Orientador: Arício Xavier Linhares / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T23:41:06Z (GMT). No. of bitstreams: 1 Franco_CarolineSiqueira_M.pdf: 1657929 bytes, checksum: 0b27568a919b921d3ae68ae3161f87fd (MD5) Previous issue date: 2014 / Resumo: A febre maculosa brasileira (FMB) é uma doença infecciosa aguda transmitida através da picada de carrapatos infectados com Rickettsia rickettsii Brumpt 1922. O carrapato Amblyomma cajennense Fabricius 1787 (Acari: Ixodidae) é um dos principais vetores no Brasil. Contudo, outras espécies de carrapatos podem ser vetores da doença e parasitar humanos. Neste estudo, procurou-se avaliar a competência vetorial de Ornithodoros mimon Kohls 1969 (Acari: Argasidae) em laboratório após ser infectado experimentalmente com a cepa Taiaçu de Rickettsia rickettsii, utilizando coelhos domésticos como modelo animal. Primeiramente, os carrapatos foram separados em grupos de acordo com seu estágio de desenvolvimento (larva, ninfa 2 e adulto). Na segunda etapa, dois coelhos foram utilizados como grupo tratamento, e foram inoculados com a Rickettsia, e um coelho foi usado como controle. Os carrapatos utilizados foram separados em lotes, de acordo com o coelho e dia em que foram alimentados. Na terceira etapa, novos coelhos sadios foram infestados com os carrapatos usados na segunda etapa, para verificar se houve transmissão. Carrapatos que tiveram contato com a bactéria foram macerados e inoculados em duas cobaias, e uma terceira cobaia foi infestada com carrapatos que foram alimentados em coelho infectado. PCR com os primers gltA e ompA (R. rickettsii), ftsZ (Wolbachia sp.), 28S (eucarioto) foi realizado, para avaliar se os carrapatos se tornaram infectados. Sequenciamento de amostras foram amplificadas com os primers ompA e 28S. Além da Reação de Imunofluorescência Indireta (RIFI), que foi realizada com o soro dos coelhos para verificar a presença de infecção. Os dois coelhos que foram inoculados com Rickettsia rickettsii apresentaram picos de febre. Um dos coelhos apresentou resultado positivo na RIFI e o outro morreu no décimo sexto dia após a inoculação. Os coelhos que foram infestados com carrapatos que foram alimentados em coelho infectado não apresentaram febre e nenhum outro sintoma da doença. As cobaias que foram inoculadas com carrapatos e a cobaia que foi infestada com carrapatos não demonstraram nenhum sintoma da doença. Das amostras amplificadas com o primer gltA apenas uma amostra foi positiva. A PCR das amostras de carrapatos usando o primer ompA não apresentou o resultado esperado, provavelmente devido a presença do DNA da bactéria Wolbachia sp. A sequência gerada, com amostras que foram amplificadas com o primer 28S, foi o DNA do carrapato. A PCR com o primer ftsZ não amplificou nenhum DNA. Portanto, conclui-se que O. mimon não se tornou infectado com a Rickettsia rickettsii, e não representa risco para a saúde pública / Abstract: The Brazilian Spotted Fever (BSF) is an acute infectious disease transmitted by the bite of ticks infected with Rickettsia rickettsii Brumpt 1922. The tick Amblyomma cajennense Fabricius 1787 (Acari: Ixodidae) is one of the main vectors in Brazil. However, other species of ticks can be vectors of this disease and parasitize humans. This study aimed to evaluate the vector competence of Ornithodoros mimon Kohls 1969 (Acari: Argasidae) in the laboratory after being experimentally infected with the Taiaçu strain of Rickettsia rickettsii, using domestic rabbits as the animal models. Firstly, the ticks were separated into groups according to their developmental stage (larva, nymph 2 and adult). Secondly, two rabbits were used as treatment group, and were inoculated with the Rickettsia, and one rabbit was used as control. The ticks were separated into batches according to the rabbit and the day they were fed. New healthy rabbits were infested with the ticks used in the second experiment, to verify the occurrence of transmission. Ticks that had contact with the bacteria were macerated and inoculated into two guinea pigs, and a third guinea pig was infested with ticks that were fed on infected rabbits. PCR with gltA e ompA primers (R. rickettsii), ftsZ (Wolbachia sp.), 28S (eukaryotic) was performed, to assess if the ticks become infected. Sequencing of the samples was amplified with the primers ompA and 28S. In addition, the Immunofluorescence Assay (IFA) was performed with serum of rabbits to verify the presence of infection. The two rabbits that were inoculated with Rickettsia rickettsii presented peaks of fever. One rabbit presented a positive result in the IFA and the other died on the sixteenth day after inoculation. The rabbits that were infested with ticks that were fed on infected rabbits, showed no fever and no other symptoms of the disease. The guinea pigs that were inoculated with ticks and the guinea pig that was infested with ticks showed no symptoms of the disease. Of the samples only one of all samples amplified with the primer gltA was positive. The PCR samples of ticks using the primer ompA did not yield the expected result, probably due to the presence of the DNA of the bacteria Wolbachia sp. The sequence generated, with samples that were amplified with the primer 28S, it was the DNA of the tick. PCR performed with the primer ftsZ did not amplify any DNA. Therefore, we concluded that O. mimon did not become infected with R. rickettsii, and do not represent risk to public health / Mestrado / Relações Antrópicas, Meio Ambiente e Parasitologia / Mestra em Biologia Animal

