R. L. Spooner

Innate and Adaptive Immune Responses Co-operate to Protect Cattle against Theileria annulata

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

Infection of bovine monocyte/macrophage populations with Theileria annulata and Theileria parva

Infection and transformation of cells of the bovine immune system by Theileria annulata and T. parva were compared. Preliminary experiments with mammary gland macrophages indicated that they were permissive to infection by T. annulata but only to a limited extent by T. parva. Further experiments involved several purified subpopulations of bovine cells including bovine monocytes, T cells and MHC class II positive and negative populations. These subpopulations were incubated with T. annulata or T. parva sporozoites in limiting dilution cultures. T. annulata preferentially infected macrophage type cells and also MHC class II positive cells, whereas the frequency of MHC class II negative cells infected by this parasite was negligible. T cells also showed a very low level of infection. In complete contrast, T. parva preferentially infected T cells and did not infect cells phenotypically defined as monocytes at all. These results suggested that class II expression was necessary for T. annulata infection and not necessary for, though not a barrier to T. parva infection. T. annulata infected cell lines all expressed class II molecules to varying degrees. Other available phenotypic markers were only expressed at very low levels or no longer expressed. The immunological significance of the different cell preferences and phenotypes of infected cell lines of T. annulata and T. parva is discussed.

A non-protective T helper 1 response against the intra-macrophage protozoan Theileria annulata

Theileria annulata is a protozoan parasite which infects and transforms bovine macrophages. Infected macrophages possess augmented antigen presentation capabilities, as they are able to activate the majority of T cells from unexposed animals. In vivo, T cells in the draining lymph node (principal site of parasite development) are activated ‘non‐specifically’ by the parasite. This event is followed by failure of the immune response to control the infection. Protective immune responses against intra‐macrophage protozoa are usually mediated by T helper 1 (Th1) T cell responses. Here we examine the cytokine responses made by T. annulata‐activated T cells. We show that the outcome of in vitro activation of T cells by parasitized macrophages is a skewing of their cytokine responses towards preferential expression of interferon‐gamma (IFN‐γ) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN‐γ protein are found in lymph efferent from infected lymph nodes, while expression of IL‐4 mRNA within the node stops. IFN‐γ production does not correlate with protection against the parasite, as infected cells flourish during peak IFN‐γ production, and only very small amounts of IFN‐γ are produced during the effective immune response of an immunized animal. Overproduction of IFN‐γ and loss of IL‐4 expression are also likely to account for the failure of B cells to reach the light zone of germinal centres, a developmental step which is tightly regulated by cytokines.

Proinflammatory cytokine expression by Theileria annulata infected cell lines correlates with the pathology they cause in vivo.

Control of Theileria annulata is currently best achieved by the use of live attenuated cell line vaccines. However, the mechanisms underlying attenuation are unclear and there is a need to rapidly produce new cell line vaccines, which could safely and effectively vaccinate cattle against tropical theileriosis. There is increasing evidence to suggest that proinflammatory cytokines produced by T. annulata infected cells play a central role in both pathology and immune evasion. This study aimed to test this hypothesis and to evaluate cytokine expression as a marker of virulence. The pathogenicity and protective efficacy of cloned T. annulata cell lines that expressed different levels of proinflammatory cytokines were compared. In two independent trials using different stocks of T. annulata, cell lines that expressed higher levels of proinflammatory cytokines induced severe reactions, and in some cases death, when used to vaccinate groups of cattle. In contrast, low cytokine expressing lines induced low post-vaccinal reactions. The results clearly demonstrated that cytokine expression by T. annulata infected cells could be used as a marker of virulence and provided strong evidence to support a role for cytokines in the induction of pathology. Both high and low cytokine expressing cell lines protected cattle against heterologous challenge infection, offering the possibility of using cytokine expression to rapidly select new safe, potent vaccines against tropical theileriosis without the need for culture attenuation.

