Isolation and propagation of an Egyptian Theileria annulata infected cell line and evaluation of its use as a vaccine to protect cattle against field challenge.

Tropical theileriosis is an important protozoan tick-borne disease in cattle. Vaccination using attenuated schizont-infected cell lines is one of the methods used for controlling the disease. This study describes the production of attenuated schizont-infected cell lines from Egypt and an evaluation of its use as a vaccine to protect calves against clinical disease upon field challenge. Two groups of exotic and crossbred male calves were divided into vaccinated and control groups. The vaccinated groups were inoculated with 4 ml (1 × 106 cells/ml) of the attenuated cell line. Three weeks after vaccination, calves of both groups were transported to the New Valley Governorate (Egyptian oasis) where they were kept under field conditions and exposed to the natural Theileria annulata challenge. All animals in the control group showed severe clinical signs and died despite treatment with buparvaquone, which was administered after two days of persistent fever due to a severe drop in packed cell volume (PCV). Animals in the vaccinated group became seropositive without developing severe clinical signs other than transient fever. Post-mortem examinations revealed enlarged and fragile lymph nodes, spleen, and liver with necrosis and hemorrhages. These findings indicate that the Egyptian attenuated cell line was successful in protecting both exotic and crossbred animals against tropical theileriosis under field conditions.

Development of a loop-mediated isothermal amplification method for detection of Theileria lestoquardi.

A loop-mediated isothermal amplification (LAMP) assay was developed for the diagnosis of Theileria lestoquardi infection. The primers were designed based on the clone-5 sequence of T. lestoquardi. The specificity and sensitivity of the assay were established. Analysis of the specificity showed that the selected LAMP primers amplified the target sequence from T. lestoquardi DNA successfully, while no amplification was seen with DNA from Theileria annulata, Theileria ovis, Babesia ovis, Anaplasma ovis, or ovine genomic DNA. The specificity of the LAMP product was further confirmed by restriction digestion and sequencing. The sensitivity of the LAMP assay was analyzed in comparison to PCR resulting in a detection limit of 10 fg/µl of plasmid DNA containing the clone-5 sequence. The suitability for utilizing the LAMP assay in the field for the diagnosis of T. lestoquardi infection was tested on 100 field samples collected in Sudan and compared with results obtained by PCR. The relative specificity and sensitivity of the established LAMP assay was determined to be 92.1% and 87.5%, respectively, indicating that it may be regarded as an alternative molecular diagnostic tool to PCR which could be used for epidemiological surveys on T. lestoquardi infection.

Identification of clone-9 antigenic protein of Theileria uilenbergi and evaluation of its application for serodiagnosis.

The pathogenic protozoan parasite Theileria uilenbergi is one of the causative agents of theileriosis in small ruminants in China. The infection results in great economical losses in the northwest part of China. Efforts are underway to establish an enzyme-linked immunosorbent assay (ELISA) based on a T. uilenbergi immunodominant recombinantly expressed protein using different approaches in order to perform epidemiological studies in the area. In this study, we describe the possible use of the clone-9 protein for this purpose, which was identified as a potential immunogenic piroplasm protein by random sequencing of cDNA library clones followed by bioinformatic analyses. The clone-9 gene was partially recombinantly expressed and used for the development of an indirect ELISA for the detection of circulating antibodies in sera of T. uilenbergi-infected sheep. No cross-reactivity was observed in serum from animals infected with Theileria lestoquardi. The cut-off was calculated at 48.6% positivity using 25 serum samples from uninfected animals. A total of 101 field samples collected from an endemic area in China were used to evaluate the clone-9 ELISA for its use in the field.

Schizonts of Theileria annulata interact with the microtubuli network of their host cell via the membrane protein TaSP.

