Poucos são os estudos acerca da saúde de onças-pintadas em vida livre. Visando obter melhores parâmetros para avaliação clínica desta espécie ameaçada de extinção, foram realizados exame clínico, hemograma, perfil bioquímico e acompanhamento de 10 onças-pintadas da região de Corumbá, no Pantanal do Mato Grosso do Sul. Além disso, buscando obter informações sobre o possível papel da espécie como suscetível, hospedeira ou sentinela de patógenos de importância em saúde pública e animal, amostras destas 10 onças foram testadas através de métodos sorológicos para verificar contato com vírus rábico, Rickettsia spp. e Ehrlichia canis. As amostras das onças e os carrapatos que as parasitavam no momento das capturas foram testados por reação em cadeia pela polimerase para a família Anaplasmataceae e os gêneros Rickettsia, Borrelia, Coxiella, Hepatozoon e Babesia. Este é o primeiro estudo a relatar os valores de hemograma e perfil bioquímico de uma população de onças-pintadas de vida livre. Dois animais, assintomáticos, apresentaram baixo título sorológico para o vírus da raiva, sugerindo contato da espécie com este patógeno. Todas as onças capturadas foram consideradas soropositivas para Rickettsia spp., e Rickettsia parkeri foi sequenciada a partir de um Amblyomma triste que estava parasitando um dos animais. Foi descoberta uma possível nova espécie do gênero Ehrlichia através do sequenciamento de DNA obtido de um Amblyomma triste e um Amblyomma cajenense que estavam parasitando onças. Quatro onças-pintadas foram consideradas soropositivas para Ehrlichia canis, possivelmente uma reação cruzada com esta outra espécie. Todas as onças-pintadas avaliadas neste estudo apresentaram DNA de Cytauxzoon sp., com 98% de similaridade a C. felis, em amostras sanguíneas. Todas as onças avaliadas neste estudo apresentaram DNA de Hepatozoon sp., com 98% de similaridade a H. felis, em amostras sanguíneas. As onças apresentavam boas condições de saúde geral. / Few studies have been conducted to investigate the health of free-ranging jaguars. In order to obtain better parameters for clinical evaluation of this endangered species, clinical exams, hemogram, biochemical tests and ecological monitoring were done for 10 jaguars in the Pantanal region - Corumbá City, Mato Grosso do Sul State. This project also evaluated the possible role of this species as susceptible, host or sentinel for pathogens of public and/or animal health importance, testing samples from these animals by serological methods to rabies virus, Rickettsia spp. and Ehrlichia canis. All samples and all ticks collected from the jaguars were also tested by polymerase chain reaction to the Anaplasmataceae family and the genera Rickettsia, Borrelia, Coxiella, Hepatozoon and Babesia. This is the first report of hemograms and biochemical profile of a free-ranging jaguar population. Two asymptomatic animals presented low seropositivity for rabies virus, suggesting contact with this pathogen. All jaguars were considered seropositive for Rickettsia spp., and Rickettsia parkeri was sequenced from an Amblyomma triste that was parasitizing one of the animals. A possible new species of the genus Ehrlichia has been identified by DNA sequencing obtained from an Amblyomma triste and an Amblyomma cajenense that were parasitizing jaguars. Four jaguars were considered seropositive for Ehrlichia canis, possibly a cross-reaction with this other species. All jaguars evaluated in this study presented DNA fragments of Cytauxzoon sp., 98% similarity to C. felis in blood samples. In addition, all jaguars presented DNA fragments of Hepatozoon sp., 98% similarity to H. felis in blood samples. In general, these jaguars presented good health.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-04-06T20:09:47Z No. of bitstreams: 1 tatianedeoliveirasouzasenra.pdf: 539017 bytes, checksum: 647e15774caf85c47318364c3b4c57e3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-04-24T04:11:36Z (GMT) No. of bitstreams: 1 tatianedeoliveirasouzasenra.pdf: 539017 bytes, checksum: 647e15774caf85c47318364c3b4c57e3 (MD5) / Made available in DSpace on 2016-04-24T04:11:36Z (GMT). No. of bitstreams: 1 tatianedeoliveirasouzasenra.pdf: 539017 bytes, checksum: 647e15774caf85c47318364c3b4c57e3 (MD5) Previous issue date: 2013-02-07 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente estudo teve como objetivo avaliar a atividade carrapaticida de formulações hidroetanólicas do carvacrol, (E)-cinamaldeído, trans-anetol e linalol sobre larvas de Rhipicephalus microplus e Dermacentor nitens. Também foram realizados testes avaliando a atividade acaricida do carvacrol, timol, eugenol e (E)-cinamaldeído em diferentes concentrações sobre larvas e ninfas de Rhipicephalus sanguineus e Amblyomma cajennense. Todas as substâncias foram testadas nas concentrações de 2,5; 5,0; 10,0; 15,0 e 20,0 μl/ml, enquanto o timol foi testado nas mesmas concentrações em mg/ml; foram feitas 10 repetições para cada tratamento. Também foram feitos grupos controle com etanol na concentração necessária para solubilizar cada substância. Os grupos experimentais foram mantidos em câmara climatizada (27±1°C e UR>80±10%) por 24 horas e após este período, a mortalidade foi avaliada. Nos testes envolvendo larvas de R. microplus e D. nitens, o carvacrol e (E)-cinamaldeído causaram mortalidade superior a 98% em todas as concentrações testadas, o trans-anetol a partir da concentração de 5,0 μl/ml também causou a morte de 