Salmonellosis is a serious problem of public health that mainly infants, young children and people with immunodeficiencies face. Human direct contact with animals is one of the possible ways of transmission of the disease. An increasing trend of keeping exotic pets, including amphibians and reptiles, has been observed for recent years in the United States and developing European countries. Most of these animals are asymptomatic carriers of Salmonella. However, in this review we introduced new and the most dangerous outbreaks of salmonellosis caused by contact with amphibians and reptiles that appeared in all continents in last ten years. It was demonstrated that Salmonella strains isolated from cold-blooded animals such as animals and reptiles differ genetically from strains isolated from humans. It means that the reason of appearance of Salmonella outbreaks caused by transmission of pathogens from amphibians or reptiles to human are genetic changes including the activation of virulence factors that cause pathogenicity in humans. It is supposed that popularity of keeping amphibians and reptiles as pets has caused an increase in the demand for these animals and in the international trade of these species. The problem is also caused by breeder's unconsciousness of proper procedures of keeping exotic animals in a household. Therefore, the World Health Organization (WHO) has censored the most important issues to minimize the risk of salmonellosis, focusing mainly on RAS salmonellosis (reptile – associated salmonellosis), because this disease is the most common in the United States. It is estimated that about 74,000 of the United States citizens are getting infected by Salmonella strains transmitted from reptiles kept as pets every year. Educating people on this topic is a key preventive method of salmonellosis. This review can help future breeders how to keep reptiles and amphibians according to recommendation of World Health Organization.

Exotic amphibians are often detrimental to native biotas. In Florida, the exotic Cuban Treefrog (Osteopilus septentrionalis) eats native frogs and may outcompete them for resources. Cuban Treefrogs thrive in disturbed areas and around buildings, and often breed in manufactured wetlands such as retention ponds and borrow pits. This study identified limnological, landscape, and biotic characteristics that discouraged pond use by Cuban Treefrogs and promoted use by native amphibian species. I sampled natural and manufactured ponds in Orange County, Florida, for one year, using standard methods to estimate the species richness and relative abundance of amphibians and their potential fish and macroinvertebrate predators. I determined the relationship between the presence of Cuban Treefrogs and twelve limnological (% vegetation, slope, pond age, pH, % dissolved oxygen, air temperature, water temperature, turbidity, conductivity, depth, perimeter, and area) and seven landscape characteristics (% canopy closure over ponds, building density, distance to nearest building, road density, distance to nearest road, distance to nearest forest stand, and % forest cover), plus five biotic factors (native amphibian richness and abundance, fish richness and abundance, and macroinvertebrate abundance). No relationship existed between native amphibian abundance or species richness and the presence or absence of Cuban Treefrogs. Ponds with a greater percentage of vegetation, large perimeters, and low pH and turbidity had greater native amphibian species richness. Cuban Treefrogs were more likely to be found in ponds with a greater percentage of aquatic vegetation and small perimeters. My results show that building large retention ponds containing low-turbidity water will restrict colonization by Cuban Treefrogs and maintain species richness of native amphibians.

Oxygen consumption can most readily be related to wet weight. However, the weight of a tadpole increases so long as it is feeding, then decreases during final non-feeding stages. Oxygen uptake may also be related to dry weight, but dry weight increases significantly from early tadpole stages to adult (Funkhouser and Mills, in press). There is change in one factor at the same time the change in the the other factor is in the opposite direction. Total nitrogen content may be used as an index of the metabolizing tissue, but is subject to the same criticism as dry weight (Moore, 1964). Ideally, the animal's respiration should be related to as many criteria as possible. The pattern of thyroid activity during amphibian metamorphosis has been described by a number of investigators using a variety of methods. Histological criteria for the functional activity of the thyroid glad include epithelial cell height, the number of epithelial cells in serial sections of gland, "vacuolization" of the colloid, the number of secretion droplets present in the follicle, the number of mitochondria, and changes in the size of the Golgi apparatus. Not all of these criteria are considered to be of equal validity (Etkin, 1930; Gorbman and Bern, 1962). The amount of iodine accumulate in the follicle has been used as an index of the