Thesis (PhD (Molecular Biology and Human Genetics))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: The growing global epidemic of human tuberculosis (TB) results in 8 million new cases of this disease and 2 million deaths annually. Control thereof will require greater insight into the biology of the causative organism, Mycobacterium tuberculosis, and into the pathogenesis of the disease. This will benefit the design of new vaccines and diagnostic assays which may reduce the degree of both disease transmission and progression. Animal models have played a vital role in the understanding of the aetiology, pathogenesis, and treatment of TB. Much of such insight has been obtained from experimental infection models, and the development of new vaccines, for example, is dependant on these. Nonetheless, studies utilising naturally occurring TB in animals, such as those which have investigated the use of interferon-gamma release assays (IGRA) for its diagnosis, have contributed substantially to the body of knowledge in this field. However, there are few such examples, and this study sought to identify and investigate naturally occuring animal TB in South Africa as an opportunity to gain further insight into this disease. During the course of this study, the dassie bacillus, a distinctly less virulent variant of M. tuberculosis, was isolated from a rock hyrax from the Western Cape Province of South Africa. This has provided new insight into the widespread occurrence of this organism in rock hyrax populations, and has given impetus to further exploring the nature of the difference in virulence between these pathogens. Also investigated was M. tuberculosis infection in dogs in contact with human TB patients. In so doing, the first reported case of canine TB in South Africa was described, v a novel canine IGRA was developed, and a high level of M. tuberculosis infection in these animals was identified. This supports human data reflecting high levels of transmission of this pathogen during the course of human disease. Additionally, the fact that infected companion animals may progress to disease and potentially act as a source of human infection was highlighted. However, an attempt to adapt a flow cytometric assay to study cell-mediated immune responses during canine TB revealed the limitations of such studies in species in which the immune system remains poorly characterised. The use of IGRAs to diagnose TB was further explored by adapting a human assay, the QuantiFERON-TB Gold (In-Tube Method), for use in non-human primates. These studies have shown that such an adaption allows for the sensitive detection of TB in baboons (Papio ursinus) and rhesus macaques (Macaca mulatta) and may be suitable for adaption for use in other species. However, they have also evidenced the limitation of this assay to specifically detect infection by M. tuberculosis. Finally, to contextualise the occurrence of the mycobacterial infections described above, and other similar examples, these have been reviewed as an opinion piece. Together, these investigations confirm that animal models will continue to make important contributions to the study of TB. More specifically, they highlight the opportunities that naturally occuring animal TB provides for the discovery of novel insights into this disease. / AFRIKAANSE OPSOMMING: Wêreldwye tuberkulose (TB) epidemie veroorsaak agt miljoen nuwe gevalle en twee miljoen sterftes jaarliks. Ingryping by die beheer hiervan vereis begrip van die biologie van die mikroörganisme Mycobacterium tuberculosis, die oorsaak van TB, asook van die patogenese van die siekte self. Hierdie kennis kan lei tot ontwerp van nuwe entstowwe en diagnostiese toetse wat gevolglik beide die oordrag- en vordering van die siekte mag bekamp. Dieremodelle speel lankal 'n rol in ons begrip van die etiologie-, patogenese- en behandeling van TB. Insig is grotendeels verkry vanaf eksperimentele infeksiemodelle, en ontwikkeling van entstowwe, onder andere, is afhanklik van soortgelyke modelle. Desnieteenstaande, studies wat natuurlike TB voorkoms in diere ondersoek, byvoorbeeld dié wat op die ontwikkeling van interferon-gamma vrystellingstoetse (IGVT) fokus, het merkwaardige bydrae gemaak tot kennis en begrip in hierdie studieveld. Daar is slegs enkele soortgelyke voorbeelde. Om hierdie rede is die huidige studie uitgevoer waarbinne natuulike