Seroepidemiological Survey of West Nile Virus Infections in Horses from Berlin/Brandenburg and North Rhine-Westphalia, Germany.

Following the introduction of the West Nile virus (WNV) into eastern Germany in 2018, increasing infections have been diagnosed in birds, equines, and humans over time, while the spread of WNV into western Germany remained unclear. We screened 437 equine sera from 2018 to 2020, excluding vaccinated horses, collected from convenience sampled patients in the eastern and western parts of Germany, for WNV-specific antibodies (ELISAs followed by virus/specific neutralization tests) and genomes (RT-qPCRs). Clinical presentations, final diagnoses, and demographic data were also recorded. In the eastern part, a total of eight horses were found WNV seropositive in 2019 (seroprevalence of 8.16%) and 27 in 2020 (13.77%). There were also two clinically unsuspected horses with WNV-specific antibodies in the western part from 2020 (2.63%), albeit travel history-related infections could not be excluded. None of the horse sera contained WNV-specific genomes. Eight horses in eastern Germany carried WNV-IgM antibodies, but only four of these showed typical clinical signs. These results underline the difficulty of detecting a WNV infection in a horse solely based on clinical signs. Thus, WNV circulation is established in the horse population in eastern Germany, but not yet in the western part.

Effects of azithromycin on bronchial remodeling in the natural model of severe neutrophilic asthma in horses.

Steroid resistance in asthma has been associated with neutrophilic inflammation and severe manifestations of the disease. Macrolide add-on therapy can improve the quality of life and the exacerbation rate in refractory cases, possibly with greater effectiveness in neutrophilic phenotypes. The mechanisms leading to these beneficial effects are incompletely understood and whether macrolides potentiate the modulation of bronchial remodeling induced by inhaled corticosteroids (ICS) is unknown. The objective of this study was to determine if adding azithromycin to ICS leads to further improvement of lung function, airway inflammation and bronchial remodeling in severe asthma. The combination of azithromycin (10 mg/kg q48h PO) and inhaled fluticasone (2500 µg q12h) was compared to the sole administration of fluticasone for five months in a randomized blind trial where the lung function, airway inflammation and bronchial remodeling (histomorphometry of central and peripheral airways and endobronchial ultrasound) of horses with severe neutrophilic asthma were assessed. Although the proportional reduction of airway neutrophilia was significantly larger in the group receiving azithromycin, the lung function and the peripheral and central airway smooth muscle mass decreased similarly in both groups. Despite a better control of airway neutrophilia, azithromycin did not potentiate the other clinical effects of fluticasone.

First clinical expression of equine insect bite hypersensitivity is associated with co-sensitization to multiple Culicoides allergens.

BACKGROUND: Insect bite hypersensitivity (IBH) is an IgE-mediated allergic dermatitis in horses incited by salivary allergens from Culicoides spp. IBH does not occur in Iceland, as the causative agents are absent, however a high prevalence is seen in horses exported to Culicoides-rich environments. AIMS: To study the natural course of sensitization to Culicoides allergens and identify the primary sensitizing allergen(s) in horses exported from Iceland utilizing a comprehensive panel of Culicoides recombinant (r-) allergens. METHOD: IgE microarray profiling to 27 Culicoides r-allergens was conducted on 110 serological samples from horses imported to Switzerland from Iceland that subsequently developed IBH or remained healthy. Furthermore, a longitudinal study of 31 IBH horses determined IgE profiles the summer preceding first clinical signs of IBH (TIBH-1), the summer of first clinical signs (TIBH) and the following summer (TIBH+1). In a group of Icelandic horses residing in Sweden, effects of origin (born in Iceland or Sweden) and duration of IBH (<4 years, 4-7 years, >7 years) on Culicoides-specific IgE was evaluated. Sero-positivity rates and IgE levels were compared. RESULTS: At TIBH, horses were sensitized to a median of 11 r-allergens (range = 0-21), of which nine were major allergens. This was significantly higher than TIBH-1 (3, 0-16), as well as the healthy (1, 0-14) group. There was no significant increase between TIBH and TIBH+1(12, 0-23). IBH-affected horses exported from Iceland had a significantly higher degree of sensitization than those born in Europe, while duration of IBH did not significantly affect degree of sensitization. CONCLUSION: Significant sensitization is only detected in serum the year of first clinical signs of IBH. Horses become sensitized simultaneously to multiple Culicoides r-allergens, indicating that IgE-reactivity is due to co-sensitization rather than cross-reactivity between Culicoides allergens. Nine major first sensitizing r-allergens have been identified, which could be used for preventive allergen immunotherapy.

