RESUMEN
Eastern equine encephalitis virus (EEEV) is one of the most virulent viruses endemic to North America. No licensed vaccines or antiviral therapeutics are available to combat this infection, which has recently shown an increase in human cases. Here, we characterize human monoclonal antibodies (mAbs) isolated from a survivor of natural EEEV infection with potent (<20 pM) inhibitory activity of EEEV. Cryo-electron microscopy reconstructions of two highly neutralizing mAbs, EEEV-33 and EEEV-143, were solved in complex with chimeric Sindbis/EEEV virions to 7.2 Å and 8.3 Å, respectively. The mAbs recognize two distinct antigenic sites that are critical for inhibiting viral entry into cells. EEEV-33 and EEEV-143 protect against disease following stringent lethal aerosol challenge of mice with highly pathogenic EEEV. These studies provide insight into the molecular basis for the neutralizing human antibody response against EEEV and can facilitate development of vaccines and candidate antibody therapeutics.
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Aerosoles/administración & dosificación , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Encefalitis Equina del Este/inmunología , Encefalomielitis Equina/inmunología , Encefalomielitis Equina/prevención & control , Adulto , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Microscopía por Crioelectrón , Modelos Animales de Enfermedad , Virus de la Encefalitis Equina del Este/ultraestructura , Encefalomielitis Equina/virología , Epítopos/química , Femenino , Glicoproteínas/inmunología , Humanos , Ratones , Modelos Moleculares , Mutagénesis/genética , Pruebas de Neutralización , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/inmunología , Virus Sindbis/inmunología , Virión/inmunología , Virión/ultraestructura , Internalización del VirusRESUMEN
Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.
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Prosencéfalo Basal/fisiopatología , Depresión/patología , Neuronas/patología , Animales , Reacción de Prevención , Prosencéfalo Basal/patología , Depresión/fisiopatología , Trastorno Depresivo Mayor/patología , Trastorno Depresivo Mayor/fisiopatología , Femenino , Técnicas In Vitro , Masculino , Mesencéfalo/metabolismo , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Parvalbúminas/metabolismoRESUMEN
Mammary organoid (MaO) models are only available for a few traditional model organisms, limiting our ability to investigate mammary gland development and cancer across mammals. This study established equine mammary organoids (EqMaOs) from cryopreserved mammary tissue, in which mammary tissue fragments were isolated and embedded into a 3D matrix to produce EqMaOs. We evaluated viability, proliferation and budding capacity of EqMaOs at different time points during culture, showing that although the number of proliferative cells decreased over time, viability was maintained and budding increased. We further characterized EqMaOs based on expression of stem cell, myoepithelial and luminal markers, and found that EqMaOs expressed these markers throughout culture and that a bilayered structure as seen in vivo was recapitulated. We used the milk-stimulating hormone prolactin to induce milk production, which was verified by the upregulation of milk proteins, most notably ß-casein. Additionally, we showed that our method is also applicable to additional non-traditional mammalian species, particularly domesticated animals such as cats, pigs and rabbits. Collectively, MaO models across species will be a useful tool for comparative developmental and cancer studies.
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Glándulas Mamarias Animales , Organoides , Animales , División Celular , Células Epiteliales/metabolismo , Femenino , Caballos , Lactancia , Mamíferos , Conejos , Células Madre , PorcinosRESUMEN
Interferons control viral infection by inducing the expression of antiviral effector proteins encoded by interferon-stimulated genes (ISGs). The field has mostly focused on identifying individual antiviral ISG effectors and defining their mechanisms of action. However, fundamental gaps in knowledge about the interferon response remain. For example, it is not known how many ISGs are required to protect cells from a particular virus, though it is theorized that numerous ISGs act in concert to achieve viral inhibition. Here, we used CRISPR-based loss-of-function screens to identify a markedly limited set of ISGs that confer interferon-mediated suppression of a model alphavirus, Venezuelan equine encephalitis virus (VEEV). We show via combinatorial gene targeting that three antiviral effectors-ZAP, IFIT3, and IFIT1-together constitute the majority of interferon-mediated restriction of VEEV, while accounting for < 0.5% of the interferon-induced transcriptome. Together, our data suggest a refined model of the antiviral interferon response in which a small subset of "dominant" ISGs may confer the bulk of the inhibition of a given virus.