Kenya has experienced severe arboviral outbreaks of public health concern in the recent past, including yellow fever (YF), Crimean Congo hemorrhagic fever (CCHF), chikungunya, and Rift Valley fever (RVF) among others. Most of these infections are under diagnosed and hence neglected due to non-specific nature of their symptoms. Often they are mistaken for endemic tropical diseases such as malaria and typhoid infections and are only recognized during major outbreaks which result in adverse public health and economic consequences to the affected communities. Ongoing inter-epidemic surveillance in RVF virus hotspots in Kenya has indicated continued intense transmission of Bunyamwera virus (BUNV) in the absence or under low level activity of RVF virus. BUNV belongs to the genus Orthobunyavirus of the family Bunyaviridae. These are segmented RNA viruses whose members have the potential for genetic reassotment and/or drift. Recently, Ngari virus (NRIV), a natural reassortant virus associated with hemorrhagic fever was documented to have emerged from BUNV, which previously was not associated with such symptoms. However, the vectors that are involved in the maintenance and transmission of BUNV and NRIV are diverse and their role in virus maintenance/dynamics is poorly known. It is thus important to investigate the dynamics of BUNV and NRIV in selected transmission foci in an effort to understand their circulation better in order to be able to control and predict outbreaks. In this study, we determined the evolutionary and phenotypic diversity of BUNV and NRIV isolates previously obtained from vectors in parts of Kenya. We have provided genetic sequences of two BUNV and three NRIV isolates which contribute to addressing paucity of genetic sequences associated with this group of viruses. Phylogenetic analysis of these sequences in addition to other sequences in GenBank revealed evidence of geographic/temporal clustering that requires further investigation. Next, we demonstrated that plaque purified phenotypes of selected BUNV and NRIV isolates differ in in vivo growth kinetics and pathogenicity in mice, possibly related to specific mutations within the genome. The phenotypic changes and identification of mutations possibly associated with these changes support further investigation of specific mutations using site directed mutagenesis. In addition, we determined the competence of some of the mosquito species implicated in their transmission, Anopheles gambiae, Aedes aegypti and Culex quinquefaciatus and evaluated the dynamics of their transmission in these vectors. We conclude that Anopheles gambiae is likely a more competent vector for NRIV than Aedes aegypti and is a moderately competent vector for BUNV, which has implications for animal movement in malaria endemic areas where the vector is present. We also report evidence of BUNV transovarial transmission in both Aedes aegypti and Anopheles gambiae with the prevalence of transmission related to the ovarian cycle. Finally, we determined the level of human exposure to these viruses in the transmission foci. Orthobunyavirus-specific antibodies were detected by plaque reduction neutralization test in 89 (25.8%) of 345 persons tested. Multivariable analysis revealed age and residence in North Eastern Kenya as risk factors. In conclusion, we propose that acute febrile disease surveillance needs to be implemented in North Eastern Kenya. This study helps identify the virus strains/populations and the vector species that play a critical role in sustenance and transmission of BUNV and NRIV in different ecosystems in the country. All these are important in understanding virus circulation, potential for emergence and risk to populations in areas of circulation, and will help in making decisions for intervention and management. Generated sequence data in this study contributes to global phylogenetic characterization of Orthobunyaviruses worldwide and their molecular epidemiology. The study also shed light/improve our knowledge on the genetic stability or diversity and evolutionary trends of Orthobunyavirus strains in Kenya. / Thesis (PhD)--University of Pretoria, 2015. / Medical Virology / PhD / Unrestricted

Dirofilaria immitis, commonly known as dog heartworm, is a dangerous filarial nematode that is transmitted by a mosquito vector. Although this parasite is historically localized to regions closer to the equator, the last few decades have experienced an increase in the amount of dog heartworm cases in Northern California, most notably in Lake and San Joaquin Counties. The primary vector of D. immitis in Northern California is Aedes sierrensis, but it is not prevalent enough in some counties to explain the increase in infections. Because of this, more prevalent and abundant species should be assessed for their ability to transmit heartworm. Culex pipiens complex (wild) and Culiseta incidens (laboratory-raised colonies) were chosen as potential vectors during this study due to their ability to meet several key vector criteria, their relative abundance in the areas of interest, and the fact that they habitually take bloodmeals from domestic dogs. Additionally, Ae. sierrensis colonies were used in this study, based on previous knowledge that they are highly competent vectors. Female mosquitoes were infected with 2.5, 5, or 10 mff/ul of D. immitis-infected blood, and decapitated at 15, 18, and 21 days post-infection. Mosquitoes were subsequently tested via qPCR for the presence and relative quantification of D. immitis DNA. We found that Ae. sierrensis and Cx. pipiens complex were both able to support the development of D. immitis to its infective stage, while Cs. incidens was not a competent vector. We also concluded that including a standard curve of known amounts of D. immitis larvae makes it possible to approximate the intensity of the infection in the mosquito. The identification of these important vectors, and the ability to assess infection intensity, may be helpful in the continued vector control efforts in Northern California and beyond.

Mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) are major threats to global public health resulting in millions of infections and hundreds of thousands of deaths annually. The presence of these viruses and their increasing emergence/spread continues to escalate. Notably, Usutu virus (USUV; Genus: Flavivirus; Family: Flaviviridae) is one such pathogen currently causing mass die-offs of avian hosts throughout Europe. USUV is categorized in the Japanese Encephalitis virus (JEV) antigenic complex and thus shares many antigenic and pathologic characteristics with fellow members, such as JEV and WNV. Respective to human infections, USUV cases are generally asymptomatic; nonetheless, acute cases have been reported. These acute cases typically cause mild symptoms, such as fevers and rashes; however, more severe cases can result in neurologic diseases, such as encephalitis and/or meningoencephalitis. In addition to these pathologic similarities, USUV shares several ecological and geographical traits with WNV, a pathogen responsible for several outbreaks during its spread from Africa, to Europe, and eventually the United States. Currently, WNV is considered endemic in areas across the United States due to its transmission via Culex spp.; mosquitoes that are ubiquitous in the United States. These parallels suggest the possible emergence of USUV into the United States and therefore, it is imperative to broaden our knowledge of USUV and assess its potential to become a major global health concern. The overall goal of this thesis was to characterize USUV and evaluate its emergence potential in the United States by: (1) developing infectious clones of recent European and African USUV isolates as tools for characterization and analysis of USUV and (2) assessing the transmission potential of several species of North American mosquitoes. In Aim 1, we show that the aforementioned infectious clones infect and replicate similarly to their parental strains in vitro in both vertebrate and invertebrate models, as well as in transiently immunocompromised CD-1 and IFNAR-/- murine models, and thus serve as useful tools for future molecular studies focusing on USUV. Furthermore, in Aim 2, we describe the ability of field-caught (Southwest Virginia, USA) Culex spp. and Aedes spp. mosquitoes to become infected with a recent European isolate of USUV; although, we report an overall limited potential for these species to transmit this virus. Altogether, these studies form a foundation for understanding the potential emergence of USUV in the United States as well as provide necessary tools needed to aid future research on USUV emergence, transmission, and pathogenesis. / Master of Science / Usutu virus (USUV) is an emerging mosquito-borne virus that was first isolated from a mosquito in 1959 in South Africa, and since then, has become a major problem throughout Africa and Europe causing acute to severe infection in dozens of patients. Additionally, this virus is causing massive die-offs in Eurasian blackbird populations. This is particularly problematic because birds play a critical role in ecosystems as they act as forms of pest control, pollinators, and seed dispersers. Depletion of these species could lead to an imbalance and, eventually, collapse of our natural ecosystem. Additionally, there is a growing concern of USUV making its way into the United States, following a similar track of emergence to WNV's introduction in New York in 1999 and its subsequent spread throughout the states. WNV's introduction to the United States was detrimental to native bird populations and humans, and has caused tens of thousands of infections and thousands of deaths since this introduction. Research has shown USUV causes similar disease symptoms to WNV. The self-limiting illness from these viruses typically includes fever and rashes but some infections can result in more severe cases causing inflammation of the brain and surrounding areas. Like many other prominent mosquito-borne viruses, there is no specific treatment or vaccine for WNV or USUV. Because USUV is so closely related to WNV, and their similar characteristics may point towards similar emergence in the United States, it is essential to garner more information on USUV. The overall goal of this thesis was to establish a reliable tool(s) for further characterization of USUV and demonstrate the potential for USUV emergence in the United States. We first developed molecular tools, known as viral clones, that are valuable to the scientific community which allows the manipulation of USUV genetic material to perform further downstream studies. Our objective for this initial study was to create a molecular tool that would behave similarly to their natural, or "parental", virus. The results from this study suggest we have successfully produced these tools. Furthermore, we sought to determine the potential for field-caught mosquitoes from Southwest Virginia, USA to transmit a recently isolated strain of USUV. These data suggest that while these mosquitoes do have the ability to become infected with USUV, they have a limited potential to transmit this virus to animal hosts. Altogether, these studies have allowed us to expand our knowledge on USUV's potential emergence in the United States and develop powerful tools to continue this essential research.

