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.

Monitoring vaccinated cattle for induction and longevity of persistent tick-transmissible infection: Implications for wider deployment of live vaccination against East Coast fever in Tanzania.

The infection and treatment (ITM) procedure remains the only available method of immunization against Theileria parva infection. One constraint to deployment is the perception that the carrier state induced by ITM could result in enhanced disease problems. More than one million cattle have been ITM vaccinated in pastoralist systems in Tanzania over the last 2 decades. We present the results of a longitudinal study of six groups of cattle in Maasai villages in northern Tanzania exposed to natural tick challenge for between 2 weeks and 14 years post-vaccination. The p104 nested PCR revealed a higher frequency of T. parva carriers among vaccinates (30%) compared with controls (8%) (OR = 4.89, p = .000), with the highest frequency of carriers found in calves vaccinated 6 months previously, although carrier state was also detected in cattle vaccinated >10 years prior to the study. Variable number tandem repeat genotype analysis revealed 6 MS7 alleles with sizes ranging from 150 bp to 500 bp, but only two alleles were detected in cattle vaccinated >4 years earlier, relative to five alleles detected in recently vaccinated cattle and controls. In terms of heterozygosity, diversity was maximal in calves vaccinated within the last 2 weeks (h = 0.776) but lowest in cattle vaccinated 4 years earlier (h = 0.375). The analysis suggested close genetic relatedness of parasites in vaccinated and unvaccinated groups and up to 96% of variation was within rather than between the groups. These results confirm that ITM leads to a long-term T. parva carrier state in cattle and the detection of vaccine component VNTR in co-grazing unvaccinated cattle suggests potential vaccine transmission by ticks. However, vaccination stocks did not totally replace local genotypes, at least in cattle populations. These findings should mitigate concerns that ITM modifies T. parva field populations in a way that enhances disease in the medium term.

Current status of tropical theileriosis in Northern Africa: A review of recent epidemiological investigations and implications for control.

Tropical theileriosis caused by the apicomplexan hemoparasite Theileria annulata is a tick-borne disease that constraints livestock production in parts of Europe, Asia and Africa. Four Hyalomma tick species transmit T. annulata in at least eight Africa countries (Mauritania, Morocco, Algeria, Tunisia, Egypt, Sudan, South Sudan and Ethiopia). The two dominant T. annulata vector ticks present in Africa, H. scupense and H. anatolicum, underlie two different patterns of transmission, which in turn greatly influence the epidemiology of tropical theileriosis. H. dromedarii and H. lusitanicum are also capable of transmitting T. annulata in North Africa, but their roles are associated with specific production systems and agro-ecological contexts. The emergence of resistance to the most widely used theilericidal compound, buparvaquone, continues to limit the effectiveness of chemotherapy. In addition, acaricide use is increasingly becoming unsustainable. Deployable T. annulata attenuated live vaccines established from local strains in Tunisia, Sudan and Egypt are available, and recent work has indicated that these vaccines can be protective under conditions of natural transmission. However, vaccination programmes may vary over space and time due to differences in the prevalence of disease amongst cattle populations, as well seasonal variation in vector activity. We review recent descriptive and analytical surveys on the epidemiology of T. annulata infection with reference to (a) demographic aspects such as breeds and ages of cattle herds previously exposed to distinct T. annulata infection pressures and (b) seasonal dynamics of tick activity and disease transmission. We then discuss how the wider endemic patterns that we delineate can underpin the development and execution of future vaccination programmes. We also outline options for integrated control measures targeting tick vectors and husbandry practices.

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.

Antigen gene and variable number tandem repeat (VNTR) diversity in Theileria parva parasites from Ankole cattle in south-western Uganda: Evidence for conservation in antigen gene sequences combined with extensive polymorphism at VNTR loci.

Theileria parva is a tick-transmitted apicomplexan protozoan parasite that infects lymphocytes of cattle and African Cape buffalo (Syncerus caffer), causing a frequently fatal disease of cattle in eastern, central and southern Africa. A live vaccination procedure, known as infection and treatment method (ITM), the most frequently used version of which comprises the Muguga, Serengeti-transformed and Kiambu 5 stocks of T. parva, delivered as a trivalent cocktail, is generally effective. However, it does not always induce 100% protection against heterologous parasite challenge. Knowledge of the genetic diversity of T. parva in target cattle populations is therefore important prior to extensive vaccine deployment. This study investigated the extent of genetic diversity within T. parva field isolates derived from Ankole (Bos taurus) cattle in south-western Uganda using 14 variable number tandem repeat (VNTR) satellite loci and the sequences of two antigen-encoding genes that are targets of CD8+T-cell responses induced by ITM, designated Tp1 and Tp2. The findings revealed a T. parva prevalence of 51% confirming endemicity of the parasite in south-western Uganda. Cattle-derived T. parva VNTR genotypes revealed a high degree of polymorphism. However, all of the T. parva Tp1 and Tp2 alleles identified in this study have been reported previously, indicating that they are widespread geographically in East Africa and highly conserved.

