O agente etiológico da Febre Maculosa das Montanhas Rochosas (RMSF), conhecida no Brasil como Febre Maculosa Brasileira, é a bactéria Rickettsia rickettsii. Essa bactéria é transmitida ao homem pela picada de diferentes espécies de carrapatos ixodídeos. No Brasil, os vetores são Amblyomma cajennense e A. aureolatum. As taxas de prevalência de R. rickettsii nas populações de carrapatos de áreas endêmicas para RMSF são baixas, em geral abaixo de 1%. Essa baixa prevalência parece estar associada a menores taxas reprodutivas e de sobrevivência de linhagens infectadas, sugerindo que R. rickettsii seja patogênica também para os seus vetores. Infecções experimentais demonstraram que 80-100% dos indivíduos de uma colônia de A. aureolatum mantida em laboratório são infectados por R. rickettsii, enquanto apenas 10-60% de A. cajennense adquirem a bactéria. Esses dados indicam que as respostas dessas duas espécies de carrapatos à infecção sejam diferentes, resultando em diferentes taxas de prevalência da bactéria. Dessa maneira, a caracterização molecular das interações entre carrapatos do gênero Amblyomma e a bactéria R. rickettsii é importante, podendo gerar informações não somente para o esclarecimento acerca dos mecanismos de patogenicidade de R. rickettsii para os carrapatos, mas também para um melhor entendimento dos mecanismos responsáveis pela aparente restringência de A. cajennense à infecção. Assim, os objetivos do presente estudo foram: (i) analisar os efeitos da infecção por R. rickettsii sobre o perfil de expressão gênica de carrapatos A. cajennense por hibridação subtrativa por supressão (SSH), (ii) validar os dados de SSH por reação em cadeia de polimerase quantitativa precedida por transcrição reversa (RT-qPCR) e (iii) caracterizar funcionalmente dois genes com expressão induzida pela infecção por RNA de interferência (RNAi). Após a análise bioinformática dos dados de SSH, 44 sequências únicas foram obtidas, das quais 36 representam genes com expressão induzida e 8 genes com expressão reprimida pela infecção. A indução dos genes codificadores da subunidade I da citocromo c oxidase (COX1), da subunidade IV da NADH desidrogenase, de uma proteína com domínio de inibidor de serina-proteases Kunitz-type (papilina-like), identificados por SSH, e de um peptídeo antimicrobiano (hebraeína), foi confirmada por RT-qPCR. O silenciamento gênico da hebraeína e da papilina-like não teve nenhum efeito na aquisição de R. rickettsii pelo vetor, indicando que, isoladamente, não são responsáveis pela proteção de A. cajennense contra a infecção. Os dados gerados pelo presente estudo abrem perspectivas para que outros genes sejam avaliados quanto ao seu papel na aquisição de R. rickettsii, os quais, no futuro, podem ser considerados como alvos para o desenvolvimento de vacinas. / The etiologic agent of the Rocky Mountain Spotted Fever (RMSF), also known as Brazilian Spotted Fever in Brazil, is the bacterium Rickettsia rickettsii. This rickettsia is transmitted to humans by the bite of various tick species. In Brazil, Amblyomma cajennense and A. aureolatum are known as vectors. The prevalence rates of R. rickettsii infected ticks in RMSF endemic areas are low, oscillating around 1%. These low prevalence rates seems to be associated with lower reproductive and survival rates of infected ticks, suggesting that R. rickettsii is also pathogenic to its vectors. Experimental infections with R. rickettsii have demonstrated that 80 to 100% of A. aureolatum ticks from a laboratory colony acquire this bacterium, whereas only 10 to 60% of A. cajennense ticks become infected. These results indicate that the responses of these two tick species against infection are different, resulting in different prevalence rates of the bacterium. Therefore, the elucidation of the interactions between ticks of the genera Amblyomma and the bacterium R. rickettsii at a molecular level is important to provide information to better understand the mechanisms of pathogenicity of R. rickettsii against ticks as well as for the elucidation of the mechanisms responsible for the apparent refractoriness of A. cajennense against infection. Therefore, the objectives of the current study were: (i) analyze the effets of the infection with R. rickettsii on the gene expression of ticks A. cajennense by suppression subtractive hybridization (SSH), (ii) validate SSH data by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and (iii) functionally characterize two genes induced by infection using RNA interference (RNAi). After bioinformatics analysis of SSH data, 44 unique sequences were obtained, among which 36 represent genes with expression induced and 8 repressed genes by infection. The induction of genes encoding subunit I of cytochrome c oxidase (COX1), the NADH dehydrogenase subunit IV, a protein containing Kunitz-type inhibitor domain (papilin-like), identified by SSH, and an antimicrobial peptide (hebraein), was confirmed by RT-qPCR. The effects of knockdown of hebraein and papilin-like encoding genes had no effect on the acquisition of R. rickettsii by the vector. Data of the current study may be used to evaluate the role of other genes in acquisition of R. rickettsii, which, in the future, may be considered as target for vaccine development.

