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SYSTEMATICS
Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Lactobacillales
Family Streptococcaceae
Genus Streptococcus
Species S. agalactiae, S.dysgalactiae, S. equi subsp zooepidemicus, S.uberis,
S. equi subsp equi, S. canis, S.suis, S. pyogenes(human)
HISTORY
Rivolta (1873) described chain forming organisms in pus from a case of strangles in
horses.
In 1878-79, Pasteur recognized this organism as a pus-forming agent.
In 1903, Hugo Schottmuller introduced blood to differentiate various types of
hemolysis.
In 1928, Rebecca Lancefield reported a serological method of grouping streptococci.
HABITAT
MORPHOLOGY
CLASSIFICATION
Group Examples
Pyogenic Streptococci S. pneumoniae, S.pyogenes, S.equi, S. dysgalactiae
Oral Streptococci S.salivarius
Enterococci S.faecalis, S.avium, S. gallinarum
Lactic acid Streptococci S.lactis
Anaerobic Streptococci S. monbillorum
Other Streptococci S.bovis, S.uberis, S.equi subsp zooepidemicus
The aerobic and facultative anaerobic streptococci are classified, based on their
haemolytic properties.
Brown (1919) established this method by employing meat infusion peptone agar with
5% horse blood. He recognized three types of reactions.
o alpha - haemolytic streptococci: They produce a greenish discolouration with
partial haemolysis around the colonies.
o The zone of lysis is small (1or2 mm wide), within which the unlysed
erythrocytes are seen.
o These ά - streptococci are generally commensals in the throat. Because of the
distinctive green color, they produce; they are called as greening or viridans
streptococci. Eg.S.pneumoniae
o Beta haemolytic streptococci produces a sharply defined, clear, colourless
zone of haemolysis, 2 or 4mm wide, within which the red cells are completely
lysed.
o Most of the pathogenic streptococci fall into the beta group and are called as
the haemolytic streptococci.
o Gamma or non-haemolytic streptococci produces no change in the medium.
o The gamma streptococci includes the faecal streptococci ( S. faecalis) and
related species. They are called the enterococcus or indifferent streptococci.
Another important way in which the beta haemolytic streptococci were classified by
Rebecca lancefield (1933) was based on the nature of a carbohydrate (C) antigen on
the cell wall. They are known as Lancefield groups, 19 of which have been identified
so far and named by the capital letters A-U (without I and J)
The M protein is acid and heat labile and the T protein is acid labile and trypsin
resistant.
Some of the Lancefield groups may be further subdivided by means of the
agglutination test and designated by Arabic numbers-Griffith typing
CULTURAL CHARACTERISTICS
BIOCHEMICAL PROPERTIES
Streptococci are catalase and oxidase negative . (Click here for visual - catalse
test)
Ferment several sugars producing acid but no gas.
They ferment sorbitol, trehalose, lactose, maltose, dextrin, and mannitol.
Gelatin not liquified.
Nitrates not reduced.
Indole is negative.
They are not soluble in 10% bile unlike pneumococci
RESISTANCE
ANTIGENECITY
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Streptococci form several exotoxins and enzymes, which contribute to its virulence.
Hemolysins
Streptolysin O
Streptolysin S
It is an oxygen stable haemolysin and so is responsible for the beta haemolysis seen
around streptococcal colonies on the surface of blood agar plates.
It is called Streptolysin S since it is soluble in serum. Addition of serum to broth
increased the yield of haemolysin.
It is protein but not antigenic. It has been shown experimentally to be nephrotoxic.
Erythrogenic toxin (Streptococcal pyogenic exotoxins/ Dick toxin)
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Four erythrogenic toxins are known and most strains of S. pyogenes produce one or
more. They are pyogenic and enhance susceptibility to lethal shock by endotoxin.
The toxin is thermostable and antigenic. The intradermal injection in rats leads to
development of erythema.
This reaction is called as “Dick test” or Schultz-charlton reaction and it is useful for
diagnosis of scarlet fever.
Enzymes
PATHOGENESIS
The natural habitat of the species of streptococci are skin, nose, throat, digestive and
urogenital tracts of man and animals.
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S.pyogenes are present in human nose and throat without causing any disease,
while S.agalactiae and S.uberis can exist in bovine udder without causing mastitis.
PATHOGENECITY
Horse
S. equi and S. equisimilis are the main causes of strangles in young horses.
It is characterized by a catarrhal discharge, with inflamation of the nasal mucous
membranes, followed by swelling of pharyngeal LN’s in which abscesses develop.
The infection spreads through lymph channels. It also causes metritis and cervicitis
in horses.
Purpura haemorrhagica, considered to be an immune mediated disease, occur in
horses 1 to 3 weeks after illness.
Bastard strangles – in which abscesses developed in many organs. It is a very serious
complication.
Chicken
Dogs
S. canis is considered to be the cause of acid milk in puppies and canine tonsillitis. It
is also associated with neonatal septicaemia and toxic shock syndrome.
Pigs
S. suis causes porcine cervical lymphadenitis and also isolated from pneumonia,
septicaemia, arthritis, endocarditis, meningitis and reproductive tract infections.
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DIAGNOSIS
Clinical examination
Microscopical examination
When long chains of organisms are detected in milk samples from chronic mastitis, it
is caused by S. agalactiae.
Bacteriological examination
When 0.1 ml of secretions inoculated on Edward’s medium (blood agar, crystal violet
and aesculin) S.agalactiae produces bluish-grey colonies and S.uberis produces dark
color colonies.
CAMP test (Christie, Atkins, Munch and Peterson, 1944)
o This test is based on the observation that ruminant red blood cells lysed by
the beta toxin of staphylococci at 370C are completely lysed in the presence
of S.agalactiae(group B).
o Differentiation between the pneumococcus and S.viridans organisms can be
achieved by bile solubility and the optochin test.
Bile solubility test
o Autolysis of pneumococcal cultures takes place within 15 minutes at 370C in
the presence of 10 per cent sodium deoxycholate.
o These substances have no effect on S. viridans organisms.
The optochin test
o The majority of pneumococcal strains are sensitive to optochin (Ethyl
hydrocuprein hydrochloride). Whereas S.viridans organisms are not.
o This test consists of placing a small circular piece of filter paper, impregnated
with 1:4000 aqueous solution of optochin, in the center of a blood agar plate
after inoculating the test cultures in streaks across the full width of the
medium.
o The growth of pneumococcal strains will be inhibited to a distance of some 5
mm from the circumference of the filter paper.
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Domain Bacteria
Phylum Firmicutes
Class Bacilli
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Order Bacillales
Family Staphylococcaceae
Genus Staphylococcus
Species S. intermedius.
S. hyicus.
HISTORY
First discovered by Scottish surgeon sir Alexander ogston (1880) in infected tissues.
He named it as staphylococcus” (Greek staphyle, bunch of grapes; KOKKAS, berry).
HABITAT
MORPHOLOGY
Spherical cells, 0.8 to 1 micrometer in diameter, on agar media the cocci are
arranged in grape like clusters.
In broth they occur as small groups, pairs, short chains of not more than four
members.
They are Gram positive, non-motile, non-acid fast, non-spore forming and have no
flagella.
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CULTURAL CHARACTERISTICS
In Nutrient agar plate the colonies are round, smooth, glistening, opaque, low,
convex, edge, entire end of a golden yellow or white colour.
In nutrient broth an uniform turbidity is present with powdery sediment.
Phenolpthaline diphosphate or tellurite agar selectively inhibits non-pathogenic
strains.
Haemolysis on blood agar. Capable of liberating ‘V’ factor into the medium, which
favours the growth of Haemophilus organism.
Purple agar, containing bromocresol purple as a pH indicator and 1% maltose, is
used to differentiate S. aureus and S. intermedius.
Most strains form pigments, hence previously classified as per the colour of the
pigment produced.
o S. aureus – yellowish or golden orange pigment.
o S. albus - white colonies.
o S. citreus - lemon yellow colour pigment
Later on it was found that pigment formation was variable. Hence such classification
was no longer followed.
In sheep or rabbit blood agar plate “double haemolysis” around colonies are formed
with incubation at 37°C it produces an incomplete haemolysis, which develops into a
complete haemolysis when held at 4°C. This is called as hot cold lysis phenomenon.
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BIO-CHEMICAL PROPERTIES
Staphylococcus aureus produces acid from glucose, maltose, mannitol, lactose, and sucrose and not from salicin,
raffinose & inulin.
The organism is indole negative, positive for NH3, methyl red and Voges - Proskauer and catalase.
Hydrolyses gelatin and coagulates serum. Negative for oxidase and H2S production.
RESISTANCE
ANTIGENICITY
It consists of
Group antigen
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Carbohydrates
o The cell wall of S. aureus contains ribitol teichoic acid.
o S. intermedius contains glycerol teichoic acid.
Protein A - Present only in S. aureus .
Type antigen
Exotoxins
o Hemolysin
Four antigenically distinct hemolysins causes hemolysis of erythrocytes. They are Alpha, Beta,
delta and Epsilon toxin, which produce partial hemolysis (hot – cold hemolysis). The alpha
toxin is the major toxin in gangrenous mastitis. It causes spasm of smooth muscle and is
necrotizing and potentially lethal.
o Leukotoxin
It has the leukocidal activity and includes a and d toxins. By doing so, the organisms may spread
more easily to other parts where they develop secondary lesions.
o Enterotoxin
It is seldom produced by animal strains. There are several antigenically distinct types of heat
stable enterotoxins. They are not destroyed at 100°C for 80 minutes. They are responsible for
food poisoning in man.
o Exfoliative or Dermonecrotoxin
This toxin causes necrosis of skin by exfoliation and intraepidermal seperation.
o Toxic shock syndrome toxin (TSST)
Induce excessive lymphokine production and results in tissue damage. Bovine and human
strains of Sta. aureus produce TSST.
Extracellular enzymes
o Coagulase
On their ability to coagulate plasma they are classified as coagulase positive staph. (CPS) and
coagulase negative staph. CPS are considered to be significant pathogens. They are resistant to
heat. The coagulation of plasma produces a fibrin film on the surface of the organisms, which
allows multiplying.
o Hyaluronidase
It hydrolyses hyaluronic acid, the mucoid ground substance of connective tissue.
o Nucleases
Deoxyribonucleases (DNAase) hydrolyze DNA and Ribonucleases hydrolyze RNA.
o Fibrinolysin
Fibrinolysin is commonly referred to as staphylokinase, is an activator of the plasma system
leading to the breakdown of fibrin.
o Lipases and esterases - They hydrolyze lipids.
o Lysozyme - Hydrolyses the peptidoglycan in the cell wall of many bacteria
PATHOGENESIS
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PATHOGENICITY
Horse: Botryomycosis: Infrequent chronic granulomatous lesions involving the udder of the mare, cow and sow
and the spermatic cord of horses.
Cattle: Mastitis: Staphylococcal bovine mastitis may be chronic, acute and peracute. Gangrenous mastitis due to a
toxin is seen in postparturient cows.
Sheep: Tick pyemia in lambs occurs in 2-5 week old lambs, which is heavily infected with Ixodes ricinus.
Periorbital eczema is an infection due to abrasions, Staphylococcal dermatitis due to scratches from vegetation.
Poultry
o Bumble foot: A pyogranulomatous process of subcutaneous tissue of foot that can involve the joints.
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o Staphylococcal arthritis and septicemia in turkeys, omphalitis – yolk sac infection, wing rot or
gangrenous dermatitis infection in poultry
Pig: Exudative epidermitis (greasy pig disease) is an acute generalized infection of suckling and weaned pigs caused
by S. hyicus. This disease is characterized by excess sebaceous secretion, exfoliation and exudation.
Dogs and Cats: Pyoderma is one of the most common skin diseases of dogs.
In addition to this, Otittis externa and other suppurative conditions are caused by S. intermedius.
Staphylococcal antigens produce intense inflammatory reaction and promote persistence of the bacteria.
Other Staphylococcal organisms
o S. aureus sub sp.anaerobius causes caseous lymphadenitis. They are anaerobic and catalase negative.
o S. caprae in goat’s milk.
o S. gallinarum and S. arlettae - skin of chickens
o S. lentus in skin of sheep and goats.
o S. equorum in skin of horses
o S. simulans and S. felis - clinical specimens in cats
o S. delphini in skin of dolphins
o S. aureus in Staphylococcal scalded skin syndrome (SSSS) and Toxic shock syndrome (TSS) in humans.
o MRSA in Methicillin resistant Staphylococcus aureus
DIAGNOSIS
Pathogenicity test
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Coagulase test: When culture added to Rabbit plasma, fibrinogen is converted to fibrin by coagulase enzymes.
In this test, a suspension of staphylococci is mixed with rabbit plasma either on a slide or in a small tube.
The slide test detects the presence of a bound coagulase or clumping factor on the bacterial surface.
A positive reaction is indicated by clumping of bacteria within 1 to2 min.
The tube test detects the free coagulase or staphylocoagulase, which is secreted by bacteria into the plasma.
It is the definitive test for coagulase production and positive reaction is indicated by clot formation in the tube
following incubation at 37ºC for 24 hrs. (Click here for visual)
Phage typing
Phage typing is carried out for epidemiological purposes, particularly for S. aureus strains of bovine mastitis.
TREATMENT
SYSTEMATICS
Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Bacilliales
Family Bacilliaceae
Genus Bacillus
Species B.anthracis, B.cereus, B.subtilis, B.mycoides, B.megaterium, B.mesentricus
B.anthracis causes Anthrax in animals and Wool sorter’s disease, hide porter’s disease, Malignant Pustule in
humans.
B.cereus causes food poisoning in humans.
Other bacilli in this group are non-pathogenic and they are called as anthracoids.
B.licheniformis is an emerging pathogen and it is implicated in sporadic abortions in cattle and sheep.
HISTORY
Discovery of the anthrax bacillus is credited to Davaine and Rayer (1863 –1868).
Considerable historic interest is attached to anthrax bacilli.
Pollender 1849 – Anthrax bacillus was the first pathogenic bacterium observed under the Microscope
Davaine 1850 – first communicable disease shown to be transmitted by inoculation of infected blood
Koch 1863 – first bacillus to be isolated in pure culture
Pasteur 1881 – used for the preparation of attenuated vaccine.
HABITAT
Bacillus anthracis spores remain viable for many years in soil, water and animal hides and products.
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Spores have been isolated from naturally infected soil as long as 60 years.
MORPHOLOGY
Members of the family are Gram +ve large rods, aerobic (facultative anaerobic), endospore forming, capsulated,
mostly catalase positive and fermentative organisms. They are motile by peritrichous flagella.
The anthrax bacillus is one of the largest pathogenic bacteria. They are Gram +ve, straight, rod shaped, non-motile
organisms measuring 4-8 μm x 1-1.5 μm.
In cultures the bacilli are arranged end to end in long chains. The ends of the bacilli are truncated or often concave
and somewhat swollen.
Chain of bacilli presents a bamboo stick appearance (and also called as Box –car bacillus - looks like linked rail
carriages).
In tissues or in blood smear, it is found singly, in pairs or in short chains, the entire bacilli being surrounded by
capsule.
The capsule is polypeptide in nature, being composed of a polymer of d-glutamic acid.
Capsules are not formed under ordinary conditions of culture, but only if the media contains serum, albumen,
charcoal, starch or bicarbonates with reduced partial pressure of carbon dioxide.
When blood films containing bacilli are stained with polychrome methylene blue for a few seconds and examined
under the microscope, an amorphous purplish material is noticed around the bacilli. This represents the capsular
material and is characteristic of the anthrax bacilli. This is called asreaction. This reaction depends on the degree of
heat employed for fixation of a blood film.
Sporulation occurs readily outside the body in the presence of oxygen. Spores are formed in culture or in the soil,
but never in the animal body during life.
Sporulation occurs under unfavourable conditions for growth and is encouraged by distilled water, 2% NaCl or
growth in oxalated agar. Sporulation takes place at an optimum temperature of 25-30°C and in atmosphere
containing low partial pressure of oxygen. Sporulation is inhibited by anaerobic conditions and by CaCl2
Spores are central, elliptical or oval in shape and are of the same width as the bacillary body. So that they do not
cause bulging of the vegetative cell. The spores do not stain by ordinary methods. But can be stained with Sudan
black B.
CULTURAL CHARACTERISTICS
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Grows readily under aerobic and as facultative anaerobic at an optimum temperature of 35-37°C.
On agar plates, irregular, round, raised, dull, opaque, grayish white, 2-3mm in diameter frosted glass appearance
colonies are produced.
Under the low power microscope, the slightly serrated edge of the colony is composed of long, interlacing chains of
bacilli, resembling locks of matted hair. This is referred to as medusa head or judge's wig or woman’s curling hair
type of growth.
B. licheniformis produces characteristic hair-like outgrowths from streaks of the organisms on agar media.
Colonies become brown with age.
The name of this species derives from the similarity of its colonies to lichen.
In medium containing iron salts, virulent B.anthracis produces pink or purple coloured pigmented colonies.
Virulent capsulated strains form rough colonies, while avirulent attenuated strains form smooth colonies.
In gelatin stab, fine filaments of growth develop laterally along the line of inoculum.
The growth nearer to the surface of the medium is the longest and then progressively shorter where there is less
oxygen, resembling inverted fir tree appearance.
On haemolysis, the B.anthracis produces slight haemolysis with capsule production, compared with anthracoid
organisms.
B. cereus produces a wide zone of complete haemolysis around the colonies.
A selective medium (PLET) consisting of polymyxin, lysozyme, EDTA and thallous acetate added to heart infusion
agar is useful for isolating B.anthracis from mixtures containing other spore-bearing bacilli.
BIOCHEMICAL PROPERTIES
RESISTANCE
ANTIGENICITY
The complex antigenic structure includes a capsular polypeptide, a somatic protein and a somatic polysaccharide
antigen.
The capsule contains poly-d-glutamic acid, which is only detectable in virulent strains, having antiphagocytic
property.
The somatic protein is protective antigen present in the edema fluid.
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B. anthracis produces an extracellular toxin which is composed of three components, Factor I, II & III namely
oedema factor (EF), protective antigen (PA) and lethal factor (LF) respectively.
Factor I consists of chelating compound containing phosphorus with protein and carbohydrate moieties.
Factor II & III consists of proteins.
All three factors are essential to exhibit their toxic properties.
They are not toxic individually but the whole complex produces local odema and generalized shock.
PATHOGENESIS OF BACILLUS
Anthrax can occur in virtually all mammalian species but birds are highly resistant.
The main routes of entry of endospores are by ingestion, from soil when grazing or in contaminated food, and by
infection of wounds.
Inhalation of spores occurs in man, but to a lesser extent in animals.
Transmission by biting insects may be important, especially during an outbreak of anthrax.
Cattle, sheep and goats are most susceptible to infection, while horses and humans occupy an intermediate
position and pigs and carnivores are comparatively resistant, but can succumb if the infective dose is high.
PATHOGENICITY
CLASSIFICATION
Symptoms
Horses
Acute form is very common and death may take place one day after edematous swelling of the throat and neck
region.
There may be symptoms of colic. In less acute, oedmatous swelling become generalized and death occurs after 2-3
days.
Cattle
Bulls are more susceptible than cows. They have a mortality rate of 90%.
There are three clinical causes of bovine anthrax.
In peracute sepeticemia death occurs within 2 hours after animal collapsing with convulsions, sudden death in
animals that appeared normal is common.
In acute septicemia death occurs within 48 to 96 hours clinical signs include fever, anorexia, ruminal stasis,
hematuria and blood tinged diarrhea.
Pregnant animals may abort and milk production often abruptly decreases.
Terminal signs include severe depression, respiratory distress and convulsions.
In chronic cases, clinical signs are manifested for more than 6 days and are rare.
B.licheniformis infection is associated with the feeding of contaminated silage and is responsible for abortion in
cattle and sheep.
Sheep
Pigs
Dogs
Lesions
The carcass of animals will putrify rapidly and develop incomplete rigor mortis.
The blood is dark,(tarry colored), clots poorly & exudes from the natural orifices.
The spleen is greatly enlarged, dark and friable. The spleen reveals black cherry jam consistency.
The LN (lymph node) at the region of initial infection site is hemorrhagic and edematous.
Ecchymotic hemorrhages on the serosal surface of the abdomen, thorax, epicardium and endocardium are
common.
Subcutaneous edematous swellings are present on the ventral aspect of the neck
Note
When suspected for anthrax care should be taken not to open the carcass. Muzzle piece or ear piece is usually sent
for examination.
DIAGNOSIS
Clinical symptoms
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Blood films from dead animals made by puncturing the superficial vein of the ear or in the region of the foot.
Care should be taken to seal the injection site by placing cotton soaked in alcohol and ignited.
The smears are heat fixed and stained by Wright's or Giemsa’s stain to reveal B. anthracis as large blue rods with
characteristic dark pink or purple coloured capsules.
In case of horses and pigs since peripheral blood contains fewer organisms, smears should be made from the
edematous fluid or LN’s.
Cultural examination
Swabs from blood are inoculated on to blood agar plates and incubated at 37°C for 24 hrs and examined for their
typical growth.
Swabs are inoculated in agar enriched with blood or serum and incubated for 6 hrs at 37°C and examined by
stained smears.
Bacteriological examination of hair, wool, hide, bone, bone meal & others
Samples are added to cold saline and shaken intermittently for 3 hours.
The supernatant fluid is then heated to 70°C for 10 minutes.
Then they are filtered through two layers of muslin cloth, added to melted agar, and poured into petridishes,
allowed to set and incubated at 37°C.
After 12 hours incubation, plates are to be examined for characteristic colony morphology.
Grind up the organ or blood of suspected animal and suspend in 5-10 parts of saline and boil for 15 minutes.
Filter through filter paper and allow it to cool. Place 0.5 ml of anti-anthrax serum (1:50) in a small test tube and
overlay with 0.5 ml of clear filtrate.
Stand at room temperature for 15 minutes. A white ring of precipitation indicates a positive reaction.
B.anthracis produces swollen round cells in chains (string of pearls) when incubated for 3-6 hrs on tryptose agar
containing 0.05 –0.5 I.U of penicillin/ml.
To 100ml of molten nutrient agar, add required Sodium beznyl penicillin and mix carefully.
Pour into petridishes and allow to set. With a scalpel cut a block about 1.6 cm 2 from the penicillin agar plate and
place it on a microscopic slide in a petridish containing a small piece of moistened absorbent cotton wool to
prevent the agar drying out.
Use a young colony to streak the center of the agar block.
Place a clean cover slip on the agar block and incubate the petridish at 370C.
After 2 hrs, remove the slide and examine the inoculum microscopically by oil immersion for the string of pearls
growth.
Differentiation from non-pathogenic Bacillus sp. It is considered reliable but needs experience.
Cherry gamma phage is used. It can be propagated on B. anthracis strain 14, which makes a suitable control.
On one half of blood agar plate, streak B. anthracis strain 14 culture as control and on the other half streak the
culture to be identified.
Add drops of phage preparation (1:10 dilution) on both halves. Incubate the plates in the upright position for 8-12
hrs.
Zones of clearing will be seen on control culture and on the suspected half if it is B. anthracis.
Animal Inoculation
Guinea pigs & mice are highly susceptible. Materials are inoculated by dermal scarification, subcutaneous or
intramuscular.
Death occurs in 2-3 days and the organisms will be readily identified in the blood & tissues.
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Apart from this laser pyrolysis, gas-liquid chromatography and mass spectrometry are used for detection of
anthrax toxin productions.
DIFFERENTIAL DIAGNOSIS
Difference between B. anthracis & Anthracoid organisms in blood smear stained with Polychrome
methylene blue stain
B. anthracis Anthracoid
Dark pink or Purple coloured capsules No capsule
Organism rod stained blue Organism rod stained blue
Ends truncated Ends bulged and rounded
Single, pair or short chain Usually long chain
Absence of spores Spores may be present
TREATMENT, IMMUNITY AND PUBLIC HEALTH
ASPECTS
Treatment
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Immunity
SYSTEMATICS
Domain Bacteria
Phylum Firmicutes
Class Clostridia
Order Clostridiales
Family Clostridiaceae
Genus Clostridium
CLASSIFIACTION
The clostridia can be divided into four major groups according to the kind of
disease they produce. They are as follows.
The Histotoxic clostridia causes a variety of tissue (often muscle) infections
frequently following wounds or other trauma (eg).
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Hepatotoxic clostridia produces their toxins in the liver, thus resulting in the
disease Bacillary haemoglobinuria and Black disease (Eg.).
The Neurotoxic clostridia cause the disease by the production of the potent
exotoxins (Neurotoxins) (eg.)
C. tetani Tetanus
C. botulinum Botulism
FAMILY CHARACTERS
Classification
They are pleomorphic, rod shaped; long filaments and involution forms are common.
Spore formation occurs with varying frequency in different species.
The shape and position of the spores vary in different species.
The clostridia are motile with peritrichous flagella except C.welchii and C.tetani typeVI. C.welchii is capsulated,
while others are not.
Clostridia are anaerobic. C.odematiens are strict anaerobes and die on exposure to oxygen.
C.histolyticum and C.welchii are aerotolerent and may even grow aerobically.
The clostridia are fermentative, oxidase negative and catalase negative organisms.
A very useful media for isolation of clostridia is Robertson’s cooked meat broth.
Clostridia grow in the medium, rendering the broth turbid most species produces gas.
Saccharolytic species turn the meat pink – C.odematiens, C.septicum, C.chauvoei and C.welchii.
Proteolytic species turn the meat black and produce foul and pervasive odour - C.tetani, C. botulinum,
C.haemolyticum.
In litmus milk medium, the production of acid, clot and gas can be detected
HISTORY
Tetanus has been known from very early times, having been described by Hippocrates.
But the knowledge of the disease was achieved only in 1884.
Rosenbach –1886 - demonstrated a slender bacillus with round terminal spores in a case of tetanus.
Kitasato –1889 – isolated C.tetani in pure culture and reproduced the disease in animals by inoculation of pure
culture.
The Greek term “tetanus” which means ‘contracture’ has been taken from the Latin medicine “rigor”.
HABITAT
Soil, especially that contaminated by animal faeces, is the natural habitat as C.tetani is often transient in the
intestines of horses and other animals.
It is ubiquitous and has been recovered from a wide variety of other sources, including street and hospital dust,
cotton wool, bandages, catgut, plaster of paris, clothing etc. It may occur as an apparently harmless contaminant in
wounds.
MORPHOLOGY
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C.tetani is a straight, slender, Gram positive rod that characteristically produces a terminal, spherical endospores
that bulges the cell giving the characteristic drumstick appearance. (The young spore may be oval rather than
spherical).
It occurs singly and occasionally in chains.
It is non-capsulated and motile by peritrichous flagella.
Cultural characters
Biochemical properties
C.tetani has feeble proteolytic property, so it does not ferment any sugars.
It forms indole. It is MR and VP negative, nitrates not reduced.
RESISTANCE
The endospores are highly resistant and while boiling kills the spores of most strains in 15mts.
Autoclaving at 121ºC for 15mts and dry heat temp of 150ºC for more than one hour is completely sporicidal.
Spores are able to survive in soil for years and they are resistant to most antiseptics.
They are not destroyed by 5% phenol or 0.1% mercuric chloride solution in two weeks or more.
Iodine (1% aqueous solution) and H2O2 kill the spores within a few hours.
ANTIGENICITY
Ten serological types have been recognized based on the flagellar antigen (types I to X). Type VI contains
non flagellated strains.
All types produce the same neurotoxin- tetanospasmin. Which can be neutralized by one common
antitoxin.
They have a common heat stable somatic antigen shared by all types.
A second somatic antigen is shared by type II, IV, V and X.
TOXINS
They are antigenically and pharmacologically distinct and their production is mutually independent.
A third toxin – a nonspasmogenic peripherally active neurotoxin has been identified recently.
It is not known whether this plays any role in the pathogenesis of tetanus.
Tetanolysin
Tetanospamin
HISTORY
The name botulism is derived from sausage (botulus, latin for sausage), an article
of food that used to be associated with the type of food poisoning.
C.botulinum was first isolated by Van Ermengam (1896) from a piece of ham that
caused an outbreak of botulism.
C. botulinum denotes a group of bacteria that produce extremely potent
neurotoxins.
These toxins cause botulism , a disease characterized by flaccid paralysis in many
animals and humans.
Botulism is most common in water birds, ruminants, horses, mink and poultry.
Botulism in animals has been called by a variety of names,
o Horses: Spinal typhus / Shaker foal syndrome
o Cattle: Lamsiekte, loin disease and contagious bulbar paralysis
o Water fowl: Limber neck, alkali poisoning and western duck sickness
Botulism is rare in domestic cats. Pigs and dogs are relatively resistant.
HABITAT
The endospores are widely distributed in soils and aquatic environment through out the world.
The disease botulism is mainly due to the ingestion of preformed toxin.
Germination of the endospores, with growth of vegetative cells and production of toxin, occurs in anaerobic
situations such as contaminated cans of meat, fish or vegetables, carcases of invertebrate and vertebrate animals,
rotting vegetation and baled silage.
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MORPHOLOGY
It is a strict anaerobe, opt.temp is 30-370 C and pH is 7-7.6. Good growth occurs on ordinary media.
Surface colonies are large, irregular, and semitransparent with fimbriate border.
Spores are produced consistently when grown in alkaline glucose gelatin media at 20 to 25 0C.
In horse blood agar, large transparent colonies with irregular edges are developed with a narrow zone of
haemolysis.
On sheep blood agar C.botulinum produce beta-haemolysis, the colonies are slightly domed with a ragged edge.
In cooked meat medium, the proteolytic strains type A, B and F produces blackening of meat and non-proteolytic
strains C, D and E do not blacken meat.
Gelatin liquefied rapidly by type A&B and slowly or not at all by type C, D and E.
RESISTANCE
Eight types of C.botulinum have been identified (Types A to G) based on the immunological difference in the toxins
produced by them.
These neurotoxins are identical in pharmacological action but differ in potency, distribution and antigenicity.
They are neutralized only by the homologus antiserum.
The toxins differ from other exotoxins in that it is not released during the life of the organism.
It is appears in the medium only on the death and autolysis of the cell.
It is believed to be synthesized initially as a nontoxic protoxin or progenitor toxin.
Trypsin and other enzymes activate progenitor toxin to active toxin.
The toxin is heat labile and its mol.wt. is 70,000.
One mg of neurotoxins contains more than 120 million mouse lethal doses.
The lethal dose for human is 1-2μg. This toxin acts slowly taking several hours to kill.
C.tetani C.botulinum
Site of toxin Wounds Carcases, decaying vegetation and
production occasionally wounds and intestine
Mode of action Centrally by blocking Peripherally by blocking neuromuscular
synaptic inhibition transmission
Type of paralysis Spastic paralysis Flaccid paralysis
Antigenic types Tetanospasmin (one Eight different toxins produced by types
of toxin antigenic type) A-G
PATHOGENESIS
C.botulinum is non invasive and virtually non infectious. Botulism is of three types.
