Characterization of Nasal Mucosal T Cells in Horses and Their Response to Equine Herpesvirus Type 1.

Equine herpesvirus type 1 (EHV-1) enters through the upper respiratory tract (URT). Mucosal immunity at the URT is crucial in limiting viral infection and morbidity. Here, intranasal immune cells were collected from horses (n = 15) during an experimental EHV-1 infection. CD4+ and CD8+ T cells were the major intranasal cell populations before infection and increased significantly by day six and fourteen post-infection, respectively. Nasal mucosal T cells were further characterized in healthy horses. Compared to peripheral blood mononuclear cells (PBMC), mucosal CD8+ T-cell percentages were elevated, while CD4+ T-cell percentages were similar. A small population of CD4+CD8+ T cells was also recovered from mucosal samples. Within the URT tissue, CD4+ cells predominantly accumulated in the epithelial layer, while most CD8+ cells resided deeper in the mucosa or the submucosa below the basement membrane. In vitro stimulation of mucosal cells from healthy horses with (n = 5) or without (n = 5) peripheral T-cell immunity against EHV-1 induced IFN-γ production in nasal T cells upon polyclonal stimulation. However, after EHV-1 re-stimulation, mucosal T cells failed to respond with IFN-γ. This work provided the first characterization of mucosal T-cell phenotypes and functions in the URT of healthy horses and during EHV-1 infection.

Aspergillus fumigatus antigen-reactive Th17 cells are enriched in bronchoalveolar lavage fluid in severe equine asthma.

Introduction: Equine asthma (EA) is a common disease of adult horses with chronic respiratory pathology and common neutrophilic airway inflammation. It presents with hyperreactivity to hay dust components such as molds, and underlying dysregulated T cell responses have been suggested. Thus far, T cells have been analysed in EA with conflicting results and the antigen reactivity of T cells has not been demonstrated. Serological and epidemiological data point to the relevance of Aspergillus fumigatus as an antigen source in EA. Here, we aimed to identify and characterise Aspergillus antigen-reactive T cells in EA. Methods: Cryopreserved bronchoalveolar lavage cells (BALC) and peripheral blood mononuclear cells (PBMC) from healthy horses (HE, n=9) and those with mild-moderate (MEA, n=3) or severe asthma (SEA, n=8) were stimulated in vitro with the recombinant A. fumigatus antigens Asp f 1, or Asp f 7 combined with Asp f 8, to assess antigen reactivity, and with phorbol-12-myristat-13-acetate and ionomycin (P/i) to assess overall T cell reactivity. Stimulated cells were analysed by flow cytometry for CD4, CD8, IL-17, IL-4, and IFN-γ. Cytokine expression in all lymphocytes, and in CD4+ or CD8+ T cells, was quantified and compared between the groups. In BAL fluid (BALF), soluble cytokines and chemokines were quantified by bead-based assays. Results: Antigen restimulation of BALC with Asp f 1 or Asp f 7/8 provoked higher frequencies of IL-17+ lymphocytes, CD4+IL-17+ Th17 cells, and CD4+IL-4+ Th2 cells in SEA than in HE, whereas MEA and HE were similar. Antigen stimulation of PBMC did not result in group differences. P/i stimulation of BALC resulted in increased IL-17+ lymphocyte and CD4+IL-17+ Th17 cell frequencies in MEA compared with HE but the limited number of horses with MEA must be considered. P/i-stimulated PBMC from MEA or SEA contained more IL-17+ lymphocytes compared with HE. Cytokines were hardly detected in BALF and similar between the groups but CCL2 and CCL5 concentrations were increased in BALF from SEA or MEA, respectively, compared with HE. Conclusion: Horses with SEA have increased Aspergillus antigen-reactive Th17 cells in their airways, emphasising local T cell responses to this mold, which were quantified in EA for the first time here.

Immune horses rapidly increase antileukoproteinase and lack type I interferon secretion during mucosal innate immune responses against equine herpesvirus type 1.

