PCR-ELISA for diagnosis of Trypanosoma evansi in animals and vector

A highly sensitive and specific polymerase chain reaction (PCR) based assay for the detection of Trypanosoma evansi present in the blood of different animals and vector was developed. A simple lysis method was used to remove of the red blood cells to facilitate direct input of samples into the PCR reactions. The primer set was designed and synthesized to amplify a single band of 257 bp PCR product that was subsequently examined by enzyme-linked immunosorbent assay (ELISA). The sensitivity limit of PCR-ELISA was 0.01 pg that was corresponded to 1 parasite/ml of blood. No cross-reactivity of the assay was observed against Babesia bovis, B. bigemina, Anaplasma marginale,Theileria sp. and host DNA. The PCR-ELISA was shown to detect 33 samples of T. evansi infected blood of animals and 10 mosquitoes from different geographical area in Thailand. The results were corresponded to those of the PCR and mouse inoculation. This implies that the technique of PCR-ELISA is not only beneficial for diagnosis of the parasite but also useful for epidemiological study and designing rational trypanosomiasis control program.     

Intraspecies variation of Brugia spp. in cat reservoirs using complete ITS sequences

The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites. GenBank accession numbers of Brugia malayi and Brugia pahangi complete ITS regions using in this study were EU373601-EU373625 and EU373626- EU373655, respectively.     

Genetic diversity of Trypanosoma evansi in beef cattle based on internal transcribed spacer region.

This study was focused on genetic diversity of Trypanosoma evansi which is a widely distributed haemoflagellate of veterinary importance that infects a variety of larger mammals including horses, mules, camels, buffalo, cattle and deer. The genetic diversity of T. evansi of beef cattle LAM19 was accomplished by using phylogenetic analysis based on internal transcribed spacer region (ITS). Blood sample was collected from a naturally infected beef cattle LAM 19 and parasitemia was raised by mouse inoculation. The parasites were collected and isolated by using DE 52 DEAE cellulose anion exchange column prior to DNA extraction. Upon PCR amplification of ITS region, the product of 1300bp in size was obtained. The ITS nucleotide sequences were analyzed and revealed that it could demonstrate the genetic diversity of T. evansi of beef cattle LAM19. Based on the ITS tree, beef cattle LAM 19 T. evansi were categorized into two main groups where the genetic diversity occurred within Group 1. The data could be applicable for the survey of parasite dynamics, epidemiological studies as well as prevention and control of the disease.