Polymorphic Immunodominant Molecule Research Articles

Prevalence of tick-transmitted pathogens in cattle reveals that Theileria parva, Babesia bigemina and Anaplasma marginale are endemic in Burundi

BackgroundTick-borne diseases (TBDs) constitute a major constraint for livestock development in sub-Saharan Africa, with East Coast fever (ECF) being the most devastating TBD of cattle. However, in Burundi, detailed information is lacking on the current prevalence of TBDs and on the associated economic losses from mortality and morbidity in cattle as well as the costs associated with TBD control and treatment. The aim of this study was, therefore, to assess the prevalence and spatial distribution of tick-borne pathogens (TBPs) in cattle across the major agro-ecological zones (AEZs) in Burundi.MethodsIn a cross-sectional study conducted in ten communes spanning the five main AEZs in Burundi, blood samples were taken from 828 cattle from 305 farms between October and December 2017. Evidence of Theileria parva infection was assessed by antibody level, measured using a polymorphic immunodominant molecule (PIM) antigen-based enzyme-linked immunosorbent assay (ELISA) and by a T. parva-specific p104 gene-based nested PCR. Antibodies against Theileria mutans infection were detected using the 32-kDa antigen-based indirect ELISA, while the 200-kDa antigen and the major surface protein 5 (MSP5)-based indirect ELISA were used to detect antibodies against Babesia bigemina and Anaplasma marginale, respectively.ResultsThe prevalence of T. parva across the ten communes sampled ranged from 77.5 to 93.1% and from 67.8 to 90.0% based on the ELISA and PCR analysis, respectively. A statistically significant difference in infection was observed between calves and adult cattle; however, T. parva infection levels were not significantly associated with sex and breed. The seroprevalence indicating exposure to T. mutans, B. bigemina and A. marginale ranged from 30 to 92.1%, 33.7 to 90% and 50 to 96.2%, respectively. Mixed infections of TBPs were detected in 82.91% of cattle sampled, with 11 different combinations of pathogen species detected .ConclusionsThe findings indicate that T. parva, A. marginale and B. bigemina infections are endemic in Burundi. Knowledge of the spatial distribution of TBPs will facilitate the design of effective targeted strategies to control these diseases. There is a need for further investigations of the distribution of tick vectors and the population structure of TBPs in order to identify the key epidemiological factors contributing to TBD outbreaks in Burundi.

Gene gun DNA immunization of cattle induces humoral and CD4 T-cell-mediated immune responses against the Theileria parva polymorphic immunodominant molecule

Theileria parva kills over one million cattle annually in sub-Saharan Africa. Parasite genetic complexity, cellular response immunodominance, and bovine MHC diversity have precluded traditional vaccine development. One potential solution is gene gun (GG) immunization, which enables simultaneous administration of one or more DNA-encoded antigens. Although promising in murine, porcine, and human vaccination trials, bovine GG immunization studies are limited. We utilized the model T. parva antigen, polymorphic immunodominant molecule (PIM) to test bovine GG immunization. GG immunization using a mammalian codon optimized PIM sequence elicited significant anti-PIM antibody and cell-mediated responses in 7/8 steers, but there was no difference between immunized and control animals following T. parva challenge. The results suggest immunization with PIM, as delivered here, is insufficient to protect cattle from T. parva. Nonetheless, the robust immune responses elicited against this model antigen suggest GG immunization is a promising vaccine platform for T. parva and other bovine pathogens.

