The molecular prevalence and MSA-2b gene-based genetic diversity of Babesia bovis in dairy cattle in Thailand.

Bovine babesiosis is an economically significant disease that affects dairy farming operations in Thailand. In the present study, 1824 blood-DNA samples prepared from cattle bred in 4 different regions of the country (North, Northeast, Central, and South) were screened using a nested PCR for the specific detection of Babesia bovis. While the overall prevalence of B. bovis was 8.8%, the Central region of Thailand was found to be a high-risk area of the country, as the prevalence of the parasite was 15.0%. The positive rate was relatively higher among the animals of 1-5 years of age. The genetic diversity among the B. bovis parasites was also studied based on their MSA-2b gene, and the findings showed that the Thai sequences were dispersed across 8 of 13 total clades observed in the phylogram. Three of these clades were formed only of Thai sequences. Similarity among the deduced MSA-2b amino acid sequences determined in the present study was 68.3-100%. In conclusion, the present study found that all the locations surveyed were infected with B. bovis and that the parasite populations in Thailand were genetically diverse. Our findings highlight the need for further studies in Thailand to generate more information before a sound control strategy could be implemented against B. bovis.

The effect of the DNA preparation method on the sensitivity of PCR for the detection of Trypanosoma evansi in rodents and implications for epidemiological surveillance efforts.

Trypanosoma evansi is responsible for the most largely distributed animal trypanosomosis, affecting a wide range of wild and domestic animals. Its surveillance requires the implementation of standardized and reliable diagnostic tools. Although the development of polymerase chain reaction (PCR) tools has greatly improved our diagnostic capacity, factors affecting their sensitivity need to be acknowledged and accounted for in the interpretation of results. The targeted gene and the primer design have already been shown to greatly affect the sensitivity of a PCR, and the best-performing sets of primers have been previously identified. However, the sensitivity of the PCR is also largely influenced by the DNA extraction or sample preparation method. In this paper, we selected 6 DNA extraction or blood sample preparation methods representative of what would be used in a budget-constrained setting: phenol-chloroform, Chelex(®), Flexigen (Qiagen(®)) kit, Genekam(®) kit and two original protocols using sodium hydroxide. We studied the effects of the preparation method on the detection limit of the subsequent PCR. Our results show that the extraction method strongly affects the PCR sensitivity. The classical phenol-chloroform extraction method allowed for the PCR with the lowest detection limit. Some combinations of extraction method and primer set had detection limits that were not compatible with their use as a reliable diagnostic test, and would severely reduce the performance of a surveillance program. Therefore, we encourage laboratories to carefully select their sample preparation and PCR protocols, depending on the aimed sensitivity, cost, safety, time requirement and objectives.

An evaluation of melarsomine hydrochloride efficacy for parasitological cure in experimental infection of dairy cattle with Trypanosoma evansi in Thailand.

Melarsomine hydrochloride can cure Trypanosoma evansi infection in camels at a dose of 0·25 mg/kg, but at that dose relapses occur in cattle. In our study, the efficacy of an intramuscular injection of melarsomine hydrochloride at 0·5 mg/kg was assessed in 3 normal and 3 splenectomized dairy cattle experimentally infected with a stock of T. evansi from Thailand. The animals were monitored for 5 months by haematocrit centrifugation, blood- or cerebrospinal fluid-mouse inoculation, polymerase chain reaction, the card agglutination test (CATT) for T. evansi, and the enzyme-linked immunosorbent assay­T. evansi. Parasitological and DNA tests became and remained negative just after treatment. By the end of the experiment, CATT was negative and ELISA scores were below or very close to the cut-off value. One of the splenectomized cattle died from anaplasmosis during the experiment, but tested negative for surra. It was concluded that the parasites had been cleared from the cattle, and melarsomine hydrochloride at 0·5 mg/kg can be recommended for treatment against T. evansi infection in dairy cattle in Thailand. Further work is necessary to validate the efficacy of the treatment in the event of confirmed CSF-infection.

Toxoplasmosis in rodents: ecological survey and first evidences in Thailand.

Domestic and wild rodents known as the most abundant and diversified order of mammals have a key role in the ecological food chain and also in the transmission of parasites and pathogens to other animals. While foraging on the ground, they can get infected by Toxoplasma gondii, a protozoan parasite, which is the causative agent of toxoplasmosis. Therefore, they serve as intermediate hosts of T. gondii and can transmit it to their predators. To assess their role in the maintenance of T. gondii lifecycle in Thailand, we sampled rodents in a range of biotopes representative of the high biodiversity and conducted a serological survey with latex agglutination test to detect anti-T. gondii antibodies. Overall, 21 of 461 (4.6%) rodents had diagnostically significant antibody titers (cutoff, 1:64). Every species with at least 37 individuals captured tested positive, confirming the wide range of potential mammalian hosts of toxoplasmosis. None of the ecological traits (sex, maturity, morphology, season, or habitat) was found significant to predict the susceptibility to T. gondii both univariately and in a multivariate analysis. However, high prevalences were reported in either forested or anthropized areas. This survey constitutes the first confirmed serological investigation of T. gondii in rodents in Thailand. The rarity of both domestic and wild felids in Thailand emphasizes the importance of rodents in maintaining T. gondii, and questions the involvement of other carnivores in the life cycle.

