山羊伪结核棒状杆菌的分离鉴定及耐药性分析

黄丽丽; 林裕胜; 江锦秀; 张靖鹏; 游伟; 肖义军; 胡奇林

doi:10.19303/j.issn.1008-0384.2019.08.009

山羊伪结核棒状杆菌的分离鉴定及耐药性分析

doi: 10.19303/j.issn.1008-0384.2019.08.009

黄丽丽^{1, 2,},
林裕胜²,
江锦秀²,
张靖鹏²,
游伟²,
肖义军¹,
胡奇林^2, ,

1.
福建师范大学生命科学学院, 福建福州 350108
2.
福建省农业科学院畜牧兽医研究所, 福建福州 350013

基金项目:

福建省科技计划项目——省属公益类科研院所基本科研专项 2018R1023-1

福建省财政专项——福建省农业科学院科技创新团队建设项目 STIT2017-1-5

详细信息

作者简介:
黄丽丽(1991—), 女, 硕士研究生, 主要从事动物传染病研究(E-mail:Huanglilixj2012@163.com)

通讯作者:
胡奇林(1963—), 男, 研究员, 主要从事动物传染病学和免疫学研究(E-mail:hql562713@163.com)

中图分类号: S855.1+2
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- 被引次数: 0
出版历程
- 收稿日期: 2019-05-28
- 修回日期: 2019-08-05
- 刊出日期: 2019-08-01

Isolation, Identification and Antibiotics Resistance of Corynebacterium pseudotuberculosis from Goats

1.
College of Life Sciences, Fujian Normal University, Fuzhou, Fujian 350108, China
2.
Institute of Animal Husbandry and Veterinary, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013, China

摘要

摘要: 目的分离、鉴定福建省某养殖场病死山羊的病原，明确山羊病死原因，为山羊伪结核棒状杆菌病的防治提供科学依据。方法以无菌采集的病羊肺脏为材料，进行细菌分离纯化，鉴定分离菌株的形态特征及培养特性；提取菌株DNA，利用特异性引物和16S rRNA通用引物进行PCR，16S rRNA扩增片段送公司进行克隆测序并对序列进行分析；并进行生化、药敏和动物攻毒试验。结果该分离菌株在含10%脱纤维绵羊血的琼脂培养基上，生长成直径1 mm左右、中央凸起、乳白色、边缘不整齐、伴有β溶血环的菌落，在营养琼脂培养基上生长不良，在麦康凯琼脂培养基上不能生长。16S rRNA测序结果经NCBI在线软件Blast后显示与GenBank上的伪结核棒状杆菌的同源性高达100%；山羊伪结核棒状杆菌特异性PCR有目的扩增条带；生化试验结果与伯杰氏细菌鉴定手册中伪结核棒状杆菌相符，表明分离菌为山羊伪结核棒状杆菌，命名为FJ-PN。小鼠攻毒试验结果显示，小鼠腹腔与皮下注射0.3 mL含量为4×10⁵CFU·mL^-1的分离菌，在40 h内全部死亡；山羊攻毒试验结果显示，山羊经皮下注射2 mL、鼻腔接种1 mL含量为4×10⁵CFU·mL^-1的分离菌3 d后死亡。药敏试验结果显示该菌对克拉霉素、环丙沙星、青霉素、四环素、左氟沙星、氧氟沙星、庆大霉素高度敏感，对红霉素、氯霉素、氨苄西林、头孢噻肟、头孢西丁、利福平低度敏感，对链霉素、甲氧苄啶表现耐药。结论本研究分离到的1株细菌为山羊伪结核棒状杆菌，具有较强的致病性，且对抗生素表现出一定的耐药性，为该病的诊断和合理用药提供了理论支持。
- 伪结核棒状杆菌 /
- 山羊 /
- 耐药性 /
- 分离 /
- 鉴定
Abstract: Objective To identify the pathogen that caused the death of goats in a farm in Fujian for prevention and treatment of Pseudotuberculosis. Method Aseptically collected specimens of lung tissues from the dead goats were used to isolate the microbes for morphological observation and strain identification. DNA of the isolates were extracted, and the specific and 16S rRNA universal primers selected for PCR. The 16S rRNA amplified fragment was cloned, sequenced and analyzed. Biochemical, drug sensitivity, and animal challenge tests were performed on the isolate for taxonomic identification. Results The isolated strain was cultured on an agar medium containing 10% defibrated sheep blood to grow into a colony with a diameter of about 1 mm, a central bulge, a milky white color, an irregular edge, and a beta hemolysis ring. It did not grow well on a nutrient agar nor a MacConkey agar medium. The 16S rRNA sequencing showed an up to 100% homology with Corynebacterium pseudotuberculosis on GenBank by the NCBI online software Blast. Like the specific PCR of C.pseudotuberculosis, that of the isolate also had the same amplified targeted bands. In addition, the biochemical test on the isolate duplicated the characteristics of C. pseudotuberculosis shown in the Berger's Bacterial Identification Manual. Hence, the isolated pathogen named as FJ-PN was verified to be C. pseudotuberculosis. A challenge test on mice with an intraperitoneal or subcutaneous injection of 0.3 mL of 4×10⁵ CFU·mL^-1 of the isolate died within 40 h. Furthermore, two goats injected subcutaneously with 2 mL of 4×10⁵ CFU·mL^-1 and intranasal inoculated simultaneously with 1 mL of 4×10⁵ CFU·mL^-1 of the isolate died within 72 h. The drug sensitivity test showed that the strain was highly sensitive to clarithromycin, ciprofloxacin, penicillin, tetracycline, levofloxacin, ofloxacin, and gentamicin, slightly sensitive to erythromycin, chloramphenicol, ampicillin, cefotaxime, cefoxitin, and rifampicin, and resistant to streptomycin or trimethoprim. Conclusion The pathogenic bacteria isolated from the diseased goats was identified as C. pseudotuberculosis. It had a strong pathogenicity with varying degrees of drug resistance to antibiotics.
- Corynebacterium pseudotuberculosis /
- goat /
- antibiotics resistance /
- isolation /
- identification