100% das larvas; entretanto, o linalol mesmo na maior concentração não foi eficiente, matando apenas 14,5 e 8,4% de R. microplus e D. nitens, respectivamente. Nos testes com larvas de A. cajennense a concentração de 5,0 μl/ml do carvacrol, timol e (E)-cinamaldeído causou a morte de 100% dos ixodídeos; para o eugenol este percentual só foi observado a partir da concentração de 15,0 μl/ml. Nos testes realizados com larvas de R. sanguineus, os grupos tratados com a menor concentração (2,5 μl/ml) de carvacrol e (E)-cinamaldeído apresentaram mortalidade de 100%; entretanto, para o eugenol, esse percentual só foi observado a partir da concentração de 10,0 μl/ml. Para ninfas desse mesmo ixodídeo, os monoterpenos carvacrol e timol a partir da menor concentração (2,5 μl/ml ou mg/ml) causaram letalidade de 100% em todas as concentrações, diferindo dos resultados obtidos para o eugenol e (E)-cinamaldeído, uma vez que mortalidade de 100% com a utilização dessas substâncias só foram observadas a partir da concentração de 10,0 μl/ml. Nos testes com A. cajennense a mortalidade das larvas tratadas com carvacrol, timol, eugenol e (E)-cinamaldeído na concentração de 2,5 μl/ml foi de 45, 62,7, 10,2 e 81,6%, chegando a 100% na concentração de 5,0 μl/ml para o carvacrol, timol e (E)-cinamaldeído. Para o eugenol esta taxa de mortalidade foi observada a partir da concentração de 15,0 μl/ml. Para ninfas os testes com carvacrol e timol causaram a morte de 100% dos indivíduos a partir das concentrações de 5,0 μl/ml e 10,0 mg/ml, respectivamente; o eugenol só atingiu mortalidade de 100% na concentração de 20,0 μl/ml, enquanto a xii mortalidade máxima no (E)-cinamaldeído não ultrapassou 64%. O conjunto de resultados obtidos neste estudo indica que os monoterpenos carvacrol e timol e os fenilpropanóides eugenol e (E)-cinamaldeído possuem atividade deletéria sobre estes ectoparasitos. Assim, existe a necessidade de novas pesquisas envolvendo outros estágios de desenvolvimento desses carrapatos, a investigação do sinergismo entre as substâncias, além de testes em condições naturais. Quanto a observação preliminar sobre a presença de ectoparasitos em aves silvestres, foram examinadas 72 aves de 17 espécies com prevalência de 29,2% de ectoparasitos. / The present study aimed to evaluate the efficiency of hydroethanolic formulations of carvacrol, (E)-cinnamaldehyde, trans-anetol and linalool against larval stages of Rhipicephalus microplus and Dermacentor nitens. It was also realized assays to evaluate the acaricide effect of carvacrol, thymol, eugenol and (E)-cinnamaldehyde in different concentrations in larvae and nymph of Rhipicephalus sanguineus and Amblyomma cajennense. All substances were tested in concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 μl/ml, thymol, tested in mg/ml; 10 repetitions were made for each treatment. It was also made with ethanol in a concentration necessary to solubilize each substance. The experimental groups were maintained in BOD incubator (27±1°C e UR>80±10%) during 24 hours and after this period the mortality was available. In the assays with R. microplus and D. nitens larvae, carvacrol and (E)-cinnamaldehyde caused mortality higher than 98% in all the tested concentrations, the trans-anetol caused a mortality of 100% in larvae from a concentration of 5.0 μl/ml; however, linalool was not efficien even at the higher concentration causing a mortality of only 14.5 and 8.4% in R. microplus and D. nitens, respectively. In assays realized with A. cajennense larvae, the concentration of 5.0 μl/ml of carvacrol, thymol and (E)-cinnamaldehyde cased a mortality of 100%; for eugenol this percentage was obtained from the concentration of 15.0 μl/ml. The test realized with R. sanguineus larvae, groups treated with the lower concentration (2.5 μl/ml) of carvacrol and (E)-cinnamaldehyde presented a mortality of 100%; nevertheless, for eugenol this percentage was observed only from the concentration of 10.0 μl/ml. For nymphs of the same species, the monoterpenes carvacrol and thymol from the lower concentration (2.5 μl/ml or mg/ml) caused a lethality of 100% in all concentrations, different from results obtained for eugenol and (E)-cinnamaldehyde, once mortality of 100% with these substances were observed only from the concentration of 10.0 μl/ml. In assays with A. cajennense, the mortality of larvae treated with carvacrol, thymol, eugenol and (E)-cinnamaldehyde in concentrations of 2.5 μl/ml of 45, 62.7, 10.2 and 81.6%, reaching 100% in the concentration of 5.0 μl/ml for carvacrol, thymol and (E)-cinnamaldehyde. For eugenol mortality rate was observed from the concentration of 15.0 μl/ml. For nymphs, tests with carvacrol and thymol caused a mortality of 100% of individuals from concentrations of 5.0 μl/ml and 10.0 mg/ml, respectively; the eugenol only reached this mortality rate at 20.0 μl/ml concentration, while the maximum mortality with (E)-cinnamaldehyde did not exceed 64%. The results indicate that monoterpenes and xiv phenylpropanoids, eugenol and (E)-cinnamaldehyde have deleterious activity in this ectoparasites. Thus, new researches are needed with other developmental stages of these tick species, the investigation of synergism between the substances, aside from tests in natural conditions. As regarding the preliminary observations of the presence of ectoparasites in wild birds, were examined 72 birds from 17 species different with prevalence of 29.2% ectoparasites.

