Veterinary Parasitology, 43 ( 1992 ) 1-14 Elsevier Science Publishers B.V., Amsterdam
1
Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization A.C. Maritim, A.S. Young, A.C. Lesan, S.G. Ndungu, D.A. Stagg and P.N. Ngumi Protozoology DivisTon, National Veterinary Research Centre, Muguga, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya (Accepted 16 December 1991 )
ABSTRACT Maritim, A.C., Young, A.S., Lesan, A.C., Ndungu, S.G., Stagg, D.A. and Ngumi, P.N., 1992. Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization. Vet. Parasitol., 43: 1-14. A sporozoite stabilate (St. 199 ) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.
INTRODUCTION
The form of Theileria parva which causes Corridor disease of cattle, is normally maintained within the African buffalo (Syncerus caffer) population (Neitz, 1957). At various times the buffalo-derived forms of T. parva have Correspondence to."A.S. Young, International Laboratory for Research on Animal Disease, P.O. Box 30709, Nairobi, Kenya.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
,) -
A.C. MARITIM
ETAL.
been described as Theileria lawrencei (Neitz, 1955) or as Theileria parva lawrencei (Uilenberg, 1976). Recent studies have shown that these forms are not genetically distinct from T. parva derived from cattle and causing East Coast fever (Conrad et al., 1989). Therefore, both forms should be described as T. parva (Anonymous, 1989). It is believed that T. parva evolved in the African buffalo in association with its vector the ixodid tick, Rhipicephalus appendiculatus (Uilenberg, 1981 ; Young, 1981 ). The form of Theileria parva causing East Coast fever (ECF) of cattle evidently developed from the buffalo form, as cattle are a relatively new introduction into East Africa. This possibility is well illustrated by a series of studies where East African isolates of T. parva derived from buffalo have been passaged using tick transmission through cattle (Barnett and Brocklesby, 1966, 1969; Young and Purnell, 1973 ). On each occasion the original buffalo-derived isolate, which kills cattle with low parasitosis of schizonts and no or few intra-erythrocytic piroplasms, behaviourally transformed after several tick/cattle passages, and became indistinguishable from the cattle form in that cattle died with high parasitosis of schizonts and many intra-erythrocytic piroplasms. It is of interest that attempts by South African scientists (Neitz, 1957; De Vos, 1982; Potgieter et al., 1988 ) have failed to transform the buffalo form into the cattle form. This p h e n o m e n o n of behavioural transformation is of importance because Young et al. ( 1973, 1977, 1978) and Radley et al. ( 1975, 1979 ) have shown that there is a lack of cross protection between stocks of T. parva derived from buffalo and cattle and Conrad et al. (1987, 1989) have demonstrated that there is more antigenic diversity in the buffalo stocks than in the cattle stocks. Hence, buffalo could represent a source of new antigenic types of T. parva which could be introduced into cattle populations. Another feature is that stocks of T. parva derived from buffalo are difficult to use in infection and treatment immunization using oxytetracycline, the only practical method of immunization against T. parva available at present (Young et al., 1988 ). The first problem is obtaining the required quantities of sporozoites from African buffalo and the second is that T. parva derived from buffalo is more variable in its reaction to drug treatment than T. parva (Dolan et al., 1984a; Mutugi et al., 1988b) as it tends to break through the drug cover. Hence in this study, attempts were made to transform buffalo-derived T. parva by tick/cattle passage so that it behaved like the cattle form and to characterize the parasite during this process of transformation. After transformation to the cattle form, this population of parasites was compared for immunogenicity with the original population in cross-immunity experiments. In this way, it was hoped to overcome the problems of using T. parva sporozoite stabilates derived from buffalo in infection and treatment immunization.
TRANSFORMATION OF THEILERI.! I~tRI;,t DERIVED FROM BUFFALO
3
MATERIALS AND METHODS
Cattle Friesian steers of about 1 year of age were used. They were purchased from farms in the Naivasha Division of Nakuru District, Kenya where strict tick control regimes are practised. Prior to experiments, all cattle were negative for antibodies to T. parva schizont antigen in the indirect fluorescent antibody test (IFAT) (Burridge and Kimber, 1972).
