Establishment And Application Of A PCR For The Detection Of Infectious Bovine Rhinotracheitis Virus In The Imported Cattle

Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis(IBR/IPV), causedby bovine herpesvirus (BHV1),is a viral disease of domestic and wild cattle.It was found primarily in beef in Colorado of the United States, IBRV was isolatedfrom the infected cattle by Madin in 1956, which was isolated from the imported cattle inChina, whereafter, seroprevalence results shown that IBRV infected friestein cow, nativescalper, buffalo and yak in Guangdong, Guangxi, Henan, Hebei, Shanghai, Shandong,Sichuan, gansu, Xinjiang, Helongjiang and Qinghai in china. The virus is distributedworld-wide, but has been eradicated from Austra, Denmark, Finland, Sweden andSwitzland and control programmes have started in some other countries.The disease is characterized by clinical signs of the upper respiratory tract,such as a(muco) purulent nasal discharge,and by conjunctivitis.Signs of general illness are fever,depression, inappetance, abortions and reduced milk yield. The virus can also infect thegenital tract and cause pustular vulvovaginitis and balanoposthitis.Mortality is low, manyinfections run a subclinical course, with latent infection and subsequent shedding of thevirus. The latent virus can be reactivated by stress conditions(such as transport, crowding)and cause clinical signs or secondary infection. IBR occurs widely distribution and hassignificant importance to milk yield, reproductive ability, labour ability and internationaltrade of the cattle industries.It is the best and most simple mean to wipe out the BHV1 antibody positive orlatent animals, which has been used to eradicate IBR by some countries, such as Austra,Denmark, Finland, Sweden and Switzland, based on the fact that the BHV1 antibody positive mean the carrier of virus.The key is to use sensitive diagnostic test enough todistinguish the positive animal. The virus neutralization test(VN) and ELISA are mostwidely used for antibody detection,the virus Can be isolated from nasal swabs taken fromthe infected animal or various organs collected at post-mortem. So far the PCR has beenused mainly to detect BHV1 DNA in semen samples. Experimentally, the PCR wasfound to be a sensitive and simple method to detect IBRV, especially to the latentinfection,which possesses widely application prospect to detect IBRV in theinternational trade of the cattle industries.During the isolation quarantine for the imported cattle in china, sera samples arecollected and detected BHV1 antibody by SN or ELISA, nasal swab samples are takenfrom the serological positive animals and inoculated into MDBK for isolating virus, thenSN was used to identified the virus that produces the CPE as BHV1 in cell cultures. Tomanipulating SN demands the skilled specialist and long time, The PCR was used as asubstitute for SN in this study, which was more simple than SN.The study succeeds in designing and synthesizing a pair of primers according to thesequence of the BHV1 gB gene, Then IBRV was inspected by polymerase chain reaction(PCR), the reaction elements was optimized, then applied to the quarantine of theimported cattle. It has shown that the PCR system has premium conditions, that is, thedenaturing temperature, the annealing temperature, the extending temperature, the circle,Mg²⁺ concentration, dNTPs concentration, primers concentration, Taq polymerase dosagewas 95℃, 64℃, 35 times, 1.6mM, 0.36 mM, 0.2 pmol/uL and 0.4U/50ul respectively.IBRV, HCV, PRRSV, PRV were amplified by the primers respectively and the specificpart of IBRV was obtained only. The inspection sensitiveness of the PCR method was1.2ng/50uL and was sensitiver than other means.In the quarantine for more than 3000 of the imported dairy cow, IBR antibodiesfrom sera were detected with ELISA and positive rate was as high as 42%(1293/3084).The nose swabs from these positive dairy cows were gathered for the virus isolation withMDBK cells, the virus isolation cultures were detected by the PCR system establishedabove,in which five cultures were found to contain the specific 362bp band of IBRVgene. The bands were cut from the gel and purified, then were sequenced. These DNAsequences were the same compared with IBR reference virus strain. These isolatedviruses were neutralized by standard BHV1 positive sera using virus neutralization testand MDBK cells didn't appear CPE. The isolated viruses with the negative sera control could make MDBK cells appear typical cytopathy: cells became round andgathered grape-like clusters, etc. These isolated viruses could be observed using electronmicroscope. The viruses appeared sphericity and partly contained envelop. The virusdiameter was about 175～200nm. The results of PCR were conformity to that ofSN,sequence detection and electron microscope test.The study shown that PCR isfeasible in the quarantine of the imported cattle.