Survey of Bovine Enterovirus in Biological and Environmental Samples by a Highly Sensitive Real-Time Reverse Transcription-PCR

Abstract

Animal enteroviruses shed in the feces of infected animals are likely environmental contaminants and thus can be used as indicators of animal fecal pollution. Previous work has demonstrated that bovine enterovirus (BEV) present in bovine feces contaminates waters adjacent to cattle herds and that BEV-like sequences are also present in shellfish and in deer feces from the same geographical area. However, little information is available about the prevalence, molecular epidemiology, and genomic sequence variation of BEV field isolates. Here we describe an optimized highly sensitive real-time reverse transcription-PCR method to detect BEV RNA in biological and environmental samples. A combination of the amplification procedure with a previously described filtration step with electropositive filters allowed us to detect up to 12 BEV RNA molecules per ml of water. The feasibility of using the method to detect BEV in surface waters at a high risk of fecal pollution was confirmed after analysis of water samples obtained from different sources. The method was also used to study the prevalence of BEV in different cattle herds around Spain, and the results revealed that 78% (78 of 100) of the fecal samples were BEV positive. BEV-like sequences were also detected in feces from sheep, goats, and horses. Nucleotide sequence analyses showed that BEV isolates are quite heterogeneous and suggested the presence of species-specific BEV-like variants. Detection of BEV-like sequences may help in the differentiation and characterization of animal sources of contamination.The environment, including surface waters, is often contaminated with enteric viruses (4, 33), a heterogeneous group of viruses that comprises enteroviruses, noroviruses (Norwalklike viruses), astroviruses, rotaviruses, enteric adenoviruses, and hepatitis A and hepatitis E viruses. Enteroviruses are characterized by their stability, both in the gastrointestinal tract and in the environment, and thus are excreted in feces in large amounts and persist in the environment for long times. As their main transmission route is the fecal-oral route, food and water contaminated with these viruses are major sources of infection. In addition, it is known that rain contributes greatly to dissemination of these viruses in the environment, and therefore, surface waters are believed to be important enteric virus reservoirs.One of the best-studied groups of enteric viruses is the human enteroviruses, in the family Picornaviridae, which have long been recognized as indicators of fecal contamination of water (1,4,5,23,25,26,33). Although less is known about animal enteroviruses, it might be expected that these viruses are also good markers of environmental fecal contamination. Moreover, as most enteroviruses have a narrow host range, usually showing a strong preference for infection of one (or a few related) animal species, their presence in water can indicate that one or a few species are the source of contamination.In this regard, we have recently shown that viruses belonging to the ...

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