Attempts to produce Theileria parva-infected mouse cells using cell fusion techniques.

Ehrlich ascites tumour (EAT) cells were fused with Theileria parva-infected bovine lymphoid (C2) cells using inactivated Sendai virus. A variety of techniques were employed to try and increase fusion percentage and harvest of heterokaryons. There was no evidence that pre-exposure of C2 cells to Sendai virus or phytohaemagglutinin improved fusion percentage, but the percentage was higher when 10(8) C2 cells were fused with 10(7) EAT cells, as compared with a ratio of 10(7) C2: 10(6) EAT. An increased yield of multinucleate cells was obtained using a calf serum gradient to separate cells of different sizes. An attempt to fuse EAT cells with free macroschizonts was not sucessful. When cells were grown in hypoxanthine/aminopterin/thymidine medium (HAT), T parva appeared to be selectively killed by the aminopterin present; this finding would seem to militate against the use of a HAT selection system to clone parasitised hybrids. C2 cells could be selected out by passage of mixed cultures through mice, but no evidence of parasitosis was detected in EAT cells which became established.     

Acquired methemoglobinemia in anemic cattle infected with Theileria sergenti.

To investigate the mechanism of anemia accompanying Japanese bovine theileriosis, we examined whether production of methemoglobin (MetHB), an indicator of erythrocyte oxidation, was associated with anemia in cattle experimentally infected with Theileria sergenti. The percentage of MetHB, which is an oxidized form of hemoglobin, increased according to the onset of anemia. During severe anemia, high levels of acquired methemoglobinemia were observed in all infected cattle. A significant correlation (r=-0.649; P<0.01) between an increase in MetHB concentration and a decrease in packed cell volume (PCV) was observed. It was considered that hemoglobin oxidation may be one of the aggravating factors of anemia in T. sergenti infection.     

Abnormality of osmotic fragility and morphological disorder of bovine erythrocytes infected with Theileria sergenti

The osmotic fragility and the surface structure of erythrocytes obtained from 3 calves infected with Theileria sergenti and from 3 phlebotomized ones were compared. As the parasitemia progressed, the osmotic fragility of the erythrocytes significantly increased in the infected calves. Particularly the hemolysis ratio in the isotonic area (21.5-94.1%) obviously increased. On the other hand, the percentage of parasitized cells in the erythrocytes did not show so much high values (16.1-21.3%). Similar phenomenon was found in each different percentage of erythrocytes suspension which was separated from density gradient centrifugation. No significant difference in the serum osmotic pressure between the infected calves and the phlebotomized calves was found. By scanning microscopy, the erythrocytes of infected calves, which were collected at the crisis period of parasitemia, were almost completely deformed and showed echinocyte form. Moreover, the appearance ratio of echinocyte form in the erythrocytes population was superior to the percentage of parasitized erythrocytes. Similar membranous alterations were also observed in the erythrocytes of grazing cattle in the crisis period of the theileriosis. It was proven that abnormality of osmotic fragility and morphological disorders of erythrocytes occurred not only in parasitized erythrocytes but also in non-parasitized ones in T. sergenti parasitemia.     

Theileria mutans in Nigeria.

Two strains of bovine Theileria from northern Nigeria were shown to be identical to Theileria mutans in the indirect immunofluorescent antibody test. One of the strains was transmitted by the tick Amblyomma variegatum; large macroschizonts, typical for T mutans, could be demonstrated in infected cattle. It is concluded from these experiments and from the literature that there is reliable evidence so far for the occurrence in Nigeria of only two bovine Tehileria species, T mutans and T velifera.     

Cultivation of Theileria. I. Attempts to complete the cycle of Theileria parva in vitro

Microschizonts and free merozoites developed in bovine lymphoblastoid cell cultures containing macroschizonts of 6 different strains of Theileria parva. Clean bovine red cells were added to the cultures, which were incubated in various ways. No penetration of red cells by merozoites was observed, not even when cultures in diffusion chambers were introduced into the peritoneal cavity of non-infected cattle.     

Erythrocytic forms of Theileria velifera.

The enigmatic veil associated with the cattle parasite Haematoxenus veliferus has been shown to be crystallike in ultrastructure and to stain with benzidine like haemoglobin. The ultrastructure of the organism is similar to that of other theilerial parasites. As less distinct veil-like structures are known to occur with some other Theileria species, and in one case, have also been shown to be crystalline in structure, it is proposed to sink the genus Haematoxenus as a synonym of Theileria.     