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: In southern Africa, the ticks Amblyomma hebraeum and Hyalomma rufipes are the main vectors of animal Heartwater and human Crimean Congo Haemorragic Fever, respectively. In order to investigate the phylogeographic structure of these ectoparasites, population genetic studies were conducted throughout southern and partly eastern Africa. Knowing the levels of genetic diversities and population connectivities throughout the species’ native ranges, may be useful for implementing control measures (cross country borders for example), evaluate the potential for drug resistance in introduced locations as well as for elucidating its vectorial capacities in the region. Mitochondrial cytochrome oxidase I (COI) and the internal transcriber spacer 2 (ITS-2) sequences were obtained from eleven sampled localities. In general, haplotype networks and molecular diversity values showed significant genetic structure between most of the sampled populations. Fu’s Fs and mismatch distributions suggested recent population expansions following a bottleneck. A Mantel regression analysis also revealed a significant isolation by distance for H. rufipes. Comparing the two species, H. rufipes showed much stronger signals of geographic genetic population structure in relation to A. hebraeum. A single taxonomic unit is suggested for A. hebraeum while H. rufipes is characterized by the existence of at least three distinct genetic assemblages that may point to incipient speciation. The difference between the two species may be explained by gene flow restriction within the region, by the number of hosts required to complete the life cycle (2 and 3 hosts, respectively), type of host species (domestic and wild animals; small and big mammals, birds) and its availability, and host species overlap. Also, a geographic break between possible host species during the Pleistocene, domestication of animals and climatic conditions in the region played a role on the genetic structure of H. rufipes. / AFRIKAANSE OPSOMMING: Die suider Afrikaanse bosluise, Amblyomma hebraeum en Hyalomma rufipes is die hoof vektore van Hartwater in diere en Crimean Kongo Haemorargiese koors in mense. Om die filogeografiese struktuur van hierdie ektoparasiete te ondersoek, is bevolking-genetiese studies uitgevoer in suidelike Afrika asook gedeeltelik in oos Afrika. Deur die vlakke van genetiese diversiteit en bevolkingsverbindings regdeur die spesies se natuurlike verspreiding te weet, sal dit dalk moontlik wees om: beheermaatreëls (byvoorbeeld oorgrens) in te stel, die waarskynlikheid van weerstandbiedendheid in nuut losgelate gebiede te verstaan, en meer insigte in die vektoriale kapasiteit te verkry. Mitochondriale sitochroom oksidae (COI) en die interne transkriberende spasieerder 2 (ITS- 2) basisnukleotiedopeenvolgings van elf bevolkings is versamel. Haplotiepe netwerke en molekulêre diversiteitwaardes het in die algemeen, betekenisvolle genetiese verkille tussen meeste van die versamelde bevolkings aangetoon. Fu’s Fs en gemiste-paringverspreidings het voorgestel dat die bevolkings onlangs uitgebrei het nadat hulle 'n bottelnek ondergaan het. 'n Mantel regressie analise het ook betekenisvolle isolasie met afstand aangetoon vir H. rufipes. As die twee spesies vergelyk word het H. rufipes baie sterker seine van genetiese geografiese bevolkingstruktuur gewys as A. hebraeum. 'n Enkele taksonomiese eenheid word voorgestel vir A. hebraeum terwyl H. rufipes gekarakteriseer word deur ten minste drie taksonomiese eenhede. Die verskil tussen die twee spesies kan moontlik verklaar word deur geen-vloeibeperkings in die gebied, die aantal gasheer spesies wat benodig word om die lewenssiklus te voltooi (2 of 3 gashere onderskeidelik), tipe gasheer (domestikeerd- of wildlewend; klein of groot soogdiere, voëls) en hulle beskikbaarheid, en die gasheerspesies se oorvleueling. Dit is ook moontlik dat 'n geografiese breek tussen moontlike gasheerspesies gedurende die Pleistoseen, domestikering van diere en klimaatstoestande in die streek 'n rol kon speel by die genetiese struktuur van H. rufipes. / The NRF and the University of Stellenbosch for funding and bursary

O protozoário Rangelia vitalii, um piroplasma patogênico para cães, foi descrito no inicio do século XX, porém, apenas recentemente, a espécie foi validada por técnicas de biologia molecular. Observações epidemiológicas têm levado a suspeitar que os carrapatos Rhipicephalus sanguineus e Amblyomma aureolatum sejam potenciais vetores de R. vitalii, muito embora, nenhum estudo tenha, até o momento, comprovado o papel de algum carrapato como vetor de R. vitalii. Desta forma, o presente projeto objetiva: 1- Avaliar a transmissão transovariana de R. vitalii em carrapatos das espécies A. aureolatum, R. sanguineus, Amblyomma ovale, Amblyomma tigrinum e Amblyomma cajennense. 