RESUMEN
Coxiella burnetii, a highly adapted obligate intracellular bacterial pathogen and the cause of the zoonosis Q fever, is a reemerging public health threat. C. burnetii employs a Type IV secretion system (T4SS) to establish and maintain its intracellular niche and modulate host immune responses including the inhibition of apoptosis. Interactions between C. burnetii and caspase-1-mediated inflammasomes are not fully elucidated. This study confirms that C. burnetii does not activate caspase-1 during infection of mouse macrophages in vitro. C. burnetii-infected cells did not develop NLRP3 and ASC foci indicating its ability to avoid cytosolic detection. C. burnetii is unable to inhibit the pyroptosis and IL-1ß secretion that is induced by potent inflammasome stimuli but rather enhances these caspase-1-mediated effects. We found that C. burnetii upregulates pro-IL-1ß and robustly primes NLRP3 inflammasomes via TLR2 and MyD88 signaling. As for wildtype C. burnetii, T4SS-deficient mutants primed and potentiated NLRP3 inflammasomes. An in vivo model of pulmonary infection in C57BL/6 mice was developed. Mice deficient in NLRP3 or caspase-1 were like wildtype mice in the development and resolution of splenomegaly due to red pulp hyperplasia, and histologic lesions and macrophage kinetics, but had slightly higher pulmonary bacterial burdens at the greatest measured time point. Together these findings indicate that C. burnetii primes but avoids cytosolic detection by NLRP3 inflammasomes, which are not required for the clinical resistance of C57BL/6 mice. Determining mechanisms employed by C. burnetii to avoid cytosolic detection via NLRP3 inflammasomes will be beneficial to the development of preventative and interventional therapies for Q fever.
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Coxiella burnetii , Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Fiebre Q , Animales , Ratones , Ratones Endogámicos C57BL , Fiebre Q/inmunologíaRESUMEN
Theileria parva is the causative agent of East Coast fever (ECF), a tick-borne disease that kills over a million cattle each year in sub-Saharan Africa. Immune protection against T. parva involves a CD8+ cytotoxic T cell response to parasite-infected cells. However, there is currently a paucity of knowledge regarding the role played by innate immune cells in ECF pathogenesis and T. parva control. Here, we demonstrate an increase in intermediate monocytes (CD14++ CD16+) with a concomitant decrease in the classical (CD14++ CD16-) and nonclassical (CD14+ CD16+) subsets at 12 days postinfection (dpi) during lethal infection but not during nonlethal T. parva infection. Ex vivo analyses of monocytes demonstrated upregulation of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) mRNA and increased nitric oxide production during T. parva lethal infection compared to nonlethal infection at 10 dpi. Interestingly, no significant differences in peripheral blood parasite loads were observed between lethally and nonlethally infected animals at 12 dpi. In vitro stimulation with T. parva schizont-infected cells or Escherichia coli lipopolysaccharide (LPS) resulted in significant upregulation of IL-1ß production by monocytes from lethally infected cattle compared to those from nonlethally infected animals. Strikingly, monocytes from lethally infected animals produced significant amounts of IL-10 mRNA after stimulation with T. parva schizont-infected cells. In conclusion, we demonstrate that T. parva infection leads to alterations in the molecular and functional phenotypes of bovine monocytes. Importantly, since these changes primarily occur in lethal infection, they can serve as biomarkers for ECF progression and severity, thereby aiding in the standardization of protection assessment for T. parva candidate vaccines.
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Monocitos/inmunología , Theileria parva/inmunología , Theileriosis/inmunología , Animales , Bovinos , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lipopolisacáridos/inmunología , Carga de Parásitos , Vacunas Antiprotozoos/inmunología , ARN Mensajero/genética , Linfocitos T Citotóxicos/inmunología , Theileriosis/parasitología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200â mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10(- )3 dilution within 15 âh. Our findings indicate that RT-QuIC was at least 10,000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.