Les maladies fébriles y compris les maladies bactériennes sont mal connues en Côte d’Ivoire et en Guinée. Tout d’abord, nous avons recherché par biologie moléculaire des bactéries pathogènes transmises par les tiques en Côte d’Ivoire. Nous avons analysé différentes espèces de tiques prélevées chez des bovins et mis en évidence des bactéries pathogènes responsables de nombreuses maladies infectieuses comme Rickettsia, Borrelia, Anaplasma, Ehrlichia, Coxiella burnetii (fièvre Q) et aussi vingt nouvelles espèces potentielles.Ensuite, notre objectif était de détecter par biologie moléculaire des micro-organismes pathogènes chez l’homme. Concernant l’étude des plaies et des peaux saines en Guinée, la plupart des patients étaient infectés par Pseudomonas aeruginosa, Staphylococcus aureus et plusieurs espèces d'Acinetobacter.Parmi les patients fébriles et les sujets apyrétiques recrutés en Guinée et en Côte d’Ivoire, Plasmodium falciparum reste le micro-organisme le plus fréquent surtout dans les échantillons de sang des patients fébriles bien que plusieurs bactéries aient été aussi identifiées. En Guinée, il s’agissait de Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Salmonella enterica Non Typhi et Non Paratyphi et R. felis. Ces bactéries ont été également identifiées ainsi que Salmonella enterica Typhi, Salmonella enterica Paratyphi, Tropheryma whipplei et une nouvelle espèce potentielle de Wolbachia en Côte d’Ivoire. Nos travaux ont permis d’établir le répertoire des bactéries transmises par les tiques en Côte d’Ivoire, celles impliquées dans les bactériémies en Côte d’Ivoire et en Guinée (Conakry). / Febrile illnesses including bacterial diseases are poorly known in Côte d'Ivoire and Guinea.In the first part of our work, we researched by molecular biology bacteria transmitted by ticks in Côte d’Ivoire. We analyzed different species of ticks collected from cattle and highlighted pathogenic bacteria responsible for many infectious diseases such as Rickettsia, Borrelia, Anaplasma, Ehrlichia, Coxiella burnetii (Q fever) and twenty potential new species. In the second part, our goal was to detect using molecular biology several microorganisms in humans in Guinea (Conakry) and Côte d'Ivoire. As regards the study of wounds and healthy skin in Guinea, most patients were infected with Pseudomonas aeruginosa, Staphylococcus aureus, several species of Acinetobacter.Among the febrile patients and healthy controls afebrile recruited in Guinea and Côte d'Ivoire, Plasmodium falciparum is the most common detected microorganism especially in blood samples from febrile patients although several bacteria were also identified. In Guinea, it was Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, non-typhoidal Salmonella spp., and R. felis. These bacteria were also identified as well as Salmonella enterica Typhi, Salmonella enterica Paratyphi, Tropheryma whipplei and a potential new species of Wolbachia in Côte d’Ivoire.This work allowed establishing the repertory of bacteria transmitted by ticks in Côte d’Ivoire, as well as those involved in bacteremia in Côte d’Ivoire and Guinea (Conakry).