Since the establishment of West Nile virus (WNV) in North America, concern has arisen that this virus may also pose a biosecurity threat to Australia. West Nile virus is maintained in an enzootic transmission cycle between birds and ornithophilic Culex spp. mosquitoes. Since its introduction into the United States in 1999, it has been responsible for over 11,000 human cases of neurological disease and over 1,000 human deaths. In addition, WNV has caused extensive equine and avian mortality. Should an exotic, virulent strain of WNV be introduced into Australia, it may have significant impact on human and animal health. This thesis examines entomological factors which may influence the potential for an exotic, pathogenic strain of WNV to become established in eastern Australia and be maintained in urban transmission cycles. Further, it aims to elucidate the most likely candidate vectors for the maintenance of WNV in natural transmission cycles. Components include: a) laboratory-based vector competence experiments to assess the ability of Australian mosquito species to become infected with and transmit a North American strain of WNV; b) identification of the vertebrate origin of mosquito bloodmeals using serological and molecular assays; c) an evaluation of the efficacy of bird-baited traps for collecting ornithophilic mosquitoes at ground level and in the tree canopy; and d) examination of the prevalence of endemic arboviruses, including Kunjin virus (KUNV; an Australian subtype of WNV) in mosquitoes collected from urban centres of eastern Australia. Vector competence experiments were conducted with field-collected Australian mosquito species. Nineteen species were were exposed to a 1999 New York strain of WNV using an artificial membrane feeding apparatus and transmission was attempted using an in vitro modified capillary tube method. A number of Culex species, including Culex quinquefasciatus and Culex gelidus, demonstrated high vector competence for WNV. Culex annulirostris, the primary KUNV vector, was the most competent vector, displaying transmission rates of 84%. Other common species, including Aedes vigilax, Aedes procax and Verrallina spp. were less susceptible to infection. Approximately 1,200 blood engorged mosquitoes were analysed using an enzyme-linked immunosorbent assay to determine host species. Overall, 90% of bloodmeals from Cx. quinquefasciatus were avian in origin, while 22% of bloodmeals from Cx. annulirostris were from birds. Molecular assays were further used to determine which bird species were commonly targeted by a selection of ornithophilic mosquito species. Mitochondrial DNA sequence data indicated that most (93%) avian bloodmeals from Cx. quinquefasciatus were obtained from passerine birds (order Passeriformes). Avian species commonly targeted by Cx. quinquefasciatus included Sphecotheres vieilloti (Australasian figbird; 39% of total identified), Sturnus tristis (common myna; 19%) and Philemon buceroides (helmeted friarbird; 17%). Alternatively, the majority (87%) of avian bloodmeals identified from Cx. annulirostris were from non-passerine birds, with most bloodmeals obtained from ducks (72%; family Anatidae). Seven field-based Latin Square trials conducted in Brisbane and Cairns showed that conventional CO2-baited CDC traps yielded higher numbers of Culex spp. than novel bird-baited traps, both at ground level and in the tree canopy. In Brisbane, CO2-baited CDC traps placed at ground level (approximately 1 m from ground) collected significantly more Cx. annulirostris than those at canopy height (5-10 m) during two trials, and more Ae. vigilax and Aedes procax during one trial each. Conversely, in Cairns, CO2-baited CDC traps placed in the tree canopy collected significantly more Culex squamosus during two trials, and significantly more Aedeomyia catasticta and Culex cubiculi during one trial each. Consistently low yields of ornithophilic species including Culex australicus and Cx. quinquefasciatus in all traps tested suggests the need to employ alternative trapping measures to target these species in urban areas. Over 1,800 mosquito pools (comprising over 65,000 individual mosquitoes collected from urban environments) were tested for evidence of arboviral infection. None of the pools showed evidence of flavivirus infection, but two alphaviruses were isolated from mosquito pools from Cairns. One isolate of Barmah Forest virus was obtained from a pool of 100 Ae. vigilax, and a pool of 89 Verrallina carmenti yielded an isolate of Ross River virus. In addition, an isolate most similar to a newly described bunyavirus, Stretch Lagoon orbivirus, was obtained from a single Aedes alternans collected from Sydney. The outcomes of this thesis demonstrate that a number of Australian mosquito species can become infected with and transmit a virulent strain of WNV, should it be introduced into Australia. Analysis of the host feeding patterns of these potential vectors in urban environments demonstrates that a number of candidate vectors, including Cx. quinquefasciatus and Cx. annulirostris, readily feed on common bird species. Further, the absence of detectable flavivirus infection in urban mosquito populations shown here suggests that endemic urban flaviviruses would have minimal influence on potential WNV transmission. Notwithstanding other ecological factors that may influence the likelihood of enzootic or epizootic transmission including susceptibility of vertebrate hosts and mechanisms of introduction, the potential establishment of an exotic, virulent strain of WNV in urban areas of Australia is certainly possible given the evidence provided in this thesis.

