Walker.indd Theileria parva is a protozoan parasite of bovids which is transmitted between cattle by the tick Rhip- i cephalus appendiculatus in East, Central and South- ern Africa. Infection of cattle causes East Coast fever with a mortality rate that constrains the profitability of cattle rearing (Irvin & Morrison 1987). When piro- plasms of T. parva in erythrocytes are ingested by the tick they differentiate into microgametes and mac ro gametes. These undergo syngamy to form zy- gotes which invade digestive cells. Then a kinete develops from each zygote and this migrates from the gut to the salivary glands (Schein, Warnecke & Kirmse 1977; Mehlhorn, Schein & Warnecke 1978; Fawcett, Büscher & Doxsey 1982). The kinete de- velops in e cells of the salivary gland by integration with the metabolism of the e cells. Few sporoblasts are produced relative to the number of piroplasms ingested and it is thought that Theileria is suscepti- ble to digestion by the tick or tick innate immunity (Pur nell & Joyner 1968). When the tick next feeds the sporoblast grows and divides to produce thou- sands of sporozoites. Tick salivation enables sporo- zoites to enter cattle and infect lymphocytes. There the schizont stage develops and undergoes further nuclear division (Shaw & Young 1995). The infec- tion of the lymphocytes induces them to divide, each daughter cell taking dividing schizonts with it. The final stage in the lymphocytes is the merozoite which develops within the schizont. Merozoites are re- leased into the blood plasma and infect erythrocytes 157 Onderstepoort Journal of Veterinary Research, 73:157–162 (2006) RESEARCH COMMUNICATION Cloned Theileria parva produces lesser infections in ticks compared to uncloned T. parva despite similar infections in cattle A.R. WALKER*, F. KATZER, D. NGUGI and D. MCKEEVER Centre for Tropical Veterinary Medicine, Royal (Dick) School of Veterinary Studies University of Edinburgh, Roslin, EH25 9RG, Scotland, UK ABSTRACT WALKER, A.R., KATZER, F., NGUGI, D. & McKEEVER, D. 2006. Cloned Theileria parva produces lesser infections in ticks compared to uncloned T. parva despite similar infections in cattle. Onder ste- poort Journal of Veterinary Research, 73:157–162 Experimental transmissions of cloned Theileria parva in cattle with Rhipicephalus appendiculatus ticks were compared to transmissions with uncloned T. parva during studies on the potential for ge- netic recombination during syngamy of Theileria to produce antigenic diversity for evasion of bovine immunity. Prevalence and abundance of T. parva infection in adult ticks, which resulted from the feed- ing of nymphs on the calves, were significantly higher in the uncloned compared to the cloned T. parva. Development of sporoblasts of T. parva in the ticks to produce infective sporozoites was simi- lar. There was no statistically significant difference in the clinical course of infection in cattle between cloned and uncloned T. parva. It was concluded that cloned T. parva has characteristics that reduce its viability during the tick stages of its life cycle. Keywords: Clone, East Coast fever, infection, Rhipicephalus appendiculatus, Theileria parva * Author to whom correspondence is to be directed. E-mail: alanw@staffmail.ed.ac.uk Accepted for publication 29 March 2006—Editor 158 Cloned Theileria parva infections in ticks compared to uncloned T. parva in which they are known as piroplasms. Successful immunity, mediated mainly by cytotoxic lympho cytes, may confine infections with potentially fatal doses of sporozoites to ones that produce few clinical signs and very low parasitaemias of piroplasms in which form the Theileria may be transmitted to more ticks (McKeever, Taracha, Innes, MacHugh, Awina, God- deeris, & Morrison 1994). Existing vaccination procedures using the infection and treatment method manipulate this cycle to pro- duce sporozoites in cryopreserved form which