The effect of dose and line on immunisation of cattle with lymphoblastoid cells infected with Theileria annulata

Thirty-one Friesian calves in Morocco susceptible to tropical theileriosis were protected against a lethal sporozoite challenge by prior infection with lymphoblastoid cell lines infected and transformed in vitro by a Moroccan stock of Theileria annulata. The challenge infection of cryopreserved sporozoites killed all four susceptible control calves within 20 days. Four schizont-infected cell cultures at Passage 3 were inoculated at four different doses, 10(8), 10(6), 10(4) and 10(2), into pairs of calves. The recipient animals showed great variation in severity of disease symptoms, which did not show a linear correlation with the cell dose inoculated. The most severe disease symptoms were recorded, prior to challenge, in the 10(2) cell dose recipients; one animal died of acute theileriosis and another had to be treated. One of the four cell lines used was more virulent than the other three. Two years after the completion of this experiment, immunised animals have shown normal productivity traits.     

Determination of adenosine deaminase activity in cattle naturally infected with Theileria annulata

The purpose of this study was to determine serum ADA activity in cattle naturally infected with Theileria annulata. In this study, a total of 37 cross-bred cattle which 27 of it showing clinical signs of theileriosis constituted infected group and 10 healthy cattle as control group were used as animal materials. Infected group divided into three groups according to their PCV values. Cattle with PCV > or = 25 were put on group I (n = 9), those with PCV 13-24 were put on group II (n = 11) and those with PCV < or = 12 were put on group III (n = 7). Microscopical diagnosis of the disease was also made. Hematological parameters, serum enzyme activities (ADA, AST, ALT and ALP) were determined in all cattle. Hematological results revealed that significant progressive decreases in HGB, PLT, PBML counts and ratios from group I onwards to group III, whereas the WBC, PBPL counts and ratios showed an increase from group I onwards to group III. The serum ADA, AST, ALT and ALP activity increased significantly in all infected groups compared to control group. However, these parameters were also observed to decrease progressively from group I to group III. Furthermore, the highest increase in enzyme activities observed in the infected group I. But, these enzyme's activities started to decrease in infected group II and III in parallel with PBML and PLT counts. Eventhough, this decrease did not reach to the values obtained from control group. On the contrary, PBPL counts and ratios increased in infected group II and III in contrast to decrease in PCV. As a result, increased serum ADA activity in tropical theileriosis may reflect the involvement of the cellular immune responses.     

Evaluation of antioxidant status and oxidative stress in cattle naturally infected with Theileria annulata

To assess the antioxidant status and oxidative stress in bovine theileriosis due to Theileria annulata blood samples were collected from 35 clinically affected cattle referred to Veterinary Teaching Hospital, School of Veterinary Medicine, Urmia University, Urmia, Iran. Complete blood count, piroplasm parasitemia percentage, erythrocyte glutathione peroxidase, superoxide dismutase, catalase and glucose-6-phosphate dehydrogenase activities, malondialdehyde concentration, osmotic fragility test and median corpuscular fragility were determined and the results were compared with those of 50 healthy controls. Of 35 affected cattle, 12 (34.28%) had severe anemia and 23 had mild to moderate anemia and parasitemia varied from 5 to 40%. The activities of erythrocyte glutathione peroxidase, superoxide dismutase and glucose-6-phosphate dehydrogenase were significantly lower (P<0.0001) and the activity of catalase was significantly higher in the affected cattle than in healthy ones (P<0.001). Malondialdehyde concentration in erythrocytes of affected cattle was significantly more than those of healthy cattle (P<0.001). The affected cattle showed increased fragility of erythrocytes, so that median corpuscular fragility (MCF) in affected group was significantly lower than those of healthy group (P<0.0001). Median corpuscular fragility showed a positive correlation with the severity of parasitemia (r=0.81, P<0.0005) and a negative correlation with the activities of GSH-Px (r=-0.78, P<0.0001), SOD (r=-0.71, P<0.0005), catalase (r=-0.53, P<0.018) and G6PD (r=-0.58, P<0.0005). The results of this study suggest that oxidative damage to RBCs may contribute to the pathogenesis of anemia in bovine theileriosis.     

Haematological profiles in pure bred cattle naturally infected with Theileria annulata in Saudi Arabia

The Abbott Cell Dyn 3500 haematology analyzer was employed to study haematological parameters in 41 adult and young Friesian cattle naturally infected with Theileria annulata in the Qassim region of Saudi Arabia. Comparison was made with clinically healthy adult and young Friesian cattle. Changes in blood parameters in T. annulata-infected cattle indicated severe macrocytic hypochromic anaemia, panleukopenia, lymphocytopenia, eosinopenia, neutropenia and thrombocytopenia but no reticulocytosis.     

