A PCR-RFLP assay to detect and type cytolethal distending toxin(cdt) genes in Campylobacterhyointestinalis

[摘要] Campylobacter hyointestinalis is considered as an emerging zoonotic pathogen. We haverecently identified two types of cytolethal distending toxin (cdt) gene in C.hyointestinalis and designated them as Chcdt-I and Chcdt-II. In this study, we developed aPCR-restriction fragment length polymorphism (RFLP) assay that can differentiateChcdt-I from Chcdt-II. When the PCR-RFLP assay wasapplied to 17 other Campylobacter strains and 25non-Campylobacter strains, PCR products were not obtained irrespectiveof their cdt gene-possession, indicating that the specificity of thePCR-RFLP assay was 100%. In contrast, when the PCR-RFLP assay was applied to 35 C.hyointestinalis strains including 23 analyzed in the previous study and 12newly isolated from pigs and bovines, all of them showed the presence ofcdt genes. Furthermore, a restriction digest by EcoT14-I revealed that29 strains contained both Chcdt-I and Chcdt-II and 6strains contained only Chcdt-II, showing 100% sensitivity. Unexpectedly,however, PCR products obtained from 7 C. hyointestinalis strains were notcompletely digested by EcoT14-I. Nucleotide sequence analysis revealed that the undigestedPCR product was homologous to cdtB but not to Chcdt-IBor Chcdt-IIB, indicating the presence of another cdtgene-variant. Then, we further digested the PCR products with DdeI in addition toEcoT14-I, showing that all three cdt genes, including a possible newChcdt variant, could be clearly differentiated. Thus, the PCR-RFLPassay developed in this study is a valuable tool for evaluating the Chcdtgene-profile of bacteria.

[学科分类] 兽医学

[关键词] cdt genes;C. hyointestinalis;PCR-RFLP