Browsing by Author "Brown, C.G.D."
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Item Adaptation and Possible Attenuation of Theileria Parva-Infected Cells Grown in Irradiated Mice(1976) Irvin, A.D.; Brown, C.G.D.; Stagg, D.A.; Kanhai, G.K.; Kimber, C.D.; Radley, D.E.; East African Veterinary Research Organisation, Muguga, P.O. Box 32, Kikuyu, KenyaTheileria parva-infected bovine lymphoid cells were taken from 8 cattle immediately after death from East Coast fever (ECF). Cells were inoculated into groups of irradiated Swiss and athymic nude mice. Cells became established in one group of Swiss mice and 2 groups of athymic mice. Development of cells in mice only occurred if cells concurrently established in culture; when establishment in culture was delayed, cells failed to develop in mice. Cells from one of the isolates in athymic mice were passaged 6 times through further mice. On inoculation of these mouse-passaged cells into cattle, the animals underwent mild reactions and subsequently resisted a lethal ECF challenge. The possibility of vaccinating cattle against ECF by means of mouse passaged cells merits further study.Item The Application of an Indirect Method of Infecting Ticks with Piroplasms for Use in the Isolation of Field Infections(1974) Cunninggham, M.P.; Purnel, R.E.; Ledger, M.A.; Irvin, A.D.; Brown, C.G.D.; Pyne, R.C.; East African Veterinary Research OrganizationNymphal R. appendiculatus ticks became infected with Theileria parva following the intraperitoneal inoculation of fresh or refrigerated infected bovine blood into rabbits on which the ticks were feeding. The method was successfully used in the isolation of field infections and as blood is the only material needed from such cases the method can be readily and conveniently applied in the isolation of different field strains of T. parva and possibly of similar blood-borne protozoal diseases.Item Attempted Transmission of Theileria Gorgonis, Brocklesby & Vidler 1961, From Blue Wildebeest (Connochaetes Taurinus) to Cattle(1973) Purnell, R.E.; Schiemann, B.; Brown, C.G.D.; Irvin, A.D.; Ledger, M.A.; Payne, R.C.; Young, A.S.; East African Veterinary Research Organization, Muguga, KenyaTwo blue wildebeest (Connochaetes taurinus) from the Serengeti National Park in Tanzania and two from Kenya were splenectomised. Of the two from Tanzania, one that had previously had detectable intra-erythrocytic piroplasms of Theileria gorgonis showed an increase in parasitaemia (percentage of erythrocytes infected) to 7%, and the other, which had had no detectable parasites, developed a low-level parasitaemia. The two animals from Kenya had severely exacerbated infections with T. gorgonis and in one case a concomitant infection with Anaplasma marginale. They developed Theileria parasitaemias of more than 70% and died. Attempts to transmit T. gorgonis to steers by means of adults of Rhipicephalus appendiculatus Neum. that had fed as nymphs on infected wildebeest were unsuccessful, although it was shown that the parasite became established in the ticks. Some 500 ticks were used on each steer.Item Attempts to Grow Tissue Culture Cells, Infected with Theileria Parva, in Laboratory Animals(1972) Irvin, A.D.; Brown, C.G.D.; Crawford, J.G.; East African Veterinary Research Organization, Muguga, P.O. Kabete, KenyaTransformed bovine lymphoid cells infected with macroschizonts of Theileria parva were inoculated into normal mice and newborn mice by the subcutaneous route and also into hamster cheek pouches and the anterior chamber of rabbits' eyes. There was no evidence of growth or persistence of parasitized cells in any of the systems, nor were tumour-like masses formed. This contrasts with previous work when inoculation of similar material into whole-body irradiated mice resulted in persistence of parasitized cells and the formation of tumour-like masses.Item Attempts to Grow Tissue Culture Cells, Infected with Theileria parva, in Laboratory Animals(1972) Irvin, A.D.; Brown, C.G.D.; Crawford, J.G.Transformed bovine lymphoid cells injected with macroschizonts of Theileria parva