Clostridium notes for veterinary microbiology notes

It is notes of veterinary microbiology

2. 
Domain : Bacteria
 Phylum : Firmicutes
 Class : Clostridia
 Order : Clostridiales
 Family : Clostridiacae
 Genus : Clostridium

4. 4
Morphology

Gram-positive, long rod shaped with rounded ends (Bacilli)
Anaerobic
Oval or spherical Spore forming
(centrally, terminally or subterminally)

 Motile by peritrichous flagella (except Cl. perfirngens &
Cl. tetani type 5)
Non capsulated (except Cl.perfirngens capsulated)

5. spores produced only under anaerobic conditions
Catalase & Oxidase-Negative
strictly fermentative mode of metabolism
5

6. Spores
Wider than bacilli giving “spindle like” appearance to
bacilli (in anthrax spore not wider than bacilli)
Oval or spherical spores produced only under anaerobic

conditions
The spores are located centrally, terminally or
subterminally.

7. 7
resistant to the physical and chemical agents
remain viable in soil and dried cultures for a long time
(many years)
Boiling for 30 minutes, dry heat at 140oC for 3 hours
and autoclaving at 121oC will destroy spores.
However, freezing temperature has no effect on the
spores.
Only prolong action of chemical disinfectants at high
concentration has killing effect on the spores - 5% phenol
for 2 days and 10-20% formalin in 10 minutes

8. Clostridium tetaniDrum stick (Terminal , spherical)
Cl.botulinum Tennis racquet (Terminal, Oval)
Cl.chauvoei Cetron or Lemon (central, oval)
Cl.septicum snow shoe shape ( sub terminal, oval)
Different type of spores

9. 9

10. 10

11. 11

12. Round terminal spores give cells a “drumstick”
appearance.

13. 13

14. Habitat
Saprophytes found in Soil,
In the soil, they survive as spores.
Under favourable conditions, these spores germinate
into vegetative cells and cause infection
Also present in water, decomposing plant, Intestine
14

15. 15
Cultural characters
Anaerobic (Strict Anaerobic- Cl.tetani)
Cl.novyi are very sensitive to oxygen, whereas Cl.perfirngens
grow in microaerophilic conditions.

Common media –
Blood agar- Haemolytic colony
(Double zone haemolytic pattern seen in Cl. perfringen .)
Egg agar,
Cooked meat medium (RCM)
Thioglycolate broth

16. The colonies are 3 mm in diameter, round or slightly
irregular, slightly raised, granular, transparent or translucent
with filamentous margins
 For anaerobic condition – Anaerobib jar, GasPak system,
McIntosh-Fildes jar or by using certain reducing substances
In broth media - organisms grow at the bottom of the media
where oxygen tension is low.

17. Cl.tetani and Cl. perfringen produces haemolysis on blood agar
Reducing substances like:Glucose, thioglycolate/ thioglycolic acid sulfite or
cysteine is used to create anaerobic condition in liquid medium.
Cooked Meat Medium contains beef heart, the muscle protein, which provide
amino acids and other nutrients. Beef heart also contains glutathione, a
reducing substance that permits the growth of obligate anaerobes.
Thioglycolate (thio) broth contains Sodium thioglycolate, a reducing agent
that creates anaerobic conditions when it reduces molecular oxygen to water.
Dyes such as resazurinor methylene blue are usually added to the broth to
provide a visual indication of the presence of oxygen.
17

18. Before inoculation RCM/CMB
 medium is boiled to make it oxygen free.
After inoculation it is covered with a layer of sterile liquid paraffin oil to
prevent entry of oxygen in the medium.
The ingredients present in the medium help to maintain the anaerobic
(reduced) environment.
Unsaturated fatty acids present in meat utilize oxygen for auto-
oxidation, this reaction is catalysed by haematin in the meat.
Glutathione and cysteine (both are reducing agents) present in meat also
utilize oxygen.
Sulphydryl compounds (present in cysteine) also contribute for a
reduced oxidation-reduction potential.

