西藏那曲市牦牛源B型多杀性巴氏杆菌的分离鉴定及基因组分析

doi:10.11843/j.issn.0366-6964.2024.07.029

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (7): 3105-3118.doi: 10.11843/j.issn.0366-6964.2024.07.029

西藏那曲市牦牛源B型多杀性巴氏杆菌的分离鉴定及基因组分析

刘博华¹(), 符汉宇¹, 王玉恒², 索朗斯珠¹, 牛家强¹, 包玉花², 李家奎¹^,³, 徐业芬¹^,*()

1. 西藏农牧学院动物科学学院西藏高原动物疫病研究自治区高校重点实验室，林芝 860000
2. 西藏自治区兽医生物药品制造厂，拉萨 850000
3. 华中农业大学动物医学院，武汉 430000

收稿日期:2023-07-24 出版日期:2024-07-23 发布日期:2024-07-24
通讯作者: 徐业芬 E-mail:484325625@qq.com;xzlzxyf@163.com
作者简介:刘博华(2000-)，男，河南三门峡人，硕士生，主要从事高原动物传染病学研究，E-mail: 484325625@qq.com
基金资助:
西藏自治区科技厅重点项目(XZ202001ZY0044N);区域科技协同创新项目(QYXTZXNQ202204);西藏农牧学院研究生教育创新计划项目(YJS2023-21);国家现代农业产业技术体系(CARS-37)

Isolation, Identification and Genome Analysis of Type B Pasteurella multocida Isolated from Yak in Tibetan Nakchu City

Bohua LIU¹(), Hanyu FU¹, Yuheng WANG², Suolangsizhu¹, Jiaqiang NIU¹, Yuhua BAO², Jiakui LI¹^,³, Yefen XU¹^,*()

1. Provincial Key Laboratory of Tibet Plateau Animal Epidemic Disease Research, College of Animal Science, Tibet Agricultural and Animal Husbandry College, Nyingchi 860000, China
2. Veterinary Biopharmaceutical Manufacturing Factory, Tibet Autonomous Region, Lhasa 850000, China
3. College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430000, China

Received:2023-07-24 Online:2024-07-23 Published:2024-07-24
Contact: Yefen XU E-mail:484325625@qq.com;xzlzxyf@163.com

摘要/Abstract

摘要：

旨在对引起西藏牦牛呼吸道感染的多杀性巴氏杆菌进行分离鉴定，明确病原菌的血清型、耐药性和致病性等生物学特性及基因组特征和遗传进化关系。本研究从西藏林芝、拉萨、那曲地区采集牦牛鼻咽拭子和组织病料498份，进行流行病学调查、细菌学分离纯化、生化试验、PCR鉴定、K-B药敏试验、动物致病性试验和分离株全基因组测序，并通过Mauve软件和MEGA 7软件进行基因共线性和系统进化分析。结果显示，经细菌分离纯化、生化试验及PCR鉴定, 发现在西藏林芝、拉萨、那曲地区巴氏杆菌阳性率为2.61%，且从病死牦牛肺组织中分离出B型多杀性巴氏杆菌菌株，命名为“Pm XZ01”株；药敏试验发现Pm XZ01株对头孢菌素类、喹诺酮类、羧苄西林、丁胺卡那、复方新诺明和氯霉素类抗生素敏感；动物试验发现感染Pm XZ01株的家兔(1.2×10⁸ CFU·mL^－1)和小鼠(1×10⁷ CFU·mL^－1)死亡率高达100%，说明在该浓度下Pm XZ01株具有较强的致病性和致死性。全基因组测序获得Pm XZ01株全基因组大小为2 331 787 bp，GC含量为40.47%，重复序列总长度为3 266 bp；含有121个非编码RNA基因，包括59个tRNA基因，19个rRNA基因，43个ncRNA基因；2个假基因，5个基因岛，3个前噬菌体，68个碳水化合物活性酶基因；CARD注释分析发现1个PBP3抗性基因和2个EF-Tu抗性基因；VFDB和PHI-base注释分析发现101个毒力因子相关基因及663个表型突变基因等；共线性分析发现Pm XZ01株与猪源HB03株(CP003328.1)、HN07株(CP007040.1)和禽源P1702株(CP097616.1)共线性最好，而与牦牛源Tibet-Pm1株(CP072655.1)共线性较差；系统进化树分析发现Pm XZ01株与中国广东牛源GDZQ201401株(KP083466.1)属于同一分支，进化关系最近。本研究完成了西藏牦牛巴氏杆菌病的流行病学调查，以及牦牛源B性多杀性巴氏杆菌的分离鉴定、生物学特性和全基因组测序分析，为探索西藏牦牛源B型多杀性巴氏杆菌病的流行规律和致病机制以及防控提供了参考。

关键词: 牦牛, B型多杀性巴氏杆菌, 分离鉴定, 药物敏感性, 致病性, 基因组

Abstract:

