Extensive polymorphism of Ra86 genes in field populations of Rhipicephalus appendiculatus from Kenya.

Commercial vaccines based on recombinant forms of the Bm86 tick gut antigen are used to control the southern cattle tick, Rhipicephalus microplus, a 1-host species, in Australia and Latin America. We describe herein sequence polymorphism in genes encoding Ra86 homologues of Bm86 in the brown ear tick, Rhipicephalus appendiculatus, isolated from four Kenyan field populations and one laboratory colony. Sequencing of 19 Ra86 sequences defined two alleles differentiated by indels, encoding 693 amino acids (aa) and 654 aa respectively, from the Muguga laboratory reference strain. Ra86 sequences were also determined from gut cDNA from four field populations of R. appendiculatus collected in different livestock production systems in Kenya. Analysis of approximately 20 Ra86 sequences from each of the four field sites in central and Western Kenya; Makuyu, Kiambu, Kakamega and Uasin Gishu, revealed three additional size types differentiated by 39-49 amino acid indels resulting in a total of 5 indel-defined genotypes. The 693 aa type 5 was isolated only from the laboratory tick stock; genotypes 1, 2 and 3 were identified in ticks from the four Kenyan field sites and appeared to be derivatives of the shorter RA86 genotype found in Muguga laboratory stock genotype 4. By contrast no large indels have yet been observed between R. microplus sequences from Australia, South America or Africa. Evidence that selection contributes to the observed sequence variation was provided by analysis of ratio of synonymous and non-synonymous substitutions and application of the selective neutrality and neutral evolution tests to the primary data. Phylogenetic analysis clustered sequences from all Ra86 size types and Bm86, into four major clades based on amino acid substitutions, but there was no evidence that these groupings correlated with geographical separation of R. appendiculatus populations.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes.

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ.

Tumor necrosis factor alpha (Tnf) plays a pleiotropic role in murine malaria. Some investigations have correlated Tnf with hypothermia, hyperlactatemia, hypoglycemia, and a suppression of the erythropoietic response, although others have not. In this study, we have evaluated parasitemia, survival rate and several pathological features in C57BL/6JTnf(-/-) and C57BL/6JTnf(+/+) mice after infection with Plasmodium chabaudi adami 408XZ. Compared to the C57BL/6JTnf(+/+) mice, C57BL/6JTnf(-/-) mice showed increased parasitemia and decreased survival rate, whereas blood glucose, blood lactate and body weight were not significantly different. However, C57BL/6JTnf(-/-) mice suffered significantly more from severe anemia and hypothermia than C57BL/6JTnf(+/+) mice. These results suggest that Tnf is an important mediator of parasite control, but not of anemia development. We hypothesize that the high mortality observed in the Tnf knock-out mice is due to increased anemia and pathology as a direct result of increased levels of parasitemia.

Comparison of pathology in susceptible A/J and resistant C57BL/6J mice after infection with different sub-strains of Plasmodium chabaudi.

Susceptible A/J and more resistant C57BL/6J mice were infected with Plasmodium chabaudi chabaudi 54X, P.c. chabaudi AS and Plasmodium chabaudi adami 408XZ. As expected, most C57BL/6J mice survived the infections with the different isolates. But in contrast to previous observations, not all A/J mice succumbed to infection: just over 50% of A/J mice survived infections with P.c. chabaudi 54X, while 80% survived P.c. chabaudi AS. The more virulent parasite, P.c. adami 408XZ, was able to kill all A/J mice and 20% of C57BL/6J mice after an intravenous infection with 10(5) pRBC. A detailed study of four parameters of pathology (body weight, body temperature, blood glucose and RBC counts) in both mouse strains after a P.c. adami 408XZ infection showed similar patterns to those previously reported after infection with P.c. chabaudi AS. These data suggest that environmental factors as well as parasite polymorphisms might influence the severity of malaria between susceptible and resistant mice.

Monoclonal antibodies identify two neutralization-sensitive epitopes in Besnoitia besnoiti endocytes.

Four monoclonal antibodies were produced against endozoite membrane and cytoplasmic antigens of B. besnoiti. In immunofluorescence antibody tests, three of the clones, designated 2M3C5, 2M1G8 and 2M9G3 recognized antigens restricted to the anterior pole of the endozoites. The fourth clone, 2M9C4, recognized a membrane-associated component in a "beaded" pattern, cytoplasmic granules and extracellular background. The staining characteristics differed from the solid diffuse staining of polyclonal serum. On Western blots of detergent-soluble extracts fractionated under non-reducing conditions in 10% SDS-PAGE gels, mAbs 2M3C5, 2M1G8 and 2M9G3 recognized a common antigen at >200 kDa. Recognition with mAb 2M3C5 was consistently different in intensity and extent. Monoclonal antibody 2M9C4 recognized a single antigen at 75 kDa. The antibodies significantly reduced infectivity of Besnoitia endozoites into cultured cells, demonstrating the potential role of the antigens in the invasion process and raising the possibility of development of a vaccine and diagnostic tests for the disease.

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells.

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP:p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Monoclonal antibody binding to a surface-exposed epitope on Cowdria ruminantium that is conserved among eight strains.

Monoclonal antibodies (MAb) binding to Cowdria ruminantium elementary bodies (EB) were identified by enzyme-linked immunosorbent assay, and surface binding of one MAb (446.15) to intact EB was determined by immunofluorescence, immunogold labeling, and transmission electron microscopy. MAb 446.15 bound an antigen of approximately 43 kDa in immunoblots of eight geographically distinct strains. The MAb did not react with Ehrlichia canis antigens or uninfected bovine endothelial cell lysate and may be useful in diagnostic assays and vaccine development.

Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina.

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina.

A 32 kDa surface antigen of Theileria parva: characterization and immunization studies.

Previous studies using monoclonal antibody (mAb) 4C9 specific for a 32 kDa antigen (p32) of Theileria parva demonstrated expression of the antigen on the surface of the sporozoite, making it a potential antigen for sporozoite neutralization. A full-length cDNA encoding the major merozoite/piroplasm surface antigen (mMPSA) of T. parva was cloned and expressed in bacteria. The expressed product reacted strongly with mAb 4C9, demonstrating identity between the p32 and mMPSA of T. parva. Using immunoblot analysis and immunoelectron microscopy with mAb 4C9 it was shown that the mMPSA is a major antigen of the merozoite and piroplasm at the cell surface, while lower levels of antigen are expressed in the sporozoite and schizont stages. Upregulation of the mMPSA occurs at merogony and can be induced by culturing schizont-infected lymphocytes at 42 degrees C. Recombinant mMPSA of T. parva induced high titres of specific antibodies in cattle but failed to confer protection against a T. parva sporozoite stabilate challenge. The pre-challenge sera also failed to neutralize infectivity of sporozoites in an in vitro assay. Possible reasons for the lack of parasite neutralization in vivo and in vitro are discussed.

A statistically derived index for classifying East Coast fever reactions in cattle challenged with Theileria parva under experimental conditions.

A statistically derived disease reaction index based on parasitological, clinical and haematological measurements observed in 309 5 to 8-month-old Boran cattle following laboratory challenge with Theileria parva is described. Principal component analysis was applied to 13 measures including first appearance of schizonts, first appearance of piroplasms and first occurrence of pyrexia, together with the duration and severity of these symptoms, and white blood cell count. The first principal component, which was based on approximately equal contributions of the 13 variables, provided the definition for the disease reaction index, defined on a scale of 0-10. As well as providing a more objective measure of the severity of the reaction, the continuous nature of the index score enables more powerful statistical analysis of the data compared with that which has been previously possible through clinically derived categories of non-, mild, moderate and severe reactions.

Reciprocal cross-protection induced by sporozoite antigens SPAG-1 from Theileria annulata and p67 from Theileria parva.

Theileria annulata and Theileria parva both possess a major surface antigen on the sporozoite stage of the life-cycle, called SPAG-1 and p67, respectively. In each case, these antigens are vaccine candidates and have been shown to induce a degree of homologous protection in earlier work. These antigens share sequence homology and are serologically cross-reactive. Here, we confirm that these antigens confer protection against homologous species challenge. More importantly, they mutually confer a degree of cross-species protection raising the prospect of a common vaccine in the future.

Enzymes of glucose and glycerol catabolism in in vitro-propagated Theileria parva schizonts.

Theileria parva schizonts propagated in vitro in peripheral blood lymphocytes were purified and assayed for key enzymes of glucose and glycerol catabolism and the citric acid cycle. The activities of glycolytic enzymes were in the range of 21-100 nmol/min/mg protein. Glycerol kinase and alpha -glycerophosphate dehydrogenase activities were more than 16 times lower than the activities of other enzymes catalysing the oxidation of the triose phosphates to lactate. It was suggested that the catabolism of glycerol is negligible and that glucose is catabolized to lactate via the Embden-Meyerhof pathway. The activities of the enzymes catalysing the section of the citric acid cycle that involves the formation of citrate to succinyl-CoA were consistently very low (less than 2.0 nmol/min/mg protein), indicating that this part of the cycle plays a minor role in this parasite. Enzyme activities of the cycle catalysing the formation of succinate from oxaloacetate were relatively higher than those catalysing other sections of the citric acid cycle, suggesting that this section of the cycle could be important to the parasite. Pyruvate carboxylase activity was more than 10 times that of phosphoenolpyruvate carboxykinase. It was suggested that pyruvate could be carboxylated to oxaloacetate. Taken together, these results suggest that the catabolism of glucose in Theileria parva schizonts is mainly via the Embden-Meyerhof pathway and that the citric acid cycle plays a minor role in energy production.

Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies.

Theileria parva is an intracellular sporozoan parasite that infects and transforms bovine lymphocytes, causing a severe lymphoproliferative disease known as East Coast fever in eastern, central and southern Africa. In this article, Declan McKeever and colleagues summarize the current understanding of immune mechanisms provoked by the parasite with regard to their role in both pathogenesis and protection. In particular, the influence of genomic polymorphism in parasite and host on the development of immunity is discussed, along with the evolution of current vaccine development strategies as a result of immunological research on the disease.

Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM).

To identify the genes encoding novel immunodominant antigens of Theileria parva a lambda gt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host-sporozoite interaction.

Proteolytic cleavage of surface proteins enhances susceptibility of lymphocytes to invasion by Theileria parva sporozoites.

A flow cytometric method using anti-parasite antibodies was developed to measure binding of Theileria parva sporozoites to the target bovine lymphocyte membrane. Parasite-host cell interactions could be inhibited by monoclonal antibodies to bovine MHC class I and partially by one of two antibodies to BoCD45R. Proteolysis of the lymphocyte surface removed CD45R but not MHC class I determinants, and enhanced sporozoite binding. These observations support the hypothesis that CD45R and CD45R antibodies may nonspecifically prevent close approximation between sporozoites and lymphocytes. Interestingly, under normal conditions, sporozoites of T. parva did not attach to lymphocytes from goats, but did so when the cells were treated with the protease, suggesting that receptor(s) for T. parva sporozoites might be present on caprine cells but are not easily accessible. These and other results indicate that proteases may be involved in binding and entry of T. parva sporozoites. Electron microscopy revealed that the process of binding and entry of sporozoites into protease-treated goat lymphocytes was very similar to that of the bovine cells. However, schizonts did not develop and lymphocyte proliferation was not induced, indicating that cell entry by sporozoites and cellular transformation are separate processes.

Delivery of the Theileria parva p67 antigen to cattle using recombinant vaccinia virus: IL-2 enhances protection.

To evaluate vaccinia virus as a delivery system for recombinant antigen in cattle, calves were immunized with a recombinant vaccinia virus (rVV) expressing the sporozoite surface antigen (p67) of Theileria parva (V-67) combined with those expressing bovine IL-4 (V-IL4) or IL-2 (V-IL2). The anti-p67 antibody levels detected in calves inoculated with the combination of V-67 and V-IL4 were higher than those produced by animals injected with V-67 alone or V-67 and V-IL2. On challenge with cryopreserved sporozoites, 5 of 7 animals receiving V-67 combined with V-IL2 were protected, while those receiving V-67 in conjunction with V-IL4 behaved like unimmunized control calves. Vaccination with a recombinant virus expressing a chimaeric p67(p583)/IL2 product gave rise to a lower level of protection, whereas V-IL2 provided no immunity. The results of this study demonstrate the potential of rVV as a delivery system for use in vaccination of cattle against Theileria parva infection.

Subunit vaccines for the control of tick-borne diseases: implications for the future.

Tick-borne parasites are a major constraint to the improvement of livestock productivity in the developing world. These parasites include Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdria ruminantium. The impact of these diseases is currently limited by the use of acaricides to decrease transmission by the tick vectors and immunization of the host animals using live vaccines. The use of acaricide is hampered by the development of acaricide resistance and live vaccines are dependent on cold chain facilities, which are generally unreliable in developing countries. There is therefore a requirement for improved vaccines that circumvent these problems. Candidate vaccine antigens have been identified for most of these parasites and are currently being evaluated for their capacity to induce solid protection.

Prospects for subunit vaccines against tick-borne diseases.

Tick-borne parasites are a serious impediment to the improvement of live-stock production in the developing world. The major parasites affecting cattle include Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdria ruminantium. The control of these infections is dependent on the use of acaricides to decrease transmission by the tick vectors, and immunization of susceptible animals with live vaccines. The use of acaricide is hampered by the development of resistance, and live vaccines require cold chain facilities, which are generally unreliable in developing countries. There is therefore a need for improved vaccines that can circumvent these problems. There is a subunit vaccine being developed for T. parva based on the major surface antigen of the sporozoite (p67). A similar antigen, SPAG 1, has been identified as a candidate for T. annulata. Although several candidate antigens have been identified for Babesia spp., progress towards development of a subunit vaccine based on these antigens has been hampered by polymorphism among isolates and between species, and lack of knowledge of the immune effector mechanisms responsible for protection. The search for protective antigens of A. marginale has focused on outer membrane proteins; immunization with a variety of these antigens alone or in combination, has yielded promising results. As with Babesia, further definition of immune effector mechanisms is needed to optimize immunization strategies. The work on identifying the protective antigens of C. ruminantium is in its embryonic stages; however, two antigens have been identified and are currently being evaluated. There is high expectancy for subunit vaccines for all these diseases; however there is need for further work to elucidate the immune mechanisms in order to select appropriate antigen delivery systems.