International Laboratory for Research on Animal Diseases
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Many Theileria parasites cause diseases in cattle, of which one of the most economically important is East Coast fever (ECF), caused by T. parva. The parasite causes high morbidity and mortality in exotic cattle, thus inhibiting the introduction of improved cattle into endemic areas. The only immunization method available against ECF is the infection-and-treatment method (Radley, 1981), in which live parasites are used. For a rational approach to ECF immunization, it is necessary to isolate and characterize T. parva stocks from the field before selecting them for immunization. Most field isolates contain a mixture of T. parva strains and some contain two or more species of Theileria. Furthermore, the immunity engendered following ECF immunization is strain/stock specific. For these reasons, it is necessary to have precise methods for identifying not only different species of Theileria, but also different strains of T. parva. This paper discusses the various methods and criteria available to differentiate Theileria species and T. parva stocks. A more comprehensive review of the subject has been published by Irvin (1987).
DIFFERENTIATION OF THEILERIA SPECIES
Theileria parva, T. annulata, T. taurotragi, T. mutans, T. velifera and T. orientalis are the six Theileria species known to infect cattle. Various criteria and methods, described below, are used to identify these parasites. In most cases it is necessary to use a combination of these methods to identify a species definitively.
The distribution of the two most important cattle species, T. parva and T. annulata, correlates well with the distribution of their vectors. Theileria parva is transmitted predominantly by the brown ear tick, Rhipicephalus appendiculatus, which is restricted to eastern, central and southern Africa; T. annulata is transmitted by several Hyalomma species, which are distributed widely over North Africa, southern Europe, the Middle East, India, southern Russia and China. There is virtually no overlap of these vectors and therefore the possibility of confusing these two parasites is low.
In many parts of Africa where more than one Theileria species occurs, the vector specificity can be used for differentiation. For example, T. mutans, T. velifera and T. orientalis are widely distributed over Africa and are transmitted by ticks of the genus Amblyomma. This group of parasites can be distinguished from T. parva and T. taurotragi, which are transmitted predominantly by R. appendiculatus. Theileria taurotragi can also be transmitted by R. pulchellus.
It is usually difficult to differentiate Theileria species solely by examining the morphology of the parasites. Parasites of different species look alike in most piroplasm and schizont stages. Two exceptions to this are T. velifera, which has a characteristic veil associated with the piroplasm, and T. taurotragi, which has bar-like structures in infected erythrocytes. However, confusion may arise if mixed infections occur. The T. mutans schizonts, which occur transiently and in low numbers, have a distinct morphology. They are larger and more diffuse than schizonts of other Theileria species and for this reason can be easily differentiated.
It is often difficult to differentiate T. parva from T. taurotragi because both species are transmitted by the same vector and mixed infections of these parasites occur in cattle. These parasites differ, however, in the range of other mammalian hosts they infect. Theileria taurotragi infects eland and sheep (A.S. Young, personal communication), and T. parva infects buffalo. Theileria taurotragi is also known to infect cells from a variety of ungulate species in vitro (Stagg et al., 1983), and therefore in vitro infectivity of sporozoites may be useful in differentiating these two species.
The virulence of different species of Theileria may vary a great deal depending on the strain of parasite, degree of host susceptibility and dose of parasite. Theileria mutans is generally known to be benign, although virulent strains have been reported from South Africa (Flanagan and Le Roux, 1957). Theileria mutans undergoes limited lymphocytic merogony; its main mode of replication occurs in erythrocytes and in virulent strains this causes a high piroplasm parasitaemia and haemolytic anaemia in the host. In contrast, T. parva replicates mainly in lymphocytes and the pathology it causes is associated with destruction of the lymphocytes. Erythrocytic merogony is limited and haemolytic anaemia is not present. Theileria annulata replicates in both lymphocytes and erythrocytes, thus causing disease with severe lymphocytopaenia, anaemia and occasionally jaundice. Theileria taurotragi, which undergoes lymphocytic and erythrocytic merogony, can be pathogenic in its eland host but is benign in cattle.
There is no evidence of cross-species protection among any of the cattle parasites. For example, cattle immunized against T. annulata are fully susceptible to T. parva and vice versa (Sergent et al., 1945; Neitz, 1957). The cross-immunity test is particularly useful in a situation where T. taurotragi and T. parva occur together. The lack of cross-protection among Theileria species contrasts with the variable degree of protection observed among different immunological stocks of T. parva (see "Cross-Immunity" under "Stock Characterization of T. parva" below).
The routine serological test used to diagnose Theileria species is the indirect fluorescent antibody test. Using this test, cross-reactions have been observed among T. parva, T. mutans, T. annulata and T. taurotragi (Burridge et al., 1974; Grootenhuis et al., 1979). Under experimental conditions and using appropriate controls, the test can be useful in identifying these Theileria species. Its usefulness in the field, however, is limited, especially in large areas of eastern, central and southern Africa where T. parva, T. taurotragi and T. mutans occur together.
