Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.

UNLABELLED: Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE: The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.

Veterinary antimicrobials in cattle feedlot environs and irrigation conveyances in a high-intensity agroecosystem in southern Alberta, Canada.

The South Saskatchewan River Basin (SSRB) is considered one of the most intensively farmed regions in Canada, with high densities of livestock and expansive areas of irrigated cropland. We measured concentrations of seven veterinary antimicrobials (VAs) in 114 surface water samples from feedlot environs and 219 samples from irrigation conveyances in the SSRB. Overall, detection frequencies in feedlot environs were 100% for chlortetracycline (CTC) and tetracycline (TC), 94% for monensin (MON), 84% for tylosin (TYL), 72% for lincomycin (LIN), 66% for erythromycin (ERY), and 23% for sulfamethazine (SMZ). For irrigation conveyances, detection frequencies for CTC and TC remained high (94-100%), but dropped to 18% for ERY, 15% for TYL, 10% for MON, and 4% for SMZ. Lincomycin was not detected in irrigation conveyance water. Maximum concentrations of VAs ranged from 1384 µg L-1 (TC) to 17 ng L-1 (SMZ) in feedlot environs while those in irrigation conveyances were 155 ng L-1 (TC) to 29 ng L-1 (ERY). High detection frequencies and median concentrations of VAs in both feedlot environs and irrigation conveyances were associated with high amounts of precipitation. However, an irrigation district (ID) with high livestock density (Lethbridge Northern) did not exhibit higher concentrations of VAs compared to IDs with less livestock, while levels of VAs in irrigation conveyances were less influenced by the degree of surface runoff. The ubiquity of CTC and TC in our study is likely a reflection of its widespread use in intensive livestock operations. Additional investigation is required to link environmental concentrations of VAs with livestock densities and increase our understanding of potential antimicrobial resistance in high-intensity agroecosystems.