Bacillus bacteriology microbiology notes.ppt

Veterinary microbiology

1. Domain : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Bacillales
Family : Bacilliaceae
Genus : Bacillus
Species:
B.anthracis, B.cereus, B.subtilis,
B.mycoides,

B.megaterium, B.mesentricus
Systematics

2. B.anthracis
Anthrax in animals
Wool sorter’s disease, hide porter’s
disease, Malignant Pustule in humans.
B.cerus
food poisoning in humans.

3. Greek
 word for
coal

4. One of the diseases of animals mentioned in ancient and
Bible history
Pollender (1849) –first pathogenic bacterium observed under
the Microscope.
Casimir Davaine (1850) – first communicable disease shown
to be transmitted by inoculation of infected blood
Koch (1863) – first bacillus to be isolated in pure culture –
used to demonstrate koch postulates
Pasteur (1881) – prepared attenuated vaccine.
Bacillus - History

5. rod shaped with truncated ends - bamboo stick
appearance, Box –car bacillus
Anthracoid - rod shaped with rounded ends
Gram positive
Long filaments in culture media,
In tissues - in pairs or short chains
Oval, centrally located endospores
Motile by peritrichous flagella except B. anthracis.
Morphological Characteristics

7. Capsules - media enriched with serum, albumen,
charcoal, starch or bicarbonates with reduced partial
pressure of carbon dioxide.
Polypeptide in nature - polymer of d-glutamic acid.
Blood films - stained with polychrome methylene blue for
a few seconds and examined, an amorphous purplish
material is noticed around the bacilli.
Represents the capsular material and is characteristic of
the anthrax bacilli - McFadeyan’s reaction.
Bacillus anthracis – capsule
Capsule production on
Bicarbonate agar medium

8. Blood films stained with polychrome methylene : amorphous
purplish material is noticed around the bacilli. Represents the
capsular material and is characteristic of the anthrax bacilli
McFadeyan’s reaction
Toludine blue

9. Endospore
Sporulation requires

Presence of oxygen

Poor nutrient conditions
1 spore per cell
Highly resistant to heat, cold, chemical disinfectants, dry
periods
Protoplast carries the material for future vegetative cell
Cortex provides heat and radiation resistance
Spore wall provides protection from chemicals & enzymes
Sporulation takes place at an opt.temp.of 25-30º C and in
atmosphere containing low partial pressure of oxygen.

10. Aerobic and facultatively anaerobic.
Grow well in medium enriched with blood or serum.
irregular, round, raised, dull, opaque, grayish white,
frosted glass appearance colonies
Under the low power microscope, the slightly
serrated edge of the colony is composed of long,
interlacing chains of bacilli, resembling locks of
matted hair - medusa head or judges wig or
women’s curling hair type of growth.
Cultural characteristics

11. Frosted glass appearanceSlight haemolysis
Tenacious---sticky consistency - like
beaten egg white
Growth on Sheep blood agar

12. Medusa head
women’s curling hair type of growth

13. PLET (polymyxin, lysozyme, EDTA
and thallous acetate in heart infusion
agar).
- selective to isolate B.anthracis
from mixtures containing other spore-
bearing bacilli
Growth on Selective medium

14. Virulent strains - rough colonies,
Avirulent attenuated strains - smooth colonies
In gelatin stab - Inverted fir tree appearance
On blood agar - slight haemolysis compared with
anthracoid organisms.
B. cereus - wide zone of complete haemolysis
around the colonies.
Guinea pigs and mice - choice for cultivation.
Cultural characteristics – cont.

15. Growing forms are only slightly resistant.
Killed by ordinary disinfectants, by pasteurization.
Spores surviving for 60 or more years.
Heat fixation of smears does not kill spores
Resist dry heat at 140
0
c for 2-3 hrs and boiling for 10 mts.
Killed at 120
0
C for 10 min and 4% KMNO4 treatment for 15
mts
Duckering :
2% solution of HCHO at 39-40
0
C for 20mts for wool.
0.25% HCHO at 60
0
C for 6 hrs – animal hair and bristles.
(Use of lime (calcium oxide) as disinfectant should be avoided. (aid
in long term preservation, providing high pH suitable for
calcium enrichment)
Use 5% HCHO or peracetic acid for farm premises.
Resistance

16. Catalase positive
Ferments glucose, maltose, saccharose,
trehalose and Dextrin produce acid but no
gas.
Nitrates reduced to nitrites.
Indole is negative.
Methylene blue reduced
Voges-Proskauer positive
Biochemical characters

17. Poly-d-glutamic acid capsule - plasmid mediated (pXO2).
Protects against phagocytosis, lytic antibodies, and
complement activity.
Toxins
Lethal factor
• pXO1
• binary toxin composed of protective antigen (PA) and LF
• PA binds to variety of cell surface receptors on a variety of cell
types
• LF is a Zinc metalloprotease
• Cytokine storm- hyper active macrophages – IL-1, 6, 8 & TNF
• increased intravascular permeability, depletion of clotting,
apoptpic death of macrophages

 
         
Virulence factors
 

18.  Edema toxin
• pXO1
• binary toxin composed of protective antigen (PA) and ET
• PA binds to variety of cell surface receptors on a variety of cell
types
•ET is a calmodulin dependant adenyl cyclase
• causes severe fluid and electrolyte imbalances –loss (odema and
shock)
Operate in combination and have little or no toxic action as single entities
Other toxins
InH – immune inhibitor
MprF- multiple peptide resistance factor
Anthrolysins – similar to phospholipase C and cholestrol binding
cytolysins – lysis of phagolysosome - liberate B.anthraces- lysis of cell
membrane – liberate into extra cellular environment
DIp- iron binding protein
AtxA – anthrax toxin activator
AcpA- anthrax capsule activator

