Development of serological (indirect ELISA) and molecular (PCR, multiplex PCR and LAMP) techniques for the detection of Theileria annulata

The aim of the present study is to develop new diagnostic methods for the detection of tropical theileriosis caused by Theileria annulata in cattle. Diagnostic approaches towards this end were based on serological and molecular methods. These were, i) Identification of recombinant proteins which can be of use in serological diagnosis of tropical theileriosis with ELISA, ii) Development of molecular diagnostic methods (PCR, multiplex PCR and LAMP) to use in the diagnosis of tropical theileriosis.In order to identify recombinant proteins which can be of use in serological diagnosis of tropical theileriosis with ELISA, bioinformatic methods were used to determine proteins specific to the macroschizont stage of T. annulata. Immun response against these proteins were then evaluated in experimentally infected animals using Western blot analysis. Following studies were aimed to determine if the TaSP, a surface antigen of Theileria annulata, is the immunodominant macroschizont antigen. TaSP along with a recombinant protein generated through immunoprecipitation (Ta9) as well as recombinant proteins generated through bioinformatic methods TA06510 and Ta9.4, a paralog gene of Ta9, were used in later studies to develop indirect ELISA.Immunodominant bands seen in Western blots were evaluated for the presence of any polymorhisms. To this end, 16 different isolates of T. annulata along with T. parva Muguga and T. lestoquardi Lahr makroschizonts as well as uninfected bovine lenfosarkoma (BL20) cell lines were used as the parasite material and protein extract. Evidence gathered from these studies demonstrated that there exists polymorphism among bands within the range of 40-42 kDa.In order to determine macroschizont stage-specific proteins of T. annulata, all proteins encoded by coding sequences in the T. annulata genome were bioinformatically screened for the presence of Signal P, GPI anchor, protein motifs such as TMD and PEST taking also the EST values and the dN/dS ratios into consideration. Genes encoding the selected proteins were then cloned, produced as recombinants and were evaluated in Western blots. It was determined that experimentally-infected animals developed an antibody response against the TA06510 recombinant protein. However, the antibody response against the TA06510 was determined to be less than the antibody response detected in previous studies against the Tams-1/2 and TaSP antigens known to be immunogenic. These results demonstrated that bioinformatic methods can be used to identify immonogenic proteins encoded by the T. annulata genome. However, functional analyses should be performed in order to determined the immunogenicity of bioinformatically-identified proteins.Western blot analyses carried out using immune sera blocked with TaSP recombinant protein revealed that 42 kDa immunodominant band detected in D7 protein extracts is not the TaSP molecule that was suggested in previous studies as the immunodominant macroschizont antigen. Immunoprecipitation experiments and proteomics analyses were performed to characterize the immunodominant bands detected in Western blot analyses. Results obtained from these studies demonstrated that one of the immunodominant bands seen in D7 protein extracts is the Ta9 protein. Subsequent studies indicated that the other immunodominant band seen in D7 protein extracts is the Ta9.4 protein belonging to the same paralog protein family with the Ta9 protein.TaSP, Ta9, Ta9.4 and TA06510 recombinant proteins were used to develop indirect ELISA tests. In tests performed with known- negative and positive serum samples, the best sensitivity (89.5%) and specificity (98.3%) was obtained with the TaSP recombinant protein. Tests performed using field serum samples indicated that a higher number of positivity was determined with the IFA test compared to those determined with the ELISA tests using the TaSP, Ta9 and Ta9.4 recombinant proteins (p<.05). However, ELISA tests developed in the present study should be re-standardized using a higher number of known negative and positive serum samples for their routine use in the field.PCR, multiplex PCR and LAMP methods were developed in the present study for molecular diagnosis of tropical theileriosis. In molecular analyses performed to this end, DNA samples obtained from twelve different isolates of T. annulata, T. parva Kenya, T. sergenti, T. lestoquardi, B. bovis, B. bigemina ve T. equi, 18 DNA samples in total, were used. Primer pairs used for the PCR, multiplex PCR and LAMP were selected based on the sequence information of relevant genes using the Clustal X program. Experimental infection was carried out in order to determine the specificity of the molecular tests.Animals with a low level of parasitemia could be identified sensitively (0.1 piroplasms in 1 ?l blood) and specifically through PCR performed using cyto b1 primer pairs amplifying the mitochondrial cytochrome b gene of T. annulata.Multiplex PCR method developed in the present study was demonstrated to be a simpleand an efficient method in simultaneous and sensitive identification of T. annulata, B. bovis veA. marginale species, all causing significant diseases in the cattle.In addition to PCR and multiplex PCR, a LAMP method was also used in the presentstudy to identify Theileria annulata in sick and/or carrier animals. The LAMP is a relativelyeasy method that enables quick amplification of numerous DNA fragments with highspecificity in isothermal conditions with no need for a thermal cycler. However, resultsobtained from LAMP studies performed using CYTOB1 primers on samples collected from thefield indicate that LAMP is less effective than the PCR in diagnosing T. annulata in sick and/orcarrier animals.