Clostridial Diseases

1
Clostridial Diseases

• Dr. M. Bashashati
• Department of Clinical Sciences, Section
• of Poultry Diseases, Faculty of Veterinary
• Medicine, University of Tehran
• E. mail mohsenbashashati_at_gmail.com

2
Introduction

• Four clostridial diseases
• Ulcerative enteritis (UE)
• Necrotic enteritis (NE)
• Gangrenous dermatitis (GD)
• Botulism
• Other clostridial species from sporadic diseases
• Clostridium chauvoei
• Comb and livers of chickens with complex diseases
  and intestines and livers of ostriches with
  neuroparalytic disease
• Clostridium difficile
• Severe enteritis and enterotoxaemia in young
  ostriches
• Clostridium sordelli
• Sporadic mortality in ostriches
• Clostridium novyi and Clostridium sporogenes
• Diseases in chicks
• Clostridium piliforme and Clostridium tertium
• Tyzzer s disease and enterotoxaemia in
  psittacines

3
Ulcerative enteritis (Quail Disease)
4
Overview

• Introduction
• Etiology
• Epidemiology
• Transmission
• Clinical Signs
• Gross Pathology
• Histopathology
• Immunity
• Diagnosis
• Intervention Strategies

5
Introduction

• Ulcerative enteritis (UE)
• Acute bacterial infection
• Young chickens, turkeys, and upland game birds
• Sudden onset and rapidly increasing mortality
• Quail disease is another name
• Worldwide distribution
• No significance public health
• 1907
• First recorded in the United States in quail

6
Etiology

• Clostridium colinum
• Gram-positive, pleomorphic, anaerobic, nonmotile,
  spore-forming rod (Oval and sub terminal)
• 13-4 µm bacillus, singly (straight or slightly
  curved rod with rounded ends)
• Closely related to c. Piliforme (subcluster
  XIV-b)
• In the past, Corynebacterium perdicum

7

• Blood smear from quail with UE

8

• Growth Requirements
• Enriched medium and anaerobic conditions
• Best medium
• tryptose-phosphate agar (Difco) 0.2 glucose
  and 0.5 yeast extract (pH7.2) 8 horse plasma
  
• Incubation time
• 1-2 days at 35-42C anaerobically
• Colony morphology
• 1-2 mm in diameter, white, circular, convex, and
  semitranslucent and have filamentous margins
• Broth medium (as mentioned above)
• Detection 1 2-1 6 hr postinoculation
• Production of gas for no more than 6-8 hr
• After 6-8 hr, growth settles to the bottom of
  the tube

9

• Biochemical Characteristics
• Fermentation of glucose, mannose, raffinose,
  sucrose, and trehalose (Acetic and formic acids)
• Weak fermentation of Fructose and maltose
• Fermentation of mannitol by some strains
• Hydrolysis of esculin, but not starch
• Nitrite, indole, Gelatinase, Catalase, urease,
  lipase, and lecithinase are not produced
• Good growth (CMC)
• Susceptibility to Chemical and Physical Agents
• Production of spores results in permanent
  contamination of premises after an UE outbreak
• Resistance to octanol and physical changes
• Yolk cultures

- 20C 70C 80C 100C
16 y 3 hr 1 hr 3 m
10
Epidemiology

• Incidence and Distribution
• Worldwide and in a wide variety of avian species
• Hosts
• Natural
• Bobwhite quail, California quail, Gambel quail,
  mountain quail, scaled quail, sharp-tailed
  grouse, ruffed grouse, domestic turkeys,
  chickens, European partridge, wild turkeys,
  chukar partridge, pigeons, pheasants, crested
  quail, robins, lories, and Eos spp.
• Experimental
• Quails infect more readily than chickens

11

• Age of Host Commonly affected
• Mostly in young birds
• Chickens 4-12 weeks
• Turkeys 3-8 weeks
• Quail 4-12 weeks
• Outbreaks in chickens often accompany or follow
• Coccidiosis, CIA, IBD, or stress conditions
• Transmission
• Horizontal
• Fecal/oral route
• It is transmitted through droppings
• Ingestion of contaminated feed, water, or litter