were inoculated into normal mice and newborn mice by the subcutaneous route and also into hamster check pouches and the anterior chamber of rabbits’ eyes. There was no evidence of growth or persistence of parasitized cells in any of the systems, nor were tumour-like masses formed. This contrasts with previous work when inoculation of similar material into whole-body irradiated mice resulted in persistence of parasitized cells and the formation of tumour-like masses.Item Autoradiographic Evidence for the Occurrence of Cell Fusion in Cultures of Theileria-Infected Bovine Lymphoid Cells(1974) Irvin, A.D.; Brown, C.G.D.; Boarer, C.D.H.; Crawford, J.G.; Kanhai, G.K.; East African Veterinary Research Organization, Muguga, Kabete, KenyaTheileria parva-infected bovine lymphoid cells, grown in tissue culture, were labelled with tritiated thymidine. Aliquots of this culture were added to equal volumes of unlabelled cultures of T. parva and T. lawrencei. Smears were prepared from the pure and mixed cultures and examined by autoradiography. Virtually all the cells in the pure labelled cultures had taken tip the tritiated thymidine, and in many cells theilerial macroschizonts were also labelled. Some of the multinucleate cells in mixed cultures appeared to be the product of cell fusion since they contained both labelled and unlabelled nuclei.Item Autoradiographic Evidence for the Occurrence of Cell Fusion in Cultures of Theileria-Infected Bovine Lymphoid Cells(1974) Irvin, A.D.; Brown, C.G.D.; Boarer, C.D.H.; Crawford, J.G.; Kanhai, G.K.Theileria parva-infected bovine lymphoid cells, grown in tissue culture, were labelled with tritiated thymidine. Aliquots of this culture were added to equal volumes of unlabelled cultures of T. parva and T. lawrencei. Smears were prepared from the pure and mixed cultures and examined by autoradiography. Virtually all the cells in the pure labelled cultures had taken up the tritiated thymidine, and in many cells theilerial macroschizonts were also labelled. Some of the multinucleate cells in mixed cultures appeared to be the product of cell fusion since they contained both labelled and unlabelled nuclei.Item Cell Fusion Using Sendai Virus to Effect Interspecies Transfer of Cell Associated Parasite(1974) Irvin, A.D.; Brown, C.G.D.; Kanhai,G. K.; Stagg, D.A.; Rowe, L.W.; Institute for Research on Animal Diseases, Compton, United Kingdom; FAO ; Central Veterinary Laboratory, United Kingdom; Central Veterinary LaboratoryBaby hamster kidney (BHK) cells were fused with Theileria parva-infecled bovine lymphoid cells, using u.v. light-inactivated Sendai virus. The resultant hamster/bovine heterokaryons were shown to be infected with T. parva. In some cases parasites were detected in cells which apparently contained only BHK nuclei.Item Cell Fusion, using Sendai virus, to Effect Inter-Species Transfer of a Cell-Associated Parasite (Theileria Parva).(1974) Stagg, D.A.; Brown, C.G.D.; Rowes, L.W.; Irvin, A.D.; Kanhal, G.K.; UNDP/FAO Tick-borne Diseases Project: East African Veterinary Research OrganizationThe production of interspecific heterokaryons by virus-induced cell fusion was first performed by Harris & Watkins (1965). Since that time the technique has been widely used in a variety of fields but its application in the field of parasitology has not so far been exploited. We have used the technique to effect inter-species, transfer of an intra-cellular host specific parasite (Theileria parva) the causative organism of East Coast fever of cattle.Item Comparative Infectivity for Cattle of Stabilates of Theileria Lawrencei (Serengeti) Derived from Adult and Nymphal Ticks(1974) Purnell, R.E.; Young, A.S.; Brown, C.G.D.; Burridge, M.J.; Payne, R.C. ; East African Veterinary Research Organization P.O Box 32, Kikuyu KenyaLarval and nymphal Rhipicephalus appendiculatus ticks were fed simultaneously on steers reacting to the inoculation of an infective stabilate of cattle-passaged Theileria lawrencei (Serengeti). The larvae and nymphs dropped engorged from the steers when their intra-erythrocytic piroplasm parasitaemias were