Amplified fragment length polymorphism based identification of genetic markers and novel PCR assay for differentiation of Campylobacter fetus subspecies.
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.
van Bergen
Marcel Ap
MA
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.
Simons
Guus
G
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.
van der Graaf-van Bloois
Linda
L
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.
van Putten
Jos Pm
JP
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.
Rombout
Jeroen
J
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.
Wesley
Irene
I
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.
Wagenaar
Jaap A
JA
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.
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
D005819:Genetic Markers