12
Clinical Signs

• Incubation Period
• 1-3 days
• Course of the Disease
• 3 weeks with peak mortality occurring 5-14 days
  postinfection
• Clinical Signs
• In birds
• No premonitory signs, well muscled and fat and
  existence of feed in the crop
• In quails
• Watery and white droppings, with progression of
  UE, Listless and hump up, closed eyes and ruffled
  feathers
• Mortality
• In young quails (100) and chickens (2-12)

13
Gross Pathology

• Acute
• Marked hemorrhagic enteritis in the duodenum
  (quail)
• Small punctate hemorrhages through the serosa in
  the intestinal wall
• Perforation of the intestines, and peritonitis
• Chronic
• Necrosis and ulceration in any portion of the
  intestine and ceca
• Small yellow foci with hemorrhagic borders on
  serosal and mucosal surfaces (As ulcers increase
  in size, the hemorrhagic border tends to
  disappear)
• Central depression of ulcers in ceca with
  dark-staining material, perforation of ulcers
  resulting in peritonitis

14

• Liver lesions vary from light yellow mottling to
  large irregular yellow areas along the edges,
  gray foci or small, yellow circumscribed foci,
  sometimes with surrounding by a pale yellow halo,
  congestion, enlargement and hemorrhage of spleen
  

15
Histopathology

• Desquamation of mucosal epithelium, edema of
  intestinal wall, vascular engorgement, and
  lymphocytic infiltration,
• Early ulcers
• Small hemorrhagic and necrotic areas (villi),
  coagulation necrosis of Cells adjacent to these
  areas (karyolysis and karyorrhexis)
• Infiltration of lymphocyte and granulocyte, small
  clumps of grampositive bacteria
• Older ulcers
• Thick masses of granular, acidophilic, coagulated
  serum proteins mixed with cellular detritus and
  bacteria,
• Infiltrations of granulocytes and lymphocytes,
  and occlusion of blood vessel
• Liver
• Foci of coagulative necrosis, with minimal
  inflammatory reaction and occasional
  intralesional, gram-positive bacterial colonies,
  scattered throughout the parenchyma

16
Immunity

• Active immunity
• In birds that recover from naturally occurring
  infections
• No noticeable effect following challenge

17
Diagnosis

• Gross postmortem lesions
• Intestinal ulcerations accompanied by necrosis of
  the liver and an enlarged, hemorrhagic spleen
• Impression smear of necrotic liver tissue
• Fluorescent antibody
• Isolation and Identification
• Sample
• Liver and intestines
• Serology
• Agar gel immunodiffusion

18
Differential Diagnosis

• Coccidiosis
• In chickens, turkeys, and pheasants
• Following UE or concurrently with UE
• Necrotic enteritis
• Histomoniasis
• Caseous cores in ceca and necrotic areas in liver
  
• In chickens, turkeys, and other gallinaceous
  birds

19
Intervention Strategies

• Management Procedures
• Removing contaminated litter and use clean litter
  for each brood
• Avoiding stresses caused by overcrowding
• Keeping coccidiosis under control
• Using preventive measures against viral diseases
• Treatment
• Streptomycin (quail, 60g/ton of feed or 1 g/gal
  of water), Bacitracin methylene disalicylate,
  furazolidone, chlortetracycline, penicillin,
  ampicillin, and tylosin

20
Necrotic enteritis
21
Overview

• Introduction
• Etiology
• Hosts
• Transmission
• Clinical Signs
• Gross Pathology
• Histopathology
• Pathogenesis
• Predisposing Factors
• Diagnosis
• Intervention Strategies

22
Introduction

• Necrotic enteritis (NE)
• Young chickens
• Clostridium perfringens type A and type C
• Sudden onset, high mortality and necrosis
• Other names
• Clostridial enteritis, enterotoxemia and rot gut
• Economic Significance
• Impairment of growth rate and feed conversion
  rate
• Cause higher condemnation rates

23

• Public Health Significance
• C. perfringens type A and type C
• Toxins and enterotoxins (foodborne)
• Type A food poisoning
• Diarrhea (outbreak report)
• Type C food poisoning
• Necrotic enteritis (very low prevalence)
• History
• 1961
• Parish