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Bioensayo/métodos , Encéfalo/metabolismo , Enfermedades de las Cabras/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Scrapie/diagnóstico , Animales , Enfermedades de las Cabras/diagnóstico , Enfermedades de las Cabras/genética , Cabras , Proteínas PrPC/genética , Proteínas PrPSc/genética , Scrapie/metabolismo , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Ebola virus (EBOV) is a Category A pathogen that is a member of Filoviridae family that causes hemorrhagic fever in humans and non-human primates. Unpredictable and devastating outbreaks of disease have recently occurred in Africa and current immunoprophylaxis and therapies are limited. The main limitation of working with pathogens like EBOV is the need for costly containment. To potentiate further and wider opportunity for EBOV prophylactics and therapies development, innovative approaches are necessary. METHODS: In the present study, an antigen delivery platform based on a recombinant bovine herpesvirus 4 (BoHV-4), delivering a synthetic EBOV glycoprotein (GP) gene sequence, BoHV-4-syEBOVgD106ΔTK, was generated. RESULTS: EBOV GP was abundantly expressed by BoHV-4-syEBOVgD106ΔTK transduced cells without decreasing viral replication. BoHV-4-syEBOVgD106ΔTK immunized goats produced high titers of anti-EBOV GP antibodies and conferred a long lasting (up to 6 months), detectable antibody response. Furthermore, no evidence of BoHV-4-syEBOVgD106ΔTK viremia and secondary localization was detected in any of the immunized animals. CONCLUSIONS: The BoHV-4-based vector approach described here, represents: an alternative antigen delivery system for vaccination and a proof of principle study for anti-EBOV antibodies generation in goats for potential immunotherapy applications.
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Ebolavirus/metabolismo , Vectores Genéticos/metabolismo , Herpesvirus Bovino 4/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Línea Celular , Codón/genética , Simulación por Computador , Cabras/inmunología , Células HEK293 , Humanos , Inmunidad Humoral , Inmunización , Cinética , Glicoproteínas de Membrana/química , Sistemas de Lectura Abierta/genéticaRESUMEN
UNLABELLED: Equine hepacivirus (EHCV; nonprimate hepacivirus) is a hepatotropic member of the Flaviviridae family that infects horses. Although EHCV is the closest known relative to hepatitis C virus (HCV), its complete replication kinetics in vivo have not been described, and direct evidence that it causes hepatitis has been lacking. In this study, we detected EHCV in 2 horses that developed post-transfusion hepatitis. Plasma and serum from these horses were used to experimentally transmit EHCV to 4 young adult Arabian horses, two 1-month-old foals (1 Arabian and 1 Arabian-pony cross), and 2 foals (1 Arabian and 1 Arabian-pony cross) with severe combined immunodeficiency (SCID). Our results demonstrated that EHCV had infection kinetics similar to HCV and that infection was associated with acute and chronic liver disease as measured by elevations of liver-specific enzymes and/or by histopathology. Although most of these animals were coinfected with equine pegivirus (EPgV), also a flavivirus, EPgV viral loads were much lower and often undetectable in both liver and blood. Three additional young adult Arabian-pony crosses and 1 SCID foal were then inoculated with plasma containing only EHCV, and evidence of mild hepatocellular damage was observed. The different levels of liver-specific enzyme elevation, hepatic inflammation, and duration of viremia observed during EHCV infection suggested that the magnitude and course of liver disease was mediated by the virus inoculum and/or by host factors, including breed, age, and adaptive immune status. CONCLUSION: This work documents the complete infection kinetics and liver pathology associated with acute and chronic EHCV infection in horses and further justifies it as a large animal model for HCV.
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Modelos Animales de Enfermedad , Hepatitis C Crónica/transmisión , Hepatitis C Crónica/veterinaria , Enfermedades de los Caballos/transmisión , Enfermedades de los Caballos/virología , Animales , CaballosRESUMEN
Equine piroplasmosis, caused by the parasites Theileria equi and Babesia caballi, is a globally important disease, affecting a large percentage of the world's horses. This article serves as a review of these divergent parasites. Discussed are the clinical presentation of disease, diagnosis, and treatment. Special attention is given to the current disease status specifically in North America.
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Babesia/aislamiento & purificación , Babesiosis/terapia , Enfermedades de los Caballos/parasitología , Theileria/aislamiento & purificación , Theileriosis/terapia , Animales , Babesiosis/tratamiento farmacológico , Babesiosis/epidemiología , Babesiosis/prevención & control , Brotes de Enfermedades/veterinaria , Equidae , Enfermedades de los Caballos/tratamiento farmacológico , Enfermedades de los Caballos/epidemiología , Enfermedades de los Caballos/prevención & control , Caballos , América del Norte/epidemiología , Theileriosis/tratamiento farmacológico , Theileriosis/epidemiología , Theileriosis/prevención & controlRESUMEN
BACKGROUND: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. RESULTS: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. CONCLUSIONS: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.