Ticks Rhipicephalus appendiculatus nymphal ticks used were obtained from the Muguga colony which has been maintained by feeding all stages on rabbits since 1952 (Bailey, 1960). Theileria parasites
Theileria parva (O1 Pejeta) stock was derived from feeding ticks on a buffalo (7014) captured in Laikipia District, Kenya. The history of this stock has been described by Mutugi et al. (1988a). The isolation of T. parva from a carrier steer (V565) after immunization by infection and treatment with the buffalo-derived sporozoites St. 199 and concurrent treatment with a longacting formulation of oxytetracycline (Terramycin LA, Pfizer Ltd., Sandwich, U K ) has been described by Maritim et al. (1989a,b). Transmission experiments Experiment 1 Two thousand clean unfed R. appendiculatus nymphs from laboratory stocks were applied to the ears of the immunized carrier steers (V565) according to the methods of Bailey (1960). The engorged nymphs were collected and incubated at 28 °C and 80% relative humidity to complete moulting. Theileria infection rates in the salivary glands of the tick were established (Young and Leitch, 1981 ). Passage ofTheileria parva in cattle Experiment 2 The adult ticks obtained from Experiment 1 were applied to individual susceptible cattle (see Table 1 and Fig. 1 ) to obtain the second passage infections in cattle. Rectal temperatures were taken and biopsy smears were prepared from the subparotid lymph nodes draining the site of tick application, stained
4
A.C. MARITIM ET AL.
TABLE 1
Theilerial reactions of cattle infected with T. parva (O1 Pejeta) during tick/cattle passage or i n o c u l a t i o n with stabilate 2 2 6 of passages through cattle
Method of infection
1
St.199
2 2 2 2
No.
No. of ticks applied
Cattle no.
Theilerial reaction days to: Schizonts
Fever
.
Recovery
.
.
Death
Maximum piroplasm (% erythrocytes infected )
-
V565
.
.
Ticks Ticks Ticks Ticks
150 100 100 80
Z061 Z360 Z369 Z639
10 11 16 13
10 26 25 16
21 28 30 25
-
3 3
Ticks St.226
200 -
Z636 Z977
9 13
14 -
35
19 -
3
St.226
-
Z978
14
32
35
-
1
Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization A.C. Maritim, A.S. Young, A.C. Lesan, S.G. Ndungu, D.A. Stagg and P.N. Ngumi Protozoology DivisTon, National Veterinary Research Centre, Muguga, Kenya Agricultural Research Institute, P.O. Box 32, Kikuyu, Kenya (Accepted 16 December 1991 )
ABSTRACT Maritim, A.C., Young, A.S., Lesan, A.C., Ndungu, S.G., Stagg, D.A. and Ngumi, P.N., 1992. Transformation of Theileria parva derived from African buffalo (Syncerus caffer) by tick passage in cattle and its use in infection and treatment immunization. Vet. Parasitol., 43: 1-14. A sporozoite stabilate (St. 199 ) of Theileria parva was obtained by feeding nymphal Rhipicephalus appendiculatus on an African buffalo (Syncerus caffer) and was used to immunize cattle by the infection and treatment method. Nymphal ticks were applied to one of the steers 90 days later and it was shown that the resultant adult tick had become infected. Using tick/cattle passage, two passage lines of T. parva were established. By the fifth tick/cattle passage, the parasite stocks had changed their behaviour to that of T. parva derived from cattle as the parasite produced relatively high schizont parasitosis and piroplasm parasitaemia in cattle, and had become highly infective to ticks. At various passage levels the parasite populations were characterized by behaviour and by monoclonal antibodies against T. parva schizonts using infected cell culture isolates from cattle during acute infections. The monoclonal antibody profile showed little evidence of antigen change of the parasite during passage through cattle, which was confirmed in a two-way cross-immunity experiment using sporozoite stabilate derived from ticks obtained from the buffalo and fourth passage in cattle. The implication of these results, particularly in relationship to immunization of cattle against T. parva derived from buffalo, is discussed.
INTRODUCTION
The form of Theileria parva which causes Corridor disease of cattle, is normally maintained within the African buffalo (Syncerus caffer) population (Neitz, 1957). At various times the buffalo-derived forms of T. parva have Correspondence to."A.S. Young, International Laboratory for Research on Animal Disease, P.O. Box 30709, Nairobi, Kenya.
© 1992 Elsevier Science Publishers B.V. All rights reserved 0304-4017/92/$05.00
,) -
A.C. MARITIM
ETAL.