English

This study was conducted to determine tick burden and immunological parameters of resistance to East Coast fever (ECF) in Tarime and Sukuma cattle. Tick load, packed cell volume (PCV), Theileria parva (T. parva)  specific antibody percent positivity (PP), and prevalence of T. parva parasites were studied in relation to dipping regime, strains, and season. A total of 50 experimental cattle were included in this study. Tick load was determined by whole body counts, antibody percent positivity was determined by the polymorphic immunodominant molecule (PIM)-based T. parva enzyme-linked immunosorbent assay (ELISA), and prevalence of T. parva parasites was detected by a nested polymerase chain reaction (PCR) based on the p104 gene. Dipping frequency on tick burden showed no statistically significant differences when cattle of either strain were dipped either once every 2 or 3 weeks in the dry and wet seasons. However, Tarime cattle had higher (p<0.05) tick count than Sukuma cattle and non dipped groups maintained high tick infestation throughout the experimental period. The PCV values were within the physiological range, although this parameter was lower in Tarime cattle (p<0.05). All cattle regardless of strain were seropositive, although Tarime cattle maintained higher PP compared to Sukuma by 15%. Conversely, the prevalence of T. parva parasites was lower in Tarime (38%) compared to Sukuma cattle (38.5%), but the difference was not significant (p>0.05). During the study period, 20% (5/25) of Sukuma cattle contracted ECF, but none of the Tarime cattle showed clinical signs for the disease. The differences between the two strains shown in terms of PP and T. parva parasite prevalence may indicate the ability of individual cattle to resist tick infestation and ECF infection under natural challenge. Higher antibody levels but lower parasite prevalence attained by Tarime cattle, suggests inherent ability of Tarime cattle to resist clinical development of ECF infection, but to remain as T. parva carriers. Key words: Carrier state, Theileria parva, ticks, seropositivity.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues ofTheileria 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.

English

This study was carried out in Fangak County, Jonglei State, South Sudan, with aim to identify the main ticks species and follow north limit of the theileriosis in Jonglei. Three localities namely; Hai, Toggar and Bichoul kun village in Phoum payam were selected as suspected area for East Coast fever (ECF), Group A) and Kuer kan in Manjang payams were selected as non suspected area for ECF (Group B). These groups A and B were based on animal movement, trade businessand intermarriages (animals paid for dowry). A total of 120 sera were collected from cattle of different age groups. The serum samples were tested using indirect polymorphic immunodominant molecule (PIM) ELISA to detect Theileria parva antibodies. The results indicated that 5/44 (11.4%) samples from non suspected area (group B) and 48/76 (63.2%) samples from suspected area (Group A) revealed antibodies. The overall positivity was 53/120 (44.17%) which was highly significant (P < 0.001) according to the locations. Three tick genera were recorded, Amblyomma, Hyalomma and Rhipicephalus. The species were A. variegatum, A. lepidum, H. rufipes, R. (B.) decoloratus, R. (B.) annulatus, R. e. evertsi and R. sanguineus. The most abundant tick species was A. variegatum, constituting 62%, while the lowest tick recorded was R. e. evertsi with prevalence rate of 2%. No R. appendiculatus tick was seen in Fangak area; while T. parva antibodies were detected. Regarding the fact that T. parva antibodies were present in the area with 44.17% prevalence, more efforts are needed to determine the extension of ECF and its vector R. appendiculatus to the northern parts of Jonglei State and this result of ECF antibodies could be an alarm to migrate cattle owners from South Sudan. Key words: Ticks, east coast fever (ECF), ELISA, South Sudan.

Molecular characterization of Theileria parva parasites from South Sudan using the PCR-RFLP approach on antigen genes

In an attempt to characterize Theileria parva parasites circulating in South Sudan cattle , polymerase chain reaction (PCR)-based assays were carried out using four single copy encoding antigen genes p104, PIM, p150 and p67 in addition to one microsatellite MS321. A total of 20 bovine DNA samples from two locations in South Sudan were included in this study, in addition to two references strains, Muguga and Katete. A total of nine alleles were identified for the polymorphic immunodominant molecule (PIM) locus using restriction fragment length polymorphism (RFLP), against 2 alleles each for the p150 and p104 loci, respectively. This confirms that differences in the polymorphic PIM genes alone could be used to characterize subdivisions in the T. parva populations in the field. On the other hand, 4 alleles were identified for the MS321 locus and 2 alleles for the p67 locus. The data indicate that the studied parasites are genetically closely related, mainly cattle derived and genetically quite distinct from the Muguga strain.