activity of the gland. Kaye (1961) using radioactive iodine (I131) and radioautography found a 12% increase in I131 accumulation during early metamorphic stages, and a 43% increase during or near the later stages of metamorphosis. The rate of development is significantly increased in normal tadpoles immersed or injected with thyroxine. Kollros (1961) was able to reproduce the normal metamorphic pattern in thyroidectomized Rana pipiens with gradually increasing concentrations of T4. All of these studies indicate that during normal metamorphosis there is a gradual increase in the concentration of thyroid hormone reaching a climax in the last stages and dropping metamorphosis has been assumed to increase because the increase in thyroxine levels accompanying metamorphosis in amphibia would be sufficient to cause a marked increase in metabolic rate in mammals. The latent period between exposure to thyroxine and maximum calorigenic effect has been described for many animals, In man this period is about two weeks (Guyton, 1964). The present study was designed to show that the small increase in oxygen uptake observed in a number of amphibians during metamorphic climax (Table II) is the result of the increase in thyroxine levels in the immediately preceding stages. Oxygen consumption of Hyla regilla was measured from hatching through metamorphosis and correlated with activity of the thyroid gland

Today, amphibian extinction rates are rising at an alarming rate. Captive assurance colonies have become a hedge against extinction, and often must employ assisted reproductive technologies (ART) in species that do not readily breed in captivity. One technique that can be utilized in assisted breeding is hormone therapy, which involves the treatment of individuals with exogenous reproductive hormones. The primary delivery method used in most breeding programs is intraperitoneal injection, but many institutions either lack the training necessary to conduct this invasive procedure, or require veterinary staff to perform them, thus delaying breeding events. Therefore, there is interest in alternate means of hormone delivery. In particular, the use of intranasaladministration. The following studies were conducted to determine the efficacy of hormones administered via alternate delivery routes, and to investigate the pathways taken by both intraperitoneal and intranasal delivery methods. Through these studies, wefound that intranasal administration gonadotropin releasing hormone (GnRH), is effective at eliciting sperm production in male anurans. In order to investigate the paths taken by intraperitoneal and intranasal GnRH, I used a treatment of hormone-conjugated quantum dot nanoparticles and employed both in-vivo fluorescence imaging techniques and histological imaging. The evidence presented here suggests that the route traveled by nasally-delivered GnRH is largely swallowed and accumulates in the GI tract, buteventually diffuses into the bloodstream in large enough concentrations to exact a reproductive response. The other hormone investigated here was arginine vasotocin (AVT), a hormone known to elicit calling and amplexus behaviors in amphibians. Though limited reproductive behaviors were observed in these studies, I found that both intranasal and intraperitoneal delivery of AVT resulted in water uptake and retention in males. Fluorescence imaging revealed that AVT, when administered nasally, is largely swallowed, similarly to GnRH. Intraperitoneally-injected AVT, however, was found to accumulate in large concentrations within the interrenal gland and kidney, where it likely stimulated the observed osmoregulatory effects. This study therefore offers insight into an effective alternate hormone delivery method (nasal) and provides compelling evidence into the organs wherein GnRH and AVT act following two different delivery routes.

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Los ovocitos ováricos totalmente crecidos de anfibios están fisiológicamente arrestados en la profase de la primera división meiótica. La progesterona, a través de un proceso denominado maduración, induce el desarresto ovocitario que se traduce en la ruptura de la vesícula germinal (GVBD). Se acepta generalmente que la hormona desencadena la maduración por un mecanismo no genómico que involucra la inhibición de la adenilil ciclasa y la disminución del AMPc intracelular a través de la unión a un receptor ligado a la membrana plasmática. Como respuesta al estímulo hormonal se sintetiza la oncoproteína c-Mos cuya actividad biológica está mediada por la cascada de las proteínas activadas por mitógenos (MAPK). En conjunto con la activación de reguladores del ciclo celular estos caminos convergen en la activación del factor promotor de la maduración (MPF). Los rafts de membrana, en particular la estructura invaginada de las caveolae, podrían proporcionar un ambiente óptimo para la interacción entre la progesterona y el ovocito en la maduración meiótica. Sin embargo, las bases moleculares y los mecanismos de la posible participación de los microdominios caveolares en la transducción de señales de la maduración aún no han