貉源支气管败血波氏杆菌分离鉴定及其生物学特性分析

doi:10.16431/j.cnki.1671-7236.2026.01.044

Abstract

Abstract:

Objective The purpose of this experiment was to clarify the cause of the death of raccoon dogs in a raccoon dog farm in Hebei province， and provide a scientific basis for the effective prevention and control of the disease. Method Sterile collection of diseased tissues and respiratory secretions from raccoon dogs was collected， and the target strains were obtained through pathogen isolation and culture. The morphology of the isolate was identified by Gram staining. The classification of isolate was clarified by using 16S rRNA gene sequence alignment. The virulence genes and drug resistance genes of the isolate were detected by PCR amplification， and further drug sensitivity tests were carried out to evaluate the drug resistance phenotypes. The pathogenicity of the isolate was detected through the mouse pathogenicity test. Result The strain isolated from raccoon dogs formed round， neatly edged grayish-white colonies on TSB agar plates and smooth， moist， transparent colonies on MAC agar plates. Gram staining revealed the strain as Gram-negative coccobacilli with bipolar staining. 16S rRNA sequence alignment analysis showed that the similarity of the isolate to other Bordetella bronchiseptica in GenBank was 97.0%-100%， and this isolate was Bordetella bronchiseptica.Detection of virulence and resistance genes demonstrated the presence of six virulence genes （prn， bvgs， fla， fhaB， cyaA and dnt） and five resistance genes （bla_TEM， bla_SHV， parC， tetC and gyrA）. The results of the drug sensitivity test showed that the isolate was resistant to penicillins， macrolides， glycopeptides， sulfonamides and nitrofurans， but sensitive to aminoglycosides and quinolones. Pathogenicity tests showed that the median lethal dose of the isolate to mice was 3.67×10⁷ CFU. Conclusion In this experiment， one strain of Bordetella bronchiseptica was isolated from dead raccoon dogs， which carried multiple virulence genes and drug resistance genes. The isolate showed resistance to penicillins， macrolides， glycopeptides， sulfonamides and nitrofurans， and was sensitive to aminoglycosides and quinolones， demonstrating multidrug resistance and strong pathogenicity. The results of this study provided a scientific basis for the diagnosis， clinical medication， and vaccine development of Bordetella bronchiseptica disease in raccoon dogs.

Key words: raccoon dog; Bordetella bronchiseptica; isolation and identification; virulence genes; drug resistance gene

CLC Number:

S855.1^＋2

WANG Qilin, CAO Runlai, LIU Peiwen, LIU Ziyu, XUE Zhuqing, DU Haixia, ZHANG Yue, ZHANG Minjing, LI Yacong, WANG Weiyang, CONG Yanfang, WANG Xiaoxu, LIU Zhijie. Isolation， Identification and Biological Characteristics Analysis of Bordetella bronchiseptica from Raccoon dog（Nyctereutes procyonoides）[J]. China Animal Husbandry & Veterinary Medicine, 2026, 53(1): 489-498.

Figures/Tables 9

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References 30

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基因 Genes	引物序列 Primer sequences （5′→3′）	片段长度 Fragment length/bp	退火温度 Annealing temperature/℃	参考文献 Reference
16S rRNA	F：AGAGTTTGATCMTGGCTCAG	1 500	56	赵梦溪等^［14］
16S rRNA	R：GGTTACCTTGTTACGACTT	1 500	56	赵梦溪等^［14］
fla	F：AGGCTCCCAAGAGAGAAAGGCTT	237	58	张瑞华等^［15］
fla	R：TGGCGCCTGCCCTATC	237	58	张瑞华等^［15］