2- Avaliar a perpetuação transestadial de R. vitalii em todos os estágios biológicos das espécies citadas acima; 3- Avaliar a capacidade de larvas, ninfas e adultos de A. aureolatum e R. sanguineus em transmitirem o protozoário R. vitalii para cães, durante o parasitismo; 4- Avaliara a presença do agente em canídeos silvestres das espécies Cerdocyon thous e Lycalopex gymnocercus; 5- Avaliar a capacidade de uma fêmea gestante de Canis lupus familiaris em transmitirem de forma vertical o agente; 6- Conhecer as áreas de distribuição da R. vitalii a partir da pesquisa de casos suspeitos; 7- Avaliar as alterações clínicas e hematológicas dos cães infectados, via carrapato ou inoculação. Para tal, cães foram experimentalmente infectados com cepas patogênicas de R. vitalii oriundas do Rio Grande do Sul. Larvas, ninfas e adultos de R. sanguineus, A. aureolatum, A. ovale, A. tigrinum e A. cajennense foram levados a infestar esses cães infectados, e posteriormente, após ecdise ou postura de ovos em incubadora, os estágios biológicos subsequentes foram testados por PCR para presença de DNA de R. viatlii e levados a infestar cães não infectados, a fim de se verificar a transmissão de R. vitalii. Além disso, foi pesquisado a presença de R. vitalii em cães domésticos e canídeos silvestres das regiões Sul e Sudeste. Os resultados obtidos permitiram adquirir uma melhor compreensão da epidemiologia da rangeliose canina, pois além de comprovar a competência vetorial de A. aureolatum para R. vitalii (e a não competência para as demais espécies de carrapatos testadas), ampliou a distribuição geográfica deste agente nas regiões Sul e Sudeste. O estudo também possibilitou relatar uma infecção natural e persistente de um C. thous por R. vitalii. Diferentemente dos cães domésticos, que manifestam severas alterações clínicas e hematológicas decorrentes da infecção por R. vitalii, foi verificado um caso de infecção 8 assintomática em um C. thous, sugerindo um possível papel de reservatório do agente na natureza, uma vez que este canídeo silvestre é o principal hospedeiro silvestre para o carrapato A. aureolatum. A distribuição geográfica dos casos de rangeliose tanto em cães domésticos com em C. thous coincidem com a distribuição geográfica deste vetor. Portanto, esta espécie de carrapato, que já se mostrou capaz de veicular o agente em condições de laboratório, é provavelmente seu vetor em condições naturais. / O protozoan Rangelia vitalii, a pathogenic piroplasmid of dogs, was described in the beginning of the 20th century; however, only recently this piroplasm species was validated through molecular analysis. Epidemiological observations have implicated the ticks Rhipicephalus sanguineus and Amblyomma aureolatum as potential vectors of R. vitalii, although no tick species has been proved to be a competent vector. Therefore, the present study aimed: 1- to evaluate transovarial transmission of R. vitalii in the tick species A. aureolatum, R. sanguineus, Amblyomma ovale, Amblyomma tigrinum and Amblyomma cajennense. 2- to evaluate transstadial perpetuation of R. vitalii among the post embryonic stages of the tick species mentioned above; 3- to evaluate the competence of larvae, nymphs, and adults of A. aureolatum and R. sanguineus to transmit the protozoan R. vitalii to dogs during parasitism; 4- to evaluate natural infection by R. vitalii in the wild canids Cerdocyon thous and Lycalopex gymnocercus; 5- to evaluate vertical transmission of R. vitalii in a pregnant female of Canis lupus familiaris; 6- to expand the distribution area of R. vitalii by searching the agent in canine clinical cases; and, 7- to evaluate clinical and hematological alterations in dogs that were infected via tick parasitism or direct inoculation. In this regard, domestic dogs were experimentally infected with pathogenic strains of R. vitalii from the state of Rio Grande do Sul, southern Brazil. Larvae, nymphs, and adults of R. sanguineus, A. aureolatum, A. ovale, A. tigrinum and A. cajennense were allowed to feed on these infected dogs, and subsequently allowed to molt or egg laying within an incubator. Part of the molted ticks or hatched larvae was tested by PCR for the presence of R. vitalii DNA, and the other part was allowed to feed on susceptible dogs, in order to verify R. vitalii transmission through tick feeding. In addition, natural infection by R. vitalii was tested in domestic dogs and wild canids from Southern and Southeastern regions of Brazil. The results contributed to a better understand of the epidemiology of canine rangeliosis, as the vector competence of R. vitalii by A. aureolatum was demonstrated (at the same time, vector incompetence was shown for the other species tested), and the geographic distribution of R. vitalii was expanded in the Southern and Southeastern regions of Brazil. In addition, natural infection by R. vitalii was verified for the first time in wild canids (C. thous) from different areas. In contrast to 10 domestic dogs, which usually developed severe clinical rangeliosis with marked hematological alterations, an assymptomatic case was observed in a naturally infected C. thous, suggesting a possible reservoir role, since this wild canid is a natural host of A. aureolatum. The geographic distribution of the natural cases of rangeliosis, either on domestic dogs or C. thous coincides with the geographic distribution of A. aureolatum, which should be considered as the main vector of R. vitalii under natural conditions.