24
Etiology

• C. perfringens (welchii) type A and Type C
• Gram-positive, spore-forming anaerobic
• Capable of producing various toxins
• C. perfringens type A alpha-toxin
• C. perfringens type C alpha-toxin and beta-toxin
• Alpha-toxin
• Is encoded by cpa gene
• Is influenced by inducers
• Production of these inducers
• Quorum sensing
• Down-regulation or up-regulation

25

• Morphology and Staining
• Blood agar plates (37C, anaerobically,
  overnight)
• an inner zone of complete hemolysis
• an outer zone of incomplete hemolysis
• Short to intermediate rods without spores
• Biochemical properties
• Fermentation of glucose, maltose, lactose, and
  sucrose (acetic and butyric acids)
• Hydrolysis of gelatin
• Digestion of milk
• No production of indole
• Growth on egg yolk agar
• Presence of lecithinase and no production of
  lipase

26
Hosts

• Natural
• Chickens 2 weeks to 6 months
• Broilers 2-5 week on litter
• Layers 3-6 month raised in floor pens
• Layers 12-16 week caged-reared
• Mature commercial layers in cages
• Turkeys 7-12 week and concurrent infection
  (ascarid infection and coccidiosis)
• Experimental
• Chickens, turkeys, and Japanese quail

27
Transmission

• Horizontal
• Fecal/oral route
• Feces, soil, dust, contaminated feed and litter
  (mostly), or intestinal contents
• Domestic flies
• Mechanical vector
• Biological vector
• Eggshells, hatchery fluff and chick box pads
• Vertical (ribotyping)

28
Clinical signs

• Often without premonitory signs
• Death
• Severe depression
• Decreased appetite
• Reluctance to move
• Diarrhea
• Ruffled feathers

29
Gross Pathology

• Small intestine (jejunum and ileum)
• Friable and distention with gas
• loosely to tightly adherent yellow to green
  pseudomembrane (Turkish towel appearance)
• Flecks of blood
• Swollen of liver, tan colored livers with
  necrotic foci and cholecystitis

30
Mottling of the serosal surface of the small
intestine
31
Early necrotic enteritis
32
Advanced lesion of necrotic enteritis
33
Histopathology

• Severe necrosis of the intestinal mucosa
• Abundance of fibrin admixed with cellular debris
  adherent to the necrotic mucosa
• At the apices of villi (initial lesion)
• Sloughing of epithelium and colonization with
  bacilli
• Coagulation necrosis
• Heterophils infiltration
• Extension of necrosis into the submucosa and
  muscular layers of the intestine

34

• Attachment of large bacilli to cellular debris
• Regenerative changes in disease survival
• Proliferation of crypt epithelial cells (mitosis)
• Cuboidal Epithelial cells and goblet and columnar
  epithelial cells decrease
• Short and flat villi
• Various sexual and asexual stages
• Liver
• Hyperplasia
• Fibrinoid necrosis
• Cholangitis
• Focal granulomatous inflammation

35
Pathogenesis

• Alpha and beta toxins released by C. perfringens
• Debate
• Toxin production by specific events
• Numbers of clostridia
• Obligate anaerobic bacterium (healthy chickens)
• sporadically and Low number in normal
  intestine(1 day to 5 months)
• Alpha-toxin(phospholipase C sphingomyelinase)
• A
• Beta-toxin
• hemorrhagic necrosis of the intestinal mucosa

Hydrolysis of phospholipids
Arachadonic acid
Inflammatory mediators
1- Contraction of blood vessels 2- Aggregation of
platelets 3- Myocardial dysfunction 4- Acute
death
36
Predisposing Factors

• Mid-intestinal species of coccidia (plasma
  proteins)
• Management factors
• high fiber litter
• bird stocking density
• programmed feed changes
• Cereal grain in the ration (wheat, barely and
  rye)
• Dietary animal protein level (glycine content)
• Seasonal effects
• Genetic resistance (MHC and background genome)

Intestinal stress
37

• Turkeys
• Coccidiosis
• Ascaridiasis
• Clinical hemorrhagic enteritis
• Gender

These factors
Secretion of intestinal mucosa
Proliferation of mucolytic bacteria
Proliferation of C. perfringens
38
Diagnosis