分类 Classification	基因 Genes	引物序列 Primer sequences （5′→3′）		片段长度 Fragment length/bp
毒力基因 Virulence genes	prn	F：CAGCGTCGATTTCCAGCAGCC	R：TCCYGCATGACGACCAGCTT	138
	bvgs	F：ATTACCTCCCATCATCCGAA	R：TCTTGACGACCGGATGGTT	351
	fla	F：CCCCCGCACATTTCCGAACTTC	R：AGGCTCCCAAGAGAGAAAGGC	164
	fhaB	F：GCGCAGAACATCACCAATG	R：TGAAATACTCCATGGCGGAC	475
	cyaA	F：CTACGAGCAGTTCGAGTTTC	R：TATTCATGTCGCCGTCGTA	377
	dnt	F：CGTTCGGCTGGGAGATGG	R：CCGGGCGGTACTTGGGAT	491
耐药基因 Drug resistance genes	bla_OXA	F：GTGAAGCGTGTTGGTTAT	R：AGCCTACTTGTGGGTCTA	274
	bla_TEM	F：TTCCGTGTCGCCCTTATTCC	R：TATCCGCCTCCATCCAGTCT	622
	bla_CTX	F：GTTGTTATTTCGTATCTTCCAG	R：ATTCGGTTCGCTTTCACT	311
	bla_SHV	F：GACCGCTGGGAAACGGAACT	R：CCCGCAGATAAATCACCACAA	307
	ermA	F：TAATGAACGCTTAATGTCAG	R：GTTTACCGCTTCTTTAGTCA	269
	ermB	F：CATTTAACGACGAAACTGG	R：CGGGTAAGTTTTATTAAGACAC	407
	parC	F：CCTTGCGCTACATGAATTTA	R：TCAATAGCAGCTCGGAATAT	380
	cmlA	F：GTACGACAGCGAGCACAATT	R：ATTCCAGCGGCGTAACACAA	544
	tetC	F：TCGGAATCTTGCACGCCCTC	R：CGCCTACAATCCATGCCAACCC	360
	catA	F：GGATCTCTTATGACCAACTA	R：GGCATCGGCTTGATTGAATT	494
	sul1	F：TTTCCTGACCCTGCGCTCTA	R：ATTGCCGATCGCGTGAAGTT	405
	floR	F：CGTTTATGCCAACCGTCCTG	R：ACGATCATTACAAGCGCGACA	402
	gyrA	F：ACGGTCTCAAGCCTGTGCATC	R：CTCCTGTTCGCTGCCGTCGTA	350

分类 Classification	抗菌药物 Antibacterial	判定标准 Judgment standards/mm			抑菌圈直径 IZD/mm	结果 Results
分类 Classification	抗菌药物 Antibacterial	R	I	S	抑菌圈直径 IZD/mm	结果 Results
青霉素类 Penicillins	苯唑西林 Oxacillin	≤10	11~12	≥13	6	R
	氨苄西林 Ampicillin	≤13	14~16	≥17	6	R
	哌拉西林 Piperacillin	≤17	18~20	≥21	6	R
头孢菌素类 Cephalosporins	头孢氨苄 Cefalexin	≤14	15~17	≥18	6	R
	头孢唑林 Cefazolin	≤14	15~17	≥18	6	R
	头孢拉定 Cefradine	≤14	15~17	≥18	6	R
	头孢呋辛 Cefuroxime	≤14	15~17	≥18	6	R
	头孢他啶 Ceftazidime	≤14	15~17	≥18	25	S
	头孢曲松 Ceftriaxone	≤13	14~20	≥21	16	I
	头孢哌酮 Cefoperazone	≤15	16~20	≥21	28	S
氨基糖苷类 Aminoglycosides	丁胺卡那 Amikacin	≤14	15~16	≥17	18	S
	庆大霉素 Gentamicin	≤12	13~14	≥15	20	S
	卡那霉素 Kanamycin	≤13	14~17	≥18	18	S
	新霉素 Neomycin	≤12	13~16	≥17	18	S
四环素类 Tetracyclines	四环素 Tetracycline	≤14	15~18	≥19	6	R
四环素类 Tetracyclines	多西环素 Doxycycline	≤12	13~15	≥16	14	I
大环内酯类 Macrolides	红霉素 Erythromycin	≤13	14~22	≥23	16	I
大环内酯类 Macrolides	麦迪霉素 Midecamycin	≤13	14~17	≥18	6	R
喹诺酮类 Quinolones	诺氟沙星 Norfloxacin	≤12	13~16	≥17	21	S
	氧氟沙星 Ofloxacin	≤12	13~15	≥16	22	S
	环丙沙星 Ciprofloxacin	≤15	16~20	≥21	27	S
林可酰胺类 Lincosamides	克林霉素 Clindamycin	≤14	15~20	≥21	6	R
糖肽类 Glycopeptides	万古霉素 Vancomycin	≤9	10~11	≥12	6	R
磺胺类 Sulfonamides	复方星诺明 Compound sulfamethoxazole	≤10	11~15	≥16	6	R
硝基呋喃类 Nitrofurans	呋喃唑酮 Furazolidone	≤14	15~16	≥17	6	R

菌液浓度 Bacterial solution concentration/（CFU/mL）	死亡数 Number of deaths/只	死亡率 Death rate/%
0	0	0
1.6×10⁵	0	0
1.6×10⁶	0	0
1.6×10⁷	2	25
1.6×10⁸	7	87.5
1.6×10⁹	8	100

Isolation， Identification and Biological Characteristics Analysis of Bordetella bronchiseptica from Raccoon dog（Nyctereutes procyonoides）

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