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20121
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Subject: Veterinary Microbiology × Author: VINOD KUMAR, N × Type: Thesis × Thesis Type: Ph.D ×
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    (Sri Venkateswara Veterinary University, TIRUPATI – 517 502,A.P, 2012-06) VINOD KUMAR, N; SREENIVASULU, D (Major); NARASIMHA REDDY, Y; SHOBHAMANI, P; ESWARA PRASAD, P
    ABSTRACT : Multiplex PCR was standardized using specific primers for alpha, beta and epsilon toxin genes of reference strains of C. perfringens. The average detection limit of the test was found to be 1- 1.5 x 104 cfu ml -1 with purified DNA extracted from the reference strains. A total of 208 clinical samples which includes 140 from ET suspected lambs and 68 from healthy slaughtered animals were subjected to multiplex PCR screening. The bacterial lysate of 24h broth culture of clinical samples were found suitable for effective screening for C. perfringens types in clinical samples. Out of 208 samples tested by multiplex PCR from ET suspected and healthy lambs, 181 were positive for C. perfringens with overall prevalence of 87.01 per cent. Out of 140 samples from ET suspected animals, 129 (92.14%) were positive, whereas 68 samples from healthy animals revealed 52 (76.47%) positives. The toxin genotyping by multiplex PCR screening showed Clostridium perfringens type A as the predominant type with prevalence of 69.61 per cent. The prevalence of 92.30 per cent of C. perfringens type A healthy animals was found to be highly significant compared with ET suspected group .C. perfringens type C is found to be the least prevalent (11.04%) among all the C. perfringens types recovered in the present study. A prevalence of C. perfringens type D was found to be 19.33 per cent with 24.82 per cent prevalence of in ET suspected animals which is significantly higher than healthy animals (5.76%). Out of 208 samples attempted for isolation, 97 (69.28%) isolates were obtained from enterotoxaemia suspected lambs and 27 (39.70%) were from healthy lambs. Among the total 124 isolates, 110 (88.71%), 4 (3.23%) and 10 ( 8.06%) belongs to C. perfringens type A type C and D, respectively. All the isolates were obtained from initial multiplex PCR positive samples. The sequence analysis and phylogenetic analysis of the amplified toxin genes of alpha, beta and epsilon reveled cent per cent homology between respective toxin genes of isolates and when compared with published sequences of respective toxin genes homology of 96 to 100 per cent was observed, all segregated in to same phylogenetic group. All were found to be showing 82.6 to 100 per cent homology with the published sequences of respective toxin genes. When all the samples collected were subjected for multiplex PCR and isolation of C. perfringens, 181sample were found to be positive for PCR reaction and 124 isolates of C. perfringens were recovered. All the isolates recovered were from PCR positive samples. Prevalence of C. perfringens types in different districts of Andhra Pradesh revealed the presence of C. perfringens type C and type D in all the districts of AP except in Viziyanagaram where only type D was noticed. Presence of type C was found to be more with the increase in the prevalence of enteritis , mortalities and case fatality rates in the enterotoxaemia suspected flocks in all the districts. Attempts for identification of most toxigenic strain from all the 10 isolates of C. perfringens type D were made. The isolate KurCpD1 from Kurnool district was found to be producing higher concentration of epsilon prototoxin both in culture supernatant (2.11± 0.03 mg/ml) and purified epsilon prototoxin (1.75± 0.04 ) among all the isolates. Plasmid profiles of six out of 10 Clostridium perfringens type D field isolates revealed similar patterns with molecular weight ranging from 1.2 MDa to 30 MDa. All C. perfringens type C isolates obtained from the outbreak of Kurnool district showed identical plasmid profile with sizes ranging from 5 MDa to 30 MDa. The plasmid profiles of type C isolates in this study could be clearly differentiated from plasmid profiles of type D isolates. Amplification of etx gene was observed from the plasmid band of size 45 kb in type D isolates . Amplification of alpha toxin genes were observed from the plasmid of 23.5 kb size in type D siolates indicating the mixing of linear chromosomal DNA with the plasmid DNA. Clostridium perfringens type C plasmids at 37 kb showed amplification of beta toxin gene. The plasmids of C. perfringens type C and D stored at - 4 0 C and -20 0 C for 24 h and 48 h revealed diffused banding patterns indicating the rapid degradation of plasmids. In conclusion, the multiplex PCR can be employed for toxin genotyping of C. perfringens. Further, the test can be used for screening of the toxin genes in the region which helps in understanding the epidemiology of C. perfringens. Unique plasmid profiles of each toxin type of C. perfringens observed in the study can be used for identification of C. perfringens toxin types based on plasmid profiles. Prevalence of C. perfringens type C was observed in the State and is found to be increasing with the severity of enterotoxaemia along with C. perfringens type D. There is a need for development of a vaccine incorporating C. perfringens type C to control clostridial infections in sheep in the region.