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Abstract
The tick-borne protozoan parasite, Theileria annulata, causes an overwhelming disease in Friesian cattle, imported to improve productivity, in a large area of the world. The parasite invades bovine macrophages and induces aberrant changes which seem pivotal in triggering disease in naïve susceptible animals: parasite infected cells acquire dendritic cell features and over-activate CD4+ and CD8+ T cells. Elevated levels of interferon-gamma (IFN-gamma) are induced and B cells are developmentally arrested in the light zone of germinal centres. Infected macrophages are refractory to the effects of IFN-gamma and indeed flourish in its presence. High levels of pro-inflammatory cytokines, as evinced by high acute phase protein responses, probably also play a role in pathology. However, animals can become immune to further challenge. Cellular immune responses involving macrophages, natural killer cells and CD8+ T cells play a major role in recovery and subsequent maintenance of immunity. The main target for immunity appears to be the parasite infected macrophage, as attenuated cell lines can protect and are used as vaccines. Cloned lines selected for low cytokine production protect with no associated pathological reactions. Theileria annulata causes a relatively mild disease in an indigenous breed of cattle, which is associated with lower acute phase protein responses (controlled by macrophage cytokines). Thus the initial host-parasite interactions must determine the balance between immunity and pathogenesis. New generation vaccines to T. annulata should both induce active immunity and suppress pathology.
... Theileria annulata Ta5- Ta9-Ta11 CD8 + T-cell responses [105] SPAG-1 Induction of IL-4-producing T-cells [106] Tams1 ...
... B cells are arrested in the draining lymph nodes and do not differentiate, probably due to the elevated levels of IFN-γ. This inhibition may be a pathogen mechanism to change the uncontrolled Th1 responses observed both in vivo and in vitro [106,126]. ...
... T. annulata-infected macrophages acquire aberrant phenotype and show dendritic cell features, including enhanced antigen-presenting capabilities [127][128][129]. Moreover, the pathogen polyclonally activates naive T cells in a non-specific, superantigen manner [106,126] with loss of CD2 expression on the T cell surface [130], disrupting the normal T lymphocyte response and subsequent protective immunity [131]. Such a process is considered as the T. annulata immune evasion strategy. ...
Tick-transmitted pathogens cause infectious diseases in both humans and animals. Different types of adaptive immune mechanisms could be induced in hosts by these microorganisms, triggered either directly by pathogen antigens or indirectly through soluble factors, such as cytokines and/or chemokines, secreted by host cells as response. Adaptive immunity effectors, such as antibody secretion and cytotoxic and/or T helper cell responses, are mainly involved in the late and long-lasting protective immune response. Proteins and/or epitopes derived from pathogens and tick vectors have been isolated and characterized for the immune response induced in different hosts. This review was focused on the interactions between tick-borne pathogenic hemoparasites and different host effector mechanisms of T- and/or B cell-mediated adaptive immunity, describing the efforts to define immunodominant proteins or epitopes for vaccine development and/or immunotherapeutic purposes. A better understanding of these mechanisms of host immunity could lead to the assessment of possible new immunotherapies for these pathogens as well as to the prediction of possible new candidate vaccine antigens.
... For example, macroschizont-infected host macrophages produce proinflamatory cytokines (IL-1α, IL-1β, IL-6, IL-10, TNF-α and INF-α) that may enhance initial establishment of the infection and cause disease [7,10,11,15,19,26], while metastasis of infected cells throughout tissues of the body is thought to involve the expression of metalloproteinases [1,2]. Infected macrophages also have the ability to stimulate an aberrant non-specific proliferation of naive T-cells leading to the production of high levels of INF-γ and IL-2, which can stimulate infected and non-infected macrophages to proliferate, but block an efficient specific immune response [3,11,15,18]. Such a disruption of the immune response is likely to result in disease pathology. ...
... Such a disruption of the immune response is likely to result in disease pathology. A more complete understanding of how T. annulata manipulates the immune response will require detailed knowledge of the molecular mechanisms Theileria parasites utilize to activate and control the gene expression profile of the infected leukocytes [18,35]. ...
... Thus, phenotypic characterization of cells for all the microscopic lesions observed indicated that they contained mature uninfected T lymphoid cells and macrophages. The presence of these cell types in the lesions could be induced by generation of the immune response against T. annulata infection [7,9,11,18], and the tissue lesions associated with acute tropical theileriosis may result from damage caused by the host immune response to different phases of infection [28,29]. For example, macroschizont-infected host macrophages produce proinflamatory cytokines (IL-1α, IL-1β, IL-6, IL-10, TNF-α and INF-α) that may enhance initial establishment of the infection and cause disease [7,10,11,15,19,26], while metastasis of infected cells throughout tissues of the body is thought to involve the expression of metalloproteinases [1,2]. ...
This study was carried out to investigate fifteen cases of acute lethal infection of calves (<or= 4 months of age) by the protozoan parasite Theileria (T.) annulata in the south of Portugal. Calves developed multifocal to coalescent nodular skin lesions, similar to multicentric malignant lymphoma. Infestation with ticks (genus Hyalomma) was intense. Theileria was seen in blood and lymph node smears, and T. annulata infection was confirmed by isolation of schizont-transformed cells and sequencing of hypervariable region 4 of the 18S rRNA gene. At necropsy, hemorrhagic nodules or nodules with a hemorrhagic halo were seen, particularly in the skin, subcutaneous tissue, skeletal and cardiac muscles, pharynx, trachea and intestinal serosa. Histologically, nodules were formed by large, round, lymphoblastoid neoplastic-like cells. Immunohistochemistry (IHC) identified these cells as mostly CD3 positive T lymphocytes and MAC387 positive macrophages. A marker for B lymphocytes (CD79alphacy) labeled very few cells. T. annulata infected cells in these nodules were also identified by IHC through the use of two monoclonal antibodies (1C7 and 1C12) which are diagnostic for the parasite. It was concluded that the pathological changes observed in the different organs and tissues were caused by proliferation of schizont-infected macrophages, which subsequently stimulate a severe uncontrolled proliferation of uninfected T lymphocytes.
... Similar resistance to a closely related parasite Theileria parva has been observed in Ankole cattle indigenous to areas endemic with the resulting East Coast Fever (Paling et al., 1991). The APP response of Sahiwals to experimental challenge with T. annulata sporozoites was considerably lower than that observed in Holsteins, especially the levels of a 1 -acid glycoprotein (Glass, 2001;Glass et al., in preparation). This suggests that pro-inflammatory cytokine expression by infected macrophages may be different in the two breeds and could account for their differences in resistance to T. annulata infection. ...
... Pro-inflammatory cytokines have been implicated as the major cause of pathology observed in susceptible B. taurus cattle infected with the protozoan parasite, T. annulata (Brown et al., 1995;Glass, 2001;Graham et al., 2001;Glass et al., 2003). Certain B. indicus breeds of cattle are relatively resistant (Preston et al., 1992;Bakheit and Latif, 2002;Glass et al., in preparation) with mild clinical responses and lower APP responses (Glass, 2001). ...
... Pro-inflammatory cytokines have been implicated as the major cause of pathology observed in susceptible B. taurus cattle infected with the protozoan parasite, T. annulata (Brown et al., 1995;Glass, 2001;Graham et al., 2001;Glass et al., 2003). Certain B. indicus breeds of cattle are relatively resistant (Preston et al., 1992;Bakheit and Latif, 2002;Glass et al., in preparation) with mild clinical responses and lower APP responses (Glass, 2001). Pro-inflammatory cytokine mRNA levels expressed in T. annulatainfected cell lines correlate with pathology induced in vivo, as measured by semi-quantitative methods (Graham et al., 2001). ...
The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance.
... Given the practical challenges, such as maintenance of the cold chain, reversion to the pathogenic form, and the occurrence of disease in immunocompromised individuals, the live vaccine has drawn less attention (13), whereas the subunit vaccines are believed to be safer and more economical (18) and carry the potential to fill this gap. Some macroschizont stage live attenuated cell line vaccines are believed to provide solid immunity against tropical theileriosis but their manufacturing time and cost for attenuations are principal limiting factors (19). Although some toxicity issues can arise, molecular vaccines are regarded as safer, economical, and identical from batch to batch with zero risk of a reversion to virulence, over-attenuation or being carriers of contaminating pathogens of culture origin. ...
... The purpose of a vaccine should be to potentiate an effective immune response and control overstimulatory mechanisms (19). Therefore, it is desirable to induce an immune response against both the sporozoite and schizont stages of Theileria for complete protection. ...
Tropical theileriosis is a lymphoproliferative disease caused by Theileria annulata and is transmitted by Ixodid ticks of the genus Hyalomma. It causes significant losses in livestock, especially in exotic cattle. The existing methods for controlling it, chemotherapeutic agents and a vaccine based on an attenuated schizont stage parasite, have several limitations. A promising solution to control this disease is the use of molecular vaccines based on potential immunogenic proteins of T. annulata. For this purpose, we selected five antigenic sequences of T. annulata, i.e. SPAG-1, Tams, TaSP, spm2, and Ta9. These were subjected to epitope prediction for cytotoxic T lymphocytes, B-cells, and helper T lymphocytes. CTL and B-cell epitopes with a higher score whereas those of HTL with a lower score, were selected for the construct. A single protein was constructed using specific linkers and evaluated for high antigenicity and low allergenicity. The construct was acidic, hydrophobic, and thermostable in nature. Secondary and tertiary structures of this construct were drawn using the PSIPRED and RaptorX servers, respectively. A Ramachandran plot showed a high percentage of residues in this construct in favorable, allowed, and general regions. Molecular docking studies suggested that the complex was stable and our construct could potentially be a good candidate for immunization trials. Furthermore, we successfully cloned it into the pET-28a plasmid and transformed it into the BL21 strain. A restriction analysis was performed to confirm the transformation of our plasmid. After expression and purification, recombinant protein of 49 kDa was confirmed by western blotting. An ELISA detected increased specific antibody levels in the sera of the immunized animals compared with the control group, and flow cytometric analysis showed a stronger cell-mediated immune response. We believe our multi-epitope recombinant protein has the potential for the large-scale application for disease prevention globally in the bovine population. This study will act as a model for similar parasitic challenges.
... Tick-borne diseases (TBD) pose major problems for the health and management of domesticated bovines of tropical and subtropical regions of the world (Jongejan and Uilenberg 1994). Bovine tropical theileriosis caused by Theileria annulata is an overwhelming haeamoprotozoan tick-borne disease in taurine and cross-bred cattle in developing countries (Glass 2001). In India, this obligate intracellular protozoan is transmitted transstadially predominantly by Hyalomma anatolicum anatolicum in dairy animals. ...
... anatolicum anatolicum) on cattle than on buffaloes (Haque et al. 2010) may be the reason for preponderance of infection in cattle population. Glass (2001) reported lower prevalence and milder form of disease in indigenous as compared to crossbred cattle which was believed to be associated with the lower acute phase protein responses controlled by macrophage cytokines in these animals. It has also been reported that mortality due to tick-borne diseases is usually low or insignificant in indigenous cattle because of enzootic stability and lower chances of tick infestation in indigenous cattle (Moll et al. 1984). ...
Bovine tropical theileriosis, caused by Theileria annulata,is one of the economically important fatal tick borne haemoprotozoandiseases of dairy animals. The aim of present investigation was to map the distribution of T. annulatain bovines of Punjab stateof India in relation to various risk factors including age, sex of animals, location and management of farms. In a cross sectionalstudy, a total of 1278 blood samples were randomly collected from twenty districts falling in five major agro-climatic zones ofPunjab. All the samples were screened by blood smear examination followed by polymerase chain reaction targeting SSU rRNAgene for Theileriaspp. PCR positive samples (n = 386) for Theileriaspp. were then analyzed for T. annulataby amplificationof Tams1gene.Overall prevalence of T. annulatawas found to be 29.26% in Punjab, with highest in western Zone (40.49%, 95% CI = 35.57–45.41) and lowest in submountain zone (18.90%, 95% CI = 13.73–24.06). The propensity of incidence of T. annulata was found to be highest in cross bred cattle (32.40%, 95% CI = 29.87–34.94), followed by indigenous cattle (19.64%,95% CI = 10.67–28.61) and buffaloes (19.2%, 95% CI = 14.99–23.41). Between the two sexes, incidence of T. annulatawashigher in female animals. Calves less than 6 months of age were found to be more prone to theileriosis.