Made available in DSpace on 2016-04-28T20:15:36Z (GMT). No. of bitstreams: 1 2009 - Tatiane Kawamura de Almeida.pdf: 416296 bytes, checksum: 10f392469ed0d8f6721023bb52beb9dd (MD5) Previous issue date: 2009-03-05 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Amblyomma parvum is a tick of wide geographical distribution which parasite wild and domestic animals. In Brazil, the literature records only data on their geographical distribution and hosts. There is no record on the action of different temperatures in Brazilian populations of this species. The objective of this work was to evaluate the effects of five different temperatures on the free living stages on the life cycle of A. parvum. The experiment was conducted under 18, 21, 24, 27 and 32?1?C and relative humidity of 80?10%. For the eggs stage, 15 animals were used for infestation in the back after previous trichotomy. The females that dropped were randomly detach at different temperatures. The eggs were packed in glass jars after the fifth day of posture and observed daily for analysis of the average pre-hatching, average hatching and mean percentage of hatching. For others stages, three rabbits were used for each temperature, totaling 15 animals for each stage. The engorged ticks that dropped were collected daily. In the laboratory, they were cleaned, weighed and observed daily. The average of pre-ecdysis of the larvae and nymphs varied inversely proportional to the temperature. The average percentage of ecdysis for the larvae was statistically similar for the five treatments. The average moulting period had the same change that the average of preecdysis period for larvae and nymphs, where longer periods corresponds to lower temperatures. The average period of larval survival was similar statistically for the treatments 18, 21 and 24?1?C and was lower in the temperatures of 27 and 32?1?C. The average ecdysis period of the nymphs were statistically different only in the treatments of 18 and 32?1?C and was much longer in the temperature of 18?1?C (19.1 days). Similar to larvae, the average percentage of ecdysis for the nymphs was statistically similar to the five treatments. The lowest average period of survival of nymphs, with average of 117.5 days, was observed at 32 ?C. The average of pre-oviposition and oviposition periods were similar. Both were inversely proportional to the increase of temperature, however, only had significant difference in the three lower temperatures. Regarding the average weight of oviposition, the heavier weight was found at 27?1?C (average of 107?48.3 mg). The average production of eggs index and the average nutritional efficiency index had the highest average at 27?1?C. The average of pre-hatching period differed significantly in five treatments, and was inversely proportional to the increase of temperature. The average hatching period and the average percentage of hatching were longer and lower in the temperature of 18?1?C. The temperature of 18?C can be used for delaying the life cycle, however, it may be deleterious to the ticks. / Amblyomma parvum ? uma esp?cie de carrapato de ampla distribui??o geogr?fica, capaz de parasitar animais silvestres e dom?sticos. No Brasil, a literatura registra apenas dados sobre sua distribui??o geogr?fica e hospedeiros. N?o existe nenhum registro sobre a a??o de diferentes temperaturas em popula??es brasileiras desta esp?cie. O objetivo deste trabalho foi avaliar os efeitos de cinco diferentes temperaturas sobre os diversos processos das fases de vida livre do ciclo biol?gico de A. parvum. Todas as fases de vida livre foram avaliadas nas temperaturas de 18, 21, 24, 27, 32?1?C e umidade relativa de 80?10%. Para a etapa dos ovos foram utilizados 15 animais para infesta??o no dorso ap?s pr?via tricotomia. As f?meas que se desprenderam foram distribu?das aleatoriamente nas diferentes temperaturas. Os ovos foram acondicionados em frascos de vidro ap?s o quinto dia de postura e observados diariamente para an?lise do per?odo m?dio de pr?-eclos?o, per?odo m?dio de eclos?o e percentual m?dio de eclos?o. Para os demais est?gios, foi realizada uma infesta??o com tr?s coelhos para cada temperatura, totalizando 15 animais para cada est?gio. Os carrapatos ingurgitados que se desprendiam eram coletados diariamente. No laborat?rio, estes foram limpos, pesados e acondicionados nas cinco temperaturas, para a observa??o cont?nua dos exemplares. Os per?odos m?dios de pr?