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Lecture note
on
Microbiology III
B.V.Sc. A.H. 5th semester
By
Dr. R.K.Bhattarai
Lecturer
Department of Microbiology and parasitology
IAAS, Rampur
August, 2010
Escherichia coli
E. coli is important pathogen of animals of genus Echerichia and family Enterobacteriaceae
Major facultative gram negative species comprising the normal flora of GIT
Septicaemic disease in foal, calves, piglet, puppies and lamb
Diarrhea in newborn farm animals
Edema dissease in pigs
It is opportunistic in almost all animal species (UTI, abscess and pneumonia)
Isolated in 1885 from feces of infant suffering from diarrhea, it is most common and
clinically important
Morphology
Gram negative cell wall composed of LPS (O-antigens) and protein
Non spore forming bacillus
Most strains motile with peritrichous flagella (non motile are from extra intestinal lesion and
posses a polysaccharide capsule)
Growth at 15 - 45˚C(37˚C)
Possess capsule (K-antigens), flagella (H-antigens) or adhesions (fimbria or pili)
They affect the control of cyclic nucleotide activity within the „target cell‟, which results in
deregulation of water and electrolyte secretion by the affected host cells.
Other enterotoxin
SLT (Shiga and Shiga like toxin because of similarity biologically, physically &
antigenically with toxin produced by Shigella dysenterica type 1): vero tissue culture
toxin because of their characteristic cytotoxic effects on vero cells(a cell lined derived
from African green monkey kidney cells in 1977).
Protein toxin similar to shiga toxin similar to shiga toxin produced by Shigella
Toxin has subunit A and subunit B. B unit is responsible for binding of the toxin to
endothelial cells whereas A unit for inhibition of protein synthesis of endothelial cells.
CNF (Cytotoxic necrotizing factor) results membrane ruffles.
Pet (Plasmid encoded toxin)
Cdt (Cytolethal distending toxin)
Hemolysins
Alpha hemolysin
Enterohemolysin
Cytolysins A
Iron acquisition
Iron is an absolute growth requirement for most, if not all, living things. Siderophores
(e.g., aerobactin) that remove iron form host iron binding proteins are necessary if a
microbe is to have invasive capabilities.
Antigenic structure
a. O-antigen
Heat stable over 165 O- antigens e.g. 1, 2, 3 and so on
Numerous cross reaction occurs between individual E. coli O-antigens & between these
and of other genera also e.g. Salmonella and Shigella
The normal colon strain belong to early O-groups (1, 2, 3 so on) & the enteropathogenic
strains belong to the later O-groups (26, 55, 86, 111, 112 etc)
b. Surface antigen
Refers to acidic polysaccharide surface (Capsular) even no detectable capsule
Two groups
o Group 1 : high molecular weight, heat stable
o Group 2: low molecular weight , heat labile
Role:
Culture character
Aerobic and facultative anaerobic
Grow well at 37˚C in 18-24 hrs, in most commonly used media, but differential media is
useful for primary isolation.
MacConkey‟s agar and tergitol 7 agar are selective and differential media for the isolation
Tryptose blood agar with 5% bovine/sheep blood can be used as primary culture medium to
support growth of E. coli.
In MacConkey‟s agar, E. coli produces 1-2 mm diameter pink colonies (lactose positive)
On turgito 7, produces 1-2 mm diameter yellow colonies with yellow zones.
In NA: Colonies are 2 – 3 mm diameter in 18 hrs, thick, colourless and moist, 2 forms are
seen Smooth (S) & Rough (R)
R forms show irregular dull surface and often autoagglutinable in saline
In BA: some strain show β hemolysis
Biochemical Reaction
Breakdown most of the sugar (lactose, glucose, mannitol, maltose) with production of acid
and gas
Typical strain of E. coli do not ferment Sucrose
Triple sugar iron agar (TSI agar)
Indole + Ve, MR +ve, VP –ve, Citrate utilization –ve
motile
Resistant
Slightly higher resistant to heat
Killed at 55˚C for 1 hour, 60˚C for 30 min, 0.5-1 ppm Cl2
Sensitive to many antibiotics
Many strains however acquire plasmids conferring resistance to one or several drugs
Pathogenesis
Predisposing Causes:
Neonates obtaining during insufficient passive immunity from colostrums quantitative or
qualitative deficiency.
Intensive husbandry practices tend themselves to a rapid transmission of the pathogenic E.
coli strains
Poor hygiene
Young neonates under 1 week of age
1. Normal flora of the intestines not fully established
2. Native immune system
3. Receptors for the adhesion of E. coli present for the 1st week of life in calves and for 6
weeks in piglets
Recently weaned pigs in stress
It is very difficult for most strains of E. coli to produce disease because they do not have the
necessary genes encoding the proteins needed to do so. If the genes are acquired (by
transduction, conjugation or transformation), the nonpathogenic strain may be changed to one
with pathogen potential. The type of disease produced would depend upon the genes acquired.
Clinical Syndrome
UTI
Gastrointestinal Disease:
Enterotoxigenic E. coli (ETEC) : ST, LT
Occurs in neonatal pigs, calves, and lambs and in weanling pigs
Also reported in dogs and horses
ETEC that produce adhesions that promote attachment to glycoproteins on the
surface of epithelial cells of the jejunum and ileum, and an enterotoxin that
affects the epithelial cell, resulting in fluid secretion and diarrhea.
Nonenterotoxigenic E. coli (EPEC): no diarrhea associated toxin
Produces diarrhea in all animal species, including human being
Produce a characteristic lesion in the intestinal tract that is described as an
attaching and effacing lesion
Characteristic lesion occurs because of the “collapse” of the microvilli of the
affected cell giving the histopathological appearance of “effacement”.
Enterohaemorrhagic E. coli (EHEC)
Some attaching and effacing strains of E. coli encodes the shiga like toxins
(SLT-I, SLT-II)
They produces hemorrhagic diarrhea
Hemolytic uremic syndrome (HUS) characterized by microangiopathic
hemolytic anaemia, glomerulonephritis, and thromocytopenia
Enteroaggregative E. coli (EAggEC): EAST 1, Pet
Weaned pigs and calves
EAggEC adheres to cells lining the small intestine by way of the AAF
adhesion. Following adhesion, EAggEC secrete a protein (encoded by a gene
termed agg for aggregation) that promotes the formation of a sheet of
microorganisms strongly adherent one to another (biofilm).
Immunity
Immunity produced by pathogenic E. coli occurs at two levels
o At the site of attachment to the target cell
o Through destruction of the bacteria or neutralization of its products.
Specific anti-adhesin antibody (Enterotoxigenic strains), specific anti-AAF antibody
(enteroaggregative strains), specific anti-Bfp antibody (Nonenterotoxigenic) and specific
antibody for SLT IIe (Edema disease) found in colostrums and milk prevents attachment
to target cells.
For invasive disease neonates acquires immunity from the dam. For the first 36 hours or
so of life, ingested IgG and IgM attach to receptors on the surface of epithelial cells of the
small intestine. Transfer across the cell into the systemic circulation follows attachment.
Laboratory Diagnosis
For enterotoxigenic strains
Presence of large number of bacteria in jejunum is highly suggestive of
enterotoxigenic E. coli (> 100 per oil immersion field implies >106/ml of contents).
Plating a portion of faecal sample onto a selective medium (MacConkey agar). As
adhesions are expressed poorly on selective media, a number of colonies are
subcultured on media that will promote the expression of the various adhesion e.g. E
medium, Minca medium. Slide agglutination tests are run on each colony using
antiserum specific for the various adhesions.
ELISA (for detection of enterotoxin. 140 pg/ml of ST (>100 times more sensitive
than the suckling mouse assay) and 290 pg/ml of LT).
PCR
FAT
For enteroaggregative diseases
Ability to associate with HEp-2 tissue culture cells in aggregative pattern (gold standard)
DNA encoding EAST1 and AAF
Histopathologically-presence of sheets of bacteria associated with the intestinal
epithelium.
For invasive
Microbiological diagnosis
Demonstration of E. coli in normally sterile sites or location (joints, bone marrow, spleen,
or blood)
In fowl, same sites are cultured, plus those grossly affected (lung, air sac), dead in-shell
embryos are cultured
Culture of the liver is to be avoided even though the Kupffer cells remove bacteria from
the blood.
Microscopy
Biochemical reaction
Agglutination test
Molecular test
Salmonella
The genus Salmonella is a member of the family Enterobacteriaceae, and is composed of two
species
S. bongori (subspecies V)
S. enterica
o S. enteric, enteric subspecies I-mammals
o S. salamae subspecies II
o S. arizonae Subspecies IIIa
o S. diarizonae (Subspeces IIIb) cold blooded vertebrate
o S. houtenae (subspecies IV, VII)
o S. indica ( (subspecies VI)
Each serovar capable of producing disease regardless of host
Numerous serotype (serovars) -----2000
Serovars names are capitalized and depicted in roman print
Some by antigenic formula
Morphology
Cell wall is typical Gram –ve
Composed of LPS and protein. Antigenic compostion of polysaccharide portion of the LPS
determine serotype. Ind and no of sugar together with the linkage between them determine
the antigenic determinant comprising the o-antigen of particular isolate.
Motile (except S. gallinarum, S pullorum (non motile) may posses fimbriae
non sporing, most member of genus are non capsulated bacilli (one capsular type, Vi (for
virulence)
size 2-4 ×0 .6µm
Reservoir
The reservoir for member of the genus Salmonella is the gastrointestinal tract of warm &
cold blooded animals.
Sources of infection include contaminated soi, vegetation, water, and components of animal
feeds (such as bone, meat, and fish meal), particularly those containing milk, meat or egg
derived constitutents, and the feces of infected individuals.
Majority of infected animals become sub clinical excretors
Survive for 9 months or more in the environment i.e soil, water, fecal materials, animal
fedds(bone meal, fish meal)
Cultural Character
Small, Circular, translucent, colourless colonies, none Lactose fermenter, Optimum
temperature 37˚ C (15-41˚C)
On selective media (Wilson & Blair bismuth sulphite)
o Colonies are jet black with metallic sheen (H2S formation), Selenite F or tetrathionate
or RVR10 are commonly used in transport media (these media inhibits the growth of
other enteric bacteria)
Biochemical Reaction
Lactose & sucrose non fermenter, Indole –ve , Ferment gelatin
Also ferment Gulcose, mannitol, maltose & dextrin with production of acid and gas (except S
typhi produce only acid not gas)
Most strain produce H2S in TSI (except S. paratyphi & S. cholerasuis)
It can utilize citrate and methyl red
Antigenic Structures:
The o-antigen, together with the antigenic determinants on the surface of the flagella (H-
antigen), which are possessed fby most salmonella helps to define an isolate serologically.
(dauffman-white scheme).
3 main types of antigens on the basis of which they are serologically classified: O, K(Vi),
H
H antigens present in either as both of two forms called phase 1 & phase 2
H antigen destroyed after a few minutes boiling or treated with alcohol but not by
formalin
Several strains posses fimbrial antigens that are not important in their identification but
till today fimbrial proteins causing confusions due to their non specific nature & wide
spread.
Antigenic Variation:
1. Phenotypic & Genotypic variation:
H O
Motile becomes non motile, may be temporary or permanent (reversible or irreversible)
Nutrient agar containing Phenol (1:800), flagella inhibited but reappears readily when the
strain sub cultured in to normal media.
Rarely lose flagella by mutation, resulting in stable or irreversible form
2. Phase variation: Phase 1 (a, b, c, d……..z & z1, z2, z3) & Phase 2(1,2,3)
3. V – W variation:
Fresh isolates of S. typhi with Vi antigen are generally inagglutinated by O antisera
because Vi antigen completely mask the O antigen. That is completely V form. Now V
forms are only agglutinated by Vi antiserum. Organism loses their V antigen either
partially or completely sfter repeated sub culture in lab.
With partially lose their Vi antigen, S. typhi agglutinate by both & Vi antisera & these
intermediate forms are called Vw forms. When there is complete loss of Vi antigen, the
strain is agglutinable only by O antiserum. These are called W form
4. S – R variation: Smooth to rough due to mutation and associated with the change of colony
morphology from smooth to rough.
5. Loss of O antigen and virulence can be maintained with Doreset‟s egg medium by
lyophillsation.
Classification
Analysis of DNA homology reveals that all Salmonella belongs to a single species S.
enteriea which has been subdivided into 7 subgroups/subspecies (Kauffman white Scheme)
The genus Salmonella is broadly divided into 4 subgroups on the basis of biochemical
test.
I. Group I : infect human & animals
II. Group II : infect reptile only
III. Group III: infect lizard only
IV. Group IV: rarely found and considered as atypical
Kauffman White Scheme: A-2, B-4, C1-7, C2-C3 8, D19, E1-3, 10, E4-13, 19
Originally classified after place of origin (S. newport, S. panama, S. poona), After the
animal (S. gallinarum), After the discoverer (S. thompson) after the name of person from whom
the first strain was isolates (S. scottmulleri)
Virulence factor:
Adhesion
o Mediate adherence to target cells in th GIT.
o Because of hydrophobicity, it promotes the associateon with the membrane of
pahgocytic cells.
o It is important virulence factor only when the microbes are on mucosal surface.
o There is at least three different adhesions for target cells(M cells, intestinal epithelium
cells)
Pef (plasmid encoded fimbriae)
Agf (aggregative fimbriae, or curli)
Lpf (long polar fimbriae)
Capsule
o No rle on salmonella since Salmonella primarlily intracellular parasite, (unlike
antiphagocytic in other organism)
o If extracellular (blocks the effects of complement system)
Cell wall
o LPS in outer membrane
o Lipid A component toxic (endotoxin)
Effector (toxin) protein- coded by pathogenicity island (cluster of gene encoding virulane
determinat)
Enterotoxin (Stn)
o Salmonella enterotoxin associate with water and electrolyte secretion by host target
cell.
o Stn differs from LT and St of E. coli by composed of subunits being peptide trather
than being compose of subunits.
Iron acquisition produces the diderophores (enterobactin) when growing in iron liniiting
condition
Stress protein- protein made when microorganism is placed under condition of stress (heat,
cold, low pH, high pH)
Virulence plasmid (spv plasmid)
Pathogenesis:
Transmission by fecal (oral route) but infection via mucus membrane of conjunctive or upper
respiratory tract is suspected. Intratracheal transmission was found to be more than oral route
(Research of Dr. Hom Bdr. Basnet)
The outcome of the interaction between host and salmonella depends upon the state of the
colonization resistance of the host, the infectious dose, and the particular species of
salmonella.
Colonization of intestinal tract and cause enteric disease.
Invasion and septicemia of different organ like liver, spleen.
The most common clinical manifestation of salmonellosis is diarrhea. In certain instances
septicaemia occurs.
The target cells are the M cells atop the lymphoid nodules and the epithelial cells of the distal
small intestine and the upper large bowel. If the flora is disrupted (stress, antibiotic), then the
infectious dose doesnot have to be as high for salmonella to gain
Access to the target cells. Following adhesion, salmonellae are internized following the
induction of membrane ruffles in the target cells triggered by Ssps and Sops subsequent to
their „injection‟ by type III secretion system.
The target cell is irreversibly damaged by this interaction, undergoing apoptosis. Salmonella
are now found within the target cells, the lymph nodule, and submucosal tissue. An
inflammatory response is initiated by release of various chemokines from affected host cells,
as well as release of proinflammatory cytokines following host interaction with cell wall
LPS-activities that result in an influx of PMN lecoytes and macrophages.
Laboratory diagnosis:
In case of intestinal infection, fecal samples are collected; in systemic disease, a blood
sample is collected for standard blood culture.
Spleedn and bone marrow are cultured for trhe salmonellae when postmortem diagnosis of
systemic salmonellosis is required.
Fresh fecal samples are placed onto one or more selective media, including MacConkey agar,
XLD agar, XLT agar, Hektoen enteric medium, and brilliant green agar
For enrichment, selenite F broth, tetrathionate or gram negative broth (GN)
Lactose non fermentativtive colonies, most serotype produce H2S. Suspected colonies can be
tested directely with polyvalent anti-salmonella antiserum or inoculated into differential
media and then tested with antisera.
To cultivate salmonella from tissue, blood agar can be used.
Isolation of organism by culture
PCR of invasion gene
Biochemical test
Serological test: widal test: agglutination test which defects serum agglutinin in patient‟s
serum suffering from S. typhi & S. paratyphi
Demonstration of circulatory antigen
Disease patterns
Ruminants
The disease affects young (usually 4-6 wks of age) as well as adult animals. Animals in feed
lots are commonly affected.
Septicaemia or be limited to the enteric tract
Pneumonia S. enteric serotype Dublin- haematogenously acquired in calvs
Abortion may follow septicaemia (S. enteric serotype Typhimurium, S. Dublin and S. enteric
serotype Newport from cattle and S. typhimurium –sheep
Swine
Acute, fulminating septicaemia or as a chronic debilitation intestinal disease
S. typhumurium and S. enteric serotype cholerasuis
Horses
Adult horses are commonly affected producing diarrhea
S. typhimurium and S. enteric serotype anatum
Dog and cat
Uncommon in dogs and cat
When outbreak occur they are usually associated with a comoon source, such as
conataminate dog food or „treat‟
Poultry
Paratyphoid
o Salmonellosis produced by any of the motile strains of salmonella (all but S. enterca
serotype pullorum and S. enterica serotype gallinarum are motile)
Pullorum disease
o Caused by S. pullorum
Fowl typpohoid
o Caused by S. gallinarum
Yersinia
The genus Yersinia is included in the family Enterobacteriaceae
There are 11 species, one of which,
Y. ruckeri affects only fish
Y. enterocolitica and Y. pseudotuberculosis associated with mesenteric lymphadenitis,
terminal ileitis, acute gastroenteritis, and septicaemia.
