fully known. Very young kittens can be infected, implying that infection can be vertically spread from the queen. As mentioned above, fighting and fleas have been implicated in transmission of infection between cats. Saliva and urine are not thought to be able to transmit the disease and non-infected and infected cats have been housed together without cross infection. Ingestion (such as with cat bites) and injection of infected blood (such as with a blood transfusion) can both result in infection. Clinical signs of anaemia include tiredness, depression, a reduced appetite and pale gums. Weight loss can occur. Such clinical signs can be seen with a variety of diseases that result in anaemia, and are not specific for FIA. Other clinical signs may include enlargement of the spleen and lymph nodes. It is believed that different strains of H.felis exist which can cause a range of clinical signs from mild non-significant disease to severe anaemia. The anaemia seen with H.felis infection is usually regenerative in type. This means that the cat is able to respond to the anaemia by producing new red blood cells, which are visible in the circulation. Some infected cats are not anaemic because they are asymptomatic carrier cats, or are infected with a strain of H.felis, which causes only mild disease. Therefore the finding of parasites in the circulation does not always mean that the cat has clinically significant FIA disease. Doxycycline has been most commonly used and is given for two to three weeks. Corticosteroids may also be used, in conjunction with antibiotics, to suppress the immune-mediated destruction of red blood cells. In cats with severe anaemia, blood transfusions may be required. Supportive cares to encourage the cat to eat, and rehydration therapy in dehydrated cases, are also important.

 

 

 

 

VMC - LECTURE # – 26

 

CHLAMYDIOSIS – PSITTACOSIS, ORNITHOSIS

 

Important species     :        Chlamydophila psittacii; Chlamydophila abortus

Synonym                 :        Parrot fever

 

Introduction: Chlamydiosis refers to infection caused by Chlamydial organisms. There are two important infections caused by Chlaymdial organisms in animals.

1.      Psittacosis is an infection caused by a microorganism called Chlamydophila psittaci (formerly referred Chlamydia psittaci).

2.      Enzootic abotion in ewes caused by Chlamydophila abortus (formerly Chlamydia ovis).

 

Classification: The family Chlamydiaceae is recently reclassified into two genera and nine species based on sequence analysis of its 16S and 23S rRNA genes (molecular taxonomy). The two genera, Chlamydia and Chlamydophila, correlate with the former species Chlamydia trachomatis and C. psittaci.

The genus Chlamydia includes C. trachomatis (human), C. suis (swine), and C. muridarum (mouse, hamster).

The genus Chlamydophila includes C. psittaci (avian), C. felis (cats), C. abortus (sheep, goats, cattle), C. caviae (guinea pigs), and the former species C. pecorum (sheep, cattle) and C. pneumonia (human).

 

C.trachomatis is a human pathogen and is also called as TRIC agent (Trachoma and inclusion conjunctivitis). The       

 

General characters:

1.     Morphology: They are gram-negative, obligate intracellular organisms. They stain as rickettsial organisms by Machiavello’s staining method. They are comparatively larger than rickettsia and viewed easily under light microscope. The organisms contain two particles elementary body and initial body. The elementary body(300 nm)  contains an electron dense core surrounded by three layers and a clear zone separates them. The elementary bodies are smaller than initial bodies. The initial body is bigger (700-1200 nm) than elementary body and has no core. The elementary body is highly infectious extracellular replicative form whereas the initial body is infectious intracellular replicative form.

2.     Resistance: They are sensitive to heat and inactivated at 60C for 10 minutes. They can remain viable for a long time under –70C.

3.     Other properties: They produce haemagglutination of mouse and hamster red blood cells.

4.     Replication: The elementary bodies gain entry into the cell and they convert themselves into initial body. The elementary body (300 nm)  contains an electron dense core surrounded by three layers and a clear zone separates them. The elementary bodies are smaller than initial bodies. The initial body is bigger (700-1200 nm) than elementary body and has no core. The elementary body is highly infectious extracellular replicative form whereas the initial body is infectious intracellular replicative form. The initial bodies multiply into smaller bodies by break down and remain as aggregates in the cytoplasm. A single initial body may fragment into five smaller bodies. Such aggregates cause lysis of the surrounding cytoplasm but the nucleus remains unchanged. Hence the infected cell continues to divide and multiply. Ultimately the particles are released by lysis of the cell.

5.     Antigens: There are two main antigens a heat stable complement-fixing antigen that is group specific and heat labile species-specific antigen.

6.     OIE Listing: Psittacosis – List B (Avian infection); Enzootic abortion – List B (Ovine infection)

7.     Human risk: Potentially hazardous organisms for humanbeings.

 

Cultivation: They are readily cultivable in embryonated eggs by yolk sac route. Mice are the laboratory animals of choice.

 

AVIAN CHLAMYDIOSIS

Psittacosis (Avian chlamydiosis) is also known as parrot fever and ornithosis and is caused by the bacterium Chlamydophila psittaci. The disease in birds was originally called psittacosis, but the term ornithosis was introduced later to differentiate the disease in domestic and wild fowl from the disease in psittacine birds. The two syndromes are currently considered to be same.

 

Pathogenesis: The types of birds affected include parrots, parakeets, pigeons, turkeys, ducks etc. C. psittaci is excreted in the feces and nasal discharges of infected birds. The organism is environmentally labile but can remain infectious for several months if protected by organic debris (e.g. litter and feces). The two routes of transmission of psittacosis are respiratory and oral. Respiratory transmission include the inhalation of infected particles of faecal, ocular, nasal, and respiratory discharges, and feather dust. Oral transmission includes the ingestion of food and water contaminated with Chlamydophila bearing faeces. Parents that are carriers can infect their nestling via the regurgitated food they feed the babies. Some infected birds can appear healthy and shed the organism intermittently. Shedding can be activated by stress factors, including relocation, shipping, crowding, chilling, and breeding

 

Symptoms : Symptoms of psittacosis are variable. They depend upon the strain of Ch. psittaci with which the bird in infected, the bird's immune system status, species, age, and the presence of other concurrent infections. Mild outbreaks of psittacosis may go unnoticed because there will be very few symptoms. Alternatively there may be very mild respiratory symptoms and diarrhoea. Symptoms are usually related to respiratory and digestive system involvement. During the acute phase the symptoms include respiratory problems (shortness of breath, noisy breathing, "runny nose," sinus infection), diarrhoea, polyuria (excess urine), lethargy, dehydration, ruffled feathers, loss of appetite and yellowish, greyish, or lime green urates. Subacute or chronic psittacosis may show the symptoms like, tremors, unusual head positions, convulsive movements, opisthotonos (neurologic disease in which the top of the head is bent over and approaches the back) and partial or complete paralysis of the legs. Other symptoms noticed are unusual tameness, lack of normal molt, poor condition in beak and nails, sneezing, swollen, infected eyelids and wasting of breast muscles.

Lesions: The important lesion is the wasting of pectoral muscle, focal liver necrosis, and enlargement of spleen, pericarditis, thickening of air sac with adhesions to the sternum, mucous plugs in digestive tract and erythematous lesions on the skin over body and legs.

 

Diagnosis:

1.     Based on the symptoms and lesions.

2.     Identification of the organisms by histochemical staining of smears of exudate and faeces, and impression smears of tissues. Giemsa, Gimenez, Ziehl–Neelsen and Macchiavello’s stains are commonly used to detect chlamydiae in impression smears of liver and spleen.

3.     Isolation of the organisms: Cell cultures and embryonated eggs are used isolate C.psittaci. The cell lines commonly used are buffalo green monkey (BGM), McCoy, HeLa, African green monkey kidney (Vero) and L cells. Chicken embryos are also used for the primary isolation of Chlamydophila. The standard procedure is to inject up to 0.5 ml of inoculum into the yolk sac of a specific pathogen free 6–7-day-old embryo. The eggs are incubated in a humid atmosphere at 39°C, as multiplication of chlamydia is greatly increased at the higher temperature. Replication of the organism usually causes the death of the embryo within 3–10 days. C.psittaci infection causes vascular congestion of the yolk sac membranes.

4.     Nucleic acid identification method - PCR

5.     Serological tests

a.      Complement fixation test: This test is performed during the infection and convalescent phase. A four fold or more increase in antibody titre is an indication of infection.

b.      Indirect complement fixation test.

c.      ELISA

 

Treatment: Penicillin, aureomycin, oxytetracyliines and doxycycline are commonly used for treatment.

 

Control: The outcome of treatment varies, depending upon the individual bird's species, age, immune status, length of illness before treatment was sought, the virulence of the strain with which it is infected, mode of treatment, and its response to that treatment. Precautions must also be taken to protect human caretakers. It is recommended that you take the following

 

1.      Isolate incoming (new) birds for thirty to forty five days. (Longer is better)

2.      Test suspicious birds (those with loose droppings, weight loss, or respiratory problems.)

3.      Treat infected birds with Doxycycline for 45 days.

4.      Thoroughly clean and disinfect cages, surroundings, and equipment used for a psittacosis bird. Quaternary ammonium disinfectants have proved very effective against this bacteria. (i.e. A-33, Barquat, Cetylcide, Floquat, Hitor, Merquat, Omega, Parvosol, Quintacide, Roccal, Zephiran. [Avian Viruses, Function and Control] As well as Roccal-D, Betadine and Environ-One-Stroke)

5.      Keep circulation of feather dust to a minimum.

6.      Droppings from an infected bird should be soaked with disinfectant and placed in a sealed plastic bag prior to disposal.

7.      Contact with infected birds by humans should be kept to an absolute minimum. Strict quarantine techniques should be used.

