Immunisation of cattle against theileriosis in Nakuru District of Kenya by infection and treatment and the introduction of unconventional tick control.

One hundred and one cross European-Boran cattle (50 cows and 51 calves), on a farm in Nakuru District, Kenya, were immunised against theileriosis using Theileria parva lawrencei and Theileria parva parva stocks from another district of Kenya. The stabilates used were T.p.lawrencei (Mara III) used at 10(-1.7) dilution and T.p.parva (Kilae) used at 10(-1.0) dilution. The stabilates were combined and inoculated simultaneously with a short-acting formulation of oxytetracycline hydrochloride given intramuscularly at 10 mg kg-1 body weight and was repeated on Day 4 after inoculation of the stabilate. Most of the theileriosis challenge on the farm was thought to be derived directly from the African buffalo (Syncerus caffer). Nine percent of the cattle had significant indirect fluorescent antibody (IFA) titres before the immunisation and 99% after immunisation. The immunised cattle were exposed to tick-borne disease challenge on the farm by withdrawal of acaricide cover. The immunised cattle were divided into five groups plus two susceptible control cows and two calves for each group. Cattle in four of the groups had acaricidal ear tags, each group having a different type, applied to both ears and the fifth group remained untagged. The animals remained without conventional acaricide application for 134 days. Ten out of 20 (50%) non-immunised control cattle became T.p.lawrencei reactors which only one out of 97 (1%) of the immunised cattle reacted. A frequent complication noted was mild infections due to unidentified Theileria sp. which required expert differentiation from T.parva infections. An additional group of ten steers whose tick load was removed by hand at weekly intervals was introduced 79 days after exposure; these had no tick control and four became T.p.lawrencei reactors. Of 12 calves born during the exposure period and without tick control, four became theilerial reactors and one died. The application of acaricidal tags however, reduced tick infestation levels considerably compared with untagged controls but did not prevent transmission of theileriosis with the possible exception of tags on Group 4. A number of transient low grade fevers were noted and attributed to Theileria sp., Ehrlichia bovis, Ehrlichia (Cytoecetes) ondiri and Borrelia theileri infections, none of which were fatal. One immunised animal died of acute dual infection of Babesia bigemina and Borrelia theileri after acaricide control by spraying was re-introduced but no Anaplasma infections were detected. An analysis of the economic effects of immunisation was made.

Evaluation of infection and treatment methods in immunization of improved cattle against theileriosis in an endemic area of Kenya.

Five experiments were carried out to determine the efficacy of immunization against theileriosis in an endemic area of Kenya using artificial infection with a mixture of stabilates of Theileria parva stock or natural infection and treatment with parvaquone or several formulations of oxytetracyclines. For the first four experiments, introduced, susceptible Sahiwal/Friesian crosses were used and in the fifth, calves of Boran/Maasai zebu crosses born on the site. Cattle were infected either artificially with sporozoite stabilates of local isolates of T. parva parva derived from cattle and T. parva lawrencei derived from African buffalo or exposed to natural tick challenge on the ranch mostly derived from buffalo. The cattle were then given various treatment regimens using either parvaquone or long- and short-acting formulations of oxytetracycline. Treatment of natural infections, although it can be effective, was not considered a practical method on a large scale because of the need for intensive monitoring in the case of parvaquone treatment and the possibility of cattle not becoming infected in the case of prolonged application of long-acting formulations of oxytetracycline. Both methods were relatively expensive. Artificial infection treatment proved more practical and methods were developed where the monitoring of cattle was not required during the immunization procedure. Out of a total of 16 drug regimens investigated, one (consisting of two treatments of a short-acting formulation of oxytetracycline at 10 mg kg-1 body weight on Days 0 and 3 or 4 after infection) was found to be the most efficacious and the cheapest, and has now been used on a routine basis. This method can be used successfully on calves greater than 1 month of age.

Immunisation of cattle against theileriosis in Coast Province, Kenya: laboratory evaluation of a Theileria parva parva stabilate for use in 'infection and treatment' immunisation in the field.