Y. enterocolitica affects mainly domestic animals and primates
Y. pseudotuberculosis affects largely birds and rodents and only occasionally
domestic animals and primates
Y. pestis plague is rodent based zoonosis
Named after Alexender Yersin - French bacteriologist sent by Institut Pasteur to
Hong Kong to study plague around 1900, first isolated plague bacillus in 1894.
Originally named the organism after his teacher (Louis Pasteur) - Pasteurella pestis
Shift in taxonomy: P. pestis, P. pseudotuberculosis, P. tularensis became Y. pestis, Y.
pseudotuberculosis, Francisella tularensis
Natural history
Growth characteristics
Yersinia grows on ordinary laboratory media, including MacConkey agar, although they
grow slower than most other member of the family enterobacteriaceae.
Colony diameter range from under 1 mm (Y. pestis) upto 1.5 mm for most other yersiniae.
They are non hemolytic when grown on BA
In biochemical activities and resistance, yersinia resembles other memer of the family
enterobacteriaceae.
Virulence factors
Plasmids
pYV (encoding type III secretion system, Yops and LcrV)
pMT1 (encoding the capsule, and Ymt)
pPCP1 (encoding pesticin, coagulase and plasminogen activator)
A chromosaomal locus called the high pathogenecity island encoding iron uptake
(yersiniabactin), Hms (for hemin storage) phenotype and encding Gsr ( glogbal stress
requirement). Colonies growing on the surface of blood agar plates that display the Hms
phenotype appear pigmented (they are not) due to the binding of hemoglobin (or Congo red
if present). Bound hemoglobin is used as a source of iron. The Hms phenotype is involved
with colonization and blockage of the proventriculus of fleas.
Capsule interfares with pahgocytosis (antiphagoctic), and the protection of the outer
membrane from the deposition of membrane attack complese generated by activation of the
complement system. The capulse of Y. pestis is called Caf1 (for capsular antigen fraction 1)
encoded by pMYT1
Cell wall of Y. pestis doesnot have O-antigen (rough phenotype). The LPS in the outer
membrane is an important virulence determinat. LPS binds to LPS-binding protein (a serum
protein), which in turn transfers it to the blood phase of CD14. The CD14-LPS complex
binds to toll like receptor proteins on the surface of macrophages cell trigerring the release of
proinflammatory cytokines.
Toxins
Y. pestis produce a number of toxins that are secreted by way of a type III secretion system.
Yops (Yersinia outer protein)- blocking phagocytosis, and down regulatin the inflammatory
resposnses. They are downregulated at 26oC, and upregulated at 37oC and low calcium.
LcrV (low calcium response virulence, also known as factor V). It reduces the excretion of
proinflammatory cytokines and inhibits the neutrophil chemotaxix. They are downregulated
at 26oC, and upregulated at 37oC and low calcium.
Ymt (Yersinia mouse toxin), phospholipass D plays a role in protecting Y. pestis from
digestive enzymes within the midgut of fleas.
Pesticin is bacteriocin for production of disease
Plaminnogen activator
Coagulase
Gsr (global stress requirement) is expressed at 37oC while Y. pestis is within the macrophage
phagolysoome.
Culture
Biochemical reaction
NLF, ferments glucose, maltose, mannitol with production of acid and no gas
MR positive VP –Ve, citrate –ve catalase +ve, aesculin +ve, and oxidase and urease -ve and
ornithine decarboxylation –ve
Variability
Serologically uniform
There are three biotypes (based on carbohydrate fermaentation and ability to reduce nitrate):
Antiqua, Medievalis, and Orientalis.
Reservoir
Pathogenesis
Yersinia pestis inside fleas proliferate until they block the fleas proventiculus (function of
Ymt and Hms). „blocked‟ fleas infest a new host and contaminate the feeding site with Y.
pestis when attempting to feed. At flea temperature, the type III secretion system, Yops,
LcrV, Caf1, and Gsr are not produced. The bacteria, thus introduce into a vertebrate host,
lack defense against the hosts innate immune system, and are killed when ingested by
neutrophils (an inflammatory response is generated due to products of the flea bite along with
the gram-negative cell wall components of Y. pestis).
In mononuclear phagocytes, at mammalian temperatures, and low calcium ion concentration,
and while protected by Gsr, Y. pestis activates type III secretion system, produces Yops,
LcrV, and Caf1 and release form the phagocytic cell following initiantion of apoptosos.
Yersinia acquire resistance to further phagocytosis and intracellular killing by neutrophil and
mononuclear phagocytes (LcrV reduces the excretion of proinflammatory cytokines, and
inhibits neutrophisl chemotaxix. Thus, early in the disease process, Y. pestis is and
intracellular parasite, and later an extracellular one.
Extracellular multiplication made possible by iron acquisition system and capsule production
elicites a hemorrhageic ainflammatory lesion, followed by local lymph node involvement
(bubo) i.e. bubonic plague
Septicaemic untreated termintes fatally (an endotoxemia aided by the function of
plasminogen activator, which accelerates initianti of disseminate intravascular coagulation,
Some develop plague pneumonia and shed Y. pestis in sputum and droplet nuclei
Immunity
Capsular antigens (F1 antigen) evoke opsonin formation. Antibody to the LcrV antigen is
protective.
Immunity following recovery is good but temporary.
Laboratory diagnosis
Samples from affected sites (i.e., edematous tissue, lymph nodes, and nasopharynx),
transtracheal spirates, CSF, and blood (for culture and serology) are collected.
Direct smears are examined followitn immmunoflourescence , wayson‟s staining or grams
staint.
Culture is done on blood or infusion agar
Identification is confirmed by immunofluorescence or bacteriophages susceptibility.
Mice or guinea pigs injected s/c with Y.pestis die within 3-8 days.
PCR
Serological tests(HA/HI, ELISA)
Therapy
a. Sanitation: controlling rat population, eliminating fleas with insecticide, eliminate crowded
living conditions/substandard housing that increases the frequency of contacts between
people and rodents
b. Immunoprophylaxis: Vx = killed cells, relatively effective, duration < 1 year
Y. pseudotuberculosis
Y. enterocolitica
pYV encoding type III secretion system, Yops and LcrV, HPI, Ail, Inv, YadA, Gsr and Yst.
Yst (Yersinia stable toxin) it is unique to Y. enterocolitica. It affects the guanylyl cyclase
system by deregulating cGMP synthesis (increase in intracellular cGMP leads to the opening
of chloride channesl with the resulatant flow of chloride and water into the intestinal lumen),
resulting in fluid and electrolyte accumaulation in the bowel lumen
There are approximately 34-O antigen and 20 H-antigen serogroups. O groups 3, 5, 27, 8 and
9 are associated with classical disease of the GIT. There are 5 biotypes.
Water, food, soil, fruits, vegetables and symptomatic aindividual from humans to mollusks
have been proposed.
Expression certain virulence determinants at 22oC to 25oC suggest thent mammals acquire Y.
enterocolitica form a cold source rather than a warm blooded animals
Infection follows ingestion of organisms expression the adhesions
Exposure is primarily through ingestion
Pathogenesis is same as other
Immunity
Laboratory diagnosis
Samples of feces, lymph node biopsy, and biopsy from affected tissues are examined
microbiologically.
Selective media contining bile salts are somewhat inhibitory to Y. enterocolitica, especially at
37oC
MacConkey agar is least inhibitory. There is special meda designated for the isolation of Y.
enterocolitica (e.g. CIN medfium) cold enrichment of the sample at 4oC aids in attempts to
isolate small numbers of Y. enterocolitica from a contaminated environment. Isolation from
tissue necessitates the use of blood agar plates incubated at 37oC
PCR
Classification:
Divided into four species or groups on the basis of differences in "O" antigens and some
biochemical reactions. They are:
Group A: Shigella dysententriae – 10 serotypes only for human
Group B: Shigella flexneri – 6 serotypes
Group C: Shigella boydile – 15 serotypes
Group D: Shigella sonnei – 1 Serotype
Manitol fermentation: Group A -ve , B, C, D +ve
Cellular structure
Cell wall-typical gram negative cell wall, aerobic, non motile, non-spore former, non
capsulated bacilli 1-3 × 0.5 µm Other characters are------similar to salmonella.
Typical for family enterobacteriacease but posses neither capules (K antigen) nor flagella
(hantigen).
Adhesins-IpaD(invasion protein antigen)
LPS (O-antigen) in outer membrane is important virulence determinant. There are four
serotypes, each of the four serotypes has been given species designation. Within each species
there are a number of serotypes.
Invasion plasmid encodes invasion proteins ipa = invasion plasmid antigens. IpaA /IpaB,
IpaC, and IpaD are probably required for invasion. Entry point used may center on Peyer's
patches (based upon location of lesions)
i) IpaB/IpaC- injection into the host target cell
ii) IcsA/IcsB intracellular spread is required for intracellular spread
Enterotoxins are two a. ShET1 b. 63KDa
Exotoxin shigatoxin produced by Shigella dysenteriae
Sig A
PiC (protein involved in internal colonization.)
Variability:
Typed based upon the makeup of the O-Antigen
Biochemical properties:
Glucose is fermented by all strains.
Mannitol is fermented by subgroups C, D and majority of B.groups
Indole may be or may not be fermented by A, B and C but never by subgroup D.
Glucose +ve with acid (Shigella flexneri type 6 acid + gas)
Salicin & Lactose – ve (Shigella sonnei late lactose fermenter)
Indole –ve Group D
H2S –ve , Citrate +ve
Catalase + ve (Shigella dysentenriae type 1)
Shigella dysenteriae is only member of the group that has the genes necessary for production
of shiga toxin. (chromosomally encoded exotoxin)
SigA (exotoxin)
Pic(protein involved in intestinal colonization)
Virulence factor:
Invasion plasmid antigens: mediate attachment, penetration in mucosal epithelial cells of
colon. Mediate escape of bacteria from phagocytosis.
Intracellular spread proteins: mediate attachment to cytoskeleton proteins & facilitate cell
to cell spread of the bacteria through membrane protusions.
Endotoxins: S. dysenteriae type 1
Exotoxins : S. dysenteriae type 1
Neurotoxin(heat labile) in S. schmitzi
Large bowel of clinically ill, recovered or asymptomatic primates are reservoir
Disease is transmitted by the fecal-oral route, but the infective dose is so small that fomites
may also play a role.
Antimicrobial drugs, stress, or dietary changes promote risk by reducing colonization
resistance (by decreasing the numbers of the normal flora resulting in a reduction of barrier),
which may lead to diseas in asymptomatic carrier animals or lowering the oral dose needed
for infection and subsequent disease.
Pathogenesis:
Trend to localize in the gut wall.
Bacterimia is uncommon with this species because that do spread in blood but are rapidly
killed by macrophages
Contaminated food: 1 – 7 day incubation period (10 –100 bacteria) can cause dysentery
Shiga toxin (like EHEC toxin) consist of A subunit-1 & B subunit- 5
B subunit bind to the host cells glycolipid & facilitates transfer of active subunit A into
the cell which disrupt protein synthesis. Primary activity of toxin is to damage the
vascular endothelia. Also cause transudation of fluid from intestinal mucosa leading to
severe diarrhea & toxaemia.
Clinical Syndrome:
Dysentery/diarrhea in man and primates. Dogs can be infected from human owners & may
excrete the bacteria for short periods without clinical signs.
Laboratory diagnosis:
Rectal swabs or samples of faeces.
Microscopic examination of stool
Direct examination of stained smear of fecal material reveals the presence of inflammatory
cells, cellular debris and red blood cells (RBC). Such finding is not diagnostic. However,
since enteritis produced by campylobacter results in the same signs. The presence of curved
rods in the direct smears suggest that campylobacter is the cause of the disease.
MacConkey agar, XLD agar, HE medium (i.e. less inhibitory than media for isolation of
salmonellae.
Culture
Biochemical test
Slide agglutination test
Colicin typing
Seveni test: to confirm the invasiveness
Cultural characteristics
Grow in ordinary media (NA, BHA)
Produce putrefactive odour (fishy, seminal)
The organism is aerobic and facultative anaerobic and grows in temperature ranges of 20-
400C
Swarming colonies of Proteus but not swarming Morganella & Providencia
On solid media, amoeboid colonies are produced which spread across the surface of media
and hence termed as 'swarming features'
The swarming phenomenon occurs in waves and therefore series of rings develop on the
surface of agar plate.
In broth, the organism produces marked turbidity.
Swarming can be reduced by
o Increasing Agar from 1-2 % to 6%
Biochemical properties:
The organism ferments glucose and sucrose forming acid and gas (except some species of
Proteus rettgeri which produce only acid
They do not ferment lactose
Urease positive
They are positive to Phenyl Pyruvic Acid (PPA) reaction i.e. able to produce PPA from
phenyl alanine. This is shown by 'proteus' only.
Indole +ve P vulgaris but –ve P mirabilis, H2S +ve, MR +ve, VP –ve, Citrate +ve
Clinical Syndrome
UTI: usually in obstructive lesions of UT follows prolonged catheterization (exogenous
infection)
Wound infection
Septicemia
Acute otitis media: Dogs & Cats
Diarrhea in young mink, lambs, calves, goats, pups
Lab diagnosis
Specimen: Urine sample, pus
Culture & Biochemical test
Morphologically Klebsiella is same as E coli except are non motile & posses a
polysaccharide capsule & is responsible for mucoid appearance as well as for virulence
factor.
Widely distributed in nature, commensal in human and animal intestine as well as
saprophytes in soil, water & vegetation.
Classification
Cultural characteristics
The organism is aerobic and facultatively anaerobic.
The organism grows well on common media at 37 0C.
On solid media, the colonies are mucoid, amorphous and opaque. The size of the colonies
varies from 1-4 mm.
The mucoid capsules is lost on repeated sub culturing
Non-capsulated strains produce smaller colonies with less mucoid consistency.
In the broth, a heavy turbidity with ropy sediment is produced.
There is no haemolysis on blood agar.
Biochemical properties
The organism produces acid and gas in glucose, lactose, sucrose and salicin.
Majority of the strains are voges proskauer positive, reduces nitrates to nitrites.
Do not liquefy gelatin and methyl red +ve except Klebsiella aerogenes and do not form indole.
Clinical Syndrome
Pneumonia:
Klebsiella pneumonia found in 10% normak individual as normal flora of respiratory
tract.
Causes pneumonia in diabetics, alcoholics and immunocompromised patients,
produce lung abscess in Foals
UTI: in dogs
Septicaemia, wound infection & meningitis
Epidemic diarrhea in new borns
Cervicitis & metritis in mares
Coliform mastitis in cattle
Lab Diagnosis
Specimen: Urine, pus, blood depending on the site of lesion
i. Culture: in MAC & blood agar (mucoid & pink colony)
ii. Biochemical test
Pasturellaceae
Pasturella
The family Pasteurellaceae contains the genera Actinobaacillus, Haemophilus and
Pasteurella.
Gram negative coccobacilli.
Facultative anaerobes
Typically oxidase positive (which sets them apart from member of family
enterobacteriacae)
Pasteurella species are primarily animal pathogens but they can produce human diseases.
This genera formerly included "Yersinia ' and Francisella" as well. The name Yersinia is given
after Alexender Yersin who discovered the bacteria causing Plague. The genus Yersinia is now
included in the family Enterobacteriacae. The genus Pasteurella is now restricted to several
related bacteria causing H.S. in animals which are now grouped under common species P.
multocida. Francisella is a new genus consisting of a single species F. tularensis named after
Francis for his contribution on tularemia, cause by the bacteria.
P. haemolytica, has undergone extensive taxonomic changes over past several years. P.
haemolytica at one time contained17 capsular type, and 2 biotypes (A for arabinose
fermaentation, and T for trehalose fermaentation). Tus , each strain of P. haemolytica was
identified by a letter (either A or T) designating the biotype, and a number (1-17) designating
capsular type. For example, P. haemolytica of the A biotype possessing a type 1 capule was
designated as P. haemolytica A1. Genetic analysis shoed A biotype comprises separate genus
Manheimia. Thus, all but one of the P. haemolytica Abiotype become Mannheimia haemolytica.
The one exception, P. haemolytica A!!, became a different species, M. glucsida. All of the T
biotype were placed into a new species, P. trehalso. Consequentely, P. haemolyticais obsolete.
Growth characterstics
Pasteurella and Mannheimia grow best in the presence of serum or blood. After overnight
incubation (35-37oC), colonies are about 1mm in diameter, clear, and smooth or mucoid,
Resistance
Culture die within 1-2 wks. Disinfectant, heat (50 oC for 30 minutes), and UV light are
promptely lethal. Pasteurella multocida survives for month in bird caracasses.
Pasteurella and Mannheimia, especially fromfood animals, have become increasingly resistant to
penicillins, tetecycline, and sulfonimade, to which they were originally susceptible.
Variability
P.multocida consists of five capsular (A,B,D,E,F) and 11 somatic(1-11) serotype occuring in 20
different combination.. Mannheimia haemolytica consists of 12 capsular type (1, 2, 5-9, 12-14,
16,17)a nd P. Trehalosi four (3, 4, 10, 15)
Pathogenesis
Pathogenesis is not fully understood. Exotoxins are important in the septicemic disease as fowl
cholera and haemorrhagic septicemia.
Infections may be endogenous or exogenous. Part of entry is usually via respiratory tract and
virulence is enhanced by animal to animal transmission as in pneumonic pasturellosis.