West Nile virus seroprevalence and associated risk factors among horses in Egypt.

Determination of the seroprevalence and risk factors that are associated with West Nile virus (WNV) in horses is essential for adoption of effective prevention strategies. Our objective in this study, therefore, was to determine the seroprevalence and to identify the risk factors associated with WNV infection in the most densely horse-populated governorates in Egypt. A cross-sectional study was conducted in 2018 on 930 horses, which were distributed over five governorates in the Nile delta of Egypt. The horses, which were randomly selected, were serologically tested through use of an ID screen West Nile competition enzyme-linked immunosorbent assay (ELISA) to detect anti-WNV immunoglobulin G (IgG) and plaque reduction neutralization tests (PRNT; gold standard) to confirm the seropositive status of animals and to avoid cross reaction with other flavi-viruses. Four variables (geographical location, breed, sex and age) were considered in the risk analysis. Univariable and stepwise forward multivariable logistic regression methods were used for risk-factor analysis. The odds ratio (OR) was used as an approximate measure of relative risk. A total of 156 (16.8%; 95% confidence interval (CI) 14.4-19.2; P < 0.001) serum samples were found to be serologically positive for WNV. The highest seroprevalence rate was detected in horses of age ≥ 15 years (68.1%; 95% CI 49.8-72.4), stallions (26.4%; 95% CI 22.7-30.4), and those of mixed breed (21.5%; 95% CI 17.7-27.5). Horses older than 15 years were found to be at increased risk of WNV infection with OR = 4.3 (95% CI 3.0-6.2, P < 0.001) compared with horses aged under 2.5 years. Also, when all the risk factors were considered, stallions were more likely than mares to be WNV seropositive (OR = 2.4, 95% CI 1.6-3.7, P < 0.001), and of the breeds, mixed-breed (OR = 1.9, 95% CI 1.2-2.8, P = 0.005) and Arabian horses (OR = 1.9, 95% CI 1.2-2.8, P = 0.005) were more likely to be seropositive. Geographical location seemed to have no impact on the seroprevalence of exposure to WNV among these horses. Due to these findings, we strongly recommend intensive surveillance and implementation of effective control and prevention strategies against WNV, especially in stallion, mixed-breed horses with ages ≥ 15 years.

The opportunistic intracellular bacterial pathogen Rhodococcus equi elicits type I interferon by engaging cytosolic DNA sensing in macrophages.

Rhodococcus equi is a major cause of foal pneumonia and an opportunistic pathogen in immunocompromised humans. While alveolar macrophages constitute the primary replicative niche for R. equi, little is known about how intracellular R. equi is sensed by macrophages. Here, we discovered that in addition to previously characterized pro-inflammatory cytokines (e.g., Tnfa, Il6, Il1b), macrophages infected with R. equi induce a robust type I IFN response, including Ifnb and interferon-stimulated genes (ISGs), similar to the evolutionarily related pathogen, Mycobacterium tuberculosis. Follow up studies using a combination of mammalian and bacterial genetics demonstrated that induction of this type I IFN expression program is largely dependent on the cGAS/STING/TBK1 axis of the cytosolic DNA sensing pathway, suggesting that R. equi perturbs the phagosomal membrane and causes DNA release into the cytosol following phagocytosis. Consistent with this, we found that a population of ~12% of R. equi phagosomes recruits the galectin-3,-8 and -9 danger receptors. Interestingly, neither phagosomal damage nor induction of type I IFN require the R. equi's virulence-associated plasmid. Importantly, R. equi infection of both mice and foals stimulates ISG expression, in organs (mice) and circulating monocytes (foals). By demonstrating that R. equi activates cytosolic DNA sensing in macrophages and elicits type I IFN responses in animal models, our work provides novel insights into how R. equi engages the innate immune system and furthers our understanding how this zoonotic pathogen causes inflammation and disease.

Antibody activities in hyperimmune plasma against the Rhodococcus equi virulence -associated protein A or poly-N-acetyl glucosamine are associated with protection of foals against rhodococcal pneumonia.

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

Equine Influenza Virus and Vaccines.