... It was also reported that TNF-α, IL-1, and IL-6 produced by infected mononuclear cells are responsible for the diverse clinical signs of theileriosis such as depression, pyrexia, anorexia, cachexia, and disseminated hemorrhages (Bielefeldt et al. 1989;Jongen-Lavrencic et al. 1996;Preston et al. 1999;Glass et al. 2003). However, the exact mechanism whereby T. annulata causes pathogenesis and clinical symptoms in susceptible cattle remains to be fully elucidated (Preston et al. 1999;Preston 2001;Glass 2001). There is also increasing evidence to suggest that IL-1, IL-6, and TNF-α are transcriptionally expressed at high levels in T. annulata-infected cells, and the pro-inflammatory cytokine mRNA expression was correlated with pathology induced in vivo; however, their individual roles in T. annulata induced pathology in cattle is still unknown (Graham et al. 2001;Glass et al. 2003). ...
... Our findings might suggest that T. annulata infections cause severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines. This finding complies with previous reports, although most of these studies are based on observation obtained in vitro experiment (Bielefeldt et al. 1989;Preston et al. 1999: Graham et al. 2001Glass 2001;Glass et al. 2005). It has been shown that inflammation in excess is detrimental, and that excessive production and release of APPs and proinflammatory cytokines may lead directly to pathology (Glass et al. 2003). ...
Abstract The present study was conducted to evaluate the
cytokine response following natural infection of Theileria
annulata in cattle. Initial survey included 173 crossbred cattle
which were examined for the presence of Theileria piroplasms.
The investigated cattle were clinically and parasitologically
examined. Blood samples were collected from all
examined cattle for microscopic examination, PCR assays
(using primers of Theileria spp., Babesia spp., and T.
annulata), and cytokines measurement. It was found that 38
cattle were positive for the presence of at least one species of
Theileria; meanwhile Babesia piroplasms were not detected
either by microscopy or PCR assay. When T. Annulata -specific
primers were used, 33 gave positive results. Twenty-two
out of 33 T. annulata-infected cattle were only included in this
study together with contemporaneous controls (n =10).
According to the severity of clinical signs, T. annulata -
infected cattle were categorized into two groups; group 1
included ten cattle with mild clinical signs and group 2 included
12 cattle with overt clinical signs. Biochemically,
tumor necrosis factor alpha, interleukin (IL)-1β, IL-6, IL-12,
haptoglobin, and Fb were significantly higher (p <0.05) in
diseased cattle compared to control and in cattle of group 2
compared to those in group 1, while interferon gamma
showed no significant variations between the two groups.
We conclude that measurements of the pro-inflammatory cytokines
following T. annulata infection provide a valuable
and quantitative assessment of the response to infection. It
seems likely that the pro-inflammatory cytokines play an
important role in the host response to T. annulata. Our findings
suggest that the pro-inflammatory cytokines are suitable
markers of inflammatory reactions in T. annulata-infected
cattle.
Keywords Pro-inflammatory cytokines . PCR . Theileria
annulata . Cattle
... It was also reported that TNF-α, IL-1, and IL-6 produced by infected mononuclear cells are responsible for the diverse clinical signs of theileriosis such as depression, pyrexia, anorexia, cachexia, and disseminated hemorrhages (Bielefeldt et al. 1989; JongenLavrencic et al. 1996; Preston et al. 1999; Glass et al. 2003). However, the exact mechanism whereby T. annulata causes pathogenesis and clinical symptoms in susceptible cattle remains to be fully elucidated (Preston et al. 1999; Preston 2001; Glass 2001). There is also increasing evidence to suggest that IL-1, IL-6, and TNF-α are transcriptionally expressed at high levels in T. annulata-infected cells, and the pro-inflammatory cytokine mRNA expression was correlated with pathology induced in vivo; however, their individual roles in T. annulata induced pathology in cattle is still unknown (Graham et al. 2001; Glass et al. 2003). ...
... Our findings might suggest that T. annulata infections cause severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines. This finding complies with previous reports, although most of these studies are based on observation obtained in vitro experiment (Bielefeldt et al. 1989; Preston et al. 1999: Graham et al. 2001 Glass 2001; Glass et al. 2005). It has been shown that inflammation in excess is detrimental, and that excessive production and release of APPs and proinflammatory cytokines may lead directly to pathology (Glass et al. 2003 ). ...
The present study was conducted to evaluate the
cytokine response following natural infection of Theileria
annulata in cattle. Initial survey included 173 crossbred cattle
which were examined for the presence of Theileria piroplasms.
The investigated cattle were clinically and parasitologically
examined. Blood samples were collected from all
examined cattle for microscopic examination, PCR assays
(using primers of Theileria spp., Babesia spp., and T.
annulata), and cytokines measurement. It was found that 38
cattle were positive for the presence of at least one species of
Theileria; meanwhile Babesia piroplasms were not detected
either by microscopy or PCR assay. When T. Annulata -specific
primers were used, 33 gave positive results. Twenty-two
out of 33 T. annulata-infected cattle were only included in this
study together with contemporaneous controls (n =10).
According to the severity of clinical signs, T. annulata -
infected cattle were categorized into two groups; group 1
included ten cattle with mild clinical signs and group 2 included
12 cattle with overt clinical signs. Biochemically,
tumor necrosis factor alpha, interleukin (IL)-1β, IL-6, IL-12,
haptoglobin, and Fb were significantly higher (p <0.05) in
diseased cattle compared to control and in cattle of group 2
compared to those in group 1, while interferon gamma
showed no significant variations between the two groups.
We conclude that measurements of the pro-inflammatory cytokines
following T. annulata infection provide a valuable
and quantitative assessment of the response to infection. It
seems likely that the pro-inflammatory cytokines play an
important role in the host response to T. annulata. Our findings
suggest that the pro-inflammatory cytokines are suitable
markers of inflammatory reactions in T. annulata-infected
cattle.
... The parasite induces the host cell to divide, and cell and parasite replicate exponentially. The schizont stage is most associated with pathogenesis, and is also likely to be the main focus of the protective immune response (Preston et al., 1999; Glass, 2001). At first, infected cells remain within the enlarging draining lymph node, until around day 6 when they exit via the efferent lymph (Nichani et al., 1999a). ...
... The kinetic analysis supports the view that host interactions with the schizont stage of the parasite are most likely to be involved in the observed breed differences. In particular macrophage responses and their regulation of lymphoproliferation and pro-inflammatory cytokines, are responsible for the down-stream generation of immunity and pathogenesis during T. annulata infections (Preston et al., 1999Preston et al., , 2002 Glass, 2001; Glass et al., 2003) and, together with phenotypic differences in infected cells (Preston et al., 2002), are likely to play a role in breed-related differences in disease resistance. As a first step to identifying the genes responsible for resistance to T. annulata, this paper has described indicator phenotypic traits that would be valuable in a genetic study, in particular the biochemical measurements of a 1 AGP levels, which are relatively straightforward to measure. ...
During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.
... In susceptible European (taurine) breeds of cattle (such as Holsteins), the parasite may overcome the immune system and often proves fatal. The pathology of the disease is associated with the presence of the intra-macrophage stage of the parasite (the schizont) but the pathogenic mechanisms remain unclear Glass, 2001;Preston, 2001). The schizont stage induces concomitant host cell division and first appears as foci of blasting cells in the medulla of lymph nodes draining the tick-bite or experimental inoculation of sporozoites . ...
... There are no previous reports on APP responses following parasite infection in cattle and relatively few reports in any other mammalian species. The exact mechanisms whereby T. annulata causes clinical responses and pathogenesis in naive susceptible cattle remain unclear Glass, 2001;Preston, 2001). However, the induction of APP supports the hypothesis that pro-inflammatory cytokines play an important role. ...
Acute phase proteins (APP) are synthesised in the liver in response to the systemic presence of high levels of pro-inflammatory cytokines. Bacteria are considered to be strong inducers of APP whereas viruses are weak or non-inducers of APP. Very few reports have been published on APP induction by parasites. Here, we report that the tick-borne protozoan parasite of cattle, Theileria annulata, induced an atypical acute phase response in cattle. Following experimental infection, serum amyloid A (SAA) appeared first, followed by a rise in alpha(1) acid glycoprotein (alpha(1)AGP) in all animals, whereas haptoglobin, which is a major APP in cattle, only appeared in some of the animals, and generally at a low level. All three APP only became elevated around or after the appearance of schizonts in draining lymph nodes and after the first observed temperature rise. Increased alpha(1)AGP levels coincided with the appearance of piroplasms. The production of SAA and alpha(1)AGP correlated strongly with each other, and also with some clinical measures of disease severity including the time to fever, development of leucopaenia, parasitaemia and mortality. These results are consistent with the hypothesis that T. annulata causes severe pathology in susceptible cattle by inducing high levels of pro-inflammatory cytokines.
... The adaptation of the Atlas Brown cattle with T. annulata over the centuries has resulted in equilibrium in the host-parasite relationship. This balance is due to a protective immunity characterised by acute-phase protein responses controlled by macrophage cytokines (Glass, 2001;Glass and Jensen, 2007). This immunity is responsible for enzootic stability and a lower risk of tick infestation (Tuli et al., 2015). ...
... 27 Many studies have shown that Theileria-infected mononuclear cells produce pro-inflammatory cytokines which are associated with clinical signs and pathological changes in different tissues, as seen in lethal tropical theileriosis. [30][31][32] The results of a study indicated a high number of CD4 + and CD8 + lymphocytes during East Coast Fever (ECF) until the final stages of the disease. 33 The high number of these cells and macrophages in the lung indicated their role in the pathogenesis of the different stages of Theileria parva infection. ...
Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.
... In animals, Theileria infections are either asymptomatic or severe, with fever, hemoglobinuria, anemia and death (Zhang et al., 2015). An animal can become infected for life, serving as a reservoir host for tick species (Glass 2001;Ahmed et al. 2008). Most cases of animal infections originate from the tropical and subtropical zones in Asia, Africa, Southern Europe and the Middle East (Sivakumar et al. 2014;Rjeibi et al. 2016;Belotindos et al. 2014;Hussain et al. 2014). ...
The risk of pathogen transmission continues to increase significantly in the presence of tick vectors due to the trade of livestock across countries. In Ghana, there is a lack of data on the incidence of tick-borne pathogens that are of zoonotic and veterinary importance. This study, therefore, aimed to determine the prevalence of such pathogens in livestock using molecular approaches. A total of 276 dry blood spots were collected from cattle (100), sheep (95) and goats (81) in the Kassena-Nankana Districts. The samples were analyzed using Polymerase Chain Reaction (qPCR) and conventional assays and Sanger sequencing that targeted pathogens including Rickettsia, Coxiella, Babesia, Theileria, Ehrlichia and Anaplasma. An overall prevalence of 36.96% was recorded from the livestock DBS, with mixed infections seen in 7.97% samples. Furthermore, the prevalence of infections in livestock was recorded to be 19.21% in sheep, 14.13% in cattle, and 3.62% in goats. The pathogens identified were Rickettsia spp. (3.26%), Babesia sp. Lintan (8.70%), Theileria orientalis (2.17%), Theileria parva (0.36%), Anaplasma capra (18.48%), Anaplasma phagocytophilum (1.81%), Anaplasma marginale (3.26%) and Anaplasma ovis (7.25%). This study reports the first molecular identification of the above-mentioned pathogens in livestock in Ghana and highlights the use of dry blood spots in resource-limited settings. In addition, this research provides an update on tick-borne pathogens in Ghana, suggesting risks to livestock production and human health. Further studies will be essential to establish the distribution and epidemiology of these pathogens in Ghana.
... Analysis of epidemiological factors revealed that cattle infested with ticks and raised with other dairy animals at farm specifically with dogs infested with ticks were found to be statistically the most infected with T. annulata (Tables 2, 3 and 4). Our results support previously published literature in Pakistan and elsewhere on tick infestation and its possible impact on T. annulata prevalence pattern as a potential risk factor (Glass et al., 2001;Zeb et al., 2019). Association between tick burden and bovine theileriosis was also reported by Inci et al. (2008), Sajid et al. (2009), Olds et al. (2018, and Parveen et al. (2021b). ...
Theileriosis is one of the most frequently reported tick borne diseases in tropical and subtropical regions and leads to annual economic losses, such as the reduced dairy products and increased casualties. Tropical theileriosis is caused by Theileria annulata and the present study was designed to improve our knowledge of Theileria annulata infection in Pakistani cattle. In order to assess the prevalence of Theileria annulata on cattle from Multan district in the Punjab province (Pakistan) according to seasons and other risk factors, a total of 1020 blood samples (340 samples each from cross, Holstein Frisian and Sahiwal breed) were collected between 2020 and 2022. Based on Tams-1 partial sequence amplification, the overall T. annulata prevalence was estimated at 11.3% (115/1020). The highest prevalence was observed in autumn season (14.1%), followed by winter (12.9%), summer (11.4%) and spring (6.7%) season. Sahiwal cattle were most susceptible to T. annulata infection (13.2%) followed by Crossbred (11.8%) and Holstein Frisian (8.8%). Epidemiological factor analysis revealed that female cattle, cattle rose with other dairy animals at farm, tick infested cattle, and cattle raised with dogs infested with ticks were associated with the prevalence of T. annulata. White blood cells, lymphocyte (%), Monocyte (%) hemoglobin, mean cell hemoglobin, mean corpuscular hemoglobin concentration, and platelet count were significantly affected blood parameters in T. annulata positive cattle of all three breeds. Representative partial Tams-1 sequences of four Pakistani T. annulata isolates revealed a single genotype genetically close to those infecting cattle from neighboring countries like Iran, Turkey and Egypt. Longitudinal survey and phylogenetic positioning of T. annulata is recommended for epidemiological correlation, diagnosis and treatment of theileriosis in such an agricultural region of Pakistan.
