Validation of Bacteroidales-based microbial source tracking markers for pig fecal pollution and their application in two rivers of North China

In China, pig feces is the predominant source of excrement produced by animal husbandry. Improper use or direct discharge of pig feces can result in contamination of natural water systems. Microbial source tracking (MST) technology can identify the sources of fecal pollution in environmental water, and contribute to the management of pig fecal pollution by local environmental protection agencies. However, the accuracy of such assays can be context-dependent, and they have not been comprehensively evaluated under Chinese conditions. We aimed to compare the performance of five previously reported pig-specific MST assays (PF, Pig-Bac1SYᴮᴿ, Pig-Bac2SYᴮᴿ, Pig-1-Bacᵀᵃqᴹᵃⁿ, and Pig-2-Bacᵀᵃqᴹᵃⁿ, which are based on Bacteroidales 16S rRNA gene markers) and apply them in two rivers of North China. We collected a total of 173 fecal samples from pigs, cows, goats, chickens, humans, and horses across China. The PF assay optimized in this study showed outstanding qualitative performance and achieved 100% specificity and sensitivity. However, the two SYBR green qPCR assays (Pig-Bac1SYᴮᴿ and Pig-Bac2SYᴮᴿ) cross-reacted with most non-pig fecal samples. In contrast, both the Pig-1-Bacᵀᵃqᴹᵃⁿ and Pig-2-Bacᵀᵃqᴹᵃⁿ assays gave 100% specificity and sensitivity. Of these, the Pig-2-Bacᵀᵃqᴹᵃⁿ assay showed higher reproducibility. Our results regarding the specificity of these pig-specific MST assays differ from those reported in Thailand, Japan, and America. Using the PF and Pig-2-Bacᵀᵃqᴹᵃⁿ assays, a field test comparing the levels of pig fecal pollution in rivers near a pig farm before and after comprehensive environmental pollution governance indicated that pig fecal pollution was effectively controlled at this location.