diere-TB geïdentifiseer en ondersoek is in Suid-Afrika om verdere kennis en insig te win aangaande TB. Die "dassie bacillus", bekend om beduidend minder virulent te wees as M. tuberculosis, is tydens hierdie studie geïsoleer vanuit 'n klipdassie (Procavia capensis) in die Wes-Kaapse provinsie, Suid-Afrika. Insig in die wydverspreide voorkoms van hierdie organisme in klipdassie bevolkings is gevolglik verkry en verskaf momentum om die aard van verskil in virulensie tussen dié patogene te bestudeer. vii Voorts is M. tuberculosis infeksie bestudeer in honde wat in kontak is met menslike TB pasiënte en word die eerste geval van honde TB dus in Suid-Afrika beskryf. In hierdie groep diere, is 'n hoë vlak van M. tuberculosis infeksie geïdentifiseer deur gebruik te maak van 'n nuut ontwikkelde IGVT vir die diagnose van honde TB. Gevolglik ondersteun dié studie bevindinge van menslike studies wat toon dat besondere hoë vlakke van M. tuberculosis oordrag voorkom gedurende die verloop van die siekte. Verder toon die studie dat geïnfekteerde troeteldiere 'n bron van menslike infeksie kan wees. 'n Poging om 'n vloeisitometriese toets te ontwikkel om die aard van selgefundeerde immuunreaksies te bestudeer in honde met TB toon die beperkings van dergelike studies in spesies waarin die immuunsisteem gebrekkig gekarakteriseer is. Die gebruik van IGVT'e in die diagnose van TB is verder ondersoek deur 'n menslike toets (QuantiFERON-TB Gold, In-Tube Method) aan te pas vir die gebruik van nie-menslike primaat gevalle. Hierdie studies toon gevolglik dat so 'n aanpassing toepaslik is vir hoogs sensitiewe deteksie van TB in chacma bobbejane (Papio ursinus) en rhesus ape (Macaca mulatta), en mag ook aangepas word vir gebruik in ander spesies. Tog word die beperkings van hierdie toets om infeksie wat spesifiek deur M. tuberculosis veroorsaak uitgelig. Ter afsluiting word hierdie studie in konteks geplaas deur 'n oorsig te gee van bogenoemde- en soortgelyke gevalle van dierlike infeksie deur mikobakterieë in Suid-Afrika. Hierdie studies bevestig dat dieremodelle steeds belangrike toevoegings maak tydens die bestudering van TB en lig veral die moontlikhede uit dat bestudering van natuulike TB in diere kan lei tot die ontdekking van nuwe insigte ten opsigte van die siekte self.

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.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Daniel U. Thomson / A series of studies were conducted in order to develop, test, implement, and utilize an objective and comprehensive gross pathology scoring system for cattle at slaughter. Individual lung, liver, and rumen gross pathology data was collected from 19,229 head of cattle and corresponding individual pre-harvest and carcass data for a subset of 13,226 head.. Across the entire population 22.6% and 9.8% of cattle displayed mild and severe lesions, respectively. Severe lung lesions at the time of slaughter were associated with a decreased ADG of 0.07 kg/ day and a carcass weight 7.1 kg less than that of their cohorts with no visible signs of pulmonary BRDC lesions (P < 0.01). Overall, 68.6 % of cattle observed had normal livers, free from abscesses and other abnormalities. Cattle with a severe liver abscess at the time of slaughter were associated with a 0.10 kg/day during the feeding period (P < 0.01). Of cattle severely affected by liver abscesses (A+, 4.6%), 14.9% also displayed severe BRDC lung lesions and 28.3 % of cattle displayed mild BRDC lung lesions. Rumenitis lesions were observed in 24.1% of the overall study population. Severe rumenitis lesions were associated with a significant decrease in average daily gain and carcass weight (0.03kg/day and 2.20 kg, respectively, P < 0.01). The system was also implemented on a population of cull cows at a commercial abattoir in the Great Lakes region of the U.S. (n=1,461; 87% Holstein, 13% other cows). Severe liver abscesses, were observed in 18.5% of cull cows at slaughter. Severe rumenitis lesions or rumenitis scars were observed in 10% and severe BRDC lesions were observed in 10.3% o of the population. A prospective study of a commercially available, direct fed microbial oral drench of Megasphaera elsdenii (NCIMB 41125) was conducted in 4,863 head of yearling feeder cattle. No significant effects of treatment were detected for final live weight (599 vs. 601 kg; P=0.79) or hot carcass weight (386 vs. 