in excess of 14 per cent.: after the ticks had moulted, they were fed on rabbits and removed after 3 and 5 days respectively. Some of the ticks were processed for histological examination of their salivary glands whilst others were ground and stabilates were prepared from them. Examination of the salivary glands indicated that there was a higher infection rate in the nymphs than in the adults although this was not statistically significant: subsequent inoculation into susceptible cattle of titrated aliquots of the stabilates, however, showed that aliquots from a certain number of adult ticks were more infective than those from the equivalent number of nymphal ticks. The duration of the prepatent period in the susceptible cattle could be correlated with the infective dose. The possibility of obtaining stabilates of nymphal material by feeding larval ticks all over the body of an infected animal is discussed, since such stabilates may be more concentrated and more easily prepared than adult tick stabilates.Item Comparative Infectivity for Cattle of Stabilates of Theileria Lawrencei (Serengeti) Derived from Adult and Nymphal Ticks(1974) Purnell, R.E.; Young, A.S.; Brown, C.G.D.; Burridge, M.J.; Payne, R.C.; East African Veterinary Research Organization, P.O. Box 32, Kikuyu, KenyaLarval and nymphal Rhipicephalus appendiculatus ticks were fed simultaneously on steers reacting to the inoculation of an infective stabilate of cattle-passaged Theileria lawrencei (Serengeti). The larvae and nymphs dropped engorged from the steers when their intra-erythrocytic piroplasm parasitaemias were in excess of 14 per cent.: after the ticks had moulted, they were fed on rabbits and removed after 3 and 5 days respectively. Some of the ticks were processed for histological examination of their salivary glands whilst others were ground and stabilates were prepared from them. Examination of the salivary glands indicated that there was a higher infection rate in the nymphs than in the adults although this was not statistically significant: subsequent inoculation into susceptible cattle of titrated aliquots of the stabilates, however, showed that aliquots from a certain number of adult ticks were more infective than those from the equivalent number of nymphal ticks. The duration of the prepatent period in the susceptible cattle could be correlated with the infective dose. The possibility of obtaining stabilates of nymphal material by feeding larval ticks all over the body of an infected animal is discussed, since such stabilates may be more concentrated and more easily prepared than adult tick stabilates.Item East Coast Fever of Cattle: 60 Co Irradiation of Infective Particles of Theileria Parva(1973) Purnell, R.E.; Brown, C.G.D.; Cunningham, M.P.; Burridge, M.J.; Musoke, A.J.; Dargie, J.D.; East African Veterinary Research Organization, Muguga, P. O. Kabete, Kenya. Faculty of Veterinary Science, University of Nairobi, Nairobi, KenyaInfective particles (IPs) of Theileria parva, the causative organism of East Coast Fever of cattle, were harvested from the tick vector, Rhipicephalus appendiculatus, using an in vitro feeding technic. In a ranging experiment, pairs of cattle were inoculated with aliquots of suspensions of IPs irradiated at doses of 4–137.6 krad. Doses of irradiation in excess of 8 krad appeared to destroy the parasite. In the 2nd and 3rd experiments, groups of 5 cattle were inoculated with aliquots of suspensions containing low and high concentrations of IPs respectively, irradiated at doses of 4–32 krad. In the 2nd experiment, doses of irradiation in excess of 10 krad appeared to destroy the parasite. In the 3rd experiment, at least 1 animal became infected when inoculated with an aliquot of a suspension irradiated at 16 krad. In all experiments, it appeared that increasing doses of irradiation destroyed increasing numbers of IPs. There was no conclusive evidence that IPs which survived irradiation were attenuated, and it appears that vaccination of cattle against ECF is unlikely to be achieved by inoculation of irradiated IPs using the