The importance of the carrier state of Theileria parva in the epidemiology of theileriosis and its control by immunization

A.C. Maritim, D.P. Kariuki, A.S. Young and J.J. Mutugi

National Veterinary Research Centre
Kenya Agricultural Research Institute
P.O. Box 32
Kikuyu, Kenya

A full understanding of the carrier state of theilerial parasites in their mammalian hosts is central for a description of the epidemiology of the disease and for assessing the implications of immunization with live parasites. The carrier state of Theileria has been defined as the ability of an infected and recovered host to infect ticks which are then able to transmit the parasite to susceptible animals (Levine, 1973; Schwabe et al., 1977; Young et al., 1986). It is possible for an infected host to develop a primary parasitosis and parasitaemia that are eventually cleared, leaving the host with-a sterile immunity. However, it is recognized that in some Theileria-infected animals, infection persists after recovery and is probably maintained by two phases of the life cycle: first, by the slow proliferation of macroschizont-infected lymphocytes, some of which develop into microschizonts and give rise to merozoites infective to erythrocytes, and, second, by the regular division of intra-erythrocyte piroplasms.

Gametocytes of Theileria, which develop in the erythrocytes, are believed to be the stage infective for the feeding tick (Gonder, 1911; Mehlhorn and Schein, 1984). Thus the presence or absence of circulating gametocytes determines whether an animal is a carrier at a given time. It is, therefore, possible for an animal with a persistent infection to act either as an initial carrier, a sporadic carrier or a continual carrier, depending on whether the gametocytes infective to ticks are present initially and then lost, or are present intermittently or continuously. Of major importance to the epidemiology of theileriosis is when ticks that became infected by feeding on a carrier have the potential to transmit the parasite to a new host.

There has been no problem in understanding how T. mutans, T. taurotragi, T. velifera, T. annulata and T. orientalis maintain their carrier state, since both division in erythrocytes and long-term persistence of the piroplasms within the erythrocytes have been demonstrated (Conrad, 1983; Conrad et al., 1985); it is possible for cattle infected with these parasites to maintain their carrier state for several years (Young, 1981). However, the carrier state of T. parva in cattle has been a subject of much controversy and is complicated by the presence of a number of sub-species of T. parva. Immunity to East Coast fever in cattle (T. parva parva infection) has until recently generally been considered to be sterile (Du Toit, 1931; Mettam and Carmichael, 1936; Henning, 1956; Neitz, 1957; Barnett, 1968), although both Bevan (1924) and Wilde (1967) considered the evidence to be inconclusive. Although the carrier state of T. p. lawrencei has been known to exist in naturally recovered African buffalo and cattle for long periods (Barnett and Brocklesby, 1966), a similar state for T. p. parva infection in cattle was only demonstrated relatively recently (Young et al., 1981).

Table 1. Maintenance of persistent infections of theilerial parasites in mammalian hosts

Theilerial species	Long term division		Blood transmission
	Schizont	Piroplasm
T. mutans	-	+	+
T. taurotragi	+	+	+
T. velifera	-	+	+
T. p. parva	+	-	-
T. p. lawrencei	+	-	-
T. p. bovis	+	-	-
T. sergenti	-	+	+
T. orientalis	-	+	+
T. annulata	+	+	+

The nature and efficiency of the carrier state in cattle of both T. p. parva and buffalo-derived T. p. lawrencei have important implications for the epidemiology of the diseases these parasites cause. For example, a carrier buffalo infected with T. p. lawrencei could be one hundred times more efficient in producing infected ticks than a group of carrier cattle (Young and Grootenhuis, 1985). Acaricide application to such cattle would increase the relative importance of the buffalo in maintaining the disease in an area.

In the South Nyanza District, Kenya, which is a theileriosis endemic area, Young et al. (1986) found that the carrier state of T. p. parva approached 100% in adult cattle. Here it was likely that carrier cattle played a greater role in the maintenance of infection than infected ticks, a complete reversal of the epidemiological concepts held for T. p. parva infection. This situation has since been shown to be common in the field in Kenya, and a high proportion of T. p. parva carriers have been found in other areas of Kenya, such as Uasin Gishu and Kajiado districts (D.P. Kariuki, personal communication). Theileria parva is unusual among theilerial species in that the persistence of infection appears to be in the schizont stage rather than by division of the piroplasm stage or both, as is the case with other species.

