RAPID COMMUNICATION: TLR4 expressed but with reduced functionality on equine B lymphocytes.

Varying susceptibility exists among mammalian species to the development of potentially fatal endotoxemia due to gram-negative bacteria molecular component, lipopolysaccharide (LPS). Toll-like receptor 4 (TLR4) is responsible for LPS-associated immune response and is expressed on numerous immune cells including B lymphocytes. TLR4 is expressed in a functional form on mouse B lymphocytes, a species much less susceptible to endotoxemia compared with humans who are highly sensitive to endotoxin. Humans possess B lymphocytes that are not responsive to LPS. Likewise, horses are highly susceptible to endotoxemia but the expression and function of TLR4 on horse B lymphocytes is not known. Colic, the major cause of mortality in horses, is often complicated by resultant endotoxemia. The objective of this study was to determine the expression and function of TLR4 on equine B lymphocytes. Lymphocytes were isolated from peripheral blood mononuclear cells that were collected from six horses, and the expression and function of TLR4 was analyzed for each horse. Flow cytometry results indicate TLR4 is expressed on horse B lymphocytes but stimulation with LPS did not alter this expression (P = 0.99) compared with unstimulated B lymphocytes after 24 h. After 72 h of in vitro LPS stimulation, analysis of cell proliferation dye by flow cytometry demonstrated that equine B lymphocytes did not proliferate, while mouse B lymphocytes predictably did. Furthermore, the total number of LPS stimulated equine B lymphocytes did not significantly differ from unstimulated cells after 72 h of culture (P = 0.92). Horse lymphocytes exhibited no significant differences in the measured TLR4 signaling pathway genes (TLR4, IL-10, IL-6, IFNß, and TNFα) when expression was compared with LPS stimulated vs. unstimulated cells. In conclusion, while TLR4 is expressed on horse B lymphocytes, it appears minimally responsive to LPS in vitro, similar to results seen in human B lymphocytes. While further studies are still needed, our work reveals a potential link between B lymphocyte TLR4 expression and endotoxin sensitivity.

Sarcocystis neurona-Induced Myeloencephalitis Relapse Following Anticoccidial Treatment.

Sarcocystis neurona is a ubiquitous parasite in the eastern United States, which is the principal causative agent in the neurologic disorder equine protozoal myeloencephalitis (EPM). While much is known about this protozoa's life cycle in its natural host, the opossum (Didelphis virginiana), little is known of how it acts in the aberrant equine host, which displays a high incidence of exposure with a relatively low rate of morbidity. For this study, we employed the popular interferon gamma knockout mouse model to determine the potential for recrudescence of S. neurona infection after treatment with the anticoccidial drug diclazuril. Mice were infected with S. neurona merozoites, and 7-days post-infection (DPI) they were treated with diclazuril for 30 or 60 days or not treated at all. All infected non-treated mice developed neurologic signs consistent with S. neurona infection within 30 DPI. All diclazuril-treated infected mice remained clinically normal while on treatment but developed neurologic signs within 60 days of treatment cessation. Histological examination of cerebella from all infected mice demonstrated characteristic lesions of S. neurona infection, regardless of treatment status. Cerebellar samples collected from infected treated mice, displaying neurologic signs, produced viable S. neurona in culture. However, cerebellar samples collected from infected and neurologically normal mice at the end of a 30-day treatment period did not produce viable S. neurona in culture. Analysis of the humoral immune response in infected mice showed that during treatment IgM antibody production decreased, suggesting the organism was sequestered from immune surveillance. The cessation of treatment and subsequent development of neurologic disease resulted in increased IgM antibody production, suggesting recognition by the immune system at that time. Based on the study results the authors propose that diclazuril was able to inhibit the replication and migration of S. neurona but not fully eliminate the parasite, suggesting recrudescence of infection after treatment is possible.