18型鸭疫里默氏杆菌的分离鉴定及致病性研究

Isolation,identification and pathogenicity of Riemerella anatipestifer serotype 18

为确定广东梅州市某养殖场病死鹅的发病原因及病原特征,本研究采集其肝脏、心脏等组织样品,经TSA血清平板培养后观察菌落形态、生化试验、PCR鉴定、血清型鉴定,确定该分离菌为血清18型鸭疫里默氏杆菌(RA-18);对分离菌进行药敏试验、毒力基因检测及马岗鹅的致病性试验;结果显示,分离菌对阿莫西林、头孢拉定、头孢唑啉、头孢噻呋、头孢曲松、大观霉素、氟苯尼考、多西环素、氧氟沙星敏感;对青霉素、新霉素、丁胺卡那、卡那霉素、庆大霉素、阿奇霉素、恩诺沙星、诺氟沙星、复方新诺明、多粘菌素B耐药;共检出6种毒力基因(Fur、lux E、tct R、luxr1、cwa H、lipid A);以实际接种量5×10~5cfu/m L的分离菌经肌肉注射感染马岗鹅,结果显示未引起马岗鹅的死亡;以实际接种量5×10^(8)cfu/m L的分离菌分别经肌肉、脚蹼注射和滴鼻感染白鸭和白鹅,在感染后7 d的观察期内观察动物的临床症状及剖检,无菌采集各组动物的脑组织、心脏、肝脏、脾脏,经相应处理后涂板,采用平板计数法测定各脏器的载菌量。结果显示,白鹅肌肉、白鹅脚蹼、白鸭肌肉接种感染可导致鹅、鸭的发病及死亡,滴鼻接种未能引起鹅、鸭死亡;对各组白鹅、白鸭的各脏器进行平板计数,结果显示各组的各种动物的心脏、肝脏、脑组织、脾脏均可测得一定的菌量。肌肉注射组动物的组织病理学观察结果显示,分离菌感染白鸭及白鹅的病理变化涉及脑组织、心脏、肝脏、脾脏等。结果表明,本研究首次在国内分离到RA-18菌株,该菌株对白鸭和白鹅具有一定的致病性,对广东地区鹅源鸭疫里默氏杆菌病(RAI)的诊断和防治有一定的参考意义。

To determine the cause and pathogenic characteristics of the disease in geese that died of illness in a breeding farm in Meizhou City,Guangdong Province.This study collected tissue samples such as liver and heart.The isolate was identified as Riemerella anatipestifer serotype 18(RA-18)after observation of colony morphology on TSA serum plate,biochemical test,PCR identification and serotyping.Antimicrobial susceptibility tests,virulence gene detection,and experimental pathogenicity tests were conducted on the isolated bacteria.The results showed that the isolate was sensitive to amoxicillin,ceftriaxone,ceftazidime,cefoperazone,ceftriaxone,gatifloxacin,levofloxacin,doxycycline,and ofloxacin,while it was resistant to penicillin,neomycin,gentamicin,kanamycin,gentamicin,azithromycin,enrofloxacin,norfloxacin,sulfa-methoxazole-trimethoprim,and polymyxin B.Six virulence genes(Fur,lux E,tct R,luxr1,cwa H,lipid A)were detected.Intramuscular injection of isolated bacteria with an actual inoculation volume of 5×10~8cfu/m L infected Magang geese did not cause any death in Magang geese;White ducks and geese were infected with the isolate at a dose of 5×10^(8)cfu/m L through muscle,footpad,and nasal drops.During the 7-day period after infection,the clinical symptoms and autopsy were observed,and the sterile collections of brain,heart,liver,and spleen tissues were plated out,and bacterial counts were determined using plate count method.The results showed that intramuscular and footpad infections in geese,and intramuscular infections in ducks,could lead to disease and death,but nasal drop infections did not cause mortality in either species;Plate counting was performed on various organs of white geese and ducks in each group,and the results showed that a certain amount of bacteria could be measured in the hearts,livers,brain tissues,and spleens of various animals in each group.Histopathological observations of the intramuscular injection group showed pathological changes of the isolate infected white geese involved brain,heart,liver,and spleen tissues.In conclusion,this is the first report that the RA-18 strain has been isolated in China,which had certain pathogenicity to white ducks,geese,and had certain significance for the diagnosis and control of Riemerella anatipestifer infection(RAI)in Guangdong region.