Predisposing factors (stress, viral infections ) lead to disease.
Themolabile dermonecrotoxin ( P. multocida type D strain) AR+ve strain have an important
role in atrophic rhinitis of pig.
P. haemolytica produces a soluble cytotoxin (leukotoxin) which plays role in breaking the
lungs primary defense mechanism by its action on the alveolar macrophages and othe
rleukocytes of ruminants.
Serogroups or types of pasturella sps
P. multocida is identified based on the differences in capsular substances (polysaccharides)
eg.A,B,D,E,F. somatic types (lipopolysaccharides) have also been determined and given
numbers.
P. haemolytica has two biotypes based on the differences of characteristics including
pathogenicity, antigenic nature and biochemical activity (e.g. biotype A ferments arabinose and
biotype T ferments trehalose.
In general, there are three manifestations of Pasteurella induced disease: respiratory tract
involvement, septicemia, and trauma-associated conditions.
1. Respiratory tract involvement is either pneumonia or upper tract disease (Atropic Rhinitis
in swine). Pneumonia is seen most frequently in Ruminants associated with P. multocida
or P. trehalosi. Microorganism is deposited in the lund, and secrete Lkt and along with
LPS from cell wall, initiate an intese inflammatory response with fibrin deposition. There
is break the lungs primary defence mechanism by its action on alveolar macropahges and
leucocytes of ruminants.
Form of Upper respiratory tract disease (Atropic rhinitis) is one in shich
Bordetella bronchosetica first adheres to the nasal mucosa, and secrete a toxic called
dermonecrtic toxin, which mildly damaged epithelium. Capule type D strains of P.
multocida adhere to theis mildely damaged epithelium and secrete Pmt and responsible
for the distruction of the nasal turbinate.
2. Septicemic disease produced by Pasteurella in ruminants (haemorrhagic septicemia in
cattle: septicemia in sheep) or avian species (avian cholera).
3. Trauma-related conditions in which mouth microorganisms (Pasteurella is most common)
are included into the site of infection.
Pasterella multocida
Pasteurella multocida is also known according to the species of animals affected like:
Pasteurella boviseptica -produces H.S. in cattle, buffaloes etc
Pasterella suiseptica -causes pneumonia infection in pigs
Pasterella aviseptica -causes Fowl Cholera.
Similarly P. ovoseptica, P. equiseptica, P. leptiseptica and P. muriseptica.
Haemorrhagic septicaemia is also called 'shipping disease/cork-stick disease in
cattle/barbone of buffaloes.
P. multocida is present as commensals in the respiratory tract of the clinically healthy
animals. They are recognized as opportunistic commensals.
Tropical and subtropical region of Nepal are affected by this organism.
Cultural characteristics
The organism is aerobe or facultative anaerobe. They prefer an environment of low O2
tension and high CO2 tension.
The optimum temperature for growth is 37 oC and PH 7.2 to 7.4.
The growth takes place on ordinary media, but is enhanced by growing best in the
presence of serum and blood.
In nutrient agar: freshly isolated strains on nutrient agar produce round, flat colonies of sticky
mucoid consistency of 2-3 mm in diameter. Such are called 'mucoid strains'
Some other strains may produce small colonies measuring 1mm in diameter, round shaped,
smooth margined, iridiscent colonies. Such strains are called-'smooth irridescent strains'
Still other strains produce colonies smaller than 1mm and are dry and rough such strains are
called 'rough strains'
Some strains are round and smooth but lack iridencence called 'smooth non-iridescent
strains'.
Smooth irridescent colonies are obtained from acute infection and rough colonies are formed
during subsequent lab culture.
In blood agar: more profuse growth takes place. The medium becomes characteristically dark. It
is very much marked in those places which are inoculated heavily. No haemolysis.
In broth: newly isolated strains produce even turbidity in 6-8 hrs in 37oC
Addition of 10 % serum increases the growth rate.
The older strains produce fine granular particles after 36-42 hrs of incubation at
37oC.
The organism doesn't grow in Mac conkey's bile salt medium.
Biochemical characteristics :
The organism produces acid but no gas in glucose, sucrose, galactose, sorbitol, fructose, xylose,
arabinase etc. Lactose, maltose, dextrin, and insulin are not fermented.
The organism procuce H2S gas and indole and reduces nitrates, gelatin liquefaction test -ve,
urease -ve.
Resistance to physical and chemical agents: The organism is readily killed by sunlight in 3-4
hours and by common physical and chemical agents
Destroyed at 60 oC in 10 minutes
Destroyed at 50 oC in 15 minutes
Destroyed by 0.5 % phenol in 15 minutes
In manure the organism remains infective for 1 month and in carcass for 3 months. The organism
is sensitive to many antibiotics. Pasteurella especially from food animals, have become
increasingly resistant to penicillins, tetracycline, and sulphonamides, to which they were
originally suceptible.
Antigen and toxin: antigenic structure differs in different types/strains. To classify antigen
groups, popularly 2-types of Ag grouping is done.
1) Robert's type- it is based on mice protection test or somatic antigen.(1-11)
2) Carter's type- it is based on capsular antigen.(A,B,D,E,F)
Nowadays real typing is done on the basis of somatic and capsular antigens(nowadays
there ate 11 somatic antigen (1 to11) and 5 capsular Ag(A,B,D,E,F) e.g. 1:A, 1:C, 2:E(2 is
Roberts and E is carters)
Toxin: a toxin composed of protein (i.e. protein toxin) has been isolated from carter's B type
strain and a lipopolysaccharide. From others, no toxin has been detected.
Pasteurella hamolytica (identicle to P. mastiditis)
Pasteurella haemolytic has a wide host range and is world wide in distribution. The organism has
been isolated from pneumonic lungs from cattle, buffaloes, sheep and goats. Like Pasteurella
multocida the organism can be carried in upper respiratory passages of animals.
In case of lambs, they have been found to be causing acute septicemia.
In ewes, one of the major causative agents of mastitis is P. mastiditis.
Morphology and staining: the organism is short, non-motile, capsulated, gram negative rods
and sometimes indistinguishable from Past. multocida. The organism measure 2.5 by 0.5µm.
Biochemical properties: the strains vary in their fermentative capacity. Acid and no gas is
produced in glucose, lactose, maltose, saccharose, mannitol, dextrin, galactose, glycerol and
insulin. Nitrates are reduced to nitrates. Indole and urease are not produced. Gelatin is not
liquified. Some strains may produce H2S.
Strains isolated from sheep have been divided into two types. Type A strains have been
mainly associates with pneumonia and ferment arabinose and not trehalose while type T strains
have been isolated more frequently from cases of septicemia than from pneumonia and ferment
trehalose but not arabinose.
Antigens and toxins: on the basis of indirect haemagglutination technique, the strains of Past.
haemolytica have been classified into 11 serological types. Exotoxin has not been reported.
The antigen adsorbs on the surface of RBC, and after the attachment, the Antigen become
insoluble and forms clumps i.e. haemagglutinate. (It is called indirect haemagglutination, since
here RBC act only as carrier of Antigens).
Disease Pattern
Cattle. Pneumonia. The most common form of bovine "pasteurellosis" involving P. multocida is
shipping fever, a fibrinous pleuropneumonia or bronchopneumonia seen wherever cattle,
especially calves, are transported, assembled, and handled under stressfull conditions.
Hemorrhagic septicemia: it is acute systemic infection with P. multocida, serotype B:2 or E:2
occuring in tropical areas as seasonal epidemics with high morbidity and mortality.
Mastitis.Mannheimia haemolytica may cause mastitis with much tissue destruction,
haemorrhage, and systemic toxic complications, which are often fatal.
Sheep and goats. Septicemia. Septicemic pasteurellosis by P. trehalosi in feeder lambs.
Enzootic pneumonia by Mannheimia haemolytica.
Mastitis: gangrenous mastitis(M.haemolytica, and P. trehalosi).
Swine. Atropic Rhinitis. Atropic rhinitis of young pigs (3 weeks to 7 months) leading to
turbinate destruction and secondary complications results from synergistic nasal infection by P.
multocida(capsule type D) and Bordetella bronchiseptica.
Pneumonia. A fibrinous pneumonia associated with P. multocida, often follows viral infection.
Rabbits. Respiratory Tract Condition. "Snuffles" a mucopurrolent rhinosinusitis of rabbits due
to P. multocida, develop under stress of pregnancy, lactation, or mismanagement. Complications
include bronchopneumonia, middle and inner ear infection, conjunctivitis, and septicemia.
Genital tract disease. Orchitis, balanoposthitis and pyometra (P. multocida)
Avian species. Avian cholera. A systemic infection due to P. multocida(capsule type A).
Dogs and cats. Mouth related conditions by P. multocida(cats) and P. canis(dogs).
Equine. Respiratory Tract conditions. Pasteurella caballi.
Laboratory Diagnosis
Direct Examination: exudates, tissue impressions, sediments of transtracheal aspirates, and, in
birds, blood smears can be stained with a polychrome stain(e.g. Giemsa, Wright's,) an examined
for bipolar staining microorganisms.
Collection of specimen- portion of pneumonic lungs from the edge of the lesion. In
septicemia pieces of liver, Spleen, kidney and lymph node and from the live animal pus,
exudates, nasal swab or bronchial lavage. In mastitis milk sampling.
Direct microscopy- small Gram –ve rod or coccobacilli. During septicemia we can observe
bipolar staining of the organism ( in Giemsa or leishman's stain)
Isolation in blood agar (sheep or ox blood). Selective medium containing clintamycin 2g\ml
(for P. multocida from nasal swab of porcine at 370c in 24-48 hr)
Identification- colony morphology ( large mucoid colony, pungent/ sweetish odor and non-
hemolytic)
P.multocida doesn‟t grow in Macconkey and are indole positive. P.haemolytica is beta
hemolytic and can tolerate bile salts so it can be grown in the MacConkey agar also. They show
pin point red colony and don‟t produce any smell and are indole negative.
Microscopically- small Gram negative rods or coccobacilli. Hanging loop observation non
motile.
Biochemical reaction- in TSI slant and butt both are yellow (acid producing, don‟t produce
H2S and gas).
Serotyping can be done for diagnosis
Animal inoculation- intraperitoneally in mice
Photobacterium damsela
Photobacterium damsela ssp. piscicida (previously classified as Pasterulla piscicida) is the agent
of fish pasteurellosis. Photobacterium damsela ssp. damselae was previously classified as Vibrio
damsela producing septicemia in mammals(dolphine and humans)
Actinobacillus lignieresi
Morphology and staining: A. lignieresi is a small rod shaped organisms often showing shorter
coccobacillary forms. The cultures give a characteristic 'morse-code' form. The bacilli are 1.15-
1.25µm by 0.4µm and are non motile, non-sporing and non acid fast, capsules are not formed.
The cells stain readily with carbon fuchsion and are gram negative: they may show bipolar
staining.
Cultural characteristics: aerobic, Grow slowly in ordinary lab. Media. The optimum
temperature for growth is 370C. on primary isolation, viscous colonies 1-2mm in diameter are
formed in nutrient or blood agar. The organism grows poorly on McConkeys agar. Growth is
enhanced by blood or serum. These organisms give confluent growth.
In broth, it produces uniform turbidity with little deposit.
In lab media the organism can't survive more than few days. So for culture, their viability should
be maintained.
Some actinobacilli (A.indolicus, A. minor, A. pleuropneumoniae biotype 1, and A.
porcinus) require nicotinamide adenine dinucleotide (NAD) for growth, while A.
pleuropneumonia biotype 2 doesnt.
Biochemical properties: A. lignieresi produces acid without gas promptly in maltose, mannitol
and sucrose, glucose, levulose and galactose. Acid is produced in litmus milk but no clot.
Oxidation reaction +ve : H2S +ve and MB reduction +ve , nitrate reduction +ve. Urease positive
but no indole is produced.
Actinobacillus equuli
Commonly present as normal inhabitant of intestinal tract of equines. They become opportunistic
when there is some stress. In foals it causes and acute septicemia with enteritis in which death
usually occurs within the first three days of life named 'sleepy foal disease'. In adults suppurative
enteritis and joints ill is caused.
A. equuli also affects pigs, resulting in fatal septicemia or chronic lesions including
arthritis, endocarditis, nephritis and hepatitis.
Morphology and staining: gram -ve, oval rods measuring about 1.5 µm. Have tendency to form
chains. Non motile, non spore former, non acid fast.
Cultural characteristic: They can grow in ordinary lab. media like nutrient agar. In nutrient
agar they produce shiny grey colored translucent colonies with a mucoid consistency. In broth,
they form slimy sediment i.e. floccules become large and settles.
Biochemical properties: They ferment lactose, maltose, sucrose, mannitol and glucose
producing acid without gas. They are nitrate +ve, H2S +ve, indole -ve, MBR-ve.
Antigens and toxins: Considerable amount of antigenic heterogenosity exists among different
strains i.e. there are many antigenic strains and serotypes. One Ag is common with A. lignieresi.
Actinobacillus suis
This species was known after the above two. The organism is able to haemolyse i.e. they produce
haemolysin, α and β. α haemolysin causes haemolysis of horse blood, haemolysis is complete i.e.
clear. B-haemolysin-causes haemolysis of sheep blood. haemolysis is incomplete.
They hydrolyse aesculin(a carbohydrate)
They produce acid without gas from cellobiose, arabinose.
A. suis affects pigs of all ages especially new-born animals resulting in septicemia pneumonia,
and nephritis and in chronic cases arthritis. A. suis has also been recognized as a pathogen of
horses.
Disease Patterns
Ruminants. "actinobacillocis" or Wooden Tongue" involving A. lignieresii occurs in ruminants
and rarely, dogs and horses.
Swine. Pneumonia. Actinobacillus pleuropneumoniae causes a primary pneumonia of swine,
particularly young pigs 2-6 months old.
Septicemia. Septicemia of young pig and arthritis, endocarditis is sometimes associated with
A.suis.
Horses. Pneumonia( A. equiuli ssp. haemolytica) asoociated with a mixture of beta hemolytic
streptococcus (S.equi subspecies zooepidemicus)
Septicaemia. Foal septicemia due to A.equuli ssp equuli(sleepy foal disease) occurs within a few
days of birth.
term Haemophilus as generic name. Various Haemophilus species are inhabitants of upper
respiratory tract, and are associated with infections of respiratory tract of animals.
Member of the genus Haemophilus, beyond sharing the family traits of the family
Pasteurellaceae, require for propagation one or both of two growth factors: porphyrins (haeme)
or nicotinamide adenine dinucleotide(NAD, NADP), originally called X(heat stable) and V
(heat-labile) factor, respectively.
Major species are:-
1. H. suis -occurs in the respiratory tract of normal pigs and is associated with atropic
rhinitis and pneumonic infections. It is a cause of the disease in pigs called "glasser's
disease".
2. H. parasuis -Glasser's disease or polyserositis and secondary respiratory disease of swine.
3. H. pleuropneumonia -causes pneumonia, infects pleura also.
4. Histophilus somni - the cause of septicemic, respiratory, and genital tract disease in cattle
and sheep. (H. somni is donated as Haemophilus somnus, Haemophilus agni, Histophilus
ovis)
5. H. agni -produces septicemic infections in lambs, bronchopneumonia in adult sheep and
meningoencephalitis in cattle.
6. H. gallinarum-produces infections like coryza, air sac infections and chronic respiratory
disease (CRD).
7. Haemophilus paragallinarum - infectious coryza in chickens.
8. H. canis: occurs on the prepuce of dogs and is nonpathogenic.
Following species are found in human beings
H. influenzae H. parainfluenza H. aegypticus.
Morphology and staining: In specimens from acute infections, the organisms are short coccoid
bacilli (1-1.5µm), sometimes occurring in short chains. In culture after repeated sub cultivation,
pleomorphic forms develop. The organisms grown on rich media have a polysaccharide capsule.
The organisms are non motile and non spore forming. The organisms stain gram negative.
Cultural characteristics: The organisms are aerobic but many species can grow anaerobically
(faculatative anerobes). Corbon dioxide enhances growth of some strains. Typically oxidase
positive and attack carbohydrates fermentatively.
On adequate media members of this genus produce
within 24-48 hrs turbidity in broth and colonies 1 mm
in diameter on agar (35-37oC). For optimum growth,
the organism needs enriched culture media such as
blood or chocolate agar because they need one or
both of two growth factors, X and V
1. Heat stable X factor: it is haemin or some
other iron containing porphyrin.
2. Heat labile V factor : it is nicotinamine
adenine dinuceotide(NAD) or phosphate
(NADP)
The V-factor remains imprisoned in blood
agar but it is released from the erythrocytes in
chocolate agar (addition of blood when melted agar is at 75-80oC rather than 50oC when
making BA). V-factor is also synthesized by staphylococci and some yeast cells. The
differential requirement for X and V factors helps to distinguish different species.
Better growth of influenza is obtained in 'Levinthal's medium' (prepared by boiling a
filtering a mixture of blood and nutrient broth) or filde's agar(prepared by adding peptic
digest of blood and nutrient broth) .The optimum temperature for growth is 370C.
When nutritional requirements are met, most Haemophilus species grow rapidly and
produce 1-2 mm translucent colonies after overnight incubation.
In 'broth media' when whole blood is added with inoculums, the organisms grow in 2-3
days and usually don't become numerous to produce turbidity.