Le virus chikungunya (CHIKV), transmis par les moustiques Aedes aegypti et Aedes albopictus, constitue un problème majeur de santé publique. Depuis 2004, des épidémies de CHIKV ont été rapportées en Afrique, en Asie, dans les îles de l'Océan Indien, et en Europe. Seule l'Amérique semblait épargnée malgré la présence de fortes densités de moustiques vecteurs et de multiples importations du virus dans le continent par des voyageurs de retour de pays où le virus circulait. Nous avons abordé dans cette thèse le risque d'émergence du CHIKV en Amérique à partir d'une évaluation de la compétence vectorielle de 35 populations d'Ae. aegypti et Ae. albopictus locaux avec différentes souches de CHIKV. Ces populations sont compétentes vis-à-vis du CHIKV avec un rôle des glandes salivaires comme "filtre" de la transmission. De plus, le génotypage des Ae. albopictus d'Amérique par microsatellites a permis d'identifier un cluster génétique de populations se caractérisant par une faible transmission des souches de CHIKV de génotype Est-Centre-Sud-africain. En octobre 2013, des souches asiatiques de CHIKV ont été signalées dans la Caraïbe. Nous avons alors évalué la réceptivité de 11 populations d'Ae. aegypti et Ae. albopictus d'Amérique vis-à-vis de CHIKV de génotype asiatique et avons mis en évidence que les deux espèces étaient compétents pour assurer la diffusion du virus sur le continent. On note aussi qu'Ae. albopictus peut faciliter la propagation du CHIKV vers l'Europe. Néanmoins, la compétence vectorielle d'Ae. albopictus de France vis-à-vis de CHIKV asiatique est affectée négativement par des températures plus basses que celles habituellement observées dans les pays tropicaux. / Chikungunya virus (CHIKV), transmitted mainly by the mosquitoes Aedes aegypti and Aedes albopictus, is a major public health problem. Since 2004, CHIKV epidemics have been reported in Africa, Asia, the Indian Ocean Islands, and Europe. Only the Americas seemed spared despite high densities of mosquitoes and multiple introductions of the virus to the continent by travelers returning from countries where CHIKV was circulating. We have assessed the risk of CHIKV emergence in the Americas by evaluating the vector competence of 35 local populations of Ae. aegypti and Ae. albopictus infected with different strains of CHIKV. These populations were shown to be susceptible to CHIKV infection, highlighting the predominant role of salivary glands as a "filter" of transmission. Genotyping of Ae. albopictus from the Americas using microsatellites allowed the identification of a genetic cluster of populations characterized by a low transmission of CHIKV strains of the East-Central-South-African genotype. In October 2013, Asian strains of CHIKV began circulating in the Caribbean. Thus, we evaluated the susceptibility of 11 populations of Ae. aegypti and Ae. albopictus to the Asian CHIKV genotype and showed that the two species were sufficiently competent to ensure dissemination of the virus throughout the continent. Furthermore, we showed that Ae. albopictus was likely to facilitate the spread of CHIKV to Europe. However, the vector competence of French Ae. albopictus to the Asian CHIKV genotype was negatively affected by temperatures lower than those usually found in tropical countries.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Natalia Cernicchiaro / Japanese encephalitis virus (JEV) is a virus of the Flavivirus genus that may result in encephalitis in vertebrate hosts. This vector-borne zoonosis occurs in Eastern and Southeastern Asia and an intentional or inadvertent introduction into the United States (US) would lead to important public health and economic consequences. The objective of this study was to gather, appraise, and synthesize primary research literature to identify and quantify vector and host competence for JEV, using a systematic review-metaanalysis (SR-MA) approach. After defining the research question, we performed a search in selected electronic databases. The title and abstract of the identified articles were screened for relevance using a defined set of exclusion and inclusion criteria, and relevant articles were subjected to a risk of bias assessment followed by data extraction. Random-effects subgroup meta-analysis models were fitted by species (mosquito or vertebrate host species) to estimate pooled summary measures as well as to compute the variance between studies. Meta-regression models were fitted to assess the association between different predictors and the outcomes of interest and to identify sources of heterogeneity among studies. Data were extracted from 171 peer-reviewed articles. Most studies were observational (59.06%) and reported vector competence (60.2%). The outcome measures reported pertained to transmission efficiency, host preference, and vector susceptibility to infection within vector competence; and susceptibility to infection within host competence. All outcome measures (JEV proportion of infection in vectors and hosts from observational studies; and JEV infection, dissemination, and transmission rates in vectors from experimental studies) had high heterogeneity. Mosquito species, diagnostic method, country, and capture method represented important sources of heterogeneity associated with the proportion of JEV infection in vectors; host species and region were considered sources of heterogeneity associated with the proportion of JEV infection in hosts; and diagnostic and mosquito capture methods were deemed important contributors of heterogeneity for the minimum infection rate (MIR) outcome. Mosquito species and administration route represented the main sources of heterogeneity associated with JEV infection rate in vectors. Quantitative estimates resulting from this SR-MA will be inputted into risk assessment models to evaluate risks associated with the introduction of JEV in the US.