methods described.Item East Coast Fever: 3. Chemoprophylactic Immunization of Cattle using Oxytetracycline and a Combination of Theilerial Strains(1975) Radley, D.E.; Brown, C.G.D.; Cunningham, M.P.; Kimber, C.D.; Musisi, F.L.; Payne, R.C.; Purnell, R.E.; Stagg, S.M.; Young, A.S.; East African Veterinary Research Organization, Kikuyu Kenya,In the first of two experiments performed, 29 animals were immunized by chemoprophylaxis against either a combination of three theilerial strains, Theileria parva (Muguga), T. parva (Kiambu 5) and T. lawrencei (Serengeti transformed), or against one or two elements of the combination separately, and then challenged with either T. parva (Kiambu 1) or T. lawrencei (Solio KB1), both of which were known to be heterologous to T. parva (Muguga). The animals immunized against the combination had mild or inapparent reactions to challenge, whilst the susceptible control cattle and many of those immunized against one or two strains either had severe reactions of died. In the second experiment, 17 cattle immunized by chemoprophylaxis against the combination were challenged with lethal doses of three theilerial strains, T. parva (Entebbe 1), T. parva (Entebbe 2) and T. parva (Ukunda), recently isolated from two widely separated areas of East Africa. They had mild or inapparent reactions whilst 13 of 15 susceptible control cattle died. Chemoprophylaxis using a combination of theilerial strains and a limited oxytetracycline regimen was shown to be an effective means of immunizing cattle against East African theilerioses. The use of this technique as a method of immunizing cattle before exposure in the field is proposed.Item East Coast Fever: 60Co-Irradiation of Theileria Parva in Its Tick Vector, Rhipicephalus Appendiculatus(1974) Purnell, R.E.; Brown, C.G.D.; Burridge, M.J.; Cunningham, M.P.; Emu, H.; Irvin, A.D.; Ledger, M.A.; Njuguna, L.M.; Payne, R.C.; Radley, D.E.; East African Veterinary Research Organization, Muguga P.O. Box 32, Kikuyu, KenyaThree experiments were carried out in which Theileria parva was irradiated in its tick vector, Rhipicephalus appendiculatus. In the first experiment, infected unfed adult ticks were irradiated at doubling doses from 4 to 32 krad. Some of the ticks were then fed for 4, 5, 6, 7 and 8 days on rabbits, and the parasites in their salivary glands examined. Five male and 5 female ticks from each irradiation dose were put onto each of a pair of susceptible cattle, whose reactions were recorded. Increasing doses of irradiation resulted in progressive destruction of the parasites. All cattle receiving ticks irradiated at doses up to and including 16 krad died of East Coast fever (ECF), and one of the cattle receiving ticks irradiated at 32 krad died.Item East Coast Fever: Challenge if Immunised Cattle by Prolonged Exposure to Infected Ticks(1975) Radley, D.E.; Brown, C.G.D.; Cunnigham, M.P.; Kimber, C.D.; Musisi, F.L.; Purnell, R.E.; Stagg, S.M.; East African Veterinary Research Organization, Muguga, PO Box 32, Kikuyu, KenyaBos taurus steers, previously treated with acaricide but washed before the trial commenced, were introduced sequentially to a paddock with a high level of T. parva infected ticks. The 12 animals exhibited macroschizonts in lymph nodes at 7-12 days, a febrile response at 9-13 days, and died at 14-21 days. Tick counts were highest on ears (the maximum level was 330), and the build-up of tick attachment correlated with time to death. Four steers immunized three months previously with tick-derived T. parva stabilate material plus Terramycin ECF were kept in the paddock throughout the experiment (60 days). Prepatent period was 10-15 days, but despite high levels of tick attachment (peaks of 500 on ears), there were minimal symptoms and only a slight transient rise in antibody titre on day 40. A few macroschizonts were detected in lymph nodes during the first week of infection and they reappeared from days 34-38. A few piroplasms were seen intermittently in blood smears.Item East Coast fever: correlation between the morphology and infectivity of Theileria