Asunto(s)
Virus de la Encefalitis Equina Venezolana , Virus , Animales , Caballos , Interferones , Línea Celular , Replicación Viral , Antivirales/farmacología , Virus de la Encefalitis Equina Venezolana/fisiologíaRESUMEN
We report fatal West Nile virus (WNV) infection in a 7-year-old mare returning to the United Kingdom from Spain. Case timeline and clustering of virus sequence with recent WNV isolates suggest that transmission occurred in Andalusía, Spain. Our findings highlight the importance of vaccination for horses traveling to WNV-endemic regions.
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Fiebre del Nilo Occidental , Animales , Femenino , Análisis por Conglomerados , Caballos , España/epidemiología , Reino Unido/epidemiología , Fiebre del Nilo Occidental/diagnóstico , Fiebre del Nilo Occidental/veterinariaRESUMEN
Herpesviruses establish a well-adapted balance with their host's immune system. Despite this co-evolutionary balance, infections can lead to severe disease including neurological disorders in their natural host. In horses, equine herpesvirus 1 (EHV-1) causes respiratory disease, abortions, neonatal foal death and myeloencephalopathy (EHM) in ~10â% of acute infections worldwide. Many aspects of EHM pathogenesis and protection from EHM are still poorly understood. However, it has been shown that the incidence of EHM increases to >70â% in female horses >20 years of age. In this study we used old mares as an experimental equine EHV-1 model of EHM to identify host-specific factors contributing to EHM. Following experimental infection with the neuropathogenic strain EHV-1 Ab4, old mares and yearling horses were studied for 21 days post-infection. Nasal viral shedding and cell-associated viremia were assessed by quantitative PCR. Cytokine/chemokine responses were evaluated in nasal secretions and cerebrospinal fluid (CSF) by Luminex assay and in whole blood by quantitative real-time PCR. EHV-1-specific IgG sub-isotype responses were measured by ELISA. All young horses developed respiratory disease and a bi-phasic fever post-infection, but only 1/9 horses exhibited ataxia. In contrast, respiratory disease was absent in old mares, but all old mares developed EHM that resulted in euthanasia in 6/9 old mares. Old mares also presented significantly decreased nasal viral shedding but higher viremia coinciding with a single fever peak at the onset of viremia. According to clinical disease manifestation, horses were sorted into an EHM group (nine old horses and one young horse) and a non-EHM group (eight young horses) for assessment of host immune responses. Non-EHM horses showed an early upregulation of IFN-α (nasal secretions), IRF7/IRF9, IL-1ß, CXCL10 and TBET (blood) in addition to an IFN-γ upregulation during viremia (blood). In contrast, IFN-α levels in nasal secretions of EHM horses were low and peak levels of IRF7, IRF9, CXCL10 and TGF-ß (blood) coincided with viremia. Moreover, EHM horses showed significantly higher IL-10 levels in nasal secretions, peripheral blood mononuclear cells and CSF and higher serum IgG3/5 antibody titres compared to non-EHM horses. These results suggest that protection from EHM depends on timely induction of type 1 IFN and upregulation cytokines and chemokines that are representative of cellular immunity. In contrast, induction of regulatory or TH-2 type immunity appeared to correlate with an increased risk for EHM. It is likely that future vaccine development for protection from EHM must target shifting this 'at-risk' immunophenotype.
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Citocinas , Infecciones por Herpesviridae , Herpesvirus Équido 1 , Enfermedades de los Caballos , Animales , Caballos , Herpesvirus Équido 1/inmunología , Femenino , Enfermedades de los Caballos/virología , Enfermedades de los Caballos/inmunología , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Citocinas/sangre , Citocinas/inmunología , Anticuerpos Antivirales/sangre , Esparcimiento de Virus , Viremia/inmunología , Viremia/veterinaria , Inmunoglobulina G/sangreRESUMEN
Equine rotavirus species A (ERVA) G3P[12] and G14P[12] are two dominant genotypes that cause foal diarrhoea with a significant economic impact on the global equine industry. ERVA can also serve as a source of novel (equine-like) rotavirus species A (RVA) reassortants with zoonotic potential as those identified previously in 2013-2019 when equine G3-like RVA was responsible for worldwide outbreaks of severe gastroenteritis and hospitalizations in children. One hurdle to ERVA research is that the standard cell culture system optimized for human rotavirus replication is not efficient for isolating ERVA. Here, using an engineered cell line defective in antiviral innate immunity, we showed that both equine G3P[12] and G14P[12] strains can be rapidly isolated from diarrhoeic foals. The genome sequence analysis revealed that both G3P[12] and G14P[12] strains share the identical genotypic constellation except for VP7 and VP6 segments in which G3P[12] possessed VP7 of genotype G3 and VP6 of genotype I6 and G14P[12] had the combination of VP7 of genotype G14 and VP6 of genotype I2. Further characterization demonstrated that two ERVA genotypes have a limited cross-neutralization. The lack of an in vitro broad cross-protection between both genotypes supported the increased recent diarrhoea outbreaks due to equine G14P[12] in foals born to dams immunized with the inactivated monovalent equine G3P[12] vaccine. Finally, using the structural modelling approach, we provided the genetic basis of the antigenic divergence between ERVA G3P[12] and G14P[12] strains. The results of this study will provide a framework for further investigation of infection biology, pathogenesis and cross-protection of equine rotaviruses.
