Epidemiology of East Coast fever
Collaboration with National Governments
Identifying Theileria Parva parasites
Developing improved tests to diagnose infections
Towards a vaccine against East Coast fever
Immunization against the sporozoite form of the parasite
Sporozoite Entry into Bovine Lymphocytes
Immunization against the schizont form of the parasite
Immunological Responses to Schizont-Infected Cells
Effects of the major histocompatibility complex
Theileriosis is a debilitating and often fatal disease that threatens over 25 million cattle in eleven countries of Africa: Burundi, Kenya, Malawi, Mozambique, Rwanda, Sudan, Tanzania, Uganda, Zaire, Zambia and Zimbabwe (Figure 2). The organism that causes theileriosis by infecting wild and domestic ruminants is a protozoan parasite belonging to the genus Theileria. Two species of this genus, Theileria parva and Theileria annulata, cause clinical disease in cattle and thus severely impede cattle farming and its improvement in Africa and many other tropical regions of the world. In eastern, central and southern Africa, the most important species is Theileria parva, which causes diseases commonly known as East Coast fever, Corridor disease and January disease. (Hereafter the disease is called East Coast fever.) In cattle that are fully susceptible to the disease, East Coast fever is nearly always fatal.
Figure 2. Reported distribution of the protozoan parasite Theileria parva, which causes theileriosis in cattle in 11 countries of eastern, central and southern Africa. (Although antibodies to T. parva are still found in South Africa and Swaziland, the disease has been fully under control in those countries for many years.)
Theileria parva is transmitted between cattle by the brown ear tick, Rhipicephalus appendiculatus. The parasite is also transmitted to cattle from Cape buffalo (Syncerus caffer), which normally show no clinical signs of theileriosis.
The types of cattle affected most severely by East Coast fever are highly productive taurine (Bos taurus) breeds, their crosses, and improved indigenous Zebu (Bos indicus) cattle that are moved from areas free of East Coast fever into endemic areas (Figure 3). A substantial and incalculable economic loss due to this disease stems from the inability of farmers to crossbreed their indigenous cattle with genetically improved taurine cattle because the latter are highly susceptible to tick-borne diseases. Among indigenous cattle where the disease is ENDEMICALLY STABLE*, East Coast fever is fatal mostly in calves, with mortality rates being as high as 50%; in areas where the disease is ENDEMICALLY UNSTABLE, such as in livestock herds first introduced the parasite, 80-100% of animals of all age groups may die. In one year alone, 1989, it is estimated that East Coast fever killed 1.1 million head of cattle and caused US$168 million in losses.
*Words appearing in SMALL CAPITAL LETTERS in this Annual Report are defined in a Glossary preceding this chapter.
Figure 3. Genetically improved taurine breeds of cattle, such as these Hereford in Kenya, are particularly desired by farmers because they are more productive than indigenous breeds. Unfortunately, highly productive grade cattle are also particularly susceptible to tick-borne diseases.
East Coast fever is controlled principally by dipping or spraying cattle with acaricide substances, which keep the animals free of ticks (Figure 4). This method, however, has several shortcomings. Acaricides are costly and must be bought with hard currency, a scarce commodity in most developing countries. In areas heavily infested with ticks, cattle herds are walked to dips or spray races as often as twice a week for treatment; this frequency damages the land, pollutes the environment with toxic residues and may be accelerating the development of tick resistance to the acaricides. In addition, proper acaricide treatment is difficult to administer in rural areas where there is little expertise in maintaining cattle dips and spray races, where water is in chronic short supply and where illegal cattle movements or civil unrest may periodically occur. Finally, because cattle regularly treated with acaricides are not exposed to T. parva, the cattle develop no immunity and thus have no protection against the parasite or other tick-borne diseases if the acaricide treatment regime is interrupted.
Figure 4. Cattle being sprayed with an acaricide on a ranch in Kenya to keep the cattle free of ticks. This is the most common tick control method used in Africa, but problems encountered in maintaining dips and spray races and the high costs of importing acaricides are forcing many governments to investigate other methods for controlling ticks and the diseases they spread.
Although curative drugs have been developed to control the parasite in infected animals, these drugs are expensive and most efficacious when the disease. which often is clinically apparent only when the infection has reached an advanced stage. is diagnosed early. For these reasons, alternative and improved methods of controlling East Coast fever are urgently needed.
