12 pp. / Mosquitoes are the most important insect pests that affect the health and well-being of humans and domestic animals worldwide. They can cause a variety of health problems due to their ability to transfer (vector) viruses and other disease-causing pathogens, including in the arid Southwest U.S. This publication describes the mosquito life-cycle, introduces common pest mosquito species and the diseases associated with them. Mosquito management for residents is covered.

This project investigates the potential of the 27 Everglades green algal strains for producing biodiesel. The five potential strains chosen by measuring the neutral lipid content using the Nile red method were Coelastrum 46-4, Coccoid green 64-12, Dactylococcus 64-10, Stigeoclonium 64-8 and Coelastrum 108-5. Coelastrum 108-5 and Stigeoclonium 64-8 yielded the same amount of lipids in both Gravimetric and Nile red method. A linear relationship between algal biomass and lipid accumulation was seen in Coelastrum 46-4, Coccoid green 64-12, Stigeoclonium 64-8 and Coelastrum 108-5 indicating that increase in algal biomass increased the lipid accumulation. Nitrogen and phosphorous stress conditions were also studied where higher lipid accumulation was observed significantly (p < 0.05) in 64-8 Stigeoclonium and 64-12 Coccoid green. Collectively, it could be summarized that Coelastrum 108-5, Coccoid green 64-12 and Stigeoclonium 64-8 were promising in some aspects and could be used for further studies.

An assay based on Nile red fluorescence was developed for quantitative analysis of triglycerides, a common cellular component with important biological functions and is routinely analyzed for diagnosis of metabolic disorders and as an important feedstock of food industry and biodiesel production. Based on studies on the Nile red fluorescence of pure, binary, and ternary systems of triglycerides, ethanol, and water, 20% ethanol aqueous solution was determined to be the most suitable solvent for lipid fluorescence measurement. Excellent linearity was established for lipid samples in the range of 0.1- 0.5 mg/ml with several different lipid standards and vegetable oils. Results also suggest that the fluorescence of triglycerides was not sensitive to the fatty acid composition of lipids. This finding is important since it implies that the assay could potentially be used for the measurement of triglyceride content of different oil crops without causing significant variations. The results of this method were then verified by comparing with the results of the conventional gravimetric methods. The results of the fluorescence assay were consistently lower than that of the gravimetric method by approximately 10%. This phenomenon was tentatively attributed to the fact that the gravimetric method measures the total amount of lipophilic materials in samples while the fluorescence assay is selective to glycerides. Attempts were also made to apply this assay to estimate the lipid content of green alga Neochloris oleoabundans. However, the results were less than ideal due to the existence of interfering components in the extract of microalga samples that could significantly repress the fluorescence of lipids.

Le virus West Nile (VWN) est un virus neurotrope principalement transmis par piqûre de moustique et dont le réservoir est constitué par la faune aviaire sauvage. Les souches circulant en Europe appartiennent à 4 lignages génétiques différents à l’origine de nombreuses épidémies d’ampleur modérée à faible et limitées géographiquement, contrairement à ce qui a été observé en Amérique du Nord. En 1998 en Israël, une importante épidémie a a été associée pour la première fois à une forte mortalité de la faune aviaire sauvage. Le virus (souche IS-98-ST1, lignage 1a) a été isolé du cerveau d’une cigogne moribonde. L’objet de cette thèse a été de construire un clone infectieux de la souche IS-98-ST1 afin d’en explorer les propriétés de neuroinvasion et de pouvoir mettre en évidence les déterminants moléculaires de sa virulence.Le virus obtenu à partir de la construction clone infectieux s’est révélé posséder les mêmes propriétés biologiques que le virus parental, que ce soit in vitro sur cellules Vero ou in vivo sur souris sensibles ou résistantes ou encore sur l’embryon de poulet. L’embryon de poulet est présenté ici comme un nouveau modèle d’évaluation de la virulence du VWN. Un modèle cellulaire neuronale (lignée de neuroblastomes humains, SK-N-SH) est aussi évalué dans ce manuscrit. En conclusion, un nouvel outil de génétique inverse a été obtenu pour le VWN. Cet outil permettra de travailler sur l’impact de mutations ponctuelles, ou de