Parasitological and clinico-pathological profiles in friesian cattle naturally infected with Theileria annulata in Saudi Arabia

Clinico-pathological profiles were studied in adult and young Friesian cattle naturally infected with Theileria annulata in the Qassim Region, Saudi Arabia. Sixty-two clinical cases of T. annulata infection in adult and young Friesian cattle were diagnosed during the period from August 1999 to July 2000. Symptoms observed were marked fever, swelling of superficial lymph nodes, inappetance, tachycardia, dyspnoea and weakness. The most prominent gross pathological features were jaundice, petechial and ecchymotic haemorrhages involving mucosal and serosal surfaces of many organs as well as body fat. A number of young and adult Friesian cattle undergoing lethal T. annulata infection developed lymphoma-like lesions in a manner similar to that of T. parva. The main histological findings were necrosis and severe lymphocytic infiltration. The spleen, lymph nodes and Peyer's patches were devoid of typical lymph nodules.     

Naturally occurring infections of cattle with Theileria lestoquardi and sheep with Theileria annulata in the Sudan

Theileria annulata is endemic in northern Sudan, hindering all efforts at upgrading cattle for milk production. T. lestoquardi clinical cases occur throughout the year and causes annual outbreaks that result in substantial losses in sheep. In the northern Sudan both cattle and small ruminants are frequently raised together and/or share common grazing grounds at river banks. In an attempt to evaluate field cross infectivity of Theileria lestoquardi and T. annulata in cattle and sheep respectively, a PCR analysis was carried out on samples collected from closely reared sheep and cattle using both T. annulata and T. lestoquardi specific primers. A total of 19 sheep out of 51 (37.3%) were positive for T. lestoquardi while four sheep (7.8%) showed T. annulata specific amplicons. A total of 38 out of 52 (73.1%) surveyed cattle were PCR positive for T. annulata and only two (3.8%) showed T. lestoquardi specific bands. These findings indicate complex epidemiology of both infections in areas where both parasites are transmitted by the same vector and call for further investigations of this phenomenon.     

Preparation of suspensions of Hyalomma excavatum ticks infected with Theileria annulata

Partially engorged adult Hyalomma excavatum ticks infected with Theileria annulata were reared for the preparation of an infective suspension. About 6000 partially engorged infected adults were obtained from 420 non-infective adult ticks. The entire cycle required about 4 months. Six rabbits were used for the production of the fully engorged females. The larvae were fed on 180 gerbils, the nymphs on 17 infected calves and the partially fed adults on 22 goats, 7 sheep and 7 rabbits The adult ticks did not show any host preference.     

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.     

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.     

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.     

Vaccination of calves with an attenuated cell line of Theileria annulata and the sporozoite antigen SPAG-1 produces a synergistic effect

The sporozoite surface antigen, SPAG-1 and the attenuated schizont infected Tunisian line CL1 of Theileria annulata have been shown, in previous studies, to induce variable levels of protection against homologous and heterologous sporozoite challenge, respectively. We report here the result of a vaccination trial comparing the protection level induced by the SPAG-1 antigen (as a recombinant full length His tagged protein) and the attenuated cell line, used singly or in combination. The results, after challenge of immunised calves with a lethal dose of sporozoites, show that SPAG-1 provides limited protection (one out of seven calves surviving), while the attenuated cell line provides moderate protection (three out of seven calves recovered). The combination of SPAG-1 and the attenuated cell line induced the best protection as indicated by the survival of all the vaccinated calves. These results, together with a range of parasitological and clinical parameters, demonstrate the enhanced protection provided by combining sporozoite and schizont antigens in vaccination against tropical theileriosis.     

Chemotherapeutic value of parvaquone and buparvaquone against Theileria annulata infection of cattle

Parvaquone (BW993C), 2-cyclohexyl-3-hydroxy-1,4-naphthoquinone, and buparvaquone (BW720C) 2-(trans-4-t-butylcyclohexyl-methyl)-3-hydroxy-1,4-naphthoquinone, were evaluated to determine their therapeutic efficacy in the treatment of theileriosis caused by Theileria annulata infection in cattle in Iran. One hundred and fifty-nine pure and crossbred Bos taurus cattle, experimentally or naturally infected with T annulata, were treated. Parvaquone was injected into 86 animals with up to three doses of 20 mg kg-1 or 10 mg kg-1 at intervals of 48 hours between doses. Buparvaquone was injected into 73 animals. Up to three doses of 2.5 mg kg-1 were injected with an interval of 48 hours between doses. The recovery rate of animals treated with parvaquone was 60.7 per cent and with buparvaquone it was 88.7 per cent. No significant side effects of relapse of disease were observed following the use of either compound. It is concluded that buparvaquone at a dose of 2.5 mg kg-1 has a satisfactory therapeutic index and is a more effective treatment of T annulata infection than parvaquone. The prophylactic use of schizont tissue culture vaccine and chemotherapy with buparvaquone could be the most promising means of controlling theileriosis in Iran.     

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.     

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.     

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.     

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.