Please read the abstract in the section 00front of this document / Thesis (DPhil (Zoology))--University of Pretoria, 2007. / Zoology and Entomology / unrestricted

Ticks are the most common external parasites of economic importance in the livestock farming sector of Southern Africa. They are vectors of tick-borne diseases and are commonly controlled using conventional acaricides, which are expensive and not readily available to resource-limited farmers. As a result many resource-limited farmers resort to use of alternative materials to control ticks on their livestock. Tick resistance to acaricide is an increasing problem in Southern Africa and poses a real economic threat to livestock and veterinary pharmaceutical industries. The objective of this study was to document and validate the ethno-veterinary remedies used as alternative methods of controlling cattle ticks by resource-limited farmers. A questionnaire survey was conducted in May 2007, at Qolora by-Sea and Nontshinga in Centane district, to determine tick control methods used by resource limited farmers in the Amathole District Municipality of the Eastern Cape Province, South Africa. Furthermore, in vitro and in vivo experiments were done to validate the acaricidal properties of ethnoveterinary remedies used by resource-limited farmers. Ticks were reported to be a major problem, transmitting diseases like anaplasmosis (89.8%), babesiosis (55.9%) and ehrlichiosis (16.9%), they also cause wounds that predispose cattle to screwworm infestation, tick worry and teat damage. All farmers used acaricides and dipping tanks provided by the government to control ticks, as the main tick control method. However, the majority (94.9%) were of the opinion that the dip wash was not effective in controlling ticks. As a result, farmers complemented the government dipping service with use of own initiatives like spraying with conventional acaricides (22%), household disinfectants such as Jeyes fluid (18.6%), used engine oil (10.2%), chickens feeding on ticks (5.1%), manual iv removal (5.1%), and pouricides (1.7%). In addition, some farmers also use plants (6.8%), mainly the leaf of Aloe ferox and the bark of Ptaeroxylon obliquum. The in vitro repellency models showed Jeyes fluid (76.8 and 100% concentrations) and P. obliquum (40%) had repellent properties that lasted 6 hours and 40 minutes respectively while that of, Tabard (35% diethyltoluamide) positive control, lasted for 4 hours when applied against nymphal stages of Rhipicephalus sanguineus ticks. In the contact bio-assay, undiluted used engine oil, T. minuta oil (50%), Ektoban® (Cymiazol 17.5% and cypermethrin 2.5%) and Jeyes fluid (76.8%) caused higher tick mortality of more than 86%. Other materials (A. ferox, Lantana camara and Tagetes minuta) had no repellence and acaricidal properties. In the in vivo study, Jeyes fluid at a concentration of 76.8% and used engine oil displayed efficacy of 71 and 76.4% respectively which was similar to that of the positive control Ektoban® at 97.8% tick load reduction. Among plant materials L. camara at 40% concentrations had an efficacy of 57% while A. ferox, P. obliquum and T. minuta were not effective at all. The remedies used by resource-limited farmers to control ticks vary in efficacy. Their ability to reduce tick load on cattle appears to be promising and a welcome development as their use could probably reduce tick burdens in cattle with less expenditure. However, further investigations need to be done before they are recommended for use. Despite being effective some of the materials have potential toxic effects in animals and also cause environmental contamination and I recommend used engine oil, Jeyes fluid and L. camara for further testing as they showed some efficacy compared to other remedies.

La spécialisation des parasites vis-à-vis de leur(s) hôte(s) et la formation de races d'hôtes sont des processus évolutifs clés dans le maintien et l'émergence de la diversité au sein des populations de parasites. Notre objectif était d'étudier ces processus chez Ixodes uriae, une tique d'oiseaux marins présentant une vaste distribution géographique et une large diversité d'hôtes, afin de mieux comprendre le rôle relatif des contraintes liées à l'hôte et aux facteurs géographiques dans l'évolution et la diversification de ce parasite. Cette question a été abordée à travers différentes approches réunissant des analyses de génétique des populations et de phylogéographie, des analyses morphologiques et une approche expérimentale sur le terrain. Les résultats obtenus montrent que le facteur spatial joue un rôle important dans la diversification d'I. uriae puisque quatre grands groupes géographiques génétiquement isolés ont été identifiés. L'évolution de races d'hôtes est également un processus récurrent de l'évolution d'I. uriae, même si la divergence entre races est plus ou moins marquée d'une région à l'autre. L'évolution des races semble impliquer une préférence d'hôte chez la tique ainsi que des contraintes adaptatives liées à l'hôte, de type mécanique ou physiologique (e.g., bec des oiseaux, digestion du sang, réponse immunitaire). L'ensemble des résultats est cohérent avec la notion d'évolution en mosaïque géographique qui prédit que les interactions entre espèces peuvent évoluer de manière différente dans un contexte spatial hétérogène ; ce qui souligne l'importance des caractéristiques écologiques des hôtes dans la diversification de la tique I. uriae. D'après nos résultats et ceux de différents collègues, la spécialisation d'hôte pourrait s'avérer être un processus commun chez les tiques et donc avoir de profondes implications épidémiologiques et évolutives pour les pathogènes qu'elles transmettent. / Host specialization and host-race formation may be key processes in maintaining and generating diversity within parasite populations. The objective of this thesis was to analyze these processes in the seabird tick Ixodes uriae, a common and widely distributed ectoparasite that exploits a great diversity of seabird host species, to better understand the relative role of host-related constraints and geographic factors in its evolution and diversification. I addressed this question using different approaches, including population genetic and phylogeographic analyses, morphometry and field experimentation. Results show that the spatial factor is a key component in shaping the diversity of I. uriae, as four geographical genetically isolated groups exist within the global distribution of this parasite. The formation of host races is also a general process in the evolution of I. uriae, but has evolved to different degrees in different geographical regions. Host preference in the tick, along with mechanical or physiological host-related constraints (e.g., beak morphology, blood digestion, immune response) are likely involved in the evolution of I. uriae host races. Overall, results fall in line with the geographic mosaic theory of evolution that predicts that the outcome of species interactions can vary across geographic landscapes, highlighting the role played by ecological characteristics of the host in the diversification of I. uriae. Given results to date, host specialization may be a common process in tick systems and thus may have strong epidemiological and evolutionary implications for the pathogens that they transmit.