Theileria parva parva Marikebuni stock, previously shown to give good protection to immunised cattle in Kilifi District, Coast Province of Kenya, was chosen for large scale immunisation in the district. A large sporozoite stabilate was prepared and evaluated for efficacy and safety in the 'infection and treatment' method, using a long or short acting formulation of oxytetracycline. Susceptible cattle were infected with selected doses of stabilate (10(0), 10(-1), 10(-1.7) and left either as untreated controls, or treated with one of the two oxytetracycline formulations. It was concluded that stabilate dilution at 10(-0.7) or 10(-1) in combination with either formulation of oxytetracycline would effect satisfactory immunisation. The short acting oxytetracycline treatment was judged to be the most efficacious in protecting cattle against homologous challenge. On heterologous challenge it was found that T p parva Marikebuni immune cattle were protected against seven T p parva stocks from Kilifi District and also against four stocks of T p parva from other areas of Kenya. In addition, the Marikebuni stock provided partial protection against challenge by T p lawrencei stocks. Furthermore, cattle immune to T p parva and T p lawrencei were protected against lethal challenge of T p parva Marikebuni stock. Thus, it appears that large scale immunisation of cattle against theileriosis in Kilifi District could be undertaken using the Marikebuni stock. With continued assessment, this stock could provide a master theilerial stock for immunisation against cattle theileriosis in areas free of buffaloes elsewhere in Kenya.

Characterization of buffalo-derived theilerial parasites with monoclonal antibodies and DNA probes.

The characteristics of intra-lymphocytic Theileria isolated from African buffalo and from cattle that were infected with buffalo-derived parasites were evaluated using anti-schizont monoclonal antibodies (mAbs) and DNA probes. Antigenic differences were revealed by the reactivities of 27 mAbs with the buffalo-derived parasites isolated from different animals. Antigenic diversity was also seen with Theileria-infected lymphoblastoid cell isolates taken from the lymph nodes and lambda gt11, showed specific hybridization to parasite DNA in Southern blots of restriction enzyme-digested, lymphoblastoid cells infected with buffalo-derived theilerial parasites. Genotypic differences between the buffalo-derived parasites were revealed by the restriction fragment length polymorphisms seen with hybridization of those probes to DNA from cloned and uncloned Theileria-infected cell lines. The evaluation of theilerial parasites derived from buffalo and from cattle which underwent typical T. p. lawrencei reactions, after being infected with buffalo-derived theilerial parasites, did not show any specific phenotypic or genotypic characteristics of these parasites that would distinguish them from T. p. parva and T. p. bovis parasites. The validity of these subspecies distinctions is discussed.

Immunization of cattle using varying infective doses of Theileria parva lawrencei sporozoites derived from an African buffalo (Syncerus caffer) and treatment with buparvaquone.

A Theileria parva lawrencei isolate in the form of a sporozoite stabilate, derived by feeding clean Rhipicephalus appendiculatus nymphal ticks on an African buffalo (Syncerus caffer) captured in the Laikipia District, Kenya, was inoculated into groups of cattle at dilutions between 10(0) and 10(-3). Groups of 3 cattle infected with 1 ml inocula at 10(0), 10(-1) and 10(-2) dilutions were treated with 2.5 mg/kg body weight of buparvaquone on day 0 and similar groups were left untreated to act as controls. An additional group, given 10(0) dilution of the stabilate, was treated with buparvaquone on day 8 post-inoculation. It was found that all control cattle inoculated with the stabilate at dilutions between 10(0) and 10(-2) became infected, but only 2 out of 3 cattle developed patent infections at 10(-3) dilution. All 3 control cattle receiving 10(0) dilution died of theileriosis, 2 at 10(-1) and 10(-2) dilutions, and 1 at 10(-3) dilution died. Buparvaquone treatment on day 0 at 10(0) dilution resulted in the survival of 2 of 3 cattle and all the cattle at 10(-1) and 10(-2) dilutions. All the surviving cattle eventually developed a significant serological response against T. parva in the indirect fluorescent antibody test, except 1 in the 10(-3) dilution group, and were immune to homologous challenge when tested 3 months later with a lethal inoculum of stabilate, except 2 cattle in the 10(-3) dilution group. As a result of a theileriosis problem at about day 60 after inoculation in 2 cattle given 10(-2) dilution of stabilate and buparvaquone treatment on day 0, an additional 5 cattle were given 10(-2) dilution of stabilate and developed a good immunity after buparaquone treatment. None was shown to develop the carrier state. Treatment with buparvaquone on day 8 after infection with 10(0) dilution of stabilate was not successful since 2 died. The stabilate used was shown to produce reproducible infection in cattle at different dilutions.