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Antígenos Virales , Diarrea , Genotipo , Enfermedades de los Caballos , Infecciones por Rotavirus , Rotavirus , Animales , Caballos , Rotavirus/genética , Rotavirus/inmunología , Rotavirus/aislamiento & purificación , Rotavirus/clasificación , Infecciones por Rotavirus/veterinaria , Infecciones por Rotavirus/virología , Infecciones por Rotavirus/inmunología , Enfermedades de los Caballos/virología , Enfermedades de los Caballos/inmunología , Diarrea/virología , Diarrea/veterinaria , Antígenos Virales/genética , Antígenos Virales/inmunología , Genoma Viral/genética , Filogenia , Línea CelularRESUMEN
IMPORTANCE: Alphavirus replicons are being developed as self-amplifying RNAs aimed at improving the efficacy of mRNA vaccines. These replicons are convenient for genetic manipulations and can express heterologous genetic information more efficiently and for a longer time than standard mRNAs. However, replicons mimic many aspects of viral replication in terms of induction of innate immune response, modification of cellular transcription and translation, and expression of nonstructural viral genes. Moreover, all replicons used in this study demonstrated expression of heterologous genes in cell- and replicon's origin-specific modes. Thus, many aspects of the interactions between replicons and the host remain insufficiently investigated, and further studies are needed to understand the biology of the replicons and their applicability for designing a new generation of mRNA vaccines. On the other hand, our data show that replicons are very flexible expression systems, and additional modifications may have strong positive impacts on protein expression.
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Alphavirus , Regulación Viral de la Expresión Génica , Interacciones Microbiota-Huesped , Replicón , Proteínas Virales , Alphavirus/genética , Alphavirus/metabolismo , Vacunas de ARNm/genética , Replicón/genética , Replicación Viral/genética , ARN Viral/biosíntesis , ARN Viral/genética , Interacciones Microbiota-Huesped/genética , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.
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Virus de la Influenza A , Gripe Aviar , Gripe Humana , Proteínas Nucleares , Animales , Perros , Humanos , Ratones , Proteínas de Ciclo Celular/metabolismo , Caballos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Gripe Aviar/genética , Gripe Humana/genética , Mamíferos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
BACKGROUND: Menopause is associated with elevated cardiovascular risk due to the loss of the cardioprotective effect of oestrogens. Postmenopausal women are often prescribed hormone replacement therapy (HRT) in order to control menopause symptoms and correct hormone imbalances; however, HRT can impact serum lipids' concentrations. At present, data on the effect of the administration of medroxyprogesterone acetate plus conjugated equine oestrogens (MPACEE) on the lipid profile in females are uncertain, as the investigations conducted so far have produced conflicting results. Thus, we aimed to clarify the impact of MPACEE prescription on the serum lipids' values in women by means of a systematic review and meta-analysis of randomized controlled trials (RCTs). METHODS: We employed a random-effects model based on the DerSimonian and Laird method to determine the combined estimates of the intervention's impact on the lipid profile. The computation of the weighted mean difference (WMD) and its corresponding 95% confidence interval (CI) relied on the mean and standard deviation values from both the MPACEE and control group, respectively. RESULTS: A total of 53 RCTs were included in the meta-analysis with 68 RCT arms on total cholesterol (TC), 70 RCT arms on low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG), and 69 RCT arms on high-density lipoprotein cholesterol (HDL-C). Administration of MPACEE resulted in a significant reduction of TC (WMD = -11.93 mg/dL; 95% CI: -13.42, -10.44; p < .001) and LDL-C (WMD = -16.61 mg/dL; 95% CI: -17.97, -15.26; p < .001) levels, and a notable increase in HDL-C (WMD = 3.40 mg/dL; 95% CI: 2.93, 3.86; p < .001) and TG (WMD = 10.28 mg/dL; 95% CI: 7.92, 12.64; p < .001) concentrations. Subgroup analysis revealed that changes in the lipid profile were influenced by several factors: body mass index (for TC, HDL-C, TG), MPACEE dosages (for TC, LDL-C, HDL-C, TG), age (for TC, LDL-C, HDL-C, TG), durations of the intervention (for TC, LDL-C, HDL-C, TG), continuous/sequential administration of MPACEE (continuous for TC; sequential for LDL-C, TG) administration of MPACEE and serum lipids' concentrations before enrolment in the RCT (for TC, LDL-C, HDL-C, TG). CONCLUSIONS: MPACEE administration can influence serum lipids' concentrations in females by raising HDL-C and TG levels and reducing LDL-C and TC values. Therefore, postmenopausal women who suffer from hypercholesterolaemia might benefit from this type of HRT.
