title: Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies.
abstract: 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.
authors.affiliation: Animal 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.
authors.forename: Marcel Ap
authors.initials: MA
authors.lastname: van Bergen
authors.affiliation: Animal 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.
authors.forename: Guus
authors.initials: G
authors.lastname: Simons
authors.affiliation: Animal 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.
authors.forename: Linda
authors.initials: L
authors.lastname: van der Graaf-van Bloois
authors.affiliation: Animal 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.
authors.forename: Jos Pm
authors.initials: JP
authors.lastname: van Putten
authors.affiliation: Animal 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.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Rombout
authors.affiliation: Animal 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.
authors.forename: Irene
authors.initials: I
authors.lastname: Wesley
authors.affiliation: Animal 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.
authors.forename: Jaap A
authors.initials: JA
authors.lastname: Wagenaar
mesh_terms: D000818:Animals; D002168:Campylobacter fetus / Q000235:genetics* / Q000302:isolation & purification*; D002417:Cattle; D005784:Gene Amplification; D005819:Genetic Markers; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012703:Serotyping; D012756:Sheep
chemical_list: D005819:Genetic Markers