The life cycle of the single-celled T. parva parasite is complex (Figure 5). In both the tick VECTOR and the mammalian host, the parasite undergoes a series of transformations into different forms. Upon ingestion by a R. appendiculatus tick (Figure 6), T. parva undergoes differentiation first in the gut of the tick, resulting in the formation of forms called kinetes (Figure 7, top right). The kinetes then migrate to the tick salivary glands, where they differentiate into sporozoite forms (Figure 7, middle right). As the tick feeds on cattle, these sporozoites. the form of the parasite that most often infects cattle. are injected into the cattle along with tick saliva. (Theileria parasites are usually transmitted to cattle by ticks that have fed as nymphs on infected animals and then feed as adults on susceptible cattle.) In the bovine host, the sporozoites attach to and enter lymphocytes, a type of white blood cell of the bovine immune system (Figure 7, bottom right). Within two to three days of invading the lymphocytes, the sporozoites develop into intracellular forms called schizonts (Figure 7, bottom left). The infected lymphocytes grow larger and begin to divide. As each enlarged lymphocyte, called a lymphoblast, divides, the schizont inside the lymphoblast also divides, ensuring infection of each of the two daughter cells produced by the dividing lymphoblast.
Figure 5. The life cycle of Theileria parva. Sporozoite forms of the parasite are transmitted from the tick to the bovine host in tick saliva as an infected tick feeds on an animal. In the animal host, the sporozoites attach to and enter lymphocytes, a type of white blood cell of the bovine immune system, where the sporozoites develop into forms called schizonts. The infected lymphocytes grow larger and begin to divide. As each enlarged lymphocyte, called a lymphoblast, divides, the schizont inside the lymphoblast also divides, ensuring infection of each of the two daughter cells produced by the dividing lymphoblast. The infected lymphocytes expand rapidly and spread throughout the lymphoid system of the animal, giving rise to widespread destruction of host cells.
Some of the schizonts develop into merozoite forms, which are released from the lymphocytes into the bloodstream, where they invade red blood cells (erythrocytes). In the red cells, the parasites develop into forms called piroplasms, which are able to infect ticks. As ticks feed on animals infected with the parasite, they ingest red blood cells containing the piroplasms. Once in the tick gut, the parasites differentiate into male and female gamonts, which fuse to form zygotes. The zygotes differentiate into kinetes, which move to the salivary gland and enter a particular cell type. Here the parasites form sporoblasts, each of which give rise to 30,000 to 50,000 sporozoites, a parasite form able to infect animals. The sporozoites are introduced into a mammalian host along with tick saliva when the tick feeds, initiating a new cycle of parasite development.
Figure 6. An unfed adult female brown ear tick (Rhipicephalus appendiculatus). Ticks feed by taking a blood meal from an animal. While feeding on cattle, brown ear ticks infected with Theileria parva transmit these parasites to the animals, causing the devastating disease theileriosis, commonly known as East Coast fever, January disease or Corridor disease.
Figure 7. Important developmental forms of Theileria parva. Clockwise from upper right: kinete forms (arrows) of the parasite, which move from the tick gut to the salivary gland; sporozoite forms (Sp), which are able to infect animal hosts, in a tick salivary gland; a sporozoite that in tick feeding has entered an animal host and invaded a white blood cell (lymphocyte); schizont forms (Sc), which develop in the bovine lymphocyte from the sporozoite forms; a schizont producing merozoite forms (arrows), which invade the red blood cells (erythrocytes) of the animal host; a piroplasm form of the parasite, which develops from the merozoite form and is able to infect ticks, in an erythrocyte. Piroplasms will be ingested by a feeding tick, develop into kinete forms in the tick gut, and the life cycle will be repeated.
In this manner, the lymphocytes that were initially parasitized expand rapidly and spread throughout the lymphoid system of the animal, giving rise to widespread destruction of cells of the host animal. In most untreated, susceptible cattle, this results in an overwhelming infection of the lymphoid system and death within three to four weeks of infection.
In the later stages of infection, some of the schizonts differentiate into merozoite forms (Figure 7, middle left). The merozoites are released from the lymphocytes into the bloodstream, where they invade red blood cells. In the red cells, the parasites change into forms called piroplasms (Figure 7, top left), which are able to infect ticks. As ticks feed on infected animals, they ingest red blood cells containing the piroplasms, and this completes the life cycle of T. parva.