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Genoma de Protozoos , Theileria/genética , Animales , Bovinos , Mapeo Cromosómico , Cromosomas/genética , Cromosomas/metabolismo , Hibridación Genómica Comparativa , Metabolismo Energético/genética , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Fosfolípidos/metabolismo , Filogenia , Proteínas Protozoarias/genética , Theileria/clasificación , Theileriosis/genética , Theileriosis/metabolismo , Theileriosis/parasitologíaRESUMEN
Mannheimia haemolytica, Pasteurella multocida, and Bibersteinia trehalosi have been identified in the lungs of pneumonic bighorn sheep (BHS; Ovis canadensis). Of these pathogens, M. haemolytica has been shown to consistently cause fatal pneumonia in BHS under experimental conditions. However, M. haemolytica has been isolated by culture less frequently than the other bacteria. We hypothesized that the growth of M. haemolytica is inhibited by other bacteria in the lungs of BHS. The objective of this study was to determine whether P. multocida inhibits the growth of M. haemolytica. Although in monoculture both bacteria exhibited similar growth characteristics, in coculture with P. multocida there was a clear inhibition of growth of M. haemolytica. The inhibition was detected at mid-log phase and continued through the stationary phase. When cultured in the same medium, the growth of M. haemolytica was inhibited when both bacteria were separated by a membrane that allowed contact (pore size, 8.0 µm) but not when they were separated by a membrane that limited contact (pore size, 0.4 µm). Lytic bacteriophages or bactericidal compounds could not be detected in the culture supernatant fluid from monocultures of P. multocida or from P. multocida-M. haemolytica cocultures. These results indicate that P. multocida inhibits the growth of M. haemolytica by a contact- or proximity-dependent mechanism. If the inhibition of growth of M. haemolytica by P. multocida occurs in vivo as well, it could explain the inconsistent isolation of M. haemolytica from the lungs of pneumonic BHS.
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Antibiosis , Mannheimia haemolytica/efectos de los fármacos , Mannheimia haemolytica/crecimiento & desarrollo , Pasteurella multocida/crecimiento & desarrollo , Pasteurella multocida/metabolismo , Neumonía Bacteriana/veterinaria , Enfermedades de las Ovejas/microbiología , Animales , Adhesión Bacteriana , Bacteriófagos/aislamiento & purificación , Medios de Cultivo/química , Mannheimia haemolytica/aislamiento & purificación , Pasteurella multocida/aislamiento & purificación , Neumonía Bacteriana/microbiología , Ovinos , Borrego Cimarrón/microbiologíaAsunto(s)
Oveja Doméstica/genética , Simportadores/genética , Animales , Oveja Doméstica/sangre , Sodio/sangreRESUMEN
The global importance of the hemoparasite Theileria haneyi to equine health was recently shown by its resistance to imidocarb dipropionate (ID) and its interference with T. equi clearance by ID in some co-infected horses. Genetic characterization of T. haneyi revealed marked genomic reduction compared to T. equi, and initial experiments demonstrated reduced clinical severity in spleen-intact horses. Furthermore, in early experiments, splenectomized horses survived T. haneyi infection and progressed to an asymptomatic carrier state, in stark contrast to the high fatality rate of T. equi in splenectomized horses. Thus, we hypothesized that T. haneyi is less virulent than T. equi. To objectively assess virulence, clinical data from nine splenectomized, T. haneyi-infected horses were evaluated and compared to published data on T. equi-infected, splenectomized horses. Seven of eight splenectomized, T. haneyi-infected horses survived. Further, in six horses co-infected with T. equi and T. haneyi, only horses cleared of T. equi by ID survived splenectomy and became asymptomatic carriers. The reduced virulence of T. haneyi in splenectomized horses instructs why T. haneyi was, until recently, undetected. This naturally occurring comparative reduction in virulence in a natural host provides a foundation for defining virulence mechanisms of theileriosis and Apicomplexa in general.
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Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.
