Tesis sobre el tema "Rickettsioses"

Des analyses d'ADN sur les restes des soldats de la Grande Armée de Napoléon (1812) ont révélé la présence entre autre de Rickettsia prowazekii. Pourtant la rickettsiologie ne commençera qu'avec les travaux de Ricketts et von Prowazek en 1910, et ne cessera de s'alimenter d'espèces et de pathologies nouvelles. En tant que premières bactéries intracellulaires strictes décrites, la taxonomie des rickettsies rassemblait initialement sur la base de ce critère, un grand nombre de genres bactériens ultérieurement reclassés avec l'avènement du séquençage et la découverte d'horloges moléculaires telle que la sous-unité 16S de ARN ribosomique ou le cytochrome C. Pour l'identification des espèces de Rickettsia, de nombreux critères phénotypiques dont la morphologie, les tests de fixation du complément, de neutralisation de toxines, de sérotypage et les profils protéiques ont longtemps été utilisés. Mais c'est la comparaison des séquences de gènes, dont ompA, ompB et sca4, qui ont permis d'identifier très précisément les espèces du genre Rickettsia et de proposer une classification phylogénique fiable. Cependant, la position phylogénique d'espèces telles que Rickettsia helvetica, Rickettsia canadensis et Rickettsia bellii n'a pu être déterminée avec certitude. Aussi, l'analyse basée sur la concaténation de plusieurs gènes, associée aux caractères phénotypiques peut constituer une meilleure alternative
The history of rickettsioses is probably as ancient as human civilisation. The first documented cases of rickettsioses dates back to 1812. In early part of the last century (1910) Ricketts and von Prowazek laid the foundation of modern rickettsiology. Their pioneering works eventually led to the recognition of new species and Rickettsiales infections. As soon as Rickettsia are the first strictly intracellular bacteria described, its taxonomy gathered on the basis of this criterion, and a great number of kinds of bacteria which will be identified only with the advent of the sequencing and the discovery of molecular clocks such as ribosomal 16S RNA and cytochrome C. Many phenotypic criterion such as morphology, tests of complement, neutralization of toxins, mousse serotyping and SDS-page proved reliable. However, gene comparison (ompA, ompB and sca4) will make it possible to very precisely determine the species containing of the genus Rickettsia and to suggest a classification supported by high bootstrap values as well as antibiotics tests. Nevertheless, the phylogenetic position of species such Rickettsia helvetica, Rickettsia canadensis and Rickettsia bellii could not be given with precision, and the polyphasic analysis of the classification of the Rickettsia species based on genes concatenation associated with phenotypic characters available might be alternatives for Rickettsia phylogeny

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Brazilian Spotted Fever and Baggio-Yoshinari Syndrome are emerging diseasest characterized as the only known to tick-borne zoonoses in Brazil. This study aimed to avaluate IgG antibodies against Borrelia burgdorferi and Rickettsia rickettsii in stray and domiciled dogs from Serop?dica-RJ and studying the fauna of ixodida these animals. To investigate the prevalence of antibodies against R. rickettsii and B. burgdorferi was performed to collect blood from 293 dogs in four areas of the city of Serop?dica-RJ. Serum samples were processed by Indirect Immunofluorescence (IFA) against crude antigens of R. rickettsii. The same animals were tested for IgG antibodies against B. burgdorferi strain of North American origin, using the Indirect Enzyme-linked immunosorbent assay (ELISA). To confirm the presence of Borrelia spp., 102 positive samples were tested by Western Blotting (WB). Ticks were also collected from the inspection of the animals. The specimens found were manually removed and stored in polypropylene bottles, and later identified. The association between seropositive animals and variables, were performed using the chi-square and analysis of variance (ANOVA) with significance level of 5%. Of the 283 sera tested by IFA, 23.67% (67/283) showed reactivity against species-specific antigens of R. rickettsii. Thus, we found a prevalence of 21.11% (19/90) in "40 km", 21.84% (19/87) in "Km 49", 25% (8 / 32) in "54 km" and 28.38% (21/74) in UFRRJ. The titles ranged from 1:64 to 1:512. On the other hand, of the 293 animals studied, 154 (52.56%) were positive for homologous antibodies anti-B. burgdorferi. It was founded a prevalence of 43.75% (14/32) in the "54 km", 51.72% (45/87) in the "49 km", 46.67% (42/90) in the "40 km" and 63.1% (53/84) in UFRRJ. It was no significant difference (p<0.05) between the prevalence of antibodies against Borrelia sp. in stray dogs and domiciled. Dogs with access to the street had a frequency against R. rickettsii significantly (p<0.05) greater than animals kept in prison. There were no statistical differences (p>0.05) the relationship between the variables: presence of ticks, animal sex, habits, frequenting pastures and age of the animals with seropositivity observed in either species. Regarding ticks, at least 64.5% (189/293) dogs were infested with ticks at the moment of collection. Only two species of ticks were identified, Rhipicephalus sanguineus and Amblyomma cajennense. The presence of antibodies anti-R. rickettsii and anti-B. burgdorferi in dogs from Serop?dica-RJ is indicative of the presence of rickettsias of the Rock Mountain Spotted Fever Group and spirochetes dogs in this area.
A Febre Maculosa Brasileira e a S?ndrome de Baggio-Yoshinari s?o enfermidades emergentes, caracterizadas como as ?nicas zoonoses conhecidamente transmitidas por carrapatos, no Brasil. O presente estudo teve como objetivo detectar anticorpos da classe IgG contra Rickettsia rickettsii e Borrelia burgdorferi e estudar a fauna de Ixod?deos nesses animais. Para investigar a preval?ncia de anticorpos contra R. rickettsii e B. burgdorferi foi efetuada a coleta de sangue de 293 c?es, em quatro ?reas do munic?pio de Serop?dica-RJ. Os soros obtidos foram processados atrav?s da Rea??o de Imunofluoresc?ncia Indireta (RIFI), contra ant?genos brutos de R. rickettsii. Os mesmos animais foram submetidos ? pesquisa de anticorpos hom?logos da classe IgG contra B. burgdorferi cepa americana G39/40, utilizando Ensaio de Imunoadsor??o Enzim?tico (ELISA) Indireto. Para confirma??o da presen?a de Borrelia spp. no munic?pio, 102 amostras positivas foram testadas pelo Western Blotting (WB). A fauna de ixod?deos foi estudada atrav?s da coleta de carrapatos a partir da inspe??o das regi?es das orelhas, dorso e coxins palmares e plantares dos animais. Os esp?cimes encontrados foram removidos manualmente e acondicionados em frascos de polipropileno, e posteriormente, identificados. O estudo da associa??o entre animais soropositivos e as vari?veis avaliadas, foram realizados por meio do teste de Qui-quadrado e An?lise de Vari?ncia (ANOVA), com n?vel de signific?ncia de 5%. Dos 283 soros testados ? RIFI, 23,67% (67/283) apresentaram reatividade contra ant?genos esp?cie-espec?fico de R. rickettsii, sendo encontrada uma frequ?ncia de 21,11% (19/90) no ?Km 40?, 21,84% (19/87) no ?Km 49?, 25% (8/32) no ?Km 54? e 28,38% (21/74) no campus da UFRRJ. A titula??o variou entre 1:64 ? 1:512. Por outro lado, dos 293 animais estudados, 154 (52,56%) foram positivos para anticorpos hom?logos anti-B. burgdorferi, sendo encontrada uma frequ?ncia de 43,75% (14/32) no ?Km 54?, 51,72% (45/87) no ?Km 49?, 46,67% (42/90) no ?Km 40? e 63,1% (53/84) na UFRRJ. Houve diferen?a significativa (p<0,05) entre as preval?ncias de anticorpos contra Borrelia spp. encontradas em caninos errantes e domiciliados. Os c?es com acesso a rua tiveram uma frequ?ncia de anticorpos contra R. rickettsii significativamente (p<0,05) maior que animais mantidos preso. N?o foram observadas diferen?as estat?sticas (p>0,05) entre a rela??o das vari?veis: presen?a de carrapatos, sexo dos animais, h?bitos de frequentar pastos e idade dos animais com a soropositividade observada em nenhuma das duas esp?cies. Quanto aos carrapatos, 64,5% (189/293) dos c?es estavam infestados por carrapatos no momento da coleta. Apenas duas esp?cies de carrapatos foram identificadas, Rhipicephalus sanguineus e Amblyomma cajennense. A presen?a de anticorpos hom?logos anti-R. rickettsii e anti-B. burgdorferi em caninos de Serop?dica-RJ ? um indicativo da presen?a de rickettsia do grupo da Febre Maculosa e espiroquetas em c?es nesta ?rea.