... multivariate: P>0.05, OR: 0.8, 95% CI: 0.6-1.2). The low prevalence rates of TT in indigenous cattle as compared to cross and exotic breeds are characterized by lower acute-phase protein responses controlled by macrophage cytokines, enzootic stability, and a lower chance of tick infestation due to a high level of resistance to ticks as a result of long term exposure to vector ticks over generations [48,57,58]. Furthermore, acclimatization of the indigenous breed to the local environment would render them more hardy and resistant to stressors that could predispose them to infection [23,28]. ...
Theileria annulata is a tick-borne hemoprotozoan parasite responsible for tropical theileriosis in the bovine population, which causes substantial economic losses to the livestock sector. The present study has investigated, characterized, and shaped epidemiologic and phylogenetic profiles of T. annulata infection in the cattle population of central Khyber Pakhtunkhwa, Pakistan. A total of 600 blood samples were collected from cattle. Microscopy and PCR (18S rRNA taxonomic marker) assays were performed to detect T. annulata infection in cattle from the study area. The overall relative prevalence rates of T. annulata in the examined cattle population were 12.8% (microscopy) and 23.7% (PCR). District-wise analysis (microscopy/PCR) showed that cattle from district Mardan were found more infected (16.0%/28.0%), as compared to cattle from district Charsadda (13.5%/25.5%) and district Peshawar (9.0%/17.5%). Based on host demographic and ecological parameters analysis, theileriosis was found to be higher in young, female, crossbred, freely grazing, tick-infested, and irregular/no acaricides treated cattle. The univariate logistic analysis showed that host age, tick infestation, acaricides use, and feeding method were significant risk factors (P<0.05) whereas multivariate analysis indicated that host age, gender, tick infestation, acaricidal application, and feeding method were potential risk factors (P<0.05) for tropical theileriosis in the cattle population. Phylogenetic and sequence analysis showed that T. annulata 18S rRNA isolates shared homology and phylogeny with other isolates from Asia and Europe. This study has addressed the epidemiology and phylogeny of T. annulata circulating in bovid in the study area where gaps were still present. These findings will serve as a baseline and will facilitate future large-scale epidemiological investigations on tropical theileriosis in the cattle population at a national level.
... India which experiences both tropical and subtropical climate is critically affected by TTBDs especially after 1960s with the government policies of upgrading the indigenous breeds with superior exotic germplasm and its indiscriminate practice all over the country; though it was already known that indigenous cattle are resistant to the clinical form of tick-borne diseases but can serve as a reservoir for the crossbred and exotic breeds of cattle. Among the TBDs babesiosis and theileriosis are important haemoprotozoan diseases affecting cattle (Glass, 2001;Ghosh et al., 2006;Preston, 2011;Shahzad et al., 2019). Tropical bovine theileriosis has got higher significance in India due to the higher incidence of clinical cases. ...
... Evidence suggests that T. parva and, to an extent, T. annulata infections in cattle and T. lestoquardi infections in sheep (the cause of malignant ovine theileriosis) are largely curbed by an effective cell-mediated immune response. 45,48,50,110,116,154,178,185 Particularly in T. parva infections, protective immunity is principally mediated by parasitespecific major histocompatibility complex class I-restricted, CD8þ cytotoxic T lymphocyte-mediated killing of infected leukocytes. 45,110,154,185 Schizont-infected leukocytes also stimulate a parasite-specific major histocompatibility complex class II-restricted CD4þ T lymphocyte response. ...
The published literature on schizont-“transforming,” or pathogenic theileriosis, in African wild artiodactyls is dated and based on limited information. Here the authors review the taxonomy, diagnosis, epidemiology, hematology, pathology, and aspects of control in various species. Molecular studies based on 18S and 16S rRNA gene sequences have shown that African wild artiodactyls are commonly infected with diverse Theileria spp., as well as nontheilerial hemoprotozoa and rickettsia-like bacteria, and coinfections with pathogenic and nonpathogenic Theileria species are often recorded. Although theileriosis is still confusingly referred to as cytauxzoonosis in many species, the validity of a separate Cytauxzoon genus in artiodactyls is debated. The epidemiology of theileriosis is complex; the likelihood of fatal disease depends on the interplay of parasite, vertebrate host, tick vector, and environmental factors. Roan calves ( Hippotragus equinus) and stressed animals of all host species are more susceptible to fatal theileriosis. Even though regenerative anemia is common, peripheral blood piroplasm parasitemia does not correlate with disease severity. Other than anemia, common macroscopic lesions include icterus, hemorrhages (mucosal, serosal, and tissue), fluid effusions into body cavities, lung edema, and variably sized raised cream-colored foci of leukocyte infiltration in multiple organs. Histopathologic findings include vasocentric hyperproliferation and lysis of atypical leukocytes with associated intracellular schizonts, parenchymal necrosis, hemorrhage, thromboembolism, and edema. Immunophenotyping is required to establish the identity of the schizont-transformed leukocytes in wild ungulates. Throughout the review, we propose avenues for future research by comparing existing knowledge on selected aspects of theileriosis in domestic livestock with that in African wild artiodactyls.
... However, Indian breeds of cattle are relatively resistant and act as reservoir hosts. Pathology of the disease is associated with the presence of intra-macrophage stage of the parasite (schizont) but the exact mechanisms remain unclear (Preston et al., 1999;Glass, 2001;Preston, 2001). Theileria parasites enter the bovine hosts during tick feeding as sporozoites, which rapidly invade mononuclear leukocytes in the lymph glands nearest to tick bite. ...
The genus Theileria includes tick-transmitted Apicomplexan parasites of ruminants with
substantial economic impact in endemic countries. Amongst the Apicomplexa phylum,
Theileria is the only genus which transforms its mammalian host cells. Theileria parva and
Theileria annulata infect leukocytes where they induce phenotypes that are shared with
some cancers such as immortalization, dissemination and hyper proliferation.
Transformation of lymphocytes is induced during the schizont stage. The mechanism of
host cell transformation induces constitutive activation of bovine transcription factors (NFkB)
and associated signal transduction pathways. P53 plays an important role in
proliferation and apoptosis and thus represents a key molecule in tumor formation.
Moreover T. annulata schizonts induce a Warburg effect in host cells by a shift in ATP
generation from predominantly oxidative phosphorylation to glycolysis and Theileria
parasites avoids autophagic clearance by directly blocking autophagy. Theileria parasites
secrete a protein, prolyl isomerase for maintaining the transformation of host cells. The
host cell transformation by Theileria parasites and the comparison between cancer biology
and host-Theileria interactions can help in revealing chemotherapeutic targets. This review
is about host cell manipulation induced by Theileria parasites and the phenotypes,
Theileria infected cells share with many cancers.
... Theileria species may overcome the immune system of the susceptible breed of the cattle and could lead to death. The pathology of the disease is related to intra-macrophage stage of the protozoa (schiznot) (Glass, 2001). The heamoprotozoan parasites such as Anaplasma, Babesia and Theileria are the highly important protozoa that cause infections in cattle and buffaloes in Pakistan. ...
... Theileria species may overcome the immune system of the susceptible breed of the cattle and could lead to death. The pathology of the disease is related to intra-macrophage stage of the protozoa (schiznot) (Glass, 2001). The heamoprotozoan parasites such as Anaplasma, Babesia and Theileria are the highly important protozoa that cause infections in cattle and buffaloes in Pakistan. ...
The prevalence of Theleria annulata in buffaloes was determined through clinical findings and microscopic examination in peri-urban and urban areas of Hyderabad, Pakistan. Out of 2400 buffaloes evaluated during the study, 1845 (76.87%) were found infested with ticks. The overall prevalence of ticks was 970 (80.8%) in peri-urban and 875 (72.91%) in urban regions of Hyderabad. Ranking of predilection sites of tick infestation in buffaloes revealed that external genitalia, udder and perineum were most infested (54%), followed by dewlap (23%), inner thighs (10%) neck and back (5%), tail (3%), ears (2%), around eyes (1%), flanks (1%) and legs (1%). Out of these 1845 tick infested bovine samples, 1680 (91.05%) were found positive for Theileria species by Giemsa-stained method. The microscopic hematological examination revealed the prevalence of 70%Theleiria annulata in the Hyderabad. The infected buffaloes showed clinical signs such as fever, anorexia, hair loss, open mouth with difficulty in breathing, lymph node enlargement, and protrusion of eyes, red skin and weakness. However, suspected buffaloes exhibited normal feed intake, urination and defecation during the survey. The occurrence of the parasitic infection was significantly higher (P<0.05) in the peri-urban areas than in urban areas. Findings of the present study revealed that peri urban buffaloes were more susceptible to theileriosis in comparison to urban buffaloes.Twenty four hundred blood samples were randomly collected from urban and peri-urban regions of Hyderabad, Pakistan. Haemotological studies revealed that Theileria annulata in buffaloes significantly produced effect (P<0.01) on erythrocyte and leukocyte indices, whereas, platelet indices remained unaffected from Theileria annulata in buffaloes.
... can result in fever, anemia, hemoglobinuria, and death in severe cases, but many species are benign and cause minor or no signs. Animals that recover from acute or primary infections usually remain persistently infected and may act as reservoirs for tick vectors [4,5]. Infected animals are found particularly in tropical and subtropical regions in Africa, the Middle East, Southern Europe, and Asia [6][7][8][9][10][11]. ...
Theileria
spp. are tick-transmitted, intracellular apicomplexan protozoan parasites infecting a wide range of animals. As there is very limited information on the prevalence of
Theileria
spp. in the Caribbean we used the recently described genus-specific pan-
Theileria
FRET-qPCR to identify infected animals in the region and a standard 18S rRNA gene PCR and sequencing to determine the species involved. We found
Theileria
spp. in 9% of the convenience samples of animals (
n
=
752
) studied from five Caribbean islands. Donkeys (20.0%: 5/25) were most commonly infected, followed by sheep (17.4%, 25/144), cattle (6.8%; 22/325), goats (5.0%; 12/238), and horses (5.0%; 1/20). Six species of
Theileria
were identified:
T
.
equi
(donkeys, cattle, goats, and sheep),
Theileria
sp. OT3 (sheep and goats),
Theileria
sp. NG-2013a (cattle),
Theileria
sp. YW-2014 (donkeys),
Theileria
sp. B15a (goats), and
Babesia vulpes
or a closely related organism (sheep and goats). Only
T. equi
has been previously reported in the Caribbean. Our findings expand the known host ranges of
Theileria
spp. and the known distribution of the organisms around the world.
... Drug therapy causes drop in the parasitaemia and hinders accurate diagnosis (Ahmed and Mehlhorn 1999;Glass 2001). In such cases, PCR assays can be used to detect low parasitaemias in the blood of carrier cattle. ...
Bovine tropical theileriosis caused by Theileria annulata is a tick-borne disease of great economic importance in tropical and subtropical regions of the world. The present study was undertaken to detect theilerosis in cattle and buffaloes by polymerase chain reaction (PCR). The diagnosis of theileriosis is usually carried out by blood smear staining technique, which is not sufficiently sensitive to detect the piroplasms in the carrier animals. In this study, a total of 116 samples were collected from infected as well as apparently healthy cattle and buffaloes. Screening of blood smears by Giemsa staining detected 15 samples (12.93 %) positive for Theileria piroplasms out of 116 samples. However, the PCR based screening using the specific primers from the major merozoite-piroplasm surface antigen sequence of T. annulata (Tams1) gene detected 74 samples (63.79 %) positive for T. annulata which included 59 samples found negative by Giemsa staining. Our study suggests that the PCR based screening is more sensitive and accurate method for diagnosis of tropical theileriosis in cattle and buffaloes.
... Indeed, the T. annulata-infected cells can activate and induce non-specifically the proliferation of non-infected autologous T cells (Glass & Spooner, 1990), known as Theileria mixed lymphocyte reaction (Theileria MLR). In MLR, the non-specifically recruited lymphocytes produce cytokines that enhance the expansion of infected cells with a dramatic increase of INFγ (gamma interferon) and TNFα (tumour necrosis factor alpha) concentrations (Glass, 2001). These cytokines increases the proliferation of young schizonts and induce the production of activated macrophages that will secrete IL-2. ...
We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to € 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.