Experimental induction of Theileria parva lawrencei carrier state in an African buffalo (Syncerus caffer).

An African buffalo (Syncerus caffer), born in captivity and demonstrated to be Theileria-free, and 2 susceptible cattle were inoculated with a Theileria parva lawrencei sporozoite stabilate. The buffalo had a very mild disease reaction, while the 2 cattle died of acute theileriosis. It was possible to isolate T. p. lawrencei from the buffalo up to 888 days after infection by the application of non-infected Rhipicephalus appendiculatus nymphs and up to 657 days after infection by the establishment of lymphoblastoid cell lines infected with T. p. lawrencei schizonts from peripheral mononuclear blood cells. The infection rate and levels of Theileria in the resultant adult ticks varied from 11 to 70% with 0.3-11 acini infected/tick. Stabilates prepared from these tick batches caused fatal T. p. lawrencei infections in cattle.

Development and survival of Theileria parva parva in Rhipicephalus appendiculatus exposed in the Trans-Mara, Kenya.

Nymphal Rhipicephalus appendiculatus (Trans-Mara) were fed on a steer infected with a Theileria parva parva (Kilae 1) stock isolated from an indigenous steer in the Trans-Mara Division, Kenya, which had a high piroplasm parasitaemia. A total of 5000 engorged nymphs which had dropped on one day were enclosed in elongated nylon bolting silk tubes in groups of 200-300 and were transported immediately to the Trans-Mara where they were suspended vertically in the grass cover with one end touching the ground. Over 98% of the nymphs moulted into adult ticks and 50% moult occurred by day 28 after exposure. The ticks showed over 80% survival up to 308 days post-exposure but thereafter showed a marked mortality so that only 22.5% of the ticks were alive after 439 days. Theileria parasites were detected in the salivary glands by day 35 post-exposure and infection rates and levels increased markedly between 180 and 235 days post-exposure. Thereafter, the infection rates and levels generally decreased. Groups of ticks were triturated and the resultant supernatant fluid inoculated into pairs of susceptible cattle, and these proved infective from day 44 to 145 after exposure. Three subsequent attempts to induce infections with supernatant fluid were unsuccessful. From 294 days after exposure, groups of 50 ticks were applied to cattle and caused lethal T. p. parva infections up to 439 days post-exposure. Climatic observations showed a relatively even monthly rainfall as well as mean maximum and minimum monthly temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Infection of African buffalo (Syncerus caffer) and cattle with Theileria parva lawrencei after serial passage in cattle.

The infectivity of a Theileria parva lawrencei stabilate, from a stock derived from an African buffalo (Syncerus caffer) in the Serengeti National Park, Tanzania, was investigated. In the first experiment a buffalo and three cattle were inoculated with a stabilate from a stock passaged three times in cattle. All cattle developed fatal theilerial infections. Isolations from the buffalo by tick feeding and cell culture isolation showed that it was infected with T p lawrencei at the time of inoculation, but the second isolation made 19 days after inoculation behaved like T p parva in cattle, developing a high parasitosis, while the third isolation made three months later behaved like T p lawrencei with low parasitosis. It was concluded that two biological types of T parva could exist in a buffalo at one time, but it was not shown that the buffalo had become a carrier of T p lawrencei adapted to cattle. In the second experiment two buffaloes and three cattle were inoculated with T p lawrencei (Serengeti) stabilate which had been passaged six times through cattle and ticks. The two buffaloes had mild theilerial infections and developed serological titres in the indirect fluorescent antibody test, but the cattle had fatal infections. Tick and cell culture isolations of T parva were possible during the clinical reactions of the buffaloes, but no carrier state was demonstrated. Theileria-infected cell lines were established from the buffaloes and the cattle and were examined using monoclonal antibodies against T parva schizonts.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of Theileria parva parva infection in an endemic area of Kenya.