been described as Theileria lawrencei (Neitz, 1955) or as Theileria parva lawrencei (Uilenberg, 1976). Recent studies have shown that these forms are not genetically distinct from T. parva derived from cattle and causing East Coast fever (Conrad et al., 1989). Therefore, both forms should be described as T. parva (Anonymous, 1989). It is believed that T. parva evolved in the African buffalo in association with its vector the ixodid tick, Rhipicephalus appendiculatus (Uilenberg, 1981 ; Young, 1981 ). The form of Theileria parva causing East Coast fever (ECF) of cattle evidently developed from the buffalo form, as cattle are a relatively new introduction into East Africa. This possibility is well illustrated by a series of studies where East African isolates of T. parva derived from buffalo have been passaged using tick transmission through cattle (Barnett and Brocklesby, 1966, 1969; Young and Purnell, 1973 ). On each occasion the original buffalo-derived isolate, which kills cattle with low parasitosis of schizonts and no or few intra-erythrocytic piroplasms, behaviourally transformed after several tick/cattle passages, and became indistinguishable from the cattle form in that cattle died with high parasitosis of schizonts and many intra-erythrocytic piroplasms. It is of interest that attempts by South African scientists (Neitz, 1957; De Vos, 1982; Potgieter et al., 1988 ) have failed to transform the buffalo form into the cattle form. This p h e n o m e n o n of behavioural transformation is of importance because Young et al. ( 1973, 1977, 1978) and Radley et al. ( 1975, 1979 ) have shown that there is a lack of cross protection between stocks of T. parva derived from buffalo and cattle and Conrad et al. (1987, 1989) have demonstrated that there is more antigenic diversity in the buffalo stocks than in the cattle stocks. Hence, buffalo could represent a source of new antigenic types of T. parva which could be introduced into cattle populations. Another feature is that stocks of T. parva derived from buffalo are difficult to use in infection and treatment immunization using oxytetracycline, the only practical method of immunization against T. parva available at present (Young et al., 1988 ). The first problem is obtaining the required quantities of sporozoites from African buffalo and the second is that T. parva derived from buffalo is more variable in its reaction to drug treatment than T. parva (Dolan et al., 1984a; Mutugi et al., 1988b) as it tends to break through the drug cover. Hence in this study, attempts were made to transform buffalo-derived T. parva by tick/cattle passage so that it behaved like the cattle form and to characterize the parasite during this process of transformation. After transformation to the cattle form, this population of parasites was compared for immunogenicity with the original population in cross-immunity experiments. In this way, it was hoped to overcome the problems of using T. parva sporozoite stabilates derived from buffalo in infection and treatment immunization.
TRANSFORMATION OF THEILERI.! I~tRI;,t DERIVED FROM BUFFALO
3
MATERIALS AND METHODS
Cattle Friesian steers of about 1 year of age were used. They were purchased from farms in the Naivasha Division of Nakuru District, Kenya where strict tick control regimes are practised. Prior to experiments, all cattle were negative for antibodies to T. parva schizont antigen in the indirect fluorescent antibody test (IFAT) (Burridge and Kimber, 1972).
Ticks Rhipicephalus appendiculatus nymphal ticks used were obtained from the Muguga colony which has been maintained by feeding all stages on rabbits since 1952 (Bailey, 1960). Theileria parasites
Theileria parva (O1 Pejeta) stock was derived from feeding ticks on a buffalo (7014) captured in Laikipia District, Kenya. The history of this stock has been described by Mutugi et al. (1988a). The isolation of T. parva from a carrier steer (V565) after immunization by infection and treatment with the buffalo-derived sporozoites St. 199 and concurrent treatment with a longacting formulation of oxytetracycline (Terramycin LA, Pfizer Ltd., Sandwich, U K ) has been described by Maritim et al. (1989a,b). Transmission experiments Experiment 1 Two thousand clean unfed R. appendiculatus nymphs from laboratory stocks were applied to the ears of the immunized carrier steers (V565) according to the methods of Bailey (1960). The engorged nymphs were collected and incubated at 28 °C and 80% relative humidity to complete moulting. Theileria infection rates in the salivary glands of the tick were established (Young and Leitch, 1981 ). Passage ofTheileria parva in cattle Experiment 2 The adult ticks obtained from Experiment 1 were applied to individual susceptible cattle (see Table 1 and Fig. 1 ) to obtain the second passage infections in cattle. Rectal temperatures were taken and biopsy smears were prepared from the subparotid lymph nodes draining the site of tick application, stained
4
A.C. MARITIM ET AL.
TABLE 1
Theilerial reactions of cattle infected with T. parva (O1 Pejeta) during tick/cattle passage or i n o c u l a t i o n with stabilate 2 2 6 of passages through cattle
Method of infection
1
St.199
2 2 2 2
No.
No. of ticks applied
Cattle no.
Theilerial reaction days to: Schizonts
Fever
.
Recovery
.
.
Death
Maximum piroplasm (% erythrocytes infected )
-
V565
.
.
Ticks Ticks Ticks Ticks
150 100 100 80
Z061 Z360 Z369 Z639
10 11 16 13
10 26 25 16
21 28 30 25
-
3 3
Ticks St.226
200 -
Z636 Z977
9 13
14 -
35
19 -
3
St.226
-
Z978
14
32
35
-
