Establishment And Applicability Of Tracer Method For Livestock And Poultry Body Water Pollution Based On Escherichia Coli And Bacteroides As Indicators

With the rapid development of the livestock and poultry breeding industry,while improving economic efficiency,it also puts pressure on ecological development.Various types of pollutant treatment technologies are relatively lagging behind,resulting in many residual pollutants.The random discharge of livestock manure has become the main cause of most water pollution,which not only damages the ecological environment,but also threatens the public safety of the people within a certain scope and turns into a public security incident.In view of the microbial contamination commonly found in surface water bodies caused by livestock manure,timely and effective control from the source is the key to solving the problem.Microbial Source Tracking(MST)technology has been developed in the research of microbial traceability of water faecal contamination.The technology is divided into two types:library dependent and non-library dependent.Mainly refers to the use of the pollution indicator microorganism itself and its unique genetic fingerprint or host-specific molecular marker as a tracer of pollution,by comparing the difference between the contaminated sample and the indicator in the suspected source of feces or the presence or absence of biological indicators Whether it is associated with a suspected source of contamination to determine the source of the contamination.In this experiment,Escherichia coli and Bacteroides were selected as polluting indicator bacteria,and bacterial colony and chicken feces contamination in the waters around the sewage mouth were carried out by means of enterobacterial repetitive intergenic consensus-PCR(ERIC-PCR),antibiotic resistance analysis(ARA)and qPCR.The situation was tracked to explore the effects of pig and chicken manure generated in the breeding process on the surrounding water sources and to analyze the regional adaptability of these three technologies,and provide technical basis for the monitoring of pig and chicken manure water pollution in the surrounding areas of water pollution.The main research contents are as follows:1.ERIC-PCR method to trace E.coli contamination of livestock and poultry manure in the waters around the sewageIn this experiment,265 strains of Escherichia coli were isolated and identified from the water samples collected from the pigs,chicken feces and surrounding sewage outlets in the farms around the sewage outlet by separation and purification,biochemical identification,Gram staining and 16S rRNA.There were 180 strains of Escherichia coli,72 strains of Escherichia coli,and 13 strains of Escherichia coli.The fingerprints of pig and chicken E.coli were established by ERIC-PCR method.From the cluster analysis of SPSS software,it was concluded that 13 water-source E.coli were derived from pig manure.The correct clustering rate obtained by cluster analysis of Pearson similarity coefficient reached 82.6%.The discriminating rate of pig source was 93.6%,and the discriminating rate of chicken source was 66.4%.The experimental results show that the method can better identify pig feces.2.Analysis of Escherichia coli contamination and drug resistance of livestock and poultry manure in the waters around the sewage outlet by ARA methodThe ARA method was applied to the isolated 180 strains of Escherichia coli isolated from Escherichia coli and 72 strains of Escherichia coli,and the resistance fingerprint was established.Using SPSS software to discriminate and analyze 13 water-borne Escherichia coli,12 strains were derived from pig feces and 1 strain was derived from chicken feces.The homologous recognition rate(RCC)was 99.4%and 13.6%,respectively.The identification accuracy of fecal contamination is better,and the recognition accuracy of chicken source is poor.The average homology recognition rate(ARCC)of the fingerprint database in the analysis mode was analyzed,and the ARCC of the source analysis was 77.8%,and the ARCC after cross-validation was 77.4%.The results showed that the antibiotics between pigs and chickens were E.coli.The resistance was significantly different(P<0.05),and the ARA method could be used to track the fecal contamination in the surrounding waters.Six antibiotic resistance rates and multi-drug resistance analysis of 265 strains of Escherichia coli were carried out by drug sensitive paper method.The results showed that E.coli was the most widely used to resist 6 kinds of antibiotics and had the highest resistance rate to doxycycline.It is 43%.The experiment showed that the surrounding water pollution mainly came from pig manure.3.Bacteroides-specific molecular markers for the detection of Bacteroides contamination and its decay in livestock and poultry manure around the sewage mouthIn this experiment,a water environment micro-system contaminated with feces was constructed from pig and chicken feces.According to the host-specific primers designed by Bacteroides,specific gene markers in pig source,chicken source and Bacteroides genus in contaminated water environment were identified.According to this,qPCR quantitative detection of fecal contamination in polluted water environment.The specific primers of 5 different porcine Bacteroides primers showed that primer 4(PS422F/Bac581R)is suitable for the specific detection of Bacteroides in pigs in this region,and it is suitable for the detection of specific primers for Bacteroides in this region.1(Bac32F/Bac303R),the specific Bacteroides primers suitable for chicken-hosting in this region were used as primer 8(chBact-F1/chBact-R16).At the same time,it was found that the illumination had no significant effect on the stability of Bacteroides-specific biomarkers,while the original microorganisms in the water promoted the degradation of specific biomarkers.Compared with the ARA method,the ARA method has a higher recognition rate for fecal contamination and a higher accuracy rate,and is suitable for tracking pollution around the sewage outlet.The Bacteroides-specific biomarker method can quantitatively analyze the water pollution,and the ambient light has less influence on the label,while the original microorganisms in the water can promote the label degradation.