In den vergangenen Jahren wurde ein Anstieg der Anzahl infektiöser Gastroenteritiden beobachtet, welche durch Viren hervorgerufen wurden. In vielen Fällen ließen sich Infektionen auf den Verzehr kontaminierter Lebensmittel zurückführen. In diesem Zusammenhang wurde der Begriff der sogenannten „food borne viruses“ geprägt. Welche Bedeutung Rohwurstprodukten bei der Übertragung humanpathogener Viren zukommt, kann derzeit nur anhand weniger objektiver wissenschaftlicher Fakten eingeschätzt werden. Die vorliegende Arbeit beschäftigte sich mit Studien zu Tenazität und Inaktivierung von Viren in Rohwurstprodukten. Dabei wurde ECHO-Virus stellvertretend für die Spezies humaner Enteroviren untersucht. Enteroviren zeichnen sich durch eine hohe Tenazität aus. Als ein wichtiger Vertreter kann Polio-Virus angeführt werden, welches bekanntlich durch Lebensmittel übertragbar ist. Im Zuge der aktuellen Problematik mit aviären Influenzaviren wurden stellvertretend zwei niedrigpathogene Isolate aviärer Influenzaviren für die Versuchsreihen gewählt. Die gewonnenen Ergebnisse sollten dazu beitragen, die Rolle des Lebensmittels Rohwurst bei der Übertragung lebensmittelassoziierter Virusinfektionen besser abschätzen zu können (hinsichtlich einer Risikobewertung) und Aussagen hinsichtlich möglicher Maßnahmen zur Risikominimierung zu ermöglichen. vorgenommen werden.

Abstract The age of an adult female mosquito is a critical determinant of its ability to transmit a range of human pathogens such as the dengue viruses. Since most pathogens require a relatively long period of development in their mosquito vector before they can be transmitted to a new host, only older insects are of epidemiological importance. It has been proposed that life-shortening strains of the inherited bacterial symbiont Wolbachia may be potentially useful to shift mosquito population age structure towards younger individuals, thereby reducing pathogen transmission without eradicating the mosquito population. However, life-shortening Wolbachia strains do not occur in mosquitoes naturally. To facilitate the evaluation of this potential strategy, the major aim of this project was to transfer a life-shortening Wolbachia strain, wMelPop, from the fruit fly, Drosophila melanogaster, to the dengue vector, Aedes aegypti. After successful transinfection, I then characterized the wMelPop infection in this mosquito species, and the associated phenotypic effects of this Wolbachia strain on several aspects of Ae. aegypti life-history. The horizontal transfer of the obligate intracellular bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. Previous attempts to directly transfer wMelPop from D. melanogaster to Ae. aegypti have been unsuccessful, presumably due to mal-adaptation of wMelPop to this novel mosquito host. In an attempt to adapt wMelPop to the mosquito intracellular environment to facilitate transfer, we serially passaged this infection for 3 years in Aedes cell culture. After long-term serial passage in mosquito cell lines, wMelPop was then initially reintroduced into its native host, D. melanogaster, by embryonic microinjection to examine the phenotypic outcome of this process. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into Drosophila, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility (CI), when compared to the original wMelPop strain. These results were suggestive of adaptation of this Wolbachia strain to the mosquito intracellular environment. Subsequently, we successfully established wMelPop in Ae. aegypti using embryonic microinjection. Two independently transinfected Ae. aegypti lines, PGYP1 and PGYP2, were generated. Analogous to infection in Drosophila, the presence of wMelPop in both of these lines halved adult mosquito lifespan under a range of laboratory conditions. The new association is stable, and wMelPop is maternally inherited with high fidelity across Ae. aegypti life span. It is capable of inducing near perfect CI that does not diminish as male Ae. aegypti age, which should facilitate its invasion into natural field populations and persistence over time. Fecundity assays using young PGYP1 females revealed that wMelPop infection induced minimal costs for reproductive fitness for females during their first gonotrophic cycle. The phenotypic effects of wMelPop on several aspects of Ae. aegypti life-history were additionally characterized. Overall, minor costs of wMelPop infection for pre-imaginal survivorship, development and adult size were observed. However, wMelPop infection decreased the viability of quiescent Ae. aegypti eggs over time. Similarly, the reproductive fitness of wMelPop-infected Ae. aegypti females declined with age. These results revealed a general pattern associated with wMelPop pathogenesis in Ae. aegypti, where host fitness costs incurred by this infection increase during aging of both immature and adult life-history stages. We also discovered that wMelPop-infected Ae. aegypti have a reduced ability to utilise blood from non-human hosts for egg development. Blood feeding by wMelPop-infected females on mouse, guinea pig or chicken hosts resulted in a near complete abolishment of reproductive output associated with both a decline in the numbers of eggs oviposited as well as the hatching rate of successfully laid eggs. In contrast, the reproductive output of wMelPop-infected females fed human blood was normal, with intermediate effects observed with other vertebrate blood sources. Removal of Wolbachia from mosquitoes using antibiotic treatment restored egg development to standard levels on all blood sources. Further blood feeding assays over two reproductive cycles definitively illustrated a nutritional interaction between host blood source and egg development in wMelPop-infected Ae. aegypti. This dramatic phenotype may provide new insights into the nutritional basis of mosquito anthropophily. The results presented in this thesis highlight several important parameters required to theoretically model the infection dynamics of wMelPop, and its potential impacts on Ae. aegypti populations. Moreover, the successful establishment of wMelPop in Ae. aegypti forms the primary basis for further field-based evaluations, which will ultimately determine the viability of this Wolbachia-based strategy as an applied tool to reduce dengue transmission.

