猪源产毒素多杀性巴氏杆菌菌株检测技术的评估

Evaluation of techniques for the detection of toxigenic Pasteurella multocida strains from pigs.

作者信息

Amigot J A, Torremorell M, Pijoan C

机构信息

Departamento de Patología Animal, Facultad de Veterinaria, Zaragoza, Spain.

出版信息

J Vet Diagn Invest. 1998 Apr;10(2):169-73. doi: 10.1177/104063879801000209.

DOI:10.1177/104063879801000209

PMID:9576345

Abstract

Recently acquired field isolates and archived isolates from our collection of Pasteurella multocida were analyzed for production of dermonecrotic toxin. Detection of the toxin was carried out using a fetal lung feline (FLF) cell line and a commercial enzyme-linked immunosorbent assay (ELISA) kit. The dermonecrotic toxin gene (ToxA) was also detected using a polymerase chain reaction (PCR) technique. Results from the 3 methods were compared. Field isolates (group 1) came from a commercial herd that had clinical signs of atrophic rhinitis. Fifty-six (17.9%) strains were isolated from 312 nasal swabs. Thirty-five of these strains belonged to serotype A and the rest (21/56), although probably serotype D, were not characterized further. All of these strains were toxin negative based on both the ELISA and FLF cell culture results. Five isolates gave faint bands in the PCR reaction, and the rest (51/56) were PCR negative. PCR and ELISA were also performed from the initial swab cultures (mixed cultures); 7 samples gave faint PCR bands, but ELISA results were all negative. Archived strains (group 2) had been isolated from clinical cases of atrophic rhinitis and from cases of pulmonary pasteurellosis. A total of 76 strains were analyzed; 46 were serotype A, and the rest (30) were serotype D. ELISA and FLF cell culture tests were negative for all serotype A strains; however, 3 strains showed faint bands in the PCR reaction. Fourteen serotype D strains showed positive results in both the ELISA and the FLF cell culture tests. PCR from these samples also gave positive results showing a strong band in the gel. However, 4 strains that were ELISA and FLF cell culture negative showed a faint band in the PCR reaction. The 3 methods gave similar results in the detection of the P. multocida dermonecrotic toxin. However, complete agreement among the tests was achieved only when strong PCR bands were considered positive. This is the first report that demonstrates the use of FLF cell line for the detection of toxigenic P. multocida.

摘要

对近期获得的多杀巴斯德菌野外分离株以及我们收集的该菌存档分离株进行了皮肤坏死毒素产生情况分析。毒素检测采用猫胎儿肺（FLF）细胞系和商用酶联免疫吸附测定（ELISA）试剂盒进行。还使用聚合酶链反应（PCR）技术检测了皮肤坏死毒素基因（ToxA）。对这3种方法的结果进行了比较。野外分离株（第1组）来自一个有萎缩性鼻炎临床症状的商业牛群。从312份鼻拭子中分离出56株（17.9%）菌株。其中35株属于A型血清型，其余（21/56）虽然可能是D型血清型，但未作进一步鉴定。根据ELISA和FLF细胞培养结果，所有这些菌株的毒素检测均为阴性。5株分离株在PCR反应中出现微弱条带，其余（51/56）PCR检测为阴性。还对初始拭子培养物（混合培养物）进行了PCR和ELISA检测；7个样本出现微弱的PCR条带，但ELISA结果均为阴性。存档菌株（第2组）从萎缩性鼻炎临床病例和肺巴氏杆菌病病例中分离得到。共分析了76株菌株；46株为A型血清型，其余（30株）为D型血清型。所有A型血清型菌株的ELISA和FLF细胞培养检测均为阴性；然而，3株在PCR反应中出现微弱条带。14株D型血清型菌株在ELISA和FLF细胞培养检测中均呈阳性结果。这些样本的PCR检测也呈阳性，在凝胶中显示出一条强条带。然而，4株ELISA和FLF细胞培养检测为阴性的菌株在PCR反应中出现微弱条带。这3种方法在检测多杀巴斯德菌皮肤坏死毒素方面结果相似。然而，只有当强PCR条带被视为阳性时，各项检测之间才完全一致。这是首次报道使用FLF细胞系检测产毒素多杀巴斯德菌。