Establishment And Application Of Traceability Method For Animal Fecal Pollution In Wanjiang River Basin

With the rapid development of large-scale livestock and poultry farming in the Wanjiang River Basin,a large number of pollutants are generated.Animal manure is the main source of agricultural surface pollution,and animal manure discharged into surface water can bring a series of ecological and public health issue,so it is important to quickly and accurately identify the source of pollution for environmental management.There may be many types of pollution in surface water sources,such as domestic and industrial sewage,untreated small-scale farm sewage,wild animal manure,etc.How to accurately identify animal manure pollution is the key to agricultural surface source pollution prevention and control.Manure contamination detection and identification technologies have evolved from fecal indicator bacteria(FIB)culture-dependent technologies to microbial source tracking(MST)technologies.Microbial source tracking technology identifies fecal contamination based on the traits or genetic characteristics of specific bacterial groups associated with the host,and can provide information on the species origin of fecal contamination.In this study,we collected a large number of livestock and poultry manure samples with 16 S r RNA high-throughput sequencing data from a variety of pollution sources in the Wanjiang basin,screened out host-specific molecular markers for pigs and chickens with good applicability to the region,and performed the establishment of marker quantification methods by q PCR,and analyzed the attenuation patterns of indicator bacteria under light and bacterial by building a water environment reactor for chicken and pig manure.The decay pattern of indicator bacteria under light and bacterial predation was analyzed by constructing chicken manure and pig manure water environment reactors.The 16 S r RNA sequencing data were used to analyze the differences of bacterial communities among different pollution sources,and a fecal taxon library was constructed.Finally,a comparison with the traditional fecal detection method of microbial source tracing was made to provide ideas for the detection of fecal pollution in water bodies in Wanjiang Basin.The main research contents are as follows.1.Tracing livestock manure pollution in water bodies based on host-specific markersThirty-six samples of chicken manure,38 samples of pig manure and 16 samples of manure of other species were collected from the Wanjiang basin,and fecal genomic DNA was extracted and used as a template for screening host-specific molecular markers by PCR.The results showed that the specificity and sensitivity of chicken-specific AV43 marker and pig-specific FUP-1 marker were both above 80%,which had good applicability in this region.Subsequently,the standard curves of the screened host-specific primers were constructed by q PCR.A manure-water mixing simulation system was constructed with chicken manure,pig manure and river water samples,and the change pattern of molecular markers under light and bacterial predation was analyzed.The experimental results showed that bacterial predation was the main cause of marker attenuation in the manure-water mixing microenvironment.2.16 S r RNA sequencing-based detection of potential pathogens and fecal contamination in water bodiesUsing 16 S r RNA sequencing,we systematically analyzed and compared the bacterial community composition of 252 samples from water bodies,sediment samples and a wide range of potential pollution sources(including village and pig farm effluents,wild bird feces,human and poultry and livestock feces)in the Wanjiang basin,and used the machine learning-based software FEAST and Source Tracker to The potential contamination sources were predicted.The results showed that the microbial diversity of water and sediment samples was significantly higher than that of fecal samples,with the highest microbial diversity in Chaohu Lake and river sediment samples,as well as the presence of a large number of unclassified species.Proteobacteria,Actinobacteria,and Bacteroidetes were widely distributed in all samples.There were some similarities in the microbial community composition of wild bird feces with goose and pig feces.A fecal source library was constructed using the 16 Sr RNA sequence dataset of pollution sources in the Wanjiang basin,and the collected water bodies in the Chaohu Lake basin were used as pollution composition prediction objects.The village outfall and sewage treatment plant outfall samples were successfully identified as the most important pollution sources of river water samples,while the sediment and lake water samples were contaminated with pig farm effluent and wild waterfowl feces.FEAST exhibits higher stability and confidence than Source Tracker.A local pathogenic 16 S database was constructed by downloading representative 16 S sequences of pathogenic bacteria from NCBI based on the pathogenic bacteria catalogs on the VFDB and PATRIC websites.After alignment of the self-built potential pathogen database,the composition of potential pathogens in all samples was obtained.Pseudescherichia,Enterococcus,Streptococcus,and Enterobacter widely distributed in fecal and aqueous samples.Finally,network analysis was performed to assess the correlations between potential pathogens and fecal indicator bacteria.The results showd that the genera Aerobacter,Escherichia coli,Prevotella and Faecalibacterium were all significantly(Pearsons’ r>0.8,p<0.05)associated with at least seven potential pathogens.3.Testing of fecal source tracking methodsWater and sediment samples were collected from the Pai River,Hangbu River and Fengle River,and fecal coliforms culture,16 S r RNA sequencing and host-specific molecular marker detection were performed.The results showed that there were some differences between traditional culture-dependent fecal contamination indication methods and fecal contamination traceability methods.q PCR results pointed out that pig feces and chicken feces contamination were prevalent in the three rivers,with the copy number of pig feces markers higher in the Pai River than in the other rivers.The prediction results based on the local 16 S pathogen database indicated that the Pai River had the highest potential pathogen level.The combined results of Source Tracker and FEAST showed that the Pai River had a higher potential contamination level than the other rivers.In conclusion,the 16 S r RNA sequencing results were consistent with q PCR results indicating that the Pai River has a higher public health risk.In summary,the combination of microbial community-based and molecular marker-based analyses can better identify potential sources of fecal pollution and characterize health risks in the rivers around the city in aggregate.It is recommended that NGS-based sequence analysis be used in subsequent water quality management to provide information on possible sources of fecal pollution,followed by quantitative detection of host-associated molecular markers to more effectively manage water quality.