Epidemiological observations of Zimbabwean theileriosis: disease incidence and pathogenicity in susceptible cattle during Rhipicephalus appendiculatus nymphal and adult seasonal activity.

Fifty-nine Hereford cattle susceptible to tick-borne diseases were used as tracer animals to assess the tick challenge and pathogenicity of Theileria parva under field conditions in Zimbabwe. They were moved periodically in groups of five to three commercial farms (one group consisted of four) during seasons of Rhipicephalus appendiculatus nymphal and adult activity. All tracer cattle were herded together with the farm cattle but were not dipped. The nymphal tick counts were high on two of the farms (up to 2000 per animal) but were very low on the third farm (less than ten per animal). On the three farms, 19 out of 24 (76%) tracers had patent Theileria schizonts. There was a range of clinical manifestations of theileriosis with acute and fatal infections occurring on one farm. The adult R. appendiculatus infestations during the wet season numbered 120-800 per animal on the three farms. The disease transmitted by the adults was very pathogenic on the three farms; 30 out of 35 (86%) had severe theileriosis infections. Cattle, which survived the nymphal diseases challenge, showed various degrees of immunity to subsequent T. parva challenge transmitted by adult ticks. Therefore, 13 out of 18 (72%) of these cattle had a second disease episode and the case fatality rate on the three farms was 46%. The factors which determined the epidemiological status of Theileria challenge on the farms, such as the farming systems and presence of wild animals, are discussed.     

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.     

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.     

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.     

Immunization of cattle with a Theileria parva bovis stock from Zimbabwe protects against challenge with virulent T.p. parva and T.p. lawrencei stocks from Kenya

Following inoculation of 34 Bos indicus (Boran) cattle with a Theileria parva bovis (Boleni) stock from Zimbabwe, 18 animals underwent mild theilerial reactions, 12 underwent moderate reactions, three suffered severe reactions and one died. When these animals were subsequently challenged with different virulent stocks of either T.p. parva (Muguga, Marikebuni or Mariakani) or T.p. lawrencei (Ngong 1 or Nanyuki) from Kenya, all except two animals resisted challenge. The two reactors were part of the group challenged with the T.p. parva (Mariakani) stock. All 12 susceptible control animals underwent severe reactions and 11 died. The results of these experiments suggest that T.p. bovis (Boleni) may be used in some situations to immunize cattle against East Coast fever without the need to provide concomitant chemotherapy as in the infection and treatment method of immunization.     

Treatment of theileriosis with parvaquone in Zambia

Parvaquone was used to treat 126 cattle with theileriosis. Theileria species schizonts were present in their lymph node biopsy smears and the majority of the animals had clinical signs of theileriosis. One hundred and fifteen treated and one untreated cattle survived the infection while 11 treated and 12 untreated animals died of the disease. Despite serological evidence of a parasite challenge during the subsequent rainy season, recovered cattle did not develop clinical signs of theileriosis but untreated cattle in the area continued to die from the disease. An intermittent low piroplasm parasitaemia (less than 1 per cent) was observed in recovered cattle for up to 14 months after detailed monitoring of cattle in the trial; this could be evidence for a carrier status for the Theileria species or strains involved.     

Immunization against East Coast fever: the use of selected stocks of Theileria parva for immunization of cattle exposed to field challenge

Two antigenically different stocks of Theileria parva parva (Kilifi and Marikebuni), previously characterized as belonging to groups A and C respectively on monoclonal antibody (MAb) profiles, were selected for immunization of different breeds of cattle against East Coast fever (ECF) by the infection and treatment method. A total of 52 immunized cattle and 33 susceptible controls of different group sizes were exposed to field challenge by ticks for periods of 42-90 days at three field sites where ECF is endemic on the Kenyan coast. All immunized cattle survived ECF challenge, but 87% of the controls died of the disease. The cattle exposed at one site had been immunized 1 year earlier and maintained tick-free in the intervening period. The level of immunity in these cattle was similar to that of cattle which had been immunized 1 or 2 months prior to exposure. Thus, immunity had not waned over the 1-year period. A study at another site showed that acaricidal treatment of immunized cattle could be safely extended from twice a week to once every three weeks, whereas in susceptible cattle even twice weekly spraying did not control ECF. The isolates made from infected controls during the trials indicated the presence of three T. p. parva stocks as defined by MAb profiles. Of the two stocks used for immunization, T. p. parva Marikebuni induced broader protection. In view of the apparent limited antigenic diversity of T. p. parva strains within the Coast Province it is suggested that the Marikebuni stock might represent a key stock for vaccination in this area.     