Equine influenza virus (EIV) is a constantly evolving viral pathogen that is responsible for yearly outbreaks of respiratory disease in horses termed equine influenza (EI). There is currently no evidence of circulation of the original H7N7 strain of EIV worldwide; however, the EIV H3N8 strain, which was first isolated in the early 1960s, remains a major threat to most of the world's horse populations. It can also infect dogs. The ability of EIV to constantly accumulate mutations in its antibody-binding sites enables it to evade host protective immunity, making it a successful viral pathogen. Clinical and virological protection against EIV is achieved by stimulation of strong cellular and humoral immunity in vaccinated horses. However, despite EI vaccine updates over the years, EIV remains relevant, because the protective effects of vaccines decay and permit subclinical infections that facilitate transmission into susceptible populations. In this review, we describe how the evolution of EIV drives repeated EI outbreaks even in horse populations with supposedly high vaccination coverage. Next, we discuss the approaches employed to develop efficacious EI vaccines for commercial use and the existing system for recommendations on updating vaccines based on available clinical and virological data to improve protective immunity in vaccinated horse populations. Understanding how EIV biology can be better harnessed to improve EI vaccines is central to controlling EI.

Immune response of adult horses, pregnant mares and foals to an experimental vaccine with recombinant EMA-2 protein of Theileria equi.

Equine theileriosis, caused by the Theileria equi protozoan, is a disease of worldwide importance. T. equi expresses surface proteins, of which the EMA-2 protein is a promising antigen for vaccine use. The aim of this study was to evaluate the immune response of adult horses, pregnant mares, and foals to an experimental EMA-2 protein of recombinant T. equi vaccine. A total of 46 horses were used in this study for vaccine trials and challenges. Twelve geldings, 14 pregnant mares, and 14 foals were divided into vaccinated and control groups. Total serum specific anti-rEMA-2 IgG, IgG subclasses, and transcription of cytokines related to the immune response were evaluated. For the vaccine challenge, six six-month-old foals were divided into vaccinated and control groups. For the challenge, blood from a horse with theileriosis was transfused to the foals. Geldings and pregnant mares maintained anti-rEMA-2 IgG levels at 130 and 140 days after vaccination, respectively. The most-detected IgG subclasses in vaccinated were IgG3/5, IgG4/7, and IgG1. IL2, IL10, IL12, IL17, IFN-γ, and TNF-α were the most-transcribed cytokines in PBMCs of vaccinated horses stimulated with rEMA-2. Challenge with T. equi demonstrated that vaccinated foals had an increase of 33% in total IgG four days after blood transfusion, while control foals had no significant response, suggesting that vaccine antibodies may have recognized EMA-2 protein of the native T. equi antigen. T. equi recombinant EMA-2 was shown to be a promising vaccine antigen by inducing humoral and cellular immunity similar to that observed in natural parasite infections.

Serum Antibody Activity against Poly-N-Acetyl Glucosamine (PNAG), but Not PNAG Vaccination Status, Is Associated with Protecting Newborn Foals against Intrabronchial Infection with Rhodococcus equi.

Rhodococcus equi is a prevalent cause of pneumonia in foals worldwide. Our laboratory has demonstrated that vaccination against the surface polysaccharide ß-1→6-poly-N-acetylglucosamine (PNAG) protects foals against intrabronchial infection with R. equi when challenged at age 28 days. However, it is important that the efficacy of this vaccine be evaluated in foals when they are infected at an earlier age, because foals are naturally exposed to virulent R. equi in their environment from birth and because susceptibility is inversely related to age in foals. Using a randomized, blind experimental design, we evaluated whether maternal vaccination against PNAG protected foals against intrabronchial infection with R. equi 6 days after birth. Vaccination of mares per se did not significantly reduce the incidence of pneumonia in foals; however, activities of antibody against PNAG or for deposition of complement component 1q onto PNAG was significantly (P < 0.05) higher among foals that did not develop pneumonia than among foals that developed pneumonia. Results differed between years, with evidence of protection during 2018 but not 2020. In the absence of a licensed vaccine, further evaluation of the PNAG vaccine is warranted, including efforts to optimize the formulation and dose of this vaccine. IMPORTANCE Pneumonia caused by R. equi is an important cause of disease and death in foals worldwide for which a licensed vaccine is lacking. Foals are exposed to R. equi in their environment from birth, and they appear to be infected soon after parturition at an age when innate and adaptive immune responses are diminished. Results of this study indicate that higher activity of antibodies recognizing PNAG was associated with protection against R. equi pneumonia, indicating the need for further optimization of maternal vaccination against PNAG to protect foals against R. equi pneumonia.

