Media for the selective recovery of the mosquito pathogens Bacillus sphaericus 1593 and B. thuringiensis serovar. israelensis were developed. Streptomycin at 100 µg/ml and 500 µg/ml in NY agar (nutrient agar with 0.05% yeast extract) successfully selected for B. sphaericus, and allowed counts equivalent to those obtained on the nutrient medium NY without antibiotics. The medium containing 100 µg/ml of streptomycin (NYST) was used to recover B. sphaericus 1593 from a mixed microbial population in pond water. Sodium chloride, penicillin G, and pH adjustment of the medium were found to be unsatisfactory selective agents. Two selective media for the recovery of B. thuringiensis serovar. israelensis gave counts equivalent to those obtained on the nonselective NY medium. One medium contained 100 µg/ml of polymyxin with 1.0 µg/ml chloramphenicol (NYPC), and the other contained 500 µg/ml of polymyxin alone. The use of the higher level of polymyxin with chloramphenicol reduced the number of viable B. thuringiensis serovar. israelensis. NYPC was used to recover B. thuringiensis serovar. israelensis from a mixed microbial population in pond water. The selective media reduced the number of pond water microorganisms on plates by 90 to 99%. A heat treatment of 50ºC for 10 minutes also reduced pond water microbiota by approximately one log. The use of heat treatment plus either NYST or NYPC reduced the pond water microbiota further. The heat treatment had little effect on sediment microbiota. A selective-differential medium for B. thuringiensis serovar. israelensis was developed for use when heat treatment of samples would be undesirable. This medium, PEMBAC, permitted the observation of peptone deamination and hydrolysis of lecithin, which are characteristic of B. thuringiensis serovar. israelensis. The medium contained 50 µg/ml of polymyxin and 1.0 µg/ml chloramphenicol as selective agents. The parasporal crystals of B. thuringiensis serovar. israelensis are the site of the mosquito larval toxin. Because the crystals are not viable, another method for their enumeration was examined. Antisera to whole crystals and to solubilized crystal antigens were prepared in rabbits for use in the indirect fluorescent antibody technique. Because of the small size and irregular shape of the parasporal crystals of B. thuringiensis serovar. israelensis, the crystals were difficult to distinguish from other small fluorescing particles. The antisera prepared precipitated several antigens in solubilized crystals, but did not adsorb to the majority of the antigens in whole crystals. / Master of Science

The possibilities of transmission of infectious synovitis were explored. Two types of tests, using mosquitoes as vectors, were employed. One type involved the feedings of mosquitoes on previously inoculated birds, then after a period of from zero to ninety-six hours refeeding the mosquitoes on assay birds. The second type of insect test comprised the feeding of mosquitoes on inoculated birds, then macerating and inoculating them into check birds at intervals of one to twenty-one days following feeding. Other experiments were conducted in which uninoculated birds were brought into close contact with inoculated chickens and their excrement. The observation of all tests indicated the absence of transmission of an infective titer of the infectious synovitis agent. These conclusions are drawn from tests using Aedes aegypti (Linn.) and Culex fatigans Wied., with methods and materials described. / Master of Science

In this project, an outgrowth and optical density (OD) measurement step was developed and validated for an established protocol for antibiotic treatment of Anopheles stephensi mosquitoes. This step allows the elimination of uncleared mosquitoes from experimental pools. Our findings suggest that, as previously demonstrated, antibiotic treatment reduced the mosquito microbiome to an extent, as evidenced by decreased bacterial growth observed through OD readings of our experimental samples. Additionally, qPCR analysis of 16S abundance provided quantitative data on bacterial abundance, which further supported the effectiveness of antibiotic treatment. Finally, a comparison of mosquito samples pooled randomly or with high OD individuals removed showed a significant improvement in bacterial clearance in samples with our refinement step included. The method established here can generate experimental samples for third-generation sequencing with minimal bacterial contamination for robust hypothesis testing. The addition of OD measurement is a rapid, cost-effective step that can potentially improve hypothesis testing on large pools of samples. This study contributes to our understanding of the impact of antibiotic treatment on mosquito microbiomes. Improvements in molecular and bench-level techniques can greatly contribute to our ability to gain insights into the role of microbes in mosquito biology.