Conrad et al. (1987a) and Allsopp and Allsopp (1988) have shown that repetitive DNA sequences from T. parva stocks, used as radio-labelled probes, hybridize specifically to T. parva parasite DNA but not to T. mutans, T. annulata and T. taurotragi DNA. To date, only T. parva DNA specific probes are available; probes against other Theileria species are urgently needed. This is a powerful and a sensitive technique and may play a significant role in identifying mixed theilerial infections, which occur commonly in cattle exposed to natural tick challenge. The DNA probes can also be used to identify mixed infections in ticks, such as T. parva and T. taurotragi in R. appendiculatus.
Restriction fragment length polymorphisms
Restriction enzymes cleave DNA at specific necleotide sequences. The DNA fragments thus produced can be resolved on agarose gel by electrophoresis and visualized under ultra-violet illumination following ethidium bromide staining. Using restriction enzymes such as Sfi I and Not I, which cut Theileria DNA infrequently, and separating the digested DNA in pulsed-field gel electrophoresis, unique and characteristic banding patterns of T. parva and T. mutans have been detected (Morzaria, 1988). This restriction fragment length polymorphism is a simple way of differentiating Theileria species. Comparative DNA profiles of other important parasites, such as T. annulata and T. taurotragi, have not been studied in this system.
STOCK CHARACTERIZATION OF THEILERIA PARVA
With the development of the infection-and-treatment method of immunization and the realization that different immunological strains existed in T. parva (Radley, 1981), precise characterization of T. parva stocks became necessary. In the last decade several tests have been developed to characterize stocks of T. parva; these are described below.
Cross-immunity was the first test developed to characterize immunological types of T. parva. The test involves immunizing cattle with a stock of T. parva using the infection-and-treatment method and challenging the immune animals with a different stock. The breakthrough stocks are classified as immunologically distinct. This test has great value because it enables the identification of a parasite stock or stocks that will provide wide immunity. Both the "Muguga cocktail" and the "Marikebuni" stocks were essentially selected on the basis of the cross-immunity tests (Radley, 1981; Morzaria et al., 1987).
A feature of the cross-immunity between two immunologically distinct stocks is that protection is frequently partial. Usually 30-40% of the immune animals are not protected when challenged with a heterologous stock. This is in sharp contrast to the results of a cross-immunity test using different species of Theileria, when no protection is observed. To obtain meaningful results from cross-immunity tests for stock characterization, many animals must be used and thus the test is expensive.
Infectivity is a rot/tine test used in the characterization of T. parva stocks. Susceptible cattle are infected with a standard dose of sporozoites and clinical and parasitological parameters - such as the length of prepatent period, the time to fever, the duration of parasitosis and fever, the time to first appearance of piroplasms and the time to death or recovery - are measured. In addition, the schizont parasitosis and piroplasm parasitaemia are estimated. These parameters define the infectivity of a particular stock for a particular breed and age of animal. For example, T. parva (Mariakani) and T. parva (Marikebuni) stocks, isolated from the Kenya coast, produce unique clinical characteristics in susceptible cattle. The Marikebuni stock usually produces a prepatent period of 8 days and death by day 17. In contrast, the Mariakani stock usually produces a prolonged clinical reaction with a prepatent period of 9 days and death between days 21 and 25. The fever almost invariably occurs biphasically.
Susceptibility to drugs
Drug sensitivity is also a rot/tine test in which groups of cattle, usually highly susceptible Bos taurus breeds, are immunized by the infection-and-treatment method: the cattle are infected with a lethal dose of sporozoite stabilate and treated with one injection of long-acting oxytetracycline. With certain parasite stocks, however, one dose of this drug formulation does not control the infection and either a diluted stabilate dose is used with the same dose of drug or two doses of drug are used with the lethal dose. This test gives additional information on the suitability of a particular stock being used for immunization.
In recent years, buparvaquone (Butalex, Coopers Animal Health) has been found to be effective in immunization against ECF. The drug is administered at 2.5 mg/kg simultaneously with the sporozoite challenge and is useful in immunization where a particular T. parva stock cannot be controlled with one dose of a long-acting tetracycline.
Depending on the results of the in vivo tests, parasites can be selected as putative immunizing stocks and tested further in pilot immunization trials. For example, the T. parva (Marikebuni) stock was found to show wide protection in cross-immunity tests and was subsequently used in immunization trials in the Coast Province, Kenya. The results of the field trials substantiated laboratory studies on its ability to provide wide protection (Morzaria et al., 1987; Mutugi et al., in press).
In vitro infectivity
Several in vitro tests are routinely used to characterize T. parva After preparing a sporozoite stabilate of a field isolate, in vivo and in vitro characterizations are performed, usually simultaneously. The test involves infecting a panel of previously characterized susceptible cloned and uncloned cells from cattle and other ungulates with a sporozoite stabilate. The cell lines obtained are a valuable source for amplification of parasite material for further in vitro characterization.