Intracellular leukoproliferative Theileria are unique as eukaryotic organisms that transform the immune cells of their ruminant host. Theileria utilize the uncontrolled proliferation for rapid multiplication and distribution into host daughter cells. The parasite distribution into the daughter cells is accompanied by a tight association with the host cell mitotic apparatus. Since the molecular basis for this interaction is largely unknown, we investigated the possible involvement of the immunodominant Theileria annulata surface protein, TaSP, in the attachment of the parasite to host cell microtubule network. Confocal microscopic analyses showed co-localization of the TaSP protein with alpha-tubulin and reciprocal immuno-co-precipitation experiments demonstrated an association of TaSP with alpha-tubulin in vivo. In addition, the partially expressed predicted extracellular domain of TaSP co-localized with the mitotic spindle of dividing cells and was co-immunoprecipitated with alpha-tubulin in transiently transfected Cos-7 cells devoid of other T. annulata expressed proteins. Pull-down studies showed that there is a direct interaction between TaSP and polymerized microtubules. Analysis of the interaction of TaSP and host microtubulin during host cell mitosis indicated that TaSP co-localizes and interacts with the spindle poles, the mitotic spindle apparatus and the mid-body. Moreover, TaSP was demonstrated to be localized to the microtubule organizing center and to physically interact with gamma-tubulin. These data support the notion that the TaSP-microtubule interaction may be playing a potential role in parasite distribution into daughter host cells and give rise to the speculation that TaSP may be involved in regulation of microtubule assembly in the host cell.

Theileria annulata surface protein (TaSP) is a target of cyclin-dependent kinase 1 phosphorylation in Theileria annulata-infected cells.

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T. annulata isolates identified a conserved CDK1 phosphorylation motif T131 PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T131 , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T. annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T. annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T. annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

The protection afforded to cattle immunized with Theileria annulata infected cell line is enhanced by subunit vaccine candidate TaSP.

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.

Serum-free in vitro cultivation of Theileria annulata and Theileria parva schizont-infected lymphocytes.

Theileriosis is a tick-borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata-infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%-20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch-to-batch variation, safety and ethical concerns. In this study, the suitability of serum-free media for the cultivation of Theileria-transformed cell lines was examined. Three commercial serum-free media (HL-1, ISF-1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS-containing RPMI-1640 medium. ISF-1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF-1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF-1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin-free, serum-free, protein-free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF-1 medium. The use of serum-free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case-by-case basis. The relevance of Theileria cultivation in serum-free media for applications such as vaccine development requires further examination.

Field validation of a competitive ELISA for detection of Theileria annulata infection.

In previous studies, Theileria annulata surface protein (TaSP) was identified as an immunodominant antigen and successfully used to develop a recombinant-protein-based indirect enzyme-linked immunosorbent assay (ELISA) for the detection of circulating antibodies in serum of T. annulata-infected animals. To increase the specificity, a competitive ELISA (cELISA) was developed using recombinant TaSP antigen and a monoclonal antibody (1C7) specifically binding to TaSP. Since the cELISA accurately differentiated T. annulata-infected from uninfected animals, a study was performed to analyse the suitability of the cELISA in the field. For this, 230 sera with unknown status from different governorates in the north of Iraq were analysed using both the indirect and competitive ELISA and were compared. There was a significant (p < 0.5 x 10(-19)) correlation (r = 0.556) between the tests, whereby the cELISA detected more sera as negative (44/230) compared to the indirect ELISA (21/270). Accordingly, less sera were determined to be positive in the competitive (186/230) than in the indirect ELISA (209/230). Sensitivity and specificity of the cELISA taking the indirect ELISA as a reference were 84.2% and 52.4%, respectively. Accordingly, the calculated prevalence of T. annulata infection was 90.9%, and the positive predictive value was determined to be 94.6%. Taken together, the cELISA proved its suitability for field application and was found qualified for use in serological surveys to monitor the prevalence of T. annulata infection and to identify carrier animals.

Molecular detection of tick-borne pathogens in cattle from Southwestern Ethiopia.

