RESUMO
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.
Assuntos
Doenças dos Bovinos/parasitologia , Theileria annulata/crescimento & desenvolvimento , Theileria parva/crescimento & desenvolvimento , Theileriose/parasitologia , Animais , Bovinos , Linhagem Celular , Meios de Cultura Livres de Soro , Leucócitos/imunologia , Leucócitos/parasitologia , Linfócitos/imunologia , Linfócitos/parasitologia , Esquizontes , Theileria annulata/imunologia , Theileria parva/imunologiaRESUMO
The protozoan parasites Theileria annulata and Theileria parva are unique amongst intracellular eukaryotic pathogens as they induce a transformation-like phenotype in their bovine host cell. T. annulata causes tropical theileriosis, which is frequently fatal, with infected leukocytes becoming metastatic and forming foci in multiple organs resulting in destruction of the lymphoid system. Exosomes, a subset of extracellular vesicles (EV), are critical in metastatic progression in many cancers. Here, we characterised the cargo of EV from a control bovine lymphosarcoma cell line (BL20) and BL20 infected with T. annulata (TBL20) by comparative mass spectrometry and microRNA (miRNA) profiling (data available via ProteomeXchange, identifier PXD010713 and NCBI GEO, accession number GSE118456, respectively). Ingenuity pathway analysis that many infection-associated proteins essential to migration and extracellular matrix digestion were upregulated in EV from TBL20 cells compared with BL20 controls. An altered repertoire of host miRNA, many with known roles in tumour and/or infection biology, was also observed. Focusing on the tumour suppressor miRNA, bta-miR-181a and bta-miR-181b, we identified putative messenger RNA targets and confirmed the interaction of bta-miR181a with ICAM-1. We propose that EV and their miRNA cargo play an important role in the manipulation of the host cell phenotype and the pathobiology of Theileria infection.
Assuntos
Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Leucócitos/metabolismo , Leucócitos/parasitologia , MicroRNAs/análise , Proteínas/análise , Theileria annulata/crescimento & desenvolvimento , Animais , Bovinos , Linhagem CelularRESUMO
Constitutive c-Jun N-terminal kinase (JNK) activity characterizes bovine T and B cells infected with Theileria parva, and B cells and macrophages infected with Theileria annulata. Here, we show that T. annulata infection of macrophages manipulates JNK activation by recruiting JNK2 and not JNK1 to the parasite surface, whereas JNK1 is found predominantly in the host cell nucleus. At the parasite's surface, JNK2 forms a complex with p104, a GPI-(GlycosylPhosphatidylInositol)-anchor T. annulata plasma membrane protein. Sequestration of JNK2 depended on Protein Kinase-A (PKA)-mediated phosphorylation of a JNK-binding motif common to T. parva and a cell penetrating peptide harbouring the conserved p104 JNK-binding motif competitively ablated binding, whereupon liberated JNK2 became ubiquitinated and degraded. Cytosolic sequestration of JNK2 suppressed small mitochondrial ARF-mediated autophagy, whereas it sustained nuclear JNK1 levels, c-Jun phosphorylation, and matrigel traversal. Therefore, T. annulata sequestration of JNK2 contributes to both survival and dissemination of Theileria-transformed macrophages.
Assuntos
Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Macrófagos/parasitologia , Proteínas de Membrana/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteínas de Protozoários/metabolismo , Theileria annulata/crescimento & desenvolvimento , Animais , Macrófagos/imunologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Modelos Teóricos , Ligação Proteica , Theileria annulata/metabolismo , Theileriose/parasitologia , Theileriose/patologiaRESUMO
The aim of this study was to identify the cell surface cluster of differentiation (CD) markers of the cell lines infected by Theileria annulata schizont. The CD molecules are very useful for the characterization of cells and different subpopulations of leukocytes. They are usually recognized by specific antibodies using flow cytometry and immunohistochemistry. In the current study, we applied reverse transcriptase-polymerase chain reaction (RT-PCR) to define the profile of cell surface markers in a cell line infected by an attenuated S15 vaccine strain of T. annulata schizont and a new laboratory-established cell line infected by a non-attenuated form. In order to determine the specific markers that can be used for excluding the non-attenuated cell lines, the characterization of the surface proteins profile of the S15 vaccine cell line is important. The RT-PCR was carried out by specifically designed primers using a panel of seven bovine CD markers, as well as beta-actin as an internal control house-keeping gene. We showed that both of the examined cell lines had a consistent expression of CD4, CD5, CD11a, CD14, CD43, and CD45 markers. However, the specific finding in this study was the expression of B-cell markers CD79a and CD5 by the newly-transformed cell line. On the other hand, CD5 as a marker for B-cell subset was expressed by S15 vaccine strain. In conclusion, we consider CD79a surface protein as a new marker for the cell lines infected by non-attenuated T. annulata schizont, while the cell lines infected by the vaccine strain do not express this marker. In addition, the identification of CD marker expression based on the RT-PCR assay might be a suitable and appropriate alternative technique for flow cytometry.
Assuntos
Antígenos CD/análise , Vacinas Protozoárias/imunologia , Theileria annulata/imunologia , Theileriose/prevenção & controle , Animais , Linhagem Celular , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Esquizontes/imunologia , Theileria annulata/crescimento & desenvolvimento , Vacinas Atenuadas/imunologiaRESUMO
Theileria annulata is an apicomplexan parasite that modifies the phenotype of its host cell completely, inducing uncontrolled proliferation, resistance to apoptosis, and increased invasiveness. The infected cell thus resembles a cancer cell, and changes to various host cell signalling pathways accompany transformation. Most of the molecular mechanisms leading to Theileria-induced immortalization of leukocytes remain unknown. The parasite dissolves the surrounding host cell membrane soon after invasion and starts interacting with host proteins, ensuring its propagation by stably associating with the host cell microtubule network. By using BioID technology together with fluorescence microscopy and co-immunoprecipitation, we identified a CLASP1/CD2AP/EB1-containing protein complex that surrounds the schizont throughout the host cell cycle and integrates bovine adaptor proteins (CIN85, 14-3-3 epsilon, and ASAP1). This complex also includes the schizont membrane protein Ta-p104 together with a novel secreted T. annulata protein (encoded by TA20980), which we term microtubule and SH3 domain-interacting protein (TaMISHIP). TaMISHIP localises to the schizont surface and contains a functional EB1-binding SxIP motif, as well as functional SH3 domain-binding Px(P/A)xPR motifs that mediate its interaction with CD2AP. Upon overexpression in non-infected bovine macrophages, TaMISHIP causes binucleation, potentially indicative of a role in cytokinesis.
Assuntos
Interações Hospedeiro-Patógeno , Macrófagos/parasitologia , Proteínas de Protozoários/metabolismo , Theileria annulata/crescimento & desenvolvimento , Animais , Bovinos , Células Cultivadas , Imunoprecipitação , Microscopia de Fluorescência , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
BACKGROUND: Differentiation of one life-cycle stage to the next is critical for survival and transmission of apicomplexan parasites. A number of studies have shown that stage differentiation is a stochastic process and is associated with a point that commits the cell to a change over in the pattern of gene expression. Studies on differentiation to merozoite production (merogony) in T. annulata postulated that commitment involves a concentration threshold of DNA binding proteins and an auto-regulatory loop. PRINCIPAL FINDINGS: In this study ApiAP2 DNA binding proteins that show changes in expression level during merogony of T. annulata have been identified. DNA motifs bound by orthologous domains in Plasmodium were found to be enriched in upstream regions of stage-regulated T. annulata genes and validated as targets for the T. annulata AP2 domains by electrophoretic mobility shift assay (EMSA). Two findings were of particular note: the gene in T. annulata encoding the orthologue of the ApiAP2 domain in the AP2-G factor that commits Plasmodium to gametocyte production, has an expression profile indicating involvement in transmission of T. annulata to the tick vector; genes encoding related domains that bind, or are predicted to bind, sequence motifs of the type 5'-(A)CACAC(A) are implicated in differential regulation of gene expression, with one gene (TA11145) likely to be preferentially up-regulated via auto-regulation as the cell progresses to merogony. CONCLUSIONS: We postulate that the Theileria factor possessing the AP2 domain orthologous to that of Plasmodium AP2-G may regulate gametocytogenesis in a similar manner to AP2-G. In addition, paralogous ApiAP2 factors that recognise 5'-(A)CACAC(A) type motifs could operate in a competitive manner to promote reversible progression towards the point that commits the cell to undergo merogony. Factors possessing AP2 domains that bind (or are predicted to bind) this motif are present in the vector-borne genera Theileria, Babesia and Plasmodium, and other Apicomplexa; leading to the proposal that the mechanisms that control stage differentiation will show a degree of conservation.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Merozoítos/crescimento & desenvolvimento , Proteínas de Protozoários/genética , Theileria annulata/genética , Proteínas de Ligação a DNA/metabolismo , Merozoítos/metabolismo , Proteínas de Protozoários/metabolismo , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/metabolismoRESUMO
Within 2 h of infection by Theileria annulata sporozoites, bovine macrophages display a two- to fourfold increase in transcription of hypoxia inducible factor (HIF-1α). Twenty hours post-invasion sporozoites develop into multi-nucleated macroschizonts that transform the infected macrophage into an immortalized, permanently proliferating, hyper-invasive and disease-causing leukaemia-like cell. Once immortalized Theileria-infected leukocytes can be propagated as cell lines and even though cultivated under normoxic conditions, both infected B cells and macrophages display sustained activation of HIF-1α. Attenuated macrophages used as live vaccines against tropical theileriosis also display HIF-1α activation even though they have lost their tumorigenic phenotype. Here, we review data that ascribes HIF-1α activation to the proliferation status of the infected leukocyte and discuss the possibility that Theileria may have lost its ability to render its host macrophage virulent due to continuous parasite replication in a high Reactive Oxygen Species (ROS) environment. We propose a model where uninfected macrophages have low levels of H2 O2 output, whereas virulent-infected macrophages produce high amounts of H2 O2 . Further increase in H2 O2 output leads to dampening of infected macrophage virulence, a characteristic of disease-resistant macrophages. At the same time exposure to H2 O2 sustains HIF-1α that induces the switch from mitochondrial oxidative phosphorylation to Warburg glycolysis, a metabolic shift that underpins uncontrolled infected macrophage proliferation. We propose that as macroschizonts develop into merozoites and infected macrophage proliferation arrests, HIF-1α levels will decrease and glycolysis will switch back from Warburg to oxidative glycolysis. As Theileria infection transforms its host leukocyte into an aggressive leukaemic-like cell, we propose that manipulating ROS levels, HIF-1α induction and oxidative over Warburg glycolysis could contribute to improved disease control. Finally, as excess amounts of H2 O2 drive virulent Theileria-infected macrophages towards attenuation it highlights how infection-induced pathology and redox balance are intimately linked.
Assuntos
Glicólise , Interações Hospedeiro-Patógeno , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leucócitos/metabolismo , Leucócitos/parasitologia , Theileria annulata/crescimento & desenvolvimento , Animais , Bovinos , Proliferação de Células , Peróxido de Hidrogênio/metabolismo , Modelos BiológicosRESUMO
There are some limiting aspects of scaling up the Neospora caninum tachyzoites in continuous cell lines, particularly as related to the absence of surface attachment. In this study, suspension cell culture of Theileria annulata-infected lymphoblastoid (TIL) was used as a host cell for the continous production of N. caninum tachyzoites. The numbers of free tachyzoites in the medium supernatant were showed regularly increased up to the day 6 post-cultivation. Transmission electron microscopy demonstrated that N. caninum tachyzoites invaded the TIL cells and multiplied intracellularly. This showed that the tachyzoites were successfully proliferated in TIL cells and were released in complete Dulbecco's modified Eagle's medium. This is a successful report of in vitro cultivation of N. caninum tachyzoites achieved by using suspension host cell culture.
Assuntos
Neospora/crescimento & desenvolvimento , Parasitologia/métodos , Theileria annulata/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Linhagem Celular , Meios de Cultura/química , Humanos , Linfócitos/parasitologia , Microscopia Eletrônica de Transmissão , Neospora/ultraestrutura , Carga Parasitária , Fatores de TempoRESUMO
The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.
Assuntos
Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Esquizontes/metabolismo , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/metabolismo , Theileriose/parasitologia , Animais , Bovinos , Espectrometria de Massas , Dados de Sequência Molecular , Proteômica , Proteínas de Protozoários/genética , Esquizontes/química , Esquizontes/crescimento & desenvolvimento , Theileria annulata/química , Theileria annulata/genéticaRESUMO
We have evaluated a new simple technique using whole blood from experimentally infected cattle for the isolation and cultivation of Theileria annulata. The study was carried out on 20 Holstein-Frisian bovines that had been experimentally infected with a virulent lethal dose of Theileria annulata. This technique has been compared to the classical peripheral blood monocyte isolation with Ficoll carried out on 22 experimentally infected Holstein-Friesian calves. The effectiveness of the reference technique was estimated to 86.4%, whilst the effectiveness of the new technique was 100%. Moreover, this new technique leads to time and money saving estimated to 3.06 per sample. It decreases the contamination risks by reducing the steps of sample manipulation.
Assuntos
Doenças dos Bovinos/parasitologia , Técnicas de Cultura/veterinária , Parasitemia/parasitologia , Theileria annulata/isolamento & purificação , Theileriose/parasitologia , Animais , Bovinos , Doenças dos Bovinos/sangue , Técnicas de Cultura/economia , Técnicas de Cultura/métodos , Técnicas de Cultura/normas , Citocinas/metabolismo , Ficoll , Linfócitos/imunologia , Parasitologia/economia , Parasitologia/métodos , Parasitologia/normas , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/imunologia , Theileriose/sangue , Fatores de TempoRESUMO
This study was carried out in two observational clinical studies. Study 1 comprised 50 adult crossbred cattle naturally infected by Theileria annulata. Infected animals were divided into 4 subgroups with different parasitaemia (<1%, 1-3%, 3-5% and >5%). Study 2 comprised 20 adult crossbred cattle naturally infected by Anaplasma marginale. Infected animals were divided into 3 subgroups with different parasitaemia (<10%, 10-20% and 20-30%). In study 1, a significant negative correlation (P<0.001) was observed between parasitaemia and superoxide dismutase (SOD). Positive correlations (P<0.001) were observed between parasitaemia and lactate dehydrogenase (LDH) and mean corpuscular fragility (MCF). In study 2 positive correlations (P<0.05) were observed among parasitaemia and MCF and LDH activity. SOD activity had a negative correlation with parasitaemia in cattle with parasitaemia lower than 10% but no significant correlation (P>0.05) was observed between SOD activity and parasitaemia in cattle with 10-20 and 20-30% parasitaemia. In comparison of both studies we came to the conclusion that in theileriosis as the severity of disease increased the anaemia, MCF and LDH activity increased and SOD activity decreased at any parasitaemia, but in anaplasmosis the anaemia, MCF and LDH activity increased at any parasitaemia but SOD activity decreased only in early but not in advanced stages of disease.
