Gillian A. Perkins

Background: Epidural injection of a low concentration of the local anaesthetic ropivacaine produces analgesia without neurological deficits or ileus in people. Objective: To determine if epidural 0.125% ropivacaine mitigates induced hindlimb lameness in horses while preserving neurological function and gastrointestinal (GI) motility. Study Design: Randomised, blinded, controlled, crossover study. Methods: Eight healthy, sound horses underwent two treatments in random order, separated by at least 3 weeks: epidural saline or epidural ropivacaine. For each treatment, an epidural catheter was inserted at the first intercoccygeal space and advanced 5 cm cranially. Correct catheter placement was confirmed radiographically after injecting contrast through it. The tibiotarsal joint was injected with 75 ng of recombinant equine interleukin‐1β to induce synovitis. Twelve hours later, baseline variables were recorded, including heart rate, respiratory rate, borborygmi, lameness score and neurological examination score. Thirty minutes thereafter [time = 0 h (t0)], 0.045 mL/kg of preservative‐free 0.9% saline or 0.125% ropivacaine was administered through the catheter over 10 min. Data were collected at t0.5, 1, 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5. Faecal piles passed the 24 h before and after t0 were counted. Continuous and categorical data were analysed using a linear mixed effects model and Fisher's exact test, respectively, with significance set at p < 0.05. Results: All horses developed moderate‐to‐severe lameness that did not improve after ropivacaine injection. No horses developed hindlimb neurological deficits. However, from t0.5–2.5, more horses in the ropivacaine (7/8) than saline (0/7) treatment had decreased to absent anus and tail tone ( p = 0.014). There were no other significant differences between treatments. Main Limitations: A single volume, concentration, and injection rate were tested. Conclusions: A single dose of 0.125% ropivacaine did not mitigate hindlimb lameness. Anus and tail tone decreased, but hindlimb neurological function and GI motility were preserved.

Background: Sepsis has been defined in humans as the concurrent proven or suspected presence of microbial infection and the systemic inflammatory response syndrome (SIRS). Sepsis is the leading cause of morbidity and mortality in neonatal foals. The clinical utility of using SIRS or its individual components to predict infection and mortality in critically ill foals is currently unknown. Objectives: Assess the ability of history and signalment, clinical findings, laboratory results, and SIRS-related indices to predict infection and mortality in critically ill foals. Animals: Retrospective, multi-center, cross-sectional study using a convenience sample of 1068 critically ill foals < 3 days of age admitted to 16 veterinary referral hospitals in 4 countries. Methods: Data were retrieved from medical records. Infection was defined as the presence of bacteremia (positive blood culture) or clinical identification of an infected focus on admission. Univariate non-parametric and categorical methods, multivariate logistic regression, and classification tree methods were used for statistical analysis. Results: Foal age at admission and presence of toxic neutrophils were independent predictors of infection, whereas SIRS-related indices were not predictive of infection. In-hospital mortality was 24%. Independent predictors for mortality were hypokinetic pulses, cold extremities, presence of seizures, blood L-lactate concentration > 6.0 mmol/L, and increased serum potassium and total bilirubin concentrations. Conclusions and Clinical Importance: The presence of infection in critically ill newborn foals was not predicted by SIRS indices. Cardiovascular dysfunction was strongly associated with mortality, suggesting that maintaining adequate perfusion and pulse pressure should be important treatment goals.

Background: Crossmatching is used to prevent life-threatening transfusion reactions in horses. Laboratory methods are laborious and technically challenging, which is impractical during emergencies. Hypothesis/Objectives: Evaluate agreement between a stall-side crossmatch kit (KIT) and a laboratory method (LAB) in horses with known and unknown blood types. Animals: Twenty-four blood-typed and alloantibody-screened healthy adult horses (Aim 1) and 156 adult horses of unknown blood type (Aim 2). Methods: Prospective, blinded study. Expected positive (n = 35) and negative (n = 36) crossmatches among 24 antibody and blood-typed horses were used to determine sensitivity and specificity of KIT and LAB against the reference method. Agreement in 156 untyped horses was evaluated by reciprocal crossmatch (n = 156). Results: Sensitivity (95% confidence interval [CI]) for LAB and KIT compared with expected reactions was 77.1% (59.9%-90.0%) and 91.4% (77.0%-98.2%), and specificity 77.8% (60.9%-89.9%) and 73.5% (55.6%-87.1%), respectively. The KIT was 100% sensitive for Aa reactions; LAB was 100% sensitive for Qab; and both were 100% sensitive for Ca. Cohen's κ agreement for LAB and KIT with expected positive and negative reactions (n = 71) was moderate (0.55 [0.36-0.74]) and substantial (0.65 [0.47-0.82]), respectively. Agreement was fair comparing LAB with KIT in Aim 1 (0.30 [0.08-0.52]) and in untyped horses in Aim 2 (0.26 [0.11-0.41]). Conclusions and Clinical Importance: Agreement between KIT and LAB with expected reactions was blood type dependent. Performance of both methods depends on blood type prevalence.

