Isoenzyme variation in piroplasms isolated from bovine blood infected with Theileria annulata and T parva.

Blood from calves infected with Theileria annulata and T parva was freed from host cell elements and the piroplasms liberated from the red cells by ammonium chloride lysis. Lysates of the purified piroplasms and control host cell material were examined electrophoretically for several enzymes. Zymograms stained for glucose phosphate isomerase showed distinct differences between the host cell enzyme pattern and parasite enzyme patterns. The isoenzyme pattern of T annulata piroplasms differed from the isoenzyme pattern of T parva piroplasms.     

Ultrastructural characteristics of in vitro parasite-lymphocyte behaviour in invasions with Theileria annulata and Theileria parva

Host-parasite relationships have been studied by electron microscopy using glutaraldehyde-OsO4-fixed pellets of lymphoid cultures infected in vitro by Theileria annulata and T. parva. Intracellular presence of the parasite resulted in a progressive and marked lymphoblastoid transformation. The schizont stage periodically provoked the formation of, and adopted an intimate association with, cytoplasmic annulate lamellae in the interphase cell. Annulate lamellae developed from the outer nuclear membrane of the host cell by a delamination process and were taken into the cytoplasmic matrix of the schizont by phagotrophy through the cytostome. Schizont nuclei themselves were seen to divide at the prometaphase stage of host cell mitosis, the division being characterized by the development of intranuclear spindle microtubules anchored in spindle pole bodies. A hypothesis is propounded that Theileria parasites, consequent on interiorization, provoke the blastoid transformation and the formation of annulate lamellae through the influence of components of their genomic material on host cell deoxyribonucleic acid (DNA) and that the annulate lamellae represent a species of messenger ribonucleic acid (mRNA) and serve as a monitoring device for the schizont, facilitating the accurate timing of the host cell cyclical events.     

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.     

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.     

Comparative autoradiographic study of parasite-host-cell cyclical relationship in lymphoblastoid cell lines infected with Theileria annulata and Theileria parva in vitro

Cyclical interactions between intracellular schizonts of the Ankara and Hissar strains of Theileria annulata and the Muguga strain of T. parva and the parasitised host lymphoblasts have been studied autoradiographically by following the incorporation of (3H) thymidine into parasite and host cell nuclei, and also by quantitating the number of schizont nuclei per lymphoblast, at various stages and phases of host cell cycle. The synthesis of DNA by Theileria schizonts and the parasitised host lymphoblasts was found to be asynchronous and to occur at different phases of the host cell interphase stage. While the lymphoblast nuclear DNA incorporates (3H) thymidine during the S phase, schizont nuclei were labelled during the G2 phase of the host lymphoblast interphase stage. The replication of schizont nuclei took place before the metaphase stage of host cell cycle, viz, prometaphase, so that the mean schizont nuclear number at host prometaphase and at anaphase--telophase was consistently more or less double the mean nuclear number at interphase.     

奶牛环形泰勒焦虫病诊治报告

２０００年６月上旬至７月下旬，我县营子、七贤等乡镇发生一种呈地方性流行或散发的急性奶牛疾病，以高热稽留、体表淋巴结肿大、贫血为主要特征。根据流行病学、临床症状、剖检变化、实验室诊断，确诊为奶牛环形泰勒焦虫（Ｔ．ａｎｎｕｌａｔａ）病，现报告如下：１流行病学从６月上旬开始陆续发病，发病率达２５％，其中死亡２头。该病以一胎母牛多发。经调查，多为带犊由外地购进，本地饲喂时间不足一年。检查牛体，能发现吸饱血或未吸饱血的豆粒大或米粒大牛蜱。２临床症状病牛初期体温上升高达４１℃，稽留热。肩前和鼠蹊淋巴结肿大，…     

Cycle of bovine lymphoblastoid cells parasitised by Theileria parva

The events were studied which occurred during different stages of the cell cycle of bovine lymphoblastoid cells infected with the parasite Theileria parva. The mean number of nuclei in macroschizonts was about 16 for cells in interphase and 30 for those in metaphase. Pulse labelling with 3H thymidine showed that macroschizonts normally incorporated thymidine when the host cell was in early mitosis. Thymidine incorporation by macroschizonts thus occurred at a different stage in the cell cycle to that when the cell nucleus incorporated thymidine in S phase. DNA synthesis by host cell nucleus and macroschizont is thus asynchronous. Division of macroschizonts appears to follow immediately after they have synthesised DNA without a G2 period. This division occurs while the host cell is in metaphase. When the cell divides each daughter cell thus contains its interphase complement of macroschizont nuclei. Some macroschizont division may occur in interphase but this is relatively insignificant when compared with that which occurs in host cell metaphase. This work suggests that T parva regulates its own DNA synthesis independently of the cell. This finding could have application in developing strategies for chemotherapeutic attack on the parasite.     

