Epidemiological Survey Of Bovine Respiratory Viruses In Jiangsu Province And Evaluation Of The Immune Efficacy Of A Recombinant BHV-1 Vaccine Expressing E2 Gene Of BVDV-2

Bovine respiratory disease complex(BRDC)is the general term for respiratory diseases in cattle caused by many factors such as viruses,bacteria or stress and so on.BRDC can cause cattle fever,loss of appetite,depression,eye and nasal secretions,cough,and different degrees of dyspnea.The major viruses causing BRDC include Bovine viral diarrhea virus(BVDV),Bovine herpesvirus 1(BHV-1),Bovine parainfluenza type-3 virus(BPIV-3)and bovine respiratory syncytial virus(BRSV).These viruses can cause cattle respiratory tract epthilial cells damage and mild clinical symptoms when cattle were infected alone,and lead to severe clinical symptoms or death when they are coinfected with bacteria or other different viruses.BRDC is common problem in cattle all over the world including China.Annual losses caused by BRDC amount to billions of dollars worldwide,so BRDC is becoming a major issue affecting and restricting the healthy development of the global cattle industry.Scientific management and vaccination are the key to prevent and control BRDC,but currently there are still lack of effective vaccine in China.This study is based on epidemiological survey of BRDC-caused viruses of cattle in Jiangsu province and evaluation of the preliminary immune efficacy of a recombinant BHV-1 vaccine expressing E2 gene of BVDV-2,and the aim is to provide reference for the scientific prevention and control of disease in the future.Study part I:Serum samples were collected from 13 large-scale dairy farms in 7 cities areas in Jiangsu provice for detecting the antibodies against IBRV and BVDV by using ELISA,and for detecting BVDV antigen by nested RT-PCR assay.The results revealed that the seropositive rate of IBRV antibody in different farms detectd was 0%～82.1%,and the average positive rate was 21.1%(114/540).The seropositive rate of BVDV antibody in different farms detected was 0%～98%,and the average positive rate was 51.5%(237/460).The positive rate of BVDV antigen in different farms was 0%～100%,and the average positive rate was 55%(55/100).The result of genotyping of BVDV revealed that the positive rate of BVDV-1 was 11%(6/55),of BVDV-2 was 78%(43/55),11%(6/55)for both BVDV-1 and BVDV-2,but BVDV-3 was not found.These results indicated that IBR and BVD were prevalent in main scale dairy herds in Jiangsu province.Study part Ⅱ:A nested polymerase chain reaction(nPCR)assay targeting a part of glycoprotein B(gB)gene of BHV-1 was developed for the detection of BHV-1.The PCR amplification system and the reaction conditions were optimized,and the sensitivity and specificity assay were done respectively.The results showed that the two sets of primers could amplify the bands of the expected size,350bp and 250bp respectively.The nPCR assay could detect DNA of BHV-1 as low as 10’6 of dilution(i.e.0.44pg),and the sensitivity of nPCR was 10,000 times of a single round of PCR.BHV-1 was detected in 20 bovine semen samples by the nPCR assay,2 of them were found positive and gB gene was sequenced with high homology compared with BHV-1 strains by BLAST analysis with the GenBank database.The results showed that compared with virus isolation method,the nPCR was more specific,sensitive,rapid,and could be successfully used for rapid detection of BHV-1 pathogens and diagnosis of clinical diseases.Study part Ⅲ:76 bovine diseased lung tissues were collected from two slaughterhouse in Yangzhou and Huaian,and 6 kinds of viruses including BHV-1,BVDV,BRSV,BPIV-3,bovine adenovirus(BAV)and bovine coronavirus(BCV)were detected from the collected lung tissues by PCR/RT-PCR methods.The results showed that the total positive rate was 58%(44/76),the infection rate of BHV-1 was 25%,the highest,BVDV 19.7%and BRSV 13.2%,but no BPIV-3,BAV and BCV was detected in the samples.The infection rate of two kinds of viruses was 18%.Virus infection rate of meat-type yellow cattle is slightly lower than that of cows.The results showed that BHV-1,BVDV and BRSV might be the prevalent viruse pathogens of BRDC in Jiangsu area.Some cattle carried the mixed infection from different viruses,which made it difficult to prevent and control BRDC in the future.Study part Ⅳ:9 head of 6 to 8-month-old Holstein cows,negative for both BVDV and BHV-1 antigens and antibodies,were randomly divided into three groups.Group I were inoculated intramuscular(IM)by routine vaccine dose of v41B(a recombinant BHV-1 vector candidate vaccine expressing E2 gene of BVDV-2)(2mL),Group II were inoculated IM by double routine vaccine doses of v41B(4mL),and Group III were inoculated EM by DMEM medium(2mL)as a control,respectively,immunized twice for interval 4 weeks.At the same time,the vaccine safety test,serum antibody test and vaccination challenge protection test were carried out.The results showed that clinical response of experimental cattle were not obvious after each immunization.Serum antibodies of anti-BHV-1 can be detected in the first week after 1st immunization by virus neutralization test(VNT)and ELISA,and serum antibodies of anti-BVDV were detected in the second week after 1st immunization by ELISA.The antibodies of anti-BHV-1 and BVDV increased rapidly after 2nd immunization and maintained at a high level until the end of experiment(13 weeks).The difference of antibodies titre between single dose vaccinated group and double dose vaccinated group was not significant.Vaccination-challenge protective test results showed that vaccinated animals presented milder clinical signs and no BVDV viremia tested after challenging with the virulent BVDV-2 strain(XJ-04)compared to control group.The results indicated that the recombinant viral vector candidate vaccine can stimulate the animal to produce a high level of anti BVDV-2 and BHV-1 antibodies elicited,had a good safety and immune protection effect,which laid the foundation for the large-scale clinical trials and application of vaccine in the future.