环形泰勒焦虫对奶牛免疫水平的影响

本试验选择5头感染环形泰勒焦虫的奶牛和5头健康奶牛,在第0天、第5天、第10天、第15天、第20天分别进行红细胞C3b受体花环率试验、红细胞免疫复合物花环试验、T淋巴细胞EA花环率试验和淋巴细胞转化率的测定。结果表明,感染环形泰勒焦虫的奶牛其红细胞免疫复合物花环、红细胞C3b受体花环率和淋巴细胞转化率明显低于健康奶牛,差异极显著。这说明环形泰勒焦虫对奶牛的免疫水平有明显的抑制作用。且随着检测时间的变化,免疫水平逐渐降低。     

Detection and characterization of Theileria sergenti proteinases.

The lysate of Theileria sergenti piroplasms was tested for proteinases using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which substrate was included in gel matrix. Six proteinases of molecular weight 330, 125, 98, 94, 67 and 58 kilodalton (kDa) were detected. From the results of the Triton X-114 phase partition, 330, 125 and 58 kDa proteinases were partitioned into aqueous phase, which indicated that they were not associated with parasite membranes. All these three enzymes were classified into metalloproteinase family because of their sensitivities to metal-ion chelating compounds, ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline. On the other hand, 98 and 94 kDa proteinases were membrane-associated metalloproteinases which were preferentially inhibited by 1,10-phenanthroline. Another metalloproteinase of 67 kDa which was inhibited by EDTA and 1,10-phenanthroline was not associated with parasite membranes. Proteinases of 98 and 94 kDa degraded heat-denatured hemoglobin.     

The serological relationship of a British Theileria with other Theileria species using the indirect fluorescent antibody test.

Antisera and antigens of a Theileria species isolated from British cattle were compared with those of six strains of Theileria mutans from geographically separated areas in East and South Africa, using the indirect fluorescent antibody (IFA) test. It was found that the British Theileria species did not react in the IFA test with these strains of T mutans. The British Theileria species was also compared with a variety of other Theileria species using the IFA test and no reactions were detected. It was concluded that the British Theileria species, which has been designated T mutans Essex, could be distinct from African T mutans.     

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.     

Establishment of optimal conditions for long-term culture of erythrocytic stages of Theileria uilenbergi

OBJECTIVE: To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi. SAMPLE POPULATION: Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi. PROCEDURES: Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled. RESULTS: The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%. CONCLUSIONS AND CLINICAL RELEVANCE: Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.     

Studies on Some Blood Parameters of Crossbred Calves with Experimental Theileria annulata Infections

Subcutaneous inoculation of 1 ml of ground Theileria annulata tick tissue stabilate (0.75 tick equivalent) into crossbred calves (n = 6, average age 53 days) resulted in the development of acute theileriosis. The percentage parasitaemia was 71.7%±3.3% on day 20 after inoculation. Macroschizonts were observed in lymphocytes and monocytes. Phagocytosed schizonts were observed in neutrophils, along with cytoplasmic vacuolation in monocytes and neutrophils. There was progressive decrease (p<0.05) in the haemoglobin and packed cell volume, along with a marked reticulocytosis. Serum analysis revealed a decrease (p<0.05) in the concentrations of calcium, cholesterol and triglycerides, while there was an increase (p<0.05) in the concentrations of blood urea nitrogen as compared to day 0 values. The total serum proteins, albumin and serum immunoglobulin concentrations and the albumin-to-immunoglobulin ratio showed marked decreases (p<0.05). Coagulopathies included thrombocytopenia and an increased prothrombin time, along with a non-significant increase in the bleeding time and activated partial thromboplastin time during the terminal stages of the disease. There was an increase in the osmotic fragility of erythrocytes during the disease. Morphological alterations in the erythrocytes were observed with the developing parasitaemia.     

Cloning of a cysteine proteinase gene of Theileria sergenti.

A cDNA encoding cysteine proteinase of Theileria sergenti was isolated from a piroplasm cDNA library and its nucleotide sequence was determined. The gene encodes a polypeptide of 402 amino acids with predicted molecular mass of 46.4 kDa. Analysis of the predicted amino acid sequence revealed a number of features common to known cysteine proteinases. Southern blot analysis showed that the cysteine proteinase gene was likely to be a single copy per genome.     

