Equine protozoal Myeloencephalitis (EPM) is a neurologic disease in horses predominantly caused by the protozoa Sarcocystis neurona. Carried by the North American opossum, Didelphis virginiana, horses are exposed to S. neurona when eating or drinking food or water contaminated with opossum feces. While exposure to the protozoa is high, only around 1% of horses develop clinical disease. While the mechanism by which S. neurona causes central nervous system damage is still unknown, this dissertation examines the histopathologic damage and potential persistence of S. neurona after anti-protozoal treatment between groups of horses with acute and chronic disease. This dissertation also examines the ability of two diagnostic techniques, immunohistochemistry (IHC) and polymerase chain reaction (PCR), to detect S. neurona. Horses were grouped based on duration of clinical signs; acutely affected horses exhibited clinical signs <6 months, while chronically affected horses exhibited clinical signs >6 months, including those previously treated for EPM. A comparison of necropsy reports revealed that chronically affected horses with EPM had more degenerative changes compared to acutely affected horses with EPM. However, when histologic changes were quantified, acutely affected horses had similar degenerative changes compared to chronically affected horses. When IHC and PCR were compared, IHC detected S. neurona presence (9/9 horses with EPM) significantly more often than PCR 4/9 horses with EPM). Our studies also show that S. neurona can be persistent in horses, as evidenced by the detection of S. neurona in the CNS of horses previously treated for EPM. Four horses had previously been treated for EPM, and all four had S. neurona present in their CNS even after anti-protozoal treatment, suggesting the ability for S. neurona to persist. In addition, this dissertation examines the possibility of using soluble CD14 (sCD14) as a supplemental assay for differentiating neurologic diseases such as EPM and cervical vertebral stenotic myelopathy (CVSM). When sCD14 levels were assessed in control EPM, CVSM, and EPM+CVSM horses, sCD14 concentrations in the cerebral spinal fluid (CSF) were significantly different between control and EPM horses and EPM horses and CVSM horses. With this information, clinicians and researchers may use sCD14 as a supplemental assay for differentiating between healthy, EPM, and CVSM horses. Finally, future directions include preliminary data that may lead to a potential for a peptide vaccine protecting horses from EPM clinical disease. Further insight into the persistence of S. neurona after anti-protozoal treatment is needed, the classification of acutely and chronically affected horses and the ability of sCD14 detection as a supplemental assay will be required; this this dissertation allows for the continuation of knowledge in combating the elusive protozoa, S. neurona. / Doctor of Philosophy / Equine protozoal myeloencephalitis (EPM) is a common neurologic disease in horses in North and South America caused predominantly by the parasite Sarcocystis neurona. This disease is carried by the North American opossum (Didelphis virginiana), and horses encounter S. neurona when eating or drinking food or water contaminated with opossum feces. Not all horses who encounter the parasite develop disease and can clear the parasite before disease occurs. One study in this dissertation found that the immunohistochemistry (IHC) test was significantly better at detecting S. neurona than polymerase chain reaction (PCR). Additionally, horses with EPM were broken into two groups: acutely affected horses with neurologic signs <6 months and chronically affected horses with neurologic signs >6 months. The study found that horses chronically afflicted with EPM had more degenerative changes compared to acutely affected horses. In addition, four horses who had previously been treated for EPM had S. neurona in their central nervous system (CNS), suggesting the ability of the parasite to persist after EPM treatment and that persistence of S. neurona may cause greater degenerative changes in horses with long-term neurological signs. The final study in this dissertation examined the potential for an assay to be used to help differentiate EPM from other neurologic diseases such as cervical vertebral stenotic myelopathy (CVSM). By measuring sCD14 concentration in the serum and cerebral spinal fluid (CSF) of control, EPM horses, CVSM horses, and EPM+CVSM horses, significant differences were found between control and EPM horses, and EPM and CVSM horses. This finding indicates the potential for sCD14 to be used to help differentiate between these two devastating neurologic diseases. The future directions include preliminary data that could lead for a potential protein vaccine capable of protecting horses from EPM disease. Overall, the results of these studies improve our knowledge of EPM and potentially improve equine health worldwide.