Assuntos
Anaplasma marginale/crescimento & desenvolvimento , Anaplasmose/parasitologia , Doenças dos Bovinos/parasitologia , Parasitemia/veterinária , Theileria annulata/crescimento & desenvolvimento , Theileriose/parasitologia , Anaplasmose/enzimologia , Anaplasmose/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/enzimologia , Doenças dos Bovinos/epidemiologia , Contagem de Eritrócitos/veterinária , Hematócrito/veterinária , Hemoglobinas/metabolismo , Irã (Geográfico)/epidemiologia , L-Lactato Desidrogenase/sangue , Parasitemia/enzimologia , Parasitemia/epidemiologia , Parasitemia/parasitologia , Superóxido Dismutase/sangue , Theileriose/enzimologia , Theileriose/epidemiologiaRESUMO
The purpose of this study was to determine serum ADA activity in cattle naturally infected with Theileria annulata. In this study, a total of 37 cross-bred cattle which 27 of it showing clinical signs of theileriosis constituted infected group and 10 healthy cattle as control group were used as animal materials. Infected group divided into three groups according to their PCV values. Cattle with PCV > or = 25 were put on group I (n = 9), those with PCV 13-24 were put on group II (n = 11) and those with PCV < or = 12 were put on group III (n = 7). Microscopical diagnosis of the disease was also made. Hematological parameters, serum enzyme activities (ADA, AST, ALT and ALP) were determined in all cattle. Hematological results revealed that significant progressive decreases in HGB, PLT, PBML counts and ratios from group I onwards to group III, whereas the WBC, PBPL counts and ratios showed an increase from group I onwards to group III. The serum ADA, AST, ALT and ALP activity increased significantly in all infected groups compared to control group. However, these parameters were also observed to decrease progressively from group I to group III. Furthermore, the highest increase in enzyme activities observed in the infected group I. But, these enzyme's activities started to decrease in infected group II and III in parallel with PBML and PLT counts. Eventhough, this decrease did not reach to the values obtained from control group. On the contrary, PBPL counts and ratios increased in infected group II and III in contrast to decrease in PCV. As a result, increased serum ADA activity in tropical theileriosis may reflect the involvement of the cellular immune responses.
Assuntos
Adenosina Desaminase/sangue , Theileria annulata/crescimento & desenvolvimento , Theileriose/enzimologia , Alanina Transaminase/sangue , Fosfatase Alcalina/sangue , Animais , Aspartato Aminotransferases/sangue , Bovinos , Hematócrito/veterinária , Hemoglobinas/metabolismo , Contagem de Leucócitos/veterinária , Contagem de Plaquetas/veterinária , Theileriose/sangue , Theileriose/parasitologiaRESUMO
Theileria annulata inhabits the cytoplasm of bovine leukocytes where it can be found as a multinucleated schizont. The schizont is the pathogenic stage of the life cycle and by interfering with host signalling pathways, it induces unlimited host cell proliferation and protection against apoptosis. In the infected animal, the schizont differentiates to the merozoite life cycle stage in a process called merogony. This takes place within the host leukocyte, resulting in the production of merozoites that are subsequently released by leukocyte lysis. In established cultures of T. annulata-transformed cells, merogony does not spontaneously occur, but the process can be activated by a shift in temperature. In this study we show that chloramphenicol induces schizont differentiation in proliferating T. annulata-transformed cells. We demonstrate that chloramphenicol-induced merogony is inherently asynchronous and has a quantitative basis. The process is accompanied by the down-regulation of schizont-specific surface proteins, de novo expression of merozoite-specific markers such as Tamr1 and Tams1 and the morphological hallmarks of merogony. Chloramphenicol-induced parasite differentiation was found to be associated with diminished proliferation potential and extensive morphological changes of the host cell, including increased numbers of pseudopodia. Significantly, chloramphenicol treatment can accelerate merogony induced by elevated temperature, supporting postulation that the differentiation event is a stochastic process that can be manipulated to alter the outcome of parasitic infection.
