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1.
Transbound Emerg Dis ; 67 Suppl 1: 8-25, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32174036

RESUMO

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.


Assuntos
Vetores Aracnídeos/parasitologia , Vacinas Protozoárias/imunologia , Theileria annulata/imunologia , Theileriose/epidemiologia , Doenças Transmitidas por Carrapatos/veterinária , Carrapatos/parasitologia , Vacinação/veterinária , África do Norte/epidemiologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/prevenção & controle , Prevalência , Estações do Ano , Theileriose/parasitologia , Theileriose/prevenção & controle , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/parasitologia , Doenças Transmitidas por Carrapatos/prevenção & controle , Vacinas Atenuadas/imunologia
2.
Transbound Emerg Dis ; 67 Suppl 1: 26-34, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32174037

RESUMO

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.


Assuntos
Doenças dos Bovinos/prevenção & controle , Imunização/veterinária , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologia , Theileria annulata/imunologia , Theileriose/prevenção & controle , Doenças Transmitidas por Carrapatos/veterinária , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Linhagem Celular , Esquizontes , Esporozoítos , Theileriose/parasitologia , Doenças Transmitidas por Carrapatos/parasitologia , Vacinas Atenuadas/imunologia , Vacinas de Subunidades/imunologia
3.
Transbound Emerg Dis ; 67 Suppl 1: 40-55, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32174040

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/parasitologia
4.
Transbound Emerg Dis ; 67 Suppl 1: 35-39, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32174041

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/imunologia
5.
Acta Trop ; 202: 105245, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31676457

RESUMO

Tropical theileriosis is a tick-borne lymphoproliferative disease of cattle caused by the apicomplexan parasite Theileria annulata, and leads to substantial economic losses to the livestock industry worldwide. Although various enzyme-linked immunosorbent assays (ELISAs) have been established to detect antibodies against T. annulata infection, a specific, rapid and reliable diagnostic assay is urgently needed for prevention and control of the disease. In the present study, a chemiluminescence immunoassay (CLIA) was developed based on the subtelomeric variable secreted protein (SVSP) of T. annulata as a sero-diagnostic antigen. Following optimization of the CLIA working parameters, the working time of the method was less than 4.5 h. The sensitivity and specificity of the established CLIA was 98.8% and 97.5%, respectively, when the cut-off value of the percent positive (PP) was 26.1% for detecting serum samples (n = 242 T. annulata positive sera, n = 158 T. annulata negative sera). After comparing 180 serum samples from Gansu province, China, the concordance rate between the CLIA and a published rSpm2 ELISA method was 72.8%. In addition, 565 serum samples of cattle collected between 2017 and 2018 from four provinces in China were detected by the CLIA, and the seroprevalence for T. annulata ranged from 53.3% to 67.3% in these regions. Our findings demonstrated that the CLIA has high specificity, sensitivity and reliability, and could be used as a rapid detection assay for epidemiological investigations of T. annulata infection.


Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Theileriose/diagnóstico , Theileriose/prevenção & controle , Animais , Bovinos , Doenças dos Bovinos/diagnóstico , Doenças dos Bovinos/parasitologia , China/epidemiologia , Medições Luminescentes/métodos , Prevalência , Estudos Soroepidemiológicos , Theileria annulata , Theileriose/parasitologia , Doenças Transmitidas por Carrapatos/diagnóstico , Doenças Transmitidas por Carrapatos/parasitologia
6.
Int J Parasitol Drugs Drug Resist ; 11: 101-105, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31794951

