Evaluation of lipid markers in surfactant obtained from asthmatic horses exposed to hay.

OBJECTIVE To evaluate the lipidomic profile of surfactant obtained from horses with asthma at various clinical stages and to compare results with findings for healthy horses exposed to the same conditions. SAMPLE Surfactant samples obtained from 6 horses with severe asthma and 7 healthy horses. PROCEDURES Clinical evaluation of horses and surfactant analysis were performed. Samples obtained from horses with severe asthma and healthy horses before (baseline), during, and after exposure to hay were analyzed. Crude surfactant pellets were dried prior to dissolution in a solution of isopropanol:methanol:chloroform (4:2:1) containing 7.5mM ammonium acetate. Shotgun lipidomics were performed by use of high-resolution data acquisition on an ion-trap mass spectrometer. Findings were analyzed by use of an ANOVA with a Tukey-Kramer post hoc test. RESULTS Results of lipidomic analysis were evaluated to detect significant differences between groups of horses and among exposure statuses within groups of horses. Significantly increased amounts of cyclic phosphatidic acid (cPA) and diacylglycerol (DAG) were detected in surfactant from severely asthmatic horses during exposure to hay, compared with baseline and postexposure concentrations. Concentrations of cPA and DAG did not change significantly in healthy horses regardless of exposure status. CONCLUSIONS AND CLINICAL RELEVANCE cPA 16:0 and DAG 36:2 were 2 novel lipid mediators identified in surfactant obtained from asthmatic horses with clinical disease. These molecules were likely biomarkers of sustained inflammation. Further studies are needed to evaluate a possible correlation with disease severity and potential alterations in the plasma lipidomic profile of horses with asthma.

Characterization of basal and lipopolysaccharide-induced microRNA expression in equine peripheral blood mononuclear cells using Next-Generation Sequencing.

The innate immune response to lipopolysaccharide contributes substantially to the morbidity and mortality of gram-negative sepsis. Horses and humans share an exquisite sensitivity to lipopolysaccharide and thus the horse may provide valuable comparative insights into this aspect of the inflammatory response. MicroRNAs, small non-coding RNA molecules acting as post-transcriptional regulators of gene expression, have key roles in toll-like receptor signaling regulation but have not been studied in this context in horses. The central hypothesis of this study was that lipopolysaccharide induces differential microRNA expression in equine peripheral blood mononuclear cells in a manner comparable to humans. Illumina Next Generation Sequencing was used to characterize the basal microRNA transcriptome in isolated peripheral blood mononuclear cells from healthy adult horses, and to evaluate LPS-induced changes in microRNA expression in cells cultured for up to four hours. Selected expression changes were validated using quantitative reverse-transcriptase PCR. Only miR-155 was significantly upregulated by LPS, changing in parallel with supernatant tumor necrosis factor-α concentration. Eight additional microRNAs, including miR-146a and miR-146b, showed significant expression change with time in culture without a clear LPS effect. Target predictions indicated a number of potential immunity-associated targets for miR-155 in the horse, including SOCS1, TAB2 and elements of the PI3K signaling pathway, suggesting that it is likely to influence the acute inflammatory response to LPS. Gene alignment showed extensive conservation of the miR-155 precursor gene and associated promoter regions between horses and humans. The basal and LPS-stimulated microRNA expression pattern characterized here were similar to those described in human leukocytes. As well as providing a resource for further research into the roles of microRNAs in immune responses in horses, this will facilitate inter-species comparative study of the role of microRNAs in the inflammatory cascade during endotoxemia and sepsis.

Effects of Experimental Sarcocystis neurona-Induced Infection on Immunity in an Equine Model.

Sarcocystis neurona is the most common cause of Equine Protozoal Myeloencephalitis (EPM), affecting 0.5-1% horses in the United States during their lifetimes. The objective of this study was to evaluate the equine immune responses in an experimentally induced Sarcocystis neurona infection model. Neurologic parameters were recorded prior to and throughout the 70-day study by blinded investigators. Recombinant SnSAG1 ELISA for serum and CSF were used to confirm and track disease progression. All experimentally infected horses displayed neurologic signs after infection. Neutrophils, monocytes, and lymphocytes from infected horses displayed significantly delayed apoptosis at some time points. Cell proliferation was significantly increased in S. neurona-infected horses when stimulated nonspecifically with PMA/I but significantly decreased when stimulated with S. neurona compared to controls. Collectively, our results suggest that horses experimentally infected with S. neurona manifest impaired antigen specific response to S. neurona, which could be a function of altered antigen presentation, lack of antigen recognition, or both.