La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, Aedes triseriatus and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. Our surveillance study demonstrated the involvement of an invasive species, Aedes japonicus, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. LACV infection did not affect the host-seeking behavior of Ae. triseriatus females. Using high performance liquid chromatography with electrochemical detection (HPLC-ED), the levels of serotonin and dopamine were measured in infected and uninfected mosquitoes. Serotonin is known to affect blood-feeding and dopamine affects host-seeking. Serotonin levels were significantly lower in LACV-infected mosquitoes but dopamine levels were unaffected by virus. A previous study found that LACV infection caused an alteration in mosquito blood-feeding in a way that could enhance virus transmission. This work showed that LACV infection can reduce the level of serotonin in the mosquito, promoting virus transmission through altered blood-feeding without impairing the vector's ability to locate a host. Standard CDC bottle assays were used to evaluate the efficacy of two pyrethroids and two essential oil sprays on LACV infected and uninfected mosquitoes. LACV-infected Ae. triseriatus females were more susceptible to both pyrethroids than uninfected ones. Infection status did not affect the susceptibility of Ae. albopictus to either pyrethroid. The essential oils were inconsistent in their effects. These results demonstrate that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virus-infected portion of the vector population. / Ph. D. / La Crosse virus (LACV) encephalitis is the most common and important endemic mosquito-borne disease of children in the U.S. with an estimated 300,000 annual infections. The disease is maintained in a zoonotic cycle involving the eastern treehole mosquito, <i>Aedes triseriatus</i> and small woodland mammals such as chipmunks and squirrels. The objectives of this study were 1) to conduct surveillance of LACV and other mosquito-borne viruses; 2) to evaluate the effect of virus infection on mosquito host-seeking and neurotransmitter levels, and 3) to determine the effectiveness of barrier sprays to control infected mosquito vectors. The surveillance study demonstrated the involvement of an invasive species, <i>Aedes japonicus</i>, in the transmission cycle of Cache Valley virus (CVV). CVV is a mosquito-borne virus that is closely related to LACV. Thus, surveillance is a critical step in public health, providing pathogen distribution and frequency data as well as identifying and incriminating new vectors. Our study of the effects of LACV infection on host-seeking and neurotransmitter levels showed that LACV can manipulate <i>Ae. triseriatus</i> females in a way that could facilitate transmission of the virus. Lastly, we showed that barrier sprays may be a viable part of a mosquito control program, not just to reduce the biting rate but to potentially reduce the virusinfected portion of the vector population.

Phlebotomine sandflies are blood sucking Dipteran and important vectors of various patogens, especially leishmania parasites (Trypanosomatida, Leishmaniinae). Apart from the genus Leishmania they also transmit variety of other parasites and they are considered to be vectors of trypanosomes (genus Trypanosoma) and hosts of some monoxenous trypanosomatids. This Master thesis is focused on the occurrence and development of mono- and dixenous trypanosomatids (other than the genus Leishmania) in sandflies. We studied experimental infections of various species of sandflies and mosquitoes by two species of trypanosomes and one species of monoxenous parasite (Strigomonas galati). All three studied trypanosomatid species were isolated from sandflies. Further concern of the thesis is the transmission of both species of trypanosomes from sandflies to vertebrate hosts and the transmission of monoxenous parasite between insect hosts and also morphological changes of parasite cells during the development in a culture and in the insect and vertebrate hosts.

