Do allogeneic bone marrow derived mesenchymal stem cells diminish the inflammatory response to lipopolysaccharide infusion in horses? A pilot study.

Endotoxemia is a leading cause of morbidity and mortality in the equine industry, with colic being the most common cause of endotoxemia in horses. The objective of this study was to evaluate the safety and potential efficacy of a single dose of allogeneic equine bone marrow derived mesenchymal stem cells (BM-MSCs) in horses after the IV administration of lipopolysaccharide (LPS). Six horses were administered an IV infusion of 30 ng/kg LPS (O55:B5 Escherichia coli) in 500 ml saline over 30 min. Immediately after infusion test horses (n = 3) were administered 100 × 106 allogeneic BM-MSCs diluted in saline IV and control horses (n = 3) were administered saline. Clinicopathological data, pro-inflammatory cytokine measurements and sCD14 concentrations were compared between groups. No adverse reactions were observed in horses administered BM-MSCs intravenously. There were no significant differences between test and control horses with regard to clinicopathological values or pro-inflammatory cytokine production. At no time point did concentrations of sCD14 exceed the reference range in any horse. Results suggest that administration of a single IV dose of freshly cultured MSCs is safe and well-tolerated in horses with induced endotoxemia. Further study to evaluate their efficacy as a potential therapeutic in a larger number of horses with clinical disease is required.

An Inter-Institutional Collaboration to "Make Teaching Matter": The Teaching Academy of the Consortium of West Region Colleges of Veterinary Medicine.

Veterinary medical education is a relatively small community with limited numbers of institutions, people, and resources widely dispersed geographically. The problems faced, however, are large-and not very different from the problems faced by (human) medical education. As part of an effort to share resources and build a community of practice around common issues, five colleges in the westernmost region of the United States came together to form a regional inter-institutional consortium. This article describes the processes by which the consortium was formed and the initiation of its first collaborative endeavor, an inter-institutional medical/biomedical teaching academy (the Regional Teaching Academy, or RTA). We report outcomes, including the successful launch of three RTA initiatives, and the strategies that have been considered key to the academy's success. These include strong support from the consortium deans, including an ongoing financial commitment, a dedicated part-time Executive Coordinator, regular face-to-face meetings that supplement virtual meetings, an organization-wide biennial conference, an effective organizational structure, and a core group of dedicated leaders and RTA Fellows. The western consortium and RTA share these processes, insights, and outcomes to provide a model upon which other colleges of veterinary medicine can build to further leverage inter-institutional collaboration.

An Inter-Institutional External Peer-Review Process to Evaluate Educators at Schools of Veterinary Medicine.

Despite its fundamental importance, the educational mission of most schools of veterinary medicine receives far less recognition and support than the missions of research and discovery. This disparity is evident in promotion and tenure processes. Despite the frequent assertion that education is every college's core mission, there is a broad consensus that faculty are promoted primarily on the basis of meeting expectations relative to publications and grant funding. This expectation is evident in the promotion packets faculty are expected to produce and the criteria by which those packets are reviewed. Among the outcomes is increasing difficulty in hiring and retaining faculty, including young clinicians and basic scientists who are drawn to academic institutions because of the opportunity to teach. The Regional Teaching Academy (RTA) of the West Region Consortium of Colleges of Veterinary Medicine initiated an inter-institutional collaboration to address the most important obstacles to recognizing and rewarding teaching in its five member colleges. Working from the medical education literature, the RTA developed an Educator's Promotion Dossier, workshops to train promotion applicants, and an external review process. Initial use has shown that the reviews are efficient and complete. Administrators have expressed strong support for the product, a letter of external review that is returned to a promotion applicant's home institution. The overall result is an evidence-based, structured process by which teaching-intensive faculty can more fully document their achievements in teaching and educational leadership and a more rigorous external review process by which member colleges can assess quality, impact, and scholarly approach.

Pharmacokinetics and competitive pharmacodynamics of ADP-induced platelet activation after oral administration of clopidogrel to horses.