Equine herpesvirus type 1 (EHV-1) is one of the most prevalent respiratory pathogens in horses with a high impact on animal health worldwide. Entry of the virus into epithelial cells of the upper respiratory tract and rapid local viral replication is followed by infection of local lymphoid tissues leading to cell-associated viremia and disease progression. Pre-existing mucosal immunity has previously been shown to reduce viral shedding and prevent viremia, consequently limiting severe disease manifestations. Here, nasopharyngeal transcriptomic profiling was used to identify differentially expressed genes following EHV-1 challenge in horses with different EHV-1 immune statuses. Immune horses (n = 4) did neither develop clinical disease nor viremia and did not shed virus after experimental infection, while non-immune horses (n = 4) did all the above. RNA sequencing was performed on nasopharyngeal samples pre- and 24 hours post-infection (24hpi). At 24hpi, 109 and 44 genes were upregulated in immune horses and non-immune horses, respectively, and three genes were explored in further detail. Antileukoproteinase (SLPI) gene expression increased 2.1-fold within 24 hours in immune horses in concert with protein secretion. Interferon (IFN)-induced proteins with tetratricopeptide repeats 2 (IFIT2) and 3 (IFIT3) were upregulated in non-immune horses, corresponding with nasal IFN-α secretion and viral replication. By contrast, neither IFIT expression nor IFN-α secretion was induced by EHV-1 infection of immune horses. Transcriptomic profiling offered a tool to identify, for the first time, the role of SLPI in innate immunity against EHV-1, and further emphasized the central role of the type I IFN response in the anti-viral defense of non-immune horses. IMPORTANCE: Equine herpesvirus type 1 (EHV-1) remains a considerable concern in the equine industry, with yearly outbreaks resulting in morbidity, mortality, and economic losses. In addition to its importance in equine health, EHV-1 is a respiratory pathogen and an alphaherpesvirus, and it may serve as a model for other viruses with similar pathogenicity or phylogeny. Large animal models allow the collection of high-volume samples longitudinally, permitting in-depth investigation of immunological processes. This study was performed on bio-banked nasopharyngeal samples from an EHV-1 infection experiment, where clinical outcomes had previously been determined. Matched nucleic acid and protein samples throughout infection permitted longitudinal quantification of the protein or related proteins of selected differentially expressed genes detected during the transcriptomic screen. The results of this manuscript identified novel innate immune pathways of the upper respiratory tract during the first 24 hours of EHV-1 infection, offering a first look at the components of early mucosal immunity that are indicative of protection.

Plasma concentration of thrombopoietin in dogs with immune thrombocytopenia.

BACKGROUND: Immune thrombocytopenia (ITP) is a common cause of severe thrombocytopenia in dogs. The pathogenesis of nonassociative, primary ITP (pITP) appears complex, with ill-defined thrombopoietic response. OBJECTIVES: Develop an immunoassay to measure plasma canine thrombopoietin (TPO) concentration and characterize TPO concentrations in dogs with pITP. ANIMALS: Forty-one healthy dogs, 8 dogs in an induced ITP model (3 control, 5 ITP), and 58 pITP dogs. METHODS: Recombinant canine TPO (rcTPO) was purchased and its identity confirmed by mass spectrometry. Monoclonal antibodies were raised to rcTPO and used to configure a sandwich ELISA using streptavidin-biotin detection. Assay performance, coefficients of variability, and healthy dog plasma TPO reference interval (RI) were determined, followed by assay of ITP samples. RESULTS: Assay dynamic range was 15 pg/mL (lower limit of detection) to 1000 pg/mL TPO, with limit of quantitation of 62 pg/mL. Plasma TPO RI was 0 to 158 pg/mL, with plasma TPO <62 pg/mL for 35/41 healthy dogs. All dogs with induced ITP developed marked increases in plasma TPO concentration. Peak values ranged from 515 to >6000 pg/mL. In contrast, only 2/58 pITP dogs had TPO values above RI. CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma TPO concentration is paradoxically low at diagnosis for most dogs with pITP. This finding suggests that ineffective thrombopoiesis contributes to thrombocytopenia in pITP dogs and supports evaluating TPO receptor agonist treatment as used for pITP in humans. The TPO assay provides a new tool to study thrombopoiesis in pITP and other thrombocytopenic syndromes in dogs.

Prostaglandin-related genes are differentially expressed in equine endometrium with different biopsy grade, degrees of inflammation, and fibrosis.