Laboratory and field investigations into the Theileria parva carrier-state in cattle in Zimbabwe.

The Theileria parva carrier-state in cattle on commercial farms on Zimbabwe was investigated using parasitological and serological methods. The proportion of cattle showing Theileria piroplasms on two farms, which had recent histories of disease outbreaks, were 64% (n = 106, total of heifers and weaned calves examined) and 71.5% (n = 60) while the proportion of T. parva antibodies for the same animals were 59% and 98.5%, respectively. On four farms where no cases of the disease occurred for over 10 years, the average proportion of animals showing piroplasms and antibodies were 55.4% (range 32-82, n = 223) and 73% (range 47-91, n = 223), respectively. However, on another three farms which had no history of theileriosis outbreaks these proportions were very low, being 11.4% (0-24, n = 157) for piroplasms and 12.2% (5-23, n = 157) for antibodies. The mean infection rate in unfed Rhipicephalus appendiculatus adults collected from farms with a high prevalence of cattle which were carriers of Theileria piroplasms during the tick activity season was 29% (range 12-60%) with 9.3 (range 2-18.7) mean infected acini per infected tick. The infectivity of different tick batches to susceptible cattle produced a wide spectrum of theileriosis reactions. Laboratory controlled experiments were carried out to study the persistence of T. parva (Boleni) piroplasms in cattle immunized with this strain as well as its infectivity for ticks and its subsequent transmissibility to cattle. Examination of the salivary glands of 15 batches of ticks collected from six immunized cattle on three different occasions over 18 months showed that none were infected with Theileria parasites. However, the infectivity of other ticks in the same batches to susceptible animals was demonstrated 6, 10 and 18 months after cattle had been immunized with Boleni stabilate.     

Identification of a target population for immunisation against East Coast fever in coastal Kenya

Two experiments were carried out to identify the target population of cattle for immunisation against East Coast fever (ECF) using the infection-and-treatment method. Firstly, a sentinel-calf study was used to determine the age window for ECF immunisation by determining ages at clinical detection of infection with Theileria parva. Six groups of five naive cross-bred (Bos taurus/Bos indicus) male calves, introduced at intervals of 2 months at a mean age of 26 days, were exposed to natural tick challenge on a high ECF-risk, small-holder farm in the coastal lowland, coconut-cassava agro-ecological zone of coastal Kenya. Secondly, a challenge study evaluated the relationship between the presence of T. parva antibodies and immunity. Ten indigenous adult Zebu cattle and nine Zebu young stock purchased from farmers in the same zone, and eight cross-bred calves (survivors of the sentinel-calf study) were challenged with 10 times the immunising dose of T. parva Marikebuni stock. Twenty-four of these 27 cattle had high antibody titres before challenge. Two cross-bred calves, obtained from an ECF-free area and seronegative to T. parva schizont antigen, also were challenged and used as susceptible controls. Twenty-five (83%) of the 30 sentinel calves contracted ECF over an age range of 36-116 days (mean 72 days). The remaining five calves died of other causes within 2 months of arrival on the farm. Fourteen of the 25 calves survived the infection and developed antibodies to T. parva. Despite tick control, seven of these 14 calves had a second episode of ECF and two died. In total, 13 of the 25 calves that contracted ECF died. Only one of 19 indigenous Zebu animals developed clinical ECF when challenged with T. parva Marikebuni (mild clinical signs with spontaneous recovery). Of the eight cross-bred survivors from the first experiment, only one succumbed to ECF when challenged and it died. Both susceptible cross-bred calves developed severe clinical signs of ECF and one died. The experimental studies show that in the high ECF-risk areas of the coconut-cassava zone of coastal Kenya, immunisation against ECF in cross-bred (B. taurus/B. indicus) cattle should be targeted at an early age (preferably within 1-2 months of birth).     