Nous avons contribue a la mise au point d'une procedure originale d'isolement et d'identification genotypique des rickettsies. Cette methode nous a permis de montrer l'existence de rickettsia rhipicephali dans notre region. Par ailleurs, nous avons etabli l'homogeneite genotypique des souches de rickettsia conorii impliquees dans differentes formes cliniques de fievre boutonneuse mediterraneenne. L'infection experimentale de cellules endotheliales humaines en culture nous a permis d'etudier les secretions enzymatiques induites par les rickettsies, transaminases et facteurs de la fibrinolyse. Enfin, nous avons contribue a l'etude des sensibilites aux antibiotiques en modeles cellulaires

Les infections rickettsiales et la fièvre aiguë indifférenciée (FAI) sont des problèmes de santé publique importants sous les tropiques. Ce projet a été réalisé dans le but d'explorer les agents de rickettsies et autres étiologies de la FAI au Vietnam.Dans la première cohorte de 67 participants se concentrant sur les patients cliniquement suspectés de typhus des broussailles, le diagnostic de typhus des broussailles a été confirmé pour 32 patients (47,8%) par qPCR ou IFA. Parmi les 20 patients pour lesquels des escarres et du sang total ont été obtenus, 17 (85%) des échantillons d'escarre et 5 (25%) des échantillons de sang total étaient positifs pour O. tsutsugamushi par qPCR. L'analyse génétique des séquences du gène TSA 56-kDa a démontré que les 14 séquences étaient apparentées à 4 génotypes: Karp, Kawasaki, Gilliam (JG-v et TG-v) et TA716. Ensuite, une analyse prospective des étiologies de la FAI au Vietnam a été réalisée avec 378 patients adultes fébriles (FAIs) et 384 patients adultes fébriles (Contrôles). L'étiologie la plus courante était le virus de la grippe (20,9% dans les FAIs versus 0% dans les Contrôles), suivi des rickettsies (11,11% vs 0,26%), virus de la dengue (7,67% vs 0,52%), Leptospira (4,76% vs 0,78%), adénovirus (4,76% vs 1,04%) et entérovirus (2,12% vs 0%) (p<0,05).Par conséquent, de telles données sur les rickettsies et autres étiologies obtenues au Vietnam pourraient être très utiles pour l’établissement d’une méthode d’approche pour les patients atteints de FAI dans les aspects de diagnostic, de gestion et de prévention au Vietnam et dans les régions tropicales dans l'avenir
Rickettsial infections and acute undifferentiated fever (AUF) are the important public health issues in the tropics. This project was performed with the aim to explore the rickettsial agents and other etiologies of AUF in Vietnam. In the first 67-participant cohort of patients who were clinically suspected of having scrub typhus, 32 (47.8%) patients were confirmed as scrub typhus by either qPCR or IFA. Among the 20 patients for whom both eschar and whole blood were obtained, 17 (85%) of the eschar specimens and 5 (25%) of the whole blood specimens tested positive for O. tsutsugamushi by qPCR. Genetic analysis of the 56-kDa TSA gene sequences demonstrated that the 14 sequences were related to 4 groups: Karp, Kawasaki, Gilliam (JG-v and TG-v) and TA716.Subsequently, a prospective case-control analysis of the etiologies of AUF in Vietnam was performed. A total of 378 febrile adult patients (AUFs) and 384 afebrile adult patients (Controls) were enrolled. The most common etiology was influenza virus (20.9% in AUFs versus 0% in Controls), followed by rickettsial agents (11.11% in AUFs versus 0.26% in Controls), dengue virus (7.67% vs 0.52%), Leptospira (4.76% vs 0.78%), adenovirus (4.76% vs 1.04%), and enterovirus (2.12% vs 0%) (p<0.05). Consequently, such data of rickettsial agents and other etiologies in Vietnam obtained from this project are extremely useful in guiding the diagnosis, management and preventive-health methods for patients with AUF in the future in Vietnam as well as in the tropics worldwide

