A comparison of susceptibilities to infection of four species of Hyalomma ticks with Theileria annulata

In this comparative study unfed nymphs of four Hyalomma tick species (Hyalomma anatolicum anatolicum, Hyalomma anatolicum excavatum, Hyalomma detritum and Hyalomma marginatum marginatum) were allowed to engorge on calves experimentally infected with Theileria annulata. The infection prevalence in the salivary glands of the adult female and male ticks of each Hyalomma species used in the study were assessed. The infection prevalence with T. annulata was high and did not vary markedly in the four Hyalomma tick species. The mean number of infected acini per tick in female and male ticks was different with female ticks having higher numbers of infected acini than the male ticks. The sex difference was more significant between H.a. anatolicum and H.a. excavatum than between H. detritum and H.m. marginatum. This study clarifies the roles of four Hyalomma tick species, and their sex, in the development of T. annulata.     

Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.     

Immunoprophylaxis against Theileria annulata with protein from plasma membrane of infected lymphoblasts

Cross-bred (Bos taurus male x Bos indicus female) calves were protected against the homologous sporozoite-induced challenge of Theileria annulata when immunised with protein from the plasma membrane of macroschizont-infected lymphoblasts of allogeneic origin. However, such protection was parasite-strain specific with the plasma membrane originating from lymphoblasts infected and transformed by the same isolate of T. annulata sporozoites. No protection ensued when the infected lymphoblasts and sporozoites were from immunologically different isolates of T. annulata. There was enhanced proliferation of cells and evidence for lymphokine, originating from antigen-sensitised lymphocytes, demonstrable as macrophage migration inhibition factor in peripheral blood lymphocytes of the calves immunised by plasma membrane protein and challenged by sporozoites of homologous origin.     

Prophylactic efficacy of buparvaquone in experimentally induced Theileria annulata infection in calves

The antitheilerial activity of buparvaquone (BW 720C) was evaluated in experimentally induced Theileria annulata infections in cross-bred male calves. T. annulata infections were induced by injecting a suspension of infected ground tick tissue suspension (GUTTS) equivalent to two ticks subcutaneously into each calf. Buparvaquone at a dose of 2.5 mg kg-1 body weight was given as a single injection (intramuscularly) on Day 0 (Group 1), Day 8 (Group 2) and Day 12 (Group 3) post-infection. The animals in Groups 4 and 5 were untreated and challenged controls, respectively. All of the recovered animals from Groups 1-4 were challenged with a lethal dose of T. annulata at 6 weeks post-infection. The immunized animals were resistant to the homologous challenge, which killed three of four control animals (Group 5); the controls showed typical antemortem and post-mortem lesions of theileriosis.     

Vaccine potential of recombinant antigens of Theileria annulata and Hyalomma anatolicum anatolicum against vector and parasite

In an attempt to develop vaccine against Hyalomma anatolicum anatolicum and Theileria annulata, three antigens were expressed in prokaryotic expression system and protective potentiality of the antigens was evaluated in cross bred calves. Two groups (grs. 1 and 4) of male cross-bred (Bos indicus × Bos taurus) calves were immunized with rHaa86, a Bm86 ortholog of H. a. anatolicum, while one group of calves (gr. 2) were immunized with cocktails of two antigens viz., surface antigens of T. annulata (rSPAG1, rTaSP). One group each was kept as negative controls (grs. 3 and 5). The animals of groups 1, 2 and 3 were challenged with T. annulata infected H. a. anatolicum adults while the animals of groups 1, 3, 4 and 5 were challenged with uninfected adult ticks. A significantly high (p<0.05) antibody responses to all the three antigens were detected in immunized calves, but the immune response was comparatively higher with rHaa86 followed by rTaSP and rSPAG1. Upon challenge with T. annulata infected ticks, animals of all groups showed symptoms of the disease but there was 50% survival of calves of group 1 while all non immunized control calves (group 3) and rSPAG1+rTaSP immunized calves died. The rHaa86 antigen was found efficacious to protect calves against more than 71.4-75.5% of the challenge infestation. The experiment has given a significant clue towards the development of rHaa86 based vaccine against both H. a. anatolicum and T. annulata.     