modifications plus importantes touchant un ou plusieurs gènes viraux sur la virulence du VWN, spécifiquement dans le contexte européen. / West Nile virus (WNV) is a neurotropic virus mainly transmitted through mosquito bites. Wild birds represent the main reservoir hosts. Strains circulating in Europe belong to four lineages and have caused numerous but limited epidemics over the last few years. In 1998, an important outbreak associated to huge bird fatalities caused by a highly neuroinvasive strain (IS-98-ST1) took place in Israel. We aimed at producing a new infectious clone, based on the lineage 1a IS-98-ST1 WNV strain, for the characterization of its neuroinvasion properties as well as the molecular determinants of European WNV virulence. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotypes of recombinant and parental WNV were indistinguishable in terms of viremia, viral load in the brain and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs, proposed as a new model for the evaluation of WNV virulence. The potential of human neuroblastoma cells (SK-N-SH) to discriminate between highly and mildly virulent WNV strains was assayed. In conclusion: a new molecular tool that is useful for the study of molecular determinants of WNV virulence has been generated. We take advantage of the high genetic stability of our one-piece infectious WNV cDNA clone to produce mutant viruses through the insertion of point mutations or the exchange of genetic fragments between WNV strains into the backbone of the IS-98-ST1 infectious clone.

Frequent measures that aim to identify the tolerance of an organism to various environmental conditions rely on the mortality of said organism. However, the effects of sub-lethal stress can be just as important to consider as they may give rise to how an organism may live in such an environment (growth, reproduction, etc.). Coping with changes in environmental conditions can have a high energy cost. Even starting a cellular stress response alone has proven to be costly. It is therefore reasonable that organisms in stressful situations will dedicate energy sources to survival mechanisms, and downregulate non-necessary activities like growth, and reproduction. As a tropical freshwater species, Nile Tilapia are subject to both Winter Stress Syndrome and saltwater intrusion as global climate change progresses, making them an ideal model organism. In order to test the physiological limits of this species, we exposed Nile Tilapia to a variety of treatments (two temperatures: 21°C & 14°C, three salinities: 0ppt, 16ppt, 34ppt) for one hour. By manipulating both temperature and salinity simultaneously the author hopes to shed light on the future of this species as global climate change progresses. Cell cycle arrest can occur at cellular checkpoints such as the ones located at G1 or G2/M. Monitoring the DNA replication process is crucial to cellular activities, and disruptions such a cell size issues or DNA damage can cause this process to stop indicating the presence of sub-lethal stress. There were no significant changes in the proportions of cells in G1, S, or G2 due to an increase of salinity in warm water. In cold water, there was a steady decrease of the percentage of cells in G1 as salinity increased suggesting cell cycle arrest is occurring at a different checkpoint. A significant increase in the number of cells in G2 in response to cold temperature was found, however, this effect was not made greater by the addition of salinity. Increasing number of cells in G2/M suggests that there is cell cycle arrest occurring at the G2/M checkpoint. The concentrations of three proteins involved in the regulation and arrest of the cell cycle were measured in gill tissue by dot blotting. Western blotting was performed to ensure the specificity of the protein antibodies. Three proteins of interest were chosen due to their roles in regulating cell cycle proliferation (Proliferating Cell Nuclear Antigen), growth arrest (Growth Arrest and DNA Damage-45) and programmed cell death (p53). Proliferating Cell Nuclear Antigen (PCNA), Gadd45, p53 all showed significant decreases in concentration in gill tissue exposed to saltwater (34 ppt) in the warm temperature treatment. There was no significant effect of salinity within the cold temperature treatment for any of the proteins tested. The CCAAT/Enhancer-binding proteins (C/EBP) are a class of transcription factors that act upon cellular proliferation and differentiation. C/EBP-𝛿 is the specific protein that is activated in response to stress stimuli. There were no significant differences observed in C/EBP-𝛿 concentrations in gill tissue.

