An Overview of Equine Enteric Clostridial Diseases.

The understanding of the pathogenesis of equine enteric clostridial organisms is an active, evolving field. Advances will improve our knowledge both from the animal welfare and human health perspectives. The zoonotic nature of this group of diseases makes them relevant in the age of One health, as a significant amount of close human-equine interactions occurs for business and pleasure. Economic and welfare reasons prompt a better understanding of enteric clostridial pathogenesis, treatment, and control of the infection in horses and ongoing efforts are needed to advance clinical outcomes.

Clostridioides difficile Infection Dysregulates Brain Dopamine Metabolism.

Gastrointestinal illnesses and dysbiosis are among the most common comorbidities reported in patients with neurodevelopmental disorders. The manuscript reports that C. difficile infection (CDI), predisposed by antibiotic-induced gut dysbiosis, causes significant alterations in dopamine metabolism in major dopaminergic brain regions in mice (P < 0.05). In addition, C. difficile infected mice exhibited significantly reduced dopamine beta-hydroxylase (DBH) activity compared to controls (P < 0.01). Moreover, a significantly increased serum concentration of p-cresol, a DBH inhibiting gut metabolite produced by C. difficile, was also observed in C. difficile infected mice (P < 0.05). Therefore, this study suggests a potential mechanistic link between CDI and alterations in the brain dopaminergic axis. Such alterations may plausibly influence the precipitation and aggravation of dopamine dysmetabolism-associated neurologic diseases in infected patients. IMPORTANCE The gut-brain axis is thought to play a significant role in the development and manifestation of neurologic diseases. This study reports significant alterations in the brain dopamine metabolism in mice infected with C. difficile, an important pathogen that overgrows in the gut after prolonged antibiotic therapy. Such alterations in specific brain regions may have an effect on the precipitation or manifestation of neurodevelopmental disorders in humans.

Evaluation of Locally Injected Mycobacterium Cell Wall Fraction in Horses with Sarcoids.

A reformulation of Mycobacterium cell wall fraction immunotherapeutic can be used to successfully treat sarcoids in horses. Sarcoids are reported to be the most common equine skin tumors with tumor type and location influencing the choice of treatment. Wide surgical excision is curative for many tumors, but may not always be feasible. Previous studies have reported sarcoid regression after injection with mycobacterial cell wall immunotherapeutics. A new formulation of the Mycobacterium phlei cell wall fraction immunostimulant (Immunocidin Equine) was used to treat cutaneous tumors in horses. Equids with skin tumors diagnosed as sarcoids were enrolled in the study. Sarcoids were injected at the initial visit with Immunocidin Equine and subsequently at approximately 2-week intervals. Of 17 cases, nine cases were completely resolved at the end of the study period evaluation or at the time of final follow-up (52.9%). Three cases were reported as improved (smaller), but not resolved (17.6%). Three cases were discontinued from the study as the respective masses were growing larger or not resolving (17.6%). One case (5.8%) with two masses had resolution of one mass, whereas the other tumor had a small regrowth 5 months after the last treatment. One case (5.8%) was lost to follow-up. All cases had mild to moderate swelling of the injection site, and some cases had discharge after the second, third, or fourth injections. No serious systemic side effects or complications were encountered during the study.

Induction of Reactive Intermediates and Autophagy-Related Proteins upon Infection of Macrophages with Rhodococcus equi.

Rhodococcus equi (R. equi) is an intracellular macrophage-tropic pathogen with potential for causing fatal pyogranulomatous pneumonia in foals between 1 and 6 months of age. In this study, we sought to determine whether infection of macrophages with R. equi could lead to the induction of autophagy. Murine bone marrow derived macrophages (BMDM) were infected with R. equi for various time intervals and analyzed for upregulation of autophagy proteins and accumulation of autophagosomes relative to uninfected controls. Western blot analysis showed a progressive increase in LC3-II and Beclin1 levels in a time-dependent manner. The functional accumulation of autophagosomes detected with monodansylcadaverine further supported the enhanced induction of autophagy in BMDM infected with R. equi. In addition, infection of BMDM with R. equi induced generation of reactive oxygen species (ROS) in a time-dependent manner. These data are consistent with reports documenting the role of ROS in induction of autophagy and indicate that the infection of macrophages by R. equi elicits innate host defense mechanisms.

Age-related variation in the cellular composition of equine bronchoalveolar lavage fluid.