Cattle that survive infection with T. parva are thereafter immune to East Coast fever for long periods. This suggests that it may be feasible to control the disease by immunizing livestock against the parasite. However, the different T. parva stocks obtained in the field and the different T. parva strains maintained in the laboratory have different antigens. those parts of the parasite recognized as foreign by the immune system of the bovine host. Immunity produced in an animal against one stock or strain of the parasite thus may not protect the animal against challenge with another. The development of an effective vaccine against East Coast fever therefore depends on determining the antigenic composition and prevalence of T. parva stocks obtained from endemic areas. Such information is being compiled in epidemiological studies at ILRAD, which are an important part of the Laboratory's theileriosis program, complementing the program's three other major areas of research, which are directed at immunization against the sporozoite and schizont forms of the parasite.
Cattle can be immunized against a stock of T. parva by infecting the animals with the sporozoite form of the parasite stock while at the same time treating the cattle with an antibiotic drug to lessen the severity of the infection (Figure 8). This infection-and-treatment technique continued to be used this year in conjunction with tick control in many regions where East Coast fever is endemic.
Figure 8. Schematic diagram of the steps involved in immunizing cattle against East Coast fever by use of an 'infection-and-treatment' method that has been refined over several years by staff from ILRAD and the Kenya Agricultural Research Institute.
Staff members conducting epidemiology studies in the theileriosis program continued to collaborate with national governments to determine the prevalence of East Coast fever in their countries, to establish and conduct immunization programs based on the infection-and-treatment method and to train personnel to apply the method. ILRAD also provided national laboratories with reagents to characterize parasite types and to diagnose East Coast fever and other important tick-borne diseases, the latter caused by Anaplasma, Babesia and Cowdria parasites.
Scientific and technical staff from laboratories in Ethiopia, Gambia, Kenya, Malawi, Mozambique, Senegal, Tanzania, Zaire, Zambia and Zimbabwe were given instruction during the year in the use of techniques to diagnose tick-transmitted diseases, to identify tick species, to maintain tick colonies in the laboratory and to prepare stabilates, which preserve live T. parva sporozoites so that these remain infective for use in future experiments and immunization projects. The institute continued to assist the Tanzanian Government in immunizing cattle against T. parva on the islands of Zanzibar, which have a complex tick-borne disease problem. Babesia bovis, Cowdria ruminantium and a pathogenic form of Theileria mutans a normally innocuous species of Theileria, have all been identified on the islands.
Success in protecting cattle against East Coast fever by immunizing them by the infection-and-treatment method depends on an accurate identification of the parasite stocks that exist in the area in which livestock will be immunized. Theileria are small parasites and the different species, and strains are often indistinguishable when viewed under a light microscope. For this reason, ILRAD researchers over the last several years have developed increasingly sophisticated laboratory procedures for parasite identification.
In the past, a cross-immunity test was the only method available for identifying a parasite stock or stocks that might provide wide immunity to East Coast fever. This test consists of immunizing cattle by the infection-and-treatment method using one stock of T. parva and subsequently challenging the animals with a different stock to discover which stocks break through, and thus which stocks are immunologically distinct. Although still very useful, cross-immunity tests are expensive and time consuming. More efficient, IN VITRO tests are now being developed to determine the immunological types of T. parva.
A panel of 20 MONOCLONAL ANTIBODIES that had been used for several years at ILRAD to distinguish T. parva stocks from each other was improved during the year. Nine monoclonal antibodies were deleted from the original panel, a few because they reacted with all parasite stocks and others because they gave only partial reactions. Two new monoclonal antibodies were raised against other stocks and added to the panel. Although none of the monoclonal antibodies in the new panel specifically recognizes only one stock, use of the panel does differentiate among the stocks used at ILRAD. Moreover, the positive and negative reactions obtained with this panel are consistent with those of stocks from East and Central Africa. These reaction patterns do not, however, correlate with cross-protection.