... Markers for genes conferring resistance could enable these genes to be specifically bred (introgression) into more productive livestock, as suggested by Hanotte et al. (2003) for trypanotolerance traits in cattle. Furthermore, discovery of such gene variants may also identify novel pathways and mechanisms that control protection and/or pathology of relevance for vaccine and therapeutic development (Glass, 2001(Glass, , 2004Gruenheid and Gros, 2010;Jarosikova, 2011). ...
Many breeds of cattle with long histories of living in areas of endemic disease have evolved mechanisms that enable them to co-exist with specific pathogens. Understanding the genes that control tolerance and resistance could provide new strategies to improve the health and welfare of livestock. Around one sixth of the world cattle population is estimated to be at risk from one of the most debilitating tick-borne diseases of cattle, caused by the protozoan parasite, Theileria annulata. The parasite mainly infects cells of the myeloid lineage which are also the main producers of inflammatory cytokines. If an infectious or inflammatory insult is sufficiently great, inflammatory cytokines produced by macrophages enter the circulation and induce an acute phase proteins (APP) response. The Bos taurus Holstein breed produces higher and more prolonged levels of inflammatory cytokine induced APP than the Bos indicus Sahiwal breed in response to experimental infection with T. annulata. The Sahiwal exhibits significantly less pathology and survives infection, unlike the Holstein breed. Therefore, we hypothesised that the causal genes were likely to be expressed in macrophages and control the production of inflammatory cytokines. A functional genomics approach revealed that the transcriptome profile of the B. taurus macrophages was more associated with an inflammatory programme than the B. indicus macrophages. In particular the most differentially expressed gene was a member of the signal regulatory protein (SIRP) family. These are mainly expressed on myeloid cell surfaces and control inflammatory responses. Other differentially expressed genes included bovine major histocompatibility complex (MHC) (BoLA) class II genes, particularly BoLA DQ, and transforming growth factor (TGF)B2. We are now exploring whether sequence and functional differences in the bovine SIRP family may underlie the resistance or tolerance to T. annulata between the breeds. Potentially, our research may also have more general implications for the control of inflammatory processes against other pathogens. Genes controlling the balance between pathology and protection may determine how livestock can survive in the face of infectious onslaught. Next generation sequencing and RNAi methodologies for livestock species will bring new opportunities to link diversity at the genome level to functional differences in health traits in livestock species.
... This pro-inflammatory cytokine is produced by CD4+ T cells during bovine neosporosis Tuo et al., 2005). However, infections with other protozoan agents in cattle have shown that CD8+ T cells also contribute to IFN-c production (Glass, 2001;Voyich et al., 2001). Furthermore, studies from in vivo protozoal infections in humans (Korbel et al., 2004) demonstrated that natural killer (NK) cells are among the first cells to produce IFN-c. ...
The intracellular protozoan parasite Neospora caninum is a cause of abortion and congenital disease in cattle worldwide. We have previously shown that natural killer (NK) cells produce IFN-gamma in response to N. caninum tachyzoites in vitro. This study aimed to investigate the role of NK cells and other cellular immune responses in an experimental N. caninum infection model in calves. Phenotyping of peripheral blood lymphocytes showed a drop in the percentage of NK cells at days 4-6 after i.v. inoculation, followed by an increase in the percentage of both NK cells and CD8+ T cells which peaked at days 11-15. A whole blood flow cytometric assay showed that CD4+ T cells were the major IFN-gamma producing cells, but in the early stages of the infection both NK cells and CD8+ T cells contributed to IFN-gamma production. We also compared the ability of two different N. caninum antigen preparations--sonicated soluble antigens and intact heat-inactivated parasites--to induce proliferation and IFN-gamma production in various cell types. Heat-inactivated tachyzoites induced a 3.7 times greater increase in the number of IFN-gamma producing NK cells compared with sonicated soluble antigens. This indicated the presence of some NK cell-stimulating antigens in the intact tachyzoite that were absent from the sonicated soluble antigens. The heat-inactivated whole tachyzoites also inhibited gammadelta T cell proliferation while the soluble antigens from N. caninum did not. We believe this is the first time NK cells have been demonstrated to be early responders in N. caninum infection in calves.
... Therefore, the speed of the innate response and the rapid recall of the CD4 + T-cell response are considered crucial in determining the efficacy of protective immunity [12]. Recently, immunization of cattle with sporozoite surface antigen 1 or attenuated schizont-infected cells induced limited protection against homologous or heterologous sporozoite challenge, whereas a combination of recombinant and live vaccine resulted in survival of all vaccinates [26]. These results provide evidence for achieving improved protective immunity by inclusion of sporozoite and schizont antigens in new generation vaccines to be developed. ...
Overwhelming evidence has accumulated of the effectiveness of immunization with live attenuated vaccines to control tick-borne diseases of livestock. Despite several disadvantages, vaccination with live attenuated organisms against tropical theileriosis, babesiosis and possibly heartwater constitutes one of the most cost-effective intervention strategies. Although great advances have been made through genomics and proteomics research, this has not yet translated into effective non-living vaccines. As a result, there is a continuing necessity to use available live vaccines in tick and tick-borne disease-control strategies adapted to conditions prevailing in many parts of the world.
Tropical theileriosis constraints the development of the dairy industry in the Sudan and vaccination using live attenuated schizont vaccines is considered a promising measure for its control. The present study was carried out to investigate the ability of recombinant T. annulata surface protein (TaSP) to improve the efficacy of the attenuated Atbara cell line in protecting calves against field challenge. To this end, 23 cross‐bred (Friesian × Kenana) calves were divided into four groups. Animals in group 1 (n = 5) were left unvaccinated. Group 2 (n = 6) received the Atbara cell line, animals in group 3 (n = 6) were immunized with three doses of TaSP on days 21, 49 and 77, while animals in group 4 (n = 6) received the cell line vaccine on day 0 and three doses of TaSP in Freund's incomplete adjuvant at days 21, 49 and 77. Twenty‐eight days after the last TaSP boost, all groups were challenged by exposing them to natural field tick infestation in a region known to be endemic for tropical theileriosis. No thermal reactions, piroplasms or schizonts were observed in the immunized animals following immunization. Upon challenge, all animals showed a range of symptoms of clinical theileriosis with variable degrees of severity. The application of TaSP alone appeared to have no effect in terms of protection. The efficacy of the cell line alone was lower than the 100% level of protection against mortality observed in the group that received the combined cell line vaccine and TaSP, suggesting a synergistic effect of this combination.
Theileria annulata is the pathogen of bovine tropical theileriosis. It is extremely harmful to the cattle industry, with huge economic losses. The toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways are crucial for resistance to infection of the protozoa, such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi. However, the role of these immune-related pathways is unclear during T. annulata infection. In the present study, peripheral blood mononuclear cells and serum were separated from blood samples of calves infected with homogenized tick supernatants carrying T. annulata sporozoites at 12 h, 24 h, 36 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h postinoculation. The Custom RT2 Profiler PCR Array was used to explore the mRNA levels of 42 TLR and NLR signaling pathway relevant genes. The TLR1, TLR6, TLR10, NLRP1, and MyD88 genes and their downstream signaling molecules significantly differed after the T. annulata infection in comparison with that of preinfection from 72 h to 168 h postinoculation. The serum concentrations of IL-6, IL-1β, and TNFα were significantly increased at 96 h and 168 h postinfection. These findings provided novel information to help determine the mechanisms of TLR and NLR signaling pathway involvement in protection against T. annulata infection.
Le présent article est une revue bibliographique de l’épidémiologie de la theilériose tropicale bovine en Tunisie. C’est une parasitose spécifique due à la présence et à la multiplication dans les phagocytes mononucléés puis dans les érythrocytes d’un protozoaire de la famille des Theileridae, Theileria annulata. Elle est transmise de manière biologique par plusieurs espèces de tiques de la famille des Ixodidae, appartenant au genre Hyalomma. L’implication de trois acteurs très éloignés sur le plan taxonomique est à l’origine d’une maladie dont l’épidémiologie est très complexe. Cette infection évolue selon trois modes enzootiques : (a) l’enzootie stable qui résulte d’un état d’équilibre entre l’hôte et le parasite, (b) l’enzootie instable modérée qui est due à la présence d’une faible population de tiques engendrant des cas cliniques chez des animaux âgés de deux à trois ans, et (c) l’enzootie instable élevée dans laquelle la population de tiques est tellement faible que la probabilité de rencontre entre une tique infectée et un hôte sensible est minime. Le type de situation épidémiologique dans lequel se trouve l’élevage permet de planifier un programme de lutte adapté.
To identify the prevalence rate of Theileria annulata, blood and lymph node biopsy smears and blood
and lymph node PCR have been developed. In 174 out of 1202 blood samples (14.478%) and in 129
out of 1202 lymph node biopsy samples (10.73%), the piroplasm forms and macroschizonts of Theileria
were observed on blood and lymph node biopsy smears, respectively. A 230 bp band in both PCR
tests that indicated the presence of T. annulata (prevalence rate) was present in 338 out of 1202 samples
(28.11%). Statistical analysis showed significant differences (P<0.01) between the ability of
biopsy smears and PCR tests to detect T. annulata. Our study entirely rejected the presence of the
other Theileria species as parasitic pathogens in southwest Iran. After analysis of data, it was recognized
that 14.47% and 8.23% of positive samples were on late and initial stages of disease, respectively.
It was estimated that 32.24% of positive samples have been treated. Application of both PCR
tests was more sensitive than the two other diagnostic assays to detect T. annulata. To our knowledge,
this study is the first report of direct identification of T. annulata in blood and lymph node
samples by evaluation of biopsy smears and PCRs tests in Iran.
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.
A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.
As part of a study of the development of hoof horn haemorrhages in first-lactation heifers, measurements were made of acute phase reactants to investigate the link between the acute phase response and the development of the haemorrhages. Over a period of two years, blood samples were taken from two separate groups of heifers, weekly in the three weeks before they calved and then twice weekly until eight weeks after calving. Plasma total protein, albumin, fibrinogen, haptoglobin, seromucoid and serum iron and caeruloplasmin were measured and the relationships between the peak concentration (or activity) or the area under the curve of each acute phase reactant and the peak scores for sole or white line haemorrhages were assessed by linear regression. The results suggested that the development of the hoof horn haemorrhages observed in the study was not accompanied by an acute phase response, and the haemorrhages were therefore not primarily caused by endotoxicosis. The diets and husbandry systems used were typical of dairy farms in the UK and the results therefore suggest that a significant proportion of hoof horn haemorrhages observed in UK dairy cows may not be caused by endotoxicosis.
Disease-resistant livestock could provide a potentially sustainable and environmentally sound method of controlling tick and tick-borne diseases of livestock in the developing world. Advances in the knowledge and science of genomics open up opportunities to identify selectable genes controlling disease resistance but first, breeds and individuals with distinguishable phenotypes need to be identified. The Bos indicus breed, Sahiwal, has been exploited in dairy breeding programmes, because it is resistant to ticks and has relatively good performance characteristics compared to other indigenous cattle breeds of tropical regions. The analyses reported here show that Sahiwal calves were also more resistant than European Bos taurus (Holstein) dairy breed calves to tick-borne tropical theileriosis (Theileria annulata infection). Following experimental infection with T. annulata sporozoites, a group of Sahiwal calves all survived without treatment, with significantly lower maximum temperatures (P<0.01) and lower rates of parasite multiplication (P<0.05) than a group of Holstein calves, which all had severe responses. Although the Sahiwals became as anaemic as the Holsteins, other measures of pathology, including enlargement of the draining lymph node and the acute phase proteins, alpha1 acid glycoprotein and haptoglobin, were significantly less in the Sahiwals than in the Holsteins (P<0.05). Additionally, the Sahiwals had significantly lower resting levels of alpha1 acid glycoprotein than the Holsteins (P<0.05). Production of a third acute phase proteins, serum amyloid A, had very similar kinetics in both breeds. Acute phase proteins are produced in response to systemic release of the kinds of pro-inflammatory cytokines that are thought to be responsible for the pyrexic, cachectic and anorexic responses characteristic of tropical theileriosis. The prolonged production of alpha1 acid glycoprotein in the Holsteins is indicative of chronic production of circulating pro-inflammatory cytokines. In contrast, Sahiwals appear able to overcome infection with T. annulata as well as limit pathology by preventing the over-stimulation of pathways involving these cytokines.
