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Introduction
Theileria annulata is an important tick-borne haemoprotozoan parasite which is transmitted by the ixodid ticks of the genus Hyalomma. It is the causative agent of an extremely debilitating and often fatal disease of cattle known as tropical theileriosis. This disease affects several hundred million cattle in the Middle East and Central Asia including the Indian subcontinent, China, North Africa, and southern Europe. In India and other developing countries, tropical theileriosis is a serious threat to cross-breeding programmes aimed at increasing the milk yield of the local zebu cattle, as high yielding exotic European cattle imported into endemic areas and their cross-breds are highly susceptible to the disease. Indigenous animals are relatively resistant, presumably because they have been living with the parasites for centuries. The macroschizont stage of the parasite has been propagated in vitro and attenuated by continuous passaging for use as a cell culture vaccine to control this disease in many countries. However, the vaccine has only been used to a limited extent to control the disease. Much progress has been made in understanding the mechanisms involved in the development of protective immunity and these are discussed and reviewed (1,2). Here we discuss the nature of the infected cells, role of cytokines in parasite induced transformation, immune responses and alterations induced by the parasite in its host that either mediate immunity or favour the progression of disease or development of immunity in tropical theileriosis.
Life cycle of the parasite
Theileria annulata enters the bovine host as a sporozoite during tick feeding. The sporozoites rapidly invade mononuclear cells and subsequently develop into intracellular schizonts in the draining lymph node. The parasite induces proliferation of the infected host cells, which become blastoid and divide in synchrony with the parasite. Lymphatic cannulation studies have shown that sporozoites migrate to the lymph node within minutes of subcutaneous infection, are trapped there and never leave the lymph node (3,4). Infected cells are probably generated from infection of lymph node cells rather than migration of infected cells from the site of sporozoite infection following in vivo infection. Macroschizont infected cells developing in the lymph nodes have been found to be of monocyte/macrophage lineage (1). The parasite infected cells start leaving the node via efferent lymph as soon as they are transformed and migrate rapidly to distant lymph nodes, spleen and thymus. The parasitized cells also spread rapidly into non-lymphoid organs viz; liver, kidney, lung, abomasum, adrenal glands and pituitary gland, brain and heart (2). After a few cycles of multiplication, merozoites are released which invade erythrocytes and subsequently mature into piroplasms. Ticks ingest parasite-infected erythrocytes during blood meals as larvae or nymphs. The parasite travels to the salivary glands and the tick becomes capable of transmitting infection in the next instars as nymph or adult.
Target cells infected by T.annulata sporozoites
Although sporozoites invade many cell types, not all transform efficiently into continuously growing cell lines. In vitro studies showed that T.annulata sporozoites infected and transformed cells of monocyte and macrophage lineage and to a lesser degree B cells, but not T cells. On the other hand, T.parva sporozoites transformed T cells and B cells, but not monocytes/macrophages (5). T.annulata preferentially infected MHC class II+ cells (6). Bovine alloreactive cytotoxic T cell lines were very easily infected by T.parva sporozoites, but not by T.annulata sporozoites (7). CD14+ (the lipopolysaccharide receptor) monocytes were transformed very efficiently by T.annulata. (8)
Immunisation with attenuated macroschizont infected cell lines
Immunisation of susceptible animals with schizont infected cell lines is the most widely used method of immunoprophylaxis against T.annulata. Schizonts were attenuated by prolonged cultivation in cell cultures and resulted in very mild clinical and parasitological reactions on inoculation into susceptible animals. With loss of virulence in cultures, the parasite lost the capacity to produce piroplasms and immunogenicity and it is necessary to get a balance where sufficient virulence is lost and enough immunogenicity retained. Animals immunised with a cell culture vaccine developed mild parasitological reactions on natural or experimental sporozoite challenge, but were usually protected. These results encouraged immunisation trials against tropical theileriosis under field conditions. Satisfactory protection from the disease was induced in young and adult cattle of all breeds and also in pregnant cows (9, 10, 11). Calves born of dams immunised with cell culture vaccine in late pregnancy were fully susceptible to theileriosis after birth. Cell culture propagated schizont vaccine has been used successfully in many T.annulata endemic countries. However, the number of doses used is small relative to the numbers of animals at risk. The vaccine has usually been subsidised by government, so in countries with private veterinarians they prefer to use treatment, which gives them more profit. In India, the usage of the only commercial vaccine has gradually fallen but this may be due to the disease becoming less important as importation of susceptible stock has been stopped. There is good cross immunity between parasite stocks from different countries and regions and as long as there is regular challenge of immunised animals, the immunity is solid.
