1
Clostridial Diseases
Dr. M. Bashashati
Department of Clinical Sciences, Section
of Poultry Diseases, Faculty of Veterinary
Medicine, University of Tehran
E. mail: mohsenbashashati@gmail.com
2
Introduction
• Four clostridial diseases:
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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
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Ulcerative enteritis (Quail Disease)
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Overview
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•
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•
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•
Introduction
Etiology
Epidemiology
Transmission
Clinical Signs
Gross Pathology
Histopathology
Immunity
Diagnosis
Intervention Strategies
5
Introduction
• Ulcerative enteritis (UE)
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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)
▫ 1×3-4 µm bacillus, singly (straight or slightly
curved rod with rounded ends)
• Closely related to c. Piliforme (subcluster XIVb)
• 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 (pH=7.2) + 8% horse plasma
 Incubation time:
▫ 1-2 days at 35-42°C 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:
- 20°C
70°C
80°C
100°C
16 y
3 hr
1 hr
3m
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
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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
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Necrotic enteritis
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Overview
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•
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Introduction
Etiology
Hosts
Transmission
Clinical Signs
Gross Pathology
Histopathology
Pathogenesis
Predisposing Factors
Diagnosis
Intervention Strategies
22
Introduction
• Necrotic enteritis (NE)
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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
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• 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
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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
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• Morphology and Staining
▫ Blood agar plates (37°C, 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
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Hosts
• Natural
▫ Chickens 2 weeks to 6 months
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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
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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)
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Clinical signs
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Often without premonitory signs
Death
Severe depression
Decreased appetite
Reluctance to move
Diarrhea
Ruffled feathers
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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
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Mottling of the serosal surface of the small intestine
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Early necrotic enteritis
32
Advanced lesion of necrotic enteritis
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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
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Hyperplasia
Fibrinoid necrosis
Cholangitis
Focal granulomatous inflammation
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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)
Hydrolysis of
▫ A
phospholipids
• Beta-toxin
Arachadonic
acid
Inflammatory
mediators
1- Contraction of blood
vessels
2- Aggregation of platelets
3- Myocardial dysfunction
4- Acute death
▫ hemorrhagic necrosis of the intestinal mucosa
36
Predisposing Factors
1. Mid-intestinal species of coccidia (plasma
proteins)
2. Management factors
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3.
4.
5.
6.
high fiber litter
bird stocking density
programmed feed changes
Intestinal stress
Cereal grain in the ration (wheat, barely and rye)
Dietary animal protein level (glycine content)
Seasonal effects
Genetic resistance (MHC and background
genome)
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These factors
Secretion of
intestinal mucosa
Proliferation of
mucolytic
bacteria
Proliferation of
C. perfringens
• Turkeys
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•
•
•
Coccidiosis
Ascaridiasis
Clinical hemorrhagic enteritis
Gender
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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
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Intervention Strategies
• Management procedures
• Vaccination
• Competitive Exclusion, Probiotics, and
Prebiotics
• Antibiotics and Anticoccidials
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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
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Competitive Exclusion, Probiotics, and
Prebiotics
• CE are Effective
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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
▫ β-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
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Botulism
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Overview
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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
1. Cultural (I-IV)
2. 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
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Morphology and Staining
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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 B<C<E
• Toxin production
▫ available water content (aw) of 0.92
51
Toxins
• Production of Type C toxin
▫ Anaerobic conditions and 10-47°C (35-37°C)
• Toxins of type C alpha
▫ C1 toxin
▫ Type D toxin
▫ C2 toxin
Bacteriophage associated
• Toxins of type C beta
▫ C2 toxin
• C1, D, A, B, E, and F toxins (nontoxic progenitor)
Protease
150-kD dichain neurotoxin
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
Loss of ACh release
Syntaxin and SNAP-25
Active metalloproteininase
HN
HC
L.
50-kD
Endocytic vesicle
receptor
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)
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▫
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Insects feeding on feces (vectors)
Carcasses of affected animals (>2000 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
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•
•
•
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
•
•
•
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•
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 < Immunogenic dose
• Carrion-eating crows and turkey vultures
▫ Resistance (antibodies to botulinal toxin)
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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)
If toxin present
▫ Two group
Inoculation with suspected serum
Signs and death
48 hr
Inoculation with suspected serum + antiserum
• Antigen capture ELISA (0.25ng/ml)
• Isolation
3-5 days
Samples
Cooked-meat medium
anaerobically
Mouse bioassay
▫ Little helpful in diagnosis 30°C
▫ Wildly distribution in gut, liver, and spleen of clinically
normal chickens
▫ In feed or environmental samples
 useful in epidemiologic studies
▫ Fluorescent antibody technique
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
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


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 37°C, 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)
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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
▫ < 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
High litter moisture
• Farm management
▫ Failing to remove dead birds
• Skin lesions
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•
•
•
▫ Overcrowding
▫ Meal time feeding
▫ Bird migration in tunnel ventilation house
Season (spring)
Nutritional deficiencies
Slow-feathering male chickens
Strains, breeds, and gender (males, > production
standards)
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
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

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