387 kg P=0.81) for Con and M.e., respectively. Fourteen point two percent and 14.0% of Con and M.e., respectively displayed a liver abscess of varying severity at the time of slaughter. Overall, 8.27 and 7.96% % of Con and M.e. cattle were observed with an altered rumen epithelial health status. The ordinal odds ratio of a M.e. treated animal having a more severe liver abscess score or rumen health score was not significant (Estimate: 0.96, 95% C.L. 0.733-1.259, P=0.771; Estimate: 1.01, 95% C.L. 0.625-1.63 P=0.96, respectively.) Comprehensive monitoring of gross pathology at slaughter is commercially plausible and provides valuable data for veterinarians, nutritionists and management personnel.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Wenjun Ma / With introduction of the 2009 pandemic H1N1 virus (pH1N1) into swine herds, reassortment between the pH1N1 and endemic swine influenza viruses (SIVs) has been reported worldwide. Recently, reassortant H3N2 and H1N2 variant SIVs that contain the M gene from pH1N1 virus and the remaining seven genes from North American triple-reassortant (TR) SIVs have emerged. These variant viruses have caused more than 300 cases of human infections and one death in the USA, creating a major public health concern. To date, the pathogenicity and transmissibility of H1N2 variant viruses in pigs has not been investigated. Through passive surveillance, we have isolated two genotypes of reassortant H1N2 viruses with pH1N1 genes from diseased pigs in Kansas. Full genome sequence and phylogenetic analysis showed that one is a swine H1N2 variant virus (swH1N2v) with the M gene from pH1N1; the other is a reassortant H1N2 virus (2+6 rH1N2) with six internal genes from pH1N1 and the two surface genes from endemic North American TR H1N2 SIVs. Furthermore, we determined the pathogenicity and transmissibility of the swH1N2v, a human H1N2 variant (huH1N2v), and the 2+6 rH1N2 in pigs using an endemic TR H1N2 SIV (eH1N2) isolated in 2011 as a control. All four viruses were able to infect pigs and replicate in the lungs. Both H1N2 variant viruses caused more severe lung lesions in infected pigs when compared to the eH1N2 and 2+6 rH1N2 viruses. Although all four viruses are transmissible in pigs and were detected in the lungs of contact animals, the swH1N2v shed more efficiently than the other three viruses in the respective sentinel animals. The huH1N2v displayed delayed and inefficient nasal shedding in sentinel animals. Taken together, the human and swine H1N2 variant viruses are more pathogenic and the swH1N2v more transmissible in pigs and could pose a threat to public and animal health.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Ying Fang / Porcine reproductive and respiratory syndrome virus (PRRSV) employs multiple functions to modulate host’s innate immune response, and several viral nonstructural proteins (nsps) are major players. In this dissertation, the research was mainly focused on identification and functional dissection of ORF1a-encoded nsps. PRRSV replicase polyproteins encoded by ORF1a region are predicted to be processed into at least ten nonstructural proteins. In chapter 2, these predictions were verified by using a panel of newly established antibodies specific to ORF1a-encoded nsps. Most predicted nsps (nsp1β, nsp2, nsp4, nsp7α, nsp7β and nsp8) were identified, and observed to be co-localized with de novo-synthesized viral RNA in the perinuclear region of the cell. Among all PRRSV proteins screened, nsp1β is the strongest type I interferon antagonist. In chapter 3, mutagenesis analysis of nsp1β was performed to knock down nsp1β’s IFN antagonist function. A highly conserved motif, GKYLQRRLQ, was determined to be critical for nsp1β’s ability to suppress IFN-β and reporter gene expression. Double mutations introduced in this motif, K130A/R134A (type 1 PRRSV) or K124A/R128A (type 2 PRRSV), improved PRRSV’s ability to stimulate the expression of IFN-α, IFN-β and ISG15. In addition to its critical roles involving in modulating host innate immune response, in the studies of Chapter 4, we demonstrated that PRRSV nsp1β functions as a transactivator to induce the -2/-1 ribosomal frameshifting in nsp2, which results in expression of two novel PRRSV proteins, nsp2TF and nsp2N. The conserved motif GKYLQRRLQ is also determined to be critical for the transactivation function of nsp1β. In chapter 5, the interferon antagonist, de-Ub and de-ISGylation activity of newly identified nsp2TF and nsp2N were evaluated. In vitro and in vivo characterization of three nsp2TF-deficient recombinant viruses indicated that all mutant viruses have improved ability to stimulate the innate immune response and provide improved protection in mutant virus-vaccinated animals. In summary, this study verified the previously predicted PRRSV pp1a processing products, further evaluated the function of nsp1β and nsp2-related proteins. These data obtained here will provide basic knowledge for future development of vaccines and control measurements.