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Enfermedades de los Caballos , Theileria , Theileriosis , Vacunas , Animales , Bovinos , Epítopos , Enfermedades de los Caballos/diagnóstico , Enfermedades de los Caballos/prevención & control , Caballos , Merozoítos , Theileriosis/prevención & controlRESUMEN
A new pathogen strain can penetrate an immune host population only if it can escape immunity generated against the original strain. This model is best understood with influenza viruses, in which genetic drift creates antigenically distinct strains that can spread through host populations despite the presence of immunity against previous strains. Whether this selection model for new strains applies to complex pathogens responsible for endemic persistent infections, such as anaplasmosis, relapsing fever, and sleeping sickness, remains untested. These complex pathogens undergo rapid within-host antigenic variation by using sets of chromosomally encoded variants. Consequently, immunity is developed against a large repertoire of variants, dramatically changing the scope of genetic change needed for a new strain to evade existing immunity and establish coexisting infection, termed strain superinfection. Here, we show that the diversity in the alleles encoding antigenic variants between strains of a highly antigenically variant pathogen was equal to the diversity within strains, reflecting equivalent selection for variants to overcome immunity at the host population level as within an individual host. This diversity among strains resulted in expression of nonoverlapping variants that allowed a new strain to evade immunity and establish superinfection. Furthermore, we demonstrated that a single distinct allele allows strain superinfection. These results indicate that there is strong selective pressure to increase the diversity of the variant repertoire beyond what is needed for persistence within an individual host and provide an explanation, competition at the host population level, for the large genomic commitment to variant gene families in persistent pathogens.
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Anaplasma marginale/genética , Variación Antigénica , Antígenos Bacterianos/inmunología , Antígenos Virales/inmunología , Genómica , Sobreinfección/genética , Virus/genética , Alelos , Anaplasma marginale/inmunología , Datos de Secuencia Molecular , Virus/inmunologíaRESUMEN
The apicomplexan hemoparasite, Theileria parva, causes East Coast fever (ECF), a frequently fatal disease of African cattle. Vaccine development has been impeded by incomplete understanding of protective immunity following natural exposure or the infection and treatment method (ITM) of immunization. This is attributable to a paucity of methods to characterize the memory T-cell repertoire following infection. To overcome this impediment, assays developed to study the immune response to other intracellular pathogens were adapted for use in studies with T. parva to enable definition of the phenotype and function of effector T cells in T. parva-immune cattle, facilitating vaccine development. As reported herein, stimulation of peripheral blood mononuclear cells (PBMC) from ITM-immunized steers with irradiated, autologous, T. parva-infected cell lines elicited a proliferative recall response comprised of CD45R0+/CCR7- CD4+ and CD8+ T cells. Subsequent co-incubation of stimulated cultures with infected cells demonstrated the presence of cytotoxic T cells (CTLs) with the ability to kill infected cells. Comparison of CTL activity in cultures depleted of CD4+ or CD8+ T cells demonstrated CTL activity was primarily attributed to CD8+ T cells. Importantly, stimulation of PBMC from vaccinated steers always elicited proliferation of CD4+ and CD8+ T cells. This was the first important observation obtained from the use of the assay described herein.
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Mycoplasma ovipneumoniae is a respiratory pathogen that impacts domestic sheep (Ovis aries; DS) and bighorn sheep (Ovis canadensis; BHS). BHS are reported to be more susceptible than DS to developing polymicrobial pneumonia associated with M. ovipneumoniae infection. Using formalin-fixed paraffin-embedded tissues, we performed a retrospective study investigating the pulmonary immune response of DS and BHS to M. ovipneumoniae infection. M. ovipneumoniae infected DS exhibited a more robust and well-organized BALT formation as compared to BHS. Digital analysis of immunohistochemical chromogen deposition in lung tissue was used to quantitate T cell marker CD3, B cell markers CD20 and CD79a, macrophage markers CD163 and Iba1, and cytokine IL-17. A significant interaction of species and infection status was identified for CD3, CD163, and IL-17. BHS had a greater increase in bronchiolar CD3 and bronchiolar and alveolar CD163 with infection, as compared to DS. BHS had an increase in bronchiolar associated lymph tissue (BALT) and alveolar IL-17 with infection, while these remained similar in DS regardless of infection status. IL-17 in respiratory epithelium of bronchi and bronchioles comparatively decreased in DS and increased in BHS with infection. These data begin to define the interspecies differential immune response to pulmonary M. ovipneumoniae infection in DS and BHS and provide the first investigations of respiratory epithelium-associated IL-17 in ovine.
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Neumonía por Mycoplasma , Enfermedades de las Ovejas , Borrego Cimarrón , Animales , Pulmón , Neumonía por Mycoplasma/veterinaria , Estudios Retrospectivos , Ovinos , Oveja DomésticaRESUMEN
Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and ß- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.