The maintenance of Theileria parva parva infection in an endemic area of Kenya on the shore of Lake Victoria was studied in the field and laboratory. High prevalences of antibodies against T. parva and T. mutans and intra-erythrocytic piroplasms were detected in local zebu (Bos indicus) cattle. The mean infection rate of Theileria parasites in the tick, Rhipicephalus appendiculatus, in field collections was 1.1%. Most of the infection was attributed to T. parva parva by application of field ticks to susceptible cattle. Five cattle, all about 1.5 years old, were purchased from local owners and transported to the laboratory. All five had oscillating antibody titres against T. parva and T. mutans and had patent theilerial infections during the subsequent 13 months. Uninfected R. appendiculatus nymphs were applied to cattle at 0, 3, 6, 9 and 13 months after transport to Muguga, and 18 out of 23 batches transmitted T. parva parva infection to cattle when 100 resultant R. appendiculatus adults were applied. Infection rates in the tick batches were usually low, with 1 salivary gland acinus infected/tick. Hence, a frequent carrier state of naturally infected cattle has been demonstrated for T. parva parva for the first time, and it is likely that this carrier state is of great importance in maintenance of T. parva parva infection in the field.

Epidemiology of theileriosis in calves in an endemic area of Kenya.

Thirty-one calves born into five Maasai zebu cattle herds over a period of 1 month in the Trans-Mara Division of Kenya, endemic for theileriosis, were recruited for an intensive study of theileriosis. No calves up to 6 months of age died but all developed Theileria infections as judged by slide examination and serology. Parasitosis by T. mutans schizonts in lymph node smears was usually higher than that of T. parva. The T. mutans schizonts usually occurred at an earlier age but persisted at a patent level for a shorter time than those of T. parva. Serological findings using the indirect fluorescent antibody test confirmed the parasitological findings. It was evident that colostral transfer of Theileria antibodies was frequent. Theileria piroplasm parasitaemia had developed in all calves by 111 days of age. The earlier parasitosis by T. mutans reflected the higher infection rates in Amblyomma spp. than in Rhipicephalus appendiculatus. The mean number of R. appendiculatus on the ears of calves during the observations was 9.1 adults and 1.5 nymphs. Clinical episodes of T. mutans and T. parva infection were associated with febrile responses, enlarged lymph nodes, anaemia and other symptoms and about 80% of calves had poor weight gains or weight losses during either clinical infection. It would appear that theileriosis is one of the most important factors in the stunting of calf development in the area.

Theileria parva: expression of a sporozoite surface coat antigen.

A monoclonal antibody specific for the Theileria parva sporozoite, which recognizes a determinant on the surface coat and blocks sporozoite infectivity, was used to investigate the presence of the determinant on other stages of the parasite lifecycle. Immunofluorescence techniques did not demonstrate this determinant on the kinete, schizont, merozoite, or piroplasm stages of the parasite. Immunoautoradiography, using a tritiated form of the monoclonal antibody, on sections of infected salivary glands collected from ticks that had fed for 0, 1, 2, 3, or 4 days revealed that the determinant recognized was synthesized predominantly during sporogony, between 2 to 3 days after the tick started feeding. Immunoelectron microscopy was performed on ultrathin frozen sections of infected tick salivary glands incubated with the monoclonal antibody followed by Protein-A--colloidal gold. The antigen or its precursor could be detected in the developing parasite. In ticks fed 2 days, the sporoblast was labeled, both in the cytoplasm and on parasite membranes, often including the nuclear envelope. In sections from ticks fed 4 days, the sporozoite surface membrane was labeled, as were membrane-bounded sporozoite organelles identified as micronemes. Observation by immunofluorescence, on sporozoites incubated with bovine peripheral blood lymphocytes, suggested that the antigen recognized by the monoclonal antibody does not enter the lymphocyte during sporozoite endocytosis. We conclude that synthesis of the antigen or its precursor(s) occurs during sporogony in the feeding tick, at the time of maximal parasite proliferation, and precedes the formation of morphologically mature sporozoites; the antigen's role in the parasite life cycle also appears to be limited to events associated with the sporozoite entry process.

Chemotherapy of East Coast fever: parvaquone treatment of clinical disease induced by isolates of Theileria parva.

Two experiments were carried out in which parvaquone was used to treat experimentally-induced acute clinical East Coast fever infections. In the first experiment, infections with Theileria parva parva (Kiambu 5) were induced by applying infected Rhipicephalus appendiculatus ticks or by inoculation of triturated infected-tick stabilate. The character of the disease was similar with both methods of infection and following a single treatment with parvaquone at 20 mg kg-1, 5 of 7 cattle in each group recovered. All untreated control cattle died. In the second experiment, 5 stabilate isolates from different locations within East Africa, and representative of the challenge likely to be met in the field, were used. Treatment was administered in 2 X 10 mg kg-1 doses 48 h apart. The isolates used were T. p. parva (Mbita), T. p. parva (Pugu), T. p. parva (Entebbe), T. p. lawrencei (Mara) and T. p. lawrencei/(Manyara); following treatment 3/7, 6/6, 6/7, 5/7 and 6/7 animals recovered, respectively. All untreated control cattle died. There was evidence of a difference in susceptibility of isolates to treatment, and some animals showed prolonged disease episodes. The nature of the response to treatment and the problems in treating a lympho-destructive disease are discussed.