19. Important species:
Clostridium tetani
Cl.botulinum
Cl.novyi, (odematiens)
Cl.chauvoei
Cl.septicum
Cl.perfringes, (welchii)
Cl.haemolyticum
19

20. previous name present name
Cl. Welchi Cl. perfringen
Cl. novyi type D Cl. haemolyticum
Cl.oedematiens Cl. novyi

21. 21

Clostridial organisms are classified into three groups
according to habitats and natural history
Many species are saprophytes closely associated with
soil and are reason for putrefaction of fruits and
vegetables.

Some are commensals that are seen in the digestive
tract
Few species are toxigenic that produce toxins to host
tissue
Classification

22. 
Toxigenic strain are classified into four groups depending
upon the tissue they affect viz,
Histotoxic clostridia – To affect muscle
(Cl.chouvoei,Cl.septicum,Cl.novyi, Cl.haemolyticum)
Enterotoxic clostridia – To digestive system
(Cl.perfringens)
Neurotoxic clostridia – To CNS
(Cl.tetani, Cl.botulinum)

23. 23
According to utilization of protein or carbohydrate in medium
Saccharolytic Proteolytic
Organisms ferment
carbohydrates of medium
Organisms utilize protein of
medium
Meat particles become pinkMeat particle becomes black
Gas bubbles are seen Gas bubbles with foul odour
1.Cl.novyi, (odematiens)
2.Cl.chauvoei,
3.Cl.septicum,
4.Cl.perfringes, (welchii)
1.Clostridium tetani,
2.Cl.botulinum
3.Cl.haemolyticum,

24. 24
Diseases in animals
Cl.chouvoei Black quarter / Black leg (B.Q.) in cattle and sheep
Cl.septicum 1. Necrotic Dermatitis in chicken
2. Braxy (Abomasitis)in sheep
3. Malignant oedema in all animals
Cl.novyi Type A - Black disease in sheep & cattle,
Type B - Big head in rams
Cl.haemolyticum Bacillary haemoglobinuria in cattle
Cl.perfringes, (welchii) 1.Type A. Necrotic enteritis in poultry,
Gas gangrene in animals,
Food poison, Enterotoximia in man
1.Type B. Lamb dysentery, haemorrhagic
enteritis in S/G
2.Type C. Struck in adult sheep
3.Type D. Pulpy kidney disease in sheep and
goats
4.Type E. Enterotoxaemia in calves/ lambs
5.Type F necrotic enteritis in man.

25. 25
Cl.tetanus Tetanus in all animals and man
Cl.botulinum 1.Type A Botulism in man

2. Type B Botullinum in man
3. Type C
a
Botullinum in duck
Limberneck in chickens
4. Type C
b Forage poisoning
5. Type D Lamzickte in cattle
6. Type E Botulism in man
Forage poisoning in horses

26. HISTOTOXIC CLOSTRIDIA

27.  Clostridium chauvoei (Large cigar shape road /
Nevicular/ Cetron)
Saccharolytic
Antigenic structure : O, H and S antigen

Spore- Oval, Subterminal
Cattle,sheep,goat,swine,deer
Ferment- Glucose, Lactose, Sucrose, Maltose
Acid and Gas produce

28. Pathogenesis
Enter through Oral or Wound infection
Multiply in the intestines
Bacteria – deposited in skeletal muscles
Dormant stage until anaerobic environment
Proliferation and release toxins (Alpha,
Beta, Gamma, Delta)
Necrosis and gas gangrene of muscles
28

29. High fever (104-107°F)
Swelling in thigh muscles
Crepitating sound on
pressure
Lameness
29

30. The affected area looks like greenish or
bluish
Oedema of subcutaneous tissue
Affected muscles have porous appearance
due to presence of gas
Black colour of muscles is due to iron
sulfide (FeS)
30