Wound botulism
Infant botulism
It occurs when spores are ingested in food and get germinate in the intestines when the normal flora has not been
fully established.
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This form is seen in human infants (Floppy baby syndrome) and as rare epidemics of type C in broiler chickens and
turkey poults.
PATHOGENICITY
Symptoms
In cattle, the incubation period varies between 2-10 days depending upon the dose of toxin ingested.
Initially there will be excitation, followed by incoordination and paralysis of the hind limbs.
There will be paralysis of muscles in the mouth, pharynx and neck, resulting in the animal being unable to swallow
and the tongue protruding from the mouth. This is followed by death.
In South Africa this condition is termed as lamsiekte in cattle caused by type D, especially in the phosphorus
deficient animals.
In poultry it results with the ingestion of type C toxin and the disease is known as duck sickness or western duck
disease and Limberneck in chicken.
The symptoms include paralysis of the wings, legs and neck, protrusion of nictitating membrane, diarrhoea and
comatase before recovering in 5-6 days time.
Lesions
Pathological changes are noticed in the CNS, especially the brain stem and 3rd ventricle, catarrhal gastroenteritis,
hepatitis and nephrosis
DIAGNOSIS
Toxin demonstration
Toxin identification
Mouse (or guinea pig) neutralization tests using a polyvalent antitoxin initially,
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HISTORY
Clostridial enterotoxaemias are acute, highly fatal intoxications that affect sheep, lambs, calves, piglets and
occasionally foals.
The diseases are caused by the major exotoxins (enterotoxins) of Clostridium perfringens types B, C, D and
occasionally types A and E.
The bacillus was originally cultivated by Achalme (1891), but it was first described in detail by Welch and
Nuttal (1892)-who isolated it from the blood and organs of cadaver.
HABITAT
Based on toxin production Clostridium perfringens organisms are classified into 5 types (A to E).
Clostridium perfringens Type A occurs in the intestinal tract of human and animals and in most soils.
Type B to E are more adapted to survival in the intestines but in outbreaks of disease they survive long enough in
soil to infect other animals
MORPHOLOGY
Gram +ve, bacillus, straight, parallel sides, rounded or truncated ends, occur either singly or in chains or small
bundles.
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It is highly pleomorphic, filamentous and involution forms are common. It is capsulated and non-motile.
The spores are oval, sub-terminal and bulge the mother cell.
They are rarely produced and their absence is one of the characteristic morphological features of C.welchii.
All the types of C.perfringens produce the alpha toxin, that is a lecithinase.
On the half of the plate without the antitoxin, the lecithin in the medium is attacked causing opalescence around
the streak.
The lecithinase reaction is neutralized on the other half of the plate with the antitoxin but the growth
of C.perfringens is unaffected.
C.welchii ferments several sugars (glucose, maltose, lactose and sucrose) and produces acid and gas. Indole –ve,
MR +ve, VP-ve, H2S +ve.
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RESISTANCE
Spores are usually destroyed within 5 minutes by boiling, but those of the food poisoning strains of type A and type
C resist boiling for 1-3 hrs.
Autoclaving temp is lethal.
TOXINS
Classification
C.welchii is one of the most prolific of toxin producing bacteria forming at least 12 different toxins , besides many
other enzymes and biological active substances.
C. Major toxins
perfringens
Type Alpha Beta Epsilon Iota
A + - - -
B + + + -
C + + - -
D + - + -
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E + - - +
Alpha toxin is produced by all types. Mostly by type A strains. It is lethal, dermonecrotic and haemolytic.
This is a lecithinase C (phospholipase) that attacks cell membranes causing cell death and destruction and also
responsible for Nagler’s reaction .
It is haemolytic for the red cells of most species except horse and goat. This toxin gives a zone of partial haemolysis
on blood agar.
The haemolysis is of hot-cold variety being best seen after incubation at 370C followed by chilling at 40C.
Beta toxin is lethal and necrotising. It is sensitive to trypsin and this explains the predilection of types B and C for
neonates as colostrum has anti trypsin activity.
It is a labile toxin and may be destroyed if there is a delay in small intestinal contents, containing the toxin,
reaching the laboratory.
Epsilon toxin is secreted as a protoxin (proto toxin) and is activated in the intestines by proteases such as trypsin.
Pulpy kidney disease is not usually seen in neonatal lambs as colostrum contains an antitrypsin factor that can
prevent the epsilon toxin being activated.
The toxin itself increases gut permeability, assuring absorption of the toxin into the blood stream.
It damages vascular endothelium (including blood vessels in the brain) leading to fluid loss and edema.
This epsilon toxin can be regarded as an enterotoxin and neurotoxin.
Iota toxin is also produced as a protoxin and is not unique to C.perfringens type E as it is also formed
by C.spiroforme and C.difficile
Besides several minor toxins are produced such as the theta (haemolysin), Kappa (collagenase), lambda
(Proteinase), Mu(hyaluronidase) and Nu(DNase) – all these may contribute to tissue damage.
Based on the type of toxin productions, Clostridium perfringens are classified into 5 types
PATHOGENESIS
The enterotoxaemias are often precipitated by certain husbandry and environmental factors such as abrupt
changes in feeding, usually to a richer diet and overeating and voracity on high protein and energy rich feeds.
This leads to slowing of peristalsis with retention of bacteria in the intestines, absorption of toxins, inadequately
digested carbohydrate and the provision of a rich medium for the proliferation of C.perfringens
The bacterium inhabits the large intestine in normal animals, but if overgrowth occurs C.perfringens can spill over
into the small intestine with the production of a large amount of toxin and enterotoxaemia.
PATHOGENECITY
Disease Clinical and postmortem signs
Pulpy kidney disease Odema of brain, glycosuria, sudden death. Excess fluid
(Over eating disease) in body cavity, focal symmetrical encephalomalacia
occurs in well-grown lambs.
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DIAGNOSIS
Gram stained smears can be made from the mucosa of the small intestine of a recently dead animal.
Large numbers of Gram-positive rods are suggestive of C.welchii.
Saccharolytic in Robertson's cooked meat media, opalescence in egg yolk agar, haemolysis on blood agar, stormy
clot fermentation and sugar fermentation tests are helpful for identification.
Histopathology on brain sections helps to demonstrate focal symmetrical encephalomalacia in pulpy kidney
disease.
Rapid kidney autolysis, pulpy cortical softening and Glucosuria are suggestive of pulpy kidney disease.
Collect 20-30ml of ileal contents from a recently dead animal and send it to the laboratory as soon as possible.
The ileal contents are centrifuged and the clear supernatant is tested for toxin.
In ileal contents, the epsilon and iota toxins are usually in the active form.
To demonstrate the toxin 0.4ml of the clarified ileal content can be inoculated i/v ly into mice.
If mouse dies within 5 mts this is probably due to shock. Death from toxin usually occurs within 10hrs.
Identification of toxin in the clarified ileal content is carried out by a neutralization test by using suitable antitoxin.
Before the lambing season the ewes are vaccinated with formalised whole culture or alum precipitated vaccine.
The resulting passive immunity, with unweaned lambs, derived from colostrum protect lambs for first 3 weeks of
life.
Similarly alum precipitated trypsin –treated toxoid is also satisfactory.
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Lambs can also be vaccinated by giving the first dose within 72 hrs of birth and repeated at 4 weeks of age.
Immunity may not last more than 6-12 months unless booster dose is given.
HABITAT
World wide in distribution. The principal habitat of C.novyi (also called as Clostridium oedematiens) is soil and
the intestine of animals.
Based on toxin production Clostridium novyi is classified into four types (A-D).
The type A is commonly found in soil. Type B is rarely found in soil, and it is common in the normal intestinal tract
of herbivores.
Strains of type A and B are recovered from the livers of normal animal. Type D (C. haemolyticum)- is found in the
ruminant digestive tract, liver and in the soil.
Large, pleomorphic, Gram+ve rods with oval to cylindrical subterminal spores are characteristic.
There is little or no swelling of the mother cell, non-capsulated, motile by peritrichous flagella.
Clostridium novyi type B and C.haemolyticum are very demanding in both their anaerobic and nutritional
requirements.
Very strict anaerobic procedures are necessary and media containing cysteine should be used. These clostridia can
die within 15mts of being exposed to atmospheric O2 .
These organisms are difficult to grow on primary culture and the growth is enhanced by agar enriched with glucose
or freshly prepared blood or fresh brain infusions.
On blood agar C.novyi produces characteristic large, irregular colonies with a rhizoid edge and a large zone of clear
haemolysis.
On moist surface of solid media after 3-4 days of incubation colonial motility develops which is characterized by
the movement of daughter colonies moving away from parent colonies in spirals or arc and few return and fuse to
the parent colony.
In horse blood agar, the colonies are haemolytic, small and usually rhizoidal in nature.
Areas of hemolysis develop beneath the colonies and develop into wider zone after 48-72hrs incubation.
In Sheep blood agar very slight haemolysis develop. In Robertson’s cooked meat medium C.novyi type D is very
strongly proteolytic.
Type A, B and C are saccharolytic. The lecithinase activity of beta toxin of type B and D, and Gamma toxin of type A
produces quite distinct opacity changes on egg-yolk agar.
C.novyi type A exhibits lipase activity on egg-yolk agar.
It will produce characteristic iridescent pearly layer on the surface of the colonies, extending on to the surface of
the medium immediately surrounding them.
C.novyi type A is the only species among clostridia that produces both a lecithinase and a lipase.
Saccharolytic type ferment glucose and maltose but not lactose.
RESISTANCE
Spores of most strains survive heating to 950C for 15mts. But are killed at autoclaving.
Spores are resistant to 5% phenol, 10% formalin or 0.1% merthiolate.
They are killed rapidly by exposure to hypochlorite.
Spores remain viable for years in soil.
TOXINS
C.novyi possess several somatic and flagellar antigens, which are not of much importance.
Based on toxin production C.novyi is classified into 4 types.
C.novyi synthesizes five major toxins, α, β, γ, δ and ε.
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Alpha Beta
C. novyi type A + -
Type B + +
Type C - -
Type D C. - +++
haemolyticum
Type A produces all toxins except beta. Type C isolates are non toxigenic.
In addition to this type B also produce zeta, eta and theta toxin.
o Alpha toxin produced by type A and B is lethal, necrotizing, causes increased capillary permeability, and
is toxic to several tissues including muscle, heart and liver.
o Beta toxin is a lecithinase and produced by type B and by C.haemolyticum in greater amounts.
o This may account for the haemolytic crisis and death in bacillary haemoglobinuria.
o Gamma toxin is a necrotising phospholipase D.
o Delta toxin is an oxygen labile haemolysin.
o Epsilon toxin is a lipolytic enzyme.
o Zeta toxin is partly haemolytic.
o Theta toxin is a lipase.
o Eta toxin is a tropomyosinase, which degrades tropomyosin and myosin and may play a role in
destruction of infected muscles.
PATHOGENESIS
In black disease and bacillary haemoglobinuria, the spores, normally present in the intestine, may reach the liver
and remain dormant in the kupffer cells.
Any destruction of liver tissue could be the initiating factor.
The tissue damage is usually due to migration of immature liver fluke (Fasciola hepatica), and anaerobic
conditions permits germination of spores, growth of vegetative cells and subsequent production of toxin.
Alpha toxin produced in the local area of necrosis and in the liver is adsorbed into the circulation and results in
systemic effects.
In case of bacillary haemoglobinuria the dominant toxin is beta toxin.
Big head in rams develop when sub cutaneous tissues traumatized during fights are subsequently invaded
by C.novyi type A.
The oedema is the result of vascular damage inflicted by the alpha toxin.
PATHOGENECITY
Symptoms
Big head
o Odematous swelling occurs in head, face and neck.
o It will be followed by collapse and death of animals.
o The mortality rate may be more than 90%.
Black disease
o Acute toxaemia leads to sudden death.
o The signs include rapidly decreasing ability to move, unsteady gait and collapse.
Bacillary haemoglobinuria
o Common in summer months, affected animals suffer from fever, abdominal pain, port-wine coloured
urine , diarrhoea and haemoglobinuria. The mortality rate is 90%.
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Lesions
Black disease
o Number of clearly defined gray-yellow foci (necrotic areas) in the liver.
o The lesion consists of a central core of necrosis surrounded by a zone of leucocytes in which there will be
masses of C.novyi. Excess fluid in body cavities.
o Straw-coloured exudates will be present in pericardial and peritoneal cavities.
o Extensive subcutaneous and bloodstained odema can be noticed in the carcass.
o Venous congestion occurs that darkens the skin(Black disease).
Bacillary haemoglobinuria:
o Number of typical anaemic infarcts in the liver.
o Pale and raised areas surrounded by a blue-red zone.
o There will be blood stained intestinal contents, dark colored urine in the bladder, marked icterus of the
carcass, widespread odema and haemorrhages in the myocardium.
Based on history
Direct Gram stained smears
o Presence of characteristic liver lesions together with large number of Gram +ve rods in liver impression
smears from a recently dead animal is suggestive of the disease.
Flourescence Antibody Test is useful for the identification of C.novyi type B and C.haemolyticum in acetone fixed
liver impression smears.
Isolation of organism from affected tissue (as like other clostridial infections) and by characteristic cultural
characters.
Animal inoculation
o Toxin in the liver can be demonstrated by intra muscular injection of homogenates into guinea pigs.
o The pathogenicity is enhanced if the homogenate is added to an equal amount of 5% CaCl2 solution before
inoculation.
o The guinea pigs die in 1-2 days with very extensive subcutaneous edema. Specific antitoxin is not readily
available for neutralization tests.
Gram +ve, highly pleomorphic, chracteristic long filamentous forms are seen in
stained smears of affected muscle.
Cigar shaped rods and citron forms are more common. Spores are oval, central
or subterminal. Non-capsulated, motile by peritrichous flagella.
Strict anaerobe, growth at an opt.temp of 370C, growth is promoted by glucose.
On ordinary media, the colonies are irregular and transparent initially, turning
opaque (large, grayish white on continued incubation).
The colonies are swarming and spreading over the entire surface.
On stiff agar, the colonies are irregular with a rhizoid edge. Some strains produce
smooth, round colonies.
In cooked meat medium meat turns pink with rancid odour, and produces
abundant gas (because, it is saccharolytic).
Like, C.perfringens, the C.septicum inoculated into litmus milk produces the
classical stormy clot or stormy fermentation reaction.
Ferment glucose, lactose, maltose and salicin but not sucrose. Acid and gas are
not produced.
Six groups have been recognized, based on somatic and flagellar antigens.
Clostridum septicum produces at least four distinct toxins and fibrinolysin.
o The α toxin is oxygen stable haemolysin, dermonecrotic and lethal.
o The β toxin is leucotoxic and DNAse.
o The γ toxin is a hyaluronidase.
o The δ toxin is an oxygen labile haemolysin.
α toxin has direct effect on cardiac muscle and is capable of causing capillary damage.
Iron is required both for growth of bacteria and for production of α toxin.
PATHOGENESIS
Braxy in sheep
In this exogenous gas gangrene infection, spores are introduced into wounds
where they may germinate in the anaerobic necrotic material and toxin is
produced by the vegetative cells.
PATHOGENECITY
Symptoms
Braxy usually occurs in well-nourished one-year-old sheep, ailing animals show signs of abdominal pain and
diarrhoea. Death occurs within few hours.
In malignat odema, infected wound, become gas gangrenous.
Fever, soft swelling around wound spreading to muscles.
Swelling odematous and wet with much exudates and gas. Muscles appear dark red to black color.
Lesions
In braxy, the lesion may be confined to the abomasum; there will be the characteristic area of haemorrhagic
inflammation in the wall of the abomasum.
There will be an extensive quantity of blood stained fluid in the peritoneal cavity.
Based on history, symptoms and characteristic lesions strongly suggestive of this disease.
FAT is to be employed for differentiate it from C.chauvoei .
Identification of specific clostridial toxin by mixing 1.2ml of culture fluid with 0.3ml of specific antitoxin, allowing
the mixture to stand for 30 minutes at 370C and inoculate two guinea pigs intra peritoneally. If specific toxin
present it will be neutralized.
Penicillin alone or with hyper immune serum can be used to treat infections.
Sheep can be effectively immunized against the disease and multi component vaccine is used.
Worldwide distribution in soil and pastures. C. chauvoei (also called as C. chauvoei type B, C. feseri) causes black
quarter or black leg in Cattle & Sheep.
Gram positive, rod shaped with rounded ends 3-8mm in length & 5mm in width.
Sometimes pleomorphic, large cigar shaped rods or citron forms occur.
Non-capsulated and motile by peritrichous flagella. Non-motile variants do occur.
Spores are oval and located centrally or subterminally. Old cultures stain Gram negative.
Strict anaerobe. Growth occurs at an optimum temperature of 370C. Growth enhanced by the addition of liver
extract or glucose.
In blood agar whitish grey colonies with irregular edges develop, surrounded by a zone of haemolysis.
In cooked meat medium growth is slow and meat is turned pink with sour odour
C. chauvoei ferments glucose, lactose, sucrose, maltose with acid & gas, but not salicin.
Classification
PATHOGENECITY
Symptoms
The disease usually occurs in young cattle of 6 months to about 2-3 years of age.
The most obvious sign is crepitating swelling particularly in the hind or fore quarter which rackels when rubbed
with the fingers as a result of gas production.
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The affected animal will become lame and the affected muscles shows trembling with violent twitching. Death
usually occurs within 24 hours.
In sheep an acute febrile condition develops within 1-2 days following an injury and a typical black quarter lesion
can be observed at the site. Death occurs suddenly
Lesion
In the central part of the lesion there is usually a well-defined area of muscle, which is dark red in colour, dry,
necrotic and filled with small gas bubbles, which give a swollen appearance to the muscles.
The lesion has a characteristic rancid odour. Surrounded this area of muscle there will be yellowish or blood
stained oedematous fluid.
In ewes there will be necrosis of the vaginal mucosa and skin with extensive oedema involving the hind limbs and
thigh muscles.
Based on History
Based on Symptoms - The most obvious sign is crepitate swelling particularly in the hind or fore quarter which
rackels when rubbed with the fingers as a result of gas production
Smears prepared from the lesions and oedmatous fluid reveal Gram positve rod.
Isolation can be done from the center of the lesion, oedematous fluid and from heart blood & spleen.
FAT used to differentiate from C. septicum
Broth cultures or oedematous fluid from the lesions can be tested for toxicity and specific neutralization by
antitoxin in mice or guinea pigs.
o The most reliable results are obtained from using a formalized alum precipitated whole culture that
confers immunity against the bacteria as well as the toxin.
o For economic reasons a multi-component vaccine containing C. chauvoei, C. septicum, C.welchii type D
and C. tetani is used.
o A stronger immunity is stimulated by two doses of vaccine at a time interval of at least 2-3 weeks
Hyper immune serum (HIS) is used to control explosive outbreaks. Penicillin along with HIS is used to treat the
disease.
Oxytetracycline & Chlortetracycline can also be employed effectively in early stages.
SYSTEMATICS
Domain Bacteria
Phylum Firmicutes
Class Bacilli
Order Bacillales
Family Listeriaceae
Genus Listeria
Listeria has been divided into seven species with two distinct groups.
Among which the Listeria monocytogenes and Listeria ivanovii are haemolytic
and pathogenic for animals.
The Listeria murrayi and Listeria grayi are nonhaemolytic, rarely isolated and
considered to be non pathogenic.
Among which the genus L. monocytogenes, is the cause of septicaemia, abortion
and CNS infections in a wide range of animal species including humans.
HISTORY
L.monocytogenes first described by Murray (1926) who named it as bacterium monocytogenes because of
characteristic monocytosis infection in laboratory animals.
It was renamed Listerella hepatolytica by Pirie (1927) and the present name given by him in 1940.
The Listeria monocytogenes was first isolated by Gill (1929) from sheep.
HABITAT
Listeria species are widely distributed in the environment and can be isolated from soil, faeces, plants, decaying
vegetation and silage (pH 5.5) in which the bacteria can multiply.
Silage is commonly implicated in outbreaks of listeriosis in cattle and sheep.
In poor quality sailage the listerial numbers may reach 107 cfu/kg of silage.
Asymptomatic faecal carriers occur in man and many animal species.
L.monocytogenes can be excreted in bovine milk.
Human foods associated with listeriosis in man include soft cheeses, milk and poultry meat
Morphology
L.monocytogenes are medium sized, Gram+ve rods, non-spore forming and non-acid fast.
Old cultures stain Gram –ve. From rapidly growing cultures or animal tissues the cells can appear coccal.
They are motile by few (1-5) peritrichous flagella. They are motile at room temperature, but not at 37°C.
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Cultural characteristics
L.monocytogenes is able to grow at temperature ranges from 4 to 45°C and grow at pH range of 5.5 to 9.6.
It is relatively resistant to high salt (10%) concentrations. They are facultative anaerobes.
The growth is enhanced by agar enriched with glucose, blood, liver extract and by 10% Co2.
They grow on nutrient agar, blood agar but not on MacConkey agar.
Small transparent colonies with smooth borders appear on blood agar in 24hrs, becoming grayish white in 48hrs.
L.monocytogenes and other non-pathogenic listeria produce narrow zones of beta haemolysis, often only under the
colony itself.
L.ivanovii produces a comparatively wide zone of haemolysis and is very similar in appearance to beta haemolytic
streptococci.
L.monocytogenes produces a CAMP reaction with the haemolysis of S.aureus.
In contrast, Listeria ivanovii is negative in the CAMP reaction with S.aureus.
On TSA or BHI agar, these colonies have a characteristic blue-green sheen when light is reflected obliquely at a 45°
angle off their surface.
In fluid medium, slimy tenacious precipitate forms after incubation for several days.
L.monocytogenes, particularly shows the characteristic tumbling motility when a 2-4 hr broth culture, incubated at
25°C, is examined by the hanging drop method.
This motility is an end-over-end tumbling of individual cells with periods of quiescence.
When grown in semisolid motility media the Listeria spp. give an unusual umbrella shaped growth in the
subsurface.
Biochemical properties
All the Listeria species hydrolyse aesculin, Catalase +ve, Oxidase –ve, Indole –ve.
They produced acid from glucose and rhamnose, but not from xylose and mannitol. Nitrates not reduced.
Resistance
It is killed by moist heat at 55°C for 40 minutes and is readily susceptible to the lethal effects of disinfectants.
Under natural conditions, in summer they survive for 1 month and in winter for 3-4 months.
Based on somatic and flagellar antigens, so far 16 serovars have been identified.
Of these 16 serovars, all cases of animal and human infections are caused by 3 serotypes. ½ a, ½ b and 4 b.
Numbers indicate O antigen and alphabet indicate H antigens.
PATHOGENESIS
In both cattle and sheep, listeriosis can manifest itself in four ways
o as a CNS infection (meningo encephalitis in adults and meningitis in the young)
o as abortion
o as a generalized septicaemia with involvement of the liver and other organs
o as mastitis in dairy cattle.
Flow Chart
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Silage is commonly implicated in outbreaks of Listeriosis in cattle and sheep. Most pathogenic bacteria require the
availability of iron in the host for metabolic activities.
High iron levels in silage that lead to elevated tissue concentrations of iron may predispose cattle and sheep fed on
silage to Listeriosis.
The pathogenic listeria can penetrate the epithelial barrier in the intestine and multiply in hepatic and spleenic
macrophages aided by the haemolysin named listeriolysin O. It leads to septicaemia.
If the pathogen penetrates through damaged mucosal surfaces to the CNS, via the trigeminal nerve or an alternate
route it may penetrate through the dental pulp (when sheep are cutting or losing teeth) to the CNS, resulting in
neural form.
PATHOGENECITY
Symptoms
Four syndromes
o Subclinical
Infections are the most common form of infection.
Usually outbreak occurs when fed with poor quality, high pH silage, particularly during cold
weather.
o Neonatal infections
Characterised as visceral infections with a septicemia.
Often gastroenteritis and bilateral meningitis.
Deaths are frequent in neonatal animals.
o Listerial abortion
It is a sporadic condition in cattle and sheep.
It occurs after 4-8 months pregnancy. Retained placenta is common.
o Neural Listeriosis (circling disease):
The incubation period ranges from 14 to 40 days.
The disease is more common in winter or early spring.
The clinical presentation of meningoencephalitis in adult ruminants may begin with signs of
depression and confusion.
The ears droop; animal holds its head to one side.
Protrusion of the tongue and salivation are common and twitching or paralysis of the facial and
throat muscles may occur.
When the animal moves, it tends to be in a single direction, giving rise to the common name of
circling disease.
In the terminal stages, the animal may fall and will be unable to rise.
o In poultry, there are signs of
Torticolis
Weakness
Incoordination of legs and
Sudden death in young birds
The disease is usually fatal in sheep, pigs and horses.
Lesions
Micro-abscess in the brain stem, usually unilateral, together with perivascular cuffing is very characteristic of
listeriosis.
The lesions are most common in the mid brain, pons and medulla oblongata.
In addition to this there will be generalized septicaemia, focal necrosis of the liver and spleen will be seen.
DIAGNOSIS
Stained smears from lesions may reveal Gram +ve rods (often coccobacillary)
Histopathological examination of brain tissue can often give a presumptive diagnosis of neural listeriosis
Isolation and Identification
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o Inoculation of specimens on selective media include blood agar with an antibiotic supplement or blood
agar containing 0.05% potassium tellurite (inhibitory to Gram –ve).
o Specimens from the visceral form of the disease or from abortion cases are inoculated directly onto the
laboratory media.
o A cold-enrichment procedure is necessary for brain tissue from neural listeriosis.
o Small pieces of spinal cord and medulla are homogenized and a 10% suspension is made in a nutrient
broth.
o The broth suspension is placed in the refrigerator at 40C and sub cultured on to blood agar once weekly
for upto 12 weeks.
Inoculation in developing chicken embryos causes development of focal necrotic lesions on the chorio allantoic
membrane.(CAM)
Anton’s test
o Inoculation of live bacterial suspension into the conjunctiva of a rabbit or guinea pig
only L.monocytogenes causes a purulent keratoconjunctivitis within 24-36hrs of inoculation.
Intra peritoneal inoculation of mice with a 24hr broth culture.
o Both L.monocytogenes and L.ivanovii are pathogenic for mice.
o They die within in 5 days with necrotic lesions present in the liver.
Specimens to be collected
Visceral form
o Material from lesions in liver, kidneys or spleen
Neural form
o Spinal fluid, brain stem, and tissue from several sites in the medulla
oblongata
Abortion
o Placenta (cotyledon), foetal abomasal contents and/or uterine discharges.
Ruminants in early stages of septicaemic listeriosis respond to systemic therapy
with ampicillin or amoxicillin.
Response to antibiotic therapy may be poor in neural listeriosis although
prolonged higher doses of ampicillin or amoxicillin combined with an
aminoglycoside may be effective.
Ocular listeriosis requires treatment with antibiotics and corticosteroids injected
subconjuctivily.
Poor quality silage should be avoided. Vaccination with killed vaccines, which do
not induce effective cell-mediated immune response, is not protective
because L.monocytogenesis an intracellular pathogen. Live, attenuated vaccines,
which contain serovars 1/2a, 1/2b, and 4b are reported to reduce the prevalence
of listeriosis in sheep.
SYSTEMATICS
Domain Bacteria
Phylum Firmicutes
Class Mollicutes
Order Incertae sedis
Family Erysipelotrichaceae
Genus Erysipelothrix
Species Erysipelothrix rhusiopathiae
HABITAT
The bactrerium is widespread in nature and has been recovered from a wide variety of wild and domestic animals
including mammals, fish (both fresh and salt water), birds, reptiles and amphibians.
It is present in the soil and can survive for 20 days or longer in alkaline soil.
The major source of infection for swine and turkeys is carrier animals of the same species.
It is reported that 30-50% of pigs carry the bacterium in their tonsils, other lymphoid tissues.
It is present in slurry of piglets and can be recovered from the faeces of carrier pigs.
MORPHOLOGY
Erysipelothrix rhusiopathiae(previously named Erysipelothrix insidiosa form S (Smooth) - form colonies and
usually from acute syndromes is a Gram-positive rod, the R (rough) form colonies usually from chronic disease is a
Gram-positive filament.
The organism is non-motile, non-spore forming, non-acid fast, occur either in singly, in groups or in chains.
CULTURAL CHARACTERISTICS
BIOCHEMICAL PROPERTIES
Erysipelothrix rhusiopathiae usually ferments lactose, glucose, levulose and dextrin. But the acid production is
poor.
To obtain good result, carbohydrate tests can be carried out in peptone water with added sterile horse serum (5-
10%) with phenol red as the indicator.
Indole, Methyl red and Voges proskauer tests are negative.
Erysipelothrix rhusiopathiae is resistant to several chemicals including sodium azide, and to drying, pickling,
salting and smoking.
It is capable of surviving for nearly a year in putrefying meat. But they are susceptible to caustic soda and
hypochlorites.
They are readily killed in moist heat at a temperature of 55ºC for 10 mts.
Based on heat labile and heat stable antigens so far 23 serotypes have been identified.
Strains of serotype 1 are subdivided into 1a and 1b. Serotypes 1a, 1b and 2 are most frequently involved in disease in
swine.
Hyaluronidase and neuraminidase are produced by some strains.
PATHOGENESIS
PATHOGENECITY
Symptoms
Erysipelas occurs in pigs of all ages, but pigs from 2 months to one year age are highly susceptible.
Four forms of clinical disease in swine have been described.
o Acute septicaemia
o Urticarial or diamond skin lesions
o Vegetative endocarditis
o Arthritis
These may occur alone or in combination. Swine erysipelas manifest in three forms.
Acute
The acute disease is characterized by high fever, inappetance, depression, a rapid course of illness, and death
within 2-3 days in untreated animals.
Some animals may show a stiff gait and reluctance to stand or move, and urticarial cutaneous lesions may develop.
The diamond shaped raised skin lesions are pathognomonic. Pregnant sows may abort.
sub acute
Sub acute disease is similar to the acute except that it is less severe and animals are likely to recover within 5-7
days.
chronic course
Specimens to be collected
Treatment
In addition to hyper immune serum, treatment with antibiotics such as penicillin and tetracyclines are effective.
Workers engaged in the fish and poultry industries are highly susceptible.
The organism enters through minor skin abrasions causing localized cellulitis referred as erysipeloid.
SYSTEMATICS
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
Subclass Actinobacteridae
Order Actinomycetales
Suborder Corynebacterineae
Family Mycobacteriaceae
Genus Mycobacterium
The genus includes animal and human pathogens as well as saprophytic members often referred to as atypical,
anonymous, opportunistic, tuberculoid and MOTT (Mycobacteria other than typical tubercle) bacilli.
CLASSIFICATION
Mycobacterium leprae
o Addition to this the unspecified acid-fast bacilli such as Mycobacterium
senegalense and Mycobacterium farcinogens were isolated from Bovine
farcy.