Les arthropodes vecteurs sont hématophages et peuvent assurer la transmission biologique active d'un agent pathogène responsable de maladies humaines ou animales. La lutte anti-vectorielle et la surveillance épidémiologique des vecteurs sont essentielles dans la stratégie de lutte contre les maladies vectorielles. Disposer d'outils d'identification précis, fiable et rapides des vecteurs et des pathogènes associés est indispensable. Ainsi dans ce projet nous avons évalué l'utilisation du MALDI-TOF MS pour identifier les arthropodes vecteurs ainsi que la détection des pathogènes associés. La première partie de notre travail consistait à utiliser MALDI TOF pour identifier les tiques, moustiques et les puces. Nous avons déterminé quelle partie du spécimen permettait d'obtenir une reproductibilité des spectres et une identification correcte par des tests à l'aveugle après création d'une base de données de référence. La deuxième partie consistait à utiliser le MALDI-TOF MS pour détecter des Rcikettsies associés aux tiques dont Rickettsia conorii et R. slovaca, deux pathogènes humains transmis respectivement par Rhipicephalus sanguineus et Dermacentor marginatus. Des variations spectrales étaient obtenues entre les spécimens infectés et non infectés, avec des masses spécifiques liés à l'infection des tiques par les rickettsies. La technique d'identification était validée par des tests à l'aveugle. Les résultats obtenus permettent de conclure que le MALDI TOF pourra être utilisé dans l'avenir pour identifier les tiques prélevées chez des patients, les arthropodes vecteurs lors des enquêtes entomologiques et préciser la prévalence d'infection de ces arthropodes. / Arthropods are vectors bloodsucking and can ensure the active biological transmission of a pathogen responsible of human or veterinary diseases. The vector control and vectors epidemiological surveillance are essential in the strategy against the vectors-borne diseases. Accurate, reliable and rapid identification of vectors and associated pathogens are essential. Thus, in this project we evaluated the use of MALDI-TOF MS for the arthropods vectors identification as well as for the detection of associated pathogens. This proteomics technology emerged since few years ago and is currently used in routine for bacteria identification in many microbiology laboratories. In the first part of our work, we used the MALDI TOF to identify the tick, mosquito and flea species. For each arthropod, we determined which part allowed obtaining reproducible spectra by MALDI TOF and correct identification by blind test, after reference database creation. The second part consisted to use the MALDI-TOF MS to detect the associated Rickettsia in ticks including Rickettsia conorii and R. slovaca, two human pathogens transmitted by Rhipicephalus sanguineus and respectively Dermacentors marginatus. The spectral variations were obtained between infected and non infected specimens with specific masses related to the tick infection by Rickettsia. The identification technique of not or infected ticks was validated by blind tests. The obtained results allowed concluding that the MALDI-TOF MS could be used in the future to identify the ticks removed from patient, the arthropods vectors and during entomological survey and determine the prevalence of infection of these arthropods.

A dirofilariose canina é uma parasitose de distribuição mundial cujo agente etiológico é um verme nemátoda filarióide. Em sua fase adulta, os vermes produzem microfilárias por meio de reprodução sexuada. Sua transmissão ocorre por meio de mosquitos que atuam como hospedeiros intermediários. No Brasil, até o momento, a dirofilariose canina foi encontrada em 15 estados de todas as regiões. A maioria dos estudos está concentrada nas regiões sudeste e sul. A prevalência nacional é de 2%, embora alguns trabalhos tenham encontrado prevalências locais mais elevadas. Neste trabalho apresentamos o primeiro registro de dirofilariose canina para o estado de Rondônia com mapas de distribuição dos cães positivos e análise de mosquitos. Amostras de sangue de 727 cães foram coletadas aleatoriamente na cidade de Porto Velho. As amostras forma analisadas em busca de microfilárias e antígenos circulantes usando duas técnicas: microscopia ótica de gota espessa corada com Giemsa e imunocromatografia de fluxo lateral (ICT). As amostras positivas foram também testadas por PCR, as negativas forma testadas na mesma técnica em pools. Mosquitos foram coletados no domicilio e peridomicílio de todos os casos de cães positivos, estes mosquitos foram testado por PCR em busca de DNA de Dirofilaria immitis. Um mapa de distribuição dos casos de cães positivos foi elaborado. Noventa e três amostras de sangue foram positivas no ICT, representando 12,8% da amostra total, nenhuma amostra foi positiva na gota espessa. O PCR das amostras de sangue resultou em 10% para as positivas no ICT e 0% nas negativas no mesmo teste. Entre os 93 cães positivos, 89 (95,7%) nasceram em Porto Velho. Nenhuma diferença estatística foi observada entre cães que moram em quintais ou intradomicílio. O mapa de distribuição indica um hotspot na região norte da cidade. O PCR dos mosquitos resultou em apenas um pool positivo. A transmissão de dirofilariose canina ocorre na cidade de Porto velho e a frequência que ocorre nos cães é considerada moderada. A técnica de imunocromatografia e PCR são mais eficazes na detecção de dirofilariose comparadas a gota espessa. A confirmação de transmissão de dirofilariose canina em Porto Velho, coloca esta doença no ranking de diagnóstico diferencial de nódulos pulmonares em Porto Velho. / Heartworm is an infectious disease with worldwide distribution. Mosquitoes acts as intermediary host and vectors. In Brazil, until now, heatworm was found in 15 states with an national average of 2%. This present work aims to report for the first time canine heartworm in the state of Rondônia and confirms the transmission of the disease in the state. Blood samples were randomly collected from 727 dogs in the municipality of Porto Velho. The samples were analyzed for the presence of microfilariae and circulating antigens using two different techniques: thick blood microscopy stained with Giemsa and immunochormatography for the detection of filarial antigens. Aiming to test the efficacy of the immunoassay test, all the positive cases were examined by PCR and pools of negative samples were also examined. Mosquitoes were collected at the domiciles presenting positive cases and analyzed by PCR. A distribution map was made with positive cases. Ninety three blood samples out of 727 (12.8%) were positive by the immunoassay technique and none by the thick smear method. All the positive cases by the immunoassay technique were examined by PCR and pools of all negatives samples were also examined resulting in 10% and 0% positivity frequency, respectively. Among the 93 positive dogs, 89 (95.7%) were born in Porto Velho. No differences in the frequency of infection were observed between dogs raised indoor or in the yards. The distribution map indicates a hotspot in the north area of the city. Mosquitoes were analyzed by PCR, resulting in only one positive pool. This result shows that the transmission of canine heartworm is occurring in the municipality of Porto Velho and has moderate prevalence among dogs. The immunoassay technique and the PCR method are more efficient in detecting D. immitis infections in dogs when compared to the blood smear technique. The confirmation of heartworm transmission in Porto Velho also includes this disease among the ranks of differential diagnosis of pulmonary nodules in humans in Rondônia.