-ecdise das larvas e das ninfas variaram de forma inversamente proporcional ? temperatura. Em rela??o ao percentual m?dio de ecdise das larvas, os tratamentos foram estatisticamente semelhantes. O per?odo m?dio de muda sofreu a mesma varia??o que o per?odo m?dio de pr?-ecdise de larvas e ninfas, onde per?odos mais prolongados correspondem a temperaturas mais baixas. O percentual m?dio de sobreviv?ncia das larvas foi estatisticamente semelhante nos tratamentos a 18, 21 e 24?1?C e diminuiu consideravelmente nas temperaturas de 27 e 32?1?C. Os per?odos m?dios de ecdise das ninfas foram diferentes estatisticamente somente nos tratamentos a 18 e 32?1?C, sendo muito mais longo na temperatura de 18?1?C (19,1 dias). ? semelhan?a do ocorrido com as larvas, o percentual m?dio de ecdise das ninfas foi estatisticamente semelhante para os cinco tratamentos. O menor per?odo m?dio de sobreviv?ncia das ninfas, com m?dia de 117,5 dias, foi observado a 32?C. Os per?odos m?dios de pr?-postura e de postura foram semelhantes. Ambos foram inversamente proporcionais ao aumento de temperatura, no entanto, somente houve diferen?a significativa nas tr?s temperaturas mais baixas. Com rela??o ao peso m?dio da postura, o maior peso foi encontrado a 27?1?C (m?dia de 107?48.3 mg). O ?ndice m?dio de produ??o de ovos e o ?ndice m?dio de efici?ncia nutricional apresentaram as maiores m?dias a 27?1?C. O per?odo m?dio de pr?-eclos?o diferiu significativamente nos cinco tratamentos, sendo inversamente proporcional ao aumento da temperatura. O per?odo m?dio de eclos?o e o percentual m?dio de eclos?o foram mais longos e menores na temperatura de 18?1?C. A temperatura de 18?C pode ser utilizada quando se pretende retardar o desenvolvimento de uma gera??o, por?m, pode apresentar um efeito delet?rio.

Made available in DSpace on 2016-04-28T20:16:23Z (GMT). No. of bitstreams: 1 2007- Valeria Castilho Onofrio-01.pdf: 1096515 bytes, checksum: 2a3f7ffa9dd8714994722615b87f96c0 (MD5) Previous issue date: 2007-03-07 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Fifty nine tick species of the genus Amblyomma are known to occur in the Neotropical region. Of these, 47 species are exclusively Neotropical, 11 occur in the Nearctic region as well and only one is not endemic to the Americas. To date, 33 species were described from Brazil, of which only two are endemic. The genus presents a wide range of hosts, including most orders of mammals. Birds are frequently infested by the immature forms of some species. Amphibians and reptiles are also among the genus' hosts. In order to revise the genus Amblyomma in Brazil, the present study was divided into two chapters. The first includes the diagnosis of each species as well as an identification key, based on electron micrographs. The second chapter presents new information on the genus Amblyomma, including data on geographic distribution and hosts for each species. A few type specimens, deposited in Brazilian and foreign institutions were examined as well as material deposited in the main Brazilian collections. The data obtained was compared to that available in literature. Of those species for which DNA sequencing was carried out, A. auricularium and A. pseudoconcolor showed 98.3% identity between them. However, the latter species showed 88.8% similarity with A. pseudoconcolor deposited in GenBank. Thus, it is necessary to repeat the analyses using a larger number of ticks from different localities and until further studies are carried out A. auricularium and A. pseudoconcolor should be treated as separate species. The taxonomic status of A. nodosum and A. calcaratum as two distinct taxa was confirmed, since the identity between sequences was 89%. Similarly, the sequences of A. parkeri and A. longirostre were different, with 87% similarity confirming the validity of both species. Based on morphological characters and on molecular biology A. parkeri was separated from A. longirostre and A. geayi and its validity confirmed. The distribution range of A. auricularium was expanded, including the states of Tocantins, Piau? and Mato Grosso. The presence of A. cajennense was confirmed for Amazonas and Amap? was cited as a new record for this species. Amblyomma