RESUMO

The tick-borne parasite Theileria annulata is the causative agent of tropical theileriosis or Mediterranean theileriosis. Infection of bovine leukocytes by the obligate intracellular parasites induces proliferative and invasive phenotypes associated with activated signaling pathways. The transformed phenotypes of infected cells are reversible by treatment with the theilericidal drug buparvaquone. Recent reports of resistance to buparvaquone in Africa and Asia highlight the need to investigate the mechanisms and prevalence of drug resistance. We screened 67 T. annulata isolates from Sudan to investigate mutations in the T. annulata prolyl isomerase I gene (TaPIN1). The secreted TaPin1 interacts with host proteins to induce pathways driving oncogenic transformation and metabolic reprogramming. We found an Alanine-to-Proline mutation at position 53 (A53P) in the catalytic loop that was previously found in Tunisian drug-resistant samples. This is the first study reporting independent confirmation of the A53P mutation in geographically isolated samples. We found several additional mutations in the predicted N-terminal signal peptide that might affect TaPin1 processing or targeting. We found that many parasites also share mutations in both the TaPIN1 and the cytochrome b genes, suggesting that these two genes represent important biomarkers to follow the spread of resistance in Africa, the Middle East and Asia.


Assuntos
Resistência a Medicamentos/genética , Peptidilprolil Isomerase/genética , Mutação Puntual , Theileria annulata/enzimologia , Theileria annulata/genética , Animais , Antiprotozoários/farmacologia , Bovinos , Naftoquinonas/farmacologia , Fenótipo , Sudão , Theileria annulata/efeitos dos fármacos , Theileriose/parasitologia
7.
Infect Genet Evol ; 75: 103972, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31344487

RESUMO

Tick-borne pathogens (TBP) are a major source of production loss and a welfare concern in livestock across the globe. Consequently, there is a trade-off between keeping animals that are tolerant to TBP infection, but are less productive than more susceptible breeds. Theileria annulata is a major TBP of bovines, with different host types (i.e. exotic and native cattle breeds, and buffalo) displaying demonstrable differences in clinical susceptibility to infection. However, the extent to which these differences are driven by genetic/physiological differences between hosts, or by different parasite populations/genotypes preferentially establishing infection in different host breeds and species is unclear. In this study, three different bovine host types in India were blood sampled to test for the presence of various TBP, including Theileria annulata, to determine whether native cattle (Bos indicus breeds), crossbreed cattle (Bos taurus x Bos indicus breeds) or water buffalo (Bubalus bubalis) differ in the physiological consequences of infection. Population genetic analyses of T. annulata isolated from the three different host types was also performed, using a panel of mini- and micro-satellite markers, to test for sub-structuring of the parasite population among host types. We discovered that compared to other host types, "carrier" crossbreed cattle showed a higher level of haematological pathology when infected with T. annulata. Despite this finding, we found no evidence for differences in the genotypes of T. annulata infecting different host types, although buffalo appeared to harbour fewer mixed parasite genotype infections, indicating they are not the major reservoir of parasite diversity. The apparent tolerance/resistance of native breed cattle and buffalo to the impacts of T. annulata infection is thus most likely to be driven by host genotype, rather than differences in the parasite population. Our results suggest that an improved understanding of the genetic factors that underpin disease resistance could help to ameliorate future economic loss due to TBP or tropical theileriosis.


Assuntos
Búfalos/parasitologia , Bovinos/parasitologia , Genótipo , Especificidade de Hospedeiro , Theileria annulata/genética , Theileriose/parasitologia , Animais , Reservatórios de Doenças/parasitologia , Reservatórios de Doenças/veterinária , Índia/epidemiologia , Theileriose/epidemiologia
8.
J Vet Med Sci ; 81(8): 1197-1200, 2019 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-31292335

RESUMO

In South Asia, Theileria annulata is known to be less pathogenic to local breeds of Bos indicus cattle comparing to Bos taurus cattle and some of mix breeds between them. Seroepidemiological surveys have revealed high sero-prevalence of T. annulata in asymptomatic local breeds of cattle in Bangladesh. Therefore, these asymptomatic infection in local breeds can be infectious sources to more sensitive breeds. In this study, 59 bloods of cattle showing no symptom were screened by species specific PCRs for hemoto-protozoan parasites, to prove the existence of T. annulata parasite in asymptomatic cattle in Bangladesh. The T. annulata infection was confirmed along with other parasitic species, and this is the first report of T. annulata DNA detection in Bangladesh.