Equine herpesvirus type 1 (EHV-1) induces respiratory infection, abortion, and neurologic disease with significant impact. Virulence factors contributing to infection and immune evasion are of particular interest. A potential virulence factor of the neuropathogenic EHV-1 strain Ab4 is ORF2. This study on 24 Icelandic horses, 2 to 4 years of age, describes the infection with EHV-1 Ab4, or its deletion mutant devoid of ORF2 (Ab4ΔORF2) compared to non-infected controls (each group n = 8). The horses’ clinical presentation, virus shedding, viremia, antibody and cellular immune responses were monitored over 260 days after experimental infection. Infection with Ab4ΔORF2 reduced fever and minimized nasal virus shedding after infection compared to the parent virus strain Ab4, while Ab4ΔORF2 established viremia similar to Ab4. Concurrently with virus shedding, intranasal cytokine and interferon α (IFN-α) production increased in the Ab4 group, while horses infected with Ab4ΔORF2 expressed less IFN-α. The antibody response to EHV-1 was evaluated by a bead-based multiplex assay and was similar in both infected groups, Ab4 and Ab4ΔORF2. EHV-1 specific immunoglobulin (Ig) G1 was induced 8 days after infection (d8 pi) with a peak on d10–12 pi. EHV-1 specific IgG4/7 increased starting on d10 pi, and remained elevated in serum until the end of the study. The intranasal antibody response to EHV-1 was dominated by the same IgG isotypes and remained elevated in both infected groups until d130 pi. In contrast to the distinct antibody response, no induction of EHV-1 specific T-cells was detectable by flow cytometry after ex vivo re-stimulation of peripheral blood mononuclear cells (PBMC) with EHV-1 in any group. The cellular immune response was characterized by increased secretion of IFN-γ and interleukin10 in response to ex vivo re-stimulation of PBMC with EHV-1. This response was present during the time of viremia (d5–10 pi) and was similar in both infected groups, Ab4 and Ab4ΔORF2. ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa. In contrast, ORF2 does not influence viremia. The immunogenicity of the Ab4ΔORF2 and parent Ab4 viruses are identical. Graphical abstract – Deletion of ORF2 reduces virulence of EHV-1 Ab4. Graphical summary of the main findings of this study: ORF2 is a virulence factor of EHV-1 Ab4 with impact on pyrexia and virus shedding from the nasal mucosa.

Summary: The development of equine immunity from the fetus to adulthood is complex. The foal's immune response and the immune mechanisms that they are equipped with, along with changes over the first months of life until the immune system becomes adult‐like, are only partially understood. While several innate immune responses seem to be fully functional from birth, the onset of adaptive immune response is delayed. For some adaptive immune parameters, such as immunoglobin ( Ig ) G1 , IgG3 , IgG5 and IgA antibodies, the immune response starts before or at birth and matures within 3 months of life. Other antibody responses, such as IgG4 , IgG7 and IgE production, slowly develop within the first year of life until they reach adult levels. Similar differences have been observed for adaptive T cell responses. Interferon‐gamma ( IFN ‐γ) production by T helper 1 ( Th1 )‐cells and cytotoxic T cells starts shortly after birth with low level production that gradually increases during the first year of life. In contrast, interleukin‐4 ( IL ‐4) produced by Th2 ‐cells is almost undetectable in the first 3 months of life. These findings offer some explanation for the increased susceptibility of foals to certain pathogens such as R hodococcus equi . The delay in T h‐cell development and in particular Th2 immunity during the first months of life also provides an explanation for the reduced responsiveness of young horses to most traditional vaccines. In summary, all immune components of adult horses seem to exist in foals but the orchestrating and regulation of the immune response in immature horses is strikingly different. Young foals are fully competent and can perform certain immune responses but many mechanisms have yet to mature. Additional work is needed to improve our understanding of immunity and immune regulation in young horses, to identify the preferred immune pathways that they are using and ultimately provide new preventive strategies to protect against infectious disease.

Objective —To evaluate metaphylactic RNA interference to prevent equine herpesvirus type 1 (EHV-1) infection in experimental herpesvirus myeloencephalopathy in horses and to determine whether horses infected with a neuropathogenic strain of the virus that develop equine herpesvirus myeloencephalopathy (EHM) have differences in viremia. Animals —13 seronegative horses. Procedures —EHV-1 strain Ab4 was administered intranasally on day 0, and small interfering RNAs (siRNAs [EHV-1 specific siRNAs {n = 7} or an irrelevant siRNA {6}]) were administered intranasally 24 hours before and 12, 24, 36, and 48 hours after infection. Physical and neurologic examinations, nasal swab specimens, and blood samples were collected for virus isolation and quantitative PCR assay. Data from the study were combined with data from a previous study of 14 horses. Results —No significant difference was detected in clinical variables, viremia, or detection of EHV-1 in nasal swab specimens of horses treated with the EHV-1 targeted siRNAs (sigB3-siOri2) versus controls. No significant differences in viremia were detected between horses that developed EHM and those that did not. Conclusions and Clinical Relevance —Administration of siRNAs targeted against EHV-1 around the time of EHV-1 infection was not protective with this experimental design. Horses infected with the neuropathogenic EHV-1 strain Ab4 that developed EHM did not have a more pronounced viremia.