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The present study was performed on antigen-presenting cells (APCs) of Theileria annulata transformed dendritic cells (TaDCs) and monocyte-derived dendritic cells (MoDCs) to compare differences in antigen presentation and stimulation of T lymphocyte proliferation. Antigen presentation for T lymphocyte proliferation was analysed by flow cytometry. Additionally, the level of mRNA transcription of small GTPases of the Rab family expressed in the TaDC cell line was analysed by quantitative real-time polymerase chain reaction (Q-RT-PCR). The endocytosis rate of TaDCs was significantly (P < 0.01) lower than in MoDCs. In contrast, when T lymphocytes were co-cultured with TaDC-APCs T cell proliferation was similar, while co-culture with MoDC-APC stimulated proliferation of CD4+ cells to a greater degree than CD8+ cells. However, the efficacy of TaDC-APCs to stimulate T lymphocytes dropped as the number of passages of TaDC-APC increased. Likewise, the transcription level of Rab family genes also significantly (P > 0.001) declined with progressive passages (>50) of the TaDC cell line. We conclude that initially the TaDC cell line efficiently presents antigen to stimulate T lymphocyte proliferation to produce a cellular immune response against the presented antigen.

This study aimed to establish a pure single-cell Theileria annulata -infected B cell line for the assessment of cytokine production in transformed and lipopolysaccharide (LPS)-stimulated cells. Several studies have aimed to identify cell surface markers in T. annulata -transformed cells; however, no information on cytokine production in these cells is available. To investigate the potential of the transformed cells to produce cytokines and their potential responses to antigen-stimulation, we purified mature B cells (CD21) from the whole blood of cattle experimentally infected with the T. annulata Kashi strain by magnetic separation. The purity and specificity of the established cell line was assessed by the identification of specific cell surface markers (CD21, IgM, and WC4) by flow cytometry analysis. The transcript levels of the cytokines IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL16, LTA, TGFB1, TNFA, IFNA, and IFNB in transformed, buparvaquone (BW720c)-treated cells, and antigen-stimulated cells were analyzed by quantitative polymerase chain reaction (qPCR) using cDNA from these cells. A T. annulata -infected bovine B cell line was successfully established with a purity of ~98.8% (CD21). IL4 and IL12A were significantly ( p < 0.01) upregulated in the transformed cells. In BW720c-treated transformed cells, IL12B, TGFB1, and IFNB were significantly ( p < 0.01) upregulated. Notably, no significant ( p > 0.05) upregulation of cytokines was observed in LPS-stimulated transformed cells. Moreover, IL1A, IL1B, IL8, and IL16 were significantly ( p < 0.01) upregulated in LPS-stimulated B cells. Our data signify the potential use of this cell line for cytokine production, observance of immunoglobulins, and production of an attenuated vaccine against tropical theileriosis., Competing Interests: The authors declare no conflict of interest.

Tropical theileriosis, caused by Theileria annulata, is distributed worldwide and causes great economic losses in dairy. The reliable diagnostic method is critical for prevention and control of the disease. In this study, a sporozoite and macroschizont gene 2 (spm2) protein from T. annulata was used to develop an indirect ELISA for tropical theileriosis. Specificity test showed that there were no cross-reactions with antibodies raised against other bovine piroplasm species using ELISA or western blotting. The specificity and sensitivity were 98.4% and 98.7%, respectively, with a threshold of 35.5% of the specific mean antibody rate (AbR). Furthermore, a total of 196 field sera samples collected from Xinjiang and Gansu provinces were detected by the spm2 ELISA and IFA. The results obtained with the spm2 ELISA and IFA in this study had the moderate agreement. The average positive rates of T. annulata sera samples detected in the present study were close to the prevalence of previous reports in these endemic areas. This indicated that the Spm2 ELISA could be used as a reliable diagnostic tool for serological survey of T. annulata infection in areas where Theileria parva is not present., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Piroplasmosis is one of the most important diseases of livestock, constraining optimal production and leading to economic loss. This study was carried out to detect Theileria annulata by using 2 different molecular techniques: recombinase polymerase amplification (RPA) and conventional polymerase chain reaction (PCR). Blood samples were collected from 274 ticks infesting asymptomatic cattle from several counties in the Chakwal, Faisalabad, and Jhang districts of Punjab Province in Pakistan by using FTA cards. After extraction of genomic DNA, each sample was subjected to RPA optimized to amplify a 281-bp fragment of the Enolase gene for T. annulata. The specificity of the test was confirmed using positive DNA samples of related piroplasm species, whereas the analytical sensitivity was calculated using different serial dilutions of a long fragment of the same gene. The RPA results were positive for 48 (17.51%) of 274 samples. All 274 samples were screened using conventional PCR, and 21 (7.66%) samples were positive for T. annulata. All the samples that were RPA positive but PCR negative were sequenced, which confirmed the results of RPA. The highest positive rate was found in Chakwal district, followed by Faisalabad and Jhang districts. This study demonstrates the application of highly sensitive and specific rapid diagnostic methods for T. annulata to a regional screening program. This is the first report of tick-borne disease from Pakistan by using RPA.

