RESUMO
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.
Assuntos
Proteína Quinase CDC2/metabolismo , Doenças dos Bovinos/prevenção & controle , Proteínas de Protozoários/metabolismo , Theileria annulata/imunologia , Theileriose/prevenção & controle , Motivos de Aminoácidos , Animais , Proteína Quinase CDC2/antagonistas & inibidores , Bovinos , Doenças dos Bovinos/parasitologia , Proliferação de Células , Ensaio de Imunoadsorção Enzimática/veterinária , Fosforilação , Purinas/farmacologia , Esquizontes , Theileria annulata/metabolismo , Theileriose/parasitologiaRESUMO
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.
Assuntos
Anticorpos Antiprotozoários/sangue , Proteínas de Protozoários/imunologia , Doenças dos Ovinos/epidemiologia , Theileria/imunologia , Theileriose/epidemiologia , Animais , China/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Proteínas Recombinantes , Reprodutibilidade dos Testes , Ruminantes , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Theileria/genética , Theileriose/imunologia , Theileriose/parasitologiaRESUMO
A growing number of studies are reporting simultaneous infections by parasites in many different hosts. The detection of whether these parasites are significantly associated is important in medicine and epidemiology. Numerous approaches to detect associations are available, but only a few provide statistical tests. Furthermore, they generally test for an overall detection of association and do not identify which parasite is associated with which other one. Here, we developed a new approach, the association screening approach, to detect the overall and the detail of multi-parasite associations. We studied the power of this new approach and of three other known ones (i.e., the generalized chi-square, the network and the multinomial GLM approaches) to identify parasite associations either due to parasite interactions or to confounding factors. We applied these four approaches to detect associations within two populations of multi-infected hosts: (1) rodents infected with Bartonella sp., Babesia microti and Anaplasma phagocytophilum and (2) bovine population infected with Theileria sp. and Babesia sp. We found that the best power is obtained with the screening model and the generalized chi-square test. The differentiation between associations, which are due to confounding factors and parasite interactions was not possible. The screening approach significantly identified associations between Bartonella doshiae and B. microti, and between T. parva, T. mutans, and T. velifera. Thus, the screening approach was relevant to test the overall presence of parasite associations and identify the parasite combinations that are significantly over- or under-represented. Unraveling whether the associations are due to real biological interactions or confounding factors should be further investigated. Nevertheless, in the age of genomics and the advent of new technologies, it is a considerable asset to speed up researches focusing on the mechanisms driving interactions between parasites.
Assuntos
Modelos Estatísticos , Parasitos , Doenças Parasitárias/parasitologia , Algoritmos , Animais , Coinfecção/parasitologia , Simulação por Computador , Estudos Transversais , HumanosRESUMO
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.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Parasitologia/métodos , Theileria/isolamento & purificação , Theileriose/diagnóstico , Medicina Veterinária/métodos , Animais , Primers do DNA/genética , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/diagnóstico , Doenças dos Ovinos/parasitologia , Sudão , Theileria/genética , Theileriose/parasitologiaRESUMO
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.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Doenças dos Bovinos/diagnóstico , Parasitologia/métodos , Theileria annulata/imunologia , Theileriose/diagnóstico , Animais , Antígenos de Protozoários , Bovinos , Doenças dos Bovinos/parasitologia , Parasitologia/normas , Sistemas Automatizados de Assistência Junto ao Leito/normas , Proteínas Recombinantes , Padrões de Referência , Sensibilidade e Especificidade , Testes Sorológicos/métodos , Testes Sorológicos/normas , Theileria annulata/isolamento & purificação , Theileriose/parasitologiaRESUMO
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.
Assuntos
Antígenos de Protozoários/química , Doenças dos Ovinos/diagnóstico , Theileria/imunologia , Theileriose/diagnóstico , Animais , Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , China , Biologia Computacional/métodos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Epitopos Imunodominantes/química , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Testes Sorológicos , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Theileria/classificação , Theileria/genética , Theileria/crescimento & desenvolvimento , Theileriose/imunologia , Theileriose/parasitologiaRESUMO
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.
Assuntos
Proteínas de Membrana/metabolismo , Microtúbulos/metabolismo , Mapeamento de Interação de Proteínas , Proteínas de Protozoários/metabolismo , Esquizontes/fisiologia , Theileria annulata/patogenicidade , Animais , Células COS , Chlorocebus aethiops , Imunoprecipitação , Microscopia Confocal , Ligação Proteica , Fuso Acromático/metabolismo , Fuso Acromático/parasitologia , Tubulina (Proteína)/metabolismoRESUMO
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.
