Short communication ‘‘HOOF-Print’’ genotyping and haplotype inference discriminates among Brucella spp. isolates from a small spatial scale Cla ´ udia Almendra a,b,1, *, Teresa Luı ´sa Silva a,1 , Albano Beja-Pereira a , Ana Cristina Ferreira c , Le ´ nia Ferra ˜o-Beck c , Maria Ina ´ cia Corre ˆa de Sa ´ c , Betsy J. Bricker d , Gordon Luikart a,e a CIBIO, Centro de Investigac ¸a˜o em Biodiversidade e Recursos Gene ´ticos, Universidade do Porto, Campus Agra ´rio de Vaira ˜o, 4485-661 Vaira ˜o, Portugal b ICBAS, Instituto de Cieˆncias Biome ´dicas, Universidade do Porto, Lg. Prof. Abel Salazar, 2, 4099-003 Porto, Portugal c LNIV, Laborato ´rio Nacional de Investigac ¸a ˜o Veterina ´ria, Estrada de Benfica, 701, 1549-011 Lisboa, Portugal d Bacterial Diseases of Livestock Research Unit, National Animal Disease Center (NADC), Agricultural Research Service, United States Department of Agriculture (USDA), 2300 Dayton Rd, Ames, IA 50010, USA e Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA 1. Introduction Brucellosis is an important zoonosis with worldwide distribu- tion and high clinical morbidity. Bacteria from the genus Brucella can infect a variety of hosts and are responsible for significant economic losses in livestock industries and serious public health problems in humans. Transmission to man can occur through many routes: foodborne, occupational, recreational and potentially through bioterrorism (Godfroid et al., 2005). Brucellosis continues to be a major problem in the Mediterranean Basin, Middle East, Latin America, Asia and Africa (Godfroid et al., 2005; Pappas et al., 2006). Portugal, like other European Union countries, employs specific regulations and measures to eradicate the disease. Regardless of the huge efforts to eliminate it, human cases still occur frequently in the country (16.1 cases per million, Direcc ¸a ˜o Geral de Sau ´ de, Divisa ˜o de Epidemiologia, 2006) and the prevalence in cattle and small ruminants herds is 0.25 and 0.70%, respectively (Direcc ¸a ˜o Geral de Veterina ´ ria, 2007). Control of brucellosis, particularly in the final stages of an eradication program, requires a rigorous program for surveillance and highly discriminatory methods for characterizing an outbreak strain, which can be used in trace back studies to determine the original source of infection and its routes of transmission. Conventional methods for subtyping of Brucella strains into species and biovars have some shortcomings, particularly, in small geographical regions where few biovars tend to predominate (e.g., most B. melitensis isolated in Portugal belong to biovars 1 and 3; LNIV, National Laboratory for Veterinary Research, unpublished data). Also, classification of Brucella relies on a large array of phenotypic tests that are prone to misinterpretation or inaccuracy (Banai et al., 1990; Ewalt and Forbes, 1987). Moreover, because of the high genetic homology among bacteria of the genus Brucella, there is a demand for the development and validation of highly polymorphic markers to increase sensitivity and resolving power. Recently, Bricker et al. Infection, Genetics and Evolution xxx (2008) xxx–xxx ARTICLE INFO Article history: Received 16 May 2008 Received in revised form 27 August 2008 Accepted 22 October 2008 Available online xxx Keywords: Brucella spp. HOOF-print assay Genotyping analysis Gametic disequilibrium Biovar discrimination Population genetics ABSTRACT We demonstrate that the ‘‘HOOF-Print’’ assay provides high power to discriminate among Brucella isolates collected on a small spatial scale (within Portugal). Additionally, we illustrate how haplotype identification using non-random association among markers allows resolution of B. melitensis biovars (1 and 3). We recommend that future studies use haplotype identification when analyzing multilocus population genetic data to help discriminate among microbial isolates such as Brucella. ß 2008 Elsevier B.V. All rights reserved. * Corresponding author at: CIBIO, Centro de Investigac ¸a ˜o em Biodiversidade e Recursos Gene ´ ticos, Universidade do Porto, Campus Agra ´ rio de Vaira ˜o, 4485-661 Vaira ˜o, Portugal. Fax: +351 252661780. E-mail address: claudia_almendra@mail.icav.up.pt (C. Almendra). 1 Both authors have equally contributed to this work. G Model MEEGID-500; No of Pages 4 Please cite this article in press as: Almendra, C., et al., ‘‘HOOF-Print’’ genotyping and haplotype inference discriminates among Brucella spp. isolates from a small spatial scale. Infect. Genet. Evol. (2008), doi:10.1016/j.meegid.2008.10.007 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 1567-1348/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2008.10.007