Assuntos
Doenças dos Bovinos/parasitologia , Bovinos/parasitologia , Theileria annulata/parasitologia , Theileriose/parasitologia , Animais , Infecções Assintomáticas/epidemiologia , Bangladesh , Doenças dos Bovinos/epidemiologia , Filogenia , Estudos Soroepidemiológicos , Theileria/genética , Theileria annulata/genética , Theileriose/epidemiologia
9.
Biochim Biophys Acta Proteins Proteom ; 1867(7-8): 732-739, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31103831

RESUMO

Theileria annulata enolase (TaENO) could be assessed as a druggable target for tropical theileriosis treatment. The parasite enzyme plays an important role in many cellular functions and carries some structural differences like dipeptide (262EK263) and pentapeptide (103EWGYC107) insertions from the host enzyme, Bos taurus enolase. In this study, the functional effects of these insertions on TaENO activity were analyzed by in vitro site-directed mutagenesis and in silico molecular docking analyses for the first time in the literature. In vitro results showed that, although the deletion of the pentapeptide insertion (TaENOΔEWGYC) reduced the enzyme activity slightly, the removal of the dipeptide insertion (TaENOΔEK) halted it. Also, molecular docking results revealed that the deletion of these insertions affected the substrate binding affinity of the mutant enzymes. The active site of TaENOΔEK exhibited a small decrease of substrate binding affinity compared to the active site of TaENOΔEWGYC relative to the wild type TaENO. Although we conclude that both regions could be evaluated as possible drug-binding sites to inhibit TaENO in further studies, these results indicate that the dipeptide insertion could be a more promising drug binding site than the pentapeptide insertion.


Assuntos
Dipeptídeos/química , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fosfopiruvato Hidratase/química , Proteínas de Protozoários/química , Theileria annulata/enzimologia , Animais , Domínio Catalítico/genética , Bovinos , Dipeptídeos/genética , Fosfopiruvato Hidratase/genética , Proteínas de Protozoários/genética , Especificidade por Substrato/genética , Theileria annulata/genética
10.
Genes (Basel) ; 10(5)2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052316

RESUMO

This study aimed to establish a pure single-cell Theileria annulata-infected B cell line for the assessment of cytokine production in transformed and lipopolysaccharide (LPS)-stimulated cells. Several studies have aimed to identify cell surface markers in T. annulata-transformed cells; however, no information on cytokine production in these cells is available. To investigate the potential of the transformed cells to produce cytokines and their potential responses to antigen-stimulation, we purified mature B cells (CD21) from the whole blood of cattle experimentally infected with the T. annulata Kashi strain by magnetic separation. The purity and specificity of the established cell line was assessed by the identification of specific cell surface markers (CD21, IgM, and WC4) by flow cytometry analysis. The transcript levels of the cytokines IL1A, IL1B, IL2, IL4, IL6, IL8, IL10, IL16, LTA, TGFB1, TNFA, IFNA, and IFNB in transformed, buparvaquone (BW720c)-treated cells, and antigen-stimulated cells were analyzed by quantitative polymerase chain reaction (qPCR) using cDNA from these cells. A T. annulata-infected bovine B cell line was successfully established with a purity of ~98.8% (CD21). IL4 and IL12A were significantly (p < 0.01) upregulated in the transformed cells. In BW720c-treated transformed cells, IL12B, TGFB1, and IFNB were significantly (p < 0.01) upregulated. Notably, no significant (p > 0.05) upregulation of cytokines was observed in LPS-stimulated transformed cells. Moreover, IL1A, IL1B, IL8, and IL16 were significantly (p < 0.01) upregulated in LPS-stimulated B cells. Our data signify the potential use of this cell line for cytokine production, observance of immunoglobulins, and production of an attenuated vaccine against tropical theileriosis.


