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Microbiology III 2015

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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)

Cellular product of medical interest/Virulence factor


 Adhesins :
 Proteins that mediate adherence to target cells (glycoprotein) on the surface of
epithelial cells of intestinal tract.
 It is important virulence factors when the microbe is on mucosal surfaces.
 F4, F5, F6 (Plasmid encoded, F41 (chromosomal)- enterotoxigenic
 F17- invasive
 AAF (aggregative adherence fimbriae)
 Bfp (bundle forming pilus)
 Capsules
 It protects the outer membrane from the membrane attack complex of the complement
cascade, and inhibits microbes from attachment to and ingestion by, phagocytic host
cells.
 Cell wall
 LPS in the outer membrane is an important virulence determinant.
 Enterotoxins : three types of plasmid encoded proteins
 Heat labile enterotoxin (LT): is a protein resembles the cholera toxin (CT) but CT is
100 times potency than LT
 Heat stable enterotoxin (ST): low molecular weight polypeptide and poorly
immunogenic.
 EAST 1 ( Enteroaggregative E. coli heat stable entterotoxin 1)

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Variability/Antigenic typing of E coli


 Antigenic makeup of the O-repeat units (types of sugar subunits, how the sub-units are
hooked together, and the length of the chain), the composition of the flagellar protein
(flagellin), and the composition of the capsule.
 There are at least 80 distinct k- antigens, approximately 165 serologically distinct O-groups,
and at least 50 serologically different flagellar (H) antigens. The O-, H-, and K-antigens are
used in serotyping a particular isolate. For example, O141:K85:H3 describes an isolate with
antigens of the 141 serogroup, capsular antigen number 85, and flagellar antigen number 3.

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:

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Inagglutinability by preventing somatic agglutination of living strain by homologous


antisera
 Act as virulence factor by impending phagocytosis and protect from killing action of
antibody and complements
c. Flagellar antigen (H antigen)
 Thermolabile
 Organism has to grow in semisolid agar for H antigen determination
d. Fimbrial antigen (F antigen)
 Cause mannose sensitive hemagglutination
 Sex pili acts as virulence factor. Type 1 mediates adhesion

Reservoirs and transmission


 Strains of E. coli capable of producing disease reside in the lower GIT and are
abundant in environments inhabited by animals
 Transmission is through the fecal-oral route

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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).

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Enteroinvasive E. coli (EIEC)


 Usually a neonate that has received inadequate amounts of colostrums or
colostrums of inadequate quality
 Association of susceptible animals with EIEC occurs by way of the
conjunctive, inadequately treated umbilicus, or ingestion.
 EIEC adheres to target cell in the distal bowel. Following adherence, EIEC
induce their own uptake by expression of either CNF1/2, resulting formation
of ruffles that entrap adhering bacteria and pull them into the intestinal
epithelial cell. Entry into the lymphatic and subsequently the blood stream
follows.
 Edema disease
 It is an acute, often fatal “enterotoxemia” of weaned pigs
 Characterized by subcutaneous and subserosal edema, caused by SLTe.
 Toxigenic strains inhabit the large bowel of normal pigs and these strains are
thought to increase in numbers during nutritional, social, or physical stress.
generalized edema of various organ and tissue (e.g. head, neck, colon,
stomach, intestine and brain)
 Colibacillosis of fowl

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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

Fig. E.coli identification scheme

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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)

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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)

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Factors involving resistant to phagocytosis


o Superoxide dismultase
o Defensins
 Factors involving resistant to acid PH (acid tolerance response (ATR) gene)

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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

Disease by Y. enterocolitica and Y. pseudotuberculosis

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Y. enterocolitica and Y. pseudotuberculosis: ubiquitous in environment, infect humans, wild


and domestic animals, acquired by ingestion of contaminated food or water
 Y. enterocolitica = most important as cause of self-limiting gastroenteritis or enterocolitis
 Particularly common among children, outbreaks in day-care centers/schools
 Symptoms: mild (diarrhea, abdominal pain) to severe (fever, severe abdominal pain,
mistaken for appendicitis) = originally identified as human pathogen after unusual
number of appendectomies performed on children, concurrent diarrhea in same age
group
 Relatively small number of strains cause disease

Natural history

 Characterized by sudden, explosive outbreaks in rodents, periods of quiescence


 Rats carry fleas naturally, pick up organism from infected person, animal
 Organism multiplies in fleas, blocks proventriculus
 Rats die of high-grade bacteremia, fleas leave for new host, may be man = accidental host in
the absence of rats
 Blocked proventriculus makes flea hungry, mad; bites more vigorously; regurgitates blood,
organisms into new host, injects the bacteria into the bloodstream.
 Incubation period 1-6 days, usually no lesion at portal of entry, organism = transported to
regional LN via lymphatics
 Ingested by fixed macrophages in LN, survive and grow in normal unactivated macrophages,
proliferate in LN
 Inflammatory response to proliferation ==> characteristic swelling, or bubo (bubonic
plague), may stop at this point; high % patients die (toxin), dissemination, usually in 3-5 days
 Organism escapes LN --> spreads via circulation (septicemic plague), becomes systemic,
lysis of bacteria releases LPS, causes septic shock.
 Often settles in lungs, parasitizes lung macrophages ==> pneumonia ==> secondary
pneumonic cases without fleas; death more rapid than with bubonic.

Morphology and staining

 Gram negative cocobacilli


 Bipolarity is common in tissue smears
 Most species are flagellated at ambient temperatures.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Yersinia pestis (Plague bacillus)


 Yersinia pestis is the cause of the rodent- based zoonotic disease called plague. In human
patient, and susceptible domestic animal species (mainly cats), plague is manifest as a local
lymphadenitis (bubonic plague), pneumonia (pneumonic plague), or septicaemia (septicemic
plague).
 Analysis of DNA sequence indicateds that Y. pestis is a subspecies of Yersinia
pseudotuberculosis.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Gsr (global stress requirement) is expressed at 37oC while Y. pestis is within the macrophage
phagolysoome.

Culture

 Aerobic and faculatative anaerobic


 Grow14-37oC over wide range of pH (5-9.6)
 Optimum temperature 27oC
 Grow on ordinary media, but slowely.
 Grow more rapidely on media containing tissue fluids or blood.
 Nutrient broth
 Flocculated growth at botton and along the side of tube with little or no turbidity
 When the organism grown on flask of broth with a little melted butter or oil on the top (ghee
broth), growth occurs in the form of „stalactites‟ (stalactactic growth) hanging down from the
under surface of the oil, which sediment after slight shaking.
 Solid media
 Very small, delicate (0.1-0.2 mm), translucent in blood agar and nutrient agar
 Non hemolytic
 MacConkey‟s agar very small colorless colonies.

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

 Tolerant rodents in endemic areas contributes the plague reservoir.


 They rarely develop fatal disese and are called maintenance or enzootic host
 Transmission
 By fleas. They carry Y. pestis to more susceptible, epizootic, or amplification hosts, such as
ground squirrels or rats. When these die, still other hosts, such as human, are attacked.
 Infected mammals may spread plague by the airborne route. Oral acquisition is by predation,
cannibalism, and scavenging.

Pathogenesis

 Fleas feed on an infected host.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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. CFR: bubonic 50-75%, pneumonic 100% without therapy


b. Infections responsive to streptomycin, chloramphenicol, tetracyclines, must be
administered early, eg, pneumonic form rarely controlled after 12-15 h fever
 5. Prevention

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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

 Yersinia pseudotuberculosis has similar in all aspects except


 Adhesin

 Ail (attachment invasion locus)


 Inv (for invasion)
 Yad (yersinia adhesion)
 Cell wall has O-antigens (smooth phenotype). And typical gram negative.
 HPI, iron acqusion, Gsr, toxinx(Yops, and LcrV) are same
 Approximately 21 serotype based on variability of the somatic (O) antigens
 Y. tuberculosis is a parasite of wild rodents, lagomorphsand birds but it infects other
mammals and reptiles and persists in the environment.
 Cat is the most commonlty infected domestic mammals minor eidemics occur among sheep,
pigs, nonhuman primates, fowl, and pet birds.
 Transmission- exposure is primarily through ingestion.
 Pathogenesis same
 Pseudotuberculosis occurs worldwide. Cases tend to cluster in the cold months.
 Natural infection leaves surviving individuals immune. Avirulent live vaccine protects
against homologous challenges but not available commercially
 Laboratory diagnosis- isolation of the agent antemortem from feces or lymph node aspirate.
Isolation from mixed culture is enhanced by cold enrichment, i.e. incubation of a 10%
mixture of inoculums in minimal medium for several weeks at 4oC.
 PCR

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.

