To screen virulent Clostridium perfringens(Cp)type G strains from chickens and establish a necrotic enteritis(NE)model in yellow-feathered broilers,a total of 292 chicken intestinal and fecal samples were collected from different regions of China,including 154 healthy chicken samples and 138 suspected NE chicken samples.Isolation was performed using TSC-egg yolk agar plates,followed by PCR amplification and sequencing of the 16S rRNA gene.Multiplex PCR was used to identify toxin genes(α,β,ε,t toxins,enterotoxin,and NetB toxin)in Cp isolates.The results showed that 86 Cp strains were obtained,of which 3 from healthy chickens and 83 from diseased chickens.PCR results of Cp toxin typing revealed the presence of 900 bp(α toxin),550 bp(β toxin),and 738 bp(NetB toxin)bands,with no amplification for ε,ι toxins,or enterotoxin.Among these,18 strains were type A(20.39％),12 were type C(13.95％),and 56 were type G(65.12％).Ten G-type strains were selected for pathogenicity testing by intraperitoneally injecting 0.2 mL(1 × 109cfu/mL)into mice,and it showed that the CpZZ2,CpDYC1,and CpZD1 strains of G-type were highly pathogenic.One-day-old yellow-feathered broilers were divided into eight groups:groups 1-6 were experimental,group 7 served as a coccidia control,and group 8 was a negative control.Chickens in the experimental groups were orally infected with sporulated oocysts of Eimeria maxima at 10 days of age,replaced with high-protein diet at 13 days of age,and orally infected with CpZZ2 and CpDYC1 strains at 14 days of age,respectively.Two groups of chickens received continuous infection with above two strains for 3 days to construct a chicken NE disease model.During a 17-day observation,the survival rate and relative weight gain rate were monitored.All chickens were sacrificed,and the intestinal lesions were evaluated according to the Dahiya's scoring criteria,and the best combination of chicken NE was screened.The results showed that an acute NE model was successfully established using a combination of Eimeria maxima(1 × 104 sporulated oocysts),a high-protein diet(35％protein),and the CpZZ2 strain.This combination resulted in up to 50％mortality,with post-mortem findings of typical NE lesions,including gas-filled,thickened intestines and reddish-brown,tofu-like contents.While the high-protein+Cp(CpZZ2)and continuous Cp infection groups showed NE-related lesions,suggesting subclinical infection,no mortality or significant symptoms were observed in other groups.In this study,G-type Cp virulent strains were isolated and screened from clinical samples,and the NE of yellow-feathered broilers was induced by combined coccidiosis infection and high protein feed,and the pathogenesis model of NE in yellow-feathered broilers was established,which provided a reference for further research into NE pathogenesis and vaccine development for yellow-feathered broilers in China.
yellow-feather broilernecrotic enteritisClostridium perfringensdisease model
维普
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