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

Abstract

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

CLC Number:

S852.612

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.

Figures/Tables 14

Fig. 1

Table 1

Table 2

Fig. 2

Table 3

Fig. 3

Table 4

Results of drug sensitivity test for isolated strains"

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

Table 4

Fig. 4

Fig. 5

Table 5

Fig. 6

Table 6

Fig. 7

Fig. 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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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
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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