Duration of immunity in the absence of reinfection after cell line immunisation has not been fully investigated. Immunity started to wane after six months in the absence of sporozoite challenge under field conditions (12). Under field conditions, animals were found to be immune for at least two disease seasons in regions where infected ticks are present (10,13). Animals might need reimmunisation if challenge is low or if they are moved from disease-free to endemic areas. During cell line immunisation, the parasite is introduced in the recipient animal within a foreign cell in the form of a graft. Therefore, histoincompatibility between cell line and the recipient influence successful reimmunisation on inoculation of a parasite infected cell line. An important feature for the development of immunity after immunisation with allogeneic infected cells is that infection has to transfer and establish into the cells of the recipient animal (14,15). Re-immunisation with the same cell line will not always be effective (15). A panel of attenuated cell lines bearing different major histocompatibility complex (MHC) phenotypes and used sequentially would provide an effective vaccination and re-vaccination strategy.
Cytokine production correlates with virulence
Several different cytokines viz; IL-1, IL-6, TNFa and IFNg play an important role in the pathogenesis of tropical theileriosis (1). There is a correlation between the cytokine profiles of the cell line and the ensuing reactions in inoculated animals. Early passage unattenuated cell lines, selected to produce low levels of cytokines, stimulated strong protective immunity without any post-vaccinal side effects (16). This technique can be exploited to select new vaccine cell lines quickly on the basis of their cytokine profiles without the need for long-term attenuation.
Immune responses against Theileria annulata
The bovine immune system is subjected to different antigenic determinants at each stage of the parasite’s life cycle. This results in a heterogeneous response comprising of humoral and cellular components. Since sporozoites are extra cellular, effective anti-sporozoite immunity is humoral and specifically mediated by neutralizing antibodies. However attempts to protect using sporozoite antigen have not been successful (2).
Cell mediated immune responses to the schizont infected cells are generally thought to be responsible for the protective immunity (2,3,17). MHC class I restricted cytotoxic T cells and macrophage mediated cytostasis have been shown to play an important role in development of protective immunity to the schizont stage. Antibodies have also been detected against this stage (18,19,20). However, these antibodies mainly seem to be of diagnostic importance and have little role in protection.
Merozoites, like sporozoites, are extra cellular and, therefore, are also a potential target for the protective humoral immune response. Antibodies against this stage can inhibit erythrocyte invasion and attempts have been made to produce a vaccine to the merozoite stage, which was not successful. However, as noted above, immunity wanes and rechallenge from the environment is essential to maintain protection. Thus merozoite challenge of cell line - vaccinated animals boosts immunity and should be encouraged. CD8+ Cytotoxic T cells (CTLs) are known to lyse schizont-infected cells in a MHC class I restricted manner (3,17,), but they also produce IFNg, which act directly on trophozoite infected cells (2). Although MHC restricted cytotoxic cells can be isolated from animals recovering from acute infection (3,17) subsequently highly immune animals show no evidence of circulating CTL. Thus other mechanisms including natural killer cells, activated macrophages, NO and TNFa may play a role and these have been discussed earlier (2).
Alterations induced by T. annulata in its host
In spite of innate and adaptive immune responses, macroschizont infected cells are not destroyed during the primary infection. The properties of the host target cells, macroschizont infected cells and their subsequent post-infection phenotypes are responsible for the alterations induced by the parasite (1).
Expression of Super-antigen: T.annulata infected cells are professional antigen presenting cells (APC) expressing high levels of MHC class II. T.annulata schizont-infected cells express super-antigen and lead to the disruption of antigen recognition and effector mechanisms including CTLs and B cells (1). MHC molecules commonly present the super-antigen but bind outside the normal antigen-binding groove of the MHC molecule and simultaneously bind to the conserved region of variable domain of the T cell receptor, thus bypassing the normal recognition site. In this way, a single antigen can activate a large number of the T cell pool non-specifically.
Altered T cell responses: Macroschizont infected cells possess an ability to activate and induce proliferation of autologous T cells from animals that have not been exposed to the parasite. Although CD8+ T cells increase greatly in number during an acute T.annulata infection, yet they exhibit no killing of parasite infected cells. Instead of the usual interaction between APCs and T cells in the lymph node paracortex, large number of CD4+ T cells are activated by the MLR capacity of infected APCs in the medulla of the node within four days. Thus T cells are activated nonspecifically by parasitized cells rather than interacting with normal APC. Instead of remaining in the node as normal, the activated T cells leave the node. A large number of IL-2 responsive, CD25+ CD4+ T cells are found in the efferent lymph from day 6 onwards. They are, therefore, no longer present at the site where an anti-parasite response is required. Thus specific T cells are not able to expand and mediate immunity (1).
Parasite protein expression: Cell proliferation is a biological process, which is controlled by a highly coordinated mechanism. There are certain proteins, which are transported from nucleus to cytoplasm, or vice-versa that can interfere with transcription or replication of eukaryotic cells. It has been postulated that parasite proteins either expressed on the surface of the schizonts or secreted into the host cell cytoplasm may interfere with the signal transduction pathway of host cells. Several transcriptional factors have been shown to be expressed in T.annulata infected cells. Transcription factor AP-1 (1), Ki67 (21) and serine/threonine kinase casein kinase II (22) are synthesized in T.annulata infected cells and are known to induce development of macroschizont infected cells.