The spotted fever group rickettsiae are emerging diseases. They cause damage in their hosts by invading the endothelium in small to medium-sized blood vessels, which results in vasculitis that can cause clinical manifestations from most organs. The present thesis describes the prevalence of Rickettsia helvetica in ticks, the incidence of rickettsial infection based on seroreactivity and seroconversion in humans and their symptoms, from different parts of Sweden and the Åland Islands in Finland. This was accomplished through serological analysis of both retrospective and prospective serum samples from confirmed and suspected tick-bitten individuals compared to individuals with no knowledge of tick exposure (blood donors). We found a comparable seroprevalence to Rickettsia spp. in different geographical areas where ticks are present; it was also comparable to the seroprevalence of Borrelia spp. Seroprevalence was also more common, as suspected, in the tick-exposed group compared to blood donors. In comparison with co-infections with other tick-borne infections (Anaplasma spp. and Borrelia spp.), we could conclude that co-infections do exist and that, based on clinical findings, it is difficult to distinguish which microorganism causes certain clinical manifestations. For reliable conclusions regarding the causative microorganism, the diagnosis should basically rely on diagnostic tests. In comparison with Borrelia spp., seroconversion to Rickettisa spp. was more common in the areas we investigated, indicating that rickettsiosis is a common tick-borne infection in Sweden and most likely underdiagnosed. When investigating patients with meningitis, we found R. felis in cerebrospinal fluid from two patients with subacute meningitis. This was the first report in which R. felis was found and diagnosed in patients in Sweden. The patients recovered without sequelae and without causal treatment. To provide guidelines on when to treat Rickettisa spp. infections, more investigations are needed. The present thesis shows that Rickettsia spp. are common in ticks and do infect humans. Rickettsial infection should be considered in both non-specific or specific symptoms after a tick bite. It was also shown in the thesis that flea-borne rickettsiosis (R. felis) occurs in Sweden and may cause invasive infections

Through a historical series of eight years (1995-2002), was analyzed in the data of the case report form (CRF) the positive cases of Brazilian Spotted Fever - BSF and other rickettsiosis for confirmation of clinical and epidemiological parameters consecrated in the literature. An exploratory study aimed st to interpret the information, objectifying the construction of the analysis of the epidemiological situation of the BSF in Minas Gerais. An analysis of the clinical symptoms of laboratory confirmed cases was carried through, in the same period, to verify the most prevalent, to improve the definition of a suspected case. A case-control study was carried through, using the (CRF) as intrument of risk factors identification.
Através de uma série histórica de oito anos (1995-2002), foi verificada a corrrelação dos dados obtidos das fichas de investigação epidemiológica (FE) dos casos positivos de Febre Maculosa Brasileira - FMB para confirmação de parâmetros clínicos e epidemiológicos consagrados na literatura nacional e ou internacional. Um estudo exploratório visou intepretar as informações, objetivando a construção da análise da situação epidemiológica da FMB em Minas Gerais. Foi realizada uma análise dos sintomas clínicos de casos confirmados laboratorialmente, deste suspeitos. Foi realizado um estudo de caso controle, utilizando as FE como instrumento de identificação de fatores de risco.

Dans ce travail nous avons compare les methodes (traditionnelles et de biologie moleculaire) permettant de differencier les rickettsies du groupe boutonneux. Cette etude, dont le cadre geographique se situe en europe et en afrique, a abouti a la description de trois nouvelles rickettsies a pathogenicite inconnue et de deux nouvelles rickettsies pathogenes pour l'homme. Le fait qu'il y ait de nouvelles rickettsies et de nouveaux vecteurs potentiels, dans des regions ou la fievre boutonneuse mediterraneenne est endemique, souleve un certain nombre de problemes d'ordre epidemiologique (specificite du rapport tiques-rickettsies) clinique (pathogenicite pour l'homme, effet de la presence de nouvelles rickettsies sur la seroprevalence des rickettsioses) et taxonomiques (definition de l'espece rickettsienne)

Rickettsiae are obligate intracellular gram-negative bacteria transmitted by arthropod vectors. Rickettsiae sometimes cause disease in humans, typically with high fever, headache and occasionally an eschar. In Sweden, Rickettsia helvetica, belonging to the spotted fever group, is the only tick-transmitted rickettsia found free in nature. The pathogenic roll of R. helvetica has not been fully investigated, but it has been implicated in aneruptive fever and cardiac disease. This thesis describes parts of the transmission pathways of rickettsiae in Sweden. Rickettsia infection rates in ticks collected from birds were analysed, and the birds’ role as disseminators and reservoirs was studied. We found that more than one in ten ticks was infected with rickettsia bacteria, predominantly R. helvetica, and that migrating birds contribute not only to long-distance dispersion of bacteria, but also to an inflow of novel and potentially pathogenic rickettsia species, in this case R. monacensis and R. sp. strain Davousti-like species, into Sweden. Further, wild and domestic animals were found to have seroreactivity against R. helvetica, which shows that they are exposed and susceptible to rickettsia. Their role as reservoirs has not been determined, yet they may indirectly be involved in transmission of rickettsia to humans by infected ticks feeding on them. The seroreactivity in humans was also studied. Patients investigated for suspected Borrelioses and blood donors had detectable antibodies against Rickettsia spp., with the highest prevalence detected in the suspected Borreliosis group. This shows that humans in Sweden are exposed to and develop an immune response against rickettsia. The suspicion that R. helvetica may cause severe symptoms was verified by a patient with subacute meningitis where the bacterium was shown for the first time to cause an invasive infection with CNS involvement and where the bacterium was isolated from the patient’s cerebrospinal fluid. Growth characteristics and morphology of R. helvetica were studied to better understand invasiveness and virulence. The findings indicate that the invasiveness is comparable with other rickettsia, though R. helvetica seems to have a stable but slightly slower growth.  Rickettsia helvetica is endemic in Sweden and therefore needs to be considered when investigating disease after a tick bite.
Rickettsia är en liten, strikt intracellulär, gramnegativ bakterie som sprids med vektorer som fästingar, löss och loppor. Bakterien kan orsaka Rickettsios hos människa, en sjukdom där de vanligaste symtomen är hög feber, huvudvärk, muskelvärk och i vissa fall ett bettmärke (eschar). I Sverige är Rickettsia helvetica, som tillhör spotted fever gruppen (SFG), den enda fästingöverförda rickettsia bakterien som hittats allmänt i naturen. Patogeniciteten för R. helvetica är ofullständigt utredd, men ”aneruptive fever” och hjärtmuskelinflammation har rapporterats. Avhandlingen beskriver delar av smittkedjan för SFG rickettsia i Sverige. Bakteriernas förekomst i fästingar plockade från fåglar har studerats, likaså det ekologiska tryck som flyttfåglars bärarskap av infekterade fästingar bidrar med när de korsar olika världsdelar. Mer än var tionde fästing var infekterad med rickettsia bakterier, i huvudsak R. helvetica. Det visade sig att flyttfåglar bidrar inte bara till långväga spridning av bakterier utan även till införsel av nya potentiellt patogena rickettsiaarter, i detta fall identifierades R. monacensis och en R. sp strain Davousti liknande art. Vidare analyserades seroreaktivitet mot Rickettsia helvetica hos både tamdjur och vilda djur, vilket visade på antikroppsutveckling, som uttryck för smittexposition, i mer än vart femte djur. Djurens roll som reservoar för bakterien är inte klarlagd, men oavsett är djuren indirekt involverade i spridningen av bakterien till människa via infekterade fästingar som suger blod. Seroreaktivitet hos människa har också studerats. Patienter, provtagna på grund av misstanke om borreliainfektion, samt blodgivare hade detekterbara antikroppar mot Rickettsiae, med högst prevalens i gruppen med misstänkt borreliainfektion. Fynden visar att människor i Sverige är exponerade för och utvecklar en immunreaktion mot rickettsia. Att R. helvetica skulle kunna ge allvarlig sjukdom verifieras av ett patientfall med subakut meningit där bakterien för första gången visats ge invasiv infektion med påverkan på nervsystemet (CNS engagemang) och där bakterien isolerats från patientens ryggmärgsvätska.  Morfologi och tillväxtegenskaper för R. helvetica undersöktes för att bättre förstå bakteriens invasivitet och virulens. Fynden indikerar att invasiviteten är jämförbar med andra rickettsiaarter men R. helvetica verkar ha en stabil men något långsammare tillväxt. Rickettsia helvetica är endemisk i Sverige och måste tas i beaktande vid sjukdomsutredning efter ett fästingbett.

