奶牛源肺炎克雷伯菌和溶血性曼氏杆菌的分离鉴定与致病性分析

doi:10.16431/j.cnki.1671-7236.2026.02.035

Abstract

Abstract:

Objective This study was conducted to identify the pathogens responsible for bovine mortality， aiming to provide a scientific reference for the prevention and control of diseases caused by co-infections with multiple pathogens in dairy cow. Method In this study， pathogenic bacteria were isolated and cultured from milk and blood samples of dead dairy cow using the plate streaking method. The isolates were subjected to species identification through Gram staining， 16S rRNA sequencing analysis， and virulence gene characterization. Antimicrobial susceptibility profiles were determined by the disk diffusion method， while pathogenicity was evaluated via animal challenge experiments. Result The bacterial isolate in the milk sample presented moist pink colonies of Gram-negative short bacilli on the McConkey medium. The results of 16S rRNA sequence analysis showed that the sequence similarity of the isolated bacteria to the representative strain of Klebsiella in GenBank was >99%， and it was clustered in the same branch as the standard strain of Klebsiella pneumoniae （K. pneumoniae） ATCC 13883. It was identified as K. pneumoniae and named BJ-B004. The results of virulence gene testing indicated that the isolate contained seven virulence genes， including fimH， uge， wabG， ureA， entB， mrkD and ycf. The results of the drug sensitivity test showed that it was resistant to streptomycin， penicillin， amoxicillin， ampicillin， and cotrimoxazole， but was sensitive to antimicrobials such as enrofloxacin， doxycycline， florfenicol， tetracycline， spectinomycin， and cefoperazone. A small Gram-negative bacillus with hemolytic properties and rounded ends was isolated from the blood sample of the dead dairy cow. It was mostly arranged singly or in pairs and showed two-level concentrated staining by Wright-Giemsa staining. The results of 16S rRNA sequence analysis showed that its sequence similarity with the Mannheimia strain was >98%， and it was clustered in the same branch as the Mannheimia strain ZY190616 of bovine origin. It was identified as Mannheimia hemolyticus （M. hemolyticus）and named BJ-B007. The results of the drug sensitivity test indicated that it was not resistant to any antibacterial drugs except gentamicin. BALB/c mice were respectively infected with K. pneumoniae， M. hemolyticus and the mixed bacterial solution at dose of 3×10⁷ CFU， with a PBS blank control group （n=5 per group）， and the results showed that all the mice in the challenge group presented symptoms of the disease. All died within 48 h in mixed infection group， 4 died in M. hemolyticus group and 1 died in K.pneumoniae group. Pathological examination revealed that M. hemolyticus caused severe congestion， hemorrhage and necrotic lesions in multiple organs such as the liver， spleen， lung and kidney. Conclusion This study successfully isolated a multidrug-resistant K. pneumoniae strain and a highly pathogenic M. haemolytica strain from pathological samples of a dead dairy cow. The mortality was determined to be primarily caused by K. pneumoniae-induced mastitis， followed by secondary bacteremia due to opportunistic infection with M. haemolytica. The results provided a scientific foundation for the integrated diagnostic strategies and comprehensive prevention protocols targeting bovine mastitis-respiratory syndrome co-infections.

Key words: dairy cow; Klebsiella pneumoniae; Mannheimia haemolytica; isolation and identification; pathogenicity

CLC Number:

S852.61

ZHANG Sui, CHENG Chunjie, YANG Chun, CHEN Mengjiao, CHENG Jing, ZHOU Linyi, LI Yongqing, WANG Xiaoying, LIU Wenxiao. Isolation， Identification and Pathogenicity Analysis of Dairy Cow-derived Klebsiella pneumoniae and Mannheimia haemolytica[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(2): 903-915.