Orientador: Cláudio Casanova / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T11:03:53Z (GMT). No. of bitstreams: 1 CollaJacques_FernandaElisa_D.pdf: 17373999 bytes, checksum: 18adb4b65dc2639da145a80ed8b6f4e2 (MD5) Previous issue date: 2014 / Resumo: Flebotomíneos (Diptera: Psychodidae) são insetos de importância médico-veterinária pois participam do ciclo de doenças tais como leishmanioses, que acometem milhares de pessoas ao redor do mundo. O conhecimento sobre a ecologia e biologia desses dípteros é de fundamental importância para determinar a capacidade e competência vetorial de suas populações, e portanto, relevantes do ponto de vista da saúde pública. Este trabalho buscou esclarecer alguns pontos com informações deficientes sobre a biologia e ecologia das espécies vetoras de Leishmania (Leishmania) infantum chagasi e Leishmania (Viannia) braziliensis, respectivamente, Lutzomyia longipalpis e Nyssomyia neivai, no estado de São Paulo. Para a leishmaniose visceral americana (LVA), objetivou-se esclarecer questões a respeito de criadouros urbanos preferenciais de Lu. longipalpis e sobre sua expansão geográfica no estado de São Paulo. Em referências às lacunas no conhecimento da ecoepidemiologia da leishmaniose tegumentar americana, a competência vetorial de Nyssomyia neivai para L. (V.) braziliensis e o estudo da composição da comunidade flebotomínica em área endêmica no nordeste do estado, bem como sua variação mensal. Criadouros de Lu. longipalpis foram encontrados predominantemente em ambientes sombreados com grande quantidade de matéria orgânica, com maior concentração de larvas associadas a galinheiros. Quanto à sua dispersão, esta parece ter seguido uma rota a partir de Mato Grosso do Sul, seguindo em direção oeste-leste e também para norte e para sul a partir dessa linha central. A ocorrência de casos de LVA em humanos frequentemente é precedida pela detecção de Lu. longipalpis e casos caninos. Nyssomyia neivai, bastante frequente em áreas endêmicas para LTA no vale do Rio Mogi Guaçu, mostrou-se competente para adquirir, manter e transmitir o parasito em condições laboratoriais, cumprindo assim mais uma etapa necessária para a sua incriminação como espécie-vetora. Ainda no vale do rio Mogi Guaçu, a comunidade de flebotomíneos mostrou-se bastante diversa, com 17 espécies, das quais cinco são importantes para o ciclo da LTA. A fauna flebotomínica encontrada era composta majoritariamente por Nyssomyia whitmani, Migonemyia migonei e Expapillata firmatoi e, mais uma vez, abrigos para animais, tais como galinheiros, foi o local preferido para estes insetos, onde a sua maioria foi capturada. Desta forma, este trabalho apresenta dados relevantes para o conhecimento a respeito desses insetos vetores, que podem ser usados como base para futuros projetos ou no desenvolvimento de novas estratégias de controle dessas doenças / Abstract: Phlebotomine sandflies (Diptera: Psychodidae) are insects of medical and veterinary importance because they take part in the cicle of diseases, such as leishmaniasis which affect thousands of people around the world. Learning about these dipteras¿s ecology and biology is fundamentaly important to define the vectorial capacity and competence of their populations, and therefore, relevant for the public health perspective. This study aimed at adressing some issues with deficiente information on the biology and ecology of vector species of Leishmania (Leishmania) infantum chagasi and Leishmania (Viannia) braziliensis, respectively, Lutzomyia longipalpis and Nyssomyia neivai, in São Paulo State. For the American visceral leishmaniasis (AVL), this paper tried to answer questions on the preferencial breeding site of Lu. longipalpis and its geographical expantion in São Paulo State. In relation to the knowledge of the ecoepidemiology of American cutaneous leishmaniasis (ACL), the vectorial competence of Nyssomyia neivai to L. (V.) braziliensis and the analysis of the sandfly community in and endemic area in the Northeast of the state were studied. Lutzomyia longipalpis breeding sites were found predominantly in shaded environments with great amount of organic matter, and the greatest concentration of larvae associated to chicken sheds. Considering its dispersion, Lu. longipalpis seemed to follow a route from Mato Grosso do Sul to São Paulo city, in a west to east direction and from that central line, an expansion to the north and south. The occurance of human AVL is generally preceded by the detection of Lu. longipalpis and canine cases. Nyssomyia neivai, frequently found in endemic áreas for ACL in the Mogi Gauçu River Valley, was shown to be competente to aquire, host and transmit the parasite under laboratory conditions, thus fulfilling another requirement to be recognized as a vector species. Still in Mogi Guaçu River Valley, the sandfly community was greatly diverse, comprised of 17 species, of which five are considered important for the ACL cicle. The sandfly fauna was mainly comprised of Nyssomyia whitmani, Migonemyia migonei and Expapillata firmatoi and, once again, animal shelters, such as chicken sheds, were the preferred site for these insects, where the greatest amount was collected. Therefore, this work presents relevant data for the knowledge on these vector insects, which can be used as basis for future projects or in the development of new control strategies against these diseases / Doutorado / Relações Antrópicas, Meio Ambiente e Parasitologia / Doutora em Biologia Animal