32. 32

33. 1. Based on symptom
Crepitating sound
2. Microscopic examination
Smear prepare from edematous fluid/Infected muscle Gram stain
Gram positive bacilli
3. Culture isolation- BHI, BA, Cooked meat medium, Thioglycolate
4.FAT
5.Animal inoculation- G.pig and Mice

34. 
Braxy is an acute fatal disease of sheep caused by Clostridium septicum and characterized by oedema, necrosis and haemorrhage in abomasum.
Antigenic structure- O, H, S (spore antigen)
Toxins - Alpha, Beta, Gamma, Delta, Fibrinolysin
Alpha toxin directly effect on cardiac muscle and it capable to damage capillary
Ferment- Glucose, Maltose, Lactose (Do not ferment Sucrose)
In litmus Milk- Stormy clot formation

Iron required for growth and production of alpha toxin
34

35. Pathogenesis

Transmission-Ingestion of spores (Over eating
frozen grass)
Occurs in enterotoxaemia vaccinated sheep
Spores localized in stomach

Germinate under anaerobic conditions
Abomasitis

36. Over filled stomach
Sudden mortality
History of enterotoxaemia vaccination
36

37.  Cl. Septicum
Cattle, sheep & pigs
Transmission : Wound (exogenous or endogenous)

Wound infection in animals are characterized by
rapidly extending swellings
Emphysematous Inflamation
No crepitant
Abortion

38. 1. History and symptom
2. Microscopic examination
Part of abomasums collected
3. Culture isolation
4.FAT

39. Gram positive, spore forming, anaerobic bacteria
Cysteine required for growth
Four type A,B,C,D based on toxin production
Lecithinase activity on egg yolk agar (due to beta toxin of type B &
D and gamma toxin of type A)
Lipase activity on egg yolk agar by type A
Ferment glucose, maltose (Not Lactose)

40.  Infectious Necrotic hepatitis
 Clostridium novyi type B
 Soil & normal intestinal tract of herbivores
 Host -Sheep & cattle
Dark discoloration of the skin caused by venous
congestion

43. Bacillary haemoglobinuria

( Haemorrhagic Disease )

Clostridium hemolyticum / cl. Novyi type D
 Cattle, sheep, pig
 The urine is a dark red or port-wine color, clear but
foamy

44. Caused by Clostridium perfringens
Enterotoxigenic clostridium
Gram positive bacilli, Capsulated, Non motile
Spore are rarely produce
BA- Narrow zone complete haemolysis due to theta toxin
Wider zone incomplete haemolysis due to alpha toxin
Acute disease of sheep, goat and camel (Clostridium perfringens type D-sheep
Clostridium perfringens type A-camel)
Characterized by endocardial haemorrhage, gastroenteritis and pulpy kidneys
44

45. Cl.perfringes
(Cl. welchii)
Type A. - Necrotic enteritis in poultry,

gas gangrene in animals
Type B. - Lamb dysentery
Type C - Struck in adult sheep
Type D- pulpy kidney in sheep and goats

Type E.- Enterotoxaemia in calves/ lambs
Type F - Necrotic enteritis in man.

46. Exotoxins Associated with
Cl. perfringens Types A-E
Major

47. Cl. perfringens Virulence Factors
M
a
jo
r
M
in
o
r

48. Alpha toxin- responsible for Nagler’s Reaction
Epsilon toxin- Acivated in intestine by protease
such as Trypsine

49. 49

51. Sample-Intestine
1. History and sign
2.Microscopic examination- Gram positive,
Capsulated, Non motile,
3.Cultural isolation- Anaerobic organism
BA-Double haemolysis
RCM-Meat become pink, Production of acid and gas
4. Nagler Reaction
5. Litmus milk test-Stromy clot reaction (Cl.septicum)

52. Nagler reaction-On Egg yolk agar alpha toxin of Cl. Perfringens
have lecithinase activity. Due to lecithinase activity of alpha
toxin clear zone around streak of cl.perfringens


53. Cl. perfringens Nagler Reaction
NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes
phospholipids in egg-yolk agar around streak on right. Antibody
against α-toxin inhibits activity around left streak.
+Ab -Ab

