Touchdown-touchup nested PCR for low-copy gene detection of benzimidazole-susceptible Wuchereria bancrofti with a Wolbachia endosymbiont imported by migrant carriers.

A novel, sensitive and specific touchdown-touchup nested PCR (TNPCR) technique based on two useful molecular markers, a Wuchereria bancrofti ß-tubulin gene involved in benzimidazole susceptibility and a Wolbachia ftsZ gene involved in cell division, was developed to simultaneously detect the parasite W. bancrofti (W1) with its Wolbachia endosymbiont (W2) from both microfilaremic and post-treatment samples of at-risk migrant carriers infected with geographical W. bancrofti isolates. The detection and characterization of authentically low-copy gene-derived amplicons revealed no false positive identifications in amicrofilaremia with or without antigenemia. The W1-TNPCR was 100-fold more sensitive than the W2-TNPCR regardless of the microfilarial DNA isolation method and compared well with the thick blood film and membrane filtration techniques. These locus-specific TNPCRs could also detect Wolbachia-carrying W. bancrofti genotype in addition to a link to benzimidazole sensitivity among those with unknown infection origins that exhibited microfilaremia responsiveness against treatment with diethylcarbamazine plus albendazole. These TNPCR methods can augment the results of microscopic detection of the parasite because these methods enhance DNA isolation and PCR amplification capabilities.

Molecular characterization of Thai Ehrlichia canis and Anaplasma platys strains detected in dogs.

Canine monocytic ehrlichiosis caused by Ehrlichia canis is of veterinary importance worldwide. In Thailand, there has been little information available on E. canis and its phylogeny. The objective of this study was to characterize and establish molecular structure and phylogeny of Thai Ehrlichia and Anaplasma strains. Genus-specific primers for Ehrlichia and Anaplasma were used to amplify the 16S rRNA gene from naturally infected canine blood samples, and these amplicon sequences were compared with other sequences from GenBank. Both homology and secondary structure analysis of 16S rRNA sequences indicated that they were novel E. canis and A. platys strains. Phylogenetic analysis revealed that the Thai E. canis strain was closely related and formed a single cluster with E. canis from different countries. A. platys found in this study showed close relationship with earlier report of A. platys from Thailand. To our knowledge this report represents the first molecular characterization of the nearly complete 16S rRNA gene from E. canis in dogs from Thailand.