Reservoirs and transmission

 Water, food, soil, fruits, vegetables and symptomatic aindividual from humans to mollusks
have been proposed.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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

 Y. enterocolitica is an extracellular microorganism.


 Phagocytic cells readily destroy it, even though the microorganism excretes proteins (Yops)
that interfares with this process.
 Most often Y. enterocolitica disease is self limiting due to the innate immune response
phagocytosis, lysis of infected epithelial cells, iron sequestration, and complement prrotens

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

The genus Shigella


Named after 'Shiga"
 Causes bacillary dysentery in primates. Almost exclusively in captive primates and appears
to be related to stressful situations( e.g. transportation, crowding)
 Gram negative, facultative, non-motile, no flagella, No H (flagellar) antigens, some
encapsulated
 Shigella sp. and E. coli are so closely related genetically = same species.
 Distribution quite narrow, natural disease rare in species other than man
 Member of enterobacteriacae i.e. normal G.I. inhabitant

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Similar virulence factors and cause infection in the same way


most extensively studied species is S. flexneri; Disease = called dysentery = (large intestine,
blood, mucus).
 Causes bacillary dysentery (Shigellosis) in human. In animals no infection has been recorded.
Some genes involved in invasion have been identified

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

Cultural characteristic: similar to salmonella


 NA: 2 mm diameter, circular, convex, smooth & translucent
 MAC: to isolate from feces: colourless
 DCA & S.S. Agar: colourless colony

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)

Antigens and Toxins:


 On the basis of somatic antigen, more than 10 serotypes have been isolated.
 Two enterotoxins have been described ShET1 (shigella enterotoxin) sen gene encoding
invasion protein.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Culture
 Biochemical test
 Slide agglutination test
 Colicin typing
 Seveni test: to confirm the invasiveness

Treatment TMP+sulphonamides, fluroquinolones.

Genus Proteus and related genera


 Proteus tribe has 3 genera: Proteus, Morganella, Providencia. All are closely related.
 Occurs naturally in the environment of animals and man particularly in the intestines, animal
manures, human sewage, soil and water. Abortion, otitis, pericarditis, dysentery and nervous
disorders in dogs.
 The most common species are Proteus vulgaris, P. morganii, P. mirabilis, P.rettgeri
 Production of phenylalanine deaminase is common ro all which helps to differentiate from
other Enterobacteriaceae
 Produce powerful Urease except some species of Provendencia
Classification:
a. Proteus: P. mirabilis, P. vulgaris
b. Morganella: M. morganii
c. Providencia: P. alcalifaciens, P. stuartii, P. rettgerii : Urease –ve
Widely distributed in nature such as rotten meat, sewage, soil & frequently in feces of man &
animals.
Morphology
 Rod shaped organism(measuring 1-3 µm in length and 0.5 µm in width Sometime short
(3µm) sometime long 10 – 30 µm
 The organism is pleomorphic and coccal form or filamentous form 10-20 µm in length.
 They are motile with peritrichous flagella, non-sporulating and capsule is not formed.
 Majority of strains produce fimbriae
 The organism is gram negative

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%

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

o Incorporating qchemicals in medium (Chloral hydrate 1 in 500, Sodium azide 1 in


5000, Boric acid 1 in 100 & sulfonamide)
 Form pale color or colorless colonies in MAC & donot swarm in MAC.

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

Resistance and toxin similar to salmonella


Antigenic structure:
 Posses OHK
 P vulgaris called X strains (OX2, OX19, OXK) that are agglutinated by cross reactive
antibodies directed against Rickettsia antigens

Pathogencity and epidemiology


Severe diarrhea and dysentery
More frequently in young animals in cattle, sheep, goat, dogs and mink. Otitis – dog

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

The genus Klebsiella

The organism is commonly found as saprophytes in intestinal tract, respiratory tract,


water and soil. The most distinctive feature is absence of motility and presence of
polysaccharides capsule.
Pathogenic species
Klebsiella aerogenes/Aerobactor aerogenes
Klebsiella pneumonia
 They are associated with pneumonia in fowl, metritis in equines, mastitis in pigs and air sac
infection in poultry.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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

Property K aerogens K oezaenae K rhinoscleromatis K pneumoni K oxytoca


Gas in glucose + +/- - + +
Acid in lactose + - - + +
M.R. - + + + v
V.P. + - - - v
Citrate + +/- - + +
Urease + +/- - + +
Malonate + - + + +
Lysine + +/- - + +
Indole - - - +

Morphology and staining


 The organism is non motile, encapsulated thick rod measuring 0.5-1.0 µm in width and 1-3 µm
in length.
 The organism stains gram negative and the capsule surrounding the organism is easily
demonstrated.
 The India ink (i.e. negative staining) method is suitable because the extra cellular slime layer
remains undistorted.
 Usually occur as single and sometime in pair and are non-spore former.
 Majority of the strains develop fimbriae

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Do not liquefy gelatin and methyl red +ve except Klebsiella aerogenes and do not form indole.

Resistance to physical and chemical agents


 The organism is killed by common disinfectants and boiling for 20 min at 60 0C. It can survive
in faecal material for month.

Antigens and Toxin


 The antigenic structure is complex consisting of somatic (O), rough(R) and capsular mucoid
(K) antigen.
 The organism does not produce exotoxin.
Virulence Factor
 Capsule: antiphagocytic & acts as major virulence factor
 Plasmid exchange: participate in plasmid exchange with other Enterobacteriaceae
 Antibiotic resistance
 Toxin: similar to heat labile & heat stable exotoxin of E coli

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Cellular product of mediacal interest


 Cell walls are typical of gram-negative microorganisms consisting mainly of LPS and
proteins. Cell wall elicite an inflammatory response.
 A type 4 fimbria (adhesin) for avian strains of P. multocida. Adhesion (adh1) is expressed
M. haemolytica.
 Capsules contain acidic polysaccharides.antiphagocytosis (antiphagocytic)the amount of
capsule produced is inversely proportional to the amount of available iron.
 Pasteurella multocida type A capsules are made of hyaluronic acid, type D capsule are
made of heparin, and type F capsules are made of Chondroitin.
 Pasteurella produce a number of proteins with toxic activity. At least two of these are
important in the pathogenesis of disease
o RTX toxin(repeat in toxin) is leucotoxin (Lkt) produced by M. haemolytica, P.
trehalosi and P. glucosidal.
o Rho activating toxin(produced by P. multocida capsule type D associated with
atropic rhinitis of swine). Pmt (P. multocida toxin) increase in intracellular
calcium.
o Miscellaneous toxin (hyaluronidase and neuraminidase). Hyalurinidase is
responsible for the microorganism‟s ability to spread through tissue.
Neuraminidase play colonization of epithelial surface by removing terminal sialic
acid residues from mucin, thereby modifying normal host innate immunity.
 Iron aquisition: iron is an absolute growth requirement. Some avian strains of P.multocida
produce a siderophore, multicidin.
The organisms belonging to this genus are usually coccoid or coccobacilli (0.2µm by
upto 2µm) with bipolar appearance on stained smears with polychrome stain (Wright stain).
They are generally capsulated but non-capsulated forms have been reported. They are non-
motile, aerobes or facultative anaerobes and grow readily on ordinary bacteriological media at
37oC. They are oxidase and catalase +ve.
The common species of this genus Pasteurella are :-
P. haemolytica, P. multocida, P. anatipestifer(in ducks), P. pneumotropica(in mice

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,

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Mannheimia haemolytica, P. Trehalosi, and P. Glucosida produce hemolysis on ruminant blood


agar. All are gram negative, non-motile coccobacilli( 2 x 0.2m. non spore forming, facultative
anaerobic. They are facultative anaerobes, typically oxidase-positive, catalase +ve (except P.
caballi), reduce nitrates, and attack carbohydrates fermentatively.