55. 55

56. Cl. TetaniCl. Tetani
Cl.botulinumCl.botulinum

57. (Lock jaw)
Caused by Caused by Cl. tetaniCl. tetani
The disease commonly attack : horse, cow, sheep, dog, pig,
chicken, rats, guinea pig & monkey.
Cl.tetani produces 2 toxins:
Tetanospasmin
Tetanolysin

58. Tetanospasmin-
Very potent neurotoxin
Responsible for tetanus
Oxygen stable and heat labile
Dimer form- Non toxic but antigenic
Monomer- Toxic
Tetanolysin
Lysis of rabbit and horse RBC
Oxygen and heat labile

60. CLINICAL SIGN
Chronic or tetanic spasm of the muscles
oLock jaw condition-Rigidity of muscle extended
from limb to the trunk, nostril get dilated and
mastication become impossible
The nostril are dilated, the tail raise

61. 61

62. 62

63. 1. Symptome- Lock jaw condition (Lumpy jaw-Actinomyces bovis)
2. Miicroscopic examination-Gram positive, Drum stick
like, Non capsulated, Motile bacilli
3. Culture examination- Strictly anaerobic
BA, RCM
4. Animal inoculation-Mice

64. ((LIMBERNECK, LAMZIEKTELIMBERNECK, LAMZIEKTE ))
Clostridium botulinum
Organism derived from sausage
Associated with food poisoning
It produce Neurotoxin
Disease due to ingestion of Toxin

65. TYPES OF BOTULISMTYPES OF BOTULISM
Infant botulism – caused by ingested spores that Infant botulism – caused by ingested spores that
germinate and release toxingerminate and release toxin
Wound botulism – spores enter through woundWound botulism – spores enter through wound
Food borne botulism – ingestion of pre-formed toxinFood borne botulism – ingestion of pre-formed toxin
65

66. PATHOGENESISPATHOGENESIS
 Incubation period is few hours to 5 daysIncubation period is few hours to 5 days
 Spores are present on foodSpores are present on food
 Potent toxin, botulinum, is releasedPotent toxin, botulinum, is released
 Toxin is carried to neuromuscular junctions and Toxin is carried to neuromuscular junctions and
blocks the release of acetylcholine, necessary for blocks the release of acetylcholine, necessary for
muscle contraction to occurmuscle contraction to occur

68. Cl.tetani Cl. botulinum
Site of toxin
production
Wounds carcases, decaying
vegetation
Mode of action centrally- blocking
Synaptic Inhibition
Peripherally –
blocking
neuromuscular
transmission
Type of paralysisSpastic Flaccid
Antigenic type of
toxin
Tetanospasmin
(one antigenic type)
8 different type of
toxins by type A-G

69. Normal resident of colon, in low numbers
Causes antibiotic-associated colitis
relatively non-invasive; treatment with broad-
spectrum antibiotics kills the other bacteria,
allowing Cl. difficile to overgrow
Produces enterotoxins that damage intestines
Major cause of diarrhea
69

71. specimens
Specimens: pus, necrotic tissue, feces, food
 Blocks of associated tissue or fluids in air free containers
Swab is keep in oxygen free gas & and after use swab is
placed in cary blair transport medium
In enterotoxemias, piece of lungs & contents of small
intestine are collected from recently dead animals

72. Direct microscopy
Gram positive rods that tend to easily decolourise when sporing
Cl.tetani - characteristic “drum stick” form may be seen in necrotic
material from wound. This is suggestive but not confirm because
Cl.tetanoides and Cl.tetanomorphum have similar morphology
Enterotoxemia - large number of gram positive rods
Cl.spiroform – curved or helical
FAT is used for Cl.chauvoei, Cl. septicum , Cl.novyi, Cl.sordellii