HISTORY
HABITAT
It has a worldwide distribution. The usual habitats of the great majority of the cultivable mycobacteria are water
and watery habitats, marshes, wet soil, streams, lakes, rivers.
The source of the pathogenic mycobacteria is usually infected animals.
Mycobacterium bovis is excreted in respiratory discharges, faeces, milk, urine and semen.
Mycobacterium avium and Mycobacterium paratuberculosis are shed in faeces and Mycobacterium
tuberculosis mainly in respiratory discharges.
The atypical mycobacteria are widespread in soil, pastures, grass and water.
A few are commensals in animals and may infect them.
MORPHOLOGY
Mycobacteria are slender rods of varying lengths that sometimes show branching filamentous form resembling
‘fungal mycelium’.
Hence, the name mycobacteria, meaning fungus like bacteria.
Although cytochemically Gram positive, the Mycobacteria do not take up the dyes of the Gram stain because the
cell walls are rich in lipids – Mycolic acid.
Once a dye has been taken up by the cells they are not easily decolourised, even by acid-alcohol. Mycobacteria are
therefore called as acid-fast bacilli.
They are usually straight or slightly curved rod occurring singly, pairs or in small groups. The morphology varies
from cells of species to species.
Mycobacterium tuberculosis is often arranged in serpentine cords.
Mycobacterium kansasi is distinct banded or beaded appearnce, while
Mycobacterium avium is often almost coccoid.
In clinical materials they may appear as bundle of faggots. They are non-motile, non-sporing and non-capsulated.
Cultural characteristics
A comparatively slow growth rate is characteristic of the mycobacteria, with generation time ranging from 14-20
hours.
Colonies appear only in about two weeks and sometimes may be delayed upto 6-8 weeks. Optimum temperature is
37°C and pH is 6.4 –7.0.
Mycobacterium tuberculosis is an obligate aerobe while Mycobacterium bovis is microaerophilic. Growth is
stimulated by 5-10% CO2.
Tubercle bacilli do not have exact growth requirements. But they are highly susceptible to even traces of toxic
substances like fatty acids in culture media.
The toxicity is neutralized by addition of serum, albumin or charcoal.
Several media, both solid and liquid, are available. The egg based Lowenstein Jensen medium and Stone Brink's
medium are most commonly used.
Malachite green dye (0.025 g/100ml) is commonly used as the selective agent.
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Mycobacterium tuberculosis, Mycobacterium avium and many of the atypical mycobacteria require glycerol for
growth. However glycerol is inhibitory to Mycobacterium bovis, while sodium pyruvate enhances its growth.
On Lowenstein Jensen medium (i.e. glycerol containing media), Mycobacterium tuberculosis giving the
characteristic rough, tough and buff colonies – is known as eugonic.
The growth of Mycobacterium avium in this medium is also described as eugonic. Mycobacterium bovis has
sparse, thin growth on glycerol containing media that is called dysgonic.
Mycobacterium bovis however grows well on pyruvate containing media without glycerol (i.e. Stone brink's
medium).
Pigment formation is tested with young, well-developed colonies on Lowenstein Jensen medium.
The cultures are exposed to a 100-watt, clear electric bulb, at a distance of 50 cm, for atleast an hour and then
incubated again in darkness for a further 1-3 days.
After this treatment the photochromogens will develop pigement.
Many of the mycobacteria produce yellow/orange pigments while Mycobacterium tuberculosis, Mycobacterium
bovis and Mycobacterium avium are non-chromogenic.
In liquid media, the growth begins at the bottom, creeps up the sides and forms a prominent surface pellicle
(mould like pellicle) that may extend along the sides above the medium.
Virulent stains tend to form long serpentine cords on liquid media, while avirulent strains grow in a more
dispersed fashion.
Supplementation of media with mycobactin (extracted from non-mycobactin dependant isolates
of M.avium subsp. paratuberculosis) is required for M.avium subsp.paratuberculosis.
Biochemical Properties
They are oxidative. Atypical mycobacteria are catalse positive, while tubercle bacilli are peroxidase positive.
Niacin production and nitrate reduction is only by Mycobacterium tuberculosis.
Urease is reduced by Mycobacterium tuberculosis and Mycobacterium bovis, but not by avian strain.
RESISTANCE
The Mycobacteria are resistance to physical influences and will retain their viability in soil and particles of dried
faeces for many months.
They are not specifically heat resistant; being killed at 60ºC in 15-20 mts .
Cultures may be killed by exposure to direct sunlight for two hours.
Bacilli in sputum may remain alive for 20-30 hrs and in droplet nuclei for 8-10 days .
They are relatively resistant to disinfectants i.e. exposure to 5% phenol, 15% H2SO4, 3% nitric acid, 5% oxalic acid
and 4% NaOH.
It is destroyed by tincture of iodine in 5mts and by 80% ethanol in 2-10 mts.
Many antigens have been identified in mycobacteria. Group specificity is due to polysaccharide and type specificity
is due to protein antigen.
They do not produce any exotoxins. The cell wall of the mycobacterium is composed of peptidoglycan,
arabinogalactan and mycolic acid.
In addition to this it contains wide range of lipids. The outer layer of the cell wall is composed of mycosides.
(Peptidoglycolipids or Phenolic glycolipids).
Mycosides are responsible for the control of cellular permeability, resistance to action of water-soluble enzymes,
antibiotics and disinfectants.
Cord factor (Trehalose – 6,6’ dimycolate) and Wax D - inhibits chemotaxis, leukotaxis, responsible for delayed
hypersensitivity
Sulfatides- sulfur containing glycolipids – promote the survival of virulent tubercle bacilli within macrophages by
inhibiting phagolysosome formation and avoiding exposure to hydrolytic enzymes present in the lysosomes.
Virulence appears to reside in the lipids of the cell wall. Mycosides, phospholipids and sulpholipids are protecting
the tubercle bacilli against phagocytosis.
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PATHOGENESIS
Lesions
Infection is usually by inhalation and ingestion. The mucociliary clearance by mucus and epithelial cilia in the
upper respiratory passages provides defense against infection.
However, microorganisms on small particles (1-4 μm in size), such as, dust and water droplets reach alveolar
spaces.
In previously unexposed animals, local multiplication of the mycobacteria occurs and the resistance to phagocytic
killing allows continued intra cellular and extra cellular replication.
Infected host cells with mycobacteria can reach local lymphnodes and from there may pass to the thoracic duct
with general dissemination.
After 10-14 days, CMI responses develop and activated macrophages are able to kill some mycobacteria.
The aggregation of macrophages contributes to the formation of a tubercle, and a fibrous layer may encompass the
lesion.
Caseous necrosis due to the cell death and tissue destruction occurs at the center of the lesion and this may proceed
to calcification or liquefaction.
Once CMI is established, the lymphatic spread is retarded but occurs via the erosion of bronchi or blood vessels to
new area.
Haematogenous spread may produce miliary tuberculosis (in deer). This involves multifocal tubercle formation in
an organ.
PATHOGENECITY
Cattle
Tuberculosis consists of a characteristic lesion – the tubercle. This is an avascular granuloma composed of a
caseous necrosis in a central area encircled by a zone of epitheloid cells, and a peripheral zone of lymphocytes,
granulocytes and fibroblasts.
Calcification may be present in the necrotic centers.
An outer boundary of fibrous tissue is usually present between the lesions and normal tissue.
Tubercle lesions are more commonly present in the lymphnode, lungs and pleura.
Military tuberculosis resembling millet seeds with similar lesions in various organs can be formed by the
haematogenous route.
Horses
Common sites are liver, spleen and lungs. In pigs, the skeleton, especially vertebrate and long bones, are common
sites.
Birds
The grayish white granulomatous lesions are found in the liver and they are also present in the intestines, spleen
and bone marrow.
Specimens to be collected
Specimens from live animals include aspirates from cavities, lymph nodes,
biopsies, tracheobronchial lavages and centrifuged deposit from about 50 ml of
milk in the case of suspected tuberculous mastitis. With dead animals, collect
fresh and fixed (10% formalin) samples of lesions.
Diagnosis
Bacteriophage typing: A, B, C, I
Tuberculin test (Intra dermal, double intra dermal, ophthalmic tests in
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MORPHOLOGY
CULTURAL CHARACTERISTICS
Mycobacterium avium subsp paratuberculosis requires mycobactin - Killed extract of M.phelei or other killed
acid-fast organism- enriched media for growth.
Slants of Herrold’s egg yolk medium with mycobactin are highly suitable for isolation of organism from specimens.
The slants are incubated at 370C for upto 16 weeks and examined weekly for evidence of growth.
They produce minute grayish white, friable, irregular colonies, less than 1 mm in d.m., in 5-16 weeks. Isolates from
sheep may be pigmented.
PATHOGENESIS
The organism is shed in the faeces, milk and semen of infected animals.
They remain viable in the environment for up to one year under suitable conditions.
Calves under one month of age are highly susceptible and they develop clinical disease than animals infected later
in life.
Infection is acquired mainly through ingestion.
The organism is an intra cellular pathogen and cell mediated reactions are mainly responsible for the enteric
lesion.
Ingested mycobacteria, engulfed by macrophages in which they survive and replicate, are found initially in Peyer’s
patches.
As the disease progresses, an immune mediated granulomatous reaction develops, with marked lymphocyte and
macrophage accumulation in the lamina propria and submucosa.
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The resulting enteropathy leads to loss of plasma proteins and malabsorption of nutrients and water.
PATHOGENICITY
Symptoms
Clinical signs develop after prolonged subclinical phase of infection. Affected cattle are usually more than 2 years of
age when signs are first observed.
In cattle, the disease is characterized by diarrhoea, initially intermittent, dark and semisolid, but becoming
persistent and profuse.
Progressive weight loss results without loss of appetite, leading to emaciation and eventually death.
The mortality rate may approach 100%. Asymptomatic carrier cattle have an increased incidence of mastitis and
infertility.
In sheep and goats, the disease is clinically evident only in mature animals. The diarrhoea is less marked and may
be absent.
Lesions
DIAGNOSIS
Specimens to be collected
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Specimens for direct microscopy from live animals include scrapings /pinch biopsies from the rectum. Faeces may
be submitted for culture.
In case of dead animals, tissues from affected region of the intestines and from regional lymphnodes are useful for
histopathology.
Diagnosis by
SYSTEMATICS
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
Subclass Actinobacteridae
Order Actinomycetales
Suborder Corynebacterineae
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Family Nocardiaceae
Genus Nocardia
History
Nocard described this organism in 1888, following its isolation from a case of bovine farcy , hence the name of the type
species is Nocardia farcinica.
Habitat
MORPHOLOGY
Nocardia has the ability to form Gram-positive, branching filaments of less than 1um in d.m.
It is closely related to Corynebacterium, Mycobacterium and Rhodococcus species.
They are obligate aerobes. Some produce true mycelia and some strains are acid-fast. All species are non-motile.
Gram stained smears from lesions revealed Gram-positive branching filaments that often showed fragmentation
into coccobacillary elements.
The modified ZN stained smears exhibit a similar morphology but most of the filaments retain the carbol-fuchsin
dye and stain red.
The Nocardia species grow very well in blood agar incubated aerobically at 370C for up to 7 days.The colonies on blood
agar are often vivid white and powdery if aerial filaments and spores are formed. Occasionally the colonies are smooth,
heaped and variably pigmented.
Inoculate the suspected colonies from blood agar into Sabouraud dextrose agar (SDA) and incubate at 370C for up to 10
days.
Both types of colonies firmly adherents to the agar surface. The colonies on SDA are dry, wrinkled and yellow, becoming
deep orange color with age.
Gram-stained smears from colonies show Gram-positive branching filaments that characteristically break up into rods or
coccobacillary elements with age.
An MZN –stained smear from young culture reveals red staining, branching filaments.
Group I strains have limited mycelia development due to early fragmentation of hyphae into coccoid forms within 2 to 14
hours of incubation.
Group II strains produce mycelia, which fragment in about 18 to 20 hours after incubation, though these mycelia break up
into mycelial fragments within two days of growth.
The pathogenic Nocardia species belong to Group III. The colonies are usually leathery in appearance and pigmented .
Extensive mycelium produced because fragmentation does not begin until after 5 days incubation.
Biochemical Properties
To differentiate Nocardia species tests such as decomposition of casein, hypoxanthine, tyrosine, urea and xanthine are
useful.
They are oxidase and Catalase positive. Reduce nitrates to nitrites. Gelatin not hydrolyzed.
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PATHOGENESIS
PATHOGENICITY
Nocardia species have been isolated from a variety of clinical situations, though the genus is in general an
opportunistic pathogen.
In primary nocardiosis, severe, suppurative or cavitary pulmonary infection simulating tuberculosis is often
observed and in some cases, show cutaneous and subcutaneous abscesses which are diagnostic of N. asteroids.
Blood stream invasion with secondary, often fatal, involvement of the internal organs and the central nervous
system are seen in N. asteroids.
In the bovine species the most important disease condition is mastitis whose presentation is in the form of
extensive fibrosis. N. asteroids is the most often isolated species.
DIAGNOSIS
Specimens to be collected
o Specimens should include exudates, aspirates, mastitic milk samples, tissue from granulomas and thin
sections from granulomas in 10% formalin for histopathology.
Based on direct microscopy
o Soft granules are not common in exudates from N. asteroides infections. Smears made from exudates,
aspirates, granulomatous tissue and from centrifuged deposits of bovine mastitic milk are stained by
Gram's and MZN stain.
o Gram-stained smears reveal Gram-positive branching filaments that often show some fragmentation into
coccobacillary elements.
o The MZN –stained smears exhibit a similar morphology but most of the filaments retain the carbol
fuchsin dye and stain red.
Based on isolation and identification
o Characteristic colonial morphology on blood and SDA agar and microscopic appearance.
Based on biochemical reactions
Differential diagnosis with Actinomyces
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Actinomyces infections respond well to penicillin and other commonly used antibiotics, nocardial infections are
often refractory to treatment and N. asteroides is susceptible only to limited range of antimicrobial agents such as
Trimethoprim-sulphamethoxazole or erythromycin.
Note
Infection with Nocardia is by inhalation from the environment, while Actinomyces infection begins in the host as a
normal flora invading damaged tissues.
Disseminated disease caused by Nocardia is more common in the dogs, while granuloma formation is the rule with
Actinomyces infection and spreads by local extension.
When there is a doubt as to whether an animal has actinomycosis or nocardiosis, precautionary measures to
preserve the anaerobic Actinomyces should be instituted, including prompt delivery to the laboratory under
anaerobic condition, culturing on brain heart infusion agar, blood plates and incubating under anaerobic,
microaerophilic, and aerobic conditions.
While Actinomyces fails to grow on Sabouraud agar, Nocardia grows uninhibited.
Acid-fast staining procedure of the sample exudate before culturing will also be helpful in the presumptive
identification of the infecting agent.
SYSTEMATICS
The actinomycetes comprise a heterologous group of prokaryotes that have the ability to form Gram positive,
branching filaments of less than 1μm in diameter.
The main animal pathogens in the actinomycetes are in the
genera Actinomyces, Arcanobacterium, Actinobaculum, Nocardia and Dermatophilus.
Non-pathogenic, prolific producers of antimicrobial substances – streptomyces are also included in Actinomycetes
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
Subclass Actinobacteridae
Order Actinomycetales
Suborder Actinomyceneae
Family Actinomycetacea
Genus Actinomyces, Arcanobacterium
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The Actinomyces species are present on mucous membrane of the host animal, often in the oral cavity, tonsils, and
nasopharynx.
The soil is the natural habitat of many Actinomyces species.
The generic name Actinomyces was first used by Harz (1879). Boestrom (1891) isolated Actinomyces bovis.
Cummins (1962) clearly demonstrated Actinomyces were bacteria and they were distinct from other branching
genera.
MORPHOLOGY
The organisms show considerable pleomorphism. Actinomyces species are usually long and filamentous although
short V, Y, and T configuration also occur.
In lesions of actinomycosis, the pus contains small pale yellow granules referred as sulfur granules .
The sulphur granule is composed of bacterial filaments and mineralized calcium phosphate of host origin.
When the granules are crushed and Gram stained, a mass of Gram-positive branching filaments about 1μm in
width, short rods, and cocci are evident.
Around this mass, a circle of club shaped bodies with their narrow ends pointing towards the centre-staining Gram
negative. Hence, called ray fungus .
They are non-acid fast, non-spore forming, nonmotile, non-capsulated and do not form endospores or conidia.
In case of Arcanobacterium pyogenes infections the pus or mastitic milk does not contain any granules.
Gram stained smears reveal large numbers of small, highly pleomorphic, Gram-positive rods, cocci and pear
shaped cells.
Occasionally short branching typical Chinese letter appearances are also seen.
CULTURAL CHARACTERISTICS
They cannot grow on Sabouraud dextrose agar. Actinomyces require enriched media for growth. They grow well on
sheep or ox blood agar.
Actinomyces bovis is capnophilic (i.e. required 5-10% CO2 for its growth).
Arcanobacterium pyogenes and Actinomyces viscous will grow aerobically but 5-10% CO2will enhance their
growth.
Actinomyces bovis and Actinomyces viscous usually require 2-4 days but the growth of Arcanobacterium
pyogenes can usually be seen in 24 hrs.
Actinomyces bovis colonies are non-haemolytic, very small (< 1nm), white, rough or smooth and adhere
tenaciously to solid medium.
Gram stained smears show Gram positive, slightly branched filaments or short forms. On subculture, the
bacterium may become diphtheroidal or coccobacillary.
Actinomyces bovis grows well in thioglycollate medium, giving a characteristic diffuse growth in about 7-10 days.
In broth cultures, it grows in coarse aggregates, which in some cases may result in a granular deposit with a
completely clear supernatant.
Arcanobacterium pyogenes produce a hazy- haemolysis after 24hrs incubation along the streak lines.
At 48 hrs incubation, the colonies are surrounded by a narrow zone of complete haemolysis.
Arcanobacterium pyogenes has the ability to pit a loeffler serum slope in 24-48 hrs. (i.e. A loopful of pure culture
of the medium is taken and a heavy inoculum is made in a small area in the center of the slope, taking care not to
break the surface of the medium. The medium is incubated at 370C for 24 –48 hrs).
Arcanobacterium pyogenes will give positive CAMP test with Staphylococcus aureus (i.e. enhancement of
staphylococcal haemolysis).
In litmus milk, the organism produces acid and clot after 3 days of growth.
Actinomyces viscous commonly produces two colonial forms, one being smooth, entire, convex and glistening and
the other is smaller, rough dry and irregular.
Neither is haemolytic. The larger colonial type yields Gram-positive diphtheroid forms and the smaller colony has
short branching filaments.
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Biochemical tests
Both Arcanobacterium pyogenes and Actinomyces bovis are catalase negative, ferments several sugars and
produce acid.
Reduction of nitrate is negative. Actinomyces viscous is catalse positive.
Resistance
Actinomyces are killed at a moist heat temperature of 600C for 20 mts and they are susceptible to various
disinfectants.
With the exception of Arcanobacterium pyogenes, Actinomyces species have not been shown to produce any toxin.
Arcanobacterium pyogenes produces a haemolytic exotoxin, which is dermonecrotic and lethal and it also
produces a protease and an extracellular neuraminidase.
PATHOGENESIS
PATHOGENICITY
Symptoms
In case of lumpy jaw in cattle there is marked swelling associated with suppurative and proliferative osteomyelitis
in the region.
Lumpy jaw produces ill health by interfering with mastication.
Arcanobacterium pyogenes infection occurs most frequently in heifer and dry cows during summer months.
Hence, it is named as summer mastitis.
The affected quarter become enlarged and firm.
Animals have fever with general toxaemia.
The mortality and morbidity rate may be high.
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Lesions
In lumpy jaw, area of suppuration accompanied by the granulation tissues, erosion of old bones and formation of
new bones.
The pus characteristically contains small sulphur granules.
In summer mastitis, abscess develops at any site containing greenish yellow foul smelling pus.
Diagnosis
Specimens to be collected
It includes pus, exudates, aspirates, tissue and scrapings from the
wall of abscesses.
If they have been incised. A volume of fluid or pus should be
collected and submitted, if possible, rather than just a small amount
on a swab.
Thin sections of granulomas in 10% formalin are useful for
histopathology.
Direct microscopy
o The pus or exudate is placed in a Petridish and washed carefully with a
little distilled water to expose the yellowish sulphur granules
of Actinomyces bovis or the softer greyish white granules of Actinomyces
viscous.
o A granule is placed on a microscopic slide in a drop of 10% KOH and
gently crushed by applying pressure on the cover slip.
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o The characteristic clubs can be examined under the low power microscope.
o If it is stained with Gram’s, the ray fungus can be demonstrated.
Isolation and Identification of organism
Fat
Pitting of loeffler serum slope and CAMP test in case of Arcanobacterium
pyogenes
SYSTEMATICS
The actinomycetes comprise a heterologous group of prokaryotes that have the ability to form Gram positive,
branching filaments of less than 1μm in diameter.
The main animal pathogens in the actinomycetes are in the
genera Actinomyces, Arcanobacterium, Actinobaculum, Nocardia and Dermatophilus.
Non-pathogenic, prolific producers of antimicrobial substances – streptomyces are also included in Actinomycetes
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
Subclass Actinobacteridae
Order Actinomycetales
Suborder Actinomyceneae
Family Actinomycetacea
Genus Actinomyces, Arcanobacterium
The Actinomyces species are present on mucous membrane of the host animal, often in the oral cavity, tonsils, and
nasopharynx.
The soil is the natural habitat of many Actinomyces species.
The generic name Actinomyces was first used by Harz (1879). Boestrom (1891) isolated Actinomyces bovis.
Cummins (1962) clearly demonstrated Actinomyces were bacteria and they were distinct from other branching
genera.
MORPHOLOGY
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The organisms show considerable pleomorphism. Actinomyces species are usually long and filamentous although
short V, Y, and T configuration also occur.
In lesions of actinomycosis, the pus contains small pale yellow granules referred as sulfur granules .
The sulphur granule is composed of bacterial filaments and mineralized calcium phosphate of host origin.
When the granules are crushed and Gram stained, a mass of Gram-positive branching filaments about 1μm in
width, short rods, and cocci are evident.
Around this mass, a circle of club shaped bodies with their narrow ends pointing towards the centre-staining Gram
negative. Hence, called ray fungus .
They are non-acid fast, non-spore forming, nonmotile, non-capsulated and do not form endospores or conidia.
In case of Arcanobacterium pyogenes infections the pus or mastitic milk does not contain any granules.
Gram stained smears reveal large numbers of small, highly pleomorphic, Gram-positive rods, cocci and pear
shaped cells.
Occasionally short branching typical Chinese letter appearances are also seen.
CULTURAL CHARACTERISTICS
They cannot grow on Sabouraud dextrose agar. Actinomyces require enriched media for growth. They grow well on
sheep or ox blood agar.
Actinomyces bovis is capnophilic (i.e. required 5-10% CO2 for its growth).
Arcanobacterium pyogenes and Actinomyces viscous will grow aerobically but 5-10% CO2will enhance their
growth.
Actinomyces bovis and Actinomyces viscous usually require 2-4 days but the growth of Arcanobacterium
pyogenes can usually be seen in 24 hrs.
Actinomyces bovis colonies are non-haemolytic, very small (< 1nm), white, rough or smooth and adhere
tenaciously to solid medium.
Gram stained smears show Gram positive, slightly branched filaments or short forms. On subculture, the
bacterium may become diphtheroidal or coccobacillary.
Actinomyces bovis grows well in thioglycollate medium, giving a characteristic diffuse growth in about 7-10 days.
In broth cultures, it grows in coarse aggregates, which in some cases may result in a granular deposit with a
completely clear supernatant.
Arcanobacterium pyogenes produce a hazy- haemolysis after 24hrs incubation along the streak lines.
At 48 hrs incubation, the colonies are surrounded by a narrow zone of complete haemolysis.
Arcanobacterium pyogenes has the ability to pit a loeffler serum slope in 24-48 hrs. (i.e. A loopful of pure culture
of the medium is taken and a heavy inoculum is made in a small area in the center of the slope, taking care not to
break the surface of the medium. The medium is incubated at 370C for 24 –48 hrs).
Arcanobacterium pyogenes will give positive CAMP test with Staphylococcus aureus (i.e. enhancement of
staphylococcal haemolysis).
In litmus milk, the organism produces acid and clot after 3 days of growth.
Actinomyces viscous commonly produces two colonial forms, one being smooth, entire, convex and glistening and
the other is smaller, rough dry and irregular.
Neither is haemolytic. The larger colonial type yields Gram-positive diphtheroid forms and the smaller colony has
short branching filaments.
Biochemical tests
Both Arcanobacterium pyogenes and Actinomyces bovis are catalase negative, ferments several sugars and
produce acid.
Reduction of nitrate is negative. Actinomyces viscous is catalse positive.
Resistance
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Actinomyces are killed at a moist heat temperature of 600C for 20 mts and they are susceptible to various
disinfectants.
With the exception of Arcanobacterium pyogenes, Actinomyces species have not been shown to produce any toxin.
Arcanobacterium pyogenes produces a haemolytic exotoxin, which is dermonecrotic and lethal and it also
produces a protease and an extracellular neuraminidase.
PATHOGENESIS
Symptoms
In case of lumpy jaw in cattle there is marked swelling associated with suppurative and proliferative osteomyelitis
in the region.
Lumpy jaw produces ill health by interfering with mastication.
Arcanobacterium pyogenes infection occurs most frequently in heifer and dry cows during summer months.
Hence, it is named as summer mastitis.
The affected quarter become enlarged and firm.
Animals have fever with general toxaemia.
The mortality and morbidity rate may be high.
Lesions
In lumpy jaw, area of suppuration accompanied by the granulation tissues, erosion of old bones and formation of
new bones.
The pus characteristically contains small sulphur granules.
In summer mastitis, abscess develops at any site containing greenish yellow foul smelling pus.
Diagnosis
Specimens to be collected
It includes pus, exudates, aspirates, tissue and scrapings from the
wall of abscesses.
If they have been incised. A volume of fluid or pus should be
collected and submitted, if possible, rather than just a small amount
on a swab.
Thin sections of granulomas in 10% formalin are useful for
histopathology.
Direct microscopy
o The pus or exudate is placed in a Petridish and washed carefully with a
little distilled water to expose the yellowish sulphur granules
of Actinomyces bovis or the softer greyish white granules of Actinomyces
viscous.
o A granule is placed on a microscopic slide in a drop of 10% KOH and
gently crushed by applying pressure on the cover slip.
o The characteristic clubs can be examined under the low power microscope.
o If it is stained with Gram’s, the ray fungus can be demonstrated.
Isolation and Identification of organism
Fat
Pitting of loeffler serum slope and CAMP test in case of Arcanobacterium
pyogenes
MORPHOLOGY
Corynebacteria are Gram positive, non-acid fast, non-motile, non-capsulated, small pleomorphic rods.
They frequently occur in rods, coccoid, cub and fiamentous shape.
The major pathogen is Corynebacterium diphtheriae, which causes diptheria in children.
Corynebacteria associated with animals are called diphtheroids.
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
Subclass Actinobacteridae
Order Actinomycetales
Suborder Corynebacterineae
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Family Corynebacteriaceae
Genus Corynebactrerium
Family Nocardiaceae
Genus Rhodococcus
MORPHOLOGY
They are Gram-positive slender rods with a tendency to clubbing at one or both ends; they are non-sporing, non-
motile, non-capsulated and non-acid fast.
They have granules composed of (high energy phosphate stores) – polymetaphosphate.
The granules are more strongly Gram positive than the rest of the bacterial cell.
Stained with Loeffler’s methylene blue, the granules take up a reddish purple color and hence they are called
metachromatic granules. They are called as volutin or Babes Ernst Granules.
They are often situated at the poles of the bacilli and are called polar bodies.
Special stains, such as Albert’s, Neisser’s and Ponder’s have been devised for demonstrating the granules clearly.
Stained smears from animal tissues often reveal groups of cells in parallel (Palisades) or cells at sharp angles to
each other (Chinese letter or Cuneiform arrangement).
This is due to the incomplete separation of the daughter cells after binary fission.
Rhodococcus equi can appear as a Gram-positive coccus or a rod or club shaped form arranged in clusters. It is
capsulated and sometimes weakly acid fast.
CUTURAL CHARACTERISTICS
Growth is scanty on ordinary media. Enrichment with blood, serum or egg is necessary for good growth. The
optimum temperature for growth is 37ºC and optimum pH is 7.2. It is an aerobe and facultative anaerobe. Sheep or
ox blood agar is used routinely along with MacConkey agar to detect any Gram-negative contaminants that may be
present.
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On blood agar, Corynebacterium ovis colonies are small, white, dry and non-haemolytic at 24hr incubation.
A narrow zone of haemolysis occurs at 72 hrs incubation. After several days incubation the colonies can reach 3mm
in d.m and appear dry, crumbly and cream in color.
Corynebacterium bovis colonies are small, white, dry and non-haemolytic.
As it is a lipophilic corynebacterium, they grow very well on media enriched with 0.5 –1% tween 80.
Rhodococcus equi colonies are small, smooth, shiny and non-haemolytic after 24 hrs incubation. But on 4-day
culture, the colonies become larger, mucoid and salmon-pink in color.
The salmon-pink pigmentation is not easily seen against a red background.
So, the mucoid colonies and salmon-pink pigmentation can easily be demonstrated on nutrient agar (4-day
culture).
Nutrient agar enriched with yeast extract and glucose is useful for enhancing the salmon pink pigmentation.
The renale groups are non-haemolytic. On nutrient agar, after 48 hrs incubation, Corynebacterium
renale produces dull yellow colonies.
The Corynebacterium pilosum produces distinct yellow and Corynebacterium cystitidis exhibit white colonies.
On milk agar, Corynebacterium renale showing casein digestion. While Corynebacterium
pilosum and Corynebacterium cystitidis do not give this reaction.
On CAMP tests, the Corynebacterium ovis, Rhodococcus equi and Corynebacterium renale interacting with beta
haemolytic strains of Staphylococcus aureus gives the following results.
Biochemical characters
They are catalase positive, oxidase negative. Except Corynebacterium bovis others are urease positive.
The renale group is very strong urease positive (less than one hour).
All diptheroids ferments sugar except Rhodococcus equi. Corynebacterium bovis and Corynebacterium renale
ferments both glucose and maltose.
Two biotypes of Corynebacterium ovis are recognized.
The ovine/caprine strains lack nitrate-reducing capacity, while the equine/bovine strains usually reduce nitrate.
Resistance
PATHOGENESIS
Corynebacterium ovis
phagocytosed.
Phagosome-lysosome fusion takes place. But Corynebacterium ovis multiplies in
the phagolysosome and phagocytic cells die.
Permeability of local blood vessels increases, encouraging the spread of infection
from the initial site to other locations, often-regional lymphnodes.
Produces toxins – Phopholipase-D. Abscesses may develop at either primary or
secondary sites, eventually rupturing and discharging a thick, caseous pus
containing large numbers of viable bacteria. In some instances, lesions become
metastatic and, as they increase in number, the thin ewe syndrome develops,
resulting in progressive debilitation and death.