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)

Reservoir and transmission


Carried on mucous membrane (most commonly in the oropharyngeal region ) of susceptible host
species (adherence to these surface is due to adhesions)
Infection is by inhalation, ingestion or bites and scratch wounds.
Many infections are endogenous.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Species Principal host Disease and


commensal status
P. multocida
Type A Cattle Shipping fever complex, enzootic
pneumonia complex in calves,
mastitis
Sheep Pneumonia, mastitis
Pigs pneumonia
rabbits Snuffles, pnwumonia,
abscesses,otitis media,
conjunctivitis, genital infection
poultry Fowl cholera
Many other domestic and wild animals Commensals in respiratory
tract and GIT
Type B Cattle, buffalom bison, yak, Haemorrhagic septicemia
other ruminants
Type D Pigs Atrophic rhinitis
Pigs and less commonly other domestic Secondary pneumonia
animal and poultry
Type E Cattle, buffalo HS ( South Africa)
Type F turkeys Not clearly known
P. haemolytica
Biotype A Cattle Shipping fever complex and
pneumonia
Sheep Enzootic pneumonia, septicemia
in lambs(3 months), gangrenous
mastitis (blue bag)
Biotype T Sheep Septicemia in lambs( 5-12 months)
P. pneumotropica rhodents Pneumonia and abbesses
Commensal in nasopharynx
Dogs, cats Commensal in nasopharynx

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

P. canis Dogs, man Isolated from res. tract


P. dogmatis Dogs, cats (man) Isolated from res. tract
P. stomatis Dogs, cats Isolated from res. tract
P. caballi Horse Respiratory infection,
pneumonia
P. aerogenes Pigs, man
P. antipestifer Ducklings, chicken, turkeys, pheasant, New duck disease ( polyserositis)
water fowl
P. anatis Ducks
P. gallinarum poultry Low grade infection ( commensal
in respiratory mucosa)
P. avium chicken isolation
P. langaa chicken isolation
P. volantum chicken isolation
P. testudinis Tortes, tortoises abscess
P. granulomatis Cattle (Brazil) Fibrogranulomatous disease

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.

Morphology and staining


 P. multocida are gram negative, non-motile and non-spore forming rods.
 Freshly, isolated strains from the carcasses of animals appear as short ovoid rods
measuring 1µm in length 0.5-0.8µm in width. After repeated culture, the organism tends
to form longer rods and becomes more pleomorphic.
 The newly isolated strains from diseased animals exhibits bipolar staining, when they are
stained with methylene blue or Leishman's stain.
 The organism usually posses a capsule when recently isolated from diseased tissues. The
capsule is composed of complex polysaccharide and Hyaluronic acid.
 The capsule is lost upon subsequent culture.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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)

Serotype Disease cause


7:A Sepsis in cattle

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

6:B & 6:E* H.S. in cattle


3:D Pneumonia in mice
1:A&1:D Pneumonia in pig and sheep respectively.
5:A&8:A Fowl cholera
*so, vaccine against H.S. should contain 6:B & 6:E Antigen

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.

Cultural characteristics: generally, they grow well in ordinary lab. media.


In nutrient agar: they form moist and translucent colonies, resembling the smooth colonies of
Pasturella multocida. Growth in broth results even turbidity.
They produce partial haemolysis. So they are recognized as weakly haemolytic. They
produce haemolysis after 48 hours of incubation in ox, sheep blood.
In lamb-blood agar: a wider zone of partial hemolysis is produced by some strains of Past.
haemolytica.
In bovine-blood agar: when Past. haemolytic is grown together with staphylococcus in bovine
blood agar, haemolytic property of the latter is increased due to secrection of extracellular
substance by the former.
In MacConke's agar: they can be grown in Mac Conkey's bile salt medium, but they don't grow
in Deoxycholate citrate agar(DCA)

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.

Resistance to physical and chemical agents: same as that of Pasteurella multocida.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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 2g\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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 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

Treatment and control


Most Pasteurella show moderate resistant to aminoglycosides, which is probably not significant
clinically. Sulphonamides, penicilline G, tetracycline are preferred.

Riemerella antipestifer/ Pasteurella anatipestifer


The organism causes a disease in ducklings known as 'long island duck disease' or 'new duck
disease'. The disease affects young duckling which produces a severe polyserositic disease shows
symptoms of depression, nasals discharge, incordination of limbs and greenish diarrhoea.
They can grow in ordinary lab media, growth is enhanced by addition of duck serum and
increased CO2 content. The organism doesnot ferment carbohydrates, and doesnot produce
indole or H2S, it is catalse +ve and liquefies gelatin.

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)

The genus Actinobacillus (Actin-rays)


The name Actinobacillus is given since the tissue in which this organism affects is formed club
shaped or ray like lesion. As in tha case with the genus Pasteurella, the genu Actinobacilllus is
undergoing a considerable amount of taxomonic changes it contains 20 species.
Major species are
A.Lignieresi - pathogenic to cattle, causes wooden tongue.(lignieres- a scientist)
A. equuli subspecies equuli (previously A. Equuli) -neonatal septicemia of foals.
A. equuli subspecies haemolytica (previously A. suis-like and occurs mainly in
respiratory tract disease of horses)
A. suis - associated with respiratory, septicemic, and localized infections of
swine). Severe in newborns, but affects pigs of all ages.
A. pleuropneumonia (the agent of porcine pleuropneumonia).
A. salpingitis - salpingitis in fowls.
The organisms are associated with granulomatous lesion in the subcutaneous tissue.
These species are distributed in nature as commensal in the oral cavity to fore stomach of
ruminates. Normal and healthy cattle usually exhibit antibodies against the organism. They affect
the soft tissues like tongue.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Antigen and toxins: A.lignieresi contain:


1.Heat stable somatic antigens
2.Heat labile surface antigens.
On the basis of heat-stable somatic antigens, A. lignieresi is divided into six types (1-6).
Most cattle strains belong to type 1 and majority of sheep strains to type 2, 3 and 4, type 5 and 6
in both.

Resistance to physical and chemical agents:


Destroyed at 60oC in 15 minutes.
Relatively susceptible to common chemical disinfectants, UV light and desiccation
Actinobacilus pleuropneumonias contain R plasmids encoding resistance to sulphonnamides,
tetracycline, and penicilline G.
Variability
 A. Lignieresii (six serotype) and A. equuli are antigenically divesrse. There are 15 somatic
serotype of A. pleuropneumonia (1-15) and two biotype (biotype 1 requires NAD for
growth, biotype 2 doesnot). A. suis has at least 2 somatic serotype (O1 and O2)
 Disease
 Ruminants Actinobacillosis or woden tongue, pneumonia ins swine by A.
pleuropneumonia, septicaemia in swine
 Horse pneumonia (A. equiuli ssp. Haemolytica), septicaemia (A.equuli ssp. Equuli) sleepy
foal disease occurs within a few days of birth

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Laboratory Diagnosis Actinobacilli can often be demonstrated in gram-stained exudates. They


grow best on blood agar under increased amounts of carbon dioxide (35-37oC). Colonies are
often sticky.

The genus Haemophilus


This is a group of small gram -ve, pleomorphic bacteria that require enriched media, usually
blood or its derivatives, for isolation. The dependence on blood for growth has given rise to the

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Antigens and toxins: Encapsulated H. influenzae contains capsular polysaccharides of 6 types.


The encapsulated H. influenzae can be typed by capsule swelling test with specific antiserum.
Four distinct types of H. suis have been distinguished. (The organism occurs in capsulate and
non-capsulate forms. They are antigenically heterogeneous and non typable)

Biochemical properties: all the members of Haemophilus reduce nitrate to nitrate(nitratase


+ve), catalase and oxidase positive, and ferment glucose and galactose.
Variability
Serotype differ in pathogenesity and geographical prevalence. There are three serotype (A-C in
page scheme, or I-III in kume scheme) of H. paragallinarum and at least semen of H. parasuis.

