| In recent years,diarrheal diseases have become one of the major diseases that endanger China's mutton sheep breeding industry.It's mainly caused by pathogenic bacteria in fattening sheep(especially lambs),leading to intestinal flora dysregulated and gastrointestinal disorders then occur diarrhea,which has caused serious economic losses to China's mutton sheep breeding industry.At present,veterinary clinics mostly use antibiotics to treat the disease.However,the unreasonable use of antibiotics not only caused the disorder of intestinal microflora in mutton sheep,affects the normal progress of rumination,but also affected the normal progress of rumination.At the same time,this disease has also induced the emergence of drug-resistant bacteria and promoted the spread of drug-resistant genes,which posing a threat to public health security.Therefore,this study aims to isolate and identify the pathogens of diarrhea-prevented sheep in Jilin Province,and to detect the fecal microbial diversity and drug-resistance abundance,in order to lay the foundation for the rational use of drugs in sheep breeding.The results are as follows:1.Isolation,identification and drug sensitivity testing of pathogenic bacteria causing diarrhea in Jilin provinceIn this study,pathogenic bacteria were isolated and identified from sheep with diarrhoea disease in Yanji and Changling,Jilin Province.The results showed that 14 pathogenic bacteria were isolated,including 2 Proteus mirabilis and 14 Escherichia coli;all isolated strains were pathogenic to BALB/c mice;the pathogenicity of the two Proteus mirabilis was more severe.The results of drug sensitivity tests showed that the isolated strains exhibited different degrees of drug resistance to drugs such as levofloxacin,gentamycin,and tetracycline.The resistance rate to ampicillin was 93% but only one isolate was moderately sensitive to it;the resistant rates of isolates to Streptomycin was 57%;the resistance rates to norfloxacin,levofloxacin and tetracycline were all 50%;the resistance rates to florfenicol,gentamycin,furazolidone,and cefotaxime were 43%,34%,29%,and 21%,respectively;all the tested strains were only sensitive to Polymyxin B.At the same time,we combined the results above with the epidemiological investigation results of diarrheal diseases in different regions in recent years to analyze the incidence rate of bacterial diseases in Jilin Province,indicating that the incidence rates of bacterial diseases was higher in the sheep farmsof Yanji(YJ)and Changling(CL),which were 12% and 7%,and the incidence rates of bacterial diseases in sheep breeding farms of Gongzhuling(GZL),Baicheng(BC),and Changchun(CC)were relatively low,which were 0.5%,0.02%,and 0.01%,respectively.2.Detection of bacterial diversity in sheep feces of different incidenceIn order to further explore the intestinal microflora of mutton sheep,16 S sequencing method was used to detect the composition and structure of fecal microorganisms and the abundance levels of different species in the five sheep farms with differents incidence rate.A total of 1592 qualified OUT sequences were obtained.The qualified sequences were compared with the microbial reference database to obtain the corresponding species classification information,followed by Alpha diversity analysis,Beta diversity analysis,species annotation and taxonomic analysis.The results showed that there were significant differences in the farms of different areas.Among them,the spatial distribution of samples in the two fields of CL(12%)and YJ(7%)which had high incidence were farther away from the field with lower incidence,and the distance between the samples within the group was larger,indicating the higher species richness of samples of these two groups.The distances between samples in the three groups with relatively low incidence rate of GZL(0.5%),BC(0.02%)and CC(0.01%)were relatively small,and species diversity was also relatively smaller.At the phylum level,the abundance of Streptomyces and Bacteroidetes were the highest among the five groups;the structures of the top ten species were obviously different at the genus level;the species with the highest abundance in the CC,BC,and GZL groups were rumenococcus;the species with the highest abundance in the CL and YJ groups were the Alistipes and the Treponema.Clinical pathogens such as Mycoplasma,Salmonella,and Streptococcus were identified in five samples by KRONA analysis,the composition of microorganisms was more complex in CL and YJ,and the number of pathogenic bacteria species was significantly larger than that of CC,BC,and GZL groups.3.Detection of resistance gene abundance in in sheep feces of different incidenceIn order to explore the distribution and prevalence of drug resistance genes,this study examined the expression of 25 drug resistance genes such as aminoglycoside antibiotic resistance gene,sulfa antibiotic resistance gene,polymyxin resistance gene(MCR-1),New Delhi metal ?-lactamase(NDM-1)etc in sheep feces in five regions including Changchun,Baicheng,Gongzhuling,Changling and Yanji.The detectionrates of various drug resistance genes were as follows: tetracycline resistance gene 21%,aminoglycoside resistance gene 20%,MLSB drug resistance gene 17%,?-lactam resistance gene 13%,sulfa drug resistance gene 6%,and quinolones resistance gene 5%;among the five groups of samples,the detection rate of drug resistance genes in the Baicheng group was the highest,accounting for 91.2%.The detection rates of the other four groups in Changchun,Yanji,Gongzhuling and Changling were 81.6%,81.6%,72% and 60% respectively.The detection rates of type 1 integrase gene,polymyxin resistance gene(MCR-1)and New Delhi metal ?-lactamase(NDM-1)were 98%,86% and 78% respectively.Tetracycline resistance gene has the highest expression level in all samples,followed by intI1.There was no direct relationship between the drug resistance genes abandance and the incidence rate of different area,showing that the production and spread of drug resistance genes are related to the medication habits in different regions,the areas with low incidence rate also use drugs to prevent the bacterial diseases of mutton sheep which has promoted the development and spread of related drug resistance genes. |
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