Historically, terrestrial food web research has focused on describing the structure of aboveground grazing webs, and determining how interactions among plants, herbivores and higher trophic levels influence primary productivity. Detrital food webs however, play a significant role in regulation of ecosystem dynamics through direct impacts on decomposition. Unraveling the complex nature of detrital food web structure is critical to developing a better understanding of ecosystem function. Therefore the primary objective of this research was to describe the structure of the leaf-litter food web in a temperate deciduous forest, with emphasis on interactions between a community of generalist predators, the forest-floor spiders, and arthropod prey. Elucidating occurrence of trophic interactions in the forest-floor food web was a formidable task due to the high diversity, small body sizes and cryptic habits of many litter-dwelling arthropods. Analysis of natural variation in consumer stable isotope ratios (δ13C and δ15N) formed the crux of this research because it simultaneously permitted quantification of the trophic positions of litterdwelling arthropods and identification of spider resources, including prey subsidies from the grazing web. A monoclonal antibody-based ELISA was employed to analyze the gut contents of spiders to quantify predation on a major arthropod taxon, the forest-floor flies. Surveys of spider distributions and prey availability in the litter layer also provided fundamental knowledge of community structure. Stable isotope analyses suggested that most spiders exhibited strong trophic connections to the detrital web, but weak links to herbivorous prey. Several lines of evidence supported a strong trophic link between large, litterdwelling collembolans (Tomoceridae) and cursorial spiders, including correlation between spider and tomocerid densities on the forest-floor, similarities in spider and tomocerid carbon signatures, and nitrogen enrichment of tomocerids relative to other prey types. Conversely, this research provided conflicting evidence regarding spider consumption of flies. Gut content assays indicated consistent predation on flies by cursorial spiders, while stable isotope models suggested that flies are likely of little importance in the spiders’ diets. This project yielded valuable insights into the role of spiders in the forest-floor food web and the potential importance of species-specific variation in prey consumption for detrital food web dynamics.

<p>The purpose of this study was to gain more knowledge about the deleterious effects of decreased muscle protein concentration on skeletal muscle function, by measuring the concentrations of myosin and actin in single pig muscle fibres. The pigs were earlier used in an experimental animal model to study the early stages of acute quadriplegic myopathy (AQM), a disease that is found in mechanically ventilated intensive care unit patients. Percutaneous biopsies were taken from these pigs and where now used in this study.</p><p>Even though the method used was accurately tested and theoretically working, certain problems arose. These problems were unexpected and caused problems to the study. The method used to measure the concentration of myosin and actin, an ELISA, gave no logical results. The reason could not be found and because of the time limit of this project no results from the AQM-pigs were gained. The efforts to make the method work is described and discussed.</p>

El objetivo del presente estudio fue determinar la seroprevalencia del virus de la artritis- encefalitis viral caprina (AEVC) en cabras de cuatro provincias del departamento de Lima. Con este fin se colectaron 754 muestras de suero de cabras mayores a 12 meses de edad entre hembras (n=698) y machos (n=56) de 89 diferentes rebaños criados en forma estabulada (n=3), semi-extensiva (n=40) y transhumante (n=46) para la detección de anticuerpos contra el AEVC mediante la prueba de ELISA de competición. El 1.3±0.8 % (10/754) de las cabras tuvieron anticuerpos contra el AEVC. Los 10 seropositivos fueron hembras de 2 a 5 años de edad clínicamente normal perteneciente a un solo rebaño de 103 animales de crianza semi-extensiva del distrito de Huaral, provincia de Huaral representando 9.7% (10/103) de seropositividad dentro del rebaño. Todos los reproductores machos resultaron seronegativos. Se concluye que la artritis encefalitis viral caprina está presente en baja prevalencia en rebaños caprinos de crianza semi-extensiva estudiados.