环形泰勒虫裂殖体表面蛋白基因PCR诊断方法的建立简

为寻求一种快速灵敏的环形泰勒虫病PCR检测方法,基于环形泰勒虫裂殖体表面蛋白（Theirelia annulata surface protein,TaSP）基因序列保守区设计特异性引物,通过PCR技术扩增出该基因长为393 bp的高免疫原性区片段。用该引物对环形泰勒虫、中华泰勒虫、瑟式泰勒虫、尤氏泰勒虫、吕氏泰勒虫、绵羊泰勒虫、马泰勒虫、驽巴贝斯虫、牛巴贝斯虫基因组模板进行特异性试验,对环形泰勒虫基因组模板进行梯度稀释后扩增,以确定试验的敏感性,同时用本试验建立的方法与常规显微镜镜检方法对150份血清样品进行检测。特异性试验结果显示,在被检测的9个样本中,只有环形泰勒虫基因组模板中扩增出了符合大小的特异核苷酸片段;敏感性试验结果表明,PCR对环形泰勒虫的扩增效率可达到10^-10;通过对150份血清样品的检测,并与血涂片方法进行比较,结果显示PCR方法具有特异性强、敏感度高等特点,适用于牛环形泰勒虫病的检测。     

Studies on the transmission of Theileria annulata to cattle by the tick Hyalomma lusitanicum

The role of the ixodid tick Hyalomma lusitanicum Koch 1844 as a vector of Mediterranean or tropical theileriosis (caused by the protozoan parasite Theileria annulata Dschunkowsky et Luhs 1904) in southern Spain was studied. Hyalomma lusitanicum was the most common tick, and the only species of the genus Hyalomma L., found on T. annulata-infected cattle from the theileriosis enzootic area studied (province of Cádiz, southern Spain). Likewise, we found that all sera of the cattle previously considered as suspected of theileriosis by clinical signs, tested for T. annulata antibodies, were positive and all blood samples of these suspected cattle examined had infected erythrocytes. Partially fed H. lusitanicum adults were collected in the field on T. annulata-infected cattle in this enzootic area and fed on an uninfected calf in an experimental farm free of theileriosis and ticks. At approximately 3 weeks post-tick feeding on the calf, this became positive for T. annulata antibodies and T. annulata merozoites were found in erythrocytes from blood smears. These results show the ability of H. lusitanicum to transmit the protozoan parasite T. annulata to susceptible cattle and indicate that H. lusitanicum is probably an important vector of T. annulata in the enzootic area surveyed.     

Prevalence and distribution of tropical theileriosis in eastern Turkey

This study was carried out to determine the prevalence and distribution of tropical theileriosis in cattle in eastern Turkey by microscopical, serological and molecular methods. A total of 1561 whole blood, 1505 serum and 1483 blood smear samples were collected from cattle of various breeds and ages in 11 towns of Eastern Turkey. Theileria annulata piroplasm DNA extracted from cattle blood was amplified by polymerase chain reaction (PCR) using species-specific primers. Serum antibodies against T. annulata were investigated by indirect fluorescence antibody test (IFAT). Blood smears were examined for Theileria piroplasms by microscopical examination (ME). In the examination of DNA extracted from 1561 blood samples, an amplicon with the size of 721bp was obtained in 37.8% (590/1561) of these samples. Serum antibodies against T. annulata and piroplasm of Theileria spp. were detected in 34.9% (526/1505) and 19.7% (293/1483) of the samples, respectively. The differences between ME and PCR results and between ME and IFAT results were statistically significant (P < 0.05). In contrast, there was no significant difference between the PCR and IFAT results. A total of 179 ticks (136 female; 43 male) belonging to Hyalomma spp. were collected from cattle from three towns. Ticks were identified to be Hyalomma anatolicum anatolicum on the basis of morphological features.     

Evaluation of indirect TaSP enzyme-linked immunosorbent assay for diagnosis of tropical theileriosis in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in Egypt

The aim of the present study was to evaluate the validity of Theileria annulata surface protein (TaSP)-ELISA, in comparison with traditional microscopic test, for the diagnosis of T. annulata infection among Egyptian baladi cattle (Bos taurus) and water buffaloes (Bubalus bubalis). Molecular confirmation of infection using T. annulata merozoite surface (Tams-1) target amplification by PCR was used as a gold standard. A total of 76 clinically suspected animals including 64 baladi cattle and 12 water buffaloes were investigated in the current study by the three methods. Based on the PCR-confirmed results, the evaluation study revealed higher sensitivity of TaSP-ELISA (72.9% and 75%) as compared to microscopic examination (58.3% and 50%) among cattle and buffaloes, respectively. On the other hand, the specificity of TaSP-ELISA in diagnosis of T. annulata infection was higher (87.5%) in baladi cattle as compared to water buffaloes (37.5%). In conclusion, TaSP-ELISA was shown to be suitable for the diagnosis of T. annulata infection in cattle under field conditions.     