Rickettsia rickettsii é o agente etiológico da febre maculosa das Montanhas Rochosas, a mais letal dentre as riquetsioses que acometem o homem. A principal espécie de carrapato-vetor de R. rickettsii na área metropolitana da cidade de São Paulo é Amblyomma aureolatum. Quando um carrapato em jejum no solo encontra um hospedeiro vertebrado e inicia a alimentação sanguínea, R. rickettsii é exposta a uma elevação da temperatura e aos componentes da refeição sanguínea. Ambos os estímulos foram previamente associados à reativação da virulência da bactéria em carrapatos, porém, os fatores responsáveis por essa conversão do fenótipo avirulento em virulento não foram completamente elucidados até o momento. Dessa forma, o presente trabalho teve como objetivo determinar os efeitos desses dois estímulos ambientais sobre o perfil de expressão gênica dessa bactéria durante a infecção de A. aureolatum. Inicialmente, estabelecemos um sistema de propagação de riquétsias para obter material genético suficiente para a padronização dos procedimentos de preparação de amostras para os experimentos de microarranjos. Para tal, estabelecemos, pela primeira vez, a infecção de uma cepa patogênica brasileira de R. rickettsii em células embrionárias do carrapato Rhipicephalus (Boophilus) microplus (BME26). Através da utilização de microarranjos de oligonucleotídeos customizados, analisamos os efeitos da elevação da temperatura em 10°C e da alimentação sanguínea sobre o perfil transcricional da bactéria infectando o conjunto de órgãos de fêmeas de A. aureolatum. Esse é o primeiro estudo da expressão gênica global de uma bactéria do gênero Rickettsia infectando um carrapato-vetor natural. Apesar de ambos os estímulos terem promovido um aumento da carga bacteriana, a alimentação sanguínea teve um efeito maior, também modulando cinco vezes mais genes que a elevação da temperatura. Dentre os genes induzidos, alguns codificam fatores de virulência, tais como componentes do sistema de secreção do tipo IV (T4SS), sugerindo que esse importante sistema de secreção bacteriano seja utilizado para secretar efetores durante a ingestão de sangue pelo carrapato. Através de análises in silico de domínios conservados das proteínas hipotéticas, identificamos outros componentes do T4SS de R. rickettsii ainda não descritos na literatura. A alimentação sanguínea também induziu a expressão de genes codificadores de enzimas antioxidantes, o que pode corresponder a uma tentativa de R. rickettsii de se proteger contra os efeitos deletérios de radicais livres produzidos pelos carrapatos alimentados. Por fim, analisamos a transcrição de uma seleção de genes de R. rickettsii em glândulas salivares e intestinos de carrapatos machos e fêmeas através de RT-qPCR microfluídica. Os resultados mostraram que a elevação da temperatura e a alimentação modulam um conjunto específico de genes em cada tecido analisado, tendo sido possível definirem-se assinaturas transcricionais tecido-específicas. Os genes diferencialmente expressos identificados neste estudo devem ser caracterizados funcionalmente, podendo ser considerados como futuros alvos para o desenvolvimento de vacinas. / Rickettsia rickettsii is the causative agent of Rocky Mountain Spotted Fever, which is the most lethal spotted fever rickettsiosis that affects humans. The main tick species that transmits R. rickettsii in the metropolitan area of São Paulos city is Amblyomma aureolatum. When an infected and starving tick begins blood feeding from a vertebrate host, R. rickettsii is exposed to a temperature elevation and to components in the blood meal. These two environmental stimuli have been previously associated with the reactivation of rickettsial virulence in ticks, but the factors responsible for this phenotype conversion have not been completely elucidated. The main aim of the present work was to determine the effects of these two environmental stimuli on the R. rickettsii transcriptional profile during A. aureolatum infection. We initially established an effective system for rickettsia propagation to generate a substantial quantity of genetic material for microarray standardization. For that, for the first time, we established an in vitro infection of the virulent Brazilian R. rickettsii strain in the BME26 tick embryonic cell line from Rhipicephalus (Boophilus) microplus. Using customized oligonucleotide microarrays, we analyzed the effects of a 10°C temperature elevation and a blood meal on the transcriptional profile of R. rickettsii infecting whole organs of Amblyomma aureolatum female ticks. This is the first bacterial transcriptome study of the Rickettsia genus when infecting a natural tick vector. Although both stimuli significantly increased the bacterial load, blood feeding had a greater effect, also modulating five-fold more genes than the temperature upshift. Among the genes induced by blood-feeding, some encode virulence factors, such as Type IV Secretion System (T4SS) components, suggesting that this important bacterial transport system is used to secrete effectors during the acquisition of the blood meal by the tick. Using an in silico conserved domain analysis of hypothetical proteins, we identified additional T4SS components of R. rickettsii that were never previously described. Blood-feeding also up-regulated the expression of antioxidant enzymes, which might correspond to an attempt by R. rickettsii to protect itself against the deleterious effects of free radicals produced by fed ticks. Finally, we studied the transcriptional profile of selected genes of R. rickettsii on the salivary glands and midguts of male and female ticks by microfluidic RT-qPCR. Results showed that temperature upshift and blood feeding modulate specific sets of genes in each tissue, allowing for the establishment of a tissue-specific transcriptional signature. The modulated genes identified in this study require further functional analysis and may have potential as future targets for vaccine development.