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A febre maculosa é uma doença transmitida por carrapatos e tem como agente etiológico bactérias do gênero Rickettsiia, envolvendo no ciclo da Febre Maculosa Brasileira, cães, roedores e marsupiais como reservatórios vertebrados e carrapatos e pulgas como seus vetores invertebrados com os humanos atuando como hospedeiros acidentais, e constituindo elo terminal no ciclo dessas bactérias. A patogenicidade atribuída a estes microorganismos é ainda desconhecida para varias espécies, bem como os fatores de risco, demográficos e sazonais relacionados às respectivas doenças ocasionadas. Para a realização deste estudo, foram selecionados dois municípios do estado de Minas Gerais com prévio histórico de ocorrência destas enfermidade em humanos, sendo dois casos recentes no município de Pingo D'água, localizado na região do Vale do Rio Doce e o município de Santa Cruz do Escalvado, localizado no Vale do Piranga, Zona da Mata Mineira, estado de Minas Gerais foco antigo para riquetsioses, que sofreu alteração recente em sua paisagem natural, devido a construção de uma usina hidrelétrica na localidade de Soberbo no ano de 2004. Assim, com o objetivo de melhor compreender a atual situação epidemiológica das riquetsioses nesses municípios, dentro de uma concepção de ocupação e transformação do espaço geográfico por ação antrópica, buscou-se avaliar o nível de contato destes agentes nas populações de animais domésticos e sinantrópicos, realizaram-se ensaios sorológicos e ferramentas da biologia molecular. Eqüinos, cães, roedores e gambás foram capturados e identificados, tendo sido ainda coletadas amostras de soro bem como ectoparasitas. Dos roedores capturados foram colhidas amostras de tecidos (fígado e baço), e dos gambás amostras de sangue total, sendo realizado ainda nestes animais colheita de material por swab anal. Das amostras de sangue, tecidos, ectoparasitas e swabs anal extraiu-se DNA, que foram amplificadas através de PCR usando oligonucleotídeos iniciadores para amplificação gênero específica do gene da citrato sintase (gltA). Os produtos amplificados obtidos foram visualizados em gel de agarose a 1,5%. Apenas amostras de ectoparasitas das espécies Amblyomma cajennense coletados de cães, eqüinos, gambás-Didelphis aurita e Ctenocefalides canis e Ctenocefalides felis em pulgas de gambás apresentaram resultados positivos à PCR. As amostras de soros de animais silvestres e domésticos foram investigadas quanto à presença de anticorpos reativos à antígenos de Rickettsia rickettsii, Rickettsia parkeri, Rickettsia felis, Rickettsia belli, Ricketssia rhipicephali e Rickettsia amblyommii pela Reação de Imunofluorescência Indireta (RIFI). Amostras que apresentaram títulos iguais ou superiores a 1:64 foram considerados positivas para Rickettsia. No município de Pingo D’ Água das 46 amostras de soros de roedores analisadas, dentre estas 31 da espécie Rattus rattus, 7 Oryzomys subflavus, 7 Nectomys squamipes e 1 Bolomys sp. 26 (83,88%) das amostras de R. rattus, seis (85,72%) das amostras de Oryzomys e Nectomys e a única amostra de soro de Bolomys analisada apresentaram resultados positivos para bacterias do grupo da febre maculosa. No município de Santa Cruz do Escalvado das 62 amostras de soro de roedores analisadas, dentre estas 32 da espécie R. rattus, 2 Oryzomys subflavus, e 23 Nectomys squamipes e 5 Akodon sp. 30 amostras de R. rattus testadas, 30 foram reativas à RIFI para Rickettsia do Grupo das Febres Maculosas, totalizando 93,75% das amostras. Os dados sorológicos obtidos nos municípios de Pingo D’ Água e Santa Cruz do Escalvado, apontam a espécie R. rattus como a mais representativa entre os roedores, o que a credencia para participação no ciclo enzoótico das riquetsioses nas referidas regiões. Levando em consideração o agravante de essa ser a espécie mais sinantrópica em relação às outras estudadas neste projeto, é importante atentar-se para o possível risco de dispersão de organismos do gênero Rickettsia por esses animais no município. Foram analisadas ainda 42 amostras de soros de eqüinos e 24 amostras de soros de cães provenientes de Pingo D’ Água, com reatividade sorológica contra riquétsias do GFM em 16 (38,09%) dentre as amostras de eqüinos e em duas (8,33%) dentre as amostras de cães. Em Santa Cruz do Escalvado foram analisadas 66 amostras de soros de eqüinos e 67 amostras de soros de cães, verificando-se uma sororeatividade contra riquétsias do GFM em dez (15,15%) dentre as amostras de soros de eqüinos e em 14 (20,89%) dentre as amostras de soros de cães. Das 38 amostras de soro de D. aurita coletados no município de Santa Cruz do Escalvado, em 16 verificou-se a presença de Rickettsia, sendo que em 42,1% dos soros foram reativos contra R. rickettsii, 39,5% contra R. parkeri, 18,4% contra de R. amblyommi e 2,6% contra R. felis. Os achados sorológicos e de biologia molecular obtidos neste trabalho recomendam a manutenção de uma vigilância epidemiológica permanente nos municípios estudados, bem como em seu entorno, buscando-se desta maneira evitar a ocorrência de novos casos humanos causada pela invasão destes focos naturais pelo homem ou pela dispersão de potenciais reservatórios e vetores de agentes riquetsiais para novas áreas do estado ___________________________________________________________________________________________________________________________________________________
Spotted fever is a disease transmitted by ticks presenting as his etiological agent bacteria of the genus Rickettsia. Involved in her natural cycle, dogs, rodents, and marsupials actuated as vertebrate reservoirs, been ticks, and fleas the invertebrates vectors. Humans actuating as accidental hosts are becoming the final link in the cycle of these bacteria. The pathogenicity attributed to these microorganisms is still unknown for several of the Rickettsia species, including the involved factors of risk and the demographic and seasonal factors related. For this study, we selected two counties in the state of Minas Gerais, Brazil, that have prior history of occurrence of this disease in humans: Pingo D'Água, located in Vale do Rio Doce, with two recent cases reported, and Santa Cruz do Escalvado, located in Vale do Piranga, Zona da Mata Mineira, a former focus for rickettsial diseases, which has suffered recent change in their natural landscape due to the construction of a hydroelectric plant in the town of Superb in 2004. Thus, in order to better understand the current epidemiological situation of the rickettsial diseases in these municipalities, under a conception of occupation and transformation of geographic space by anthropic actions, we sought to evaluate the level of transmission of these agents in populations of domestic and synanthropic animals, realizing serological and molecular biology assays. Horses, dogs, rodents and opossums were captured and identified, from whom were collected serum samples and ectoparasites. From rodents captured were collected samples of tissues (liver and spleen). From opossums were collected samples of whole blood and anal swab. From blood, tissues, ectoparasites, and anal swabs were extracted DNA samples, which were subsequently amplified by PCR using primers for amplification of Rickettsia genus-specific citrate synthase gene (gltA). The amplified products obtained were visualized in agarose gel 1.5%. Only samples of parasites of the species Amblyomma cajennense collected from dogs, horses, and opossums, and fleas (Ctenocefalides canis and Ctenocefalides felis) collected from opossums tested positive by PCR. Serum samples from wild and domestic animals were investigated by Immunofluorescence Assay (IFA) for the presence of antibodies reactive to antigens of Rickettsia rickettsii, Rickettsia parkeri, Rickettsia felis, Rickettsia belli, Rickettsia rhipicephali and Rickettsia amblyommii. Samples that had titers equal or greater than 1:64 in the IFA were considered positive for Rickettsia. In the municipality of Pingo D’Água, from 46 serum samples rodents tested, among these 31 species of Rattus rattus, seven Oryzomys subflavus, seven Nectomys squamipes, and one Bolomys sp., 26 (83.88%) samples of R. rattus, six (85.72%) samples of Oryzomys and Nectomys, and the single serum sample of Bolomys examined were positive for bacteria of the spotted fever group (SFG). In Santa Cruz do Escalvado, serum samples from 62 rodents tested, among these 32 species of R. rattus, two Oryzomys subflavus and five Nectomys squamipes, and 23 Akodon sp., 30 samples of R.rattus were reactive by IFA for Rickettsia agents from the SFG, totaling 93.75% of the samples. The serological data obtained in the municipalities of Pingo D'Água and Santa Cruz do Escalvado, indicated that the species R. rattus is the most representative among the rodents, which accredits to participate in the enzootic cycle of rickettsial diseases in these regions. Taking into account the aggravating factor that is the most synanthropic species over all others studied in this project, it is important to pay attention to the possible risk of dispersal of the genus Rickettsia by these animals in the municipalities. We also analyzed serum samples of 42 horses and 24 serum samples of dogs from Pingo D'Água, which showed serological reactivity against rickettsiae from SFG. Sixteen (38.09%) among the samples from horses and two (8, 33%) among the samples of dogs tested showed positive results. In Santa Cruz do Escalvado were we analyzed 66 serum samples from horses and 67 serum samples from dogs, were found a seroreactivity against rickettsiae organisms of SFG in ten (15.15%) among the serum samples of horses and in 14 (20 89%) among the serum samples of dogs tested. Of 38 serum samples from D. aurita collected in Santa Cruz do Escalvado, in 16 were verified the response against Rickettsia, been 42.1% of sera reactive against R. rickettsii, 39.5% against R. parkeri, 18.4% against R. amblyomma and 2.6% against R. felis. The findings of serological and molecular biology in this work recommend the maintenance of effective epidemiological surveillance in the municipalities studied, as well as its surroundings, searching in this way avoid the occurrence of new human cases of outbreaks caused by invasion by man or by natural dispersal of potential reservoirs and vectors for Rickettsia to new areas of the state.

