SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH 30 Vol 40 No. 1 January 2009 RESEARCH NOTE MOLECULAR GENETICS ANALYSIS FOR CO-INFECTION OF BRUGIA MALAYI AND BRUGIA PAHANGI IN CAT RESERVOIRS BASED ON INTERNAL TRANSCRIBED SPACER REGION 1 Supatra Areekit, Sintawee Khuchareontaworn, Pornpimon Kanjanavas, Thayat Sriyapai, Arda Pakpitchareon, Paisarn Khawsak, and Kosum Chansiri Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand Abstract. This study described the diagnosis of a mixed infection of Brugia malayi and Brugia pahangi in a single domestic cat using the internal transcribed spacer 1 (ITS1) region. Following polymerase chain reaction amplification of the ITS1 region, the 580 bp amplicon was cloned, and 29 white colonies were randomly selected for DNA se- quencing and phylogenetic tree construction. A DNA parsimony tree generated two groups of Brugia spp with one group containing 6 clones corresponding to B. pahangi and the other 23 clones corresponding to B. malayi. This indicated that mixed infection of the two Brugia spp, B. pahangi and B. malayi, had occurred in a single host. INTRODUCTION Lymphatic filaria, Brugia malayi , is mainly distributed in many Asian countries. It has been reported that B. malayi can infect not only humans but also animals such as cats, monkeys, and dogs (Laing et al, 1960; Mak et al, 1982; Mak, 1984). These animal res- ervoirs play an important role as disease car- riers and lead to problems with eradication in endemic areas. Basically, B. malayi and B. pahangi mi- crofilaria cannot be distinguished morpho- logically by using traditional Giemsa stain- ing. Although acid phosphatase staining is effective but it is not reproducible and the procedure is complicated (Yen and Mak, 1978). This makes it difficult for diagnosis of mixed infection of B. malayi and B. pahangi in a single cat reservoir. Molecular methods based on polymerase chain reaction (PCR) have been introduced for discrimination of B. malayi and B. pahangi (Thanomsub et al, 2000; Chansiri et al, 2002; Fischer et al, 2002; Nuchprayoon et al, 2005; Rishniw et al, 2006). However, because there is more than 95% homology in nucleotide sequences of vari- ous genes in the two species, it is difficult for their application in diagnosis of mixed infection of these two closely related species. In this study, molecular phylogenetic analysis for identification of mixed Brugia spp infection based on internal transcribed spacer region 1 (ITS1) was explored in a single cat reservoir. ITS region has been routinely used for investigation of closely related species of Correspondence: Dr Kosum Chansiri, Depart- ment of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand. Tel: 66 (0) 2260 2122 Ext 4605; Fax: 66 (0) 2260 0125 E-mail: kosum@swu.ac.th