| Poultry morbidity and mortality caused by bacterial infection has always been a key problem to be solved by the breeding industry.In recent years,with the extensive exploration of various bacteria,some new pathogenic bacteria have been gradually discovered,and because of the lack of research on a large number of newly discovered bacteria,the non-standard use of antibiotics and other reasons,some of these bacteria seriously threaten public health security.In this study,bacteria were isolated from diseased and dead poultry samples sent to the Animal Hospital of Yangzhou University from Yangzhou and its surrounding areas.Based on the epidemiological investigation of bacterial diseases,the bacterial prevalence and two new isolates of goose-derived pathogenic bacteria were investigated during 2020-2022.The study of Rothia nasimurium from goose and Plesiomonas shigelloides from goose was further explored according stain microscopy,16S rDNA amplification,antibiotic resistance test and mouse pathogenicity test.It was found that the two strains of bacteria were pathogenic to goslings,and the pathogenicity of the Plesiomonas shigelloides was stronger and could kill mice.Subsequently,the Rothia nasimurium,which received little attention in the past,was taken as the main research object,genome analysis,cooperative hemolysis experiment,gosling regression experiment,and exploration of the influence on the intestinal flora of mice were carried out.The details are as follows:1.Epidemiological investigation of bacterial infection in poultry in Yangzhou and surrounding areas from 2020 to 2022In order to understand the epidemiological situation of bacterial infection in poultry in Yangzhou and its surrounding areas,this epidemiological survey was conducted.A total of 267 samples of various poultry disease samples were collected and sent to the Animal Hospital of Yangzhou University.Detailed records were made for each case,followed by bacterial isolation,purification culture,staining microscopy,biochemical experiments,MALDI-TOF MS identification,preservation,and statistical analysis of the bacterial isolation of 267 samples.The results showed that 86 of the 267 samples contained bacteria,and 91 strains of different bacteria were isolated,among which 46 strains of Escherichia coli were the most isolated,with a isolation rate of 50.55%,accounting for more than half of all bacteria,and Salmonella was the second most isolated,with a total of 11 strains,with a isolation rate of 12.09%.In addition,for the first time,Rothia nasimurium and Plesiomonas shigelloides were isolated from geese.In this study,the epidemiological situation of bacterial diseases in poultry in Yangzhou and its surrounding areas during 2020-2022 was analyzed statistically,and two strains of newly isolated bacteria from geese were selected to provide reference for bacterial diseases in poultry breeding.2.Preliminary study on two isolate strains of goose pathogenic bacteriaBased on the epidemiological investigation in the first chapter,two strains of rare bacteria were selected from the dead geese as the research objects:Rothia nasimurium and Plesiomonas shigelloides.The two strains of bacteria were stained and observed,and biochemical experiments were carried out.Two methods of identification,MALDI-TOF MS and 16S rDNA sequencing identification.16S rDNA sequences were used to establish an evolutionary tree,and then effective antibiotics were screened by drug sensitivity test.The bacteria were injected into mice to observe their pathogenicity on mice,and pathological sections were made for diseased organs of dead mice.In addition,the co-hemolysis experiment and gosling regression experiment were carried out to explore the characteristics and effects of Rothia nasimurium on gosling.The results showed that Rothia nasimurium was gram-positive coccus,which could ferment glucose,maltose and sucrose.Among the 20 antibiotics tested,it was only sensitive to cefzolin,amicacin,fosfomycin and ampicillin/sulbactam.Mice did not die in the pathogenicity test,and goslings showed hair loss in the regression test.Plesiomonas shigelloides is gram-negative short bacillus,which can ferments glucose,maltose,citrate and urea,and is sensitive to 10 antibiotics such as levofloxacin and meropenem.The LD50 of mice is 5.0×106.5 CFU,and there are obvious lesions in the liver and intestinal tract of mice,showing strong lethality.In this study,we explored and reported the Rothia nasimurium and Plesiomonas shigelloides from goose,which enriched the types of pathogenic microorganisms in geese,suggesting