• Gross and microscopic lesions
• Isolation
• Intestinal contents
• Intestinal wall scrapings
• Hemorrhagic lymphoid nodules
• Identification
• Sandwich ELISA technique
• PCR

39
Differential Diagnosis

• UE
• Focal necrosis and ulceration in the distal small
  intestine and ceca and liver
• Eimeria brunetti
• E. maxima

40
Intervention Strategies

• Management procedures
• Vaccination
• Competitive Exclusion, Probiotics, and Prebiotics
• Antibiotics and Anticoccidials

41
Management procedures

• Addition of NaCl to poultry house dirt floors
• Placing birds on acidified litter
• Cleaning and disinfection of live haul containers
  
• 5 sodium hypochlorite solution or 0.4
  quaternary ammonia solution

42
Vaccination

• Immunity
• Active
• Passive
• Virulent strain of C. perfringens followed by an
  antibiotic treatment
• Live alpha-toxin-deficeint isolate of C.
  perfringens
• Alpha-toxin vaccines

43
Competitive Exclusion, Probiotics, and Prebiotics

• CE are Effective
• lowering numbers of C. perfringens
• Reducing the number of gross lesions
• Reducing the mortality
• Reducing the performance losses
• Probiotics
• Lactobacillus acidophilus, Streptococcus faecium
  and Bacillus subtilis
• Prebiotics
• Lactose, mannaoligosaccharide
• Other compounds
• ß-mannanase, Essential oil blends derived from
  plants

44
Antibiotics and Anticoccidials

• Prevention
• Virginiamycin, tylosin, penicillin, ampicillin,
  bacitracin, and furazolidone in the feed
• Treatment
• Lincomycin, bacitracin, oxytetracycline,
  penicillin, and tylosin tartrate in the water
• Anticoccidials
• Monensin (altering the microbial ecology of the
  ileum)
• Reducing ileal lactobacilli populations
• Increasing C. lituseburense and C. irregularis
  populations

45
Botulism
46
Overview

• Introduction
• Etiology
• Morphology and Staining
• Toxins
• Hosts
• Transmission
• Clinical Signs
• Gross Pathology
• Histopathology
• Immunity
• Diagnosis
• Intervention Strategies

47
Introduction

• Botulism
• C. botulinum
• Limberneck and western duck sickness
• Free-ranging and confinement-reared poultry and
  feral birds
• Public health significance
• Minimal (nonhuman primates and captive monkeys)

48

• History
• 1900s
• Western duck sickness
• 1917
• First report in chickens
• Incidence and Distribution
• Worldwide
• Ducks, broiler chickens, and pheasants
• Warmer months

49
Etiology

• C. botulinum
• Gram-positive and spore-forming bacterium
• Two grouping methods
• Cultural (I-IV)
• Toxigenic (A, B, C alpha, C beta, D, E, F and G)
• Mainly in human by A,B, E, and F
• Mainly in birds A, C, and E
• Natural cases in chickens, ducks, pheasants, and
  turkeys
• By type C toxigenic group

50
Morphology and Staining

• Singly or in short chains
• Motility in vegetative cells
• Subterminal or occasional terminal endospores
• Rapid autolysis and gram-variable staining
• Cell-wall lysin
• Resistance
• Heat inactivation
• A and BltCltE
• Toxin production
• available water content (aw) of 0.92

51
Toxins

• Production of Type C toxin
• Anaerobic conditions and 10-47C (35-37C)
• Toxins of type C alpha
• C1 toxin
• Type D toxin
• C2 toxin
• Toxins of type C beta
• C2 toxin
• C1, D, A, B, E, and F toxins (nontoxic
  progenitor)
• 150-kD dichain neurotoxin

Bacteriophage associated
Protease
52
Hemaggluting pr.
A
Nontoxic non-hem.
Inactive 150-kD
B
C
D
E
Protease-sensitive loops
Active 150-kD
Mild alkaline pH
F
H. 100-kD
HN
HC
L. 50-kD
Loss of ACh release
Syntaxin and SNAP-25
receptor
Endocytic vesicle
Active metalloproteininase
Presynaptic membrane
53
Hosts