Tests based on monoclonal antibodies distinguish differences in the physical characteristics of T. parva stocks. New techniques developed in molecular biology have enabled ILRAD research workers to make DNA PROBES with which to identify GENOMIC differences among parasite stocks as well (Figure 9). Molecular biology research at ILRAD on the genetic material of T. parva led scientists to identify fragments of DNA each of which is characteristic of a single T. parva stock or strain. Having determined the NUCLEIC ACID sequences of these fragments, the scientists then made smaller DNA probes (oligonucleotides). single strands of the double stranded parasite DNA fragment. each of which combines, or hybridizes, only with genetic material obtained from T. parva of the same subspecies or strain. By screening field samples of T. parva with a panel of DNA probes made for different subspecies and strains, laboratory workers can now identify these with unequalled precision.
Figure 9. Diagram of the nucleic acid hybridization process. Single-stranded DNA from an organism of interest is allowed to attach itself to a membrane. A single-stranded DNA probe binds to its immobilized complementary strand. This binding can be detected by labelling the probes with radioisotopes or with non-radioactive reporter molecules, such as the biotin-streptavidin-enzyme complex shown here.
In 1990, use of the polymerase chain reaction technique continued to improve nucleic acid probes being made at ILRAD for both diagnostic and epidemiological work. The polymerase chain reaction is a simple but revolutionary technique for copying a defined stretch of DNA in the laboratory with readily available reagents. Because the number of copies increases exponentially, billions of copies can be made in a few hours. By using appropriate primers, the polymerase chain reaction can amplify a variable sequence of DNA located between two conserved regions in the parasite GENOME so that this sequence may be characterized. ILRAD scientists used the polymerase chain reaction in several ways during the year to identify genomic differences among Theileria species and stocks.
In 1990, for example, scientists at Cambridge University and ILRAD collaborated on work that identified species-specific regions in the small ribosomal subunit DNA of five Theileria species. DNA probes that specifically recognize these species were synthesized using the polymerase chain reaction. Four of these probes--T. parva, T. annulata, T. mutans and T. lawrencei--were tested on cultures of lymphocytes infected with different Theileria species and were shown to be species specific. The probes are now being adapted to identify parasites in the tissues of infected livestock and the salivary glands of infected ticks.
The polymerase chain reaction technique was also used to study the parasite carrier state in cattle. Using primers derived from a major DNA repetitive sequence in the T. parva genome, parasite-specific products obtained using the polymerase chain reaction were generated in whole blood of cattle subclinically infected with T. parva. In view of the importance of the carrier state in the epidemiology of East Coast fever, the ability to detect carrier animals without having to perform time-consuming and expensive parasite transmission experiments will facilitate epidemiological work done by national programs, leading to improved control of East Coast fever.
A third use of the polymerase chain reaction technique was to investigate whether there is a sexual cycle in T. parva. Sexual stages have been detected when the parasite is in the tick gut and these are thought to contribute to the antigenic complexity of T. parva in the field. Such genetic recombination has important implications both for the epidemiology of East Coast fever and for the selection of parasite strains for use in immunizing animals against the disease.
DNA from sporozoites obtained from ticks simultaneously infected with T. parva Muguga and T. parva Uganda was amplified by the polymerase chain reaction and the products probed with stock-specific oligonucleotides; results obtained in 1990 show that genetic recombination does indeed occur between these two stocks. A new physical map of the T. parva genome, described below, will facilitate studies in this area, as well as studies of the possible effects of genetic recombination on the structure of potentially'protective antigens'. MOLECULES that upon inoculation into the host animal induce protective immune responses.
A major achievement at ILRAD in 1989 was the construction of a physical map of the genome of T. parva (Muguga) (made using Sfi I linking clones) that showed the parasite has four chromosomes (Figure 10). In 1990, putative locations were assigned for all known T. parva GENES. By the end of the year, only one Sfi fragment of the entire genome remained to be assigned.
Figure 10. The four chromosomes that make up the genome of Theileria parva, as revealed using field-inversion gel electrophoresis. The sizes of the chromosomes range from 2.9 to 1.9 megabases.
Accurate diagnosis of tick-borne cattle diseases is essential for the success of disease control projects in endemic areas. Serological diagnosis of tick-borne infections in national diagnostic laboratories, however, is hampered by the lack of standardized reagents. For this reason, results of diagnostic tests often may be either inaccurate or incomparable among laboratories.