OBJECTIVE: To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses. ANIMALS: 6 healthy adult horses. PROCEDURES: Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model. RESULTS: Compared with results for preadministration samples, platelet aggregation in response to 12.5µM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25µM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20µM) and prostaglandin E1 (3.3µM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505-512).

Frequency of shedding of respiratory pathogens in horses recently imported to the United States.

BACKGROUND: Imported horses that have undergone recent long distance transport might represent a serious risk for spreading infectious respiratory pathogens into populations of horses. OBJECTIVE: To investigate the frequency of shedding of respiratory pathogens in recently imported horses. ANIMALS: All imported horses with signed owner consent (n = 167) entering a USDA quarantine for contagious equine metritis from October 2014 to June 2016 were enrolled in the study. METHODS: Prospective observational study. Enrolled horses had a physical examination performed and nasal secretions collected at the time of entry and subsequently if any horse developed signs of respiratory disease during quarantine. Samples were assayed for equine influenza virus (EIV), equine herpesvirus type-1, -2, -4, and -5 (EHV-1, -2, -4, -5), equine rhinitis virus A (ERAV), and B (ERBV) and Streptococcus equi subspecies equi (S. equi) using quantitative PCR (qPCR). RESULTS: Equine herpesviruses were detected by qPCR in 52% of the study horses including EHV-2 (28.7%), EHV-5 (40.7%), EHV-1 (1.2%), and EHV-4 (3.0%). Clinical signs were not correlated with being qPCR-positive for EHV-4, EHV-2, or EHV-5. None of the samples were qPCR-positive for EIV, ERAV, ERBV, and S. equi. The qPCR assay failed quality control for RNA viruses in 25% (46/167) of samples. CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical signs of respiratory disease were poorly correlated with qPCR positive status for EHV-2, -4, and -5. The importance of γ-herpesviruses (EHV-2 and 5) in respiratory disease is poorly understood. Equine herpesvirus type-1 or 4 (EHV-1 or EHV-4) were detected in 4.2% of horses, which could have serious consequences if shedding animals entered a population of susceptible horses. Biosecurity measures are important when introducing recently imported horses into resident US populations of horses.

Curricular Revision and Reform: The Process, What Was Important, and Lessons Learned.

Beginning in 2005, the Doctor of Veterinary Medicine program at the University of California underwent major curricular review and reform. To provide information for others that follow, we have documented our process and commented on factors that were critical to success, as well as factors we found surprising, difficult, or problematic. The review and reform were initiated by the Executive Committee, who led the process and commissioned the committees. The planning stage took 6 years and involved four faculty committees, while the implementation stage took 5 years and was led by the Curriculum Committee. We are now in year 2 of the institutionalizing stage and no longer refer to our reform as the "new curriculum." The change was driven by a desire to improve the curriculum and the learning environment of the students by aligning the delivery of information with current teaching methodologies and implementing adult learning strategies. We moved from a department- and discipline-based curriculum to a school-wide integrated block curriculum that emphasized student-centered, inquiry-based learning. A limit was placed on in-class time to allow students to apply classroom knowledge by solving problems and cases. We found the journey long and arduous, requiring tremendous commitment and effort. In the change process, we learned the importance of adequate planning, leadership, communication, and a reward structure for those doing the "heavy lifting." Specific to our curricular design, we learned the importance of the block leader role, of setting clear expectations for students, and of partnering with students on the journey.

Duration of serum antibody response to rabies vaccination in horses.