Assuntos
Antígenos de Protozoários , Parasitologia/métodos , Proteínas de Protozoários , Doenças dos Ovinos/diagnóstico , Theileria/química , Theileriose/diagnóstico , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , DNA de Protozoário/química , DNA de Protozoário/genética , Ensaio de Imunoadsorção Enzimática/métodos , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA , Ovinos , Doenças dos Ovinos/parasitologia , Theileria/imunologia , Theileriose/parasitologiaRESUMO
In an ad hoc survey conducted during 2006, the epidemiology of tropical theileriosis in Kurdistan Region, Iraq, was addressed. For this purpose, a total of 299 blood samples were collected from female cattle older than 1 year reared under open system management in Duhok (n = 99), Sulaimanyia (n = 100) and Erbil (n = 100) governorates. The samples were subjected to TaSP indirect ELISA as well as polymerase chain reaction (PCR) and nested PCR assays. The results indicated that the seroprevalence was 77.9%, and PCR reported an infection rate of 68.9% in the Kurdistan Region of Iraq. The implication of the results in the epidemiology of tropical theileriosis in the region is discussed with emphasis on comparisons between the two tests used and recommendations for the future work are outlined.
Assuntos
Doenças dos Bovinos/epidemiologia , Theileria annulata/isolamento & purificação , Theileriose/epidemiologia , Animais , Bovinos , DNA de Helmintos/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Iraque/epidemiologia , Reação em Cadeia da Polimerase/métodos , Prevalência , Estudos SoroepidemiológicosRESUMO
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.
Assuntos
Anticorpos Monoclonais , Anticorpos Antiprotozoários , Antígenos de Protozoários , Doenças dos Bovinos/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Theileria annulata/isolamento & purificação , Theileriose/diagnóstico , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Feminino , Iraque , Prevalência , Sensibilidade e Especificidade , Theileria annulata/imunologia , Theileriose/epidemiologiaRESUMO
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid detection method in which the target deoxyribonucleic acid (DNA) can be efficiently amplified with high specificity and sensitivity under isothermal conditions using a set of either four or six specific primers. In this study, we have identified a conserved sequence for Theileria luwenshuni (UTRlu8) and for T. uilenbergi (UTRu6) suitable for designing a set of six primers for the simultaneous detection by LAMP of these pathogens causing theileriosis in sheep and goats in China. LAMP was performed at 63 degrees C, and the amplified DNA was detectable within 15 min. The specificity of the reaction was confirmed through EcoRI restriction enzyme digestion analysis and sequencing. The assay was proven sensitive since specific amplification was obtained from 0.1 pg DNA of T. luwenshuni or T. uilenbergi. The LAMP assay was evaluated by testing 86 field samples in comparison to the reverse line blot method, showing a sensitivity and specificity of 66.0% and 97.4%, respectively. These results indicate that the LAMP assay is rapid and simple to run, cost effective, sensitive, and specific and has potential usefulness for application in diagnostics of and epidemiological studies on T. luwenshuni and T. uilenbergi infection of small ruminants.
Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Doenças dos Ovinos/diagnóstico , Theileria/isolamento & purificação , Theileriose/diagnóstico , Animais , China , Primers do DNA , Microscopia Eletrônica de Transmissão , Sensibilidade e Especificidade , Ovinos , Doenças dos Ovinos/patologia , Theileria/classificação , Theileria/genética , Theileriose/parasitologia , Fatores de TempoRESUMO
The intracellular protozoan parasite Theileria annulata causes a severe, and often fatal, disease of pure and cross-bred cattle in tropical and subtropical countries. The present review refers to the importance of innate immunity as far as it is known to date in this infectious disease. Specifically, macrophages and the mediators produced by these cells are outlined. In addition, the latest findings concerning cattle breed differences in susceptibility to T. annulata infection in relation to macrophage activation are discussed.
Assuntos
Imunidade Inata , Ativação de Macrófagos , Theileria annulata , Theileriose/imunologia , Animais , Bovinos , Proliferação de Células , Predisposição Genética para Doença , Interações Hospedeiro-Parasita , Fatores Imunológicos/metabolismo , Ativação de Macrófagos/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Modelos Imunológicos , Óxido Nítrico/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Especificidade da Espécie , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/parasitologia , Theileriose/genética , Theileriose/fisiopatologia , Theileriose/prevenção & controleAssuntos
Babesiose/prevenção & controle , Hidropericárdio/prevenção & controle , Theileriose/prevenção & controle , Doenças Transmitidas por Carrapatos/prevenção & controle , Vacinas Atenuadas/imunologia , Animais , Animais Domésticos , Babesiose/imunologia , Babesiose/parasitologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Ehrlichia ruminantium , Hidropericárdio/imunologia , Hidropericárdio/microbiologia , Vacinas Protozoárias/administração & dosagem , Vacinas Protozoárias/imunologia , Theileria/classificação , Theileria/imunologia , Theileriose/imunologia , Theileriose/parasitologia , Doenças Transmitidas por Carrapatos/imunologia , Doenças Transmitidas por Carrapatos/microbiologia , Doenças Transmitidas por Carrapatos/parasitologia , Vacinas Atenuadas/administração & dosagemRESUMO
In this study potential molecular markers for identification of attenuation in a Theileria lestoquardi-infected cell line to be used in vaccination trials were identified. Two markers associated with attenuation in Theileria annulata vaccine strains were analyzed (metalloproteinase activity and TNF? mRNA expression). The result showed a decreased activity of MMP 9 and decreased mRNA expression of TNF? with increasing passage number. Suppression subtractive hybridization was used to identify potential new markers of attenuation. Random screening revealed nine differentially expressed genes, one from the parasite and eight from the host. Quantitative real time-PCR confirmed mRNA expression of the parasite vacuolar H+ATPase to be downregulated at higher passages.