Em algumas regiões, como nos municípios de Corumbá e Ladário, no Estado de Mato Grosso do Sul, existem evidências ecológicas e epidemiológicas de que Lutzomyia (Lutzomyia) cruzi (Mangabeira, 1938) seja a principal responsável pela transmissão do protozoário Leishmania (Leishmania) infantum Nicolle, 1908 (ou subespécie de L. (L.) infantum chagasi Cunha & Chagas, 1937 segundo alguns autores), agente etiológico da leishmaniose visceral (LV). A ausência de Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912), principal vetor do parasito, reforçam esta hipótese. Este estudo teve por objetivo avaliar os parâmetros e estimar a capacidade vetorial de Lu. cruzi para L. (L.) infantum e Leishmania (Leishmania) amazonensis Lainson & Shaw, 1972. Para este último, apenas foram avaliados os parâmetros, sem a estimativa numérica da capacidade vetorial. A avaliação da capacidade vetorial foi realizada a partir de experimentos laboratoriais (infecção experimental) e de campo (atratividade aos flebotomíneos). Por intermédio da infecção experimental de Lu. cruzi pelo parasito, foi possível estimar a expectativa de sobrevida diária de fêmeas infectadas (estimativa vertical e laboratorial), avaliar o período de incubação extrínseco do parasita e obter duração do ciclo gonotrófico. Para a avaliação da competência vetorial do inseto, foram realizadas tentativas de transmissão experimental e natural de Leishmania, a partir de fêmeas provenientes de colônia cujos indivíduos foram alimentados durante o xenodiagnóstico e de fêmeas selvagens capturadas em campo, respectivamente. A distribuição sazonal de Lu. cruzi foi avaliada por meio da instalação semanal de armadilhas luminosas no peridomicílio de cinco residências na área urbana do Município de Corumbá. Variáveis meteorológicas obtidas junto ao Centro de Monitoramento do Tempo, do Clima e dos Recursos Hídricos de Mato Grosso do Sul, índices radiométricos calculado a partir de imagens de resolução espacial (GeoEye) e o percentual de cobertural vegetal foram utilizados neste estudo. Os resultados obtidos permitiram estimar a capacidade vetorial de Lu. cruzi para L. (L.) infantum, que foi de 0,24, ou seja, espera-se que a população de fêmeas da área produzam 0,24 novas infecções viii por dia de exposição de uma infecção. A competência vetorial de Lu. cruzi para L. (L.) infantum e L. (L.) amazonensis, via picada, foi demonstrada por meio de transmissão natural e experimental dos parasitos, respectivamente. Também foi identificada a infecção natural de Lu. cruzi por L. (L.) amazonensis. Com relação à distribuição mensal, embora não tenha sido observada a presença de associação entre essas espécies e as variáveis ambientais de vegetação e clima, foi possível observar picos elevados populacionais na estação chuvosa e picos menores na estação seca. O padrão da distribuição sazonal das espécies de flebotomíneos demonstrado neste estudo foi determinado basicamente pelos espécimes de Lu. cruzi capturados, uma vez que eles representam 93,94 por cento . A variação mensal demonstrou que a espécie Lu. cruzi tem grande plasticidade, tendo sido observada em todos os meses de coleta. / In some regions such as in the municipalities of Corumbá and Ladário in Mato Grosso do Sul state, there are ecological and epidemiological evidence that Lutzomyia (Lutzomyia) cruzi (Mangabeira, 1938) is the vector of Leishmania (Leishmania) infantum Nicolle, 1908 (or subspecies of L. (L.) infantum chagasi Cunha & Chagas, 1937 according to some authors), the etiologic agent of visceral leishmaniasis (VL). The absence of Lutzomyia (Lutzomyia) longipalpis (Lutz & Neiva, 1912), the main vector of the parasite, supports this hypothesis. This study aimed to evaluate the parameters and estimate the vectorial capacity of Lu. cruzi for L. (L.) infantum and Leishmania (Leishmania) amazonensis Lainson & Shaw, 1972. For the latter, only parameters was evaluated without numerical estimation of the vectorial capacity. The evaluation of the vectorial capacity was carried out from laboratory experiments (experimental infection) and field (attractiveness to sandflies). Through experimental infection by the parasite, it was possible to estimate the expected daily survival of infected females (vertical and laboratory estimate), evaluate the extrinsic incubation period of the parasite and get the length of gonotrophic cycle. To evaluate the insect vector competence, attempts have been made of experimental and natural transmission of Leishmania from females from colony whose subjects were fed for xenodiagnosis and wild females captured in the field, respectively. Monthly and seasonal distribution of Lu. cruzi was evaluated by weekly installation of light traps in the peridomicile of five residences in the urban area of the Municipality of Corumbá. Meteorological variables obtained from the Weather Monitoring Center, Climate and Water of Mato Grosso do Sul Resources, radiometric indices calculated from spatial resolution images (GeoEye) and the percentage of plant cobertural were used in this study. Results allowed estimating the vectorial capacity of Lu. cruzi for L. (L.) infantum, which was 0.24, i.e., it is expected that the female population in the region produce 0.24 new infections per day of exposure to an infection. Vector competence of Lu. cruzi for L. (L.) infantum and L. (L.) amazonensis by biting, was demonstrated by natural and experimental transmission of both parasites, respectively. Natural infection of Lu. cruzi by L. (L.) amazonensis was identified. Regarding the monthly x distribution, there was no significant association between of sandflies and the environmental and climate variables. It was observed high peaks population in the rainy season and lower peaks in the dry season. The pattern of seasonal distribution of species of sand flies demonstrated in this study was determined primarily by Lu. cruzi specimens, since this species represent 93.94 per cent of the total captured. The monthly change showed that Lu. cruzi species has great plasticity and has been observed in all months of collection.