parva developing in its tick vector(1973) Purnell, R.E.; Brown, C.G.D.; Cunningham, M.P.; Burridge, M.J.; Kirimi, L.M.; Ledger, M.A.Rhipicephalus appendiculatus ticks, infected with Theileria parva, were fed on rabbits and removed at daily intervals for 9 days. Half of the ticks removed were dissected and their salivary glands processed and examined for parasites. The remaining ticks which had been removed were ground up and aliquots of the supernatant fluid thus obtained were injected into East Coast fever susceptible cattle. Cattle injected with supernatant fluids from ticks fed for 4-9 days became infected, and this could be correlated with the histological detection of mature parasites in the corresponding ticks. The most highly ineffective supernatant fluids were prepared from 5-day-fed ticks. In a second experiment it was shown that female ticks of a variety of weights contained ineffective particles of T.parva.Item East Coast Fever: The Infectivity for Cattle of Infective Particles of Theileria Parva Harvested in Various Substrates(1973) Cunningham, M.P.; Brown, C.G.D.; Burridge, M.J.; Joyner, L.P.; Purnel, R.E.; East African Veterinary Research Organization, Muguga, P.O. Kabete, KenyaFemale Rhipicephalus appendiculatus ticks infected with Theileria parva were pre-fed on rabbits for 4 days before being removed and restrained on ‘plasticine’. They were then fed for 2-h periods on capillary tubes containing various substrates, the contents of which were subsequently inoculated into East Coast fever-susceptible cattle. Using this technique, precolostral calf serum and Eagle's Minimum Essential Medium, with the addition of Bovine Albumin Powder, were selected as substrates suitable for further laboratory investigation on East Coast fever as they were acceptable to the ticks and supported the viability of the parasites.Item Establishment of Theileria Parva Infected Bovine Tissue Culture in Swiss and Athymic Mice(1977) Irvin, A.D.; Brown, C.G.D.; Stagg, D.A.; East African Veterinary Research Organisation; Institute of Research on Animal Diseases; Central Veterinary LaboratoryTheileria parva-infected bovine lymphoid cells, grown in culture, were inoculated by different routes into neonatal and adult Swiss mice immunosuppressed by irradiation, thymectomy or inoculation of anti-lymphocyte serum. Tumour-like masses, composed of parasitized bovine lymphoid cells, formed at the site of subcutaneous inoculation in immunosuppressed neonatal and adult mice, but consistent establishment of cells following intra-peritoneal inoculation occurred only in neonatal mice. In all cases the degree of cellular establishment was proportional to the degree of immunosuppression. The best “take” was in irradiated neonatally thymectomized mice. Cells underwent short-term multiplication in mice but, as immune competence returned, the cells were rejected. There was no evidence that cells, on passage, became more adapted to grow in mice, nor that mouse cells became parasitized. Culture-derived cells were also inoculated subcutaneously into irradiated and non-irradiated nu/nu, nu/+ and Swiss mice. Tumour-like masses, composed of parasitized bovine lymphoid cells, developed at the site of inoculation in all irradiated mice. In nu/+ and Swiss mice these masses regressed after 2–3 weeks, but in the athymic nu/nu mice there was generally no rejection or cellular degeneration and parasitized cells became widely disseminated in the host's tissues and organs, in some cases causing death. T. parva-infected cells could not be established in non-irradiated nu/nu mice, nor when irradiated nu/nu mice were inoculated by the intra-peritoneal route. “Take” in irradiated neonatal nu/nu mice was also poor. Cells were passaged three times in irradiated nu/nu mice inoculated subcutaneously and it seems probable that indefinite passage of T. parva in mice can now be achieved.Item Establishment of Theileria Parva-Infected Bovine Tissue Culture in Swiss and Athymic (Nude) Mice(1977) Irvin, A.D.; Brown, C.G.D.; Kanhai, G.K.; Stagg, D.A.; East African Veterinary Research