sido completamente dilucidados. En este trabajo de tesis se analizó el efecto de la maduración inducida por la progesterona sobre el contenido y la composición de los lípidos neutros y polares de las plaquetas vitelinas, organelas del ovocito de Bufo arenarum que cumplen un rol importante durante la embriogénesis. Se realizó un análisis cuantitativo de los lípidos y de las proteínas de las membranas de baja densidad aisladas de ovocitos ováricos y la identificación bioquímica y el estudio biofísico de los microdominios tipo-caveolares, como un requisito para comprender mejor sus funciones regulatorias. La metil-β-ciclodextrina (MβCD) se usó como herramienta para modular el colesterol celular con el fin de evaluar la participación de los rafts de membrana en la maduración inducida por progesterona y en la inducida por ceramida, disparador de la reiniciación de la meiosis en otras especies de anfibios. En particular, se indagó la vía de las MAPK en la señalización de la maduración meiótica. Se demostró que los lípidos de las plaquetas vitelinas están involucrados activamente en la reiniciación del ciclo meiótico lo que apoya la hipótesis de un rol dinámico de estas organelas. La maduración produjo una disminución en el contenido total de fosfolípidos fundamentalmente por la caída de fosfatidilcolina, un fosfolípido considerado esencial para que se complete la meiosis. Los principales cambios en el perfil de ácidos grasos se observaron en esfingomielina, en ácido fosfatídico y en los diacilgliceroles, lípidos bioactivos implicados en caminos de señalización celular. El tratamiento hormonal disminuyó el nivel de la esfingomielina plaquetaria lo que podría vincularse con su rol como precursor de ceramidas. Se pusieron a punto distintos métodos de aislamiento de microdominios de membrana y se logró obtener una fracción de membranas livianas en ausencia de detergentes. Se determinó que dicha fracción deriva de la membrana plasmática, está enriquecida en colesterol y en el gangliósido GM1 y presenta un nivel importante de esfingomielina. Las membranas livianas muestran un enriquecimiento en una caveolina de 21 kDa indicando la existencia de estructuras tipo-caveolares en el ovocito de Bufo arenarum. Además, están asociadas significativamente con las moléculas señales, c-Src y H-Ras. En estas membranas se encontró una banda proteica que, por espectrometría de masa, se identificó como la cadena pesada de la miosina no muscular planteando la posibilidad de una relación con el citoesqueleto. La depleción de colesterol, mediada por MβCD, afectó principalmente el nivel de colesterol de las membranas livianas alterando el orden lipídico y la localización de los marcadores moleculares de rafts, caveolina, c-Src y GM1, e inhibiendo la maduración de manera dosis-dependiente lo que sugiere que estos microdominios de membrana están involucrados en la inducción hormonal. La repleción de colesterol indicó una recuperación de la habilidad para madurar de los ovocitos tratados especialmente en la concentración de MβCD 25 mM en la cual la reversibilidad fue cercana al valor control. Se demostró que la ceramida es un inductor efectivo de la maduración que afecta la distribución de los marcadores moleculares de rafts en las fracciones de membrana. Por el contrario, la progesterona no parece afectar la integridad de los microdominios de membrana. En concordancia con la inhibición de la GVBD, el tratamiento con MβCD retardó la fosforilación en tirosinas y la activación de la p42 MAPK en la maduración inducida por progesterona. La presencia de los marcadores de rafts, caveolina, GM1, c-Src y H-Ras, y el hallazgo de moléculas señales de la cascada de las MAPK funcionalmente asociadas a las membranas livianas, sugieren que esta fracción enriquecida en microdominios tipo-caveolares puede, en parte, recrear eficazmente la señalización de la maduración. / Amphibian full-grown ovarian oocytes are physiologically arrested at the first meiotic prophase. Progesterone, through a mechanism called maturation, induces meiotic resumption represented by germinal vesicle breakdown (GVBD). It is generally accepted that progesterone hormone triggers maturation through a nongenomic mechanism that involves the inhibition of adenylyl cyclase and the reduction of intracellular cAMP by association with a plasma membrane receptor. As a response to the hormonal stimuli oncoprotein c-Mos is synthesized and its biological activity is mediated by the mitogen-activated protein kinase (MAPK) cascade. Together with the activation of cell cycle regulators, both pathways converge in the activation of the M-phase promoting factor (MPF). Membrane rafts, particularly the invaginated structure of caveolae, seems to provide an optimal environment for hormone binding leading to meiotic maturation. However, the molecular bases and the mechanisms of the posible caveolar microdomain involvement in maturation signal transduction pathways have not been fully elucidated to date. In the present thesis, the effect of progesterone-induced maturation on the quantity and composition of neutral and polar lipids of yolk platelets, organelles from Bufo arenarum