Comparison of ELISA and RT-PCR versus immune electron microscopy for detection of bovine torovirus (Breda virus) in calf fecal specimens.
(1/11)
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. (+info)
Detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle.
(2/11)
The objectives of this study were to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (< or = 6 months old), 27 young adults (52 years), 125 adults (> or = 2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV. Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves (P < 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV-positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent. (+info)
Phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events.
(3/11)
Toroviruses (family Coronaviridae, order Nidovirales) are enveloped, positive-stranded RNA viruses that have been implicated in enteric disease in cattle and possibly in humans. Despite their potential veterinary and clinical relevance, little is known about torovirus epidemiology and molecular genetics. Here, we present the first study into the diversity among toroviruses currently present in European swine and cattle herds. Comparative sequence analysis was performed focusing on the genes for the structural proteins S, M, HE, and N, with fecal specimens serving as sources of viral RNA. Sequence data published for animal and human torovirus variants were included. Four genotypes, displaying 30 to 40% divergence, were readily distinguished, exemplified by bovine torovirus (BToV) Breda, porcine torovirus (PToV) Markelo, equine torovirus Berne, and the putative human torovirus. The ungulate toroviruses apparently display host species preference. In phylogenetic analyses, all PToV variants clustered, while the recent European BToVs mostly resembled the New World BToV variant Breda, identified 19 years ago. However, we found ample evidence for recurring intertypic recombination. All newly characterized BToV variants seem to have arisen from a genetic exchange, during which the 3' end of the HE gene, the N gene, and the 3' nontranslated region of a Breda virus-like parent had been swapped for those of PToV. Moreover, some PToV and BToV variants carried chimeric HE genes, which apparently resulted from recombination events involving hitherto unknown toroviruses. From these observations, the existence of two additional torovirus genotypes can be inferred. Toroviruses may be even more promiscuous than their closest relatives, the coronaviruses and arteriviruses. (+info)
First isolation of cytopathogenic bovine torovirus in cell culture from a calf with diarrhea.
(4/11)
A cytopathogenic virus (designated the Aichi/2004 strain) was isolated in a human rectal adenocarcinoma cell line (HRT-18) from the ileum contents of a calf with diarrhea. Oval and elongated particles, approximately 100 to 170 nm in diameter, with club-shaped projections were seen in the infected culture supernatant, and torovirus-like (tubular and torus nucleocapsid) structures were seen in the infected cells by electron microscopy. An antiserum against bovine torovirus (BToV) reacted with the infected cells by immunofluorescence and neutralized the isolate. However, antisera against bovine coronavirus (BCV) failed to react with the infected cells by immunofluorescence or did not neutralize the isolate. Further, the isolate was positive for BToV by reverse transcription-PCR (RT-PCR) targeting fragments of the nucleocapsid (N), membrane (M), and spike (S) genes. Comparison of the nucleotide sequences of the PCR products with those of the published N, M, and S genes (476 to 497, 672, and 687 to 690 nucleotides, respectively) of toroviruses showed high sequence identities (up to 99.4%, 98.7%, and 94.9% for the N, M, and S genes, respectively) between the isolate and BToVs. In contrast, the isolate was negative for BCV by RT-PCR. In a serological survey of serum samples from 355 calves at 33 farms, 92% of calves were positive for neutralizing antibodies to the isolate. These results indicate that the isolate in this study was BToV and that BToV infection might be common in cattle in Japan. To our knowledge, this is the first isolation of BToV in tissue culture. (+info)
Genetic and antigenic characterization of newly isolated bovine toroviruses from Japanese cattle.
(5/11)