Le typhus épidémique, une maladie infectieuse ré-émergente due à Rickettsia prowazekii, est transmis à l'homme par le pou de corps humain. L’infection peut demeurer sous forme latente chez l’homme pendant des années. Sa réactivation, à la suite d’un stress par exemple, déclenche une forme modérée de typhus épidémique, la maladie de Brill-Zinsser, qui peut être source de nouvelles épidémies en présence de poux. R. Prowazekii, un agent potentiel de bioterrorisme, est une bactérie intracellulaire qui infecte l'endothélium vasculaire, ce qui conduit à une vascularite et des lésions inflammatoires constituées d'infiltrats de leucocytes mononucléés. La pathogénèse de l'infection à R. Prowazekii est mal connue, en partie par manque de modèles animaux adéquats. L'objectif de ma thèse a été de mettre au point un modèle murin d’infection à R. Prowazekii et d'étudier certains aspects de la physiopathologie de cette infection. J’ai montré que l’infection des souris dépend de leur fond génétique. Les souris BALB/c miment par bien des aspects l’infection humaine: sont ainsi trouvés, chez les souris infectées, des pneumonies avec consolidation des alvéoles, des hémorragies au niveau pulmonaire et cérébral, de multiples granulomes hépatiques. L’infection des souris BALB/c est associée à une réponse inflammatoire au sein des tissus atteints, comme l’illustre l’augmentation de l'expression de cytokines inflammatoires telles que l’IFN-γ, le TNF et RANTES. J’ai également réussi à réactiver l'infection à R. Prowazekii chez les souris BALB/c soumises à une corticothérapie, ce qui conforte notre modèle expérimental de typhus épidémique et offre, de surcroît, le premier modèle de maladie de Brill-Zinsser. J’ai enfin posé quelques jalons dans l’étude de la pathogénèse de l’infection à R. Prowazekii. L’infection des cellules endothéliales par des bactéries virulentes augmente la migration transendothéliale des leucocytes, alors que les bactéries atténuées n'ont pas d'effet. Ce phénomène de migration transendotheliale implique la production de cytokines inflammatoires par les cellules endothéliales infectées, ce qui rend inflammatoires les leucocytes ayant transmigré. Le profil inflammatoire induit par R. Prowazekii participe probablement aux lésions caractéristiques du typhus épidémique.