8.      Any Flu-like symptoms in human caretakers should be monitored and a physician should be contacted. Just as with birds, human psittacosis is treatable, but can develop into a serious problem without proper treatment.

Zoonotic importance: The infection spreads from birds to bird keepers. It may also be found in farmers and slaughterhouse workers who process turkeys and ducks. Psittacosis is usually spread by inhaling dust from dried bird droppings and by handling infected birds in slaughterhouses. Some birds infected with psittacosis may appear healthy, but can still spread the infection to other birds or humans. Human-to-human spread is very rare. Psittacosis can cause fever, headache, chills, muscle aches, and sometimes pneumonia with a relatively non-productive cough. The period between exposure and the beginning of symptoms may range from 7 to 28 days but is usually 10 days. Infection does not provide permanent immunity from this disease. Antibiotics such as tetracycline are usually prescribed. Erythromycin is sometimes used in young children. The disease is usually mild in humans, it may be severe, and can uncommonly result in death especially in untreated older people.

OVINE CHLAMYDIOSIS

Ovine chlamydiosis is also called as chalmydial abortion of ewes and is caused by Chlamydophila abortus (formerly Chlamydia psittaci immunotype-1). Goats, cattle and deer are also affected to a lesser extent. It is a zoonosis and the organism must be handled with biosafety precautions. Pregnant women are particularly susceptible. In sheep, abortion in late pregnancy with expulsion of necrotic fetal membranes are the important symptoms. The infection is more commonly seen in flocks that are closely congregated during the parturient period. Infected animals show no clinical illness prior to abortion. Pathogenesis commences around day 90 of gestation when rapid fetal growth occurs. At this point of time chlamydial invasion of placentomes produces a progressively diffuse inflammatory response, thrombotic vasculitis and tissue necrosis. Milder changes occur in the fetal liver and lung and, in cases in which placental damage is severe, there may be evidence of hypoxic brain damage. Abortion results from a combination of impairment of materno-fetal nutrient and gaseous exchange, disruption of hormonal regulation of pregnancy and induced cytokine aggression.

 

The infections should be differentiated from infectious causes of abortion such as brucellosis, coxiellosis or other bacterial pathogens like Campylobacter, Listeria and Salmonella.

 

Infected ewes shed vast numbers of infective C. abortus at the time of abortion or parturition, particularly in the placenta and uterine discharges. Human infection may be acquired from such sources or from carelessly handled laboratory cultures of the organism, with effects that range from subclinical infection to acute influenza-like illness. Appropriate precautions should be taken when handling cultures and potentially infected tissues. Human placentitis and abortion caused by C. abortus of ovine origin indicate that pregnant women are at special risk and should not be exposed to sources of infection

 

OTHER INFECTIONS:

Chlamydial organisms also cause catarrhal pneumonia in sheep and goats, respiratory disorders in calves, fibrinous pericarditis in swines and numerous other respiratory infections in animals.

VMC 311 LECTURE # 27

HIGHER BACTERIA - DERMATOPHILUS

 

Dermatophilosis (also known as streptothrichosis) is an exudative, pustular dermatitis affecting cattle, sheep, horses, goats, dogs, cats, many wild mammals, reptiles and, occasionally, humans. The severe disease in ruminants is promoted by immunomodulatory effects induced by infestation with the tick, Amblyomma variegatum.

 

Dermatophilosis in animals is caused by Dermatophilus congolensis. Various infections caused by this organism includes Streptothricosis (rain scald) in cattle, lumpy wool disease of sheep and strawberry of foot rot of sheep.

 

General characteristics:

1.     Morphology: The organism is Gram positive and its morphology is more readily appreciated in smears stained with Giemsa. It has two characteristic morphologic forms—filamentous hyphae and motile zoospores. The hyphae are characterized by branching filaments (1-5 µm in diameter) that ultimately fragment by both transverse and longitudinal septation into packets of coccoid cells. The organisms appear as rows of eight cells due to division pattern. The coccoid cells mature into flagellated ovoid zoospores (0.6-1 µm in diameter). It is also referred as higher bacteria since it shows extensive filamentation, aerial hyphae with asexual spores or conidia. They produce zoospores that are motile have four or six flagella.

2.     Habitat: These organisms are pathogenic and are predominantly seen in back area.

3.     Classification: Eubacteriae is a class of unicellular or mycelial organisms with rigid cell walls, flagella and form conidia or endospores. This class is further divided into unicellular Eubacteriales and mycelial Actinomycetes. Under this order, the family Dermatophilaceae is proposed with Dermatophilus congolensis as type species.  Three species had been proposed under this genus that include

                                                              i.      D.congolensis           -        Mycotic dermatitis of cattle

                                                            ii.      D.pedis,                  -        Strawberry foot-rot

                                                          iii.      D.dermatonomus      -        Lumpy wool disease of sheep

But now only species D.congolensis has been proposed deleting the other two.

4.     OIE Classification: List B disease (Bovine disease)

5.     Human risk: The infection can spread from affected animals to humanbeings.

 

Cultural characters: It is difficult to cultivate these organisms under laboratory conditions. They require a temperature of 37C and 10% CO₂. Generally shreds from underside of the scab are put in distilled water and they are kept in candle jar at a temperature of 30-35C and 10% CO₂ for 2 hours. This step will allow the liberation of zoospores. A small loopful of zoospores are streaked over blood agar and incubated overnight at 37C. The colonies are very small and can been with hand lense. The colonies appear as tiny adherent waxy and doomed. They colonies may be yellow or orange in colour. The incomplete haemolysis is completed in association with Corynebacterium equi or Streptococcus agalactiae.  In glucose or serum broth, small fluffy spherical colonies form the bottom of the medium or attach to the walls of the container. Smears prepared from fluid media will show organisms with characteristic morphology.

 

Biochemical reactions: They produce acid without gas, do not reduce nitrates and methylene blue and produce negative reaction for indole, methyl red and Voges-Prostaur tests.

 

Pathogenesis:

It is mainly infection of the epidermis, and is seen worldwide but more prevalent in the tropics. The infection is wrongly called mycotic dermatitis. The lesions are characterized by exudative dermatitis with scab formation. Dermatophilus congolensis has a wide host range. Among domestic animals, cattle, sheep, and goats are affected most frequently; horses occasionally; and pigs, dogs, and cats rarely. It is commonly called cutaneous streptothrichosis in cattle, goats, and horses; in sheep, it is termed lumpy wool when the wooled areas of the body are affected. Deaths occasionally occur, particularly in calves and lambs, because of generalized disease with or without secondary bacterial infection and secondary fly or screwworm infestation. The incidence of the infection has got close relationship with heavy rainfall, tick bites and scratches caused by thorns. During heavy rainfall season the coat of the animal is drenched and the keratin layer is spongy and allows easy entry of organisms through sites of tick bite, cut wounds, scratches etc. Blood sucking flies are play a role in the pathogenesis of the infection.  The organisms can also remain in dry skin and hide for a long time. The severe disease in ruminants is promoted by immunomodulatory effects induced by infestation with adults of the tick species Amblyomma variegatum. The primary economic consequences are damaged hides in cattle, wool loss in sheep, and lameness and loss of performance in horses when severely affected around the pastern area.

 

Symptoms and lesions

1.     Streptothricosis (cattle): Cattle are mostly affected and infection is more common in countries with tropical climate (high temperature and high rainfall). The back regions particularly hump is an important site affected by this bacteria. It affects an inverted triangular area of each side of back immediately beneath the hump. Other parts affected include face, neck, dewlap, flank, udder, scrotum, escutcheons, axilla and groins. Eruptive eczema followed by thickening of the skin and formation of exuding pimples is the common lesion observed. The serous exudates form a crust at the base of hairs. In cattle, the lesions can be observed in three stages:

a.      hairs matted together as “paintbrush” lesions,

b.      crust or scab formation as the initial lesions coalesce,

c.      accumulations of cutaneous keratinized material forming “wart-like” lesions that are 0.5-2 cm in diameter.

The entire skin is covered with white powdery crusts, which fall during handling. Secondary bacterial infection results in extensive cellulites and death of affected animal.

2.     Streptothricosis (Horses): Lesions on horses are seen in saddle region and similar to those of cattle, developing with matted hair and “paintbrush” lesions leading to crust or scab formation with yellow-green pus present under larger scabs. Matting and scab formation is uncommon; loss of hair with a fine “paintbrush” effect can be extensive. Persistent wetting in yards, stables, or at pasture leads to lower limb infection. White legs and the white-skinned areas of the lips and nose are more severely affected. Generalized disease is also associated with prolonged wet weather. Outbreaks occur on farms with previously affected horses.

3.     Lumpy wool disease: The lesions are confined to wool-covered areas of skin especially the lumbar and flank region. The lesions are also occasionally seen in hairy parts of face, ears, scrotum and legs. The incidence of the infection is associated with mild wet weather. The infection starts are papule formation and exudation resulting in matting of the wool. The wool may appear yellow or golden coloured. Scab formation is also noticed at the base of wool fibres. The affected wool is graded less resulting in economic loss.