Equine Protozoal Myeloencephalitis (EPM) is arguably the most common and costly equine neurologic diseases nationwide. The national seroprevalence is >50%, but only 0.5-1% of all horses develops disease during their lifetimes. Some EPM affected horses have decreased immune response. A cell-mediated immune response has been shown to be protective for development of EPM after infection with Sarcocystis neurona in mouse models. Levamisole has been proposed as an adjunctive therapy for EPM to upregulate the cell-mediated immune response based on positive results in other species, but there are very limited studies in equids. We hypothesized that levamisole will upregulate the equine cell-mediated macrophage (M1) dendritic cell (DC1) CD4 T-helper 1 (Th1) CD8 Tc1 immune response in vitro. The first aim was to determine optimal conditions and effects of levamisole on cellular proliferation. Equine PBMCs were harvested from ten horses seronegative for S. neurona. The cells were cultured alone, or with one of the mitogens: concanavalin A (ConA) or phorbol 12-myristate 13-acetate and ionomycin (PMA/I), or with a combination of the above mitogens and levamisole at several conditions. Cellular proliferation was assessed using a colorimetric bromodeoxyuridine ELISA assay. The second aim was to determine the ability of levamisole, under optimized conditions, to upregulate the M1 DC1 CD4Th1 CD8 Tc1 response in vitro based on activation and function. PBMCs from the same 10 horses were cultured with each of the following: no stimulation, conA, and levamisole with and without ConA. To determine proliferation of each specific subset, cells were labeled with a fluorescent dye, CellTrace. Proliferation was determined based on dye dilution using flow cytometry. To determine the effects of levamisole on the specific immune response, cell subsets were labeled with fluorescent antibodies for cell surface markers (CD4, CD8, CD21, CD172a, CD14) and dendritic and macrophage activations markers (MHC Class II, CD86). Induction of T-regs was based on FoxP3 expression. Immune phenotypes were determined based on intracellular cytokine expression (IFNɣ, IL4, IL10). Study results indicate that levamisole alone did not significantly alter PBMC proliferation compared to the response of unstimulated cells. Cells cultured with either ConA or PMA/I resulted in a statistically significant increase (P<0.05) in proliferation compared to unstimulated cells. Cells cultured with ConA and levamisole at 1µg/mL resulted in a significant decrease (P<0.05) in proliferation compared with cells cultured with ConA alone. Flow cytometry data failed to elucidate the specific immune phenotype that is affected by levamisole. Subjectively, there appeared to be a trend for inceased IFNɣ production by CD14 and CD172a positive cells (macrophages and dendritic cells) and a decrease in IFNɣ production by CD4 and CD8 positive cells (T-lymphocytes). These results demonstrate that levamisole downregulates ConA stimulated PBMC proliferation. Based on these in vitro results, further studies to determine the effectiveness of levamisole on modulating the equine immune system in vivo and to more specifically evaluate the immune cell subets affected by levamisole are warranted. / Master of Science

Equine protozoal myeloencephalitis (EPM) is a progressive neurological disease of horses caused by Sarcocystis neurona. Two projects were conducted to identify factors involved in the development of EPM. The first study explored a possible genetic susceptibility to EPM by attempting a genome-wide association study (GWAS) on formalin-fixed, paraffin-embedded (FFPE) tissue from 24 definitively-positive EPM horses. DNA extracted from tissues older than 14 months was inadequate for SNP analysis on the Illumina Equine SNP50 BeadChip probably due to degradation and formalin cross-linking. Results were inconclusive as analysis was not possible with the small sample set. The second study evaluated an artificial infection method in creating a reliable equine EPM model. Five horses were injected intravenously at 4 time points with autologous blood incubated with 1,000,000S. neurona merozoites. Challenged horses progressively developed mild to moderate clinical signs and had detectable S. neurona serum antibodies on day 42 post challenge. Horses appeared to have produced a Th1 immune response and cleared the infection by the conclusion of the study on day 89. No histopathological evidence of S. neurona infection was found within central nervous system tissue. This artificial infection method was not effective in replicating the severe clinical EPM seen in natural infections.