Glucose phosphate isomerase isoenzyme patterns in bovine lymphoblastoid cell lines infected with Theileria annulata and T parva, with an improved enzyme visualisation method using meldola blue

Five strains of Theileria annulata from three geographically different areas and one strain of T parva were grown in bovine lymphoblastoid cell lines. Lysates of the parasitised cells were examined by thin layer starch gel electrophoresis for multiple forms of the enzyme glucose phosphate isomerase. A reported difference between the glucose phosphate isomerase isoenzyme patterns of T annulata and T parva was confirmed. Cultures of one strain of T annulata grown in cells derived from four different cattle showed similar host and parasite isoenzyme patterns. Two strains of T annulata and one strain of T parva grown in lymphoid cells derived from the same animal showed identical host cell isoenzyme patterns whereas the isoenzyme pattern associated with each parasite was different. Strains of T annulata from different geographical areas showed major differences in their isoenzyme patterns, but no differences were detected between strains from the same geographical area. Meldola blue was found to be superior to phenazine methosulphate as an intermediate electron acceptor in the visualisation of enzyme activity.     

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.     

Factors affecting the capacity of Theileria annulata sporozoites to invade bovine peripheral blood lymphocytes

The number of T. annulata sporozoites invading bovine peripheral blood lymphocytes (PBL) under different conditions (in vitro) was determined. Heat-inactivation of T. annulata sporozoites for 45 min, in a thermostatically controlled, shaking water bath preset and stabilised at 60 degrees C resulted in an almost total lack of invasion of fresh, normal PBL by the sporozoites, indicating that the interiorization process is parasite-effected. The mean number of T. annulata sporozoites interiorization (per 1000 lymphocytes) in cultures set up using sporozoites and PBL, mixed and incubated at 0 degrees C for 1 h in melting ice, was highly significantly reduced (P less than 0.01), indicating the invasion of bovine lymphocytes by T. annulata sporozoites is an active process dependent on active metabolism which is markedly affected by temperature. Pre-treatment of PBL with trypsin significantly reduced the number of invading sporozoites thus incriminating proteins or glycoproteins as constituents of receptors involved in sporozoite-lymphocyte recognition.     

Evaluation of an enzyme immunoassay for serodiagnosis of infections with Theileria parva and T annulata

An enzyme linked immunosorbent assay (ELISA) was used to determine antibody levels in cattle infected with Theileria parva and T annulata, using antigens prepared from the intra-erythrocytic piroplasm stage of the parasites. Antibody levels in calves infected with T parva increased from the 16th day after infection to reach peak values at days 28 to 35 and then declined rapidly, but in calves infected with T annulata antibody levels rose steadily up to day 40. Similar patterns of antibody production were shown by indirect fluorescent antibody tests. Sera from animals infected with T parva gave higher ELISA values with the antigen prepared from the homologous parasite species than with the antigen prepared from T annulata, but sera from cattle infected with T annulata gave similar high ELISA values with antigens prepared from both T parva and T annulata. Sera from animals infected with T mutans, T sergenti, T velifera, Babesia divergens, B major and B bovis gave only slight or no cross reactions with the piroplasm antigens, but serum from a calf infected with B bigemina cross reacted at a significant level with both piroplasm antigens.     

Common and stage-specific antigens of Theileria annulata.

Western blot analysis of Theileria annulata antigens was carried out using sera collected from cattle which had been immunised and challenged with either T. annulata sporozoites or schizont-infected cells. Three antigens between 71 and 73 kDa proved to be common to the three stages of parasite studied: sporozoites, schizonts and piroplasms. An antigen was found at 32 kDa which was specific to T. annulata piroplasms. Results were reproducible using sera from Morocco and the UK. At least one of the proteins at 71-73 kDa, but not that at 32 kDa were also recognised by sera from animals infected with Babesia species.     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: II. In vitro studies.

In the studies previously reported, the tick-borne protozoan parasites Theileria lestoquardi and Theileria annulata were shown to differ in their capacity to infect sheep and cattle. In the studies presented here, these findings were further supported. In vitro infectivity of T. lestoquardi and T. annulata sporozoites for peripheral blood mononuclear cells of sheep and cattle were determined by analysis of cell cultures for cell proliferation, the detection of parasites in Giemsa-stained cytospin smears and the establishment of continuously growing schizont-infected cell lines. In the same way, the development of schizont-infected cells into continuously growing cell lines was studied with material isolated ex vivo from the sheep and cattle undergoing primary infections described elsewhere. Comparisons were also made between development of ex vivo cell lines from animals undergoing primary infections with those of the animals undergoing challenge infection with the other parasite species. Theileria species specific primers were used in a PCR to determine the identity of the parasites in the cell lines. These in vitro studies confirmed earlier observations that T. lestoquardi was unable to infect cattle, whereas infection of all sheep with T. annulata was proven. Moreover, earlier indications of the development of partial cross-immunity in sheep of T. annulata to T. lestoquardi and vice versa were strengthened. These findings may thus have consequences for the understanding of the epidemiology of T. lestoquardi infections of sheep. On the other hand. since piroplasms were not demonstrated in sheep infected with T. annulata, such sheep will not be infective to ticks and will consequently be unlikely to play a role in the maintenance and transmission of T. annulata to cattle.     

