Distinction Between Persistent and Transient Infection in a Bovine Viral Diarrhoea (BVD) Control Programme: Appropriate Interpretation of Real-Time RT-PCR and Antigen-ELISA Test Results

Abstract

Control of bovine viral diarrhoea (BVD) in Belgium is currently implemented on a voluntary basis at herd level and mainly relies on detection and culling of persistently infected (PI) animals. The present field study was conducted during the winter of 2010/2011 to assess the performances of diagnostic assays used in the testing scheme for BVD as proposed by the two Belgian regional laboratories. Individual blood samples were collected from 4972 animals, and individual samples from the same herd were pooled (maximum of 30 individual samples per pool) and screened for the presence of Bovine Viral Diarrhoea Virus (BVDV)-specific RNA using a commercial real-time RT-PCR test (ADIAGENE). Individual samples from positive pools were then tested in parallel with the same RT-PCR test and with an antigen-capture ELISA test (IDEXX) to detect viremic animals. This study demonstrated that individual results differed according to the type of assay used (P < 0.001): 140 animals (2.8%) were positive by RT-PCR and 72 (1.4%) by antigen-ELISA. A second blood sample was taken 40 days later from 74 PCR positive animals to detect persistent viremia: 17 (23%) of these were still PCR positive and considered to be PI and the 57 that no longer tested positive were assumed to be transiently infected (TI) animals. All PI animals were positive also by antigen-ELISA at both time points. Among TI animals, 10 (16%) were positive by antigen-ELISA at the first but none at the second sampling. A highly significant difference in cycle threshold (Ct ) values obtained by RT-PCR was observed between PI and TI animals. ROC analysis was performed to establish thresholds to confirm with high probability that an animal is PI, based on the result of RT-PCR test performed on a single individual blood sample.

Circulation of bovine viral diarrhea virus – 1 (BVDV-1) in dairy cattle and buffalo farms in Ismailia Province, Egypt

“…Optimization of the real time RT-PCR protocol is a crucial step in ensuring high assay sensitivity and consistent results. The advantage of using real-time RT-PCR for BVDV screening is relatively high sensitivity in comparison to other diagnostic assays that enable detection of transiently infected animals where the viral load is relatively low [28,39] The clinical examination of those three calves revealed stunted growth and curly hair coat as expected in PI calves. Due to feasibility reasons, we could not collect additional samples from those animals three weeks later to confirm their infection status.…”

“…Due to feasibility reasons, we could not collect additional samples from those animals three weeks later to confirm their infection status. A commercially available real-time RT-PCR assay has been previuosly used to differentiate between BVDV transient infection and persistent infection [39]. Based on the study, a Ct cut-off value of 24.79 was suggested for the differentiation between the transiently infected and PI animals.…”

Evaluation of diagnostic methods to distinguish between calves persistently and transiently infected with bovine viral diarrhoea virus in respect to the presence of maternal antibodies

“…The loads of virus shed by the PIs into the environment are so high that even short contact with such animals may lead to infection in the herd (20). BVDV persistent infection is observed in 1%-2% of cattle population (13,18,19,24). In Poland, prevalence of PI animals is similar to other geographic regions of Europe.…”

“…The number of animals, which needed retesting was limited to 17.2% of PIs and 11.5% of TIs, which gave Ct values between 29.10 and 32.06 corresponding to10 4 and 10 4.87 copies/μL. Similar observation was made recently by Belgian researchers (13). Their study, however, did not include measurement of the impact of colostral antibodies.…”

“…High sensitivity of real-time RT-PCR (16) allows the testing of pooled samples (13) while reducing the costs of testing with respect to Ag ELISA on an individual sample basis (21). However, problems such as sample quality, cross-contamination (13), sensitivity and specificity of the tests (11), and the need for validation of the methods under individual laboratory conditions should be also considered when selecting RT-PCR-based method.…”