Assuntos
Regulação da Expressão Gênica , Metaloproteinase 9 da Matriz/metabolismo , Proteínas de Protozoários/genética , Theileria/crescimento & desenvolvimento , Theileria/patogenicidade , Fator de Necrose Tumoral alfa/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Bovinos , Linhagem Celular , Interações Hospedeiro-Patógeno , Metaloproteinase 9 da Matriz/genética , Proteínas de Protozoários/metabolismo , Inoculações Seriadas , Ovinos , Fator de Necrose Tumoral alfa/genética , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Cattle which survive an infection with Theileria annulata become effectively immune to challenge with the same parasite strain, and are thought to be protected against a heterologous strain of the parasite. T-cells play a crucial role in both induction and maintenance of immunity to T. annulata. The generation of cytotoxic T lymphocytes (CTL) is closely related to the control of the infection - macroschizont-infected cells are killed in an MHC class I restricted manner. Any strain-specificity induced by immunisation is likely to be manifested by CTL. Besides CTLs, CD4+ T-cells also play an important role in protective immunity to T. annulata infection.
Assuntos
Vacinas Protozoárias/imunologia , Linfócitos T Citotóxicos/imunologia , Theileria annulata/imunologia , Theileriose/imunologia , Animais , Bovinos , Citotoxicidade Imunológica , Ativação Linfocitária , Vacinas Protozoárias/administração & dosagem , Theileriose/parasitologia , Theileriose/prevenção & controle , VacinaçãoRESUMO
Sheep and goats in northwest China suffer from theileriosis from infection with Theileria sp. (China), resulting in large economic losses. To investigate the immune response to infection with Theileria sp. (China), parameters of cellular and humoral immunity of experimentally infected sheep against two recombinantly expressed Theileria proteins were investigated. The in vitro proliferative response of blood mononuclear cells to a recombinant T. annulata membrane protein and a recombinant Theileria sp. (China) homologue to T. annulata surface protein, both putative membrane proteins, was significantly elevated and significant amounts of specific immunoglobulins were produced against both.
Assuntos
Proteínas de Protozoários/imunologia , Ovinos/imunologia , Theileriose/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/sangue , Proteínas Recombinantes/imunologiaRESUMO
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.
Assuntos
Genes de Protozoários , Splicing de RNA , RNA Mensageiro/genética , Theileria/genética , Animais , Sequência de Bases , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Theileria/classificaçãoRESUMO
Recently, Theileria sp. (China) has been designated as T. luwenshuni[formerly Theileria sp. (China 1)] and T. uilenbergi[formerly Theileria sp. (China 2)]. A cDNA library of T. uilenbergi merozoites was constructed and subjected to random sequencing. Among the obtained sequences were three highly identical cDNA clones, indicating a gene family. Bioinformatic analyses indicated these genes contain signal peptides and encode potential immunogenic proteins. The presence of tandemly arranged and additional variants of these genes was shown. Analysis of one recombinantly expressed clone revealed immunoreactivity for serum from Theileria-infected animals. No cross-reaction with serum of T. lestoquardi-, Babesia motasi-, or Anaplasma ovis-infected animals was observed, indicating a potential antigen for development of serological diagnostic tools.
Assuntos
Antígenos de Protozoários/genética , Etiquetas de Sequências Expressas , Genes de Protozoários , Theileria/genética , Animais , DNA Complementar , Theileria/imunologiaRESUMO
The polymorphic region of the Theileria annulata surface protein (TaSP) was cloned and sequenced from different isolates of cattle and cell lines from different areas of Sudan. Amino acid sequence alignment revealed a high diversity showing amino acid and length polymorphism, both within and between parasite isolates. The generation of TaSP diversity may allow the evasion of host immunity by the parasite since TaSP is a highly antigenic parasite protein.