As espécies reativas de oxigênio (ERO) e de nitrogênio (ERN) são essenciais na resposta imune dos artrópodes aos microrganismos. Este estudo teve como objetivo a caracterização do estresse oxidativo das células embrionárias BME26 do carrapato R. microplus em resposta à infecção por A. marginale. Mostrou-se que A. marginale invade as células BME26 durante a primeira hora. No perfil de expressão de diversos genes codificadores de proteínas que estão envolvidas na produção e destoxificação de ERO e ERN após desafios com A. marginale e R. rickettsii, foi observado que A. marginale inibiu os transcritos das enzimas oxidantes 72 horas após a infecção. Em contrapartida os genes codificadores das enzimas antioxidantes foram todos induzidos. O perfil de expressão gênica pela infecção por R. rickettsii foi diferente do obtido com A. marginale uma vez que se detectou uma maior indução dos genes oxidantes e repressão dos antioxidantes. O silenciamento de quatro genes antioxidantes por RNAi indicaram o envolvimento da resposta redox no controle da infecção por A. marginale. Com isto se demonstra que o estresse oxidativo é importante no controle da infecção pela bactéria nas células BME26 podendo afetar a capacidade vetorial do carrapato na transmissão desse patógeno. / The production of reactive oxygen species (ROS) and nitrogen (ERN) is essential in the immune response of the arthropods. The aim of this study was the characterization of the oxidative stress of the embryonic cell line BME26 from the cattle tick R. microplus in response to A. marginale infection. It was shown that A. marginale invade BME26 cells during the first 1 hour. The expression profile of sixteen genes encoding proteins involved in either production or detoxification of ROS and RNS in response to A. marginale e R. rickettsii, was shown A. marginale caused a down-regulation of the genes encoding the oxidant enzymes in 72 hours after infection. In contrast the genes encoding the antioxidant were induced. The gene expression pattern in response to R. rickettsii was different from that triggered by A. marginale challenge, with induction of oxidant genes and down-regulation of antioxidant genes. The knockdown of four genes encoding the antioxidant enzymes by RNAi suggests that redox response play a role in the control of infection by A. marginale. Our dataset have shown the importance of the oxidative stress to control A. marginale infection by BME26 cells and might have implications on the vectorial capacity of R. microplus in transmission of this pathogen.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Roman Reddy R. Ganta / Ehrlichia and Anaplasma species are rickettsial organisms which infect a variety of mammalian species. The organisms are transmitted from ticks and are maintained in reservoir hosts. Several pathogens have been identified in recent years as the causative agents for emerging infections in people. One of the primary reservoir hosts for the pathogens is the white tailed deer. In this study, 147 deer blood samples and 37 ticks were evaluated for the prevalence of Ehrlichia/Anaplasma species by TaqMan-based real time amplification assay and DNA sequence analysis. One hundred and thirteen (74%) samples tested positive with the Ehrlichia/Anaplasma genera-specific probe. Further analysis of the samples with the probes specific for human ehrlichiosis agents, E. chaffeensis and E. ewingii identified 4 (2.7%) and 7 (4.7%) positives, respectively. Test positives from 24 randomly selected samples were further evaluated by sequence analysis targeting to a 450 bp segment of 16S rRNA gene. All 24 samples were confirmed as positive for the Ehrlichia GA isolate # 4 (GenBank #U27104.1). DNAs from 37 pools of ticks collected from the white tailed deer were also evaluated. The TaqMan-based real time PCR assay with Anaplasma/Ehrlichia common probe identified 29 (78%) tick pools as positives whereas E. chaffeensis- and E. ewingii-specific probes identified three (8%) and one (3%) positives, respectively. The PCR and sequence analysis of tick samples identified Gram-negative bacteria species which included one endosymbiont of Rickettsia species (one tick pool), one Alcaligenes faecalis strain (three tick pools), five different Pseudomonas species (9 tick pools) and five different uncultured bacteria organisms (7 tick pools). Although the pathogenic potential of the white-tailed deer isolates of Anaplasma and Ehrlichia agents remains to be established, their high prevalence and the presence of human ehrlichiosis pathogens in white-tailed deer is similar to earlier findings. The high prevalence of the deer isolates of Anaplasma and Ehrlichia species demonstrates the need for further assessment of the pathogenic potential of these organisms to people and domestic animals.

