Dolan, T.T.

Grootenhuis, J.G.

Kariuki, D.P.

Leitch, B.L.

Linyonyi, A.

Maritim, A.C.

Mining, S.K.

Morzaria, S.P.

Mutugi, J.J.M.

Ndungu, S.G.

Ngumi, P.N.

Stagg, D.A.

Young, A.S.

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Parasite stocks used for East Coast feverimmunization in KenyaJ.J. Mutugi, A.S. Young, A.C. Maritim, S.G. Ndungu, A. Linyonyi,P.N. Ngumi, S.K. Mining, B.L. Leitch, D.A. Stagg, J.G. Grootenhuis,*D.P. Kariuki, S.P. Morzariat and T.T. DolantProtozoology DivisionNational Veterinary Research CentreKenya Agricultural Research InstituteP.O. Box 32Kikuyu, Kenya*Wildlife Diseases SectionNational Veterinary Research CentreKenya Agricultural Research InstituteP.O. KabeteNairobi, Kenyatlnternational Laboratory for Research on Animal DiseasesP.O. Box 30709Nairobi, KenyaFollowing the development of the infection-and-treatment method of immunization against East Coast fever (ECF) by the United Nations DevelopmentProgramme/Food and Agriculture Organization of the United Nations group atthe East African Veterinary Research Organisation, now the National VeterinaryResearch Centre (NVRC), in Muguga, Kenya, between 1967 and 1977, theKenya Government was reluctant to sanction the extensive field use of themethod. The following concerns were expressed.a) Immunized cattle might show a reduction in productivity.b) Insufficient information was available on the various Theileria parva parasites prevalent in the country.c) Theileria parva parasites from cattle would not protect cattle against buffalo-derived parasite stocks.d) Immunized animals might become carriers and thus introduce alien strainsof parasites into previously uninfected regions of the country.IMMUNIZATION REPORTSe) The infection-and-treatment immunization method might be impracticaland/or unsafe.Since then, work in various laboratories has been undertaken to address theseconcerns. Presently, a Kenya/British project based at NVRC, Muguga, has beencharged with the safe implementation of large-scale ECF immunization in thecountry. We report here on some laboratory studies carried out, focussing onsome of the concerns listed above.SAFETY STUDIES ON THE INFECTION-AND-TREATMENT IMMUNIZATIONStudies to determine safe and optimal immunizing sporozoite doses for a number of T. p. parva and T. p. lawrencei stocks were carried out, as well as investigations on the efficacy of treatment with the available immunizing drugs. Thestudies involved inoculating groups of susceptible Friesian cattle with variousdoses of T. parva sporozoite stabilate dilutions, either singly or in combination.The infections were then treated with one of three anti-theilerial drugs:Medamycin 100 (TechAmerica Group, Inc.), a short-acting oxytetracycline;Terramycin LA (Pfizer Ltd., U.K.), a long-acting oxytetracycline; or a newchemotherapeutic drug, buparvaquone (ButalexR, Coopers Animal Health).Medamycin was given at 10 mg/kg on days 0 and 4 of the immunization.Terramycin LA was given at 20 mg/kg at the same time as stabilate inoculation,and buparvaquone was given at 2.5 mg/kg also at the same time as stabilateinoculation. All drugs were injected intramuscularly.Following sporozoite stabilate inoculation and appropriate drug treatment, theimmunization reaction was monitored using daily clinical and parasitologicalobservations (theilerial schizont parasitosis). On days 28 and 35 after immunization, surviving cattle were examined for T. parva antibodies using the indirect fluorescent antibody test (Burridge and Kimber, 1972). On about day 60after immunization, the surviving cattle were challenged with a lethal dose ofhomologous parasite. These cattle were later challenged with heterologous parasites.The results for five titration experiments involving various T. parva stocksfrom geographically separated areas of Kenya are summarized in Table 1 . In thetitration involving T. p. lawrencei (01 Pejeta) stabilate 199, the highest concentration of 1.0 ml of undiluted stabilate could not be satisfactorily controlled byeither Terramycin LA or Medamycin 100. However, at lower concentration(1:100), it was possible to induce subclinical theileriosis with the developmentof antibodies to T. parva. These cattle were immune to homologous challenge.Bupavaquone controlled higher concentrations of the stabilate better than thetwo oxytetracycline formulations. In contrast, both oxytetracyclines controlled40PARASITE STOCKS USED IN KENYAundiluted and 1:80 stabilate concentrations of the T. p. parva (Marikebuni)stock. However, cattle inoculated with the lower dilutions of this parasite stock,as well as surviving controls, were not immune to homologous challenge. Thesignificance of this is discussed later.Table 1 . Reactions of cattle receiving titrated doses of various Theileria parva stabi-lates and treatment with either Medamycin 100 at 10 mg/kg on days 0 and4, Terramycin LA at 20 mg/kg on day 0 or buparvaquone at 2.5 mg/kg onday 0ParasitestockDrugtreatmentsCattle reaction(survived immunization)/D28Undiluted 1:10 1:100 1: 1000T. p. lawrencei (01 Pejeta)(Stabilate 1 99)No drugTerramycin LAMedamycin 100Buparvaquone0/31/30/31/31/32/33/33/31/3 3/3(2)3/33/38/81:5 1:10 1:100T. p. lawrencei (Mara III)(Stabilate 202)andNo drug 0/3 1/3T. p. parva (Kilae)(Stabilate 1 87)Terramycin LA 2/3 2/3 3/3Medamycin 100 2/3 3/3 3/3Undiluted 1:10 1:100T. p. lawrencei (Mara III)(Stabilate 202)No drug — — 2/3(1)T. p. parva (Kilae)(Stabilate 187)No drug — — 3/3 (3)Undiluted 1:10 1:807. p. parva (Marikebuni) No drug 0/5 4/4 (2) 5/5 (4)(Stabilate 3014) Terramycin LAMedamycin 1005/55/55/5(1)+5/55/5 (2)*5/5(1)*( ) Not immune to homologous challenge.( )* Negative serology after immunization (D35).( )+ Positive serology after immunization.41IMMUNIZATION REPORTSImmunization using mixed T. parva stocks, T. p. parva (Kilae) stabilate 187and a T. p. lawrencei (Mara III) stabilate 202, worked well. Mixed concentrations of these stabilates at 1:10 and 1:100 dilutions were controlled satisfactorilyby both formulations of oxytetracyclines. In a separate titration, stabilate 202was shown to be more virulent than stabilate 187. Theileria parva carrier stateswere demonstrated in some oxytetracycline-immmunized cattle, but not in bu-parvaquone-treated animals. Both T. p. parva and T. p. lawrencei stocks wereshown to produce the carrier state.CROSS-IMMUNITY STUDIESSince the advent of the infection-and-treatment immunization method, researchers have searched for a T. parva stock capable of conferring a wide protection against challenge with other theilerial parasites. An alternative approachwas to combine several theilerial parasite stocks to form an "immunization unit"with wide protection. Such a combination of parasites exemplified by the"Muguga cocktail" can be used with a measure of success, as demonstrated inseveral countries. Recently, Irvin et al. (1983) isolated a theilerial parasite stock,referred to as T. p. parva (Marikebuni), from Kilifi District, Kenya, which wasshown to provide good protection against severe challenge with other stocksisolated from the district. As the stock is well characterized, it was decided touse this isolate in cross-immunity studies with other T. parva isolates fromwidely separated areas of Kenya.Table 2 lists the T. parva isolates from Kenya used in the studies. Eight ofthese isolates (all T. p. parva) were from Kilifi District, Coast Province, Kenya.Five other stocks, two of T. p. parva and three of T. p. lawrencei, were isolatesfrom the Rift Valley Province, Kenya. One isolate, T. p. parva (Mbita), wasfrom Nyanza Province and three new isolates, Kil, Ki3 and Ki4, were fromKiambu District, Central Province. The isolation location for each isolate isindicated on the map of Kenya (Figure 1). The isolates were used in cross-immunity studies to challenge cattle immune to the T. p. parva (Marikebuni) stock.The experimental studies required the generation of T. p. parva (Marikebuni)immune cattle by immunizing groups of Friesian steers with selected doses ofthe stabilate and treating the steers with either Medamycin or Terramycin LA.Groups of the Marikebuni immune cattle were challenged in the following ways:a) challenge of Marikebuni immune cattle by other coastal T. p. parva stocksfrom Kilifi Districtb) challenge of Marikebuni immune cattle with T. p. parva and T. p. lawrencei stocks from elsewhere in Kenya42PARASITE STOCKS USED IN KENYAc) challenge of cattle immune to T. p. parva and T. p. lawrencei stocks fromelsewhere in Kenya with a lethal dose of T. p. parva (Marikebuni)The reactions of the experimental cattle on challenge were described as"inapparent" where no macroschizonts were detected, "mild" where low numbers of schizonts were detected transiently and where a transient fever may ormay not have been observed, and "severe" where prolonged schizont parasitosisoccurred, usually in high numbers for several days and accompanied by fever."Very severe" reactions were those where high schizont parasitosis wasrecorded with a marked development