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HDL-Colesterol , LDL-Colesterol , Estrógenos Conjugados (USP) , Acetato de Medroxiprogesterona , Ensayos Clínicos Controlados Aleatorios como Asunto , Triglicéridos , Femenino , Acetato de Medroxiprogesterona/farmacología , Acetato de Medroxiprogesterona/administración & dosificación , Humanos , Estrógenos Conjugados (USP)/farmacología , Estrógenos Conjugados (USP)/administración & dosificación , Triglicéridos/sangre , HDL-Colesterol/efectos de los fármacos , HDL-Colesterol/sangre , LDL-Colesterol/efectos de los fármacos , LDL-Colesterol/sangre , Colesterol/sangre , Lípidos/sangre , Terapia de Reemplazo de Estrógeno/métodos , Posmenopausia/efectos de los fármacos , Persona de Mediana EdadRESUMEN
Variation in mesenchymal stromal cell (MSC) function depending on their origin is problematic, as it may confound clinical outcomes of MSC therapy. Current evidence suggests that the therapeutic benefits of MSCs are attributed to secretion of biologically active factors (secretome). However, the effect of donor characteristics on the MSC secretome remains largely unknown. Here, we examined the influence of donor age, sex, and tissue source, on the protein profile of the equine MSC secretome. We used dynamic metabolic labeling with stable isotopes combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify secreted proteins in MSC conditioned media (CM). Seventy proteins were classified as classically secreted based on the rate of label incorporation into newly synthesized proteins released into the extracellular space. Next, we analyzed CM of bone marrow- (nâ =â 14) and adipose-derived MSCs (nâ =â 16) with label-free LC-MS/MS. Clustering analysis of 314 proteins detected across all samples identified tissue source as the main factor driving variability in MSC CM proteomes. Linear modelling applied to the subset of 70 secreted proteins identified tissue-related difference in the abundance of 23 proteins. There was an age-related decrease in the abundance of CTHRC1 and LOX, further validated with orthogonal techniques. Due to the lack of flow cytometry characterization of MSC surface markers, the analysis could not account for the potential effect of cell population heterogeneity. This study provides evidence that tissue source and donor age contribute to differences in the protein composition of MSC secretomes which may influence the effects of MSC therapy.
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Células Madre Mesenquimatosas , Secretoma , Animales , Caballos , Espectrometría de Masas en Tándem , Cromatografía Liquida , Médula Ósea/metabolismo , Células Madre Mesenquimatosas/metabolismo , Medios de Cultivo Condicionados/farmacologíaRESUMEN
BACKGROUND AIMS: The prevalence of chronic wounds continues to be a burden in human medicine. Methicillin-resistant Staphylococcus aureus (MRSA) is commonly isolated from infected wounds. MRSA infections primarily delay healing by impairing local immune cell functions. This study aimed to investigate the potential of mesenchymal stromal cell (MSC)-secreted bioactive factors, defined as the secretome, to improve innate immune responses in vivo. MSCs were isolated from the bone marrow of horses, which serve as valuable translational models for wound healing. The MSC secretome, collected as conditioned medium (CM), was evaluated in vivo using mouse models of acute and MRSA-infected skin wounds. METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load. RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds. CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.
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Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.