In 1990, ILRAD assisted national animal disease control workers to screen blood for Theileria. The Laboratory also continued work to develop standardized tests for the diagnosis of Theileria infections based on enzyme-linked immunosorbent assays (ELISAs). ILRAD scientists have developed antigen- and antibody-trapping assays that detect infection with T. mutans. An ELISA has advantages over the routinely used indirect fluorescent antibody test for extensive epidemiological studies of T. parva: the ELISA has greater sensitivity and is applicable in the field. To validate the utility of the ELISA, stocks of T. parva were examined on Western blots with schizont antigens to determine if the antigen used in the ELISA is conserved in the species. The information obtained was used to develop two microELISAs for T. parva, which are being compared and further evaluated.
Three other parasite antigens were identified and used in antibody-detection ELISAs: an antigen of Anaplasma marginale having a molecular mass of 54 kilodaltons, a 200-kilodalton antigen of Babesia bigemina and a 32-kilodalton antigen of T. mutans. Using two monoclonal antibodies. one that captures circulating antigen and another that recognizes a different epitope as an indicator. ILRAD staff have developed capture microELISAs for A. marginale and T. mutans. The antibody-and antigen-capture ELISAs are now being evaluated on field samples.
ILRAD aims ultimately to produce a panel of high-quality, standardized ELISA reagents for the diagnosis of all the tick-borne cattle diseases that occur in the endemic regions. The availability of such a comprehensive panel would considerably improve the diagnostic and disease-monitoring capacities of national laboratories.
The major disadvantage of the only immunization method now available for East Coast fever. the infection-and-treatment method. is its reliance on live parasites. These must be cryopreserved to remain infective, cattle immunized with the live parasites must be treated simultaneously with therapeutic drugs, and after immunization the cattle may remain carriers of infection. A vaccine based on antigens of parasites or on whole dead parasites would be a great improvement over the current vaccines based on live parasites.
Two developmental forms of T. parva are of particular interest to ILRAD regarding vaccine development. The first are sporozoites, transmitted to cattle by the bite of infected ticks and thus the first form of the parasite encountered by an animal's immune system. The second are schizonts, which develop from sporozoites soon after the latter enter the host's white blood cells. ILRAD researchers have identified two proteins displayed on the surface of these forms that could be the bases of improved vaccines.
Electron microscopic studies were conducted during the year on the processes by which sporozoite forms of T. parva bind and enter bovine lymphocytes. Theileria parva sporozoites are nonmotile and thus the initial sporozoite-lymphocyte interaction is a chance event, which can occur at 0. 2 ° C. It was discovered that all subsequent stages in the entry process are temperature-dependent and require the participation of live intact parasites and host cells. Scientists began to identify the molecular interactions involved in this process, which occurs in a series of steps that in total take only a few minutes (internalization of the parasite occurs in less than 3 minutes at 37 ° C).
First, the parasite binds to a host lymphocyte, with the outer membranes of the two cells attaching in a zipper-like manner. The parasite then enters/is internalized by the lymphocyte. Inside the host cell, the parasite retains both its original surface membrane and, on top of this, another membrane comprising the section of lymphocyte membrane that enclosed the parasite during the entry process. Once inside the lymphocyte, the parasite rapidly escapes from the encapsulating host cell membrane and, now free in the cytoplasm, differentiates into schizont forms, which, in a way as yet unknown, induces division of the host cell and thus clonal expansion of infected cells. The molecules involved in this elaborate binding and entry process are targets for immunological intervention and are thus being studied further.
Of the two T. parva surface antigens ILRAD scientists found to be likely candidates for vaccine material, one. designated p67 because its molecular mass is about 67 kilodaltons. occurs only on the surface of sporozoites. This molecule has been found in all stocks of T. parva tested. It has been shown to induce antibodies in cattle that neutralize sporozoite infectivity to lymphocytes in vitro. Researchers are now investigating whether p67 also induces cell-mediated responses, which involve T cells rather than B cells.
In 1989, ILRAD workers cloned the gene coding for p67 and filed a patent in the USA covering its potential use for vaccination against East Coast fever. In collaboration with researchers at SmithKline Beecham, Animal Health Department (Philadelphia), ILRAD produced large quantities of the protein using RECOMBINANT DNA TECHNOLOGY. In 1990, to test its effectiveness as a potential vaccine, researchers at ILRAD inoculated calves with the recombinant protein. The protein induced high levels of antibodies in the cattle. When infected with a T. parva sporozoite stabilate, the immunized cattle showed a significant degree of protection while nonimmunized control cattle developed fatal disease.