West Nile Virus (WNV), a vector-borne disease continues to be a serious threat to public health in the United States, particularly in the Southwest region. While all the states in the U.S. experienced a decreasing trend of WNV disease in 2010, the state of Arizona experienced a sharp increase from 20 in 2009 to 166 cases the following year. This dissertation endeavored to develop forecasting models to predict future cases of disease and identify counties with increased propensity for WNV. Furthermore, this study aimed to identify environmental and economic factors that contributed to the increase in WNV cases in Maricopa County, Arizona. A spatiotemporal stochastic regression model was developed using Bayesian principles and was successful in calculating the annual mean cases of disease from 2003 to 2011 for all counties. The model was also able to predict future cases of disease by fitting historical data. The model-based inference identified counties in the southern region of Arizona as having an elevated propensity for disease compared to counties in the northern region. A Seasonal Autoregressive Integrated Moving Average (SARIMA) model was developed and effectively forecasted monthly cases of human WNV in Maricopa County, Arizona. By fitting the SARIMA model to monthly historical disease data from 2005 to 2011, the temporal model presented a decreasing trend of monthly incidence of disease for 2012. The impact of home foreclosures, climate variability, and population growth on the resurgence of human WNV disease cases in Maricopa County during the 2010 epidemic was investigated. These factors were found to have contributed to the resurgence of the disease by creating the optimal environmental conditions that allowed the amplification of mosquito populations, thus increasing the risk of disease transmission to humans. As spatiotemporal disease data become readily available, forecasting models can be an important and viable risk assessment tool for public health practitioners. Forecasting models allow the mobilization and distribution of limited resources to areas with elevated propensity for disease, and the timely deployment of intervention programs to reduce the overall risk of disease. / Graduation date: 2013

The present thesis explored ethological and geographical approaches for the investigation of vector-borne parasites. In the first part, the role of associative learning on vector preferences for hosts was investigated through a comprehensive series of behavioural experiments using the vector of dengue and yellow fever diseases, the mosquito Aedes aegypti. To this end, the possibility that the mosquitoes were able to associate unconditional stimuli with particular odours and visual patterns to which they were responsive was explored, but no evidence supporting the hypothesis that associative learning abilities are present in adults of this species was found. A critical review of the literature on learning in mosquitoes conducted afterward allowed the reinterpretation of findings in the field, narrowing the scope of evidence suggesting the existence of these cognitive abilities in some species. In the second part of the thesis, the distribution and evolution of mosquito-borne viruses was investigated with the use of geo-coded environmental data and spatial statistics. Initially, the eco-climates associated with the distribution of Japanese encephalitis virus were described and modelled, allowing the production of a worldwide predictive map defining the probability of each region to develop this disease in the future. Predominating amongst those areas shown to be under high risk were the equatorial regions of South America and Africa. The methodology used to infer such patterns – non-linear discriminant analysis – was subsequently explored with a number of simulations. Overall, differences in the choice of parameters required for the analysis were shown to lead to differences in the final outputs produced, basically in those cases where the environmental range for which predictions are generated is not rigorously limited. Finally, eco-climate surrogates for the evolution of the Japanese encephalitis serocomplex were investigated, but the current environmental distances between the viruses did not seem to be associated with the events leading to their speciation.

Thesis (Ph.D.) - University of Queensland, 2004. / Program shared between two schools. Includes bibliography.