A comparison of six primer sets for detection of Trypanosoma evansi by polymerase chain reaction in rodents and Thai livestock.

To face the worldwide threat of Surra caused by Trypanosoma evansi, international organizations have stressed the need to evaluate and standardize diagnostic tools. PCR detection of T. evansi has known a great expansion during the last 20 years, but primer sets are often insufficiently assessed and compared. In this work, we compared the performances of six primer pairs-TBR1/2 (Masiga et al., 1992), ESAG6/7 (Holland et al., 2001a, b), TEPAN1/2 (Panyim et al., 1993), pMUTEC F/R (Wuyts et al., 1994), TRYP1 R/S (Desquesnes et al., 2001) and TRYP4 R/S (Desquesnes et al., unpublished)-tested with purified T. evansi DNA serial dilutions, T. evansi-infected rat blood serial dilutions and Thai dairy cattle samples. TBR1/2 primer set was able to detect 0.01 pg of purified DNA, and a parasitaemia below one parasite per ml in rat blood. They presented the highest sensitivity in cattle samples as well as a high specificity, without non-specific products nor false positive reactions out of 84 negative cattle samples tested. ESAG6/7 showed equivalent results with purified DNA and rat samples but presented non-specific products with Thai dairy cattle samples, leading to non interpretable results. TEPAN1/2 was not able to detect less than 0.1 pg of purified DNA or 50 trypanosomes/ml in rat blood. In cattle, TEPAN1/2 primers detected only 36% of the positives detected by TBR1/2. Given the parasitemic level detected, pMUTEC F/R, TRYP1 R/S and TRYP4 R/S were not more sensitive than classical microscopic examination of the buffy coat. TBR1/2, TEPAN1/2, pMUTEC F/R and TRYP4 R/S did not cross-reacted with Babesia sp., Trypanosoma theileri and Anaplasma marginale. TBR1/2 was the most sensitive primer set to detect T. evansi in purified DNA, rodent blood and cattle blood, and did not show cross reaction with the other pathogens tested: it should be therefore preferred for epidemiological surveys. These results confirmed that TBR1/2 primers remain the reference for the detection of Trypanozoon DNA and should therefore be included in subsequent evaluations of new diagnosis tools based on DNA detection.

Antibody-ELISA for Trypanosoma evansi: application in a serological survey of dairy cattle, Thailand, and validation of a locally produced antigen.

Trypanosoma evansi is generally considered a mild pathogen in bovines. However, in Asia, acute and chronic signs have been observed in cattle, with high levels of parasitaemia, abortion and death. Investigations in Asian cattle are needed to better understand this epidemiological situation. To generate comparable data at a regional level, development and standardization of an antibody-enzyme linked immunosorbent assay for T. evansi (ELISA/T. evansi) was initiated and applied in an epidemiological survey carried out in dairy cattle in Thailand. A batch of 1979 samples was collected from dairy farms located throughout the country's four regions. Soluble T. evansi antigens initially produced in France were also produced in Thailand for comparison and technology transfer. Screening of 500 samples allowed us to identify reference samples and to determine the cut-off value of the ELISA. Seropositive animals - some of them confirmed by PCR - were found in the four regions, in 12 out of 13 provinces, in 22 out of 31 districts, in 56 farms out of 222 (25%, 95%CI+/-6%) and in 163 animals out of 1979 (8.2, 95%CI+/-1.2%). Estimated seroprevalence in 35 farms ranged between 1% and 30%, and in 21 farms it was >30%. Approximately 25% of survey cattle were exposed to the infection, in various situations. A sub-sample of 160 sera was tested on both antigens. Wilcoxon's (Z=1.24; p=0.22) and McNemars's tests (CHI2=3.55; p=0.09) did not show any significant differences, showing that the locally produced antigen is suitable for further evaluation in the surrounding countries. Use of this standardized serological method will broaden knowledge of the prevalence and impact of the disease at the regional level in South-East Asia. Further validation of this ELISA will be necessary in other host species such as buffalo, horse and pig.

Diagnosis of a Trypanosoma lewisi-like (Herpetosoma) infection in a sick infant from Thailand.

Trypanosomes were observed in a peripheral blood smear from a 45-day-old Thai infant displaying fever, anaemia, cough and anorexia. Human trypanosomiasis is not endemic to Thailand, so parasite identification was undertaken to determine likely sources of the infection. Several morphological parameters of the trypanosomes were similar to those of Trypanosoma evansi and statistically different from those of Trypanosoma lewisi-like parasites from a naturally infected indigenous rat. However, duplicate PCR assays with primers flanking trypanosome rRNA internal transcribed spacer 1 (ITS1) resulted in amplicons of approximately 623 bp that corresponded to the expected size for T. lewisi-like parasites. The ITS1 sequence from the infant's blood was 98 and 49 % identical to T. lewisi and T. evansi sequences, respectively. Based on molecular results, it was concluded that the infant was infected with a T. lewisi-like (Herpetosoma) species.