Source: Masters Abstracts International, Volume: 47-06, page: 3501. Adviser: Kristy O. Murray. Includes bibliographical references.

This thesis project addresses Islamic water management by presenting case studies on regional water issues and analyzing the extent to which Muslim-majority states behave in a way consistent with Islamic shariah law. The case studies presented in this thesis address both international cooperation related to the management of trans-boundary water basins (the Nile and Tigris-Euphrates River Basins) and domestic water management strategies employed by Muslim-majority states in the MENA region (Jordan and Yemen). In each case, it is not clear that there is consistency between the Islamic ideals discussed by academics and the actual techniques employed by various states. In international attempts at managing the shared waters of the Nile and Tigris-Euphrates Basins, the fact that many riparian states have Muslim-majority populations does not appear to make the management of trans-boundary resources any easier or more successful. The implications for Islamic water management at the domestic level is also unclear – with shariah playing a positive role in Jordanian attempts at water conservation but promoting the over-exploitation of resources in Yemen. Although shariah appears to play a limited role in the management of trans-boundary water resources, it seems to be better suited for informing how states internally manage their endowments of freshwater resources. / text

Up until 1969 Nile Crocodiles (Crocodylus niloticus) were considered as vermin in South Africa and were actively persecuted throughout the country. In an effort to re-establish viable populations within protected areas in Zululand a restocking program was initiated in the late 1960‟s and early 1970‟s. Ndumo Game Reserve (NGR) in north eastern KwaZulu-Natal was one of the reserves involved in the restocking program and the Nile Crocodile population in the reserve increased from a minimum absolute abundance of 348 (SE ± 3.39; N = 3) in the early 1970‟s to a minimum absolute abundance of 992 (SE ± 58.70; N = 4) in the 1990‟s. However, in recent years there has been some concern that the NGR Nile Crocodile population may be on the decline, initiating the current investigation into the ecology and conservation of the NGR population. We examined changes in relative abundance using aerial survey data from 1971 – 2009. The precision and accuracy of population estimates was affected by water level, season, aircraft type and the use of different observers. A correction factor was applied to survey data and the current NGR Nile Crocodile population is estimated at an absolute abundance of 846 (± 263). Distribution data from the aerial surveys were also used to examine habitat use over the last 40 years and revealed that Nile Crocodiles were not evenly distributed in NGR and that crocodiles favoured the Phongola over the Usuthu floodplain systems. NGR is characterised by a floodplain mosaic landscape and crocodile distributions between the various habitat patches were influenced by landscape physiognomy and composition as well as connectivity and corridor quality. Anthropogenic disturbances influenced the functionality of the floodplain landscape negatively with impacts on habitat use and connectivity. To quantify the effects of environmental conditions on crocodile habitat use we conducted 40 diurnal counts at Lake Nyamithi between 2009 and 2012 and related changes in crocodile numbers here to temperature, rainfall and water level. Crocodile density in Lake Nyamithi was significantly and negatively related to average maximum ambient temperature and numbers increased in the lake over the cool, dry winter season. Water level and rainfall had strong but not significant (p >0.05) negative influence on crocodile density in Lake Nyamithi. Environmental variables influenced different size class of Nile Crocodiles differently and the density of crocodiles in the 1.5 – 2.5 m Total Length (TL) size class were significantly influenced by rainfall and average minimum monthly temperature. Movement patterns of 49 Nile Crocodiles between 202 – 472 cm total length (TL) were followed over 18 months using mark-resight (n = 36), radio (n = 10) and satellite (n = 3) telemetry. The duration of radio transmitter attachment (131 days, SE ± 11.35) was significantly related to TL and reproductive status. Satellite transmitters stopped functioning after 15 (SE ± 12.53) days and home