ORIGINAL ARTICLE Prevalence of Escherichia coli O157 and Salmonella spp. in solid bovine manure in California using real-time quantitative PCR Z. Chen 1,2 , S. Biswas 1 , P. Aminabadi 2 , J.W. Stackhouse 3 , M.T. Jay-Russell 2 and P.K. Pandey 1 1 Deparment of Population Health and Reproduction, School of Veterinary Medicine, Davis, CA, USA 2 Western Center for Food Safety, University of California, Davis, CA, USA 3 University of California Cooperative Extensions, Eureka, CA, USA Significance and Impact of the Study: Our findings suggested that the presence of Escherichia coli O157 and Salmonella spp. in solid bovine manure may pose potential risks if untreated manure is applied as biological soil amendments. Considering the large-scale sampling used in this study, the observations provide a holistic assessment in terms of pathogen prevalence in solid bovine manure. Keywords biological soil amendments, Escherichia coli O157, pathogen, qPCR, Salmonella spp., solid bovine manure. Correspondence Pramod K. Pandey, Deparment of Population Health and Reproduction, School of Veteri- nary Medicine, Davis, CA 95616, USA. E-mail: pkpandey@ucdavis.edu 2018/2303: received 9 December 2018, revised 20 March 2019 and accepted 25 March 2019 doi:10.1111/lam.13156 Abstract The occurrence of Escherichia coli O157 and Salmonella spp. in solid bovine manure was investigated through a multi-county survey in California. Solid bovine manure samples (n = 91) were collected from 13 dairy farms located in multiple counties in California between June 2016 and August 2017. To quantify pathogens, DNA was extracted from bacteria in manure samples. Afterwards, the prevalence and levels of E. coli O157 and Salmonella spp. in solid bovine manure were determined by real-time quantitative PCR (qPCR). The prevalence of E. coli O157 and Salmonella spp. in solid bovine manure was 15Á4 and 6Á6% respectively. Escherichia coli O157 and Salmonella spp. levels in positive samples ranged from 3Á1 to 5Á3 log CFU per g and from positive (the population was <3 log CFU per g) to 5Á2 log CFU per g respectively. Surface samples of manure piles had higher prevalence and levels of E. coli O157 and Salmonella spp. than subsurface samples, while no seasonal effects on pathogen occurrence were observed. Our results indicated that solid bovine manure is a source of E. coli O157 and Salmonella spp. and the application of untreated manure as biological soil amendments may pose potential risks to public health. Introduction California is the largest dairy producer in the US. In 2014, there were 1Á8 million dairy cattle that produced over 35 million tons of manure (Californians Against Waste 2018). In California, manure in dairy barns is flushed and subsequently passes through a separator, which separates solid fractions from liquid fractions (Chastain et al. 2001). Solid fractions of manure are stacked in the form of piles before they are applied to cropland as biological soil amendments and liquid frac- tions are stored in lagoon systems (ponds). The common agricultural practice of using stacked bovine manure as biological soil amendments presents potential risks to public health if untreated manure containing human pathogens, such as E. coli O157 and Salmonella spp., is applied to cropland (Bicudo and Goyal 2003). Direct con- tamination of produce via land application of raw man- ure and indirect contamination by irrigation water that has accidental contact with raw manure has been reported (Guan and Holley 2003). Two outbreaks of Escherichia coli O157:H7 associated with lettuce were traced back to organic farms that may have used bovine manure (Nelson 1997). Hence, this emphasizes that understanding the prevalence and levels of human pathogens in bovine Letters in Applied Microbiology 69, 23--29 © 2019 The Society for Applied Microbiology 23 Letters in Applied Microbiology ISSN 0266-8254