Stage-specific responses following infection withTheileria annulata as evaluated using ELISA

An enzyme-linked immunosorbent assay (ELISA) using sporozoite, schizont and piroplasm antigens was developed to study the immune response of animals that had been immunised with eitherTheileria annulata sporozoites or schizont-infected cells and then challenged with sporozoites. The aim was to identify the most suitable antigen for a routine screening test and to compare the sensitivity of the latter with that of the indirect fluorescent antibody test (IFAT). As determined by ELISA, cattle produced antibodies to all three antigens, regardless of the method of immunisation. The schizont antigen was the least sensitive, whereas the sporozoite antigen displayed high pre-inoculation values. In contrast, the piroplasm antigen exhibited low non-specific pre-infection levels and high post-immunisation and post-challenge values according to both ELISA and IFAT. Therefore, the latter was thought to be the most appropriate antigen for use in ELISA.

Evidence for strain specificity in cytotoxic T-lymphocyte-mediated, major histocompatibility complex class I-dependent killing of Theileria annulata-infected cells

Abstract Cattle immunised against Theileria annulata with one parasite strain have been found to be immune to re-challenge with different strains of the parasite. However, recent evidence of apparent strain specificity has been documented in cattle immunised with attenuated parasite-infected cells. In this study the strain specificity of major histocompatibility complex class I-restricted cytotoxic T-lymphocytes (CTL), a major anti-parasite effector mechanism, was examined. CTL generated following challenge with the Hissar (Indian) strain effectively lysed autologous cells infected with this strain of the parasite. However, CTL were less effective against cells infected with the Gharb (Moroccan) strain and showed virtually no reactivity against the Ankara (Turkish) strain, providing the first direct evidence for strain specificity in immune responses against T. annulata.

Characterization of efferent lymph cells and their function following immunization of cattle with an allogenic Theileria annulata infected cell line.

Immunization of cattle with in vitro propagated bovine mononuclear cells infected with Theileria annulata induces a protective immune response. Activation and effector function of T cells exiting the lymph node draining the site of cell line immunization were investigated to understand the mechanisms involved in the generation of immunity. Immunized animals exhibited a biphasic immune response in efferent lymph as well as peripheral blood. The first phase corresponded to allogenic responses against MHC antigens of the immunizing cell line and the second was associated with parasite specific responses. An increase in the output of CD2(+) cells and MHC class II(+) cells in efferent lymph was observed after cell line immunization with a corresponding decrease in WC1(+) cells. Although the percentage of CD4(+) T cells did not change significantly over the course of the experiment, they became activated. Both CD25 and MHC class II expressing CD4(+) T cells were detected from day 7 onwards, peaking around day 13. Efferent lymph leukocytes (ELL) exhibited sustained responses to IL-2 in vitro following cell line immunization. Antigen specific proliferation was also detected first to the immunizing cell line and then to parasite antigens. The two peaks of CD2(+) cells were observed, which corresponded to similar peaks of CD8(+) cells. The increase in CD8(+) cells was more pronounced during the second parasite specific phase than the first allogenic phase. Activated CD8(+) T cells mainly expressed MHC class II and some expressed CD25. Significantly the peak of activated CD4(+) T cells preceded the peak of activated CD8(+) T cells, highlighting the role of T. annulata specific CD4(+) T cells in inducing parasite specific CD8(+) cytotoxic responses. A biphasic cytotoxic response also appeared in efferent lymph and peripheral blood, the first directed against MHC antigens of the immunizing cell line followed by MHC class I restricted parasite specific cytotoxicity. The cytotoxic responses in efferent lymph appeared earlier than peripheral blood, suggesting that activated CD8(+) cells exiting the draining lymph node following immunization with T. annulata infected schizonts play an important role in the development of protective immune responses.

PROTECTIVE IMMUNE RESPONSES TO THEILERIA ANNULATA OF RELEVANCE TO VACCINE DEVELOPMENT

SummaryA series of projects onTheileria annulata funded by the European Union (STD1/STD2/STD3) have provided convincing evidence that macrophage and natural killer (NK) cell-dependent immune mechanisms may directly control the proliferation of different stages ofT. annulata in cattle. The evidence for this conclusion and the implications for vaccine development are discussed in the following paper.