range was calculated for 7 crocodiles ranging in size from 202 cm TL – 358 cm TL. Sub-adults (1.5 - 2.5 m TL) occupied smaller, more localized home ranges than adults (> 2.5 m TL). Home ranges overlapped extensively suggesting that territoriality, if present, did not cause Nile Crocodiles to maintain spatially discrete home ranges in NGR during the dry season. A single large scale migration event occurs every year between October and November whereby the majority of the NGR crocodile population leaves the reserve and enters the Rio Maputo floodplain in adjacent Mozambique and only return in April/May. Nesting effort (19 – 21 %) in NGR was comparable to other populations of Nile Crocodile in southern Africa. Nests are completely destroyed by floods once every 10 years and predation rates may range from 20 – 86 % per year. In addition to aerial surveys, nesting surveys and movement studies crocodiles (n = 103) were caught opportunistically to collect demographic data on population structure. The population structure of Nile Crocodiles in NGR is currently skewed towards sub-adults and adults suggesting an aging population that may decline naturally in the future. This could be due to low recruitment levels in NGR that are not able to sustain the artificially high population size created by the restocking program. Sex ratios were skewed towards females in the juvenile and sub-adult size classes and towards males in the adult size class while the overall sex ratio was even between males and females. It is predicted that the NGR Nile Crocodile population will decline in the future and that this decline should be considered as a natural process. However, the rate of decline will be accelerated at an unnatural speed and to an unnatural extent due to poaching, uncontrolled harvesting and destruction of nesting habitat within NGR. Based on the findings of the current study, management recommendations for the conservation of the combined NGR – Rio Maputo Nile Crocodile population were made. It is important that further research takes place in the Rio Maputo floodplain in Mozambique to better quantify the nesting ecology of the NGR Nile Crocodile population and to identify possible threats facing Nile Crocodiles in this region. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Thesis (M.S.) -- University of Tennessee, Knoxville, 2005. / Title from title page screen (viewed on July 13, 2005). Thesis advisor: Reid R. Gerhardt. Document formatted into pages (xii, 77 p. : ill. (some col.), maps (some col.)). Vita. Includes bibliographical references (p. 67-76).

Les flavivirus ayant un impact en médecine vétérinaire sont largement distribués dans le monde (à l’exemple de la fièvre West Nile (WNF) présente sur les cinq continents ou de l’Encéphalite japonaise (EJ) en Asie du Sud-Est) et sont responsables de maladies à dominante neurologique chez l’homme et/ou le cheval.La virémie étant généralement brève lors de ces infections virales, les méthodes de diagnostic utilisées sont essentiellement sérologiques. Or le chevauchement fréquent des aires de répartition des flavivirus complique le diagnostic sérologique. En effet, des réactions sérologiques croisées entre flavivirus sont observées lors de l’utilisation de méthodes de diagnostic usuelles telles que l’ELISA et l’immunofluorescence (IF). Les résultats sérologiques doivent donc être confirmés par la méthode fastidieuse de séroneutralisation (SNT) virale avec les différents flavivirus existants dans la région. De plus, le risque d’émergence sur un territoire donné de nouveaux flavivirus comme le virus Zika au Brésil ou en Amérique du Nord ne peut être exclu.Nous avons donc développé dans la première partie de ce travail une nouvelle stratégie de diagnostic sérologique multiplexe à l’aide de la méthode d’immuno-essai (MIA) sur billes. Sachant que la glycoprotéine d’enveloppe soluble (sE) des flavivirus est divisée en 3 domaines (D) structuraux, DI, DII et DIII et que les épitopes du DIII sont spécifiques de chaque flavivirus, des protéines EDIII recombinantes de différents flavivirus d’intérêt ont été synthétisées en système d’expression Drosophila S2 et leur antigénicité a été testée sur sérums équins et ovins. Nous avons obtenu des résultats très encourageants en démontrant que l’utilisation des EDIII associée avec la capacité de multiplexage de la méthode MIA apparaît comme une réponse adaptée au défi du diagnostic sérologique des infections à flavivirus.Nous avons en outre utilisé la même méthode de