of fever, usually resulting in death. Cattlewith inapparent and mild reactions were considered immune. Those with severeand very severe reactions were regarded as not immune, irrespective of whetherthe animal died.ETHIOPIA Figure 1. The locations in Kenya where Theileria parva stocks were isolated for use in cross-immunity studies. The key to the locationsis given in Table 2.43IMMUNIZATION REPORTSThe results of the cross-immunity experiments are shown in Table 2. Thei-leria p. parva (Marikebuni) immune cattle were protected against challenge withseven T. p. parva stocks from Kilifi District and four T. p. parva stocks fromother areas of Kenya and showed partial protection against challenge by T. p.lawrencei stocks. Furthermore, cattle immune to various T. parva stocks wereprotected against lethal challenge with T. p. parva (Marikebuni).Table 2. Theileria parva stocks used in cross-immunity experimentsCoast Province1. 7. p. parva (Mariakani) Stabilate 30292. T. p. parva (Utange) Stabilate 2233. T. p. parva (Mtwapa) Stabilate 22624. T. p. parva (Kilifi) Stabilate 10155. T. p. parva (Kibarani) Stabilate 24486. T. p. parva (Kiswani) Stabilate 22407. T. p. parva (Magarini) Stabilate 23658. T. p. parva (Junju) Stabilate 10869. T. p. parva (Marikebuni) Stabilate 3014Rift Valley Province10. T. p. parva (Uasin Gishu 6) Stabilate 21611. T. p. parva (Kilae) Stabilate 1 8712. T. p. lawrencei (Mara III) Stabilate 20213. T. p. lawrencei (Ngongl) Stabilate 230614. T. p. lawrencei (01 Pejeta) Stabilate 1 99Nyanza Province15. T. p. parva (Mbita) Stabilate 1 69Central Province16. T. p. parva (Ki1) Stabilate 21017. T. p. parva (Ki3) Stabilate 21318. T. p. parva (Ki4) Stabilate 214Note: See Figure 1 showing location of isolation site.From the titration and cross-immunity studies the following observationswere made.a) There was an optimal range of sporozoite dose for each stabilate that couldbe controlled satisfactorily by the antitheilerial drugs, which produced subclinical theilerial reactions, and the cattle were immune to homologouschallenge. Sporozoite concentrations above the optimal dose produced severe theilerial reactions and those below either did not infect cattle (asshown by lack of T. parva antibodies) or infected cattle with only a propor44PARASITE STOCKS USED IN KENYAtion of the antigenic components of the stabilate. Such cattle were subsequently shown to be susceptible to homologous challenge. This was probably the result of certain parasite components not being present at thesevery high dilutions.b) It was possible to immunize cattle with virulent T. p. lawrencei stocks(stabilate 199 killed cattle at 1:1000 dilution), provided the right dilutionwas selected together with the right combination of drug dose and treatment regimen.c) Mixed T. p. parva and T. p. lawrencei parasites could be combined andused in various concentrations in immunization.d) The use of buparvaquone in immunization with the more difficult T. p.lawrencei stocks may be justified in special cases, but for routine T. p.parva immunization, especially on a large scale, oxytetracyclines are thedrugs of choice. Two doses of Medamycin were comparable to one dose ofTerramycin LA, but in the T. parva (Marikebuni) titration, the short-actingdrug gave slightly superior results. The Medamycin treatment regimen wasalso cheaper, but required that the animals be mustered twice.e) Cattle immunized with the T. p. parva (Marikebuni) stock were protectedagainst parasites from the Rift Valley, Central and Nyanza provinces anddid not break through the immunity provided by T. parva stocks fromgeographically separated areas of Kenya. Theileria p. parva (Marikebuni)could provide a master immunizing stock for cattle-derived theileriosis inKenya.REFERENCESBurridge, MJ. and Kimber, CD. (1972). The indirect fluorescent antibody testfor experimental East Coast fever (Theileria parva infection of cattle):evaluation of a cell culture schizont antigen. Research in VeterinaryScience 13: 451-455.Irvin, A.D., Dobbelaere, D.A.E., Mwamachi, B.M., Minami, T.P. and Ocama,J.G.R. (1983). Immunization against East Coast fever: correlation betweenmonoclonal antibody profiles of Theileria parva stocks and cross-immunityin vivo. Research in Veterinary Science 35: 341-344.45

Parasite stocks used for East Coast Fever immunization in Kenya

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Parasite stocks used for East Coast Fever immunization in Kenya

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