Comparison of recombinant Culicoides allergens produced in different expression systems for IgE serology of insect bite hypersensitivity in horses of different origins.

Insect bite hypersensitivity (IBH) is an allergic dermatitis of horses, an IgE-mediated reaction to Culicoides midges. Causative Culicoides spp. are not indigenous in Iceland resulting in high prevalence of IBH in horses born in Iceland and exported as compared to Icelandic horses born in a Culicoides rich environment. The aims were (i) to compare IgE levels in sera of IBH-affected horses born in Iceland (n = 47) with horses of the Icelandic breed (n = 23) and of other breeds (n = 27) born in Culicoides infested area; (ii) to investigate if barley could be a useful production system of allergens for IBH immunoassays. IgE binding in sera was tested by ELISA on two recombinant Culicoides allergens, rCul n 3 and rCul n 4, each produced in E. coli, insect cells and barley. Significantly more IgE was detected against all allergens in sera from IBH-affected compared to healthy horses. Icelandic-born Icelandic horses stand out with higher IgE levels against the allergens and higher area under the curve (AUC) on rCul n 4 as compared to the European-born horses. The barley and E.coli produced allergens had very similar performance in distinguishing between IBH-affected and healthy horses.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis.

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

Studies of molecular pathways associated with blood neutrophil corticosteroid insensitivity in equine asthma.

Severe equine asthma is characterized by airway hyperresponsiveness, neutrophilic inflammation and structural alterations of the lower airways. In asthmatic horses with neutrophilic inflammation, there is insensitivity to corticosteroids characterized by the persistence of neutrophils within the airways with therapy. We hypothesized that hypoxia or oxidative stress in the microenvironment of the lung contributes to this insensitivity of neutrophils to corticosteroids in asthmatic horses. Blood neutrophils isolated from horses with severe asthma (N = 8) and from healthy controls (N = 8) were incubated under different cell culture conditions simulating hypoxia and oxidative stress and, in the presence, or absence of dexamethasone. The pro-inflammatory gene and protein expression of neutrophils were studied. In both groups, pyocyanin-induced oxidative stress increased the mRNA expression of IL-8, IL-1ß, and TNF-α. While IL-1ß and TNF-α were downregulated by dexamethasone under these conditions, IL-8 was not. Simulated hypoxic conditions did not enhance pro-inflammatory gene expression in neutrophils from either group of horses. In conclusion, oxidative stress but not hypoxia may contribute to corticosteroid insensitivity via a selective gene regulation pathway. Equine neutrophil responses were similar in both heathy and asthmatic horses, indicating that it is not specific to asthmatic inflammation.

Viral infection and allergy - What equine immune responses can tell us about disease severity and protection.

Horses have many naturally occurring diseases that mimic similar conditions in humans. The ability to conduct environmentally controlled experiments and induced disease studies in a genetically diverse host makes the horse a valuable intermediate model between mouse studies and human clinical trials. This review highlights important similarities in the immune landscape between horses and humans using current research on two equine diseases as examples. First, equine herpesvirus type 1 (EHV-1) infection initiates a series of innate inflammatory signals at its mucosal entry site in the upper respiratory tract. These inflammatory markers are highly synchronized and predictable between individuals during viral respiratory infection and ultimately lead to adaptive immune induction and protection. The timing of early inflammatory signals, followed by specific adaptive immune markers correlating with immunity and protection, allow accurate outbreak tracking and also provide a foundation for understanding the importance of local mucosal immunity during other viral respiratory infections. Second, rare peripheral blood immune cells that promote allergic inflammation can be analyzed during Culicoides hypersensitivity, a naturally occurring type I IgE-mediated allergic disease of horses. Rare immune cells, such as IgE-binding monocytes or basophils, can be studied repeatedly in the horse model to unravel their larger mechanistic role in inflammation during allergic and other inflammatory diseases. We conclude with a survey of all other common equine inflammatory conditions. Together, this review serves as a reference and rationale for the horse as a non-rodent model for immunological research.

IgE-Binding Monocytes Have an Enhanced Ability to Produce IL-8 (CXCL8) in Animals with Naturally Occurring Allergy.