Prostaglandins have many roles in the equine reproductive tract, including but not limited to luteolysis, luteal support, ovulation, transport through the uterine tube, uterine contraction, embryonic mobility, inflammation, and fibrosis. Altered secretion of inflammatory proteins are likely to disrupt the balance of endometrial function and could impair fertility. Our overall goal was to measure the expression of several prostaglandin- and inflammation-related genes in mares with different degrees of endometrial histological changes. Our hypothesis was that mares with neutrophilic and lymphocytic plasmocytic inflammation, fibrosis, or different biopsy grades would have altered concentrations of prostaglandin E2 (PGE2) and F2α (PGF2α), as well as altered expression of inflammation- and prostaglandin-related genes, compared to mares with minimal to no histological changes on biopsy evaluation. Forty-five endometrial biopsies from estrous mares were assessed by a reproductive pathologist for the degree of neutrophilic inflammation, lymphocytic and plasmocytic inflammation, and fibrosis, and a biopsy grade was assigned based on the Kenney-Doig system. A low-volume uterine lavage was collected from a subset of twenty-six mares prior to biopsy collection and was used to measure PGE2 and PGF2α concentrations via ELISA. Total RNA was extracted from biopsies and mRNA expression was evaluated for twenty-five genes of interest. A restricted maximum likelihood linear model was used to compare differences of mRNA expression, with a statistical significance set at P < 0.05. There was no difference in the abundance of PGE2 or PGF2α between any of the variables tested. Mares with endometrial biopsy grade I had lower expression of NF-kB, PTGS1 and HPGD compared to grade IIA or IIB (P < 0.05). Mares with neutrophilic inflammation had decreased expression of NF-kB, PTGS1, PTGER4, CBR1, mPGES2 and PTGIS compared to mares without inflammation. Mares with mild or minimal endometrial fibrosis had increased expression of mPGES2 and PTGIS, compared to mares with moderate endometrial fibrosis. In conclusion, several genes were identified to be differentially expressed in mares with histological changes compared to mares with no to minimal histological changes. The presence of inflammation and fibrosis may alter the concentration of prostaglandins in endometrial tissue, which could impair many of the uterine reproductive and immune functions during estrus, affecting early embryo survival.

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.

Introduction: Equine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF). Methods: Serum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A. fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays. Results: MEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig. Discussion: A. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

Monoclonal antibodies for equine IL-1ß enable the quantification of mature IL-1ß in horses.

Interleukin-1ß (IL-1ß) is one of the key mediators of inflammation during innate immune responses. Mature bioactive IL-1ß mediates essential host defense mechanisms but also has a mechanistic role in several autoinflammatory and degenerative diseases. In horses, specific and sensitive assays for IL-1ß are crucial for immunological research on inflammatory processes and diseases. In this article, we describe the development of four monoclonal antibodies (mAbs) against equine IL-1ß. The specificity of the new IL-1ß mAbs was confirmed using a panel of equine recombinant cytokines and chemokines. The mAbs were validated for detection of native mature IL-1ß in a fluorescent bead-based assay and for staining of IL-1ß-producing immune cells by flow cytometry. The bead-based assay for equine IL-1ß had a linear quantification range between 60 pg/ml to 960 ng/ml. Horse peripheral blood mononuclear cells (PBMC) secreted IL-1ß after lipopolysaccharide (LPS) stimulation in time and dose dependent manner as quantified by the new equine IL-1ß bead-based assay. A comparison of two commercial equine IL-1ß ELISA kits with the new IL-1ß fluorescent bead-based assay revealed that the bead-based assay improved the quantification of native equine IL-1ß in LPS stimulated PBMC supernatants by detecting it with high intensity and a broad linear quantification range, while both ELISAs resulted in low signals and poor native IL-1ß recognition. Intracellular staining and flow cytometric analysis confirmed that the main cellular source of IL-1ß in equine PBMC after LPS stimulation were CD14+ monocytes. IL-1ß secretion from PBMC was inhibited by a caspase inhibitor but protein translation within the cells was not, supporting the accumulation of pro-IL-1ß within the cells even when proteolytic cleavage for IL-1ß activation is missing. This confirmed the importance of specific mAbs for analyzing the biologically active, mature IL-1ß in horses.