The success of such a vaccine will depend on an effective means of delivering it to the bovine immune system. ILRAD scientists used a vaccinia virus to express the gene that encodes p67, which produced recombinant antigen that was demonstrated to induce bovine antibodies that neutralized sporozoites (see `Antigen Delivery Systems', below). These exciting developments provide the first opportunity to test a recombinant parasite antigen as a vaccine for T. parva.
The other potentially protective protein identified at ILRAD. an 85-kilodalton antigen (p85). appears on both sporozoites and intracellular schizonts. The form of p85 expressed on the surface of schizonts appears to be polymorphic, varying in size from 79 to 105 kilodaltons, which suggests that the antigen is processed differently in the sporozoite and schizont stages of the parasite. The p85 antigen has been found to be immunodominant, that is, the immune response to p85 is stronger than that to other antigens of the parasite.
The schizont is the pathogenic stage of T. parva and causes disease through destruction of lymphocytes. The death or recovery of an animal depends greatly on the ability of the animal's immune system to control the rapidly dividing infected lymphocytes. Several features of immunity to T. parva infection suggest that protective responses against natural challenge are directed largely at the schizont stage of the parasite. These include the frequent observation of a schizont parasitosis in immune animals at the time of remission of infection and the fact that immune animals are solidly protected against challenge with large numbers of schizont-infected cells grown in the laboratory. Over the past decade, scientists at ILRAD have determined that these responses are likely to be mediated by a population of bovine white blood cells known as T lymphocytes. The identification of the parasite components that induce these responses is a major priority of the institute.
There is strong evidence that cell-mediated immune responses are essential for the recovery and protection of cattle from T. parva infection. ILRAD scientists have determined that the effector cell that mediates these responses is likely to be a subpopulation of T lymphocytes known as cytotoxic T lymphocytes. Parasite-specific cytotoxic T lymphocytes can be cultured in vitro and are capable of killing schizont-infected lymphocytes in the laboratory.
Results of an experiment conducted in 1990 provided conclusive evidence that cytotoxic T lymphocytes help protect immune cattle challenged with T parva by killing schizont-infected cells. Lymphocytes emanating from a bovine lymph node at peak response to challenge with the parasite were collected by surgical cannulation and purified using lineage-specific monoclonal antibodies to give a subtyped cytotoxic T-cell population. The purified cytotoxic T cells were transferred to an identical (split-embryo) twin of the immune animal, which had been infected with a lethal dose of T. parva. The transferred cells were capable of high levels of parasite-specific killing in vitro and conferred protection on the naive twin. In further experiments, scientists cloned the demonstrably protective cytotoxic T cells derived from the cannulated lymph node and are using these to identify other T. parva schizont antigens with vaccine potential.
Vaccine development is a new project area in ILRAD's theileriosis program initiated to characterize and exploit the immunization and protective properties of candidate vaccine antigens. This work includes studies to determine the inductive requirements of the bovine immune response and work on the development of antigen delivery systems.
ILRAD's approach to the development of new vaccines includes detailed studies of the bovine immune system so as to provide a rational basis for vaccine development. The protective immune response that has been identified and examined in most detail is the cell-mediated response to schizont-infected lymphocytes. Theileria parva has contributed much, as a model, to an understanding of the bovine immune response. Areas of basic bovine immunology that continued to be studied in 1990 are the definition of subpopulations of white blood cells (leucocytes), T-cell receptors, major histocompatibility complex antigens and antigen-presenting cells.
T lymphocytes cannot recognize free antigens, but require that antigen first be processed to peptide fragments by an antigen-presenting cell and expressed on the cell surface in association with major histocompatibility molecules. The latter are glycoproteins encoded by a family of genes known as the major histocompatibility complex (MHC). Cytotoxic T lymphocytes recognize foreign antigens that have been synthesized within the target cell (for instance by a virus or intracellular parasite) only when these are found in association with class I MHC molecules (Figure 11). In contrast, helper T cells, the other major subpopulation of T lymphocytes, recognize external soluble antigens that have been taken up and processed by antigen-presenting cells before being expressed on their surface in association with class II MHC molecules.