Equine Protozoal Myeloencephalitis (EPM) is a serious neurologic disease of horses in the United States. The primary etiologic agent is Sarcocystis neurona (S. neurona). Currently, there is limited knowledge regarding the protective or pathologic immune response to infection to the intracellular protozoa S. neurona. The objective of these studies was to determine the effects of S. neurona infection on the immune response of horses that had EPM due to natural infection (experiment 1) and experimental infection (experiment 2). In experiment 1, twenty-two horses with naturally occurring cases of EPM, which were confirmed positive based on detection of antibodies in the serum and/or CSF and clinical signs, and 20 clinically normal horses were included to determine whether S. neurona altered the immune responses, as measured by immune cell subsets (CD4, CD8, B-cell, monocytes, and neutrophils) and leukocyte proliferation (antigen specific and non-specific mitogens). Our results demonstrated that naturally infected horses had significantly higher percentages of CD4 and neutrophils (PMN) in peripheral blood mononuclear cells (PBMCs) than clinically normal horses. Leukocytes from naturally infected EPM horses had a significantly lower proliferation response, as measured by thymidine incorporation, to a non-antigen specific mitogen phorbol 12-myristate 13-acetate (PMA) / ionomycin (I) than did clinically normal horses (p=0.04). The implications of these findings will be discussed. In experiment 2, 13 horses were randomly divided into two groups. Baseline neurologic examinations were performed and all horses were confirmed negative for S. neurona antibodies in the CSF and serum. Then, one group with 8 clinically normal seronegative horses was inoculated intravenously with approximately 6000 S. neurona infected autologous leukocytes daily for 14 days. All the challenged horses showed neurologic signs consistent with EPM. PBMCs were isolated from the control and infected horses to determine how S. neurona alters the immune responses based on changes in immune cell subsets and immune function. There were no significant differences in the percentage of CD4 cells in peripheral blood lymphocytes or IFN-γ production by CD4 and/or CD8 cells. PMA/I stimulated proliferation responses in PBMCs appeared suppressed compared to that of uninfected controls. Additional studies are necessary to determine the role of CD4 and CD8 cells in disease and protection to S. neurona in horses, as well as to determine the mechanism associated with suppressed in vitro proliferation responses. This project was funded by Patricia Stuart Equine grants and paramutual racing funds from Virginia Tech. / Master of Science

Equine Protozoal Myeloencephalitis is the most frequently diagnosed neurologic disorder of horses in the united states, which is caused by the protozoan organism Sarcocystis neurona. The disease has a profound impact on the American Horse Industry. This impact includes prolonged and expensive treatment without a guaranteed return to a previous level of use for the individual horse. Poor respponse to and prolonged duration of treatment may suggest an immune mediated impariement of host response. There is limited information about the direct interaction between the pathogen and the host. In two in vitro experiments we investigated a) whether the presence of the protozoan organism can influence mitogen-stimulated peripheral blood mononuclear cells (PBMCs), suggesting a direct influence of the protozoan organism on cells of the immune system, and b) if cerebrospinal fluid (CSF) from horses with EPM has an effect on mitogen-stimulated PBMCs, suggesting that the microenvironment of the site of infection influences the course of disease. Experiment 1: Mitogen simulated PBMCs from EPM affected and control horses were co-cultured with fragments of freeze thawed bovine turbinate cells that were infected with S. neurona merozoites. Compared to controls PBMCs co-cultured with S. neurona fragments were the only cells that showed a decreased proliferation (p<0.05). A difference between EPM affected and control horses could not be detected (p>0.05). These