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

No / The indoloquinoline alkaloid cryptolepine 1 has potent in vitro antiplasmodial activity, but it is also a DNA intercalator with cytotoxic properties. We have shown that the antiplasmodial mechanism of 1 is likely to be due, at least in part, to a chloroquine-like action that does not depend on intercalation into DNA. A number of substituted analogues of 1 have been prepared that have potent activities against both chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum and also have in common with chloroquine the inhibition of ß-hematin formation in a cell-free system. Several compounds also displayed activity against Plasmodium berghei in mice, the most potent being 2,7-dibromocryptolepine 8, which suppressed parasitemia by 89% as compared to untreated infected controls at a dose of 12.5 mg kg-1 day-1 ip. No correlation was observed between in vitro cytotoxicity and the effect of compounds on the melting point of DNA (¿Tm value) or toxicity in the mouse¿malaria model.

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.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Brazil has a sanitation service with greater deficit coverage, this factor, related to water pollution that receives the discharge of effluents untreated, mainly from domestic sources, as the collection and treatment do not exist or are inadequate. Discharging effluents directly into the ground without proper treatment can lead to contamination of water resources, soil and groundwater, according to local characteristics. Amid this reality, the degradation of natural resources on the campus of UFSM, due to increased academic population, has drawn attention of researchers and managers of the institution. There is a considerable extension of plants as weeds very close to launch domestic Campus of the University, such as Typha domingensis, which are responsible for much of retention of microorganisms in waste water releases and the study location does not exist until the when a microbiological control. This work is justified by the importance of analysis of environmental samples such releases and advocate methodologies for proving the presence of helminth eggs and oocysts of protozoa present in the rhizome of these plants. This factor becomes important by the fact that helminths and protozoa are great cause of gastrointestinal diseases and may, depending on habitat conditions, health and age, cause the individual to death. The study area was next to two buildings in the student's home with release of wastewater. The aim of this study was to develop a methodology to verify the presence of helminth eggs and oocysts of protozoa in the rhizosphere of weeds in natural environment, with release of sewage. The study was conducted in two phases, with the first phase analysis of helminth eggs and oocysts of protozoa present in twelve samples of waste water releases through Bailenger method that occurred during a period of three months, with analysis performed weekly. The second phase was to detect the presence of helminth eggs and oocysts of protozoa with a modified methodology in this paper, called Roberts & Wolff, for analysis of the rhizosphere of the rhizome of macrophyte Typha domingensis in 10 specimens of the plant, also with an approximate time of analysis of three months. The results of the first stage coincide with the data of some studies with wastewater. The results of the second phase even being positive for the presence of helminth eggs and oocysts of protozoa do not make it possible to compare, since there is the analysis results literature helminth eggs and oocysts of protozoa present in the rhizosphere of macrophyte Typha domingensis. As a result of the analysis of the first phase gave an average value of the presence of helminth eggs 112.4 eggs/ L FP35 and 113.5 eggs/ L FP50. Samples of Cryptosporidium sp all samples tested positive for the presence of oocysts and Giardia sp all were negative. In the second phase, we obtained a greater number of eggs found in a sample of one of the analyzed samples and other samples remained very close values of helminth eggs. In Cryptosporidium sp samples only the last three specimens was negative and Giardia sp only a sample of a specimen was positive. / O Brasil possui um serviço de saneamento com maior déficit de cobertura, fator esse, relacionado a poluição das águas que recebe o lançamento de efluentes sem tratamento, principalmente os de origem doméstica, uma vez que a coleta e o tratamento não existe ou é inadequado. O lançamento de efluentes diretamente no solo, sem tratamento adequado pode acarretar a contaminação dos cursos hídricos, do solo e da água subterrânea, de acordo com as características do local. Em meio a essa realidade, a degradação de recursos naturais no Campus da UFSM, devido ao aumento da população acadêmica, tem chamado atenção de pesquisadores e gestores da instituição. Há uma considerável extensão de plantas como macrófitas muito próximas aos lançamentos domésticos no Campus da Universidade, tais como a Typha domingensis, que são responsáveis por uma boa parte de retenção de microrganismos presentes em lançamentos de águas residuais e que no local estudado não existe até o momento um controle microbiológico. O presente trabalho justifica-se pela importância de análises de amostras ambientais desses lançamentos e de preconizar metodologias que possibilitem comprovar a presença de ovos de helmintos e oocistos de protozoários presentes no rizoma dessas plantas. Esse fator torna-se importante pelo fato que helmintos e protozoários são grande causadores de doenças gastrointestinais, podendo, em função das condições de habitat, saúde e idade, levar o indivíduo a morte. A área estudada foi próxima a dois prédios da casa do estudante com lançamento de águas residuais. O objetivo geral desse trabalho foi desenvolver metodologia para verificar a presença de ovos de helmintos e oocistos de protozoários na rizosfera de macrófitas em ambiente natural, com lançamento de efluentes sanitários. O estudo foi desenvolvido em duas fases, tendo como primeira fase a análise de ovos de helmintos e oocistos de protozoários presentes em doze amostras de lançamentos de águas residuais através do método de Bailenger que ocorreu durante um período de três meses, com análise realizadas semanalmente. A segunda fase foi detectar a presença de ovos de helmintos e oocistos de protozoários com uma metodologia modificada neste trabalho, denominada de Rodrigues & Wolff, para análise da rizosfera do rizoma da macrófita Typha domingensis em 10 exemplares da planta, também com um tempo aproximado de análise de três meses. Os resultados da primeira fase coincidem com dados de alguns estudos realizados com águas residuais. Os resultados da segunda fase, mesmo sendo positivos para a presença de ovos de helmintos e oocistos de protozoários não tornam possível a comparação, visto que não existe na literatura resultados de análise de ovos de helmintos e oocistos de protozoários presente na rizosfera da macrófita Typha domingensis. Como resultado das análises da primeira fase obteve-se um valor médio da presença de ovos de helmintos de 112,4 ovos/L na FP35 e 113,5 ovos/L na FP50. Nas amostras de Cryptosporidium sp todas as amostras deram positivas para a presença desse oocisto e Giardia sp todas deram negativas. Na segunda fase, obteve-se um maior número de ovos encontrados em uma das amostras de um dos exemplares analisados e as demais amostras mantiveram valores muito próximos de ovos de helmintos. Nas amostras de Cryptosporidium sp somente as dos últimos três exemplares deu negativo e Giardia sp somente em uma amostra de um exemplar deu positivo.

