Development and Evaluation of a One-Step Multiplex Real-Time TaqMan\u3csup\u3e®\u3c/sup\u3e RT-qPCR Assay for the Detection and Genotyping of Equine G3 and G14 Rotaviruses in Fecal Samples

Background: Equine rotavirus A (ERVA) is the leading cause of diarrhea in neonatal foals and has a negative impact on equine breeding enterprises worldwide. Among ERVA strains infecting foals, the genotypes G3P[12] and G14P[12] are the most prevalent, while infections by strains with other genomic arrangements are infrequent. The identification of circulating strains of ERVA is critical for diagnostic and surveillance purposes, as well as to understand their molecular epidemiology. Current genotyping methods available for ERVA and rotaviruses affecting other animal species rely on Sanger sequencing and are significantly time-consuming, costly and labor intensive. Here, we developed the first one-step multiplex TaqMan® real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes for the rapid detection and G-typing directly from fecal specimens. Methods: A one-step multiplex TaqMan® RT-qPCR assay targeting the NSP3 and VP7 genes of ERVA G3 and G14 genotypes was designed. The analytical sensitivity was assessed using serial dilutions of in vitro transcribed RNA containing the target sequences while the analytical specificity was determined using RNA and DNA derived from a panel of group A rotaviruses along with other equine viruses and bacteria. The clinical performance of this multiplex assay was evaluated using a panel of 177 fecal samples and compared to a VP7-specific standard RT-PCR assay and Sanger sequencing. Limits of detection (LOD), sensitivity, specificity, and agreement were determined. Results: The multiplex G3 and G14 VP7 assays demonstrated high specificity and efficiency, with perfect linearity. A 100-fold difference in their analytical sensitivity was observed when compared to the singleplex assays; however, this difference did not have an impact on the clinical performance. Clinical performance of the multiplex RT-qPCR assay demonstrated that this assay had a high sensitivity/specificity for every target (100% for NSP3, \u3e 90% for G3 VP7 and \u3e 99% for G14 VP7, respectively) and high overall agreement (\u3e 98%) compared to conventional RT-PCR and sequencing. Conclusions: This new multiplex RT-qPCR assay constitutes a useful, very reliable tool that could significantly aid in the rapid detection and G-typing of ERVA strains circulating in the field

Mieloencefalopatía por Herpesvirus Equino 1, una amenaza para la hípica mundial

Ante las noticias que nos han llegado a través de los medios y de colegas que se desempeñan en Europa, sobre el grave y extendido brote de enfermedad neurológica por la infección con Herpesvirus equino 1 (HVE-1) nos hacemos las siguientes preguntas...Fil: Carossino, Mariano. Universidad del Salvador. Escuela de Veterinaria; Argentina. Louisiana Tech University; Estados UnidosFil: Vissani, María Aldana. Universidad del Salvador. Escuela de Veterinaria; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; ArgentinaFil: Barrandeguy, María Edith. Universidad del Salvador. Escuela de Veterinaria; Argentina. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Virologia E Innovaciones Tecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Virologia E Innovaciones Tecnologicas.; Argentin

Steroidogenic Enzyme and Steroid Receptor Expression in the Equine Accessory Sex Glands

The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3β-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD), P450,17α hydroxylase, 17–20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-β, and 3βHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p \u3e 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p \u3c 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3β-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed

Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare

Background: The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. Results: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR. Conclusion: This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse

Kinetics of the Chromosome 14 MicroRNA Cluster Ortholog and Its Potential Role During Placental Development in the Pregnant Mare

Background: The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. Results: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR. Conclusion: This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse

Relevamiento de la ocurrencia de síndromes clínicos en caninos y felinos en la práctica veterinaria

The information available regarding disease occurrence in companion animals is limited. The purpose of this study is to generate a surveillance system that would allow estimating the incidence of certain diseases in companion animals as well as identifying gaps in the diagnostic methodology that could lead to the development of rapid diagnostic techniques to use in clinical settings. From a total of 167 submitted forms from 5 veterinary clinics, 140 where canine and 27 feline cases. The distribution by dog breed included 48 mixed—breed, 14 Poodles, 8 Golden Retriever, 7 Pitbull, 7 German Shepherds, 7 Labrador, 7 Boxers, and other minor breeds. From the canine cases, a 40 % where included in a vaccination program and 44 % had anti-helminthic treatment under veterinary supervision. A 30 % have had both. Among the reasons for attending the veterinary clinic, we found 27 % associated with a gastrointestinal syndrome, 11 % genito-urinary, 28 % dermatological, 6 % ophthalmological, 17 % cardio-respiratory, 7 % tumoral, and 4 % neurological. The gastrointestinal cases were primarily associated with diarrhea, including parasitic disease and presumptive parvoviral and canine viral hepatitis infections. Regarding the feline cases, 11 were mixed-breed, 5 Siamese, 9 European, 1 Persian, and 1 Burmese. A 22 % had current vaccinations (triple and rabies) and 44% had anti-helminthic treatment under veterinary supervision. Only 15 % have had both. Among the reasons for attending the veterinary clinic, we found 46 % due to genito-urinary syndrome, 7 % gastrointestinal, 26 % cardio-respiratory, 8 % dermatological, 7 % ophthalmological and 6 % tumoral. A single case presented both gastrointestinal and dermatological signs. The data collected here demonstrates that only a small percentage of companion animals (<50 %) are under vaccination and anti-helminthic programs monitored by veterinarians. This highlights the need for intensifying the outreach and educational efforts to pet owners for the prevention of zoonotic diseases.La información sobre la frecuencia de enfermedades en animales de compañía es muy limitada. El propósito del presente proyecto es generar un sistema de vigilancia epidemiológica que permita determinar la incidencia/prevalencia de ciertas patologías en animales de compañía e identificar aquellos síndromes en los que hacen falta mayores esfuerzos para el desarrollo de técnicas de diagnóstico rápido y aplicable en la clínica diaria. De las 167 encuestas realizadas en 5 clínicas veterinarias, 140 fueron consultas caninas y 27 felinas. La distribución por raza en caninos incluyó 48 mestizos, 14 Caniches, 8 Golden Retriever, 7 Pitbull, 7 Ovejeros Alemanes, 7 Labradores, 7 Boxers, y los restantes correspondían a variadas razas. El 40 % estaban incluidos en un programa de vacunación y un 44 % contaban con tratamiento antiparasitario controlado. Un 30 % contaba con ambos. El motivo de la consulta incluyó: 27 % por problemas digestivos, 11 % genito-urinarios, 28 % dermatológicos, 6 % oftálmicos, 17 % cardio-respiratorios, 7 % tumorales y 4 % neurológicos. Los signos digestivos estuvieron asociados mayoritariamente al síndrome diarreico, incluyendo parasitosis y presunta parvovirosis y hepatitis viral canina. En cuanto a los felinos, 11 eran mestizos, 5 Siameses, 9 Común Europeo, 1 Persa y 1 Sagrado de Birmania. El 22 % tenían registro de vacunaciones al día (triple felina y antirrábica) y el 44 % un programa de desparasitación controlado. Un 15 % contaban con ambos. El motivo de la consulta incluyó: 46 % por problemas genitourinarios, 7 % por trastornos digestivos, 26 % cardiorespiratorios, 8 % dermatológicos, 7 % oftálmicos y 6 % oncológicos. Un único caso presentó signología digestiva y dermatológica simultáneamente. Los datos obtenidos demuestran que solo un pequeño porcentaje de animales de compañía (<50 %) se encuentran incluidos en programas de vacunación y desparasitación controlada, lo que evidencia la necesidad de intensificar los programas de extensión sobre tenencia responsable de mascotas, considerando la existencia de enfermedades zoonóticas

Expression profile of the chromosome 14 Microrna Cluster (C14MC) ortholog in equine maternal circulation throughout pregnancy and its potential implications

Equine chromosome 24 microRNA cluster (C24MC), the ortholog of human C14MC, is a pregnancy-related miRNA cluster. This cluster is believed to be implicated in embryonic, fetal, and placental development. The current study aimed to characterize the expression profile of this cluster in maternal circulation throughout equine gestation. The expression profile of miRNAs belonging to this cluster was analyzed in the serum of non-pregnant (diestrus), pregnant (25 d, 45 d, 4 mo, 6 mo, 10 mo), and postpartum mares. Among the miRNAs examined, 11 miRNAs were differentially expressed across the analyzed time-points. Four of these miRNAs (eca-miR-1247-3p, eca-miR-134-5p, eca-miR-382-5p, and eca-miR-433-3p) were found to be enriched in the serum of pregnant mares at Day 25 relative to non-pregnant mares. To further assess the accuracy of these miRNAs in differentiating pregnant (25 d) from non-pregnant mares, receiver operating characteristic (ROC) analysis was performed for each of these miRNAs, revealing that eca-miR-1247-3p and eca-miR-134-5p had the highest accuracy (AUCROC = 0.92 and 0.91, respectively; p &lt; 0.05). Moreover, eca-miR-1247-3p, eca-miR-134-5p, eca-miR-409-3p, and eca-miR-379-5p were enriched in the serum of Day 45 pregnant mares. Among those miRNAs, eca-miR-1247-3p and eca-miR-409-3p retained the highest accuracy as shown by ROC analysis. GO analysis revealed that these miRNAs are mainly implicated in nervous system development as well as organ development. Using in situ hybridization, we localized eca-miR-409-3p in the developing embryo (25 d) and extra-embryonic membranes (25 and 45 d). In conclusion, the present study is the first to elucidate the circulating maternal profile of C24MC-associated miRNAs throughout pregnancy and to suggest that serum eca-miR-1247-3p, eca-miR-134-5p, and eca-miR-409-3p could be used as pregnancy-specific markers during early gestation (25 and 45 d). Overall, the high abundance of these embryo-derived miRNAs in the maternal circulation suggests an embryo-maternal communication during the equine early pregnancy

Landscape of overlapping gene expression in the equine placenta

Increasing evidence suggests that overlapping genes are much more common in eukaryotic genomes than previously thought. These different-strand overlapping genes are potential sense-antisense (SAS) pairs, which might have regulatory effects on each other. In the present study, we identified the SAS loci in the equine genome using previously generated stranded, paired-end RNA sequencing data from the equine chorioallantois. We identified a total of 1261 overlapping loci. The ratio of the number of overlapping regions to chromosomal length was numerically higher on chromosome 11 followed by chromosomes 13 and 12. These results show that overlapping transcription is distributed throughout the equine genome, but that distributions differ for each chromosome. Next, we evaluated the expression patterns of SAS pairs during the course of gestation. The sense and antisense genes showed an overall positive correlation between the sense and antisense pairs. We further provide a list of SAS pairs with both positive and negative correlation in their expression patterns throughout gestation. This study characterizes the landscape of sense and antisense gene expression in the placenta for the first time and provides a resource that will enable researchers to elucidate the mechanisms of sense/antisense regulation during pregnancy

Viral infections in horses in Argentina : an overview based on laboratory results

Inst.de VirologíaFil: Vissani, Aldana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Olguin Perglione, Cecilia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Miño, Samuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Carossino, Mariano. University of Kentucky. Department of Veterinary Science. Maxwell H. Gluck Equine Research Center; Estados Unidos. Universidad del Salvador. Escuela de Veterinaria; Argentina.Fil: Micheloud, Juan Francisco. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Salta. Grupo de Trabajo de Patología, Epidemiología e Investigación Diagnóstica. Área de Sanidad Animal; ArgentinaFil: Russo, S. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Departamento de Rabia y Enfermedades de Pequeños Animales, Virología Dilab-SENASA; ArgentinaFil: Tordoya, Maria Silvia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología: ArgentinaFil: Becerra, María Luciana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; ArgentinaFil: Zabal, Osvaldo Alfredo. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología: Argentina. Universidad del Salvador. Escuela de Veterinaria. Cátedra de Enfermedades Infecciosas; ArgentinaFil: Barrandeguy, Maria Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología; Argentina. Universidad del Salvador. Escuela de Veterinaria. Cátedra de Enfermedades Infecciosas; Argentin

Equine Arteritis Virus Long-Term Persistence Is Orchestrated by CD8\u3csup\u3e+\u3c/sup\u3e T Lymphocyte Transcription Factors, Inhibitory Receptors, and the CXCL16/CXCR6 Axis

Equine arteritis virus (EAV) has the unique ability to establish long-term persistent infection in the reproductive tract of stallions and be sexually transmitted. Previous studies showed that long-term persistent infection is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistence is maintained despite the presence of local inflammatory and humoral and mucosal antibody responses. Here, we performed transcriptomic analysis of the ampullae, the primary site of EAV persistence in long-term EAV carrier stallions, to understand the molecular signatures of viral persistence. We demonstrated that the local CD8+ T lymphocyte response is predominantly orchestrated by the transcription factors eomesodermin (EOMES) and nuclear factor of activated T-cells cytoplasmic 2 (NFATC2), which is likely modulated by the upregulation of inhibitory receptors. Most importantly, EAV persistence is associated with an enhanced expression of CXCL16 and CXCR6 by infiltrating lymphocytes, providing evidence of the implication of this chemokine axis in the pathogenesis of persistent EAV infection in the stallion reproductive tract. Furthermore, we have established a link between the CXCL16 genotype and the gene expression profile in the ampullae of the stallion reproductive tract. Specifically, CXCL16 acts as a “hub” gene likely driving a specific transcriptional network. The findings herein are novel and strongly suggest that RNA viruses such as EAV could exploit the CXCL16/CXCR6 axis in order to modulate local inflammatory and immune responses in the male reproductive tract by inducing a dysfunctional CD8+ T lymphocyte response and unique lymphocyte homing in the reproductive tract