Figure 11. Interaction of a cytotoxic T lymphocyte (T cell) that specifically recognizes the parasite Theileria parva and a lymphoblast infected with this parasite. Inside the lymphoblast, parasite antigen is degraded in the cytosol to peptide fragments. The parasite fragments associate with nascent molecules of the class I major histocompatibility complex (MHC) en route to the surface of the lymphoblast. On the cell surface, the peptide-MHC complex is recognized by an antigen receptor on the surface of the cytotoxic T cell. This interaction is stabilized by CD8 accessory molecules and leads to activation of the cytotoxic T cell killing mechanism, which destroys the infected cell.
The antigen receptor on T cells recognizes a combination of antigenic peptide and self-MHC molecules, and cytotoxic T lymphocytes from an immune animal will identify and kill only infected cells carrying MHC molecules identical to its own. This phenomenon is known as MHC restriction (Figure 12). Continuing work in this area indicates that parasite antigens associate preferentially with certain MHC molecules, suggesting that the MHC type of an animal may influence the specificity of its cytotoxic T lymphocyte responses to the parasite. Such MHC-related effects may be relevant to any future immunization of large populations of cattle with subunit parasite components.
Figure 12. Schematic diagram of the role of MHC restriction in bovine immune responses to cells infected with the parasite that causes East Coast fever. A host cytotoxic T cell has recognized and attached itself to a site on the surface of a another host lymphocyte cell that has been infected with Theileria schizonts. When a lymphocyte cell is parasitized by Theileria schizonts, parasite-related antigens become embedded in the host cell membrane. Host cytotoxic T cells specifically recognize such foreign molecules only when these are displayed on a cell surface in combination with proteins that identify the hosT. These latter proteins are class I proteins of the major histocompatibility complex (MHC), a group of genes responsible for immune responses. The cell infected by the parasite is killed by the cytotoxic T cell when the parasite molecules and host MHC proteins are located together on the cell surface. However, if the cytotoxic T cell and parasitized T cell do not share the same MHC antigens, the cytotoxic T cell will not kill the parasitized cell. This phenomenon is known as MHC restriction. (From an illustration in Readings from Scientific American: The Molecules of Life, New York: W.H. Freeman, 1985, p. 74.)
The pivotal role of MHC molecules in T-cell recognition has made the characterization of these molecules in cattle a priority of scientists at ILRAD. These studies have concentrated on typing cattle according to their class I MHC PHENOTYPE and using this information in studies of bovine T-cell responses. The function of MHC molecules in antigen presentation is also being investigated.
Afferent lymph, which drains the various mammalian tissues into lymph nodes, contains a population of dendritic cells known as veiled cells. Scientists at ILRAD have shown that afferent lymph veiled cells are more efficient than blood monocytes (another type of antigen-presenting cell) in the presentation of soluble antigen to immune T lymphocytes. The mechanisms by which afferent lymph veiled cells process and present antigen continued to be characterized during the year with a view to exploiting these cells in immunization strategies based on soluble antigenic components of T. parva.
The results of this work demonstrated that the afferent lymph veiled cells play an important role in the generation of primary and secondary T-cell responses in cattle. The results also indicate that afferent lymph veiled cells may be important in antigen delivery strategies. The cannulation of afferent lymphatics appears to be a useful technique for investigating the effects of adjuvants on the antigen-presenting function of these cells.
An important part of research in vaccine development is the identification of antigen delivery systems that will help induce protective immune responses in the host animal. Delivery vehicles based on recombinant organisms have several advantages over conventional systems. Recombinant vaccinia viruses have been shown in several experimental systems to give rise to potent antibody and cytotoxic-T-lymphocyte responses.
A recombinant vaccinia virus was constructed in 1989 that incorporates the gene encoding the p67 sporozoite antigen of T. parva (Figure 13). Inoculations of this virus into rabbits elicited antibody capable of neutralizing parasite invasion in vitro. The vaccinia virus continued to be characterized this year, during which it was shown to produce similar neutralizing responses in guinea pigs and cattle. In cattle, T-cell proliferation in response to the p67 antigen was observed in vitro. In all instances, however, the antibody levels produced were low and unlikely to protect the host. Further studies of the immune responses of cattle to recombinant virus are in progress.
Figure 13. This figure gives a broad outline of ILRAD's use of genetic engineering to produce large quantities of a protein molecule identified at ILRAD that appears on the surface of Theileria parva parasites. This molecule, termed p67, shows promise as a base for a vaccine to protect livestock against theileriosis.