OBJECTIVE To investigate the impact of age and inferred prior vaccination history on the persistence of vaccine-induced antibody against rabies in horses. DESIGN Serologic response evaluation. ANIMALS 48 horses with an undocumented vaccination history. PROCEDURES Horses were vaccinated against rabies once. Blood samples were collected prior to vaccination, 3 to 7 weeks after vaccination, and at 6-month intervals for 2 to 3 years. Serum rabies virus-neutralizing antibody (RVNA) values were measured. An RVNA value of ≥ 0.5 U/mL was used to define a predicted protective immune response on the basis of World Health Organization recommendations for humans. Values were compared between horses < 20 and ≥ 20 years of age and between horses inferred to have been previously vaccinated and those inferred to be immunologically naïve. RESULTS A protective RVNA value (≥ 0.5 U/mL) was maintained for 2 to 3 years in horses inferred to have been previously vaccinated on the basis of prevaccination RVNA values. No significant difference was evident in response to rabies vaccination or duration of protective RVNA values between horses < 20 and ≥ 20 years of age. Seven horses were poor responders to vaccination. Significant differences were identified between horses inferred to have been previously vaccinated and horses inferred to be naïve prior to the study. CONCLUSIONS AND CLINICAL RELEVANCE A rabies vaccination interval > 1 year may be appropriate for previously vaccinated horses but not for horses vaccinated only once. Additional research is required to confirm this finding and characterize the optimal primary dose series for rabies vaccination.

Multiple intravenous injections of allogeneic equine mesenchymal stem cells do not induce a systemic inflammatory response but do alter lymphocyte subsets in healthy horses.

INTRODUCTION: Intravenous (IV) injection of mesenchymal stem cells (MSCs) is used to treat systemic human diseases and disorders but is not routinely used in equine therapy. In horses, MSCs are isolated primarily from adipose tissue (AT) or bone marrow (BM) and used for treatment of orthopedic injuries through one or more local injections. The objective of this study was to determine the safety and lymphocyte response to multiple allogeneic IV injections of either AT-derived MSCs (AT-MSCs) or BM-derived MSCs (BM-MSCs) to healthy horses. METHODS: We injected three doses of 25 × 10(6) allogeneic MSCs from either AT or BM (a total of 75 × 10(6) MSCs per horse) into five and five, respectively, healthy horses. Horses were followed up for 35 days after the first MSC infusion. We evaluated host inflammatory and immune response, including total leukocyte numbers, serum cytokine concentration, and splenic lymphocyte subsets. RESULTS: Repeated injection of allogeneic AT-MSCs or BM-MSCs did not elicit any clinical adverse effects. Repeated BM-MSC injection resulted in increased blood CD8(+) T-cell numbers. Multiple BM-MSC injections also increased splenic regulatory T cell numbers compared with AT-MSC-injected horses but not controls. CONCLUSIONS: These data demonstrate that multiple IV injections of allogeneic MSCs are well tolerated by healthy horses. No clinical signs or clinico-pathologic measurements of organ toxicity or systemic inflammatory response were recorded. Increased numbers of circulating CD8(+) T cells after multiple IV injections of allogeneic BM-MSCs may indicate a mild allo-antigen-directed cytotoxic response. Safety and efficacy of allogeneic MSC IV infusions in sick horses remain to be determined.

Pre-analytical stability of adrenocorticotrophic hormone from healthy horses in whole blood, plasma and frozen plasma samples.

The stability of equine adrenocorticotrophic hormone (ACTH) in blood samples is not fully known. The study objectives were to determine ACTH stability (1) in whole blood and plasma over 72 h at either 4 or 21 °C, and (2) in plasma frozen at either -20 or -80 °C over 30 days. Nine horses were sampled and ACTH concentration were measured after storage as whole blood or plasma, at 4, 21, -20 and -80 °C for up to 30 days. The ACTH concentration was significantly reduced at 24 h but remained stable when plasma was frozen at -20 and -80 °C for 30 days. Beyond 24 h, samples stored at 21 °C showed a greater reduction in ACTH concentrations than those stored at 4 °C. Therefore, samples can be stored for 8 h without centrifugation, or frozen for 30 days without appreciable reductions in ACTH concentrations.

Incidence, management, and outcome of complications of castration in equids: 324 cases (1998-2008).