Monoclonal antibodies for equine CD25 improve detection of regulatory T cells in horses.

CD25, the interleukin-2 receptor α-chain, is expressed on cell surfaces of different immune cells and is commonly used for phenotyping of regulatory T cells (Tregs). CD25 has essential roles in the maintenance of hemostasis and immune tolerance and Treg cell involvement has been shown in human diseases and murine models for allergy, autoimmunity, cancer, chronic inflammation, and many others. In horses, a cross-reactive anti-human CD25 antibody has previously been used for characterizing Tregs. Here, we developed monoclonal antibodies (mAbs) to equine CD25 and compared their staining pattern with the anti-human CD25 antibody by flow cytometry. The comparison of the two reagents was performed by two separate analyses in independent laboratories. Overall, similar staining patterns for equine peripheral blood lymphocytes were obtained with the anti-human CD25 antibody and equine CD25 mAb 15-1 in both laboratories. Both reagents identified comparable CD4+CD25+ and CD4+CD25+FOXP3+ percentages after stimulation of peripheral blood mononuclear cells (PBMC) with pokeweed mitogen. However, when compared to the anti-human CD25 antibody, the equine CD25 mAb 15-1 resulted in a better staining intensity of the equine CD25+ cells and increased the percentages of Tregs and other CD25+ cells ex vivo and after culturing of PBMC without stimulation. In summary, the equine CD25 mAbs provide new, improved reagents for Tregs and CD25+ cell phenotyping in horses.

Neonatal and maternal upregulation of antileukoproteinase in horses.

Introduction: The end of gestation, ensuing parturition, and the neonatal period represent highly dynamic phases for immunological changes in both mother and offspring. The regulation of innate immune cells at the maternal-fetal interface during late term pregnancy, after birth, and during microbial colonization of the neonatal gut and other mucosal surfaces, is crucial for controlling inflammation and maintaining homeostasis. Innate immune cells and mucosal epithelial cells express antileukoproteinase (SLPI), which has anti-inflammatory and anti-protease activity that can regulate cellular activation. Methods: Here, we developed and validated new monoclonal antibodies (mAbs) to characterize SLPI for the first time in horses. Peripheral blood and mucosal samples were collected from healthy adults horses and a cohort of mares and their foals directly following parturition to assess this crucial stage. Results: First, we defined the cell types producing SLPI in peripheral blood by flow cytometry, highlighting the neutrophils and a subset of the CD14+ monocytes as SLPI secreting immune cells. A fluorescent bead-based assay was developed with the new SLPI mAbs and used to establish baseline concentrations for secreted SLPI in serum and secretion samples from mucosal surfaces, including saliva, nasal secretion, colostrum, and milk. This demonstrated constitutive secretion of SLPI in a variety of equine tissues, including high colostrum concentrations. Using immunofluorescence, we identified production of SLPI in mucosal tissue. Finally, longitudinal sampling of clinically healthy mares and foals allowed monitoring of serum SLPI concentrations. In neonates and postpartum mares, SLPI peaked on the day of parturition, with mares returning to the adult normal within a week and foals maintaining significantly higher SLPI secretion until three months of age. Conclusion: This demonstrated a physiological systemic change in SLPI in both mares and their foals, particularly at the time around birth, likely contributing to the regulation of innate immune responses during this critical period.

Monoclonal antibody development advances immunological research in horses.

Host immune analyses require specific reagents to identify cellular and soluble components of the immune system. These immune reagents are often species-specific. For horses, various immunological tools have been developed and tested by different initiatives during the past decades. This article summarizes the development of well characterized monoclonal antibodies (mAbs) for equine immune cells, immunoglobulin isotypes, cytokines, and chemokines.

Equine herpesvirus type 1 (EHV-1) replication at the upper respiratory entry site is inhibited by neutralizing EHV-1-specific IgG1 and IgG4/7 mucosal antibodies.

