Morakot Kaewthamasorn

A preliminary survey of gastrointestinal and haemoparasites of beef cattle in the tropical livestock farming system in Nan Province, northern Thailand

Parasitism is a primary cause of production losses in most cattle-producing countries of the world, including Thailand. A cross-sectional study was designed to determine baseline data on the prevalence of gastrointestinal and haemoparasites of beef cattle in Nan Province, northern Thailand and to investigate the factors associated with the prevalence of parasitic infections. A total of 207 faecal and 162 blood samples were collected during the summer of 2005. The basic data of management were recorded, including the number of animals on the farm or in the village, major animal health problems, deworming programme and veterinary service. The overall prevalence of gastrointestinal parasitic infections was 61% (126/207). The most common helminth infections in this study were rumen flukes 28% (58), followed by strongyles 27% (55), and the rest were due to Strongyloides 1% (2) and Trichuris 1% (2). The common protozoan infections were Buxtonella cysts 2% (5) and coccidia oocysts 2% (4). Of the 162 blood samples examined, 50% (81) contained Theileria sp., 2% (4) trypanosome, and 1% (1) microfilariae. The high rate of parasitic infections in these areas might be related to the poor management by the farmers, such as sharing the same grazing pasture.

The development of malaria diagnostic techniques: a review of the approaches with focus on dielectrophoretic and magnetophoretic methods

The large number of deaths caused by malaria each year has increased interest in the development of effective malaria diagnoses. At the early-stage of infection, patients show non-specific symptoms or are asymptomatic, which makes it difficult for clinical diagnosis, especially in non-endemic areas. Alternative diagnostic methods that are timely and effective are required to identify infections, particularly in field settings. This article reviews conventional malaria diagnostic methods together with recently developed techniques for both malaria detection and infected erythrocyte separation. Although many alternative techniques have recently been proposed and studied, dielectrophoretic and magnetophoretic approaches are among the promising new techniques due to their high specificity for malaria parasite-infected red blood cells. The two approaches are discussed in detail, including their principles, types, applications and limitations. In addition, other recently developed techniques, such as cell deformability and morphology, are also overviewed in this article.

Ungulate malaria parasites

Haemosporida parasites of even-toed ungulates are diverse and globally distributed, but since their discovery in 1913 their characterization has relied exclusively on microscopy-based descriptions. In order to bring molecular approaches to bear on the identity and evolutionary relationships of ungulate malaria parasites, we conducted Plasmodium cytb-specific nested PCR surveys using blood from water buffalo in Vietnam and Thailand, and goats in Zambia. We found that Plasmodium is readily detectable from water buffalo in these countries, indicating that buffalo Plasmodium is distributed in a wider region than India, which is the only area in which buffalo Plasmodium has been reported. Two types (I and II) of Plasmodium sequences were identified from water buffalo and a third type (III) was isolated from goat. Morphology of the parasite was confirmed in Giemsa-reagent stained blood smears for the Type I sample. Complete mitochondrial DNA sequences were isolated and used to infer a phylogeny in which ungulate malaria parasites form a monophyletic clade within the Haemosporida, and branch prior to the clade containing bird, lizard and other mammalian Plasmodium. Thus it is likely that host switching of Plasmodium from birds to mammals occurred multiple times, with a switch to ungulates independently from other mammalian Plasmodium.

Canine dirofilariasis and concurrent tick-borne transmitted diseases in Bangkok, Thailand

A retrospective study in dogs presented to the Chulalongkorn Small Animal Teaching Hospital, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand, from January 2001 to December 2003 was carried out. A total of 917 dogs were diagnosed with canine dirofilariasis and other concurrent tick-borne transmitted diseases by the Veterinary Diagnostic Laboratory, Faculty of Veterinary Science, Chulalongkorn University. The highest occurrence within each year was observed in November 2001 (40 cases), April 2002 (41 cases), and July 2003 (36 cases), respectively. Of the total 917 positive cases, a single infection of dirofilariasis was detected in 869 dogs (94.8%; group 1), while 37 dogs (4.0%; group 2) were diagnosed with dirofilariasis and ehrlichiosis, 4 dogs (0.4%; group 3) with dirofilariasis and hepatozoonosis, and 7 dogs (0.8%; group 4) with dirofilariasis, ehrlichiosis, and babesiosis, respectively. Laboratory data comprising hematological and blood chemistry profiles were evaluated and compared among groups. Group 4 was defined as moderate microcytic anemia and was significantly different with regard to hematological profiles from others (P < 0.05). In addition, severe thrombocytopenia was observed in both groups 2 and 4 (65.4 × 103 and 59.0 × 103 cells/μl, respectively). The hepatobiliary profiles in all groups revealed increases in serum alanine aminotransferase and serum alkaline phosphatase activities indicative of hepatocellular damage. These epidemiological results serve as baseline information for preventive strategies against blood parasites in the endemic area. Moreover, both biochemical and hematological abnormalities should be considered as appropriate monitors during disease interventions.