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图 1 16S rRNA基因的进化分析

Figure 1. Phylogenetic tree analysis on 16S rRNA gene of FJ-PN

下载: 全尺寸图片幻灯片

图 2 山羊伪结核棒状杆菌PCR验证

注：M为2000 DNA Ladder；1为分离菌株；2为阴性对照。

Figure 2. PCR verification of C. pseudotuberculosis from diseased goats

Note: M=2000 DNA ladder; 1=isolated strain; 2=negative control.

下载: 全尺寸图片幻灯片

图 3 小鼠攻毒试验结果

注：A为腹腔注射；C为皮下注射；B、D为空白对照。

Figure 3. Results of challenge test on mice

Note: A=intraperitoneal injection; C=hypodermic injection; B, D=blank control.

下载: 全尺寸图片幻灯片

图 4 山羊攻毒试验结果

Figure 4. Results of challenge test on goats

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

靶基因 Target gene	引物 Primer	引物序列(5′ → 3′) Primer sequence(5′ → 3′)	扩增片段大小 Amplified fragment size/bp
16S rRNA	F1	AGAGTTTGATCCTGGCTCAG	1500
	R1	CGGCTACCTTGTTACGACTT
PLD基因	F2	GTGAGAAGAACCCCGGTATAAG	291
	R2	TACCGCACTTATTCTGACACTG

下载: 导出CSV

表 2 GenBank中公布的参比序列

Table 2. Reference sequence published in GenBank

菌株 Strain	登录号 Login ID	宿主 Host	分离地点 Separation location	分离时间 Separation time
3/99-5	CP003152.1	sheep	United Kingdom: Scotland	1999年
OVI2C	CP026374.1	Ovis aries	Brazil:Petrolina	2009年
E7	CP036535.1	Goat	Brazil:Pernambuco	2010年
Cap8W	CP036257.1	goat	Brazil:Pernambuco	2010年
MEX9	CP014543.1	Goat	Mexico: Guanajuato	2012年
42/02-A	CP003062.1	Sheep	Australia	2014年
PAT10	CP002924.1	sheep	Argentina	2014年
N1	CP013146.1	Sheep	Equatorial Guinea: Mongomo	2014年
P54B96	CP003385.1	wildebeest	South Africa	2014年
PO222/4-1	CP013698.1	Goat	Portugal	2015年
KM01	CP024995.1	Goat	China: Yunnan, Kunming	2017年
262	CP012022.2	Bos taurus	Belgium	2017年
I19	CP002251.2	Bovine with severe clinical mastitis in two quarters	Israel	2017年

下载: 导出CSV

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