Satellitism: After inoculating suspected H.influenzae in a blood agar plate, Staph. aureus as
feeder bacterium is streaked across the same plate and incubated overnight at 370C. In such
mixed culture the colonies of H. influenzae are large and well developed towards the streak
whereas those further away from staphylococcal streak are smaller. This feature is called
'satellitism" and demonstrates the staphylococci synthesize V factor in high concentration near
the staphylococcal growth.
Resistance: readily killed by heat and die rapidely in culture and storage unless freeze dried or
stored at minus 70oC. At cool temperature, H. paragallinarum survives in exudates for several
days.. The organism is killed at 550C in 30 minutes. Destroyed by physical and chemical agents.
Reservoir
Member of hamophilus and histophilus live the URT or Lower genital tract.
H. parasuis- lives in nasopharynx of normal swine
H. paragallinarum- RT of sick or recovered poultryHistophilus somni –normal cattle both in
lower genital tract (prepuce, vagina) and URT.
Transmission by airborne or by close contact.
Determinant of pathogenicity
Capsular polysaccharide: constituted by phosphoribosryl ribotol phosphate (PRP) which has
antiphagocytic properties is the prime virulence factor of H. influenza type b
Membrane lipooligosaccharide: similar to LPS of E. coli less toxic than E. coli, may be
responsible for attachment, invasiveness and paralysis of ciliated respiratory epithelium
IgA protease: IgA protease probably inactivates secretary antibodies
Pathogenesis:
Clinical syndrome:
Invasive infection
Meningitis: 6 months to 2 years of age in whom anti PRP antibodies are absent
Acute epiglottitis (2 – 4 years of age)
Bacterimia
Suppurative lesion
Pneumonia
Non invasive infection: produce local infection underlying physiological as anatomical
abnormality. Eg otitis media, sinusitis
Disease pattern
Swine. Pneumonia. Haemophilus parasuis can cause bronchopneumonia secondary to virus
infections( e.g. swine influenza).
Septicemic disease. In young weaned pigs, H.parasuis also cause Glassers
diseases(polyserositis), and acute inflammation affection pleura, peritonium, mediastinum,
pericardium, joints, and meninges.
Poultry. Infectious coryza. Infectious coryza ( cause by H. paragallinarum) is and acurte
contagious upper respiratory infection of chickens.
Dog and cat: dogs H. haemoglobinophilus, a commensal of the canine lower genital tract,
sometimes cystitis and neonatal infections.
Cats. Haemophilus felis (conjunctivitis).
Species Animal Disease
H. somnus Cattle Infection thrombolic meningeioencephalolitis,
septicaemia, respiratory disease, genital infection
endometritis & abortion
Sheep Epididimytis,orchitis in Ram, pneumonia, mastritis,
polyarthiritis, mengitis & septicaemia
H. parasuis Pig Glasser‟s diseas(polyserositis & meningitis), arthritis
& pneumonia
H. paragallinarum Poultry Infectious coryza of chicken
H. haemoglobinophilus Dog Lower genitalia
H. aphrophilus Human Oral flora
H. ovis Sheep Bronchopneumonia
H. paracuniculus Rabbit Intestine
H. influeuzaemurium Mice Respiratory infection, conjuctivitis
H. piscium Trout Ulcer disease of gills & mouth
H. influenzae Human Repiratory
H. parainfluenza
Lab Diagnosis
Specimen: very frazil must be protected from drying
Direct microscopy: Gram –ve rod, FAT
Bacteriological investigation:
Bordetella bronchoseptica
Morphology-
Short pleomorphic rods showing coccoid shapes 0.5-1.0 µm and bacillary forms 0.5-1.5 µm in
size. It usually occurs singly but pairs are found and in fluid media chains may be observed. Due
to peritrichous flagella, the organism is motile but B. pertussis and B. parapertussis are non-
motile.. Capsules are not found in culture (B. bronchseptica produces a capsule) and spores are
not produced. It is gram negative but may show bipolar staining. (B. pertusissis and B.
prapertussis are non motile). Most if not all members of the genus produce fimbrial adhesins (
pili). All are catalase +ve and oxidase +ve. B. bronchoseptica and B. avium will grow on
MacConkey
Changes in nomenclature
Natural habitat
Upper respiratory tract of human, animal and birds. B. pertussis and B. parapertussis are human
pathogens (whooping cough). B. bronchoseptica present on upper resp. tract of pigs, dogs, cats,
rabbits, guinea pigs, rats, horses and other animals too. B. avium on respiratory tract of infected
turkey, poultry. Mammalian infections are mainly transmitted by aerosols but in turkeys indirect
spread can be via water and litter.
Virulence factors:
Pathogenesis
Resistance
Bordetella spp are killed by heat or disinfectants. They are suceptible to broad-spectrum
antibiotics and polymyxin, but not to penicillin.
Disease pattern
Swine. Atropic Rhinitis. The progressive form of Atrophic Rhinitis is due to combined
infection with P. multocida and B. bronchiseptica.
Pneumonia
Dogs and cat : canine infectious Traccheobronchitis (Kennel cough) and pneumonia
Laboratory diagnosis
Culture: media is sheep blood agar, Mac. Agar. 24-48 hrs at 370C aerobically grown.
Bordetella pertussis
Morphology and physiology: It is an extremely small, strictly aerobic, Gram negative,
non-motile cocobacillus (short rod). Compared to other Bortdetella species, B. pertussis
does not grow on common laboratory media. Selected media include Bordet-Gengou
medium or potato-glycerol-blood agar. B. pertussis can be distinguished from B.
parapertussis in that B. pertussis is oxidase positive but urease negative, while B.
parapertussis is oxidase negative and urease positive. B. bronchosepticus is positive for
both enzymes.
Pathogenesis: The symptoms following the infection are due to many factors. In addition
to the attachment to and growth on ciliated cells, the organism produces a number of
exotoxins which contribute to these symptoms.
Pertussis toxin (pertussigen): It is an oligopeptide AB-type exotoxin (A: subunit1; B:
subunit 2-5 complex) which is the major cause of pertussis (abnormal cough). It causes
T cell lymphocytosis and has adjuvant properties. It also causes hypoglycemia, increased
IgE synthesis, and increased histamine and endotoxin sensitivity. The toxin inhibits many
leukocyte functions, including chemotaxis, phagocytosis and respiratory burst and
impairs NK cell killing. It exerts many of its effects by covalent addition of ADP-ribose
to the GTP-binding Gi-protein (ADP-ribosylation) and thereby preventing the
deactivation of adenylate cyclase. This results in the accumulation of large amounts of
cAMP and consequently increased mucus secretion and interferes with many cellular
functions.
Adenylate cyclase toxin: This exotoxin penetrates the host cells, is activated by
calmodulin and catalyzes the conversion of ATP to cAMP. Like pertussigen, it also
inhibits phagocyte and NK cell functions. However, the cAMP increase caused by this
toxin, in contrast with pertussigen, is shortlived.
Tracheal cytotoxin: It is a peptidoglycan-like molecule (monomer) which binds to
ciliated epithelial cells, thus interfering with ciliary movement. In higher concentrations,
it also causes extrusion and destruction of
ciliated epithelial cells. The destruction of
these cells contributes to pertussis.
Dermonecrotic (heat-labile) toxin: It is a very
strong vasoconstrictor and causes ischemia and
extravasation of leukocytes, and in association
with tracheal cytotoxin, it causes necrosis of
the tracheal tissue.
Natural habitat
Obligate parasite. Each sps. has preferred natural host that serve as a reservoir of
infection. Predilection for ungulate placentas, fetal fluids and testes of bulls, rams, boars
and dogs. B. abortus excreted in bovine milk and can remain live in milk, water, damp
soil for upto 4 months.
Morphology and staining : brucella species are small, coccobacillary measuring about
0.6-2µm in length and 0.3-0.5µm in breadth. They are gram negative rods. They are non
motile, non acid fast and non-spore forming.
Brucella differeantial staining technique:
Although brucellas are not acid fast, they can resist decolorization with 5 % acetic acid.
This property differentiates brucella from other species.
Brucella stained with carbol fuchsion(1 min)-----> rinse with 5 % acetic acid(15
sec)----> stained with methylene blue(1 min)-----> if stained with carbol fuchsion the
organism is Brucella and if stained with MB, other than Brucella.
The allantoic fluid factors stimulate the growth of brucellae. The outer membrane protein
porine are to stimulate delayed typed hypersensitivity and account for the varying
susceptibility to dyes observed for the different species. Production of adenine and
guanine monophosphate by Brucella inhibits phagolysome fusion and activation of the
myeloperoxidase- halide system. Brucella is able to inhibit apoptosis in infected
macrophage, thereby preventing host cell elimination. It is the soluble protein products
inhibit TNF-alpha production. The virus operon encodes a type IV secretion system
which appears to be involved with intramacrophage survival.
Cultural characteristics: Growth requires an aerobic condition and enriched media such
as trypticase soy or blood agar. grows in ordinary lab. Media but slowly. After 48 hrs of
incubation visible colonies may be seen. Generally it requires 72 hrs to produce
macroscopic colonies. B. abortus and B. suis requires an atmosphere containing 5-10 %
CO2 . Brucella colonies are translucent and semitransparent. Optimum growth can be
obtained in 24 hours by using serum agar, liver infusion serum agar, tryptose agar,
dextrose potato, or glycerol potato. While using liver infusion serum, agar should be
checked for the presence of inhibitory factors.
In nutrient agar, in a 'humidified CO2-incubator", they produce 0.5 mm sized
round convex, translucent colonies with glistening surface, in case of smooth strain. The
rough strains produce larger colonies which are flattened, pale yellow with granular
appearance.
In broth medium, they grow slowly (in 2-3 wks) producing turbidity, then
granular deposit is observed on the lower part. Upon further incubation, the medium
become viscous.
On dextrose agar the colonies appear small and delicate, 1-2mm in dm in 24-48 hrs and if
continued incubation may become 8-9mm. Brucella colonies are translucent and
semitransluscent. The normal colony is smooth(S) and other types are mucoid (M) and
rough(R).
Biochemical properties : Brucella utilize carbohydrates but produce neither acid nor gas
insufficient amounts. Catalase and oxidase are produced by some strains. Nitrates are
reduced to nitrites. American strains of Brucella suis produce H2S, but Danish strains of
Br.suis do not produce H2S. The production of H2S by Br.abortus is less marked but
Br.meletinsis usually don't produce H2S. Smooth strains of Brucella are more virulent
than rough strains. Variation in co2 requirement, H2S production, urease production,
susceptibility to differing concentration of certain dyes (thionin and basic fuschin), and
susceptibility to naturally or mutagen- derived bacteriophages account for diversity
among species and biovars within species.
Serum dextrose agar slant facilities to determine the amount of gas produced. The
organisms are inoculated in the slant, 10 % lead acetate is inserted in the lid. After 48
hours of incubation, if the filter paper becomes black, H2S is produced.
Urease activity is high with both American and Danish strains of B. suis but is
low with B. abortus and variable with B. malitensis. The organism doesn't produce
indole, dont liquify gelatin, Voges-proskauer and methylene red reduction are negative.
Resistance to physical and chemical agents: Brucellae survive freezing and thawing.
Under proper environmental condition they survive for up to 4 months in milk,
urine,water,and damp soil. the organism is destroyed at 60oC in 10 minute. So
pasteurization is effective to kill there. They are susceptible to acidic pH, most of the
disinfectants and sunlight. The organism can survive for years in aborted fetus. In lab.
They can be stored at -40 oC for considerable period of time.
Most disinfectants active against gram- negative bacteria kill Brucella. Pasteurization
effectively kills Brucella in milk.
Virulent factors:
1. Ability to resist phagocytosis: low molecular wt substances in cell wall inhibit fusion
of lysosomal granules with the phagosomes and hence survive and multiply.
2. Ability of intracellular survival: survive and multiply in RE cells of lymph nodes,
liver, spleen and bone marrow. Very difficult for elimination by immune system.
3. antigenic structure: they have 2 main somatic antigen: A and M. mostly they are
present in 3 main sps- A type prevalent in B. abortus, M type prevalent in B.
melitensis. B. suis are of intermediate type.
Pathogenesis:
easy to multiply there and transported to the regional lymph node and via the thoracic
duct from the thoracic duct again goes to blood stream then to parenchymatous organs
and other tissue plus joints., produce granulomatous foci and they are suppurative.
Produce disease of sexually mature animal. Predilection site is the reproductive tract of
male and female specially pregnant uterus (allantoid factors stimulate the growth of most
of Brucella). Allantoic factors include erythritol, possibly steroid hormone and other
substances. Females usually abort only once after which a degree of immunity develops
and animal remains infected. Permanent infertility may occur in male dogs. Human can
be infective and can develop undulant fever.
Specimen: whole fetus is possible. Fetal stomach content, some lesions, cotyledons,
uterine discharges, semen and tissue from epididymus or testes from the males.
Direct microscopy can be done by staining with acid fast ( Ziel Neelson) or even with
Gram staining.
Isolation: specially done in any media enriched with (5-10%) or serum. Grow well on 5-
10% blood agar.
Identification: colony morphology- after 3-5 days it shows pin point, smooth, glistening,
and bluish, translucent colonies when aged turns into opaque form. FAT can be used.
Disease pattern
B. abortus
Most important due to disease in cattle, causes abortion.and disease of respiratory tract in
cattle, pigs, goat and sheep.similar disease is caused in Dog but less frequently, and
occasionlly in horses.
Transmission by ingestion (contact with aborted material, infected milk)
Causes mammary infection (not mastitis), transmission in milk
Disease in humans : human being are equally suceptible. The condition is known as
'Brucellosis' a 'undulant fever' or malta fever'. It is charcterized by acute septicemia,
intermittent fever, muscle and joint pain, hypersensitivity.(male gonad is affected), it is a
zoonotic disease and mostly occupational disease as well transmitted via milk, meat or
direct contact. Treatment involves use of antibiotic for 40 days. For human, major source
of infection are: abortus fetus, placenta, clostrum and genital secretion.
B. melitensis
Sheep, goats
Early localization in mammary gland, spread to humans (gastroenteritis,
Malta/Mediterranean fever)
Disease in goat similar to abortus in cattle
B. ovis
Epididymitis in rams, abortion in ewes
Least pathogenic of all brucellae
Brucella suis
Transmitted by ingestion, venereal also
In sows: granulomatous inflammation of entire endometrium
In boars: seconary infections in bones/joints, spleen, liver, kidney, brain
Prolonged bacteremia
Control by gradual depopulation after pos tests
B. canis
Dogs, oral and venereal transmission
Enlarged retropharngeal LN or superficial inguinal/external iliac LN
Bacteremia, abortion, epididymitis, testicular degeneration
Morphology and staining: - Moraxella is genus of coccobacillary, gram -ve rods. The
organisms are small bacilli measuring about 2 µm in length and 1.0 µm in breadth. They are
usually paired end to end (dipplobacilli) or short chains, capsule may be demonstrable in newly
isolated strains. They are non motile and non- spore forming.
Structure and composition: the cell wall is typical of gram negative bacteria being composed of
LPS and protein. The LPS of moraxellae does not contain O-repeat units, in contrast to many
other gram-negative mocroorganisms. The fimbrial adhesins (pili) of M. bovis are virulence
determinants and can be lost in subculture.
Cellular products of Medical interest: Moraxella bovis produces a type 4 pilus (fimbria) that
adheres to conjunctival and corneal epitheliaal cells. This pilus is similar to those of
Pseudomonas aeruginosa, Neiseria gonorrhoeae, Dicholobacter nodosus, Pasteurella
multocida, and Vibrio cholera. Mutant unable to produce this adhesin are avirulent.
Exotoxins : RTX( repeats in toxin) type of cytotoxin. (Pasteurella leukotoxin, actinobacillus
haemolysin, bordetella adenyl cyclase toxin, E.coli haemolysin).this cytoxin, sometimes reffered
as 'hemolysin' due to its behaviour on blood agar plates, has been termed Mbx(Moraxella bovis
toxin).Mbx is pore forming toxin with specificity for conjunctival and corneal epithelial cell, and
neutrophils.
Cultural characteristics: Moraxella bovis grows best at 35oC in the presence of serum and
blood. No grow occurs on MacConkey agar or anaerobically. In 48 hours, fresh isolates produce
flat, hemolytic, friable colonies, about 1 mm in size that corrode the agar and autoagglutinate
when suspended in saline. Different according to the species.
M. Bovis * Aerobic, forms small circular translucent grayish colony in 5% bovine agar
blood, measuring about 3-4 mm in 48 hours of incubation.
* Hemolytic characteristics present.
M. lacunata * Grows in nutrient agar containing blood or serum, even in less amount.
* Colonies are inconsequent and very small.
The above 2-species can be differentiated by liquefaction of gelatin, where M. bovis gives - ve
test. (Liquefied slowly). Moraxella bovis is oxidase-positive, nonfermenting, and catalase-
variable. Nitrates and urea are not attacked, but proteins are digested.
Pathogenesis : disease produce by M.bovis is closely linked to cytoxin (Mbx) and pili.
Attachment(pili) to conjunctival epithelium is followed by destruction of conjunctival and
corneal cell.
Laboratory Diagnosis
The agent may be demonstrated in smears of exudate, exudate is cultured on blood agar and
Moraxella are identified by colonial characterstics, oxidase activity, hemolysis, proteolysis, and
failure toferment carbohydrates.
Pseudomonas
1. Medium sized (1.5-5 x 0.5-1m) gram-negative, strict Aerobic, catalase and oxidase +, require
oxygen or some other inorganic electron acceptor (grow anaerobically in presence of nitrate or
some similar substance), not fermentative. Motile by way of polar flagella (except Psedomonas
mallei). Surrounded by a carbohydrate containing capsule. produce soluble pigments, grow on
MacConkey.And pili are produced.