BACKGROUND: Previous reports reveal variation in the cellular composition of equine bronchoalveolar lavage fluid (BALF). OBJECTIVES: The purpose of this study was to compare the profiles of BALF from horses to assess age-related differences. Serial BALF samples were collected from the same individuals over a one-year period to identify changes in individual animals as they aged. METHODS: Collection of BALF was performed on horses aged one week and one, 2, 6, and 12 months. Total nucleated cell count (TNCC), protein concentration, and cytology were assessed. Longitudinal analysis was performed and compared to healthy adults. RESULTS: Foals at one week and 6 months of age had significantly higher TNCC than adults (medians: 320/µL, 285/µL, and 90/µL, respectively); no differences in total protein were found. Foals at one month had the highest proportion of macrophages (median: 87.3%), differing significantly from both yearlings and adults (medians: 45.5% and 48.7%, respectively). Foals aged one week and one month had significantly lower proportions of lymphocytes than yearlings and adults (medians: 3.2% and 4.7% vs 43.2% and 45.8%, respectively). Eosinophil percentage was lowest in foals aged one week, one month, and 2 months (median: 0.0%) and highest in foals aged 6 months (median: 2.2%). Mast cell percentages were highest in yearlings and adults (medians: 2.2% and 3.3%, respectively) and neutrophil percentage was highest in foals aged one week (13.7%). CONCLUSIONS: Cytologic profiles of BALF from foals and adult horses differed considerably. Significant changes in TNCC and percentages of lymphocytes, macrophages, and eosinophils occurred with age.

Macrophage effector responses of horses are influenced by expression of CD154.

Reactive intermediates contribute to innate immunity by providing phagocytes with a mechanism of defense against bacteria, viruses and parasites. To better characterize the role of CD154 in the production of reactive intermediates, we cloned and expressed recombinant equine CD154 (reqCD154) in Chinese Hamster Ovary (CHO). In co-culture experiments, CHO cells ectopically expressing reqCD154 elicited superoxide production in monocyte-derived macrophages (MDM). Collectively, our results indicate that regulation of CD154 expression plays a role in innate host defenses.

CD47 expression in cryopreserved equine cutaneous masses and normal skin.

We investigated CD47 expression in cryopreserved sections of equine cutaneous masses and normal skin. CD47 is a cell surface protein expressed on many cell types and overexpressed in some tumors. Interaction of CD47 and signal regulatory protein-alpha (SIRPα) inhibits phagocytosis by macrophages. Formalin-fixed tissues from horses prospectively enrolled in the study were used to establish a histologic diagnosis. Immunohistochemical assays were performed on cryopreserved tissues using anti-CD47 antibodies or IgG control antibodies. CD47 was not expressed on equine normal skin but positivity to CD47 was present in 13 of 24 (54%) masses. Immunotherapy with anti-CD47 antibodies for equine cutaneous tumors that express CD47 warrants further investigation.

Effects of intravenous administration of polymyxin B in neonatal foals with experimental endotoxemia.

OBJECTIVE: To evaluate the effect of IV administration of polymyxin B on clinical and serum biochemical variables in foals with experimental endotoxemia. DESIGN: Prospective experimental study. ANIMALS: 14 healthy neonatal foals. PROCEDURES: Foals were randomly assigned to a treatment or control group and were administered a single dose of lipopolysaccharide (0.5 µg/kg [0.23 µg/lb]) IV over 30 minutes. The treatment group received polymyxin B (6,000 U/kg [2,727 U/lb], IV) immediately after completion of lipopolysaccharide infusion; the control group was administered an equal volume of saline (0.9% NaCl) solution. Subsequent doses of polymyxin B or saline solution were administered IV at 8 and 16 hours. Blood was collected at various time points, and outcome variables, including heart rate, respiratory rate, rectal temperature, attitude score, WBC count, neutrophil count, lymphocyte count, monocyte count, platelet count, Hct, blood lactate concentration, blood glucose concentration, serum tumor necrosis factor-α concentration, and plasma thromboxane B2 concentration, were measured. Urine was collected prior to and after experimentation to determine whether nephrotoxicosis was associated with treatment. RESULTS: The treatment group had significantly lower blood lactate concentration and serum tumor necrosis factor-α and plasma thromboxane B2 concentrations and had higher blood glucose concentrations and better attitude scores, compared with the control group, at various time points during the study. No other significant differences and no evidence of overt nephrotoxicosis were detected. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of polymyxin B IV in healthy neonatal foals challenged with lipopolysaccharide attenuated some clinical and serum biochemical derangements associated with endotoxemia.

Mechanisms of equine infectious anemia virus escape from neutralizing antibody responses define epitope specificity.

Determining mechanisms of viral escape to particular epitopes recognized by virus-neutralizing antibody can facilitate characterization of host-neutralizing antibody responses as type- versus group-specific, and provides necessary information for vaccine development. Our study reveals that a single N-glycan located in the 5' region of the Wyoming wild-type equine infectious anemia virus (EIAV) principal neutralizing domain (PND) accounts for the differences in neutralization phenotype observed between PND variants, while variations in charged amino acids within the PND do not appear to play a key role in viral escape. Site-directed mutagenesis and peptide mapping of a conserved epitope to neutralizing antibody in the 3' region of the PND showed rapid selective pressure for acquisition of a 5' PND N-glycan responsible for defining the specificity of the neutralizing-antibody response.