Occurrence of Theileria parva infection in cattle on a farm in the Ladysmith district, KwaZulu-Natal, South Africa

Theileria parva causes widespread morbidity and mortality in cattle in endemic regions. An outbreak of theileriosis occurred on a farm near Ladysmith in KwaZulu-Natal, South Africa, which is not a declared Corridor disease-infected area. A survey of Red Brangus cattle from all age groups and areas of the farm was performed. Transmission of the parasite from infected animals on the farm to susceptible animals by tick transmission and tick-stabilate injection, was attempted. The survey indicated high numbers of animals with antibody titres to T. parva but only 6 infected animals, based on real-time PCR and RLB analysis. The transmission experiments failed to transmit the parasite. The study shows the difficulty in elucidating a source of infection and determining the dynamics of new infections in a herd where multiple possible sources are present and treatment with tetracyclines has taken place.     

East coast fever: 2. Cross-immunity trials with a Kenya strain of Theileria lawrencei

Cross-immunity trials were carried out on cattle which were immunized against theileriosis either by chemoprophylaxis or by sub-lethal challenge. In the first of two experiments, animals were immunized against either one of two strains of Theileria parva or one of two strains of Theileria lawrencei. They were then challenged with a Kenya strain of T. lawrencei (T. lawrencei KB 5). The former animals were not protected against the challenge, whereas the latter were either partially protected, when a Tanzanian strain of T. lawrencei had been used for immunization, or completely protected when another Kenyan strain of T. lawrencei had been used.In the second experiment, animals were immunized by chemoprophylaxis against T. lawrencei (KB5) and challenged with T. parva (one strain) or T. lawrencei (five strains). The cattle were protected against challenge with the identical or related T. lawrencei strains, but only partially protected against the T. parva strain and a Tanzanian strain of T. lawrencei.It appears that cattle cannot be immunized for field exposure to theileriosis throughout East Africa by chemoprophylaxis using T. lawrencei (KB5) alone, but may be able to withstand field exposure in a variety of situations if a combination of different theilerial strains is used in the vaccination procedure.     

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.     

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.     

Non-specific immunization against bovine tropical theileriosis (Theileria annulata) using killed Corynebacterium parvum

Killed Corynebacterium parvum was used as an adjuvant for the production of non-specific resistance against Theileria annulata in cattle. Groups of cross-bred (Bos indicus X Bos taurus) calves were administered C. parvum adjuvant subcutaneously and were then challenged with T. annulata-infected ticks on 45, 60 or 90 days later. The challenge caused mild reactions in the protected calves. None of the 10 immunized calves died due to theileriosis, whereas all three paris of susceptible control calves died due to theileriosis. It appears from this pilot study that cattle can be protected non-specifically with C parvum adjuvant against T. annulata.     

Further evaluation of the use of buparvaquone in the infection and treatment method of immunizing cattle against Theileria parva derived from African buffalo (Syncerus caffer).