Corynebacterium renale
Rhodococcus equi
PATHOGENICITY
Symptoms
Corynebacterium ovis
Caseous lymphadenitis in sheep is characterized by skin wounds, enlarged lymph glands and abscesses distributed
throughout the body.
But the disease can be a mild infection and it is unnoticed until postmortem is done.
Ulcerative lymphangitis in horses are similar to glanders.
Initially there is a pain and swelling of the hind limbs and enlargement of lymphatic vessels.
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Ulcers develop, discharging purulent material. In severe cases it spreads to abdomen, forelegs and neck, leading to
death.
Corynebacterium renale
In pyelonephritis, characteristically frequent passage of turbid or blood stained urine by animals, which are
pregnant or recently calved.
Restlessness and kicking at the abdomen may indicate renal pain. The urine contains red blood cells, pus cells and
albumin.
Ulcerative balanoposthitis (Pizzle rot), particularly common in Merino sheep and Angora goats, is characterized by
ulceration around the preputial orifice, with a brownish crust developing over the lesion
Rhodococcus equi
In suppurative bronchopneumonia, the foals develop cough, fever and increased respiratory rate.
In severe cases purulent discharge from nose just before death.
Lesions
Corynebacterium ovis
In caseous lymphadenitis the superficial lymphnode contains a mass of greenish yellow caseation in concentric
layers, which have an onion ring appearnce in, cross section (hence, named it as Corynebacterium
pseudotuberculosis). In advanced cases, similar lesions are seen in lungs, kidney, liver and spleen.
Corynebacterium renale
The kidneys are enlarged, necrosis and suppuration in the medulla. Wedge shaped suppurative foci in the cortex.
The kidney pelvis contains blood and pus
The bladder contains blood and pus with petechial haemorrhages and ulceration of the tract.
Rhodococcus equi
Characterized by pyogranulamatous lesions with abscesses in the lung, associated lymphnodes and pus in the
bronchi.
Diagnosis
Specimens
o Pus or exudates are collected from suppurative conditions and mid stream urine for isolation of
the Corynebacterium renale.
o A tracheal wash technique with infusion of saline, can be used for the recovery of Rhodococcus equi from
affected foals.
Diagnosis is mainly based on history, symptoms and lesions
Isolation and identification of the organism
o Based on microscopical appearance, colonial morphology on blood agar, CAMP tests and Biochemical
tests.
Diphtheroids are susceptible to penicillin, tetracycline, erythromycin, lincomycin, neomycin and gentamicin.
LEARNING OBJECTIVE
Coliforms, white scours, watery mouth, mushy-yolk disease and Hjarre's disease
Diseases caused by E.coli in domestic animals
Antigens, toxins and virulence factors of E.coli
IMViC test
Classification of pathogenic E.coli
Role of Mac Conkey agar in diagnosis of Enterobacteriaceae
General approaches used to isolate, identify and serotyping of E.coli
HABITAT
E. coli is a natural inhabitant of the large intestine and lower small intestine of all
mammals.
It is usually present in large numbers in carnivores, omnivores than in
herbivores.
E .coli is voided in faeces and can survive in faecal particles, dust and water for
weeks or months.
The presence of E .coli in water samples, being tested for potability, is taken as
evidence of faecal pollution.
This genus is named after Theobald Escherich, who was first to describe the
Colon bacillus under the name Bacterium coli commune (1885).
Morphology
Gram negative, rods, non-spores, non-acid fast, occurs either in singly or in pairs.
Usually motile with peritrichous flagella. Some strains possess a polysaccharide capsule and fimbriae.
Cultural characteristics
It is an aerobe and facultative anaerobe, optimum temp. is 37oC, good growth occurs on ordinary media.
Colonies are large, thick, grayish white, moist, smooth and opaque.
The 'S' forms are seen on fresh isolation.
The 'R' forms seen on repeated subculture.
On blood agar, many strains, especially those isolated from pathological conditions, are Beta haemolytic.
As they are very strong lactose fermenters, the colonies on MacConkey agar are bright pink. Click here for visual
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Because of its ability to ferment sugars, it gives yellow - green colonies on Brilliant green agar (BGA) and yellow
colonies on XLD agar.
On Eosin methylene blue agar (EMB), E.coli colonies have a unique and characteristic metallic sheen. Click here
for visual
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In nutrient broth, growth occurs as general turbidity and a heavy deposit, which
disperses completely on shaking.
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Biochemical properties
The four-biochemical tests- IMViC- (Indole, Methyl Red, Voges- Proskauer, Citrate utilization) are widely
employed to identify E.coli.
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Resistance
The capsular antigens (K) are polysaccharides and the cell wall or somatic (O) antigens are determined by the sugar
side chains on the LPS of the outer membrane.
The Flagellar (H) and Fimbrial (F) antigens are proteins.
Three kinds of K antigens L, A, B have been described based on the effect of heat on the agglutinability, antigenicity
and antibody binding power.
The O, H and K antigens can be used to serotype strains of E.coli. So far, 164 types of O antigens, 103 K antigens
and 75 H antigens have been recognized.
The antigenic pattern of a strain is recorded as the number of particular antigen it bears for eg: 0157: K85: H19.
Capsule
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o The capsular polysaccharide is antiphagocytic and also protects the cell wall from the damaging effects of
complement.
Cell wall
o The endotoxin (lipid A) is the toxin associated with colisepticaemias and the toxaemia in coliform
mastitis.
o Lipid A also interferes with the complement components that are responsible for the attack on the outer
membrane of E.coli.
o Endotoxin is released when bacteria die and lyse.
o The effects of endotoxin in the animal body include fever, leukopenia followed by leukocytosis and
hyperglycemia with a subsequent fall in blood sugar and lethal shock after a latent period.
Fimbriae/ Pili
o Fimbrial antigens are adhesins (i.e. they confer the adhesive properties on the organism).
o Several major specific adhesins Type I (mannose sensitive), mannose resistant K88 (F4) and K99 (F5), F6
and F 41 allow the attachment of E.coli to RBC's or glycoproteins on the surface of the epithelial cells of
jejunum and ileum with consequent colonization.
Enterotoxins
o Both heat labile (LT) and heat stable (ST) enterotoxins are produced by the ETEC strains that also have
the K88, K99 or other colonizing agents.
o The LT enterotoxin is a large molecular weight protein and is antigenically related to cholera toxin, which
causes stimulation of asenylate cyclase activity.
o The ST enterotoxin is also a protein and there are two types, STa and STb. STa toxin increases the
guanylate cyclase system.
Verotoxins or shiga like toxins or Odema disease toxin
o It inhibits protein synthesis in host cells following interaction with 60S ribosomal subunit.
o There are two types,SLT I / VT-1- that are neutralized by shiga antitoxin, SLT 2 / VT2e - which causes
odema disease in pigs and haemorrhagic colitis in man and it is not neutralized by shiga antitoxin.
o Shiga like toxin can cause destruction of intestinal epithelial cells where there is dense adherence of the
pathogenic E.coli.
Haemolysins
o E.coli strains produce both alpha and beta haemolysin.
o The alpha haemolysin is a protein that damages host cell membranes and also increases the availbility of
iron, which is required for invading organisms in the host.
Siderophores
o Siderophores – iron-binding molecules are particularly important for the invasive strains.
o In response to low level of iron, the siderophores – aerobactin and enterobactin are synthesized and
released which allows the bacteria to multiply in an environment of limited concentration of free iron.
Colicins
o Colicins are distinctive class of proteinaceous antibiotic substances produced by strains of E.coli, which
was described, by Gratia and Frederiq in 1946.
o They are 40-60 Kda, plasmid encoded proteins that are released extracellularly.
o Characteristically, they become attached to specific receptors on the surface of susceptible bacteria,
causing the death of these organisms.
o Colicin V (COLv) is found primarily among virulent bacteria.
o It is plasmid encoded, small molecule and is released by an export mechanism.
o The Col V plasmid also encode for virulence, increased serum survival, altered motility, changes in
hydrophobicity and facilitates attachment to appropriate host cells.
PATHOGENESIS
Predisposing factors
They are paramount importance for establishing the disease. Young neonates, particularly one week of age, are
particularly susceptible because,
o Normal flora of the intestine is not fully established
o Have a naive immune system
o Receptors for the adhesions of E.coli (i.e. ETEC strains) are present for the first week of life only in calves
and for the first 6 weeks of life in piglets.
In addition to this, poor husbandry practices, insufficient passive immunity, change of environment and healthy
grain diets in weaned pigs leads to massive colonization of the intestine by E.coli.
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PATHOGENICITY
Symptoms
Horse
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Newborn foals suffer from a disease known as Joint ill, navel ill or sleepy foal disease. There will be a rise in
temperature, general weakness, diarrhoea, lameness and death.
Cattle (K99)
It causes white scour or colisepticaemia in calves. In adult cases the symptoms are scouring, weakness, prostration
and death within hours after initial symptoms.
In less acute cases the calves become listless, fail to suck and develop diarrhoea.
The faeces are grayish white in colour (hence the term white scour) with fetid odour.
Other signs include swollen joints and pneumonia. Faeces with blood stained mucus, subnormal temperature,
animal becomes comatose and dies.
E.coli is also associated with acute bovine mastitis.
The udder becomes swollen, hot and painful. Milk production falls rapidly and cease with systemic disturbances.
E.coli infection causes piglet diarrhoea and edema disease. Piglets 4 days old to 3 weeks of age are susceptible.
There will be diarrhoea, listlessness. They may collapse and die.
There may be odematous lesions in various parts of the body.
They will show twitching of the ears and trembling staggering gait.
Poultry (O2,O78,O1)
E.coli infection causes colibacillosis, airsacculitis, Hjarre’s disease or coligranuloma and yolk sac infection.
Death of some birds occurs rapidly while some birds show loss of appetite, respiratory distress, diarrhoea and
weakness.
Coligranuloma is a chronic condition characterized by granulomatous lesions in the epithelium of the intestine and
other organs. In broilers it causes swollen head syndrome.
Dogs (O42)
Similar lo that of calves. The watery mouth is characterized by severe depression, loss of appetite, profuse
salivation and abdominal distension.
Lesions
Cattle
In cases of pneumonia the lungs may show areas of congestion and necrosis.
The spleen and mesenteric lymph nodes are enlarged and congested. Joint infections develop as synovitis.
Pigs
The intestine shows area of congestion and the stomach is filled with clotted milk.
In oedema disease the carcass shows edematous areas of eyelids, ears and face.
The abdominal cavity, pleural and pericardial sac may contain clear fluid containing fibrin.
Poultry
Dogs
The lesions include petechial haemorrhages throughout the carcass, congested lungs and haemorrhagic
gastroenteritis.
DIAGNOSIS
Note
Passive immunization
Passive immunization can be achieved by immunizing sows with E.coli K88 antigen during gestation.
This results in the production of anti K-88 antibodies in the colostrum and milk.
This when ingested by piglets prevent the adhesive capacity of K88 antigen on the bacteria.
In cattle the mother should not be moved to new environment shortly before calving.
Active immunization
Three types of vaccines are available. They are live E.coli K88 antigen (oral vaccine), killed E.coli bacteria with K88
antigen (bacterin) and bacteria free K88 antigen (subunit vaccine).
Vaccination of pregnant cows with purified E.coli K99 fimbrial or whole cell preparations, often combined with
rotavirus antigen, can be used to enhance colostral protection.
Antibiotics have been used in the prevention and treatment of colibacillosis, particularly oxytetracycline,
chlortetracycline, streptomycin, ciprofloxacin, and enrofloxacin. But this has resulted in the development of
resistant strains of E.coli.
HISTORY
Salmonella has long been recognized as important zoonotic pathogen of worldwide economic significance in
humans and animals.
Infections of animals with various species of Salmonella sometimes result in serious disease.
The interplay of Salmonella with its host takes a variety of forms, including remarkable host specificity, inapparent
infection, recovered carriers, enteritis, septicaemia, abortion and combination of disease syndromes.
The typhoid bacillus Salmonella typhi was first observed by Eberth (1880) in the mesenteric nodes and spleen of
fatal cases of typhoid fever and was isolated by Gaffky (1884).
It came to be known as Eberth – Gaffky bacillus or Eberthella typhi.
Salmon and Smith (1885) described a bacillus, which was believed to cause hog cholera in swine.
This bacillus, now called Salmonella cholera suis was the first of a series of similar organisms to be isolated from
animal and man- the genus Salmonella
Salmonellae currently comprise about 2400 serotypes, of which 50 of them are potentially pathogenic.
HABITAT
The reservoir for salmonellae is the intestinal tract of warm blooded and cold-blooded animals.
The majority of infected animals become sub clinical excretors resulting in contamination of water, food and the
environment.
In Poultry some serotypes, such as Salmonella enteritidis infect the ovaries and be transmitted through eggs.
The undercooked egg dishes may result in human food poisoning.
The salmonellae can survive for 9 months or more in moist soil, water, faecal particles and animal feeds, especially
in blood, bone and fish meal.
MORPHOLOGY
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Cultural characteristics
They are aerobic and facultative anaerobic, growing readily on simple media over a pH range of 6.8. Optimum
temperature for growth is 370C.
On nutrient agar the colonies are large, circular, low convex and smooth.
The selective enriched media for salmonellae are tetrathionate broth, selenite broth and rappaport vasiliadis
medium.
The host adapted serotypes from pigs and poultry are more fastidious than others.
They do not tolerate selenite broth and tetrathionate broth. In this case, Rappaport is highly suitable.
After 24-48 hr incubation on selective broth the subculture will be made on MacConkey agar, Brilliant green agar,
XLD and Salmonella Shigella (SS) agar.
The majority of salmonellae, except some strains of S.arizonae, are non-lactose fermenters and produce pale or
colorless colonies on MacConkey agar.
Most salmonellae give an alkaline reaction in brilliant green agar and have red colonies.
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On XLD medium, they produce H2S and have red colonies with a black center (Black center with red skirt).
On Salmonella and Shigella agar they produce colorless colonies with black center.
The typical reaction for salmonellae in TSI (triple sugar iron) agar is an alkaline slant (red), acid butt (yellow) and
superimposed H2S (black) production (R/Y/H2S+). Click here for visual
The test for lysine decarboxylation is positive.
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Biochemical properties
Salmonella gives IMViC test -, +,-,+. They ferment maltose, mannitol, mannose and glucose and produce acid and
gas. But do not ferment lactose, sucrose and salicin.
Urease negative. Most salmonellae produce H2S except S.cholera suis and S.paratyphi A.
Salmonella pullorum ferments glucose and rhamnose while S.gallinarum ferments dulcitiol and maltose.
Resistance
Antigens
Flagellar antigen
It is heat labile protein when mixed with H antisera, agglutinates rapidly producing large, loose fluffy clumps.
The H antigen is strongly immunogenic. They have the unique character of diphasic variation.
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Somatic antigen
It is a heat stable phospholipid protein polysachharide complex, which forms an integral part of the cell wall.
It is identical with endotoxin.
Boivin is extracted from the bacterial cell by treating with trichlor acetic acid hence, it is called as Boivin antigen.
When mixed with O antiserum, it forms compact chalky granular clumps. The O antigen is less immunogenic than
H antigen.
Vi antigen
Many strains of S.typhi, S.paratyphi and S.dublin fail to agglutinate with O antiserum due to the presence of Vi
antigen enveloping the O antigen.
The Vi antigen is related to virulence and may be lost by serial subculture. The Vi antigen is poorly immunogenic.
In addition to endotoxin, enterotoxins similar to heat labile entero toxin of E.coli and cholera toxin are produced.
The cytotoxin similar to shigella cytotoxin is also produced.
Antigenic variations
H-O variation
This variation is associated with loss of flagella. When Salmonella are grown in agar containing phenol (1 in 800),
flagella are inhibited. This change is phenotypic and temporary.
To attain a population of Salmonella rich in H antigen Craigie’s tube or U tube on soft agar can be employed.
Phase variation
Phase 1 antigen is either specific for a species or shared by a few species only. Hence termed as specific phase.
Phase 2 antigens are widely shared and hence termed as nonspecific or group phase.
Phase 1 antigens are designated as a, b, c…Z and after Z as Z1, Z2, Z3…etc. Phase 2 is designated as 1,2…etc.
The flagellar antigens of salmonellae occur in 1 or 2 phases.
Strains that posses both phases are called diphasic and strains having 1 phase are called as monophasic.
V-W variation
Fresh isolates of S. typhi carry a surface Vi antigen that completely masks the O antigen.
Such bacillus does not agglutinate with O antiserum but agglutinable with Vi antiserum. This is called as V form.
After repeated sub culturing the Vi antigen is completely lost and does not agglutinate with Vi antiserum but
agglutinate with O antiserum.
This is called as W form. Intermediate stages which agglutinate with both Vi and O antisera is called VW form.
S-R variation
The smooth to rough variation is associated with the change in colony morphology, loss of O antigen and virulence.
The colonies become large, rough and irregular.
R forms occur by conversion into mutation.
It can be prevented by maintaining the cultures by lyophilisation.
Variation in O antigens
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Changes in the structure of O antigens may be induced by lysogenisation with certain phages resulting in alteration
of serotypes.
CLASSIFICATION OF SALMONELLA
O, H and Vi antigens
Based on biochemical reactions,(Reeves, et.al., 1989), the genus Salmonella is divided into two
species, S.enterica and S.bongori.
S.enterica is subdivided into 6 subspecies. Enterica (I), Salamae (II), Arizonae (III a), Diarizonae (III b), Indica
(IV) and Houtenae (V).
Sub genus enterica is the largest and most important, containing all the species that commonly cause human and
animal infections.
Members of this sub genus are given a name like S.enterica sub sp. enterica serovar typhimurium.
Serological classification is done by Kauffmann – white scheme. It depends on the identification by O and H
antigens of the strain and agglutination
Example
O * Serotype Antigens
serogroup
O H
Phase Phase
1 II
2 A Paratyphi A 1,2,12 a -
4 B Paratyphi B 1,4,5,12 b 1,2
Typhimurium 1,4,5,12 c 1,2
7 C Cholera suis 6,7 c 1,5
Paratyphi C 6,7 (W) c 1,5
9 D Typhi 9 ,12 d -
Enteritidis (W) g, m 1,7
Pullorum and 1,9,12
gallinarum 1,9,12 - -
Dublin
1,9,12 g, p -
(Vi)
Primary subdivision is into O serogroups, each of which shares a common somatic antigen.
Where more than one O antigen is present, one of them is the major O Ag and is regarded as determining the group
to which the strain shall be allocated.
O serogroups were formerly designated by letters of the alphabet (A-Z).
But now are indicated by numbers 1,2,3… The series of O antigens numbered O1 –67 is not continuous because
some O antigens were originally assigned to bacteria that proved subsequently not to be salmonellae. Thus, only 46
O serogroups are defined by the 67 O antigens described.
PATHOGENESIS
Salmonella
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Ingestion
Gastroenteritis form Enteric fever form
Colonize intestine Colonize intestine
Penetrate epithelial cells of intestinal villi Penetrate epithelial cells of intestinal villi
Produce enterotoxins Enter blood stream
Gastroenteritis Endotoxaemia
Abortion
Following adherence with fimbriae to the surface of the intestinal mucousal cells, the bacteria induce ruffling of cell
membranes.
The ruffles facilitate uptake of the bacteria in membrane bound-vesicles, which often coalesce.
The org. replicate in these cells and eventually released from the cells.
The virulence of salmonellae relates to their ability to invade host cells and replicate in them.
The O-antigen chains of LPS resist phagocytosis and long chains of LPS prevent complement action, which will
facilitate the spread of organisms.
The LPS is also responsible for endotoxin activity.
Salmonellae often localize in the mucosae of the ileum, caecum and colon, and in the mesenteric lymphnodes of
infected animals. Subclinical infection may persist with small numbers of salmonellae in the faeces. Latent
infections, in which salmonellae are present in the gall bladder but are not excreted, also occur.
Stress factors such as inter-current infections, transportation, overcrowding, pregnancy, extreme ambient
temperature, sudden changes in rations etc may activate latent or sub clinical salmonellosis.
PATHOGENICITY
Pathogenesis
Cattle
Horses
Pigs
Poultry
Lesions
Cattle
There are areas of haemorrhagic inflammation and necrosis of the large intestine.
The mesenteric lymph nodes are oedematous and haemorrhagic.
Areas of necrosis occur in the liver and spleen.
Horse
Petechial haemorrhages occur in the heart, spleen and lungs of the aborted
fetuses.
Fetal membranes are oedematous, haemorrhagic with areas of necrosis.
The abomasum and small intestine have areas of congestion and haemorrhage.
Haemorrhages occur in the myocardium and kidney cortex with oema of the
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Pigs
In acute cases the mucosa of stomach, intestine, kidney cortex and myocardium
reveals haemorrhagic spots.
Mesenteric lymph nodes and spleen are enlarged and hyperaemic. There may be
discoloration of the skin.
Poultry
DIAGNOSIS
Specimens to be collected
It includes faeces and blood from live animals, intestinal contents and tissue lesions from dead animals, abomasal
contents from aborted fetuses and parenchymatous organs from septicaemic conditions.
Diagnosis based on
Whole blood agglutination test: 1 drop of colored or plain pullorum antigen is mixed with 1 drop of blood and
allowed for 2 minutes. In positive cases there will be clumps.
Tube agglutination test: In this test the sample serum is diluted serially to which 0.5 ml of constant amount of
antigen is added and kept at 37°C in a water bath overnight.
Agglutinations in dilutions at a titre of 1 in 40 indicates a positive test.
In horses, cattle, sheep and goat live vaccines give rise to solid immunity but the animal may remain as carrier
throughout its life.
Killed vaccines are safe but can stimulate only a temporary resistance.
For cattle attenuated live culture of S. dublin vaccine has been developed.
In poultry either killed or live attenuated vaccine stimulates good immune response.
Various chemotherapeutic agents have been used to treat salmonellosis including Chloromycetin, Terramycin,
neomycin, furazolidone and sulfasuxidine.
The majority of serotypes are potential pathogens for both man and animals.
The important route of transmission is through milk, meat and egg.
This will result in Salmonella food poisoning with symptoms of severe gastroenteritis.
Yersinia species are non-lactose fermenters and, with the exception of Y. pestis are motile.
Although there are more than 10 Yersinia species, only Yersinia pestis , Yersinia enterocolitica , Yersinia
pesudoteuberculosis are pathogenic for animals and man.
They characteristically demonstrate bipolar staining in Giemsa-stained smears from animal tissues.
Yersinia pesudotuberculosis and Yersinia enterocolitica are found in the intestinal tracts of a wide range of wild
mammals, birds and domestic animals.
All these animals may be reservoirs of infection. Many avian species may act as amplifier hosts and may also
transfer the organisms mechanically.
They able to grow in a wide temperature range (5 to 420C) and survive for long periods in cool wet conditions.
In endemic areas, wild rodents are important reservoirs of Yersinia pestis.
Fleas, especially Xenopsylla cheopis, the oriental rat flea, transmit the infection to man and other animals.
Diseases caused by Yersinia
YERSINIA PSEUDOTUBERCULOSIS
Distribution
The organisms has a world wide distribution and is particularly associated with a disease known as
pseudotuberculosis in guinea pigs and to a less extent in turkeys, pigeons, rats, rabbits and horses.
It has occasionally been isolated from sheep, goats, pigs, cattle, cats & man.
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Morphology
Cultural characters
It can grow on MacConkey agar, which is a point of differentiation between Pasturella multocida and Yersinia.
Indole is not produced & nitrates are reduced to nitrates.
Of the ten serotypes of Yersinia pseudotuberculosis, serotypes I,II, III contain the majority of pathogens.
Yersinia posses a common flagellar antigen and a common somatic antigen.
Symptoms
The acute form of the disease develops as a sudden and acute septicaemia.
The septicaemic form is called as pseudotuberculosis.
Deaths may be sudden and mortality may be high.
In chronic cases there will be intermittent diarrhoea with emaciation before death.
Lesions
The spleen and mesenteric lymph nodes are enlarged and congested.
In chronic cases necrotic and caseous lesions in the spleen, liver and mesenteric Lymph nodes will be noticed,
resembling pseudotuberculosis.
Diagnosis
By isolation & identification of the organism from the liver, spleen, heart blood and bone marrow.
A cold enrichment procedure may facilitate recovery of yersiniae from faeces, especially if they are present in very
low numbers.
A 5% suspension of faeces in PBS, held at 40C for 3 weeks, is subcultured weekly on MacConkey agar.
Y. pseudotuberculosis in man produce chronic infection of the mesenteric lymph nodes and produce symptoms
suggestive of an appendicitis.
YERSINIA PESTIS
This organism causes bubonic plague or pneumonic plague in man. It is carried by rats & other rodents.
They are Gram-negative short coccobacilli showing bipolar staining with Leishmans stain.
It can grow aerobically and anaerobically in the presence of serum and blood.
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It grows on MacConkey agar and capsule formation is noticed at 37°C. Y.pestis produces a capsular antigen and a
somatic antigen.
It causes pneumonic or bubonic plague and the lesions include swollen and haemorrhagic lymphatic glands and
spleen with multiple necrotic areas in the spleen and liver.
Diagnosis is done by isolation & identification of the organism.
Live and killed vaccines are used for prevention and plague cases are treated with oxytetracycline & other
antibiotics.
Three clinical forms of feline plague are recognized; bubonic, septicaemic and pneumonic.
The most common form of the disease is characterized by enlarged lymphnodes (Buboes) associated with
lymphatic drainage (serosanguineous fluid or pus) from the site of infection.
Septicaemia may occur without lymphadenopathy and is potentially fatal.
Pneumonic lesions may result from haematogenous spread.
Cats with pneumonic lesions are potential source of human infection through aerosol generation.
YERSINIA ENTEROCOLITICA
Y. enterocolitica has been identified from pigs, horse, ox, sheep and goat, dogs, cats, rodents etc. 57 O-factors, 6 K -
antigens and 19 H - antigens have been identified.
Based on this there are five biotypes and more than 50 serotypes have been identified.
Somatic antigens 2,3,5,8 and 9 are present in isolates from clinical infections caused by this species.
Serotype O:9 is of particular importance because it shares common antigens with Brucella species and it may
induce false-positive reactions in brucella agglutination tests.
They produce either generalized infections or enteritis.
It can grow on blood agar and the optimum temperature for growth is 28°C.
A preceding cold enrichment improves the isolation rate.
It produces gastroenteritis in human infants and terminal ileitis, mesenteric lymphadenitis and pseudo
appendicitis in adolescents.
The pig is the natural reservoir for Yersinia enterocolitica serotype O3 biotype 4, which is an important pathogen
in humans
SHIGELLA
Four species (S. boydii, S. dysenteriae, S. flexneri and S. sonnei) are etiological
agents of diarrhoea and dysentery in man and subhuman primates.
They are Gram –ve, non-motile, rod shaped organisms found in the intestinal
flora of animals.
Shigella are facultatively anaerobic and grows on blood agar and MacConkey
agar at 37oC without haemolysis.
Shigella produces an enterotoxin which acts on the epithelial lining of the
terminal illeum and large intestine, producing bloody mucus stained stool.
CITROBACTER
Citrobacter freundi forms part of the normal intestinal flora of domestic animals and are also found in soil and
water.
They are Gram negative, facultatively anaerobic, motile, rod shaped organisms, which grows on blood agar and
MacConkey agar at 37°C producing no haemolysis.
In domestic animals, Citrobacter are opportunistic pathogens in a variety of extra intestinal infections.
EDWARDSIELLA
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Edwardsiella tarda appears to have a worldwide distribution and has been isolated from all classes of vertebrates,
fish, amphibia, reptiles, birds and mammals.
They are Gram negative, motile rods. They are facultatively anaerobic and grow on blood agar and MacConkey agar
at 37°C.
They are non-haemolytic and lactose negative organisms.
E. tarda has been implicated as the cause of diarrhoea,wound infections and sepsis in animals and humans.
ENTEROBACTER
KLEBSIELLA
Kelbsiella species occur naturally in soil and water and are normal intestinal flora of domestic animals.
They are non-motile, encapsulated rods arranged singly, in pairs or short chains.
The Klebsiella are non haemolytic on blood agar and lactose positive on MacConkey agar.
Klebsiella pneumoniae has O antigen (11 types) and K antigen (70 types).
In horses Klebsiella causes inflammation of the genital mucosa and abortion and generalized infections of foals.
In cattle it causes mastitis, generalized infections and enteritis of calves.
In pigs it causes atrophic rhinitis and in poultry it causes air sac and yolk sac infections.
K. pneumoniae and K. oxytoca are the most commonly isolated species from domestic animals.
MORGANELLA
PROTEUS
Proteus mirabilis and P.vulgaris are normal intestinal flora of domestic animals and also are found in soil and
polluted water.
Proteus is Gram-negative rod shaped organism but often show pleomorphism.
All strains are normally motile with peritrichous flagella and majority produce fimbriae. Spores and capsules are
not produced.
These organisms are aerobic and facultatively anaerobic and growth takes place at 37°C growth on solid media
shows the feature of swarming.
All species are positive to the phenylpyruvic acid. They are responsible for endometritis, gastroenteritis and
arthritis.
In pigs it causes diarrhoea and urinary tract infections.
In dogs and cats it causes urinary tract infections, cystitis, otits externa and sepsis. Click here for visual
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PROVIDENCIA
Providencia rettgeri and P.stuartii are found in the normal intestinal flora of
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domestic animals.
They are Gram-negative rods and motile by peritrichous flagella.
They are facultatively anaerobic and growth on blood agar and MacConkey agar
at 37°C produces no haemolysis and is lactose negative.
P.rettgeri is frequently isolated in pure culture from urinary infections.
SERRATIA
SYSTEMATICS
Domain Bacteria
Phylum Proteobacteria
Class Gamma proteobacteria
Order Pasteurellales
Family Pasteurellaceae
Genus Pasteurella
Genus Mannheimia
Genus Haemophilus
Genus Actinobacillus
Genus Lonepinella
History
The first significant report of the P.multocida was made by Bollinger (1878) and Perroncito (1878) and it was
extensively investigated in 1880 by Pasteur as the cause of Fowl cholera.
The P.haemolytica was isolated from disease of calves and sheep by Jones (1921).
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Habitat
Pasteurella organisms are world wide in distribution with a wide spectrum of hosts.
They infect practically all domesticated and many wild animals and birds.
They occur as commensals in the mucous membrane of the Upper respiratory and intestinal tract of animals.
MORPHOLOGY
They are of small Gram –ve rods or coccobacilli that showbipolar staining particularly when fresh isolates stained
with Leishman’s or Wrights or Giemsa. They are non motile and non spore forming. ,
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Cultural characteristics
Growth takes place at an optimum temperature of 37oC. Growth is enhanced by the addition of blood or serum. On
serum agar Pasteurella exhibit 3 different colonial forms
o Smooth form: Virulent for rabbits, growing diffusely in broth. Forming smooth, moderately opaque
iridescent colonies on serum agar. It contains a type specific polysaccharide capsular antigen.
o Rough form: Completely avirulent for rabbits, giving a granular deposit in broth and forming translucent
bluish colonies. It has neither a capsular nor a mucoid antigen.
o Mucoid form: Intermediate virulence. It is rich in hyaluronic acid.