oocyte that play an important role during embryogenesis, were analyzed. A quantitative analysis of lipids and proteins of low-density membranes isolated from ovarian oocytes and the biochemical identification and a biophysical study of caveolae-like microdomains were performed as a requisite to further understand how these domains carry out their regulatory functions. Methyl-β-cyclodextrin (MβCD) was thus used for cellular cholesterol modulation in order to assess the membrane raft involvement in maturation induced by progesterone and by ceramide, the latter being trigger of meiosis reinitiation in other amphibian species. We demonstrated that lipids from yolk platelets are actively involved in the resumption of the meiotic cell cycle supporting the hypothesis of a dynamic role for these organelles. Phospholipid content decreased mainly as a result of a fall at the level of phosphatidylcholine, a phospholipid considered crucial for the completion of meiosis. Fatty acid composition registered significant changes in sphingomyelin, phosphatidic acid and diacylglycerols, bioactive lipids involved in cellular signaling pathways. Hormonal treatment induced a decrease at sphingomyelin level that could be related to its role as ceramide precursor. Different isolation methods were assayed to obtain membrane microdomains and a light membrane fraction was obtained in the absence of detergents. Light membranes derive from the plasma membrane, show an enrichment in cholesterol and GM1 ganglioside, and evidence an important level of sphingomyelin. The finding of a 21 kDa caveolin enriched in light membranes indicates the presence of caveolae-like structures in Bufo arenarum oocytes. In support of this finding, signaling molecules as c-Src and H-Ras are significantly associated to this fraction. A protein band was found in these membranes and it was identified as a non-muscle myosin heavy chain by mass spectrometry suggesting possible membrane-cytoskeleton interactions. Cholesterol depletion mediated by MβCD affected mainly light membranes cholesterol level disturbing lipid order and localization of rafts markers, caveolin, c-Src, and GM1 and inhibiting maturation in a dose-dependent manner, thus suggesting that these membrane microdomains are involved in hormonal induction. Cholesterol repletion showed a recovery of the ability of MβCD-treated oocytes to mature, particularly at the 25 mM concentration at which reversibility was close to control level. We also demonstrated that ceramide is an effective inducer of maturation that affects the distribution of raft markers among membrane fractions. On the contrary, progesterone seems not to affect membrane microdomain integrity. In agreement with GVBD inhibition, MβCD treatment delayed tyrosine phosphorylation and p42 MAPK activation in progesterone-induced maturation. The presence of the rafts markers, caveolin, GM1, c-Src, and H-Ras, and the finding of signaling molecules from the MAPK cascade functionally associated to light membranes suggest that this fraction enriched in caveolae-like microdomains could efficiently recreate, at least in part, maturation signaling.

Assessing ecological risk to wildlife exposed to anthropogenic contamination in soil has traditionally been problematic. Attempts to standardize an approach to evaluate risk for various community types in North America have been challenging, given the variation in terrestrial communities and the values in which policy makers are bound to protect. This has resulted in vague, yet flexible guidance from the U.S. Environmental Protection Agency and other interested parties (e.g., the U.S. Army Corps of Engineers, and the Tri-Service Ecological Risk Assessment Working Group). Interpretation of these and other guidance has been variable, often resulting in conflicting opinions on how best to address the question of ecological risk to receptors that are exposed to xenobiotics in a soil matrix. This work reports the results of research designed to address the question of ecological risk to terrestrial vertebrates. Objective, ecologically-relevant criteria were used in the selection and development of models in this research. Several lines of logic were considered: 1) substance sensitivity, 2) ecological sensitivity (i.e., the species importance to the system; e.g., keystone species); and, 3) probability and extent of exposure. A primary soil contaminant at many U.S. Army installations is 2,4,6-trinitrotoluene (TNT). This was a result of the mass manufacturing, storing, and assembly of weapons from the early 1900's until the 1950s. The Army has reported soil concentrations of TNT ranging from 0.12 to 38,600 ug/g (Walsh and Jenkins 1992) and 0.08 to 64,000 ug/g (Hovatter et al. 1997). The chemical-physical properties of TNT result in a relatively unique compound, not easily amenable to current modeling techniques to estimate exposure to terrestrial wildlife. Moreover, there are few data describing the effects of exposure to TNT in other than mammals, fish, and specific invertebrates. In this research, the pathways of exposure and selected potential toxic effects from TNT exposure were