Assuntos
Linfócitos B/metabolismo , Citocinas/genética , Theileria annulata/efeitos dos fármacos , Theileriose/genética , Animais , Antígenos/genética , Antígenos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/parasitologia , Bovinos , Citocinas/classificação , Citometria de Fluxo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Naftoquinonas/farmacologia , Análise de Célula Única , Theileria annulata/patogenicidade , Theileriose/sangue , Theileriose/parasitologia
11.
Ticks Tick Borne Dis ; 10(4): 815-821, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30952580

RESUMO

The water buffalo industry is a vital part of the Philippine livestock economy and is an essential contributor to the developing local dairy industry. Although relatively less susceptible to diseases, water buffaloes can still be infected and can act as reservoirs of tick-borne pathogens (TBPs). However, limited information is available regarding the prevalence of tick-borne infections in water buffaloes in the Philippines. This study was conducted to identify TBPs harbored by water buffaloes and to characterize these pathogens molecularly. One hundred water buffalo blood samples collected from three areas in Bohol, Visayas region, Philippines were screened for various TBPs using pathogen-specific PCR assays. TBPs were detected in 46% of the samples (39% singly infected, 7% coinfected). The pathogens detected were Anaplasma marginale (29%), Babesia bovis (21%), and B. bigemina (3%). None of the blood samples were positive for Theileria annulata, T. orientalis, and B. ovata. A. marginale infection rates were significantly higher (37.5%) among water buffaloes aged ≤6 years (P = 0.046) than those >6 years old (18.2%) and was detected only in Bulgarian Murrah (36.1%) and US Murrah (25.9%) breeds. Phylogenetic analyses revealed that groEL sequences of A. marginale were 100% identical with isolates from the Philippines (Batangas and Cebu) and China. Two B. bigemina RAP-1a gene sequences were identical to each other and were homologous with previous isolates from Thailand, Indonesia, Uruguay, and the Philippines. Moreover, four B. bovis SBP-2 partial sequences obtained in this study had 92.4-99.7% identities. This study is the first molecular detection and characterization of A. marginale, B. bigemina and B. bovis in water buffaloes in the Visayas region, and the first molecular confirmation of B. bovis infection in water buffaloes in the country. The findings presented in this study may serve as baseline data for crafting effective tick-borne disease surveillance and prevention programs in Bohol and in the Philippines.


Assuntos
Anaplasmose/epidemiologia , Babesiose/epidemiologia , Búfalos/microbiologia , Filogenia , Theileriose/epidemiologia , Doenças Transmitidas por Carrapatos/veterinária , Anaplasma marginale/genética , Anaplasma marginale/isolamento & purificação , Animais , Babesia/genética , Babesia/isolamento & purificação , Babesia bovis/genética , Babesia bovis/isolamento & purificação , Búfalos/parasitologia , DNA de Protozoário/genética , Feminino , Variação Genética , Filipinas/epidemiologia , Reação em Cadeia da Polimerase , Theileria annulata/genética , Theileria annulata/isolamento & purificação , Doenças Transmitidas por Carrapatos/microbiologia , Doenças Transmitidas por Carrapatos/parasitologia , Carrapatos/microbiologia , Carrapatos/parasitologia
12.
Vet Parasitol ; 265: 63-73, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30638522