Diagnosis:

1.      Examination of smears from scabs: Impression smears are made from the moist, concave undersurfaces of freshly removed scabs. Alternatively, scabs can be soaked overnight in sterile water or saline to sufficiently moisten them so that the undersurface of the scab can be used to make effective impression smears by firmly pressing this surface on to a microscope slide. Smears are then air-dried, fixed by heating or immersion in methanol for 5 minutes, and stained. The organism stains well in dilute carbol fuchsin or methylene blue stain, but Gram’s stain or, preferably, a 1 in 10 dilution of Giemsa stain for 30 minutes, gives better differentiation in thick smears, the darkly stained D. congolensis contrasting with the paler or pink counterstained background of keratinocytes and neutrophils

2.     Cultural examination:

a.      Haalstra method: Small pieces of scab are placed in a bijou bottle containing 1 ml of sterile distilled water and allowed to stand at room temperature for 3–4 hours. The open bottle is then placed for 15 minutes in a candle jar. Samples of the surface liquid are removed with a bacteriological loop and cultured. The method depends on the release from the scab of the motile cocci of D. congolensis and their chemotropic attraction towards the carbon-dioxide-rich atmosphere of the candle jar. A small loopful of zoospores are streaked over blood agar and incubated overnight at 37C. Growth is accelerated under microaerophilic conditions; rough, usually haemolytic, greyish-yellow colonies, about 1 mm in diameter, are seen pitting the medium after 24 hours. Incubation in air produces similar pinpoint colonies at 24 hours that grow to about 1 mm at 48 hours. The rough colonies are formed by the branching filaments, but continued growth in air stimulates the production of the cocci, which are commonly yellow in colour. Colonies take on a smooth, often yellowish, appearance. The cocci are normally vigorously motile when taken from young cultures. The colonies are very small and can been with hand lense.

b.      The other method include macerating the scab material with dry glass slide and after removing the coarse particles the finer ones are scattered over the surface of media. The colonies appear as tiny adherent waxy and doomed. They colonies may be yellow or orange in colour.

c.      The incomplete haemolysis is completed in association with Corynebacterium equi or Streptococcus agalactiae.

d.      In glucose or serum broth, small fluffy spherical colonies form the bottom of the medium or attach to the walls of the container. Smears prepared from fluid media will show organisms with characteristic morphology.

3.      Fluorescent Antibody Test on smears to identify D.congolensis.

4.      Based on symptoms and lesions.

5.      Based on biochemical characters after confirming by culturing.

6.      Serological tests are not currently used in the diagnosis of infection. However, ELISA is used in certain laboratories.

 

Treatment: Treatment is not very effective and recovery is spontaneous. Arsenicals, Copper sulphate and quaternary ammonium compounds can be used as eternal therapeutic agents. Penicillin, streptomycin and intravenous iodide therapy are also effective.

 

Strawberry foot rot: The signs of infection vary from mild reddening (inflammation) of the interdigital skin (skin between the digits or toes) to complete separation of the horn of the hoof. Infection commences when bacteria lodge on the interdigital skin causing inflammation; the skin-horn junction then begins to erode and the horn starts to lift. From this point, the bacteria move under the horn causing separation of horn around the heel, sole, toe and eventually to the outer wall. Infected feet may also have a characteristic foul smell. Removal of scabs reveals a raw surface with numerous bleeding points resembling strawberry fruit. Accurate diagnosis is essential, as early outbreaks of footrot can be difficult to distinguish from other diseases.Many feet must be examined. Examining only one or two sheep can be misleading and may result in unnecessary or incorrect treatment.

 

To describe footrot at its various stages, a scoring system has been developed called Modified Egerton Scoring System

 

      Score                    Description               

0          Normal foot - No lesion (sign of disease)

1          A limited mild interdigital dermatitis (scald)

2          More extensive interdigital dermatitis.

3          Severe interdigital dermatitis and under-running of the horn of the heel and sole.

4          Severe interdigital dermatitis and under-running of the horn of the heel and sole but with the under-running extending to the walls of the hoof.

5          Under-running of the hard horn of the outside walls of hoof.

 

VMC 311 LECTURE # 28

DERMATOPHYTES (DEMATOMYCOSIS)

 

Dermatophytosis otherwise known as ringworm is a cutaneous infection caused by a variety of different genera of fungi called the "dermatophytes." The term dermatophytes refer to "plants that live on the skin". The name ringworm comes from the characteristic circular red lesions that occur commonly in humans. However, ringworm lesions in animals are much less circumscribed, even presenting as a diffuse folliculitis, a thickened, gray colored area of alopecia; or, a non-specific ulcerated lesion due to licking by the animal. The pathogenic ringworm fungi are divided into 2 major groups. The so-called "anthrophilic" dermatophytes are those for which human beings are the primary reservoir. These fungi are only very rarely isolated from domestic animals. The "zoophilic" dermatophytes are those for which domestic and wild animals are the major reservoirs. However, several of the zoophilic dermatophytes. In animals Dermatophytes consist of three important genera Microsporum, Trichophyton and Epidermophyton. In animals there are 3 specific fungi responsible for causing the infection

• Microsporum canis - The source of this species of Ringworm is almost always a cat, especially long-haired cats.
• Microsporum gypseum - This species of Ringworm is usually from dogs and cats that dig into contaminated soil.
• Trichophyton mentagrophytes - This species infects dogs and cats when they are exposed to rodents or the burrows they live in.

General characters:

1.     Morphology: All these genera possess a thread like, branching septate hypha forming the mycelium. The type of spores formed varies in parasitic phase and in artificial cultures. In parasitic phase, the septate hyphae forms number of asexual spores called arthrospore along the shaft of hair arranged as mosaic or in rows. These arhthrospores are called endothrix if they are arranged inside the hair shaft and called ectothrix if they are arranged out side the hair shaft. In artificial cultures they produce macroconidia and microconidia. Microconidia are small asexual spores of various shapes and sizes that occur alongside the hyphae. When they are arranged alongside the hyphae it is called en thyrse and when arranged in clusters or in long chain it is called en grappe. Macroconidia is also called fuseaux that are larger imperfect spores. Macroconidia are usually elongated, multiseptate, fusiform or spindle shaped structures with thick walls. The shape of macroconidia is of importance in diagnosing dermatophytes. The structure and shape of macroconidia of three important dermatophytes are given below. Beside the macroconidia and microconidia, the hyphae formed are also of diagnostic importance. They produce racquet hyphae, pectinate hyphae, spiral hyphae, nodular organs and chlamydospores. Racquet hyphae are composed of individual hyphal cells that are elongated at one end resembling tennis racquets. Pectinate hyphae possess irregular projections at one end of the hyphae resembling teeth of a comb or with irregular projections along one side of the hyphal elements that resemble stag-horns that is also called favic chandeliers.

a.      Microsporum: Elongated, multiseptate, fusiform or spindle shaped structures with thick walls which are some times wrinkled and wart-like.

b.      Trichophyton: Long, thin, multiseptate, smooth walled and cigar shaped.

c.      Epidermophyton: Oval or pear shaped with few septae.

2.     Classification: The ringworm group of fungi are classified into three types as already mentioned.

a.      Anthropophilics: These are pathogenic only for man. Some times thy may cause infection in animals. Eg. M.furrugineum.

b.      Zoophilics: These are pathogenic only for animals. Some times they may cause infection in man. Eg. M.canis, T.gallinae, T.equinum and T.mentagrophytes. 

c.      Geophilics: They are free-living dermatophytes in soil and cause infection in man and animals only in certain conditions.

3.     Habitat and distribution: They are found throughout the world basically as saprophytes.

4.     Risk to humanbeings: Zoonotic infection. Humanbeings get the infection from affected pet animals.

 

Cultural characters: The commonly used media for cultivation are Sabouraud’s dextrose agar or malt agar with chloramphenicol and cyclohexamide. These two substances suppress bacterial growth and saprophytic moulds. Addition of potassium tellurite will also reduce the bacterial growth. The clinical material are embedded in the agar and incubated between 22-28C for two to three weeks. The cultures can be examined every 3-4 days for growth. After growth is noticed, a small amount of growth may be transferred to a glass slide stained by lactophenol cotton blue will reveal the structure of hyphae and macro and microconidia. The type of growth and colour are also of importance. Trichophyton will produce orange colouration of the plate behind white cottony mycelial growth where as Epidermophyton will produce brown colour.

 

Pathogenesis:

Direct contact with infected animals is the common method of spread of the fungi. The infection can also spread from spore contaminated veterinary and farm equipment that have not been properly cleaned. Spores germinate and attack the shafts of the hair and the surface layers of the skin. Dermatophytes have an affinity for keratin-bearing tissues like skin, hair, feathers, nails and horns. They produce enzyme called keratinase that is capable of breaking keratin and keratin is used as source of nutriment. They do not involve the tissues beneath keratin layers or any other organs. The infection is restricted to stratum corneum and adjoining skin. The dermatophytes that affect hair parasitise superficial layers of newly keratinised cells above the root bulb. Exudate oozes from the damaged skin and mixes with debris from skin and hair, thereby forming a crusty scab. The scab is grey-white and noticeably higher than the surrounding skin. Infection spreads from the center outwards and results in the circular lesion 1 to 1.5 inches in diameter. Adjacent lesions may overlap and create larger infected areas. Lesions are most frequent on the head, tail and neck, but they may be found over the entire body in severe cases. Scabs may fall from older lesions and leave a hairless area in the center. Lesions appear as circular, scaly areas of alopecia with or without crust formation. The infection is also characterised by inflammatory and delayed hypersensitivity responses.

 

Symptoms and lesions:

1.     Cattle: The important species-affecting cattle are T.verrucosum var discoides, and T.mentagrophytes. The hair in the affected area falls out with large discrete, circular confluent raised scaly lesions appear on the head, neck and less frequently the back, flanks, escutcheon and limbs.

2.     Horses: The important species is T.equinum. Oval or irregular, focal inflamed oedematous lesions with loss of hair and formation of soft crust. The withers, saddle and girth regions are mostly affected.

3.     Sheep and goat: T.verrucosum is the common species. Generally the infection is rare in sheep and goat. The encrusted lesions are noticed in ears, horns, nose, tail, back and chest.

