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Applied and Environmental Microbiology, July 2005, p. 3536-3543, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3536-3543.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Miguel Angel Jiménez-Clavero,{dagger} Estela Escribano-Romero, Carmen Mansilla, Nuria Gómez, Laura Córdoba, Neftal Roblas, Fernando Ponz, Victoria Ley, and Juan-Carlos Sáiz*

Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ctra. A Coruña Km 7.5, 28040 Madrid, Spain

Received 14 July 2004/ Accepted 7 January 2005

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.

* Corresponding author. Mailing address: Environmental Virology, Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Ctra. A Coruña Km 7.5, 28040 Madrid, Spain. Phone: 34-91-347-1497. Fax: 34-91-357-2293. E-mail: jcsaiz{at}inia.es.

{dagger} Present address: Laboratorio Central de Veterinaria, Ctra. Algete Km 8, 28110 Algete, Madrid, Spain.

Applied and Environmental Microbiology, July 2005, p. 3536-3543, Vol. 71, No. 7
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.7.3536-3543.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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