are inoculated to mimic natural infection. To develop syn thetic antigen vaccines it is necessary to under- stand how Theileria evades cattle immunity (Mor- zaria, Nene, Bishop & Musoke 2000). This evasion is thought to be mediated by continual generation of antigenically diverse strains during recombination in the tick (McKeever 2001). Cloning Theileria is a tech- nique for studying the genetics of strain diversity. This communication is from a project which used transmissions with cloned T. parva to produce re- combined stocks with hypothetical potential for im- mune evasion. We found abnormally low levels of infection in ticks with cloned Theileria. Therefore re- sults of previous transmission experiments with un- cloned Theileria of the same strains were compared with the performances of cloned Theileria in both cattle and tick infections in order to determine what levels of transmissibility can be expected when plan- ning experiments. Theileria parva of two strains which had been iso- lated from two sites in Kenya (Muguga and Mar ike- buni) were used. Both strains have been maintained as cryopreserved stabilates used for cattle to tick transmissions. The uncloned Muguga strain consists of a fairly homogeneous parasite population which can be distinguished by four genetic satellite mark- ers (MS7, MS14, MS16 and MS25) developed by Oura, Odongo, Lugega, Spooner, Tait & Bishop (2003). The uncloned Marikebuni strain, however, is very diverse, and at least 48 out of the 60 markers described by Oura et al. (2003) reveal polymor- phism between its constituents. Clones of these two strains were produced by infecting 1 x 107 periph- eral blood mononuclear cells (PBMC) in vitro with a dose of T. parva stabilate equivalent to that carried by one infected tick. At 48 h after infection the cells were plated out by limiting dilution with non-infected PBMC in U bottom 96-well plates (Morzaria, Dolan, Norval, Bishop & Spooner 1995). The wells were scored for growth after 14 days and cells were har- vested for DNA extraction and typing by satellite markers. Male calves were Friesian at the Centre for Tropical Veterinary Medicine (CTVM) and Boran at the Inter- national Livestock Research Institute (ILRI) in Kenya. All were without previous exposure to T. parva. They were kept in isolation pens (20 °C at CTVM) and mon- itored daily during infections for temperature, schiz- onts in the lymph node draining the site of infection and piroplasms in venous erythrocytes. The Theileria used as inocula were either stabilated spor ozoites from infected adult ticks that had been partially fed for 4 days, or cell culture containing schiz onts in PBMC, or in infected ticks. The doses of Theileria were calculated from previous in vivo and in vitro titra tions to give patent piroplasm para sita emias (0.1 % or more of erythrocytes infected). Infec tions were controlled when necessary with tetra cyc line (short acting) injected intramuscularly at 10 mg/kg for up to 5 days starting from first pyrexia and appear- ance of schizonts. Experience has shown that this has no effect on infections in the ticks. The ticks used were R. appendiculatus of a stock originating in the 1960s from Muguga in Kenya which have been maintained at CTVM since then. In addi- tion, one experiment at ILRI used a stock desig nated as Muguga, of the same origin as above but main- tained at ILRI since the 1970s. Transmissions were done by application of uninfected nymphal ticks to calves at a time corresponding with expected peak of piroplasm parasitaemia in red blood cells. Engorged nymphs detaching from the cattle were maintained at 28 °C, 85 % relative humidity for 28 days until com- pletion of post moult development. Adults of both sexes in equal numbers (minimum sample of 32 ticks) that had detached on the day of peak piroplasm parasitaemia were assessed for infection with T. par- va by incubation for 5 days