4.     Pigs: T.mentagrophytes is the common species. The less important species are T.verrucosum and M.nanum. The lesions appear as circular reddened areas on neck and trunk. These areas are covered with brown or reddish brown crusts.

5.     Dogs: M.canis, M.gypseum and T.mentagrophytes are the common species that affect dogs. The lesions are more commonly seen in head, base of he ear, abdomen and scrotal area. Encrusted scaly lesions are commonly seen. The skin lesions that appear with Ringworm are variable, and do not necessarily form a ring. There will be hair loss, usually in small patches at first. As time goes on the patches may disappear or appear at other locations on the skin. There might be scratching due to itchiness.

6.     Cats: M.canis is the most common species that affects cats. The lesions are well pronounced in long haired breeds. Typical skin lesions are discrete, roughly circular, areas of hair loss, particularly on the head, ears or extremities of the paws. The hairs surrounding affected areas appear broken. The affected skin is often scaly and look inflamed.

7.     Poultry: T.gallinae is the most common species. The infection is also called as favus. The lesions appear as thick white crusts on the comb, wattles and other featherless areas. The infection may also involve the base of the feathers called circular favus. In severe infection the entire head is covered with crusts, loss of feathers, emaciation and death.

 

Diagnosis:

1.     Based on symptoms and lesions.

2.     Examination under Wood’s light: In this method the affected hairs are viewed directly by UV rays. In Wood’s lamp, the light from a mercury vapour lamp is filtered through sodium-barium-silicate glass containing nickel oxide. The arthrospores that are found externally (ectothrix) on the shaft of the hairs produce bright greenish fluorescence in dark room under Wood’s lamp. However, the endothrix do not produce any fluorescence.

3.     Processing of hairs, nails and skin scrapings and examined in 10 or 20% KOH under a cover slip for arthrospores and hyphae. Before examination the materials should be warmed in KOH for 10 minutes and cooled for 30 minutes. Addition of 36% DMSO will enhance the clarity of observation. A drop of lactophenol cotton blue stain will also increase clarity.

4.     Cultural methods: Sabouraud’s dextrose agar or malt agar with chloramphenicol and cyclohexamide is commonly used. The clinical material are embedded in the agar and incubated between 22-28C for two to three weeks. The cultures can be examined every 3-4 days for growth. After growth is noticed, a small amount of growth may be transferred to a glass slide stained by lactophenol cotton blue will reveal the structure of hyphae and macro and microconidia. The type of growth and colour are also of importance. Trichophyton will produce orange colouration of the plate behind white cottony mycelial growth where as Epidermophyton will produce brown colour. Block slide culture and agar sausage method can also be used.

 

Treatment:

1.     Oral administration of griseofulvin is useful in treatment at a dose rate of 20-40mg/kg body weight.

2.     Topical application of salicylic acid, benzoic acid, sulphur containing compounds like ichthammol, tars, paraffin etc.

 

Dermatophytosis in humans:

Dermatophytosis in humans appear as circular patches of thickened, inflamed skin or hair loss with scaling. These may be itchy. Lesions may occur anywhere on the skin or scalp. Ringworm in humans usually responds well to treatment.

VMC 311 LECTURE # 28

 

PHYCOMYCETES

 

Important genus       :        Absidia – A. corymbifera, A.ramousa

Mucor – M.pusillus, M.spinosus, M.recimosus

Rhizops – R.suinus, R.microsporus, R.equinus

 

Important disease     :        Mucormycosis

 

General characters:

1.     Morphology: Phycomycetes are characterised by non-septate hyphae, asexual spores (sporangiospores) produced in thin walled sporangium and sexual zygospores and oospores.

2.     Classification:  Mucoraceae is one of the important families under the order zygomyctes that are characterised by zygospores, sporangia bearing clearly defined columella that includes Mucor, Rhizopus and Absidia. The important difference between them is Mucor does not possess root like rhizoids in the pseudomycelium, Rhizopus have rhizoids opposite to the base of aerial sporangiophores and in Absidia the rhizoids are found between the sporangiophores. The other order oomycetes include the genus Saprolegina that are characterised by formation of sexual spores (oospores) within an oosphere and elongated club shaped (clavate) sporangia opening by a single pore at the tip or side.

3.     Habitat: They are saprophytes.

4.     Reproduction: They reproduce by both sexual and asexual methods.

a.      Sexual method: Perfect spores are produced when the tips f two suitable adjacent hyphae come together and form short side branches called suspensors. Gametangium forms at this area. This gametangium enlarges and secretes a thick horny wall, which forms the resting spores or zygospores. Oospores are formed when male portion of hypha fuses with female portion. The structure that contains oospores is termed as oosphere.

b.      Asexual method: Phycomycetes produce long non-septate modified aerial filaments called sporangiophores which terminate in large thin walled structure called sporangium which contains asexual spores – the sporangiospores. Two types of sporangiospores are seen - the non-motile multinucleated aplanospores and motile zoospores.

 

Pathogenesis: The natural hosts are man and may species of domestic animals. Susceptible animals get the infection by ingesting or inhaling the spores. Debilitating conditions, over usage of corticosteroids, diabetes mellitus and leukaemia are some of the predisposing factors. The organs mostly affected are the lymph nodes, alimentary tract, respiratory tract etc. where they produce granulomatous lesions with ulcerative characters. In guinea pigs a pseudo tuberculosis type infection is produced. The genus Rhizopus is also associated with placental infection in cattle resulting in abortion. In poultry and sheep the infection is mostly limited to respiratory system. In dogs all the three-genus cause otitis. Placental infection is also noticed in swine.

 

Diagnosis: Generally the infection goes unnoticed without

proper exhibition of symptoms. Only during post mortem examination, histopathological examination of granulomatous lesions will reveal coenocytic hyphal elements. In aborted animals, histopathological examination of placental membranes and foetal stomach contents will reveal coenocytic hyphae.

 

Treatment: Not effective. Intravenous iodide therapy may be followed.

 

ASCOMYCETES (ASCOMYCOTA)

 

Important genus       -        Aspergillus

 

Important diseases    -        Brooder pneumonia, bovine mycotic abortion

 

Morphology:

The members of the genus ascomycetes are characterised by presence of a hyphae that is septate. The hyphae are well developed, profusely branched, and septate and their cells are multinucleate. They multiply by through sexual and asexual spores. They possess fruiting bodies called ascocarp (scletoium). This ascocarp contains number of sac like structure called ascus that is filled with sexual spores – ascospores. The structure of asexual spores is also significant. The hyphal cell that branches to give conidiophore is called foot cell. The conidiophores are long erect structures that terminate in bulbous structures called vesicle. The development from vesicle is of two types biseriate and monoseriate. In biseriate, a layer of cells called metulae cover the vesicle and from this metulae conidiogenous cells or phialides arise. In uniseriate the structure metulae are absent.  

 

Reproduction:

1.     Asexual: The conidiogenous cells mature and form conidia in basipetal chains. The conidium development is blastic. Each conidium develops as spherical wall protrusions from phialide tips. The phialide nuclei undergo mitotic division and one of the nuclei enters into developing conidium. A septum then forms between conidium and phialide cell and separates both. The next conidium develops beneath this and pushes the already developed conidium above. Due to physical connection between each conidia they remain in chain for some time.

2.     Sexual: The sex organs antheridia and ascogonia are produced close to each other on somatic hyphae. Both are multinucleate and elongate structures often coil around each other. Pairing of nuclei in ascogonium takes place irrespective of the entry of antheridia nuclei. However, if antheridial nuclei enter into ascogonia pairing takes place between antheridial and ascogonial nuclei and produce number of ascogenous hyphae that branch within the developing ascocarp. The asci are formed at the tips of such hyphae at different levels.

 

Habitat:

Most of the members of this group are saprophytes and non pathogenic. They are the common laboratory contaminants. Few genera like Aspergillus, Claviceps contain pathogenic species. They cause respiratory infection or food poisoning.

 

Cultural characters:

Members of this group are aerobic and grow well in ordinary laboratory media at an incubation temperature of 25-37C. Blood agar and Sabouraud’s agar, potato dextrose agar are commonly used to study these fungi. The colonies develop very fast that appear first as white, fluffy, low filamentous growth later turning into granular and spreads across the media. The colour of the colonies changes into green. This green colouration is due to conidia.

 

Pathogenesis:

There are more than 200 species of the genus Aspergillus and it is difficult to differentiate between them. Hence the infections produced by this genus are collectively called as Aspergillosis. The spores of the genus are commonly found in dust, soil, rotting plants and animal matters. The spores are abundantly found on hay, straw and grain. The settling of spores on these feed occurs during storage. When these spores are inhaled or ingested they settle in lungs or other organs and produce infection. Apart from the lung infection that is very common, Aspergillus is also associated with lesions on skin, external ear, nasal sinuses and occasionally bones and meninges. They also cause placental infection in cattle resulting in abortion. In horses, it causes equine guttural pouch mycosis. Of all the infections produced by Aspergillus, avian aspergillosis (brooder pneumonia) caused by Aspergillus fumigatus is most important.

 

Symptoms and lesions:

1.     Avian aspergillosis (Brooder pneumonia): The affected chicks die suddenly within 24-48 hours without any symptoms. The symptoms if present are generally anorexia, listlessness, high temperature (pyrexia), increased respirations, diarrhoea and convulsions. The clinical symptoms and post mortem lesions are indistinguishable from pullorum disease. In post mortem the affected birds show number of small, discrete, military type, yellow white granulomata scattered throughout the lung. Small firm nodules are seen in air sacs. The air sacs consist of masses of densely packed hyphae and mycelium. In chronic cases, the fungus grows in the mucous membranes of bronchi and air sac filling them with dense dark green fluffy mass of hyphae and spores. Some times caseous exudation is also noticed.

