Clostridial Diseases

Dr. M. BashashatiDepartment of Clinical Sciences,

Section of Poultry Diseases, Faculty of

Veterinary Medicine, University of Tehran

E. mail: mohsenbashashati@gmail.com

1

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

2

Ulcerative enteritis (Quail Disease)

3

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

4

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

5

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 XIV-b)

•In the past, Corynebacterium perdicum

6

Blood smear from quail with UE

7

•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

8

•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 3 m

9

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

10

•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

11

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%)

12

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

13

▫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

14

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

15

Immunity

•Active immunity

▫In birds that recover from naturally occurring infections

▫No noticeable effect following challenge

16

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

17

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

18

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

19

Necrotic enteritis

20

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

21

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

22

•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

23

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

24

•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

25

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

26

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)

27

Clinical signs

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

28

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

29

30

Mottling of the serosal surface of the small intestine

31

Early necrotic enteritis

32

Advanced lesion of necrotic enteritis

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

33

•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

34

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 vessels2- Aggregation of platelets 3- Myocardial dysfunction4- Acute death

35

Predisposing Factors1. Mid-intestinal species of coccidia (plasma

proteins)2. Management factors

• high fiber litter• bird stocking density• programmed feed changes

3. Cereal grain in the ration (wheat, barely and rye)

4. Dietary animal protein level (glycine content)5. Seasonal effects 6. Genetic resistance (MHC and background

genome)

36

Intestinal stress

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

These factors

Secretion of intestinal mucosa

Proliferation of mucolytic bacteria

Proliferation of C. perfringens

37

Diagnosis

•Gross and microscopic lesions •Isolation

▫Intestinal contents ▫Intestinal wall scrapings ▫Hemorrhagic lymphoid nodules

•Identification •Sandwich ELISA technique•PCR

38

Differential Diagnosis

•UE▫Focal necrosis and ulceration in the distal

small intestine and ceca and liver •Eimeria brunetti•E. maxima

39

Intervention Strategies

•Management procedures

• Vaccination

•Competitive Exclusion, Probiotics, and Prebiotics

•Antibiotics and Anticoccidials

40

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

41

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

42

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

▫β-mannanase, Essential oil blends derived from plants

43

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

44

Botulism

45

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

46

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)

47

•History ▫1900s

Western duck sickness ▫1917

First report in chickens •Incidence and Distribution

▫Worldwide ▫Ducks, broiler chickens, and pheasants ▫Warmer months

48

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

49

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 B<C<E

• Toxin production ▫available water content (aw) of 0.92

50

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

•Toxins of type C beta ▫C2 toxin

•C1, D, A, B, E, and F toxins (nontoxic progenitor)

150-kD dichain neurotoxin

Bacteriophage associated

Protease

51

52

A

B C D E

F

Inactive 150-kDInactive 150-kD

Active 150-kDActive 150-kD

H.100-kD

L.50-kD

Protease-sensitive loops

Nontoxic non-hem.

Hemaggluting pr.

Mild alkaline pH

HN HC

receptorPresynaptic membrane

Endocytic vesicle

Active metalloproteininase

Syntaxin and SNAP-25

Loss of ACh release

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)

53

Transmission•Fecal/oral route(toxins)

▫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)

54

•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)

55

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

56

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

57

Immunity

•No immunity ▫Toxigenic dose < Immunogenic dose

•Carrion-eating crows and turkey vultures ▫Resistance (antibodies to botulinal toxin)

58

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

59

• 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

Inoculation with suspected serum

Inoculation with suspected serum + antiserum

If toxin present

48 hr

Signs and death

Samples Cooked-meat medium anaerobically

3-5 days

30°C

Mouse bioassay

60

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

61

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)

62

Gangrenous Dermatitis

63

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

64

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

65

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

66

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

67

Transmission

•Clostridia ▫Soil, feces, dust, contaminated litter or feed

and intestinal contents•Staphylococci

▫Ubiquitous▫Common inhabitants of skin and mucous

membranes of poultry

68

Clinical signs

•Depression•Incoordination•Inappetence•Leg weakness•Ataxia•Course of disease

▫< 24 hr •Mortality

▫1-60%

69

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)

70

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

71

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, > production

standards)

72

High litter moisture

Diagnosis

•Gross and microscopic lesions •Isolation

▫Sample Exudates of skin and subcutaneous

• Identification

73

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

74

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

75

•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

76