鸭源沙门菌的分离鉴定、耐药性及致病性分析

【目的】为防控鸭源沙门菌感染，本试验针对四川德阳地区鸭源沙门菌的感染情况开展研究。【方法】从该地区3个种鸭场和1个鸭孵化场共采集不同类型样本222份，按国标GB 4789.4－2016方法对沙门菌进行分离鉴定，进一步通过K-B法检测分离菌对14种抗菌药物的敏感性，并对鉴定出的稀有血清型沙门菌进行致病性研究。【结果】疑似沙门菌分离株在BS琼脂上呈黑色、灰色或棕褐色、有金属光泽菌落，在XLD琼脂上呈无色透明、或中心黑色或几乎全黑的菌落。将疑似沙门菌株接种三糖铁琼脂斜面进行生化鉴定，疑似沙门菌分离株均能使三糖铁斜面变为红色，底部变为黄色带黑色，符合沙门菌生化特性。通过PCR对沙门菌特异性基因invA进行扩增，经琼脂糖凝胶电泳后可在500 bp左右观察到目的条带，确认共分离到17株沙门菌，总分离率为7.7%（17/222），包括鼠伤寒沙门菌、哈托沙门菌和波恩沙门菌3种血清型，其比例分别为47.1%（8/17）、29.4%（5/17）和23.5%（4/17），优势血清型为鼠伤寒沙门菌。分离菌对β-内酰胺类和氨基糖苷类抗菌药耐药率最高，对氨苄西林和链霉素的耐药率分别为82.4%（14/17）和88.2%（15/17），对喹诺酮类抗菌药最敏感，其中对萘啶酸100%（17/17）敏感。分离菌存在严重的多重耐药现象，其中耐10种以上（包括10种）抗菌药的6株，占比35.2%，且10种抗菌药分别来自至少6类不同种类抗菌药；耐8种抗菌药物的2株，占比11.8%；耐5~7种抗菌药的6株，占比35.3%；耐2~3种抗菌药物的共3株，占比17.6%。通过寇氏改良法测得稀有血清型菌株H4、B2的LD₅₀分别为3.98×10⁶和1.58×10⁶ CFU，致病性试验结果表明，哈托和波恩沙门菌均能引起小鼠急性死亡，脏器充血、出血，细胞变性等。【结论】本研究成功分离到17株鸭源沙门菌，从鸭中分离到哈托沙门菌在国内外属首次。分离菌具有严重的耐药性和多重耐药现象，2株稀有血清型沙门菌对小鼠的致病力均较强，且致病作用相似。本研究结果可为鸭场沙门菌病的防治提供参考。

Isolation,Identification, Drug Resistance and Pathogenicity Analysis of Salmonella from Duck

【Objective】 In order to prevent and control duck Salmonella infection, this test was conducted to study the infection of duck Salmonella in Deyang, Sichuan.【Method】 222 samples of different types were collected from 3 duck breeding farms and 1 duck incubator in the region, and Salmonella was isolated and identified according to GB 4789.4－2016. Further, the sensitivity of the isolated bacteria to 14 antibiotics was detected by K-B method, and the pathogenicity of the identified rare Salmonella serotype was studied.【Result】 The suspected Salmonella isolates were black, gray or brownish, metallic colonies on BS agar and colorless, transparent, or black or almost all black colonies on XLD agar.The suspected Salmonella isolates were inoculated with trisaccharide iron agar slant for biochemical identification.The suspected Salmonella isolates could make the trisaccharide iron slant turn red and the bottom turn yellow with black, which conformed to the biochemical characteristics of Salmonella.The specific gene invA of Salmonella was amplified by PCR.After agarose gel electrophoresis, the target band was observed above 500 bp.It was confirmed that 17 Salmonella strains were isolated, with a total isolation rate of 7.7% (17/222).The 17 strains of Salmonella included three serotypes, namely Salmonella Typhimurium, Salmonella Hato and Salmonella Bonn, with the proportions of 47.1% (8/17), 29.4% (5/17) and 23.5% (4/17) respectively.The predominant serotype is Salmonella Typhimurium.The isolates were the most resistant to β-lactams and aminoglycosides, and the resistant rates to ampicillin and streptomycin were 82.4% (14/17) and 88.2% (15/17), respectively.The isolates were the most sensitive to quinolones, and 100% (17/17) to nalidixic acid.The isolated bacteria had serious multi-drug resistance, six strains were resistant to more than 10 kinds of antibiotics (including 10 kinds), accounting for 35.2%.And the 10 kinds of antibiotics were from at least 6 different kinds of antibiotics.Two strains were resistant to 8 kinds of antibiotics, accounting for 11.8%.Six strains were resistant to 5-7 kinds of antibiotics, accounting for 35.3%.A total of 3 strains were resistant to 2-3 antibiotics, accounting for 17.6%.The LD₅₀ of rare serotype strains H4 and B2 were determined to be 3.98×10⁶ and 1.58×10⁶ CFU respectively by Karber’s modified method.The results of pathogenicity test showed that both Salmonella Hato and Salmonella Bonn could cause acute death, organ congestion, hemorrhage, cell degeneration and other pathological changes in mice.【Conclusion】 In this study, 17 strains of Salmonella from ducks were successfully isolated, it was the first time that Salmonella Hatto was isolated from ducks at home and abroad.The isolates had serious drug resistance and multiple drug resistance, the two rare serotypes of Salmonella had strong pathogenicity to mice, and the pathogenic effects were similar.This study could provide a reference for the prevention and treatment of salmonellosis in duck farms.