Development Of Indirect ELISA For Detection For Specific Antibodies Against Infectious Bovine Rhinotracheitis Virus And Application In Epidemiological Investigation

Infectious bovine rhinotracheitis(IBR) caused by bovine herpersvirus-1(BHV-1),was an acute, pyrexic and contagious disease, and incurred great economic loss to theglobal cattle husbandry. It is a major pathogen of cattle causing respiratory and genitaltract infections such as infectious bovine rhinotracheitis, abortion, infectious pustularvulvovaginitis, and systemic infection in neonates (Muylkens et al., 2007). SinceBHV-1 was firstly demonstrated in 1928 by Reisinger and Reimarm, IBR has occurredon all continents, although the prevalence and incidence are different. Therefore,control programs were rapidly developed in North America and Europe. However,only a small number of countries have achieved the goal of IBR-eradication includingAustria, Denmark, Finland, Norway, Sweden and Switzerland.IBR was listed as one of B diseases by OIE, and was the main object quarantined inthe international trade. At present, main measure to control the IBR is to inoculatevaccines and eradicate IBR is to slaughter the infected cattle after detection. Therefore,it is very important to establish a sensitive, specific and rapid detection method.The genome of IBRV is a linear double-strand DNA of approximately 138 kb, withthe content of G+C 71-72%. It consists of two unique parts called the long unique(UL) and the short unique(US), and the two reverse repeat units are located at internaland terminus respectively. IBRV contains 54 to 59 genes, encoding 11 glycoproteinsand other structural and non-structural proteins. IBRV gG is a secretedglycosaminoglycan, which is nonessential for virus growth (Nakamichi et al., 2000),but it is required for efficient cell-to-cell transmission in bovine kidney cells. It wasspeculated that gG functions in maintaining the cell-to-cell junctional adhesion duringviral infection. Furthermore, gG was found to elicit a strong antibody response andtherefore, was widely exploited for use as a diagnostic reagent for detection of otherherpesvirus infection. This dissertation was mainly targeted to stablish a diagnosticmethod by using gG of IBRV to detect the antibodies. This novel method was furtherutilized to determine the nationalwide seroprevalence status of the cow population. Itwould be of significance to make a potential surveillance program. The research results are summarized as follows:1. Taking the genome DNA of infectious bovine rhinotracheitis virus (IBRV) as thetemplate, the gG gene was amplified with PCR and was cloned into the T cloningvector pMD18-T. After being identified by restriction digestion and DNA sequencing,the insert was subcloned into the expression vector pGEX-KG. Sodium docecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot assayshowed that this gene was expressed as both soluble form and inclusion body by thetransformed E. coli BL21 strain (DE3).2. The fusion protein was purified and used as the coating antigen to develop theindirect Enzyme-Linked Immunosorbent Assay (iELISA). The optimal conditions forthe ELISA were first established by matrix titration. The amount of gG protein forcoating ELISA plates were determined to be 0.585μg/well. The sera dilution wasdetermined to be 1:50. The cut-off value expressed as OD₆₃₀ was established to be0.299 calculated as mean(X)±0.025 based on the results of 60 negative sera samples.Comparison between this gG-ELISA and commercial IBRV gB-ELISA Kit (IDEXX)was made by testing 380 cow serum samples. The results demonstrated a goodagreement of 92%. At the same time, comparison between this gG-ELISA and thevirus neutralization test with Bartha Nu/67 IBRV strain was made by detecting 38cow serum samples. The results demonstrated an agreement of 74%.To further evaluate the specificity of the test, the gG-ELISA was used to test apanel of irrelative reference antisera, including antisera against cows with foot andmouth disease(FMD), bovine viral diarrhea-mucosal disease (BVD), BovineTuberculosis (BT), Babesia Bovis (BB), bovine Eperythrozoon disease (BED), bovinebrucellosis (Br) by using reference positive antisera against IBR as the positivecontrol. As a result, only the IBR positive control was detected to be positive bygG-ELISA, indicating the gG-ELISA was highly specific.To test reproducibility of the gG-ELISA, 6 serum samples with different antibodylevels, ranging from negative to highly positive were tested repeatedly (6 times) withELISA plates coated with two batches of gG protein. Each serum sample was assayedsix times with each batch of coated plates. Data showed there were no significant differences among the six repetitions for each sample.3. Serological survey of infectious bovine rhinotracheitis in ChinaTo understand the nationwide seroprevalence of IBR in China, the stratified randomsampling was carried out. 1344 sera of dairy cows from 29 provinces were collected.In addition, we collected 765 sera from 6 herds representing different farm sizes andlocations in two main dairy farming areas in Hubei province. Another 483 sera werefrom imported cows for entry inspection. The results demonstrated that the overallseroprevalence is 35.8% (481/1344) and each province has positive samples. However,the prevalence rates vary greatly in different provinces from 12.1%-77.8%. Among765 sera in Hubei Province, the overall seroprevalence rate was 22.2%(170/765)which is similar to the result obtained from the national sampling. However, theprevalence rates vary greatly from farms to farms, ranging from 0.0% to 41.5%. Thesera from imported cows had a prevalence of 21.7% (105/483).