Comparison of ELISA and RT-PCR versus immune electron microscopy for detection of bovine torovirus (Breda virus) in calf fecal specimens

Abstract

Bovine Torovirus (BoTV) is an uncultivable enteric pathogen of cattle. Its failure to grow in vitro limits epidemiological studies, characterization of the virus, and development of diagnostic techniques. The objectives of this study were to develop and standardize an antigen-capture enzyme-linked immunosorbent assay (ELISA) and a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the detection of BoTV in fecal specimens. These assays were compared with immunoelectron microscopy (IEM) to evaluate their sensitivity, specificity, and efficiency as well as their advantages and limitations. Additionally, several methods to calculate ELISA cutoff values were used and compared using a statistical approach to obtain the optimal cutoff value for the ELISA. A plate cutoff ELISA value was determined to be the best method to calculate the cutoff value. The ELISA and RT-PCR assays developed in this study identified BoTV antigen and viral nucleic acids in feces without cross-reactions with the other calf enteric viruses examined. Both assays showed good agreement with IEM, with a Kappa value of 0.86 for ELISA and 0.85 for RT-PCR. The latter exhibited the higher analytical sensitivity. On the basis of the results obtained in this study, it is recommended that no single test should be used alone in an epidemiological survey because of the observed limitations of each assay. The fast and inexpensive ELISA combined with the highly specific and sensitive RT-PCR are a practical approach for future epidemiological studies of BoTV. These results should provide other researchers with the information needed to develop similar diagnostic assays for the study of BoTV.