两株兔源多杀性巴氏杆菌分离鉴定及荚膜血清分型

doi:10.3969/j.issn.1004-1524.20230705

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (5): 1041-1046.DOI: 10.3969/j.issn.1004-1524.20230705

两株兔源多杀性巴氏杆菌分离鉴定及荚膜血清分型

季权安(), 徐翔飞, 鲍国连, 黄叶娥, 崔雪梅, 刘燕^*()

浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2023-06-01 出版日期:2024-05-25 发布日期:2024-05-29
作者简介:季权安(1967—),男,浙江庆元人,主要从事畜禽传染病诊断与防治研究。E-mail:jiquanan@outlook.com
通讯作者: * 刘燕,E-mail:ly-liuyan@163.com
基金资助:
浙江省自然科学基金(LTGD24C180001);国家兔产业技术体系(CARS-43-C-2)

Identification and capsule serotyping of two strains of Pasteurella multocida isolated from rabbit

JI Quan’an(), XU Xiangfei, BAO Guolian, HUANG Ye̍e, CUI Xuemei, LIU Yan^*()

Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2023-06-01 Online:2024-05-25 Published:2024-05-29

摘要/Abstract

摘要：

为探明浙江省规模兔场主要传染病流行现状,明确其致病菌及血清型,开展了家兔流行病学调查与病原分离鉴定。调研发现,不少兔场存在以打喷嚏和鼻腔流出浆液性、黏液性或脓性分泌物为主要临床特征的呼吸道传染病,进而对浙江省家兔主产区发病严重兔场进行采样,从嵊州、宁波三家患病兔鼻腔中分离到两株病原菌,进行了病原分离鉴定、荚膜血清分型及药敏试验。根据分离菌的菌落形态、革兰氏染色特征、特异性PCR以及荚膜血清分型PCR鉴定结果,确定两株致病菌均为荚膜血清A型多杀性巴氏杆菌,分别命名为ZJNB0321和ZJSZ0322。构建系统发育树,结果表明,两分离株属于同一分支,且与多杀性巴氏杆菌处于同一分支,并拥有独立的分支。药敏试验结果表明,不同地区分离株药敏特性不尽相同。研究结果为兔场合理用药及有效防控提供了科学依据。

关键词: 兔, 呼吸道疾病, 多杀性巴氏杆菌, 荚膜血清型, 药敏试验

Abstract:

To investigate the prevalence of major infectious diseases in rabbit farms in Zhejiang Province, the epidemiological survey was carried out to isolate, identify the serotype of pathogenic bacteria. It was found that respiratory infectious diseases, mainly characterized by sneezing and nasal discharge of serous, mucous or purulent secretions, existed in most rabbit farms. Thus, rabbits with severe respiratory diseases from two farms in Ningbo and Shengzhou were sampled for bacterial isolation and identification, serotyping, and drug sensitivity test. Based on the colonial morphology, characters of gram-staining, specific PCR, and capsule seroptyping, the bacterium were identified as capsule seroptype A Pasteurella multocida, which were named as ZJNB0321 and ZJSZ0322, respectively. The evolutionary tree results showed that the two isolates belonged to the same clade, and were in the same clade as Pasteurella multocida, but they had their own separate clade. The results of the drug sensitivity test indicated that the bacterium showed different drug sensitive characteristics. This study provides scientific basis for rational drug use, and development of effective control measures for rabbit farms.

Key words: rabbit, respiratory disease, Pasteurella multocida, capsule serotype, drug sensitivity test

中图分类号:

S858.291

季权安, 徐翔飞, 鲍国连, 黄叶娥, 崔雪梅, 刘燕. 两株兔源多杀性巴氏杆菌分离鉴定及荚膜血清分型[J]. 浙江农业学报, 2024, 36(5): 1041-1046.

JI Quan’an, XU Xiangfei, BAO Guolian, HUANG Ye̍e, CUI Xuemei, LIU Yan. Identification and capsule serotyping of two strains of Pasteurella multocida isolated from rabbit[J]. Acta Agriculturae Zhejiangensis, 2024, 36(5): 1041-1046.