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection. / Grand Challenges Canada / Revisión por pares

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

Horses that are exposed to Sarcocystis neurona, a causative agent of equine protozoal myeloencephalitis, produce antibodies that are detectable in serum by western blot (WB). A positive test is indicative of exposure to the organism. Positive tests in young horses can be complicated by the presence of maternal antibodies. Passive transfer of maternal antibodies to S. neurona from seropositive mares to their foals was evaluated. Foals were sampled at birth (presuckle), at 24 hours of age (postsuckle), and at monthly intervals. All foals sampled before suckling were seronegative. Thirty-three foals from 33 seropositive mares became seropositive with colostrum ingestion at 24 hours of age, confirming that passive transfer of S. neurona maternal antibodies occurs. Thirty-one of the 33 foals became seronegative by 9 months of age, with a mean seronegative conversion time of 4.2 months. These results indicate that evaluation of exposure to S. neurona by WB analysis of serum may be misleading in young horses. Cerebrospinal fluid (CSF) samples from 15 neonatal (2-8 day) foals were examined for the presence of antibodies to S. neurona by WB analysis. Twelve of 13 foals that were seropositive were also CSF positive, suggesting that maternal antibodies to S. neurona cross the blood-CSF barrier in neonatal foals resulting in a positive CSF WB. Repeat taps were performed on 5 of the foals which showed that the immunoreactivity of the western blot decreases over time. Two of the 5 foals were CSF negative at 83 and 84 days of age, with 1 foal still positive at 90 days, and 2 foals positive at 62 days. These results indicate that maternal antibodies to S. neurona in the CSF can confound WB results in neonatal foals up to several months of age. / 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