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is associated with formation of autoantibodies, activation of inflammatory cascade and up-regulation of several cytokines. These processes lead to persistent synovial inflammation, joint damage and systemic manifestations. The aim of this diploma thesis is to characterize the role of a novel cytokine interleukin-20 (IL-20) in the pathogenesis of RA and to investigate its involvement in different stages of the disease as a potential surrogate biomarker. In this work, several methods including Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry and Real-Time quantitative Polymerase Chain Reaction (RT-qPCR) have been employed. We demonstrated increased expression of IL-20 in the synovial tissue of RA compared with control osteoarthritis (OA) patients. Along with the up-regulation at sites of inflammation, concentrations of IL-20 were higher in the synovial fluid compared with circulating levels of IL-20. Furthermore, serum and synovial fluid IL-20 levels significantly correlated with RA disease activity. Synthesis of IL-20 was significantly increased in peripheral blood mononuclear cells (PBMCs) and synovial fibroblasts upon stimulation with some TLR ligands and pro-inflammatory cytokines. Although not regulating PBMCs functions in...

Doctor of Philosophy / Department of Animal Sciences / Barry Bradford / The transition into lactation is a period of primary concern to dairy producers because of the tremendous incidence of health disorders observed during this time. Two common disorders that lead to decreases in production and retention within the herd include fatty liver disorder (FL) and ketosis. These two disorders have been commonly associated with negative energy balance, yet recently it has been hypothesized that inflammation is a contributor to the etiology of these disorders. Three individual projects were completed for this dissertation, all involving inflammation. The role of endogenous inflammation was determined by administration of sodium salicylate (SS) to cows for 7 d after parturition, and metabolites and production responses were evaluated. Overall it appears that SS induced hypoglycemic conditions and increased triglyceride accumulation in the liver (while administered), increased lipid mobilization and ketones (2 weeks after administration ended), and increased whole lactation milk production in older cows. A sensitive, specific sandwich ELISA for bovine tumor necrosis factor-[alpha] was developed, which provided the ability to measure “normal” circulating levels of this cytokine. The final study involved inducing inflammation by daily injections of the TNF[alpha] to the early lactation dairy cow. In this model, cows receiving TNF[alpha] had a reduction in dry matter intake, water intake, and decreases in milk production and milk components. Overall, it appears that inflammation is involved in the normal biology of the transition dairy cow and disrupting this can lead to interesting negative effects and some improvements of production; however, when inflammation is much greater it can lead to negative production effects.

Master of Public Health / Department of Diagnostic Medicine and Pathobiology / M. M. Chengappa / Rabies is the most deadly disease on earth and has a 99.9% human fatality rate. Rabies kills 61,000 humans annually and results in an economic burden of $124 billion USD annually. Each day 3.3 million people live with the risk of rabies. It is estimated that 95% of human rabies cases are a result of coming in contact with an infected canine, majority of these cases being children 15 years and younger. It is estimated that 1 person every 8 minutes dies of rabies. Rabies is a highly neurotropic disease which attacks the brain and central nervous system. Once clinical symptoms are presented, death is invariably the outcome as no cure exists for rabies. Rabies is 100% preventable in humans by proper wound management and proper administration of prophylaxis. Rabies can be adequately controlled in animal populations by contraception and animal rabies vaccine efforts. Whilst it is known that rabies can be prevented in humans and controlled in animal populations, further scientific efforts are still warranted to fully understand this deadly virus so that a cure can one day be discovered. As human and animal populations continue to grow, so does the cost and burden of this horrific disease. As a result, the importance of prophylaxis and passive immunity are critical in the event of medically managing an exposure, and preventing exposures. The World Health Organization has defined global recommendations for individuals and animals who have received prophylaxis to be adequately protected. Measuring this protection is performed using a variety of approved testing methodologies, virus-neutralizing assays and antigen-binding assays. Whilst the WHO recommendations were defined from clinical studies performed with virus-neutralizing assays, the assumption that these recommendations are suitable for the antigen-binding assays is inaccurate. The testing methodologies, virus-neutralization and antigen-binding, share similarities, as they are measuring an immune response to the rabies virus. However; enough differing characteristics are presented such that exact comparisons cannot be made. Establishing the same standards and recommendations for both testing methodologies will never be sufficient.