The routine medium for isolation of Pasteurella species is Brain heart Infusion agar and Ox or Sheep blood agar.
P. multocida has a characteristic sweetish odour, is non-haemolytic, and does not grow on MacConkey agar
Mannheimia haemolytica and P. trehalosi are beta haemolytic. On sheep blood agar, colonies are surrounded by a
single narrow zone of hamolysis, but in lamb blood agar a double zone haemolysis may be seen. Mannheimia
haemolytica and P. trehalosi usually tolerate the bile salts in MacConkey agar to grow as pin point pink red
colonies. On TSI produce Y/Y/H2S-ve .
P. anatipestifer produces dewdrop colonies within 48 hrs.
Biocehmical properties
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They are oxidase +ve, catalase +ve and fermentative (except P. trehalosi and R.anatipestifer). P.multocida is
indole positive.
Mannheimia haemolytica is indole negative. They ferment several sugars (glucose, sucrose, galactose and sorbitol)
with acid and no gas.
P.anatipestifer is non fementative, non haemolytic, does not grow on MacConkey agar, indole –ve, urease –ve.
Based on capsular polysaccharide antigens, using PHA, Carter classified P.multocida into 5 serotypes (A-F). A type
C is not a valid capsular serotype.
The capsular types may be subdivided further into 16 somatic types based on heat stable somatic antigen, using
AGPT (i.e. serologic differences in LPS). The capsular type followed by the number representing the somatic type
designates a serotype
For e.g. Type A:5, A:8, A:9 produce fowl cholera
Type B:6 cause haemorrhagic septicaemia in Asia
Type E:6 cause haemorrhagic septicaemia in Africa
The toxin produced by P.multocida (mostly type A and D strain) is mainly cell associated, polypeptide, heat stable,
dermonecrotic, lethal and immunogenic.
In case of Mannheimia haemolytica/ P. trehalosi, on the basis of soluble surface antigens so far 17 serotypes have
been recognized.
There are two distinct biotypes, A and T, based on fermentation of arabinose and trehalose respectively.
Serotypes 3,4,10 and 15 are P. haemolytica biotype T - P. trehalosi, all others are A - Mannheimia haemolytica.
Biotype A strains are associated with ruminant pneumonias, septicaemia in young lambs and ovine mastitis,while
biotype T strains are usually associated with septicaemic infection in older lambs and sheep.
All strains of Mannheimia haemolytica elaborate a soluble cytotoxin (Leukotoxin).
It is a protein of relatively large mol.wt, thermolabile, resembles the alpha haemolysin in E.coli and is
immunogenic.
Resistance
They are readily killed at 550C for 15mts or if exposed to sunlight for 3-4 hrs.
They are also killed by 0.5% phenol in 15 mts.
They are susceptible to penicillin but resistant to sulphonamides.
PATHOGENESIS
Lysis of platelets results in pulmonary vascular thrombosis and fibrin exudation typically associated with shipping
fever pneumonia.
Speices Disease
P.multocida (Based on Capsular polysaccharide Fowl cholera
typing) Haemorrhagic
septicaemia /
Type A Barbone ( Asia and
Type B Australia )
Type D Atrophic rhinits of
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Type E pigs
Type F Haemorrhagic
septicaemia ( Africa )
Disease in turkey
PATHOGENECITY
Symptoms
Cattle
In acute cases of haemorrhagic septicemia the symptoms include a rise in temperature, a sudden drop in milk
yield, signs of abdominal pain, severe diarrhoea and dysentery.
Respiration becomes rapid and shortly before death the mucous membranes appear cyanotic.
In less acute cases there will be odema development in the region of the head, neck and brisket.
The nasal discharge may be blood stained or purulent. Death occurs within 2-4 days.
P.haemolytica give rise to symptoms of bronchopneumonia with pleuritis. This condition is referred as transit or
shipping fever.
Sheep
Pigs
Pasteurella type D infection causes atrophic rhinitis in pigs and is a secondary invader in respiratory infections.
Poultry
The acute form of fowl cholera can affect many species of wild and domesticated birds including chicken, turkey,
ducks and geese causing high mortality.
Death occurs suddenly without any predominant symptoms.
In ailing birds, breathing is rapid through the open beak, the feathers are ruffled and the comb and wattles become
cyanotic.
In chronic cases swollen wattles and comb, hot and painful joints are characteristic.
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Lesions
Cattle
In acute form, the lesions include multiple haemorrhages on the serous membranes and organs together with
blood stained exudates in the thorax and abdomen.
There will be severe gastroenteritis with enlargement of mesenteric lymphnodes.
In edematous form the striking lesion is odema occurring in the subcutaneous tissues in the region of throat, and
brisket region.
In pectoral form, the lesions are confined to the lungs and pleural cavity with enlargement of bronchial and
mediastinal lymph glands.
In acute cases there may be exudates in the pleural cavity and pericardial sac.
Poultry
The liver and spleen are enlarged, hyperaemic with necrotic foci.
There will be peritonitis, fluid in the pericardial sac and petechial haemorrhages on the serous surfaces.
In chronic infections there will be swelling of the comb and wattles and accumulation of serofibrinous fluid in the
joints.
Diagnosis
Isolation and Identification of the organisms from heart blood and fluid exudates in blood agar and brain heart infusion
agar will show dewdrop like colonies after 18hrs incubation.
In putrified material bone marrow of long bones are useful to get pure cultures.
Animal inoculation is done by inoculating the suspected material or bacterial culture in mice and rabbits by scarification
or subcutaneous route.
The animal dies in 24-72 hrs with haemorrhagic tracheitis.
Immunity is predominantly humoral. Pasteurella multocida P52 strain have proved useful. It gives protection for 6
months to 1 year.
Vaccines for fowl cholera are not successful. So vaccines should be prepared from types prevalent in that area for
protection.
Pasteurella is amenable to Penicillin-G, streptomycin, chloramphenicol, chlortetracycline, sulpha and tripmethoprim,
enrofloxacin and oxytetracycline.
It is one of the few Gram-negative bacteria sensitive to penicillin.
Haemophilus
History
Habitat
Morphology
Gram negative, small, medium sized coccobacilli or rods, often markedly pleomorphic, sometimes filamentous,
non-motile, non-spore forming and non-acid fast.
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Capsules can be produced. H.inflenzae, H.parasuis and H.paragallinarum require one or both of two accessory
growth factors X and V.
Cultural characteristics
H. influenzae + +
H. aegyptius + +
H.parasuis - +
H.paragallinarum - +
H.parinfluenzae - +
H.somnus - -
In addition to X and V factors, the growth of the many of the Haemophilus species is enhanced by 10% C02.
Haemophilus grows on blood agar, but growth is scanty, as the V factor is present mainly intra cellularly in red
cells.
The chocolate agar is the most suitable medium for isolation of Haemophilus.
In chocolate agar, the V factor is released from the red cell, and the heat stable X factor is still present.
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On chocolate agar, H.paragallinarum produces typical dewdrop like colonies and H.somnus shows characteristic
yellow tinge colonies.
Sattelitism
When Staphylococcus aureus is streaked across a plate of blood agar on which a specimen
containing Haemophilus has been inoculated, after 18-24hrs incubation at 370C under 5-10% Co2, the colonies of
the Haemophilus will be large, well developed alongside the streak of Staphylococcus, and smaller farther away.
This phenonmenon is called sattelitism and demonstrate the dependence of Haemophilus on V factor, which is
available in high concentration near the staphylococcal growth and only in smaller quantities away from it. Click
here for visual
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Media supplemented with yeast extract, Levinthals medium (clear transparent media may be prepared by boiling
and filtering a mixture of blood and nutrient broth) or Filde’s agar (by adding a peptic digest of blood to nutrient
agar) are also suitable for the primary isolation of Haemophilus.
MORPHOLOGY
Biochemical properties
Resistance
Haemophilus species are fragile. They are readily destroyed by heating (550C for 30mts), refrigeration (00C to 40C),
drying and disinfectants.
In cultures the cells die within 2 to 3 days due to autolysis. For long-term preservation the cultures may be
lyophilized.
Antigens
Non-capsulated strains are antigenically heterogenous. Some somatic antigens have been identified.
The capsular antigens are polysaccharide in nature and they resemble pneumococcal capsular antigens.
It is immunochemically similar to the K antigens of E.coli.
Based on immunodiffusion test, using heat stable antigens 15 serovars of H.parasuis, 9 serotypes
of H.paragallinarum and 15 serotypes of H.somnus have been recognized.
PATHOGENESIS
Young or previously unexposed animals are highly susceptible. Stress factors contribute to the development of
disease.
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The capsule and cytotoxic factor are thought to be virulence factors and endotoxin may play a role in the disease
process.
During stress, the bacteria may invade the mucosal barrier. The invasive mechanism is not known.
In respiratory tract initially nasopharyngitis. If this infection is not checked, it may lead to sinusitis, otitis media
and pneumonia.
If a bacteraemia develops, joint infections and meningitis may follow.
Diseases caused by the Haemophilus species
PATHOGENICITY
Symptoms
Lesions
In Glasser's disease during acute death conditions, the PM reveals large deposition of fibrin in joints and on any or all of
the serosal surfaces (poly serositis) of the body.
In addition there will be fibrinous pericarditis, pleuritis and peritonitis. Catarrhal inflammation of the infra orbital sinus is
characteristic of infectious coryza.
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Diagnosis
Specimens
In H.somnus infections, the organism can be demonstrated in brain lesions, they can be recovered from semen
samples and preputial washings of healthy bulls.
Haemophilus species are highly delicate and do not survive long when removed from the host.
Clinical material is best frozen (dry ice preferred) and delivered to the laboratory within 24 hrs.
Refrigeration and transport media may not assure viability.
A presumptive identification of Haemophilus can be made based on the host species, clinical signs and lesions,
colony characters, X and V factor requirements, oxidase and catalase reactions and whether or not C02 enhances
growth.
Serological tests including agglutination, AGPT, ELISA are used to
detect H.paragallinarum and H.somnus infection.
Treatment
Haemophilus species are susceptible to gentamicin, tetracycline, sulfonamides, chloramphenicol, neomycin and
erythromycin.
Vaccines prepared from H.paragallinarum grown in egg yolk, if inoculated intramuscularly reduces the incidence
of infectious coryza.
The Actinobacillus species are Gram negative, very small, non-motile, non-sporing and non acid-fast bacilli.
Small coccal elements are often lying at the pole of a larger form, giving a characteristic ‘Morse-code’ appearance
History
Habitat
Actinobacillus species is worldwide in distribution. They are commensals of the respiratory, alimentary or genital
mucosa.
A.lignieresii is a commensal in the oral and rumen of cattle and sheep.
A.equuli occurs as a commensal in the equine intestinal tract and in the mouth.
A.suis is present in the tonsil and upper respiratory tract of healthy pigs.
Actinobacilli cannot survive in the environment, carrier animals play a major role in transmission.
Morphology
Gram –ve, small, rod shaped organism. They are non-motile, non-sporing and non-acid fast.
They are non capsulated (except A.pleuropneumoniae) but extracellular slime is present in three major species
(A.lignieresii, A.equuli and A.suis)
In media containing fermentable carbohydrates, the occurrence of rather long, almost filamentous forms is seen.
Small granules are found scattered along the bacilli, often lying at the pole of a bacillary or filamentous form, giving a
characteristic ‘Morse code’ form.
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In lesion in the animal body small grayish white granules are present.
If these granules are crushed on a slide and stained, club colonies are seen consisting of club-like processes of calcium
phosphate, with Gram-negative rods of A. lignieresii in the center.
Both bacilli and club forms are Gram negative.
They can be distinguished with ZN stain in which the club appears red and the bacilli blue.
Cultural characteristics
Biochemical properties
They are catalase positive (except A.pleuropneumoniae). Oxidase and urease positive.
Ferment several sugars, produce acid and gas. IMViC negative, H2S positive.
Resistance
They are rapidly killed by heating at 620C for 10 mints and by drying.
Culture lose their viability rapidly and should be subcultured every 5-7 days.
In A.lignieresii, heat stable somatic and heat labile surface antigens are described.
Six antigenic types (1-6) and two subtypes (1a and 1c) have been demonstrated.
In A.equuli, as in A.lignieresii, both heat labile and heat stable antigens can be demonstrated.
In A.suis, the antigens have not been studied in any detail.
In A.pleuropneumoniae, 12 serotypes and 2 biotypes have been described,.
Biotype 1 requires NAD for its growth while biotype 2 is NAD independent.
In A.pleuropneumoniae, capsule, LPS, outer membrane proteins and toxins (haemolytic and cytotoxic) play a
major role in pathogenicity.
Exotoxin is not formed in A.lignieresii.
PATHOGENESIS
Bovine actinobacillosis is spread by the lymphatics. The specific disease is wooden (timber) tongue, but
granulomatous lesions can also involve the head, neck and limbs.
Less commonly, the lungs and other internal organs are also affected.
A.suis : Infection occurs via the aerosal route by close contact or through skin.
Once the organism has entered the blood stream it spreads rapidly throughout the body.
Several factors, LPS, cytotoxin etc are responsible for gross lesions and they are usually seen in the lungs, kidney,
heart, spleen, intestines and skin.
The lungs may also be filled with serous or serofibrinous exudates with pleuropneumonia.
A.pleuropneumoniae : The organism enters the lungs, multiplies rapidly.
During growth the organism releases a large quantity of OMP, LPS, cytokines and other factors which causes
destruction of neutrophils that is likely to be responsible for the massive and tissue damage.
Diseases caused by the pathogenic actinobacilli
PATHOGENICITY
Symptoms
Cattle
o Chronic pyogranulomatous lesions occur on tongue and other soft tissues.
o Enlargement and protrusion of the tongue that interferes feeding. The pus
contains soft grayish white granules.
Pigs
o A.suis can infect pigs of all ages. But infection is most serious in very
young animals.
o In neonates and suckling pigs, A.suis can cause an acute and rapidly fatal
septicaemia.
o Death occurs within 15hrs. Affected animal may show signs of cyanosis,
petechial haemorrhages, fever, respiratory distress, neurologic
disturbances and arthritis.
o In older animals, the disease is less severe and may be characterized by
fever, anorexia and persistent cough. The mortality is also much lower.
o A.pleuropnemoniae can cause acute and rapidly fatal pleuropneumonia.
o The acute form is characterized by extensive haemorrhage and fibrin
deposit in the lungs.
o Affected animals show signs of severe respiratory distress, cyanosis, fever
and vomiting.
o In chronically infected animals the organism may be sequestrated in the
lungs in the necrotic lesions, tonsils and URT and may responsible for
spread of infection.
Sheep
o Actinobacillus seminis is a common cause of epididymitis in young rams.
o The organism is found in the prepuce. The infection occurs probably
following an ascending opportunistic infection.
o Abscesses and purulent discharge through fistulae on the scrotal skin are
most commonly seen.
Horse
o Sleepy foal disease is an acute, potentially fatal septicaemia of newborn
foals caused by Actinobacillus equuli.
o Occasionally it causes abortion and peritonitis in adult horses.
o The organism is found in the reproductive and intestinal tracts of mares.
o Foals can be infected in utero and after birth via the umbilicus.
o Affected foals are febrile and recumbent. Death usually ocuurs in 1 to 2
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days.
Diagnosis
Specimens to be collected
o Pus, biopsy material and tissues in case of wooden tongue.
o Tracheal washings or affected portions of lung in pleuropneumonia cases.
Based on history and symptoms
Isolation and identification of organism
o Club colonies in tissue sections, growth pattern on blood and MacConkey agar and biochemical test are
highly useful.
Treatment
Polyvalent bacterins may induce protective immunity but fail to prevent transmission or the development of a
carrier state.
SYSTEMATICS
The Pseudomonas is Gram –ve, aerobic, medium-sized rods, motile by one or several polar flagella
(except P.mallei).
Catalase and oxidase positive and some species produce water-soluble pigments and most will grow on MacConkey
agar.
Domain Bacteria
Phylum Proteobacteria
Class Gamma proteobacteria
Order Pseudomonadales
Family Pseudomonadaceae
Genus Pseudomonas
History
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Habitat
Most of the species in this genus are saprophytes, including P.aeruginosa and P.pseudomallei.
The P.aeruginosa is ubiquitous in the environment and is found in water, soil and on plants, as well as on skin and
mucous membrane of healthy animals.
The P.pseudomallei mainly is present in the tropics and it is considered to be a soil organism.
Wild rodents act as important reservoir of this organism. P.mallei is an obligate parasite.
MORPHOLOGY
Gram negative, rod shaped bacteria. They have parallel sides and rounded ends.
They are arranged singly, in small bundles, in short chains or in filaments.
All except P.mallei are motile by means of polar flagella at one or both ends.
Some strains are lopotrichate and some are monotrichate. P.pseudomallei is peritrichous.
Non spore forming and non acid fast.
It is non capsulated but when grown in the absence of sucrose an extracellular polysaccharide slime layer may be
formed.
Most of the strains possess fimbriae.
CULTURAL CHARACTERISTICS
Biochemical properties
In addition to pigment production (P.aeruginosa only) and characteristic odour the pseudomonads are strongly
oxidase positive, reduce nitrate to nitrite, liquefy gelatin, IMViC -,-,-,+.
All ferment glucose and produce less acid. P.pseudomallei ferments lactose. OF test is positive for P.aeruginosa.
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The Pseudomonas is highly heterogenous. Serotyping, immunotyping, pyocin typing and phage typing are all used
for characterization of isolates.
Based on somatic antigens 17 serotypes have been described in P.aeruginosa.
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P.aeruginosa produces numerous extracellular toxins and enzymes. Those that may play role in pathogenesis are
fibrinolysin, elastase, lecithinase, lipase, protease, haemolysin (heat stable and heat labile), leucocidin, alginate,
phospholipase C, esterase and exotoxin A.
In addition to this, the pigment produced by P.aeruginosa exhibits antimicrobial activities against a wide range of
bacteria and some fungi. P.pseudomallei produces very potent exotoxin (similar to exotoxin A), protease, lipase
and a lecithinase.
No exotoxin has been described in Pseudomonas mallei.
RESISTANCE
They are more resistant to high dilutions of quaternary ammonium compounds and phenolic compounds.
P.aeruginosa can survive for long periods on water faucets, utensils, floors, instruments, baths, humidifiers and
respiratory equipments.
They grow very well in antiseptic lotions kept for use in hospitals. Pseudomonas is susceptible to ethylene oxide
and heat (550C for one hour).
Pseudomonads are resistant to wide range of antimicrobial drugs.
Much of this intrinsic resistance is attributed to the outer membrane porins, which restrict passage of many
antimicrobial agents into the periplasm.
Resistance to penicillin, Ampicillin, tetracycline, first and second-generation cephalosporin, sulfonamides,
neomycin, streptomycin, kanamycin, chloramphenicol, nitrofuran and trimethoprim-sulfonamide.
Susceptible to gentamicin, amikacin, colistin, Polymyxin B, carbenicillin, cefoxamine, third generation
cephalosporins and Ciprofloxacin.
PATHOGENESIS
Depends upon the degree and site, the P.aeruginosa produces wide variety of suppurative infections.
In localized lesions there will be yellowish green pus.
The acute form of P.pseudomallei occurs more often in young animlas.
The chronic form is characterized by nodules in the lungs, liver, spleen, lymphnodes and subcutis, the lesions
resembling those of caseous lymphadenitis and numerous visceral abscesses.
Three forms of glanders are seen in horse. They are classified as
Nasal form
Nasal form initially there is reddening of the nasal mucosa and mucoid discharge from nostrils.
It will become purulent, blood stained and adherent as brown crust. Ulcers may form on the nasal mucosa.
DIAGNOSIS
Specimens
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Urine, pus, affected tissues and swab from infected tissue surfaces are suitable for P.aeruginosa.
In case of P.mallei, collect tissue containing early nodules or pus from ulcers.
Diagnosis
SYSTEMATICS
Domain Bacteria
Phylum Proteobacteria
Class Alpha proteobacteria
Order Rhizobiales
Family Brucellaceae
Genus Brucella
History
B.melitensis was identified by Bruce in Malta in 1887. B.abortus was first recognised by Bang in 1897. B.suis was
discoverd by Traum in 1914.
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Alice Evans (1918) identified the first Brucella of human origin in the USA. Buck (1930) developed live attenuated
strain 19 vaccine.
Habitat
Brucella species are obligate parasites and each species has a preferred natural host that serves as a reservoir of
infection.
Brucellae have a predilection for ungulate placentas, foetal fluids and testes of bulls, rams, boars and dogs.
Morphology
Brucellae are coccobacilli or short rods, arranged singly or in short chains. They are non-motile, non-capsulated,
non-sporing and partial acid fast.
They are stained by Grams, MZN and Koster’s stain.
Koster’s stain is mostly useful in demonstrating Brucella in smears from the cotyledons in bovine abortion (Cells of
chorion are packed with organism).
MZN is specially useful to stain smears from foetal membranes, uterine swabs, smear or stomach contents of
aborted foetus.
Cultural characteristics
Biochemical properties
Brucellae are catalse positive, oxidase positive (except B.ovis and B.neotomae), and urease +ve (except B.ovis and
B.melitensis), reduce nitrate, IMViC, -,-,-,-. (indole, methyl red, voges proskauer and citrate utilization negative).
Resistance
Brucellae are destroyed by heat at 60ºC in 10mts and by 1% phenol in 15mts. They survive in soil and manure for
several weeks.
Remain viable for 10 days in refrigerated milk, one month in ice cream, 4 months in butter and many weeks in
meat.
B.abortus will survive for 41/2 hrs when exposed to direct sunlight, 4 days in urine, 5 days in cloth at room
temperature and 75 days in an aborted fetus.
Antigens
The somatic anigens of brucellae contain two main antigenic determinants designated as A and M, which are
present on the LPS proten compelx.
Brucella abortus contains more A antigen than M (20:1).
B.melitensis has more M than A (20:1). B.suis has an intermediate pattern.
B.canis and B.ovis are antigenically rough and do not possess the A and M antigens. Both possess a surface antigen
R.
Like many other Gram-negative bacteria, the change from S to R is associated with loss of virulence, a tendency to
auto agglutination and loss of antigen.
The vaccine strain, B.abortus 19 is an intermediate mutant that, although low in virulence, is antigenic.
Antigenic cross-reactions exist between brucellae with several bacteria like Yersinia enterocolitica, Vibrio
cholerae and some strains of E.coli and Salmonella (Heterophile antigens).
No exotoxins have been described. In addition to endotoxin, the surface cell wall carbohydrate is responsible for
binding to B-lymphocytes and play a major role in pathogenesis.
Brucella biotyping
Based on CO2 requirement, H2S production (B.abortus biotype 1,2,3,4 and B.neotomae), urease activity, growth in
the presence of dyes (thionin, basic fuchsin), agglutination with monospecific sera (B.canis and B.ovis –
agglutinate only with anti R sera) and Phage typing, three biotypes have been recognized in B.melitensis, eight
in B.abortus and four in B.suis.
B.abortus biotype 3 is commonly present in India.
PATHOGENESIS
The common routes of infection in animals and humans are via the mucous membrane of the digestive tract,
genital tract and skin.
Veneral transmission is main route for B.ovis. Less commonly infection may occur through inhalation or by
conjuctiva.
Organisms may penetrate the mucosa of nasal or oral cavities. Soon after entry into the host the brucellae are
engulfed by phagocytic cells, in which they survive, multiply and are transported to the regional lymphnodes.
After multiplication, the organism pass to the thoracic duct and then via blood stream to parenchymatous organs
and other tissues such as joints, granulomatous foci develop in lymphatic tissues, liver, spleen, bone marrow and
other locations. On occasion it will become abscess.
Brucellosis is essentially disease of the sexually mature animal, the predilection sites being the reproductive tracts
of males and females especially the pregnant uterus.
Allantoic factors such as erythritol (Polyhydric OH), steroid hormones and other substances favour the growth of
most brucellae.
Erythritol is present in the placenta and male genital tract of cattle, sheep, goats and pigs but not humans.
Erythritol does not stimulate the growth of B.ovis and inhibits B.abortus strain 19, the attenuated vaccine strain.
A pyogranulomatous reaction occurs in affected placentae and abortion occurs from 6 month onwards.
Note: Females usually abort only once, after which a degree of immunity develops, and the animals remains
infected and large numbers of brucellae can be excreted in foetal fluids at subsequent parturitions. Permanent
infertily may occur in male dogs infected with B.canis.
Diseases caused by Brucella species
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PATHOGENECITY
Symptoms
Cattle
Sheep
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B.melitensis infections results in abortion at about 3rd or 4th month of pregnancy. Other signs are lameness and
mastitis.
Swine
Swine brucellosis is characterized by abortion, sterility, and birth of stillborn or weak pigs, focal abscesses in
various organs, spondylitis and lameness.
Abortion may occur at any time during gestation.
Dogs
Expelled tissues and vaginal discharges of aborted bitches and the urine of infected males are primary sources of
the infectious agent.
The incubation period is 6 to 21 days. Infected bitches usually aborted in the last trimester.
Following abortion there is yellow brown to dark brown discharges that persist for 1 to 6 weeks.
Epididymitis and testicular atrophy with decreased spermatogenesis are common in the male and may result in
irreversible sterility.
Lesions
The gravid bovine uterus infected with B.abortus develops a necrotic placentitis.
The cotyledons become swollen, hyperaemic and surrounded by brownish exudates.
The inter cotyledonary spaces are thickened and have a characteristic leathery appearance.
Orchitis in bull result in abscess formation or areas of necrosis in the testicles, surrouned by fibrous tissues.
Brucella suis causes placentitis, metritis in ewes and epididymitis and orchitis in boars.
In horses, B.abortus infection associated with fistulous withers (a chronic inflammatory condition of the
supraspinous bursa) and poll evil.
DIAGNOSIS
Specimens to be collected
In abortion cases a full range of specimens should be collected and submitted for a differential diagnosis.
A whole foetus should be sent, if feasible.
Alternatively foetal stomach contents, any foetal lesions, cotyledons, uterine discharges, urine, coloustrum, paired
serum samples and sections of cotyledons and foetal lesions in 10% formalin for histopathology. Semen and tissue
from epididymis or testes from males could be examined.
Direct examination
Modified ZN and Koster’s stain are useful in demonstrating brucellae in smear from the placenta (cotyledons) in
bovine abortion.
Cells of the chorion are packed with organism, which stain red against a blue background.
Organism can also be demonstrated directly in smears from vaginal mucous, semen and various tissues. Direct and
indirect FAT is also used.
Based on biotyping
Animal inoculation
Immunological tests
The immunity acquired from natural infection is not always sufficient to prevent reinfection.
The general basis for elimination of brucellosis is testing and removal of reactors from the herd.
Because the cattle will be less susceptible to reinfection, calf hood vaccination is recommended.
The attenuated live vaccine (Strain 19 B.abortus biotype 1) is used in female calves 4 to 12 months of age.
Because strain 19 cause infertility in some male calves, its use is restricted to females.
The adjuvant bacterins eg: vaccine 45/20 is used as booster vaccine. B.melitensis Rev.1 live attenuated vaccine has
been used with success to immunize rams against B.ovisinfection.
SYSTEMATICS
Domain Bacteria
Phylum Proteobacteria
Class Epsiloproteobacteria
Order Campylobacterales
Family Campylobacteraceae
Genus Campylobacter
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The most important animal pathogens are C. fetus subsp. fetus, C. fetus subsp. venerealis, C. jejuni and possibly C.
mucosalis and C. hyointestinalis.
The Campylobacter species were once placed in the genus Vibrio and some of the diseases are still occasionally
referred to as 'vibriosis'.
HABITAT
Morphology
The Campylobacter species are thin, curved, Gram-negative, microaerophilic, motile rods.
The cells are 0.2-0.5 u.m in width and when daughter cells remain joined, S-shaped, seagull-shaped and
sometimes long spiral forms may be seen.
They are motile by means of polar flagella. Small, curved or seagull-shaped rods can be demonstrated by using
DCF-stained (dilute carbol fuchsin for 4 minutes) smears prepared from colonies. Wet mounts under phase
contrast or darkfield microscopy reveal the characteristic curved forms with darting motility.
Cultural characterstics
The organisms grow at 25 °C or 45 °C. They grow best on nutritious basal media supplemented with 5-10 per cent
blood under reduced oxygen tension.
The Campylobacter fetus (both subspecies) grows very well on nutritious base (Brucella, Columbia or brain heart
infusion agars) supplemented with 5-10 per cent blood.
The medium can be made selective by the addition of polymyxin B sulphate (2 units/ml), novobiocin (2 µg/ml) and
cycloheximide (20 µg/ml).
Incubate the plates at 37°C for 4-6 days in a microaerophilic atmosphere containing 6 per cent O2, 10 per cent CO2
and 84 per cent N2.
These atmospheric conditions must be adhered to strictly. Both subspecies of C. fetus have small (1 mm), round,
slightly raised, smooth, translucent colonies- said to have a 'dewdrop' appearance.
C. jejuni grows very well on charcoal-cefopera-zole-deoxycholate agar or Blaser's Campy-BAP medium.
This latter medium is Brucella agar with 5 per cent sheep blood and vancomycin (10 ug/ml), polymyxin B sulphate
(2-5 units/ml), trimethoprim lactate (5 ug/ml) cephalothin (15 ug/ml), and amphotericin B (2 ug/ml).
The plates should be incubated under the atmosphere described above for 2-3 days at 42° C.
The colonies of C. jejuni are usually flat, grey, and larger than those of C. fetus and can be spreading and watery on
moist plates. C. mucosalis / C. coli grows on Columbia blood agar with and without 1:60,000 brilliant green.
Incubate the plates anaerobically at 37°C for 2-5 days. After primary isolation the bacterium will grow well
microaerophilically.
C. coli produces a pink-tan pigment and the growth of C. mucosalis may appear as a dirty-yellow colour.
Biochemical properties
Resistance
Antigencity
C. fetus subsp. venerealis has "0" and "W' cell wall antigens. "0" antigen is stable, whereas "W" antigen is altered
once specific (sIgA) antibodies are produced.
Now "W" antigen is called Surface Array protein. As sIgA immunologic memory is poor, the bacterium can shift
between antigens.
C. fetus subsp. fetus has"0" A-2 and B. But the protein antigens are important
PATHOGENESIS
Little is known of the pathogenic mechanisms of most Campylobacter species. C. jejuni produces an adhesin, a
cytotoxin and a heat-labile toxin similar to that of Escherichia coli.
Transmission of many of the Campylobacter species, including C. fetus subsp. fetus, is by the faecal-oral route. C.
fetus subsp. venerealis is transmitted by coitus and infection of the female genital tract may lead to metritis with
resulting death and resorption of the embryo (infertility), or occasionally to abortion.