In the last few years, microarray technology has emerged as the method of choice for large-scale gene expression studies. It provides an efficient and rapid method to investigate the entire transcriptome of a cell. No research field has benefited more from microarray technology than the study of the exquisite interplay between pathogens and hosts. Numerous microarray studies have now been published in this field, which have provided insights into the mechanisms of host defence and the tactics employed by pathogens to circumvent these protection strategies. These studies have led to a more comprehensive understanding of the host immune response and identified new avenues of research for potential control strategies against pathogens. In the past, research has concentrated on human and mouse microarrays to investigate host-pathogen interactions, regardless of the host species. This trend is changing with the ever-expanding sequence resources now available for many pathogen and host species, including livestock animals. The use of species-specific microarrays has furthered our understanding of host-pathogen interactions for particular organisms and aided in the annotation of unknown genes. Macrophages play a central role in the host's innate and adaptive immune responses to pathogens. These cells are in the first line of defence and interact with a wide range of pathogens; many of which have evolved strategies to circumvent the macrophage defence mechanisms and survive within these cells. In this report, we review the wealth of studies using microarray technology to investigate the response of macrophages to pathogens. These studies illustrate how microarray technology has expanded our understanding of the dialogue between macrophage and pathogen and provide examples of the benefits and pitfalls of using this technique. Furthermore, we discuss the resources available to use microarray analysis to study the immune response of a non-human, non-rodent species, the cow.
Disease is a major source of economic loss to the livestock industry. Understanding the role of genetic factors in immune responsiveness and disease resistance should provide new approaches to the control of disease through development of safe synthetic subunit vaccines and breeding for disease resistance. The major histocompatibility complex (MHC) has been an important candidate locus for immune responsiveness studies. However, it is clear that other loci play an important role. Identifying these and quantifying the relative importance of MHC and non-MHC genes should result in new insights into host-pathogen interactions, and information that can be exploited by vaccine designers. The rapidly increasing information available about the bovine genome and the identification of polymorphisms in immune-related genes will offer potential candidates that control immune responses to vaccines. The bovine MHC, BoLA, encodes two distinct isotypes of class II molecules, DR and DQ, and in about half the common haplotypes the DQ genes are duplicated and expressed. DQ molecules are composed of two polymorphic chains whereas DR consists of one polymorphic and one non-polymorphic chain. Although, it is clear that MHC polymorphism is related to immune responsiveness, it is less clear how different allelic and locus products influence the outcome of an immune response in terms of generating protective immunity in outbred animals. A peptide derived from foot-and-mouth disease virus (FMDV) was used as a probe for BoLA class II function. Both DR and DQ are involved in antigen presentation. In an analysis of T-cell clones specific for the peptide, distinct biases to particular restriction elements were observed. In addition inter-haplotype pairings of DQA and DQB molecules produced functional molecules, which greatly increases the numbers of possible restriction elements, compared with the number of genes, particularly in cattle with duplicated DQ genes. In a vaccine trial with several peptides derived from FMDV, BoLA class II DRB3 polymorphisms were correlated with both protection and non-protection. Although variation in immune responsiveness to the FMDV peptide between different individuals is partly explainable by BoLA class II alleles, other genetic factors play an important role. In a quantitative trait locus project, employing a second-generation cross between Charolais and Holstein cattle, significant sire and breed effects were also observed in T-cell, cytokine and antibody responses to the FMDV peptide. These results suggest that both MHC and non-MHC genes play a role in regulating bovine immune traits of relevance to vaccine design. Identifying these genes and quantifying their relative contributions is the subject of further studies.
Cattle infected with the tick-borne protozoan, Theileria annulata, usually undergo severe morbidity, and mortality ensues in a high proportion of animals. However, we have shown that a Bos indicus breed, the Sahiwal, which originates in a T. annulata endemic area, is more resistant to the parasite. Although Sahiwals become infected, the breed exhibits fewer clinical signs and recovers from a dose of parasite which is fatal in the Holstein B. taurus breed. The Sahiwals have a significantly lower fever response, and lower levels of parasite than the Holsteins. One unusual feature of this disease is the production of acute phase proteins (APP), indicating that the parasite induces high systemic levels of pro-inflammatory cytokines. In the Holsteins there is prolonged production of the APP, α1-glycoprotein, which, in contrast, is only slightly elevated in the Sahiwals. As the parasite infects macrophages (mϕ), our hypothesis is that the Sahiwals can control the excessive production of pro-inflammatory cytokines in response to infection, and that this control is expressed at the level of the mϕ.
B cells convert what are normally conditions for Th1 differentiation into an environment suitable for Th2 development. This capacity is dependent on CD40 as B cells from CD40-/- mice do not elicit Th2 differentiation. To elucidate the basis of this effect, we surveyed cytokine RNA made by naive B cells after activation with anti-Ig and anti-CD40. Resting B cells make TGF-beta message only, however, 4 days after activation, RNA encoding IL-6, IL-10, and TNF-alpha was found. The expression of these messages was accelerated by 2 days in the presence of IL-12. The relevance of these observations to T cell differentiation was investigated: addition of OVA peptide to splenic cells from DO.11.10 transgenic mice causes most T cells to make IFN-gamma. Coactivation of B cells in these cultures reduces the number of IFN-gamma-producing T cells and increases the number synthesizing IL-4. Abs to IL-6 and IL-10 block the IL-4 enhancement. Dissection of the component APC demonstrated that interaction of B cells with IL-12-producing dendritic cells is crucial for B cell-mediated IL-4 enhancement: Thus, B cells preactivated in the presence of dendritic cells from IL-12-/- mice show little IL-4-inducing activity when used to activate T cells. This immune regulation is initiated by IL-12 and therefore represents a feedback loop to temper its own dominant effect (IFN-gamma induction).
Cachectin/tumor necrosis factor-alpha (TNF), a protein produced by macrophages upon stimulation, has been implicated as an important mediator of inflammatory processes and of clinical manifestations in chronic infectious diseases. In order to study further the potential role of TNF in infectious diseases, a homologous system was employed in which recombinant Escherichia coli (E. coli) derived bovine TNF (rBoTNF) was injected in cattle, either as a single bolus or in a repetitive treatment-regime. No clinical signs were observed, although changes occurred in hematologic and immunologic parameters when less than 0.5 mg of TNF/100 kg body weight was administered twice daily for 18 days. Prolonged treatment with 0.05-0.5 mg/100 kg induced histologic but no gross changes in the kidneys and liver. When doses were increased above 0.5 mg/100 kg, depression, anorexia, cachexia, and diarrhea appeared rapidly. Pathologic changes were apparent in various tissues including liver, kidneys, and lymphoid organs; body fat depots were depleted. Most of these changes appeared to be reversible; return to normal tissue-morphology occurred within 3 weeks of withdrawal of rBoTNF. The clinical and pathologic changes induced by prolonged rBoTNF administration resembled those observed in some chronic parasitic and viral infections of cattle in which macrophage-activation characteristically occur. Our finding may be relevant to the elucidation of the pathogenesis of these and other chronic infections.
Theileria parasites infect and transform bovine leukocytes. We have analyzed laboratory-established Theileria sp.-infected leukocyte lines and observed that transformed macrophages express CD5. Low-level expression of CD5 by macrophages was further confirmed on three independent Theileria annulata clinical isolates from Tunisia. Interestingly, the fourth CD5(+) clinical isolate (MB2) was morphologically different, expressed surface immunoglobulin M (IgM) and BoLA class II, and had rearranged Ig light-chain genes. To demonstrate that MB2 did indeed contain CD5(+) B cells, individual clonal lines were obtained by limiting dilution, and CD5 expression and Ig gene rearrangement were confirmed. This suggests that in natural infections T. annulata can invade and transform CD5(+) B cells.
For many years, innate immunity has been considered as a separate entity from the adaptive immune response and has been regarded to be of secondary importance in the hierarchy of immune functions. For the past few years, however, interest in innate immunity has grown enormously, so that now it is studied intensively in many laboratories that seek to integrate these two distinct types of immune function. Our intent in this review is to point out the similarities and differences in these two types of host response to infection, and to indicate our present level of understanding of how these can be integrated into a more complete description of the immune response.
B cells convert what are normally conditions for Th1 differentiation into an environment suitable for Th2 development. This capacity is dependent on CD40 as B cells from CD40-/- mice do not elicit Th2 differentiation. To elucidate the basis of this effect, we surveyed cytokine RNA made by naive B cells after activation with anti-Ig and anti-CD40. Resting B cells make TGF-beta message only, however, 4 days after activation, RNA encoding IL-6, IL-10, and TNF-alpha was found. The expression of these messages was accelerated by 2 days in the presence of IL-12. The relevance of these observations to T cell differentiation was investigated: addition of OVA peptide to splenic cells from DO.11.10 transgenic mice causes most T cells to make IFN-gamma. Coactivation of B cells in these cultures reduces the number of IFN-gamma-producing T cells and increases the number synthesizing IL-4. Abs to IL-6 and IL-10 block the IL-4 enhancement. Dissection of the component APC demonstrated that interaction of B cells with IL-12-producing dendritic cells is crucial for B cell-mediated IL-4 enhancement: Thus, B cells preactivated in the presence of dendritic cells from IL-12-/- mice show little IL-4-inducing activity when used to activate T cells. This immune regulation is initiated by IL-12 and therefore represents a feedback loop to temper its own dominant effect (IFN-gamma induction).
Studies of two distinct human T-cell systems have provided the exciting finding that T cells are able to recognize non-peptide antigens: γδ T cells have been shown to recognize isopentenyl pyrophosphate and related structures and human CD1 has been shown to present microbial lipids and lipoglycans such as mycolic acids and lipoarabinomannan to T cells. T-cell responses to these non-peptide antigens should provide a strategic target for immunologic intervention in infectious disease.
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.
The proliferation of Theileria annulata macroschizont-infected cell lines in vitro was significantly inhibited by nitric oxide (NO) generated by S-nitroso-N-acetyl-DL-penicillamine (SNAP). Incubation with SNAP caused the macroschizonts to disappear and host cells to become apoptotic. SNAP-derived NO also significantly inhibited the incorporation of tritiated thymidine by cultures of cells in which the schizonts had been induced to differentiate into merozoites by maintenance at 41 degrees C instead of 37 degrees C, the temperature used for culturing macroschizont-infected cells. These results point to NO as the mediator of macrophage anti-T. annulata activity and provide new evidence that the protective immune mechanisms which allow cattle to recover from primary infection and resist challenge may be attributed principally to the products of activated macrophages. These findings indicate that effective inactivated vaccines against T. annulata should include antigens able to stimulate the type of CD4+ T cell response which elicits macrophage activation and NO synthesis.
This work extends basic knowledge of tropical theileriosis in taurine and crossbred cattle. Infection of Bos taurus and Bos taurus cross Bos indicus (Sahiwal) calves with graded doses of sporozoites of Theileria annulata (Hissar), an Indian stock of the parasite, showed the following to be dose dependent in both cattle types: the time to appearance and population size of macroschizonts, microschizonts and piroplasms, time and severity of pyrexia, anaemia manifested by erythrocyte counts and haematocrit. All infections were accompanied by a prompt and severe panleucopenia. This effect was dose related in both the taurine and the Sahiwal crossbred calves. Lymphocyte counts returned to preinfection levels in the blood of animals which recovered, but death from theileriosis was characteristically accompanied by a persistent and severe lymphocytopenia. Flow cytometry using monoclonal antibodies to bovine mononuclear cells was used to identify the lymphocyte subsets involved in lymphocytopenia. The outcome of infection was dose dependent in the crossbred calves but not in taurine calves. Although the results obtained did not differ qualitatively between the two cattle types, they provided some preliminary evidence for resistance to tropical theileriosis in Sahiwal crossbred calves.
The following bovine (Bo) and human (Hu) cytokines--Bo rTNF-a, Bo rIFN-g, Hu IFN-a, Hu rIL-1, Hu rIL-2--significantly inhibited the in vitro development of trophozoite-infected cells of three stocks of Theileria annulata and of Theileria parva (Muguga). However, none of these cytokines inhibited the proliferation of established T. annulata or T. parva macroschizont-infected cell lines. Indeed, Bo rTNF-a and Hu rIL-2 consistently enhanced the proliferation of macroschizont-infected cell lines of both species and the blastogenesis of uninfected lymphocytes in trophozoite-infected cultures. These results suggest that cytokines could help in resistance to challenge infections by preventing the further development of trophozoite-infected cells but provide no evidence that any of the above cytokines directly help to resolve primary infections by inhibiting the growth of macroschizont-infected cells. These findings also suggest that both TNF-a and IL-2 could play a role in the pathogenesis of Theileria infections by promoting the proliferation of macroschizont-infected cells and the associated lymphoid hyperplasia.
Three bovine cell lines and four ovine cell lines infected with Theileria parva or Theileria annulata were examined for the production of interferon (IFN). Biologically active IFN was detected in the tissue culture supernatants of four of the cell lines. Only one, a bovine cell line infected with T. parva, produced IFN-gamma as measured by specific neutralization with a monoclonal antibody to bovine IFN-gamma. This observation was confirmed by analysing RNA from the cell lines on Northern blots using an IFN-gamma cDNA probe. The other three cell lines which produced IFN were infected with T. annulata. The IFN produced by those lines was not IFN-gamma.
Theileria annulata, a protozoan parasite of cattle, infects major histocompatibility complex (MHC) class II+ cells, particularly macrophages, and transforms them into continuously growing cell lines. We examined the effects of parasitism by T. annulata on antigen-presenting cell function. T. annulata-infected cells (TaH) presented ovalbumin (as measured by [3H]thymidine incorporation) to both resting autologous bovine T cells and ovalbumin-specific bovine CD4+ T cell lines. However, the former cells were also stimulated by TaH without exogenous antigen although to a lesser degree than in the presence of antigen. This "nonspecific" proliferation was not seen with the ovalbumin-specific T cell lines. The magnitude of response by resting T cells in the presence of antigen, to TaH or purified peripheral blood monocytes, was essentially similar. However, on a per cell basis fewer TaH were required. Considerably greater proliferation to antigen was seen with the ovalbumin-specific T cell lines in the presence of TaH compared to monocytes and again fewer TaH were required to elicit a response. The kinetics of processing did not appear to be substantially altered in TaH and the increased proliferation may be due to the elevated MHC class II expression of these cells. Genetic restriction studies with the T cell lines indicated that the restricting elements used to present ovalbumin by TaH were the same as those used by monocytes, as identified by an isoelectric focusing technique. The continuously growing cell lines provide us with a unique model for investigating parasite-accessory cell interactions in theileriosis. The augmented antigen presenting cell activity of TaH may play an important role in the pathogenesis of the disease. TaH will also provide us with a valuable resource for our antigen presentation studies. In particular, the enhanced antigen presentation by TaH enabled us to detect responses to lower levels of antigen, often an important consideration for experiments where the quantity of antigen available is the major limiting factor.
Two groups of animals were immunized with either 10(6) autologous or 10(6) allogeneic Theileria annulata-infected lymphoblastoid cells cultured in vitro. The development and specificity of cytotoxic cells generated in vivo were measured throughout immunization and challenge using a panel of target cells that were either Theileria-infected or uninfected blast cells of known bovine lymphocyte antigen (BoLA) specificities. After inoculation of the cell lines the two groups showed distinct differences in both their clinical responses and the target specificity of the cytotoxic cells detected. The allogeneic T. annulata cell line recipients showed a very mild clinical response, and on day 9 after inoculation a strong cytotoxic response was detected. The response appeared to be directed against the allogeneic major histocompatibility complex (MHC) antigens of the inoculated cell line in some form of graft rejection response. By day 23 the predominant cytotoxic response was directed against the recipient animals' own cells infected with the parasite. In contrast, the autologous T. annulata cell line recipients showed very severe clinical reactions, and low levels of cytotoxicity were detected. The cytotoxicity was directed against parasite-infected targets but did not appear to be MHC restricted until day 20. Both groups were immune to a heterologous sporozoite challenge that proved lethal to two susceptible control animals, and on day 10 after challenge a peak of cytotoxicity was detected which was directed against the autologous infected target cell. This would suggest that this cytotoxic response was MHC restricted and was also cross-reactive between the heterologous parasite stocks used.
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.
Bovine peripheral blood mononuclear cells (PBMC) were labelled with monoclonal antibodies recognizing bovine MHC class II, sIgM, monocyte, T-helper and T-cytotoxic cell phenotypes. They were sorted into positive and negative populations with a fluorescence-activated cell sorter (FACS). The cell populations were infected in vitro with sporozoites of either Theileria annulata or T. parva, and the degree of infection and transformation determined. The results showed that despite the many similarities between these two parasites, they infected different cells of the immune system. T. annulata preferentially infected MHC class II-positive cells but did not infect T cells. Monocytes were infected very efficiently by T. annulata but were uninfectable with T. parva. B cells were infected much more efficiently by T. annulata than T. parva. Cell lines derived from infections with T. annulata were analysed phenotypically. Virtually all reactivity was lost for the anti-sIgM and the anti-monocyte monoclonal antibodies post-infection and no T-cell markers were detected.
Immunization with either sporozoites or macroschizont-infected cell lines protected calves against challenge with lethal doses of sporozoites of Theileria annulata. Stocks from India, Turkey and Morocco all conferred protective immunity to each other, irrespective of the immunizing regime. Although heterogeneous clinical responses were induced by the two immunizing regimes, both stimulated similar patterns of macrophage cytostasis as expressed as an inhibition of proliferation of macroschizont-infected cell lines. Macrophage cytostasis was detected consistently after immunization and after challenge, arising at the same time as macroschizonts were detectable. Its expression was sustained and inhibited the proliferation of both autologous and allogeneic (BoLA-mismatched) cell lines. In contrast, these two immunizing regimes differed in their ability to stimulate the production of cytotoxic cells. Calves immunized with autologous cell lines or sporozoites developed very transient populations of cytotoxic cells expressing only a low level of specific lysis for autologous infected cells; agglutinating antibodies for immunizing or autologous cell lines were not detected in these calves. Calves immunized with allogeneic cell lines produced cytotoxic cells which were specific only for the immunizing cell lines; these calves also produced antibodies which agglutinated the immunizing cell lines.
Recovery of calves from tropical theileriosis was accompanied by the disappearance of macroschizonts from lymph nodes and the appearance of cytotoxic cells in the blood and lymph nodes. Acute, fatal disease was associated with incremental parasitosis and parasitaemia and, in general, an absence of detectable cytotoxic cells in the blood or lymph nodes. After recovery from infection, calves were resistant to challenge. Challenge with sporozoites was followed sometimes by an immediate reappearance or by a later peak, or sometimes by twin peaks of cytotoxic cells but macroschizonts were not detected. Histocompatibility (BoLA) typing indicated that calves produced two sequential populations of cytotoxic cells during recovery from primary infection with Theileria annulata. The expression of lysis by the first appeared to be BoLA restricted. In contrast, both the peaks of lysis manifest after challenge appeared to be BoLA restricted. Results suggest that BoLA restricted cells are established in the immunological memory and are probably analogous to cytotoxic T cells, while non-BoLA restricted cytotoxic cells are natural killer like cells. The results suggest a role for cytotoxic cells in recovery from primary infection, in the inhibition of proliferation of macroschizonts which evade mechanisms of acquired resistance and in the lysis of macroschizont infected cells deriving from challenge sporozoites which have evaded serum-mediated inhibition.
Bovine peripheral blood mononuclear cells (PBM) infected in vitro with Theileria annulata sporozoites have previously been characterized as MHC class II+ mature macrophages. The ability of T. annulata sporozoites to infect different subpopulations of MHC class II+ bovine monocytes was investigated. Cells were labelled with monocyte specific monoclonal antibodies (MoAb) and isolated using magnetic cell sorting (MACS). Sporozoites infected both immature and mature monocytes, but more readily infected the mature population. A potential ligand for sporozoite entry is the elastin receptor which is expressed mainly on the immature population of monocytes and not on B cells or T cells. T. annulata sporozoites infected elastin receptor positive and negative cell populations equally well. Infected immature cells lost the expression of elastin receptors and the immature marker, subsequently expressing the mature marker. All monocytes lost the expression of CD14 (the LPS receptor) upon infection with sporozoites. The infection of specific populations and subsequent alterations in phenotype may alter the function of these cells and play an important role in disease pathogenesis.
Bovine macrophage-derived tumour necrosis factor-alpha/cachectin (TNF-alpha) was synthesized when peripheral blood mononuclear cells (PBMC) and purified adherent PBMC from naive and Theileria annulata-infected cattle were incubated in vitro with concanavalin A (Con-A) or bovine recombinant interferon gamma (Bo rIFN-gamma). TNF-alpha production was also induced when adherent PBMC were cultured with T. annulata macroschizont-infected cells. In contrast, non-adherent PBMC from sublethally infected cattle produced interferon (IFN) when incubated with Hu rIL-2, Con-A, phytohaemagglutinin (PHA) or T. annulata macroschizont-infected cells growing as cell lines in vitro. Whilst PBMC from lethally infected cattle spontaneously produced IFN-gamma during advanced stages of infection, the sera of such animals contained type 1 IFN (alpha/beta). IFN was also produced by T. annulata macroschizont-infected cell lines maintained in vitro. This work suggests that cytokines serve as crucial links between proliferating Theileira-infected cells and the characteristic clinical symptoms of tropical theileriosis.
The protozoan parasite of cattle, Theileria annulata, causes a severe lymphoproliferative disease, developing initially in the draining lymph node, which is often fatal in naive animals. Infection of macrophages with T. annulata leads to an augmentation of their antigen-presenting capability in vitro and infected cells can induce proliferation of autologous resting T cells from naive animals. This inappropriate activation of T cells may play an important role in the failure of the host to mount an effective immune response in vivo. To investigate this hypothesis we characterized further the response of T cells from naive cattle to infected cells in vitro, and also examined the development of the immune response in lymph nodes draining the sites of T. annulata infection. Both CD4+ and CD8+ T cells from naive peripheral blood mononuclear cells (PBMC) were induced to proliferate and express the activation markers IL-2R and MHC class II when cultured with infected cells. This effect was seen in both 'naive' and 'memory' T cells, and was dependent upon contact with infected cells. In vitro, infected cells are therefore capable of activating T cells irrespective of their antigen specificity or memory status. In draining lymph nodes, although large numbers of IL-2R+ cells developed following infection, these activated cells were only associated with areas of parasite-induced proliferating cells, and subsequently disappeared from the node. Cells expressing IL-2R were not present in recognized sites for T cell development. Germinal centres were severely affected, losing T cell-dependent zones followed by a total destruction of morphology. T cell function is therefore severely disrupted within draining nodes. This study has shown that parasitized cells supply sufficient signals in vitro to activate T cells irrespective of specificity. T cells also are not stimulated in a conventional manner in vivo, and this may play an important role in preventing an effective immune response from being generated.
A major feature of the pathology induced by Theileria annulata is acute lymphocytic proliferation, and this study investigates the mechanisms underlying the intrinsic ability of T. annulata-infected monocytes to induce naive autologous T cells to proliferate. Different T. annulata-infected clones expressed different but constant levels of MHC class II, varying from < 1.0 x 10(5) to 1.5 x 10(6) molecules/cell, as measured by saturation binding. However, no correlation was found between the level of MHC class II expression and levels of induced T cell proliferation. Theileria annulata-infected cell lines and clones were assayed for cytokine mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). The infected cells assayed produced mRNA specific for IL-1 alpha, IL-1 beta, IL-6, IL-10 and tumour necrosis factor-alpha (TNF-alpha), but not IL-2 or IL-4. One clone (clone G) did not produce mRNA for TNF-alpha. The degree of T cell proliferation induced by infected cells was directly correlated with the amount of mRNA produced for the T cell stimulatory cytokines IL-1 alpha and IL-6, as assessed by a semiquantitative technique. In contrast, cells infected with the related parasite T. parva produced mRNA for IL-1 alpha, IL-2, IL-4, IL-10 and interferon-gamma (IFN-gamma). Since T. parva-infected cells also induce naive autologous T cell proliferation, it seems likely that the production of IL-1 alpha by cells infected with either parasite is a major signal for the induction of non-specific T cell proliferation.
A C terminal fragment (SR1) of SPAG-1, a sporozoite surface antigen of Theileria annulata, has been expressed as a fusion protein in the e1 loop of hepatitis B core antigen (HBcAg). This recombinant antigen (HBcAg-SR1) is produced in the form of self-assembling polyhedral particles which have been visualised under the electron microscope. Cattle immunised with HBcAg-SR1 produced high titres of neutralising antibodies. A significant T cell response to both the HBcAg and SR1 determinants was observed but evidence of a T suppressor determinant in SR1 was also revealed. Immunised cattle showed some evidence of protection to sporozoite challenge as assessed by severity of the disease. The significance of these findings for the development of a sub-unit vaccine against T. annulata is discussed.
Kept in continuous in vitro culture, the protozoan parasite Theileria annulata gradually loses virulence when inoculated into cattle. These attenuated cell lines form the basis of the in vitro live vaccines which have been used successfully to control tropical theileriosis in several endemic regions. In the study reported here, events occurring during in vitro culture of an Indian (Hisar) cell line, which may be associated with the reduction in virulence, have been investigated. Hybridization with two polymorphic DNA probes following Southern blotting showed that selection of particular parasite genotypes occurs very rapidly with culture; a novel hybridization pattern is observed with both probes after 50-100 passages in vitro. In addition to this selection process, immunofluorescence studies using a monoclonal antibody which specifically recognizes virulent T. annulata revealed alterations in antibody reactivity following in vitro culture. This loss of reactivity was observed in three cloned cell lines derived from the early, virulent Hisar line and implies that phenotypic changes resulting from alterations to parasite gene expression are taking place during the attenuation process. When considered with the results from in vivo infections with serial passages of this cell line, it can be proposed that both altered gene expression and selection may be involved in the loss of pathogenicity of T. annulata during continuous in vitro culture.