2.     Bovine mycotic abortion: Along with Mucor, Absidia, Rhizopus and Penicillium, Aspergillus fumigatus also cause abortion in cattle. This infection is characterised by placentitis. There is gross thickening and focal necrosis of affected cotyledons. Abortion usually occurs during second half of pregnancy. Ingestion and inhalation of spores through contaminated feed are the important modes of spread of infection. Infection generally occurs during winter months due to confinement. Affected animals do not show any symptoms. The fungus does not remain in uterus after infection.   

 

Diagnosis:

1.      Diagnosis of aspergillosis based on symptoms and lesions alone is very difficult. Demonstration of fungal hyphae by lactophenol cotton blue in affected tissue is a easy and reliable method. However it is difficult to arrive at the species based on either cultural or microscopical methods.

2.      Diagnostic tests like complement fixation test and AGID are done to identify the fungal antigens or antibodies in the serum

 

Other miscellaneous infections:

1.      Equine guttural pouch mycosis: This infection is characterised by epistaxis or pharyngeal paralysis.

2.      Green mould of eggs.

3.      Small encapsulated lesion of cattle.

VMC 311 LECTURE # 28

OCTOBER 10, 2000

 

FUNGI IMPERFECTI

 

This group consists of fungi that do not have sexual stage of development. This groups consists of fungi ranging from yeast and yeast like organisms to dimorphic fungi and dermatophytes. Some of the common pathogenic organisms are Cryptococcus sp., Candida sp. (yeast and yeast like organisms), Blastomyces, Histoplasma, Sporotrichum and Coccidiodes (Dimorphic fungi) and Microsporum, Trichophyton, Epidermophyton (Dermatophytes).

 

Yeast and yeast like organisms:   

 

Morphology: Yeasts are a large group of organisms with three groups based on their mode or replication. Generally yeasts are unicellular that reproduce vegetatively by budding. Generally the yeast cell is haploid except the zygote that is diploid. Certain yeasts produce a series of buds in succession that remain joined for a period or time forming a pseudo-mycelium. However, some yeast does produce true mycelium. The hypha of yeasts is septate with single or many pores in the septal wall (perforate septum). The septal wall grows centripetally and almost closes the pores forming a closure line. They also have an electron dense structure called Woronian bodies found in the hypha near the septal wall.

 

Reproduction: Budding and formation of conidia and arthrospore are the usual methods of asexual reproduction. Formation of bud is initiated at the time of duplication of spindle pole body. An area of enzymatic wall softening within a newly developed chitinous ring allows a portion of the cell contents along with newly synthesised wall to come out through the ring. At this stage a plate like structure called abscission plate begins to form at the isthmus between the two cells. Cell wall materials are deposited on either side of the plate and thus the mother and daughter cells are separated. A bud from the mother usually leaves a scar on the surface of the mother cell that resembles like a crater. The sexual reproduction of yeasts is through production of ascospores. These ascospores are found inside ascus, which in turn are located inside the ascocarp. The ascospores otherwise called as ascus are multishaped. The process of formation of new yeast from ascus is called as ascosporogenesis.

 

Classification:

1.      Certain yeasts however, form spores and the spore that develops from mother cell is called blastospore. Some species of yeast produce pseduo-mycelium or mycelium that forms arthrospore and rarely into chlamydospore.  However, certain yeasts reproduce sexually by forming ascospore.

2.      Certain yeasts produce aerial spores called ballistospores that forcibly project into the air.

3.      The third group consists of yeasts that do not produce ascospores or ballistospores and reproduce exclusively by vegetative process. This group of fungi is called as fungi imperfecti. The family Cryptococcaceae that contains many pathogenic types of yeast come under this group. The further classification of this family into genera is based on the morphology of asexual spore, presence or absence of pseudo-mycelium, fermentation of sugars etc.

 

Habitat: The yeasts in general are saprophytes.

 

Yeast infections:

1. Cryptococcosis

 

a.      This infection is otherwise called as European blastomycosis or torulosis. The causative organism is Cryptococcus neoformans.

b.      Morphology: The fungi appear as spherical, thick walled, single budding yeast surrounded by a wide gelatinous transparent capsule.

c.      Habitat: Generally they are found in the soil, fruit, milk etc. The main source of this fungus is pigeon droppings. In pigeons this fungi is found in the intestine but does not multiply due to higher body temperature of pigeons.

d.      Cultural characters: They grow well in Sabouraud’s dextrose agar, malt agar, blood agar and other laboratory media. The young cultures appear as white or cream-coloured later turning in to pale brown coloured. This fungus lacks the ability to ferment sugars and is urea positive.

e.      Pathogenesis:  It is a non-contagious, chronic, fatal systemic infection that involves lungs, skin or other parts of the body. This fungus has got marked predilection for brain and meninges. The hosts affected by this infection are cattle, pigs, dogs, man, monkey, dog etc.  The infection initially starts as lung infection followed by infection of other organs particularly brain and meninges. The fungus spreads via blood stream. The infection may take a long time even years to set in. In horses, it causes myxoma like lung infection with respiratory distress and nasal discharge. In cattle, it causes mastitis. The udder becomes swollen and hard coupled with enlargement of regional lymph nodes and drop in milk yield. In dogs, it causes CNS infection resulting in incoordination, hyperaesthesia and nasal discharge. Granulomatous lesions are also noticed in lungs, various organs and areas around ears, face and feat. Extension of infection from nasal cavity to optic nerve results in blindness.

f.       Diagnosis:

                                                              i.      Direct microscopical examination on specimens stained by Giemsa stain or India ink reveals spherical, thick walled, single budding yeast surrounded by a wide gelatinous transparent capsule.

                                                             ii.      The fungus can also be viewed by immunofluorescense.

                                                           iii.      Isolation of fungi by culturing: This fungi grow well in Sabouraud’s dextrose agar, malt agar, blood agar and other laboratory media. The young cultures appear as white or cream-coloured later turning in to pale brown coloured.

                                                          iv.      Biochemical reaction: This fungus do not sugars and is urea positive.

                                                            v.      Animal inoculation: This is the only fungus under the family Cryptococcaceae that can grow in mice. Mice are infected by intra-peritoneal or intra-cerebral route. Fungus can be seen as budding cells in abdominal tissues, lungs and brain. This fungus also produces fatal infection in pigeons and chick embryos by intra-cerebral or intra-venous route.

g.      Treatment: Generally the treatment is ineffective. However, amphotericin B is found to be effective.

LECTURE – MIB221 – 11 A

OCTOBER 17, 2000

 

FUNGI IMPERFECTI – II

(Continuation)

 

2. Pityrosporum

 

The important species and type species of this genus under the family Cryptococcacea is Pityrosporum ovale. It is also called as bottle bacilli. The organisms are gram-positive, oval or non-mycelial asporogenous yeasts without fermentative ability. It is associated with dandruff scales and is also associated with sebhorric dermatitis and psoriasis. The classification of this genus is based on cell shape that varies from oval to egg-shaped to spherical and budding which may occur either at the broad base end of the cell or at narrow end. Of the four important species P.ovale, P.orbiculare, P.pachydermatis and P.canis the later two are pathogens of rhinoceros and canines respectively. P.canis is oval or flask shaped with double contoured. Budding cells are approximately 2-3 x 4-8 µm in diameter. The blastospore develops at one of the cell. Many yeast cells develop as lateral projections from mature blastospore resembling as protoplasmic collars.

 

Cultivation: They can be cultivated in malt agar, blood agar and variety of other media. The colonies appear within 48 hours of incubation at 37^oC. The colonies are small, circular, doomed, buff and white in colour. Later on drying these colonies become brittle and appear as frosted glass and dimpled appearance-resembling skin of an orange. In fluid media the growth settles at the bottom. All the species hydrolyse urea. They vary in requirement of oleic acid and olive oil as growth factors. While P.ovale and P.orbiculare require these substance P.pachdermatis and P.canis do not require these. Cultivation in mice, guinea pig and rabbits are less significant.

 

Candida

 

The genus Candida is also known as Monilia. It is classified under the order Moniliales. The important species under this genus are C.albicans, C.parapsilosis, C.tropicalis, C.krusei and C.stellatoidea.

 

1.      Habitat: The Candida organisms are found as saprophytes in mucous membranes of digestive, uro-genital and respiratory systems.

2.      Morphology: The budding cells appear as gram-positive. They are characterised by formation of pseudohyphae. The yeast cells repeatedly divide and form a chain like structure called pseudohyphae. Each cell serves as spore. The species C.albicans is characterised by the production of thick walled chlamydospores (resting spores).

3.      Cultural characters: The Candida species readily grow in bacteriological media at 25-37^oC producing smooth, creamy bacterial like surface colonies. The pseudohyphae formed extended into the medium. This can be well demonstrated in agar slope culture. These colonies become sub-surfaced and furrowed after prolonged incubation. The commonly used media are malt agar or blood agar or Raulin’s media with or without antibiotics. The resting spores – chlamydospores are well formed in corn-meal agar. The organisms produce severe kidney, liver and spleen lesions in rabbits and mice by intravenous route. This organisms ferment glucose, sucrose, maltose and galactose and produce acid and gas.