Alloimmune neonatal neutropenia and neonatal isoerythrolysis in a Thoroughbred colt.

A 3-day-old Thoroughbred colt was originally presented for treatment of neonatal isoerythrolysis, which was treated with a blood transfusion. However, persistent neutropenia was observed despite the absence of detectable infection. Subsequently, a granulocyte agglutination test was performed by incubating the colt's neutrophils with the mare's serum; results were positive, leading to a clinical diagnosis of alloimmune neonatal neutropenia. The diagnosis was further supported via flow cytometric analysis. The colt was hospitalized and treated prophylactically with antimicrobials and 4 separate doses of recombinant human granulocyte colony-stimulating factor (rhG-CSF; 1.4-3.5 µg/kg, subcutaneously) in attempts to maintain the neutrophil count within reference intervals over a 4-week period. The colt's neutrophil count increased after administration of rhG-CSF and eventually stabilized within reference intervals by day 20. The colt maintained normal neutrophil counts after discharge and was reportedly healthy at 6 months of age. Alloimmune neonatal neutropenia should be considered in foals with persistent neutropenia in the absence of infection. Alloimmune neonatal neutropenia can be treated with prophylactic antimicrobials combined with rhG-CSF with a favorable outcome.

Agreement between arterial partial pressure of carbon dioxide and saturation of hemoglobin with oxygen values obtained by direct arterial blood measurements versus noninvasive methods in conscious healthy and ill foals.

OBJECTIVE: To determine agreement between indirect measurements of end-tidal partial pressure of carbon dioxide (PetCO(2)) and saturation of hemoglobin with oxygen as measured by pulse oximetry (SpO(2)) with direct measurements of PaCO(2) and calculated saturation of hemoglobin with oxygen in arterial blood (SaO(2)) in conscious healthy and ill foals. DESIGN: Validation study. ANIMALS: 10 healthy and 21 ill neonatal foals. PROCEDURES: Arterial blood gas analysis was performed on healthy and ill foals examined at a veterinary teaching hospital to determine direct measurements of PaCO(2) and PaO(2) along with SaO(2). Concurrently, PetCO(2) was measured with a capnograph inserted into a naris, and SpO(2) was measured with a reflectance probe placed at the base of the tail. Paired values were compared by use of Pearson correlation coefficients, and level of agreement was assessed with the Bland-Altman method. RESULTS: Mean ± SD difference between PaCO(2) and PetCO(2) was 0.1 ± 5.0 mm Hg. There was significant strong correlation (r = 0.779) and good agreement between PaCO(2) and PetCO(2). Mean ± SD difference between SaO(2) and SpO(2) was 2.5 ± 3.5%. There was significant moderate correlation (r = 0.499) and acceptable agreement between SaO(2) and SpO(2). CONCLUSIONS AND CLINICAL RELEVANCE: Both PetCO(2) obtained by use of nasal capnography and SpO(2) obtained with a reflectance probe are clinically applicable and accurate indirect methods of estimating and monitoring PaCO(2) and SaO(2) in neonatal foals. Indirect methods should not replace periodic direct measurement of corresponding parameters.

Syringohydromyelia in horses: 3 cases.

Syringomyelia and hydromyelia are cavitary lesions of the spinal cord that may be acquired or congenital. These lesions are not frequently reported in large animal species. The presenting complaints, clinical, gross pathological, and histopathologic findings of 2 cases of syringomyelia and 1 case of hydromyelia in horses are described.

Influenza A pandemic (H1N1) 2009 virus infection in domestic cat.

Influenza A pandemic (H1N1) 2009 virus continues to rapidly spread worldwide. In 2009, pandemic (H1N1) 2009 infection in a domestic cat from Iowa was diagnosed by a novel PCR assay that distinguishes between Eurasian and North American pandemic (H1N1) 2009 virus matrix genes. Human-to-cat transmission is presumed.

Cell-mediated immunity evaluation in foals infected with virulent equine herpesvirus-1 by multi-parameter flow cytometry.