19. Spores are acquired from the environment.
Phagocytosed by macrophages.
Spores germinate within the phagolysosome compartment.
Bacteria produce the regulatory proteins AtxA and AcpA
Regulate the production of LeTx, EdTx, capsule and other toxins
InH and MprF aid in the survival of B.anthracsis - in the phagolysosome.
Anthrolysins permit escape from the phagolysosome and macrophage
Production of lethal toxin triggers the hyper production of cytokines
(IL-1,6 & 8, TNF)
Multiple organ dysfunction, shock and depletion of clotting factors
Edema factor produces severe fluid and electrolyte imbalances
Resulting in localized odema and systemic shock.
Pathogenesis

20. Pathogenesis in Human

21. Cattle, Sheep and Goat
Incubation period : 1 to 5 days
Per acute form: occur in herbivores; terminate fatally in 1-2 hours,
sick animals are rarely seen.
Rapidly-developing cerebral anoxia, pulmonary edema
Death often reported as due to lightning strike.
Acute form: is fatal in less than 24 hours, the first sign is a rise in
temp. to about 104-108ºF.
Excitement follows, affected cows sometimes charge the
attendant.
Excitement stage is followed by depression, respiratory
distress, trembling staggers, convulsions, and death.
Rumen stasis and a great reduction in the amount of milk
produced.
Bloody discharge from the natural openings, particularly the
anus – most important feature.
Organism found in the excretion or the blood in fairly large
numbers at the time of death.
Anthrax

22. Rapid putrification of the carcass is characteristic.
Blood is very dark and tarry, clots poorly.
Rigor mortis is never complete.
Gas forms in the intestine, and they are generally
distended.
Mucosa is cyanotic, and blood exudes from the body.
Postmortem Lesions

26. Horses
Acute form is very common
Odematous swelling of the throat and neck region.
Colic and diarrhoea.
Death due to asphyxiation
In less acute: oedmatous swelling become generalized
and
death occurs after 2-3 days.
Spleen (black cherry jam appearance)
usually show enlargement, particularly in the horse.
 
Severe lymphoid depletion with necrosis and collapse of
the white pulp characterized by lymphocytolysis.
Multifocally, endothelial cells are hypertrophied and there
are intravascular bacilli.
Diffuse intracapsular hemorrhage and expansion of the
pericapsular fibroadipose tissue by hemorrhage and fibrin,
admixed with numerous bacilli.

27. Pigs
Have great natural resistance than herbivores.
Localization in pharyngeal tissues is typical.
Odematous swelling of the throat and neck region
Ulcerative haemorrhagic enteritis
Dogs
oThe throat will be inflamed and swollen with
gastroenteritis

28. Clinical symptoms
Microscopical examination of peripheral blood films from
dead animals
Cultural examination
Bacteriological examination of hair, wool, hide, bone, bone
meal & others
Ascoli’s Precipitation test
String of Pearl’s test ( TSA- 0.05 – 0.5 i.u. of penicillin/ml)
Fluorescent antibody technique
Bacteriophage identification
Animal Inoculation : Guinea pigs & mice are highly
susceptible.
Differential diagnosis should be made with anthracoids
both in culture and in blood smear
Diagnosis

30. B. anthracis Anthracoid organisms
1. Non motile generally motile
2. Capsulated non capsulated
3. Grows in long chains Grows in short chains
4. No turbidity in broth Turbidity in broth
5. Inverted fir tree in gelatin Atypical or absent
6. Methylene blue reduced weakly Reduced strongly
7. Haemolysis weak Strong
8. Liquefaction of gelatin is slow Rapid
9. Lecithinase reaction is weak Rapid
10. Ferments salicin slowly Rapid
11. Produces toxin neutralised by not neutralized
B. anthracis antitoxin
12. Pathogenic to g.pigs & mice non pathogenic
13. Susceptible to gamma phage not susceptible.

31. Difference between B. anthracis & Anthracoid organisms in blood
smear stained with Polychrome methylene blue stain

B. anthracis Anthracoid organisms
•Dark pink or purple coloured capsules No capsule
•Organism rod stained blue Organism rod stained blue
•Ends truncated Ends bulged and rounded
•Single, pair or short chain Usually long chain

32. Post-mortem on anthrax-suspects should not be done
Carcasses incinerated on site - Burning must be complete
Buried -well below ground level - Burying 6 feet under with
peracetic acid or 5% HCHO.
Flesh, blood, offal's and discharges from an anthrax-
infected carcass are full of anthrax germs, and thus
dangerous to animals and humans
Treatment of infected pasture should be done
Barns and fences are disinfected with lye -10% sodium
hydroxide
Boiling for 30 minutes will kill spores in utensils
Surface soil should be treated with 3% peracetic acid @ of
8 litres per square meter
Sanitation in Anthrax

33.  Penicillin has a marked therapeutic value in curing anthrax. Not less
than 1 million units daily until recovery should be given.
 Sulfonamides (sulfamerazine and sulfathiazole) @ of 1 gram/lb/day
have also been used.
 Aureomycin and terramycin are also effective.
 Immune serum has been shown to be of value.
 
 Sterne strain spore vaccine (avirulent live non encapsulated strain)
is effective- 10 million viable spores per dose.
Treatment