A new multiple-locus variable-number tandem repeat analysis reveals different clusters for Anaplasma phagocytophilum circulating in domestic and wild ruminants.

Thibaud Dugat,Amélie Chastagner,Fabien Corbière,Benoît Durand,Xavier Bailly,Simon Thierry,Henri-Jean Boulouis,Hélène Verheyden,Renaud Maillard,Elisabeth Petit,Gwenaël Vourc’H,Anne-Claire Lagrée,Luc Chabanne,Nadia Haddad,Agnès Leblond

doi:10.1186/1756-3305-7-439

Abstract

BackgroundAnaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium. It is the causative agent of tick-borne fever in ruminants, and of human granulocytic anaplasmosis in humans, two diseases which are becoming increasingly recognized in Europe and the USA. However, while several molecular typing tools have been developed over the last years, few of them are appropriate for in-depth exploration of the epidemiological cycle of this bacterium. Therefore we have developed a Multiple-Locus Variable number tandem repeat (VNTR) Analysis typing technique for A. phagocytophilum.MethodsFive VNTRs were selected based on the HZ human-derived strain genome, and were tested on the Webster human-derived strain and on 123 DNA samples: 67 from cattle, 7 from sheep, 15 from roe deer, 4 from red deer, 1 from a reindeer, 2 from horses, 1 from a dog, and 26 from ticks.ResultsFrom these samples, we obtained 84 different profiles, with a diversity index of 0.96 (0.99 for vertebrate samples, i.e. without tick samples). Our technique confirmed that A. phagocytophilum from roe deer or domestic ruminants belong to two different clusters, while A. phagocytophilum from red deer and domestic ruminants locate within the same cluster, questioning the respective roles of roe vs red deer as reservoir hosts for domestic ruminant strains in Europe. As expected, greater diversity was obtained between rather than within cattle herds.ConclusionsOur technique has great potential to provide detailed information on A. phagocytophilum isolates, improving both epidemiological and phylogenic investigations, thereby helping in the development of relevant prevention and control measures.Electronic supplementary materialThe online version of this article (doi:10.1186/1756-3305-7-439) contains supplementary material, which is available to authorized users.

Highlights

Anaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium
Variable number tandem repeat (VNTR) selection Using the tandem repeats database developed by Le Flèche et al [48], we pre-selected 721 VNTR candidates according to the criteria described above
Following amplification optimization, only those VNTRs that were amplified and appeared to be polymorphic when tested on five of our samples, were selected and used in this study. These VNTRs corresponded to those listed as Anaplasma phagocytophilum VNTR (APV)-A, APV-B, APV-C, APV-D and APV-E

Summary

Introduction

Anaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium. It is the causative agent of tick-borne fever in ruminants, and of human granulocytic anaplasmosis in humans, two diseases which are becoming increasingly recognized in Europe and the USA. For each pathogenic species, it remains difficult to determine whether different animal species are randomly infected, or whether certain strains circulate in more or less distinct epidemiological cycles [3,4]. Many pathogens transmitted by Ixodes ricinus typically exploit multiple different host species, as vector ticks can feed on diverse numbers of vertebrates [6]. This multiplicity of hosts is one of the main reasons why it is so difficult to identify the precise role of hosts involved in the epidemiological cycle(s) of certain tick-borne pathogens

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