No início de 2010, uma nova riquetsiose foi descrita em um paciente humano, que foi infestado por carrapato em Barra do Una, no litoral sul do Estado de São Paulo. Técnicas moleculares indicaram que esta nova doença foi causada por uma nova cepa de riquétsia, que foi denominada de Rickettsia parkeri cepa Mata Atlântica. Estudos mais recentes demonstraram que 10 a 15% dos carrapatos Amblyomma ovale, coletados em áreas de Mata Atlântica nos estados de São Paulo (incluindo a área do caso índice da infecção humana em Barra do Una) e Santa Catarina estavam infectados com R. parkeri cepa Mata Atlântica. Desta forma, o presente estudo iniciou-se a partir de fêmeas ingurgitadas de A. ovale, coletadas de cães naturalmente infestados em Barra do Una. No laboratório, foi constatado por PCR que parte dessas fêmeas (6,25%) estavam naturalmente infectadas por R. parkeri cepa Mata Atlântica; os ovos dessas fêmeas foram utilizados para formar uma colônia de A. ovale naturalmente infectada por R. parkeri. Ovos de fêmeas não infectadas, foram utilizados para formar uma colônia não infectada. As duas colônias foram estudadas de forma paralela no laboratório, visando analisar e quantificar a transmissão transestadial e transovariana de R. parkeri cepa Mata Atlântica e a competência vetorial do A. ovale. As infestações por larvas foram realizadas em roedores (Calomys callosus), enquanto ninfas e adultos foram alimentados em Oryctolagus cunicullus (coelho doméstico). Amostras de 10 indivíduos de cada uma das fases (larvas, ninfas e adultos F1, ovos, larvas e ninfas F2) foram testadas individualmente por um sistema de taqman real-time PCR, para presença de Rickettsia spp. Os soros sanguíneos de todos animais infestados foram testados por imunofluorescência indireta com antígeno de R. parkeri, no dia zero e 21 dias após a infestação por carrapatos, a fim de verificar soroconversão para antígenos de Rickettsia. Os resultados obtidos demonstraram 100% de transmissões transestadial (larva para ninfas e ninfas para adultos) e transovariana de R. parkeri em A. ovale, uma vez que todas as amostras de ovos, larvas, ninfas e adultos do grupo infectado foram positivas na PCR. Larvas e ninfas de A. ovale demonstraram alta competência vetorial, pois todos animais infestados por esses estágios infectados soroconverteram para R. parkeri. Por outro lado, adultos foram parcialmente competentes, pois apenas metade dos coelhos soroconverteu após ser infestada com carrapatos adultos infectados. Nenhum carrapato do grupo controle foi positivo na PCR, assim como nenhum animal deste grupo soroconverteu para R. parkeri. Fêmeas infectadas por R. parkeri cepa Mata Atlântica tiveram parâmetros reprodutivos inferiores aos das fêmeas não infectadas, indicando algum efeito deletério da infecção por este agente sobre os carrapatos. Os resultados sugerem a importância do carrapato A. ovale na epidemiologia desta nova riquetsiose brasileira, assim como sugerem uma capacidade vetorial de A. ovale para R. parkeri cepa Mata Atlântica, uma vez que este carrapato é frequentemente encontrado infestando humanos no bioma de Mata Atlântica. / In early 2010, a novel rickettsiosis was described in a human patient that had been infested by a tick in Barra do Una, southern coast of the state of São Paulo, Brazil. Molecular tests indicated that the disease was caused by a new rickettsial strain, named Rickettsia parkeri strain Atlantic rainforest. More recent studies demonstrated that 10 to 15% of Amblyomma ovale ticks, collected in Atlantic rainforest areas in the states of São Paulo (including the human index case area in Barra do Una) and Santa Catarina were infected with R. parkeri strain Atlantic rainforest. Within this context, the present study started with A. ovale engorged females collected from naturally infested dogs in Barra do Una. In the laboratory, it was shown by PCR that part of these females (6.25%) were naturally infected with R. parkeri strain Atlantic rainforest; the eggs of these females were used to form a lab colony of A. ovale, naturally infected by R. parkeri. Eggs laid by uninfected females were used to form an uninfected tick colony. Both colonies were evaluated in parallel in the lab, aiming to verify and quantify the transstadial and transovarian transmissions of R. parkeri strain Atlantic rainforest in A. ovale ticks, and the vector competence of the different parasitic stages of this tick. Larval infestations were done on rodents (Calomys callosus), while nymphal and adult infestations were done on rabbits (Oryctolagus cunicullus). Samples of 10 individuals of each tick stage (F1 larvae, nymphs and adults; F2, eggs, larvae, and nymphs) were tested individually by a taqman real-time PCR targeting the genus Rickettsia. Blood serum samples from all infested animals were tested through immunofluorescence assay employing R. parkeri antigens, on zero and 21 days post tick infestation, in order to verify seroconversion to rickettsial antigens. The results demonstrated 100% of transstadial (larvae to nynphs; nymphs to adults) and transovarian transmissions of R. parkeri in A. ovale ticks, since all egg, larval, nymphal, and adult samples from the infected colony were PCR-positive. A. ovale larvae and nymphs demonstrated high vector competence because all animals infested by these tick infected stages seroconverted to R. parkeri. On the other hand, adult ticks were partially competent since only half of the animals seroconverted after being infested by infected adult ticks. None tick of the uninfected control colony was PCR positive, as well as no animal from this group seroconverted to R. parkeri. Tick females infected by R. parkeri strain Atlantic rainforest had lower reproductive parameters than uninfected females, indicating some deleterious effect of the infection on these ticks. The results suggest an important role of A. ovale ticks in the epidemiology of this novel Brazilian rickettsiosis, and also suggest that A. ovale are capable to transmit R. parkeri strain Atlantic rainforest under natural conditions, since this tick is frequently found infesting humans on the Atlantic rainforest biome.