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Among the previously established enzyme-linked immunosorbent assays (ELISAs), an ELISA using the full length of a recombinant thrombospondin-related adhesive protein of Babesia gibsoni (rBgTRAPf) is considered as the most sensitive diagnostic method for the detection of an antibody to B. gibsoni in dogs. However, the expression of rBgTRAPf in high concentration is poor and, thus, limits its usefulness as a diagnostic antigen. To improve its expression level, we have truncated BgTRAPf into two fragments having either an N- or a C-terminus (BgTRAPn or BgTRAPc, respectively). The expression of BgTRAPc protein in Escherichia coli yielded adequate recombinant protein. The specificity and sensitivity of ELISAs with the truncated proteins were determined using dog sera experimentally infected with B. gibsoni and specific pathogen-free (SPF) dog sera. A total of 254 field dog sera were examined by the ELISA with rBgTRAPn, rBgTRAPc, and rBgTRAPf as well as by an indirect fluorescent antibody test (IFAT). The specificity of rBgTRAPc was the highest (97.15%), and its kappa value was more (0.8003) than rBgTRAPn (0.7083). With a sufficient level of expression as well as higher specificity and reliable sensitivity, rBgTRAPc appears to be a potential candidate antigen for the serodiagnosis of B. gibsoni infection in dogs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this study, we describe the first survey in Thailand of Trypanosoma theileri, a widespread and prevalent parasite of cattle that is transmitted by tabanid flies. Investigation of 210 bovine blood samples of Thai cattle from six farms by hematocrit centrifuge technique (HCT) revealed 14 samples with trypanosomes morphologically compatible to T. theileri. Additional animals were positive for T. theileri by PCR based on the Cathepsin L-like sequence (TthCATL-PCR) despite negative by HCT, indicating cryptic infections. Results revealed a prevalence of 26 ± 15% (95% CI) of T. theileri infection. Additionally, 12 samples positive for T. theileri were detected in cattle from other 11 farms. From a total of 30 blood samples positive by HCT and/or PCR from 17 farms, seven were characterized to evaluate the genetic polymorphism of T. theileri through sequence analysis of PCR-amplified CATL DNA sequences. All CATL sequences of T. theileri from Thai cattle clustered with sequences of the previously described phylogenetic lineages TthI and TthII, supporting only two major lineages of T. theileri in cattle around the world. However, 11 of the 29 CATL sequences analyzed showed to be different, disclosing an unexpectedly large polymorphic genetic repertoire, with multiple genotypes of T. theileri not previously described in other countries circulating in Thai cattle., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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 assay‑T. 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.

Trypanosoma lewisi is a mild or non-pathogenic parasite of the sub-genus Herpetosoma transmitted by fleas to rats. In a previous study we described pan-trypanosome specific primers TRYP1 which amplify the ITS1 of ribosomal DNA by hybridizing in highly conserved regions of 18S and 5.8S genes. These primers proved to be useful for detecting T. lewisi DNA in laboratory rats, but a recent large scale survey in wild rodents demonstrated a lack of specificity. In the present study, we designed and evaluated mono-specific primers LEW1S and LEW1R, for the detection and identification of T. lewisi by a single-step PCR. These primers were designed inside the highly variable region of the ITS1 sequence of T. lewisi ribosomal DNA. The product size of 220 bp is specific to T. lewisi. The sensitivity limit was estimated between 0.055 and 0.55 pg of DNA per reaction, equivalent to 1-10 organisms per reaction. All the PCR products obtained from 6 different T. lewisi isolates were more than 98% similar with each other and similar to the sequences of T. lewisi already published in Genbank. All DNA of 7 T. lewisi stocks from China gave the specific 220 bp product. We showed that LEW1S and LEW1R primers enabled sensitive detection and identification of T. lewisi infection in laboratory and wild rats. This assay is recommended for monitoring T. lewisi infections in rat colonies or for studying infections in the wild fauna. An absence of cross reaction with human DNA means that these primers can be used to investigate atypical trypanosome infections in humans. Given the risk of T. lewisi infection in human, we believe that these primers will be beneficial for public health diagnosis and rodents investigation programmes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright 2010 Elsevier B.V. All rights reserved.)

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.