Memoria para optar al Título Profesional de Médico Veterinario / La Paratuberculosis o Enfermedad de Johne es una enteritis granulomatosa crónica de los rumiantes, causada por Mycobacterium avium subsp. paratuberculosis, comunmente conocido como Mycobacterium paratuberculosis. Esta enfermedad se caracteriza por poseer un período de incubación de más de dos años, infectándose el animal en los primeros meses de vida, principalmente en el período neonatal. La gran mayoría de los animales infectados la cursan en forma subclínica, lo que dificulta inmensamente su diagnóstico, debido a la baja sensibilidad de los métodos existentes. El objetivo de este trabajo fue comparar y relacionar dos métodos diagnósticos, uno bacteriológico y otro serológico, en animales de predios infectados de la Región Metropolitana, con el fin de aportar con información para la implementación de un adecuado método de control de esta enfermedad en el país. Es así como se recolectó muestras de sangre y de deposiciones de 136 animales sintomáticos y asintomáticos procedentes de 2 rebaños lecheros infectados. Las muestras de suero fueron sometidas a una prueba de ELISA comercial y las muestras de deposiciones fueron cultivadas en medio de cultivo Herrold modificado con yema de huevo con y sin Micobactina J. Las muestras de deposiciones fueron procesadas previamente utilizando como decontaminante fecal el cloruro de hexadecilpiridinium y luego se realizó una incubación con los antibióticos, vancomicina, amphotericina B y ácido nalidíxico según el protocolo descrito por Whitlock y Rosenberger (1990). El 10,29% de las muestras de deposiciones resultaron positivas al cultivo, siendo confirmadas como tal, por su lento crecimiento sólo en el medio con Micobactina J (a partir de la octava semana) y mediante la técnica de tinción de Ziehl Neelsen. El diagnóstico serológico mediante la prueba de ELISA detectó un 83,8% de animales positivos. En este estudio hubo un alto porcentaje de muestras de cultivo contaminadas con hongos (44,85%), lo que resultó en una falta de asociación entre la prueba del cultivo fecal y la prueba de ELISA. El análisis estadístico mostró que no existe asociación entre las técnicas empleadas (P < 0,0001; kappa = 0,029). La alta tasa de contaminación de los cultivos se debió principalmente a que en algunas muestras se utilizó tubos, que en vez de tapa rosca, poseían un tapón de algodón cardé y que estaban además sellados con cinta “Parafilm”. El 90% de las muestras contaminadas correspondió a este tipo de tubos. Además un grado alto de contaminación siempre es esperable, debido a que se trabaja con material fecal. Para mejorar la técnica, se sugiere probar el método propuesto por Stabel (1997), quien propone el uso de antibióticos (vancomicina y ácido nalidíxico) en el medio de cultivo

L'Organisation Mondiale de la Santé définit l'infertilité comme une maladie et un échec de l'appareil reproducteur à parvenir à une grossesse après 12 mois ou plus de rapports sexuels réguliers non protégés. De nos jours, l'infertilité est devenue un phénomène commun affectant 1 couple en âge de procréer sur 5. Une origine idiopathique est le plus souvent associée à un système immunitaire actif qui pourrait produire des niveaux élevés d'anticorps anti-liquide séminal ou anti-sperme. L'auto-immunisation, aussi bien que l'iso-immunisation, joue un rôle significatif dans jusqu'à 30% des cas signalés. Le liquide séminal, qui est défini comme un fluide complexe contenant le sperme, les vésicules cellulaires et autres cellules et composantes, pourraient immuniser l'appareil génital féminin. Cette thèse est liée à l'infertilité féminine immune, en particulier à l'iso-immunisation féminine. Une meilleure compréhension de cette manifestation physiopathologique consiste en (1) la détermination des isotypes d'anticorps jouant un rôle significatif dans cette maladie, puis en (2) la caractérisation et l'identification des antigènes de liquide séminal ou de sperme reconnus par ces anticorps, (3) la proposition de marqueurs diagnostiques potentiels afin d'adapter des thérapies spécifiques, et, en outre, la conception d'un outil de diagnostic miniaturisé basé sur les marqueurs sélectionnés, (4) la suggestion d'une éventuelle immuno-intervention. En se fondant sur la distribution des isotypes d'anticorps spécifiques au liquide séminal/sperme, nous suggérons que les immunoglobulines