Vector-borne diseases are an increasing global threat to humans due to climate changes, elevating the risk of infections transmitted by mosquitos, ticks, and other arthropod vectors. Ixodes ricinus, a common tick in Europe, transmits dangerous tick-borne pathogens to humans. Tick-borne encephalitis (TBE) is a vector-borne disease caused by TBE virus (TBEV). Climate change has contributed to increased tick abundance and incidence of tick-borne diseases, and between 10,000 and 15,000 human TBE cases are reported annually in Europe and Asia. TBEV shows a patchy geographical distribution pattern where each patch represents a natural focus. In nature, TBEV is maintained within the tick-rodent enzootic cycle. Co-feeding is the main route for TBEV transmission from infected to uninfected ticks and for maintenance within the natural foci. The increasing number of TBE cases in Scandinavia highlights the importance of characterizing additional TBEV sequences and of identifying novel natural foci, and in this work we sequenced and phylogenetically characterized four TBEV strains: Saringe-2009 (from a blood-fed nymph), JP-296 (from a questing adult male), JP-554 (from a questing adult male), and Mandal-2009 (from a pool of questing nymphs, n = 10). Mandal-2009 represents a TBEV genome from a natural focus in southern Norway. Saringe-2009 is from a natural endemic focus in northern Stockholm, Sweden, and JP-296 and JP-554 originate from a natural focus “Torö” in southern Stockholm. In addition, we have studied the effect of different biotic and abiotic factors on population dynamics of I. ricinus in southern Stockholm and observed significant spatiotemporal variations in tick activity patterns. Seasonal synchrony of immature stages and total tick abundance are important factors for the probability of horizontal transmission of TBEV among co-feeding ticks. We found that the probability of co-occurrence of larvae, nymphs, and female adults was highest during early summer whereas increasing vegetation height and increasing amounts of forest and open water around the study sites had a significant negative effect on co-occurrence of larvae, nymphs, and female adults. The proximal part of the 3 ́non-coding region (3 ́NCR) of TBEV contains an internal poly(A) tract, and genomic analysis of Saringe-2009 revealed variability in the poly(A) tract indicating the existence of different variants within the TBEV pool of Saringe-2009. Like other RNA viruses, TBEV exists as swarms of unique variants called quasispecies. Because Saringe-2009 came from an engorged nymph that had been feeding on blood for >60 h, we propose that Saringe-2009 represents a putative shift in the TBEV pool when the virus switches from ectothermic/tick to endothermic/mammalian environments. We investigated the role of poly(A) tract variability in replication and virulence of TBEV by generating two infectious clones of the TBEV strain Toro-2003, one with a short/wild-type (A)3C(A)6 poly(A) tract and one with a long (A)3C(A)38 poly(A) tract. The infectious clone with the long poly(A) tract showed poor replication in cell culture but was more virulent in C57BL/6 mice than the wild-type clone. RNA folding predictions of the TBEV genomes suggested that insertion of a long poly(A) tract abolishes a stem loop structure at the beginning of the 3 ́NCR. Next generation sequencing (NGS) analysis of the TBEV genomes after passaging in cell culture and/or mouse brain revealed molecular determinants and quasispecies structure that might contribute to the observed differences in virulence. Our findings suggest that the long poly(A) tract imparts instability to the TBEV genome resulting in higher quasispecies diversity that in turn contributes to TBEV virulence. Phylogenetic analysis of Saringe-2009, JP-296, JP-554, and Mandal-2009 predicted a strong evolutionary relationship among the four strains. They clustered with Toro-2003, the first TBEV strain from Torö, demonstrating a Scandinavian clade. Except for the proximal part of the 3 ́NCR, TBEV is highly conserved in its genomic structure. Genomic analysis revealed that Mandal-2009 contains a truncated 3 ́NCR similar to the highly virulent strain Hypr, whereas JP-296 and JP-554 have a genomic organization identical to Toro-2003, the prototypic TBEV strain from the same natural focus. NGS revealed significantly higher quasispecies diversity for JP-296 and JP-554 compared to Mandal-2009. In addition, single nucleotide polymerphism (SNP) analysis showed that 40% of the SNPs were common between quasispecies populations of JP-296 and JP-554, indicating the persistence and maintenance of TBEV quasispecies within the natural focus. Taken together, these findings indicate the importance of environmental factors for the occurrence pattern of the different life-stages of the tick vector, which are important for the persistence of TBEV in nature. Our findings also show that the selection pressure exerted by specific host also affects the population structure of the TBEV quasispecies. In addition, our results further demonstrate that the evolution of quasispecies has effect on TBEV virulence in mice. / Vektorburna sjukdomar är ett växande globalt hot mot både människor och djur. De pågående klimatförändringarna kan leda till förhöjda risker för infektioner överförda av myggor, fästingar och andra leddjursvektorer. Ixodes