A histocompatibility barrier to immunization against East Coast fever using Theileria parva-infected lymphoblastoid cell lines.

Histocompatibility may be a barrier to the infection of cattle when Theileria parva parva-infected tissues or in vitro cultured macroschizont-infected lymphoblastoid cell lines are used for immunization. By inoculating 10(3) and 10(5) infected cells into autologous recipients infection was achieved and immunity engendered. Cell lines inoculated into BoLA matched recipients did not produce patent infections but some recipients developed antibodies to the parasite and 3/5 were immune to challenge. No evidence of infection or immunity was found in BoLA half matched or mismatched cattle. This result suggests that there is an histocompatibility barrier to infection using T. p. parva-infected lymphoblastoid cells.

Infection of mammalian cells with Theileria species.

Experiments were carried out to determine the susceptibility of mammalian cells to infection with different species of Theileria in vitro. Sporozoites of Theileria parva (parva), Theileria parva (lawrencei) and Theileria taurotragi were isolated from Rhipicephalus appendiculatus ticks by grinding infected ticks in medium, filtering the suspension and concentrating by centrifugation. The sporozoites were used in attempts to infect in vitro peripheral blood leucocytes harvested from 16 different mammalian species which included 12 species of Bovidae from 6 different sub-families. The technique was shown to be both sensitive and reproducible. The sporozoites of T. parva (parva) infected and transformed cells from 2 species of the sub-family Bovinae, the two cattle types and African buffalo. Theileria parva (lawrencei) infected and transformed cells from the two cattle types, African buffalo and Defassa waterbuck. Theileria taurotragi sporozoites infected in vitro cells from 11 different species of Bovidae which were members of 6 sub-families; Bovinae, Tragelaphinae, Reduncinae, Alcelaphinae, Antilopinae and Caprinae. Transformed lymphoblastoid cell lines were established from 7 of the species infected. Sporozoite attachment and infection was not observed with non-susceptible bovid host cells, nor were any of the non-bovid leucocytes infected by the parasites. The host range observed in this study corresponded to the known host range in vivo.

Transmission of Theileria parva by a population of Rhipicephalus appendiculatus under simulated natural conditions.

A 2 hectare paddock on the Muguga Estate, Kiambu District of Kenya (altitude 2100 m) free of Theileria parva-infected ticks was seeded by applying Rhipicephalus appendiculatus nymphs to cattle infected with Theileria parva (Kiambu 4). It was estimated that over 50 000 engorged nymphs fell onto the pasture from 4 cattle with high parasitaemias during the cold season (June). Samples of these ticks were placed in plastic cylinders under the vegetation in the paddock and sexual stages of T. parva were detected in gut lumen smears in these samples up to 21 days after repletion. Zygotes were first observed to transform into kinetes on day 55 and parasites were first detected in salivary glands of adults on day 64. Moulting of the nymphs started on day 60 after repletion and was completed by day 87. Cattle introduced into the paddock showed their first infestation with adult R. appendiculatus on day 64 after repletion of the nymphs and the infestation level gradually increased. On day 76 after repletion, 17% of a sample of adult ticks infesting cattle showed salivary gland infections with T. parva and this increased to over 70% by day 150. From day 360 onwards, a decrease in both the percentage of ticks infected and the number of salivary gland acini infected/tick was noted. In addition, T. parva infections within the salivary glands required a longer period of feeding in the older ticks before they developed into sporozoites. Cycling of Theileria through ticks was prevented by the removal of female ticks before they completed repletion. Lethal challenge levels of T. parva for cattle existed in the paddock up to day 547 after repletion, after which introduced cattle showed no infection or only a sub-lethal infection. Cattle introduced into the paddock on day 808 after repletion showed no evidence of T. parva infection. The tick infestation of introduced cattle decreased markedly from day 368 and infestation had virtually ceased by day 808.