2. Pseudomonads have very diverse metabolic capabilities and can grow on a wide range of
carbon sources.
3. An experienced diagnostic microbiologist can often identify pseudomonas by its smell, which
is somewhat like grapes.
4. Pseudomonads are widely distributed in soil and water, and are found in the gut of about 10%
of healthy humans; they are, thus readily available for nosocomial infections. Many
Pseudomonads can grow in distilled water to greater than 106 per ml. Pseudomonas aeruginosa
is a water loving organism commonly transmitted through the air. However, they are very
susceptible to drying, making airborne spread less likely.
5. Some pseudomonal infections are caused by organisms in the pseudomallei group
a. P. mallei causes glanders in horses and can be transmitted to man. It is rare in the West,
usually occurring in Asia, Africa, and the Middle East. Disease is characterized by necrosis of
nasal mucus membranes, lymphatic, lymph nodes, and skin. Acute or chronic pneumonia may
also occur.
b. Burkholderia pseudomallei cause melioidosis. This may take the form of a rather benign
pulmonary disease (like certain mycoses), but may also be a rapidly fatal septicemia with
occurrence of disseminated abscesses. The infection may become latent, leading to
recrudescence. It is usually seen only in SE Asia, and a significant number of cases were
diagnosed in returning Vietnam veterans.
6. the most important species in this genus is P. aeruginosa.
a. Disease caused by this organism is rare in healthy individuals, in whom it usually only causes
chronic external otitis
Natural habitat
Most of the species are exclusively saprophytes. Two species are important animal pathogen ( P.
aeruginosa and P. pseudomallei) which are present in soil and water.P aeruginosa is world wide
and P. pseudomallei mainly in tropical region. Found on skin, mucous membrane and in faeces
of animals . P. fluorescens present in soil and water associated in food spoilage and can cause
lesions in reptile and fish.
They are fairly heat resistant (killing @ 550C for 1 hr). Resistant to most of the antiseptic and
disinfectants as well as to most of the antibiotics. They are sensitive to acid, strong phenolic
disinfectant and silver salts.
Pathogenesis.
P. aeruginosa produce numbers of protein exotoxins and enterotoxins responsible for diarrhea.
An endotoxin and numerous exracellular products e.g. protease and haemolysins may play role
in pathogenesis. Pili facilitate adherence to epithelial cells. Capsules (some strain) is
antiphagocytic. Bacteriosins (pyocins) and pigment exhibit antimicrobial activity. Blue green
pigment (pyocyanin) can colour pus and stain wool a greenish hue. P. aeruginosa is
opportunistic and cause primary disease only and rarely. Predisposing cause includes trauma to
tissue (burns and wound), debilitation due to malignancy or immunodeficiency and reduced
numbers of normal flora often cause by antibiotic therapy.
P. pseudomallei have a wide host range including man. Infections usually systemic. Lesions are
nodules that may suppurate and can form in any tissue. Most infections are chronic but acute
form with terminal septicemia also seen. Toxin includes a lethal factor with anticoagulent
activity and skin necrotizing prteolytic agent. P. mallei cause glanders or farcy in equines.
Sometimes P. mallei infection in cat, dog, sheep, goat and camels possible. However, cattle,
pigs, rats and birds are resistant.
Determinats of pathogenicity
1. Alginate capsules (formed by polymer of manuronic acid and glucuronic acids) – enables
the bacteria to survive in aquatic environment and also protects the organism from phagocytosis.
It acts as adhesion factor.
2. Pilin and non- pilus adhesions- colonization
3. Haemolysin – phospholipase C, breaks down lipids and lecithin facilitating tissue
destruction
4. Elastse- two enzymes, Las A (serine protease) and Las B (zinc metalloprotease) act
synergistically to degrade elastin that cause damage of lung parenchyma and haemorrhagic
lesions.
5. Pyocyanin- catalyses the production of superoxide and H2O2.
6. Endotoxin- sepsis syndrome.
7. Exotoxins- exotoxin A is heat labile and interferes phagocytosis. It is the most important
virulence factor which blocks protein synthesis in eukaryotic cells. Exotoxin S is heat stable and
also interferes phagocytosis.
8. Antibiotic resistant
9. Enterotoxins
Lab diagnosis
P. mallei and P. pseudomallei are among the most dangerous bacteria to work with in a lab. A
biohazard cabinet must be used and all necessary safety procedures should be taken.
Isolation: can grow in ordinary media and can be enhanced by adding 1% glycerol. Selective
media can be made by adding 1000 units polymyxin D, 1250 units bacitracin and 0.25mg
actidione to 100ml TSA. P. aeruginosa can grow in other media intended for other
enterobacteriaceae family. E.g. Mac, BGA, XLD agar. Growth occurs @ 370C for 24-48 hrs.
Identification: P. aeruginosa: large (3-4 mm), flat, grayish blue colonies with characteristic
fruity, grapelike odor. Beta hemolytic on blood agar. Pyocyanin unique to P. aeruginosa. S form
to R form. In MacConkey, large, pale colonies unable to utilize lactose, with greenish blue
pigment superimposed. BGA and XLD: red, medium sized colonies. No H2S in XLD.
P. pseudomallei- colony smooth mucoid to rough with a dull, wrinkled, corrugated surface. In
some smooth form colonies are round, low convex, entire shiny and grayish yellow. After
several days' colonies become opaque, yellowish brown and cumbonate. It has earthy or musty
order. Initially partial and later complete haemolysis. Lactose fermenter, colony on MacConkey
but no growth in DCA and SS agar.
P. mallei: slow growth, 1-2mm colonies in 24-48 hours; smooth and white to cream; in ageing
they become granular and yellowish or brown. Unable to grow in MacConkey.
Immunological tests, animal inoculation and AST can be done for identification.
Clinical manifestations:
UTI, wound infections, chronic otitis media and otitis externa, eye infection, meningitis,
respiratory infection, septicemia, necotising vasculitis.
Psedomonas aeruginosa
The organism is worldwide in distribution and occurs as a part of bacterial flora of intestines of
man and animals. The organism is also found in water and soil. The organism is associated with
a wide variety of infections in animals and man and is frequently the cause of suppurative
lesions.
Morphology: slender rod, motile due to 3 polar flagella, doesnot produces spores or capsule.
The organism is gram -ve.
Cultural characteristic: P. aeruginosa is aerobic and grows readily on ordinary nutrient media
over a wide temperature range (20-40oC) the colonies produced on agar are larger, irregular,
translucent and grayish with white centre and an entire or undulate edge. An abundant growth
occurs in broth with the formation of a thick pellicle and dense turbidity.
P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment
pyoverdin and the blue pigment pyocyanin. The pyocyanin is produced abundantly in media of
low-iron content and functions in iron metabolism in the bacterium. Pyocyanin (from
"pyocyaneus") refers to "blue pus", which is a characteristic of suppurative infections caused by
Pseudomonas aeruginosa. A bluish green water soluble pigment diffuses throughout the
medium. The blue pigment "Pyocyan" is only produced by P. aeruginosa. "fluorescein" a yellow
pigment that fluoresces under ultraviolet light is also produced but it is not unique of P.
aeruginosa.
Grow well on blood agar medium, the colonies are somewhat large, >1mm in diameter, gray
(gunmetal), rough, usually with a zone of hemolysis.
Iron is an absolute growth requirement for all living things. P. aeruginosa produces the
iron acquiring siderophores pyochelin and pyoverdin, as well as using the siderophores produced
by other bacteria living in its environments (e.g. enterobactin and aerobactin). These products are
used to remove iron from host iron-binding proteins.
Biochemical properties: acid without gas is produced glucose. Gelatin is liquefied and urea is
slowly hydrolyzed. It doesn't reduce nitrates to nitrites, doesn't produce H2S.
Resistance: the organisms are susceptible to lethal effect of heat and are killed at 55oC in 1 hour.
Antigen and toxins:
Several toxins and enzymes produced by P. aeruginosa enhances it's virulence.
1. Extracellular product,
2. Extracellular enzymes and haemolysin
3. exotoxin: Exotoxin A, Exotoxins S and T, Exotoxin U, Exotoxin Y.
4. Miscellaneous products : bacteriocins(pyocins) and
pigments(pyocyanins).Pyocyanin reacts with oxygen to form reactive oxygen
radicals that are toxic to eukaryotes and prokaryotes organism.
Alginate (polysaccharide) ==> viscous gel around bacteria --> coating may protect from
phagocytosis in lung infections, c. Adhesion
1. Pili: type 4, similar to those of N. gonorrheae, also resemble those of V. cholerae
f. Elastase: Enzyme system which degrades elastin = major component of lung
g. At least two phospholipases C are produced - one hemolytic and one nonhemolytic
h. Leukocidin lethal for white blood cells, but nonhemolytic
i. Ninety percent of pseudomonads form pigments
Diagnosis
1. commonly used as diagnostic feature
2. involved in iron acquisition
3. Pyocyanin is antibacterial (suppresses growth) and thus may aid in colonization; damages
endothelial tissue in vitro.
4. Fluorescein is another pseudomonal pigment, flouresces in tissue.
8. Many pseudomonads are broadly resistant to antibiotics, may be plasmid-mediated.
Morphology : P. mallei is a slender rod with rounded ends and normally measures 0.3 to 0.5µm
in breadth and 1.5 to 5.0 µm in length. The organism is non capsule producing, non motile and
nonspore producing. The organism is gram negative and is not stained with common dyes.
Cultural characterstics : the organism is aerobic and facultative anaerobic. An acidic pH 6.6 is
most conductive for growth at 37oC. The organism grows well on most common bacteriologic
media but the addition of serum and glycerine yields more rapid and abundant growth. After 48
hrs. of incubation of serum and glycerine agar, the organism produces a small, round, amorphous
translucent colony. The growth on solid media is slimy and tenacious in consistency. The surface
colonies become yellowish brown as the culture ages.
Biochemical properties: the organisms do not ferment carbohydrates (but some strains produce
slight acid reaction in glucose). The organism does not produce indole, doesn't reduce nitrates.
Resistance: the organism is highly susceptible to moist heat. It is killed at 55oC in 10 mins.
Natural desiccation and sunlight does not allow the organism to live long outside the body.
Organism in pus remains protected to some extent form the action of disinfectants. Phenol is less
effect against the organism.
Antigen and toxins: P.mallei strains have been divided into three antigenic groups on the basis
of antigens which are specific to this organism. The organism does not produce exotoxin.
Pathogenicity : P. mallei primarily pathogenic to horses, mules and donkey, cattle, pigs and
poultry are resistant.
The clinical form of the disease is commonly classified into three types:-
1. Pulmonary glander's: characterized by the formation of round grayish, firm, encapsulated
nodules embedded throughout the lung tissue. Many of nodule contain yellowish, cheesy
pus surrounded by a zone of inflammation. Some of the nodules are composed of fibrous
form of tissue.
2. Nasal glander's: develop initially as a reddening of nasal mucosa and mucoid discharge
from one or both nostrils. The noduled develop on the nasal septum. The nodules rupture
and liberate mucopurrolent exudates.
3. cutaneous glander's or farcy: typical noduled or farcy buds form along the lymph vessels
between affected lymph nodes. The noduled rupture and discharge yellowish pus and
form into ulcers which heal slowly.
Glanders in man is rare. The organism localized in the respiratory organs or in the skin
and subcutaneous tissues.
Diagnosis : the most commonly used test is Mallein Test " for diagnosis.
It is an allergic test similar to the tuberculin test. Mallein is and autoclave whole culture
of P.mallei. Various sites are used for incubation. These days intradermal palpebral test is most
often used. The concentrated mallein in 0.1 ml quantity is inoculated into the skin of lower eyelid
near its edge. A positive reaction is characterized by tender swelling of eyelid with congestion of
conjunctiva and mucous discharge from the eye. The reaction begins 9-10 hours after inoculation
and reaches its height between 24-36 hours. The mallein test is fairly specific and sensitive but
may be negative in acute and advanced stages of the diseases.
Identification includes (a) pigments: pyocyanin (blue-green) and fluorescein (green-
yellow, fluorescent) and (b) biochemical reactions. Cultures have fruity smell.
5. Slime layer is anti-phagocytic.
6. Toxin A - ADP ribosylates EF2 similar action to diphtheria toxin
Rickettsias and chlamydiae are two unrelated groups of bacteria that are obligate
intracellular parasites of eukaryotic cells. Rickettsias cannot grow outside of a host cell
because they have leaky membranes and are unable to obtain nutrients in an extracellular habitat.
Chlamydiae are unable to produce ATP in amounts required to sustain metabolism outside of a
host cell and are, in a sense, energy-parasites.
Rickettsias occur in nature in the gut lining of arthropods (ticks, fleas, lice, etc.). They
are transmitted to vertebrates by an arthropod bite and produce diseases such as typhus fever,
Rocky Mountain Spotted Fever, Q fever and ehrlichiosis.
Chlamydiae are tiny bacteria that infect birds and mammals. They may colonize and
infect tissues of the eye and urogenital tract in humans. Chlamydia trachomatis causes several
important diseases in humans: Chlamydia, the most prevalent sexually transmitted disease in the
U.S., trachoma, a leading cause of blindness worldwide, and lymphogranuloma venereum.
Chlamydia pneumoniae is a cause of pneumonia and has been recently linked to atherosclerosis
The rickettsiae are small (0.3-0.5 x 0.8-2.0 um), Gram-negative, aerobic, coccobacilli
that are obligate intracellular parasites of eukaryotic cells. They may reside in the cytoplasm or
within the nucleus of the cell that they invade. They divide by binary fission and they metabolize
host-derived glutamate via aerobic respiration and the citric acid (TCA) cycle. They have typical
Gram-negative cell walls, and they lack flagella. The rickettsiae frequently have a close
relationship with arthropod vectors that may transmit the organism to mammalian hosts. The
rickettsiae have very small genomes of about 1.0-1.5 million bases.
Rickettsiae must be grown in the laboratory by co-cultivation with eukaryotic cells, and
they have not been grown by in axenic culture. The basis of their obligate relationship with
eukaryotic cells has been explained by rickettsial possesion of "leaky membranes" that require
the osmolarity and nutritional environment supplied by an intracellular habitat.
The rickettsiae, in spite of their small size and obligate intracellular habitat, are a group
of alphaproteobacteria, which inlcude many well-known organisms such as Acetobacter,
Rhodobacter, Rhizobium and Agrobacterium. Very few of the alphaproteobacteria are pathogens
of humans. Brucella, Bartonella, Rickettsia, and a related intracellular parasite, Ehrlichia, are the
main exceptions.
Taxonomy
The genus Rickettsia is included
in the bacterial tribe Rickettsieae,
family Rickettsiaceae, and order
Rickettsiales. This genus
includes many other species of
bacteria associated with human
disease, including those in the
spotted fever group and the
typhus group (figure1).
Figure 1. Taxonomic classification of
the order Rickettsiales
The rickettsiae can be subdivided into two or three major groups, depending on your taxonomic
point of view (1. spotted fever; 2. typhus; and 3. scrub typhus groups) based on clinical
characteristics of disease and phylogenetic relationships.
Spotted Fever Group (SFG)
Rickettsia rickettsii is the cause of Rocky Mountain spotted fever (RMSF) and is the
prototype bacterium in the spotted fever group of rickettsiae. Rickettsia rickettsii is found in the
Americas and is transmitted to humans through the bite of infected ticks. The bacterium infects
human vascular endothelial cells, producing an inflammatory response. The pathogenesis of
RMSF is discussed in some detail below.
Other spotted fever group rickettsiae that produce human rickettsioses include R. conorii,
R. mongolotimonae and R. slovaca (bouteonneuse fever and similar illnesses), R. akari
(rickettsial pox), R. japonica (Japanese spotted fever), R.sibirica (North Asian tick typhus), R.
africae (African tick bite fever), R. helvetica (perimyocarditis), R. australis(Queensland tick
typhus) and R. honei (Flinders Island spotted fever). The spotted fever rickettsiae have been
found on every continent except Antarctica.
Typhus Group (TG)
Rickettsia prowazekii is the cause of epidemic or louse-borne typhus and is the
prototypical bacterium from the typhus group of rickettsiae. R. prowazekii infects human
vascular endothelial cells, producing widespread vasculitis. In contrast to RMSF, louse-borne
typhus tends to occur in the winter. Infection usually is transmitted from person to person by the
body louse and, therefore, tends to manifest under conditions of crowding and poor hygiene. The
southern flying squirrel is apparently the reservoir in the United States, but the vector involved in
transmission from the flying squirrel to humans is unknown. The disease has a worldwide
distribution.
Other rickettsiae in the typhus group include R. typhi and R. felis. Murine typhus is
caused by transmission of R. typhi from rats, cats and opossums to humans via a flea vector.
Murine typhus is found worldwide and is endemic to areas of Texas and southern California in
the United States. Although R. felis is phylogenetically more closely related to the spotted fever
group of rickettsiae than the typhus group, it shares antigens with R. typhi and produces a murine
typhus-like illness. Rickettsia felis has been detected in cat fleas and opossums.
Scrub Typhus Group (STG)
Orientia (Rickettsia) tsutsugamushi is the cause of scrub typhus. Originally called
Rickettsia tsutsugamushi, this organism was given its own genus designation because it is
phylogenetically distinct from the other rickettsiae, though closely related. Orientia
tsutsugamushi is transmitted to humans by the bite of trombiculid mites (chiggers), which are the
vector and host. Scrub typhus occurs throughout much of Asia and Australia.
