CHARACTERIZATION OF BETA-2 TOXIN PRODUCING Clostridium perfringens ISOLATED FROM ANIMALS, FOODS OF ANIMAL ORIGIN AND HUMAN
Loading...
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
AAU, Khanapara
Abstract
The present work was undertaken with a view to study the prevalence of C. perfringens in animals, birds, foods of animal origin and human. A total of 661 samples were collected from different sources, viz. animal faecal samples (433), bird faecal samples (93), human stool samples (49) and food of animal origin (86), which revealed 119 isolates of C. perfringens. The isolates were confirmed by the detection of C. perfringens alpha toxin (cpa) gene and were characterized in respect to beta 2 toxin (cpb2) gene and beta 2 toxin (CPB2). Sample wise distribution of C. perfringens was 16.17, 27.96, 32.65 and 8.14 percent in animals, birds, human stool and foods of animal origin, respectively. Irrespective of health status, isolation of C. perfringens was done from different animal and bird species. However, none of the samples collected from buffalo and horse yielded any C. perfringens isolate. All the human stool samples positive for C. perfringens were found to have a history of diarrhoea. Characterization of C. perfringens isolates in respect to the major toxin genes revealed presence of cpa (alpha), cpb (beta), etx (epsilon) and ιA (iota) toxin gene, either alone or with different combinations. Based on distribution of major toxin genes, a total of 25 isolates were identified to be of type A, one isolate as type B and 93 isolates to be of type C. Among them, type A was isolated from animal (16), birds (7) and human (2), while the only type B isolate belonged to animal. The remaining type C isolate of C. perfringens were recovered from animal (53), birds (19), human (14) and foods of animal origin (7). In addition to the major virulence genes, a total of 86 isolates of C. perfringens were found to be positive for beta2 (cpb2) toxin gene, while only 6 were found to bear the enterotoxin (cpe) gene. Out of the cpb2 positive isolates, 39 (55.71%) belonged to animals and 24 (92.31%) to birds. All the isolates of C. perfringens recovered from human and foods of animal origin were found to possess cpb2 gene. Among the cpe bearing isolates, 2 (2.86%) were of animal origin, 1 (3.85%) of birds, 2 (12.5) of human and 1 (14.29%) of foods of animal origin. Four randomly selected cpb2 positive C. perfringens isolates representing different sources were studied for their genetic diversity by PFGE, PCR-RFLP and gene sequencing. The PFGE of the cpb2 positive isolates of C. perfringens representing different sources revealed that the isolates of foods of animal origin and poultry faeces were closely related. On the other hand, the isolates of human stool and poultry faeces were found to be unrelated. Similar type of genetic diversity was observed between C. perfringens isolates of human stool and foods of animal origin. However, the human stool and animal faeces were found to be possibly related. The PCR-RFLP results revealed that isolates belonging to foods of animal origin and animal faeces were of same type. Similar restriction pattern was also exhibited by the cpb2 positive C. perfringens isolates recovered from poultry faeces and human stool. Gene sequencing results revealed that the cpb2 positive isolates of C. perfringens representing different sources were clustered into four main clusters. Cluster I contains cpb2 positive C. perfringens isolated from various sources. Similar is the case with the other two clusters. The fourth cluster contains only a faecal sample of poultry origin from NCBI. This indicates that C. perfringens have no specific host specificity and there may be interspecies transmission of the organisms. Protein profiling of the partially purified beta-2 toxin of randomly selected cpb2 positive strains of C. perfringens representing different sources was done to detect the presence of C. perfringens beta 2 toxin (CPB2) in the culture supernatant. The result revealed the presence of 28 kDa protein in all the isolates representing different sources besides the presence of various other protein bands. The presence of 43 kDa protein represents the alpha toxin and the presence of 35 kDa protein represents the beta toxin in all the isolates. Western blotting of the cpb2 positive C. perfringens isolates revealed that all the isolates were immunogenic.
Description
Keywords
null