• Type C botulism
• Chickens, turkeys, ducks, pheasants, and
  ostriches
• Wild life 117 avian species in 22 families
• Mammalian species
• Mink, ferrets, cattle, pigs, dogs, horses, and a
  variety of zoo mammals
• Fish
• Type C botulism in ruminants fed poultry manure
• Serious economic loss
• Laboratory rodents
• Fully susceptible
• Bioassay for toxin detection and typing (mice)

54
Transmission

• Fecal/oral route(toxins)
• Insects feeding on feces (vectors)
• Carcasses of affected animals (gt2000 MLD/gr)
• Fly larvae feeding on carcasses (104-105 MLD)
• Small crustaceans and insect larvae, In aquatic
  environments (oxygen depletion)
• Lakes with shallow sloping banks (fluctuations in
  water level)

55

• Litter and feces from infected flocks
• Potential source of infection for other animals
• Presence of organisms in the gastrointestinal
  tract of wild and domestic birds
• Type A and E (rarely)
• Consumption of spoiled human food products
• Botulism in Sea gulls, loons, and grebes
• By eating dead or dying fish
• Site of toxin production (cecum)

56
Clinical signs

• Incubation period (toxin doses)
• Several hours-2 days
• Similar (chickens, turkeys, pheasants, and ducks)
• Sitting and reluctant to move, ruffled feather
• Flaccid paralysis of legs, wings, neck, and
  eyelids
• Progression of paralytic signs
• Cranially from the legs to include wings, neck,
  and eyelids
• Drooping of wings
• Limberneck
• Comatose
• Gasping
• Death results from cardiac and respiratory
  failure
• In broiler chickens
• diarrhea with excess urates in the loose
  droppings

57
Mortality and Morbidity

• Depend on
• Amount of toxin ingestion
• Mortality up to 40 in broiler flocks
• Very high in wild birds and in pheasants reared
  on game farms
• Pathology
• Lacking gross or microscopic lesions
• Maggots or feathers in the crop of affected birds

58
Immunity

• No immunity
• Toxigenic dose lt Immunogenic dose
• Carrion-eating crows and turkey vultures
• Resistance (antibodies to botulinal toxin)

59
Diagnosis

• Differential diagnosis
• Clinical signs and lack of gross and microscopic
  lesions
• Advanced stages
• Obviuos
• Mild intoxication
• Marek s disease, drug and chemical toxicity,
  appendicular skeletal problems (mouse bioassay)
• In water fowl
• Fowl cholera and chemical toxicities (lead)
• Definitive diagnosis
• Detection of toxin in serum, crop, or
  gastrointestinal washings from morbid birds

60

• Mouse bioassay (0.12ng/ml)
• Sensitive and reliable method (serum)
• Two group
• Antigen capture ELISA (0.25ng/ml)
• Isolation
• Little helpful in diagnosis
• Wildly distribution in gut, liver, and spleen of
  clinically normal chickens
• In feed or environmental samples
• useful in epidemiologic studies
• Fluorescent antibody technique

If toxin present
Inoculation with suspected serum
Signs and death
48 hr
Inoculation with suspected serum antiserum
3-5 days
anaerobically
Samples
Cooked-meat medium
Mouse bioassay
30C
61
Treatment

• Providing water and feed for sick birds
• Sodium selenite and vitamins A, D3, and E
• Antibiotics
• Bacitracin, streptomycin, or periodic
  chlortetracycline
• Inoculation with specific antitoxin
• Valuable birds

62
Prevention and Control

• Prevention
• Disposal of dead birds
• Culling of sick birds
• Removal contaminated litter and thorough
  disinfection
• Calcium hypochlorite, iodophor or formalin
• Disinfection of areas around poultry houses
• Fly control
• Control
• Feeding lower energy diets
• Acidification of drinking water with citric acid
• Lowering gut pH
• Promotion of normal flora growth
• Inhibition of C. botulinum growth
• Heavy metal chelator (iron)
• Immunization
• Inactivated bacterin-toxoids (pheasants)