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:

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Portal entry: enters into body through upper RT particularly nasopharynx


 Dissemination: through blood stream or spreads directly to meninges

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:

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 Specimen: nasopharyngeal swabs, pus, blood & CSF


 Microscopy: pleomorphic gram –ve coccobacilli
 Culture: in meningitis, CSF in chocolate agar, Blood culture: in laryngotracheal
bronchitis
 Biochemical test: indole +ve, Urease +ve, Ornithine & arginine test +ve, X & V
requirement
 Antigen detecton: polysaccharide antigen
 Serology: H paragallinarum antibody detection after 1-2 weeks of infection

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

The Genus Bordetella


Bordetella pertussis is the only organism of major clinical significance in this genus, causing
whooping cough in infants and young children. However, a closely related organism, B.
parapertussis can also cause a milder form of bronchitis. B. bronchosepticus, another member of
the genus Bordetella, is the causative agent of respiratory diseases in cats and swine, but can
cause broncho-pulmonary symptoms in severely immunosuppressed individuals.
The name given to this group of organism was in memory of 'Bordet' who first described
Bordetella pertussis the cause of whooping cough in children.
Major species:
B. bronchoseptica :- infect dogs(canine kennel cough) and in porcine Atropic
Rhinitis, causes bronchopneumonia in many species.
B. pertussis :- whooping cough in children.
B. parapertussis :- whooping cough of mild form in human .
B. avium :- the cause of rhinotracheitis of birds( mainly turkeys)

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

Alkaligenes faecalis - B. avium (strain causing turkey coryza)

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.

Celluar product of medical interest


Fimbriae Cell wall LPS
Filamentous haemagglutinatinin Tracheal colonization factor
Peractin (Prn) Pertussis toxin (Ptx)
Cell wall (LPS, bordetella resistance to killing(brk) i.e. outer membrane protein

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Exotoxin(Tracheal cytotoxin, dermonecrotic toxin(DNT), adenylyl cyclase toxin,


pertussis toxin)
Cultural characteristics: Nutritionally fastidious, normally cultivated on medium
containing blood . Grows under aerobic condition at 370C in a medium enriched with
animal tissues or plasma. After 48 hrs of incubation tiny circular dew drop like colonies
appear. In broth the organism produces a uniform turbidity with granular sediment.

Biochemical properties: Bordetella avium and B. bronchoseptica are catalase and


oxidase positive. The organism doesn't ferment carbohydrates. In litmus milk after 24 hrs
of incubation a deep blue ring appears. The organism doesn't form indole or H2S. It is
urease +ve, reduces nitrates, utilize citrate as an organic carbon source, B. avium urease –
ve, nitrate negative.
The organism causes secondary infections in lungs of dogs suffering
from 'canine distemper'. In young pigs it is associated with pneumonia concurrently with
viral diseases.

Virulence factors:

1. adhesions: most important adhesion is filamentous haemagglutinin which also


causes agglutination of red cells
2. toxins: i) pertussis toxin (PT), ii) adenylate cyclase like toxin (ACT), iii) tracheal
cytotoxin (TCT), iv) endotoxin LPS.

Pathogenesis

A. bronchoseptica attacks firmly to ciliated respiratory epithelium – rapid proliferation


occurs – ciliary paralysis – inflammatory response. Virulent strains produce pili and
extracellular enzyme, adenylate cyclase. Pili help for adherence and adenylate cyclase
has antiphagocytic activity and protects the bacteria from intracellular destruction. A
dermatonecrotising toxin is also found responsible for nasal turbinate atrophy and may
play role in pneumonia and other respiratory infections. B. infections depress the
respiratory clearance mechanism facilitating invasion by other organisms. Infectivity of
B. avium is associated with a plasmid, depress the cell mediated immune reactions and
produce a histamine sensitizing factors resembling toxin of B. pertussis. Cytotoxin,
haemagglutinin and dermonecrotizing toxin involve in disease process in birds.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Acts. Bordetella bronchiseptica produces a mild upper respiratory tract infection.


Poultry. Turkey coryza

Laboratory diagnosis

Nasal swabs (Atrophic Rhinitis), sediment of transtracheal washes (canine


tracheobronchitis), and tracheal swabs (coryza of turkeys) are cultured on blood and
MacConkey agars.

Direct microscopy: Gm staining – Gm –ve , small, coccobacilli. FAT.

Culture: media is sheep blood agar, Mac. Agar. 24-48 hrs at 370C aerobically grown.

Identification: colony morphology. Sheep or horse blood agar: B. broncoseptica forms


very small convex colony. Some strains may be haemolytic. B. avium similar with
bronchoseptica but not haemolytic. In MacConkey small, pale colony with pinkish hue
and amber discoloration at the underlying media.

Haemagglutination test: HA positive with sheep RBC (B. bronchoseptica).

Serology: tube agglutination, microagglutination, CFT, immunofluoroscence technique


and ELISA procedure have been developed for B. avium and B. bronchoseptica. In direct
immunoflluoroscence examination of smear of nasopharyngeal exudates is done using
fluorescence labeled antibodies.

Animal inoculation: Dermonecrotising toxin of B. avium and B. bronchoseptica are


thought to be important virulence factors. (There is no cross reactivity between the two
toxins, intracellular, heat labile toxins, Lethal in mice if inoculated intraperitoneal,
necrosis of skin when injected intradermally).

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Figure . Binding of pertussis toxin to the cell


Filamentous haemagglutinins (agglutinogens): These are not exotoxins but are
filament associated lipo-oligo-saccharides which are implicated in the binding of the
organism to ciliated epithelial cells. Antibodies against these molecules are protective,
probably by preventing bacterial attachment and hence constitute a component of
acellular vaccine.

Lipopolysaccharide (LPS): Like LPS of other Gram-negative bacteria, it causes


induction of a number of cytokines and other inflammatory products ( TNF, IL1, IL6,
prostaglandins, etc.) and generates complement-activation products which result in a
variety of patho-physiological effects. When released in relatively large quantities
following bacterial cell lysis, it causes irreversible shock and cardiovascular collapse.

Diagnosis: Symptoms are characteristic. Laboratory diagnosis is made by obtaining a


nasopharyngeal aspirate and primary culture in Bordet-Gengou medium or potato-
glycerol-blood agar. Growth is inhibited by peptones, unsaturated fatty acids, sulphides,
etc. found in ordinary media. The organism grows as small transparent hemolytic
colonies on blood agar. It can be serologically distinguished from B. parapertussis and B.
bronchosepticus.
Prevention and treatment: An acellular vaccine consisting of filamentous hem-
agglutinins and detoxified pertussigen is a component of recommended routine
immunization.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

The Genus Brucella


Gram negative, coccobacilli, hallmark is intracellular survival (facultative intracellular
parasites) .
Cause zoonoses worldwide, six species: B. abortus, B. suis, B. melitensis, B. canis, B.
ovis and B. neotomae
Classification
A. major species
1.B. melitensis: goat and sheep ( malta fever)
2. B. abortus: cattle, human (undulant fever)
3. B. suis: pigs, important cause of human brucellosis
B. additional species
1. B. canis: dogs ( humans occasionally ilfected)
2. B. ovis: ram
3. B. neotomae: desert rodents.

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.

Cellular products of medical Interest


1. Cell wall
The genus of the memberss have gram negative cell wall. The LPS in outer membrane is
an important virulence determinant. LPS binds to LPS- binding protein (a serum protein)
which transfer to the blood phage of CD14. The CD14-LPS complex binds to toll like
receptor proteins on the surface of macrophage cells triggering the release of
proinflammatory cytokines. The cell wall of brucella aid in its survival within
macrophages.
2. Erythritol
Ertthritol (a four carbon alcohol) is one of several “allantioc fluid factors” found in the
gravid uterus and affinity to localization to the reproductive tract of the pregnant animals.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Antigens and Toxins: different species of brucellae contain surface protein-


lipopolysacharides surface antigens designated A(abortus) and M(melitensis) in different
proportions. The A and M antigens of B. abortus occur in a ratio of 20:1, where as in B.
melitenis, the proportion is 1:20. and can be distinguished by agglutination reaction. A
superficial L antigen has been demonstrated that resembles the Vi antigen of salmonellae.
The different species of Brucella vary in host preference and degree of virulence within and
among animal species.beside, there are other minor antigens.
No Extracellular toxins have been identified.
Typing of Brucella by the use of bacteriophage:
Phages specific for B.abortus have been developed. By using a dilution of phage known
as routine test dilution (RTD), there is complete lysis of bacteria. With RTD, the smooth
cultures of B. abortus are lysed but a higher concentration of phage (10,000 RTD) also
lyses strains of B.suis but not of B. melitensis.
A variety of serological tests are used to diagnose brucellosis because no single
method is satisfactory. Some are:
1. serum agglutination test(SAT)
2. rose Bengal plate test(RBPT)
3. complement fixation test(CFT)
4. milk ring test(MRT)