Thesis (MSc (Veterinary Science)--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (MSc (Production Animal Studies)--University of Pretoria, 2007. / Includes bibliographical references.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 284-323.

A research program was developed to investigate basic and applied aspects of toxicity, both lethal and sublethal, of hemoxidants, particularly nitrite, on fish, non-fish aquatic vertebrates, and crayfish. The major objectives of this research were to determine A) acute and sublethal toxicity of nitrite to selected aquatic organisms: 1. aquatic salamander larvae, Ambystoma texanum, 2. swamp crayfish, Procambarus simulans, 3. bluegill, Lepomis macrochirus, 4. bullfrog, tadpoles, Rana catesbiana, 5. channel catfish, Ictalurus punctatus, B) the influence of environmental chloride on acute and sublethal exposures to hemoxidants: 1. on acute nitrite toxicity to salamander larvae, crayfish, and bluegill, 2. on nitrite-induced methemoglobinemia in bullfrog tadpoles, Rana catesbian, C) the effect of environmental hydrogen ion concentrations (pH) on acute nitrite toxicity 1. to the crayfish, Procambarus simulans, 2. to the bluegill, Lepomis macrochirus, D) the effect of temperature in sublethal exposures to nitrite 1. methemoglobin formation in channel catfish exposed at different acclimation temperatures, 2. recovery from methemoglobinemia at different acclimation temperatures, E) the effect of the fish anesthetic TMS-222 on nitrite-induced methemoglobinemia in channel catfish 1. supression of nitrite-induced methemoglobinemia, 2. dose-response curve for TMS-222 induced methemoglobinemia, and F) if a methemoglobin reductase system is present in channel catfish.