小亚璃眼蜱对牛巴贝斯虫未定种和环形泰勒虫传播的试验研究

利用蜱传播试验确定小亚璃眼蜱对中国新分离的牛的巴贝斯虫未定种和环形泰勒虫的传播能力与传播方式，进而明确其在中国牛梨形虫病传播中的流行病学意义。试验结果表明：小亚璃眼蜱可在雌虫阶段受到巴贝斯虫未定种的感染，并可在次代若虫（2／2）和成虫（3／3）阶段将病原传播给试验牛；小亚璃眼蜱幼虫和若虫吸入环形泰勒虫，饱血脱落的幼虫和若虫所蜕化发育的饥饿若虫（2／2）和成虫（2／2）均可将病原传递给敏感动物；感染巴贝斯虫未定种的小亚璃眼蜱饱血雌虫所孵育出的次代幼虫和若虫仍可被环形泰勒虫感染，所发育出的若虫（2／2）和成虫（2／2）可在一次传播试验中将环形泰勒虫和巴贝斯虫未定种同时传播给试验动物。     

In vitro infection of bovine alloreactive cytotoxic T cell lines with sporozoites of Theileria annulata and T parva

Bovine alloreactive cytotoxic lymphocyte (CTL) lines of known target specificity were infected in vitro with sporozoites of Theileria annulata and T parva and cultured in limiting dilution. The phenotypes of the CTL lines both pre- and post infection were assessed using a panel of monoclonal antibodies specific for defined bovine lymphocyte subpopulations. The effector function of the resultant infected cell lines was determined using a Cr51 release assay and compared to the uninfected control CTL line. The results indicated that T parva sporozoites consistently infected and transformed the CTL lines very efficiently even at the lowest cell doses. In contrast the T annulata sporozoites were largely unable to infect and transform the alloreactive CTL except at the very highest cell and sporozoite doses. A factor which appeared to influence susceptibility to T annulata infection was an increased level of class II expression on the CTL line. None of the cell lines showed cytotoxic effector function after infection with either T annulata or T parva sporozoites.     

Isolates of Theileria annulata collected from different parts of India show phenotypic and genetic diversity

Phenotypic and genetic polymorphism was studied amongst four Theileria annulata isolates collected from three different parts of India. Amongst various markers studied for the comparison of growth characteristics of schizont cell lines established from these isolates, viability, non-viability counts and nitric oxide (NO) production showed significant variation. A negative correlation was observed between NO production and mRNA expression for TNF-alpha, a potent proinflammatory cytokine related to the pathogenesis of the disease. Phenotypic polymorphism was also revealed by T. annulata schizont-specific monoclonal antibodies (Mabs), viz. 1C7, 1E11, 2G2 and EU-106, which recognized variable number of cells in indirect fluorescent antibody and indirect immunoperoxidase tests, when tested against the four T. annulata isolates collected from India. Genetic polymorphism was recognized amongst the four isolates by restriction digestion analysis of Tams-1 gene PCR products. These observations revealed that the four isolates of T. annulata are different from each other and might be expressing different antigenic determinants on their cell surface.     

Metabolic energy pathways in Theileria annulata sporozoites and their significance in sporozoite-bovine lymphocyte interactions in vitro

The existence of metabolic energy pathways has been studied in extracellular T. annulata sporozoites using chemicals known to inhibit specific energy-generating pathways, and their role during invasion of bovine peripheral blood lymphocytes (PBL) by the sporozoites determined in an in vitro system. An inverse relationship was depicted between the dose of various chemicals and the number of T. annulata sporozoites invading PBL: as the concentrations of the inhibitor drugs increased, the number of T. annulata sporozoites within the lymphocytes decreased. An ultracytochemical study demonstrated the presence of the respective pathway marker enzymes, i.e., lactic dehydrogenase (LDH) in the cytosol and within mitochondria, succinic dehydrogenase (SDH) on the mitochondrial membranes and in the contiguous matrix, and cytochrome oxidase (CO) between the inner and outer mitochondrial membranes, in infective T. annulata sporozoites fixed in situ within whole salivary glands of 3-day fed Hyalomma anatolicum anatolicum ticks.     

Prevalence of blood parasites among cattle at the central area of Saudi Arabia

The present study aimed to investigate the parasites infecting cattle blood at Al-Qassim region. Examination of 307 blood samples revealed that 235 (76.5%) and 3 (0.98%) of cattle were infected with Theileria annulata and Anaplasma marginale, respectively. T. annulata was the prevalent blood parasite among cattle while A. marginale was scarcely encountered. The monthly incidence of T. annulata ranged from 38.5% in March to 94.7% in October. Also, the infection rate reached a maximum (84.3%) in both autumn and summer seasons, while it decreased to reach (59.4%) in spring. It has been found that there was no difference between the infection rate of the laboratory and the abattoir collected samples. However, the intensity of infection was different between the two groups.