An important question is why it took so long for the widespread carrier state of T. p. parva to be recognized in the field, while the carrier state of T. p. lawrencei and T. p. bovis has been recognized for such a long time. In Kenya it appears that too much emphasis has been placed on the results of studies of a laboratory stock, T. p. parva (Muguga), one of the few stocks not shown to produce a carrier state (Barnett, 1968). However, it is a fact that in South Africa and Zimbabwe, T. p. parva infection has been eradicated by means that certainly would not have eradicated the infection in Kenya. Since the parasite was introduced into South Africa by cattle imported from East Africa, it is possible that by chance the introduced stocks did not produce the carrier state. Alternatively, stocks that did not produce the carrier state may have been selected in a fully susceptible cattle population. It is of considerable interest that in Zimbabwe it has not been possible to eradicate T. p. bovis, which has a well-developed carrier state.

An increase in the prevalence of the carrier state of T. parva has undoubtedly occurred since the introduction of the curative drugs parvaquone (Clexon, Wellcome) and halofuginone (Terit, Hoechst); Dolan (1986a and 1986b) demonstrated a high prevalence of the carrier state after treatment with these drugs. Mutugi et al. (1988a) have indicated that buparvaquone (Butalex, Coopers Animal Health) treatment used for immunization could reduce the incidence of the carrier state of T. p. lawrencei in cattle.

In recent studies, a start has been made to characterize the parasites inducing the carrier state of T. p. parva in buffalo and cattle. The carrier state of T. p. lawrencei has been characterized by Grootenhuis et al. (1987 and 1987b) and Conrad et al. (1987 and 1989). Conrad et al. (1987) using monoclonal antibodies showed that one cell culture isolate from a carrier buffalo contained at least 5 antigenically different T. p. lawrencei schizonts, which demonstrated the antigenic diversity possible in carrier buffalo. It is possible that in the future buffalo-derived T. p. lawrencei will be used to immunize cattle in the field. It is important to determine how the introduction of this parasite into an area could influence the epidemiology of theileriosis, given that it can induce a carrier state in immune cattle (Mutugi et al., 1988b) and could result in the production of new antigenic types.

Studies were performed to elucidate the nature of T. p. lawrencei in carrier cattle after immunization using the infection-and-treatment method to help predict whether Corridor disease immunization in the field could be hazardous to cattle. These studies focussed on the relationship between the T. p. lawrencei isolated from carrier cattle and the original immunizing T. p. lawrencei stock using cattle immunized with T. p. lawrencei on its own or in combination with T. p. parva and treated with different formulations of oxytetracycline.

Groups of cattle were immunized with 1:10 dilutions of sporozoite stabilates of either T. p. lawrencei (stabilate 199) or T. p. parva (stabilate 187) combined with T. p. lawrencei (stabilate 202) and treated with oxytetracycline. The animals were examined for persistent infection by cell culture isolation and for carrier state by tick application and examination of tick salivary glands. Of eleven animals examined, six were shown to be infective for nymphal ticks three to four months after immunization by applying the resultant adults to susceptible cattle. Two were carriers, shown by examination of adult tick salivary glands, and two were shown to have a persistent schizont infection by cell culture isolation.

Adult ticks infected with the carrier parasites derived from stabilate 199 or a combination of stabilates 187 and 202 were applied to individual susceptible cattle on nine occasions. All the cattle became infected and showed clinical theilerial reactions with the development of febrile responses. Seven out of nine were treated with parvaquone and recovered. The other two animals died in spite of treatment, showing that the carrier state parasite can be highly pathogenic. The survivors developed significant antibody titres in the indirect fluorescent antibody test. The seven recovered cattle were challenged with a lethal dose (1 ml undiluted) of the homologous immunizing stabilates (199, or 202 and 187, as appropriate), together with control cattle. Six out of seven carrier cattle and all the controls died of theileriosis, while none of the cattle originally immunized with the same stabilates died or showed clinical disease on challenge (Mutugi et al., 1988b). The seventh animal that survived following challenge underwent an unapparent theilerial reaction. The cattle that died on challenge generally showed a longer prepatent period to schizonts and a longer time to febrile response and death than controls, suggesting that some degree of protection may have been provided. When a carrier parasite was isolated and prepared as a sporozoite stabilate (226) and then used to challenge cattle immunized with T. p. lawrencei (stabilate 199), the cattle showed unapparent or mild theilerial reactions while the controls died from acute theileriosis (Table 2).