C. fetus subsp. venerealis - is an obligate parasite of bovine genitalia.
Encounter is usually through coitus or artificial insemination - in bulls the infection is usually unapparent and
involves the eptihelium of the penis and the fornix of the prepuce.
The organism localizes in the anterior vagina and cervix during the ovulatory phase but does not invade the uterus
and oviducts until progesterone release - and then causes endometritis and salpingitis for several weeks to a few
months during which the animal is infertile - animals usually regain fertility within 5 months.
C. fetus subsp. fetus - Ovine abortions, however organism enters via ingestion, (venereal transmission does not
occur) causes bacteraemia and finally localizes in the placenta causing placentitis and abortion near the end of
gestation.
Infection of ewes in the first half of the gestation period does not result in abortion.
Occasionally causes abortion during latter half of gestation in cattle - organism enters through ingestion.
C. sputorum subsp. mucosalis - found in mouth of piglets for about 2 weeks following weaning - if conditions
favour (stress, overcrowding, underlying gastroenteritis), the organisms can invade intestinal mucosa and multiply
intracellularly.
The result is excessive proliferation of functionally (absorptive capacity) immature epithelial cells which carry the
organism.
Affected pigs do not thrive; lesions are in the lower ileum and cecum (proliferating epithelia) with loss of villi and
development of polypoid (polyp-like) masses.
Animals usually recover in 6 weeks. The diseases caused by Campylobacter species are
abortion
PATHOGENECITY
Symtpoms
Cattle
Affected cattle usually show signs of acute endometritis with in 2 weeks after exposure.
Failure to implant or early abortions, Abortions at 5 -7 months, retained placenta and increase number of services
per conception resulting in repeat breeders are most commonly seen.
Sheep
Pregnant ewes that become infected may lose weight and appear unthrifty. Diarrhea may be present. They usually
abort late in their pregnancy.
They may also deliver stillborn or weak lambs. In unvaccinated flocks, the abortion rate may reach 70% of the
ewes.
The aborted fetus is autolyzed (already showing signs of decomposition).
This is in contrast to a fresh fetus in Chlamydial/Enzootic Abortion of Ewes (EAE), which is another common cause
of ovine abortion.
The placenta is often hemorrhagic (bloody), necrotic (decomposition), and edematous (swollen, leathery).
Blood tinged fetal fluids. Characteristic necrotic spots in the fetal liver.
Following the abortion, the ewe may develop an infection of the uterus (metritis) and require additional medical
attention.
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Mortality in the ewes may exceed 5%. Surviving ewes may become carriers.
Swine
Proliferative enteritis in swine with adenomatous changes due to excessive proliferation of immature epithelial
cells. 6 to 20 week old pigs are affected mostly.
DIAGNOSIS
Both subspecies of Campylobacter fetus can be demonstrated in foetal abomasal contents using the DCF stain.
Fluorescent antibody staining of smears from foetal abomasal contents, cervical mucus and preputial washings is
most reliable, especially when small numbers of the bacterium are present.
C. jejuni can be seen in wet mounts of faces by phase contrast or darkfield microscopy.
The typical darting motility of corkscrew-like organism is suggestive of Campylobacter species.
Characteristic slender, curved rods can be demonstrated in DCF-stained smears, or by phase contrast of ovine
foetal abomasal contents and in bile from chickens with hepatitis.
C. mucosalis can be visualised using the modified Ziehl-Neelsen (MZN) stain on heat-fixed smears from mucosal
scrapings.
Large numbers of pink-staining, slender, curved rods, located intracellularly are seen.
Inoculate the above said specimens on to any one of the selective agar and identification of the organism is based
on colonial morphology as described earlier.
Campylobacter fetus (both subspecies): cervical mucus and preputial washings can be passed through a 0.65 µm
membrane filter to reduce contamination.
Foetal abomasal contents and filtrates are useful for isolation and identification.
C. jejuni: rectal swabs or faeces are the most sutiable specimens.
C. mucosalis: Homogenates of mucosal scrapings from the affected intestine are useful.
Susceptibility or resistance to nalidixic acid or cephalothin, hydrogen sulphide production, nitrate reduction
growth at 25º C or 45º C and the catalase reaction, are some of the criteria on which a definitive identification of
the Campylobacter species is made.
Hippurate test
By Serology
The cervical mucus agglutination test for C. fetus subsp. venerealis is accurate if carried out 2-7 months post-
infection.
A vaginal mucus agglutination test has been found useful but the serum agglutination test is unreliable.
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As C. jejuni is present in the intestines of many normal animals, its isolation from faeces may not necessarily be
significant.
A four-fold increase in an agglutinating antibody titre to the bacterium would suggest involvement of the organism
in the diarrhoea.
Fluorescent antibody-stained smears can be used to identify C. fetus, but the technique does not distinguish
between the subtypes.
To detect infected bull, virgin heifers are inseminated with semen and preputial washings from suspected animals
and examined the heifer’s vaginal mucous during the following 3-4 weeks.
This test is effective but expensive and time consuming.
This disease is very contagious and spreads rapidly among the remaining animals unless very strict hygiene is
practiced.
The fetus, placenta, birth fluids, vaginal discharge, and feces from the affected animals are all sources of infection.
If the water or feeding areas become contaminated with these materials, the abortion rate can be very high.
Isolate the aborting animals immediately, along with proper disposal of the aborted fetus/placenta, and
disinfection procedures.
Prevent the disease from spreading by limiting access to the aborted materials by wild birds and wild or domestic
mammals, which can carry the bacteria to other lots or ranches. Cleanliness is absolutely essential.
Many Campylobacter species produce beta-lactamase, thus accounting for their resistance to penicillin and
ampicillin.
Killed bacterins are effective both prophylactically and therapeutically. Heifers and cows should be vaccinated 1 to
4 months before breeding.
Segregate affected bulls and test new additions. If infected, suspend breeding for 90 days and use antibiotics and
artificial insemination.
In Sheep, vaccinate all incoming and unvaccinated ewes thirty days prior to breeding season and again sixty to
ninety days later.
Follow up with a booster every year at the onset of breeding season.
While some immunity is obtained following an outbreak, immunity against one strain of Campylobacter is not
cross-protective against the other strain.
This false sense of security combined with the presence of carrier animals can result in further abortion storms.
Campylobacter has potential public health significance. C. jejuni - a very important zoonotic pathogen - Milk,
minced meat, dogs, cats, and poultry are important reservoirs of infection for humans.
It is widely distributed in the intestine of domestic and wild animals but is rarely isolated from the feces of normal
humans.
In human even 500 c.f.u./ml in milk are infective, the signs are fever, abdominal pain, nausea, vomiting, and
bloody stools. Some may become persistently infected.
MORPHOLOGY
Domain Bacteria
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Phylum Firmicutes
Class Mollicutes
Order Mycoplasmatales
Family Mycoplasmataeceae
Genus Mycoplasma
Order Acholeplasmatales
Family Acholeplasmataeceae (not required cholestrol)
Genus Acholeplasma
Order Entomoplasmatales
Family Spiroplasmataeceae
Genus Spiroplasma
History
The first mycoplasmal species, the causative agent of bovine pleuropneumonia, was first isolated by Nocard and
Roux (1898).
The species discovered later were called PPLO, because of their resemblance to the organisms causing
pleuropneumonia.
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Habitat
MORPHOLOGY
Mycoplasmas are highly fastidious organisms and most require specific growth factors, an isotonic medium and the
absence of inhibitory substances for growth.
Mycoplasma and Ureaplasma require reduced sterol or cholestrol for their growth.
Because the Mycoplasma are unable to synthesise purines and pyrimidines, they require complex media.
The agar which contains bovine heart infusion, 20% horse serum, (Pooled serum from several animals), 10% yeast
extract, 20ml of adenine dinucleotide, 50 units of penicillin and 0.25 mg of thallous acetate (inhibitory to Gram –
ve and fungi) and the optimal pH is 7.5.
Most grow aerobically but some require N2 with 5% to 10% Co2 after 2 to 6 days of aerobic incubation at 370C,
colonies on solid media are 0.1 to 0.6 mm in d.m.
under low power magnification, the colonies appear transparent, flat and often resemble a fried egg.
Colonies grow into the medium and are difficult to remove from the agar surface.
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This is then put, stain-slide downwards on the microcolonies on the agar block then examined under low power of
microscope.
The dense center of the microcolonies, which grow down into the agar, stain dark blue, the less dense peripheral
zone, resembling surface growth, stains light blue.
Most mycoplasmas are haemolytic in swine blood agar. Mycoplasmas may grow in chicken embryos (yolk sac
route) and cell cultures.
Ureaplasma produce tiny colonies (T-mycoplasma) than the conventional mycoplasmas
Biochemical characters
Resistance
PATHOGENESIS
Transmissions are usually veneral, vertical or by aerosols and many important avian mycoplasmas are egg
transmitted.
Various stress factors predispose the mycoplasmal infection. They parasitic mycoplasma tends to adhere firmly to
the host’s mucous membranes with specific attachment structures.
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The organisms are extra cellular and produce haemolysins, proteases, nucleases and other toxic factors like
capsular carbohydrates, ammonia that can lead to death of host cells or to a chronic infection.
Some pathogenic species have a predilection for mesenchymal cells lining joints and serous cavities.
The respiratory tract and lungs are most frequent sites of infection.
Mycoplasmas are capable of destroying the cilia of cells in the respiratory tract, thus predisposing secondary
bacterial invasion.
The fibrinous exudates frequently present in infections protects them from antibody and antimicrobial drugs.
AIR SACCULITIS
DIAGNOSIS
Specimens
o Mycoplasmas are fragile and specimens must be kept refrigerated and delivered to laboratory within 24-
48 hrs of collection.
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o The samples may include mucousal scrapings, tracheal exudates, aspirates, and pneumonic tissue from
the edge of the lesion, cavity or joint fluids and mastitic milk.
o Swabs should be submitted in transport medium.
Diagnosis
CFT for CCPP and enzootic pneumonia. FAT, ELISA, LAT, species specific DNA probes are highly useful in the
identification of mycoplasmas.
Tetracycline, gentamycin, kanamycin, tylosin, erythromysin and quinolones are effective in some infections.
Dipping hatching eggs in an antibiotic solution has been effective in producing chicks free of M.gallisepticum.
Cattle are vaccinated with live attenuated M.mycoides subsp mycoides strain are useful to prevent CBPP.
Live, attenuated and inactivated vaccines give partial protection against losses in egg production and infections due
to M.gallisepticum.
SYSTEMATICS
Domain Bacteria
Phylum Spirochaetes
Class Spirochaetes(Spira- coil, Chaite- hair)
Order Spirochaetales
Family Spirochaetaceae
Genus Borrelia
Family Serpulinaceae
Genus Serpulina
Family Leptospiraceae
Genus Leptospira(Leptos-fine or thin) Leptonema
The spirochaetes are slender, motile, unicellular, helically coiled, bacteria ranging from 0.1 -3µm in width.
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Based on pathogenicity, DNA composition, and inability to grow at 13ºC the genus Leptospira has traditionally
been divided into two groups
o The pathogenic species - L.interrogans
o Free-living non-pathogenic strains - L.biflexa
The common Leptospira interrogans serovars are L.interrogans serovar Pomona, L.interrogans serovar
Hardojobovis, L.interrogans serovar Icterohaemorrhagiae,L.interrogans serovar Canicola, L.interrogans serovar
Grippotyphosa, L.interrogans serovar Bratislava, and L.interrogans serovar Tarassovi.
History
Synonyms of leptospirosis
Weil’s disease, Seven-day fever, Japanese autumnal fever, Rice field fever, Sugar cane fever and Stuttgart disease
(dogs).
Natural habitat
Leptospirosis occurs worldwide. Leptospires are present in the proximal convoluted tubules of mammalian kidneys
(Sometimes genital tract) and are excreted in urine, often for several months. Characteristically, a reservoir host
shows minimal or no clinical signs.
Several animals act as carrier. Rats are particularly important and they carry most pathogenic serotypes.
Animal carriers often excrete upto 100 million leptospirae per ml of urine.
A warm moist environment with the presence of ground water with a neutral or slightly basic pH favours the
survival of the Leptospira outside the host.
In India the disease has been more frequently reported from southern states than rest of the country.
MORPHOLOGY
Leptospires are actively motile, helically shaped, slender spirochaetes possessing large number of light and fine
spirals.
They are 0.2 –0.3 µm in d.m. and 6-20 µm in length. Characteristically, Leptospira have hooked ends and two
periplasmic flagella (PF), also known as axial filament and endoflagella.
Rotation of the PF results in the distinct spinning mobility of Leptospira.
They stain weakly or not at all with both aniline and Romanowsky stains. They may be stained with giemsa stain.
But they can be best viewed under dark field illumination microscope and can be stained by silver impregnation
technique of Fontana. Leptospira divide by binary fission
CULTURAL CHARACTERISTICS
Leptospira are obligate aerobes that use long chain fatty acids or fatty alcohols rather than CHO and aminoacid as
their energy and carbon sources.
In addition to this it also requires vitamins B1, B12 and purines. Optimum temperature for growth is 25ºC to 35ºC.
They are highly fastidious.
They grow very well in media enriched with rabbit plasma (rabbit plasma contain high concentration of bound
vitamin B12).
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Several liquid and semisolid media are available. Korthof’s modified medium, Stuart’s liquid medium, Fletcher’s
semi solid medium, EMJH (Ellinghausen, McCullough (1965), Johnson and Harris (1967), protein free medium
(commomly used for preparation of vaccine) and Ellis medium (mainly for isolation of leptospires from the genital
tract of cows).
Among these, the EMJH medium contains bovine albumin (fraction V), Tween 80 and rabbit plasma and is most
commonly used for isolation of Leptospira.
The generation time in laboratory media is 12 –16 hrs. The cultures are incubated at 30ºC for upto 8 weeks.
In semisolid media, growth occurs characteristically a few millimeters below the surface.
Addition of 5-flurouracil (100 mgm/ml) in medium is inhibitory for most of the microorganism but not for
leptospires.
The colonies of Leptospira strains appear colorless and below the surface of the agar. They may not be visible until
held against opaque light.
The leptospires are identified on the basis of their typical morphology and motility under dark field microscopy.
In fluid medium, the leptospires appear to rotate alternatively along their axis, moving backward and forward with
no apparent polar differentiation.
In semisolid media, flexing, boring and serpentine movements are seen.
Resistance
Leptospira are highly susceptible to heat, being killed in 10 mts at 50ºC and in 10 sec at 60ºC.
They are also sensitive to acid and are destroyed by gastric juice in 30 mts. Bile destroys them rapidly.
They are also readily destroyed by most antiseptics and disinfectants.
They can survive for several days in alkaline water and only 12 –14 wks in sewage.
Dogs may shed leptospires in their urine for 2 to 6 months, cattle for 3 months and rats for longer periods.
Leptospira have remained viable for atleast 6 days in coagulated blood and also they remain viable in unfrozen
kidneys for several days after the death of the animal.
PATHOGENESIS
The accidental (incidental) hosts are infected by direct transmission through infected urine, placental or fetal
tissues or indirectly through contact with a contaminated environment.
Venereal transmission plays a major role in pigs. Vertical transmission from the mother to the foetus may also
occur in cattle.
Leptospires gain entry through mucous membrane (nasal, genital, ocular, intestinal) or through abraded or water-
softened skin.
After epithelial penetration there is haematogenous spread (Leptospires able to invade the blood stream more
rapidly than other bacteria), with localization and proliferation in parenchymatous organs, particularly the liver,
kidneys, spleen and sometimes meninges for upto 16 days.
It causes damage to endothelium of small blood vessels, leading to extravasion of blood and secondary ischaemia
result in damage to liver, kidney and adrenals.
In the kidneys, the organism reaches and localize in the lumen of proximal convoluted tubules.
Penetration and multiplication in the fetus leads to fetal death and resorption, abortion or weak-off spring. (In
foetus, if infection occur at 3rd trimester, can produce specific antibodies and may overcome the infection).
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The leptospires tend to persist in sites such as renal tubules, eyes and uterus where antibody activity is minimal.
Leptospira causes following disease syndromes in domestic animals.
PATHOGENECITY
DIAGNOSIS
Specimens
Direct microscopy
Leptospires can be demonstrated in urine, other body fluids, and tissues by darkfield microscopy (DFM) and by
FAT.
To detect under DFM, one probably needs 10,000 to 20,000 leptospires/ml in the sample to view atleast
one Leptospira in the high power field.
Urine is centrifuged to concentrate the leptospires. Unclotted blood is centrifuged at low speed to sediment the
RBC after which the plasma can be removed and centrifuged at high speed.
After inoculating the suitable media incubate at 30ºC for upto 8 weeks. A drop of the culture is examined by DFM.
Animal inoculation
Guineapigs, hamsters and weaning gerbils can be inoculated i/p with 0.5 to 1 ml of neutralized urine, unclotted
blood or a 10% tissue suspension in EMJH or 1% BSA.
Cardiac blood is taken aseptically when a temperature rise is detected or at 5,8,10 and 14 days post infection.
Media are inoculated with 2-3 drops of the freshly collected blood.
By serology
Macroscopic agglutination test: it is a screening test and uses dead antigen (lack of specificity).
Microscopic agglutination test (MAT): it uses live leptospires as antigen and is highly sensitive and serovar specific.
CFTand ELISA are also useful for detection of leptospiral antibodies in serum.
To identify the serovar, MAT, restriction endonucleae, DNA analysis and monoclonal antibodies are useful.
Immunity appears to be mainly humoral in that the organisms are not intracellular and bacterins give protection
for short duration.
o Dogs: Bacterins usually contain serovars, Canicola and Icterohaemorrhagiae.
o Cattle: Bacterins usually contain serovars Hardjo, Grippotyphosa, Canicola and Icterohaemorrhagiae.
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Combined penicillin, streptomycin are highly effective. The tetracycline and macrolide antibiotics are also effective.
SYSTEMATICS
Domain Bacteria Bacteria Bacteria
Phylum Proteobacteria Bacteroidetes Fusobacteria
Class Gammaproteobacteria Bacteroidetes Fusobacteriales
Order Cardiobacteriale Bacteroidales Fusobacteriales
Family Cardiobacteriaceae Bacteroidaceae Fusobacteriaceae
Genus Dichelobacter Bacteroides -
Habitat
B. nodosus are obligate anaerobic bacteria of the digital epidermis of sheep under normal climatic conditions.
They will not survive on pastures for more than a week.
Other Bacteroides species can be normal inhabitants of the skin, mucous membrane and G.I. tract of domestic
animals.
Fusobacterium species occurs as a commensal in the alimentary tract and mucous membranes of a variety of
animals.
Morphology
Bacteroides nodosus appear as Gram-negative, fairly large (1.7 x 3-6 µm), slightly curved and non-motile rods.
Often swollen at one or both ends. They occur singly or occasionally in pairs
Fusobacterium necrophorum is Gram-negative, long and filamentous but does not branch.
Filaments can be up to 100 µm in length and 0.5-0.7 µm in diameter.
May have tapered or rounded ends. Irregular staining is characteristic
CULTURAL CHARACTERISTICS
Anaerobic jars with a catalyst, an anaerobic indicator and an atmosphere free of oxygen (10% Hydrogen, 5% Co2
and 85% Nitrogen) is essential for the culture of these strict anaerobes.
Enriched blood agar is highly suitable for these fastidious anaerobes.
Eugon, Columbia, trypticase soy or brain-heart infusion agar enriched with 0.5 per cent yeast extract, vitamin K (10
µg/ml), haemin (5 µg/ml), kanamycin (100 µg/ml) and vancomycin (7.5 µg/ml) are most commoly used.
Bacteroides spp. (except B. ureolyticus) are resistant to kanamycin but the Fusobacterium spp. is sensitive to this
antibiotic.
A 'Fastidious Anaerobe agar' is available commercially with various antibiotic supplements, depending on the
anaerobe that is being sought.
Eugon agar base with 0.2 per cent (w/v) yeast extract, 10 per cent defibrinated horse blood and one µg/ml
lincomycin is the selective medium for Bacteroides nodosus.
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Lemco agar containing pulverized hoof powder can also be used. Members of the B. fragilis group will grow on bile
aesculin medium with 5 per cent sheep blood.
Agar media should be pre-reduced in an anaerobic jar for 6-24 hours before use. The plates are incubated at 35-
37°C immediately after streaking for 4-8 days.
Liquid media such as Cooked meat broth with 0.4 per cent glucose or thioglycollate medium is also suitable with
the addition of the vitamin K-haemin supplement.
The cellular morphology, and sometimes the colonial morphology, can be very variable depending on the strain,
medium and cultural conditions.
Bacteroides nodosus
B. nodosus in a Gram-stained smear from enriched blood agar, appears as straight or slightly curved rods with the
characteristic terminal knobs on one or both ends of the cells
Three basic colonial types are described
o B-type: papillate or beaded (most pathogenic) from ovine foot rot.
o M-type: mucoid (less pathogenic) from non-invasive infections of sheep and cattle.
o C-type: circular (non-pathogenic) and resulting from repeated passage in media.
o The colonies, generally, are greyish-white and 0.5-3.0 mm, diameter, in 3-7 days.
Bacteroides melaninogenicus
Bacteriodes asaccharolyticus
Colonies are 0.5-1.0 mm in diameter, round, convex, opaque and light grey after 48 hours' incubation.
In 6-14 days the colonies may become black. Some strains are haemolytic on rabbit blood agar
Bacteroides fragilis
Fusobacterium necrophorum
F. necrophorum produces grey to yellowish shiny colonies on blood agar, that are about 2-3 mm in diameter after
48 hours' incubation.
Haemolysis is variable.
A Gram-stained smear from the colonies shows long Gram-negative filaments that are less characteristic than
those from direct microscopic examination of specimens.
Lipase, but not lecithinase, activity is exhibited by many strains of F. necrophorum on egg yolk agar.
Biochemical characters
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All pathogenic Bacteroides species are catalase negative except B. fragilis. B. nodosus does not ferment
carbohydrates.
F. necrophorum is indole and H2S positive. Gelatin is not liquefied.
They ferment glucose and maltose with production of acid and gas but not lactose.
Resistance
Toxins
Pathogenesis
The infections are often endogenous, arising from normal flora at the site or by wounds contaminated by nearby
flora.
For these strict anaerobes multiply at a focus in animal tissue if the redox potential of the area is lowered.
This can occur through trauma and necrosis, ischaemia, parasitic invasion or concomitant multiplication of
facultative anaerobes.
B. nodosus produces keratinolytic enzymes and F. necrophorum produces leukotoxin – which
protects Corynebacterium pyogenes from phagocytosis. C. pyogenes produces a diffusible factor that stimulates
the proliferation of Fusobacterium in tissue.
The conditions caused by these non-sporing anaerobes include soft tissue abscesses and cellulitis, post-operative
wound infections, periodontal abscesses, aspiration pneumonia, lung and liver abscesses, peritonitis, pleuritis,
myometritis, osteomyelitis, mastitis and foot rot. The excessive odour is due to production of volatile fatty acids.
Diseases caused by Bacteroides and Fusobacterium species
Symptoms
Infections in animals generally occur when animals are kept in filthy, manure-laden surroundings.
Among the Fusobacterium only necrophorum regularly causes disease in animals.
It is frequently a secondary invader (e.g., liver abscesses in cattle often found with Corynebacterium pyogenes and
is characterized by a necrotic process, commonly causing diseases collectively referred to as necrobacilloses and
present as necrosis, abscess formation, and putrid odour (most common fermentation product is butyric acid).
Cattle
Sheep
Foot rot (interdigital dermatitis, infective bulbar necrosis and heel abscess), mouth lesions and abortions (rare).
Swine
Principal cause of bull nose or Ulcerative stomatitis via injury from fitting nose rings.
Cats
Opportunistic. Highly suppurative. Involves nasal passages, oral cavity and bone.
Secondary invaders to tissue damage.
Dental tartar leads to gingivitis and periodontal disease.
DIAGNOSIS
Choice of specimens
As the non-sporing anaerobes constitute a major portion of the normal flora, the specimens must be collected with
care to avoid contamination from the normal anaerobic flora, situated mainly on mucous membranes and in the
intestinal tract. The following samples are suitable for culture of the non-spore-forming anaerobes.
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Collection of specimens
Specimens for the isolation of these strict anaerobes should be placed immediately in an oxygen-free container,
especially small pieces of tissue or material taken on swabs.
Larger pieces of tissue (over 2 cm3) usually maintain an anaerobic microenvironment deep in the tissue and can be
placed in an air-tight jar for transportation.
Fluid specimens can be collected in a sterile syringe, the air expelled and the needle bent over or plugged. However,
if the specimen cannot be processed within an hour, a fluid specimen should be placed in an oxygen-free tube or
vial.
All specimens for anaerobic culture should be processed within a few hours of collection.
It is best to keep the specimens at ambient temperature rather than in the refrigerator, as oxygen absorption is
greater at lower temperatures.
o Direct examination
Gram-stained smears of the specimens are useful as a screening process, although many of
these anaerobes are not morphologically distinctive.
Dilute carbol fuchsin (4-8 minutes) stained smears are more useful
for Bacteroides and Fusobacterium species as they tend to stain faintly with the Gram-stain.
Fusobacterium necrophorum in clinical specimens is long and filamentous (about 1µm in
diameter) and characteristically stains in an irregular manner.
Bacteroides nodosus is a large rod characterised by the presence of terminal enlargements at
one or both ends (barbell or club shaped).
o FAT is reported as being specific and sensitive
o By Isolation and identfication in anerobic media
o By animal inoculation
Mice, rats and guinea pigs inoculated with cultures of B. nodosus will produce abscess and
sepsis.
Inoculate rabbit subcutaneously with material suspected for F. necrophorum - produces lesions
throughout the body.
Differential diagnosis should be made with strawberry foot rot caused by Dermatophilus species, FMD and
pyogenic infections associated with Corynebacterium pyogenes.
In B. nodosus infection the affected hooves should be trimmed and treated with
10% formalin or chloramphenicol or tetracycline.
Parental treatment with penicillin and streptomycin may be of value.
In Fusobacterium infections, 5-10% CuSO4 is recommended for the treatment of
foot lesions.
For early treatment sulphonamides, tetracycline and erythromycin are useful.
SYSTEMATICS
Domain Bacteria
Phylum Actinobacteria
Class Actinobacteria
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Subclass Actinobacteridae
Order Actinomycetales
Suborder Micrococcineae
Family Dermatophilaceae
Genus Dermatophilus
Dermatophylaceae is a group of bacteria with mycelial filaments which divide transversely and in at least two
longitudinal planes to form masses of coccoid or cuboidal cells, which characteristically become motile.
They are Gram positive, non acid fast, aerobic and produce aerial mycelium when their growths are stimulated by
10% CO2.
Dermatophilus congolensis, Dermatophilus dermatonomus and Dermatophilus pedis are the pathogens causing
variety of skin lesions in mammals, including man.
History
Bovine disease was first described by Van Saceghem in 1915 in the Congo now known as Zaire in Africa.
Habitat
Dermatophilus congolensis is the only species in the genus thought to maintain itself in small foci of infection on a
carrier animal or within scab particles in dust.
It can survive in scab material for periods upto 3 years.
MORPHOLOGY
D. congolensis is Gram positive, non acid fast and aerobic to facultatively anaerobic.
It is filamentous and branching. Mature filaments are composed of motile, coccal zoospores, in parallel lines, at
least two abreast, resulting in a 'tram-track'-like appearance.
The zoospores are about 1 um in diameter. If the flakes of scab (collected from infections) are treated too roughly,
when the smears are made, the filaments will disintegrate and only Gram-positive cocci (zoospores) will be seen.
Life cycle
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Transverse, horizontal and vertical septa form in the immature filaments dividing it into coccal zoospores.
When mature, these zoospores are motile by polar flagella and are infective.
Cultural characteristics
The bacterium is comparatively easy to culture and grows well on sheep or ox blood agar.
An atmosphere of 5-10 per cent CO2 enhances the growth of the organism, especially on primary isolation.
The inoculated plates are incubated at 37°C (Optimum temp. 37oC.) for up to 5 days, although colonies may be seen
after 24-48 hours incubation.
Colonial morphology
Small (about 1 mm) greyish-yellow, distinctly haemolytic colonies can be seen after 24-48 hours incubation.
They are firmly adherent to the medium and are embedded in the agar.
After 3-4 days, isolated colonies can be 3 mm in diameter and are rough, wrinkled with a golden-yellow colour.
Older colonies can become mucoid.
No growth occurs on Sabouraud dextrose agar.
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Microscopic appearance
Gram-stained smears from colonies do not show the characteristic 'tram-track' appearance seen on direct
microscopy.
Usually the smears reveal uniformly staining, Gram-positive, branching filaments but sometimes coccal forms
predominate.
Biochemical characters
D. congolensis is catalase-positive, urease-positive, gelatin-positive and produces acid from glucose, fructose and
maltose.
It is indole-negative, does not reduce nitrate and non-fermentative although acid is produced from certain
carbohydrates.
PATHOGENESIS
Dermatophilus congolensis causes very severe clinical disease and its infection is most common in tropical and
subtropical regions.
Dermatophilus congolensis mainly affects Cattle, horses, sheep and goats, but many animal species and man can
be infected.
The disease has many names
o Cattle : Rain scald, Streptothricosis, Dermatophilosis
o Sheep: Mycotic dermatitis (general infection), lumpy wool (wool- covered skin) and strawberry foot rot
(skin of lower leg and coronet)
The bacterium produces disease in many animal species. It is also a zoonosis.
The common name for the disease is dermatophilosis or streptothricosis.
As far as is known the bacterium is considered an obligate parasite, living only on animals.
Infection is spread by contact, biting insects, fomites and by other unknown means.
Moist conditions and high relative humidity are known to promote the prevalence of the disease.
D. congolensis causes skin infections most commonly seen in cattle, sheep, goats, horses and polar bears in
zoological collections.
The infection is characterised by the formation of thick crusts which come away easily with a tuft of hair, leaving a
moist, depressed area with bleeding points from capillaries.
Infections can be localised but have a tendency to spread over large areas of the body and the morbidity and
mortality can be high, especially in tropical regions.
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Infections have been reported in dogs and cats. In dogs, spontaneous dermatophilosis has been confined to the
intugementary structures, whereas deeper tissue lesions in the form of abscesses have been reported in the felines.
Lymph node enlargements with draining fistulas of the subcutaneous and submucosal tissues have also been
reported in cats.
Diagnosis
A tuft of hair that is plucked from the lesion usually detaches with scab material adhering to it.
Grind up a small amount of scab material and place a little in 2 ml distilled water in a container and incubated for
3.5 hours at room temperature.
Place the container, with lid removed, in a candle jar at room temperature for 15 minutes.
The motile zoospores are chemotactically attracted to the carbon dioxide-enhanced atmosphere in the candle jar
and move to the surface of the distilled water.