4.      Pathogenesis: The infection caused by Candida organisms are called and candidiasis or moniliasis. C.albicans is the most important species. The infection caused by C.albicans may be acute, subacute or chronic. The organisms are generally found as saprophytes and are non-infectious. However, the produce infection in hosts when the host is having malignant diseases or nutritional deficiency. Candida infection is common in individuals undergoing prolonged antibiotic therapy. Candida infection occurs as cutaneous and on mucous membranes. The infection in mucous membranes is referred as thrush. In human beings, the mucous membranes oral cavity and vagina are commonly affected. Oral thrush occurs in babies and patients at the terminal stages of wasting diseases. The infection in poultry is called as avian candidiasis or moniliasis or thrush. Candidiasis in poultry occurs in young birds and acute infection is characterised by rapid death and high mortality. Characteristic lesions develop in the inner lining or crop. In acute cases, the lesions appear as tiny, discrete, yellow-whine or grey-white pustules loosely adherent to the mucous membrane. In chronic infections, the crop wall is thickened and covered by a corrugated false membrane of yellowish-grey necrotic material resembling Turkish towelling. Necrotic exudate may be seen in the gizzard and intestines with miliary abscesses in lung and liver. C.albicans cause stomatitis and gastric ulcers in pigs, mastitis and abortion in cattle and otitis in dogs.

5.      Diagnosis:

a.      Based on symptoms and lesions.

b.      Microscopical examination culture stained by Giemsa or Gram’s stain.

c.      Isolation of C.albicans by culturing.

d.      Isolation in rabbits or mice.

6.      Treatment: No satisfactory treatment is available. However, nystatin in drinking water, amphotericin B and iodides may give some results.

 

 

   LECTURE – MIB221 – 12

OCTOBER 13, 2000

 

PATHOGENIC DIMORPHIC FUNGI

 

Dimorphic fungi are characterised by two forms that they exhibit during their lifetime. At 37^oC they exhibit yeast like growth and at 20^oC they exhibit mycelial growth. The parasitic phase is characterised by absence of mycelial growth and in tissues they appear like yeast and multiply by budding. Generally they appear as yeast like cells in infected tissues and mycelial colonies in artificial media. Most of parasitic fungi that cause infections in human beings and animals belong to this category. There are four important groups under this category. They are Histoplasma, Blastomyces, Coccidiodes and Sporotrichum.

 

A. Histoplasmosis:

Histoplasmosis is the general term given for infection caused by species of the genus Histoplasma. The two important species of Histoplasma genus are H.capsulatum and H.farciminosum. H.capsulatum causes fatal granulomatous infection of lung and lymphatic system mostly in man and canines. H.farcimonosum causes epizootic lymphangitis or pseudo glanders or Japanese farcy in equines, which is a chronic infection of lymph glands and lymph nodes.

 

H.capsulatum: This species is worldwide in distribution and occurs sporadically in many countries. It causes fatal progressive granulomatous disease of reticuloendothelial system or a mild asymptomatic infection of the lung. The infection is non-contagious and spreads through inhalation of the infected dust particles. The species of animals affected are cattle, horses, cats, dogs and rodents. Generally the infection is asymptomatic and diagnosed after post-mortem. In dogs the infection is characterised by chronic cough and dysentery.

 

Morphology: The yeast bodies appear as small, capsulated oval cells that are usually found in macrophages. Budding is not a common feature of these fungi and rarely they are found out side the cell. In artificial cultures, the mycelial growth consists of fine cottony aerial hyphae bearing lateral conidia. The chlamydospores are thick walled bearing spiny projections from outside.

 

Diagnosis

1.      Identification of non-budding yeast like cells in the tissues of reticuloendothelial system

2.      Demonstration of conversion to mycelial phase at 22oC in Sabouraud’s dextrose agar and reconversion of mycelial phase to yeast like colonies at glucose blood agar. The mycelial colonies appear as white coloured that later changes into tan coloured. Presence of aerial hyphae, lateral conidia and horny chalamydospores are or diagnostic importance. The yeast like colonies appears as wrinkled and creamy. Budding which is not seen in tissues during parasitic phase is common in artificial media.

3.      Demonstration of yeast phase in mice through intraperitoneal inoculation.

4.      Demonstration of yeast phase in HeLa cells.

5.      An intradermal histoplamin test is done in dogs to identity canine histoplasmosis.

Treatment:    Not successful. Amphotericin B at 0.1% intravenously may provide some relief.

 

H.farciminosum: This infection (Epizootic lymphangitis) is more common in the horse breeding areas of Asia, Africa and Europe. It is chronic infection characterised by granulomata and suppurating ulcers on the skin with associated lymph glands and lymphatics. It is also known as Pseudo glanders, and Japanese farcy. Horses, mules and donkeys are the normal host. However, man, pigs and camels are also affected by the infection. The important modes of spread are through direct contact, through contaminated utensils, saddlery, grooming utensils (fomites) or through flies particularly biting flies. Ingestion and inhalation are other important modes of transport.

 

The infection is characterised by pus containing lesions on the exposed surface of skin, associated lymphatic vessels particularly limbs and neck. The infected lymphatics become dilated at intervals forming lines of hard abscesses. This is called cording. Cording is more common in limbs and neck. Later, this abscesses rupture-releasing blood stained pus. The granulating ulcers have a concave, bright red, glistening surface. A scab forms over the ulcer, which later sloughs off leaving a scar. The mucous membranes of lungs, tracheal and nasal cavity are affected. There is also a watery discharge from the nostrils and eyes. Conjunctivitis and papules on the conjunctiva and nictitating membrane are the other signs.

 

          Diagnosis:

1.      Demonstration of thin walled spherical, oval or pear shaped yeast like bodies in pus form unopened abscess or from nasal or conjunctival discharges.

2.      Isolation of the causative organism in Sabouraud’s agar or Harley digest agar containing 10% horse serum (pH 7.4). The colonies appear as tiny grey flakes after 2-8 weeks of incubation.

3.      Demonstration of antibodies against the infection by complement fixation test (CFT).

4.      Allergic intradermal test.

5.      Differential diagnosis with glanders: Mallein test, isolation of causative organisms and their cultural characters will differentiate between glanders and psudoglanders.

 

Treatment:

1.      Disinfection and cauterisation of the open lesion and removal of affected lymphatic cords.

2.      Intravenous iodide therapy

 

Control:

1.      Isolation and slaughter of the affected animal.

2.      Use of hygienic saddlery, grooming equipment, other utensils etc.

3.      Installation of fly proof stalls.

4.      Avoiding saddle galls and other injuries to horses.

 

B. Blastomycosis: Blastomycosis is a chronic non-contagious infection of dogs and horses caused by Blastomyces dermatitidis. It is characterised by formation of suppurative and granulomatous lesion in any part of the body. The fungus multiplies in lungs, skins and bones.

 

Morphology: The yeast like cells appear as thick walled with an attached blastospore. They are found inside the phagocytic cells. The mycelial phase consists of septate aerial hyphae with large number of spherical or pear shaped conidia and many smooth walled chlamydospores.

 

Pathogenesis: Canine blastomycosis is a chronic, debilitating and highly fatal disease associated extensive pulmonary lesions. Nodules are also commonly seen in dogs. The chest cavity is filled with nodules and the lungs are highly congested. Bronchial and mediastinal lymph nodes show enlargement. In horses, the infection is characterised by series of long standing abscesses in the anus and vulva associated with debility. In cattle the infection is characterised by mastitis.

 

Diagnosis:

1.     Based on symptoms and lesions.

2.     Isolation of fungi and demonstration of yeast phase with wrinkled, creamy or waxy colonies on blood agar at 37oC. The mycelial phase in Sabouraud’s agar at 22oC exhibit fluffy white mycelia that later turns into tan or brown. The mycelial phase consists of septate aerial hyphae with large number of spherical or pear shaped conidia and many smooth walled chlamydospores.

3.     Radiographic examination.

 

Treatment: Not very effective. Iodide therapy or intravenous amphotericin B provides some relief.

LECTURE – MIB221 – 12 A

OCTOBER 24, 2000

 

PATHOGENIC DIMORPHIC FUNGI – II

 

C. Coccidioidomycosis (Coccidiodes immites): It is an extremely infectious but non-contagious disease of man, monkeys, horses, cattle, dogs, sheep, rodents etc. The infection occurs as an acute, benign and self-limiting disease of pulmonary system. It may also occur as chronic infection involving cutaneous, visceral and osseous tissues.

 

Morphology: The fungus appears in several phases in infected tissues. They appear as spherical thick walled bodies called spherules that contain large number of irregularly shaped endospores. These endospores are released into the surroundings. Hyphal elements develop from the wall of spherules. The hyphal segments also break and forms arthrospore.

 

Habitat: These fungi are generally found in the soil and inhalation of the dust particles is the important source of spread among hosts.

 

Cultural characters: The media commonly used are Sabouraud’s dextrose agar and Littman Oxgall agar. The growth initially appears as white surface colonies, moist and membranous. Later, these surface colonies turn into cottony mycelial growth. Though the fungus is a dimorphic fungus, no differences are found between colony characteristics at 22C and 37C. The arthrospore formation is very conspicuous in artificial culture. Hence, the cultures should be handled very carefully.

 

Pathogenesis: The organisms are commonly found in the soil and inhalation of the dust particles is the most important route of spread. The arthrospores are also introduced through the skin as a result of injury. The incidence is more during dry and dusty months. The infection remains sub clinical with automatic recovery. In dogs the infection remains for a period between 2 and 5 months and is extensive than man and cattle. The infection is characterised by granulomata of lungs, pleura, heart, spleen, liver, kidneys, bones and joints. However, granulomatous lesions are mostly restricted to bronchial and mediastinal lymph nodes and occasionally lungs.

 

Diagnosis:

1.     Based on symptoms and lesions.

2.     Examination of sputa, biopsy materials as wet mounts stained by lactophenol cotton blue reveals the spherules containing endospores and broken hyphal elements (arthrospore).

