Parasitology

Research Article

Development of multiple-locus variable-number tandem-repeat analysis for rapid genotyping of Ehrlichia ruminantium and its application to infected Amblyomma variegatum collected in heartwater endemic areas in Uganda

RYO NAKAOa1, LIAM J. MORRISONa2, LIJIA ZHOUa1, JOSEPH W. MAGONAa1a3, FRANS JONGEJANa4a5 and CHIHIRO SUGIMOTOa1 c1

a1 Department of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan

a2 Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, 464 Bearsden Road, Glasgow G61 1QH, UK

a3 National Livestock Resources Research Institute (NaLIRRI), P.O. Box 1026, Tororo, Uganda

a4 Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584CL Utrecht, The Netherlands

a5 Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa

SUMMARY

The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater, a serious tick-borne disease in ruminants. The genetic diversity of organisms in the field will have implications for cross-protective capacities of any vaccine developed, and for an effective vaccine design strategy proper genotyping and understanding of existing genetic diversity in the field is necessary. We searched for variable-number tandem-repeat (VNTR) loci for use in a multi-locus VNTR analysis (MLVA). Sequencing analysis of 30 potential VNTRs using a panel of 17 reference strains from geographically diverse origins identified 12 VNTRs with allelic profiles differing between strains. Application of MLVA to 38 E. ruminantium-infected Amblyomma variegatum collected from indigenous cattle in 6 different districts of Uganda identified 21 MLVA types. The discriminatory power of MLVA was greater than that of map1 PCR-restriction fragment length polymorphism analysis, with which only 6 genotypes were obtained. The high discriminatory power as well as cost- effective performance of MLVA provide the potential for this technique to be applied in the future with respect to optimizing vaccine trials by identifying local strain diversity, and also raise the possibility of exploring the association between E. ruminantium genotypes and phenotypes such as pathological outcome in the ruminant host.

(Received April 22 2011)

(Revised July 26 2011)

(Revised August 17 2011)

(Accepted August 18 2011)

(Online publication October 05 2011)

Correspondence:

c1 Corresponding author: Research Center for Zoonosis Control, Hokkaido University, Kita 20, Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0020, Japan. Tel: +81 11 706 5297. Fax: +81 11 706 7370. E-mail: sugimoto@czc.hokudai.ac.jp

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