Os sedimentos aquáticos podem fornecer importantes subsídios na avaliação dos ecossistemas aquáticos lênticos, uma vez que grande parte dos nutrientes se encontra no estado sólido. A ocorrência de uma floração de cianobactérias tóxicas originada no Blang, segundo de três reservatórios em cascata do Sistema Salto de Hidrelétricas, localizado no município de São Francisco de Paula (RS), motivou a investigação das possíveis fontes de nutrientes, especialmente de fósforo (P) que foi identificado como limitante para a eutrofização. Foram investigadas fontes de origens difusas (solos, águas e sedimentos dos tributários) e pontuais (uso urbano e a carga interna representada pelo sedimento do leito dos reservatórios). Os resultados mostraram características oligotróficas nas fontes externas e a presença de P disponível em excesso no sedimento do leito, indicando que a carga interna poderia ser uma fonte significativa de nutrientes. A possibilidade de circulação da água hipolimnética foi investigada através de números adimensionais e características físicas do corpo de água, demonstrando que dificilmente ocorreria, uma vez que somente eventos climáticos extremos poderiam provocar a inversão da massa líquida. Excluídas as fontes externas, os resultados sugerem que a fertilização do reservatório Blang tenha sido causada pela abertura da comporta de fundo do reservatório Divisa, localizado imediatamente à montante, durante uma estiagem. O segundo objetivo desta pesquisa foi avaliar a importância dos agregados de partículas e da seleção granulométrica para a análise química de nutrientes em sedimentos aquáticos. Resultados obtidos em sedimentos de ambientes lóticos e lênticos (Sistema Salto, RS), demonstraram que os agregados maiores (465 - 63 μm) concentram nitrogênio nos ambientes lênticos, por isso a análise deve ser realizada, preferencialmente, na fração ≤ 465 μm em sedimentos de ambientes lênticos e na fração ≤ 63 μm dos ambientes lóticos. O último objetivo deste estudo foi testar os efeitos da oxidação sobre a capacidade e a velocidade da sorção de fosfato em sedimentos aquáticos orgânicos, identificando as melhores condições para retenção. Foram utilizados sedimentos aquáticos siliciclásticos de origens distintas, principalmente no que se refere ao clima e à origem do conteúdo orgânico, alóctone e autóctone, representados pelos tipos Dy (Reservatório Divisa, RS) e Sapropel (Reservatório de Tapacurá, São Lourenço da Mata, PE) respectivamente. O experimento foi realizado em suspensões de sedimentos mantidos sob níveis de potencial redox compreendidos entre - 200 mV e + 400 mV. Resultados demonstram que a sorção de fosfato é maior no sedimento tipo Dy em condições reduzidas e no Sapropel em condições oxidadas. / Water-borne sediments can provide important information for evaluating lentic aquatic ecosystems because a large proportion of their nutrients are found in the solid phase. A toxic algal bloom in the Blang Reservoir, the second of three in the Salto System chain of hydroelectric dams located in Sao Francisco de Paula in Rio Grande do Sul, Brazil, motivated the investigation of possible nutrient sources, especially phosphorus, which was identified as the limiting nutrient for eutrophization. Non-point sources, including soil, water and sediment carried by tributaries, and point sources, including urban areas and the internal load from the reservoir bottom, were examined. Results showed oligotrophic characteristics in the external sources and excess P availability in bottom sediments, indicating that the internal load may be a significant source of nutrients. The possible circulation of hypolimnic water was investigated using adimensional numbers and physical characteristics of the body of water, demonstrating that this is unlikely to occur, since only extreme climatic events could cause inversion of the liquid mass. By excluding external sources, results suggest that the fertilization of the Blang Reservoir was most likely caused by the opening of the floodgates from the Divisa Reservoir immediately upstream during a drought period. The second goal of this study was to evaluate the importance of particle aggregates and particle size selection for chemical analyses of nutrients and water-borne sediments. Results from sediments in lentic and lotic environments from the Salto System show that the larger aggregates (465 - 63 μm) concentrate nitrogen under lentic conditions and that, therefore, analyses should be performed on the fraction smaller than 465 μm in lentic environments and in the fraction ≤ 63 μm in lotic environments. The final objective of this study was to test the effects of oxidation on the phosphate sorption capacity and rate in organic aquatic sediments, identifying the best conditions for retention. Silicate aquatic sediments from different origins in terms of climate and source of organic content (allochtonous or autochtonous) were used, and were represented by the types Dy (Divisa Reservoir, RS) and Sapropel (Tapacurá Reservoir, São Lourenço da Mata, PE), respectively. The experiment was carried out in suspended sediment maintained under levels of redox potential between –200mV and +400mV. Results demonstrated that phosphate sorption is greatest in sediment type Dy under reduced conditions and in Sapropel under oxidized conditions.

Our study was focused on the effect of tick-borne encephalitis virus infection on global gene expression in two human neural cell lines (neuroblastoma and glioblastoma). Changes of gene expression were determined using microarray approach. We identified several genes with up-or down-regulated expression in neural cells following the infection. The changes in expression of some of them were similar in both cell lines,other exhibited different pattern.

The cattle tick, Rhipicephalus (Boophilus) microplus is a tick of veterinary and health importance globally, transmitting Babesia bovis and B. bigemina. Tick control is important and needed to prevent livestock diseases caused by tick-transmitted pathogens. Traditionally, tick control methods have resulted in development of acaricide-resistant ticks, environmental pollution and meat and milk contamination. Therefore, there is a need for alternative method and vaccines directed against tick feeding. The aim of this study was to identify proteins involved in tick feeding, tickhost-pathogen interactions and tick reproduction. Consequently, these will help in identification of antigens with the ultimate goal of developing anti-tick vaccines. R. (B.) microplus female ticks were collected at five different feeding stages. RNA was isolated from the salivary gland extracts (SGEs). The cDNA libraries were synthesized and sequenced with the Illumina MiSeq technology. Transcriptome data was analyzed with CLC Genomics Workbench, Trinity and Minia. The SGEs were also used to isolate the fractions: membrane, soluble and pellet protein for proteomic analysis. The proteomics data was analysed with Mascot, X!Tandem and Scaffold. Both the transcriptome and proteome analysis revealed the presence of major secretory protein families such as Kunitz, lipocalins, serpins, cement proteins and metalloproteases, while the majority of transcripts coded for housekeeping genes. / National Research Foundation (South Africa) / Life and Consumer Science / M. Sc. (Life Science)