Pathogenesis:

Transmission by direct or indirect contact with tnfected excretions. Route often by


ingestion but veneral transmission may occur and is the main route for Bruceella ovis.
Less transmission possible via conjunctiva, utero or via inhalation. It possesses an
endotoxin that contributes to the pathogenesis and surface cellas all carbohydrate
responsible for binding to B- lymphocytes and survive inn phagocytic cell and its very

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Public health hazards: the sources of infection may be-


1. milk and milk products
2. meat
3. infected foetus for vets and lab worker, farmers
Though B. spps is suceptible to various antibiotics, treatment is not so easy. In
man antibiotic course for about 40 days is recommended.
Immune mechanisms
 Susceptibility to infection depends on age, sex, and breed and pregnancy status.
 Younger animals are more resistance than sexually matured.
 Infected animals shed organisms in the milk, raw milk products of anials are sources
of infection in humans.
 Marine mammal Brucella species have caused intrcerebral granulomas in humans.
 The antibodies IgM appears after infection and low level of IgG will cause
complement- mediated lysis of Brucella.
 Elevated IgG to act as blocking antibodies that modulate the ability of the
complement membrane attack complex to lyse cells.
 The blocking antibodies are opsonizing and promote uptake byy phagocytes where
Brucella have developed mechanisms for survival and proliferation.
 Phagocytic cells unable to eliminate Brucella play a role in dissemination of
organisms to other parts of the body and in persistence of infection.
 IgA autoantibody has been demonstrated in dogs infected with B.canis and observed
effect of fertility.

Lab diagnosis: extreme care must be exercised while working.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Biochemical tests: slide agglutination test.

Animal inoculation – guinea pigs used. 0.5-1ml of suspected tissue homogenate is


injected. Then within 2-3 days isolate from guinea pigs and after 3-5 days shows
symptoms (pustule like lesions at various organ). Immunological tests for detecting
antibodies to B. abortus like milk ring test, plate agglutination test, Rose Bengal Plate
Test, ELISA, complement fixation and serum agglutination test.

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.

Long incubation period (weeks, months)


Malaise, chills, fever, sweats, weakness, myalgia, headache
Nervous symptoms (psychoneurosis)
Difficult diagnosis due to vagueness of symptoms

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

The genus Moraxella

This organism was formerly included in Haemophilus but as it does not


require X or V factors, it is placed in a new genus, Moraxella are gram negative rods and cocci
belonging to family Moraxellaceae. It was described by 'Morax' and Axenfeld independently in
sub acute conjunctivitis in M. lancunata.
Two subgenera of moraxella :
Moraxella(rod-shaped members) and Branhamella ( the coccoid members)
Other species:
M. bovis -cause infectious bovine keratoconjunctivitis(IBK),"New forest disease"
M. Liquefacians - cause human conjunctivitis and corneal ulcers.
All the species infect different structures of the eye eg. Keratitis,
conjunctivitis, corneal ulceration etc.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

* pitting on the medium can be studied using 'loeffler's coagulated serum".


M. bovis doesnot produce acid in carbohydrates.
Resistance : it is usually suceptible to commonly used antibiotics.

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 0.5-1m) 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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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. aeruginosa is resistant to many commonly used antibiotics.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Specimen: depends upon the clinical signs and lesions.

Direct microscopy: little diagnostic use.

A fluorescent antibody technique can be useful for P. mallei and P. pseudomallei.

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.

Pigments: P. aeruginosa strains produce-

Pyocyanin(blue), pyoverdin(yellow), pyorubin (red), pyomelanin (dark brown).

Microscopically- medium sized Gm –ve rods.

Biochemical characteristics; colonial appearance, including pyocyanin production and odor,


strong oxidase reaction.

Immunological tests, animal inoculation and AST can be done for identification.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Burkholderia mallei/ Psedomaona mallei


The causal organism was called as Actinobacillus mallei, laefferala mallei, pfeifferella mallei,
mallomyce mallei and bacilllus mallei. The organism is the cause of glanders in equines species
and man.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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

The genus Aeromonas


Cultures of bacteria which are commonly found in water and soil are sometimes found to
be pathogenic for man and animals. Such an organism is aeromonas hydrophilia formerly called
Proteus hydrophilus.
A. hydrophila is a short, plump, aerobic rod 0.6µ by 1.3 µ in size and is motile by means
of polar flagella, although peritrichous flagella have been observed by some workers. It is gram-
negative. Grayish-white, stippled, translucent, moist colonies are formed on and agar plate and a
heavy turbidity and thick pellicle develop in broth. Optimum temperature for growth is 30oC. At
that temperature acid and gas are produced in glucose, maltose, sucrose, mannitol, trehalose,
arabinose, sorbitol and salicin. Lactose is not quickly fermented but some strains will produce
acid in a few days. Nitrates are reduced to nitrates; indole is formed; gelatin is liquefied; milk is
coagulated and peptonized; catalase and oxidase are produced; aesculin is hydrolysed; H2S is
produced in cysteine broth; most strains are Voges-Proskauer positive; ammmonia is produced
from arginine.
In animals A. hydrophila is found in frogs where it produces a condition known as
"redleg" due to the haemorrhagic erosion found in the skin and between toes and limb
attachments. The condition is also found in salamanders and fish which may be kept in tanks in
laboratories or sales rooms without the circulation of cool, running water. It has been isolated
from stored eggs where it cause a condition characterized by the term "black rot". It has been
isolated from man, guinea pigs, mice, and rabbit. Pure cultures of the organism produce a tetanic
condition in organism produce a tetanic condition in frogs. And alcohol precipitate toxin causes
effects similar to those produce on muscles by caffein, digitilis, and feratrin. Some strains
produce β haemolysin.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Rickettsias and chlamydiae

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Figure 2. Helicobacter pylori from


Helicobacter pylori Causing Adenocarcinoma,

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

The genus Borrelia


The grooup of organism are comparatively large, irregularly coiled organisms which are stained
easily and are gram negative. In this genus, there are large number of species some are
commensal and other pathogenic for man, animals and birds.
Strains of Borrelia
Recently, the Borrelia causing Lyme disease were divided into several "genospecies", three of
which have been firmly established and are well accepted:
I. Borrelia burgdorferi sensu stricto
II. Borrelia garinii
III. Borrelia afzelii
Others are
IV. B. anserina-a pathogen of birds
V. B. theileri- cause a benign disease in catttle and horses
VI. B. recurrents/duttoni- causes relapsing fever in man

Borrelia burgdorferi
Borrelia burgdorferi, like the human pathogen Treponema
pallidum, is a spirochete.

B. burgdorferi dark field illumination


When Borrelia burgdorferi is Gram-stained, the cells stain a weak Gram-negative by default, as
safranin is the last dye used. Borrelia, like most spirochetes, does have an outer membrane that
contains an LPS-like substance, an inner membrane, and a periplasmic space which contains a
layer of peptidoglycan. Therefore, it has a Gram-negative bacterial type cell wall, despite its
staining characteristics.
Cultivation
Unlike Treponema pallidum, Borrelia burgdorferi can be cultivated in vitro. However, the
bacterium is fastidious and requires a very complex growth medium. The medium used to grow
Borrelia burgdorferi is called Barbour-Stoenner-Kelly (BSK) medium. It contains over thirteen
ingredients in a rabbit serum base. Borrelia burgdorferi has an optimal temperature for growth of
32 C, in a microaerobic environment. Even under optimal conditions, the generation time is
slow, about 12-24 hours.
Borrelia from ticks and from the blood, skin, and cerebrospinalfluid of Lyme disease patients
have been successfully cultivated in BSK medium. BSK solidified with 1.3% agarose allows the
production of colonies from single organisms.
Outer Surface Proteins
The outer membrane of Borrelia burgdorferi is composed of various unique outer surface
proteins (Osp) that have been characterized (Osp A through OspF). They are presumed to play a
role in virulence. Osp A and Osp B are by far the most abundant outer surface proteins.
Pathogenicity
Borrelia burgdorferi invades the blood and tissues of various infected mammals and birds. The
natural reservoir for Borrelia burgdorferi is thought to be the white-footed mouse. Ticks transfer
the spirochetes to the white-tailed deer, humans, and other warm-blooded animals after a blood
meal on an infected animal. In humans, dogs, and many other animals, infection with Borrelia
burgdorferi results in the pathology of Lyme Disease.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Campylobacter (campylo-curved)