Spirochetes
The spirochetes are a phylogenetically distinct group of bacteria which have a unique cell
morphology and mode of motility. Spirochetes are very thin, flexible, spiral-shaped procaryotes
that move by means of structures called axial filaments or endoflagella. The flagellar filaments
are contained within a sheath between the cell wall peptidoglycan and an outer membrane. The
filaments flex or rotate within their sheath which causes the cells to bend, flex and rotate during
movement. Most spirochetes are free living (in muds and sediments), or live in associations with
animals (e.g. in the oral cavity or GI tract). When the filaments rotate within this space, the
spirochetes move in cork-screw fashion. This type of movement may be an adaptation to viscous
environments such as aquatic sediments, biofilms, mucosal tissues and the intestinal tracts of
animals. For pathogens, this allows the spirochetes to hide their flagella, which are normally
antigenic, from the host immune defenses.
A few are pathogens of animals Treponema pallidum is the agent of syphilis, a sexually
transmitted disease, and Borrelia burgdorferi causes Lyme Disease. which is transmitted by the
bite of the deer tick.
Figure 1. Spirochetes: A. Cross section of a spirochete showing the location of endoflagella between the inner
membrane and outer sheath; B. Borrelia burgdorferi, the agent of Lyme disease; C. Treponema pallidum, the
spirochete that causes syphilis.
Spirochetes are usually much longer than they are wide, and often their width is below the
resolving power of the light microscope. For example, Borrelia may have a length of 20-30um
but a width of only .2-.3um. Hence, most spirochetes cannot be viewed using conventional light
microscopy. Dark-field microscopy must be used to view spirochetes. Dark field microscopy
utilizes a special condenser which directs light toward an object at a angle, rather than from the
bottom. As a result, particles or cells are seen as light objects against a dark background. The
spirochetes are not classified as either Gram-positive or Gram-negative.
Spirilla and other curved bacteria
Spirilla are Gram-negative bacteria with a helical or spiral shape.
Their metabolism is respiratory and never fermentative. Unlike
spirochetes, they have a rigid cell wall and are motile by means of
ordinary polar flagella. Two important pathogens of humans are
found among the spiral forms. Campylobacter jejuni is the cause of
bacterial diarrhea, especially in children. The bacterium is
transmitted via contaminated food, usually undercooked poultry or
shellfish, or untreated drinking water. Helicobacter pylori is able to
colonize the gastric mucosal cells of humans, i.e., the lining of the
stomach, and it has been well established as the cause of peptic
ulcers and there is strong evidence for its involvement in
adenocarcinoma.
Borrelia burgdorferi
Borrelia burgdorferi, like the human pathogen Treponema
pallidum, is a spirochete.
Borrelia anserina
Morphology and staining:B.anserina is a long, spiral organism, 10-12µm long and
approximately 0.3µm in breadth. Usually 4-6 cells has been observed. They are actively motile
with serpentine or flexous movement. The organism are gram-ve, but are easily seen after
staining with giemsa or leishman's or wright's staining of blood.
Cultural characteristic: the organism are difficult to grow in ordinary lab media. They grow
under aerobic conditions (i.e. O2 is neither utilized, nor toxic) at 37 oC with difficulty. It can be
cultivated in complex media contining pieces of rabbit's kidney or heart muscles and ascitis fluid.
The organism grows in chicken embryo by choroallantoic membrane route. Usually 5 days of
chick embryo is used.
Borrelia are highly suceptible to changes in their environment. The organisms remain
alive in citrated blood for 3 months at 4 oC.
Antigenicity: the monst characteristic antingenic property of Borrelia is its mutability. In
borrelia anseina, there is evidence of existance of different antigenic types.
Epidemiology: borrelis anserina infects chicken, gees, turkeys, ducks, sparrows and several
species of bird. The avian spirochaetosis is transmitted from one birds to another by fowl tick
argas persicus. It is probable that apart from Argas persicus and Argas reflexes, the organisms
may be transmitted by red mite and rarely moquitoes.
The genus Treponema
None of the species in the genus Treponema is of any significace to animal health.
Treponema are spiral filament, anaerobic, not easily stained with ordinary stainig techniques.
They are gram negative.
Some pathogenic speces
Treponema pallidum cause syphilis in man
Treponema cuniculi is the cause of rabbit syphilis
Treponema suis cause preputial infection in pigs
Treponema hydrodysenteriae cause swine dysentry.
The genus spirillum
The genus includes number of saprophytic bacteria found in putrefying material or stagnat water.
Only one species: Spirillum minus is pathogenic. S. minus cause rat bite fever in ams. The
organism is carried by rats, mice and possibley other animal species and is transmitted to man by
rat bite. The disese is prevlaent in far eastern countries.
Campylobacter fetus
Campylobacter fetus is prevalent throughout the world and causes abortions in sheep and
infertility and abortions in cattle. It is natural inhabitant of genital and intestinal tract of sheep &
cattle.
Morphology: C. fetus in young cultures is comma shaped and S-shaped measuring 0.2 - 0.5 µm
in width and 1.5 - 5 µm length (sometimes they give seagull like appearance). In older cultures
spiral forms are seen in which the organisms cling together. In tissues shorter forms are seen.
The organism are motile and Gram -ve, non-spore former.
Cultural characteristics: the organism grows best in microaerophilic conditions. This can be
provided by 10% CO2. the optimum temperature for grwth is 37 oC. a semisolid agar medium
known as 'thiol' medium is superior to either media, it is used both for isolation and maintenance
of sultures. On blood agar, the organism forms fine, pin point bluish areas, raise above the
surface of the medium. In fluid media, it produces a faint clouding.
The organism grwos in smooth(S), rough(R) and mucoid(M) colonies. The smooth
colonies are composed of virulent cells and rough colonies have avirulent organisms.
BIochemical properties: the organism doesnot ferment sugars, doesnot form, it is nitrate
positive, doesnot change indole.
Resistance: the organism is suceptible to heat and is killed in 5 minutes at 60 oC , it is readily
killed by drying, sunlight and chemical disinfectants. It survives in hay, soil and manure for 10-
20 days.
Antigenicity: the organism possesses somatic(O), flageller(H) and capsular(K) antigens
Human being are suceptible to C. fetus, cause abortion, prenatal birth, undulant fever and
headache. Animals not only the source of injection i.e. environment etc. also acts as the source of
infection.
L. grippotyphosa
L. icterohaemorrhagiae
Cultural characteristics: leptospirae grow best in aerobic conditions at 29-32 oC. the organism
requires O2 for growth but some strains also require small quantities of CO2. pathogenic strains
require the addition of serum to the medium. Rabbit serum is commonly used. All serum samples
are tested to ensure absence of leptospiral antibodies. The media commonly used for growing
leptospirae are korthof's medium, fletcher's agar, and ringen and gillespie medium.
Biochemical properties: leptospirae derive energy form long chain fatty acids and conanot use
aminoacids or carbohydrates as a major source of energy.
Resistance: leptospira donot withstand dryness or heat. Killed at 60 oC for 15 sec.multiplication
outside the host is rare. Alkaline surface water with a pH 7.0 to 8.0 favours survival of these
organism in nature for weeks.
In tissue at -20 oC, they survive for 3 months and at 5-7 oC, they survive for 7-14 days.
Antigenicity: on the basis of their antigenic structure, members of the group have been placed in
18 serogroups and more than 150 serotypes. The pathogenic leptospirae produce toxic substances
which have hemolytic and lipolytic properties.
Listeria
monocytogenes and Listeriosis
Listeria monocytogenes is a Gram-positive rod-shaped bacterium. It is
the agent of listeriosis, a serious infection caused by eating food
contaminated with the bacteria. Listeriosis has recently been
recognized as an important public health problem. The disease affects
primarily pregnant women, newborns, and adults with weakened
immune systems. It causes myocardial degeneration in fowls.in
Listeria monocytogenes
domestic animals meningo encephalitis and abortion is found.
In cattle, mastitis also develops.
Listeriosis is a serious disease for
humans; the overt form of the disease has a
mortality greater than 25 percent. The two
main clinical manifestations are sepsis and
meningitis. Meningitis is often complicated by
encephalitis, a pathology that is unusual for
bacterial infections.
The actinomycetes are not thought of as pathogenic bacteria, but two of their relatives
are among the most important pathogens of humans, these being the agents of tuberculosis and
diphtheria. Actinomycetes are a large group of Gram-positive bacteria that usually grow by
filament formation, or at least show a tendency towards branching and filament formation. Many
of the organisms can form resting structures called spores, but they are not the same as
endospores. Branched forms superficially resemble molds and are a striking example of
convergent evolution of a procaryote and a eukaryote together in the soil habitat. Actinomycetes
such as Streptomyces have a world-wide distribution in soils. They are important in aerobic
decomposition of organic compounds and have an important role in biodegradation and the
carbon cycle. Actinomycetes are the main producers of antibiotics in industrial settings, being
the source of most tetracyclines, macrolides (e.g. erythromycin), and aminoglycosides (e.g.
streptomycin, gentamicin, etc.).
Two genera of bacteria that are related to the actinomycetes, Corynebacterium and
Mycobacterium, contain portant pathogens of humans: Otherwise, many nonpathogenic
mycobacteria and corynebacteria live in normal associations with animals.
Species: In the past, staphylococci were differentiated into three types based on pigment
production; staph. Aureus, staph. Albus and staph. Citreus. Since pigment production is not
constant and an uncertain chararacter, this classification is now obsolete. Pathogenic strains
exhibit some characteristics which include production of coagulage, phosphatase,
deoxyribonuclease, possession of protein A antigen and ability to ferment mannitol. Production
of coagulage and to certain extent formation of mannitol with acid production, are related to
virulence of the strains.
Distribution: the animal forms part of bacterial environments of animals and man throughout
the world makes normal flora of skin and mucous membrane of man and animals. It is present in
many animal products like eggs, meat and milk. Staph. Pyogenes produces several toxins and
cause many pathological conditions like mastitis in animals yolk sac infection and arthrits in
poultry, tick pyemia in lambs, skin lesions and pyemic infection in man and animals. The
organism is opportunistic.
Morphology and staining: they are gram positive, nonspore forming, non motile, aerobic and
normally faculatively anaerobic cocci(1µm in diameter), characteristically arranged in striking
clusters, due to division in 3 successive planes. They may be arranged singly, in pairs, tetrads or
short chains of 3-4 cells. The organisms may become gram negative in aging cultures In films
prepared on slides, they are broken (i.e. clusters) and occurs in 2-3 or more cells.
Cultural characteristics: Facultative anaerobes, grow better aerobically, some = helped by CO2
within a temperature ranges of 10-42 oC, optimum temperature being 37 oC.
Golden pigment = produced by many strains (aureus = gold), especially with extended
incubation. Nearly all isolates are hemolytic (> 4 hemolysins = produced); most common = and
ß; hemolysis = frequently double-zone).
a. In nutrient agar: after 24 hrs incubation, large (2-4mm diameter), round, smooth, convex and
glistening colonies are developed, with entire edges. The colonies are sometimes pigmented
and resemble a drop of oil paint.
b. In milk agar: the pigmentation develop intensely and rapidly on milk agar when incubated at
22-25oC. the pigmentation is not a constant feature. If they form a clear zone, it suggests that
the cells can digest casein.
c. In blood agar: on blood agar most of the strains, but not all, show B-hemolysis surrounding.
The hemolysis is best seen with rabbit or sheep blood. The haemolysis become well
developed when incubated in an atmosphere of 10% CO2
d. Mac conkey's agar:(a differential media): the colonies on Mac Conkey's agar are small and
pinkish yellow or deep red or purple.
e. Broth medium: in broth medium, a uniform turbidity is present with powdery sediment.
f. CAMP Test: This test named after the discovers (Christie, Atkins and Munch-Peterson) is
based on the observation that ruminants RBC partially haemolysed by beta-toxin of
Staphylococcus at 370C are lysed completely in the presence of Stre. Agalactiae. A culture of
Staph. aureus with a wide zone of partial haemolysis (beta haemolysis), is streaked across the
centre of sheep or ox blood agar plates. A streak of suspected group B streptococci is made at
right angle to, and taken to with in 1-1.5 mm of the staph. Streak. The plate is incubated at
370C for 24 hrs. A +ve CAMP test is indicated by an arrow-head of complete haemolysis.
The group B streptococci produce a diffuable metabolites that partially haemolysed by B
haemolysin of Staph.
Biochemical properties: Stpaph. Aureus produce acid from glucose, maltose, mannitol, lactose,
sucrose and glycerol. The organism is indole negative, NH3 positive, methylene red positive, and
voges-proskauer positive; reduces nitrates to nitrites; reduces methylene blue, forms slight H2S,
hydrolyses gelatin and coagulated serum; urease positive. In contrast to other staphylococci
strains, staph. Pyogenes, in the medium containg 7.5% sodium chloride, ferments mannitol to
yield organic acids and acid reaction.
Since, varieties of toxins, some acting as enzymes are produced. Some of the toxins associated
with virulence are:-
A. Protein A
1. S. aureus only
2. Most = cell-bound, some = released extracellularly
3. Reacts nonspecifically with Fc portion of Igs
B. Capsules
1. Few strains = encapsulated in vitro
2. More may be encapsulated in vivo, lost on cultivation
3. Encapsulated = more virulent for animals
4. Anticapsular antibodies = protective
5. Capsular antigens = antiphagocytic
C. Toxins:
The extracellular free coagulase is a heat labile enzyme secreted free in the culture
medium. It requires the corporation of plasma factor i. e; coagulase reacting factor (CRF)
for its clotting action. CRF is present in human and rabbit plasma. The free coagulase has
thrombin like activity and converts fibrinogen into fibrin.
Bound coagulase is called clotting factor is a heat stable constituent of cell wall which
treat directly with the fibrinogen and causes aggregation of the Staphylococcus.
Slide coagulase test: it is done for detection of bound coagulase produced on
culture. For that; A loopful of the staph. Culture is emulsified in a drop of saline
or water on the slide and mixed with a drop of undiluted human or rabbit plasma.
Clumping occurs with in 1-2 minutes in the positive reactions.
Tube coagulase test; it is done for detection of free coagulase produced on culture.
For that; 0.5 ml of rabbit plasma is placed in a small test tube. Two drops of an
overnight broth culture of staphylococcus are added and tube is mixed gently then
incubated at 370C for 3-6 hrs, clotting (gel formation) of plasma indicating
positive reactions.
Based on coagulase production, staphylococci are classified into two groups-coagulase +ve and -
ve. Since most of the coagulase +ve strains produce golden yellow colonies, though some may
be white or cream colored, they are known as staph. Aureus (also known as staph. Pyogenes).
They produce toxins. The coagulase-ve strains are usually non-pathogenic and are called staph.
Epidermidis(often known as staph. Albus). They are non-toxigenic. Most coagulase negative
strains form white colonies.
normal flora, S. pyogenes can infect when defenses are compromised or when the organisms are
able to penetrate the constitutive defenses. When the bacteria are introduced or transmitted to
vulnerable tissues, a variety of types of suppurative infections can occur.
It produces a wide array of virulence factors and a very large number of diseases. Virulence
factors of Group A streptococci include: (1) M protein, fibronectin-binding protein (Protein F)
and lipoteichoic acid for adherence; (2) hyaluronic acid capsule as an immunological disguise
and to inhibit phagocytosis; M-protein to inhibit phagocytosis (3) invasins such as
streptokinase, streptodornase (DNase B), hyaluronidase, and streptolysins; (4) exotoxins,
such as pyrogenic (erythrogenic) toxin which causes the rash of scarlet fever and systemic
toxic shock syndrome.
Classification: Streptococci are subdivided into groups with antibodies that recognize surface
antigens. These groups may include one or more species. The most important groupable
streptococci are A, B and D. Among the groupable streptococci, infectious disease (particularly
pharyngitis) is caused by group A. Streptococcus pneumoniae (a major cause of human
pneumonia) and Streptococcus mutans and other so-called viridans streptococci (among the
causes of dental caries) do not possess group antigens.
Hemolysis on blood agar: Three types of hemolysis reaction are seen after growth of
streptococci on sheep blood agar (alpha, beta, and gamma). α refers to partial hemolysis with a
green coloration (from production of an unidentified product of hemoglobin) seen around the
colonies; β refers to complete clearing and γ means there is no lysis. Group A and group B
streptococci are β hemolytic, whilst D are usually α or γ. Streptococcus pneumoniae and viridans
("green") streptococci are α hemolytic. The hemolysis reaction along with one physiologic
characteristic is sufficient for A presumptive clinical identification.
The type of hemolytic reaction displayed on blood agar has long been used to classify the
streptococci. Beta -hemolysis is associated with complete lysis of red cells surrounding the
colony, whereas alpha-hemolysis is a partial or "green" hemolysis associated with reduction of
red cell hemoglobin. Nonhemolytic colonies have been termed gamma-hemolytic. Hemolysis is
affected by the species and age of red cells, as well as by other properties of the base medium.
Group A streptococci are nearly always beta-hemolytic; related Group B can manifest alpha,
beta or gamma hemolysis. Most strains of S. pneumoniae are alpha-hemolytic but can cause ß-
hemolysis during anaerobic incubation. Most of the oral streptococci and enterococci are non
hemolytic. The property of hemolysis is not very reliable for the absolute identification of
streptococci, but it is widely used in rapid screens for identification of S. pyogenes and S.
pneumoniae.