ricinus är en vanlig fästing i Europa som överför fästingburna patogener som är farliga för människor. Fästingburen encefalit (TBE) är en vektorburen sjukdom som orsakas av TBE-virus (TBEV). De pågående klimatförändringarna har bidragit till en ökning både av vektorn och sjukdomsfrekvensen. Mellan 10 000 och 15 000 mänskliga TBE-fall rapporteras årligen i Europa och Asien. Den geografiska fördelningen av TBEV visar ett ojämnt fördelningsmönster där viruset är koncentrerat till vissa fokusområden. TBEV återfinns i naturen i en livscykel där viruset hela tiden överförs mellan fästingar och däggdjur. Spridningen sker dels från en infekterad fästing till ett ryggradsdjur när fästingen äter på värddjuret. Spridning mellan fästingar sker troligen främst genom så kallad “co-feeding”, det vill säga att flera fästingar suger blod samtidigt från samma värddjur. Viruset kan då passera från en infekterad fästing, genom värddjuret till oinfekterade fästingar. Virus kan identifieras och studeras med genetiska metoder. Det ökande antalet TBE-fall i Skandinavien styrker vikten av att hitta och karakterisera ytterligare TBEV-stammar och identifiera nya naturliga fokusområden. Vi har sekvenserat och fylogenetiskt beskrivit fyra TBEV-stammar: Saringe-2009 (blodfylld nymf), JP-296 (födosökande vuxen hane), JP-554 (födosökande vuxen hane) och Mandal-2009 (födosökande nymfer, n = 10). Mandal-2009 är ett TBEV från ett naturligt fokusområde i södra Norge. Saringe-2009 kommer från ett naturligt fokusområde i norra Stockholms län, Sverige. JP-296 och JP-554 härstammar från Torö som är ett naturligt fokusområde i södra Stockholms län, Sverige. Förutom den genetiska sekvenseringen av TBEV har vi också studerat effekten av olika biotiska och abiotiska faktorer på populationsdynamik av I. ricinus i södra Stockholm och observerade variation i fästingsaktivitetsmönster både temporalt och spatialt. Förekomstmönster av fästinglarver, nymfer och vuxna honor, och det totala antalet fästingar är viktiga faktorer för sannolikheten för horisontell överföring av TBEV mellan fästingar. Vi fann att sannolikheten för synkron förekomst av larver, nymfer och honor var högst under försommaren. Vegetationshöjd, mängden skog och mängd öppet vatten runt undersökningsområden hade signifikanta negativa effekter på sannolikheten för att larver, nymfer och honor skulle förekomma samtidigt. Den variabla delen av den icke-kodande 3 ́regionen (3'NCR) av TBEV-genomet innehåller ofta en intern poly(A)-sekvens. Liksom andra RNA-virus, förekommer TBEV som så kallade ”quasispecies” vilka definieras som grupper av olika genetiska varianter av virus. Genom analysen av TBEV-stam Saringe-2009 avslöjades variation i poly(A)-sekvensen vilket indikerar förekomst av ”quasispecies”. Eftersom Saringe-2009 kom från en blodfylld nymf som hade sugit blod i > 60 timmar, föreslår vi att Saringe-2009 visar en förändring i ”quasispecies”-poolen när viruset överförs från exoterm fästingmiljö till endoterm däggdjursmiljö. Vi undersökte poly(A)-ekvensens variabilitet och dess roll vid replikering och för virulens hos TBEV, genom att skapa två infektiösa kloner av Torö-2003 stammen; en med en kort/vild-typ (A)3C(A)6 poly(A)-sekkvens, och en med en lång (A)3C(A)38 poly(A)-sekvens. Den infektiösa klonen med lång poly(A)-sekvens replikerade sämre än vildtypklonen i cellkultur, men (A)3C(A)38 poly(A) var mer virulent i C57BL/6-möss än (A)3C(A)6 poly(A). Datasimulering av TBEV-genomets sekundär-RNA-struktur visade att de längre poly(A)-sekvenserna påverkar veckningen av en specifik sekundärstruktur (SL14) i början av 3 ́NCR. Djupsekvenseringsanalys av TBEV-gnomen avslöjade skillnader för specifika gener och ”quasispecies”-strukturen efter passering i cellkultur och/eller mushjärna. Dessa förändringar föreslås bidra till de observerade skillnaderna i virulens. Våra resultat indikerar att den långa poly(A)-sekvensen ger instabilitet i TBEV-genomet, vilket resulterar i ökad mångfald av ”quasispecies”-populationen som i sin tur kan bidra till TBEV-virulens. Fylogenetisk analys av Saringe-2009, JP-296, JP-554 och Mandal-2009 visade på ett nära släktskap mellan de fyra skandinaviska TBEV-stammarna. De nya stammarna formerade ett kluster med en tidigare TBEV-stam identifierad på Torö (Toro-2003), vilket skapade ett skandinaviskt klad. Genetisk analys visade att Mandal-2009 innehåller en trunkerad 3 ́NCR som liknar den högvirulenta stammen HYPR. JP-296 och JP-554 hade däremot samma genetiska struktur som den längre Torö-2003 stammen från samma fokusområde. Djupsekvensering visade höge mångfald av ”quasispecies”-populationen för JP-296 och JP- 554 jämfört med Mandal-2009. Analys av enkel nukleotid polymorfism (SNP) visade att 40 % av alla SNP var gemensamma mellan ”quasispecies”-populationen för JP-296 och JP-554. Detta indikerar att TBEV-”quasispecies”-strukturen kan vara konserverad för närbesläktade virus vilken kan leda till att den bevaras inom specifika fokusområden. Sammantaget så visar dessa studier att miljöfaktorer påverkar förekomsten av fästingvektorn och dess olika livsstadier, vilket är en bakomliggande faktor för utbredning av TBEV i naturliga fokusområden. Det visar även på att värdmiljön påverkar strukturen för ”quasispecies”-populationen. Dessutom visar våra studier att evolution och utveckling av ”quasispecies”-strukturen kan påverka virulensen för TBEV i möss.