multiplexage mais avec des antigènes NS1 du WNV pour mettre en place un test DIVA (Differentiating Infected from Vaccinated Animals) permettant de différencier les chevaux infectés par le WNV des chevaux vaccinés avec un vaccin recombinant WNV.Un autre écueil en médecine vétérinaire est le traitement des affections à flavivirus. En effet l’arsenal thérapeutique est limité et le traitement est avant tout symptomatique. Nous avons dans la seconde partie de ce travail testé in vitro une molécule antivirale à large spectre, le sr1057 sur nos virus d’intérêt (WNV et JEV). Cette molécule qui provient d’une stratégie de criblage développée par l’Institut Pasteur a probablement pour cible la cellule de l’hôte car elle est capable d’inhiber des virus très différents, à génome à ARN de polarité positive ou négative et ADN.Les résultats que nous avons obtenus avec ce composé sont en demi-teinte pour les flavivirus testés avec une efficacité démontrée pour le JEV mais plus modeste pour le WNV. Ils n’excluent cependant pas une possible utilisation de cet antiviral en médecine vétérinaire équine car une activité inhibitrice in vitro sur les virus de l’Herpes équin de type I et de l’artérite virale a été confirmée par d’autres collaborateurs. / Flaviviruses with a major impact in veterinary medicine are widely distributed (e.g. West Nile fever (WNF) has spread across the five continents and Japanese Encephalitis (JE) is reported in South-East Asia) and are mainly responsible for neurological diseases in humans and/or horses.After flavivirus infection, viremia in mammal hosts is generally short and consequently indirect methods are mostly used to diagnose flavivirus infections. However, frequent spatial overlapping in their circulation areas renders the interpretation of serological assays difficult. Indeed, cross-reactions between flaviviruses are observed in rapid serological tests such as in ELISA and immunofluorescence assays (IFA). Serological assay results should thus be confirmed by the tedious comparative virus neutralization test (VNT) using a panel of viruses known to circulate in the area. Moreover, the risk of emergence of new flaviviruses such as reported recently with the Zika virus in Brazil or in North America should be considered when studying flavivirus epidemiology.In the first section, a new strategy aiming at improving the serological diagnosis of flavivirus infections was developed using the multiplexing capacity of microsphere immunoassays (MIA). The flavivirus soluble envelope (sE) glycoprotein ectodomain is composed of three domains (D), e.g. DI, DII and DIII, with EDIII containing virus-specific epitopes. Recombinant EDIIIs of different flaviviruses were synthesized in the Drosophila S2 expression system. The microspheres coupled with rEDIIIs were assayed with equine and ovine sera from natural and experimental flavivirus infections or non-immune samples. Very encouraging results have been obtained and this innovative multiplex immunoassay based on flavivirus rEDIIIs appears to be a powerful alternative to ELISAs and VNTs for veterinary diagnosis of flavivirus-related diseases.MIA with WNV nonstructural 1 protein were also implemented to differentiate Infected from Vaccinated Animals (DIVA). Such a DIVA approach was only successful when horses had been immunized with a recombinant canarypox vaccine, while horses receiving inactivated WNV vaccine developed immune responses close to the ones induced after natural infection.Another pitfall in veterinary medicine is the lack of therapeutics for viral diseases and specifically for flaviviruses. The therapeutic arsenal is indeed rather limited and animals are generally administered supportive treatments only. In the second part, the results of the in vitro testing of a broad spectrum antiviral named sr1057 on WNV and JEV replication are presented. This chemical, identified from a unique screening strategy developed by Pasteur Institute, is probably targeting the host cell and was found to inhibit the replication of varied RNA and DNA viruses belonging to different virus families. The sr1057 compound was not as efficient at inhibiting the replication of flaviviruses as for other RNA+ viruses, with a modest antiviral effect demonstrated for WNV and a higher efficacy on JEV. This antiviral presents however potentials for applications in equine veterinary medicine because it efficiently inhibited equine herpes virus-1 and equine arteritis virus in vitro, as clearly shown by other collaborators.