Equine herpesvirus type 1 (EHV-1) is a contagious respiratory pathogen that infects the mucosa of the upper respiratory tract (URT). Mucosal immune responses at the URT provide the first line of defense against EHV-1 and are crucial for orchestrating immunity. To define host-pathogen interactions, we characterized B-cell responses, antibody isotype functions, and EHV-1 replication of susceptible (non-immune) and clinically protected (immune) horses after experimental EHV-1 infection. Nasal secretion and nasal wash samples were collected and used for the isolation of DNA, RNA, and mucosal antibodies. Shedding of infectious virus, EHV-1 copy numbers, viral RNA expression, and host B-cell activation in the URT were compared based on host immune status. Mucosal EHV-1-specific antibody responses were associated with EHV-1 shedding and viral RNA transcription. Finally, mucosal immunoglobulin G (IgG) and IgA isotypes were purified and tested for neutralizing capabilities. IgG1 and IgG4/7 neutralized EHV-1, while IgG3/5, IgG6, and IgA did not. Immune horses secreted high amounts of mucosal EHV-1-specific IgG4/7 antibodies and quickly upregulated B-cell pathway genes, while EHV-1 was undetected by virus isolation and PCR. RNA transcription analysis reinforced incomplete viral replication in immune horses. In contrast, complete viral replication with high viral copy numbers and shedding of infectious viruses was characteristic for non-immune horses, together with low or absent EHV-1-specific neutralizing antibodies during viral replication. These data confirm that pre-existing mucosal IgG1 and IgG4/7 and rapid B-cell activation upon EHV-1 infection are essential for virus neutralization, regulation of viral replication, and mucosal immunity against EHV-1.IMPORTANCEEquine herpesvirus type 1 (EHV-1) causes respiratory disease, abortion storms, and neurologic outbreaks known as equine herpes myeloencephalopathy (EHM). EHV-1 is transmitted with respiratory secretions by nose-to-nose contact or via fomites. The virus initially infects the epithelium of the upper respiratory tract (URT). Host-pathogen interactions and mucosal immunity at the viral entry site provide the first line of defense against the EHV-1. Robust mucosal immunity can be essential in protecting against EHV-1 and to reduce EHM outbreaks. It has previously been shown that immune horses do not establish cell-associated viremia, the prerequisite for EHM. Here, we demonstrate how mucosal antibodies can prevent the replication of EHV-1 at the epithelium of the URT and, thereby, the progression of the virus to the peripheral blood. The findings improve the mechanistic understanding of mucosal immunity against EHV-1 and can support the development of enhanced diagnostic tools, vaccines against EHM, and the management of EHV-1 outbreaks.

Optimization of a bovine cytokine multiplex assay using a new bovine and cross-reactive equine monoclonal antibodies.

Cytokines are important markers for immune activation, regulation, and homeostasis. The lack of monoclonal antibodies (mAbs) and sensitive assays to evaluate cytokine secretion has hindered research of bovine inflammation and immune regulation. We recently developed a fluorescent bead-based multiplex assay (multiplex assay) for bovine IL-10, TNF-α, and IFN-γ. Although the original assay covers a broad concentration range for the 3 targets, analytical sensitivity for IL-10 and IFN-γ could be improved to facilitate detection of these cytokines in their physiological low pg/mL range. To optimize the multiplex assay, we generated a new bovine IL-10 mAb and explored its use for the detection of intracellular and secreted bovine IL-10. The new bovine IL-10 mAb 130 recognized recombinant bovine IL-10 fusion protein and did not react with the fusion protein tag, or the TNF-α and IFN-γ standards in the multiplex assay. For improving IFN-γ detection, we explored cross-reactivity of anti-equine IFN-γ mAbs by intracellular staining of bovine stimulated peripheral blood mononuclear cells (PBMC). Equine IFN-γ mAb 3 showed excellent cross-reactivity with bovine IFN-γ by intracellular detection. Adding IL-10 mAb 130 and IFN-γ mAb 3 to the bovine multiplex assay substantially improved the analytical sensitivity with lower limits of detection in the low pg/mL range for all analytes. The detection ranges for the optimized multiplex assay were determined as 2 - 134,000 pg/mL for IL-10, 8 - 127,000 pg/mL for IFN-γ, and 12 - 193,000 pg/mL for TNF-α. The assay was next used to measure cytokine concentrations in cell culture supernatants from PBMC stimulated in plasma from whole blood stimulation to confirm native IL-10, TNF-α, and IFN-γ recognition and to explore the upper detection limits of the assay. In PBMC stimulation with a mix of phorbol myristate acetate (PMA) and ionomycin resulted in highest cytokine concentrations, while in plasma from whole blood stimulation, highest concentrations were observed in samples stimulated with a mix of lipopolysaccharide (LPS), phytohemagglutinin (PHA), and the TLR-2/6 agonist Pam2Csk4. PBMC and whole blood stimulation protocols showed that the optimized multiplex assay covers a wide linear detection range for measuring cytokine concentrations in bovine samples. For whole blood stimulation, a cocktail of pathogen associated molecular patterns elicited a stronger cytokine response than a mix of PMA and ionomycin, but response varied considerably between individual cattle. In conclusion, optimizing the bovine cytokine assay with new reagents improved the lower detection limits and widened the linear detection ranges while lowering the background of the multiplex assay.

Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.

Introduction: Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA. Methods: Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses. Results and discussion: For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

SARS-COV-2 SEROSURVEY OF HEALTHY, PRIVATELY OWNED CATS PRESENTING TO A NEW YORK CITY ANIMAL HOSPITAL IN THE EARLY PHASE OF THE COVID-19 PANDEMIC (2020-2021).

SARS-CoV-2, the cause of the ongoing COVID-19 pandemic, not only infects humans but is also known to infect various species, including domestic and wild animals. While many species have been identified as susceptible to SARS-CoV-2, there are limited studies on the prevalence of SARS-CoV-2 in animals. Both domestic and non-domestic cats are now established to be susceptible to infection by SARS-CoV-2. While serious disease in cats may occur in some instances, the majority of infections appear to be subclinical. Differing prevalence data for SARS-CoV-2 infection of cats have been reported, and are highly context-dependent. Here, we report a retrospective serological survey of cats presented to an animal practice in New York City, located in close proximity to a large medical center that treated the first wave of COVID-19 patients in the U.S. in the Spring of 2020. We sampled 79, mostly indoor, cats between June 2020 to May 2021, the early part of which time the community was under a strict public health "lock-down". Using a highly sensitive and specific fluorescent bead-based multiplex assay, we found an overall prevalence of 13/79 (16%) serologically-positive animals for the study period; however, cats sampled in the Fall of 2020 had a confirmed positive prevalence of 44%. For SARS-CoV-2 seropositive cats, we performed viral neutralization test with live SARS-CoV-2 to additionally confirm presence of SARS-CoV-2 specific antibodies. Of the thirteen seropositive cats, 7/13 (54%) were also positive by virus neutralization, and two of seropositive cats had previously documented respiratory signs, with high neutralization titers of 1/1024 and 1/4096; overall however, there was no statistically significant association of SARS-CoV-2 seropositivity with respiratory signs, or with breed, sex or age of the animals. Follow up sampling of cats showed that positive serological titers were maintained over time. In comparison, we found an overall confirmed positive prevalence of 51% for feline coronavirus (FCoV), an endemic virus of cats, with 30% confirmed negative for FCoV. We demonstrate the impact of SARS-CoV-2 in a defined feline population during the first wave of SARS-CoV-2 infection of humans, and suggest that human-cat transmission was substantial in our study group. Our study provide a new context for SARS-CoV-2 transmission events across species.

Representative Public Health Surveys Pose Several Challenges: Lessons Learned Across 9 Communities During the COVID-19 Pandemic.

Community surveillance surveys offer an opportunity to obtain important and timely public health information that may help local municipalities guide their response to public health threats. The objective of this paper is to present approaches, challenges, and solutions from SARS-CoV-2 surveillance surveys conducted in different settings by 2 research teams. For rapid assessment of a representative sample, a 2-stage cluster sampling design was developed by an interdisciplinary team of researchers at Oregon State University between April 2020 and June 2021 across 6 Oregon communities. In 2022, these methods were adapted for New York communities by a team of veterinary, medical, and public health practitioners. Partnerships were established with local medical facilities, health departments, COVID-19 testing sites, and health and public safety staff. Field staff were trained using online modules, field manuals describing survey methods and safety protocols, and in-person meetings with hands-on practice. Private and secure data integration systems and public awareness campaigns were implemented. Pilot surveys and field previews revealed challenges in survey processes that could be addressed before surveys proceeded. Strong leadership, robust trainings, and university-community partnerships proved critical to successful outcomes. Cultivating mutual trust and cooperation among stakeholders is essential to prepare for the next pandemic.

