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Lala Lajpat Rai University of Veterinary & Animal Sciences, Hisar

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Author: Agrawal, Anshul × End date: 2009 × Start date: 2004 ×
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    Epidemiology of enteritis in broilers and its association with α and β toxins of Clostridium perfringens
    (LUVAS, 2009) Agrawal, Anshul; Narang, Gulshan
    The present work was carried out to study the epidemiology of enteritis in broiler chickens in parts of Haryana as well as to look for its association with alpha and beta toxin of Clostridium perfringens. The data related to enteritis cases for the period from March, 08 to February, 09 were analyzed. During this period, 484 cases of enteritis were recorded in broiler chickens with overall morbidity of 3.15%. The disease was observed through out the year but maximum number of cases were recorded during winter season. Maximum cases of enteritis (158) were recorded in birds of 29-35 days of age. Maximum number of enteritis cases were associated with cocccidiosis, indicating that coccidiosis is a predisposing factor for enteritis. A total of 68 intestinal samples of poultry were screened for isolation of C. perfringens and biochemical characterization. Of the 68 samples, 49 intestinal samples (72.05%) were positive for C. perfringens. All the 49 samples gave black wool like colonies on Tryptose Sulphite Cycloserine (TSC) agar enriched with thioglycollate broth. The bacteria isolated were gram positive rod, non-motile with square ends. On TSC agar colonies were round yellowish-gray, semi-translucent and smooth with entire edge. Screening of 49 C. perfringens isolates revealed that majority of isolates (89.79%) expressed lecithinase activity and 83.60% isolates showed haemolysis on sheep blood agar. Antibiogram studies of 30 C. perfringens isolates with 11 antibiotics showed highest sensitivity to ciprofloxacin (93.3%), followed by enrofloxacin (86.6%) and moderate sensitive to chloramphenicol (76.6%), oxytetracycline (70%) and co-trimaxazol (63.3%). The organism showed 100% resistance to streptomycin and neomycin. A total of 28 C. perfringens isolates were subjected to vero cell assay to evaluate the expression of cytopathic effect (CPE). It was observed that 22 isolates (78.5%) expressed CPE in vero cell culture. Percent cytotoxicity of toxin on vero cells varied from 50.2% to 77.3% and percent dilution of toxin which showed CPE varied from 1:2 to 1:64 dilution. A total of seventy (70) intestinal fluid samples (62 from cases of necrotic enteritis and 8 cases of necrotic enteritis) were collected from dead birds and were tested by ELISA. The results revealed that five samples (7.14%) were positive for alpha toxin alone, two (2.87%) samples were positive for beta toxin alone and three (4.28%) samples were positive for both alpha and beta toxin. Out of these eight necrotic enteritis cases, four (50%) samples were positive for alpha toxin alone and three (37.5%) samples were positive for alpha and beta toxin, and one sample was negative for both of them. A total of 28 isolates of C. perfringens were tested for presence of cpa gene (alpha toxin) and cpb2 gene (beta2 toxin) by polymerase chain reaction (PCR). Number of isolates positive for alpha toxin alone were eleven (39.3%) indicates type ‘A’ Clostridium perfringens. Number of isolates positive for both alpha and beta2 toxin were nine (32.1%) indicates type ‘C’ Clostridium perfringens. Eight isolates (28.6%) were negative both for alpha and beta2 toxin indicating some other type of toxin producing Clostridium perfringens. Nucleotide sequence results for cpa gene (α toxin) revealed that three isolates of present study had 100% similarity with positive control (type ‘C’ Clostridium perfringens), whereas 98.2-100% similarity with cpa/plc gene of strains used for comparison. Nucleotide sequence results for cpb2 gene (β2 toxin) revealed that three isolates of present study had 100% similarity with positive control (type ‘C’ Clostridium perfringens).