E, M, A1,2, et G3 ne sont pas impliquées dans la sensibilisation physiopathologique chez les femmes. Les IgG4, semblent constituer la sous classe majeure interagissant avec les protéines de sperme. A l’inverse, les IgG1 semblent être principalement impliquées dans la réactivité vis-à-vis des protéines séminales. Nous avons également élargi le groupe déjà existant d’IgGs liés aux protéines de sperme à d’autres protéines, parmi lesquelles la protéine de choc thermique 70 1A/1B, la protéine apparentée aux protéines de choc thermique 71kDa et l’alpha-énolase ont été reconnues, pour la première fois, être liés à l’iso-immunisation féminine. Nous avons mis en évidence le rôle des protéines séminales dans l’iso-immunisation et pas seulement dans l’hypersensibilité au sperme par l’intermédiaire d’IgE. En particulier, l’antigène spécifique de la prostate, la phosphatase acide prostatique et la protéine à doigt de zinc 778 ont été décrites comme immuno-dominants parmi les protéines séminales reconnues par des IgGs liés aux protéines de sperme. La détermination des sous classes d’IgG de sérum féminin, spécifiques au liquide séminal/sperme, pourrait rendre le diagnostic des patients plus complet. Les IgG1 et IgG4 anti-liquide séminal/sperme pourraient présenter un intérêt pour l’immunothérapie. Par ailleurs, les protéines décrites, dans notre étude, pourraient se révéler être des bio marqueurs utiles pour de telles pathologies. Le dispositif miniaturisé pourrait être de type LFIA (Lateral Flow Immuno Assay), se basant sur la détection immuno-chimique d’anticorps spécifiques. L’immuno-intervention envisagée pourrait reposer sur l’effet d’immunoglobulines intraveineuses. / The World health Organization reports infertility as a disease and a failure of reproductive tract to achieve pregnancy after 12 months or more of regular unprotected sexual intercourse. Nowadays, infertility has become a common life phenomenon affecting 1 out of 5 couples at reproductive age. Idiopathic cause is mostly associated with active immune system which may produce high levels of anti-seminal and/or anti-sperm antibodies. Auto-immunization as well as iso-immunization has a significant role in up to 30% of reported cases of infertility. Semen that is defined as a complex fluid containing sperm, cellular vesicles and other cells and components, could immunize the female genital tract. This thesis is related to female immune infertility, in particular to female iso-immunization. The better understanding of this pathophysiological event consists of (1) the determination of antibody isotype playing a significant role in this disease, then (2) the characterization and identification of semen antibody-binding proteins, seminal and/or sperm, (3) the proposal of potential diagnostic markers to adapt specific therapy and, in addition, the design of miniaturized diagnostic tool based on the selected markers, (4) the suggestion of potential immuno-intervention. Based on the distribution of seminal/sperm-specific antibody isotypes, we suggest that immunoglobulins E, M, A1,2, G3 are not involved in the primary pathophysiological female sensitization. IgG4 appears to be the major subclass interacting with sperm proteins. On a contrary, IgG1 seems to be the one mainly involved in the reactivity towards seminal proteins. We have also extended the existing group of IgG-binding sperm proteins, among which heat shock protein 70 1A/1B, heat shock cognate protein 71 kDa and alpha-enolase have been shown, for the first time, to be related to female iso-immunization. We have put the emphasis on the role of seminal proteins in iso-immunization and not only in the IgE-mediated semen hypersensitivity as known so far. In particular, prostate-specific antigen, prostatic acid phosphatase and zinc finger protein 778 have been determined as immunodominant among IgG-binding seminal proteins. The determination of female serum seminal/sperm-specific IgG subclasses could make the patient diagnoses more comprehensive. Anti-seminal/sperm IgG1,4 might be of interest for immunotherapy. Furthermore, the herein described proteins could be useful biomarkers of such pathology. The miniaturized chip could be a lateral flow immunoassay-based device acting on the immunochemical detection of specific antibodies. The intended immuno-intervention could consist of the effect of intravenous immunoglobulins.