RESUMO

Tropical theileriosis is a tick-borne haemoparasitic disease of cattle caused by the protozoan parasite Theileria annulata. Globally, the economic impact of the disease is immense and enhanced control measures would improve livestock production in endemic regions. Immunisation with a live attenuated vaccine is an effective and widely used control method, however, the repeated use of live vaccines may have an impact on the field parasite population at a genetic level. Additionally, there has been an increasing number of reports of vaccine breakthrough cases in recent years. Thus, the present study was designed to evaluate the genetic composition of a parasite population over a disease season in a locality where live cell line vaccination is practised. A diverse range of parasite genotypes was identified and every T. annulata positive cattle blood sample harboured multiple parasite genotypes. An alteration in the major genotype and an increasing multiplicity of infection in individual animals was observed over the course of the disease season. Vaccination status was found not to effect within-host multiplicity of infection, while a significantly higher number of genotypes was detected in grazed cattle compared to non-grazed ones. A degree of genetic isolation was evident between parasite populations on a micro-geographic scale, which has not been reported previously for T. annulata. Analysis of parasite genotypes in vaccinated animals suggested only a transient effect of the vaccine genotype on the genetic diversity of the T. annulata population. The vaccine genotype was not detected among clones of two vaccine 'breakthrough' isolates and there is no suggestion that it was responsible for disease. The obtained data indicated that in the system studied there is no apparent risk of introducing the vaccine genotype into the population with only a transient effect on the genetic diversity of the parasite population during the disease season.


Assuntos
Doenças dos Bovinos/prevenção & controle , Vacinas Protozoárias/imunologia , Theileria annulata , Theileriose/prevenção & controle , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/parasitologia , Clonagem Molecular , Genótipo , Repetições de Microssatélites , Theileria annulata/genética , Theileriose/epidemiologia , Turquia/epidemiologia
13.
Free Radic Biol Med ; 134: 282-287, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30639613

RESUMO

Theileria annulata is a protozoan parasite that infects and transforms bovine macrophages causing a myeloid-leukaemia-like disease called tropical theileriosis. TGF-ß2 is highly expressed in many cancer cells and is significantly increased in Theileria-transformed macrophages, as are levels of Reactive Oxygen Species (ROS), notably H2O2. Here, we describe the interplay between TGF-ß2 and ROS in cellular transformation. We show that TGF-ß2 drives expression of catalase to reduce the amount of H2O2 produced by T. annulata-transformed bovine macrophages, as well as by human lung (A549) and colon cancer (HT-29) cell lines. Theileria-transformed macrophages attenuated for dissemination express less catalase and produce more H2O2, but regain both virulent migratory and matrigel traversal phenotypes when stimulated either with TGF-ß2, or catalase to reduce H2O2 output. Increased H2O2 output therefore, underpins the aggressive dissemination phenotype of diverse tumour cell types, but in contrast, too much H2O2 can dampen dissemination.


Assuntos
Adenocarcinoma de Pulmão/secundário , Catalase/metabolismo , Neoplasias do Colo/secundário , Peróxido de Hidrogênio/metabolismo , Macrófagos/imunologia , Fator de Crescimento Transformador beta2/metabolismo , Adenocarcinoma de Pulmão/metabolismo , Animais , Catalase/genética , Bovinos , Neoplasias do Colo/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Oxidantes/metabolismo , Oxirredução , Fenótipo , Theileria annulata/isolamento & purificação , Theileriose/parasitologia , Fator de Crescimento Transformador beta2/genética , Células Tumorais Cultivadas
14.
Acta Trop ; 191: 128-132, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30599177

RESUMO

Drug resistance is one of the emerging and re-emerging epidemics affecting both veterinary and public health sectors. Buparvaquone provides the most satisfactory means in the treatment of bovine tropical theileriosis. However, recently there has been widespread reports of development of resistance of Theileria annulata to buparvaquone. To investigate the situation in Sudan where bovine tropical theileriosis is endemic, fifty blood samples from T. annulata-positive cattle. were used for DNA extraction, PCR and cytochrome b gene nucleotide sequencing. Analysis of the two buparvaquone binding site regions Q01 (130-148) and Q02 (244-266), revealed three non- synonymous mutations at codon 146; alanine (GCT) to threonine (ACT) within the Q01 region across all 50 isolates and the other mutation at codon 129; serine (AGC) to glycine (GGC) in 18 isolates which is very close to the Q01 binding site. However, we documented another mutation at position 227; valine (GTG) to methionine (ATG) close to the close to the Q02 binding site, in three isolates with mutation at codon 129. We concluded that this study has provided evidence of point mutations in the cytochrome b gene of T. annulata that might be associated with buparvaquone treatment failure in Sudan.


Assuntos
Antiprotozoários/uso terapêutico , Citocromos b/genética , Resistência a Medicamentos/genética , Naftoquinonas/uso terapêutico , Theileria annulata/genética , Theileriose/terapia , Animais , Bovinos , Mutação Puntual , Sudão , Falha de Tratamento
15.
Cell Microbiol ; 21(1): e12969, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30370674

RESUMO

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 Celular
16.
Transbound Emerg Dis ; 66(1): 526-536, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30383917

RESUMO

Tick-borne diseases (TBDs) have a large impact on animal health and the livelihood of livestock owners, particularly in developing countries. Although climatic and ecological conditions in Pakistan may favour the transmission of tick-borne pathogens (TBPs), only a few systematic studies have been carried out on TBPs and the diseases that they cause in this country. To improve our understanding of the distribution of TBPs, 3,807 ticks were collected from ruminants (n = 369) on 108 livestock farms (semi-arid zone = 36, arid zone = 72) in Punjab Province. After morphological identification ticks were pooled into 405 pools (Hyalomma anatolicum = 300, Rhipicephalus microplus = 89, Hyalomma dromedarii = 9, Rhipicephalus turanicus = 7) based on their species, locality of collection, and the host. DNA from each pool was screened by a Reverse Line Blot (RLB) hybridization assay for the presence of Anaplasma, Ehrlichia, Rickettsia, Babesia, and Theileria species. DNA from at least one TBP was found in 142 (35.1%) pools. Among the positive pools, 91 (64.1%) had a mixed infection with two or more TBPs, whereas 51 (35.9%) pools were infected with a single TBP. The detected pathogens not only included species that were known to be endemic in Pakistan, such as Theileria annulata (6.7%), Theileria orientalis (3.5%), Anaplasma marginale (5.7%), Anaplasma centrale (2.7%), Anaplasma ovis (1.5%), Babesia bigemina (0.7%), and Babesia bovis (0.2%), but also several TBPs that had not been reported to occur in Pakistan before. This included Ehrlichia minasensis (3.2%), an Anaplasma platys-like organism (1.2%), Babesia occultans (0.2%), and Rickettsia massiliae (0.2%), as well as two previously uncharacterized species: Ehrlichia sp. Multan (18.0%) and Anaplasma sp. (BL099-6) (2.22%). The pathogenicity of these novel species remains to be examined. This study shows that a much broader spectrum of TBPs is present in Pakistan than previously thought, including several zoonotic pathogens.


Assuntos
Doenças dos Bovinos/epidemiologia , Gado , Doenças Transmitidas por Carrapatos/veterinária , Carrapatos/microbiologia , Carrapatos/parasitologia , Anaplasma/genética , Anaplasma/isolamento & purificação , Animais , Babesia/genética , Babesia bovis/genética , Babesia bovis/isolamento & purificação , Bovinos , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/parasitologia , DNA Bacteriano/genética , DNA de Protozoário/genética , Ehrlichia/genética , Ehrlichia/isolamento & purificação , Feminino , Gado/microbiologia , Gado/parasitologia , Paquistão/epidemiologia , Reação em Cadeia da Polimerase/veterinária , Rickettsia/genética , Rickettsia/isolamento & purificação , Theileria annulata/genética , Theileria annulata/isolamento & purificação , Doenças Transmitidas por Carrapatos/epidemiologia , Doenças Transmitidas por Carrapatos/microbiologia , Doenças Transmitidas por Carrapatos/parasitologia
17.
Cell Microbiol ; 21(3): e12973, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30412643

RESUMO

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/patologia
18.
Rev Sci Tech ; 38(3): 703-709, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-32286574

RESUMO

Bovine tropical theileriosis caused by Theileria annulata is an overwhelming haemoprotozoan tick-borne disease in taurine and cross-bred cattle in Punjab, India. However, there seems to be no report from India of cutaneous nodules associated with the disease. This report describes a five-year-old cross-bred cow presented to a university clinic with a history of fever, inappetence and malaise for the past six to seven days. Clinical examination revealed normal vital parameters, pale mucous membranes, mild enlargement of the prescapular lymph nodes and multiple subcutaneous nodular masses (2-4 cm) on the neck and abdomen. Haematology revealed mild anaemia and leucopenia with 48% neutrophils, 48% lymphocytes and 4% eosinophils. Romanowsky-stained smears of fineneedle aspiration biopsy samples from swollen lymph nodes and subcutaneous masses showed an increased number of lymphoid cells, suggesting cutaneous lymphomatosis. However, a critical examination of the smears from subcutaneous nodules showed a large number of Koch's blue bodies in macrophages and lymphoblasts, and several piroplasms were also noticed within the red blood cells in lymph node smears. A peripheral blood smear revealed mild to moderate parasitaemia. Extracted DNA from the parasitologically positive blood sample was subjected to nested polymerase chain reaction (nPCR) using T. annulata speciesspecific primers encoding the 30-kiloDalton major sporozoite surface antigen. The desired 572-base pair amplified product of the nPCR was comparable to the positive control. This seems to be a rare case of T. annulata in an adult cross-bred cow, showing cutaneous nodular involvement.


Assuntos
Doenças dos Bovinos/diagnóstico , Theileriose/diagnóstico , Animais , Bovinos , Feminino , Índia , Theileria annulata
19.
Arch Razi Inst ; 74(4): 433-438, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31939260

RESUMO

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/imunologia
20.
Acta Parasitol ; 63(4): 759-765, 2018 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-30367760

RESUMO

This study describes development and evaluation of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bigemina and Anaplasma marginale infections in bovines. The assay was developed using parasites specific genomic DNA and three sets of PCR primers targeting the Tams1, 18S rRNA and 16S rRNA genes of T. annulata, B. bigemina and A. marginale, respectively. Blood samples collected from a total of 461 bovines, suspected for haemoparasitic infections, were examined microscopically to record the status of infection and simultaneously, genomic DNA extracted from these blood samples were utilized for the optimization and validation of multiplex PCR assay. Microscopic examination of blood samples revealed presence of single and multiple species of haemoparasites in 25.8% and 2.4% samples, respectively. Results of multiplex PCR revealed the presence of single haemoparasitic species infection in 159 cases (34.5%), whereas mixed infection was recorded in 82 (17.8%) samples. Occurrence of individual species infection detected by mPCR in the study was 26.03% (120/461) for T. annulata, 3.25% (15/461) for B. bigemina and 5.20% (24/461) for A. marginale. The detection limit of multiplex PCR assay was at the template dilutions of 10-6, 10-6 and 10-4, which corresponded to 0.1 pg, 0.1 pg and 10.0 pg of DNA for T. annulata, A. marginale, and B. bigemina, respectively. Based on the high diagnostic sensitivity and throughput, multiplex PCR assay developed in the present study could be exploited as a tool to conduct large-scale epidemiological survey for tick-borne haemoparasitic infection of bovines.


Assuntos
Doenças dos Bovinos/diagnóstico , Reação em Cadeia da Polimerase Multiplex/veterinária , Doenças Transmitidas por Carrapatos/veterinária , Anaplasma/genética , Anaplasma/isolamento & purificação , Anaplasmose/diagnóstico , Animais , Antígenos de Protozoários/genética , Babesia/genética , Babesia/isolamento & purificação , Babesiose/diagnóstico , Babesiose/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Clonagem Molecular , DNA Bacteriano/sangue , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , DNA de Protozoário/sangue , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Theileria annulata/genética , Theileria annulata/isolamento & purificação , Theileriose/diagnóstico , Theileriose/parasitologia , Doenças Transmitidas por Carrapatos/diagnóstico
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