The organisms were previously named as vibrio. The genus comprises microaerophilic vibrios.
Ther are a number of species which are associated with disease in animals. The pathogenic
species includes:
C. fetus - causes infertility and abortion among cattle and abortion in sheep.
C. jejuni - cause acute enteritis in cattle.
C. coli - causes sever dysentery in young pigs.
C. bubulus - cause porcaine intestinal adenomatous in pig.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Leptospira


Leptospira are widely distributed in nature and produce disease in variety of animal species and
can be transmitted to man, usually through water contaminated with urine.
The genus leptospira is now classified into two disease
L. interrogans- potential pathogen of man and animals
L.biflex-contain saphrophytic leptospiras
Morphology: the leptospira are 8-12µm in width. They are closely spiralled with one end
commonly bent into the shape of a hook. There is active rotational motion but no flagella has
been reported. Under electrone microscope, individual filament consist of 3-portions,
outermost....middle and central axial filament.
The leptospira are delicate and in dark field microscope appear as a chain of cocci. The
organsim doesnot stain readily.
Pathogenic species for human:
L. interohaemorrhagiae
L. hebdomadis
L. autumnalis
In case of animal diseases, different species are responsible given as follows.
L. canicola
L. pomona

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Vibrio cholerae and Asiatic Cholera


The genus Vibrio consists of Gram-negative straight or
curved rods, motile by means of a single polar flagellum.
Vibrios are capable of both respiratory and fermentative
metabolism. O2 is a universal electron acceptor; they do not
denitrify. They have structural and metabolic properties
that overlap with both the enterics and the pseudomonads.
Vibrios are facultative (grow in the presence or absence of
Vibrio cholerae
O2), like enterics, but they have polar flagella, are oxidase-
positive, and degrade sugars in the same manner as the pseudomonads. Most species are oxidase-
positive. In most ways vibrios are related to enteric bacteria, but they share some properties with
pseudomonads a well. The Family Vibrionaceae is found in the "Facultatively Anaerobic Gram-
negative Rods" in Bergey's Manual (1986), on the level with the Family Enterobacteriaceae. In
the revisionist taxonomy of 2001 (Bergey's Manual), based on phylogenetic analysis,
Vibrionaceae, Pseudomonadaceae and Enterobacteriaceae are all landed in the
Gammaproteobacteria. Vibrios are distinguished from enterics by being oxidase-positive and
motile by means of polar flagella. Vibrios are distinguished from pseudomonads by being
fermentative as well as oxidative in their metabolism. Of the vibrios that are clinically significant
to humans, Vibrio cholerae,the agent of cholera, is the most important. The cholera toxin, which
is the classic model of a bacterial enterotoxin, is also produced by some strains of E. coli.
Cholera Toxin isCholera toxin activates the adenylate cyclase enzyme
Most vibrios have relatively simple growth factor requirements and will grow in synthetic media
with glucose as a sole source of carbon and energy. However, since vibrios are typically marine
organisms, most species require 2-3% NaCl or a sea water base for optimal growth. Vibrios vary
in their nutritional versatility, but some species will grow on more than 150 different organic
compounds as carbon and energy sources, occupying the same level of metabolic versatility as
Pseudomonas. In liquid media vibrios are motile by polar flagella that are enclosed in a sheath
continuous with the outer membrane of the cell wall. On solid media they may synthesize
numerous lateral flagella which are not sheathed.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Vibrios are one of the most common organisms in


surface waters of the world. They occur in both
marine and freshwater habitats and in associations
with aquatic animals. Some species are
bioluminescent and live in mutualistic associations
with fish and other marine life. Other species are
pathogenic for fish, eels, and frogs, as well as other
vertebrates and invertebrates.
V. cholerae and V. parahaemolyticus are pathogens
of humans. Both produce diarrhea, but in ways that
are entirely different. V. parahaemolyticus is an
invasive organism affecting primarily the colon; V.
cholerae is noninvasive, affecting the small
intestine through secretion of an enterotoxin. Vibrio
vulnificus is an emerging pathogen of humans. This organism causes wound infections,
gastroenteritis, or a syndrome known as "primary septicemia."
Campylobacter jejuni (formerly Vibrio fetus), is now moved to the class Epsilonproteobacteria
in the the family Campylobacteraceae. Campylobacter jejuni has been associated with
dysentery-like gastroenteritis, as well as with other types of infection, including bacteremic and
central nervous system infections in humans. Another vibrio-like organism, Helicobacter pylori
causes duodenal and gastric ulcers and gastric cancer. It is also reclassified into the class
Epsilonproteobacteria
family Helicobacteraceae.
Vibrio vulnificus is a Gram-
negative, motile curved
bacterium

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Morphology and staining


Microscopically Listeria species appear as small, Gram-positive rods with rounded ends,
which are sometimes arranged in short chains. In direct smears they may be coccoid, so they can
be mistaken for streptococci. Longer cells may resemble corynebacteria. (Indeed, as palished
shaped, Gram-positive, nonsporeforming, catalase-positive rods, the genus Listeria was
classified in the family Corynebacteriaceae through the seventh edition of of Bergey's Manual).
Flagella are produced at room temperature but not at 37° C. Hemolytic activity on blood agar has
been used as a marker to distinguish Listeria monocytogenes among other Listeria species, but it
is not an absolutely definitive criterion. Further biochemical characterization may be necessary
to distinguish between the different Listeria species.The organism is nonporeformer, non acid
alchol fast, gram +ve, but old cultures are decolorized easily and stain gram -ve.
Cultural characteristics: L. moncytogenes is aerobic and mocroaerophilic. Growth on ordinary
media is not abundant(i.e. can support their, but only moderate). Some strains require increase
CO2 in the environment. Prsence of
serum, blood, glucose or liver extract
enhances the growth. The smooth
form produces round translucent
colonies with smooth edge, buish red
color-measuring 1-3mm. the rough
type is 3-6mm with uneven surface
and roughened edge.
In blood agar the colonies are small and Listeria monocytogenes Gram Stain
surrounded by a narrow zone of beta
haemolysis. In broth, a slight turbidity is observed and granular sediment is formed.
Biochemical properties: gelatin and serum not liquified. Nitrate reduction test-ve, indole -ve,
urease -ve, the organism ferments-glucose, maltose, levulose, ramnose, trehalose and solicin.
Resistance: the organism is destryoed at 58 oC in 10 minutes(55 oC in 40 min.) it is easily killed
by usual disinfectants.
Antigen and toxins: L. monocytogenes have both somatic(o) and flagellar(H) antigen. The
organism doesnot produce toxin.
Public heath aspect: human infectionl are rare. The disease is transmitted by diseased animals
and birds. Th source of infection may be meat/milk.
Infection occurs as an occupational hazard as a result of direct contact such as, in
butchers, abottoir workers, farmers and veterinarians. Meningitis accounts for the 75% cases of
listeriosis. Other forms of listeriosis include-typhoidal listeriosis, listoirial endocarditis, ocular
infections, dematitis, infection of serous cavities and absess in different organs.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Staphylococci


Staphylococci are spherical, ovoid gram positive cocci arranged in grape-like clustures. Some are
members of the normal flora of the skin and mucous membrane of man and animals and others
are the commonest cause of suppuralation. Many species are associated with animal disease.
Broadly there are 2 spp. of staph. Different strains of staph pyogenes have specific names.
Staph. Aureus or Staph. Pyogenes(produce yellow color)
Staph. Albus(produce white pus)
Staph. Citreous(lemon/greenish color).

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).

