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Marc Desquesnes Ketsarin Kamyingkird Mathieu Pruvot Chanya Kengradomkij Geraldine Bossard Nachai Sarataphan Sathaporn Jittapalapong 《Preventive veterinary medicine》2009,90(3-4):233-241
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. 相似文献
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Mathieu Pruvot Ketsarin Kamyingkird Marc Desquesnes Nachai Sarataphan Sathaporn Jittapalapong 《Veterinary parasitology》2013,191(3-4):203-208
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. 相似文献
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Garcia HA Kamyingkird K Rodrigues AC Jittapalapong S Teixeira MM Desquesnes M 《Veterinary parasitology》2011,180(3-4):363-367
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. 相似文献
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