investigated in a terrestrial salamander: Ambystoma tigrinum (tiger salamanders). A. tigrinum was chosen since they are exclusively carnivorous, relatively long-lived, have a thin integument, and are large enough to investigate individual effects. These investigations were designed to mimic natural conditions as closely as possible, though maintain a degree of homogeneity in a laboratory environment. All studies exposed salamanders to soil and food (earthworms) in identical preparations. As such, these exposures were considered complete, eliminating assumptions made regarding daily food consumption, systemic dermal dose, etc. The first study examined the relative contribution of dermal or oral exposures to the whole-body burdens of TNT and primary metabolites. A poly-chlorinated biphenyl (PCB) mixture (Aroclor7 1260) was used with TNT to simultaneously to assist in the evaluation of each pathway, since the fate and transport of PCBs are well characterized. Tiger salamanders were exposed 28 days in situ. The dermal route of exposure contributed the most to the final burdens of TNT in salamanders, with the primary reduction products, 2-amino-4,6-dinitrotoluene and 4-amino, 2,6-dinitrotoulene reaching higher concentrations than of parent compound. Other TNT metabolites were found in insignificant quantities. The concentrations of PCBs were higher in the oral treatment, as expected. These results were corroborated in a subsequent study using Ambystoma maculatum (spotted salamanders). The second series of investigations evaluated the potential toxic effects from TNT exposure. Two treatments consisting of TNT and a control were used to evaluate these effects to A. tigrinum. The salamanders were exposed in situ for 14 days to TNT in soil and food (earthworms of which were exposed to TNT in the soil in similar preparations). Non-specific immune effects were evaluated through the characterization of splenic phagocytes in their ability to: 1) phagocytize foreign particles, and 2) digest (through oxygen radicals) phagocytized material. This was conducted using fluorescent microspheres and a fluorescent chemical probe specific to hydrogen peroxide, measured per each cell using flow cytometry. Other data collected included histological examination (e.g., liver, kidney, and other miscellaneous organs), blood differentials, weight changes over time, organ/ body weight comparisons, and an analysis of organ-specific metabolism. No significant effects were noted in salamanders exposed to these conditions. Coordinated with the preceding study included a search for biomarkers of exposure and an investigation of the metabolites of TNT in situ. Biotransformation products of TNT were found including primary (e.g., 2-amino-4,6-dinitrotoluene) and secondary (e.g., 2,4-diamino-6-nitrotoluene) in relative concentrations in skin, liver, and kidney. Biomarkers of exposure included an analysis of cytochrome p450, b5, and the glutathione antioxidant enzymes in liver, kidney, skin, lung, and serum, respectively. Traces of parent compound were found in the skin and liver only. Levels of 2,4-diamino-6-nitrotoluene were found only in the liver and kidney, suggesting that TNT is reduced primarily in or on the skin. Levels of p450 were higher in TNT exposed salamanders than controls. Glutathione and related enzyme levels are reported. This work suggests that salamanders have levels of detoxification enzymes capable of the biotransformation of anthropogenic substances in soil rivaling that of mammals. Another investigation evaluated these same immunological parameters in white-footed mice (Peromyscus leucopus). This species was chosen based on the relative importance of small mammals to the community structure in many North American ecosystems. Mice were exposed to TNT in the feed at 0.264, 0.066, 0.033, and 0.017%, where actual daily dose estimates for males were 604, 275, 109, and 65; and for females was 544, 282, 143, and 70 mg/kg/d. An investigation to evaluate the specificity of commercially-available monoclonal antibodies specific to cell surface markers for thymocytes and splenocytes in inbred mice was unsuccessful. These results suggest the recognition epitopes of monoclonal antibodies prepared against Old-World mice are not conserved into Peromyscus, a New-World species. However, high dose males and females had larger spleens consistent with the hemolytic effects previously reported for mammals exposed to TNT. Further, males exposed at all levels had reduced phagocytic activity of splenocytes, and reduced hydrogen peroxide production associated with the two highest doses relative to controls. Females showed no response relative to treatment. This research has shown the feasibility for these types of investigations, and provides toxicity information valuable for modeling estimates of ecological risk. Further, the in situ exposures have provided media concentrations that are or are not toxic for species of concern. This type of information reduces the uncertainty associated with ingestion modeling estimates, dermal exposure estimates, and other factors not traditionally considered in toxicity studies. / Ph. D.