Metalloproteinase expression: Infection of bovine leukocytes with T.annulata is associated with the expression of nine proteinase activities belonging to metalloproteinase (MMP) class. The infected cells produce MMP, which causes lesion formation by digestion of the extra cellular matrix and are postulated to be responsible for various pathological features of theileriosis. It has been reported that cell lines with a low level of MMP activity exhibit reduced metastasis (23). However, recent studies revealed no correlation of MMP profiles with virulence (16).
Non-protective cytokine response: Several cytokines were tested for their capacity to enhance or inhibit the proliferation of macroschizont-infected cells. T. annulata infected cells constitutively express mRNA for a range of proinflammatory cytokines i.e. IL-1, IL-6, IL-10, TNF-a (16,24). Parasitized cells express IL-2R (CD25), through which the exogenous IL-2 may exert its growth enhancing effect. IL-2 seems to play an important role in the pathogenesis of theileriosis by supporting the initiation of the host cell transformation.
Macroschizont-infected cells induce activation of neighbouring T cells in the lymph node via IL-12 and cause T cells to secrete IFNg. At this stage, there is a danger of apoptosis to parasite-infected cells. But IFNg induces IL-1 and TNFa production by the developing infected cells, which act as autocrine growth/apoptosis rescue factors, thus stabilizing the developing macroschizont-infected cells (1). Greatly elevated amounts of IFNg coinciding with the peak of activated T cells during a primary infection with T.annulata are not capable of protecting cattle, rather help infected cells to proliferate in the draining lymph node (1). In the case of other macrophage resident protozoan parasites such as Toxoplasma and Leishmania, IFNg activation of infected macrophages induces production of NO, which damages intracellular parasites and leads to their rejection (1).
Altered CTL responses: MHC class I restricted CTLs are very important for parasite clearance during T.annulata infection, but no CTL activity is found in lethally infected animals undergoing primary infection (25). Activated CD8+ T cells are present in the lymph nodes and efferent lymph of acutely infected animals and also express activation markers in the form of CD25 and MHC class II (3,26). These CD8+ cells have altered surface expression of CD2, which is an important adhesion molecule and is involved in contact of effector CTLs to target cells, activation of CTL activity and target lysis. These cells were MHC class II+ but had lost CD25 expression suggesting an inappropriate activation of T cells in response to the parasite. The cells were less responsive to Con A or exogenous IL-2 stimulation in vitro during the later stages of infection. The cells did not proliferate in vitro in response to autologous parasite infected cells and did not kill autologous parasite infected cells suggesting lymphocyte unresponsiveness. Further, expression of CD2 on CTLs was restored after treatment of sick animals with buparvaquone, a drug that effectively kills the parasite (26). This suggests that the loss of CD2 epitopes from CD8+ T cells is associated with the growth of macroschizont–infected cells. The mechanisms involved in the down regulation of CD2 expression on CD8+ T cells are yet to be investigated.
Future prospects
For control of this disease, which is our ultimate goal, a suitable vaccine against the parasite is required. It should be cheap, stable and safe with short selection time and provide efficient CTL responses. The current control method employed is the vaccination of the animals with live attenuated T.annulata cell culture vaccines. These are highly efficient and cheap to produce. The problems are more in logistics. A panel of candidate vaccine lines has to be established, which can now be selected on the basis of their capacity to produce low levels of proinflammatory cytokines in vitro (16). Low passage low cytokine expressing clonal cell lines may provide enhanced vaccine efficacy as compared to the existing vaccines, which are high passage lines and with low immunogenicity. Studies on improved delivery of the vaccine to make it suitable for field use are very important.
Research is continuing on the development of sub-unit vaccine(s). Two stage specific antigens i.e. sporozoite antigen SPAG-1 and a major merozoite surface antigen, Tams-1 have been tested with poor results (2). Immunization with the sporozoite antigen SPAG-1 can slow the onset of the disease and time to death but animals still die. Immunization against merozoites does not protect and could prevent field challenge, which is needed to boost immunity. Much of the effort on molecular vaccines has been justified by the need to produce a vaccine that cannot ‘revert to virulence’. This concept is a misunderstanding of the epidemiology of the disease. Vaccinated animals have to be exposed to constant virulent challenge to maintain immunity. This is not to say that a molecular vaccine which stimulated cell mediated immunity could be very much easier to deliver and remove the dangers of passing on other diseases which are always a problem with a live vaccine.
Finally we must consider variation in genetic susceptibility. Local breeds in endemic areas are relatively resistant to the disease and as the pressure to crossbreed wanes the disease is becoming less important. Another aspect of genetic variation in susceptibility is that when European breeds are imported into endemic areas some die but others do not. May be these resistant animals carry resistance genes similar to those in the indigenous breeds and as a result general susceptibility falls. If one could identify these resistance genes one could select suitable stock for importation.
Certainly in India it is not only the vaccine usage that has fallen, Butalex, the only treatment is also little used suggesting that the importance of the disease is falling. Perhaps Theileria annulata in the future will provide a model for studying how to produce vaccines to intracellular parasites in man such as Malaria as much as its economic importance to the cattle industry.
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