Role of the Polymorphic Immunodominant Molecule in Entry of Theileria parva Sporozoites into Bovine Lymphocytes

Theileria parva is a tick-transmitted apicomplexan parasite that infects cattle and African buffalo. In cattle, it causes a fatal lymphoproliferative disease called East Coast fever. The polymorphic immunodominant molecule (PIM) is expressed by two stages of the parasite: the sporozoite, which is inoculated by the tick to infect mammalian lymphocytes, and the schizont, the established intralymphocytic stage. Here, we demonstrate that monoclonal antibodies (MAb) to PIM can reduce the ability of sporozoites to infect bovine lymphocytes in vitro. This reduction appears to be due to blocking of sporozoite attachment by binding of the MAb to several regions of PIM. Interestingly, one MAb, which recognizes an epitope in the central variable region of PIM, did not inhibit sporozoite infectivity. We also demonstrate that PIM antigen, as a recombinant molecule, can also reduce sporozoite infectivity in vitro by blocking both attachment and internalization of sporozoites. Electron microscopic studies showed that PIM is present in microspheres below the sporozoite surface and is transported to the parasite surface soon after contact with bovine lymphocytes. The results suggest that at least two sporozoite molecules, PIM and the previously described p67, are involved in the entry of T. parva into mammalian lymphocytes.

Analyses of genes encoding Theileria parva p104 and polymorphic immunodominant molecule (PIM) reveal evidence of the presence of cattle-type alleles in the South African T. parva population

Restriction fragment length polymorphism analysis of PCR products (PCR-RFLP) and sequencing of the variable region of the p104 and PIM genes was performed on samples obtained from South African T. parva parasites originating from cattle on farms with suspected theileriosis and from buffalo. p104 and PIM PCR-RFLP profiles similar to those of the T. parva Muguga stock, an isolate that causes ECF in Kenya, were obtained from three of seven cattle samples collected on a farm near Ladysmith in KwaZulu-Natal Province. Amino acid sequences of the p104 and PIM genes from two of these samples were almost identical to the T. parva Muguga p104 and PIM sequences. This result supports findings from a recent p67 study in which p67 alleles similar to those of the T. parva Muguga stock were identified from the same samples. While these results suggest the presence of a cattle-derived T. parva parasite, reports of cattle-to-cattle transmission could not be substantiated and ECF was not diagnosed on this farm. Although extensive diversity of p104 and PIM gene sequences from South African T. parva isolates was demonstrated, no sequences identical to known cattle-type p104 and PIM alleles were identified from any of the buffalo T. parva samples analyzed. ‘Mixed’ PIM alleles containing both cattle- and buffalo-type amino acid motifs were identified for the first time, and there appeared to be selection of cattle-type and ‘mixed’-type PIM sequences in the cattle samples examined.

Loop-mediated isothermal amplification (LAMP) assays for detection of Theileria parva infections targeting the PIM and p150 genes

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1 fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries.

Orientation of bovine CTL responses towards PIM, an antibody-inducing surface molecule of Theileria parva, by DNA subunit immunization

East Coast fever, an acute lymphoproliferative disease of cattle, is caused by the apicomplexan parasite Theileria parva. Protective immunity is mediated by CD8 + cytotoxic T lymphocytes directed against schizont-infected cells. The polymorphic immunodominant molecule, although an antibody-inducing surface molecule of the schizont, has been hypothesized to play a role in protective immunity. In order to evaluate the immunogenicity of PIM for inducing CTL, cattle were immunized with PIM in isolation from other T. parva antigens, forcing the presentation of PIM-derived epitopes on the MHC class I molecules. Although parasite-specific cytotoxicity was induced in both vaccinated animals, their immune response was clearly different. One animal generated MHC-restricted parasite-specific CTL against PIM while the other calf exhibited a strong PIM-specific proliferative response but non-MHC-restricted parasite-specific cytotoxicity. Only calf 1 survived a lethal sporozoite challenge. This DNA immunization technique with an antigen in isolation of CTL-immunodominant antigens might open possibilities for directing CTL responses against predefined antigens, such as strain cross-reacting CTL antigens.