The cell-mediated immune (CMI) response of foals to virulent equine herpesvirus-1 (EHV-1) infection was evaluated by multi-parameter flow cytometry (FCM). Ten 7-8-month-old EHV-1 seronegative foals were infected intranasally with virulent EHV-1 and 10 foals served as uninfected controls. Blood samples were collected 6 and 7 weeks after infection to test for specific CMI responses to live heterologous EHV-1 recall antigen. The activation markers included major histocompatibility complex class II (MHC II), intracellular interferon gamma (IFN-gamma) and interleukin 4 (IL-4). The results from both tests were averaged before statistical analysis. Following EHV-1 stimulation, the MHC II expression index (EI) increased significantly in CD2+CD4+CD8- and CD2+CD4-CD8+ subsets of the infected group. At 4 days after incubation, the non-antigen stimulated CD2+CD4-CD8- subset of the infected group expressed a high percentage (61.1%) of MHC II. When stimulated with EHV-1, the MHC II expression declined significantly but remained at a relatively high percentage (34.4%). The IFN-gamma EI was significantly higher in infected foals in all major T cell subsets (CD2+) while only the CD2+CD4+CD8- subset showed a significant increase in intracellular IL-4 EI. The FCM results showed strong specific CMI responses to EHV-1 by all three tested parameters compared to the control group (p<0.01). The high MHC II expression in the CD2+CD4-CD8- subset suggests that this T cell subset may represent a gammadelta TCR repertoire and thereby plays an important role as antigen presenting cells in the horse, as reported in other species. Being able to simultaneously quantify the frequency of specific lymphocyte subsets and the expression of cytokines that characterize activation of lymphocytes and protective CMI by multi-parameter FCM enables evaluation of subset-specific CMI responses to EHV-1 infection. This system can be applied to measure CMI responses to other equine vaccines and pathogens.

Adrenocorticotropic hormone stimulation tests in healthy foals from birth to 12 weeks of age.

The purpose of this study was to investigate total baseline plasma cortisol and adrenocorticotropic hormone (ACTH) concentrations, and ACTH-stimulated cortisol concentrations in foals from birth to 12 wk of age. Plasma (baseline) cortisol and ACTH concentrations were measured in 13 healthy foals at birth and at 1, 2, 3, 4, 5, 7, 10, 14, 21, 28, 42, 56, and 84 d of age. Each foal received cosyntropin (0.1 microg/kg) intravenously. Plasma cortisol concentrations were measured before (baseline), and 30, and 60 min after cosyntropin administration at birth and at 3, 5, 7, 10, 14, 21, 28, 42, 56, and 84 d of age. Compared with baseline, cortisol concentration increased significantly 30 min after administration of cosyntropin on all days. Cortisol concentration was highest at birth, measured at 30 and 60 min after cosyntropin administration, compared with all other days. With the exception of birth measurements, cortisol concentration was significantly higher on day 84, measured at 30 and 60 min after cosyntropin administration, when compared with all other days. Baseline plasma ACTH was lowest at birth when compared with concentrations on days 2, 3, 4, 5, 7, 10, 14, 42, 56, and 84. Administration of 0.1 microg/kg of cosyntropin, IV, reliably induces cortisol secretion in healthy foals. Differences in the magnitude of response to cosyntropin are observed depending on the age of the foal. These data should serve as a reference for the ACTH stimulation test in foals and should be useful in subsequent studies to evaluate the hypothalamic-pituitary-adrenal axis in healthy and critically ill foals.

Baseline plasma cortisol and ACTH concentrations and response to low-dose ACTH stimulation testing in ill foals.

OBJECTIVE: To evaluate baseline plasma cortisol and ACTH concentrations and responses to low-dose ACTH stimulation testing in ill foals. DESIGN: Cross-sectional study. ANIMALS: 58 ill foals. PROCEDURES: Baseline cortisol and ACTH concentrations and cortisol concentrations after administration of a low dose of cosyntropin were determined within 6 hours after admission. Foals were assigned to 4 groups on the basis of age (

Immune selection of equine infectious anemia virus env variants during the long-term inapparent stage of disease.

The principal neutralizing domain (PND) of equine infectious anemia virus (EIAV) is located in the V3 region of SU. Genetic variation in the PND is considered to play an important role in immune escape and EIAV persistence; however, few studies have characterized genetic variation in SU during the inapparent stage of disease. To better understand the mechanisms of virus persistence, we undertook a longitudinal study of SU variation in a pony experimentally inoculated with the virulent EIAV(Wyo). Viral RNA isolated from the inoculum and from sequential sera samples was amplified by RT-PCR, cloned, and individual clones were sequenced. Of the 147 SU clones obtained, we identified 71 distinct V3 variants that partitioned into five major non-overlapping groups, designated PND-1 to PND-5, which segregated with specific stages of clinical disease. Genotypes representative of each group were inserted into an infectious molecular clone, and chimeric viruses were tested for susceptibility to neutralization by autologous sera from successive times post-infection. Overall, there was a trend for increasing resistance to neutralizing antibody during disease progression. The PND genotype associated with recrudescence late in infection was resistant to both type-specific and broadly neutralizing antibody, and displayed a reduced replication phenotype in vitro. These findings indicate that neutralizing antibody exerts selective pressure throughout infection and suggest that viral strategies of immune evasion and persistence change in the face of an evolving and maturing host immune response.