Epidemiological Investigation Of BVD,IBR And PR On Cattle Farms In Shandong Province During 2017-2019 And Study On Pathogenic Mechanism Of Co-infection Of BVDV And BHV-1

Bovine viral diarrhea(BVD)is an infectious disease that mainly infects ruminants caused by bovine viral diarrhea virus(BVDV).BVDV is a member of the genus Pestivirus of the Flaviviridae family.Bovine viral diarrhea(BVD)can cause diarrhea,decreased fecundity,decreased milk production,slow growth of fetus,respiratory symptoms,continued infection of calves,and also cause abortion,fetal deformity,mummification,stillbirth and other reproductive disorders.The persistent infection of cattle will maintain the state of viremia and continue to induce excretion of the virus.The disease is prevalent all over the world,which brings serious economic losses to the cattle industry.Bovine infectious rhinotracheitis(IBR)is an acute,hot,and contagious disease of cattle caused by bovine infectious rhinotracheitis virus(IBRV,also known as BHV-1).IBRV belongs to the herpesvirus family,?-herpesvirus subfamily,varicella virus genus member.IBR mainly manifests as nasal tracheal inflammation,dyspnea,runny nose and other symptoms,which can cause pustular vulvovaginitis,balanitis,conjunctivitis,calf meningoencephalitis,mastitis,abortion.The mortality caused by IBR is not high,but it affects the growth,milk production and international trade of cattle,and seriously affects the healthy development of cattle industry.Both BVDV and BHV-1,can cause immunosuppression,making animals more likely to be infected by other pathogens,causing more serious clinical symptoms.These two viruses are closely related to the ‘shipping fever' of cattle,are the two main pathogens causing the respiratory syndrome of cattle(BRDC),and are the epidemic that the cattle industry pays close attention to and pays attention to.Pseudorabies(PR),also known as Aujeszky's disease(AD),is caused by pseudorabies virus(PRV).PRV is a linear double stranded DNA virus.Like BHV-1,PRV belongs to herpesviridae,?-herpesvirus subfamily and varicella virus genus.PRV can infect ruminants,carnivores and rodents,and it has also been reported that PRV has a high mortality rate and a certain threat to cattle industry.From 2017 to 2019,this study continued to track the epidemic trends of the abovementioned virus diseases on large-scale cattle farms in Shandong Province.Blood samples were collected from large-scale cattle farms in different regions of Shandong Province and clinical cases were collected.The epidemiologic investigation of BVD,IBR and PR was carried out by means of serology,etiology and pathology.The co-infection status of BVD and IBR was investigated and analyzed,and the clinical situation and pathological characteristics was observed in detail.The molecular biological characteristics and genetic evolution of PRV,BVDV and BHV-1 strains isolated from cattle in Shandong Province were analyzed.A total of 333 serum samples were collected from 10 cattle farms in 10 regions of Shandong Province in 2017,451 serum samples were collected from 11 farms in 11 regions of Shandong Province in 2018,and 514 serum samples were collected from 22 farms in 16 regions of Shandong Province in 2019 for seroepidemiological investigation.The results show that the positive rate of the two diseases in Shandong Province remained high from 2017 to 2019.The positive rate of the two diseases in Shandong cattle farm was over 90% or even up to 100% in these three years.On the other hand,there was a negative field with a positive rate of 0.From 2017 to 2019,the average individual positive rate of BHV-1 in three years was 44.20%,48.56%,61.67% respectively,and the farm positive rate of BHV-1 in three years was 100%,100%,77.27% respectively;the average individual positive rate of BVDV in three years was 61.60%,59.42%,61.33% respectively,and the farm positive rate of BVDV in three years was 80%,100%,95.45% respectively,indicating that the epidemic situation of two diseases in Shandong Province was relatively serious.The above results indicate that the epidemic situation of the two diseases is serious in Shandong Province.In addition,it is worth noting that the positive rates of coinfection of the two epidemics are relatively high.The average positive rates of co-infection in the three years are 37.10%,35.92%,and 46.33%,respectively.Because the investigated