Theileriosis is a disease of domesticated animals in tropical and subtropical countries and causes significant reductions in livestock productivity. The arid region of Punjab in Pakistan is notorious for the presence of the vector tick (Acari: Ixodidae) and tick-borne diseases, such as theileriosis and babesiosis. The distribution of Theileria annulata and T. orientalis in the Chakwal district of Punjab was determined by developing a multiplex recombinase polymerase amplification (RPA) assay as a scientific basis for formulating control strategies for bovine theileriosis. Specificity was evaluated using DNA from related piroplasm species, while analytical sensitivity was calculated using a long fragment of the enolase gene. A total of 188 blood samples were collected on FTA cards (Whatman ® ) from tick-infested asymptomatic breeds of cattle (Bos indicus, Bos taurus, and Bos indicus × Bos taurus) in the study district. Finally, infections with of T. annulata and T. orientalis were detected using the multiplex RPA and compared with the conventional multiplex polymerase chain reaction (PCR). The multiplex RPA specifically amplified 282-bp and 229-bp fragments of the enolase gene from T. annulata and T. orientalis and had no cross-reaction with other piroplasm species. It was determined that 45 (23.9%) and 5 (2.6%) out of 188 blood samples were positive for T. annulata and T. orientalis, respectively, when examined using RPA. Multiplex PCR detection indicated that 32 (17.0%) and 3 (1.6%) blood samples were positive for T. annulata and T. orientalis, respectively. In the present study, a specific RPA method was developed for simultaneous differentiation and detection of T. annulata and T. orientalis infections and used for the first time for the detection of the two bovine Theileria infections., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Bovine theileriosis, a tick-borne protozoan disease caused by Theileria annulata, Theileria orientalis and Theileria sinensis, is widespread in China and is a serious economic problem for the Chinese livestock industry. In this study, recombinant major piroplasma surface proteins (MPSP) of T. annulata, T. orientalis and T. sinensis based on MPSP genes were expressed in Escherichia coli BL21(DE3). The immunogenicity and specificity of the three purified recombinant MPSP proteins were evaluated with the reference positive sera of T. annulata, T. orientalis, T. sinensis, Babesia bovis, B abesia bigemina, Babesia major, Babesia motasi, Theileria luwenshuni, Theileria uilenbergi and Anaplasma ovis using an enzyme-linked immunosorbent assay (ELISA) or western blotting. The results showed that all three of the rMPSP proteins had a strong reaction with the sera from cattle infected with T. annulata, T. orientalis and T. sinensis via western blotting but not with other piroplasma and Anaplasma species. Then, the rMPSP protein of T. sinensis was used to develop an iELISA for detecting the three Theileria species infections. The specificity and sensitivity were 95.7 and 95.5 %, respectively, with a threshold of 28.8 % of the specific mean antibody rate (AbR). Finally, 2473 field-collected bovine sera, from 42 prefectures of 17 provinces in China, were tested using the ELISA to evaluate the prevalence of bovine theileriosis, and the average positive rate was 43.6 %. The developed iELISA could be a suitable tool to detect the three bovine Theileria species, and the data also provided important information regarding the current prevalence of bovine theileriosis in China.