IL-8 is a potent chemokine that recruits neutrophils and basophils to promote inflammation in many species. IL-8 is produced by many cell types, including monocytes. In this study, we report a novel role for IgE-binding monocytes, a rare peripheral immune cell type, to promote allergic inflammation through IL-8 production in a horse model of natural IgE-mediated allergy. We developed a mAb with confirmed specificity for both recombinant and native equine IL-8 for flow cytometric analysis. Equine IL-8 was produced by CD14+/MHC class II+/CD16- monocytes, including a subpopulation of IgE-binding monocytes, following stimulation with LPS. In addition, IgE cross-linking induced IL-8 production by both peripheral blood basophils and IgE-binding monocytes. IL-8 production was compared between healthy horses and those with a naturally occurring IgE-mediated skin allergy, Culicoides hypersensitivity. Allergic horses had significantly higher percentages of IL-8+ IgE-binding monocytes after IgE cross-linking. In contrast, frequencies of IL-8+ basophils after IgE cross-linking were similar in all horses, regardless of allergic disease, highlighting IgE-binding monocytes as a novel source of IL-8 during allergy. We concluded that IgE-binding monocytes from allergic individuals have an increased capacity for IL-8 production and likely contribute to the recruitment of innate immune cells during IgE-mediated allergy and promotion of inflammation during repeated allergen contact.

Association of inbreeding and regional equine leucocyte antigen homozygosity with the prevalence of insect bite hypersensitivity in Old Kladruber horse.

Inbreeding depression is the reduction of performance caused by mating of close relatives. In livestock populations, inbreeding depression has been traditionally estimated by regression of phenotypes on pedigree inbreeding coefficients. This estimation can be improved by utilising genomic inbreeding coefficients. Here we estimate inbreeding depression for insect bite hypersensitivity (IBH) prevalence, the most common allergic horse disease worldwide, in Old Kladruber horse. In a deep pedigree with 3214 horses (187 genotyped), we used a generalised linear mixed model with IBH phenotype from 558 horses examined between 1996 and 2009 (1368 records). In addition to the classical pedigree information, we used the single-step approach that enabled joint use of pedigree and genomic information to estimate inbreeding depression overall genome and equine leucocyte antigen (ELA) class II region. Significant inbreeding depression was observed in all models fitting overall inbreeding coefficients (odds ratio between 1.018 and 1.074, P < 0.05) with the exception of Kalinowski's new inbreeding (P = 0.0516). The increase of ELA class II inbreeding was significantly associated with increased prevalence of IBH (odds ratio 1.018; P = 0.027). However, when fitted jointly with the overall inbreeding coefficient, the effect of ELA class II inbreeding was not significant (odds ratio 1.016; P = 0.062). Overall, the higher ELA class II and/or overall inbreeding (pedigree or genomic) was associated with increased prevalence of IBH in Old Kladruber horses. The single-step approach provides an efficient use of all the available pedigree, genomic, and phenotype information for estimation of overall and regional inbreeding effects.

Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton.

Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and ß- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

Equine respiratory viruses, airway inflammation and performance in thoroughbred racehorses.

Equine asthma is a common cause of poor performance in racehorses but it is unclear if respiratory viruses contribute to its etiology. The objective of the study was to determine if respiratory viruses were associated with clinical signs and bronchoalveolar lavage fluid (BALF) cytology in Thoroughbred racehorses. Equine herpesviruses (EHV-1, 2, 4, 5) and equine rhinitis A and B viruses (ERBV, ERAV) genomes were quantified by qPCR in nasopharyngeal, tracheal, and BALF samples collected after racing. The relationships between virus detection and load and clinical signs, performance, BALF cytology, and environmental exposures were examined with generalized linear mixed models. Ninety-two samples were collected from 31 horses. EHV-1 and ERAV were not found; EHV-4 was detected in only one sample. EHV-2, EHV-5 and ERBV were more likely to be detected in upper airway samples than in BALF (P < 0.0001). Neither respiratory virus detection nor load was associated with clinical signs or performance. Nasopharyngeal detection and load of ERBV and tracheal detection and load of EHV-5 were associated with increased proportions of neutrophils in BALF (P < 0.003). However, nasopharyngeal detection and load of EHV-5 was not (P = 0.11). Nasopharyngeal detection and load of EHV-2 were associated with decreased BALF mast cell proportions. Respirable dust exposures were significantly higher in horses with detection of ERBV when compared to horses with no detectable ERBV (P < 0.001). Our results suggest that ERBV, EHV-2 and EHV-5 are commonly present in upper airways of healthy racehorses; however, the role they play in the etiology of equine asthma remains unclear.