Immunostimulatory CpG oligodeoxynucleotides enhance the induction of bovine CD4 + cytotoxic T-lymphocyte responses against the polymorphic immunodominant molecule of the protozoan parasite Theileria parva

Enhancement of the induction of cytotoxic T-cell responses by immunostimulatory CpG oligodeoxynucleotides has been described in humans and mouse models. The present study attempted to address whether CpG has a similar effect in cattle. Immunisation of cattle with a recombinant form of the polymorphic immunodominant molecule from Theileria parva emulsified with immunostimulatory CpG oligodeoxynucleotides in adjuvant had no effect on the induction of antibody responses including the isotype profile, but significantly enhanced the induction of cytolytic responses that were mediated by CD4 +CD3 + T cells utilizing the perforin-granzyme pathway.

Post-translational signal peptide cleavage controls differential epitope recognition in the QP-rich domain of recombinant Theileria parva PIM

The presence of the schizont stage of the obligate intracellular parasites Theileria parva or T. annulata in the cytoplasm of an infected leukocyte results in host cell transformation via a mechanism that has not yet been elucidated. Proteins, secreted by the schizont, or expressed on its surface, are of interest as they can interact with host cell molecules that regulate host cell proliferation and/or survival. The major schizont surface protein is the polymorphic immunodominant molecule, PIM, which contains a large glutamine- and proline-rich domain (QP-rd) that protrudes into the host cell cytoplasm. Analyzing QP-rd generated by in vitro transcription/translation, we found that the signal peptide was efficiently cleaved post-translationally upon addition of T cell lysate or canine pancreatic microsomes, whereas signal peptide cleavage of a control protein only occurred cotranslationally and in the presence of microsomal membranes. The QP-rd of PIM migrated anomalously in SDS–PAGE and removal of the 19 amino acids corresponding to the predicted signal peptide caused a decrease in apparent molecular mass of 24 kDa. The molecule was analyzed using monoclonal antibodies that recognize a set of previously defined PIM epitopes. Depending on the presence or the absence of the signal peptide, two conformational states could be demonstrated that are differentially recognized, with N-terminal epitopes becoming readily accessible upon signal peptide removal, and C-terminal epitopes becoming masked. Similar observations were made when the QP-rd of PIM was expressed in bacteria. Our observations could also be of relevance to other schizont proteins. A recent analysis of the proteomes of T. parva and T. annulata revealed the presence of a large family of potentially secreted proteins, characterized by the presence of large stretches of amino acids that are also particularly rich in QP-residues.

Characterisation of Theileria parva isolates from Kiambu district, Kenya

Four Theileria parva isolates from Muguga area of Kiambu district, Kenya, were used to establish schizont-infected cell lines. Their protein antigens were then separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS page). The isolates were subsequently subjected to protein analysis and characterisation by the western immunoblotting technique. Probing for the polymorphic immunodominant molecule (PIM) was done using monoclonal antibody no. 4. SDS page detected up to 20 protein antigens of molecular mass 35,000–180,000 Da. The western blot analysis revealed a greater heterogeneity in the molecular mass ( M r) of PIM than previously thought. The M r of PIM varied between 80 and 90 kDa. The isolates further revealed different densities of surface epitopes with variable reaction to the monoclonal antibody. The implications of these findings to the epidemiology of east coast fever and immunisation programmes are discussed.

An unusual mosaic structure of the PIM gene of Theileria parva and its relationship to allelic diversity.

Genetic diversity and structural organisation of the polymorphic immunodominant molecule (PIM) gene of the protozoan parasite Theileria parva was studied in isolates from sympatric and allopatric areas. The analyses revealed a mosaic structure consisting of highly conserved regions shared among some of the isolates from geographically different areas and homologous sequence runs shared among isolates from one area. The specific pattern of diversity in which large insertions and deletions were observed, giving a mosaic structure to the PIM locus, is quite exceptional for single-locus genes. The polymorphic middle region of the gene was characterised by large deletions or insertions in many isolates. There was no correlation between the copy number of the tetrapeptide repeats in this region and the total length of the sequence. The gene was highly polymorphic when compared with sequences from other known T. parva antigenic regions. The findings support the concept that as yet unidentified mechanisms are generating extensive diversity and shaping the PIM locus. The relevance of this finding for diagnosis and the relationship between these mechanisms and the possible role of this protein in host immune responses is discussed.

Characterization of a polymorphic Theileria annulata surface protein ( TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane.

Molecular epidemiology of Theileria parva in the field.

