Cell-mediated immune responses to sporozoites and macroschizonts of Theileria annulata.

The nature of cell-mediated immune (CMI) responses was studied in cross-bred bovine calves, immunised by attenuated and allogeneic macroschizonts of Theileria annulata. The CMI responses were also investigated in calves, destined to survive or die of tropical theileriosis (Theileria annulata) induced by a virulent dose of sporozoites or macroschizont-infected lymphoblasts. Calves suffering fatal theileriosis showed poor CMI response. Microcytotoxicity assay revealed an enhanced population of specific cytotoxic cells amongst the peripheral blood lymphocytes (PBL) of calves resolving the infection successfully. The E rosette assay showed proliferation of T cells and the assay for macrophage migration inhibition factor (MIF) demonstrated antigen sensitised cells in the PBL. Calves, immunised by allogeneic and attenuated macroschizont-infected lymphoblasts or those recovering from virulent macroschizont-induced infection, showed protective CMI responses with patterns similar to those appearing after non-fatal sporozoite infection.     

Nitric oxide causes the macroschizonts of Theileria annulata to disappear and host cells to become apoptotic

The proliferation of Theileria annulata macroschizont-infected cell lines in vitro was significantly inhibited by nitric oxide (NO) generated by S-nitroso-N-acetyl-DL-penicillamine (SNAP). Incubation with SNAP caused the macroschizonts to disappear and host cells to become apoptotic. SNAP-derived NO also significantly inhibited the incorporation of tritiated thymidine by cultures of cells in which the schizonts had been induced to differentiate into merozoites by maintenance at 41°C instead of 37°C, the temperature used for culturing macroschizont-infected cells. These results point to NO as the mediator of macrophage anti-T. annulata activity and provide new evidence that the protective immune mechanisms which allow cattle to recover from primary infection and resist challenge may be attributed principally to the products of activated macrophages. These findings indicate that effective inactivated vaccines against T. annulata should include antigens able to stimulate the type of CD4+ T cell response which elicits macrophage activation and NO synthesis.     

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.     

群牛暴发牛泰勒虫病的诊治

牛泰勒虫病在南方地区较为罕见。2001年7月，湖南省衡阳县樟树乡某村牛场所养的93头牛暴发牛泰勒虫病，经笔者诊治及时控制了疫情，现报告如下：     

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.     

Investigation of lectin activity in Theileria annulata piroplasms

Adhesion to target cells is an essential step in the pathogenesis of many protozoal infections. Some protozoa have been reported to have a lectin activity involved in their attachment to the cell surface. The ligand-receptor interaction involved in Theileria annulata infection is unclear at present, in spite of the fact that some aspects of the process have been investigated. To this end, T. annulata piroplasms have been screened for lectin activity. Blood taken from infected cattle was first depleted of leukocytes and then subjected to ammonium chloride lysis in order to isolate the piroplasms. The piroplasms were homogenised and a crude membrane extract was prepared by centrifugation. To investigate lectin activity in piroplasm proteins, a simple screening procedure was employed for analysing piroplasm proteins binding to various lectin ligands. Numerous immobilised lectin ligands (L-fucose-sepharose, N-acetyl-neuraminic acid-sepharose, N-acetyl-D-galactosamine-agarose, N-acetyl-D-glucosamine-agarose, D-mannose-agarose, beta-D-glucose-agarose, alpha-methyl-D-mannoside-agarose) were incubated with T. annulata piroplasm crude membrane extract. The ligand-bound proteins were eluted and separated by a brief centrifugation and determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The present study suggests that a 32 kDa protein of piroplasm binds to D-galactosyl residues of the agarose matrix and that the binding is inhibited by galactose and not by the other monosaccharides tested.     

Antigenic diversity of Theileria major piroplasm surface protein gene in Jeju black cattle

Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B.     

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.     

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.     

Cross-reaction among four isolates of Theileria annulata from India

A close identity in virulence and cross-protection of four isolates of Theileria annulata was observed when infection was produced in naive, crossbred (Bos taurus male X B. indicus female) male calves. The evidence that strains of T. annulata in India differ in virulence and immunogenicity is equivocal at present.     

牛环形泰勒焦虫裂殖体胶冻细胞苗应用效果观察

河南省桐柏县是牛环形泰勒虫病流行的多发区,对养牛业危害十分严重,1985年该县从宁夏农林科学院畜牧兽医研究所引进“虫苗”2万头份,在区域试验的基础上在全县16个乡（镇）进行了大面积防疫注射,经年终统计表明：虫苗安全性100%,注苗15000头（其中有水牛45000头）,发病3头,发病率0.02%;未注苗36000头,发病2060头,发病率5.7%,实际有效保护率达99.98%,取得了明显的社会经济效益。     

Stage-specific antigenicity in Theileria annulata: a case report

Blood from sick cattle in Bahrain transmitted piroplasms of Theileria annulata to a splenectomized calf. Larvae of Hyalomma anatolicum anatolicum were infected on the calf and, after moulting, induced clinical theileriosis, associated with numerous schizonts, in the same calf. The animal was cured by specific treatment. Antigenic differences thus shown between piroplasms on the one hand, and sporozoites and schizonts on the other hand, were confirmed in the indirect fluorescent antibody test, as a significant titre to T. annulata piroplasm antigen developed after the inoculation of blood, but to schizont antigen only after the infective ticks had induced the appearance of schizonts.     

Treatment of Theileria annulata infection in calves with parvaquone

Fifteen calves were infected by the injection of stabilate of a suspension of Hyalomma anatolicum anatolicum ticks infected with the Ankara strain of Theileria annulata. Three were kept untreated, as controls, and they all died of theileriosis. Three groups of four calves were treated intramuscularly with parvaquone (Clexon; Wellcome) when early signs of theileriosis were clinically apparent. One group received 20 mg (kg bodyweight)-1 of parvaquone 10 days after infection. Two of these calves were clinically cured and two died of theileriosis. The remaining two groups of four calves received two doses of parvaquone, each of 10 mg (kg bodyweight)-1, either on days 10 and 11 or days 10 and 12. Three calves in each group were clinically cured while one in each group died of theileriosis. Total parasitological cure was not achieved in any of the calves. No symptoms of toxicity due to parvaquone treatment were observed.     

牛环形泰勒虫enolase基因的原核表达及生物信息学分析

【目的】获得牛环形泰勒虫(Theileria annulata)新疆株enolase基因,并分析其生物学特性及反应原性。【方法】对牛环形泰勒虫enolase基因进行扩增和克隆,构建原核表达载体pGEX-4T-1-enolase,诱导表达enolase重组蛋白并进行蛋白纯化,通过Western blotting验证enolase重组蛋白反应原性。利用生物信息学方法对enolase基因编码蛋白的理化性质、亲疏水性、跨膜区、信号肽、磷酸化、亚细胞定位及蛋白互作网络进行预测分析。【结果】PCR扩增出大小为1 248 bp的牛环形泰勒虫enolase基因片段,enolase重组蛋白大小约70 ku; Western blotting结果表明,该重组蛋白与牛环形泰勒虫阳性血清发生反应。enolase基因编码416个氨基酸,理论等电点为5.91,有38个磷酸化位点;二级结构主要由α-螺旋(43.03%)和无规则卷曲(33.17%)组成;具有17个B细胞抗原表位,亚细胞定位主要位于细胞质中;enolase蛋白与磷酸甘油酸变位酶(PGAM)、二磷酸核苷激酶(NDK)、磷酸甘油酸激酶(PGK)、热休克蛋白...