at 37 °C and 100 % rela- tive humidity, followed by dissection and staining of their salivary glands with methyl green and pyronin (Walker, McKellar, Bell & Brown 1979). Counts were made by microscopy of sporoblasts per pair of sali- vary glands. Data were not normally distributed thus analysis used Chi square, Mann Whitney U test and Kendall’s rank correlation. Percentages were con- verted by arcsin and abundances of Theileria spor o- blasts in ticks were transformed to log10(n +1) to nor- malize the overdispersion of this variable (Büscher & Otim 1986). Two batches of uncloned and cloned Theileria in equal infection levels in ticks were com- pared for degree infectivity of the sporozoites for bovine cells after the Theileria were matured by par- tially feeding the ticks. Batches of ten ticks were sur- face sterilized and then ground in commercial me- dium using tissue grinders to release sporozoites. The suspensions of sporozoites were used to infect 159 A.R. WALKER et al. cultures of bovine peripheral blood mononuclear cells in an infectivity titration (Wilkie, Kirvar & Brown 2002). Historical data on transmissions using uncloned Thei leria could not be standardized for use in the comparisons with cloned Theileria of this study. Three of the ten transmissions reported here repre- senting cloned Theileria were of mixed infections using two different clones of Theileria sufficiently dis- tinguishable by genetic markers for analysis of ge- netic recombinations. The outcome of these mixed infections was examined by polymerase chain reac- tion (PCR) using a sub-set of published satellite markers (Oura et al. 2003) which distinguish between the parent Theileria using their specified PCR con- ditions. The DNA used for this was extracted from lymph node and blood sampled repeatedly during the infections. From lymph node cells DNA was ex- tracted using the Promega Wizard SV Genomic DNA Purification System and for blood samples the Qiagen QIAamp DNA Blood Mini Kit was used. The Theileria types in the ticks were also determined by PCR using the same satellite markers on DNA that was extracted either from dissected salivary glands or from whole ground ticks, with the same method as for lymph node cells. The analysis of the mixed infections in Fig. 1 shows that the infections of animals BZ114 and BZ115 were dominated by the T. parva Marikebuni clone and that the T. parva Muguga clone was barely detectable during the infection (Lanes 11 and 12). This was also seen in the infections in the ticks, where ticks fed on animal BZ114 did not pick up T. parva Muguga and for BZ115 Muguga alleles were only barely detect- able (data not shown). We considered these mixed infections were more characteristic of infections from single sources and did not exclude them from further comparisons with single source infections. Table 1 shows data from ten transmissions using cloned T. parva that met the criteria of producing patent infection with pyrexia at 40.0 °C and piro- plasms detectable by microscopy. These are com- pared with seven transmissions using uncloned T. parva meeting the same criteria. Sufficient data for a statistical comparison were obtained by combin- ing results from infections with both strains of T. parva. Experience at CTVM and ILRI have shown that although these strains are different in immuno- logical cross reactivity, they produce similar infec- tions in cattle and ticks. Moreover, the five clinical variables measured in days were used to compare the Muguga and Marikebuni strains by Chi square tests between different pairs of calves. No signifi- cant differences were found between these pairs. The grouping of this data was considered justified for further comparison using Mann Whitney U test. The threshold for recording pyrexia was strict, at Lane 1 Negative control Lane 2 Stabilate 64 (Marikebuni) Lane 3 Stabilate 70 (Marikebuni) Lane 4 Stabilate 81 (Muguga) Lane 5 Stabilate 72 (Marikebuni) Lane 6 Calf 211 infected with stabilate 72 Lane 7 Clone A3 Lane 8 Clone 13 Lane 9 Muguga clone 1, ILRI stabilate 3308 Lane 10 Marikebuni clone 1, ILRI stabilate 4210 Lane 11 Calf BZ114, Day 21 of infection with stabilates 3308 + 4210 Lane 12 Calf BZ115, Day 21 of infection with stabilates 3308 + 4210 1 650 bp 1 000 bp 850 bp 650 bp 500 bp 400 bp 1 2 3 4 765 12111098 FIG. 1 Polymorphism in PCR size detected within the PIM gene of T. parva 1 6 0 C lo n e d T h e ile ria p a rva in fe ctio n s in ticks co m p a re d to u n clo n e d T . p a rva TABLE 1 Infection characteristics of cloned compared to uncloned T. parva in cattle and Ticks. Ten transmissions with cloned T. parva and seven transmissions with uncloned T. parva, all meeting the criteria of patent disease with pyrexia and production of piroplasms. Analysis by Mann-Whitney U test Strain of T. parva Clone number Infection method Day 1st pyrexia Day 1st schizont Day 1st piroplasm Day max. piroplasm Days pyrexic Max. % piroplasms Prevalence of sporoblasts per tick (%) Abundance of sporoblasts per tick Muguga Muguga Marikebuni Marikebuni Marikebuni Marikebuni Marikebuni Marikebuni Muguga + Marikebuni Muguga + Marikebuni 3308 3308 3262 3262 13 + 17 4210 4210 A3 3308 + 4210 3308 + 4210 Stabilate Ticks Ticks Ticks cells Stabilate Stabilate Cells Stabilates Stabilates 8 12 9 6 8 12 7 9 10 10 10 10 11 6 6 11 8 6 7 6 12 17 15 14 10 14 12 10 12 11 17 18 17 16 12 18 15 14 18 19 11 3 9 9 4 5 9 4 9 9 5.6 0.1 1.2 0.1 0.01 1.2 2.0 0.01 5.8 2.6 60 1.7 35 0 0.9 52 38 14 19 6 17.9 0.3 4.5 0 0.3 13.0 4.2 0.7 0.9 0.01 Medians 9.0 7.5 12.0 17.0 9.0 1.2 16.5 0.8 Muguga Muguga Muguga Muguga Muguga Marikebuni Marikebuni Not Not Not Not Not Not Not Stabilate Stabilate Stabilate Stabilate Stabilate Stabilate Stabilate 12 6 8 8 6 12 9 11 5 5 6 6 10 6 13 10 10 12 12 14 12 14 16 16 15 16 16 17 6 11 9 7 11 6 10 0.1 7.9 5.5 3.2 7.3 0.2 6.0 95 51 88 59 58 95 100 121 17.9 63.7 29.7 7.9 78 44 Medians 8.0 6.0 12.0 16.0 9.0 5.5 88.0 58.0 Probability 0.65 0.20 0.56 0.25 0.27 0.15 0.003* 0.002* * Significantly different at P < 0.01 161 A.R. WALKER et al. 40.0 °C, and the number of days pyrexic was re- corded up to Day 18. When ticks were used to in- fect, Day 0 of cattle infection was counted as Day 4 of tick feeding. The course of infection of the calves using cloned Theileria was similar to that using uncloned Theileria. The median values of the characteristics measured in days were the same or representing milder dis- ease in the cloned Theileria infections. The number of days pyrexic is the best single measure of these infections; it was 9.0 in both the infections with cloned T. parva and with uncloned T. parva. None of the comparisons of these characters between cloned and uncloned T. parva were significantly different. Although the level of piroplasm parasitaemia was more than twofold higher in the transmissions of un- cloned Theileria, this difference was not significant. In contrast, the differences of infection with Theileria of the ticks from the transmission with cloned com- pared to uncloned Theileria were large and highly significant for both characteristics of prevalence of infection and abundance of infection. The relation between piroplasm parasitaemia and tick infection was examined using data from the uncloned T. par- va transmissions. Kendall’s rank correlations showed an inverse relationship, with negative and nearly sig- nificant correlations (piroplasms against prevalence –0.5, P = 0.06; piroplasms against abundance –0.49; P = 0.06). The results obtained at CTVM contrasted greatly with those from ILRI where, in totals of 29 transmis- sions with uncloned T. parva Muguga and separately T. parva Marikebuni, compared to 17 similar trans- missions with cloned T. parva of both strains, the un- cloned transmissions resulted in median tick infec- tions having prevalence of 60.0 % ticks infected and abundance of 20.8 sporoblasts per tick compared to cloned transmissions with prevalence of 72.0 % and abundance of 22.0 (Paul Spooner, personal com- munication 2005). Table 2 shows that similar high levels of infectivity of sporozoites for bovine cells were found with the batches of cloned and uncloned T. parva Marikebuni strain. There was no significant difference when compared by Chi square test. It was important that only simple conclusions were drawn from the analysis because of the highly vari- able clinical course of infections with Theileria. We con sider that we have found a characteristic of cloned T. parva that may have an influence on studies us- ing clones and that this needs to be reported so that it can be incorporated into experimental procedures. Cloned T. parva produced a course of disease in calves with a clinical pattern without significant dif- ferences from that produced by uncloned T. parva. The cloned T. parva produced an acute infection that was potentially fatal and with sufficient piro- plasms in the peripheral blood to expose feeding ticks to the typical massive numbers of the microg- amete and macrogamete stage of Theileria in the tick’s gut. Cloned T. parva infections in ticks were characterized by prevalences and abundances of infection that were both significantly lower than ob- tained with uncloned T. parva. It can be considered that this difference was due to the cloned Theileria infections producing a median of maximum piro plasm parasitaemias of 1.2 % compared to 5.5 % for the un cloned Theileria. However, these data are not sig- nificantly different and it is clear from Table 1 that low piroplasm parasitaemias can give rise to high prev- alence and abundance of infection in some cases. Young, Dolan, Morzaria, Mwakima, Norval, Scott, Sherriff & Gettinby (1996) demonstrated, with trans- missions in 113 calves of uncloned T. parva Muguga to R. appendiculatus, that there is a significant and positive linear regression of piroplasm parasitaemia with both prevalence and abundance of infection in ticks. However, this relationship was not simple; a large increase in piroplasm parasitaemia related to only a small increase in infections in the ticks. The data we present do not support a proposition that the difference in tick infections between cloned and uncloned T. parva was due directly to the difference in piroplasm parasitaemias. The infections of the ticks with cloned T. parva in the tick’s salivary glands TABLE 2 In vitro assessment of infectivity of sporozoites of T. parva Marikebuni uncloned and clone 13. Data are mean numbers of wells with growth out of a total of 96 available wells for each concentration of cells per well 9 000 cells per well 3 000 cells per well 1 000 cells per well 300 cells per well Total 4 day fed ticks Uncloned Cloned Cloned 92 96 96 67 91 88 37 61 56 14 28 22 210 262 262 5 day fed ticks Uncloned Cloned 96 93 91 71 80 29 38 14 305 207 162 Cloned Theileria parva infections in ticks compared to uncloned T. parva matured normally to produce sporozoites of infectiv- ity to cattle cells that were similar to uncloned T. parva. These results indicate that there was a characteris- tic of cloned T. parva that either reduced the rate of syngamy or viability of the zygotes and kinetes in the tick. It may be important that the meiotic reduc- tion division of T. parva is thought to occur in the kinete stage (Gauer et al. 1995). The tick gut and haemolymph between the gut and salivary glands are potentially hostile from the likely effects of an innate immune system of this arthropod. Theileria parva in high levels of infection in R. appendiculatus can harm the salivary glands and thus it is likely that this species of tick has evolved immune defences against it (Watt & Walker 2000). Genetic experiments using cloned Theileria should be adjusted to this anomaly. ACKNOWLEDGEMENTS The work at