ORIGINAL ARTICLE Quantitative PCR assays to survey the bovine adenovirus levels in environmental samples K. Wong and I. Xagoraraki Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA Introduction Animal manure is often land applied without prior treat- ment, and there is a growing concern regarding transmis- sion of pathogens by land application because of the increase in concentrated animal feeding operations (CAFOs). According to USEPA (2006), faecal contamina- tion from livestock manure handling and storage facilities is one of the most prevalent sources of groundwater microbiological pollution. A recent outbreak in Canada indicates that the potential of human infections caused by zoonotic pathogens is real. More than 2300 people in the town of Walkerton, Ontario, Canada suffered gastrointes- tinal illness where seven people perished when the town’s shallow water supply was contaminated by manure patho- gens from a nearby farm after more than 5 inches of rain fell over a 5-day period in May, 2000 (Hrudey and Hrudey 2004). Bovine adenoviruses (BAdV) and enteroviruses (BEV) were suggested to be suitable agents for animal faecal indication, because they were reported to be environmen- tally stable (Pell 1997) and have been detected in surface Keywords bovine adenoviruses, faecal indicator, quantitative PCR. Correspondence Irene Xagoraraki, Department of Civil and Environmental Engineering, Michigan State University, A124 Engineering Research Complex, Michigan State University, East Lansing, MI 48824, USA. E-mail: xagorara@msu.edu 2009 ⁄ 1335: received 27 July 2009, revised 8 January 2010 and accepted 11 January 2010 doi:10.1111/j.1365-2672.2010.04684.x Abstract Aims: Previous studies suggested bovine adenoviruses (BAdVs) could be used as cattle faecal indicators. The main aim of this study was to survey the levels of BAdV in environmental samples using quantitative polymerase chain reac- tion (qPCR). Methods and Results: Two qPCR assays were developed to identify and quan- tify BAdVs in environmental samples. BAdVs were detected in all dairy manure, and in most cases, the sample concentrations were around 10 3 – 10 4 copies ml )1 . Farm tile drainage samples were also detected, but the concentrations were about 1–3 log 10 lower than the BAdV concentrations in the manure samples. The genome equivalent copy (GEC) levels of BAdV and the phage forming unit (PFU) levels of somatic phage in manure samples were comparable. Four of twenty individual cattle faeces were positive with concen- trations similar to that found in the manure samples. Sequencing results con- firmed the presence of BAdV in the environmental samples, and phylogenetic analysis indicated that BAdV 2 and 4 were the most prevalent serotypes in all the manure samples tested. The qPCR assays developed in this study showed higher sensitivity in detecting BAdV 1 and 2 than the previous published nested assay. Conclusion: The high levels of BAdV in the environmental samples may sug- gest it could be used for bovine faecal indicator. The significant levels of BAdV in the drainage samples may indicate the potential of surface water pollution by the manure applied to farm fields. Significance and Impact of the Study: This is the first study that reports the quantitative level of BAdV in environmental samples. These results could be useful when it comes to determining whether BAdV could be utilized as a bovine faecal indicator. Journal of Applied Microbiology ISSN 1364-5072 ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 605–612 605