图/表 9

表1 试验所用引物

Table 1 The primers used in the experiment

类型 Type	引物序列 Primer sequences	片段大小 Fragment size/bp
Pm	5'-ACGGGTGAGTAATGCTTGGG-3'	172
	5'-GCAGGCTTGGTAGGCCTTTA-3'
PmA	5'-TGCCAAAATCGCAGTCAG-3'	1 044
	5'-TTGCCATCATTGTCAGTG-3'
PmB	5'-CATTTATCCAAGCTCCACC-3'	760
	5'-GCCCGAGAGTTTCAATCC-3'
PmD	5'-TTACAAAAGAAAGACTAGGAGCCC-3'	657
	5'-CATCTACCCACTCAACCATATCAG-3'
PmE	5'-TCCGCAGAAAATTATTGACTC-3'	511
	5'-GCTTGCTGCTTGATTTTGTC-3'
PmF	5'-AATCGGAGAACGCAGAAATCAG-3'	851
	5'-TTCCGCCGTCAATTACTCTG-3'

图1 临床症状和病理变化 A,患病兔鼻孔周围潮湿;B,患病兔鼻腔脓性分泌物;C,病死兔气管充、出血;D,病死兔肺有脓肿。

Fig.1 Clinical symptom and pathological change A, The area around the nostrils is moist; B, Purulent secretion of nasal cavity of sick rabbit; C, The congestion and bleeding in the trachea of dead rabbits; D, The dead rabbit had an abscess in its lung.

图2 细菌分离及革兰氏染色 A,细菌划线纯化;B,分离菌革兰氏染色。

Fig.2 Bacterial isolation and gram staining A, Bacterial purification by scribing culture; B, Bacterial purification by scribing culture.

图3 分离菌特异性PCR扩增结果

Fig.3 Results of specific PCR amplification of isolated bacteria M, DL 2000 DNA marker; 1, ZJNB0321; 2, ZJSZ0322.

图4 ZJNB0321荚膜血清型PCR扩增结果 M,DL 2000 DNA marker;A、B、D、E、F分别为荚膜血清A、B、D、E、F型。下同。

Fig.4 PCR amplification of ZJNB0321 capsule serotype M, DL 2000 DNA marker; A, B, D, E, F were capsule serotype A, B, D, E, F, respectively.The same as below.

图5 ZJSZ0322荚膜血清型PCR扩增结果

Fig.5 PCR amplification of ZJSZ0322 capsule serotype

图6 基于ZJNB0321和ZJSZ0322构建进化树

Fig.6 Evolutionary tree based on ZJNB0321 and ZJSZ0322

表2 ZJNB0321药敏试验结果

Table 2 Results of drug sensitivity test of ZJNB0321

抗生素Name of antibiotic	敏感性Sensitivity
青霉素Penicillin	R
链霉素Streptomycin	R
红霉素Erythromycin	S
庆大霉素Gentamicin	S
林可霉素Lincomycin	R
甲氧嘧啶Methoxymidine	R
多黏菌素Polymyxin	S
克林霉素Clindamycin	R
头孢拉定Cefradine	R
左氟沙星Levofloxacin	S
四环素Tetracycline	S
环丙沙星Ciprofloxacin	S
卡那霉素Kanamycin	S
氨苄西林Ampicillin	I
恩诺沙星Enrofloxacin	S
氟苯尼考Flufenicol	S
阿莫西林Amoxicillin	S

表3 ZJSZ0322药敏试验结果

Table 3 Results of drug sensitivity test of ZJSZ0322

抗生素Name of antibiotic	敏感性Sensitivity
青霉素Penicillin	S
链霉素Streptomycin	S
红霉素Erythromycin	R
庆大霉素Gentamicin	R
林可霉素Lincomycin	R
甲氧嘧啶Methoxymidine	R
多黏菌素Polymyxin	I
克林霉素Clindamycin	R
头孢拉定Cefradine	S
左氟沙星Levofloxacin	R
四环素Tetracycline	R
环丙沙星Ciprofloxacin	R
卡那霉素Kanamycin	R
氨苄西林Ampicillin	S
恩诺沙星Enrofloxacin	R
氟苯尼考Flufenicol	R
阿莫西林Amoxicillin	S

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两株兔源多杀性巴氏杆菌分离鉴定及荚膜血清分型

Identification and capsule serotyping of two strains of Pasteurella multocida isolated from rabbit

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