Establishment And Application Of A Duplex PCR Assay For Detection Of Theileria Luwenshuni And Anaplasma Phagocytophilum In Sheep And Goats

Ovine Theileriosis is an ageneraldesignation of a class of diseases caused by the parasitic protozoa belonged to Theileriidae,Theileria,which is parasitic in erythrocytes,lymphocytes or macrophages of sheep and goats.Anaplasmosis is a kind of rickettsiosis caused by pathogens belonged to Anaplasmataceae,Anaplasma in human and animal blood cells.Theileriosis and Anaplasmosis belong to tick-borne diseases,causing human and animals to have very similar clinical symptoms such as high fever,anemia,jaundice,and weight loss,which can lead to death when severe infection occurs.Theileria luwenshuni is a kind of malignant species,which has a strong pathogenicity.And Anaplasma phagocytophilum,as a zoonoses pathogen,severely threatens the health of humans and animals.In clinical practice,the mixed infection of these two pathogens is more common.At present,the detection method of these two pathogens is mainly a conventional PCR,but it is limited to the examination of one specific pathogen at one time,and it consumes manpower and material resources in clinical applications.To date,no report has been published on the establishment of a PCR diagnostic method for the simultaneous detection of T.luwenshuni and AP.The purpose of this study was to establish a duplex PCR assay with high specificity and high sensitivity and good repeatability in order to be able to simultaneously detect T.luwenshuni and AP,providing a technical method for rapid detection of both pathogens and rapid diagnosis of disease caused by these two pathogens.1.Establishment and application of a PCR assay for detection of Theileria luwenshuniIn order to establish a sensitive and specific method for detection of T.luwenshuni in sheep and goats,a set of primers T.lu7F/T.lu7 R was designed based on the 18 S r RNA gene sequence of published T.luwenshuni(Accession No.: KC735166.1)in Gen Bank to establish a PCR detection method.The optimum reaction and reaction system conditions were screened.Moreover,the specificity,sensitivity and reproducibility of test as well as detection of clinical samples were carried out.The results showed that this method can specifically amplify the 962 bp target fragment.There was no cross reaction with Theileria uilenbergi,Theileria ovis,Theileria annulata,Babesia motasi and Toxoplasma gondii.The maximum dilution of T.luwenshuni DNA was 10-7,in other words,the minimum detection of T.luwenshuni DNA was 29.13 fg·?L-1 and this method had good repeatability.76 sheep clinical blood samples were detected using the established and reported methods,respectively.The 77.63%(59/76)positive rate by the PCR assay was higher than the positive rate of 60.53 %(46/76)by the reported PCR method.What is more,the difference in detection rate was statistically significant(0.01<p<0.05).The results indicate that the established PCR method can be used for clinical sample detection.2.Establishment and application of a duplex PCR assay for Detection of Theileria luwenshuni and Anaplasma phagocytophilum in Sheep and GoatsThe above-mentioned primers that can specifically amplify the 18 S r RNA gene of T.luwenshuni were combined with primers that have been reported to specifically amplify the 16 S r RNA gene of AP to establish a duplex PCR assay for simultaneous detection of these two pathogens by optimizing the reaction system,reaction conditions and performing the specificity,sensitivity,repeatability tests.The results of verification experiments showed that the targeted gene fragment was 962 bp for T.luwenshuni and 641 bp for AP in length.This detection method had no cross reaction with T.uilenbergi,T.ovis,T.annulata,Anaplasma ovis,Anaplasma bovis,Anaplasma marginale,B.motasi and T.gondii.The sensitivity reached to 29.13 fg·?L-1 and 1.534fg·?L-1 for T.luwenshuni and AP,respectively.This detection method was stable and had good repeatability.54 clinical blood samples were tested using single PCR and the duplex PCR,respectively.The results showed that the positive rates of single PCR detection in T.luwenshuni and AP were 87.04%(47/54)and 83.33%(45/54),respectively,and the mixed infection rate was 72.22%(39/54).The positive rates of the duplex PCR detection in T.luwenshuni and AP were 87.04%(47/54)and 68.52%(37/54),respectively,and the mixed infection rate was 55.56%(30/54).There was no difference in sensitivity between the single PCR assay and the duplex PCR to test T.luwenshuni.However,the sensitivity of the single PCR assay for the detection of AP tended to be greater,when compared with the duplex PCR.But the method can be used to detect T.luwenshuni and AP of clinical samples and provide a new technical method for the detection of these two pathogens.This test method is of great significance for the early diagnosis,epidemiological investigation of Ovine Theileriosis and and Anaplasmosis.