A necessidade de investigar a relação entre as doenças de animais domésticos e animais silvestres da fauna brasileira motivou a realização deste trabalho. Tais doenças podem afetar de forma irreversível populações de espécies silvestres e, sobretudo comprometer do ponto de vista sanitário, produtivo e comercial a criação de animais domésticos de produção. A nova concepção da Organização Mundial de Saúde Animal sobre a importância da introdução das espécies silvestres nos sistemas de vigilância de doenças notificáveis, o significativo aumento de eventos de doenças emergentes cujo reservatório é um animal silvestre e a necessidade de preservação da nossa biodiversidade levou ao estudo de um modelo teórico de sistema de monitoramento de doenças de animais silvestres e domésticos em uma unidade de conservação de uso sustentável. Setenta por cento das unidades de conservação criadas pelo Estado de São Paulo são de uso sustentável. Como ali a permanência de residentes é permitida, acredita-se que o desafio da convivência entre seres humanos, animais domésticos e silvestres é ainda maior. Além disso, a corrente neo conservacionista atual tende a defender a permanência de comunidades tradicionais em áreas naturais protegidas. Com todos esses fatores, decidiu-se tomar como modelo de estudo a unidade de conservação de uso sustentável da Serra do Japi, pois ela constitui uma área de transição de ecossistemas e, portanto, possui uma riquíssima fauna e mais de 100 propriedades rurais com criação de animais domésticos. Inicialmente, a estratégia foi fazer um diagnóstico da situação sanitária dos animais domésticos de produção e ao mesmo tempo analisar as possibilidades de implantação de um monitoramento das doenças de animais silvestres e domésticos na Serra do Japi. O grande desafio identificado no estudo foi definir a metodologia a ser empregada para que seja possível estabelecer uma rotina de coleta de amostras e introduzi-las em um sistema de monitoramento permanente visando posteriormente a criação de um sistema de vigilância. Outro desafio será reunir entidades afins, porém heterogêneas em sua concepção e organização, com o propósito de tornar factível a implantação de um sistema futuro de vigilância de doença de animais silvestres para o Estado de São Paulo. Tal situação sugere-nos a imprescindível necessidade de regulamentar uma parceria entre o serviço veterinário oficial e demais serviços ligados à saúde animal e ao meio ambiente e organizações não governamentais. A sorologia para brucelose nos animais domésticos da região foi negativa para todas as amostras. Enquanto a prevalência para leptospirose foi de 43,4% [37,5-49,4] para bovinos e 63, 7 % [57, 3-70, 5] para equinos. / What motivated the accomplishment of this work was the need to investigate the link between wild and domestic animals diseases of the Brazilian fauna. Such diseases can irreversibly affect the wild species populations and especially compromise (from the sanitary, productive and commercial point of view) the breeding of domestic production animals. The new concept of the World Animal Health Organization regarding the inclusion of wild species in the health surveillance systems, the significant increase of new cases of diseases which originated from wild species and also the need to preserve our biodiversity led to the study of a theoretical model of a disease monitoring system for wild and domestic animals in a conservation area of sustainable use.Seventy percent of the conservation areas established by the São Paulo State are of sustainable use. Since human beings are allowed to reside in these sites, the challenge of the coexistence among human beings, wild and domestic animals is even greater. Besides