G型产气荚膜梭菌的分离鉴定及黄羽肉鸡发病模型的建立

lsolation and identification of Clostridium perfringens type G strains and establishment of a disease model in yellow-feather broilers

为筛选鸡源G型产气荚膜梭菌(Cp)强毒菌株并建立黄羽肉鸡坏死性肠炎(NE)的发病模型,本研究从国内不同地区采集292份鸡肠道及粪便样品,其中健康鸡样品154份,疑似患NE鸡样品138份,经TSC-卵黄平板分离及16S rRNA基因的PCR扩增及测序鉴定,采用多重PCR扩增分离Cp的毒素基因(α、β、ε、ι毒素、肠毒素和坏死性肠炎毒素B(NetB),以确定分离株的毒素分型。结果显示,共获得86株Cp,其中3株从健康鸡样品中获得,83株从患病鸡样品中获得。毒素分型的PCR结果显示,分别出现约900 bp(α毒素)、550 bp(β毒素)、738 bp(NetB毒素)的目的条带;未扩增到ε毒素、ι毒素及肠毒素(506 bp);经统计后结果显示,18株Cp为A型(20.39%),12株为C型(13.95%),56株为G型(65.12%)。选择其中10株生长较好的G型Cp,通过腹腔注射0.2 mL(1×109cfu/mL)感染小鼠以评估其致病性,结果显示G型Cp分离株CpZZ2、CpDYC1和CpZD1的致病性均较强。将一日龄黄羽肉鸡分成8组,1组~6组为实验组,7组为球虫对照,8组为阴性对照,相应组别黄羽肉鸡于10日龄时经口灌服感染巨型艾美耳球虫(Eimeria maxima)孢子化卵囊,13日龄更换为高蛋白饲料,14日龄分别经口灌服感染G型Cp CpZZ2和CpDYC1株,其中两组鸡连续3 d感染上述两株G型Cp,以构建鸡NE发病模型。在17 d的观察期内统计各组肉鸡的存活率、相对增重率。同时剖杀所有鸡,根据肠道病变评分标准评价各组鸡的肠道病变,筛选致鸡NE的最佳组合。结果显示,通过上述标准判定使用巨型艾美耳球虫(1×104个孢子化卵囊)+高蛋白饲料(蛋白含量35%)+梭菌(CpZZ2)组的鸡急性NE发病模型成功构建,该组鸡因NE引起的死亡率可达50%,剖检后该组鸡肠道出现充气增粗、肠道内容物为红褐色豆腐渣样物等NE典型病变,而高蛋白饲料+Cp(CpZZ2)组及连续感染Cp组鸡虽不致死鸡,但该两组鸡小肠肠道也出现NE相关病变,可能为亚临床感染,其余各组鸡均无死亡和明显临床症状。本研究从临床分离并筛选的G型Cp强毒菌株,联合球虫感染和高蛋白饲料,诱发了黄羽肉鸡的NE,建立了黄羽肉鸡NE的发病模型,为进一步研究我国黄羽肉鸡NE的发病机制及疫苗研发奠定了基础。

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,ιtoxins,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(αtoxin),550 bp(βtoxin),and 738 bp(NetB toxin)bands,with no amplification forε,ιtoxins,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.