The entry of Theileria parva sporozoites into bovine lymphocytes: evidence for MHC class I involvement.

We have examined the process of Theileria parva sporozoite entry into susceptible bovine lymphocytes and have begun to identify one of the possible molecular interactions involved in the process. The entry process involves a defined series of events and we have used a number of experimental procedures in combination with a method of quantitation to examine various aspects of this process. T. parva sporozoites are nonmotile organisms and the initial sporozoite-lymphocyte interaction is a chance event which can occur at 0-2 degrees C. All subsequent stages in the process are temperature dependent, require the participation of live intact sporozoites and host cells, and involve some cytochalasin-inhibitable rearrangement of the host cell surface membrane or cytoskeleton. Sporozoite entry can be inhibited by antibodies (mAbs) reactive with major histocompatibility complex (MHC) class I molecules (IL-A 19, IL-A 88) and with beta 2 microglobulin (B1G6), whereas mAbs reactive with MHC class II molecules (IL-A 21, J 11), and a common panleucocyte surface antigen, (IL-A 87; a bovine equivalent of CD 11a) have no effect. These results indicate that MHC class I molecules play a role in the process of T. parva sporozoite entry into bovine lymphocytes although as yet the precise role has not been determined. Once internalized within the lymphocyte, a process that takes less than 3 min at 37 degrees C, the sporozoite rapidly escapes from the encapsulating host cell membrane; a process which occurs concurrently with the discharge of the contents of the sporozoite rhoptries and microspheres. The intracytoplasmic parasite is covered by a layer of sporozoite-derived fuzzy material to which host cell microtubules rapidly become associated.

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.

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.

Characterisation of the gene encoding a candidate vaccine antigen of Theileria parva sporozoites.

We have cloned and characterised the gene encoding the 67-kilodalton stage-specific surface antigen, p67, of Theileria parva (Muguga) sporozoites. The gene which is present in a single copy, is divided into 2 exons by an intron 29 bp long and is transcribed into mRNA of about 2500 nucleotides. The gene is present in all stocks of T. parva and there is a related gene in Theileria annulata. The deduced amino acid sequence of 709 residues predicts that p67 is a membrane protein and that it lacks tandemly repeated sequences. Recombinant p67 has been expressed in Escherichia coli as a fusion protein with Sj-26, a glutathione-S-transferase of Schistosoma japonicum. Antibodies to purified recombinant proteins containing residues 9-316 or 397-709 of p67 bind to p67 in immunoblots and neutralise sporozoite infectivity in vitro. Recombinant p67 is, therefore, a candidate antigen for development of an anti-sporozoite vaccine for East Coast fever in cattle.

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis.

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4-7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9-10 X 10(6) base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7-8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G + C content than T. parva or the presence of highly reiterated G + C-rich regions.

Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins.

Monoclonal antibodies binding to the surface of live Mexico isolate Babesia bigemina merozoites have defined 4 parasite-encoded surface antigens (36, 45, 55, and 58 kDa) that are potential targets for immune-mediated neutralization of merozoites. In this study, we have characterized the post-translational modification, antigenic polymorphism, and immunogenicity of these 4 proteins. Monoclonal antibody immunoaffinity-purified 36- and 55-kDa polypeptides were identical in gel electrophoresis to immunoprecipitated radiolabeled proteins, while the purified 45-kDa protein consisted of 2 closely spaced polypeptides with relative molecular weights of 45 and 43 kDa. The 36-, 45-, and 55-kDa proteins were post-translationally modified by incorporation of [3H]glucosamine and [3H]myristic acid, suggesting they are integral membrane proteins secured by a phosphatidylinositol anchor. Cross-reactivity studies with monoclonal and monospecific polyclonal antibodies revealed marked antigenic polymorphism of these 3 glycoproteins among diverse geographic isolates. In contrast, none of the polypeptides bound by anti-p58 monoclonal antibody were glycosylated or myristilated. Both monoclonal and monospecific polyclonal antibodies recognizing p58 bound to similar molecular weight proteins in 4 additional isolates of B. bigemina from Mexico, Puerto Rico, St. Croix, and Kenya, suggesting widespread conservation of p58 immunogenic epitopes among geographic isolates. Calves immunized with immunoaffinity purified gp45, gp55, or p58 antigens were able to neutralize the infectivity of merozoites as indicated by significant reductions in the peak parasitemia after experimental challenge. Precise definition and appropriate presentation of neutralization sensitive epitopes on gp45, gp55, or p58 may enhance the merozoite neutralizing immune response in immunized cattle.

The chromosome profiles of Trypanosoma congolense isolates from Kilifi, Kenya and their relationship to serodeme identity.

Chromosomal DNA from 117 Trypanosoma congolense clones from 54 stocks, isolated from cattle introduced onto a ranch in Kilifi in the coastal area of Kenya, was fractionated by the orthogonal field alternation gel electrophoresis technique. The technique resolved chromosomes in the size range of 100 kb-1 Mb. The chromosome profile for cloned trypanosome populations was relatively stable with regard to number and size of the chromosome bands following transmission in mice, cattle, goats or tsetse flies. Only in one clone was a shift observed in the position of one medium-sized chromosome band following cyclical development in tsetse. On the basis of their chromosome profiles, the 117 clones could be divided into 18 distinct groups. Representative clones, randomly selected from 7 of the 18 chromosome profile groups were inoculated into steers and goats in order to raise variable antigen type (VAT) repertoire-specific infection sera. Cross-neutralization assays demonstrated that recovery sera from animals infected with a clone neutralized all the clones with an identical chromosome profile. This suggests that clones having an identical chromosome profile also express an identical VAT-repertoire (serodeme).

Sequence and expression of a 90-kilodalton heat-shock protein family member of Theileria parva.

A Theileria parva specific full-length cDNA clone, T7, which encodes a protein with more than 60% homology to heat shock protein 90 (hsp90) of other organisms, has been identified. T7 appears to be a single copy gene. The gene is expressed as a protein of 87 kDa in both the sporozoite and schizont stages of T. parva. The protein was not found in the piroplasm stage, although the corresponding transcript was detected, suggesting post-transcriptional regulation of the gene. In the schizont stage the T7 protein is upregulated upon heat shock and localized in the cytoplasm.

Characterisation of the gene encoding a 104-kilodalton microneme-rhoptry protein of Theileria parva.

A neutralizing antiserum, C16, raised against sporozoites of Theileria parva parva was used to screen a lambda gt11 expression library of T. parva parva (Muguga) genomic DNA fragments. Proteins encoded by one phage clone, lambda TpS-17, were reactive with the C16 antiserum. Detailed characterisation of the DNA insert showed it to encode determinants found on four theilerial antigens of approximately 104, 90, 85 and 35 kDa. The sequence encoded by the clone is expressed during sporogony as a single RNA transcript of about 3000 nucleotides. On sequencing a portion of the 5000-bp insert, an open reading frame of 2772 bp was revealed that encoded a 104-kDa protein. Immunoscreening a library of subfragments of the DNA insert with the original antiserum localised sequences encoding the dominant antigenic determinants to an 800-bp stretch of DNA at the 3' end of the open reading frame. Sequence data from three subclones spanning this region show portions of the antigenic domains to be unusually rich in proline residues which are repeated every three amino acids. These repeats often take the form X-S(T)-P or X-K(R)-P. Antibodies directed against each of the three subclones recognize the 104- and 35-kDa antigens and different combinations of the 90- and 85-kDa kDa antigens, suggesting that the smaller proteins are derived from the 104-kDa antigen by limited proteolysis occurring at the carboxyl terminus end of the protein. In immunoelectron micrographs the antigen is associated with the microneme/rhoptry complexes of the sporozoite.

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.

Comparison between bloodstream and procyclic form trypanosomes for serological diagnosis of African human trypanosomiasis.

Trypanosoma brucei brucei MiTat 1.2 bloodstream and corresponding procyclic forms, as well as procyclics of T. b. gambiense and T. b. rhodesiense, were fixed, in suspension, using a mixture of 80% acetone and 0.25% (v/v) formalin in saline and used as antigens for diagnosis of African human trypanosomiasis by the indirect immunofluorescent antibody test. T. b. brucei bloodstream forms detected 41/42 and 37/41 parasitologically diagnosed cases of T. b. rhodesiense and T. b. gambiense trypanosomiasis, respectively, while the procyclic stages detected the same number (41/42) of T. b. rhodesiense, but fewer (29/41) T. b. gambiense infections.

Failure of trypanosomal membrane antigens to induce protection against tsetse-transmitted Trypanosoma vivax or T. brucei in goats and rabbits.

A purified protein, relative molecular weight 83 kilodalton (kD), and plasma membranes from Trypanosoma brucei were tested as potential vaccines against tsetse-transmitted T. vivax and T. brucei in goats and rabbits. The 83 kD protein was found in lysates of all clones of T. brucei examined, as well as in lysates of T. vivax, T. congolense and T. rhodesiense. Rabbits and goats were immunized with various amounts of antigen in Freund's complete adjuvant and boosted twice with antigen in Freund's incomplete adjuvant. Two weeks after the last inoculation, the goats were challenged with T. vivax-infected and the rabbits with T. brucei-infected Glossina morsitans morsitans. Although high antibody levels were detected in all the animals immunized with either antigen as measured by radioimmunoassay and immunodiffusion, they became infected and the course of disease was the same as that in unimmunized controls.

Clone-specific immune colostrum induces increased resistance in goat kids challenged with Trypanosoma congolense.

The course of infection and the humoral immune response to Trypanosoma congolense clone ILNat 3.1 were studied in test goat kids receiving colostrum from dams immunized with the surface coat of ILNat 3.1 and control kids that received colostrum from nonimmunized dams. At 24-48 h after birth, all test kids had detectable serum antibodies to the trypanosome clone. There was no difference in the prepatent period between the test and control kids following challenge with 10(3) T. congolense ILNat 3.1 trypanosomes 8 days after birth. After the first 7 days of the experimental period, significantly lower parasitemia was recorded in test kids than in control kids. The mean packed red cell volume of test kids was not significantly different from that of control kids 7 days after infection. The test kids gained as much weight as noninfected control kids; both groups gained twice as much weight as infected control kids. Following infection, all kids developed antibodies against the infecting trypanosome clone. Fifteen test kids had titers equal to or greater than 1280 compared to only two control kids. The test kids survived longer after infection compared to control kids. The results suggest that colostrum from does immunized with the surface of a T. congolense clone did not prevent infection, but decreased parasitemia and prolonged survival of kids challenged with the same clone.

Differences between N'Dama and Boran cattle in the ability of their peripheral blood leucocytes to bind antibody-coated trypanosomes.

Investigations were undertaken to evaluate the immune response of trypanotolerant N'Dama (Bos taurus) and susceptible Boran (Bos indicus) cattle to two Trypanosoma congolense variable antigen types (VATs) expressed in both breeds following tsetse-transmitted challenge. The VAT-specific antibodies of both IgM and IgG1 isotypes produced by both breeds had similar neutralizing titres. The interaction between immune sera, trypanosomes and freshly isolated peripheral blood leucocytes (PBL) from uninfected N'Dama and Boran animals was studied. It was found that both N'Dama and Boran immune sera were able to induce adherence of trypanosomes to the N'Dama PBL, but not to Boran PBL. The adherence-inducing activity was exhibited by both IgM and IgG1 antibodies, but IgG1 antibodies were more efficient in this respect. These results suggest that there are qualitative and/or quantitative differences in the immunoglobulin receptor function of PBL between the two breeds of cattle.

Analysis of the variable antigen composition of Trypanosoma brucei brucei metacyclic trypanosomes using monoclonal antibodies.

The metacyclic trypanosomes of a Trypanosoma brucei brucei clone (ILTat 2.1) were analysed with regard to their variable antigen (VAT) composition using monoclonal antibodies. The metacyclic population was antigenically heterogeneous. Despite the heterogeneity, however, the overall VAT composition of the metacyclic population appeared to be limited in number. A similar pattern of reactivity was observed when the monoclonal antibodies were tested on metacyclics of another clone (IL Tat 2.2) derived from a rabbit 30 days after infection with IL Tat 2.1 as well as those of the parent stock (STIB 247). The VAT characteristics of the metacyclics of this serodeme were consistent regardless of whether they were transmitted by Glossina morsitans morsitans or G.m. centralis. The monoclonal antibodies also reacted with some of the bloodstream VATs isolated within 72 h from cyclically infected mice. None of the monoclonal antibodies, however, reacted with metacyclics of a different stock (LUMP 227).

