| Bovine respiratory infectious diseases usually manifest as bovine respiratory disease complex (BRDC) and are a major problem for cattle and continue to cause serious economic losses for the global cattle industry annually. BRDC is caused by stress and one or more virus infections. Bovine adenovirus type3(BAV-3) is considered one of the most important respiratory tract pathogens of cattle, and commonly involved in BRDC in feedlot cattle. BAV-3, a member of the Mastadenovirus genus in the family Adenoviridae,,is a type of non-enveloped virus with an icosahedral nucleocapsid containing a double stranded DNA genome. Bovine adenoviruses (BAVs) cause a variety of clinical signs including conjunctivitis, pneumonia, diarrhea, and polyarthritis. BAV-3infections have been reported in the USA, Canada, Finland, Belgium, Australia, Turkey and Japan since this virus was firstly isolated by Darbyshire and coworkers in Britain. Generally, BAV-3is involved in the respiratory and enteric infections of calves. The experimental infections of calves with BAV-3revealed that BAV-3was pathogenic to calves and the experimentally infected calves showed a few of clinical signs related with bovine respiratory disease, such as fever and anorexia, and consolidation of the infected lungs at necropsy were observed. As well, naturally occurring BAV-3infections were also determined and shown to cause respiratory diseases in feedlot calves. Recently, the serological investigation of BAV-3infection indicated that up to92.3%positive rate was calculated from the cattle farms. A virus strain was isolated from a nasal swab collected from a diseased feedlot cattle in Heilongjiang Province, China in2009for the first time. The isolate was confirmed as BAV-3and named as HLJ0955. However, the detection, isolation or serological evidence of BAV-3was not reported in China before2009. Subsequently, BAV-3is frequently isolated or detected from calves with respiratory diseases and has been isolated from Liaoning Province and Shandong Province in China. So far, the research on BAV-3was just preliminary and no vaccine and diagnostic reagents were available for BAV-3in China.Hexon protein is located in the surface of adenovirus and the most abundant of the structural proteins, accounting for63%of the total protein mass. Therefore the hexon protein was selected as the target viral antigen for detection of BAV-3and polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) against BAV-3were produced and used to develop a double antibody enzyme-linked immunosorbent assay (DAS-ELISA), which might prove to be useful for detecting BAV-3. To prepare monoclonal antibodies (MAbs) against hexon protein of BAV-3, the SP2/0cells were fused with the splenic cells from BALB/c mice immunized with truncated hexon protein of HLJ0955expressed in E.coli and14hybridomas secreted MAbs against BAV-3were obtained after screening by indirect ELISA based on BAV-3. All the MAbs reacted with BAV-3in Western blotting and immunofluorescence staining. Further specific assays indicated that14MAbs were specific to BAV-3, but did not cross-react with bovine parainfluenza virus type3, bovine viral diarrhea virus and bovine herpesvirus type1by ELISA. In addition, the MAb1F8had a higher antibody affinity which was suitable for detecting BAV-3in laboratory animal tissue samples by immunohistochemistry staining. The preparation of the MAbs provided the basis for further epitope identifications of BAV-3hexon protein and immunohistochemistry staining for BAV-3detection.Meanwhile, a DAS-ELISA for detection of viral antigens of bovine adenovirus type3was developed based on rabbit PAbs against BAV-3and the MAb1B2, The cut-off value was calculated from OD values of fifty identified negative samples for BAV-3as6.29. When OD492nm value was greater than0.29, the sample was qualified as positive, otherwise the samples would be considered as negative. The specificity test shows that only the BAV3can be detected by the established DAS-ELISA, the minimum detection of the virus was1018TCID50per50μL. Furthermore,21positive samples were detected via this method among102nasal swabs, and the positive rate was20.6%, which indicated that the natural BAV-3infections and virus shedding occurred in cattle and needed further investigations.In summary,14hybridomas secreting MAbs against BAV-3were obtained and one MAb1F8could be used for immunohistochemistry staining for BAV-3in experimentally infected laboratory animals. Meanwhile, a DAS-ELISA for detection of viral antigens of BAV-3was developed based on rabbit PAbs against BAV-3and a MAb1B2. The detection results of102bovine nasal swabs showed that the established DAS-ELISA could be used to detect samples and pathogen investigations for BAV-3. This study provided a basis for the pathogen diagnostics for BAV-3. |
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