coelebs and A. dissimile were recorded for the first time from Roraima, A. dubitatum from Amazonas and A. naponense from Acre, Bahia, Tocantins and Esp?rito Santo. The states of Roraima and Amazonas were included in the distribution range of A. oblongoguttatum, and Paran? and Roraima in the distribution range of A. scalptutarum. The occurrence of A. ovale was confirmed from the state of Acre and A. triste from Mato Grosso do Sul. After many years, A. parkeri was again recorded from Brazil and it's distribution range expanded to the states of Rio de Janeiro and Paran?. The states of Esp?rito Santo, Sergipe and Tocantins were considered new locality records for A. rotundatum. The record of A. dissimile from Rio Grande do Sul was not considered since the host was already infested when it reached the state. The record of A. humerale from S?o Paulo was also not considered since the specimen was actually A. rotundatum. The occurrence of A. geayi in the state of Paran? was considered doubtful. / Na regi?o Neotropical, ocorrem 59 esp?cies de carrapatos do g?nero Amblyomma, das quais 47 s?o exclusivas, 11 ocorrem tamb?m na regi?o Ne?rtica e apenas uma n?o ? aut?ctone das Am?ricas. Atualmente no Brasil, existem 33 esp?cies descritas, das quais duas s?o end?micas. Neste g?nero, a variedade de hospedeiros ? grande, compreendendo a maioria das ordens de mam?feros. Aves s?o freq?entemente parasitadas pelas formas imaturas de algumas esp?cies. Anf?bios e r?pteis tamb?m est?o entre os hospedeiros deste g?nero. Com a finalidade de revisar o g?nero Amblyomma no Brasil, o presente estudo foi dividido em dois cap?tulos. O cap?tulo 1 ? composto pela diagnose de cada esp?cie e por uma chave de identifica??o, baseada em microscopia eletr?nica de varredura. O segundo cap?tulo teve por objetivos apresentar novas informa??es sobre o g?nero Amblyomma, acrescentando dados de distribui??o geogr?fica e de hospedeiros para cada esp?cie. Foram examinados alguns tipos depositados nas cole??es brasileiras e estrangeiras, assim como o material das principais cole??es brasileiras. Os dados obtidos foram comparados ?queles citados na literatura dispon?vel. Das esp?cies em que foi realizado o sequenciamento g?nico, A. auricularium e A. pseudoconcolor mostraram 98,3% de identidade entre si, por?m esta ?ltima esp?cie mostrou 88,8% de similaridade com A. pseudoconcolor depositada no GenBank. Dessa forma ? necess?rio repetir as an?lises, utilizando um n?mero maior de exemplares de carrapatos de diferentes localidades. Assim, at? que mais estudos sejam realizados A. auricularium e A. pseudoconcolor devem ser mantidas como esp?cies distintas. As esp?cies A. nodosum e A. calcaratum tiveram sua posi??o taxon?mica confirmada como dois t?xons distintos, uma vez que a identidade entre as seq??ncias g?nicas foi de 89%. Igualmente as seq??ncias de A. parkeri e A. longirostre foram diferentes apresentado 87% de similaridade confirmando a validade das duas esp?cies. Com base em caracter?sticas morfol?gicas e na biologia molecular, A. parkeri foi separada de A. longirostre e A. geayi e sua validade foi confirmada. A distribui??o geogr?fica de A. auricularium foi ampliada, incluindo os estados do Tocantins, Piau? e Mato Grosso. A presen?a de A. cajennense no Amazonas foi confirmada e o Amap? foi citado como um novo relato de localidade para esta esp?cie. Amblyomma coelebs e A. dissimile foram relatadas pela primeira vez para o estado de Roraima, A. dubitatum para o Amazonas e A. naponense para o Acre, Bahia, Tocantins e Esp?rito Santo. Para A. oblongoguttatum foram acrescentados os estados de Roraima e Amazonas, e para A. scalpturatum os estados do Paran? e Roraima. Foi confirmada a ocorr?ncia de A. ovale no estado do Acre e de A. triste no Mato Grosso do Sul. Ap?s muitos anos, A. parkeri foi novamente reportada para o Brasil e sua ?rea de ocorr?ncia ampliada com os registros para o Rio de Janeiro e Paran?. Os estados do Esp?rito Santo, Sergipe e Tocantins foram considerados novos relatos de localidade para A. rotundatum. O registro de A. dissimile para o Rio Grande do Sul foi desconsiderado, uma vez que o hospedeiro j? se encontrava parasitado quando chegou ao estado e o de A. humerale para S?o Paulo, tamb?m, pois se tratava de A.rotundatum. A ocorr?ncia de A. geayi foi considerada duvidosa para o estado do Paran?.