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Membrana Eritrocítica/metabolismo , Enfermedades de los Caballos/parasitología , Proteínas Protozoarias/inmunología , Proteínas Protozoarias/metabolismo , Theileria/patogenicidad , Theileriosis/parasitología , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Antígenos de Protozoos/inmunología , Proteínas Sanguíneas/metabolismo , Claudinas , Epítopos de Linfocito B/inmunología , Eritrocitos/parasitología , Enfermedades de los Caballos/inmunología , Caballos/sangre , Caballos/parasitología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Merozoítos/genética , Merozoítos/metabolismo , Pruebas de Neutralización , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Theileria/crecimiento & desarrollo , Theileria/inmunología , Theileria/metabolismo , Theileriosis/inmunologíaRESUMEN
The apicomplexan parasite Theileria haneyi is one of two known causative agents of equine theileriosis. It causes milder clinical disease than its more virulent counterpart, Theileria equi, in experimentally infected horses, and can superinfect T. equi-positive horses. The current equi merozoite antigen 1 (EMA1)-based competitive enzyme-linked immunosorbent assay (ELISA)used in the U.S. to detect equine theileriosis detects T. equi but not T. haneyi, and the complexity of molecular assays precludes widespread use for epidemiologic studies. In order to facilitate urgently needed studies on the prevalence of T. haneyi, the goal of this study was to develop a sensitive and specific serologic assay for the diagnosis of T. haneyi based on the equi merozoite antigen 11 (ThEMA11). To achieve this objective, ThEMA11 was recombinantly expressed in eukaryotic cells and its antigenicity assessed using sera from T. haneyi-experimentally infected horses. Confirmation of sera reactivity enabled design and optimization of an indirect ELISA. Specificity of the ELISA for T. haneyi was assessed using a cohort of sera from horses experimentally infected and confirmed PCR-positive for either T. equi or T. haneyi. Data from field samples further demonstrate that the ThEMA11 ELISA is capable of identifying T. haneyi antibodies in horses from multiple continents around the world.
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BACKGROUND: Vector-borne diseases pose an increasing threat to global food security. Vaccines, diagnostic tests, and therapeutics are urgently needed for tick-borne diseases that affect livestock. However, the inability to obtain significant quantities of pathogen stages derived from ticks has hindered research. In vitro methods to isolate pathogens from infected tick vectors are paramount to advance transcriptomic, proteomic, and biochemical characterizations of tick-borne pathogens. METHODS: Nymphs of Rhipicephalus appendiculatus were infected with Theileria parva by feeding on a calf during an acute infection. Isolation of sporozoites was accomplished by feeding infected adult ticks on an in vitro tick feeding system. Sporozoite viability was tested using in vitro bovine lymphocytes. RESULTS: We isolated infectious T. parva sporozoites secreted into an in vitro tick feeding system. Infected adult R. appendiculatus ticks attached to and successfully fed on silicone membranes in the in vitro tick feeding system. Bovine blood in the receptacle was replaced with cell-free medium and the ticks were allowed to feed for 3 h to collect secreted T. parva sporozoites. Secreted sporozoites infected in vitro bovine lymphocytes, demonstrating that isolated sporozoites remained viable and infectious. CONCLUSIONS: This work is the first to report the isolation of mature infectious T. parva sporozoites using an in vitro tick feeding system, which represents a significant step towards the development of a more efficient control strategy for T. parva. Isolation of infectious tick-stage parasites will facilitate the examination of the vector-pathogen interface, thereby accelerating the development of next-generation vaccines and treatment interventions for tick-borne pathogens.
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Rhipicephalus/parasitología , Theileria parva/fisiología , Animales , Interacciones Huésped-Parásitos , EsporozoítosRESUMEN
Anaplasma marginale subsp. centrale is a naturally attenuated subtype that has been used as a vaccine for a century. We sequenced the genome of this organism and compared it to those of virulent senso stricto A. marginale strains. The comparison markedly narrows the number of outer membrane protein candidates for development of a safer inactivated vaccine and provides insight into the diversity among strains of senso lato A. marginale.
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Anaplasma marginale/genética , Genoma Bacteriano/genética , Datos de Secuencia MolecularRESUMEN
This report describes the genome sequences of Mannheimia haemolytica serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated ovine (O), and the other from Bos taurus, designated bovine (B).