La mobilité actine-dépendante des rickettsies dans le cytosol des cellules eucaryotes a longtemps été considérée comme l’une des caractéristiques permettant la différenciation des rickettsies du groupe boutonneux (SGF) avec celles du groupe typhus (TG), bactéries intracellulaires strictes à l’origine de fièvres éruptives et du typhus. De fait, la protéine RickA, dont le gène est absent dans le TG, a été décrite comme étant responsable de la mobilité des rickettsies du SFG. Certaines observations récentes suggèrent toutefois que le schéma est plus complexe que ce que l’on imaginait. Cette thèse a donc eu pour objectif de mieux caractériser la mobilité des rickettsies du genre Rickettsia. Dans un premier temps, et en suivant des protocoles standard, nous avons produit un anticorps monoclonal dirigé contre la protéine RickA recombinante de R. Conorii. En western-blot cet anticorps reconnaît spécifiquement RickA pour toutes les rickettsies testées classées dans le SFG. La seconde partie de notre travail a concerné l’étude de la mobilité d’une rickettsie du SFG récemment isolée, soit R. Raoultii. La particularité de ces bactéries est que leur capacité à se mouvoir varie en fonction de leurs cellules hôtes. Nous avons montré que l’absence de mobilité de R. Raoultii dans les cellules L929 n’était pas corrélée avec l’enfermement des bactéries dans la vacuole de phagocytose, ni avec un défaut d’expression de RickA. Ces données démontrent que l’expression de RickA ne suffit pas à la mobilité des rickettsies, ce qui renforce l’hypothèse d’un autre facteur, évoqué antérieurement. Enfin, en utilisant comme support de culture différentes lignées de cellules eucaryotes, nous avons mis en évidence une réplication exacerbée de R. Conorii dans les L929. Ces données ont une importante toute particulière dans le contexte de l’analyse post-génomique des bactéries qui requiert de grandes quantités de matériel. Globalement, nous pensons que ces résultats contribuent à une meilleure connaissance de la pathogénicité des rickettsies.

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.

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Universidade Estadual Paulista (UNESP)
A Febre Maculosa Brasileira (FMB) é causada por bactérias do gênero Rickettsia e é uma zoonose de grande importância em Saúde Pública. Transmitida por carrapatos do gênero Amblyomma sp., é uma riquetsiose de ocorrência reconhecida no país. Casos humanos têm sido descritos desde a década de 20, principalmente na região sudeste do país. Sua ocorrência vem aumentando progressivamente nos últimos anos e já abrange grande parte do território nacional. Entretanto há pouca informação sobre a epidemiologia da doença em áreas não endêmicas, o que pode ser explicado por falhas no diagnóstico e subnotificação. Com letalidade de até 80% em casos não tratados, a doença é considerada um desafio do ponto de vista clínico, pois exige precocidade no diagnóstico e tratamento adequado. A doença passou a ser de notificação compulsória no estado de São Paulo a partir de 2002. De 2003 a 2008 foram confirmados 240 casos de FMB em seres humanos em território paulista, com 71 óbitos e letalidade que variou de 21,9% a 40,0%. Com o objetivo de iniciar estudos da vigilância da FMB no município de Botucatu, Estado de São Paulo, considerada área não endêmica, foram colhidas amostras de sangue de cães que vieram para campanha anual de vacinação antirrábica no ano de 2009, através de adesão voluntária dos proprietários. As amostras de um total de 640 cães foram encaminhadas ao Centro de Controle de Zoonoses no município de São Paulo (CCZ/SP) e processadas pela técnica de reação de imunofluorescência indireta, que é considerada padrão para diagnóstico sorológico da FMB. Dentre as amostras analisadas, seis apresentaram reagentes para a FMB frente à técnica realizada, resultado este, que contribuirá de forma a consubstanciar informações junto aos órgãos de saúde do município, servindo para futuras estratégias de controle e prevenção desta enfermidade
Brazilian spotted fever (BSF) is caused by bacteria of the genus Rickettsia and is a zoonosis of great importance in public health. Transmitted by Amblyomma sp., is a recognized rickettsial disease occurring in the country. Human cases have been described since the '20s, especially in the southeast of the country. Its occurrence has been increasing steadily in recent years and now covers much of the country. However there is little information on the epidemiology of the disease in non-endemic areas, which can be explained by failures in diagnosis and underreporting. With a fatality rate of up to 80% in untreated cases, the disease is considered a challenge from a clinical standpoint, it requires early diagnosis and appropriate treatment. The disease became notifiable in the state of São Paulo from 2002. From 2003 to 2008 were 240 confirmed cases of BSF in humans in the state territory, with 71 deaths and mortality rates ranged from 21.9% to 40.0%. In order to initiate studies of BSF surveillance in Botucatu, São Paulo, considered non-endemic area, blood samples were collected from dogs that came to the annual campaign rabies vaccination in 2009, through the voluntary owners. Samples from a total of 640 dogs were sent to the Zoonosis Control Center in São Paulo (CCZ / SP) and processed by the technique of indirect immunofluorescence, which is considered standard for serological diagnosis of BSF. Among the samples analyzed, six-reactive to the front of the BSF technique performed, a result which will help to substantiate the information from health agencies in the city, serving for future strategies for control and prevention of this disease