3.     Isolation of the fungi is Sabouraud’s dextrose agar and Littman oxgall agar. The growth initially appears as white surface colonies, moist and membranous. Later, these surface colonies turn into cottony mycelial growth.

4.     Hypersensitivity test (Coccid Odin skin test): 0.1 ml of the extract of fungus (coccid Odin) when given intradermally produce erythematous lesion at the site of injection in patients having coccidiodes infection after 20-48 hours.

5.     Demonstration of complement fixing antibodies through complement fixing antibodies.

6.     Other serological test like agar gel immuno diffusion (AGID) and immunofluorescense.

7.     Inoculation of the suspected material into mice through intraperitoneal injection produces the specific granulomatous lesions in mice after 1-2 weeks.

 

Treatment: Intravenous amphoterecin B therapy is useful.

 

D. Sporotrichosis (Sporotrichum schenekii): It is a non-contagious granulomatous infection of skin, lymphatics and less frequently the internal organs of man and animals.

 

Morphology: They are gram-positive, spindle or cigar shaped yeast like bodies. They possess septate hyphae bearing clusters of pear-shaped condia radiating from tips of unbranched conidiophores. The yeast phase is prominent at 37C and mycelial phase is prominent at 22C.

 

Habitat: These fungi are saprophytes that grow on decaying plants, wood and in the soil.

 

Cultural characters: These fungi can be cultivated in Sabouraud’s dextrose agar, blood agar and brain heart infusion agar. The colonies at 37C are moist, flat and leathery cream coloured colonies containing budding yeast cells. At 22-28C cottony mycelial growth is observed.

 

Pathogenesis: These fungi are saprophytes that grow on decaying plants, wood and in the soil. The infection is more common among gardeners and agricultural workers as a result of entry of the fungi through cut wounds at the extremities caused by the splinters and thorns of plants. The infection is also found in dogs, horses, donkeys, mules, cattle, camels and poultry. The infection is characterised by small nodule at the site of injury that gradually increases in size becomes indurate and breaks to form inactive ulcers. Since the infection spreads via lymphatics, the lymphatics become thickened and cord like. Multiple nodules or abscesses develop throughout the course of lymphatic ducts, which may rupture and ulcerate. The nodules formed initially may also turn into a hairless area releasing exudates and form a scab. In horses, the condition resembles pseudo glanders. In dogs the infection also involves liver and lungs.

 

Diagnosis:

 

1.     Based on symptoms and lesions.

2.     Isolation of the organism in blood agar, Sabouraud’s dextrose agar or in brain heart infusion agar. The colonies at 37C are moist, flat and leathery cream coloured colonies containing budding yeast cells. At 22-28C cottony myceial growth is observed.

3.     Inoculation into mice intraperitoneally produces peritonitis and intratesticularly produces orchitis. The oval or cigar shaped yeast bodies can be demonstrated in macrophages.

 

Treatment: Iodide therapy produce effective results. Oral administration of potassium iodide and intravenous administration of sodium iodide produce effective results.

 

Rhinosporidiosis (Rhinosporidium seeberi): It is a chronic non-fatal mycotic infection of man and animals. It is primarily disease of fishes with man and animals are accidental hosts. The infection is characterised by formation of polyps in the mucous membranes of eye, nose, skin, ears, larynx and genital organs. The polyps are pedunculate or sessile, pink in colour with white spots of sporangia that may contain endospores. They are soft to touch and bleed readily. The sporangia are much larger than that or Coccidiodes spp. The organisms are difficult to cultivate in the laboratory and exact mechanism of pathogenesis is also not known. In man, antimony compounds together with surgical excision or cautery is successful.

 

 

 

 

 

LECTURE – MIB221 – 13

OCTOBER 20, 2000

DERMATOPHYTES (DEMATOMYCOSIS)

 

The infection produced by dermatophytes is called ringworm. The term ringworm denotes a clinical entity than an infection produced by specific dermatophyte. Dermatophytes consist of three important genera Microsporum, Trichophyton and Epidermophyton. Of theses three genera, Epidermophyton usually does not produce any infection in human beings.

 

General characters:

5.     Morphology: All these genera possess a thread like, branching septate hypha forming the mycelium. The type of spores formed varies in parasitic phase and in artificial cultures. In parasitic phase, the septate hyphae forms number of asexual spores called arthrospore along the shaft of hair arranged as mosaic or in rows. These arhthrospores are called endothrix if they are arranged inside the hair shaft and called ectothrix if they are arranged out side the hair shaft. In artificial cultures they produce macroconidia and microconidia. Microconidia are small asexual spores of various shapes and sizes that occur alongside the hyphae. When they are arranged alongside the hyphae it is called en thyrse and when arranged in clusters or in long chain it is called en grappe. Macroconidia is also called fuseaux that are larger imperfect spores. Macroconidia are usually elongated, multiseptate, fusiform or spindle shaped structures with thick walls. The shape of macroconidia is of importance in diagnosing dermatophytes. The structure and shape of macroconidia of three important dermatophytes are given below. Beside the macroconidia and microconidia, the hyphae formed are also of diagnostic importance. They produce racquet hyphae, pectinate hyphae, spiral hyphae, nodular organs and chlamydospores. Racquet hyphae are composed of individual hyphal cells that are elongated at one end resembling tennis racquets. Pectinate hyphae possess irregular projections at one end of the hyphae resembling teeth of a comb or with irregular projections along one side of the hyphal elements that resemble stag-horns that is also called favic chandeliers.

a.      Microsporum: Elongated, multiseptate, fusiform or spindle shaped structures with thick walls which are some times wrinkled and wart-like.

b.      Trichophyton: Long, thin, multiseptate, smooth walled and cigar shaped.

c.      Epidermophyton: Oval or pear shaped with few septae.

6.     Classification: The ringworm group of fungi are classified into three types.

a.      Anthropophilics: These are pathogenic only for man. Some times thy may cause infection in animals. Eg. M.furrugineum.

b.      Zoophilics: These are pathogenic only for animals. Some times they may cause infection in man. Eg. M.canis, T.gallinae, T.equinum and T.mentagrophytes. 

c.      Geophilics: They are free-living dermatophytes in soil and cause infection in man and animals only in certain conditions.

7.     Habitat and distribution: They are found throughout the world basically as saprophytes.

 

Cultural characters: The commonly used media for cultivation are Sabouraud’s dextrose agar or malt agar with chloramphenicol and cyclohexamide. These two substances suppress bacterial growth and saprophytic moulds. Addition of potassium tellurite will also reduce the bacterial growth. The clinical material are embedded in the agar and incubated between 22-28C for two to three weeks. The cultures can be examined every 3-4 days for growth. After growth is noticed, a small amount of growth may be transferred to a glass slide stained by lactophenol cotton blue will reveal the structure of hyphae and macro and microconidia. The type of growth and colour are also of importance. Trichophyton will produce orange colouration of the plate behind white cottony mycelial growth where as Epidermophyton will produce brown colour.

 

Pathogenesis: Dermatophytes have an affinity for keratin-bearing tissues like skin, hair, feathers, nails and horns. They produce enzyme called keratinase that is capable of breaking keratin and keratin is used as source of nutriment. They do not involve the tissues beneath keratin layers or any other organs. The infection is restricted to stratum corneum and adjoining skin. The dermatophytes that affect hair parasitise superficial layers of newly keratinised cells above the root bulb. The infection spread from between host either by direct contact or through contaminated materials. In animals, the lesions generally produced by dermatophytes are referred as “ringworm” and Epidermophyton spp seldom cause infection in animals. The lesions in domestic animals are characterised by circular, scaly areas of alopecia with or without crust formation. Head and tail are the most important areas where the lesions are noticed in horses and cattle. In dogs and cats the lesions are mainly noticed in extremities. The infection is characterised by inflammatory and delayed hypersensitivity responses.

 

Symptoms and lesions:

8.     Cattle: The important species-affecting cattle are T.verrucosum var discoides, and T.mentagrophytes. The hair in the affected area falls out with large discrete, circular confluent raised scaly lesions appear on the head, neck and less frequently the back, flanks, escutcheon and limbs.

9.     Horses: The important species is T.equinum. Oval or irregular, focal inflamed oedematous lesions with loss of hair and formation of soft crust. The withers, saddle and girth regions are mostly affected.

10. Sheep and goat: T.verrucosum is the common species. Generally the infection is rare in sheep and goat. The encrusted lesions are noticed in ears, horns, nose, tail, back and chest.

11. Pigs: T.mentagrophytes is the common species. The less important species are T.verrucosum and M.nanum. The lesions appear as circular reddened areas on neck and trunk. These areas are covered with brown or reddish brown crusts.

12. Dogs: M.canis, M.gypseum and T.mentagrophytes are the common species that affect dogs. The lesions are more commonly seen in head, base of he ear, abdomen and scrotal area. Encrusted scaly lesions are commonly seen.

13. Poultry: T.gallinae is the most common species. The infection is also called as favus. The lesions appear as thick white crusts on the comb, wattles and other featherless areas. The infection may also involve the base of the feathers called circular favus. In severe infection the entire head is covered with crusts, loss of feathers, emaciation and death.

 

Diagnosis:

5.     Based on symptoms and lesions.

6.     Examination under Wood’s light: In this method the affected hairs are viewed directly by UV rays. In Wood’s lamp, the light from a mercury vapour lamp is filtered through sodium-barium-silicate glass containing nickel oxide. The arthrospores that are found externally (ectothrix) on the shaft of the hairs produce bright greenish fluorescence in dark room under Wood’s lamp. However, the endothrix do not produce any fluorescence.