Molecular tools based on seminested RFLP-PCR techniques to characterize field parasites in bloodspots dried on filter paper permitted investigation of the extent and the dynamics of diversity of Theileria parva populations in the field. Parallel molecular studies explored the long-term genome stability of various isolates by probing Southern blots of EcoRI digested total genomic DNA with four different reference nucleic acid probes. Three polymorphic single copy loci encoding for antigen genes were developed for seminested PCR detection in order to apply them for a multilocus approach in population genetic studies. Seven alleles were identified for the polymorphic immunodominant molecule (PIM) locus by using restriction enzymes, and 4 alleles each for the p150 and p104 loci. A simple DNA extraction method gave good results in amplifying these loci from carrier animals using samples of blood dried on filter papers. Results from probing Southern blots of cultures taken at sequential timepoints indicate relative genome stability in T. parva in comparison to other parasitic protozoa such as Plasmodium. Comparatively homogeneous profiles in sympatric isolates from Zambia were identified using all four probes and PCR amplified products which contrasted with the variety found amongst Kenyan stocks. Preliminary characterization of T. parva field samples from the Southern Province of Zambia strongly suggest clonal expansion of one of the components of a non-Zambian trivalent vaccine used on a limited scale in the Province from 1985 until 1992.

Immmunohistochemical detection of parasite antigens in Theileria parva-infected bovine lymphocytes

Theileria parva is the causal agent of East Coast fever (ECF), a fatal disease of cattle characterized by pyrexia, transient lymphadenopathy and panleukopenia. We have evaluated monoclonal antibodies (mAb) against three distinct antigens (p67, PIM and p32) of the parasite as immunohistological reagents for monitoring the kinetics of infection in cattle. Bovine lymphocytes were stained with the mAb at various intervals after infection in vitro and in vivo. The p67 sporozoite surface antigen was detected in only a small percentage of both, in vitro and in vivo infected cells. In contrast, expression of the polymorphic immunodominant molecule (PIM) of the parasite proved a useful indicator of infection and staining was correlated with the results of Giemsa analysis. PIM was detected from day 3 in in vitro-infected cells, but was not detected until day 5 in vivo after challenge with a 70% lethal dose of stabilized sporozoite. The p32 antigen was expressed only late in infection in vivo and its expression was associated with the development of merozoites. Less than 20% of in vitro-infected cells expressed p32. The immunohistochemical staining with anti-PIM mAb was found to be a useful tool for analysis of T. parva infection kinetics in cattle.

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.

An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule.

Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of > 99% and a specificity of between 94% and 98%.

Identification of neutralization and diagnostic epitopes on PIM, the polymorphic immunodominant molecule of Theileria parva.

The polymorphic immunodominant molecule (PIM) of Theileria parva is expressed by the schizont and sporozoite stages of the parasite. We have recently cloned the cDNA encoding the PIM antigen from two stocks of the parasite: the cattle-derived T. parva (Muguga) stock and a buffalo-derived stock. The cDNAs were used in transient-transfection assays to assess the reactivity of the antigen with monoclonal antibodies (MAb) previously raised against schizont-infected cells and used for parasite strain identification. We demonstrate that 19 of the 25 MAb are specific for PIM. Antibody reactivities with deletion mutants of a fusion protein containing PIM and Pepscan analysis of the Muguga version of the molecule with 13 of the MAb indicate that there are at least 10 different epitopes throughout the molecule. None of the MAb react with a tetrapeptide repeat present in the central region of the molecule, probably because of an inability of BALB/c mice to produce antibodies to this repeat. In contrast, sera from infected cattle react strongly with the repeat region, suggesting that this region alone may be useful as a diagnostic reagent. Previous studies showed that MAb to PIM inhibit sporozoite infectivity of bovine lymphocytes in vitro, which suggests that the antigen may be useful in immunizing cattle against T. parva infection. Pepscan analysis revealed that sera from infected cattle reacted with peptides recognized by the neutralizing MAb, as did sera from cattle inoculated with a PIM-containing recombinant protein. The latter sera did not, however, neutralize sporozoite infectivity in vitro. These results will be useful in exploiting the strain identification, diagnostic, and immunizing potentials of this family of antigens.