Antigenic types: The cell surface structure of Group A streptococci is among the most studied of
any bacteria. The cell wall is composed of repeating units of N-acetylglucosamine and N-
acetylmuramic acid, the standard peptidoglycan. Historically, the definitive identification of
streptococci has rested on the serologic reactivity of "cell wall" polysaccharide antigens as
originally described by Rebecca Lancefield. Eighteen group-specific antigens (Lancefield
groups) were established. The Group A polysaccharide is a polymer of N-acetylglucosamine
and rhamnose. Some group antigens are shared by more than one species. This polysaccharide is
also called the C substance or group carbohydrate antigen.
Morphology and Staining: The individual organisms are spherical or ovoid measuring about
1µm in diameter and usually occurs in chains of 2-12 cells. The length of the chain depends on
the species and growth conditions. Capsules are occasionally demonstrable in some species after
growth in tissues or on primary isolation, but after continuous cultivation on lab. Media, they are
not observed. The organism does not form spores. Motile strains occasionally occur, the motility
is apparent for a limited period of time during growth. Gram reaction is positive, although some
cells in older cultures appear gram negative.
Cultural characteristics: streptococcus grows best in media enriched with serum or defibrinated
blood under aerobic conditions. The organisms are facultative anaerobes. The optimum
temperature for growth is 37oC.the maximum temperature for growth is 42oC, but some
thermophiles grow up to 45oC or even higher.
In nutrient agar: they give a poor growth. When blood serum or fermentable carbohydrates are
added to the media, they grow well.
In blood agar: after 24 hrs of incubation in blood agar, they form colonies of 2 mm diameter,
circular and grayish in color but some of the colonies are mucoid or give a matt appearance and
this is often characteristic of virulent strains. A distinctive feature of streptococcus growth on
blood agar by many species is α (green) or β (clear) haemolysis. Most of the pathogenic species
are hemolytic. Quality and quantity or hemolysis differs from strain to strain or condition of
growth. Generally, the haemolytic strains cause maximum haemolysis within 8-hours of their
incubation. So, pathogenic strains are usually β-haemolytic strains. Some pathogenic strains are
α -hemolytic. α haemolytic is divided into two types:
In Edwrd's medium: they can grow readily (but staph. Can't), in MacConkey's agar, majority
of the streptococcal species donot grow, In broth medium, the growth is slow and an evenly
dispersed faint opacity is produced with fluffy deposit adherent to the side of the tube, and a
clear supernant fluid.
Biochemical properties: they grow rapidly in fermentable carbohydrate. They ferment lactose,
sorbitol, mannitol, insulin, raffinose and trehalose.
Resistance to physical and chemical agents: The organisms are usually killed after exposure
for 30 minutes at 56oC, but some strains resist this temperature. In the absence of direct sunlight,
the organisms can survive for weeks and months in dust and in animal houses. The organisms are
susceptible to most of the disinfectants, but the effect is reduced by the presence of pus or
degraded protein.
Antigen and Toxins: On the basis of the lancefield's grouping (i.e. based on the CHO
constituent of cell wall) 20 different antigens has been detected. On the basis of the difference of
other antigenic structures (except CHO) e.g. protein antigen, different types are: "M type" having
M antigen (alcohol soluble), 'T type' having T antigen (alcohol insoluble) 'R type' having R
antigen (neither type nor associated with virulence)
The cell surface of Streptococcus pyogenes accounts for many of the bacterium's determinants
of virulence especially those concerned with colonization and evasion of phagocytosis and the
host immune responses. The surface of Streptococcus pyogenes is incredibly complex and
chemically-diverse. Antigenic components include capsular polysaccharide (C-substance), cell
wall peptidoglycan and lipoteichoic acid (LTA), and a variety of surface proteins, including M
protein, fimbrial proteins, fibronectin-binding proteins, (e.g. Protein F) and cell-bound
streptokinase.
The capsule of S. pyogenes is non antigenic since it is composed of hyaluronic acid, which is
chemically similar to that of host connective tissue. This allows the bacterium to hide its own
antigens and to go unrecognized as antigenic by its host. The Hyaluronic acid capsule also
prevents opsonized phagocytosis by neutrophils or mancrophages.
Adhesins
It is now realized that S. pyogenes (like many other bacterial pathogens) produces multiple
adhesins with varied specificities. There is evidence that Streptococcus pyogenes utilizes
lipoteichoic acids (LTA), M protein, and multiple fibronectin-binding proteins in its
repertoire of adhesins. The fibronectin-binding protein, Protein F, has also been shown to
mediate streptococcal adherence to the amino terminus of fibronectin on mucosal surfaces.
For the most part, streptococcal invasins and protein toxins interact with mammalian blood and
tissue components in ways that kill host cells and provoke a damaging inflammatory response.
The soluble extracellular growth products and toxins of Streptococcus pyogenes (see Figure 2,
above), have been studied intensely. Streptolysin S is an oxygen-stable leukocidin; Streptolysin
O is an oxygen-labile leukocidin. NADase is also leukotoxic. Hyaluronidase (the
original”spreading factor") can digest host connective tissue hyaluronic acid, as well as the
organism's own capsule. Streptokinases participate in fibrin lysis. Streptodornases A-D
possess deoxyribonuclease activity; Streptodornases B and D possess ribonuclease activity as
well. Protease activity similar to that in Staphylococcus aureus has been shown in strains
causing soft tissue necrosis or toxic shock syndrome. This large repertoire of products is
important in the pathogenesis of S. pyogenes infections. Even so, antibodies to these products are
relatively insignificant in protection of the host.
Streptococcal invasins lyse eukaryotic cells, including red blood cells and phagocytes; they lyse
other host macromolecules, including enzymes and informational molecules; they allow the
bacteria to spread among tissues by dissolving host fibrin and intercellular ground substances.
Pyrogenic Exotoxins
Three streptococcal pyrogenic exotoxins (SPE), formerly known as Erythrogenic toxin, are
recognized: types A, B, C. these toxins act as superantigens by a mechanism similar to those
described for staphylococci.
In 1872, Ferdinand Cohn, a student of Robert Koch, recognized and named the bacterium
Bacillus subtilis. The organism was made to represent a large and diverse genus of Bacteria,
Bacillus, and was placed in the family Bacillaceae. The family's distinguishing feature is the
production of endospores, which are highly refractile resting structures formed within the
bacterial cells. Since this time, members of the genus Bacillus are characterized as Gram-
positive, rod-shaped, aerobic or facultative, endospore-forming bacteria. The organism of this
groups rod shaped measuring approximately 0.7*3-10 µm.
The ubiquity of Bacillus species in nature, the unusual resistance of their endospores to chemical
and physical agents, the developmental cycle of endospore formation, the production of
antibiotics, the toxicity of their spores and protein crystals for many insects, and the pathogen
Bacillus anthracis, have attracted ongoing interest in the genus since Koch's time. Spores are
round or oval and may be central, subterminal or terminal depending on the species of the
organisms.
There is great diversity in physiology among members of the genus, whose collective features
include degradation of most all substrates derived from plant and animal sources, including
cellulose, starch, pectin, proteins, agar, hydrocarbons, and others; antibiotic production;
nitrification; denitrification; nitrogen fixation; facultative lithotrophy; autotrophy; acidophily;
alkaliphily; psychrophily; thermophily; and parasitism. Spore formation, universally found in the
genus, is thought to be a strategy for survival in the soil environment, wherein the bacteria
predominate. Aerial distribution of the dormant spores probably explains the occurrence of
Bacillus species in most habitats examined.
Flagella
Most Bacillus species are motile by means of peritrichous flagella. Chemotaxis has been studied
extensively in B. subtilis. The flagellar filament of B. firmus, an alkaliphile, has a remarkably
low content of basic amino acids, thought to render it more stable in environmental pH values up
to 11.
Capsules
The capsules of many bacilli, including B. anthracis, B. subtilis, B. megaterium, and B.
licheniformis, contain poly-D- or L-glutamic acid. Other Bacillus species, e.g., B. circulans, B.
megaterium, B. mycoides, and B. pumilus, produce carbohydrate capsules. Dextran and levan are
common, but more complex polysaccharides are produced, as well.
Some of the Bacillus polysaccharides cross react with antisera from other genera of bacteria
including human pathogens. For example, B. mycoides with Streptococcus pneumoniae type III;
B. pumilus with Neisseria meningitidis group A; B.alveli with Haemophilus influenzae type B.
The capsules are easily observed by light microscopy, especially if the bacteria are prepared
ahead of time by growth on media that enhance capsule production. Heavily encapsulated strains
may form a mucoid or slimy colony on agar.
Most Bacillus species are versatile chemoheterotrophs capable of respiration using a variety of
simple organic compounds (sugars, amino acids, organic acids). In some cases, they also ferment
carbohydrates in a mixed reaction that typically produces glycerol and butanediol. The majority
are mesophiles, with temperature optima between 30 and 45 degrees, but the genus also contains
a number of thermophilic species with optima as high as 65 degrees. In the laboratory, under
optimal conditions of growth, Bacillus species exhibit generation times of about 25 minutes.
Anthracoides: a non pathogenic strains of bacillus, similar to anthrax bacteria. Anthracoids are
scattered everywhere in nature. They are differentiated from anthrax bacilli as follows:
Bacillus anthracis is very large, Gram-positive, sporeforming rod, 1 - 1.2µm in width x 3 - 5µm
in length.They are non-motile. The bacterium can be cultivated in ordinary nutrient medium
under aerobic or anaerobic conditions. Genotypically and phenotypically it is very similar to
Bacillus cereus, which is found in soil habitats around the world, and to Bacillus thuringiensis,
the pathogen for larvae of Lepidoptera. The three species have the same cellular size and
morphology and form oval spores located centrally in a nonswollen sporangium.
Bacillus cereus is a normal inhabitant of the soil, but it can be regularly isolated from foods such
as grains and spices. B. cereus causes two types of food-borne intoxications (as opposed to
infections). One type is characterized by nausea and vomiting and abdominal cramps and has an
incubation period of 1 to 6 hours. It resembles Staphylococcus aureus food poisoning in its
symptoms and incubation period. This is the "short-incubation" or emetic form of the disease.
The second type is manifested primarily by abdominal cramps and diarrhea with an incubation
period of 8 to 16 hours. Diarrhea may be a small volume or profuse and watery. This type is
referred to as the "long-incubation" or diarrheal form of the disease and it resembles food
poisoning caused by Clostridium perfringens. In either type, the illness usually lasts less than 24
hours after onset.
Cultural characters
Bacillus anthracis grows on common laboratory media under aerobic and also in partial anerobic
atmosphere (they requires low O2 and high CO2). Sporulation occurs in an atmosphere of low
partial pressure of O2.
In nutrient agar: they produce peculiar type of colonies at 35-37oC, incubated for 24hrs. the
colony formed is rough with frosted appearance, measuring 3-5 mm. the colonies are dull,
opaque, greyish white with an irregular border from which long strands of cell are seen in
parallel arrangement resembling a 'Medusa head'. As the colony ages 'vesicles' may appear on
the surface giving it a contoured appearance.
For sporulation, optimum temperature is 25-30 oC. During incubation to minimize the chances of
spore formation, partial pressure of CO2 in the environment is increased. Colony under
magnifying glass seems to be made up of scattered chains. When grown on 50% serum agar, in
and atmosphere of 65% CO2, the colonies are smooth, mucoid, which after long incubation
develop rough edges.
In gelatin stabculture: the growth pattern is inverted fir tree. From the line of inoculum a
number of fine filaments develop laterally. Those nearer the surface are longest and become
progressively shorter the further they are from the surface. (Stab cultures are prepared by taking
solid medium in test tube e.g. gelatin) the liquifaction of gelatin is slow.
In nutrient broth: growth give rise to turbidity with a flocular growth on the surface which
sinks to the bottom in 24 hrs resembling to a piece of wood being suspended in the medium,
more marked in newly isolated strains.
Biochemical properties: B. anthracis forms acid but no gas from glucose, sucrose, maltose,
fructose, trehalose and dextrin but doesnot ferment lactose, galactose, mannitol etc. indole and
H2S is not formed and nitrates are reduced to nitrites. Vages-proskauer reaction is positive and
methylene blue is reduced.
Resistance to physical and chemical agents: the vegetative cells are killed at a temperature of
60oC in 30 minutes but the spores of the organism are highly resistant. Spore remains infective
for years. The spores survive boiling at 100oC for 5 minutes but are killed at 120oC, for 10
minutes.(moist heat). At dry heat they require 1400C for 2-3 hrs. heat fixation of slides maynot
be lethal for spores. 10% formaldehydraded at 40 oC killes the spres in 15 minutes. At lower
temperature more time is allowed for disinfection; a 5% solution of sodium hydroxide is a
satisfactory disinfecting agent.
To disinfectant wool and hairs, 0.25% formaldehyde(HCHO) is used at 60oC for 6hrs.
Several nonselective and selective media for the detection and isolation of Bacillus anthracis
have been described, as well as a rapid screening test for the bacterium based on the morphology
of microcolonies. Table 1 provides the differential characteristics that are used to distinguish
Bacillus anthracis from most strains of Bacillus cereus and Bacillus thuringiensis but not
necessarily from other saprophytic species of Bacillus. Otherwise, it is not the intent of this to
provide information on the growth of the bacterium in the laboratory.
Anthrax
The most common form of the disease in humans is cutaneous anthrax, which is usually
acquired via injured skin or mucous membranes. A minor scratch or abrasion, usually on an
exposed area of the face or neck or arms, is inoculated by spores from the soil or a contaminated
animal or carcass. The spores germinate, vegetative cells multiply, and a characteristic gelatinous
edema develops at the site. This develops into papule within 12-36 hours after infection. The
papule changes rapidly to a vesicle, then a pustule (malignant pustule), and finally into a necrotic
ulcer from which infection may disseminate, giving rise to septicemia. Lymphatic swelling also
occurs within seven days. In severe cases, where the blood stream is eventually invaded, the
disease is frequently fatal.
Another form of the disease, inhalation anthrax (wool sorters' disease), results most commonly
from inhalation of spore-containing dust where animal hair or hides are being handled. The
disease begins abruptly with high fever and chest pain. It progresses rapidly to a systemic
hemorrhagic pathology and is often fatal if treatment cannot stop the invasive aspect of the
infection.
Gastrointestinal anthrax is analogous to cutaneous anthrax but occurs on the intestinal mucosa.
As in cutaneous anthrax, the organisms probably invade the mucosa through a preexisting lesion.
The bacteria spread from the mucosal lesion to the lymphatic system. Intestinal anthrax results
from the ingestion of poorly cooked meat from infected animals. Gastrointestinal anthrax is rare,
but may occur as explosive outbreaks associated with ingestion of infected animals. Intestinal
anthrax has an extremely high mortality rate.
Meningitis due to B. anthracis is a very rare complication that may result from a primary
infection elsewhere.
Bacillus anthracis clearly owes its pathogenicity to two major determinants of virulence: the
formation of a poly-D-glutamyly capsule, which mediates the invasive stage of the infection, and
the production of the multicomponent anthrax toxin which mediates the toxigenic stage.
Poly-D-glutamyl capsule
Anthrax Toxin
One component of the anthrax toxin has a lethal mode of the action that is not understood at this
time. Death is apparently due to oxygen depletion, secondary shock, increased vascular
permeability, respiratory failure and cardiac failure. Death from anthrax in humans or animals
frequently occurs suddenly and unexpectedly. The level of the lethal toxin in the circulation
increases rapidly quite late in the disease, and it closely parallels the concentration of organisms
in the blood.
Factor I is the edema factor (EF) which is necessary for the edema producing activity of the
toxin. EF is known to be an inherent adenylate cyclase, similar to the Bordetella pertussis
adenylate cyclase toxin.
Factor II is the protective antigen (PA), because it induces protective antitoxic antibodies in
guinea pigs. PA is the binding (B) domain of the anthrax toxin which has two active (A)
domains, EF (above) and LF (below).
Factor III is known as the lethal factor (LF) because it is essential for the lethal effects of the
anthrax toxin. Apart from their antigenicity, each of the three factors exhibits no significant
biological activity in an animal. However, combinations of two or three of the toxin components
yield the following results in experimental animals.
Immunity to Anthrax
Vaccines composed of killed bacilli and/or capsular antigens produce no significant immunity. A
nonencapsulated toxigenic strain has been used effectively in livestock. The Sterne Strain of
Bacillus anthracis produces sublethal amounts of the toxin that induces formation of protective
antibody.
The anthrax vaccine for humans, which is used in the U.S., is a preparation of the protective
antigen recovered from the culture filtrate of an avirulent, nonencapsulated strain of Bacillus
anthracis that produces PA during active growth. Anthrax immunization consists of three
subcutaneous injections given two weeks apart followed by three additional subcutaneous
injections given at 6, 12, and 18 months. Annual booster injections of the vaccine are required to
maintain a protective level of immunity.
The vaccine is indicated for individuals who come in contact in the workplace with imported
animal hides, furs, bone, meat, wool, animal hair (especially goat hair) and bristles; and for
individuals engaged in diagnostic or investigational activities which may bring them into contact
with anthrax spores. Otherwise, of course, it has been indicated for the military during the
curerent era of biological warfare.
The vaccine should only be administered to healthy individuals from 18 to 65 years of age, since
investigations to date have been conducted exclusively in that population. It is not known
whether the anthrax vaccine can cause fetal harm, and pregnant women should not be vaccinated.
The inhalation of anthrax spores can lead to infection and disease. The possibility of creating
aerosols containing anthrax spores has made B. anthracis a chosen weapon of bioterrorism.
Several powers may have the ability to load spores of B.anthracis into weapons. Domestic
terrorists may develop means to distribute spores via mass attacks or small-scale attacks at a
local level.