Inflammatory markers for differentiation of endometritis in the mare.

BACKGROUND: Endometrial biopsy is required to diagnose mares with chronic endometritis and endometrial degenerative fibrosis. An increase in understanding of equine reproductive immunology could be utilised to create less-invasive, time-efficient diagnostic tools especially when evaluating mares for chronic endometritis. OBJECTIVES: To evaluate inflammatory cytokine and chemokine concentrations in uterine fluid samples collected by low-volume lavage (LVL) as a potential screening diagnostic biomarker for endometritis. STUDY DESIGN: Prospective cross-sectional clinical study. METHODS: Forty-six mares underwent a LVL and subsequently endometrial biopsy. Mares were split in three groups: healthy, acute endometritis, and chronic endometrial fibrosis (CEF) based on cytological and histological evaluation. A fluorescent bead-based multiplex assay for IFN-γ, IFN-α, IL-1ß, IL-4, IL-10, IL-17, sCD14, TNF-α, CCL2, CCL3, CCL5 and CCL11 were carried out on the LVL fluid. The endometrial biopsy was utilised for histology and qPCR of IFN-γ, IL-1ß, IL-6, IL-8, IL-17, TNF-α, CCL2 and CCL3 genes. Statistical analyses examined differences in inflammatory markers and predictive modelling for diseased endometrium. RESULTS: Secreted concentrations of IFN-γ were lower in LVL fluid from reproductively healthy mares compared with acute endometritis (p = 0.04) and CEF (p = 0.006). Additionally, IL-17, IL-10, IL-1ß, TNF-α, CCL2, CCL3, CCL5 and CCL11 were significantly increased (p ≤ 0.04) in LVL from CEF mares compared with healthy mares. Mares with CCL2 concentrations ≥550 pg/mL (14/14) had 100% probability of having CEF and/or acute endometritis. Healthy mares had lower relative abundance of IL-17 mRNA compared with mares in CEF group [median (interquartile rage) = 14.76 (13.3, 15.3) and 12.4 (10.54, 13.81)], respectively (p = 0.02). MAIN LIMITATIONS: Limited sample size: larger numbers of mares with and without endometritis are required and reference intervals in LVL samples have to be established. CONCLUSIONS: Inflammatory chemokines and cytokines concentrations differed between healthy mares and mares with acute endometritis or CEF in LVL.

Comparison of antibody and antigen response to intranasal and intramuscular EHV-1 modified-live vaccination in healthy adult horses.

During neurological EHV-1 outbreaks, modified-live vaccines (MLV) are often administrated intranasally in an off-label fashion to healthy cohort horses in order to achieve rapid mucosal immunity. Thus, the goal of the present study was to determine if a commercially available EHV-1 MLV given intranasally to healthy horses would trigger a measurable systemic and/or mucosal antibody response. Eight healthy adult horses were given the EHV-1 MLV vaccine intranasally, while 8 healthy adult horses received the vaccine intramuscularly. An additional 8 healthy horses served as unvaccinated controls. EHV-1 specific antibodies (total IgG, IgG4/7, IgG1 and IgA) were measured in blood and nasal secretions prior to vaccine administration and 14- and 30-days post-vaccine administration. Further, nasal secretions and whole blood were tested for the presence of EHV-1 DNA by qPCR prior to and 5 days after vaccine administration. EHV-1 was detected by qPCR for the first 48 hours post-intranasal vaccine administration in nasal secretions in a total of three horses. Total EHV-1 IgG and IgG4/7 antibody values in serum increased only in horses receiving the intramuscular MLV. Antibody values at 14- and 30-days post vaccine administration were not different from values prior to vaccine administration in horses receiving the intranasal vaccine. The results support the intramuscular use of the EHV-1 MLV as recommended by the manufacturer. Intranasal vaccination with the study-specific EHV-1 MLV did not induce an increase in systemic or nasal antibodies, therefore, this vaccine route seems suboptimal and should not be used to vaccinate adult horses that have received multiple EHV-1 vaccinations and have pre-existing antibodies against EHV-1.