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.

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.

A review of recent research on Theileria parva: Implications for the infection and treatment vaccination method for control of East Coast fever.

The infection and treatment (ITM) live vaccination method for control of Theileria parva infection in cattle is increasingly being adopted, particularly in Maasai pastoralist systems. Several studies indicate positive impacts on human livelihoods. Importantly, the first detailed protocol for live vaccine production at scale has recently been published. However, quality control and delivery issues constrain vaccination sustainability and deployment. There is evidence that the distribution of T. parva is spreading from endemic areas in East Africa, North into Southern Sudan and West into Cameroon, probably as a result of anthropogenic movement of cattle. It has also recently been demonstrated that in Kenya, T. parva derived from cape buffalo can 'breakthrough' the immunity induced by ITM. However, in Tanzania, breakthrough has not been reported in areas where cattle co-graze with buffalo. It has been confirmed that buffalo in northern Uganda national parks are not infected with T. parva and R. appendiculatus appears to be absent, raising issues regarding vector distribution. Recently, there have been multiple field population genetic studies using variable number tandem repeat (VNTR) sequences and sequencing of antigen genes encoding targets of CD8+ T-cell responses. The VNTR markers generally reveal high levels of diversity. The antigen gene sequences present within the trivalent Muguga cocktail are relatively conserved among cattle transmissible T. parva populations. By contrast, greater genetic diversity is present in antigen genes from T. parva of buffalo origin. There is also evidence from several studies for transmission of components of stocks present within the Muguga cocktail, into field ticks and cattle following induction of a carrier state by immunization. In the short term, this may increase live vaccine effectiveness, through a more homogeneous challenge, but the long-term consequences are unknown.

Comparison of protectiveness of recombinant Babesia ovis apical membrane antigen 1 and B. ovis-infected cell line as vaccines against ovine babesiosis.

Babesiosis is a disease complex caused by unicellular Babesia parasites and among them, malignant ovine babesiosis caused by B. ovis has a devastating economical impact on the small ruminant industry. The control of disease is mainly based on chemotherapy and preventing animals from tick infestation and to date no vaccine is available against ovine babesiosis. The requirement for vaccination against B. ovis infection in endemically unstable regions is necessary for implementation of effective disease control measures. The aim of the present study was to evaluate the effectiveness of different immunisation protocols against disease in sheep experimentally vaccinated with recombinant B. ovis apical membrane antigen-1 (rBoAMA-1) and/or live, a B. ovis-infected cell line. Sheep were divided into four experimental groups, plus a control group. Animals were immunised either with the B. ovis stabilate, or with rBoAMA-1, or with both rBoAMA-1 and the B. ovis stabilate. Western blots and ELISAs indicated that immunisation with rBoAMA-1 resulted in generation of a specific response against the recombinant protein, but the degree of antibody response did not correlate with the level of induced protection against challenge. The strongest immune response was induced in animals co-immunised with the live B. ovis stabilate plus rBoAMA-1. Both the hematological and parasitological findings indicated that this co-immunisation regimen has vaccine potential to limit losses incurred by ovine babesiosis in endemic countries.

Epidemiological study on tropical theileriosis (Theileria annulata infection) in the Egyptian Oases with special reference to the molecular characterization of Theileria spp.

Theileria annulata infection is a tick-borne disease known as Egyptian fever since 1947. It is a destructive obstacle for the livestock production in the Egyptian Oases (EL-Wady EL-Geded Province). The present study was conducted on 1068 cattle, ranged from below one year to more than eight years old; belonged to different farms and villages in EL-Wady EL-Geded Province. The infection was confirmed by blood smears, Tams-1 target based polymerase chain reaction (Tams-1 PCR), 18Ss rRNA polymerase chain reaction and semi nested-polymerase chain reaction (nPCR) followed by DNA sequencing and phylogenetic analyses, in addition to tick identification. Molecular techniques confirmed the infection in 63.6% (679/1068) of the examined animals while Giemsa-stained blood smears confirmed it in 36.8% (393/1062). Male and female animals showed molecular confirmed infection rates of 64.5 and 62.7%, respectively. Animals less than one year old were more infected (83.33%, 400/480) followed by animals less than three years (57.31%, 149/260) and animals less than five years (42.45%, 90/212), respectively. On the other hand, animal of five years old or above were less infected and the infection rate in this group was estimated to be 34.48% (40/116). Two tick species were identified during the present study: Hyalomma anatolicum and Rhipicephalus annulatus. Theileria annulata was the only Theileria species found in the Egyptian oases in respect to phylogenetic analysis of the obtained sequences.