results may imply that the persistence of S. neurona infection in the horses CNS is, in part, due to a pathogen-derived mechanism that attentuates the hosts immune response. Experiment 2: Mitogen stimulated PBMCs from a horse affected with EPM and a control were co-cultured n the presence of CSF from EPM affected and uninfected controls. Prior to co-culture the CSF was fractionated by a filtration process over two microfilter units. An identical volume of NaCl (0.9%) served as a control for the volume of CSF that was added. The proliferation assay revealed a deviation of the response depending on cell donor and CSF fraction used. The effect was independant of the protein concentration of the CSF fraction, and a decrease in lymphocyte proliferation was not caused by increased cellular death. This suggests the presence of subsets within the CSF which have a stimulatory of suppressive influence on the cells in culture. The effect was cell donor dependant which implies a difference in lymphocyte subsets between the two horses that were used. / Master of Science

Previous studies have demonstrated differences in CD4+, CD8+ and B-cell populations between EPM affected and normal horses. The overall goal of our project was to further define the immune deficiencies associated with S. neurona infection. We hypothesized that PMA/I stimulated suppression in EPM horses is due to decreased proliferation of monocytes, CD4+ and CD8+ cells. Our objectives were 1) to determine whether S. neurona infection causes an increase in apoptosis of a particular immune subset, and 2) to determine whether S. neurona causes a decrease in the number of cellular divisions (proliferation) of a particular immune cell subset. For this study, nine S. neurona antibody negative, immunocompetent horses were obtained. Baseline neurologic examinations, SnSAG1 (S. neurona Surface Antigen 1) ELISAs on cerebrospinal fluid (CSF) and serum, and baseline immune function assays were performed. Horses were randomly divided into groups. Five horses were challenged for ten days via intravenous injection of autologous lymphocytes infected with S. neurona. Neurologic parameters of all horses were assessed for 70 days following infection. Immune function was based on proliferation responses to mitogens, as assessed through thymidine incorporation. Enumeration of cellular subsets, degree of apoptosis and number of cellular divisions were assessed through flow cytometry. SnSAG1 ELISA of serum and CSF samples performed post-infection confirmed infection and disease. All infected horses displayed moderate neurologic signs on clinical examination. Some significant differences in cellular activities were noted. Additionally, this is the first time the method using S. neurona infected lymphocytes has been reproduced successfully by different investigators. / Master of Science

Equine protozoal myeloencephalitis (EPM), caused by the protozoan parasite Sarcocystis neurona, is one of the most important neurological diseases of horses in the Americas. While seroprevalence of S. neurona in horses is high, clinical manifestation of EPM occurs in less than 1% of infected horses. Factors governing the occurrence and severity of EPM are largely unknown, although horse immunity might play an important role in clinical outcome. We hypothesize that EPM occurs due to an aberrant immune response, which will be discernable in the equine IgG subisotypes a, b, and (T) that recognize S. neurona in infected diseased horses versus infected but clinically healthy horses. Based on previously-established serum antibody concentrations for IgG subisotypes in healthy horses, standard curves were generated and served to establish the concentration of antigen-specific IgG subisotypes in equine serum and CSF in infected diseased and infected normal horses. The subisotype concentrations and ratios between subisotypes were analyzed to assess whether neurological disease is associated with detectable differences in the antibody response elicited by infection. Results indicate a type I biased immune response in infected diseased horses, implicating the role of immunity in the development of EPM.