The aim of this study was to isolate and identify Pasteurella multocida which caused respiratory tract infection in Tibetan yaks, and to identify the biological characteristics, such as serotype, drug resistance, pathogenicity, genome characteristics and genetic evolution of the pathogenic bacteria. In this study, 498 nasopharyngeal swabs and tissue samples of yaks were collected from Nyingchi, Lhasa and Nakchu regions of Tibet. Epidemiological investigation, bacteriological isolation and purification, biochemical tests, PCR identification, K-B susceptibility test, animal pathogenicity test and whole genome sequencing of the isolates were conducted. Gene collinearity and phylogenetic analysis were carried out by Mauve software and MEGA7 software. The results showed that the positive rate of Pasteurella multocida was 2.61% in Nyingchi, Lhasa and Nakchu regions of Tibet, and a strain of Pasteurella multocida type B was isolated from the lung tissue of dead yaks, named "Pm XZ01" strain. Drug susceptibility tests showed that Pm XZ01 strain were sensitive to cephalosporins, quinolones, carbenicillin, bucaricana, cotrimoxazole and chloramphenicol antibiotics; Animal experiments showed that the mortality of rabbits (1.2×10⁸ CFU·mL^－1) and mice (1×10⁷ CFU·mL^－1) infected with Pm XZ01 strain was as high as 100%, indicating that Pm XZ01 strain had strong pathogenicity and lethality at this concentration. The total genome size of the Pm XZ01 strain obtained by whole genome sequencing was 2 331 787 bp, the GC content was 40.47%, and the total length of the repeats was 3 266 bp. It contained 121 non-coding RNA genes, including 59 tRNA genes, 19 rRNA genes and 43 ncRNA genes. There were 2 pseudogenes, 5 gene islands, 3 prophage, 68 carbohydrate active enzyme genes; CARD annotation analysis found out one PBP3 resistance gene and two EF-Tu resistance genes. VFDB and PHI-base annotation analysis revealed 101 virulence factor related genes and 663 phenotypic mutant genes. The collinearity analysis showed that Pm XZ01 strain had the best collinearity with pig HB03 (CP003328.1), HN07 (CP007040.1) and avian P1702 (CP097616.1) strains, but poor collinearity with yak Tibet-Pm1 (CP072655.1) strain. Phylogenetic tree analysis showed that Pm XZ01 strain belonged to the same branch as GDZQ201401 strain (KP083466.1) from Guangdong cattle in China, and had the closest evolutionary relationship. This study completed the epidemiological investigation of pasteurellosis in Tibetan yaks, as well as the isolation, identification, biological characteristics and whole genome sequencing analysis of Pasteurellosis multocida type B of yak origin, providing a reference for exploring the epidemic rule, pathogenic mechanism and prevention and control of type B pasteurellosis of Tibetan yaks.

Key words: yak, Pasteurella multocida type B, isolation and identification, drug sensitivity, pathogenicity, genome

中图分类号:

S852.612

刘博华, 符汉宇, 王玉恒, 索朗斯珠, 牛家强, 包玉花, 李家奎, 徐业芬. 西藏那曲市牦牛源B型多杀性巴氏杆菌的分离鉴定及基因组分析[J]. 畜牧兽医学报, 2024, 55(7): 3105-3118.

Bohua LIU, Hanyu FU, Yuheng WANG, Suolangsizhu, Jiaqiang NIU, Yuhua BAO, Jiakui LI, Yefen XU. Isolation, Identification and Genome Analysis of Type B Pasteurella multocida Isolated from Yak in Tibetan Nakchu City[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(7): 3105-3118.

图/表 14

图 1

表 1

表 2

图 2

表 3

图 3

表 4

分离菌株药物敏感性试验结果"