Cattle ticks and tick-borne infections (TBIs) undermine cattle health and productivity in the whole of sub-Saharan Africa (SSA) including Nigeria. In this West African country, two thirds of the cattle population are reared in the central-northern regions, kept under the traditional pastoral husbandry of Fulani herders. Under the Fulanis’ management, cattle are grazed extensively, being exposed to infestation by several tick genera (i.e. Amblyomma, Hyalomma, and Rhipicephalus spp., sub-genus Boophilus spp. included), vectors of the causative agents of the most important bovine TBIs in West Africa: anaplasmosis, babesiosis and ehrlichiosis (cowdriosis). Nevertheless, the Fulani pastoralists do not usually employ chemicals to control ticks in their cattle, merely relying on traditional methods (i.e. manual removal of the most conspicuous specimens). This approach, however, does not prevent cattle from being re-infested, leaving the animals challenged by a broad variety of other tick species, most of which are vectors of economically relevant TBIs. Knowledge of tick and TBIs occurrence is an essential pre-requisite to assist field diagnosis and devising effective control strategies for a given area. Existing information on tick infestation of cattle in Nigeria is rather out-dated, mostly derived from studies carried out in the south of the country. Similarly, all studies published to date on cattle TBIs in the country do not include any molecular analysis, being based instead on cytological and/or serological diagnostics. Therefore, the aim of the present thesis was to assess the presence of cattle ticks and TBIs occurring in an area of Central Nigeria (i.e. Plateau State). This is a densely populated area with traditionally managed cattle, where no acarides have historically been employed on livestock. The work undertaken herein firstly reviews the information available to date on ticks and TBIs known to be endemic in Nigerian cattle, identifying gaps present in the existing knowledge, leading to the rationale of this study. An initial survey was conducted documenting the tick species infesting cattle in Central Nigeria, in order to assess the infestation rate of surveyed animals at the time of the year when the tick load on the host is known to be most abundant (i.e. the wet season). The survey provided novel information on tick populations in cattle in Nigeria disclosing the presence of a broad variety of species, most of which are vectors of hazardous TBIs. In order to conduct a molecular diagnosis of the TBIs within the study area, a novel methodology was developed (i.e. reverse line blotting, RLB). The application of this approach was based on a thorough review of its application to the diagnosis of TBIs worldwide as well as in SSA. The optimisation of the RLB at the University of Edinburgh to enable the detection of a broad-spectrum of TBIs in Nigeria, caused by an array of five genera of microorganisms (i.e. Ehrlichia and Anaplasma, Theileria and Babesia, Rickettsia spp.) is presented. The assessment of the analytical sensitivity of this technique for the detection of Anaplasma marginale, a highly endemic tick-borne pathogen in SSA, demonstrated a detection threshold of ≥ 7 infected cells (keeping the limit of a natural infection). The occurrence of TBIs in cattle in the study area was assessed during a large-scale epidemiological survey through the application of the validated RLB. This study disclosed the occurrence of a high prevalence of several bovine TBIs in Central Nigeria, some of which are of great veterinary and zoonotic concern. The RLB enabled the detection of carrier status as well as of numerous multiple infections (69.5%, 95% CI: 65.5–73.6%). Based on the findings presented, endemic stability for highly prevalent haemoparasites (i.e. Theileria mutans, Theileria velifera, Theileria taurotragi, Anaplasma marginale, Ehrlichia species Omatjenne) is postulated, whereas a more instable epidemiological scenario is hypothesized for other microorganisms (i.e. Anaplasma centrale and Babesia bovis), which might be connected with outbreaks of clinically apparent disease, sporadically seen in the study area. The effect of a monthly tsetse-borne trypanosomiasis-focused control programme (based on the application 0.005% deltamethrin spray formulation, applied only to the lower quarters of cattle) on the kinetics of bovine TBIs was assessed at the village level. Longitudinal monitoring of control and treated cattle was conducted over the period of eleven months. Results generated provide input to the improvement of future control strategies to be rolled out across SSA, aiming to achieve an integrated control of both trypanosomiasis and TBIs. The present thesis contributes to a better understanding of the epidemiology of bovine TBIs in Nigeria as well as in the rest of West Africa, using a highly sensitive tool of wide applicability. These findings will be shared with the local pastoralist communities to further promote effective yet sustainable, vector control, in tune with the traditional long-established practices.