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Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies

Marcel AP van Bergen¹, Guus Simons², Linda van der Graaf-van Bloois^1,3, Jos PM van Putten³, Jeroen Rombout², Irene Wesley⁴ and Jaap A Wagenaar^1,3

¹Animal Sciences Group, Division of Infectious Diseases, and OIE Reference Laboratory for Campylobacteriosis, PO Box 65, 8200 AB, Lelystad, The Netherlands ²Keygene N.V., Department of Microbial Genomics, Wageningen, The Netherlands ³Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, and OIE Reference Laboratory for Campylobacteriosis, Utrecht University, Utrecht, The Netherlands ⁴Pre-Harvest Food Safety and Enteric Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, Iowa, USA

Correspondence Jaap A. Wagenaar jaap.wagenaar{at}wur.nl

Received 3 June 2005
Accepted 9 August 2005

Differentiation of Campylobacter fetus into C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) is important for both clinical and economic reasons. In the past, several molecular typing methods have been used for differentiation, including amplified fragment length polymorphism (AFLP). In this study, AFLP was employed to identify C. fetus subspecies specific markers that can serve as a basis for design of novel PCR primer sets for Cfv. Four groups of C. fetus strains with different phenotypic or genotypic traits were examined by AFLP using 22 different DdeI/MboI primer combinations. Specific AFLP fragments were deduced and sequenced resulting in 41 sequences. Based on the obtained sequences, five potential subspecies-specific PCR assays were developed. Extensive evaluation of the five selected PCRs with a set of 65 diverse C. fetus strains identified primer set Cf C05 as subspecies Cfv-specific. This newly developed PCR is fully consistent with the AFLP subspecies differentiation results. The data indicate AFLP as a powerful tool for comparing closely related genomes and for exploiting this information to develop a specific PCR with extensive typing potential.

Abbreviations: AFLP, amplified fragment length polymorphism; Cff, Campylobacter fetus subsp. fetus; Cfv, Campylobacter fetus subsp. venerealis.

The GenBank accession number for the target gene sequence of PCR Cf C05 is DQ146479.

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