Remove a loopful of fluid from the surface and inoculate a blood agar plate.
Incubate the inoculated plate at 37°C for 72 hours under 5-10 per cent CO2.
Identification of this bacterium does not seem to pose any problems. Lesion and typical morphology are quite
diagnostic.
The organism grows well on blood agar plates within 24 to 48 hours as small grayish white colonies turning yellow
to orange upon further incubation.
Although the isolation of D. congolensis may not be necessary for a diagnosis of streptothricosis, Scab material
contains many contaminants and Haalstra's method was developed to overcome this problem.
Small pieces of material are shaved from the scab with a scalpel and the flakes of scab are softened in a few drops of
distilled water on a microscope slide.
A smear is made, taking care to leave a few flakes of scab material intact. The smear can be stained by either
Giemsa or Gram stains.
Giemsa is the better stain to show the characteristic morphology of the bacterium.
Segmenting filaments and coccoid spores stain deep purple. The spores are seen in packets.
Immunity
There is no known vaccine for immunization against the disease produced by Dermatophilus.
Recovery from the disease seems to confer permanent immunity to reinfection.
SYSTEMATICS
o Domain : Bacteria
o Phylum : Proteobacteria
o Class : 1. Alphaproteobacteria
o Order : 1. Rickettsiales
o Family : 1. Rickettsiaceae
o Family : 2. Ehrichiaceae
o Genus : Ehrlichia
o Genus : Anaplasma
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o Genus : Cowdria
o Genus : Neorickettsia
o Genus : Aegyptianella
o Order : 2. Rhizobiales
o Family : Bartonellaceae
o Genus : Bartonella
o Class : 2. Gammaproteobacteria
o Order : Legionellales
o Family : Coxiellaceae
o Genus : Coxiella
o Phylum : Firmicutes
o Class : Mollicutes
o Order : Mycoplasmatales
o Family : Mycoplasmataceae
o Genus : Haemobartonella
o Genus : Eprythrozoon
They are minute obligate intra cellular parasites requiring living cells for multiplication.
They were formerely considered closely related to virus.
But based on their characters like
o cell walls similar to those of other Gram negative bacteria.
o divide by binary fission
o possessing cell wall containing muramic acid
o metabolic enzymes independanat of the host cell
o posess both DNA and RNA
o large enough to be seen under the light microscope
o held back by bacterial filters
o susceptible to antibiotics
o They are considered true bacteria, specially adapted to obligate intra cellular parasitism.
CLASSIFICATION
Depending on the diseases they produce, the vectors that transmit them, antigenic relationships, growth properties
and resistance to physical and chemical agents.
Rickettsiales are usually parasites of alimentary tract of arthropods such as fleas, lice, ticks and mites.
Transmission is from artropod to animal.
The principal diseases, hosts, mode of transmission of pathogens in the Rickettsiales are:
History
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The name Rickettsiales has been given in honor of American Pathologist Howard Taylor Rickets (1909) who first
observed these microorganisms in Rocky Mountain spotted fever.
Derrick (1935) investigated cases of fever occurring in abattoir workers in Brisbane, Australia.
As the etiology of the disease was unknown, it was referred to as Querry or Q fever.
The causative agent was identified by Burnert (Australian) and Cox (American)- so, it named as Coxiella burnetti.
Habitat
The Rickettsiales are essentially parasites of arthropods, replicating in the cells of gut.
Some can be passed transovarially, in ticks and mites but others such as Cowdria and Ehrlichia, are passed
transtadially.
They do not survive the outside the living cells (host or vector) with the exception of Coxiella burnetii, which
produce endospore like forms, that can survive in dust particles for 50 days or more.
Several Rickettsiales may persist in the host in a latent form.
Morphology
Rickettsiae are small, non-motile, non-capsulated pleomorphic, coccobacillary (0.3 – 0.6 x 0.8-2µm in size) forms
existing as obligate intra cellular parasite.
Under the EM, the rickettsiae are seen to have a 3 layered cell wall and trilaminar plasma membrane, thus
resembling Gram –ve bacteria.
They stain reasonably well with Giemsa, Castaneda, (bluish purple), Gimenez, Machiavello (deep red), and
Leishman stains, but poorly with Gram’s stain.
Cultivation
Rickettsiae multiply by simple binary fission. They have cytochromes and their metabolic reactions are aerobic.
They possess many of the metabolic functions of bacteria but require exogenous co- factors from animal cells.
Rickettsiae can genrate their own energy, but they also depend on their host for some energy.
Rickettsiales require living cells for replication. They are readily cultivated in the yolk sac of developing chicken
embryo (first shown by Cox), or in cell lines like mouse fibroblast, HeLa and HEp2.
Growth generally occurs in the cytoplasm of the infected cells or in some cases (spotted fever) in the nucleus.
E.canis can be propagated very well in dog monocytes culture.
Rochalimae quintana –the only rickettsiae which have the ability to grow on blood agar.
Guinea pig and Mice are useful for primary isolation.
Resistance
PATHOGENESIS
o Adherence-, which is facilitated by the surface receptors of the host cell
o Endocytosis
o Phagosome destruction- Rickettsiae destroy the phagosomal mambrane by phospholipase
o Multiply within the cytoplasm or in certain cases (spotted fever) nucleus.
Infection begins in the vascular system, organism proliferate in the endothelial and phagocytic cells and are
disseminated via blood stream.
There is obstruction of small blood vessels because of hyperplasia of infected endothelial cells and resulting
thrombi.
If capillary endothelium is affected, producing thrombi that result in haemorrhagic skin rashes.
Q fever organism has prediliction for mammary gland and placentae in cattle and sheep. Occasionally
asymptomatic infections occur.
Q fever infection causes abortion in sheep, goats and cattle and bronchopneumonia in sheep.
Fever, hamorrhagic rash, stupor, shock and patchy gangrene of subcutis and skin are the common signs and lesions
noticed in rickettsial infections
In case of Ehrlichiosis, the affected animals show symptoms of congested mucous membrane, purulent discharge
from eyes and nose, gastritis, oedema of the hind legs and enlarged lymphnodes.
The mortality rate is 100% in acute cases. On P.M., the lesions are pulmonary oedema, haemorrhages of lung,
hydrothorax, splenomegaly and hyperplasia of lymphnodes.
In Salmon poisoning, the mortality reaches 90%, affected dogs become weak, with vomition, depression and
diarrhoea.
The important lesion is hyperplasia of lymphnodes with necrosis.
In mammals, by direct penetration of skin as a result of feeding by an infected arthropod (tick, louse, flea or mite)
In arthropods as the result of ingestion of blood of infected animals
From arthropod to progeny by infected ova.
In Q fever, wild animals such as bandicoot may constitute the primary reservoir, the infection being transmitted
among them by Ixodes tick.
Transovarial transmission has been demonstrated in Ixodes tick. The rickettsiae are abundant in tick faeces and
survive in them for long periods in the dry state.
Ticks transmit the disease to cattle, sheep and poultry. The rickettsiae are shed large numbers in the milk of
infected cattle, uterine discharges, after-birth and other secretions.
The infected material gets dried up in the atmosphere and becomes suspended in the air and transported to long
distances.
In human, the principal route of infection is mainly by inhalation of contaminated dust particle, ingestion of
infected milk, and contact with contaminated material.
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In Salmon poisoning in dogs, the disease is transmitted by eating raw salmon fish, which contained the infected
fluke (Nanophytes salminicola).
Infected metacercariae encysted in the muscles of fish are ingested by dogs; flukes will get mature and release
invasive rickettsiae.
The fluke eggs are passed in the dog intestine. These eggs develop into meracids, which infect the snail.
The cercaria develops within the snail and passes from snail and infects susceptible species of fish.
DIAGNOSIS
It vary with the disease, but usually include unclotted blood for blood smears, affected tissues (such as brain in
heart water), paired serum samples for serology are appropriate.
In Q fever, besides blood, the sputum and less often the urine, may yield the causative agent.
By Direct microscopy
Both blood and tissue smears, stains such as Giemsa, Gimenez, Machiavello and Leishman as well as FAT are
useful.
By Isolation and cultivation
This is often difficult and is not usually necessary for a laboratory diagnosis.
Guineapigs and mice are useful for primary isolation. Suspected materials are inoculated i/peritoneally and the
animals have to be observed for 3-4 weeks for raising their temperature. (Rochalimae quintana will not grow in
guinea pigs and mice).
Disease Diagnosis
Q-fever Detection of organisms in
o Giemsa or FAT stained smears from ruminant
placentas
o Inoculation of chicken embryos, guinea pigs and
hamsters
o Paired sera samples for agglutination, CFT and
ELISA
o Allergic test
The cattle and horses are inoculated in the lower eyelids
with the antigen. After 3-4 days, there is acute swelling of
the eyelid in the positive case with rise in temperature.
Weil-felix reaction
Immunity is both cellular and humoral. Vaccines are not available for the prevention of the rickettsial diseases of
animals.
Tetracyclins and chloramphenicol are effective in control of Q-fever Requires adequate pasteurisation of milk and
care in the handling of animals an their products.
Eradication of ticks is very helpful in control of rickettsial infection.
SYSTEMATICS
Domain Bacteria
Phylum Chlamydiae
Class Chlamydiae
Order Chlamydiales
Family Chlamydiaceae
Genus Chlamydia
Chlamydophila
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Chlamydia are derived from the characteristic appearance of the inclusion bodies produced by these agents, which
are seen close to the nuclei of infected cells as a cloak or mantle (chlamys meaning mantle).
Chlamydiae are obligate intra cellular parasites, filterable, requiring living system for multiplication.
They differ from viruses in
o They possess cell wall, resembling Gram-negative bacteria but lacking muramic acid
o Do not have an eclipse phase following an infection.
o Produce basophilic intracytoplasmic inclusion bodies in infected cells (Hence, referred as basophilic
virus).
o Pocesses both DNA and RNA
o Multiply by binary fission.
o Sensitive to antibiotics.
Therefore, they occupy a position intermediate between rickettsia and viruses.
o Chlamydiae are more dependant on host cell for high energy compounds such as ATP. Hence, they are
called as energy parsites.
o C.psittaci causes variety of infections in animals such as gastritis, diarrhoea, pneumonia, enzootic
abortion in ewes, abortion, orchitis, epididymitis, seminal vesiculitis, feline pneumonitis, sporadic bovine
encephalomyelitis (BUSS disease), conjuctivitis, rhinitis, hepatitis, and polyserositis and poly arthritis.
o Chlamydia infection in psittacine birds such as parrots called as psittacosis and this infection in non-
psittacine birds such as pigeons, sparrows, turkey and domestic poultry called as ornithosis.
History
The preliminary study in psittacosis was carried out by Sir Samuel Bedson.
Hence, the psittacosis agents are also termed as Bedsoniae.
Natutal habitat
In both birds and mammals the G.I.tract appears to be natural inhabitant of C.psittaci.
The infection with prolonged faecal shedding is characteristic.
C.psittaci is commonly carried in the spleen and kidney of normal appearing birds.
MORPHOLOGY
o They have a unique developmental cycle with alternating, morphologically distinct, infectious and
reproductive forms.
o The elementary bodies (EB) are small, spherical 200-300nm in d.m., infectious and represents the
extracellular form of the organism.
o The elementary bodies enters a cell by endocytosis and differentiate into the large (500 –1000nm size),
non infectious, but metabolically active reticulate body (RB) inside an expanding vacuole.
o The RB multiplies by binary fission producing further RB’s.
o At about 20hrs following infection, some of the RB’s start to condense and mature within the inclusion to
form EB’s.
o In general, release of infectious EB’s begins at about 40hrs post infection due to lysis of the cell.
Chlamydiae are Gram negative; the EBs can be demonstrated by the use of either chemical stains or FAT and IPT.
In modified ZN stain the EBs tend to occur in clumps and stain red against a blue background.
When the modified ZN stained smears examined under darkfield microscopy, the EBs appears as bright green,
coccal structures.
In methylene blue stain under dark field illumination the EBs show autofluorescence, they are revealed as
refractile, yellow green bodies surrounded by a halo.
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In machiavello stain, the EBs stain red against a blue background. In castanedas stain, the EBs are stained blue
with a reddish background.
Giemsa stain: Particularly useful in smears from conjuctival swab of feline pneumonitis cases.
Infected conjuctival epithelial cells contain basophilic intracytoplasmic aggregates of C.psittaci.
CULTIVATION
RESISTANCE,ANTIGENSAND TOXINS
Resistance
PATHOGENESIS
Chlamydial organisms may be shed in faeces of carrier animals. Chlamydial elementary bodies are shed in the
semen.
Young one gets infection mainly through milk of the dam.
Animals and humans are infected by the inhalation of infectious dust and droplets.
In case of enzootic abortion and enteritis in ewes, the infection may take place by ingestion.
The severity of the disesase depends on
o Strain and virulence of the agent
Ovine and bovine type 1 isolates are more frequently associated with abortion, genital infections
and enteritis.
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DIAGNOSIS
Chalmydiae can be isolated from the blood during the early stage and from the sputum on later stage.
Specimens
In case of abortion, smears from affected cotyledons or chorion, prepared from vaginal swabs and from the wet
surface of aborted fetuses.
Uterine discharges, fetal membranes, fetal tissues are also useful.
Aspirated synovial fluid in polyarthritis, conjuctival swab, samples of lung, liver, spleen and paired serum samples
are mostly helpful.
Demonstration of Chlamydophila psittaci elementary bodies on smears by using either
chemical stains or Immunofluorescence staining.
In stained smears, Brucella species may look very similar to Chlamydophila psittaci. But can be
differentiated by serology, isolation and immunological staining methods.
Isolation and cultivation
By inculation into mouse, yolk sac route of embryonated eggs, cell culture and demonstration of
LCL bodies.
Serological tests like ELISA, CFT, IFAT, LAT
Cross reactivity between Chlamydiae species and other Gram negative bacteria will complicate
the interpretation of serological test.
PAGE, Restriction endonuclease analysis and monoclonal antibody typing will help in
diagnosis.
It varies in size and complexity, ranging from the single cell microscopic yeast to multicellular molds, puff balls and
mushrooms.
Fungi are usually aerobic (Particularly Molds), some yeast, however, are facultatively anaerobic and can obtain
energy by fermentation.
Obligate or strict anaerobic fungi are found in the rumen of the cattle.
Fungi grow in wide range of temperature between 10°C and 40 °C. Some fungi can grow at temperature of 50°C or
even in freezing temperature.
Optimum temperature is 22 to 30°C (Saprophytic fungi) and for parasitic fungi 30 -37°C.
Mostly fungi prefer an acidic pH for their growth. The optimum pH range is between 3.8 to 5.6.
However, some fungi can grow even in salt water and some can grow in high concentration of solute.
Some fungi that exist in the mycelial form in nature at room temperature will convert to a yeast form at 37°C in the
tissues of animals.
These fungi are called as dimorphic and the shift is called as YM (Yeast to Mold) shift.
Macroscopic Morphology
Microscopic Morphology
Type of Reproduction
Fungi reproduce asexually or sexually or both depending upon the species and
environmental conditions.
o If a fungus species demonstrate sexual reproduction alone or sexual and
asexual reproduction, the fungus is called perfect fungus
eg. Saccharomyces cerevisiae.
o If a fungus species demonstrates only asexual reproduction, this fungus is
called an imperfect fungus, also known as fungi-imperfecti and these
belong to the classDeuteromycetes.
Taxonomy
Class Phycomycetes
o The Phycomycetes are the most primitive class of fungi.
o They produce broad, aseptate hyphae and reproduce asexually by forming
sporangia that contain sporangiospores.
o Sexual reproduction occurs by means of thick walled resting spores, which
can be zygospores or oospores.
Class Ascomycetes
o Ascomycetes are represented by two morphologically distinct types.
o The first type has unicellular, round or oval forms reproducing asexually
by budding of blastospores.
o The perfect yeast, genus Saccharomyces, represents this type. If
conditions are favourable, sexual ascospores are formed.
o Four or eight ascospores develop within each sac-like enclosure called an
ascus. The asci break open to release the ascospores.
o The second type of Ascomycetes have septate hyphae, producing
filamentous forms, which reproduce asexually by spores called conidia and
sexually by ascopores.
o In this type, the asci are usually enclosed within a tightly meshed network
of hyphae (mycelia) called perithecium.
Class Basidiomycetes
o Basidiomycetes develop sexual basidiospores from specialized club shaped
structures called basidia.
o Each basidium usually bears four exogenous basidiospores resembling
toes on a foot.
Class Deuteromycetes
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Mycosis
Mycoses can be divided into four categories according to the tissues usually
invaded.
Superficial
o The etiological agents of the superficial mycoses are confined to the
outermost layers of skin and hair.
o The superficial mycoses are less serious than the other mycoses
e.g. Trichosporon cutaneum.
Cutaneous
o Most of the etiological agents of the cutaneous mycoses possess the special
ability to invade and destroy keratin in skin, hair and nails, e.g. Candida
albicans, Trichophyton, Microsporum, & Epidermophyton spp.
Subcutaneous
o The agents responsible for subcutaneous mycoses are more serious than
the cutaneous mycoses and they invade primarily the muscle tissue,
e.g. Rhinosporidium seebri, Sporothrix schenckii.
Systemic
o The agents of systemic mycoses invade deep tissue and create symptoms
resembling other diseases of the particular tissues or organ invaded.
o Systemic mycoses may also have cutaneous manifestations.
o The systemic mycoses are the most serious of the mycoses.
o E.g. Candidiasis, cryptococcosis, histoplasmosis, geotrichosis,
blastomycosis etc.,
Prolonged antibiotic therapy. Antibiotics may lower host resistance in some ways, but the best known effect is
through alteration of the normal bacterial flora.
The most common fungal infection resulting from antibiotic treatment is intestinal candidiasis.
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Immunosuppressive treatment and drugs. Drugs that interfere with inflammation and humoral and cellular
immunity, make animals particularly more susceptible to opportunistic fungal infections.
Radiation therapy is particularly toxic for antibody-producing cells.
Infectious and noninfectious diseases (e.g. cancer), and pregnancy can reduce host resistance and make animals
more susceptible to fungal infections.
Genetic immune deficiencies.
Environmental factors, such as trauma, general lowered resistance due to stress, a moist environment and
exposure to a large number of organisms.
REFERENCE BOOKS
INTRODUCTION
Dermatophytosis is an infection produced by molds capable of parasitizing only keratinized epidermal structures:
superficial skin, hair, feathers, horn, hooves, claws and nails.
Those that have a sexual reproductive phase belong to the Ascomycetes. Dermatophyte infections are called
ringworm (tinea).
The dermatophytes, more than any other group of fungi, have developed a close host-parasite relationship with
animals.
Many dermatophytes have evolved into obligate parasites.
Dermatophyte infections are contagious and a zoonotic source of infections for humans.
Infections from animal to human are referred to as zoophilic, while those from soil to animal or human are referred
to as geophilic.
The most common animal dermatophytes are
o Zoophilic pathogens
mentagrophytes
Trichophyton verrucosum Cattle and sheep
Trichophyton gallinae Fowl
Trichophyton equinum Horses
Microsporum canis predominately dogs and cats primates and
horses also
Microsporum nanum Swine and humans
o Geophilic Pathogen: Microsporum gypseum – mainly affects dogs, cats, horses, and humans.
Rare causes of animal dermatophytes are the anthropophilic, globally prevalent Trichophyton
rubrum, Microsporum audouinii, Trichophyton schoenleinii, Microsporum
cookei,Microsporum distortum, Trichophyton megnini.
History
Ringworm, the common name for dermatophyte infections, has been described from the earliest historical times.
The name came about due to the fact that the fungus grows equally in all directions and forms lesions with circular
or ring forms.
The Romans associated the disease with insects and referred to it as tinea, meaning small insect.
Tinea is still used to refer to different clinical settings of the disease (e.g., tinea pedis - ringworm of the feet).
In 1910 Sabouraud published a detailed work on systematic and scientific studies of the dermatophytes. There are
currently 37 species of dermatophytes.
In 1959 the sexual state of some of the dermatophytes was identified, and they are now classified in the
class Ascomycetes.
In 1958 Gentles published the first report on the oral administration of griseofulvin, which cured experimental
dermatophytosis in a guinea pig.
Natural habitat
In their nonparasitic phase, including culture, dermatophytes produce septate, branching hyphae collectively called
mycelium.
The asexual reproductive units (conidia) are found in the aerial mycelium. These units may be either macroconidia
or microconidia.
Shape, size, structure, arrangement and abundance of conidia are diagnostic criteria.
A general rule that can be applied (but not always) is that most species in the genus Microsporum produce
predominately macroconidia, and most Trichophyton species produce predominately microconidia with few or no
macroconidia.
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Hyphal peculiarities — spirals, nodules, rackets, chandeliers and chlamydoconidia — are more common in some
species than others, but they are rarely diagnostic.
Pigmentation is useful in dermatophyte differentiation.
In tissue sections, arthroconidia can often be identified. This parasitic phase arthrospore can remain infectious for
years.
Except in size ranges, which overlap among dermatophyte species, arthroconidia are indistinguishable from
species to species.
Chlamydoconidia are also commonly seen in some dermatophytes in vitro and their presence may be of use
diagnostically in the absence of other conidia.
Sexual spores (ascospores) are absent in the parasitic phase.
CULTURAL CHARACTERISTICS
The most common media for propagating dermatophytes are dermatophyte test medium (DTM) or Sabouraud's
dextrose agar, a 2% agar containing 1% peptone and 4% glucose.
Its acidity (pH 5.6) renders it mildly bacteriostatic and selective.
The selectivity is enhanced by addition of cycloheximide (500µg/ml), which inhibits other fungi, and gentamicin
and tetracycline (100 µg/ml of each), or chloramphenicol (50 µg/ml).
Dermatophytes are aerobes-and nonfermenters. Some attack proteins and deaminate amino acids.
They grow optimally at 25°C to 30°C and require several days to weeks of incubation.
Some dermatophytes in skin and hair (but not in culture) produce a green fluorescence due to a tryptophan
metabolite that is visible under a Wood's light. Of animal dermatophytes, only Microsporum canis produces this
reaction.
PATHOGENESIS
Proteolytic enzymes (elastase, collagenase, keratinase) may determine virulence, particularly in severe
inflammatory disease.
The dermatophytes are highly specialized for utilizing keratin as food source. Localization in keratinized epidermis
has been attributed to the lack of sufficient available iron elsewhere.
Keratinase is therefore a clearly recognized virulence factor for dermatophytes.
It is thought that those dermatophytes that are highly species-specific have a keratinase that can only hydrolyze
keratin from a particular animal species, whereas those with more broad acting keratinases can invade the skin of
many different species (e.g. T. mentagrophytes).
The infectious unit conidia enters the skin through an abrasion, germinate and hyphae begin to grow in the
stratum corneum.
Portions of mycelium differentiate into arthroconidia. This growth pattern in the hairless skin predominates with
some dermatophytes (M. nanum, T. rubrum).
Hair invasion, which is prominent in most animal ringworm, begins with germination of a spore near a follicular
orifice.
The hyphae invade the hair follicle and enter the cortex of the hair by dissolving the keratin.
The hyphae and conidia are carried to the surface by the growing hair, which often breaks off.
Hair invasion may be endothrix (arthroconidia develop within the hair shaft only and the cuticle remains intact) or
ectothrix (arthroconidia develop outside the hair shaft and hyphae are within the hair shaft; the cuticle is
destroyed).
PATHOGENICITY
Symptoms
Although not fatal, dermatophytosis can be a cause of significant economic loss and a source of infection for man.
One of the first clinical signs is loss of hair, followed by an inflammatory reaction of the skin due to the host response.
Dermatophytosis occurs more commonly in very young, old, or sick animals and most often in stabled rather than
pastured animals.
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The peak incidence occurs in the winter. The characteristic lesion is a hyperkeratosis with septate hyphae and
arthroconidia in the stratum corneum.
Invasion of the hair causes the shaft to become weak and break, resulting in circular, scaly areas of alopecia with or
without crust formation.
Arthroconidia within or outside the hairshaft are also referred to as arthrospores.
Manifestations range from erythema to vesiculopustular reactions and suppuration.
Mild forms are seen in T. verrucosum infection of calves. Severe reactions are typical in T. mentagrophytes infection of
dogs and M. gypseum infection of horses.
Local plaques (kerion) may resemble certain skin tumors, especially in dogs.
The inflammatory reaction may arrest the mycotic infection but become the primary problem through secondary
suppurative bacteria! infection.
The roughly circular pattern of the lesions and their inflamed margins suggested the terms ringworm and tinea (Latin for
worm). Different tineas are
o Tinea barabe – beard
o Tinea capitis – scalp
o Tinea corporis – body
o Tinea cruris – groin
o Tinea favosa – favus
o Tinea imbricata and Tinea manum – hands
o Tinea pedis – feet
o Tinea unguium – nails
Lesions
DIAGNOSIS
Diagnosis
Differential diagnosis
Treatment
Ringworm generally regresses spontaneously within a few weeks or months, unless complicated by secondary
infections or constitutional factors.
The agents may persist after clinical cure.
Combined topical and systemic treatment is often preferable. Of two systemic agents available, griseofulvin and
ketoconazole, the latter is more costly and less proven.
Both drugs are given orally and are relatively well tolerated.
For small animals, griseofulvin given orally is most beneficial for severe infections.
The drug is incorporated into the keratin of the tissue and renders the skin resistant to infection.
The fungus is shed with the dead skin, and therefore requires prolonged therapy (given for at least a month, or 2
weeks), particularly if the infection is in the nail. Some toxicity may occur.
Ketoconazole and fluconazole may also be used, but would be very expensive.
Topical treatments can be used for large animals and for skin infections, but are not useful for nail infections.
These treatments include salicylic and benzoic acids, iodine, natamycin-s, and imidazole derivatives. Infections can
recur.
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Prevention
Wood light screening of scalps and suspected animal reservoirs. (Cats and dogs), hair brush technique for culture
of none fluorescing scalps, improve hygiene and discourage sharing of clothing and accessories.
In cattle, vaccines for T. verrucosum have been reported to be successful for 3-5 years. However, this has not been
shown with vaccines to other dermatophytes.
INTRODUCTION
Candidiasis is a general term covering diseases caused by yeasts of the genus Candida, especially C.albicans.
Candidiasis can occur either as a superficial or as a systemic infection.
There are more than 150 species of Candida, but only C.albicans is commonly associated with disease in animals.
Diseases and main hosts of Candida albicans
HABITAT
PATHOGENESIS
Most infections are endogenous in origin and predisposing causes such as immunosuppression, prolonged
antibiotic therapy and malnutrition.
Disseminated candidiasis (or) systemic candidiasis is more common in immunosuppressed animals.
Candida possesses adhesions consisting of fibrillar peptide, Mannans, which have an affinity for the fibronectin on
the surface of cells.
The yeast forms are responsible for tissue damage.
Inhibition of yeast cell division results in hyphal elements that invade tissues.
Possible virulence factors are cell wall glycoprotein, proteases, neuraminidase, chitin, mannoprotein and lipids.
The cell-wall glycoproteins have an endotoxin like activity.
Infection caused by C.albicans frequently involves mucous membranes. Granulomatous lesions are rare.
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PATHOGENECITY
Lesions
In acute cases the lesions appear as tiny, discrete yellowish white (or) grayish white pustules which loosely adhere
to the mucous membranes and rather resemble a small quantity of curdled milk.
In acute cases, the wall of the crop is thickened and covered by a corrugated pseudomembrane of yellowish grey
necrotic material giving the characteristic ‘turky-towelling’ appearance.
In avian moniliasis (or) thrush lesion are confined to the crop, less frequently invade the mouth, oesophagous,
proventriculus, gizzard and intestine.
Symptoms
DIAGNOSIS
Specimens
Scrapings from lesions, centrifuged milk samples, biopsy or tissue samples in 10% formalin for histopathology.
Based on morphology
o C.albicans grows as oval, budding yeast cell on agar cultures & in animal tissues.
o Pseudohyphae are also produced in animal tissue by elongation of yeast cells that fail to separate.
o In Gram stained smears C.albicans appear as purple-blue yeast cell.
o It can also be demonstrated in specimens by 10% KOH (or) by lacto phenol cotton blue.
o The tissue sections stained by PAS-haematoxylin (or) methaneamine silver stains, the C.albicans appear
as thin walled oval, budding yeast cells and/or in the form of pseudohyphae.
Based on isolation and identification
o C.albicans grows well on blood agar or SDA without inhibitors (Candida spp may be inhibited by
cycloheximide).
o The plates are streaked with a small inoculum as for bacteria. The cultures are incubated at 37ºC,
aerobically, for upto 5 days.
o Colonies of C.albicans are white to cream, shiny, high convex and have a pleasant beery smell.
o Smears from the colonies stained with Gram's or lactophenol cotton blue or methylene blue stain reveal
thin walled budding yeast cell and pseudohyphae.
o BiGGy agar (Bismuth-sulphite-glucose- glycine- yeast agar) can also be used for the isolation and
identification of C.albicans.
o Most bacterial contaminants are inhibited by the Bismuth sulphite. C.albicans and C.tropicalis strongly
reduce the Bismuth sulphite to Bismuth sulphide.
o C.albicans gives smooth, circular, brownish colonies and no color diffusion into the surrounding
medium.
o The colonies of C.tropicalis are similar but there is diffuse blackening of the medium after 72 hrs.
Germ tube or serum tube test
o A small inoculum from an isolated colony is suspended in 0.5 ml of sheep, bovine, rabbit or human serum
and incubated at 370C for 2-3 hrs.
o A drop of the preparation is examined under phase contrast or high objective of the light microscope.
o Small, thin walled tubes will be seen projecting from some of the yeast cells. This is charcteristic
of C.albicans.
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Animal inoculation
o Rabbits and mice are susceptible to intra venous and intra peritoneal inoculation respectively. Abscesses
develop in the kidney.
The majority of the candidiasis cases are associated with predisposing diseases, unsanitary conditions or
medication with antibiotics.
Correction of this condition is the first step in therapy.
Nystatin (Mycostatin) administered in the feed to treat candidiasis in chickens, turkeys, swine, dogs and cats.
It has been administered in the mammary gland to treat mastitis in cattle.
Amphotericin B is the most effective drug for the treatment of systemic candidiasis.
Ketoconazole and clotrimazole have been effective in the treatment of mucocutaneous candidiasis.
Rhinosporidiosis is mycosis of cattle, horses, mules, dogs and humans and is characterized by large polyps, tumors
or wart like lesions on the nasal and ocular mucous membrane.
The causative agent is Rhinosporidium seeberi.
History
The natural habitat of the organism is thought to be associated with stagnant water.
The disease has worldwide distribution but its occurrence is most common in India and Srilanka.
The disease has been reported in India more frequently in human beings.
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PATHOGENESIS
PATHOGENICITY
Rhinosporidiosis in dogs
It most commonly occurs in the nose and nostrils, but can also take hold in the
nose and eyes. Rhinosporidiosis belongs to the zoonotic class of fungal infections,
meaning that it can be transmitted to humans.
