Profiling&Molecular Typing Of Clostridium Perfringens Isolated From Broiler Chicken In China And Pakistan

Clostridium perfringens is the causative agent of avian Necrotic Enteritis(NE).NE is characterized by the necrosis of intestinal mucosa epithelial cells and threat to poultry industry globally.Recent reports demonstrated that the genetic characteristics were diverse among the strains isolated from NE chickens and healthy individuals and between different areas.In this study,feces and intestine samples of NE and healthy chickens from the different areas of China and Pakistan were collected.C.perfringens strains were isolated from these samples and identified by biochemical tests and 16s rRNA specific PCR.Meanwhile,toxin genes of isolated strains were profiled by specific toxin PCR.In addition,pulsed-field gel electrophoresis(PFGE)analysis was also performed to evaluate the genetic diversity of these isolates.Seventy-nine C.perfringens strains,confirmed by 16s rRNA sequencing,were isolated successfully from samples collected from different provinces of China and Pakistan.The toxin typing results showed that 50 out of 50(100%)isolates from Chinese samples were type A(?-toxin),and 23 out of 29(79%)isolates from Pakistan were type A(?-toxin),and 6 out of 29(21%)from Pakistan were type G(?-toxin,NetB).10 out of 50(20%)from China and 6 out of 29(21%)isolates from Pakistan were ?-2 toxin positive.The genetic backgrounds of 79 C.perfringens strains were classified by PFGE.Strains with similar band pattern were grouped into same genetic cluster,and 98%similarity means the same genotype.The results indicated that all the 79 strains were grouped into 5 clusters(A to E)and 45 PFGE profile(pulsotypes).Our results suggested that the genetic relationship of the strains is correlated to a geographical location but irrelevant to strain toxin types.The healthy chicken carried several different C.perfringens clones both within a flock and even individual birds,whereas flocks suffering from NE carry one or two clones.A multilocus Sequence Typing(MLST)scheme was developed for C.perfringens with the aim of grouping isolates with respect to disease presentation and/or host preference.Sequence data were obtained from one virulence and seven housekeeping genes for 50 C.perfringens isolates that comprised all type A and type G toxin types and were isolated from broiler chicken of China and Pakistan.Forty-nine types(STs)were identified,Minimum Spanning tree(MST)and dendrogram were constructed to identify Clonal complex analysis identified six clonal complexes,Clonal complex(CC)? and ? contained 32%,predominantly type A and type G isolates from diverse host species.Clonal complex ?,? and ? contained 68%of type A isolates examined in this study.The data obtained indicates that MLST may be used to identify host species relationships of C.perfringens isolates from geographically diverse.According to best of our knowledge this is first study to decrypt the genetic diversity of C.perfringens in China and Pakistan.These results represent a primary and significant step in understanding the epidemic characters of C.perfringens in from China and Pakistan and fundamental data sets for the disease control.