63
Gangrenous Dermatitis
64
Overview

• Introduction
• Etiology
• Hosts
• Transmission
• Clinical Signs
• Gross Pathology
• Histopathology
• Predisposing Factors
• Diagnosis
• Intervention Strategies

65
Introduction

• Gangrenous dermatitis (GD)
• Sudden onset of acute mortality
• Necrosis of the skin and subcutaneous tissue
• Necrotic dermatitis, gangrenous cellulitis,
  gangrenous dermatomyositis, avian malignant
  edema, gas edema disease, wing rot, and blue wing
  disease
• Public health significance
• Minimal

66
Etiology

• C. perfringens type A, C. septicum or
  Staphylococcus aureus
• C. septicum
• Blood agar (2.5 agar)
• Incubation (1-2 days at 37C, anaerobically)
• Oval and subterminal spores
• Fermentation of glucose, maltose, lactose, and
  salicin (acetic and butyric acids)
• Hydrolysis of gelatin
• No digestion of milk and production of indole
• Growth on egg yolk agar
• No lecithinase and lipase production

67
Host

• Mostly
• Chickens (17 days-20 weeks of age)
• Broiler chickens (4-8 week old)
• Layers (6-20 week old)
• Broiler breeder (20 week old)
• Following caponization
• Turkeys
• Commercial and breeder hens

68
Transmission

• Clostridia
• Soil, feces, dust, contaminated litter or feed
  and intestinal contents
• Staphylococci
• Ubiquitous
• Common inhabitants of skin and mucous membranes
  of poultry

69
Clinical signs

• Depression
• Incoordination
• Inappetence
• Leg weakness
• Ataxia
• Course of disease
• lt 24 hr
• Mortality
• 1-60

70
Gross Pathology

• Mostly
• Wings, breast, abdomen, or legs
• Dark reddish-purple and Weepy areas of the skin
  (devoid of feathers)
• Extensive blood-tinged edema, with or without gas
  (emphysema)
• Discoloration of Underlying musculature
• Emphysema and serosanguineous fluid in
  subcutaneous tissue
• No internal lesions (focal necrosis of liver and
  flaccid bursae of Fabricius)

71
Histopathology

• Edema and emphysema
• Numerous large, basophilic bacilli or small cocci
  within subcutaneous tissues
• Severe congestion hemorrhage and necrosis of
  underlying skeletal muscle
• Discrete areas of coagulation necrosis in liver
  with intralesional bacteria
• Extensive follicular necrosis and atrophy of
  bursae of Fabricius

72
Predisposing factors

• Immunosuppressive agents
• Environmental factors
• Poor drinker management
• Poor ventilation
• Farm management
• Failing to remove dead birds
• Skin lesions
• Overcrowding
• Meal time feeding
• Bird migration in tunnel ventilation house
• Season (spring)
• Nutritional deficiencies
• Slow-feathering male chickens
• Strains, breeds, and gender (males, gt production
  standards)

High litter moisture
73
Diagnosis

• Gross and microscopic lesions
• Isolation
• Sample
• Exudates of skin and subcutaneous
• Identification

74
Differential Diagnosis

• Contact or ulcerative dermatitis (broiler)
• Plantar pododermatitis (turkeys)
• Infectious or inflammatory process (market age
  broilers)
• Scabby hip dermatitis (broilers)
• Squamous cell carcinoma (keratocanthoma)
• Fungal dermatitis
• Vesicular lesions

75
Intervention Strategies

• Management Procedures
• Cleaning and disinfection
• Phenolic disinfectants (1500 gallons/20,000 ft2)
• Salt (60-100 pounds/1,000 ft2)
• Litter
• Improving litter condition
• Reducing litter moisture
• Acidifying litter pH
• Reducing bacterial levels
• Minimizing trauma

76

• Vaccination
• Mixed clostridial bacterin (1 day of age)
• Mixed E. coli, S. aureus and C. perfringens
  bacterin (5 weeks of ages)
• Treatment
• Chlortetracycline, oxytetracycline, erythromycin,
  penicillin, and copper sulfate in the water
• Chlortetracycline and furoxone in the feed
• Water acidification
• Citric acid
• Proprionic acid