that, the modern neo-conservationist current tends to defend the permanence of traditional communities in natural areas which are protected. Taking these factors into account, we decided to use as a study model the conservation area of sustainable use in the Japi Mountain Range, since it is an area of ecosystem transition and; thus, it has an invaluable fauna and it has more than 90 rural properties which raise domestic animals. At first, the strategy was to make a diagnostic of the sanitary situation of domestic production animals and at the same time to analyze the means for implanting a disease monitoring system for wild and domestic animals in the Japi Mountain Range.The great challenge identified in this study was to define the methodology to be employed in order to establish a routine of collection of samples and to introduce them in a permanent monitoring system so that later a surveillance system could be created. Another challenge was to bring together entities, which were similar but at the same time heterogeneous in its organization and conception, in order to make the implantation of a future disease monitoring system for wild animals in the State of São Paulo feasible. Such situation indicates that there would be a need to have a partnership among the official veterinary services, other services related to animal health, and with environmental and nongovernmental agencies. The serology for undulant fever (brucellosis) in domestic animals in the region was negative in all samples. While the prevalence of leptospirose was of 43, 4% [37.5 - 49, 4] for bovine and 63, 7 % [57, 3-70, 5] for equine.

A necessidade de investigar a relação entre as doenças de animais domésticos e animais silvestres da fauna brasileira motivou a realização deste trabalho. Tais doenças podem afetar de forma irreversível populações de espécies silvestres e, sobretudo comprometer do ponto de vista sanitário, produtivo e comercial a criação de animais domésticos de produção. A nova concepção da Organização Mundial de Saúde Animal sobre a importância da introdução das espécies silvestres nos sistemas de vigilância de doenças notificáveis, o significativo aumento de eventos de doenças emergentes cujo reservatório é um animal silvestre e a necessidade de preservação da nossa biodiversidade levou ao estudo de um modelo teórico de sistema de monitoramento de doenças de animais silvestres e domésticos em uma unidade de conservação de uso sustentável. Setenta por cento das unidades de conservação criadas pelo Estado de São Paulo são de uso sustentável. Como ali a permanência de residentes é permitida, acredita-se que o desafio da convivência entre seres humanos, animais domésticos e silvestres é ainda maior. Além disso, a corrente neo conservacionista atual tende a defender a permanência de comunidades tradicionais em áreas naturais protegidas. Com todos esses fatores, decidiu-se tomar como modelo de estudo a unidade de conservação de uso sustentável da Serra do Japi, pois ela constitui uma área de transição de ecossistemas e, portanto, possui uma riquíssima fauna e mais de 100 propriedades rurais com criação de animais domésticos. Inicialmente, a estratégia foi fazer um diagnóstico da situação sanitária dos animais domésticos de produção e ao mesmo tempo analisar as possibilidades de implantação de um monitoramento das doenças de animais silvestres e domésticos na Serra do Japi. O grande desafio identificado no estudo foi definir a metodologia a ser empregada para que seja possível estabelecer uma rotina de coleta de amostras e introduzi-las em um sistema de monitoramento permanente visando posteriormente a criação de um sistema de vigilância. Outro desafio será reunir entidades afins, porém heterogêneas em sua concepção e organização, com o propósito de tornar factível a implantação de um sistema futuro de vigilância de doença de animais silvestres para o Estado de São Paulo. Tal situação sugere-nos a imprescindível necessidade de regulamentar uma parceria entre o serviço veterinário oficial e demais serviços ligados à saúde animal e ao meio ambiente e organizações não governamentais. A sorologia para brucelose nos animais domésticos da região foi negativa para todas as amostras. Enquanto a prevalência para leptospirose foi de 43,4% [37,5-49,4] para bovinos e 63, 7 % [57, 3-70, 5] para equinos. / What motivated the accomplishment of this work