73. 73

74. Isolation
 Freshly prepared or prereduced blood agar is suitable for fastidious
anaerobes Like Cl.chauvoei, Cl.hemolyticum & Cl. novyi type B &Cl.
 liquid or semisolid media with low redox potential such as cooked meat
or thioglycollate broth are used for grow & maintain pure culture
All should be grown in strict anaerobic condition under 5-10 % co2

75. 75

76. Biochemical reaction
 on egg agar medium , closridia with lecithinase activity Produce
opalescence around Colonies due to enzymatic action on lecithin in the
medium while those producing lipase cause a pearly layer conering
colony and even medium
Cl.Perfringen , Cl.sordelli & Cl. novyi type A,B & D produces
opalescence due to lecithinase which is inhibited by specific
antiserum
Cl.perfringen:-
“Storming fermentation”-- clot torn by gas in 24 hrs
Lecithinase test-- precipitate formed around colony on
egg yolk media

77. 77

78. Cl. perfringens Nagler Reaction
NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes
phospholipids in egg-yolk agar around streak on right. Antibody
against α-toxin inhibits activity around left streak.
+Ab -Ab

79. 79

80. Species Spores Lecithinase Lipase Indole Urease
Cl. novyi A ST + + V -
Cl. perfringens - + - - -
Cl. septicum ST - - - -
Cl. botulinum
(types A,   B, F
proteolytic)
ST - + - -
Cl. tetani T - - V -
Cl.
sordellii/biferme
ntans
C/ST + - + +/-
Spore position: ST, sub-terminal; T, terminal; C, central; V, variable.
Simple tests to differentiate the pathogenic species of clostridia
associated with wound infection

81. Animal inoculation
 Young guinea pig or mice are used in two ways:
1.As “ Biological filter “ for contaminated specimen
contain small number of bacilli
1.In neutralization or protection test to specifically
identify the toxin

82. Prevention and control

Isolation of sick animalsIsolation of sick animals

Hygienic conditionsHygienic conditions

QuarantineQuarantine

VaccinationVaccination

83. Tetanus-Poultry are not susceptible
Serological type of Cl. tetani dist. By –H Ag
Canned food responsible for food poisoin due
to.- Cl,botulinum toxin

86. C. tetani
Control
Active immunization with tetanus toxoid
(toxin detoxified with formalin)
Aluminum salt-adsorbed toxoid
DPT vaccine
Course of immunization: as mentioned in
C. diphtheriae.
Narcotics addicts are a high-risk group.

87. penicillin or tetracycline; and muscle relaxants
 Hyperimmune serum
Symptomatic treatment
Tetanus
TIG: binds to and eliminates unbound toxins from body but
cannot affect already bound toxins, as this is an irreversible
event
87

88. 88
gas gangrene group
The organisms are highly invasive and infect when
the tissues are injured and devitalized (necrosis and
anaerobic condition).
There is septicaemia as well as toxaemia
Gas gangrene in man- Cl.septicum, Cl.edematus,
Cl.perfinges
Examples:
-B.Q., Enterotoxaemia,
-Bacillary Haemoglobinuria,
-Black disease
Histotoxic clostridia
On the basis of pathogenisity

89. 89
Tetanus group:
The organism is slightly invasive
Organism multiply at local wound and produce toxin which
spread in the body
There is only toxaemia but no septicaemia
Example: Tetanus
Botullinum group:
The organism is non invasive
Infection is present in food (not in animals) and toxin produced in
food by organisms.
Consumption of such food produce disease.
Example: Botullinum
NEUROTROPIC CLOSTRIDIA

90.  Enterotoxins are formed in the intestine
Absorbed into blood stream and producing generalised
toxemia
 Eg.- Cl. perfringen type A-E
Cl.difficile
Cl. spiroforme

91. Infection Entoxication
Bacteria multiply within tissue &
produce toxemia
Bacteria forming powerful toxin which
is absorb by living tissues
Black Leg- Cl.chauvoei
Malignant Edema- Cl.septicum
Pulpy Kidney- Cl.perfringens
Tetanus - Cl.tetani
Botulism - Cl.botulinum