the Centre for Tropical Veterinary Med- icine was supported by the Wellcome Trust. We thank the Royal (Dick) School of Veterinary Studies, and the International Livestock Research Institute for provision of facilities and encouragement for such clinical investigations. We are specially grateful for the considerable experimental contributions to this study of Paul Spooner at ILRI. REFERENCES BÜSCHER, G. & OTIM, B. 1986. Quantitative studies on Theileria parva in the salivary glands of Rhipicephalus appendiculatus adults: quantitation and prediction of infection. International Journal of Parasitology, 16:93–100. FAWCETT, D.W., BÜSCHER, G. & DOXSEY, S. 1982. Salivary gland of the tick vector of East Coast fever. 3: the ultrastruc- ture of sporogony in Theileria parva. Tissue and Cell, 14:83– 206. GAUER, M., MACKENSTEDT, U., MEHLHORN, H., SCHEIN, E., ZAPF, F., NJENGA, E., YOUNG, A. & MORZARIA, S. 1995. DNA measurements and ploidy determination of develop- mental stages in the life cycles of Theileria annulata and Thei- l eria parva. Parasitology Research, 81:565–574. IRVIN, A.D. & MORRISON, W.I. 1987. Immunopathology, immu- nology and immunoprophylaxis of Theileria infections, in Immune responses in parasitic infections: immunology, im- munopathology and immunoprophylaxis, edited by E.J.L. Souls by. Boca Raton, Florida: CRC Press. McKEEVER, D.J., TARACHA, E.L.N., INNES, E.L., MACHUGH, N.D., AWINA, E., GODDEERIS, B.M. & MORRISON, W.I. 1994. Adoptive transfer of immunity to Theileria parva in CD8+ fraction of responding efferent lymph. Proceedings of the National Academy of Sciences of the United States of America, 91:1959–1963. McKEEVER, D.J. 2001. Cellular immunity against Theileria par- va and its influence on parasite diversity. Research in Vet- erinary Science, 70:78–81. MEHLHORN, H., SCHEIN, E. & WARNECKE, M. 1978. Electron microscopic studies on the development of kinetes of Thei- leria parva in gut of vector ticks Rhipicephalus appendicula- tus. Acta Tropica, 35:123–136. MORZARIA, S.P., DOLAN T.T., NORVAL, R.A.I., BISHOP, R.P. & SPOONER, P.R. 1995. Generation and characterization of cloned Theileria parva parasites. Parasitology, 111:39–49. MORZARIA, S.P., NENE, V., BISHOP, R. & MUSOKE, A. 2000. Vaccines against Theileria parva. Tropical Veterinary Dis- eases, 916:464–473. OURA, C.A.L., ODONGO, D.O., LUGEGA, G.W., SPOONER, P.R., TAIT, A. & BISHOP, R.P. 2003. A panel of microsatel- lite and minisatellite markers for the characterisation of field isolates of Theileria parva. International Journal for Parasit ol- ogy, 33:1641–1653. PURNELL, R.E. & JOYNER, L.P. 1968. Development of Theileria parva in the salivary glands of the tick Rhipicephalus appen- diculatus. Parasitology, 58:725–732. SCHEIN, E., WARNECKE, M. & KIRMSE, P. 1977. Development of Theileria parva in gut of Rhipicephalus appendiculatus. Parasitology, 75:309–316. SHAW, M.M. & YOUNG, A.S. 1995. Differential development and emission of Theileria parva sporozoites from the salivary gland of Rhipicephalus appendiculatus. Parasitology, 111: 153–160. WALKER, A.R., McKELLAR, S.B., BELL, L.J. & BROWN, C.G.D. 1979. Rapid quantititative assessment of Theileria infection in ticks. Tropical Animal Health and Production, 11:21–26. WATT, D.M. & WALKER, A.R. 2000. Pathological effects and reduced survival in Rhipicephalus appendiculatus ticks in- fected with Theileria parva protozoa. Parasitology Research, 86:207–214. WILKIE, G.M., KIRVAR, E. & BROWN, C.G.D. 2002. Validation of an in vitro method to determine infectivity of cryopreserved sporozoites in stabilates of Theileria parva. Veterinary Para- sitology, 104:199–209 YOUNG, A.S., DOLAN, T.T., MORZARIA, S.P., MWAKIMA, F. N., NORVAL, R.A.I., SCOTT, J., SHERRIFF, A. & GETTINBY, G., 1996. Factors affecting infections in Rhipicephalus ap- pendiculatus ticks fed on cattle infected with Theileria parva. 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