Evaluation of neutrophil apoptosis in horses with acute abdominal disease.

OBJECTIVE: To quantify peripheral blood neutrophil apoptosis in equine patients with acute abdominal disease (ie, colic) caused by strangulating or nonstrangulating intestinal lesions and compare these values with values for horses undergoing elective arthroscopic surgery. ANIMALS: 20 client-owned adult horses. PROCEDURES: Peripheral blood was collected from horses immediately prior to and 24 hours after surgery for treatment of colic (n = 10) or elective arthroscopic surgery (10), and neutrophils were counted. Following isolation by means of a bilayer colloidal silica particle gradient and culture for 24 hours, the proportion of neutrophils in apoptosis was detected by flow cytometric evaluation of cells stained with annexin V and 7-aminoactinomycin D. Values were compared between the colic and arthroscopy groups; among horses with colic, values were further compared between horses with and without strangulating intestinal lesions. RESULTS: Percentage recovery of neutrophils was significantly smaller in preoperative samples (median, 32.5%) and in all samples combined (35.5%) for the colic group, compared with the arthroscopy group (median, 66.5% and 58.0%, respectively). No significant differences in the percentages of apoptotic neutrophils were detected between these groups. Among horses with colic, those with strangulating intestinal lesions had a significantly lower proportion of circulating apoptotic neutrophils in postoperative samples (median, 18.0%) than did those with nonstrangulating lesions (66.3%). CONCLUSIONS AND CLINICAL RELEVANCE: The smaller proportion of apoptotic neutrophils in horses with intestinal strangulation suggested that the inflammatory response could be greater or prolonged, compared with that of horses with nonstrangulating intestinal lesions. Further investigations are needed to better understand the relationship between neutrophil apoptosis and inflammation during intestinal injury.

Increased apoptosis of CD4 and CD8 T lymphocytes in the airways of horses with recurrent airway obstruction.

Recurrent airway obstruction (RAO, also known as equine heaves) is an inflammatory condition similar to human asthma caused by exposure of susceptible horses to poorly ventilated stable environments. The disease is characterized by neutrophilic airway inflammation, mucus hypersecretion and reversible bronchoconstriction. This inflammatory process is mediated by several factors, including antibodies, cytokines, resident cells of the airway and inflammatory cellular components that arrive in the respiratory tract. An increasing body of evidence has lent support to the concept that a dysregulation of T cell apoptosis may play a central role in the development of airway inflammation and the associated asthma. Therefore, the aim of this study was to investigate early and late apoptosis of CD4 and CD8 T cell subpopulations obtained from the airways of acute RAO-positive animals after exposure to hay/straw. The percentages of CD4 and CD8 T cells and their associated frequencies of apoptosis were quantified using flow cytometry. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and increased airway mucus production in RAO-positive horses. In addition, allergen exposure increased the percentage of CD4 T cells in RAO-positive horses as well as the frequency of early and late apoptosis in both CD4 and CD8 lymphocyte subpopulations. These results suggest that the higher frequency of lymphocyte apoptosis may play a role in disease progression of horses afflicted with RAO and may partially explain the characteristic remission of this pathological condition once the allergen source is removed. However, further studies are needed to clarify the role of T cell apoptosis in RAO-affected horses.

Surfactant alterations in horses with recurrent airway obstruction at various clinical stages.

OBJECTIVE: To evaluate the phospholipid composition and function of surfactant in horses with recurrent airway obstruction (RAO) at various clinical stages and compare these properties with findings in horses without RAO. ANIMALS: 7 horses with confirmed RAO and 7 without RAO (non-RAO horses). PROCEDURES: Pairs of RAO-affected and non-RAO horses were evaluated before, during, and after exposure to hay. Evaluations included clinical scoring, lung function testing, airway endoscopy, and bronchoalveolar lavage fluid (BALF) absolute and differential cell counts. Cell-free BALF was separated into crude surfactant pellet and supernatant by ultracentrifugation, and phospholipid and protein concentrations were determined. Phospholipid composition of crude surfactant pellets and surface tension were evaluated with high-performance liquid chromatography and a pulsating bubble surfactometer, respectively. Findings were compared statistically via mixed-effects, repeated-measures ANOVA. RESULTS: Total phospholipid concentration in BALF was lower in RAO-affected versus non-RAO horses at all sample collection times. In the RAO-affected group, total phospholipid concentration was lower during exposure to hay than before or after exposure. There were no significant differences in BALF protein concentration, percentages of phospholipid classes, or surface tension between or within groups of horses. CONCLUSIONS AND CLINICAL RELEVANCE: All clinical stages of RAO-affected horses were characterized by low surfactant concentration in BALF. Exacerbation of RAO led to an additional decrease in surfactant concentration. Causes for low surfactant concentration in RAO-affected horses remain to be determined. Low phospholipid concentration may render RAO-affected horses more susceptible than unaffected horses to surfactant alterations and contribute to clinical disease status and progression.

