Identification of campylobacteria isolated from Danish broilers by phenotypic tests and species-specific PCR assays M. Wainø 1 , D.D. Bang 1 , M. Lund 1 , S. Nordentoft 1 , J.S. Andersen 2 , K. Pedersen 1 and M. Madsen 1 1 Department of Poultry, Fish and Fur Animals, Danish Veterinary Institute, A ˚ rhus N, and 2 Danish Veterinary Institute, Danish Zoonosis Centre, Bu¨lowsvej 27, 1790 Copenhagen V, Denmark 2002/465: received 22 November 2002, revised 17 March 2003 and accepted 26 March 2003 ABSTRACT M. WAINØ, D.D. BANG, M. LUND, S. NORDENTOFT, J.S. ANDERSEN, K. PEDERSEN AND M. MADSEN. 2003. Aims: To validate a phenotypic Campylobacter species identification method employed to identify campylo- bacters in broilers by comparison with campylobacterial species identification using various species-specific PCR analyses. Methods and Results: From a collection of 2733 phenotypically identified campylobacterial cultures, 108 Campylobacter jejuni cultures and 351 campylobacterial cultures other than Camp. jejuni were subjected to various species-specific PCR assays. On the basis of the genotypic tests, it was demonstrated that Camp. jejuni and Camp. coli constituted approx. 99% of all cultures, while other species identified were Helicobacter pullorum, Camp. lari and Camp. upsaliensis. However, 29% of the 309 Camp. coli cultures identified by phenotypic tests were hippurate-variable or negative Camp. jejuni cultures, whereas some Camp. lari cultures and unspeciated campylobacter cultures belonged to H. pullorum. It was also notable that 2–6% of the cultures were, in fact, mixed cultures. Conclusions: The phenotypic identification scheme employed failed to appropriately differentiate Campylobacter species and particularly to identify the closely related species, H. pullorum. Significance and Impact of the Study: Future phenotypic test schemes should be designed to allow a more accurate differentiation of Campylobacter and related species. Preferably, the phenotypic tests should be supplemented with a genotypic strategy to disclose the true campylobacterial species diversity in broilers. Keywords: Campylobacter, Helicobacter pullorum, identification, polyphasic, poultry. INTRODUCTION Human gastroenteritis caused by Campylobacter spp. sur- passed the incidence of human salmonellosis in Denmark in 1999 reaching a peak of 86 cases per 100Æ000 inhabitants in the year 2001 (Anon. 1999, 2002). A growing trend in the incidence of human campylobacteriosis has indeed been noted in many western countries in the past few decades (e.g. Nielsen et al. 1997; Rohner et al. 1997; Anon. 2001). However, an effort to manifest the rationale underlying this phenomenon is hampered by the difficulty in tracking the sources of human campylobacter strains. Nevertheless, a number of case–control investigations points at consumption of undercooked poultry as one of the major risk factors for human campylobacteriosis (e.g. Nor- krans and Svedhem 1982; Kapperud et al. 1992; Neimann et al. 1998). Also, interventions aimed to reduce the flow of campylobacters from poultry to humans in Iceland from 1999 to 2000 led to a 72% decline in domestically acquired human campylobacteriosis (Reiersen et al. 2001) and studies show- ing a certain level of clonal identity between human and chicken isolates (On et al. 1998; Duim et al. 1999; Hudson et al. 1999) corroborate these notions. Given that poultry thus might be an important risk factor for human campylobacterial gastroenteritis, initiatives were launched to reduce the human exposure to campylobacters in Denmark. These included an assessment of the risk factors Correspondence to: Michael Wainø, Novo Nordisk, DBP Microbiology Lab, Hallas Alle´, DK-4400, Kalundborg, Denmark (e-mail: mwai@novonordisk.com). ª 2003 The Society for Applied Microbiology Journal of Applied Microbiology 2003, 95, 649–655 doi:10.1046/j.1365-2672.2003.01996.x