| Objective: Calf diarrhea is the most serious disease that harms calves,and the high morbidity and high mortality of this disease is a major obstacle to the development of the cattle industry.Bovine coronavirus(BCo V)is one of the main pathogens causing diarrhea in calves.At present,BCo V-related vaccines have been developed abroad,but there is no vaccine in my country,and there are few detection reagents,which seriously affects the diagnosis,treatment and prevention of the disease.The purpose of this experiment is to identify the pathogens of diarrhea in calves in farms(free-range households)in some areas of Xinjiang.To establish an indirect ELISA detection method for BCo V to provide reference for the diagnosis,treatment and prevention of BCo V.Methods:(1)178 calves with diarrhea were collected from large-scale farms(farmers)in some areas of Xinjiang.Five common viral nucleic acid detections were carried out by RT-PCR technology.(2)For the diarrhea disease of calves in Kashgar Cattle Farm D,after the pathogen is determined,symptomatic treatment,cause treatment and adjuvant treatment should be given to the diarrhea calves.(3)Download the complete sequences of bovine coronavirus N and S genes from Gen Bank,and use Oligo software to design specific primers for the two genes.The expression vector p ET-30a(+)-N/S was constructed to express the S/N protein of bovine coronavirus.(4)Purify the BCo V-N protein and use the purified protein as an antigen to establish an indirect ELISA method.Results:(1)The detection results of the 5 viruses were: the BCo V positive rate was 25.8%(46/178),The BRV positive rate was 56.7%(101/178),The BEV positivity rate was 0(0/178),The positive rate of BVDV was 28%(50/178),The BNo V positive rate was 0(0/178).BCo V and BRV were simultaneously detected in 24 stools,BCo V and BVDV were simultaneously detected in 5stools,BVDV and BRV were simultaneously detected in 11 stools.(2)Virus pathogen detection and bacterial isolation and culture test showed that in addition to bovine coronavirus,8 strains were pathogenic Escherichia coli.Drug susceptibility test showed that the pathogenic Escherichia coli was sensitive to 4 antibiotics,ciprofloxacin,florfenicol,amikacin and norfloxacin,and resistant to9 other antibiotics.After active and comprehensive treatment of the calves on the farm,all the calves with diarrhea were cured.(3)Prokaryotic expression of bovine coronavirus N and S genes.The size of the N gene fragment is 1347 bp,and the relative molecular mass of the recombinant protein BCo V-N is 60 k Da.It exists in both the supernatant and the pellet,and the expression levels in the supernatant and the pellet are almost equal;The size of the S gene fragment is 4092 bp,and the relative molecular mass of the recombinant protein BCo V-S is 164 k Da,which mainly exists in the supernatant.The optimal induction and expression conditions of recombinant protein BCo V-N were determined: the induction temperature was 32 °C,the final concentration of IPTG was 2 mmol/L,and the induction time was 16 h.(4)The established indirect ELISA method for bovine coronavirus antibody has good repeatability,specificity and sensitivity.Conclusion:(1)Bovine coronavirus,bovine rotavirus,and bovine viral diarrhea/mucosal virus were detected in 178 samples,and there was mixed infection in all the tested cattle farms.(2)The calf diarrhea in Kashi Cattle Farm D was caused by the mixed infection of bovine coronavirus and pathogenic Escherichia coli.All calves with diarrhea were cured,indicating that the treatment measures are effective and can be promoted in farms.(3)Both prokaryotic expressed N and S proteins of BCo V can be expressed in a soluble manner without renaturation,which is convenient for recovery,and can be recognized by bovine coronavirus immune serum,indicating that these two proteins have good specificity and reactivity.(4)The clinical test results show that: N protein can be used as a diagnostic antigen for epidemiological investigation of bovine coronavirus disease.The indirect ELISA method established in this experiment can lay a foundation for the development,prevention and treatment of bovine coronavirus antibody diagnostic reagents. |
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