乳牛产后发生疑似环形泰勒焦虫病

引进乳牛产犊后出现以体温升高稽留为特征的疾病;通过发病情况、临床检查、实验室检验和治疗效果,诊断为乳牛产后发生疑似环形泰勒焦虫病. 1发病情况临朐县饲养乳牛专业户刘××为扩大饲养规模,于2000年4月23日,由××省引进18头初孕黑白花乳牛.     

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.     

Observations on the in vitro multiplication of bovine lymphoid cells infected with Theileria annulata schizonts

The multiplication of a bovine lymphoid cell line infected with Theileria annulata schizonts was studied. In cells dividing mitotically, the schizont occupied a central position during the late stages of the division and was shared by the two newly formed cells. Binucleated cells with schizonts located between the nuclei were also observed. No definitive conclusions can be drawn as to whether these cells are stages in amitotic division or result from fusion of infected cells. Wide variations in the number of schizont nuclei per infected cell occurred with an average of 12.2 nuclei per schizont. The great majority of the cells contained 4 to 16 schizont nuclei.     

新疆牛环形泰勒虫Tams1基因的克隆与表达

采用PCR方法扩增环形泰勒虫Tams1基因,将扩增产物与pUcm-T载体连接,重组质粒经PCR和双酶切鉴定、测序确认后被亚克隆于pGEX-4T-2表达载体中.构建的表达重组质粒经IPTG诱导表达后,进行SDS-PAGE、Western blot分析.结果显示,克隆的Tams1基因片段长846 bp,为一个完整的开放阅读框,编码281个氨基酸,与GenBank中的Tams1基因核苷酸同源性在93.6%～99.8%之间;系统进化树分析表明新疆分离株与印度株、毛利塔尼亚株和北非共和国株进化途径一致,与苏丹株、巴林株进化途径相差较远;推测的蛋白质抗原决定簇预测表明,Tams1基因编码的氨基酸具有13个交叉的抗原决定簇;表达的融合蛋白为56 ku,能够被环形泰勒虫多克隆血清所识别,表明该融合蛋白具有较好的反应原性和免疫原性,为进一步开展牛环形泰勒虫的分子生物学诊断、亚单位疫苗和核酸疫苗的研究奠定了可靠的基础.     

Antigenic diversity of Theileria annulata macroschizonts

A series of monoclonal antibodies has been produced which reacts with the intracellular macroschizont of Theileria annulata, and this series has been used to examine the level of antigenic diversity between and within stocks of the parasite in addition to species specificity within the genus. The majority of the monoclonal antibodies (six of eight) reacted with all stocks tested and four were species-specific. Two monoclonal antibodies detected variation between stocks and within stocks. The variation within a stock was manifest as only a proportion of the macroschizonts reacting with the monoclonal antibody. This was demonstrated as being due to the strain containing more than one antigenic type of parasite by cloning the infected lymphocyte cell line and showing that the resulting cloned cell lines were antigenically homogeneous. The variation between stocks allowed them to be divided into three groups on the basis of antigenic profile.     

Presence of Theileria annulata in Mauritania

A fatal case of bovine theileriosis by Theileria annulata is reported from Mauritania in a Friesian cow born in a dairy farm in Nouakchott. The farm was originally established with cattle imported from France. Specific diagnosis was based on piroplasm morphology and on high specific antibody titres in some of the animals. The only tick found in the herd was Hyalomma dromedarii. Four of 49 local zebus sampled at random in the south of the country had high specific antibody titres to T. annulata, while 14 had lower or doubtful titres. The infection therefore appears to be autochthonous.     

Comparative pathogenicity of Theileria annulata strains

Twenty three susceptible crossbred calves were inoculated with five Indian strains of Theileria annulata, collected from natural cases of tropical theileriosis at Ludhiana, Hissar, Jaipur, Uruli-Kanchan and Bangalore. All the strains produced clinical disease with typical symptoms and lesions of acute theileriosis. The strains were considered highly virulent and equally pathogenic.     

Comparative pathogenicity of Theileria annulata strains

Summary

Twenty three susceptible crossbred calves were inoculated with five Indian strains of Theileria annulata, collected from natural cases of tropical theileriosis at Ludhiana, Hissar, Jaipur, Uruli‐Kanchan and Bangalore. All the strains produced clinical disease with typical symptoms and lesions of acute theileriosis. The strains were considered highly virulent and equally pathogenic.