Inactivation of indicators and pathogens in cattle feedlot manures and compost as determined by molecular and culture assays.

Accurate and conservative information about pathogen inactivation rates is needed as the basis for safe manure management on beef cattle feedlots. The survival of indicators and pathogens in faecal pen manure, stockpiled manure and manure compost was measured with autochthonous indicator bacteria (Escherichia coli, Clostridium perfringens, enterococci, total coliforms) and pathogens (Listeria monocytogenes, Campylobacter jejuni) using culture and/or real-time quantitative PCR (qPCR) methods. Additionally, the manures were incubated at 20, 37, 50 and 60 °C in microcosms to quantify the persistence of autochthonous microorganisms and selected process performance surrogates (Clostridium sporogenes, green fluorescent protein-labelled E. coli and L. monocytogenes). Estimated qPCR cell counts indicated that up to four orders of magnitude more target cells were present compared with the culturable counts. Corresponding T(90) estimates were up to sixfold higher. This study demonstrates the benefits of nucleic acid-based quantification of pathogen inactivation in cattle manures and concludes that the concurrent analysis of microorganisms by molecular and culture methods provides complementary value.

Diversity and abundance of zoonotic pathogens and indicators in manures of feedlot cattle in Australia.

The occurrence of 10 pathogens and three fecal indicators was assessed by quantitative PCR in manures of Australian feedlot cattle. Most samples tested positive for one or more pathogens. For the dominant pathogens Campylobacter jejuni, Listeria monocytogenes, Giardia spp., Cryptosporidium spp., and eaeA-positive Escherichia coli, 10² to 107 genome copies g⁻¹ (dry weight) manure were recovered.

Monitoring bacterial indicators and pathogens in cattle feedlot waste by real-time PCR.

Quantitative microbial health risk assessment requires accurate enumeration of pathogens in hazard-containing matrices as part of the risk characterization process. As part of a risk management-oriented study of cattle feedlot waste contaminants, we investigated the utility of quantitative real-time PCR (qPCR) for surveying the microbial constituents of different faecal wastes. The abundance of Escherichia coli and enterococci were first estimated in five cattle feedlot waste types from five localities. Bacteria were quantified using two culture methods and compared to the number of genome copies detected by qPCR targeted at E. coli and Enterococcus faecalis. Bacterial numbers detected in the different wastes (fresh faeces, pen manure, aged manure, composted manure, carcass manure compost) ranged from 10-(7) to 10(2)g(-1) (dry weight). Both indicator groups were detected by qPCR with a comparable sensitivity to culture methods across this range. qPCR measurements of E. coli and E. faecalis correlated well with MPN and spread plate data. As a second comparison, we inoculated green fluorescent protein (GFP) labeled reference bacteria into manure samples. GFP labeled E. coli and Listeria monocytogenes were detected by qPCR in concentrations corresponding to between 18% and 71% of the initial bacterial numbers, compared to only 2.5-16% by plating. Our results supported our selection of qPCR as a fast, accurate and reliable system for surveying the presence and abundance of pathogens in cattle waste.