that doctors and veterinarians should pay more attention to these two kinds of bacteria.3.A preliminary study on pathogenic mechanism of Rothia nasimurium from gooseIn order to explore the drug resistance of Rothia nasimurium and whether is indeed the cause of gosling hair removal and the mechanism of gosling hair removal,this study intends to explore these two problems further.First,all the drug-resistant genes contained in it were found through the combination of second-generation sequencing and third-generation sequencing,and then the genome was analyzed.After that,the goslings were infected with bacteria again,and the infection methods were intraperitoneal injection or oral administration.The state of the goslings was observed,and trace elements Fe and Zn were detected for the diseased goslings.Blood routine and blood biochemical tests were performed on mice to explore the effects on mammals.The results showed that this strain of Rothia nasimurium contained macrolide resistance genes,trimethoprim resistance genes,streptomycin resistance genes and other drug-resistant genes,and part of the genome function was related to the acquisition and metabolism of iron.The goslings suffered from hair loss again after infection,and the goslings in the intraperitoneal injection group lost hair faster and more obviously.Trace element detection showed that Fe increased significantly in liver,kidney and serum,and Zn was also affected,but the degree of influence was smaller than that of Fe.The high dose infection group was affected more than the low dose infection group,the goslings had different degrees of liver and intestinal lesions,and the blood routine of mice showed that the WBC,Mon#and Gran#were mostly increased after infection.The Lymph%and MCHC after infection were lower than that of negative control,showing a decrease,and the degree of rise and fall was positively correlated with the dose of infection.Blood biochemistry showed that AST,CREA and LDH increased after infection,while P decreased after infection,and the degree of rise and fall was also positively correlated with the dose of infection.The study confirmed that the Rothia nasimurium of goose carries a variety of drug resistance genes,and can indeed cause goslings to lose hair,which is related to the changes of Fe and Zn in the body.At the same time,the Rothia nasimurium of goose can infect mammals and cause certain harm to them,suggesting that the threat to public health security should not be ignored.4.Study on the effect of Rothia nasimurium from goose on the intestinal microflora of miceBased on the findings above,oral administration of the bacteria caused changes in the intestinal villi of goslings.What is the effect of the presence of the bacteria in the intestinal tract of mammals?In order to investigate the effects of Rothia nasimurium on mammalian intestinal flora,mice were inoculated with Rothia nasimurium at 1.0×109 CFU and 1.0×107 CFU,and feces were collected at 0 h,12 h,24 h,36 h,48 h and 72 h after inoculation.Intestinal flora was detected by sequencing the 16S v3-v4 region of all bacteria in fecal samples.Through Alpha diversity analysis,Beta diversity analysis and structural diversity analysis of mice intestinal flora,it was found that the composition of group H and group NC was significantly different at the OTU level,and the species richness was ranked as Group H>Group L>Group NC.PCA analysis and PCoA analysis were performed on samples from the three groups at 12 h.It was found that the samples of mice in group H and group L were more discrete than those in group NC.The histogram of relative abundance of species showed that the average abundance of Bacteroidetes in different groups were 68.70%in NC group,55.35%in H group and 63.75%in L group.The average abundance of Firmicutes in different groups was 20.35%in NC group,30.90%in H group and 24.45%in L group,respectively.It can be concluded that after the intestinal tract of mice is infected with the bacteria,the proto-intestinal microecological environment of mice is changed,and the number of intestinal OTUs of mice is increased.In addition,the bacteria of the Rothia nasimurium has the ability to change the composition of the intestinal microecological environment,inhibit Bacteroidetes and promote the bacteria of Firmicutes.Bacteroidetes are mostly probiotics,while Firmicutes are mostly the condition pathogenic bacteria.It is speculated that it can change the health state of animals by changing the composition of intestinal flora. |
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