OBJECTIVE: To determine the incidence of complications and identify risk factors associated with development of complications following routine castration of equids. DESIGN: Retrospective case series. Animals-311 horses, 10 mules, and 3 donkeys. PROCEDURES: Medical records of equids undergoing routine castration were reviewed. Age, breed, surgical techniques (closed vs semiclosed castration and use of ligatures), anesthesia method (general IV anesthesia vs standing sedation with local anesthesia) and repeated administration of IV anesthetic agents, administration of antimicrobials and anti-inflammatory drugs, and details regarding development, management, and outcome of complications were recorded. Odds ratios and 95% confidence intervals were determined. Associations between additional doses of anesthetic agents during surgery and development of complications were analyzed with a Jonckheere-Terpstra test. RESULTS: 33 of 324 (10.2%) equids developed a complication after surgery; 32 recovered and 1 was euthanized because of eventration. Equids that underwent semiclosed castration had significantly higher odds of developing a complication (OR, 4.69; 95% confidence interval, 2.09 to 10.6) than did those that underwent closed castration. Equids that received additional doses of anesthetic agents to maintain adequate general anesthesia developed complications more frequently than those that did not require this treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Incidence of complications was low, and most evaluated variables were not significantly associated with development of complications following castration in equids. However, findings suggested that the choice of surgical technique (closed vs semiclosed) is an important factor in this regard. Future studies should investigate whether duration of surgery is associated with complications following castration in equids.

Development of a live, attenuated, potential vaccine strain of R. equi expressing vapA and the virR operon, and virulence assessment in the mouse.

Pneumonia caused by Rhodococcus equi remains a significant problem in foals. The objective of this study was to develop a safe and efficacious attenuated strain of R. equi for eventual use in oral immunization of foals. The approach involved expression of vapA in a live, virulence plasmid-negative, strain of R. equi (strain 103-). PCR-amplified fragments of the vapA gene, with and without the upstream genes virR, orf5, vapH, orf7 and orf8 (orf4-8), were cloned into a shuttle vector pNBV1. These plasmids, named pAW48A and pAWVapA respectively, were electroporated into strain 103-. The presence of the recombinant vectors in the attenuated strain (103-) and the integrity of the inserted genes were confirmed, and both constructs expressed VapA. The virulence of the two strains was compared to that of wild type R. equi 103+ and negative controls by their intravenous inoculation into mice, followed by examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection, whereas strain 103-/pAW48A persisted in 47% of mice.

Intradermal injections of equine allogeneic umbilical cord-derived mesenchymal stem cells are well tolerated and do not elicit immediate or delayed hypersensitivity reactions.

BACKGROUND AIMS. The use of allogeneic mesenchymal stem cells (MSC) to treat acute equine lesions would greatly expand equine cellular therapy options; however, the safety and antigenicity of these cells have not been well-studied. We hypothesized that equine allogeneic umbilical cord tissue (UCT)-derived MSC would not elicit acute graft rejection or a delayed-type hypersensitivity response when injected intradermally. METHODS. Six Quarterhorse yearlings received 12 intradermal injections (autologous MSC, allogeneic MSC, positive control and negative control, in triplicate) followed by the same series of 12 injections, 3-4 weeks later, at another site. Wheals were measured and palpated at 0.25, 4, 24, 48, 72 h and 7 days post-injection. Biopsies were obtained at 48 and 72 h and 7 days post-injection. Mixed leukocyte reactions were performed 1 week prior to the first injections and 3 weeks after the second injections. RESULTS. There were no adverse local or systemic responses to two intradermal injections of allogeneic MSC. MSC injection resulted in minor wheal formation, characterized by mild dermatitis, dermal edema and endothelial hyperplasia, that fully resolved by 48-72 h. No differences were noted between allogeneic and autologous MSC. The second injection of MSC did not elicit more significant physical or histomorphologic alterations compared with the first MSC injection. Neither allogeneic nor autologous UCT-derived MSC stimulated or suppressed baseline T-cell proliferation in vitro prior to or after two MSC administrations. CONCLUSIONS. Equine allogeneic UCT MSC may be safely administered intradermally on multiple occasions without eliciting a measurable cellular immune response.