Genetics of disease resistance and the potential of genome mapping

Evidence for genetic variation in resistance to disease has been accumulating for many years but it is only recently that much direct use is being made in commercial animal production. Although there has been evidence of local breeds being more resistant than imported breeds to a variety of pathogens little use has been made of this. It is perhaps because of the immense success over the past 100 years of bacteriology and parasitology, coupled with advances in therapy and vaccine development, that there has been reduced interest in genetic resistance. There are many economically important diseases for which there is no cost effective vaccine. Trypanosomiasis, African swine fever, Theileria sergenti infection are three. With some of these there is no treatment either, but even where there is a therapy, drug resistance becomes a problem. Development of drug resistance to many parasites, ticks and intestinal nematodes is widespread and increasing. It is a serious problem in many countries and as resistance develops the new acaricide or anthelminthic to overcome this resistance will be more expensive than the one it replaces. With tropical theileriosis where a new drug, Butalex, has been only recently marketed, there has not yet been time for resistance to develop. But develop it almost certainly will and as there are no other drugs in the pipeline at the moment this route for protecting against the disease will only have a finite life. In Zimbabwe for instance there had been, preindependence, very intensive acaricide application, enforced by laws. During the independence struggle one of the ways to fight against the government was not to obey those laws and dips were filled in and dipping stopped. East Coast fever in particular killed thousands of cattle during this time as the cattle included many European breeds, highly susceptible to East Coast fever. If they had had more indigenous breeds then the effect would have been less marked and some in Zimbabwe advised against returning to a blanket dipping regime in the future. An alternative means of control is by genetic means. Genetic resistance to disease is the sustainable control measure par excellence. Because once established it does not require high management inputs to maintain it. Look for instance at the situation with myxomatosis in rabbits. It was introduced to the rabbit population here and in Australia. Initially it was highly lethal but gradually resistant animals developed and now the disease is a non event. But one cannot generally expose agricultural animals to disease as happened with rabbits. Poultry provide an example of a species where genetic resistance has been used and is used commercially. It was shown many years ago that there were genetic components to resistance against a variety of protozoal, bacterial, fungal and viral diseases. It was shown that one could breed chickens resistant to Mareks disease by exposing birds to the virus. But then a vaccine was developed and most of the industry forgot about genetic resistance. It was then found that some animals were not well protected by the vaccine and succumbed to disease before commercial slaughter age. Genetic resistance was revisited and it was found that the vaccine worked better in genetically resistant animals. Now both vaccines and genetic resistance play a part in the poultry industry. Although there has been suggestive evidence for many years that there are breed differences in disease resistance in cattle, there has been too little use of this fact in the

Parasite-mediated steps in immune response failure during primary Theileria annulata infection

Summary“Exotic” European cattle are highly susceptible toT. annulata infection. In immunised animals, several effective anti-parasite responses can be demonstrated, such as anti-macroschizont cytotoxic T cells (CTL), and nitric oxide killing of parasites. The failure of infected animals to mount an effective primary immune response suggests that the presence of the parasite directly interferes with the development of immunity. When the activation pathways of CD4+ T cells in draining lymph nodes were examined during the course of a primary infection it was found that the development of this essential arm of the immune response was altered. Instead of interacting with antigen presenting cells in the paracortex, the majority of CD4+ T cells were rapidly activated by developing infected cells in the medulla of the node. Activation of T cells by infected cells also drastically alters the cytokines produced by the T cells. During effective immune responses, the principal cytokine involved appears to be IL-2, with only small, controlled “bursts” of IFNγ production. However, IL-2 responsiveness is only transient in animals undergoing primary infection, while IFNg production is greatly elevated. IFNγ does not appear to control parasitised cells, and may even aid the growth of infected macrophages—large numbers of macrophages enter the cell cycle during the peak period of IFNγ production. Uncontrolled parasite-induced IFNγ production is also likely to account for the local failure of antibody responses. Germinal centres in infected lymph nodes lose normal morphology, with IFNγ sensitive zones failing to develop. A third strategy which the parasite uses to evade immune response destruction is through affecting CTL activity. CTL in infected draining lymph nodes lose expression of the adhesion molecule CD2—a molecule is essential in adherence to target cells for lysis. CD2− CTL are unable to lyse macroschizont infected cells.