Signs and symptoms of rhinosporidiosis include the following: sneezing,
bleeding, wheezing, or labored breathing; an infection of the nostrils with a
cauliflower-like growth; a polyp or other growth located near or on the nostril -
this growth may be white or yellowish in color and may appear speckled or
spotted because of the fungus associated with the growth
Rhinosporidium seeberi has not been grown in culture and no laboratory animals are available for cultivation.
Only method of diagnosis is demonstration of spores and sporangia in wetmount preparations of nasal discharge
and section of polyps.
Spores are 6 - 7 µm in d.m. Spores increase in size and attaining a size of approximately 100 µm become
transformed into sporangia by the deposition of a layer of cellulose within the chitinous wall. Numerous nucleoid
division occur and it attains 200 -300µm in d.m.
A sporangium contains approximately 16,000 to 20,000 spores.
At one point, the sporangium thins to form a pore and the spores are escaped.
In the tissue sections stained by H &E stain, various forms of sporangia are seen
Young trophic forms: 10 to 100 µm in d.m. with single central basophilic karyosomes and amorphous cytoplasm.
Mature forms 100 -300µm in d.m. containing sporangiospore.
Empty and collapse form of sporangia.
Treatment
INTRODUCTION
Cryptococcosis also known as European blastomycosis or Torulosis is a subacute or chronic mycotic infection of
man and various species of animals involving the CNS, the respiratory system and eye.
It is caused by encapsulated yeast – Cryptococcus neoformans. Of the 19 species of Cryptococcus,
only C.neoformans is pathogenic for animals and humans.
Diseases and main hosts of Cryptococcus neoformans
There are two varieties Cryptococcus neoformans var neoformans (serotypes A and D) and Cryptococcus
neoformans var gattii (Serotypes B and C).
C. neoformans is a member of the fungi imperfecti. But Filobasidiella neoformans is the teleopmorphic (sexual
stage) state of serotypes A and D, Filobasidiella bacillispora is the teleomorphic state of serotypes B and C.
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C. neoformans is a spherical to oval, thin walled, budding yeast that varies greatly in d.m.
The cells are surrounded by a mucoid polysaccharide capsule that varies in thickness, but in animal tissues it is
usually very large, the width of the capsule exceeding the d.m. of the parent cell.
Daughter cells are usually single and bud from the parent cell by a thin neck.
Yeast cells are Gram positive. Lactophenol cotton blue or nigrosin stains are commonly used to demonstrate the
spherical budding yeast surrounded by a capsule.
HABITAT
C. neoformans is present in dust and has been isolated from the skin, mucous
membrane and intestinal tract of normal animals and birds.
The reservoir of types A and D is the faeces of birds, particularly pigeons and soil contaminated by avian excreta.
The pigeon is not infected, the organism colonise the faeces after they have been passed.
The organisms are concentrated in pigeon faeces due to their high content of creatinine.
The creatinine inhibits many other micro organism but can be utilized by C. neoformans.
It can survive in pigeon droppings for more than a year. C. neoformans has a worldwide distribution
Cultural characters
Biochemical characters
PATHOGENESIS
Infections are exogenous and are usually acquired by inhalation. Animal to animal transmission is not known to
occur.
The route of infection is usually respiratory, often with localization in the nasal cavity or paranasal sinuses and
later extension to the brain and meninges.
The virulence of C. neoformans is largely associated with the antiphagocytic and immunosuppressive capsule and a
unique enzyme diphenol oxidase.
The cryptococcal lesions, on gross examination, resemble myxomatous neoplasm; infection of the meninges can
resemble tubercular meningitis.
Infections extend to the optic nerve resulting in blindness. Subcutaneous granuloma is often occurring in cervical
or pedal regions.
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PATHOGENECITY
In cryptococcal mastitis cows with mild infections often show no clinical signs except swelling of the affected
glands.
In severe infections, animal exhibits typical clinical signs of bacterial mastitis (fever, swelling and firmness of the
udder).
Milk secretion gradually diminishes. Milk will appear grey, white, highly viscid and mucoid.
In dogs, it affects the CNS, causing incoordination, hyperaesthesia and nasal discharge.
Subcutaneous granuloma around the ear, face and feet. In cats, chronic nasal and ocular discharge, granulomas
and blindness are common.
DIAGNOSIS
Diagnosis
Specimens: CSF, lesions or exudates, mastitic milk, biopsies and tissues should be collected.
Direct microscopy
o Demonstration of budding yeast with a large capsule by India ink, Nigrosin and LPCB staining methods.
Histological sections on biopsies of tissue from lesions can be stained by the PAS-haemotoxylin stain. This will
stain or outline, the yeast cell but not the capsule, which appear as a clear area surrounding the cell.
Based on isolation and identification: production of brown pigment on birdseed agar.
Based on urease production
By Animal inoculation
o Mice are susceptible to pathogenic strains.
o When intra cerebral or intra peritoneal inoculation of suspected material, the mice will die within 4 days
to two weeks.
o PM reveals gelatinous lesions in the abdominal cavity and lungs.
o The budding encapsulated yeast can be demonstrated from the lesions.
By serology
o Slide LAT, tube agglutination, CFT, ELISA, IFAT are useful to detect antibodies.
Treatment
Amphotericin B is the drug of choice. Imidazole derivatives such as ketoconazole and fluconazole are also useful.
INTRODUCTION
Synonyms of this disease are small form histoplasmosis, Darling’s disease and reticuloendothelial cytomycosis.
Histoplasmosis is a chronic, granulomatous disease caused by Histoplamsa capsulatum var
capsulatum (Teleomorph : Ajellomyces capsulatus).
The organisms become heavily concentrated in the feces of birds (particularly black birds, sea gulls, starlings and
pigeons); bats are also important vectors of this disease. Thus, the fungus has been isolated from soil in bat caves,
bird roosts, chicken houses, and silos inhabited by pigeons.
The organisms are facultative, intracellular parasites of macrophages.
The tissue form cells of H. capsulatum var.capsulatum appear as small, oval yeast cells with or without buds.
Daughter cells are attached to mother cells by a narrow attachment point.
The yeast cells are relatively small (2-4 um). A clear halo is seen around darker-staining central material.
CULTURAL CHARACTERISTICS
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H. capsulatum may be cultured on the Sabouraud agar with chloramphenicol but without cycloheximide at 25° C,
or on brain heart infusion agar with blood at 37° C. At 25° C, the mycelial phase has two types of conidia: small,
round microconidia and large (7-18 µm) thick-walled, macroconidia with knob-like projections. The yeast phase
grows at 37° C on blood agar.
EPIDEMIOLOGY
Prevalent in America, parts of Africa and Asia. Rare in Australia and Europe.
In the United States, histoplasmosis occurs throughout the midwest and much of the eastern half of the United
States.
Infection usually occurs through inhalation of spores of the dimorphic fungus Histoplasma capsulatum.
Infections may also occur through ingestion. Most infections are subclinical or benign.
Histoplasmosis may occur commonly in animals in endemic areas.
Dogs are particularly susceptible, but the disease has also been reported in cattle, cats, swine, horses, sheep and
wild animals.
The disease has not been reported in birds. Histoplasma capsulatum is not contagious.
PATHOGENESIS
The disease may vary from small granulomatous nodules to an acute, disseminating, rapidly fatal form.
Usually, the disease manifests itself either as a pulmonary or intestinal infection, and may be inapparent or
subclinical, mild, acute, chronic, or disseminated.
Ulcerations and tuberculosis-type lesions may occur in many of the organ systems.
Mice are highly susceptible to infection, whereas other laboratory animals vary in susceptibility.
Following inhalation of spores, macrophages phagocytize the organisms and an inflammatory response ensues.
The fungus is either killed, or local granulomas form with calcification.
Host immunity and the number of spores inhaled determine which form the disease manifests itself as.
The macrophages may carry the organisms to various body sites and actually help to disseminate it.
Thus, histoplasmosis has been referred to as a disease of the reticuloendothelial system.
Enlargement of the liver and spleen, and nodules on the tongue, ocular involvement, and abortion have also been
reported.
PATHOGENECITY
Clinical signs
Lesions
become calcified.
DIAGNOSIS
Diagnosis
The organisms are rarely seen extracellularly. Specimens (CSF, biopsies, bone marrow, lymph nodes, or buffy coat)
stained with a variety of histological stains (Gomori methanamie or Periodic acid-schiff stains) reveal the
organisms within macrophages. The yeast cells are relatively small (2-4 um).
A clear halo is seen around darker-staining central material.
The skin test becomes positive after exposure to the fungus and remains so for the life of the animal.
Thus, the skin test is of little usefulness in detecting active disease.
Furthermore, skin testing may induce antibody formation, thereby, interfering with more useful serological tests.
The complement fixation test for detection of specific antibodies is useful for diagnosis.
Animals develop a rapid rise in titer following infection; the titer falls off gradually and disappears by nine months.
However, cross-reactivity to antigens of the other systemic fungal pathogens may occur.
The immunodiffusion test is a useful adjunct to the CF test; the development of 2 bands may indicate active or past
infection, respectively.
A latex agglutination test and fluorescent antibody test are useful screening tests, but complement fixation is
considered the confirmatory test.
Treatment
Amphotericin B is the drug of choice, but ketoconazole, sulfonamides, and ethyl vallinate may also be effective.
CULTURAL CHARACTERISTICS
Both yeast and mycelial forms can be cultivated if suitable media, temperature of incubation and carbon dioxide
tenson are provided.
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The organism grows slowly when the yeast phase is grown on media rich in protein and in an atmosphere enriched
with CO2.
Several culture media have been used, but the most satisfactory were Sabouraud's dextrose agar enriched with
2.5% glycerol; brain heart infusion agar enriched with 10% horse blood; nutrient agar supplemented with 2%
dextrose; mycobiotic agar and mycoplasma-like organism medium.
Growth on all media is very slow and appears after four to eight weeks of incubation at 25°C.
Colonies of the mycelial form are a yellowish/light brown to deep brown, convoluted, waxy and cauliflower-like.
In body tissues, the ability of H. farciminosum to convert from the mycelial form to the yeast form appears to be
dependent on temperature and nutrition as well as the strain, However, in vitro, conversion of the mycelial form to
the yeast form of H. farciminosum can be achieved by incubating at 35°C to 37°C.
Complete conversion to the yeast form is achieved only after four to five repeated serial transfers onto fresh media
every eight days.
Biochemical characteristics
The biochemical characteristics of the mycelial form include positive reactions to catalase and urease tests as well
as the assimilation of ammonium sulphate as the sole source of nitrogen.
No Fermentation of carbohydrate sugars, liquefaction of gelatine or reduction of nitrate occurs.
Resistance
The organism is highly resistant to the effects of physical and chemical agents and can survive for at least a month
in the dust of stables or kraals.
This pathogen is also known to be viable and virulent after desiccation in the laboratory for 25 months.
H. farciminosum may survive for up to ten weeks in non-sterile water at 26°C.
EPIDEMIOLOGY
PATHOGENESIS
PATHOGENECITY
Clinical signs
The cases of epizootic lymphangitis can be grouped into four different forms, namely: cutaneous, respiratory,
ocular and asymptomatic carriers.
The cutaneous form of the disease, after which the disease was named, is the most common.
The initial lesion is an open granulomatous wound along the course of a lymphatic vessel, which has a tendency to
ulcerate, or to undergo alternating periods of discharge and closure for some weeks before healing with residual
scar formation.
Lesions are most common in the forelimbs, the chest wall and the neck. In severe cases, skin over the entire body
may be affected.
The lesions begin as indolent, chancre-like papules, becoming larger over the course of weeks and eventually form
irregular pyogranulomatous nodules, which frequently ulcerate.
Mortality does not usually exceed 10% to 15% and the main loss results from the inability of animals to work for
several weeks because of extremely painful lesions.
The ophthalmic form of the disease is less frequent. Infection may occur as conjunctivitis or a naso-lachrymal
infection.
The infection rarely becomes generalised. Initial infection is characterised by a watery discharge from one or both
eyes and some swelling of the eyelids, followed by the development of papules and ulcerating button-like growths
on the conjunctiva and/or on the nictitating membrane .
The respiratory form of the disease is characterised by lesions which are mostly confined to the upper respiratory
tract.
This form usually occurs as a late development in the cutaneous form of the disease.
On the nasal mucosa, the lesions begin as yellowish papules or nodules and these soon form crater-like granulating
ulcers that bleed easily.
The lesions are usually found near the external nares. These lesions may also occur in the lungs.
Asymptomatic carriers can be identified clinically by the identification of fibrocalcific skin lesions at previous sites
of infection. Such horses will give a positive result to an intradermal sensitivity test and positive reactions to
serological tests.
Lesions
Gross lesions are manifested by pyogranulomas, purulent discharge of thickened superficial lymphatic vessels and
enlargement of regional lymph nodes.
Histopathologically, a typical granulomatous tissue reaction occurs with a predominance of the large macrophages,
many of which contain oval organisms in the cytoplasm.
Affected tissues stained by Gram stain revealed the presence of ovoid double-contoured yeast-like cells.
Periodic-acid Schiff or Gomori's methanamine silver stains are very useful to demonstrate the presence of the
organisms.
Typical nodules of liquefied foci have also been recorded in the pleura, spleen, liver and bone marrow.
DIAGNOSIS
Diagnosis
Laboratory tests used in the diagnosis of epizootic lymphangitis include isolation of the causative agent by culture
and tests for the presence of antibodies in the blood.
Haemalological picture showed leucocytosis, neutrophilia and an increase in the erythrocyte sedimentation rates.
Direct smear examination and culture technique
o Diagnosis is usually based upon demonstration of the typical yeast-like, double-contoured cells in pus
collected aseptically from the lesion and confirmed by culturing the pathogen.
o H. farciminosum is a Gram-positive organism and is successfully cultivated on a vanety of media.
o Growth is relatively slow; most isolates require from four to eight weeks for development of characteristic
colonies.
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Serological tests
o In the absence of positive culture of H. farciminosum, a presumptive diagnosis is usually made, based on
the presence of antibodies in the serum.
o Although several serological tests have been used for the diagnosis of epizootic lymphangitis, none of the
tests are sufficiently sensitive or specific to confirm diagnosis.
o The four serological tests such as FAT, AGID, ELISA and serum agglutination are relevant.
Electron microscopic examination
o Tissues taken from cutaneous lesions revealed the presence of oval bodies.
o Most of the details of the fine internal structures could be observed.
Animal inoculation
o Experimental transmission of H. farciminosum has been attempted in mice, guinea-pigs and rabbits.
o Imnunosuppressed mice were highly susceptible to experimental infection and can be used for diagnostic
purposes.
o Intra dermal test
o An accurate and reliable method of skin testing is the intradermal test.
o This consists of intradermal injection of 0.1 ml of soluble antigen prepared from H. farciminosum.
o An increase in the thickness of the skin of 8 mm to 20 mm, 24 h after injection of the antigen can be
regarded as a positive result.
Differential diagnosis
A number of diseases may be confused with epizootic lymphangitis (e.g. glanders, strangles, ulcerative
lymphangitis and sporotrichosis), especially when these diseases occur under the same environmental conditions.
TREATMENT
Epizootic lymphangitis is a chronic disease, although some cases may heal spontaneously a few weeks after the
development of clinical signs.
The intravenous injection of 100 ml of sodium iodide of a 10% solution, repeated weekly for four weeks, gives good
results.
The infected horses were treated with an intravenous injection of amphotencin B at a dose of 0.2 mg/kg body
weight three times on alternate days.
The scabs were removed and the areas cleaned daily with an iodine solution for seven days.
Administration of griseofulvin, repeated if necessary, has given good results when combined with iodides and local
surgical treatment.
The surgical treatment usually consists of opening the nodules and packing with gauze soaked in 7% tincture of
iodine.
Outbreaks in non-endemic areas are probably best controlled by the slaughter of affected animals.
The long incubation period of the disease, the high resistance of the causative agent and the presence of clinically
healthy carriers make control of the disease difficult in endemic areas.
Control of the disease depends upon elimination of the infection by culling infected horses and preventing spread
by hygieneic precautions.
Cleaning and disinfection will help to prevent the disease from spreading.
This method of control is the most satisfactory and proven to be mandatory for large breeding companies in
endemic areas.
A killed formalised vaccine prepared from the yeast form of the fungus, administered subcutaneously in a dose of 5
ml once a year, has given good results.
An attenuated vaccine was developed by exposure of the causative agent to high temperature.
Horses inoculated subcutaneously with 3 ml in a single dose have given a higher protection rate.
INTRODUCTION
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Coccidioidomycosis is usually a benign, inapparent, or mildly severe upper respiratory infection that resolves
naturally.
On occasion, the disease may become an acute or chronic, disseminating, fatal mycosis.
The etiologic agent, Coccidioides immitis, is the most virulent of the fungal pathogens.
The disease is not uncommon among laboratory workers who isolate the agent.
It is endemic in the soils of the Southwest United States and Central South America.
Dust storms increase the incidence of disease.
Of domestic animals, dogs are most frequently affected, although horses are occasionally affected as well.
Infections also occur in cats, swine, sheep, cattle, human and nonhuman primates, and some 30 species of non-
domestic mammals.
MORPHOLOGY
In the soil, C. immitis is a mould made up of slender septate hyphae that give
rise, on thicker secondary branches, to chains of infectious arthroconidia
(arthrospores, arthroaleuriospores, arthroaleurioconidia).
These are bulging, thick-walled cells, separated by empty cells, through which
breaks occur when arthroconidia are dispersed.
In tissue, arthroconidia grow into spherical sporangia with birefringent walls,
"spherules", which by internal cleavage produce several hundred "endospores".
The walls disintegrate, allowing dissemination of endospores, each of which may
repeat the cycle or, on a nonliving substrate, give rise to mycelial growth.
Though only arthroconidia are naturally infectious, endospores can
experimentally initiate disease. Sexual spores are not known.
"Coccidioidin" in supernatants of mycelial C. immitis broth cultures is largely
polysaccharide, but contains some amino acid nitrogen.
It is used in cutaneous hypersensitivity and serologic tests.
"Spherulin," a lysate of cultured spherules, is also used in skin tests. Both are
leukotactic.
Cultural characteristics
This dimorphic fungus grows more quickly than the other dimorphic pathogens (1-2 weeks), but the same
cultivation media are used.
On Sabouraud's or blood agar, at 25° C or 37° C, a moist white colony develops that later is covered with a fluffy
mycelium. Bovine blood agar is hemolyzed.
Arthroconidia are produced in 5 to 7 days. Mycelial growth should be evident within a week and is examined for
presence of arthroconidia in a lactophenol cotton blue wet mount.
Thick-walled, barrel-shaped arthroconidia alternating with empty disjunctor cells is characteristic.
The isolate can be reconverted to the sporangial phase by animal inoculation or cultivation in a spherule medium.
The sporangial phase is produced at 40°C in media containing casein hydrolysate, glucose, biotin, glutathione and
a salt mixture.
Resistance
Arthroconidia resist drying and tolerate heat and salinity better than do competing soil organisms.
In summer heat, C. immitis survives in soil layers nearer the surface than its competitors.
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When conditions favor growth again after rains, C. immitis repopulates the superficial soil layers first, ensuring its
widespread dispersal.
PATHOGENESIS
Infection is mainly by inhalation of dust. Primary cutaneous infections are rare. Infection is initiated by inhalation
of the arthrospores.
Relevant cell products include proteases, T suppressor cell activator, and leukotactic agents. Leukocytes in
vitro encourage arthroconidial metamorphosis to spherules.
The agent triggers an inflammatory response in the lung, is engulfed but not killed by phagocytes and is conveyed
to the lymph node, where another inflammatory focus develops.
Inflammation is stimulated in part by a potent serine protease, which is liberated during the growth of the
fungus in vivo (digests elastin, collagen, and immunoglobulins).
Normally, cell-mediated immune responses arrest the process at this stage following stimulation of TH -1,
lymphocytes that activate macrophages.
With inadequate cell-mediated immunity, dissemination can occur to bones, skin, abdominal viscera, heart, genital
tract, and eye (and rarely in animals to brain and meninges).
Clinical Signs
In all species, overt disease is the exception. Highest prevalence of canine systemic coccidioidomycosis is observed
in male dogs, 4 to 7 years of age.
Young Boxer dogs and Doberman pinschers are highly susceptible.
Pulmonary disease may be asymptomatic, symptomatic of variable degree, benign and chronic, or progressive.
Dissemination may occur, but only in the dog and human, and depends on host resistance and the level of
exposure.
There may be respiratory signs (including cough), fever, lameness due to bone involvement, or discharging sinuses
from deep lesions.
The disease is most common in Boxers and Doberman Pinschers.
The disease has not been reported in cats.
In cattle, sheep, and swine, the disease is usually asymptomatic, limited to lungs and regional lymph nodes and
undiagnosed until slaughter.
Lesions
Gross lesions are white granulomas varying from miliary nodules to irregular masses. Peritoneal, pleural, and
pericardial effusions occur.
The initial lesions are found in the lungs. Systemic disease may involve the meninges, bones, joints, and
subcutaneous and cutaneous tissues.
Lesions may also occur in the lung, brain, liver, spleen, and kidney.
In acute cases, burrowing abscesses are common.
In chronic and slowly advancing cases, focal and suppurative granulomatous lesions are common without
caseation or calcification.
Severe, disseminated disease is usually seen only in dogs and humans.
Cattle and swine are often infected, but the disease is restricted to a few tuberculous-like lesions in the lymph
nodes and sometimes the lung.
Diagnosis
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SPOROTRICHOSIS
yeast phase.
Single-celled, cigar-shaped yeasts may or may not be seen in pus from lesions.
Fluorescent antibody enhances visualization and confirmation of the disease.
Serological diagnosis can be made by demonstration of a rise in complement-
fixing antibody.
Potassium iodide, Amphotericin B, ketoconazole and micoconazole are effective
for treatment.
BLASTOMYCOSIS
INTRODUCTION
When compared to infection in avian species, the intensity of infection in other species is less.
Aspergillosis is an economically important disease because of its high mortality and morbidity in brooder chicks.
CULTIVATION
The Aspergillus grows very well in ordinary Sabouraud's Dextrose Agar with chloramphenicol.
Cycloheximide should never been incorporated in the media.
Morphology of Single, usually one on Double, cover entire Single and double,
conidiospore upper half of the vesicle, form radiate cover entire vesicle,
and sterigmata vesicle, parellel to axis head point out in all
of stalk. directions
PATHOGENECITY
Acute Aspergillosis
Chronic Aspergillosis
Cattle
Conditions of high humidity and temperature encourage the growth of molds when hay and straw is stored and this
constitutes the source of infection for cattle.
Aspergillus fumigatus is considered as the primary cause of mycotic abortion, however many
other Aspergillus species, A.flavus, A.nidulans, A.niger, A.terreus and A.versicolorare also found to be associated
with abortion.
Infections mainly occur by inhalation into lungs or by ingestion, and then carried to the placenta in the blood
stream from lesions in the respiratory tract or ulcers, mycotic ruminits or other lesions of the digestive tract.
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This results in a slowly developing fungal placentitis (one to two months) and intefere with the nutrition of the
foetus, resulting in foetal death and abortion.
Chronic form may lead to purulent vaginitis, cervicitis and endometritis, resulting in infertility.
Abortion most commonly occurs in 6-7 months of gestation.
The aborted foetus shows discrete, raised ringworm type lesions on the skin of head and neck.
The placenta is found thickened, haemorrhagic, odematous and necrotic.
The cotyledons will be grey in color, inter cotyledonary area will be leathery, grey and tan in color.
On necropsy grayish or yellowish gaseous exudates with mycelia are commonly seen in the lung and airsac.
Sometimes the organism colonise the bronchi, forming a compact spherical colony, which is called fungus ball.
The fungal balls are produced most frequently by A.niger than A.fumigatus.
Characteristic nodular lesions are also seen in alimentary canal, kidneys and ovaries.
DIAGNOSIS
Diagnosis required continuous effort because it is one of the common contaminant of laboratory and it can be
cultured routinely from the skin and URT of healthy animals.
For confirmatory diagnosis, consider the following points:
o Repeated isolation
o Absence of any other pathogen
o Recovery from unexposed tissue and demonstration of hyphae.
Method of diagnosis
Prevention
Vaccines
Several types of vaccines are used involving different parts of the fungal elements. i.e. whole cell filterate, spores,
mycelial fragments, germinating cells and they are inactivated with the use of heat, phenol, formalin etc. some
vaccines of live cell origin are also available.
Treatment
HISTORY
Fungal toxins are referred to as mycotoxins and the diseases they produced are termed as mycotoxicosis.
The epidemics of ergot poisoning caused by eating cereal grains infected with the parasitic fungus Claviceps
purpura have been recorded since the middle ages, the toxin compounds responsible for this ergotism were
identified in 1875. During 1942 – 1947, the disease alimentary toxic aleukia (ATA) outbreak occurred due to the
consumption of mouldy cereal grains.
One of the first, well-documented outbreaks of animal mycotoxicosis (aflatoxicosis) occurred in East Anglia,
England in 1960 when more than 1,00,000 turkey poults died of an unknown disease (Turkey X disease).
Subsequently, the examination of the incriminated grounnut meal revealed the presence of A.flavus mycelia and
the toxic metabolites revealed by thin layer chromatography (TLC) were called aflatoxins.
Many of the toxigenic fungi, over 100 known species, are capable of elaborating mycotoxins.
The same mycotoxin can be produced by different fungi and the same fungus can produce different mycotoxins.
Toxin production occurs only under specific conditions of moisture, temperature, suitability of substrate and
appropriate oxygen tension.
The optimum conditions for toxin production are relatively specific for each fungus.
For e.g. Fusarium elaborates its toxin at freezing temperature, while A. flavus requires a temperture of 250C.
The susceptibility of different crops to mould infection is goverened by the presence of suitable substrates.
Damage to the seed coat by insects, mechanical harvesting, severe frost or other factors may predispose crops to
fungal attack.
Insects may also serve as carriers of fungal spores.
The fungi associated with cereal grains have been divided mainly into two types.
o Field fungi which invade the grains before harvest and required greater water activity for growth
e.g. Fusarium, Helminthosporium and Cladosporium
o Storage fungi which invade the grains after harvest during drying and in storage
e.g. Aspergillus, Penicillium
The main types of toxigenic fungi, which produce mycotoxins of animal/ poultry importance, are
Species Toxins
A.flavus and A.parasiticus Aflatoxins
A. ocheraceus Ochratoxin
Fusraium roseum Trichothecane (t-2) toxin
Penicillium citrinum Citrinin
A.nidulans and A.versicolor Sterigmatocyosin
CHARACTERISTICS OF MYCOTOXINS
The term mycotoxin is derived from the Greek word – ‘mykes’ meaning ‘fungus’ and the Latin word – ‘toxicum’
meaning ‘poison’.
Mycotoxins are group of compounds produced by some strains of certain fungi that cause illness or death when
ingested by man or animals.
They are low molecular weight, non-antigenic, heat stable secondary fungal metabolites.
They can activate at low concentrations. They have a wide spectrum of toxic effects, like carcinogenic, mutagenic,
teratogenic and immunosuppressive.
Acquired immunity does not occur following exposure.
Each toxin affects specific target organs or tissues.
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AFLATOXICOSIS
The name aflatoxin derives from Aspergillus flavus toxin. Afalatoxins are a group of approx.
20 related toxic compounds produced by some strains of A. flavus and A.parasiticus during growth on a variety of
cereal grains and food stuffs such as maize, cotton seed and groundnuts.
High humidity and high temperature during pre-harvesting, harvesting, transporatation and storage, as well as
damage to feed crops by insects, drought and mechanical injury during harvesting, favours the growth and toxin
production of Aspergillus flavus.
Mould growth and toxin formation require a moisture content of the substrate greater than 15%, temp.25 0C and
adequate aeration.
Aflatoxins are a group of related bisfuranocumasin compounds with toxic, carcinogenic, teratogenic and mutagenic
activity.
The four major aflatoxins are B1, B2, G1 and G2. These mycotoxins are named according to their position and
fluorescent colour on thin layer chromatography (TLC).
B1and B2 produce blue colour and G1, G2 gives green fluorescence.
Aflatoxins M1, M2 are hydroxylated metabolites of B1 and B2 that are excreted in the milk of lactating animals such
as dairy cows.
Acute toxicity
Hepatic injury and nervous signs such as ataxia and convulsions. Death may occur suddenly.
Chronic toxicity
There is reduction in efficiency of food conversion, depressed daily weight gain, decreased milk production in dairy
cattle and enhanced susceptibility to intercurrent infections due to immunosuppression.
PATHOGENECITY
Symptoms
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Young animals, particularly young pigs, calves, turkey poults and ducklings are highly susceptible.
Aflatoxin B1 produce the most hepatogenic, carcinogenic, teratogenic and embryotoxic effects.
Prominent signs in calves include blindness, circling, grinding of teeth, diarrhoea, tenesmus and convulsions.
Aflatoxicosis has ben described in goats. But sheep are highly resistant.
In dairy cattle, afalatoxin M1 and M2 are excreted in the milk.
In pigs, signs include drowsiness, inappetance, jaundice, weight loss and yellow urine.
Ducklings are considered to be the most susceptible avain species to aflatoxins.
Signs include anorexia, poor growth rate, ataxia and opisthotonus, followed by death.
In birds over three weeks of age, subcutaneous haemorrhages of legs and feet.
Lesions
Principle target organ is liver. Depending on the severity of intoxication, hepatomegaly with necrosis and marked
bile duct hyperplasia will occur.
Acute hepatic failure and massive haemorrhage due to impaired blood clotting, increased capillary fragility leading
to death may occur with higher doses.
In chronic toxicity, in additon to liver damage, degenerative changes in the kidney, thymus cortical aplasia leading
to decreased cell mediated immune response will occur.
DIAGNOSIS
Chemical identification of mycotixins in food samples and biological assays for toxicity are important confirmatory
steps.
Demonstration of toxigenic strains of Aspergillus flavus and Aspergillus fumigatus and of potentially toxic levels
of mycotoxins in the food, tissues, secretions are helpful for diagnosis.
Concentration of aflatoxin B1 in excess of 100μg /kg of feed are considered toxic for cattle.
Thinlayer chromatography and HPLC are more sensitive analytical methods for determing afaltoxins levels in the
food.
Radio immuno assay and ELISA methods are also available.
Biological assays
Ducklings are mostly susceptible. Bile duct proliferation in one-day-old ducklings and chick embryo bioassay are
highly useful.
Prevention of contamination at all stages of food production, storage and use is the preferred method of preventing
aflatoxicosis.
Decontamination procedures like physical removal and chemical treatment of aflatoxin contaminated feeds such as
with acids, alkalies, aldehydes, oxidizing agents of selected gases (ammonia) have been used for degrading
aflatoxins.
High affitnity inorganic compounds such as benzoic and propionic acid have been widely used as preservatives for
stored agricultural products.