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Enfermedades de los Caballos , Células Madre Pluripotentes Inducidas , Fiebre del Nilo Occidental , Virus del Nilo Occidental , Animales , Caballos , Humanos , Fiebre del Nilo Occidental/veterinaria , Fiebre del Nilo Occidental/epidemiología , Encéfalo , Antivirales/farmacología , Antivirales/uso terapéutico , Enfermedades de los Caballos/tratamiento farmacológicoRESUMEN
Equine influenza virus (EIV) remains a threat to horses, despite the availability of vaccines. Strategies to monitor the virus and prevent potential vaccine failure revolve around serological assays, RT-qPCR amplification, and sequencing the viral hemagglutinin (HA) and neuraminidase (NA) genes. These approaches overlook the contribution of other viral proteins in driving virulence. This study assesses the potential of long-read nanopore sequencing for fast and precise sequencing of circulating equine influenza viruses. Therefore, two French Florida Clade 1 strains, including the one circulating in winter 2018-2019 exhibiting more pronounced pathogenicity than usual, as well as the two currently OIE-recommended vaccine strains, were sequenced. Our results demonstrated the reliability of this sequencing method in generating accurate sequences. Sequence analysis of HA revealed a subtle antigenic drift in the French EIV strains, with specific substitutions, such as T163I in A/equine/Paris/1/2018 and the N188T mutation in post-2015 strains; both substitutions were in antigenic site B. Antigenic site E exhibited modifications in post-2018 strains, with the N63D substitution. Segment 2 sequencing also revealed that the A/equine/Paris/1/2018 strain encodes a longer variant of the PB1-F2 protein when compared to other Florida clade 1 strains (90 amino acids long versus 81 amino acids long). Further biological and biochemistry assays demonstrated that this PB1-F2 variant has enhanced abilities to abolish the mitochondrial membrane potential ΔΨm and permeabilize synthetic membranes. Altogether, our results highlight the interest in rapidly characterizing the complete genome of circulating strains with next-generation sequencing technologies to adapt vaccines and identify specific virulence markers of EIV.
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Enfermedades de los Caballos , Subtipo H3N8 del Virus de la Influenza A , Infecciones por Orthomyxoviridae , Vacunas , Animales , Aminoácidos/genética , Genómica , Caballos , Subtipo H3N8 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/veterinaria , Reproducibilidad de los Resultados , Análisis de Secuencia/veterinaria , Factores de VirulenciaRESUMEN
Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.
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Interleucina-13 , Nematodos , Animales , Caballos , Interleucina-13/metabolismo , Interleucina-4 , Células Caliciformes , Mucosa IntestinalRESUMEN
Epizootic lymphangitis (EL) is a highly prevalent and contagious infectious disease affecting horses in many parts of Ethiopia caused by Histoplasma capsulatum sensu lato ('var. farciminosum'). In this study, 12 suspected isolates of H. capsulatum sensu lato or yeasts unidentified by conventional biochemical tests isolated from Ethiopian horses with EL were characterised by ITS sequencing. Six of the 12 isolates were identified to be members of H. capsulatum sensu lato and the other six were Pichia kudriavzevii (synonym: Candida krusei) (n = 3), Trichosporon asahii (n = 1), Geotrichum silvicola (n = 1) and Moesziomyces aphidis (n = 1), respectively. The six H. capsulatum sensu lato isolates were further characterised by multilocus sequence analysis. Four distinct gene loci [arf (462 bases), H-anti (410 bases), ole1 (338 bases) and tub1 (272 bases)] of these six isolates as well as those of two H. capsulatum sensu lato ('var. farciminosum') reference strains (ATCC 58332 and ATCC 28798) were PCR-amplified and sequenced. Phylogenetic analyses of their concatenated nucleotide sequences showed that three of the isolates and the reference strain ATCC 58332 were identical and belonged to the Eurasia clade within Latin American (LAm) A (H. suramericanum), and those of the other three isolates and the reference strain ATCC 28798 were identical and belonged to the Africa clade. At least two distinct phylogenetic clades of Histoplasma capsulatum sensu lato were circulating in Ethiopian horses with EL. Advanced molecular technologies and bioinformatics tools are crucial for accurate identification and typing of pathogens as well as discovery of novel microorganisms in veterinary microbiology.
Using multilocus sequence analysis with four concatenated housekeeping gene loci, at least two distinct phylogenetic clades, namely Eurasia clade and Africa clade, of Histoplasma capsulatum sensu lato were confirmed to be circulating in Ethiopian horses with epizootic lymphangitis.