Monoclonal antibody profiles
Several T. parva antischizont monoclonal antibodies (MAb) have been generated and used in an indirect fluorescent antibody (IFA) test against schizont-infected cells derived from in vitro cultures to demonstrate stock-specific diversity. Minami et al. (1983) showed that the presence or absence of binding to MAbs 2 and 3 and to 15 and 16 was a convenient way of dividing T. p. parva stocks into three groups. However, extensive studies with more schizont MAbs and several other T. parva stocks have revealed that the diversity is much greater than was originally thought. Most T. p. lawrencei stocks react with MAb 19, which does not react with T. p. parva, and most of the T. p. bovis stocks isolated from Zimbabwe do not react with MAb 7.
Minami et al. (1983) showed that the percentage of schizont-infected cells reacting with a particular MAb agrees closely with the percentage of infected cells as identified by Giemsa staining of the antigen slide. However, Conrad et al. (1987b) found that with many T. p. lawrencei isolates the percentage of infected cells reacting to certain MAbs was often lower than the percentage of schizont-infected cells. This was subsequently shown to be due to heterogeneity in the stocks studied. Similar findings have also been observed in T. p. parva isolates (P.R. Spooner, personal communication).
Irvin et al. (1983) showed in limited cross-immunity tests that if cattle were immunized with a T. parva stock and challenged with another parasite stock with an identical MAb profile, good cross-protection was recorded. If the cattle were challenged with a stock of a different MAb profile, the protection was variable. To make a general statement with regard to the relationship between the in vitro and in vivo tests, more extensive cross-immunity tests need to be performed because it is clear that the MAbs recognize schizont-specific antigens but not necessarily those antigens that may be involved in immunity.
At present monoclonal antibody profiles demonstrate antigenic diversity and are a useful way of characterizing T. parva stocks. The characteristic profiles of parasite stocks provide a useful laboratory check where contamination with other parasites is suspected. However, it is important to recognize that the test is performed on parasites that have been cultured in vitro and passaged several times. Parasite selection may occur during isolation and in vitro maintenance. The MAb profile obtained thus relates to the cultured parasite and may not reflect the true characteristics of the original stock.
The monoclonal antibody test is essentially a qualitative test. The more precise nature of parasite antigens can be determined by probing western blots of the relevant antigens with the panel of MAbs. Shapiro et al. (1987) showed that MAb 5 identifies a polymorphic schizont antigen in T. parva stocks. Certain antischizont MAbs may therefore provide stock-specific markers. This area needs further investigation.
Two-dimensional gel electrophoresis has been used to characterize infection-specific proteins of various T. parva stocks (Sugimoto et al., in press). Stock-specific differences have been detected but the technique is difficult to perform and often difficult to interpret. It remains a useful laboratory technique for stock identification.
Most of the characterization methods described above recognize phenotypic characteristics of T. parva. With the advent of recombinant DNA technology, several workers have attempted to identify polymorphisms in T. parva stocks at the DNA level. Conrad et al. (1987a) and Allsopp and Allsopp (1988) have developed DNA probes that differentiate certain T. parva stocks. Polymorphism in T. parva has also been detected using rare cutter restriction enzymes such as Sfi I and Not I and by separating DNA fragments on modified pulsed-field gel electrophoresis systems (Morzaria, 1988). Characteristic and unique DNA banding patterns have been detected in several T. parva stocks, thus enabling workers to differentiate stocks.
To conduct epidemiological studies and plan rational immunization programmes against ECF, Theileria parasites must be precisely identified and characterized. Several criteria and methods are available to achieve this and with the advent of new biochemical and molecular biological techniques, the task of identifying Theileria species is becoming easier. These new techniques are being used increasingly as routine tests to characterize T. parva stocks.
A recent development in the field of molecular biology is the polymerase chain reaction technique, which allows amplification of very small (picogram) quantities of DNA to a detectable level. This technique is now being used routinely in diagnostic research, especially to diagnose acquired immune deficiency syndrome (AIDS) in carriers that cannot be easily detected by conventional means. In future the polymerase chain reaction technique will be an important tool in studies of the epidemiology of theileriasis because it can be used to detect Theileria carrier animals and it may enable diagnosis at species and strain level.
One of the main reasons for developing in vitro characterization tests is to identify markers that will correlate with immunity in vivo. To date, none of the modern techniques used fulfil this requirement. However, biochemical and genetic characterization of T. parva provides an understanding of the parasite at the molecular level, thus enhancing our knowledge of the basic biology of the parasite. At present, characterization of T. parva is being performed on stocks; the aim should be to characterize cloned T. parva parasites to define the nature of polymorphisms more accurately. Research should also continue to develop an in vitro test to identify the antigenic nature of parasite isolates and stocks. Such a test would simplify the establishment of immunization programmes by doing away with expensive and time-consuming in vivo cross-immunity tests.
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