Human Monocytic Ehrlichiosis (HME) is an emerging disease first described in 1987 and is transmitted by the bite of Amblyomma americanum. Over the past 10 years, the CDC has documented increasing ehrlichiosis case reports nationwide. Our study site is a golf-oriented retirement community located in the Cumberland Plateau of Tennessee. In 1993, four men at the study site had symptoms consistent with HME which prompted a CDC outbreak investigation and led community managers to mitigate ticks feeding on deer. The objectives of this study were to measure the efficacy of current tick mitigation attempts, to determine the level of infection and composition of tick-borne disease in the study area, and to assess which wildlife species are potentially acting as reservoirs for disease. Ticks were sampled in the community at eight sites of ‘4-poster’ acaricide applicator utilization and at seven untreated sites. Close to the ‘4-poster’ devices, larval, nymphal, and adult tick abundances were reduced by 90%, 68% and 49% respectively (larval p<0.001, nymphal p<0.001, adult p=0.005) relative to the untreated areas. We extracted DNA from A. americanum ticks collected at the treatment and non-treatment sites and tested for Ehrlichia spp. infections. Of 253 adult and nymphal A. americanum tested, we found 1.2% to be positive for Ehrlichia chaffeensis, 4.7% positive for Ehrlichia ewingii, and 1.6% positive for Panola Mountain Ehrlichia; in combination this prevalence is similar to that reported in other Ehrlichia-endemic areas of the eastern U.S.. We also performed blood meal analysis on DNA from A. americanum ticks and the results suggest that the most significant reservoir hosts for Ehrlichia spp. are white-tailed deer, turkeys, grey squirrels, and Passeriformes. We conclude that while the ‘4-poster’ acaricide applicators reduce the number of ticks close to treatment, at the density at which they are currently being used (8 applicators per 52.6 km2, average distance between applicators = 6.6km) they will have no large-scale effect on the community’s tick population. In order to accomplish area-wide reduction of A.americanum and Ehrlichia spp. in this locale, community managers should develop an integrated management strategy that utilizes other techniques in addition to ‘4-poster’ devices.

Amblyomma americanum is a hard tick species found in southern Indiana. Once a notorious pest to humans and livestock, A. americanum has now taken on a role as vector to pathogenic organisms. This study aimed to detect and quantify three microbes in A. americanum: Rickettsia amblyommii, Ehrlichia chaffeensis, and Borrelia lonestari. A primary objective of this study was to determine microbial interaction within a single A. americanum tick through quantification of each microbe within a co-infected tick. A second objective was to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. Infection rates were 44%, 1%, and 0% for R. amblyommii, E. chaffeensis, and B. lonestari respectively. This study found no co-infected ticks, therefore no microbial interaction was determined. This study also found multiple drawbacks with utilizing quantitative real-time PCR to determine the density of R. amblyommii within the salivary glands of A. americanum ticks. / Department of Biology

Human monocytotropic ehrlichiosis (HME) is a tick-borne infectious disease caused by the bacterium Ehrlichia chaffeensis and transmitted by the lone star tick, Amblyomma americanum. The disease was recognized in Indiana for the first time in 1994. Since 1999, 11 cases have been confirmed in Indiana and two additional cases are under investigation. In the past five years, the cases have been reported from Crawford, Harrison, Warrick, Martin, Perry, Spencer, and Madison counties.A total of 2765 adult Amblyomma americanum ticks were collected from eight counties in southern Indiana during two field trips in May 2000. Ticks were pooled and examined for the presence of Ehrlichia chaffeensis using nested PCR with primers HE1 and HE3, specific for the 16S rRNA gene of the pathogen. Ninety-six pools of A. americanum specimens tested positive for E. chaffeensis DNA. This represented a minimum infection rate (MIR) of 3.5%.To identify different genetic forms (strains) of E. chaffeensis, the positive tick pools were probed for the Variable Length PCR Target (VLPT) gene of E. chaffeensis. The data were used to develop a geographic map of the distribution of the different strains of the pathogen. Overall, nine different genetic variants (91HE17, Arkansas, Jax, Liberty, Osceola, Sapulpa, St. Vincent, Wakulla, West Paces) of E. chaffeensis were identified from pools of ticks collected in four counties (Harrison, Perry, Pike, Warrick). All samples positive for the 16S rRNA were also positive for the VLPT gene.E. chaffeensis isolates are polymorphic in the number of repetitive sequences within the genes encoding the VLPT, and the isolates obtained illustrate this phenomenon. The high concordance rate between the 16S rRNA and the VLPT gene reveals that the VLPT gene is a very sensitive tool for detecting E. chaffeensis in the lone star ticks. We found no clear correlation between geographic distribution of different genetic variants of E. chaffeensis and the genetic polymorphism of the VLPT gene. Further study with a relatively larger sample size from a wider geographical area might be able to detect such a pattern. / Department of Physiology and Health Science

