| Père David’s deer(Elaphurus Davidianus)is the first-grade state protected animal in China,and is an important wild animal resource.In Shishou Shishou Milu National Nature Reserve,the population of Père David’s deer has been expanding,the population density is increasing,the environmental capacity is approaching the threshold,and it has a high risk of disease.Clostrida is a kind of microflora with high correlation with the diseases of Père David’s deer,so it is urgent to monitor.To monitor the health dynamics of the deer herd,76 fecal samples were collected from October 2021 to September 2022 and analyzed for the prevalence of Clostrida and the biology of Paraclostridium bifermentans.Main results were as follows:1.107 strains were isolated from fecal samples,26 of them belong to Clostridium and Paraclostridium,and the main isolation season is in winter.The results of Quantitative Real-time-PCR showed that the concentration of Clostridium perfringens in the faeces of Père David’s deer was very low,and the positive rate of Paraclostridium bifermentans was 12%,and the detection frequency of samples in the winter of 21 years is higher.2.Sequencing results of the microbime of Père David’s deer in the nature reserve showed that the microbime of Père David’s deer in the disease outbreak year and the healthy year of are quite different;Theintestinal microbime of deer collected in 2021December,which is the closest time to the outbreak of the disease,showed significant changes in the structure of the microbiome,in particular the explosive growth of Clostrida.In formation of Clostridia,the main change is the proportion of Lachnospiraceae family and Peptostreptococcaceae family,which leads to disorder of the structure of the intestinal microbiome.Peptostreptococcaceae,is supposed to be regarded as an important object of study in Clostrida,for example,the isolated Paraclostridium bifermentans.Cluster analysis of fecal samples from each month showed that the gut microbiota structure of Père David’s deer changed significantly in December 2020 and 2021,and December is the month to focus on for winter monitoring of deer intestinal health3.The virulence genes,drug resistance genes and drug resistance of 16 isolates of Paraclostridium bifermentans were detected.One of the 16 isolates,B0221a,was found to have one virulence gene,6 kinds of drug resistance genes were detected in 3strains B1221b,B1213b,S1161b,15 strains(except for B1021a,B1251a)of Paraclostridium bifermentans are low sensitive to 8 kinds of drugs,which confirmed that Paraclostridium bifermentans had poor invasion ability and fine stress resistance.4.The environmental tolerance test,enzyme-producing ability test,antibacterial test and genome sequencing analysis of Paraclostridium bifermentans were carried out.Results showed that 16 strains of Paraclostridium bifermentans showed uniform environmental tolerance,two strains B1021a and B1251a showed non-toxic,low resistance and strong antibacterial activity.Genome sequencing showed that B1251a had strong probiotic potential.5.LD50 of Str.CX1-5,a strain of Clostridium perfringens that cause a large number of death in Père David’s deer in winter from 2020 to 2021,was 1.12×10~7CFU in mouse model.Strain B1021a and B1251a were used to prevent and treat the infection of mice.The results showed that both strains were protective against infection in mice,and the protective effect of strain B1251a was superior to that of strain B1021a.In conclusion,we should pay attention to the prevalence of Clostrida in winter,especially in December,for the monitoring of intestinal health of Père David’s deer in Shishou Milu National Nature Reserve.The focus is on the abundance of Peptostreptococcaceae family in Clostridia and its ratio to Lachnospiraceae family.Paraclostridium bifermentans isolated from the faeces of Milu deer are characterized by low toxicity,high resistance,good enzyme production and antibacterial activity,Strains B1021a and B1251a could inhibit the virulence of Clostridium perfringens in the mouse infection model,and strains B1251A had a strong probiotic potential. |