Orientador: José Rafael Modolo
Coorientador: Cassiano Victória
Banca: Luis Carlos de Souza
Banca: Samir Issa Samara
Resumo: A Febre Maculosa Brasileira (FMB) é causada por bactérias do gênero Rickettsia e é uma zoonose de grande importância em Saúde Pública. Transmitida por carrapatos do gênero Amblyomma sp., é uma riquetsiose de ocorrência reconhecida no país. Casos humanos têm sido descritos desde a década de 20, principalmente na região sudeste do país. Sua ocorrência vem aumentando progressivamente nos últimos anos e já abrange grande parte do território nacional. Entretanto há pouca informação sobre a epidemiologia da doença em áreas não endêmicas, o que pode ser explicado por falhas no diagnóstico e subnotificação. Com letalidade de até 80% em casos não tratados, a doença é considerada um desafio do ponto de vista clínico, pois exige precocidade no diagnóstico e tratamento adequado. A doença passou a ser de notificação compulsória no estado de São Paulo a partir de 2002. De 2003 a 2008 foram confirmados 240 casos de FMB em seres humanos em território paulista, com 71 óbitos e letalidade que variou de 21,9% a 40,0%. Com o objetivo de iniciar estudos da vigilância da FMB no município de Botucatu, Estado de São Paulo, considerada área não endêmica, foram colhidas amostras de sangue de cães que vieram para campanha anual de vacinação antirrábica no ano de 2009, através de adesão voluntária dos proprietários. As amostras de um total de 640 cães foram encaminhadas ao Centro de Controle de Zoonoses no município de São Paulo (CCZ/SP) e processadas pela técnica de reação de imunofluorescência indireta, que é considerada padrão para diagnóstico sorológico da FMB. Dentre as amostras analisadas, seis apresentaram reagentes para a FMB frente à técnica realizada, resultado este, que contribuirá de forma a consubstanciar informações junto aos órgãos de saúde do município, servindo para futuras estratégias de controle e prevenção desta enfermidade
Abstract: Brazilian spotted fever (BSF) is caused by bacteria of the genus Rickettsia and is a zoonosis of great importance in public health. Transmitted by Amblyomma sp., is a recognized rickettsial disease occurring in the country. Human cases have been described since the '20s, especially in the southeast of the country. Its occurrence has been increasing steadily in recent years and now covers much of the country. However there is little information on the epidemiology of the disease in non-endemic areas, which can be explained by failures in diagnosis and underreporting. With a fatality rate of up to 80% in untreated cases, the disease is considered a challenge from a clinical standpoint, it requires early diagnosis and appropriate treatment. The disease became notifiable in the state of São Paulo from 2002. From 2003 to 2008 were 240 confirmed cases of BSF in humans in the state territory, with 71 deaths and mortality rates ranged from 21.9% to 40.0%. In order to initiate studies of BSF surveillance in Botucatu, São Paulo, considered non-endemic area, blood samples were collected from dogs that came to the annual campaign rabies vaccination in 2009, through the voluntary owners. Samples from a total of 640 dogs were sent to the Zoonosis Control Center in São Paulo (CCZ / SP) and processed by the technique of indirect immunofluorescence, which is considered standard for serological diagnosis of BSF. Among the samples analyzed, six-reactive to the front of the BSF technique performed, a result which will help to substantiate the information from health agencies in the city, serving for future strategies for control and prevention of this disease
Mestre

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A Febre Maculosa é uma zoonose causada por bactérias do gênero Rickettsia. A relação natural entre vetores e seus hospedeiros vertebrados, durante o ciclo enzoótico, pode variar no tempo e espaço, evidenciando uma complexidade epidemiológica que deve ser investigada e monitorada como principal indicador para as ações de políticas públicas na prevenção e controle de casos humanos. Nesse aspecto, cães e equinos podem atuar como sentinelas, sinalizando a circulação de riquétsias em uma região, bem como servindo como dispersores de potenciais vetores, permitindo, assim, a amplificação de um foco já estabelecido ou o estabelecimento de novos focos. Com o objetivo de melhor entender a circulação de riquétsia através de pesquisa sorológica no perímetro urbano do município de Juiz de Fora, Minas Gerais, Brasil, considerado área endêmica para febre maculosa, sangue de cães e equinos de diversas regiões foram testados através da Reação de imunofluorescência indireta (RIFI) e Reação em Cadeia da Polimerase (PCR). As amostras resultaram 12,75% (31/243) de cães e 23,68% (9/38) de equinos pela RIFI com títulos ≥ 1:64. Na PCR foram utilizados iniciadores para amplificar fragmentos dos genes gltA e ompA. Nenhum resultado positivo foi encontrado. O maior número de cães positivos ocorreu na região leste da cidade, área com casos humanos descritos entre 2007 e 2008. Entretanto, a maior prevalência e concentração de títulos elevados em cães foi assinalada na região norte, área com casos recentes confirmados (2012), resultado semelhante àquele obtido para equinos, confirmando uma maior atividade dos focos naquela região. Áreas sem caso humano confirmado apresentaram animais com altos títulos, sugerindo um potencial risco para a população. Os resultados confirmam o papel de cães e equinos no ciclo enzoótico de riquétsias na região estudada e seu potencial como indicadores epidemiológicos para a investigação de casos e vigilância de ambiente na febre maculosa.
Spotted Fever is a zoonotic disease caused by bacteria of the genus Rickettsia. The natural relationship between vectors and their vertebrate hosts during the enzootic cycle, may vary in time and space, showing an epidemiological complexity that must be investigated and monitored as a key indicator for the actions of public policy in the prevention and control of human cases. This way, dogs and horses are sentinels, indicating the movement of Rickettsia sp. in a region and serving as dispersers of vectors, allowing the amplification of a focus or establishing new foci. To better understanding of the movement of Rickettsia sp. in the urban area of Juiz de Fora, Minas Gerais, Brazil , endemic area for spotted fever, blood of dogs and horses from different regions were tested by indirect immunofluorescence assay (IFA ) and Polymerase Chain Reaction ( PCR ). Samples resulted 12.75% (31/ 243) of dogs and 23.68 % ( 9/38 ) equine with titers ≥ 1:64. PCR primers were used to amplify fragments of the gltA and ompA genes, no positive results. The highest number of positive dogs occurred in the east region, an area with human cases reported between 2007 and 2008. However, the highest prevalence and concentration of high titers in dogs was reported in the northern region, an area with confirmed recent cases ( 2012 ). Similar results were obtained from equine, confirming a higher activity in the foci of the region. Areas with no human case presented animals with high titers, suggesting a potential risk for the population. The results confirm the role of dogs and horses in the enzootic cycle of Rickettsia in the region studied and its potential as epidemiological indicators for the investigation of cases and monitoring of spotted fever in the environment.