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Resistance to physical and chemical agents:


 Staphylococci are the most resistant of the cocci.
 They are more resistant to heat and dehydration than most of non-sporulating organism.
 Majority of the strains are killed at 60 oC in 30 minutes, but come strains are resistant.
 Disinfectant-they are readily killed by common disinfectants including (HgCl2, phenol,
Na-hypochlorite solution), salt (7.5 - 10%) in the absence of pus, mucus and serum,
chlorine. Staph. Present in the normal environment, can accumulate in higher numbers as
saprophytes are killed by disinfection ==> ready source for food poisoning.
 They survive for many months in lab culture media, dust and variety of media in the
absence of sunlight.
 They are susceptible to crystal violet. In a selective medium called Edward's medium,
containing 1:500000 dilution of crystal violet, staph. Can't grow but streptococcus can.
 Antigens and toxins: different polysaccharide antigens have been identified and on their
basis, different types i.e. AgA, AgB, AgC has been classified.
 AgA is present in pathogenic strains. AgB is presnt in saphrophytic stains
Staphylococcus is one of the typical members of toxin producing bacteria. (Cellular
products of Medical Interest)

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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:

1. Coagulase: it is not a toxin, but it probably plays a role in the pathogenesis or


staphylococcal infections. It coagulates the blood plasma producing fibrin from
fibrinogen and can be demonstrated by Coagulase Test; this test is used to determine the
pathogencity of Staphylococcus isolates. Staph. Aureus produces coagulase. It is an
enzyme-like protein and cause clotting human and rabbit plasma. It is not a toxin, but it
probably plays a role in the pathogenesis or staphylococcal infections. There are 7
antigenic types of coagulase. Most of them exists in two forms; free and bound forms.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

2. DNase: Enzyme for pathogenicity.


3. Hyaluronidase: it digests the intracellular substance of tissue, and helps the bacteria to
invade the tissue.
4. Lipase: degrades cidal fatty acids of skin during entry.
5. Staphylokinase: dissolves clots.
6. phosphatase: it is the phenopthalein test +ve.
7. Fibrinolysin: digests fibrin. This is thermostable as well as antigenically distinct for
staphylococcus.
8. Necrotoxin: causes necrosis of cells. Function same as alpha hemolysin.
9. Lethal toxin: similar to necrotoxin but more toxic.
10. Leucodidin: it is and exotoxin and is lethal to polymorphonuclear leucocytes and is
distinctive from alpha-haemolysin. There are 3 distinct types of leucocidins. Type I, Type
II, and Type III.
11. Enterotoxins: Staphylococcal food poisoning for over 40% of all cases of food
poisoning. The disease is characterized by vomiting and diarrhea commencing 1 - 6 h
after consumption of contaminated food, especially dairy produce. Symptoms usually last
no longer than 24 h and death is extremely rare. Like botulism, staphylococcal food
poisoning is commonly caused by the ingestion of food containing preformed
enterotoxins.
12. Hemolysins:
 Alpha hemolysin: Rabbit RBCs = much more sensitive than other species; eg, human
RBCs = 1000 x less sensitive. They are secreted as a water-soluble protein which
undergoes self-induced oligomerization to form pores. Its Importance in vivo is
unknown. They produce a partial hemolysis with an ill-defined greenish discoloration
around the colony. The zone of lysis is small (1-2 mm wide). It is not how RBC is spread.
greenish discoloration is due to formation of an unidentified reductant Hb. It is seen in
various Streptococci and Pneumococci.
 Beta hemolysin: sphingomyelinase C; significance in pathogenesis of disease but still
unknown; apparently more common in animal strains of S. aureus than in human strains.
It prdduces wide (2-4 mm) with clear zone of complete hemolysis around the colony in
which no red cell are visible on microscopic examination.
 Gamma haemolysin: They produce no haemolysins but cytopathic some tissue.
 Delta hemolysin: Heat-stable peptide, It is 26 amino acid residues molecule.

The genus Streptococcus

Streptococcus pyogenes (Group A streptococcus) is a Gram-positive, nonmotile,


nonsporeforming coccus that occurs in chains or in pairs of cells. Individual cells are round-to-
ovoid cocci, 0.6-1.0 micrometer in diameter. Streptococci divide in one plane and thus occur in
pairs or (especially in liquid media or clinical material) in chains of varying lengths. The
metabolism of S. pyogenes is fermentative; the organism is a catalase-negative (staphylococci are
catalase positive) aerotolerant anaerobe (facultative anaerobe), and requires enriched medium
containing blood in order to grow. Group A streptococci typically have a capsule composed of
hyaluronic acid and exhibit beta (clear) hemolysis on blood agar.
It is one of the most frequent pathogens of humans. It is estimated that between 5-15% of normal
individuals harbor the bacterium, usually in the respiratory tract, without signs of disease. As

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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:

α1-haemolysis-partial hemolysis with hazy (claudy) appearance, α haemolysis- partial


haemolysis with greenish color.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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 Hyaluronic Acid Capsule

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.

 Extracellular products: invasins and exotoxins

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

S. Parameters Staphylococcus species Streptococcus species


N.
1. Catalase Test Positive (pathogenic) Negative
2. Coagulase Test Pathogenic strains are positive Negative
3. Phosphatase Produced +ve
4. KOH Test -ve -ve
5. Oxidase test -ve -ve
6. B-haemolysis Pathogenic Alpha,non-pathogenic B. Non pathogenic B-haemolysis
7. Esculin hydrolysis +ve
8.

The Genus Bacillus

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Flagellar stains (Leifson's Method) of various species of Bacillus from CDC

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.

Nutrition and Growth

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:

Table: Differential Characteristics of B. anthracis and Anthracoid bacilli

Characteristic B. anthracis Anthracoid bacilli


Growyh in N Agar Medusa head Not so
growth requirement for thiamin Required Non required
hemolysis on sheep blood agar Not or slight Yes
Gelatin stab culture Inverted fir tree appearance Liquefaction
0
Growth at 45 C No growth Usually grow
lysis by gamma phage Yes not

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Motility Non motile Motile


Glutamyl-polypeptide capsule Present Absent
Chain formation Grow in long chains Grow in short chains
Turbidity in broth No turbidity Uniform turbidity
growth on chloralhydrate agar Absent Present
Ascoli‟s penicillin test Strongly positive Negative
McFadyean‟s reaction Positive Negative
Anthrax phage susceptible to it non susceptible
Location of spore within the bacillus centre, spore don‟t bulge out central, subterminal and may bulge out

Bacillus anthracis and anthrax


The anthrax bacillus, Bacillus anthracis, was the first bacterium shown to be the cause of a
disease. In 1877, Robert Koch grew the organism in pure culture, demonstrated its ability to form
endospores, and produced experimental anthrax by injecting it into animals.

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 thuringiensis is distinguished from B. cereus or B. anthracis by its pathogenicity for


Lepidopteran insects (moths and caterpillars) and by production of an intracellular parasporal
crystal in association with spore formation. The bacteria and protein crystals are sold as "Bt"
insecticide, which is used for the biological control of certain garden and crop pests.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

On blood agar: Organisms produce slight haemolysis in comparision to anthracoid organisms


but usually non-hemolytic colonies.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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

Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous


animals, such as cattle, sheep, horses, mules, and goats. Humans become infected incidentally
when brought into contact with diseased animals, which includes their flesh, bones, hides, hair
and excrement.

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.

Pathogenicity of Bacillus anthracis

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

Bacillus anthracis forms a single antigenic type of capsule consisting of a poly-D-glutamate


polypeptide. All virulent strains of B. anthracis form this capsule. Production of capsular
material is associated with the formation of a characteristic mucoid or "smooth" colony type.
"Smooth" (S) to "rough" (R) colonial variants occur, which is correlated with ability to produce
the capsule. R variants are relatively avirulent. Capsule production depends on a 60 megadalton
plasmid, pX02; its transfer to nonencapsulated B. anthracis via transduction produces the
encapsulated phenotype.

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.

Production of the anthrax toxin is mediated by a temperature-sensitive plasmid, pX01, of 110


megadaltons. The toxin consists of three distinct antigenic components. Each component of the
toxin is a thermolabile protein with a mw of approximately 80kDa.

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

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

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

PA+LF combine to produce lethal activity


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

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Anthrax and Biological Warfare

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.

