Detection Of Pathogens From Beef Cattle With Bovine Respiratory Disease Complex And Establishment And Application Of An Insulated Isothermal PCR For On-site Detecting IBRV

Bovine respiratory disease complex?BRDC?is a common health problem for the cattle industry,which result in serious economic damage.The etiology of BRDC is complex,and the infection of virus,bacteria and mycoplasma is mainly cause in BRDC.Meanwhile,stresses caused by transport and environmental changes also play an important role in the occurrence of this disease.Now,BRDC has become a serious threat to beef cattle industry in China.However,the epidemiological data on BRDC is still limitation.The purpose of this study were to detect the pathogens from clinical samples collected from beef cattle with BRDC in Sichuan,isolate infectious bovine rhinotracheitis virus?IBRV?and establish a fluorescence PCR for detecting IBRV on-site.The results obtained are as follows:1.Detection of pathogens from beef cattle with bovine respiratory disease complex.One hundred and eight nasal swab were collected from beef cattle with BRDC in the11 beef cattle farms in Sichuan from 2016 to 2018.Total 10 pathogens,named Infectious Bovine Rhinotracheitis Virus?IBRV?,Bovine Viral Diarrhoea Virus?BVDV?,Bovine Coronavirus?BCoV?,Bovine Parainfluenza Virus type 3?BPIV3?,Bovine Respiratory Syncytial Virus?BRSV?,Bovine Adenovirus type 3?BAV3?,Mycoplasma bovis?M.Bovis?,Mannheimia haemolytica?M.haemolytica?,Pasteurella multocida?P.multocida?and Histophilus somni?H.somni?were detected by specific PCR assays.The capsular serotype of M.haemolytica was further identified by serotype specific PCR assay.The results showed that the positive rates of IBRV,BVDV,BCoV,BPIV3,BAV3,M.Bovis,P.multocida,M.haemolytica and H.somni were 37%,16.7%,4.6%,5.6%,6.5%,24.1%,6.5%,46.3%and 5.6%respectively,and BRSV was detected as negative.Differential detecting rate of individual pathogen was found among farms.However,the individual positive rates and farm positive rates of IBRV,M.Bovis and M.haemolytica were higher than that of other pathogens,and co-infection of the three pathogens was common.The main capsular serotypes of M.haemolytica was identified as type 6 and 1?38/50?.In conclusion,9 pathogens were detected from clinical samples of BRDC,and co-infection of IBRV,M.Bovis and M.haemolytica was common in BRDC of beef cattle in Sichuan province.To the best of our knowledge,this is the first detection of H.somni from BRDC in China.The results will contribute to the prevention and control of BRDC in China.2.Isolation of infectious bovine rhinotracheitis virusTen IBRV-positive samples were used to inoculated MDBK cells in order to isolate IBRV.After 4 generations of blind transmission,one cell culture was observed the typical cytopathic effect,With characteristics as cells shrinks,pulls the net,and aggregates into a grape-like.Stable cytopathy was observed from 5 to 7 generations.Then,the isolate,named IBRV/2018/SMU6352,was identified as IBRV by PCR and indirect immunofluorescence assay.After plaque purification,the virus titer of the isolate was determined to be 10⁸.25.25 TCID₅₀/mL.The complete gB and gC gene sequences?GenBank ID are MK654723 and MK654724 respectively?of the isolate were obtained by PCR amplification.Based on the genetic evolution of the gB gene sequences,the IBRV/2018/SMU6352 was identified as IBRV 1.2 type.Compared with all complete 55IBRV gB gene sequence of IBRV available in GenBank before 18 March 2019,including the sole gB gene sequence?JN787952?from China,a insertion of 6 nucleotides?GCGACG?was found in 1576-1577 of gB gene nucleotide sequence of the IBRV/2018/SMU6352,which result in 2 amino acid?GD?insertion in 525-526 of the gB amino acid sequence.The insertion amino acid is located in the extracellular domain of gB protein beyond the T cell and B cell recognition site of the extracellular domain of gB protein.This is the first description the variation in gB gene in Chinese IBRV.Compared with all complete 56 gC gene sequence of IBRV available in GenBank before 18 March2019,including the sole gC gene sequence?JN787953?from China,5 non-synonymous nucleotide substitutions?T614C,C712T,A833G,A1190G,A1247G?were found in gC gene sequence of the IBRV/2018/SMU6352,which result in 5 amino acid mutation?V205A,R238C,Q278R,E397G,E416G?.In conclusion,the IBRV-1.2 strain with the unique variations in the gB and gC amino acid sequences was firstly isolated in China,contributing to better understand of the genetic evolution of IBRV and further development of IBRV vaccine.3.Establishment and application of an insulated isothermal PCR?iiPCR?for on-site detecting IBRVIBRV is a pathogen for quarantine in import and export trade asked by OIE and China government.However,PCR assay for on-site detecting IBRV has not been reported still now.After selection of a pair of primers and a fluorescent TaqMan probe according to previous report,and optimizing the reaction system,the iiPCR assay was successfully established for the first time.The results exhibited that the assay only specially amplify the target fragment of IBRV,and do not amplify BVDV,BPIV3,BRSV,BAV3,BCoV,M.Bovis,P.multocida and M.haemolytica,indicating that this assay has a good specificity.Meanwhile,the iiPCR assay has good sensitivity and reproducibility,and the detection limits for IBRV nucleic acid reach 2.4copies/?L.The detection ration for clinical samples was same as that of a real-time quantitative PCR recommended by OIE.Notably,combined with the PetNAD nucleic acid extraction kit,it takes only 1 hour from nucleic acid extraction to report test result,with the characteristics of easy operation,fast and on-site.Furthermore,29 commercial frozen semen collected from 9 provinces were detected as 24%IBRV positive,indicating that IBRV in commercial frozen semen was common in China.In conclusion,this study provides a reliable method for on-site detecting of IBRV.