Theileria orientalis in Iran

A benign species of Theileria of cattle in northern Iran proved to be indistinguishable from T. orientalis in the indirect fluorescent antibody test as well as in the morphology of its piroplasms. It was transmissible transstadially by Haemaphysalis punctata.     

Infectivity and cross-immunity studies of Theileria lestoquardi and Theileria annulata in sheep and cattle: I. In vivo responses.

In a series of experiments, sporozoite stabilates of a Theileria lestoquardi (Lahr) and a T. annulata (Ankara) stock prepared from Hyalomma anatolicum anatolicum ticks, were used to examine the infectivity of both parasite species for sheep and cattle and to study the development of cross-immunity between these parasite species. In the first experiment sheep and cattle were inoculated with T. lestoquardi sporozoites. Surviving animals and naive sheep and cattle were, in the second experiment, inoculated with T. annulata. In the third experiment, naive sheep and sheep previously infected with T. annulata, were inoculated with T. lestoquardi. The following responses to inoculations were monitored: clinical and haematological signs of infection, appearance of parasitic stages of the parasites in lymph node biopsies and in peripheral blood and serological response to T. lestoquardi and T. annulata schizont antigens. While T. lestoquardi readily infected sheep and caused severe disease, it did not infect cattle. On the other hand, T. annulata infected both cattle and sheep. However, whereas cattle became severely affected, infected sheep showed mild clinical symptoms only and piroplasms did not develop. Despite their different behaviour in the host species examined, cross-immunity studies suggested that the parasite species are very closely related. Experiments in sheep indicated that T. lestoquardi infection protected against subsequent T. annulata infection. On the other hand, recovery from T. annulata infection did not prevent infection by sporozoites of T. lestoquardi, resulting in the establishment of schizonts and their subsequent development into piroplasms, although it protected against the major clinical effects of T. lestoquardi infection.     

Theileria taurotragi in Zambia

Summary

Theileria sp. (Bwengwa) of low virulence was isolated by feeding R. appendiculatus ticks collected from the field on a susceptible calf and subsequently transmitted between cattle by R. appendiculatus ticks‐ Theileria sp. (Bwengwa) was shown to be T. taurotragi on parasitological, clinical and serological grounds. T. taurotragi is the fourth Theileria sp. shown to be present in Zambia.     

Theileria taurotragi in Zambia

Theileria sp. (Bwengwa) of low virulence was isolated by feeding R. appendiculatus ticks collected from the field on a susceptible calf and subsequently transmitted between cattle by R. appendiculatus ticks- Theileria sp. (Bwengwa) was shown to be T. taurotragi on parasitological, clinical and serological grounds. T. taurotragi is the fourth Theileria sp. shown to be present in Zambia.     

Exposure of cattle immunized with different stocks of Theileria parva to buffalo-associated Theileria challenge on two game parks in Zimbabwe.

Eight cattle immunized with cattle-derived Theileria parva Boleni stabilate together with six susceptible controls were released in Dombawera Game Park on the Highveld of Zimbabwe. This coincided with Rhipicephalus appendiculatus nymphal activity. The cattle grazed together with African buffaloes (Syncerus caffer) and were not treated against tick infestation. The nymphal tick infestation was high, and seven of the eight immunized cattle and three of the controls had severe and fatal reactions. Subsequently, two stocks of Theileria parva to be tested for their immunizing abilities were prepared-one from adult ticks which were fed as nymphs on one of the sick control animals (Dom 268) and the other from adult ticks collected from pastures grazed by buffaloes (Bv-1). Two groups of cattle were immunized with either the Dom 268-derived strain (eight animals) or the Bv-1-derived strain (four animals). These together with three non-immunized controls, were released in Bally Vaughaun Game Park in the Highveld, where buffaloes are present, during the season of nymphal tick activity. A third group of five cattle, immunized with stabilate Bv-1, and three non-immunized controls were released at the same site during the season of adult tick activity. The nymphal and adult tick infestations of the cattle were large and more than 2000 nymphs and 1000 adult ticks were counted per animal. Cattle were treated with a pyrethroid pour-on preparation to control the tick infestation and screw-worm strike. The immunized cattle in the three groups survived the theileriosis challenge for a period of 18 months, but the non-immunized control cattle suffered a severe and fatal theileriosis 19-23 days after being placed on the pasture.