As an agent of biological warfare it is expected that a cloud of anthrax spores would be released
at a strategic location to be inhaled by the individuals under attack. Spores of B. anthracis can be
produced and stored in a dry form and remain viable for decades in storage or after release.
Anthrax spores are killed by boiling (100C or 212F) for 30 minutes (the actual reported time is
considerably less). If boiling as a means of disinfection, the spores must be in liquid suspension
(to ensure killing) and in a sealed container (to avoid aerosolization or vaporization of droplet
nuclei containing spores).
An infection of local animal populations such as sheep and cattle could follow a biological attack
with spores. Infected animals could then transmit the disease to humans through the cutaneous,
intestinal or inhalation route by spores from a contaminated animal, carcass or hide.
A segment of the U.S. military population has been vaccinated against anthrax. Anthrax vaccine
consists of a series of six doses with yearly boosters. The first vaccine of the series must be given
at least four weeks before exposure to the disease. This vaccine protects against anthrax that is
acquired through the skin and it is believed that it would also be effective against inhaled spores
in a biowarfare situation. Of course, a vaccinated military population would be needed to
respond to a terrorist attack with anthrax spores.
Endotoxin producers: These organisms grow and multiply in food stuff. Toxin present on the
cell is released "after cell lysis". The production of toxin takes place in carcass besides dead and
decaying material outside the body. E.g. clostidium botulinum is a notorious food poisoning
organism, condition resulted by it is ' botulism'. The toxin liberated is regarded as one of the
most potent toxin known, nature of toxin is neurotoxin. In botulism, the animal dies without any
symptoms.
Exotoxin producers: e.g. clostridium tetani causes tetanus in man and animals. The disease is
due to exotoxin produced by living bacteria. The organism produces different lethal toxins like
fibrolysin, tetanospasmin etc.
Mild toxin producer: most of the disease are due to these organisms e.g. clostridium
perfrigens(syn. Clostrdium welchii). Type A causes gas gangrene and food poisoining, type B
causes dysentery in lambs, type C cause struck and Type D causes enterotoxemia.
Clostridium haemolyticum causes bacillary hemoglobinuria in cattle.
Cl. chauvoei causes black quarter.
Cl. septicum cause Braxy.
Cl. novyi causes Black disease/bighead disease of Rams/Malignant oedema.
In most of the case 'gas' is formed at the site of infection. This is due to production of acid and
gas by the saccharolytic member from sugars. Other utilize proteins and are called proteolytic
e.g. cl.haemolyticum, cl. tetani. Cl botulinum etc. proteolytic are more dangerous.
These two can be differentiated as follows:
In cooked broth medium, saccharolytic strains form pink color, whereas black color is formed by
the proteolytics.
Clostridium tetani
Cl.tetani causes tetanus in man and animals. The spores of the organism are widely distributed in
nature. The spores are found in soil, dust, faeces of man and animals particularly horses.
Morphology and staining: the organism is a long slender rod measuring 1-4 µm in length and
0.5 µm in width. The ends of the organism are rounded. The spores are situated terminally and
are 2-3 times the diameter of the cell giving a drumstick appearance. The organism takes gram
+ve stain but may become gram -ve in culture which are more than 48 hrs old. The organism is
non capsulated.
Cultural characteristics: strictly anaerobic, can grow between 35-37oC (optm). The organism
grows well in liquid media in which meat particles have been added. The colonies on agar are
irregularly and often spreading. In gelatin stabs, typical 'fir tree' growth occur and gelatin is
slowly liquefied.
Biochemical properties: the carbohydrates are not fermented. Litmus milk is usually
unchanged. Nitrate reduction test is negative.
Resistance: the spores are highly resistant. Boiling kills the spore in 15 minutes, but spore of
some strains are resistant and survive 1-3 hrs boiling. At 120 oC, spores are killed in 20 minutes.
Spore is also resistant to chemical treatment. Some strains resist action of 5 % phenol for longer
than 10 days and are also resistant to 1:1000 dilution of per chloride of mercury.
Antigens and toxins: on the basis of heat-labile flagellar antigens, the organism has been
divided into ten types. The neurotoxins produced by all types are antigenecially similar. Some
strains are toxigenic while others are non-toxingenic. The two exotoxin produce are
tetanospasmin and tetanolysin. Tetanospasmin is most important neurotoxin. It is heat labile
antigenic protein which is readily neutralized by antitoxin and destroyed by intestinal proteases.
Treatment with formalin converts it into non-toxic product known as toxoid. Toxoid retains its
antigenicity and stimulates the production of antitoxin. The horse is very susceptible to
tetanospasmin while birds are relatively resistant. Tetanolysin is the other toxin and causes lysis
of RBC. It is produced during active growth of organism and then becomes inactive. It is heat
labile and oxygen labile.
Public health aspect: human is susceptible to contamination of wounds by spores (in dust, soil
etc). Tetanus is major health hazard. Sometimes spores are present at the surface of the skin.
Alpha toxin (Lecithinase) splits lipoprotein complexes in serum or egg yolk preparation in the
presence of free Ca++ and Mg++ ions. Lecithine is split into phosphoryl choline and a
diglyceride(Lipid). The lipid diposites around the bacterial colonies resulting in opalescence. The
effect of lecithinase is especially neutralized by antitoxin.
C. perfringens are inoculated on egg yolk agar medium in a plate of 10-12cm diameter. To one
half of the plate, antitoxin is layered on the surface. The inoculated media is in cubated at 37OC
for 24 hrs. the growth in one half of the medium without antitoxin shows opalescence(holes)
surrounding the colonies while colonies on the other half with antitoxin (anti-gas gangrene
serum) shows no opalescence. When neomycin is incorporated in the culture medium, it
becomes more selective due to inhibition of coliform and aerobic spore bearers. The alpha toxin
hydrolyses phospholipid in serum and yolk producing an opaque precipitate the contain 55 egg
yolk. Egg yolk in the medium may be substituted by 20% human serum.
Morphology and staining: since mycoplasma lack cell wall, they have tendency towards
pleomorphism. They occur as cocci, club, filament or 'tear drop form' Mycoplasmas cannot be
studied by the usual bacteriological method because of the small size of their colonies. A variety
of bizarre shapes develop during their life cycles such as granules or cocci which are often called
elementery bodies, clubs, filaments, rings and stellate forms. Mycoplasma donot have cell wall
but are bounded by a single triple layered membrane which contain sterol. Mycoplasma contain
both RNA and DNA (in the ratio of DNA: RNA=2:1). They do not have nuclear membrane and
their cytoplasma contains ribosomes.
Muycoplasma stain poorly with aniline dyes but are gram -ve. They are usually stained with
'Giemsa' after fixation with methyl alcohol. Negative staining (with cotton blue, india ink) gives
satisfactory results.
1. Formation of elementary body-to the end of filaments they produce small elementary
bodies, released by breaking the filaments e.g. in M. mycoplasmatales.
2. Tear drop formation e.g. in M. gallisepticum
3. Binary fission. E.g. M. agalactiae
Usually 1 elementary body gives rise to 4.
Cultural characteristics: Mycoplasma is fastidious organisms (i.e. they have special nutritional
requirement) and require cholesterol of related sterol for growth and membrane synthesis. Most
of the species grow aerobically but some requires an atmosphere of 10 % CO2. One of the media
supporting mycoplama will contain per ml (heart infusion peptone broth)
20 % horse serum (unheated)
10 % yeast extract (not heated beyond 75oC)
20µg of DNA
50 units of penicillin and
0.25 mg thallous acetate
The medium should have a pH of 7.8 (adjusted by using K2HPO4)after incubating in the above
medium at 38oC, for 3-5 days, visible yellowish colonies translucent periphery, size 0.5-1.0 mm
can be seen. This growth when observed with a magnifying lens gives a poached egg
appearance.
Resistance to physical and chemical agents: Mycoplasma are easily destroyed by disinfectants
but are relatively susceptible to moist heat and are killed by temperature of 55oC in 15 min, 60oC
in 5 minutes.(resistant to penicillin, sensitive to tetracycline or erythromycin)
Both mycoplasma and cell wall deficient L- forms of bacteria show the extreme pleomorphism.
These are wall defective microbial forms that can replicate serially as nonrigid cell and form
colonies on solid media. Some L- forms are stable while other are unsstatble and revert to
bacterial parental forms. These are not genetically related to mycoplasma and result from
spontaneous mutation or from the effect of chemicals. Treatment of eubacteria with cell wall
inhibiting drugs or lysosomes, produce L- forms. Protoplasts are L- form usually derived from
gram positive organisms. Spheroplasts are L- form derived from gram negative bacteria.
Corynebacterium pyogenes
C. pyogenes is world wide in distribution. The organism occurs in healty cattle pigs, sheep and
goats and it is in these species that the organism causes a variety of suppurative conditions.
Morphology and staining : non motile, gram +ve rods, non spore former, non capsulated, rod
club shaped or pleomorphic, tendency to form clumps, pallisade arragement.
Young cultures, the organism is gram +ve, but old cultures are usually decolorized.
Cultural characterstics: it is aerobic and facultative anaerobe. The optimum temperature for
growth is 37 oC.in blood aga, they form round greyish colonies of about 1mm size with a zone of
haemolysis. On serum agar, the colonies are like dewdrops, resembling the colonies of
streptococci. In coagulated serum, small colones are formed, whch sink in few days in the
medium as the result of liquifaction of the medium. Loquifaction also occurs on gelatin or
inspissated egg medium. In serum broth the organisms form a light powdery sediment along with
the wall of the tube as well as in the bottom.
Biochemical properties: acid but no gas is produced in glucose, maltose and lactose. Indole is
not formed, nitrates are not reduced. Litmus milk is acidified and coagulated in 3 days and there
after the clot is digested and within a week the medium becomes clear.
Antigen and toxins: when grown in milk or cooked meat, produce a haemolytic toxin. Usually
the pus is taken for diagnosis and tested in a medium containing blood and serum.
Corynebacterium renale
The organism causes polynephritis and cystitis in cattle. The organism has also been isolated
from vagianal mucous membrane of healthy cows as well as from genital tract of bulls.
Morphology and staining: C.renale is pleomorphic, non motile and non-capsular former. The
organism occurs singly but clumps and palisade formations are common. Metachromatic
granules are formed when the organism is grown on loeffer's medium. The organism is Gram
+ve and metachromatic granules are seen when stained by 'albert's or 'neissers' stain.
Cultural characteristics: C. renale is aerobic and facultative anerobe. The optimum temperature
for growth is 37 oC. in blood agar, they produce small moist grayish white colonies without
haemolysis. Gelatin and serum agar can not liquified. When grown in litmus milk, they produce
alkali. They can liquify casein when grown in a medium containing 10 % milk.
Biochemical properties: glucose is fermented with the production of acid. Catalase +ve, indole
test -ve, H2S test -ve, nitrate reduction test -ve, urease test +ve.
Erysepelothrix insidiosa
The organism causes swine erysepelas and infection in other animal in many part of the world.
Morphology and staining : the organism is variable in form. In smears from infected tissue, it
appears as short slender rods, straight or bent, occuring singly, in groups or in chains. In chronic
lesion, the orgainsm develops a mass of long filaments. On culture media, a mixture of short rods
and elongeted thick ended filaments are seen.
It is non-motile and non spore forming. The organism stains well with ordinary dyes and is gram
positive.
Biochemical properties: most of the strains ferment lactose, fructose and galactose with
production of acid. It does not form indole, does not reduce nitrates.
Resistance: the organism is resistant and survives for about a year in putrefying meat and
smoked ham. The organism is killed at 70 oC in 5-10 mins. It is comparatively resistant to
alcohol, hydrogen peroxide, formladehyde, phenol and brine and is susceptible to caustic soda
and bichloride of murcury.
Antigen and Toxin: antigenic structure is complex. Both heat labile and heat stable antigens
have been identified. On the basis of somatic antigens, several serotypes can be distinguished.
There is no antigenic distinction between serotypes isolated from different host species.
Thses organisms are nonmotile, non capsulated, nonspore forming and mostly very slow
growing. The genus includes obligate parasites pathogenic to man, mammals, birds and reptiles,
opportunistic pathogens and saphrophytic varities.
The disease produced include tuberculosis in man and animals cause by human, bovine, avian,
murine and cold blooded types of Myco. Tuberculosis; jone's disease in cattle and sheep due to
myco. Paratuberculosis, leprosy in man due to Myco. Leprae.
A group of miscelleneous bacteria, distinct from human or bovine tubercle bacilli are grouped
together under the loose term 'atypical' mycopbacteria. These are also callled 'anonymous' or
unclassified mycobacteria. Most of these bacteria occur in the environment. E.g. M. phlei(in
grass) and M. smegmatis( in smegma). They are mostly oppurtunistic and are not transmitted
from person to person.
Disease caused by mycobacteria develops slowely, follows a chronic course and elicicts a
granulomatous response. The mycobacteria donot produce classic exotoxins or endotoxins and
the disease process are the result of delayed type hypersensitivity reaction to mycobacterial
proteins.
To obtain their visible colones within 1week is required. Granulomatous lesion is associated with
necrosed tissue. Sometimes inside the granulomatous lesion calcium is deposited called 'calcified
granulomatous lesion' and if caseous material is present it is called ' caseous granulomatous
lesion'.
The bovine type: M bovis: causes disease in cattle, pigs, cats, dogs, horses and man
The avian type: M. avium: causes disease in birds and sometimes mammals.
M. marinum: has been isolated from fish, swiming pools and skin lesions in man.
M. piscium: pathogenic for frogs, toads, turtles, lizards, snakes, carp etc.
M. murium: only rodents are affected
Type Disease of susceptibility(in decreasing order)
Human type: Human-being(primary host), guinea pigs, cattle, buffalo, sheep, goat, horse,
dog, cat and birds
Bovine type: Bovine, pig, dog, cat, horse, rabbit, man, sheep, goat.
Avian type: Birds, other mammals except man, man (infection with this type is found less)
The organism is not easily stained by aniline dyes. The organisms are stained pink with hot
carbol fuchsin and then they resist decolorization with 3% HCl in 95 % ethanol. This is due to
waxy substance known as mycolic acid.
Cultural characteristic: human type, bovine type and avian type, all requre aerobic conditions
for growth. For human and bovine type:
They are aerobic which grows well at 37-38oC.
The organism donot grow in ordinary lab media. The growth takes place in media
containing serum, potato extract or egg e.g. 'Dorset egg medium'. In this medium, the
generation time is 8 hrs and time required for visible colony formation 10-14 days.
Glycerine is commonly added in many media used to cultivate the organisms. Glycerine
stimulates the growth of human and avian type but retards the growth of bovine type.
Complex media are used for primary culture e.g. ' lowenstein-jense' medium comprising
60 % homogenized egg in nutrient base containing low concentration of malachite
green as inhibitor of non-mycobacterial contaminant.(Lowwnstein-jensen medium=
serum+potato extract+egg+glycerine)
The growth of bovine type is improved if glycerol is replaced by pyruvic acid. It is called
'stonebrink's medium", which contains Na- pyruvate, malachite green and inspissated
serum besides serum potato and egg.
Mycobacterium cells are found to be closely adherent to each other giving a tenacious
and friable bacterial mass. So it is difficult to prepare a homogenous suspension. '
tween-80 (polyoxyethylene sorbitan mono-oleate, water soluble ester of fatty acid) is a
detergent that can be used to reduce surface tension and so helps to preparation of
homogenous suspension.
For avian type:
The optimum temperature for growth is 40oC , but it can grow at 42oC-43 o
Doesnot growth in ordinary lab media.
Growth takes place on Dorset egg medium containing glycerine.
The growth rate is more rapid than mamalian types and growth occurs i.e. 4-5 days.
Antigens and Toxins: Myco tuberculosis has a highly complex antigenic structure. The
serological tests show that close relationship exists between various types. The mammalian type
and additional antigen specific for avian type. Mycobacteria donot produce classic exotoxins or
endotoxins.
People at risk : Franers (Aerosol infection) Lab. Workers and technicians examining the
diseaseed animal.
So, control of tuberculosis in animals is a part of controlling T.B. in man.
Mycobacterium Paratuberculosis
Mycobacterium paratuberculosis was originally known as Mycobacterium johnie after johne.
John's disease causes chronic contagious enteritis in cattle, sheep, goats, camels and buffaloes.
Morophology and staining: Myco. Paratuberculosis is a shot rod measuring 0.5 um in width
and 1-2 um in length. The organism is stained with acid-fast stains and is acid alcohol fast. The
organism is considered to be gram positive, non motile and non spore former.
Cultural characteristics: The organism grows with difficulty on artificial media. They are
obligate parasite. It grows slowly on media containing killed acid-alcohol-fast organisms or
extracts prepared from them. Egg yolk agar containing glycerine, extract of myco. phlei is
recommended for isolation of myco. Paratuberculosis. The primary cultures require 4-8 wks of
incubation for the growth of the organism. Minute, grayish white, friable, irregular colonies
appear and become visible to naked eye. As the culture ages, the colonies increases in size and
become pale yellow in colr. The subcultres produce improved growth. The optimum temperature
for growth is 38-39 oC.
Resistance: the resistance to heat and chemical disinfectants of the organism is simirla to myco.
tuberculosis
Biochemical properties: donot produce changes in the various substances that are used in the
study of bacteria. Similar to other acid fast bacteria.
Antigens and Toxins: little is known concerning the antigenic structure of this organism. Toxins
are not produced by this organism
Pathogenecity: myco. Paratuberculosis produce chronic bacillary dysentery in cattle and to some
extent in sheep, goats and other ruminant.