The monoclonal antibody profiles of infected cell lines isolated from the experimental animals were examined as described by Conrad et al. (1987). The stocks used for immunization and the carrier parasites contained mixed antigenic populations. Interestingly, the carrier parasites appeared to be similar to the original immunizing parasites (for example, stabilates 199, 202 and 187) except for the cell line isolated from animal Z639, which was immunized with carrier parasite derived from T. p. lawrencei (stabilate 199). This parasite had a markedly different monoclonal antibody profile from other parasites isolated from cattle immunized in the same manner and showed a very restricted pattern of reactivity with the panel of 20 monoclonal antibodies.

The evidence from this study appears to support the hypothesis that in many cases the carrier state is induced by only a part of the immunogenic population in the stocks, because the carrier parasite immunized cattle were shown to be immune only to homologous challenge while the cattle immunized against the original T. p. lawrencei stock were immune to challenge with T. p. lawrencei (carrier state) parasite.

Table 2. Results of in vivo cross-immunity studies

Previous history	Challenge stock	Stabilate no.	Animal no.	Results
				Classification of reaction
				I*	MR*	SR**	VSR**
Recovered from T. p. lawrencei carrier parasite	T. p. lawrencei (0I Pejeta)	199	Z061	-	-	-	+
			Z065	-	-	-	+
			Z639	-	-	-	+
			Z360	+	-	-	-
Controls	T.p. lawrencei (0I Pejeta)		Z060	-	-	-	+
			Z071	-	-	-	+
			Z649	-	-	-	+
			Z638	-	-	-	+
Recovered from T. p. lawrencei and T. p. parva carrier parasite	T. p. lawrencei (Trans-Mara) and T. p. Parva (Kilae)	202/187	Z058	-	-	-	+
			Z059	-	-	-	+
			Z091	-	-	-	+
Controls			Z074	-	-	-	+
			Z075	-	-	-	+
T. p. lawrencei (0I Pejeta) immune St. 199	T. p. lawrencei (carrier state)	226	Z884	-	+	-	-
			Z885	-	+	-	-
			Z887	-	+	-	-
			Z888	-	+	-	-
			Z889	-	+	-	-
			Z890	+	-	-	-
Controls			Z863	-	-	-	+
			Z894	-	-	-	+
T. p. lawrencei (carrier state) immune St. 226	T. p. lawrencei (0I Pejeta)	199	Z809	-	+	-	-
			Z817	-	+	-	-

* Immune: I = inapparent reaction, MR = mild reaction.

^** Not immune: SR = severe reaction but recovered, VSR = very severe reaction usually resulting in death.

The next series of experiments is to determine the antigenic nature of carrier parasites of T. p. parva and T. p. lawrencei in cattle and buffalo infected with cloned parasites to determine whether antigenic variation occurs in carrier hosts and how it is expressed after tick transmission from infected animals.

In Kenya a high prevalence of the T. parva carrier state occurs in endemic areas, approaching 100% in adult immune cattle (D.P. Kariuki, personal communication). This prevalence is lower in epidemic areas but even here may reach as high as 10%, as has been shown on farms in some areas, such as Nakuru District (Young et al., this meeting). The effects of immunization against theileriosis using infection and treatment may make all the cattle immune and the majority of the cattle carriers. In such a situation, the carrier state may be beneficial because it maintains immunity both in individual animals and, by tick transmission, in cattle populations.

ACKNOWLEDGEMENTS

We would like to thank Dr. J.G. Grootenhuis, of the National Veterinary Research Centre, Kabete, and Drs. P.A. Conrad, T.T. Dolan and S.P. Morzaria, of the International Laboratory for Research on Animal Diseases, for their help in these studies. We are also grateful for the comments of Dr. S.M. Williamson. Dr. A.S. Young is employed by the Overseas Development Administration of the United Kingdom.

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