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.

A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic® cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex® resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent.

Assessment of the First Commercial ELISA Kit for the Diagnosis of Theileria annulata.

The present study assesses the efficacy of SVANOVIR Theileria annulata-Ab, the first commercial ELISA kit for the diagnosis of Theileria annulata infection in cattle based on a recombinant protein known as T. annulata surface protein (TaSp). As a reference test, a polymerase chain reaction (PCR) assay depending on T. annulata merozoite surface antigen (Tams-1) was applied. A total of 468 blood samples as well as serum samples were randomly collected from cattle and tested in the PCR as well as in the ELISA developed in this study. Moreover, all samples were also analyzed by conventional Giemsa-stained blood smear. The results of this study revealed a good correlation between the results obtained by PCR and the ELISA, whereas all PCR positive samples scored correctly positive in the ELISA and 73 of the 125 PCR negative samples scored correctly negative. Taken together, a sensitivity of 91.25% and a specificity of 78.4% were recorded, when compared to the PCR data. In conclusion, the SVANOVIR Theileria annulata-Ab is a suitable diagnostic assay for use in the diagnosis and epidemiological surveys of Theileria annulata infection in chronic and carrier animals.

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.

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.

Development and laboratory evaluation of a lateral flow device (LFD) for the serodiagnosis of Theileria annulata infection.

Several DNA-based and serological tests have been established for the detection of Theileria annulata infection, including polymerase chain reaction, reverse line blot and loop-mediated isothermal amplification, indirect enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. In this study, we have applied knowledge from the development and application of a recombinant protein-based indirect ELISA and competitive ELISA to establish a rapid test for point-of-care diagnosis of T. annulata infection in the field to be used by the veterinarian. For the development of a lateral flow test, the recombinantly expressed T. annulata surface protein (TaSP) was applied as the test antigen and anti-TaSP antiserum as the control line. TaSP antigen conjugated to colloidal gold particles was used as the detection system for visualization at the test line for the binding of anti-TaSP antibody present in the serum of infected animals. The developed test specifically detected antibodies in the serum of animals experimentally infected with T. annulata and showed no cross-reactivity with serum from animals infected with other tested bovine pathogens (Trypanosoma brucei, Anaplasma marginale, Babesia bigemina, Babesia bovis, and Theileria parva). Testing of field samples was compared to results obtained by other serological tests, resulting in a sensitivity and specificity of 96.3% and 87.5% compared to indirect fluorescence antibody test, 98.7% and 81.8% compared to indirect ELISA, and 100% and 47.6% compared to competitive ELISA. In conclusion, a rapid test for the detection of T. annulata infection (T. annulata lateral flow device, Ta-LFD) has been developed, which is easy to perform, delivers results to be read by the naked eye within 10 min, and is suitable for the detection of infection in field samples.

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.

Identification of Theileria uilenbergi immunodominant protein for development of an indirect ELISA for diagnosis of ovine theileriosis.

Theileriosis of small ruminants in the northwest of China is a protozoan disease that restricts the development of the livestock industry. The disease is caused by infection with Theileria uilenbergi and Theilerialuwenshuni, both of which are transmitted by ixodid Heamaphysalis ticks. The development of serological tools as a means of integrated control of the disease is an urgent and important requirement. Here we describe the identification and partial recombinant expression of a T.uilenbergi immunodominant protein (TuIP), which was identified by immunoscreening of a merozoite cDNA library. Using the recombinant TuIP (rTuIP), a novel indirect ELISA was established using 329 negative serum samples to determine the cut-off value. The internal quality control revealed satisfactory stability and repeatability of the assay. Preliminary validation using 128 positive and 48 negative reference samples demonstrated that the rTuIP ELISA is able to detect T. uilenbergi infection with high sensitivity and specificity. No cross-reactivity was found in sera from animals infected with Theileria lestoquardi, Babesia sp. China or Anaplasma ovis. Furthermore, circulating antibodies were detected in sera collected from endemic regions in China. Analyses of the antibody responses of experimentally infected animals demonstrated that tick infestation resulted in a sharply rising and stronger production of specific antibodies against TuIP while inoculation with infected blood induced an earlier production of TuIP-specific antibodies. The persistence of the TuIP-specific antibodies lasted more than 100days p.i. These data indicate the usefulness of the TuIP antigen for the development of diagnostic methods and as a potential candidate for vaccine design.