7.     Processing of hairs, nails and skin scrapings and examined in 10 or 20% KOH under a cover slip for arthrospores and hyphae. Before examination the materials should be warmed in KOH for 10 minutes and cooled for 30 minutes. Addition of 36% DMSO will enhance the clarity of observation. A drop of lactophenol cotton blue stain will also increase clarity.

8.     Cultural methods: Sabouraud’s dextrose agar or malt agar with chloramphenicol and cyclohexamide is commonly used. The clinical material are embedded in the agar and incubated between 22-28C for two to three weeks. The cultures can be examined every 3-4 days for growth. After growth is noticed, a small amount of growth may be transferred to a glass slide stained by lactophenol cotton blue will reveal the structure of hyphae and macro and microconidia. The type of growth and colour are also of importance. Trichophyton will produce orange colouration of the plate behind white cottony mycelial growth where as Epidermophyton will produce brown colour. Block slide culture and agar sausage method can also be used.

 

Treatment:

3.     Oral administration of griseofulvin is useful in treatment at a dose rate of 20-40mg/kg body weight.

4.     Topical application of salicylic acid, benzoic acid, sulphur containing compounds like ichthammol, tars, paraffin etc.

LECTURE – MIB221 – 14

OCTOBER 23, 2000

MYCOTOXICOSIS

 

DEFINITION: Certain strains of fungi (moulds) growing in feed or feed ingredients can produce toxins that, when eaten by man or animals, can cause a very lethal disease called mycotoxicosis. The fungal metabolites that cause mycotoxicosis are called as mycotoxins. These toxins can accumulate in maturing corn, cereals, soybeans, sorghum, peanuts, and other food and feed crops in the field and in grain during transportation. The toxins may occur in storage under conditions favourable for the growth of the toxin-producing fungus or fungi.

 

1. AFLATOXICOSIS: The most important mycotoxicosis is aflatoxicosis. Aspergillus flavus is a common mould that grows on many substances, and grows especially well on grain and nuts. The other important fungus is A.parasiticus. The toxin produced by these fungi causes aflatoxicosis in animals and poultry. The condition occurs worldwide. All species of animals are susceptible, especially younger poultry, swine and calves; adult animals are more resistant to the acute form of the disease. The infection was first called as Turkey X disease since it was first noticed in young turkeys. The aflatoxins include four closely related metabolites of A. flavus known as B₁, B₂, G and G₂. The B₁ toxin is the most toxic and is of greatest concern to the poultry industry. Aflatoxins M_l and M₂ are found in milk from animals fed aflatoxin-contaminated feeds. Infection is most common after the kernels have been damaged by insects, birds, mites, hail, early frost, heat and drought stress, windstorms, and other unfavourable weather.

All animal species are susceptible to aflatoxicosis as mentioned earlier, although sensitivity varies considerably from species to species. For example, birds, fish, dogs, and swine appear to be more susceptible than mature cattle.

1.      In poultry, the aflatoxins under certain conditions cause death, reduced growth, reduced egg production, reduced hatchability, signs associated with "physiological stress" and impaired ability to develop immunity to infectious agents.  Besides fatty liver and kidney disorders, leg and bone problems can develop as well as outbreaks of coccidiosis. Aflatoxins may cause vaccines to fail, increase the birds' susceptibility to disease, and result in suppression of the natural immunity to infection. The birds become susceptible to infection by bacteria such as Salmonella and to various viruses and other infectious agents commonly found around the farmyard, feedlot, or poultry house. Decreased blood clotting results in a greater downgrading and condemnation of the birds because of massive bleeding and bruises. Less carcass pigmentation is exhibited and egg yolks are paler. The hatchability of eggs can drop, and reduced production may be noted as well as smaller eggs with shell problems. Growth is restricted and mortality increases, especially during the growing period.

2.      In animals there is decreased feed consumption, poor feed conversion, stunting, and decreased flesh growth. Decreased productivity may be accompanied by damage to the liver, haemorrhaging into the muscles or body cavities, and suppression of natural immunity to parasites and pathogens always present in the environment. Once the damage has been done, the animals will not fully recover, even if returned to a toxin-free ration.

3.      In human beings it causes “Farmer's lung" - a disease that affects grain handlers and is frequently associated with skin irritation, fever, wheezing, breathlessness, cough, and ulcers. Farmer's lung is thought to be caused by an allergic reaction to fungal spores and other material in grain dust.

In both animals and birds, the liver is the principal organ affected; high doses of aflatoxins cause severe cellular necrosis. Prolonged low-level exposure can result in liver enlargement and slow growth

DIAGNOSIS OF AFLATOXIN:

a.                           Visual inspection of the grain that may locate lots presumed to be contaminated with aflatoxin (blacklight test)

b.                           Rapid screening procedures to determine the presence or absence of aflatoxin that include the fluorometric iodine rapid screening and minicolumn tests;

c.                           Laboratory procedures to find out the actual amounts of toxin present (thin-layer chromatography, gas-liquid chromatography, high-pressure liquid chromatography, fluorometric iodine, or ELISA tests).

d.                           Traditional method of identification of fluid cultures with UV light and ascertain by the fluorescence emitted.

 

2. DEOXYNIVALENOL (VOMITOXIN): This toxin is produced by the fungus Fusarium graminearum. Corn is the most important feed ingredient that contains this toxin. Pigs are mainly affected by this toxin. Clinical signs and lesions in affected swine included feed refusal, a few instances of vomiting, lack of weight gain, poor feed efficiency, failure of mature sows to return to oestrus, reduced efficiency, high mortality of nursing pigs, intestinal tract inflammation, and acute diarrhoea in young pigs. Autopsies of young pigs revealed haemorrhaging into the abdominal cavities and pale, friable livers. The infection is not very common in cattle and poultry.

 

3. FUMONISIN - BLIND STAGGERS IN HORSES: Blind staggers (equine leucoencephalomalacia) occasionally occurs in horses, mules, or donkeys foraging corn left standing in the field after harvest or fed grain or screenings heavily infected with F. moniliforme. The toxins fumonisin B₁ and B₂ are produced only by certain strains of F. moniliforme. F. moniliforme is common even in food-grade corn and is often abundant in ground feeds and in silage. Research on the fumonisin toxins began only recently, and current thought is that concentrations of more than 5 to 10 ppm are necessary for mycotoxicosis in horses and more than 10 to 20 ppm for swine. As with other mycotoxins, various strains of this fungus vary greatly in their toxin producing ability.

 

4. OCHRATOXIN, CITRININ, AND PENICILLIC ACID (PA) (NEPHROTOXINS): Ochratoxin A, produced primarily by members of the Aspergillus ochraceus group and a number of species of Penicillium, especially P. viridicatum have been found in some samples of food and feed grains. Frequently, citrinin or PA is produced by these same fungi simultaneously. In the field, however, injury from ochratoxin poisoning has occurred chiefly (or only) in poultry and swine. Listlessness, huddling, diarrhoea, tremors, and other neural abnormalities are often encountered in broiler poultry. Ochratoxin damage to the kidneys of swine is called "porcine nephropathy”. Regular consumption of a ration containing several hundred ppb of ochratoxin results in poor feed conversion, reduced growth rate, and general unthriftiness, accompanied by reduced immunity to infection by bacteria and viruses. Other prominent features of ochratoxin poisoning are increased water consumption and increased urine production because of kidney damage. The increased urine production in pigs results in the floor of the pig pen being constantly wet and needing to be cleaned daily.

 

5. ERGOTISM: Ergot toxicity, caused by the fungus Claviceps purpurea, differs from other mycotoxicoses, since it results from the consumption of considerable amounts of fungal tissue. In other mycotoxicoses the toxins are secreted into plant tissues in which the fungus is growing, and very little fungal material is consumed. The ergot fungus infects the flowers of cereals and many grasses when flowering occurs during predominantly cool, moist weather Infected florets show characteristic black, spur-like sclerotia that replace the seed. The sclerotia or ergot bodies contain a variety of ergopeptine and clavine alkaloids that, when consumed regularly in small amounts, result in a complex of signs collectively called ergotism. Symptoms of ergotism are poor hair condition, gangrene or loss of extremities, and poor performance. Additional symptoms include persistent diarrhoea, agalactia and abortion in cattle

 

6. FUSARIUM TOXICITY - TIBIAL DYSCHONDROPLASIA IN POULTRY:  Tibial dyschondroplasia (TDP) is a common and economically important bone deformation in growing broiler chickens and turkeys. The lesion appears in a cone of cartilage extending distally from the proximal tibiotarsalphysis. The most likely cause of this deformation is a toxin called fusarochromanone produced by Fusarium equiseti. It also kills chick embryos in fertilized eggs.

 

7. OTHER INFECTIONS:

 

INFECTION	IMPORTANCE
Aspergillotoxicosis	It causes hyperkeratosis in cattle. Aspergillus chevalieri and A.clavatus are the causative fungi. It is basically a respiratory infection. There is also gross thickening of skin due to hyperkeratinisation
Malt sprout poisoning	A.oryzae and A.clavatus
Facial eczema	Pithomyces charterum
Stachybotryotoxicosis	Stachybotrys alternans – sooty fungus

 

CONTROL:

1.     Harvest at maturity and as soon as the moisture content allows minimum grain damage.

2.     Adjust the harvesting equipment for minimum seed or kernel damage and maximum cleaning.

3.     Dry all grain to at least 15-percent moisture as rapidly as possible. Cool the grain after drying and maintain dry storage conditions.

4.     Thoroughly clean the grain and all bins before storage to remove dirt, dust, and other foreign matter, crop debris, chaff, and cracked or broken seeds and kernels.

5.     Where feasible, choose varieties of grain that are resistant to insects, diseases, and mechanical damage.