cattle farms are both non immune fields of the two diseases,the serological positive rate reflects that the cattle individuals are infected or have been infected with the two viruses.The high coinfection rate of the two viruses prompts us to pay attention to the pathogenic effect and mechanism of the co-infection of the two viruses in addition to the pathogenicity of a single pathogen.Through pathogenic epidemiological investigation,a total of five BVDV strains were isolated,all of which were NCP biotype,which were subtypes 1b,1d,1f,1m and 1n respectively;one BHV-1 strain was isolated,which was BHV-1.2 subtype.In order to investigate the infection of PR in cattle,we randomly sampled the PRV gE antibody of 246 serum samples from nine cattle farms for PRV gE antibodies.No PRV gE antibody-positive cattle were found,that is to say,no surviving cattle infected with PRV were found.However,during the epidemiological investigation,a case of PRV naturally infected cattle was found.All 9 infected cattle died,and the remaining uninfected cattle were urgently eliminated.This case made us deeply appreciate the risks brought by PRV to the cattle industry.We conducted a pathological examination of the dead cow in this case and collected tissue materials such as brain,liver,heart,etc.,and carried on the research of histopathological observation and virus isolation and identification.The autopsy of dead cattle only showed obvious hyperemia of the meninges,and no obvious ocular lesions were seen in other tissues and organs.Histopathological observation showed that the brain of dead cattle had typical viral encephalitis lesions,and subsequently a PRV strain(SDLY-China-2018)was isolated from the brain tissue.The genetic evolution analysis,virulence and pathogenicity of the PRV strains were carried out,and it was finally confirmed that the PRV strains isolated were closely related to the PRV strains which broke out in pig farms in China since 2012,and also had strong virulence and pathogenicity.Before the emergence of PRV variants,the cattle industry has been in a stable state of PR for a long period time in China,so it reminds us that the PRV mutation not only increases the pathogenicity of pigs,but also poses a threat to the health of animals other than pigs and even humans.In view of the high co-infection positive rate of BVD and IBR,BVDV can form a persistent infection,BHV-1 can form a latent infection,and both of them can cause immunosuppression in infected organisms.To study whether the two viruses have synergistic pathogenicity and co-infection pathogenesis,we carried out experiments of co-infection of BVDV and BHV-1 in vitro.It was found that co-infection inhibited the replication of the two viruses in MDBK cells and BT cells compared with single infection.In addition,the coinfection of BVDV and BHV-1 decreased the expression of LC3-? compared with BVDV alone,that is,the level of autophagy decreased compared with BVDV alone.Flow cytometry analysis found that co-infection increased the level of apoptosis caused by BVDV,but decreased the level of apoptosis caused by BHV-1.Since BVDV can induce autophagy and virus replication is enhanced when autophagy is enhanced,the autophagy level of MDBK cells induced by BVDV decreases when the two viruses are co-infected,which may be one of the reasons for the inhibition of BVDV proliferation during co-infection.BHV-1 can induce apoptosis of MDBK cells and cause the cells to die quickly,so that the progeny virus can be released quickly and infect more cells,thereby promoting the proliferation of BHV-1.Coinfection reduces the level of apoptosis of MDBK cells.Therefore,the release of the BHV-1 progeny virus is blocked,so this may be one of the reasons for the co-infection causing the proliferation of BHV-1 to be weakened.In addition,in order to study the relationship between the co-infection and single infection of the two viruses and the innate immune response,we used qPCR technology to detect the changes of IFN-related genes(IFN?Mx1?ISG-15)mRNA levels in MDBK cells during co-infection and single infection of the two viruses.The results showed that the natural immune response induced by co-infection of the two viruses was stronger than that induced by single infection,which may be one of the reasons why the proliferation of co-infection of the two viruses was weaker than that of single infection.