Figures/Tables 8

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References 49

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目的基因 Target genes	引物序列 Primer sequences （5′→3′）	产物长度 Product length/bp	退火温度 Annealing temperature/℃	参考文献 References
16S rRNA	27-F： AGAGTTTGATCCTGGCTCAG	1 542	55	Weisburg等^［13］
	1492-R： GGTTACCTTGTTACGACTT
Khe	F： TGATTGCATTCGCCACTGG	428	60	Yin-Ching等^［14］
	R： GGTCAACCCAACGATCCTG
16S rRNA	Mh-F： ACGGTAGCACATAGGAGT	453	56	Griffin^［9］
（溶血性曼氏杆菌 M.haemolytic）	Mh-R：TATTACCGCGGCTGCTG
rmpA2	F： GTGCAATAAGGATGTTACATTA	430	50	Russo等^［15］
	R： GGATGCCCTCCTCCTG
iuc	F： GCTTATTTCTCCCCAACCC	583	59
	R： TCAGCCCTTTAGCGACAAG
rmpA	F： GAGTAGTTAATAAATCAATAGCAAT	332	50
	R： CAGTAGGCATTGCAGCA
magA	F： GGTGCTCTTTACATCATTGC	1 282	59	Candan等^［16］
	R： GCAATGGCCATTTGCGTTAG
wcaG	F： GGTTGGKTCAGCAATCGTA	169	54
	R： ACTATTCCGCCAACTTTTGC
allS	F： CCGAAACATTACGCACCTTT	508	57
	R： ATCACGAAGAGCCAGGTCAC
wabG	F： ACCATCGGCCATTTGATAGA	683	58
	R： CGGACTGGCAGATCCATATC
fimH	F： GCTCTGGCCGATACCACCACGG	423	60	邹鹏等^［17］
	R： GCGAAGTAACGTGCCTGGAACGG
mrkD	F： CCACCAACTATTCCCTCGAA	226	62	El-Domany等^［18］
	R： ATGGAACCCACATCGACATT
kfu	F： GAAGTGACGCTGTTTCTGGC	797	54
	R： TTTCGTGTGGCCAGTGACTC
uge	F： TCTTCACGCCTTCCTTCACT	534	54
	R： GATCATCCGGTCTCCCTGTA
entB	F： ATTTCCTCAACTTCTGGGGC	371	57	El Fertas-Aissani等^［19］
	R：AGCATCGGTGGCGGTGGTCA
ycf	F： ATCAGCAGTCGGGTCAGC	160	55
	R： CTTCTCCAGCATTCAGCG
ybtA	F： ATGACGGAGTCACCGCAAAC	960	55	徐水宝等^［20］
	R： TTACATCACGCGTTTAAAGG
ureA	F： GCTGACTTAAGAGAACGTTATG	337	50	和晋渝^［21］
	R： GATCATGGCGCTACCTCTA
iroN	F： GTCCGGCGGTAACTTCAGCC	829	58	高淩^［22］
	R： TCAGAATGAAACTACCGCCC

抗菌药 Antimicrobials	含量 Content/ （μg/片）	肺炎克雷伯菌 K. pneumoniae		溶血性曼氏杆菌 M. haemolytica
抗菌药 Antimicrobials	含量 Content/ （μg/片）	抑菌圈直径IZD/mm	结果Results	抑菌圈直径IZD/mm	结果Results
恩诺沙星 Enrofloxacin	5	30.67±0.29	S	24.51±0.45	S
环丙沙星 Ciprofloxacin	5	24.03±0.05	I	22.72±0.24	S
左氧氟沙星 Levofloxacin	5	25.00±0.00	S	23.19±0.25	S
萘啶酸 Nalidxic Acid	30	13.05±0.05	R	21.37±0.56	S
诺氟沙星 Norfloxacin	10	27.05±0.90	S	21.18±0.80	S
氟苯尼考 Florfenicol	30	25.01±0.39	S	22.25±0.55	S
阿奇霉素 Azithromycin	15	15.10±0.40	S	20.25±0.16	S
强力霉素 Doxycycline	30	18.83±0.14	S	19.14±0.12	S
四环素 Tetracycline	30	21.50±0.50	S	21.49±0.15	S
大观霉素 Spectinomycin	100	19.00±0.00	S	12.63±0.07	I
丁胺卡那 Amikacin	30	19.00±0.24	I	13.93±0.21	I
庆大霉素 Gentamicin	10	21.50±0.80	S	8.76±0.08	R
链霉素 Streptomycin	10	6.00	R	12.13±0.23	I
新霉素 neomycin	30	18.00±0.33	S	24.64±0.43	S
头孢克肟 Cefixine	5	25.16±0.44	S	26.56±0.21	S
头孢克洛 Cefaclor	30	26.67±0.68	S	22.02±0.36	S
头孢哌酮 Cefoperazone	75	27.00±0.95	S	25.96±0.24	S
头孢拉定 Cefradine	30	18.00±0.00	S	17.86±0.33	S
头孢他定 Ceftazidime	30	27.50±1.06	S	22.10±0.18	S
头孢唑林 Cefazolin	30	24.01±0.31	S	18.80±1.47	S
氨苄西林 Ampicilllin	10	10.03±0.61	R	21.09±0.30	S
阿莫西林 Amoxicillin	10	8.00±0.00	R	25.27±0.15	S
青霉素 Penicillin	10 IU	6.00	R	17.82±0.11	S
氯霉素 Chloramphenicol	30	25.00±0.48	S	11.03±0.18	S
复方新诺明Co-trimoxazole	1.25/23.75	6.00	R	16.55±0.11	S

Isolation， Identification and Pathogenicity Analysis of Dairy Cow-derived Klebsiella pneumoniae and Mannheimia haemolytica

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