| Ovine and caprine theileriosis is a kind of blood protozoosis that widely distributed in theregion of Northwestern China. It causes significant economic losses to the sheep industry. Thedisease is mainly caused by two Theileria species, T. luwenshuni and the T. uilenbergi (earlyregarded as the T. lestoquardi). Most cases of ovine and caprine theileriosis are the mixed infectionof T. luwenshuni and T. uilenbergi. However, traditional diagnostic methods are difficult todifferentiate infection of the2Theileria species. Therefore，it’s necessary to develop a fast andaccurate method to perform differential diagnosis of T. luwenshuni and T. uilenbergi infection.In this study,3sets of primers of loop-mediated isothermal amplification (LAMP), each setincluding four primers that specificially recognizate six regions of hypervariable region, weredesigned based on18S rRNA gene sequences for differential and simultaneous detection ofT.luwenshuni and T. uilenbergi. The reaction conditions were optimized, such as amplificationtemperature and time. And then it was evaluated by the samples from experimental infected sheep.A LAMP method was developed for differential diagnosis of T. luwenshuni and T. uilenbergiinfection. Finally, the method was used in a small-scale epidemiological survey via detection of272samples from sheep or goats in Guangdong, Guizhou, Chongqing, Gansu and Guangxiprovinces/municipality. The results showed that:The universal primers designed according to the18S rRNA gene sequence couldsimultaneously detect T. luwenshuni&T. uilenbergi and was no cross-reaction with other ovineand bovine piroplasms. The primers TC1and TC2were specific for T. luwenshuni&T. uilenbergi,respectively and no cross-reaction with other dominant piroplasms infected ruminants. It coulddetect2pg genomic DNA of T. luwenshuni or T. uilenbergi.Amplified products were sequenced. Results showed that the sequences of amplified targetfragment were exactly consistent with18S rRNA gene sequences of T. luwenshuni and T.uilenbergi submitted in GenBank.The LAMP methods could detect T. luwenshuni and T. uilenbergi parasites on14thor13thdaypost infestation and thewith field-collected Haemaphysalis qinghaiensis from epidemic foci, Lintancounty, Gansu Province. That is earlier than that result of microscopic examination., whichsuggests the method can be used in early diagnosis.The data epidemiological survey showed that the animals were mainly infected by T. luwenshuni in Guangdong, Guizhou, and Chongqing provinces and mixed infection by T.luwenshuni and T. uilenbergi in Gansu province.The LAMP method established in this study were estimated with sensitivity, specificity andthe field test. It is confirmed that the LAMP method can be used to early diagnosis, differentialdiagnosis diagnosis and epidemiological survey of T. luwenshuni and T. uilenbergi. It could be usedto detect T. luwenshuni and T. uilenbergi infection and epidemiological investigation primaryveterinary stations. |
PDF Full Text Request