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The purpose of this study was to examine kinematic and neuromuscular responses of the head and body to pelvis perturbations with different intensities and frequencies during sitting astride in children with CP. Sixteen children with spastic CP (mean age 7.4 ± 2.4 years old) were recruited in this study. A custom designed cable-driven robotic horse was used to apply controlled force perturbations to the pelvis during sitting astride. Each participant was tested in four force intensity conditions (i.e., 10%, 15%, 20%, and 25% of body weight (BW), frequency = 1 Hz), and six force frequency conditions (i.e., 0.5 Hz, 1 Hz, 1.5 Hz, 2 Hz, 2.5 Hz, and 3 Hz, intensity = 20% of BW). Each testing session lasted for one minute with a one-minute rest break inserted between two sessions. Kinematic data of the head, trunk, and legs were recorded using wearable sensors, and EMG signals of neck, trunk, and leg muscles were recorded. Children with CP showed direction-specific trunk and neck muscle activity in response to the pelvis perturbations during sitting astride. Greater EMG activities of trunk and neck muscles were observed for the greater intensities of force perturbations (P < .05). Participants also showed enhanced activation of antagonistic muscles rather than direction-specific trunk and neck muscle activities for the conditions of higher frequency perturbations (P < .05). Children with CP may modulate trunk and neck muscle activities in response to greater changes in intensity of pelvis perturbation during sitting astride. Perturbations with too high frequency may be less effective in inducing direction-specific trunk and neck muscle activities.
Asunto(s)
Parálisis Cerebral , Postura , Sedestación , Niño , Preescolar , Humanos , Electromiografía , Músculo Esquelético/fisiología , Postura/fisiologíaRESUMEN
Equine infectious anemia (EIA) is a contagious disease of horses caused by the equine infectious anemia virus (EIAV). The clinical signs at the acute phase include intermittent high fever, thrombocytopenia, hemorrhage, edema, and anemia. The clinical signs at chronic and relapsing subclinical levels include emaciation and progressive weakness. Surviving horses become lifelong carriers because of the integration of the viral genome into that of the host, and these horses can produce and transmit the virus to other animals. This increases the difficulty of imposing practical control measures to prevent epidemics of this disease. Serological tests measuring the antibodies in equine sera are considered to be a reliable tool for the long-term monitoring of EIA. However, the standard serological tests for EIV either have low sensitivity (e.g., agar gel immunodiffusion test, AGID) or are time consuming to perform (e.g., ELISA and western blotting). The development of a rapid and simple method for detecting the disease is therefore critical to control the spread of EIA. In this study, we designed and developed a colloidal gold immunochromatographic (GICG) test strip to detect antibodies against EIAV based on the double-antigen sandwich. Both the p26 and gp45 proteins were used as the capture antigens, which may help to improve the positive detection rate of the strip. We found that the sensitivity of the test strip was 8 to 16 times higher than those of two commercially available ELISA tests and 128 to 256 times higher than AGID, but 8 to 16 times lower than that of western blotting. The strip has good specificity and stability. When serum samples from experimental horses immunized with the attenuated EIAV vaccine (n = 31) were tested, the results of the test strip showed 100% coincidence with those from NECVB-cELISA and 70.97% with AGID. When testing clinical serum samples (n = 1014), the test strip surprisingly provided greater sensitivity and a higher number of "true positive" results than other techniques. Therefore, we believe that the GICG test strip has demonstrated great potential in the field trials as a simple and effective tool for the detection of antibodies against EIAV. KEY POINTS: ⢠A colloidal gold immunochromatographic (GICG) fast test strip was developed with good specificity, sensitivity, stability, and repeatability ⢠The test strip can be used in point-of-care testing for the primary screening of EIAV antibodies ⢠Both the p26 and gp45 proteins were used as the capture antigens, giving a high positive detection rate in the testing of experimentally infected animal and field samples.