The efficacy of imiquimod 5% cream (Aldara® in the treatment of aural plaque in horses: a pilot open-label clinical trial.

Aural plaques affect at least 22% of horses and can be asymptomatic or cause ear sensitivity. Immunohistochemical and electron microscopy studies have shown a strong association between aural plaques and papilloma virus. The purpose of this study was to investigate the efficacy of imiquimod 5% cream, an immune response modifier with potent antiviral activity, in the treatment of equine aural plaques. Twenty-one horses were enrolled and 16 completed the study. Imiquimod 5% cream was applied three times a week, every other week. When both ears were affected only the worst affected ear was treated. Adverse effects in all horses included marked local inflammation, exudation and thick crust formation at the site of treatment and the adjacent skin. Removal of the crust before treatment was painful and required sedation in most horses. Complete resolution of lesions was noted in all horses immediately post-treatment and the long-term resolution rate was 87.5%. Duration of therapy ranged from 1.5 to 8 months (median: 2.9 mean: 3.5). All horses were followed-up for 12-22 months after treatment was discontinued and only two horses had a recurrence of lesions. Clinical signs related to the aural plaques prior to treatment were reported in 11 of 16 (68.8%) horses and included resistance to touching the ears and bridling. Complete resolution of these signs was reported by the owners in all of the horses followed-up for at least 12 months. In conclusion, the topical application of imiquimod 5% cream is an efficacious treatment for aural plaques in horses.

Equine herpesvirus-4 kinetics in peripheral blood leukocytes and nasopharyngeal secretions in foals using quantitative real-time TaqMan PCR.

Based on the hypothesis that the viral load of cells infected with EHV-4 will likely change during the course of disease, TaqMan PCR was used to investigate and characterize the kinetics of EHV-4 viral DNA load (glycoprotein B gene) and transcriptional activity (glycoprotein B and latency-associated transcripts) in peripheral blood leukocytes (PBLs) and nasopharyngeal secretions (NSs) collected from 11 foals during a field outbreak of respiratory disease. The EHV-4 DNA load in PBLs was low and of short duration after onset of clinical signs. In contrast, the EHV-4 load in NSs remained high for the majority of the foals over a period of 4 weeks. Viral replication determined by detection of mRNA expression of the structural glycoprotein B was detected only in NSs during the first 7 days after onset of clinical signs for most foals. The majority of foals expressed latency-associated transcripts in NS sonly during the first 7 days after onset of clinical signs. Persistence of the expression of latency-associated transcripts in NS, as a reflection of a latent viral state, was not documented during the 28-day study period. Based on these results, it was concluded that lytic infection with EHV-4 can be diagnosed either by high EHV-4 DNA load of glycoprotein B gene or by detection of transcriptional activity of glycoprotein B.

IL-4 induced CD23 (FcepsilonRII) up-regulation in equine peripheral blood mononuclear cells and pulmonary alveolar macrophages.

The objectives of this study were to quantify the induction of equine CD23 transcripts in equine peripheral blood mononuclear cells (PBMCs) and pulmonary alveolar macrophages cultured with recombinant equine IL-4 (rEq IL-4). PBMCs were isolated from blood drawn from four healthy horses. Bronchoalveolar lavage (BAL) fluid was collected from three healthy horses and alveolar macrophages were purified using adherence to plastic for 120 min. PBMCs and alveolar macrophages were cultured using four different conditions: rEq IL-4 and LPS, LPS alone, rEq IL-4 alone and a media control. Total RNA was isolated from cells cultured for 24 or 48 h. Reverse transcribed mRNA was amplified and quantified in real-time polymerase chain reaction (RT PCR) using a fluorescein labeled internal TaqMan probe for CD23 expression. Without exception, the relative value for CD23 mRNA transcripts from equine PBMCs and pulmonary alveolar macrophages cultured with rEq IL-4 for 24 and 48 h were higher than those cultured with LPS alone or the untreated control. Furthermore, morphologic changes were noted in alveolar macrophages cultured with rEq IL-4 prompting an investigation of cytokine expression levels. Alveolar macrophages cultured with LPS exhibited increased IL-8 and IL-12 p40 expression when compared to rEq IL-4, rEq IL-4 + LPS or the untreated control. These findings support two conclusions, (1) equine CD23 has a role in IL-4 mediated immune responses in the horse and (2) rEq IL-4 can modulate LPS-induced, pro-inflammatory cytokine production by equine pulmonary alveolar macrophages.

