Brett A. Sponseller

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 (<or=24 hours vs 1 to 56 days) and presence of septicemia (yes vs no). Values were compared among groups and with values previously reported for healthy foals. RESULTS Plasma cortisol concentrations 30 and 60 minutes after cosyntropin administration in foals<or=24 hours old were significantly higher than corresponding cortisol concentrations in older foals. In all 4 groups, plasma cortisol concentration 30 minutes after cosyntropin administration was significantly higher than baseline cortisol concentration or concentration 60 minutes after cosyntropin administration. No differences in baseline cortisol or ACTH concentration or in the ACTH-to-cortisol ratio were detected between groups or when ill foals were compared with healthy foals. A small number of ill foals had low baseline cortisol and ACTH concentrations or low responses to cosyntropin administration, compared with healthy foals. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that most ill foals in the present study population had adequate responses to cosyntropin administration. However, a small subset of ill foals appeared to have dysfunction of the hypothalamic-pituitary-adrenal axis.

Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses.

Foals are particularly vulnerable to infection by Rhodococcus equi during the first 2 weeks of life whereas mature horses are not. While an innate immunodeficiency likely accounts for this clinically relevant vulnerability, the factors that contribute to infection by R. equi have not been fully elucidated. In this study, we demonstrate that cells of the monocyte lineage, including monocytes, macrophages, and dendritic cells, that have been activated with LPS and IFN-gamma, respond with a statistically significant, greater amount of cytokine mRNA production of IL-10, IL-12p35, and IL-12p40 than unstimulated control cells. Interestingly, activation of neonatal cells resulted in a twofold log increase in baseline cytokine mRNA expression of IL-10 compared with adult cells. In contrast, no significant differences in mean cytokine mRNA expression of IL-12p35 and IL-12p40 were detected, suggesting that the defect in chromosomal remodeling that prevents IL-12p35 gene transcription as a cause for decreased IL-12 synthesis in human neonates is not a likely occurrence in equine neonates. Collectively, these differences indicate that in vivo activation of equine cells of the monocyte lineage may result in different autocrine and paracrine cellular responses that vary according to age, with potential impact on regulation of adaptive and innate immune responses.

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.

Clinical and Immunomodulating Effects of Ketamine in Horses with Experimental Endotoxemia

BACKGROUND Ketamine has immunomodulating effects both in vitro and in vivo during experimental endotoxemia in humans, rodents, and dogs. HYPOTHESIS Subanesthetic doses of ketamine will attenuate the clinical and immunologic responses to experimental endotoxemia in horses. ANIMALS Nineteen healthy mares of various breeds. METHODS Experimental study. Horses were randomized into 2 groups: ketamine-treated horses (KET; n = 9) and saline-treated horses (SAL; n = 10). Both groups received 30 ng/kg of lipopolysaccharide (LPS, Escherichia coli, O55:B5) 1 hour after the start of a continuous rate infusion (CRI) of racemic ketamine (KET) or physiologic saline (SAL). Clinical and hematological responses were documented and plasma concentrations of tumor necrosis factor-α (TNF-α) and thromboxane B(2) (TXB(2)) were quantified. RESULTS All horses safely completed the study. The KET group exhibited transient excitation during the ketamine loading infusion (P < .05) and 1 hour after discontinuation of administration (P < .05). Neutrophilic leukocytosis was greater in the KET group 8 and 24 hours after administration of LPS (P < .05). Minor perturbations of plasma biochemistry results were considered clinically insignificant. Plasma TNF-α and TXB(2) production peaked 1.5 and 1 hours, respectively, after administration of LPS in both groups, but a significant difference between treatment groups was not demonstrated. CONCLUSIONS AND CLINICAL IMPORTANCE A subanesthetic ketamine CRI is well tolerated by horses. A significant effect on the clinical or immunologic response to LPS administration, as assessed by clinical observation, hematological parameters, and TNF-α and TXB(2) production, was not identified in healthy horses with the subanesthetic dose of racemic ketamine utilized in this study.

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.

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.

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.

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.

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.