抗生素 Antibiotic	药物名称 Antimicrobial agents	判定标准Criterion			平均直径/mm Mean diameter	结果 Result
抗生素 Antibiotic	药物名称 Antimicrobial agents	R	I	S	平均直径/mm Mean diameter	结果 Result
青霉素类 Penicillins	青霉素Penicillin	≤10	10~15	≥15	5.96	R
	苯唑西林Oxacillin	≤14	15~22	≥23	6.45	R
	氨苄西林Ampicillin	≤13	14~16	≥17	13.31	I
	羧苄西林Carbenicillin	≤13	14~16	≥17	21.11	S
	哌拉西林Piperacillin	≤17	18~20	≥21	18.5	I
头孢菌素类 Cephalosporins	头孢氨苄Cephalexin	≤14	14~18	≥19	17.09	I
	头孢唑林Cefazolin	≤14	14~18	≥19	22.13	S
	头孢拉定Cefradine	≤14	14~18	≥19	14.62	I
	头孢呋辛Cefuroxime	≤14	14~18	≥19	19.41	S
	头孢他啶Ceftazidime	≤14	15~17	≥18	19.96	S
	头孢哌酮Cefoperazone	≤15	16~20	≥21	23.76	S
	头孢曲松Ceftriaxone	≤13	14~20	≥21	26.62	S
大环内酯类 Macrolides	红霉素Erythromycin	≤13	14~22	≥23	6.62	R
大环内酯类 Macrolides	麦迪霉素Medemycin	≤10	11~15	≥16	5.95	R
氨基糖苷类 Aminoglycosides	阿米卡星Amikacin	≤14	15~16	≥17	20.66	S
	庆大霉素Gentamicin	≤12	13~14	≥15	8.19	R
	卡那霉素Kanamycin	≤13	14~17	≥18	17.92	I
	新霉素Neomycin	≤17	18~21	≥22	18.92	I
四环素类 Tetracycline	四环素Tetracycline	≤11	12~14	≥15	6.47	R
	多西环素Doxycycline	≤9	10~12	≥13	8.37	R
	米诺环素Minocycline	≤12	13~15	≥16	9.42	R
喹诺酮类 Quinolones	诺氟沙星Norfloxacin	≤12	13~16	≥17	28.27	S
	氧氟沙星Ofloxacin	≤13	14~16	≥17	31.03	S
	环丙沙星Ciprofloxacin	≤15	16~20	≥21	31.55	S
多肽类 Polypeptide	万古霉素Vancomycin	≤10	11~16	≥17	15.26	I
多肽类 Polypeptide	多黏菌素B Polymyxin B	≤11	―	≥12	11.91	I
磺胺类 Sulfonamides	复方新诺明 Cotrimoxazole	≤9	10~16	≥17	22.93	S
呋喃类Furans	呋喃唑酮Furazolidone	≤13	14~22	≥23	14.50	I
氯霉素类 Chloramphenicol	氯霉素 Chloramphenicol	≤12	13~17	≥18	26.30	S
林可酰胺类 Lincoamides	克林霉素 Clindamycin	≤10	11~15	≥16	6.44	R

表 4

图 4

图 5

表 5

图 6

表 6

图 7

图 8

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基因 Gene	引物名称 Primer name	引物序列(5′→3′) Primer sequence (5′→3′)	产物大小/bp Product size	退火温度/℃ Annealing temperature
16S rRNA	Pm-16s	F: AGAGTTTGATCATGGCTCAG R: AAGGAGGTGATCCAACC	1 534	55
Kmt1	Pm-Kmt1	F: ATCCGCTATTTACCCAGTGG R: GCTGTAAACGAACTCGCCAC	460	55
hyaD-hyaC	A型	F: TGCCAAAATCGCAGTCAG R: TTGCCATCATTGTCAGTG	1 044	53
bcbD	B型	F: CATTTATCCAAGCTCCACC R: GCCCGAGAGTTTCAATCC	760	53
dcbF	D型	F: TTACAAAAGAAAGACTAGGAGCCC R: CATCTACCCACTCAACCATATCAG	657	53
ecbJ	E型	F: TCCGCAGAAAATTATTGACTC R: GCTTGCTGCTTGATTTTGTC	511	53
fcbD	F型	F: AATCGGAGAACGCAGAAATCAG R: TTCCGCCGTCAATTACTCTG	852	53

项目 Project	结果 Result	项目 Project	结果 Result	项目 Project	结果 Result
甘露醇Mannitol	+	乳糖Lactose	-	山梨醇Sorbitol	+
蔗糖Sucrose	+	鼠李糖Rhamnose	-	尿素Urea	+
麦芽糖Maltose	-	葡萄糖Glucose	+	硫化氢Hydrogen	+
明胶Gelatin	-	硝酸盐Nitrate	+	枸橼酸盐Citrate	-

数据库 Archive	基因数量 Gene number
NOG	1 974
GO	1 749
KEGG	1 585
NR	2 138
Pfam	1 961
Swiss-prot	1 811
TrEMBL	2 121
CAZy	68
CARD	3
PHI	663
VFDB	101

抗生素耐药基因 Antibiotic resistance genes	数量 Number	抗性 Resistances
PBP3	1	碳青霉；头孢菌素；青霉素；单菌霉素；头霉素 Penicillium carbonum; Cephalosporins; Penicillin; Monomycin; Cephalomycin
EF-Tu	2	芬尼法霉素Fenifamycin

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西藏那曲市牦牛源B型多杀性巴氏杆菌的分离鉴定及基因组分析

Isolation, Identification and Genome Analysis of Type B Pasteurella multocida Isolated from Yak in Tibetan Nakchu City

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采样年份 Sampling year	地区 District	样本数量/头 Sample number	阳性数/头 Positive number
2020	西藏自治区林芝市	59	0
2020	西藏自治区拉萨市	63	0
2020	西藏自治区那曲市	61	6
2021	西藏自治区林芝市	77	0
2021	西藏自治区拉萨市	62	0
2021	西藏自治区那曲市	87	5
2022	西藏自治区林芝市	30	0
2022	西藏自治区那曲市	24	2
2023	西藏自治区林芝市	35	0
总计Total		498	13