Diagnosis of bovine viral diarrhea virus (Proceedings)
Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of cattle worldwide. Because of the insidious and complex pathogenesis of BVDV, laboratory diagnosis is critical in preventing and controlling BVDV infections. These same characteristics often make laboratory diagnosis challenging. A firm understanding of the disease pathogenesis is required to select the appropriate samples for diagnostic submission and then make sound interpretations of the results.
Virus isolation has historically been the most common method for identifying cattle infected with BVDV. The virus is relatively easy to isolate from a variety of specimens including serum, buffy coats (WBC's), nasal swabs, and tissue samples.Several factors must be considered in selecting the appropriate sample and method for virus isolation. Since BVDV appears to replicate best in lymphoid cells, samples that contain these cell types should be considered, especially when attempting to identify acute infections. These samples would include whole blood from which buffy coats can be isolated and lymphoid tissue such as peyer's patches, mesenteric lymph nodes, spleen and thymus from postmortem cattle or aborted fetuses. In cattle persistently infected with BVDV, virus can usually be isolated from serum, buffy coats and a majority of tissues (although lymphoid tissues are preferable). Sample condition is important in the success of isolating BVDV and should be considered when submitting samples. The method of virus isolation may depend on the type of BVDV infection being considered. The immunoperoxidase microtiter plate assay (IPMA) is commonly used for rapidly screening herds to identify cattle persistently infected (PI) with BVDV but is less appropriate for identifying cattle acutely infected with BVDV. Techniques involving multiple cell passage may be necessary to identify low virus titers often associated with acute infections.
Virus isolation is useful for differentiating cytopathic from noncytopathic virus. It also allows for further genotypic and antigenic characterization of the virus.
Antigen detection consists of using labeled antibodies to directly detect the presence of BVDV antigens in submitted samples. The most common application of antigen detection is the enzyme-linked immunosorbent assay (ELISA). Sensitivity and specificity of this assay can vary depending on the antibodies used and the conditions that it is performed under.
Another common use of antigen detection is directly identifying BVDV in tissues. The fluorescent antibody (FA) test is most commonly used because of the rapidity that it can be performed. This assay is often used with smear preparations made from samples such as nasal swabs, lymph nodes and spleen and is often performed on gross necropsy samples as a first line screening assay for virus presence. The sensitivity and specificity of this test varies widely.
Immunohistochemistry (IHC) identifies BVDV antigen in frozen or formalin fixed tissues. This has clear advantages as tissue morphology is maintained which allows virus to be identified in conjunction with histopathological findings. IHC is useful when investigating disease outbreaks that involve the respiratory, gastrointestinal or reproductive system where BVDV is suspected. Using immunohistochemistry, BVDV can be detected in properly fixed tissues for an extended period of time whereas the ability to isolate virus from fresh tissues can dissipates rapidly with time. This is especially advantageous when field samples cannot immediately be submitted to a diagnostic lab. IHC can also be used to look retrospectively for BVDV or other pathogens of interest in properly fixed tissues.
Recently, immunohistochemistry has been used to detect BVDV in skin samples of cattle persistently infected with BVDV. The amount and distribution of antigen in skin biopsies from PI cattle appears to be much different than those undergoing acute infection, making it a useful tool for identifying PI animals
Nucleic Acid Detection
Nucleic acid detection assays involve the direct identification of BVDV viral genomic RNA or a synthesized copy of the RNA called cDNA. Nucleic acid detection systems can be very sensitive and specific. However, technical and costly protocols have limited their use as a common diagnostic tool. The polymerase chain reaction (PCR) is the most commonly used nucleic acid detection assay. It is estimated that PCR amplification of BVDV nucleic acids is 10 to 1000 times more sensitive than virus isolation. Detection of BVDV by PCR requires that genomic RNA be extracted from the sample of interest. This can be problematic as RNA is rapidly degraded if exposed to Rnases, which are ubiquitous in nature. Careful sample handling is necessary to reduce this pitfall. Samples in which BVDV RNA has been detected include serum, buffy coats, nasal swabs, homogenized tissues, semen, and milk.
Specific PCR primers have been designed to differentiate between the reported genotypes of BVDV. This may be useful in designing vaccine programs aimed at controlling different genotypes of BVDV.