Virulent and avirulent strains of equine arteritis virus induce different quantities of TNF-alpha and other proinflammatory cytokines in alveolar and blood-derived equine macrophages.

Equine arteritis virus (EAV) infects endothelial cells (ECs) and macrophages in horses, and many of the clinical manifestations of equine viral arteritis (EVA) reflect vascular injury. To further evaluate the potential role of EAV-induced, macrophage-derived cytokines in the pathogenesis of EVA, we infected cultured equine alveolar macrophages (AMphi), blood monocyte-derived macrophages (BMphi), and pulmonary artery ECs with either a virulent (KY84) or an avirulent (CA95) strain of EAV. EAV infection of equine AMphi, BMphi, and ECs resulted in their activation with increased transcription of genes encoding proinflammatory mediators, including interleukin (IL)-1beta, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha. Furthermore, the virulent KY84 strain of EAV induced significantly higher levels of mRNA encoding proinflammatory cytokines in infected AMphi and BMphi than did the avirulent CA95 strain. Treatment of equine ECs with the culture supernatants of EAV-infected AMphi and BMphi also resulted in EC activation with cell surface expression of E-selectin, whereas infection of ECs with purified EAV alone caused only minimal expression of E-selectin. The presence of TNF-alpha in the culture supernatants of EAV-infected equine AMphi, BMphi, and ECs was confirmed by bioassay, and the virulent KY84 strain of EAV induced significantly more TNF-alpha in all cell types than did the avirulent CA95 strain. Thus, the data indicate that EAV-induced, macrophage-derived cytokines may contribute to the pathogenesis of EVA in horses, and that the magnitude of the cytokine response of equine AMphi, BMphi, and ECs to EAV infection reflects the virulence of the infecting virus strain.

Cutaneous and ocular habronemiasis in horses: 63 cases (1988-2002).

OBJECTIVE: To describe clinical manifestations of cutaneous and ocular habronemiasis in horses and evaluate outcome of treatment. DESIGN: Retrospective study. ANIMALS: 63 horses. PROCEDURE: The diagnosis was made on the basis of history, clinical signs, and identification of calcified concretions (sulfur granules) in lesions. Histologic examination of biopsy specimens was used to confirm the diagnosis. Case horses were compared with a control population of 12,720 horses examined during the same period. RESULTS: Arabians, gray horses, and horses with diluted coat colors were overrepresented; Thoroughbreds were underrepresented. Lesions were identified most often during the summer and early fall. The medial canthus of the eye, male genitalia, third eyelid, and distal portions of the extremities were the most commonly affected locations. Twenty-five lesions were biopsied, and results of histologic examination were consistent with a diagnosis of habronemiasis. However, nematode larvae were seen in only 11 (44%) biopsy specimens. Treatment consisted of surgical removal (7 horses) or medical treatment (56) consisting of debulking granulation tissue and topical, intralesional, or systemic treatment with corticosteroids. All horses were treated with ivermectin. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that cutaneous and ocular habronemiasis should be considered when examining a horse during the summer months with a proliferative, moist, granulomatous lesion. Treatment should be aimed at decreasing the size of the lesion, reducing inflammation, and preventing recurrence. In general, the prognosis was good, and healing occurred within a few weeks. Fly control and regular deworming with ivermectin are recommended to reduce the incidence of habronemiasis.