Asunto(s)
Virus de la Anemia Infecciosa Equina , Animales , Caballos , Anticuerpos Antivirales , Ensayo de Inmunoadsorción Enzimática , Western Blotting , Oro ColoideRESUMEN
BACKGROUND: Swimming has been used empirically for rehabilitation and conditioning of horses. However, due to challenges imposed by recording physiological parameters in water, the intensity of free swimming effort is unknown. OBJECTIVES: Measure the physiological workload associated with untethered swimming in horses. Five fit Arabian endurance horses were assessed while swimming in a 100 m-long indoor pool. Horses were equipped with a modified ergospirometry facemask to measure oxygen consumption (VÌO2) and ventilatory parameters (inspired/expired volumes, VI, VE; peak inspiratory/expiratory flows, PkVI, PkVE; respiratory frequency, Rf; minute ventilation, VE; inspiratory/expiratory durations and ratios, tI, tE, tI/ttot, tE/ttot); and an underwater electrocardiogram that recorded heart rate (HR). Postexercise venous blood lactate and ammonia concentrations were measured. Data are reported as median (interquartile ranges). RESULTS: Horses showed bradypnea (12 breaths/min (10-16)) for the first 30 s of swimming. VÌO2 during swimming was 43.2 ml/(kg.min) (36.0-56.6). Ventilatory parameters were: VI = 16.7 L (15.3-21.8), VE = 14.7 L (12.4-18.9), PkVI = 47.8 L/s (45.8-56.5), PkVE = 55.8 L/s (38.3-72.5), Rf = 31.4 breaths/min (20.0-33.8), VE = 522.9 L/min (414.7-580.0), tI = 0.5 s (0.5-0.6), tE = 1.2 s (1.1-1.6), tI/ttot = 0.3 (0.2-0.4), tE/ttot = 0.7 (0.6-0.8). Expiratory flow tracings showed marked oscillations that coincided with a vibrating expiratory sound. HR was 178.0 bpm (148.5-182.0), lactate = 1.5 mmol/L (1.0-1.9) and ammonia = 41.0 µmol/L (36.5-43.5). CONCLUSIONS: Free (untethered) swimming represents a submaximal, primarily aerobic exercise in horses. The breathing pattern during swimming is unique, with a relatively longer apneic period at the beginning of the exercise and an inspiratory time less than half that of expiration.
Asunto(s)
Frecuencia Cardíaca , Consumo de Oxígeno , Espirometría , Natación , Animales , Caballos/fisiología , Natación/fisiología , Consumo de Oxígeno/fisiología , Frecuencia Cardíaca/fisiología , Espirometría/veterinaria , Masculino , Condicionamiento Físico Animal/fisiología , Ácido Láctico/sangre , Femenino , Amoníaco/sangreRESUMEN
BACKGROUND: Studies on equine air transport practices and consequences are scarce. This prospective study aimed to describe horse and air journey details and practices, document how horse behavior and health changed during the air transport phases, quantify the occurrence of welfare issues, and identify possible associations between horse and journey details, air transport practices, and welfare issues. RESULTS: Data were collected from before departure to five days after arrival on 118/597 horses traveling on 32 commercial air journeys on different routes, varying in duration and conditions. Most horses were middle-aged warmblood females, 26% of which were pregnant, and being moved by air for sales. Before flying, most were quarantined (median: 18; IQR: 9-53 days), and their fitness for travel was certified by veterinarians. At the departure airports, external temperatures varied from - 6 °C to 33 °C, and horses were loaded by experienced flight grooms (median: 35; IQR: 15-40 years) into jet stalls (three-horse: 87%, two-horse: 13%). During the flights, horses were regularly watered (water intake median: 14 L) and fed ad libitum (feed consumption median: 8 kg). At the arrival airport, horses were unloaded from the jet stalls, and external temperatures ranged from - 5 °C to 32 °C. Then, all horses were transported to arrival quarantine by road. Air transport phases affected horses' health status and behavior; increased heart and respiratory rates and behaviors, such as pawing, head tossing, and vocalization, were mainly identified at departure and arrival. Horse interaction, nasal discharge, increased capillary refill time (CRT), and abnormal demeanor were observed more often one hour before landing while resting and normal capillary refill time were more often displayed five days after arrival (all P < 0.01). One hour before landing, horses with bad temperament and horses of unknown temperament were more likely to develop nasal discharge when transported in winter and autumn (P < 0.001). The likelihood of an increased CRT was associated with shorter flights in horses of unknown travel experience (P < 0.001). Ten horses were injured, and 11 developed pleuropneumonias (i.e., shipping fever). CONCLUSIONS: Air transport is a complex procedure with several different phases affecting horse health and behavior. Therefore, experienced staff should carefully manage each horse before, during, and after air journeys to minimize welfare hazards.