Memoria para optar al Título Profesional de Médico Veterinario. / La Enfermedad de Chagas (ECh) es una zoonosis causada por el parásito Trypanosoma cruzi. En Chile, se estima que existen aproximadamente 160.000 personas infectadas. La ECh cursa con dos etapas clínicas, la etapa aguda y la crónica; un 30% de los pacientes desarrolla cardiopatía, megasíndromes digestivos o ambos. La mayoría de los individuos se encuentran en la fase crónica indeterminada, donde los resultados de tratamiento farmacológico no han sido concluyentes, debido a que la negativización total de las pruebas serológicas convencionales requiere años de seguimiento. Nifurtimox (NF) y Benznidazol (BZ) son los únicos fármacos autorizados para el tratamiento etiológico. El objetivo fue evaluar la eficacia de NF en el tratamiento de la ECh crónica en seguimiento prolongado utilizando parámetros serológicos, determinando si existen diferencias estadísticamente significativas entre dos grupos de estudio, utilizando las técnicas de Inmunofluorescencia Indirecta (IFI) y ELISA. La población en estudio estuvo conformada por 100 individuos con ECh crónica tratados (6,6 años promedio), 100 individuos con ECh crónica no tratados y 100 individuos negativos para ECh. Se realizó el análisis estadístico utilizando pruebas no paramétricas en ELISA (Mann Withney: p= 0,0003) y paramétricas en IFI (t Student: p=0,0001). Se logró determinar que existen diferencias estadísticamente significativas (p<0,05) en ambas pruebas. Concluyéndose que los niveles de IgG en individuos con ECh son menores en individuos tratados con NF, respecto a los no tratados, lo que indica eficacia quimioterapéutica en seguimiento prolongado / Human American Trypanosomiasis or Chagas disease (ChD) is caused by the protozoan parasite Trypanosoma cruzi. Probably affects 160.000 people in Chile. ChD has two successive phases: acute and chronic. Several years of starting the chronic phase, 30% of infected individuals develop irreversible lesions of the autonomous nervous system in the heart, digestive system, or both. Mostly infected are in the indeterminate form of human chronic ChD, without conclusive results, because the total negativization of conventional serological tests requires years of follow-up. Nifurtimox (NF) and benznidazole (BZ) are the availables drugs for the etiological treatment. The aim was to evaluate the efficacy of NF in the treatment of chronic ChD using serological parameters, determining if there are statistically significant differences between three study groups, using Indirect Immunofluorescence (IFA) and ELISA techniques. Study population consisted of 100 individuals with treated chronic ChD (6.6 years average), 100 individuals with chronic untreated ChD and 100 individuals negative for ChD. Statistical analysis was performed using nonparametric tests in ELISA (Mann Withney: p = 0.0003) and parametric tests in IFA (Student t test: p = 0.0001), which allowed determining that there are statistically significant differences (p <0.05) in both tests. It is concluded that the levels of IgG in individuals with ChD are lower in individuals treated with NF, compared to those not treated, indicating chemotherapeutic efficacy in prolonged follow-up / Financiamiento: Proyecto Fondecyt No. 1161485.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA