Equine Arteritis Virus Elicits a Mucosal Antibody Response in the Reproductive Tract of Persistently Infected Stallions

Equine arteritis virus (EAV) has the ability to establish persistent infection in the reproductive tract of the stallion (carrier) and is continuously shed in its semen. We have recently demonstrated that EAV persists within stromal cells and a subset of lymphocytes in the stallion accessory sex glands in the presence of a significant local inflammatory response. In the present study, we demonstrated that EAV elicits a mucosal antibody response in the reproductive tract during persistent infection with homing of plasma cells into accessory sex glands. The EAV-specific immunoglobulin isotypes in seminal plasma included IgA, IgG1, IgG3/5, and IgG4/7. Interestingly, seminal plasma IgG1 and IgG4/7 possessed virus-neutralizing activity, while seminal plasma IgA and IgG3/5 did not. However, virus-neutralizing IgG1 and IgG4/7 in seminal plasma were not effective in preventing viral infectivity. In addition, the serological response was primarily mediated by virus-specific IgM and IgG1, while virus-specific serum IgA, IgG3/5, IgG4/7, and IgG6 isotype responses were not detected. This is the first report characterizing the immunoglobulin isotypes in equine serum and seminal plasma in response to EAV infection. The findings presented herein suggest that while a broader immunoglobulin isotype diversity is elicited in seminal plasma, EAV has the ability to persist in the reproductive tract, in spite of local mucosal antibody and inflammatory responses. This study provides further evidence that EAV employs complex immune evasion mechanisms during persistence in the reproductive tract that warrant further investigation

Evaluation of mass spectrometric methods for detection of the anti-protozoal drug imidocarb

Imidocarb [N,N\u27-bis[3-(4,5-dihydro-1H-imidazol-2-yl)phenyl]urea, C19H20N6O1, m.w. 348.41] is a symmetrical carbanilide derivative used to treat disease caused by protozoans of the Babesia genus. Imidocarb, however, is also considered capable of suppressing Babesia-specific immune responses, allowing Babesia-positive horses to pass a complement fixation test (CFT) without eliminating the infection. This scenario could enable Babesia-infected horses to pass CFT-based importation tests. It is imperative to unequivocally identify and quantify equine tissue residues of imidocarb by mass spectrometry to address this issue. As a pretext to development of sensitive tissue assays, we have investigated possibilities of mass spectrometric (MS) detection of imidocarb. Our analyses disclosed that an unequivocal mass spectral analysis of imidocarb is challenging because of its rapid fragmentation under standard gas chromatography (GC)-MS conditions. In contrast, solution chemistry of imidocarb is more stable but involves distribution into mono- and dicationic species, m/z 349 and 175, respectively, in acid owing to the compound\u27s inherent symmetrical nature. Dicationic imidocarb was the preferred complex as viewed by either direct infusion-electrospray-MS or by liquid chromatography (LC)-MS. Dicationic imidocarb multiple reaction monitoring (MRM: m/z 175 → 162, 145, and 188) therefore offer the greatest opportunities for sensitive detection and LC-MS is more likely than GC-MS to yield a useful quantitative forensic analytical method for detecting imidocarb in horses

Clones of \u3cem\u3eStreptococcus zooepidemicus\u3c/em\u3e from Outbreaks of Hemorrhagic Canine Pneumonia and Associated Immune Responses

Acute hemorrhagic pneumonia caused by Streptococcus zooepidemicus has emerged as a major disease of shelter dogs and greyhounds. S. zooepidemicus strains differing in multilocus sequence typing (MLST), protective protein (SzP), and M-like protein (SzM) sequences were identified from 9 outbreaks in Texas, Kansas, Florida, Nevada, New Mexico, and Pennsylvania. Clonality based on 2 or more isolates was evident for 7 of these outbreaks. The Pennsylvania and Nevada outbreaks also involved cats. Goat antisera against acutely infected lung tissue as well as convalescent-phase sera reacted with a mucinase (Sz115), hyaluronidase (HylC), InlA domain-containing cell surface-anchored protein (INLA), membrane-anchored protein (MAP), SzP, SzM, and extracellular oligopeptide-binding protein (OppA). The amino acid sequences of SzP and SzM of the isolates varied greatly. The szp and szm alleles of the closely related Kansas clone (sequence type 129 [ST-129]) and United Kingdom isolate BHS5 (ST-123) were different, indicating that MLST was unreliable as a predictor of virulence phenotype. Combinations of conserved HylC and serine protease (ScpC) and variable SzM and SzP proteins of S. zooepidemicus strain NC78 were protectively immunogenic for mice challenged with a virulent canine strain. Thus, although canine pneumonia outbreaks are caused by different strains of S. zooepidemicus, protective immune responses were elicited in mice by combinations of conserved or variable S. zooepidemicus proteins from a single strain

Brewing of filter coffee

We report progress on mathematical modelling of coffee grounds in a drip filter coffee machine. The report focuses on the evolution of the shape of the bed of coffee grounds during extraction with some work also carried out on the chemistry of extraction. This work was sponsored by Philips who are interested in understanding an observed correlation between the final shape of the coffee grounds and the quality of the coffee. We used experimental data gathered by Philips and ourselves to identify regimes in the coffee brewing process and relevant regions of parameter space. Our work makes it clear that a number of separate processes define the shape of the coffee bed depending on the values of the parameters involved e.g. the size of the grains and the speed of fluid flow during extraction. We began work on constructing mathematical models of the redistribution of the coffee grounds specialised to each region and on a model of extraction. A variety of analytic and numerical tools were used. Furthermore our research has progressed far enough to allow us to begin to exploit connections between this problem and other areas of science, in particular the areas of sedimentology and geomorphology, where the processes we have observed in coffee brewing have been studied

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

Equine arteritis virus long-term persistence is orchestrated by CD8+ 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

Quantum Gates and Memory using Microwave Dressed States

Trapped atomic ions have been successfully used for demonstrating basic elements of universal quantum information processing (QIP). Nevertheless, scaling up of these methods and techniques to achieve large scale universal QIP, or more specialized quantum simulations remains challenging. The use of easily controllable and stable microwave sources instead of complex laser systems on the other hand promises to remove obstacles to scalability. Important remaining drawbacks in this approach are the use of magnetic field sensitive states, which shorten coherence times considerably, and the requirement to create large stable magnetic field gradients. Here, we present theoretically a novel approach based on dressing magnetic field sensitive states with microwave fields which addresses both issues and permits fast quantum logic. We experimentally demonstrate basic building blocks of this scheme to show that these dressed states are long-lived and coherence times are increased by more than two orders of magnitude compared to bare magnetic field sensitive states. This changes decisively the prospect of microwave-driven ion trap QIP and offers a new route to extend coherence times for all systems that suffer from magnetic noise such as neutral atoms, NV-centres, quantum dots, or circuit-QED systems.Comment: 9 pages, 4 figure

Downregulation of MicroRNA Eca-Mir-128 in Seminal Exosomes and Enhanced Expression of CXCL16 in the Stallion Reproductive Tract Are Associated with Long-Term Persistence of Equine Arteritis Virus

Equine arteritis virus (EAV) can establish long-term persistent infection in the reproductive tract of stallions and is shed in the semen. Previous studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S) and that persistent infection is maintained despite the presence of a local inflammatory and humoral and mucosal antibody responses. In this study, we demonstrated that equine seminal exosomes (SEs) are enriched in a small subset of microRNAs (miRNAs). Most importantly, we demonstrated that long-term EAV persistence is associated with the downregulation of an SE-associated miRNA (eca-mir-128) and with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. The findings presented here suggest that SE eca-mir-128 is implicated in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. This is a novel finding and warrants further investigation to identify its specific mechanism in modulating the CXCL16/CXCR6 axis in the reproductive tract of the EAV long-term carrier stallion

Equine Arteritis Virus Uses Equine CXCL16 as an Entry Receptor

Previous studies in our laboratory have identified equine CXCL16 (EqCXCL16) to be a candidate molecule and possible cell entry receptor for equine arteritis virus (EAV). In horses, the CXCL16 gene is located on equine chromosome 11 (ECA11) and encodes a glycosylated, type I transmembrane protein with 247 amino acids. Stable transfection of HEK-293T cells with plasmid DNA carrying EqCXCL16 (HEK-EqCXCL16 cells) increased the proportion of the cell population permissive to EAV infection from \u3c 3% to almost 100%. The increase in permissiveness was blocked either by transfection of HEK-EqCXCL16 cells with small interfering RNAs (siRNAs) directed against EqCXCL16 or by pretreatment with guinea pig polyclonal antibody against EqCXCL16 protein (Gp anti-EqCXCL16 pAb). Furthermore, using a virus overlay protein-binding assay (VOPBA) in combination with far-Western blotting, gradient-purified EAV particles were shown to bind directly to the EqCXCL16 protein in vitro. The binding of biotinylated virulent EAV strain Bucyrus at 4°C was significantly higher in HEK-EqCXCL16 cells than nontransfected HEK-293T cells. Finally, the results demonstrated that EAV preferentially infects subpopulations of horse CD14+ monocytes expressing EqCXCL16 and that infection of these cells is significantly reduced by pretreatment with Gp anti-EqCXCL16 pAb. The collective data from this study provide confirmatory evidence that the transmembrane form of EqCXCL16 likely plays a major role in EAV host cell entry processes, possibly acting as a primary receptor molecule for this virus

Identification of a Ruminant Origin Group B Rotavirus Associated with Diarrhea Outbreaks in Foals

Equine rotavirus group A (ERVA) is one of the most common causes of foal diarrhea. Starting in February 2021, there was an increase in the frequency of severe watery to hemorrhagic diarrhea cases in neonatal foals in Central Kentucky. Diagnostic investigation of fecal samples failed to detect evidence of diarrhea-causing pathogens including ERVA. Based on Illumina-based metagenomic sequencing, we identified a novel equine rotavirus group B (ERVB) in fecal specimens from the affected foals in the absence of any other known enteric pathogens. Interestingly, the protein sequence of all 11 segments had greater than 96% identity with group B rotaviruses previously found in ruminants. Furthermore, phylogenetic analysis demonstrated clustering of the ERVB with group B rotaviruses of caprine and bovine strains from the USA. Subsequent analysis of 33 foal diarrheic samples by RT-qPCR identified 23 rotavirus B-positive cases (69.69%). These observations suggest that the ERVB originated from ruminants and was associated with outbreaks of neonatal foal diarrhea in the 2021 foaling season in Kentucky. Emergence of the ruminant-like group B rotavirus in foals clearly warrants further investigation due to the significant impact of the disease in neonatal foals and its economic impact on the equine industry