Diversity and population structure of Plasmodium falciparum in Thailand based on the spatial and temporal haplotype patterns of the C-terminal 19-kDa domain of merozoite surface protein-1

BackgroundThe 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum (Pf MSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of Pf MSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, Pf MSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown.MethodsSixty-three isolates of P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of Pf MSP-119 haplotypes in individual populations. The Pf MSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite.ResultsFive conserved polymorphic positions, which accounted for five distinct haplotypes, of Pf MSP-119 were identified. Differences in the prevalence of Pf MSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual Pf MSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries.ConclusionThe major MSP-119 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.

Stable allele frequency distribution of the polymorphic region of SURFIN4.2 in Plasmodium falciparum isolates from Thailand

Plasmodium falciparum SURFIN₄.₂ (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN₄.₂ using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN₄.₂ was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN₄.₂ sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajimas D test, and Fu and Lis D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988-1989 still circulated 14 years later, suggesting low recombination of the analyzed surf₄.₂ sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN₄.₂ Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported.

The rediscovery of malaria parasites of ungulates.

Over a hundred years since their first description in 1913, the sparsely described malaria parasites (genus Plasmodium) of ungulates have been rediscovered using molecular typing techniques. In the span of weeks, three studies have appeared describing the genetic characterization and phylogenetic analyses of malaria parasites from African antelope (Cephalophus spp.) and goat (Capra aegagrus hircus), Asian water buffalo (Bubalus bubalis), and North American white-tailed deer (Odocoileus virginianus). Here we unify the contributions from those studies with the literature on pre-molecular characterizations of ungulate malaria parasites, which are largely based on surveys of Giemsa-reagent stained blood smears. We present a phylogenetic tree generated from all available ungulate malaria parasite sequence data, and show that parasites from African duiker antelope and goat, Asian water buffalo and New World white-tailed deer group together in a clade, which branches early in Plasmodium evolution. Anopheline mosquitoes appear to be the dominant, if not sole vectors for parasite transmission. We pose questions for future phylogenetic studies, and discuss topics that we hope will spur further molecular and cellular studies of ungulate malaria parasites.

Stable Allele Frequency Distribution of the Plasmodium falciparum clag Genes Encoding Components of the High Molecular Weight Rhoptry Protein Complex

Plasmodium falciparum Clag protein is a candidate component of the plasmodial surface anion channel located on the parasite-infected erythrocyte. This protein is encoded by 5 separated clag genes and forms a RhopH complex with the other components. Previously, a signature of positive diversifying selection was detected on the hypervariable region of clag2 and clag8 by population-based analyses using P. falciparum originating from Thailand in 1988–1989. In this study, we obtained the sequence of this region of 3 clag genes (clag2, clag8, and clag9) in 2005 and evaluated the changes over time in the frequency distribution of the polymorphism of these gene products by comparison with the sequences obtained in 1988–1989. We found no difference in the frequency distribution of 18 putatively neutral loci between the 2 groups, evidence that the background of the parasite population structure has remained stable over 14 years. Although the frequency distribution of most of the polymorphic sites in the hypervariable region of Clag2, Clag8, and Clag9 was stable over 14 years, we found that a proportion of the major Clag2 group and one amino acid position of Clag8 changed significantly. This may be a response to a certain type of pressure.

Molecular detection of the avian malaria parasite Plasmodium gallinaceum in Thailand.

Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds.

Genetic diversity of the merozoite surface protein-3 gene in Plasmodium falciparum populations in Thailand

BackgroundAn effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown.MethodsThe msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (Fst) and neutrality tests (Tajima’s D, Fu and Li D* and Fu and Li’ F* tests) to determine any signature of selection.ResultsThe sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia.ConclusionThis study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.