There is no evidence of person-to-person transmission of anthrax. Quarantine of affected


individuals is not recommended. Anthrax spores may survive in the soil, water and on surfaces
for many years. Spores can only be destroyed by steam sterilization or burning. Chemical
disinfection of buildings is problematic. The U.S. Navy Manual on Operational Medicine and
Fleet Support entitled Biological Warfare Defense Information Sheet states "Disinfection of
contaminated articles may be accomplished using a 0.05% hypochlorite solution (1 tbsp. bleach
per gallon of water). Spore destruction requires steam sterilization."

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Clostridium


This group includes organisms which are anaerobes, spore forming bacilli and are gram positive.
The natural habitat of the majority of the species is soil and intestinal tract of animals. In soil,
they complete their lifecycle in about 3 months. Vegetable, fruits and grass become
contaminated with clostridia and are ingested by man and animals and gain come back through
faeces. So faeces are also sources of them. For all species of clostridia, sporulation is an
important part of their lifecycle, though is not necessary

There are three types of clostridia:


Saprophytes: responsible for putrefaction of dead animals or vegetables.
Commensals: present in intestinal tract of man, animals and birds.

Pathogenic strains are of 3 types on the basis of toxin producing ability.


 Food poisoning (endotoxin)
 Producing potent toxins (exotoxin)
 Mild toxin producers (Endo+Exotoxin)

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:

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Negler reaction for Clostridium perfringens:

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Mycoplasma/Acholeplasma


Mycoplasma and acholeplasma are 2 groups of bacteria. Mycoplasma requires sterol (e.g.
cholestterol) for their growth e.g. cholesterol, but acholeplasma donot require sterols

 Mycoplasma occupy a place between bacteria, virus and L-forms


 Most important property is that they lack cell wall, but they are not derived from parent
bacteria having cell wall i.e. do not revert to bacterial forms.
 These are smallest free-living organisms.
 The smallest reproductive unit has a size of 150-250 nm, highly pleomorphic because
they lack rigid cell wall and instead are bound by a membrane which contains a sterol.
 Mycoplasma has been allocated in a new class known as mollicutes (mollis-soft, cutis-
skin).

Major species are associated with 'Pleuropneumonia'


 In cattle- Mycoplasma mycoides var. mycoides- contagious bovine
pleuropneumonia(CBPP)
 In goat- Mycoplasma mycoides var. capri causes contagious caprine
pleuropneumonia(CCPP)
 Mycoplasma agalactiae cause contagious agalactia of sheep and goat
 Mycoplasma gallisepticum causes chronic respiratory disease (CRD) in chickens.
 Mycoplasma synoviae causes synovitis in chickens and turkeys.

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.

Reproduction: broadly, there can be three types of reproduction.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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)

Antigen and toxins: the antigenic structure is very complex.


 Mycoplasma mycoides produces a diffusible tosin.
 Mycoplasma gallisepticum produces a soluble exotoxin
 Mucoplasma neurolyticum produces a neurotoxin.

L- phase variant of Mycoplasma

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.

The genus 'Corynebacterium' (diptheroids)


These are non acid fast, non-sporing, catalase +ve, slender bacilli, average size 3*0.3 µms. They
are frequently club shaped (coryne means club shaped) due to presence of meta chromatic
granules at the ends. The cells often remain attached after division which gives an arrangement
of 'chinese letter'. The organism is non-motile.
Major species:

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 pyogenes-mastitis and suppurative lesions in cattle and sheep.


 bovis-do
 ovis-purrulent lymphadenitis(sheep), ulcerative lymphadenitis(equine)
 C. renale- pyelonephritis and cystitis
 C. diphtheriae-human diptheria
 C. equi-purulent pneumonia (fowl, sheep, goat and pig)
 C. suis- pyelonephritis and cystitis (sow)
 C. haemolyticum- pneumonia
 C. ulcerans-tonsilitis

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.

Resistance: the organism killled at 60 oC and is very suceptible to disinfectants.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Erysipelothrix


The organisms of this genus are rod shaped with a tendency to form long filaments. They are
non-motile, non-capsule forming and non-sporing. They are gram +ve. The organism causes
disease in animals and man. The organism is widely distributed among animals and in decaying
organic matter. For many years the genus Erysipelothrix included three species:
 E.rhusiopathiae-found in man
 E.muriseptica-found in mice
 E. erysepeloides- found in swine causes 'erysepelas'
The 1957 edition of bergy's mannual has included all three in single species which has been
given the name Erysepelothrix insidiosa.
'erysepelas' is characterized by urticaria-skin eruption, arthritis, endocarditis and dyspnoea. In
human 'Erysepeloid' is and inflammatory lesion of the skin usually of the fingers and hand
resembling erysepelas. Erysepeloid is and occupational mazard of meat and fish handlers and
veterinary surgeons.

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.

Cultural characterstics: the organism is microaerophilic in primary isolation but grows as


aerobe or facultative anaerobe on subculture. Optimum temperature-37 oC. the growth takes
place an ordinary culture media but growth is enhanced by the addition of serum or blood. There
is slight haemolysis on blood agar. Certain unidentified growth factors are required for rpaid and
abundant growth.
Growth occurs in smooth or rough form on agar, the colonies of smooth strain are small,
glistening, translucent measuring 0.5 to 1mm in diameter. The rough colonies have serrated edge
and measure 1-2 mm but increase in size 3-4mm on further incubation. The colonies have
granular appearance. (Smooth strains form colonies with entire edges). In broth, smooth form of
growth leads to a uniform turbidity with no pellicle and little sediment; the rough type produce a
heavy turbid growth with sediment in broth.
In gelatin stab, the growth radiates from the line of inoculation giving a characteristic bottle
brush appearance'

Biochemical properties: most of the strains ferment lactose, fructose and galactose with
production of acid. It does not form indole, does not reduce nitrates.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

The genus Mycobacterium


Mycobacteria are sender bacilli and sometimes exhibit filamentous form resembling gungal
mycelium (Myces=fungus) and hence they are so named. They are difficult ot stain by ordinary
stains because of the presence of waxy material in their cell-walls. Although they are gram +ve,
they stain poorly, if at all by gram's stain. They are better stained by hot carbol-fuchsion, and
once stained; they resist decolorization with 1 % HCl and also with 95 % ethanol and are
therefore reffered to as 'acid-fast' or 'acid-alcohol fast organism'

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'.

Mycobacterium tuberculosis (tubercle bacilli)


Mycobacterium tuberculosis 'human type' is the cause of human tuberculosis but also cause
disease in cattle, pigs, dogs, monkeys, parrot and canaries. The other types of the organism are:

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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)

Morphology and staining: different types are indistinguishable morphologically. Their


morphology also differs in tissue and lab cultures slightly. In tissue, they occur as slender rods
ranging 1-4 µm in length and 0.2-0.3µm in diameter. They vary from coccobacillus slender rods,
cub-shaped or curved and can occur. Singly in pairs or in bundles.
In lab culture, they are non-capsulated and non-motile, and non flagellate.

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

 The growth is less and more rapidely emulsified.


 The avian strains produce yellow or slight pink pigment.
Resistance: The tubercle bacilli of various types are quite sensitive to heat and are killed at
60degree c in 15 minutes. The organism is usually resistant to drying, to most disinfectants and
to acids and alkalies. This resistance is due to hydrophobic lipid surface. The organism is
susceptible to ionic detergents and sunlight. The organism remains viable for long period in the
dark, putrefying material, faces and sputum.

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.

Public health aspect:


 It is a zoonotic disease
 The bovine type can also infect human beings. Practically both human and bovine
type are equally infective to human begins. Humans acquire the infection from
bovines as bovine types.
 The organism is present milk i.e. milk is highly potential source of transmission to
human beings. (Pasteurization destroys it.)
 For the prevention of disease from mother to young ones, it is better to feed the milk
only after pasteurization.

Human health aspect:


Transmission : Human bovine i.e. 2 way transmission sources of transmission:
1)Milk
2) Meat: having tubercles-which can be seen during meat inspection, but small
nodules can't be seen. Boiled meat will be safe.

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

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)
Microbiology III 2015

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.

Dr Rebanta Kumar Bhattarai, lecturer


B.V.Sc. & A.H., M.V.Sc.(Microbiology)

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