Effects of Experimental Sarcocystis neurona

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

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

Establishment of a Gene Expression System in Ochrobactrum anthropi

Genetic studies of Ochrobactrum anthropi are hindered by the lack of a suitable gene expression system. We constructed a set of vectors containing several promoters and a His tag fusion in the N terminus to facilitate protein detection and purification. The new vectors should significantly enhance the genetic manipulation and characterization of O. anthropi

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.\ud \ud Animals — 7 horses with confirmed RAO and 7 without RAO (non-RAO horses).\ud \ud 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.\ud \ud 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.\ud \ud 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

Organization and conservation of the miR-155 precursor gene.

<p>The structure of the human precursor gene is shown, with the location of the pre-miRNA-encoding region and promoter regions as described in the current assembly of the human genome. Comparison to the equine gene, and alignment with both human and murine genes is shown. Only predicted transcription factor binding sites conserved between humans and horses are indicated. Conservation is defined here as 70% nucleotide identity across a 100bp reading frame.</p

Interactions between miR-155, its targets, cytokines and transcription factors in innate immune pathways.

<p>Pathways have been simplified. Targets shown here (indicated by red arrows) are predicted in the horse and have supportive experimental evidence in other species.</p

Expression changes with time in miR-146a, miR-146b and miR-155.

<p>A, C and E: Expression changes at 2 and 4 hours as determined by NGS. Expression data are presented as fold change in normalized read count from unstimulated samples at time 0. B, D and F: Expression changes from 2–8 hours as determined by qRT-PCR. Data have been normalized to the endogenous controls miR-26a and miR-103, and are expressed as fold change from unstimulated samples at time 0 (2^-ΔΔCT). The whiskers denote twice the interquartile range, and outliers are indicated with a ‘+’ symbol. Mean is indicated with a diamond symbol, and median with a horizontal line. <i>P</i>-values for all significant differences between LPS-stimulated and control cells are given. For effects of time, significant differences from the 2-hour time point within each treatment group are indicated by # (<i>p</i> < 0.05) or ## (<i>p</i> < 0.001).</p

Correlation matrix of associations between miRNA expression (on qRT-PCR) and supernatant cytokine concentration.

<p>PCR data have been normalized to the endogenous controls miR-26a and miR-103. Cytokine data have been square-root transformed. The best fit line is indicated in blue. Pearson’s correlation coefficient (<i>r</i>) and Bonferroni-adjusted <i>p</i>-value are displayed for each pair.</p

Details of miRNA expression changes in cultured equine PBMCs.

<p>Details of miRNA expression changes in cultured equine PBMCs.</p