Tick-borne diseases (TBDs) cause significant losses among livestock and impact the livelihoods of resource-poor farming communities worldwide. In Ethiopia, detailed studies on the epidemiology of tick-borne pathogens (TBPs) in cattle using sensitive molecular detection methods are scarce. The objective of this study was to determine the prevalence and species composition of bovine TBPs of veterinary significance in local cattle populations. A comprehensive cross-sectional epidemiological study was conducted in cattle populations of Illubabor zone in Southwestern Ethiopia from June to August 2013. For this purpose, blood samples were collected from 392 cattle. A combination of polymerase chain reaction (PCR) and a Reverse Line Blot (RLB) hybridization assay was employed for the detection of TBPs in these samples. The PCR/RLB results of the 392 blood samples indicated a high overall prevalence of 96.9% for TBPs, including Theileria mutans (66.1%), Theileria orientalis (51.8%), Anaplasma sp. Omatjenne (25.5%), Anaplasma marginale (14.5%), Babesia bigemina (14.0%) and Theileria velifera (13.0%) and minor occurrences of Ehrlichia ruminantium (0.5%) and Ehrlichia minasensis (0.26%). Moreover, three novel Anaplasma genotypes were detected in bovine blood samples. A phylogenetic analysis revealed that they most likely represent three, but at least two, new species. The prevalence of the three novel Anaplasma species, preliminary designated as Anaplasma sp. Hadesa, Anaplasma sp. Saso and Anaplasma sp. Dedessa, was 12.5%, 14.3% and 5.6%, respectively. Overall, a total of 227 cattle (57.9%) were found to be co-infected with two or more TBPs simultaneously and 86 different species combinations were observed. The findings show a very high burden of infection of cattle with TBPs in Ethiopia. The high frequency of co-infections suggests that clinical manifestations might be complex. Further research is required to determine the pathogenicity, host cell types and vector of the three novel Anaplasma species identified in this study.

Identification of potential antigenic proteins of Theileria lestoquardi.

A PCR strategy was used to identify potential antigenic proteins of T. lestoquardi suitable for the development of an ELISA by searching for homologous proteins previously identified in other theilierial parasites to be antigenic.

Investigation of MAP kinase activation in Theileria-infected cell lines.

A study on different Theileria annulata and Theileria parva infected cell lines was performed in order to evaluate the general relevance of the MAP-kinase activation status for Theileria- mediated transformation.

Purification of macroschizonts of a Sudanese isolate of Theileria lestoquardi (T. lestoquardi [Atbara]).

Research on malignant theileriosis is affected by the limited access to biological materials required for studies aiming at controlling the disease through the establishment of diagnostic tools and vaccines. The main aims of this work were to isolate, establish, and characterize a Theileria lestoquardi-infected cell culture (line) as a source of biological material and to generate a schizont cDNA library for further studies aiming at the identification of antigenic proteins. The T. lestoquardi isolate used originated from a sheep showing typical signs of malignant theileriosis in Atbara town in northern Sudan, and was maintained as an infected cell culture. A high-quality representative schizont cDNA library was established by isolating and purifying the schizonts using a nocodazole/aerolysin protocol followed by Percoll gradient ultracentrifugation. As a parameter to assess the quality of the schizont library, a provisional estimation of the percentage of recombinant phage clones originating from T. lestoquardi (Atbara) was undertaken. Ten clones with inserts ranging in size between 600 and 1200 bp were selected randomly, sequenced, and subjected to BLAST similarity searches. As 6 of the 10 sequenced clones showed similarities to T. parva, T. annulata, and other apicomplexan genes, it was concluded that the majority of the library phage clones originated from the parasite and not from host cell transcripts. The cDNA library will be used for screening of antigenic proteins using sera from infected sheep.

Identification and characterization of merozoite antigens of a Theileria species highly pathogenic for small ruminants in China.

A new pathogenic Theileria species transmitted by Haemaphysalis qinghaiensis was identified in the Northwestern part of China and was shown to be highly pathogenic for small ruminants. The present article aimed at identifying merozoite antigens that might be suitable for developing diagnostic methods and designing a potential vaccine. Absence of other theilerial or babesial infections was confirmed by reverse line blot in all antigen samples used. Extensive Western blot analyses using serum from infected and noninfected animals led to the identification of four potential merozoite immunoreactive proteins at different molecular weights. Further protein characterization using peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching resulted in two significant hits that identified two proteins of parasite origin, one homologous to a possible MO25-family protein from Cryptosporidium parvum and the other with an HSP70 from Theileria annulata. Another protein was also identified as a parasite protein but without significant homology. Immunization of rabbits with selected proteins produced antisera that reacted specifically on Western blots with merozoite antigens of the corresponding sizes. This article represents the first identification and characterization of potential antigenic proteins of Theileria sp. (China) for veterinary purposes.

Epidemiology of Theileria annulata infection of dairy cattle in the Sudan using molecular techniques.

This study provides the first epidemiological data regarding T. annulata infection of diary cattle in Sudan using a combination of routine microscopic examination and two molecular techniques, PCR and reverse line blot (RLB).

