Background and Aim: Dog blood parasites are important tick-borne diseases causing morbidity and mortality in dogs worldwide. Four dog blood parasites species are commonly found in Thailand: Babesia canis, Hepatozoon canis, Ehrlichia canis, and Anaplasma platys. They are transmitted easily by tick species. However, there is little prevalence data available in Thailand. Diseases presentation of blood parasites infection is similar, but the treatment of each species is different. Current diagnosis mainly relies on microscopic examination of a stained blood smear, which has low sensitivity. Therefore, accurate diagnosis is important. This study aims to evaluate the efficacy of the conventional polymerase chain reaction (PCR) method and routine blood smears in the detection of four blood parasites species in dogs from Buriram Province, Thailand. Materials and Methods: In total, 49 EDTA-blood samples were collected from dogs in Buriram Province, Thailand. Blood parasite infection was compared using the Giemsa-stained blood smear technique to identify the parasite under a 100× oil immersion with PCR amplification of the 18S rDNA gene of B. canis and H. canis and the 16S rDNA gene of E. canis and A. platys. Results: Only one dog out of 49 was positive for H. canis based on microscopic examination whereas the PCR results showed that 2.04% (1/49), 4.08% (2/49), 36.73% (18/49), and 30.61% (15/49) of dogs were positive for B. canis, H. canis, E. canis, and A. platys, respectively. Moreover, coinfection was found in 16.33% (8/49) of dogs. Conclusion: This study is the first report to demonstrate the molecular prevalence of blood parasites in domestic dogs in Buriram Province. The results indicated that the PCR method exhibited much higher sensitivity and reliability for blood parasites diagnosis in dogs. Therefore, our data support serious concern regarding the diagnostic technique used in routine blood testing and also provide prevalence data for the management and control of blood parasites in this area.

In this study, microsatellite markers were developed for the genetic linkage mapping and breeding program of the black tiger shrimp Penaeus monodon. A total of 997 unique microsatellite-containing expressed sequence tags (ESTs) were identified from 10 100 EST sequences in the P. monodon EST database. AT-rich microsatellite types were predominant in the EST sequences. Homology searching by the blastn and blastx programs revealed that these 997 ESTs represented 8.6% known gene products, 27.8% hypothetical proteins and 63.6% unknown gene products. Characterization of 50 markers on a panel of 35-48 unrelated shrimp indicated an average number of alleles of 12.6 and an average polymorphic information content of 0.723. These EST microsatellite markers along with 208 other markers (185 amplified fragment length polymorphisms, one exon-primed intron-crossing, six single strand conformation polymorphisms, one single nucleotide polymorphism, 13 non-EST-associated microsatellites and two EST-associated microsatellites) were analysed across the international P. monodon mapping family. A total of 144 new markers were added to the P. monodon maps, including 36 of the microsatellite-containing ESTs. The current P. monodon male and female linkage maps have 47 and 36 linkage groups respectively with coverage across half the P. monodon genome.

Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in folate biosynthesis, and a major malarial drug target. Point mutations in P. falciparum TS-DHFR have caused widespread global antifolate resistance, and yet the most effective antifolate known to overcome drug-resistance, WR99210, has poor oral bioavailability. More specific, less toxic therapies are urgently needed. Antifolates target the conversion of methylene tetrahydrofolate to dihydrofolate by TS, and that of dihydrofolate to tetrahydrofolate by DHFR. In humans, TS and DHFR are two discrete enzymes. In P. falciparum, however, TS-DHFR is a bifunctional enzyme, with TS and DHFR encoded within a single protein, and tethered together with by a ‘linker’ region. This linker is not homologous to any other known TS or DHFR enzymes, and is essential for enzyme activity. This bifunctional enzyme thus presents different design approaches for developing novel inhibitors against drug-resistant mutants: developing active-site inhibitors equally effective against wildtype and drug-resistant parasites, or targeting unique non-active site regions for parasite-specific inhibitors. As a first step in identifying unique inhibitors, we performed a high-throughput in silico screen of a database of diverse, drug-like molecules against a non-active site pocket within the linker region of TS-DHFR. The top compounds from the virtual screen were evaluated by enzymatic and cellular assays. In vitro enzymatic studies and cell culture studies of wildtype and drug-resistant P. falciparum parasites identified three compounds active to 20 μM IC50s in both wildtype and antifolate-resistant enzymatic studies, as well as in P. falciparum cell culture. Moreover no inhibition of human DHFR enzyme was observed indicating the inhibitory effects appeared to be parasite-specific. Notably, all three compounds had a biguanide scaffold. Further computational analysis was utilized to determine the relative free energy of binding and these calculations suggested that the compounds might preferentially interact with the active site over the screened ‘linker’ region. To resolve the two possible modes of binding, co-crystallization studies of the compounds complexed with TS-DHFR enzyme were performed to determine the three-dimensional structures. Surprisingly, the structural analysis revealed that these novel, biguanide compounds, distinct from WR99210, do indeed bind at the active site of DHFR, and additionally revealed the molecular basis by which they overcome drug-resistance. To our knowledge, these are the first co-crystal structures of novel, biguanide, non-WR99210 compounds that are active against folate-resistant malaria parasites in cell culture. These studies reveal how serendipity coupled with computational and structural analysis can identify unique compounds as a promising starting point for rational drug design to combat drug-resistant malaria.

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