| Salmonella is one of the most important zoonotic pathogens,usually parasitizing in intestines of human and animals,causing food poisoning of human.In this study,the occurrence and prevalence of salmonellosis in duck farms,poultry outpatient clinics,duck meat and products in Xuzhou and surrounding areas were investigated.The distribution of Salmonella serotypes was analyzed and molecular typing was performed by ERIC-PCR.The drug resistance and phenotype of Salmonellar were also detected.A fimA-LAMP rapid detection method was established and validated clinically.In addition,we screened the traditional Chinese medicine compound for duck salmonellosis.This study provides theoretical and technical support for the prevention and control of duck salmonellosis.1.Isolation,identification,serotype typing,and ERIC-PCR molecular typing of salmonellaIn this study,380 rectal swab samples from 6 duck farms,50 tissue samples of clinical diseased ducks,and 300 samples of duck meat or by-products in markets were collected from Xuzhou and surrounding areas.A total of 56 strains of salmonella were isolated.26 strains of Salmonella were isolated from the duck farms,with the separation rate of 6.84%.Among them,24 Salmonella belong to four serotypes:2 S.Typhimurium(8.33%),4 S.Indiana(16.67%),2 S.duck(8.33%),16 S.Enteritidis(66.67%)and the dominant serotype of S.Enteritidis.24 strains of Salmonella were isolated from the clinical diseased ducks,with the separation rate of 48%.There were 6 serotypes,3 S.typhimurium(12.5%),2 S.Agona(8.33%),2 S.Potsdam(8.33%),10 S.Indiana(41.67%)and 1 S.duck(4.17%)and 6 S.enteritidis(25.0%).The dominant serotype was S.Indiana.6 strains of Salmonella were isolated from markets,with the separation rate of 2%.Among them,5 strains were divided into 2 serotypes,4 S.enteritidis(80%)and 1 S.Indiana(20%).The dominant serotype was S.Enteritidis.21 Salmonella were classified into 18 genotypes by ERIC-PCR,and the genetic similarity ranged from 59%to 100%.The results showed good polymorphism and fingerprint data.2.Detection and analysis of antibiotic resistant genes in salmonellaThe resistance of 56 strains of salmonella to 20 antibiotics was determined by Kirby Bauer(k-b)method,and 19 pairs of primers were designed to detect 6 kinds of drug-resistant genes.Antibiotic resistance results showed that the separation of salmonella to 20 antibiotics have different percentages and multiple drug resistance phenomenon.Single antibiotics to streptomycin resistance rate was the highest(98.2%),followed by ampicillin(94.6%).The resistant phenotypes are mainly AStAC,ANStOCSx and AAKDOCSx.The detection rate of resistant gene blaTEM-1,aadAl,aadA2,aph(3 ')II a,tetA,gyrA,Sul2,dr? was more than 50%,and aadAl gene showed the highest detection rate(91.1%),followed by blaTEM-1 gene(83.9%).TetC gene was not detected.The results of antibiotic resistance gene detection were basically consistent with the phenotype of antibiotic resistance.3.Establishment and application of LAMP rapid detection method based on fimA gene of salmonella2 primers targeted the fimA gene were designed to establish a rapid loop-mediated isothermal amplification method for detecting Salmonella.The reaction conditions were optimized,including reaction temperature,detection specificity and sensitivity.LAMP was compared with conventional PCR to detect clinical samples.The results showed that the optimum reaction temperature for LAMP was 64 C,the concentration of Mg2+ was 8 mol/L,the concentration of dNTPs was 0.8 mmol/L,and the ratio of internal and external primers was 1:8.Specificity test showed that 6 related intestinal bacterium were all negative,only Salmonella showed positive.Sensitivity test indicated that the detection limit of LAMP was 8.6×101 cfu/mL,and 10 times more sensitive than PCR.Compared with the traditional method,the coincidence rate of the LAMP method was 100%.The LAMP method has strong specificity,high sensitivity,visible results,simple operation,and no expensive instruments,which is more suitable grass-roots laboratory applications.4.Screening a traditional Chinese medicine for treatment of duck salmonellosisWe investigated the antibacterial effect of the compound preparation of the pulsatilla in vitro,and the therapeutic effect on artificial infected salmonella ducklings.In vitro antibacterial experiment,the antibacterial ring of compound preparation 4 was the highest with 17.6mm,and the minimum inhibition concentration was 166.7 mg/mL,significantly higher than the other groups,indicating that salmonella was highly sensitive to compound preparation 4.In vivo,the results showed that the treatment effect of high dose group was the best,the protection rate can reach 80%,which was close to that of ceftiofur group.After challenge,the number of red cells decreased significantly,and the number of white cells increased significantly(P<0.01).The number of red blood cells and white blood cells close to normal level after treatment of traditional Chinese herbal medicine 9?12d.It concluded that compound preparation 4 with pulsatilla as monarch medicine has a good therapeutic effect on salmonellosis in ducklings. |
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