Assuntos
Antibacterianos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Cloranfenicol/farmacologia , Leucócitos/parasitologia , Merozoítos/crescimento & desenvolvimento , Theileria annulata/parasitologia , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Immunoblotting , Theileria annulata/crescimento & desenvolvimentoRESUMO
Tropical theileriosis, a tick borne disease of cattle caused by the protozoan Theileria annulata, was widely distributed in northern China causing great economical losses before the 1980's. In the 1960's blood passaging, irradiation and in vitro culture of parasite were used for the development of attenuated vaccine. Two lines of the schizont stage were obtained which both provided sufficient protection against tick infestation or blood challenge in experimental and field trials. A gelatin protection technique allowed storage at 4 degrees C, significantly prolonged shelf life and permitted transport without a cold chain. The vaccines were commercialized and widely administered in epidemic areas, leading to control of T. annulata infection in China.
Assuntos
Doenças dos Bovinos/prevenção & controle , Vacinas Protozoárias/administração & dosagem , Esquizontes/imunologia , Theileria annulata/imunologia , Theileriose/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , China/epidemiologia , Vacinas Protozoárias/imunologia , Theileria annulata/crescimento & desenvolvimento , Theileriose/epidemiologia , Theileriose/imunologia , Theileriose/parasitologia , Vacinas Atenuadas/imunologiaRESUMO
Hyalomma anatolicum anatolicum, a three host tick vector transmitting the causative agent of bovine tropical theileriosis, is widely distributed throughout India. As a component of integrated control measures against the tick vector, attempts have been made to identify candidate protein molecules for development of an anti-tick vaccine in the different stages of this tick species. By strategic methods of isolation of the targeted molecules using affinity purification of proteins showing reactivity with immunoglobulins of animals previously immunized with different sources of tick antigen, six proteins were isolated in a significantly pure form. The recovery percentage of the candidate proteins was very low in the range of 1.8-8.0%. The protective potentiality of the antigens was tested in immunization and challenge trials and maximum potential was observed in the proteins isolated from total larval extracts, nymphal extracts and in larval glycoprotein. One of the antigens with a molecular weight of 37kDa isolated from larvae of H. a. anatolicum was found to have some adverse effect on development of Theileria annulata in the vector tick. The progress in the development of immunoprophylactic measures against H. a. anatolicum is discussed.
Assuntos
Doenças dos Bovinos/prevenção & controle , Desenho de Fármacos , Ixodidae/imunologia , Infestações por Carrapato/veterinária , Vacinas , Animais , Anticorpos/sangue , Antígenos/imunologia , Vetores Aracnídeos/imunologia , Vetores Aracnídeos/parasitologia , Vetores Aracnídeos/fisiologia , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/parasitologia , Imunização/métodos , Imunização/veterinária , Índia , Ixodidae/parasitologia , Ixodidae/fisiologia , Larva/imunologia , Masculino , Ninfa/imunologia , Glândulas Salivares/parasitologia , Theileria annulata/crescimento & desenvolvimento , Theileriose/parasitologia , Theileriose/prevenção & controle , Theileriose/transmissão , Infestações por Carrapato/imunologia , Infestações por Carrapato/parasitologia , Infestações por Carrapato/prevenção & controleRESUMO
Intracellular leucoproliferative 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 equal distribution of the schizont into the daughter cells is thought to be accomplished by a tight association with the host cell mitotic apparatus. In this study, we describe a highly conserved novel 37 kD Theileria annulata protein (TaSE). TaSE was found to be localized inside the parasite, the parasite membrane and within the host cell cytoplasm. Moreover, it co-localized at distinct points with host cell microtubules, which was especially apparent during mitosis, where co-localization was found with the centromere, the mitotic spindle and the midbody. Association of TaSE with the host cell tubulin network was corroborated by coimmunoprecipitation and transient transfection experiments. This is the first description of a theilerial protein co-localizing and potentially interacting with a host cell protein. The distribution of TaSE during mitosis makes it a protein to consider as playing a potential role for parasite distribution into daughter host cells.