Identification of Host Factors Associated with the Development of Equine Herpesvirus Myeloencephalopathy by Transcriptomic Analysis of Peripheral Blood Mononuclear Cells from Horses.

Equine herpesvirus-1 is the cause of respiratory disease, abortion, and equine herpesvirus myeloencephalopathy (EHM) in horses worldwide. EHM affects as many as 14% of infected horses and a cell-associated viremia is thought to be central for EHM pathogenesis. While EHM is infrequent in younger horses, up to 70% of aged horses develop EHM. The aging immune system likely contributes to EHM pathogenesis; however, little is known about the host factors associated with clinical EHM. Here, we used the "old mare model" to induce EHM following EHV-1 infection. Peripheral blood mononuclear cells (PBMCs) of horses prior to infection and during viremia were collected and RNA sequencing with differential gene expression was used to compare the transcriptome of horses that did (EHM group) and did not (non-EHM group) develop clinical EHM. Interestingly, horses exhibiting EHM did not show respiratory disease, while non-EHM horses showed significant respiratory disease starting on day 2 post infection. Multiple immune pathways differed in EHM horses in response to EHV-1. These included an upregulation of IL-6 gene expression, a dysregulation of T-cell activation through AP-1 and responses skewed towards a T-helper 2 phenotype. Further, a dysregulation of coagulation and an upregulation of elements in the progesterone response were observed in EHM horses.

Apoptosis in epithelial cells and its correlation with leukocyte accumulation in lamellar tissue from horses subjected to experimental sepsis-associated laminitis.

Inflammation and apoptosis in the hoof lamellar interface both contribute to the early stages of sepsis-associated laminitis, but it is not clear whether apoptosis is occurring before the onset of inflammation or is being provoked by inflammation. Apoptosis and inflammation were therefore measured in lamellar tissues obtained at different time points throughout the early stages of experimentally induced laminitis. Apoptotic cells and leukocyte were enumerated in archived paraffin embedded lamellar tissue samples from previous experiments in which acute laminitis was induced using Black Walnut Extract (BWE) or starch (CHO). BWE-derived samples from 20 horses were allocated into four groups: Control (CON = 5); Early Time Point (ETP, 1.5 h after induction, n = 5); Developmental Time Point (DTP, 3-4 h after induction, n = 5); Obel Grade 1 (OG1, Onset of Lameness, n = 5). CHO-derived samples from 25 horses were allocated into four groups: CON (n = 8); DTP (10-12 h after induction, n = 6); OG 1 (n = 6); Obel 3 (OG3, lameness progression, n = 5). Apoptotic cells were enumerated using a horse validated TUNEL technique. Compared to controls, significant increases in apoptotic cell counts were not detected in lamellar epithelial cells during the developmental phase or at the onset of lameness during laminitis induction. A negative correlation between apoptosis and leukocyte infiltration was detected in the BWE model (P < 0.05). In conclusion, apoptosis does not play an important role in the initial stages of sepsis-related laminitis.

Prevalence of heterophilic antibodies in serum samples from horses in an equine hospital, and elimination of interference using chicken IgY.

BACKGROUND: Heterophilic antibodies in serum and plasma can interfere with mammalian antibodies in immunoassays and result in false test results, usually false positive. Although studies screening for heterophilic antibodies as well as elimination studies have been conducted in dogs and cats, knowledge of the presence of heterophilic antibodies in other species in veterinary medicine is limited. In this study, a 2-site sandwich-type interference assay that detects anti-mouse antibodies was used to detect heterophilic antibodies in a population of horses treated in an animal hospital. RESULTS: A total of 194 serum samples from 127 individual horses were analyzed. There were 11/127 (8.7%) interference-positive horses, and these were analyzed in an assay exchanging the capture mouse IgG with chicken IgY. The positive samples were negative in the chicken IgY assay, indicating elimination of a possible interference, with the chicken-based assay. Four interference-positive samples were from geldings, and anti-Müllerian hormone (AMH) was analyzed from these samples. AMH concentrations were negative in these samples as expected in geldings, indicating that the heterophilic antibodies did not cause interference in the AMH assay. CONCLUSION: The present study shows that there are heterophilic antibodies in horse serum samples like in samples from humans, dogs, and cats. The use of chicken-based reagents, such as chicken IgY, which do not cross-react with mammalian IgG, eliminates the effects of interfering antibodies in the samples. Equine heterophilic antibodies do not necessarily cause interference in commercial immunoassays.