Orientador: Rogerio Martins Amorim / Resumo: Estudos com células estromais mesenquimais multipotentes (MSCs) estão em crescente progresso devido às suas propriedades imunomoduladoras, antiinflamatórias, antiapoptóticas e de regeneração tecidual, tornando essa modalidade de terapia celular promissora no tratamento de diversas doenças. Devido à limitada capacidade regenerativa do sistema nervoso central (CNS), causando sequelas funcionais, as MSCs estão sendo investigadas como uma alternativa terapêutica para condições neurológicas inflamatórias, vasculares, traumáticas e degenerativas em diversas espécies animais. A Mieloencefalite protozoária equina (EPM) causada por ambos os protozoários do filo Apicomplexa, Sarcocystis neurona e Neospora hughesi, permanece como uma importante doença neurológica dos equinos nas Américas, embora a maioria dos casos seja devida à infecção por S. neurona. A aplicação da proteômica com sua gama de ferramentas na clínica de equinos pode contribuir significativamente para o entendimento de processos patológicos e facilitar a descoberta de novos alvos terapêuticos ou marcadores diagnósticos. Neste contexto, os objetivos deste estudo foram avaliar o perfil proteômico do líquido cefalorraquidiano (CSF) antes e após múltiplos transplantes intratecal de MSCs em equinos hígidos e o perfil proteômico do CSF de equinos cronicamente afetados pela EPM. Doze cavalos adultos clinicamente saudáveis foram divididos aleatoriamente em três grupos experimentais: grupo DPBS (DPBS ou control; n = 4) onde a sol... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Multipotent mesenchymal stromal cell (MSCs) studies are under increasing progress because of their immunomodulatory, anti-inflammatory, antiapoptotic and tissue regeneration properties, making this modality of cell therapy promising in the treatment of various diseases. Due to the limited regenerative capacity of the central nervous system (CNS), causing functional sequelae, MSCs are being investigated as a therapeutic alternative for inflammatory, vascular, traumatic and degenerative neurological conditions in various animal species. Equine protozoal myeloencephalitis (EPM) caused by both protozoa of the Apicomplexa phylum, Sarcocystis neurona and Neospora hughesi, remains an important neurological disease in horses in the Americas, although most cases are due to S. neurona infection. The application of proteomics with its range of tools in the equine clinic can contribute significantly to the understanding of pathological processes and facilitate the discovery of new therapeutic targets or diagnostic markers. In this context, the objectives of this study were to evaluate the proteomic profiling of cerebrospinal fluid (CSF) before and after multiple intrathecal transplantations of MSCs in healthy horses and the CSF proteomic profiling of horses chronically affected by EPM. Twelve clinically healthy adult horses were randomly divided into three experimental groups: DPBS (DPBS or control; n = 4), in which intrathecal "transplants" with Dulbecco's phosphate buffered saline (DPB... (Complete abstract click electronic access below) / Doutor

Sarcocystis neurona is a protozoan parasite that causes the serious neurologic disease equine protozoal myeloencephalitis (EPM). The life cycle of S. neurona progresses through multiple developmental stages that differ morphologically and molecularly. The S. neurona merozoite surface is covered by multiple related proteins, which are orthologous to the surface antigen (SAG) gene family of Toxoplasma gondii. The SAG surface antigens in T. gondii and another related parasite Neospora caninum are life cycle stage-specific and seem necessary for parasite transmission and persistence of infection. The present research was conducted to explore the gene family of SnSAGs in S. neurona. Specifically, the project identified new SnSAGs in the draft genome sequence of S. neurona and examined the stage-specific expression and potential function of these surface antigens. For the first part of the study, expression of the S. neurona merozoite surface antigens was evaluated in the sporozoite and bradyzoite stages. The studies revealed that SnSAG2, SnSAG3 and SnSAG4 are expressed by sporozoites, while SnSAG5 appeared to be downregulated in this life cycle stage. In S. neurona bradyzoites, SnSAG2, SnSAG3, SnSAG4 and SnSAG5 were either absent or expression was greatly reduced. For the second part of the study, the draft sequence of the S. neurona genome was searched for potential new SnSAGs. Multiple searches revealed sixteen potential new SnSAG genes, and bioinformatic analyses of the sequences revealed characteristics consistent with the SAG gene family. Two of the new SnSAGs, designated SnSAG7 and SnSAG8, have been characterized in detail. The studies showed that SnSAG7 is expressed by the merozoite stage, while SnSAG8 is expressed by the bradyzoite stage. The third part of the study assessed the role of SnSAGs in host cell attachment and/or invasion by S. neurona. Serum neutralization assays using polyclonal serum raised against SnSAG1, SnSAG2, SnSAG3, and SnSAG4 suggested that SnSAG1 and SnSAG4 play a role in host cell attachment and/or invasion; treatment with antibodies against SnSAG2 and SnSAG3 were inconclusive. The information acquired about the stage-specific expression of the SnSAGs, identification of new SnSAG paralogues, and their functional characterization will help to understand the importance of the SnSAG proteins for parasite survival and could lead to improved methods for EPM prevention and/or treatment.