Evaluation of the precision of intradermal injection of control substances for intradermal testing in clinically normal horses.

OBJECTIVE: To evaluate the precision of intradermal testing (IDT) in horses. ANIMALS: 12 healthy adult horses. PROCEDURE: IDT was performed on the neck of each horse by use of 2 positive control substances (histamine and phytohemagglutinin [PHA]) and a negative control substance. An equal volume (0.1 mL) for each injection was prepared to yield a total of 20 syringes ([4 concentrations of each positive control substance plus 1 negative control substance] times 2 positive control substances times 2 duplicative tests) for each side of the neck. Both sides of the neck were used for IDT; therefore, 40 syringes were prepared for each horse. Hair was clipped on both sides of the neck, and ID injections were performed. Diameter of the skin wheals was recorded 0.5, 4, and 24 hours after ID injection. RESULTS: Intra- and interhorse skin reactions to ID injection of histamine and PHA resulted in wheals of uniform size at 0.5 and 4 hours, respectively. Significant intra- and interhorse variation was detected in wheals caused by PHA at 24 hours. CONCLUSIONS AND CLINICAL RELEVANCE: ID injection of histamine and PHA caused repeatable and precise results at 0.5 and 4 hours, respectively. Concentrations of 0.005 mg of histamine/mL and 0.1 mg of PHA/mL are recommended for use as positive control substances for IDT in horses. This information suggests that consistent wheal size is evident for ID injection of control substances, and variation in wheals in response to ID injection of test antigens results from a horse's immune response to specific antigens.

Comparison of results for intradermal testing between clinically normal horses and horses affected with recurrent airway obstruction.

OBJECTIVE: To evaluate differences in response to ID injection of histamine, phytohemagglutinin (PHA), and Aspergillus organisms between clinically normal horses and horses with recurrent airway obstruction (RAO). ANIMALS: 5 healthy adult horses and 5 adult horses with RAO. PROCEDURE: Intradermal testing (IDT) was performed on the neck with 2 positive control substances (histamine and PHA) and a mixture comprising 5 Aspergillus species. Four concentrations of each test substance plus a negative control substance were used. Equal volumes (0.1 mL) of each test substance were prepared to yield 15 syringes ([4 concentrations of each test substance plus 1 negative control substance] times 3 test substances) for each side of each horse (ie, 30 syringes/horse). Intradermal injections were administered; diameter of wheals was recorded 0.5, 4, and 24 hours after injection. RESULTS: Hypersensitive responses to ID injection of histamine were detected 0.5 hours after injection, and a delay in wheal formation after ID injection of Aspergillus mixture 24 hours after injection was detected in RAO-affected horses but was not observed in clinically normal horses. No differences were detected between the 2 groups after ID injection of PHA. CONCLUSIONS AND CLINICAL RELEVANCE: RAO-affected horses are hypersensitive to histamine, suggesting that RAO is associated with a heightened vascular response to histamine. Higher concentrations of Aspergillus mixture may be needed to detect horses that are sensitive to this group of antigens. Wheal reactions to Aspergillus may be a delayed response, suggesting that IDT results should be evaluated 0.5, 4, and 24 hours after ID injection.

Nutritional support for neonatal foals.

In recent years, equine neonatal medicine has made significant advances. The importance of nutritional support for the sick neonatal foal has been recognized, and methods of providing that sup-port have been developed. Today, the clinician has many options when designing a nutritional plan for the neonatal foal. When the foal's gut permits, enteral diets are an inexpensive source of nutrients. Under conditions where the gut requires rest, methods for delivering nutrients by the parenteral route have also been developed. In this article, the nutrition of the normal and sick foal is described. Guidelines for designing a nutritional plan are also reviewed.