IgE-binding monocytes upregulate the coagulation cascade in allergic horses.

IgE-binding monocytes are a rare peripheral immune cell type involved in the allergic response through binding of IgE on their surface. IgE-binding monocytes are present in both healthy and allergic individuals. We performed RNA sequencing to ask how the function of IgE-binding monocytes differs in the context of allergy. Using a large animal model of allergy, equine Culicoides hypersensitivity, we compared the transcriptome of IgE-binding monocytes in allergic and non-allergic horses at two seasonal timepoints: (i) when allergic animals were clinical healthy, in the winter "Remission Phase", and (ii) during chronic disease, in the summer "Clinical Phase". Most transcriptional differences between allergic and non-allergic horses occurred only during the "Remission Phase", suggesting principal differences in monocyte function even in the absence of allergen exposure. F13A1, a subunit of fibrinoligase, was significantly upregulated at both timepoints in allergic horses. This suggested a role for increased fibrin deposition in the coagulation cascade to promote allergic inflammation. IgE-binding monocytes also downregulated CCR10 expression in allergic horses during the "Clinical Phase", suggesting a defect in maintenance of skin homeostasis, which further promotes allergic inflammation. Together, this transcriptional analysis provides valuable clues into the mechanisms used by IgE-binding monocytes in allergic individuals.

A linear SARS-CoV-2 DNA vaccine candidate reduces virus shedding in ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused more than 760 million cases and over 6.8 million deaths as of March 2023. Vaccination has been the main strategy used to contain the spread of the virus and to prevent hospitalizations and deaths. Currently, two mRNA-based vaccines and one adenovirus-vectored vaccine have been approved and are available for use in the U.S. population. The versatility, low cost, and rapid production of DNA vaccines provide important advantages over other platforms. Additionally, DNA vaccines efficiently induce both B- and T-cell responses by expressing the antigen within transfected host cells, and the antigen, after being processed into peptides, can associate with MHC class I or II of antigen-presenting cells (APCs) to stimulate different T cell responses. However, the efficiency of DNA vaccination needs to be improved for use in humans. Importantly, in vivo DNA delivery combined with electroporation (EP) has been used successfully in the field of veterinary oncology, resulting in high rates of response after electrochemotherapy. Here, we evaluate the safety, immunogenicity, and protective efficacy of a novel linear SARS-CoV-2 DNA vaccine candidate delivered by intramuscular injection followed by electroporation (Vet-ePorator™) in ferrets. The linear SARS-CoV-2 DNA vaccine candidate did not cause unexpected side effects. Additionally, the vaccine elicited neutralizing antibodies and T cell responses on day 42 post-immunization using a low dose of the linear DNA construct in a prime-boost regimen. Most importantly, vaccination significantly reduced shedding of infectious SARS-CoV-2 through oral and nasal secretions in a ferret model.

IgE+ plasmablasts predict the onset of clinical allergy.

Introduction: IgE+ plasmablasts develop following allergen exposure and B cell activation. They secrete IgE and therefore are directly linked to maintain the mechanisms of IgE-mediated allergies. Here, we show that the presence of IgE+ plasmablasts in peripheral blood not only coincides with clinical allergy, but also predicts the upcoming development of clinical disease. Methods: Using an equine model of naturally occurring allergy, we compared the timing of allergen exposure, arrival of IgE+ plasmablasts in peripheral blood, and onset of clinical disease. Results: We found that IgE+ plasmablasts predict the development of clinical allergy by at least 3 weeks and can be measured directly by flow cytometry or by IgE secretion following in vitro culture. We also compared the IgE secretion by IgE+ plasmablasts with total plasma IgE concentrations and found that while IgE secretion consistently correlates with clinical allergy, total plasma IgE does not. Discussion: Together, we describe IgE+ plasmablasts as a reliable and sensitive predictive biomarker of allergic disease development.