Organization, Muguga, P.O. Box 32, Kikuyu KenyaTheileria parva-infected bovine lymphoid cells, grown in culture, were inoculated by different routes into neonatal and adult Swiss mice immunosuppressed by irradiation, thymectomy or inoculation of anti-lymphocyte serum. Tumour-like masses, composed of parasitized bovine lymphoid cells, formed at the site of subcutaneous inoculation in immunosuppressed neonatal and adult mice, but consistent establishment of cells following intra-peritoneal inoculation occurred only in neonatal mice. In all cases the degree of cellular establishment was proportional to the degree of immunosuppression. The best “take” was in irradiated neonatally thymectomized mice. Cells underwent short-term multiplication in mice but, as immune competence returned, the cells were rejected. There was no evidence that cells, on passage, became more adapted to grow in mice, nor that mouse cells became parasitized. Culture-derived cells were also inoculated subcutaneously into irradiated and non-irradiated nu/nu, nu/+ and Swiss mice. Tumour-like masses, composed of parasitized bovine lymphoid cells, developed at the site of inoculation in all irradiated mice. In nu/+ and Swiss mice these masses regressed after 2–3 weeks, but in the athymic nu/nu mice there was generally no rejection or cellular degeneration and parasitized cells became widely disseminated in the host's tissues and organs, in some cases causing death. T. parva-infected cells could not be established in non-irradiated nu/nu mice, nor when irradiated nu/nu mice were inoculated by the intra-peritoneal route. “Take” in irradiated neonatal nu/nu mice was also poor. Cells were passaged three times in irradiated nu/nu mice inoculated subcutaneously and it seems probable that indefinite passage of T. parva in mice can now be achieved.Item Fine Structure and Invasive Behaviour of the Early Developmental Stages of Theileria Annulata In Vitro(1983) Jura, W.G.Z.O.; Brown, C.G.D.; Kelly, B.J.V.P.; Centre for Tropical Veterinary Medicine, Easter Bush, Roslin, Midlothian, EH25 9RG Gt. BritainThe interaction, in vitro, between bovine peripheral blood by lymphocytes and sporozoites of Theileria annulata (Ankara) was studied by light and electron microscopy. Beginning five minutes following incubations, samples were taken for Giesma-stained smears and glutaraldehyde-fixed pelletes, for light and electron microscopy, respectively. Sporozoites of T. annulata measure an average of 0.9 μm long, 0.8 μm broad and posses a limiting unit membrane, the pellicle; a round-to-ovoid, eccentrically situated, non-chromocentric nucleus, double-membraned, tubular, acristate mitochondria, varying numbers of anisocytic, densely osmiophilic and pleomorphic organelles, the rhoptries which together with the polar ring form the apical complex; and numerous, loosely scattered, electron-dense ribosomal particles. As early as 5 min of incubation, sporozoites had made contact with, and penetrated, lymphocytes. Sporozoites consistently attached to the lymphocyte plasmalemma by their basal end, possibly at specific receptor sites. Apparently only a proportion of lymphocytes (up to 40% and more commonly 10–20%) were susceptible. Two subpopulations of the susceptible lymphocytes were observed; one which appeared to have receptor sits localized on one pole of the plasmalemma and the other subpopulation in which the receptor sites were distributed evenly around the plasmalemmal surface. With individual susceptible lymphocytes, the number of interiorized sporozoites increased from 1 to 3 at 5–10 min to as many as 15 or more parasites at around 60 min of incubation. Theileria annulata sporozoites were interiorized by the invagination of the host cell palsmalemma which remained intact throughout the process but the later fragmented. Within 30 min of interiorization, each sporozoite underwent dedifferentiation by the loss of its rhoptries and transformed into a trophozoite. Around 24 h, the trophozoite, a uninucleate, motile and feeding stage of the parasite, developed into a schizont an acentric, closed mitosis.
