| Salmonellosis is a zoonotic disease,whose prevention and control are becoming more and more complicated due to the antibacterial drug abuse and drug resistance enhancement.Based on the fact that chicken salmonellosis is common and difficult to control in Zhongshan District of Liupanshui City,this study carried out the isolation and identification,drug sensitivity test and drug resistance gene analysis of salmonellosis on the basis of epidemiological investigation of salmonellosis in Zhongshan District of Liupanshui City.1.Epidemiological investigation of chicken salmonellosis in Zhongshan District and isolation and identification of pathogenic bacteria.In this study,from January 2017 to December 2019,chicken flocks of six chicken farms with two different breeding methods were regularly sampled(serum samples were collected every two months),and the specific antibody of Salmonella was detected by plate agglutination test.Preliminary diagnosis of the diseased chickens was made through clinical symptom observation and systematic pathological autopsy,and then liver,spleen,and heart tissue samples of diseased chickens were collected for Salmonella isolation,and the isolated bacteria were respectively cultured on ordinary nutrient agar medium,nutrient broth,and Mac Conkey agar to observe the growth status of bacteria.Morphology of the bacteria isolates was observed by Gram's stain;the biochemical characteristics were identified using 14 kinds of biochemical assays;PCR amplification,sequencing and genetic evolution analysis of 16 S r DNA genes of the 3 isolates were also performed.The results of the plate agglutination test showed that the average positive rate of Salmonella in the investigated chicken farms was22.3%,of which the average positive rate in ground-level chicken farms was 26.4%,and the average positive rate in caged chicken farms was 18.4%.It could be seen that salmonellosis in chicken flocks raised on the ground is more serious than that in caged chickens.The clinical diagnosis of the diseased chicken showed that clinical symptoms of the sick chicken was listlessness,afraid of the cold and reluctant to walk,loose feathers,wings drooping,diarrhea,and white paste-like feces around the cloaca;necropsy showed that the liver and spleen were enlarged and congested,and there were nodules on the surface of the spleen,ascites in the abdominal cavity,and intestinal inflammation.The symptoms and necropsy lesions are consistent with chicken salmonellosis.The results of isolation and culture showed that the colonies of the isolates on the ordinary nutrient agar medium were 2?4 mm in diameter,white translucent,moist and smooth,round and neat,and the colonies on the Mac Conkey medium were colorless,transparent and round.The shape bulge is consistent with the culture characteristics of Salmonella.The results of Gram's stain showed that the isolates were Brevibacterium rubrum with uniform size,blunt ends,no capsules,and no spores,which was in line with the typical Salmonella Gram staining characteristics.The biochemical test results showed that the isolates showed positive reactions in the biochemical tests of hydrogen sulfide,methyl red,citrate,glucose,lysine,ornithine,sorbitol,xylose,etc.,and showed positive reactions with the biochemical characteristics of Salmonella Match.The PCR results showed that after the PCR products of the three isolates were electrophoresed,the specific target band appeared at about 1 550 bp,which was consistent with the expected target gene size,while no band appeared in the negative control.The comparison between the sequencing results and the Salmonella reference strains showed that although the 16 S r DNA genes of the three isolates were different from those of the Salmonella reference strains,they were generally highly similar.Genetic tree analysis showed that the three isolates were on the same clade as Salmonella.The above results indicated that all the three strains were Salmonella.2.Study on the resistance of Salmonella isolates.The sensitivity of the three isolated and identified Salmonella strains(named as strains 1,2 and 3)to 10 antibacterial drugs was determined by the drug-sensitive paper method?At the same time,PCR amplification,sequencing,homology and genetic evolution analysis of 10 pairs of resistant genes of Salmonella were carried out.The results of the drug susceptibility test showed that the three Salmonella strains were resistant to ?-lactam drugs,amoxicillin,penicillin and macrolide drugs erythromycin,and were resistant to aminoglycoside drugs streptomycin,gentamicin,and erythromycin.Namycin,tetracycline drugs doxycycline,?-lactam drugs carbenicillin,quinolone drugs enrofloxacin and sulfa drugs compound sulfamethoxazole sensitive.The analysis of drug resistance genes showed that the ?-lactam blatem-1 drug resistance gene showed specific purpose bands in the 3 Salmonella strains,and the chloramphenicol Cml A drug resistance gene only showed specific purpose in the No.3 strain.Strips,other drug resistance genes were not detected.The homology analysis of blatem-1 resistance genes showed that the blatem-1 resistance genes of the three Salmonella strains had homology between 98.3% and 98.6% with the reference strain.The phylogenetic tree analysis of blatem-1 drug resistance genes showed that the three Salmonella strains blatem-1 drug resistance genes have a certain degree of difference,but the genetic evolutionary relationship is relatively close in general.The chloramphenicol Cml A resistance gene carried by strain 3 has 100% homology with that of the reference strain.The phylogenetic tree analysis of Cml A resistance gene shows that strain 3 and the reference strain are in the same branch,indicating that strain 3 The Cml A resistance gene is highly conserved..In summary,this experiment was based on the epidemiological investigation of Salmonella in Zhongshan District,and successfully isolated and identified 3Salmonella strains.The results of the isolated bacterial resistance study showed that the 3 Salmonella strains were partly ?-lactams and macrolides are resistant to drugs,and are sensitive to aminoglycosides,tetracyclines,quinolones,and sulfonamides.?-lactam blatem-1 resistance genes are present in the three Salmonella strains.There is a chloramphenicol-like Cml A resistance gene in strain No.3. |
PDF Full Text Request