An in vitro model system has been developed in which freshly isolated resting WC1+ gamma/delta TcR+ T cells proliferate in response to cells transformed by the protozoan parasite Theileria annulata, providing a strategy in which the basis of activation of naive gamma/delta T cells can be investigated. Irradiated parasite-transformed cells stimulate the proliferation, but not cytolytic activity, of autologous peripheral blood mononuclear cells (PBMC) from non-immune animals. The proliferating cells are mainly WC1+ gamma/delta T cells. The majority of WC1+ gamma/delta T cells in freshly isolated PBMC express CD25 at a low level that increases when stimulated with T. annulata-infected cells. Purified WC1+ gamma/delta T cells fail to proliferate when cultured with irradiated T. annulata-infected cells and produce a small proliferative response to IL-2, but proliferate strongly to irradiated or lightly fixed Theileria-infected cells in combination with IL-2. The Theileria-infected cells express cytokine transcripts encoding IL-1 alpha, IL-1 beta, IL-6 and IL-10, but not IFN gamma, IL-2, IL-4 and IL-7. Purified WC1+ gamma/delta T cells stimulated with T. annulata-infected cells with or without IL-2 fail to produce IL-2 transcripts, but do produce those for TNF alpha. These experiments show that WC1+ gamma/delta T cells recognize a surface determinant on T. annulata-infected cells, that together with a second signal, which can be provided by exogenous IL-2, stimulates their proliferation.
For many years, innate immunity has been considered as a separate entity from the adaptive immune response and has been regarded to be of secondary importance in the hierarchy of immune functions. For the past few years, however, interest in innate immunity has grown enormously, so that now it is studied intensively in many laboratories that seek to integrate these two distinct types of immune function. Our intent in this review is to point out the similarities and differences in these two types of host response to infection, and to indicate our present level of understanding of how these can be integrated into a more complete description of the immune response.
Theileria annulata is a tick-transmitted protozoan parasite of cattle, which transforms cells of macrophage (Mphi) or B cell lineage. Bone marrow cells, bone marrow cell-derived, and monocyte-derived Mphi were infected with T. annulata sporozoites, and the resulting cell lines were assessed for surface marker expression and function. Transformed lines expressed histocompatibility complex (MHC) class-I and II, CD44, CD45, and the myeloid marker DH598-surface markers CD14, CD11b, M-M7, TH57A, and to a lesser extent CD11a/CD18, CD11c, and ACT(B), were down-regulated. Likewise, transformed cells failed to express Mphi functions (Fc-receptor-mediated phagocytosis, phorbol myristate acetate-induced oxidative burst, lipopolysaccharide-induced tumor necrosis factor alpha, and nitric oxide generation and procoagulant activity up-regulation). Mphi origin was assured by homogeneity of the starting population, cloning of cells by limiting dilution, and repeated microscopic and flow cytometric monitoring of the cell lines. Elimination of the parasite by treatment with BW720c resulted in the re-acquisition of monocyte lineage properties, as evidenced by up-regulation of CD14, and by re-acquisition of the capacity to ingest opsonized sheep red blood cells and bacteria. Thus, Mphi transformed by T. annulata appear to undergo a process of parasite-induced dedifferentiation but reassume the differentiated phenotype upon elimination of the parasite.
Forsyth, L.M.G., Jackson, L.A., Wilkie, G., Sanderson, A., Brown, C.G.D. and Preston, P.M., 1997. Bovine cells infected in vivo with Theileria annulata express CD11b, the C3bi complement receptor. Veterinary Research Communications, 21 (4), 249-263
Bovine cells from cattle infected with Theileria annulata were phenotyped with monoclonal antibodies recognizing bovine leukocyte antigens. Macroschizont-infected, transformed cell lines prepared from peripheral blood mononuclear cells of cattle, infected with sporozoites, were assessed by flow cytometry; parasitized cells in tissues from infected cattle were examined by immunocytochemical techniques. Co-expression of markers for different cell lineages by the cell lines precluded a definite conclusion as to their phenotypic origins. For, while the pattern of leukocyte antigens expressed by these in vivo-derived schizont-infected cells, which included CD11b, was indicative of a myeloid origin, the possibility that they were NK cells could not be excluded. The monoclonal antibody (MAb) IL-A15, which recognizes CD11b, reacted with a high proportion of parasitized cells in sections of tissues from infected cattle at all stages of acute disease. Mononuclear cells infected with parasites at all stages of differentiation, from macroschizont to microschizont, expressed CD11b. Such parasitized cells occurred throughout the lymphoid tissues, being found in the thymus, spleen and lymph nodes, particularly the prescapular node draining the site of infection, the hepatic, mesenteric and precrural nodes, as well as in the reticulo-endothelial tissue of the liver, kidney, lung, abomasum, adrenal and pituitary glands. These observations provided the first evidence for a myeloid origin for the parasitized T. annulata cells found in infected bovine tissues and blood and suggested a mechanism whereby schizonts could transfer from cell to cell during mechanical infection with schizont-infected cells.
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-gamma) mRNA. The in vitro response is mirrored during in vivo infection, as greatly elevated amounts of IFN-gamma protein are found in lymph efferent from infected lymph nodes, while expression of IL-4 mRNA within the node stops. IFN-gamma production does not correlate with protection against the parasite, as infected cells flourish during peak IFN-gamma production, and only very small amounts of IFN-gamma are produced during the effective immune response of an immunized animal. Overproduction of IFN-gamma 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.
We investigated with three-color flow cytometry the expression of perforin (Pf) in normal human lymphocyte subpopulations identified by means of activation and differentiation-related antigens. Interestingly, Pf could be detected in a substantial subset (13 +/- 2%) of memory CD4+ CD45RO+ cells, on relevant percentages of memory (CD45RO+) and naive (CD45RA+) CD8+ cells and on virtually all CD3- CD16+, CD3- CD56+ and NKB1+ natural killer cells, as expected. The analysis of fluorescence intensity showed higher levels of Pf expression on CD8dim and NK cells compared to CD8bright and CD4+ lymphocytes, Pf and CD69, HLA-DR, CD95 and CD25 activation/differentiation-related antigens were never co-expressed. On average, 15 +/- 3% of CD3+ CD28+ cells were found to be Pf+, in line with a previously activated or memory cell type. Comparable percentages of CD8+ CD11b- (cytotoxic) and CD8+ CD11b+ (suppressor) T cells were Pf+. Multiparameter flow cytometry is a powerful tool to detect minute fractions of Pf-expressing cells in heterogeneous populations.
Theileria are tick-transmitted protozoans causing often fatal diseases in ruminants. Theileria sporozoites immortalize and transform host cells of haematopoietic origin. Transformation is associated with profound functional alterations. For example, bovine cells infected by Theileria annulata or T. parva. constitutively produce interferon (IFN). In this study, the type and family of IFN produced by a panel of T. annulata-transformed cell lines and a T. parva-transformed cell line was investigated, using molecular probes specific for the members of the IFN-alpha, IFN-beta, IFN-gamma and IFN-omega family. T. parva-transformed cells produced IFN-gamma exclusively, whereas T. annulata-infected cells expressed type I IFN only. Analysis of mRNA expression showed that this type I IFN was confined to IFN-beta, regardless of the cellular origin of the transformed cells. When cells were exposed to double-stranded RNA (poly (I:C)) which induces IFN production in other systems, a 10-5,000 fold increase in IFN activity was noted. The amounts of IFN-beta mRNA were increased, but mRNA coding for IFN-alpha, IFN-omega or IFN-gamma was not detected. In contrast, primary macrophages, from which many of the tested lines were derived, expressed IFN-alpha, IFN-beta and IFN-omega mRNA to similar degrees when stimulated by LPS or poly (I:C). Thus, T. annulata appears to constitutively turn on IFN-beta gene transcription while silencing the genes coding for IFN-alpha and IFN-omega.
The first part of this study of the biological mechanisms underlying attenuation of virulent Theileria annulata macroschizont-infected cell lines screened four pairs of T. annulata (Hisar) in vivo- and in vitro-derived macroschizont-infected cell lines (lines) and identified a single in vivo-derived line, which induced lethal tropical theileriosis. The other seven lines were relatively avirulent. Analysis of the clinical, hematological, and parasitological responses of cattle immunized with different passages of the virulent line after in vitro culture showed that it was partly attenuated by passage (p) 50 and avirulent by p130. Clones representing the three glucose phosphate isomerase (GPI) isotypes, which constituted the newly isolated virulent culture, were obtained from p3 by limiting dilution; p50 and p130 consisted of one isotype. The second part of the study raised monoclonal antibodies (MAbs) against macroschizont-infected cells, as reagents for detecting antigenic differences between virulent and avirulent parasites, and identified two MAbs that recognized the surface of infected cells as well as macroschizonts. MAb EU1 recognized an antigen expressed by all the lines tested, whether in vitro- or in vivo-derived, whether uncloned or cloned, and irrespective of extent of subpassage in culture. MAb EU106 recognized an antigen whose expression by the virulent line and its clones disappeared on passage in culture. This antigen was not expressed at all by the avirulent in vitro-derived line prepared with cells from the same calf. Both antigens were expressed by lines infected with other stocks of T. annulata, including two lines known to induce lethal disease. The different profiles of expression of the two novel antigens, recognized by MAbs EU1 and EU106, by the line undergoing attenuation suggest (1) that the two antigens interact differently with the bovine immune system; and (2) that there are two, very different, potential roles for these antibodies in the development of vaccines against T. annulata infections.
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.
Host defense against intracellular pathogens is thought to require cytotoxic T cells. Recent studies have investigated the impact of host cell lysis and cytokine production by cytotoxic T lymphocytes on the fate of intracellular pathogens. The identification of two mechanisms of lysis induced by cytotoxic T lymphocytes--the granule exocytosis pathway and the Fas-FasL interaction--have provided new insight into the role of cytotoxic T lymphocyters in immunity to infection.
A large number of changes, distant from the site or sites of inflammation and involving many organ systems, may accompany inflammation. In 1930 interest was focused on these changes by the discovery of C-reactive protein (so named because it reacted with the pneumococcal C-polysaccharide) in the plasma of patients during the acute phase of pneumococcal pneumonia.1 Accordingly, these systemic changes have since been referred to as the acute-phase response,2 even though they accompany both acute and chronic inflammatory disorders. New acute-phase phenomena continue to be recognized, and the mechanisms mediating them are becoming better understood. This review summarizes much of . . .
The distribution of schizont-infected cells in six calves undergoing acute, lethal sporozoite-induced infections with Theileria annulata was examined, the calves being killed in the early, middle or late stages of disease. A combination of histological and immunocytochemical techniques showed that schizont-infected cells became disseminated rapidly through the lymphoid tissues from the prescapular lymph node draining the site of inoculation to distant lymph nodes (e.g., precrural, mesenteric and mediastinal) and to the spleen and thymus. The parasitized cells also spread rapidly into non-lymphoid organs, being found in the liver, kidney, lung, abomasum, adrenal glands and pituitary gland by day 7, in the brain by day 12 and in the heart by day 14 after infection. As infection progressed, the schizonts differentiated into merozoites. By the late stages of disease, the cells containing merozoites greatly out-numbered schizont-infected cells. The parasitized mononuclear cells were labelled by antibodies to bovine interferon-alpha1 and tumour necrosis factor-alpha and, during the later stages of the disease, contained erythrocytes parasitized by piroplasms. The results suggested that the parasitized mononuclear cells themselves played a role in the development of clinical disease and in tissue damage. These findings provide new evidence that tropical theileriosis can no longer be viewed as a lymphoproliferative disease resulting from the uncontrolled multiplication and metastasis of lymphoid cells infected with T. annulata schizonts, but is caused by a parasite that lives in, and is disseminated by, cytokine-secreting, proliferating mononuclear phagocytes.
The protozoan parasite Theileria annulata is the causative agent of the tick-borne disease tropical theileriosis, responsible for morbidity and mortality of cattle in many developing countries. Here, John Campbell and Roger Spooner discuss how the parasite might evade immune destruction during an acute primary infection. Theileria annulata macroschizont-infected macrophages act as over-efficient antigen-presenting cells within the infected draining lymph node. Infected cells activate CD4+ and CD8+ T cells abnormally, giving rise to a cascade of cytokine production. This altered immune response does not reject the parasitized cells, and might actively participate in the growth of the developing parasite.