Phylogenetic position of small-ruminant infecting piroplasms.

Theileria and Babesia are tick-transmitted protozoa that cause great economical losses in livestock. Recently, interest has risen in sheep-infecting piroplasms and a number of previously unidentified pathogens were described, particularly in China. To address the phylogenetic relationship of Theileria and Babesia species infecting sheep, the complete sequences of the 18 S small subunit ribosomal RNA genes of a panel of piroplasm isolates, including T. lestoquardi, T. ovis, T. separata, B. ovis, B. motasi, B. crassa, and several novel species, were compared. The classification based on the established phylogenetic tree corresponded with traditional systematics and revealed that sheep/goat piroplasm species are of a polyphyletic origin. In addition, these studies revealed the existence of at least two novel sheep/goat piroplasm species, designated Theileria sp. (China 1) and Theileria sp. (China 2).

Identification of homologous genes of T. annulata proteins in the genome of Theileria sp. (China).

Homologues to previously described Theileria (T.) annulata genes (T. annulata surface protein [TaSP], putative T. annulata membrane protein [TaD]) were successfully amplified by polymerase chain reaction (PCR) from Theileria sp. (China) merozoite cDNA, with 88% identity to TaD; TcSP partial cDNA, 94% identity to TaSP. Moreover, homologues to a secretory protein of T. annulata (TaSE), with a sequence identity of 99% on the cDNA level (TcSE partial cDNA) and to a potential membrane protein of T. lestoquardi (Clone-5), with a sequence identity of 100% on the genomic level (Tc Clone-5) but lacking an intron at positions 1894-1928 were identified.

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.

Identification of antigenic proteins of a Theileria species pathogenic for small ruminants in China recognized by antisera of infected animals.

The antigenic proteins of the piroplasm stage of Theileria species (China), the causative agent of theilerosis of small ruminants in China, were analyzed by Western blot, revealing several specific immunoreactive proteins of different predicted molecular weights. Furthermore, sera from Theileria species (China)-infected animals were probed for reactivity with the TaSP protein of T. annulata, for which a homologue has been described in Theileria species (China). Affinity chromatography demonstrated the presence of TaSP-reactive antibodies, and the majority of the sera showed reactivity with this protein both in Western blots and in ELISA. The identified parasite antigens and TaSP will be assessed for their suitability for developing diagnostic methods as well as evaluated for their capacity to stimulated host immune competent cells.

Validation of a recombinant protein indirect ELISA for the detection of specific antibodies against Theileria uilenbergi and Theileria luwenshuni in small ruminants.

An enzyme-linked immunosorbent assay (ELISA) based on a recombinant Theileria uilenbergi immunodominant protein (rTuIP) was validated for detection of antibodies in 188 positive and 198 negative reference serum samples, respectively. The cut-off value was determined at 32.7% with 95% and 90% accuracy levels by two-graphic receiver-operating characteristic (TG-ROC). The equal diagnostic sensitivity (Se) and specificity (Sp) were calculated to be 98.4%. Further validation of the repeatability with positive and negative reference samples indicated the reliable performance of the assay. Monitoring the antibody dynamics of sheep experimentally infected with Theileria luwenshuni showed the efficient detection of antibody response against the pathogen at the early infection stage and up until two months post infection. Application of this assay for detection of antibody in field sera from previous unknown Theileria endemic regions in Suizhou and Guiyang showed 17.8% and 11.6% seroprevalence, respectively, and presence of the pathogen was confirmed by identification of the 18S rRNA gene in the corresponding blood of the seropositive animals. These data support that the rTuIP ELISA could be a useful tool to study the epidemiology of theileriosis caused by T. uilenbergi and/or T. luwenshuni.

Existence of splicing variants in homologues of Theileria lestoquardi clone-5 gene's transcripts in Theileria annulata and Theileria parva.

Clone 5 has been described as an immunogenic protein and was used to establish an ELISA for malignant theileriosis. Molecular characterization of the gene product revealed alternative splicing at the single intron resulting in two mRNA transcripts, translating into a long and a short protein form. Homologues of clone 5 exist in Theileria annulata and T. parva according to the available annotated GenBank sequences, showing however only the long protein forms in these parasites (GenBank accession numbers CAI73679, EAN33624). The present study aimed to determine whether two splice variants of homologues of clone 5 occur in T. annulata and T. parva.