was the need to investigate the link between wild and domestic animals diseases of the Brazilian fauna. Such diseases can irreversibly affect the wild species populations and especially compromise (from the sanitary, productive and commercial point of view) the breeding of domestic production animals. The new concept of the World Animal Health Organization regarding the inclusion of wild species in the health surveillance systems, the significant increase of new cases of diseases which originated from wild species and also the need to preserve our biodiversity led to the study of a theoretical model of a disease monitoring system for wild and domestic animals in a conservation area of sustainable use.Seventy percent of the conservation areas established by the São Paulo State are of sustainable use. Since human beings are allowed to reside in these sites, the challenge of the coexistence among human beings, wild and domestic animals is even greater. Besides that, the modern neo-conservationist current tends to defend the permanence of traditional communities in natural areas which are protected. Taking these factors into account, we decided to use as a study model the conservation area of sustainable use in the Japi Mountain Range, since it is an area of ecosystem transition and; thus, it has an invaluable fauna and it has more than 90 rural properties which raise domestic animals. At first, the strategy was to make a diagnostic of the sanitary situation of domestic production animals and at the same time to analyze the means for implanting a disease monitoring system for wild and domestic animals in the Japi Mountain Range.The great challenge identified in this study was to define the methodology to be employed in order to establish a routine of collection of samples and to introduce them in a permanent monitoring system so that later a surveillance system could be created. Another challenge was to bring together entities, which were similar but at the same time heterogeneous in its organization and conception, in order to make the implantation of a future disease monitoring system for wild animals in the State of São Paulo feasible. Such situation indicates that there would be a need to have a partnership among the official veterinary services, other services related to animal health, and with environmental and nongovernmental agencies. The serology for undulant fever (brucellosis) in domestic animals in the region was negative in all samples. While the prevalence of leptospirose was of 43, 4% [37.5 - 49, 4] for bovine and 63, 7 % [57, 3-70, 5] for equine.

Pituitary pars intermedia dysfunction (PPID) is one of the most common endocrine diseases affecting senior horses. PPID causes abnormally high concentrations of adrenocorticotropic hormone (ACTH) in the plasma and a very distinct, long, shaggy haircoat (hypertrichosis). At present, the recommended treatment for PPID is daily oral administration of pergolide mesylate. Due to the increased ACTH levels associated with PPID, it is commonly thought that these horses are immunosuppressed and at increased risk of opportunistic infections, although current research in this area is sparse. Additionally, it is not well-understood how treatment with Prascend® (pergolide tablets) affects endocrine measures other than ACTH and if it also impacts the immune response. To better understand how PPID influences endocrine and immune function in the horse, Non-PPID horses (n=10), untreated PPID horses (n=9), and PRASCEND-treated PPID horses (n=9) were followed over 15 months. Endocrine measures assessed included basal ACTH, ACTH responses to thyrotropin-releasing hormone (TRH) stimulation tests, basal insulin, insulin responses to oral sugar tests (OST), total cortisol, and free cortisol. Systemic immune function measures included basal and stimulated whole blood and peripheral blood mononuclear cell (PBMCs) cytokine and receptor expression, plasma myeloperoxidase levels, and complete blood counts. Localized immune function measures within the lung included cytokine and receptor expression after stimulation of cells obtained via bronchoalveolar lavage (BAL), myeloperoxidase levels in BAL fluid, and BAL fluid cytology. We hypothesized that PPID would affect immune function, but that any alterations would be corrected by treatment with PRASCEND. Results for the endocrine analyses