Theileria annulata is a tick-borne protozoan parasite which causes the disease bovine tropical theileriosis. In immunized or drug-treated animals, the pathogenic macroschizont stage of the parasite is destroyed by MHC class I-restricted cytotoxic T lymphocytes (CTL). Here we show that although CD8+ T cells increase greatly in number and display activation markers during an acute infection, they exhibit no killing of infected cells. During the ineffectual response, efferent lymph cells' ability to proliferate to IL-2 drops, coinciding with loss of MoAb binding to CD2 by CD8+ cells. When animals were treated with the anti-parasite drug 'Butalex', IL-2 responses, anti-CD2 antibody binding by CD8+ cells and strong CTL activity were restored within 24 h. The initial activation of CD4+ T cells by parasite-infected cells altering the IL-2 production in the draining lymph node is the likely cause of the failure of CTL responses.
The object of these experiments was to study the pathogenesis and kinetics of Theileria annulata infection in the efferent lymph of the draining lymph nodes of calves. Efferent lymphatics of calves were cannulated prior to infection with T. annulata sporozoite or an allogeneic schizont cell line. Potentially lethal sporozoite challenge induced cell shut-down from days 4-6 and then a massive increase in output of blasting cells (both infected and non-infected) in the efferent lymph. The rate of lymph flow and total cell output increased to 5 to 10-fold from day 6 onwards. Sporozoites were never isolated from the efferent lymph. However, large numbers of parasite-infected cells were seen in efferent lymph from the sixth day of infection. The animals inoculated with an allogeneic T. annulata-infected cell line exhibited only a small increase in flow rate and cell output. Parasite-infected cells of recipient origin were seen in efferent lymph from day 11 onwards. However, cells of donor origin were never isolated either from efferent lymph or peripheral blood. Thus the parasite transferred from the inoculated donor cell line to the cells of the recipient before schizonts appeared in efferent lymph.
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.
The intracellular protozoan parasite Theileria annulata causes a severe and often fatal disease of pure and crossbred cattle in tropical and subtropical countries. Animals that recover from the infection are immune against challenge with homologous parasite strains. In the present review we refer to the role of immunocompetent cells and their products in containing the infection or in facilitating the progress of the disease. Parasite-infected host cells produce cytokines, which, depending on their concentration and timing of production, may enhance the establishment of the infection. Thus, cell lines producing high levels of proinflammatory cytokines cause severe postvaccinal reactions when inoculated into cattle. This may be supported by an aberrant non-specific activation of naive T-cells, leading to the production of high levels of gamma-interferon (IFN-γ). Under these circumstances development of the specific immune response may be inhibited. At this stage, innate immune reactions are operating to contain the infection. Natural killer cells and macrophages may represent the most important part of this immunity. Antibodies and specific T-lymphocytes, CD4+ T-cells and cytotoxic T-lymphocytes (CTLs), play the most important role in a challenge infection. In this context, CD4+ T-cells produce cytokines required for the clonal expansion of CTLs that kill their target cells in a major histocompatibility complex (MHC) class I-restricted manner. In addition, CD4+ T-cells produce macrophage-activating cytokines such as IFN-γ. Such activated macrophages produce mediators such as NO, which destroy the intracellular schizonts.
Attempts have been directed toward the identification of parasite antigens involved in the induction of immunity. To date, only a limited number of sporozoite and merozoite antigens have been identified and examined for their immunogenicity, and the protection achieved is partial. An effective vaccine must include schizont proteins, notably, those proteins that are secreted into the host cell cytoplasm because these may have access to the MHC class I and II compartments to be presented to CTLs and CD4+ T-cells, respectively. Several schizont proteins have been identified and these are now under investigation.
Host responses to infectious challenges include initial events elicited directly by agent structures distinct from host determinants, activation of innate immune system components by the products of initial events, and the shaping of downstream adaptive immunity by these initial/innate responses. The picture emerging from viral infections is that viral structures interact with intracellular signaling pathways to induce expression of the type 1 interferons, IFN-alpha/beta. In addition to mediating direct antiviral effects, these cytokines play dominant roles in regulating innate and adaptive immune responses to infection. In particular, IFN-alpha/beta acts to inhibit interleukin-12 (IL-12) expression and IL-12 activation of innate natural killer (NK) cell IFN-alpha production, while inducing NK cell cytotoxicity and proliferation, and promoting adaptive T cell IFN-alpha responses. Although certain viral infections do elicit initial/innate IL-12 and NK-cell-produced IFN-alpha, endogenous IFN-alpha/beta also controls the magnitudes of these responses. Thus, the pathways activated, to dominantly regulate innate and adaptive immune responses during viral infections, are being defined.
Attenuated vaccines are an important means of controlling Theileria annulata infection of cattle. Production is by prolonged cultivation of macroschizont-infected cells. The mechanism of attenuation remains unclear. There are three general nonmutually exclusive possibilities: Selection of avirulent subpopulations, genome rearrangements and alterations in gene expression. Several groups, including ours, have provided evidence that the population structure usually tends to simplify during attenuation. Our data on the T. annulata (Ta) Ankara cell line show that attenuation is not necessarily accompanied by the population becoming clonal. We have been unable to detect large DNA rearrangements. Evidence for alterations in host and parasite gene expression during attenuation is available. With respect to the host we have shown that attenuation is accompanied by loss of expression of parasite induced matrix metalloproteinases (MMPs). However, in different lines different protease activities are involved. In the T. annulata Ode line we have shown that 8 activities (including MMP9) are downregulated and that this correlates with a loss of metastatic behaviour. This has previously been shown in vitro using reconstituted basement membrane (Matrigel) and is demonstrated in vivo using scid mice in this study. Thus part of the pathology, namely the ability to disseminate, mediated by host MMPs, is lost upon attenuation. Re-isolation experiments have shown that the reduction/loss of MMP is a stable transferable trait. A logical extension is that loss of MMP activity (and virulence in general) must be at the most fundamental level a genetic trait of the parasite. Evidence for loss of parasite gene expression is implied by the loss of the ability to differentiate into merozoites on attenuation. Specific evidence for loss of parasite gene expression has been obtained using differential RNA display. We view virulence as a multifactorial phenomenon involving interacting subpopulations of cells and attenuation is a threshold effect whereby the number of virulence factors is reduced below a critical level. On this basis there will be many different ways to achieve attenuation.
The major sporozoite surface antigen of Theileria annulata (SPAG-1) is a candidate for inclusion in a subunit vaccine. In this paper we summarize the results of 4 vaccination experiments using recombinant SPAG-1 expressed in different systems and presented in different adjuvants. The antigen has been presented as either a C terminal 108 amino acid peptide (called SR1) expressed as both beta-galactosidase and hepatitis B core antigen fusions or as a full-length form expressed as a GST fusion with an N terminal His6 tag. We used different adjuvants, namely Freund's, saponin, ISCOMs and a proprietary adjuvant supplied by SmithKline Beecham, which we call SKBA. The data point to the conclusion that SPAG-1 can elicit partial protection and is therefore suitable for inclusion in an eventual multicomponent subunit vaccine.
Theileria parva and T. annulata provide intriguing models for the study of parasite-host interactions. Both parasites possess the unique property of being able to transform the cells they infect; T. parva transforms T and B cells, whereas T. annulata affects B cells and monocytes/macrophages. Parasitized cells do not require antigenic stimulation or exogenous growth factors and acquire the ability to proliferate continuously. In vivo, parasitized cells undergo clonal expansion and infiltrate both lymphoid and non-lymphoid tissues of the infected host. Theileria-induced transformation is entirely reversible and is accompanied by the expression of a wide range of different lymphokines and cytokines, some of which may contribute to proliferation or may enhance spread and survival of the parasitized cell in the host. The presence of the parasite in the host-cell cytoplasm modulates the state of activation of a number of signal transduction pathways. This, in turn, leads to the activation of transcription factors, including nuclear factor-kappa B, which appear to be essential for the survival of Theileria-transformed T cells.
There are three economically important bovine Theileria species: Theileria annulata, which causes tropical theileriosis and occurs across north Africa and most of central Asia; Theileria parva, which causes East Coast fever and is found in East and Central Africa; and Theileria sergenti, which is predominantly a problem in Japan and Korea. Theileria annulata preferentially infects macrophages in vivo. It is controlled largely by means of live, attenuated vaccines, which are produced by prolonged tissue culture of the schizont-infected cells. The immunity induced in animals, which have either recovered from an infection or have been vaccinated (with an attenuated vaccine), is broad, solid and cell mediated. It is considered that the main effector cells are cytostatic macrophages that produce nitric oxide. Subsidiary roles for bovine leucocyte antigen (BoLA)-restricted, transiently appearing, cytotoxic T cells, and possibly also natural killer (NK) cells, have been identified. Cytokines such as tumour necrosis factor alpha (TNF-alpha) may have important roles, particularly in the induction of pathology. Matrix metalloproteinases have been implicated in the metastatic behaviour of schizont-infected cells. The nature of the protective schizont target antigens remains unknown. Attempts to develop a subunit vaccine have focused upon a sporozoite antigen (SPAG-1) and a merozoite antigen (Tams1). Both SPAG-1 and Tams1 have given partial protection using different delivery systems and adjuvants, but further vaccine development will probably require identification of a range of other antigens, especially from the schizont stage. Theileria parva has a tropism for T cells. Vaccination is currently by the 'infection and treatment' method, which involves challenging with a controlled dose of sporozoite stabilate and the simultaneous administration of long-acting tetracyclines. The immunity thus induced is mediated by BoLA-restricted cytotoxic T cells, which recognize polymorphic schizont antigens. These antigens have not been characterized at the molecular level. However, the polymorphic nature of the target antigens underlies the fact that the immunity is very strain specific--a situation that distinguishes T. parva from T. annulata. Interestingly, it is not possible to produce an attenuated vaccine to T. parva, as T. parva requires up to two orders of magnitude more schizonts in order to achieve transfer to the new host. A suggested reason for this is that the macrophage targets of T. annulata are phagocytes and thus the schizont has a natural, efficient route of entry whilst the preferred host of T. parva is the non-phagocytic T cell. Analysis of the cytotoxic T-cell response has revealed evidence of BoLA haplotype dominance plus competition between parasite epitopes. Subunit vaccination using a recombinant sporozoite antigen (p67) has proved very promising, with levels of protection of the order of 70% being achieved. A proportion of the protected calves exhibits complete sterile immunity. Interestingly, the basis for this immunity is not clear, since there is no correlation between the titre of antibodies that inhibit sporozoite penetration of lymphocytes and protection. Similarly, there is no significant T-cell response that distinguishes the protected and susceptible animals. These data are very encouraging, but other components, particularly those derived from the schizont, need to be identified and characterized. The mild Theileria species of Japan and Korea (termed T. sergenti in the literature) cause fever and severe chronic anaemia. The schizont stage of the life cycle is very rare and the host cell type is not known. The pathology is associated with chronic piroplasm infection. Immunity can be induced by immunizing with crude piroplasm extracts. Serological analysis of immune sera reveals that the immunodominant antigen is a polypeptide of 30-33 kDa, which corresponds to the protective T. annulata polypeptide Tams1. (ABSTRACT T
There is growing interest in carbohydrate-recognizing receptors of the innate immune system. Among them are members of the C-type lectin family, which include the collectins and the selectins and which operate by ligating exogenous (microbial) or endogenous carbohydrates. De novo assignments of the sequences of ligands for carbohydrate-recognizing receptors are among the most challenging topics in cell biology. This is because of the heterogeneity of oligosaccharides on proteins and lipids, and their availability only in limited amounts. To address the need for a microprocedure for direct binding studies with oligosaccharides derived from glycoproteins, we introduced the neoglycolipid technology for generating solid phase oligosaccharide probes for binding experiments. The technology has enabled assignments of unsuspected oligosaccharide ligands for the selectins and given valuable insights into those for the collectins. The ligands so far identified appear not to be unique for a given receptor system; there are considerable cross-reactions. Specificity can be created, however, through different modes of oligosaccharide presentation on macromolecular carriers, or the expression of a particular oligosaccharide sequence on a selected cell type in a given body compartment, and the regulated expression of the receptor protein at the desired location. The existence of unique ligand structures is not ruled out, however. Co-ligation of a receptor may also occur to a second carbohydrate or even to a non-carbohydrate ligand to create a unique assembly. A further group of C-type lectin-like proteins occurs on natural killer (NK) cells and NK T cells, and is associated with activation or inhibition of the cell effector functions. An important challenge is to determine whether carbohydrates are among physiological ligands for this important group of receptors.
The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.
Upon infection with Theileria annulata, bovine leukocytes are induced to express eight novel metalloproteinase activities. In this article, Rachel Adamson and Roger Hall suggest that these enzymes are virulence factors and their presence may explain some of the features of the pathology of the disease. Specifically, they discuss the possibility that the metastatic properties of infected cells, the 'cigarette burn' ulcers and the cachexia characteristic of tropical theileriosis are associated with metalloproteinase expression. Furthermore, they propose that loss of metalloproteinase activity during the generation of a vaccine line could explain the attenuated phenotype.
Resistance to theileriosis of imported and local breeds in Morocco
- OUDICH