Three experiments were undertaken to determine the efficacy of different doses of buparvaquone in the infection and treatment immunization of cattle against Theileria parva derived from African buffalo (Syncerus caffer). Two of these experiments also compared buparvaquone with standard doses of long- and short-acting formulations of oxytetracycline. In addition, different dilutions of stabilates were used in the experiments. In the first experiment, a 10(-1.0) dilution of stabilate was used to infect groups of cattle treated with buparvaquone at doses of between 5 and 0.625 mg kg-1 body weight (bwt) on Day 0 after infection. All control cattle developed severe theileriosis and none of the treatment regimes (including those utilizing long-acting oxytetracycline) prevented the development of theileriosis. Treatment with buparvaquone at 2.5 mg kg-1 bwt or oxytetracycline gave the most satisfactory results. In the second experiment when the sporozoite dose was reduced to 10(-2.0) dilution, buparvaquone treatment at 5 and 2.5 mg kg-1 bwt and short- and long-acting formulations of oxytetracycline reduced reactions greatly. While all the oxytetracycline treated animals produced a serological response and were immune to a 50-fold higher challenge with the immunizing stabilate, several animals in the buparvaquone groups did not show a serological response and were not immune to challenge. In the third experiment, groups of cattle were infected with 10(-1.2), 10(-1.4) and 10(-1.6) dilutions of stabilate and were treated with 2.5 mg kg-1 bwt of buparvaquone. No animals developed severe theileriosis and all seroconverted. On homologous challenge, however, two out of 14 cattle showed severe reactions. It was concluded that further work on immunization using buparvaquone treatment at 2.5 mg kg-1 bwt and 10(-1.6) dilution of the stabilate would have to be carried out before such a system could be used in the field.     

Epidemiological observations on theileriosis following field immunisation using infection and treatment

Thirty-seven high grade cattle were immunised against Corridor disease (Theileria parva lawrencei infection) on a farm with a history of heavy and often lethal theilerial challenge. Nineteen cattle were immunised by treating with two doses of long-acting oxytetracyclines given at 20 mg/kg on days 0 and 4 after sporozoite stabilate inoculation, while the other 18 were treated with naphthoquinone buparvaquone, given as a single dose of 2.5 mg/kg simultaneously with stabilate inoculation. All the cattle underwent subclinical theilerial reactions with all but two developing high antibody titres on the IFAT test against T. parva schizont antigen by day 35 after the immunisation. Both buparvaquone and long-acting oxytetracycline appeared equally effective in the immunisation. To date, 26 months later, only two cases of theileriosis parasitologically characteristic of T. p. parva have been reported in the immunised cattle. Following the two cases, investigations showed that when uninfected Rhipicephalus appendiculatus nymphal ticks were deliberately fed on healthy resident cattle on the farm, the resultant adult ticks transmitted acute and lethal theilerial infections to five out of five susceptible cattle. The resultant infections were parasitologically characteristic of T. p. parva infections. Furthermore, the monoclonal antibody profiles of schizont infected cell lines from these infections appeared to be characteristic of T. p. parva. It was thus concluded that resident cattle on the farm could be a potential source of T.p. parva infection which had broken through the immunity of T.p. lawrencei immunised cattle and could constitute a reservoir of theilerial infection for ticks and hence to susceptible stock on the farm.     

Field evaluation of parvaquone against naturally occurring East Coast fever

Parvaquone (Clexon Cooper) was clinically tested for efficacy as a treatment for East Coast Fever (Theileria parva parva infection) in naturally infected cattle. The drug showed a suppressive effect on Theileria schizonts and piroplasms. A recovery rate of 79% was recorded. Best results were obtained when Clexon treatment was initiated in the early stage of the disease, before many red blood cells were invaded and before respiratory distress was evident. A carrier state in animals recovered from East Coast Fever is suspected.     

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.     

Theileria parva: the immune status of calves born of dams immunised against East Coast fever

Eight Friesian cross cows three months pregnant to a single Friesian bull were immunised against East Coast fever by infection with Theileria parva (Muguga) sporozoite stabilate and treatment with pyrrolidino-methyl tetracycline. They were challenged with the homologous stock four times before calving and a fifth time after calving, and resisted all five challenges which killed all of the five groups of five susceptible controls. Calves born to these hyperimmunised dams were fully susceptible on challenge with the same stabilate, as were susceptible cows from the same farm and their calves. In both instances the calves died three to seven days earlier than the cows which were approximately 10 times heavier. These results show that one- to two-month-old taurine calves from artificially immunised dams are not protected from experimental T parva sporozoite challenge and that there is no inherent calfhood resistance to East Coast fever.