Cerebral trypanosomiasis in cattle with mixed Trypanosoma congolense and T. brucei brucei infections.

Six Boran steers were infected simultaneously with Trypanosoma congolense and T. brucei brucei while another group of 3 was inoculated with T. b. brucei one year after infection with T. congolense. Three further steers were infected with T. b. brucei alone. Whereas, the six animals which received simultaneous infections developed clinical signs of cerebral trypanosomiasis as evidenced by depression, ataxia and occasional circling, those infected with T. b. brucei alone did not. At necropsy, 4 out of the 6 simultaneously infected animals had a mild to severe disseminated non-suppurative meningoencephalitis. Trypanosoma b. brucei was isolated from the cerebrospinal fluid (CSF) of three out of the four animals with histological lesions. Two of the cattle superinfected with T. b. brucei one year after infection with T. congolense also developed both clinical and histological evidence of cerebral trypanosomiasis. Trypanosoma congolense was isolated from the CSF of one of these 2 animals. Specific antibodies to the variable surface glycoproteins (VSGs) of the infecting T. b. brucei and T. congolense clones were found in the CSF of the 8 animals that developed cerebral trypanosomiasis. In these animals however, there was neither temporal nor quantitative correlation between VSG-specific antibodies in serum and in CSF, implying a de novo synthesis of antibodies to the infecting trypanosomes in the CSF.

Production and evaluation of specific antisera against sera of various vertebrate species for identification of bloodmeals of Glossina morsitans centralis.

Specific antisera against sera of 46 species of vertebrates were prepared. The antisera to 21 Bovidae species were raised in goats except the antiserum to goat serum which was raised in sheep. The antisera to 3 Suidae species were produced either in domestic pigs or warthogs, while antisera to most of the other vertebrate species were raised in rabbits. The antisera were used in an enzyme-linked immunosorbent assay (ELISA) to identify the source of bloodmeals ingested by teneral and non-teneral tsetse at different time intervals after feeding. The bloodmeal donors were identifiable in 100% of the teneral tsetse up to 40 h post-feeding and in 87.5% in those tested up to 74 h post-feeding. Non-teneral tsetse digested the species-distinguishing bloodmeal components faster than the tenerals. Bloodmeals could be identified in 100% non-tenerals at 20 h post-feeding but only 67.5% and 50% of the bloodmeals could be identified 40 h and 74 h post-feeding, respectively. The antisera were also able to identify mixed bloodmeals from closely related species.

Complement activation and fibrinolysis during infection with Theileria parva (East Coast fever) in cattle.

The role played by complement and fibrinolysis in the pathogenesis of pulmonary oedema and the disseminated, subendothelial, petechial haemorrhages in cattle infected with Theileria parva was investigated. There was a marked reduction in total haemolytic complement in animals that died of this disease whereas the survivors showed only transient changes. The reduction in C3 levels in animals with severe disease, however, was equivalent to that in the survivors. Neither circulating soluble immune complexes nor free antibody against macroschizonts were detected in sera from cattle with severe infection. In addition to the complement changes, high levels of fibrinogen degradation products (FDPs) were detected in sera from the infected animals. The rate of production of FDPs and reduction in total haemolytic complement paralleled the clinical course of the disease. It is, therefore, suggested that the pulmonary oedema and disseminated petechial haemorrhages seen in East Coast fever may be the direct result of the combined effects of complement activation and consumption of fibrinogen and fibrin.

Biologic activities of bovine IgG subclasses.

With regard to our studies on the functional properties of bovine IgG1 and IgG2 antibodies, the differences were minimal when homologous systems were evaluated. Both IgG1 and IgG2 fixed bovine complement by the classical pathway, caused homologous passive cutaneous anaphylaxis, caused phagocytosis of coated erythrocytes by cultured monocytes and precipitated with an antigen having multiple unique determinants (ovalbumin). In contrast to IgG2, IgG1 caused neither adherence nor phagocytosis by freshly isolated neutrophils and monocytes. Additionally, IgG2 antibodies to DNP precipitated with DNP19ovalbumin while IgG1 antibodies formed soluble complexes with this antigen.