showed that basal ACTH was reduced in the PRASCEND-treated horses to the levels of the Non-PPID horses, but ACTH in response to TRH stimulation was only reduced in the PRASCEND-treated horses at non-fall timepoints. PPID did not affect basal insulin, insulin responses to OSTs, total cortisol, or free cortisol, and PRASCEND treatment did not appear to have an impact on these measures either. These results suggest that PPID and hyperinsulinemia/insulin dysregulation are distinct endocrine conditions, and that the excess ACTH in horses with PPID is inactive, as it is unable to stimulate a normal cortisol response. In the immune function analyses, PPID horses had decreased expression of interferon gamma (IFNγ) from PBMCs stimulated with Rhodococcus equi and Escherichia coli and increased transforming growth factor beta (TGFβ) expression from the E. coli-stimulated PBMCs. TGFβ was also increased in PPID horses in the unstimulated whole blood samples. These results suggest that PPID horses are unable to mount an appropriate Th1 response, and that the regulatory subset of T-lymphocytes may be contributing to this decreased Th1 response. Results for the localized immune function analyses may indicate altered Th2 responses within the lung of PPID horses, although these results were severely limited by the sample size available for analyses. PRASCEND did not appear to affect immune function as measured in this study. In summary, PRASCEND successfully reduces basal ACTH in PPID horses and remains the best choice for veterinarians in monitoring dosage and response to PRASCEND treatment. Insulin, total cortisol, and free cortisol were not affected by PPID status or PRASCEND treatment in this study. Immune function was altered in horses with PPID, and it is likely that these horses are indeed at increased risk of opportunistic infection. PRASCEND treatment did not correct the differences in immune function in this study. Additional research is needed to further understand which mechanisms are driving the alterations in immune function for horses with PPID.

The extracellular matrix protein Fibronectin (FN) plays an important role in cell contractility, differentiation, growth, adhesion, and migration. The 12th -14th Type III repeats of FN (FNIII 12-14), also referred to as the Heparin-II domain, comprise a highly promiscuous growth factor (GF) binding region. This binding domain aids in cellular signaling initiated from the ECM. Additionally, FN has the ability to assemble into fibrils under certain conditions, mostly observed during cell contractile processes such as those that initiate due to upregulation of Transforming Growth Factor Beta 1 (TGF-β1) [1], [2]. Previous work from our lab has shown that self-assembly of FN into insoluble fibrils is crucial for Epithelial-Mesenchymal Transition (EMT) [3]. The transition from epithelial to mesenchymal cell type has been implicated as an early event in tumor formation and breast cancer. We were previously able to find that upregulation of FN fibrils drive EMT through contractility due to the increase of the GF latent TGF-β complex concentration at the cell membrane [3]. The challenge in the current work is to exploit the role of Heparin-II binding domain and to concentrate growth factors of interest, such as those that are pro-EMT or anti-EMT at the signaling sites of the cell membrane. Initially, we investigated the localization of the fragments FNIII 12-14 delivered to cell membrane using FITC conjugated protein. We then investigated the effects of exogenous FNIII 12-14 on EMT using breast epithelial cells (MCF10A) in the presence or absence of TGF-β1 to determine whether FNIII 12-14 alters EMT signaling. Quantification of mRNA expression, for EMT markers such as Slug, Snail, Twist, and ZEB1 were analyzed. Results showed that dosage increase of FNIII 12-14 appears to inhibit EMT transcription factors. This study will develop a new understanding of disease and gene control using ECM proteins. The exploitation of ECM natural protein interactions could become a new method in turning on/off genes of interest. While we are currently investigating this as a mechanism of blocking EMT, it could also have implications in wound healing, fibrosis, and tissue engineering, where EMT is an important aspect of the physiologic progression.

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.