Assuntos
Citoplasma/metabolismo , Interações Hospedeiro-Parasita , Proteínas de Protozoários/metabolismo , Theileria annulata/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Bovinos , Biologia Computacional , Cricetinae , Cricetulus , Mitose , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Análise de Sequência de DNA , Theileria annulata/genética , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/metabolismo , Tubulina (Proteína)/metabolismoAssuntos
Apicomplexa/genética , Genoma de Protozoário , Theileria annulata/genética , Theileria parva/genética , Animais , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Apicomplexa/crescimento & desenvolvimento , Apicomplexa/imunologia , Apicomplexa/fisiologia , Criptosporidiose/parasitologia , Criptosporidiose/transmissão , Cryptosporidium/genética , Cryptosporidium/fisiologia , Genes de Protozoários , Humanos , Estágios do Ciclo de Vida , Linfócitos/citologia , Linfócitos/parasitologia , Malária/parasitologia , Malária/transmissão , Organelas/fisiologia , Organelas/ultraestrutura , Plasmodium/genética , Plasmodium/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Análise de Sequência de DNA , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/imunologia , Theileria annulata/fisiologia , Theileria parva/crescimento & desenvolvimento , Theileria parva/imunologia , Theileria parva/fisiologia , Theileriose/parasitologia , Theileriose/transmissão , Toxoplasma/genética , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Toxoplasmose/transmissãoRESUMO
Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.
Assuntos
Genoma de Protozoário , Proteínas de Protozoários/genética , Theileria annulata/genética , Theileria parva/genética , Motivos de Aminoácidos , Animais , Bovinos , Proliferação de Células , Mapeamento Cromossômico , Cromossomos/genética , Sequência Conservada , Genes de Protozoários , Estágios do Ciclo de Vida , Metabolismo dos Lipídeos , Linfócitos/citologia , Linfócitos/parasitologia , Dados de Sequência Molecular , Família Multigênica , Filogenia , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Proteoma , Proteínas de Protozoários/química , Proteínas de Protozoários/fisiologia , Análise de Sequência de DNA , Especificidade da Espécie , Sintenia , Telômero/genética , Theileria annulata/crescimento & desenvolvimento , Theileria annulata/imunologia , Theileria annulata/patogenicidade , Theileria parva/crescimento & desenvolvimento , Theileria parva/imunologia , Theileria parva/patogenicidadeRESUMO
Disease-resistant livestock could provide a potentially sustainable and environmentally sound method of controlling tick and tick-borne diseases of livestock in the developing world. Advances in the knowledge and science of genomics open up opportunities to identify selectable genes controlling disease resistance but first, breeds and individuals with distinguishable phenotypes need to be identified. The Bos indicus breed, Sahiwal, has been exploited in dairy breeding programmes, because it is resistant to ticks and has relatively good performance characteristics compared to other indigenous cattle breeds of tropical regions. The analyses reported here show that Sahiwal calves were also more resistant than European Bos taurus (Holstein) dairy breed calves to tick-borne tropical theileriosis (Theileria annulata infection). Following experimental infection with T. annulata sporozoites, a group of Sahiwal calves all survived without treatment, with significantly lower maximum temperatures (P<0.01) and lower rates of parasite multiplication (P<0.05) than a group of Holstein calves, which all had severe responses. Although the Sahiwals became as anaemic as the Holsteins, other measures of pathology, including enlargement of the draining lymph node and the acute phase proteins, alpha1 acid glycoprotein and haptoglobin, were significantly less in the Sahiwals than in the Holsteins (P<0.05). Additionally, the Sahiwals had significantly lower resting levels of alpha1 acid glycoprotein than the Holsteins (P<0.05). Production of a third acute phase proteins, serum amyloid A, had very similar kinetics in both breeds. Acute phase proteins are produced in response to systemic release of the kinds of pro-inflammatory cytokines that are thought to be responsible for the pyrexic, cachectic and anorexic responses characteristic of tropical theileriosis. The prolonged production of alpha1 acid glycoprotein in the Holsteins is indicative of chronic production of circulating pro-inflammatory cytokines. In contrast, Sahiwals appear able to overcome infection with T. annulata as well as limit pathology by preventing the over-stimulation of pathways involving these cytokines.