榆林部分地区湖羊乳房炎病原菌的分离鉴定及耐药性和毒力基因检测

doi:10.16431/j.cnki.1671-7236.2024.04.042

摘要/Abstract

摘要： 【目的】明确规模化湖羊场乳房炎的主要病原菌种类及其耐药性和毒力基因分布情况,指导临床治疗湖羊乳房炎合理用药。【方法】在6个规模化羊场采集临床型乳房炎乳样43份,采用细菌形态学鉴定、生化鉴定及16S rDNA序列分析的方法确定引起湖羊乳房炎的主要病原菌,采用纸片扩散法检测其耐药性,并通过PCR方法检测相关毒力基因。【结果】革兰染色镜检显示获得了革兰阳性球菌和革兰阴性杆菌。革兰阳性球菌在Baird-Parker琼脂平板上呈现出边缘圆润,中间呈黑色的菌落;生化试验鉴定结果显示,分离株对甘露醇、过氧化氢和兔血浆凝固试验为阳性;经16S rDNA测序比对,与金黄色葡萄球菌高度相似的分离株共31株。革兰阴性杆菌在伊红-美蓝琼脂平板上呈现出黑紫色泛有金属光泽的菌落,在麦康凯琼脂平板上呈现出粉红色的菌落;生化试验鉴定显示,分离株对吲哚试验和甲基红试验为阳性,对VP试验和枸橼酸盐利用试验呈阴性;经16S rDNA测序比对,与大肠杆菌高度相似的分离株共12株。药敏试验结果显示,分离的金黄色葡萄球菌对环丙沙星、左氧氟沙星和头孢噻肟具有较高的敏感性,对青霉素、红霉素和链霉素耐药;分离的大肠杆菌对环丙沙星和左氧氟沙星亦有较高的敏感性,对青霉素和红霉素耐药。在金黄色葡萄球菌中检测出7种毒力基因,分别为PVL、Hlb、seb、clfA、clfB、fnbB和icaD基因,未检测到Hla、sea和fnbA基因;在大肠杆菌中检测出6种毒力基因,分别为hlyA、etrA、fasA、eaeA^a、exhA^a和stx1基因,未检测到cnf1、aer、estA和stx2基因。【结论】引起该规模化湖羊场乳房炎的主要病原菌为金黄色葡萄球菌和大肠杆菌,且携带丰富的毒力基因,建议优先使用环丙沙星和左氧氟沙星治疗,本研究结果为临床治疗该病提供了科学依据。

关键词: 湖羊; 乳房炎; 病原菌; 耐药性

Abstract: 【Objective】 The purpose of this experiment was to clarify the distribution of the main pathogenic bacteria,their resistance and virulence genes,and guide the rational use of drugs in the clinical treatment of mastitis in Hu sheep farms.【Method】 43 clinical mastitis milk samples were collected from 6 large-scale sheep farms.Bacterial morphology identification,biochemical identification and 16S rDNA sequence analysis were used to determine the main pathogenic bacteria causing mastitis in Hu sheep.Drug resistance was detected using paper diffusion method,and related virulence genes were detected using PCR method.【Result】 The Gram staining results showed that Gram positive cocci and Gram negative bacilli were obtained. On the Baird Parker agar plate,Gram positive cocci exhibited rounded edges and black colonies in the middle.Biochemical tests showed that the isolated strains were positive for mannitol,hydrogen peroxide and rabbit plasma coagulation tests.After 16S rDNA sequencing comparison results showed that a total of 31 strains were highly similar to Staphylococcus aureus.Gram negative bacilli presented black purple colonies with metallic luster on the Eosin Methyl Blue agar plate,while on the MacConkey agar plate,pink colonies appeared.Biochemical tests showed that the isolated strains were positive for indole and methyl red tests,and negative for VP and citrate utilization tests.After 16S rDNA sequencing comparison,a total of 12 strains were highly similar to Escherichia coli.The drug sensitivity test results showed that the isolates of Staphylococcus aureus were sensitive to drugs such as ciprofloxacin,levofloxacin and cefotaxime,but resistant to penicillin,erythromycin and streptomycin.The isolates of Escherichia coli were sensitive to drugs such as ciprofloxacin and levofloxacin,and were resistant to penicillin and erythromycin.Seven virulence genes were detected in Staphylococcus aureus,namely PVL, Hlb, seb, clfA, clfB, fnbB and icaD genes,but Hla, sea and fnbA genes were not detected.Six virulence genes were detected in Escherichia coli,namely hlyA, etrA, fasA, eaeA^a, exhA^a and stx1 genes,but no cnf1, aer, estA and stx2 genes were detected.【Conclusion】 The main pathogens causing mastitis in large-scale sheep farms were Staphylococcus aureus and Escherichia coli,which carried rich virulence genes.It was recommended to prioritize the use of ciprofloxacin and levofloxacin for treatment.This article provided scientific basis for clinical treatment of this disease.

Key words: Hu sheep; mastitis; pathogenic bacteria; drug resistance

中图分类号:

S852.61

王玉, 石嵩, 靳盼豪, 李金昕, 张洺溪, 李托, 李陇平, 屈雷, 董书伟. 榆林部分地区湖羊乳房炎病原菌的分离鉴定及耐药性和毒力基因检测[J]. 中国畜牧兽医, 2024, 51(4): 1746-1756.

WANG Yu, SHI Song, JIN Panhao, LI Jinxin, ZHANG Mingxi, LI Tuo, LI Longping, QU Lei, DONG Shuwei. Isolation,Identification and Detection of Resistance and Virulence Genes of Pathogenic Bacteria Causing Mastitis in Some Areas of Yulin[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(4): 1746-1756.

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