Orientador: Maria Izabel Camargo Mathias / Banca: Celeste Paola D'Alessandro / Banca: Bruno Rodrigues Sampieri / Banca: Caio Márcio de Oliveira Monteiro / Banca: Fabio Rau Akashi Hernandes / Resumo: Os carrapatos de corpo duro (Acari: Ixodidae) contam com 724 espécies descritas, sendo 120 espécies registradas oficialmente na região neotropical. No entanto, na Colômbia o registro e distribuição das espécies da família Ixodidae é escasso e pouco se sabe sobre a interação destes ectoparasitas com as diferentes espécies de Rickettsia na região, deixando uma lacuna em estudos e em conhecimento ixodológico e zoonótico no país. Desta forma, o presente trabalho buscou: a) realizar o levantamento e identificação de espécies de carrapatos duros associados a hospedeiros domésticos em regiões da Colômbia, utilizando caracteres morfológicos externos e marcadores moleculares; b) aferir a existência de caracteres morfohistológicos do sistema reprodutor masculino de carrapatos do gênero Amblyomma e c) verificar a ocorrência de bactérias Rickettsia spp. em carrapatos duros em regiões da Colômbia. Durante o período de agosto de 2014 e maio de 2016, foram coletados 1.745 carrapatos diretamente de hospedeiros domésticos e em ativa alimentação, em 17 municípios de 10 departamentos da Colômbia. Os indivíduos coletados foram identificados com base em sua morfologia externa e preparados para aplicação de técnicas de microscopia, bem como técnicas de análise molecular dos genes ITS2, citocromo oxidase I (COI) e 16S rDNA. Os resultados encontrados permitiram o registro de três gêneros e oito espécies de carrapatos duros nas áreas de estudo, sendo registrada pela primeira vez a espécie Amblyomma m... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Hard-bodied ticks (Acari: Ixodidae) comprise 724 known species, with 120 species officially registered for the Neotropical region. However, in Colombia, species registry and distribution for the family Ixodidae is scarce, and little is known of the interaction of these ectoparasites with the different Rickettsia species in the region, leading to gaps regarding the ixodological and zoonotic study and knowledge in the country. In this context, this study aimed to: a) establish and identification hard-bodied ticks associated with domestic hosts in various regions of Colombia, using external morphological characters and molecular markers; b) assess the existence of morphohistological characters of the male reproductive system of Amblyomma ticks; and c) verify the occurrence of Rickettsia spp. in hard-bodied ticks in various regions of Colombia. During August of 2014 and May of 2016, 1745 ticks were collected actively feeding on domestic hosts in 17 municipalities of 10 departments of Colombia. The specimens were identified based on their external morphology and prepared for microscopy, as well as for molecular analyses of genes ITS2, cytochrome oxidase I (COI) and 16S rDNA. The results show the presence of three genera and eight hard-bodied tick species in the municipalities studied, including the first register of the species Amblyomma mixtum for Colombia. In addition, we report an unprecedented case of gynandromorphism in A. mixtum, which also constitutes the first report for t... (Complete abstract click electronic access below) / Doutor

The aim of the study was to analyze the infectious potential of the newly described species, B. americana and B. carolinensis, studied on the laboratory model mammals mice. Our goal was to analyze and compare the vectorial capacity of two different tick vectors, Amblyomma americanum and Ixodes ricinus, in acquiring and transmition of both spirochete species to the host. The results of this study confirmed that ticks A. americanum and I. ricinus are capable to maintain and transmit B. americana and B.carolinensis.We confirmed that both analysed spirochete species, B. carolinensis and B. americana, showed the potential to develop the disease in laboratory model mammal, which indirectly support the fact that both spirochete species might be concidered as the risk factors in the area where they are distributed. Our results shows that A. americanum is able to transmit both spirochete species, which increases that risk of acquiring the Lyme disease to human population in the area of distribution of A. americanum