Orientador: Prof. Marcelo Beltrão Molento
Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Programa de Pós-Graduação em Ciencias Veterinárias. Defesa: Curitiba, 2007
Inclui bibliografia
A Febre Maculosa Brasileira (FMB) é uma zoonose de caráter endêmico, com casos humanos registrados em grande parte do território nacional. Entretanto o Paraná teve o primeiro registro da doença em abril de 2005, no município de São José dos Pinhais, região metropolitana de Curitiba. Com o objetivo de iniciar estudos da epidemiologia da FMB na região supracitada, foram colhidas amostras de sangue de eqüinos de carroceiros da região, através de adesão voluntária dos proprietários, e também de eqüinos e cães provenientes de 4 áreas consideradas foco (um foco humano e três focos animais). As amostras de um total de 83 eqüinos e 16 caninos foram processadas pela técnica de Reação de Imunofluorescência Indireta (RIFI), que é considerada padrão ouro para diagnóstico sorológico da FMB. Para as amostras dos eqüinos de carroceiros foram utilizados antígenos de Rickettsia rickettsii, e para as amostras dos animais dos focos, R. rickettsii e R. parkeri. Os resultados foram de soropositividade em 9,33% dos eqüinos de carroceiros. Dos eqüinos dos focos, houve positividade em 25% de soros para ambos os antígenos testados, e dos cães, 12,5% de soros positivos, todos apenas para R. rickettsii. Estes resultados representam necessidade de avaliação constante da FMB na região e devem ser complementados com pesquisa de positividade nos vetores do gênero Amblyomma sp. por meio de biologia molecular
Brazilian spotted fever (BSF) is an endemic zoonosis and human cases have been registered in almost all country. However, the first registered case in Paraná State was in April of 2005, in São José dos Pinhais, suburb area of Curitiba. In order to begin the studies about BSF epidemiology in this area, it was obtain serum samples of cart horses, by owners volunteer adhesion, and of horses and dogs in 4 focus (a human focus and three animal focus). Eighty three horses and sexteen dogs samples were prossessing by Indirect Immunofluorescence Assay (IFA), the gold standart test for serologic diagnostic of BSF. Cart horse samples were done using an antigen against Rickettsia rickettsii, and for the focus animals it was used R. rickettsii and R. parkeri antigens. It was determined soropositivity in 9,33% of cart horses. In focus horses 25% was positive to both antigens, and 12,5% of dogs were positive to R. rickettsii. The results demonstrate the necessity for constant evaluation of BSF in this area, and should be complemented whit positivity vectors of Amblyomma sp. gennus investigation by molecular biology

Les rickettsies du groupe boutonneux sont considérées comme des pathogènes émergents ou réémergents, transmis pour la plupart par des tiques à travers le monde. Durant les vingt dernières années, 16 nouvelles espèces ou sous espèces de rickettsies ont été caractérisées grâce aux outils épidémiologiques, microbiologiques et moléculaires. De nombreuses inconnues subsistent sur les relations entre les rickettsies et les tiques vectrices ainsi que sur l’éco-épidémiologie des maladies transmises à l’homme. Nous avons utilisé une approche moléculaire pour préciser l’épidémiologie des rickettsioses transmises par les tiques en France, au Maroc, au Tchad, à Djibouti, en Ethiopie, en Ouganda et en Guyane Française. Dans une deuxième partie de nos travaux, nous avons mis en place un protocole d’élevage de tiques, infectées ou non par des rickettsies. Des modèles de Rhipicephalus sanguineus infectés par Rickettsia conorii, Amblyomma variegatum par R. Africae et Dermacentor marginatus par « Rickettsia sp. DmS1 » ont pu ainsi être étudiés. La transmission verticale des rickettsies a été quantifiée. Les points forts de notre travail sont les résultats obtenus sur la relation de Rh. Sanguineus et R. Conorii l’agent pathogène de la fièvre boutonneuse méditerranéenne (FBM) endémique dans les pays du pourtour méditerranéen. Rh. Sanguineus est un vecteur et pourrait aussi jouer le rôle de réservoir de R. Conorii d’après nos résultats. La faible prévalence des tiques trouvées naturellement infectées dans la nature n’est cependant pas expliquée. Nous avons débuté l’étude des facteurs extrinsèques, comme la température sur l’écoépidémiologie de la FBM ainsi que sur les relations Rh. Sanguineus – R. Conorii. Nous avons montré que le réchauffement de la température du milieu environnemental rend ces tiques plus agressives pour l’homme, lors d’une enquête effectuée à Nîmes avec une approche épidémiologique, clinique, microbiologique, entomologique, complétée par des modèles expérimentaux. Le deuxième cas d’infection à R. Massiliae a été rapporté à cette occasion. La température semble être essentielle sur l’écologie et l’épidémiologie des rickettsioses transmises par les tiques et des expérimentations sont en cours pour étudier son influence sur les relations tiques-rickettsies
Tick borne rickettsioses have limited geographic distributions that are determined by their tick vectors. During the last 20 years, 16 additional rickettsial species or subspecies were identified and characterized by epidemiologic, microbiological and molecular tools, but there are many unknown questions on the relation between the rickettsiae and their vectors. We used a simple and effective molecular approach to study the epidemiology of the tick borne rickettsioses in France, in Morocco, in Chad, in Djibouti, in Ethiopia, in Uganda and French Guiana. The same approach can be extended to other countries. Three colonies of the naturally infected ticks: Rhipicephalus sanguineus infected with R. Conorii, Amblyomma variegatum infected with R. Africae and Dermacentor marginatus infected with Rickettsia sp. DmS1 and some colonies of not infected ticks were established in the laboratory. Studies of transmission, maintenance, infectivity, virulence, and pathogenicity of tick borne agents require the use of large numbers of live laboratory-raised ticks. A protocol of maintenance of laboratory ticks was established. The vertical transmission of these rickettsiae was studied and these infected ticks could be used to study other aspects of the relations ticks-rickettsiae. One of important point of our work is the results obtained on the relation of Rh. Sanguineus and R. Conorii, agent of the Mediterranean spotted fever (MSF) endemic in the countries of the Mediterranean area. Rh. Sanguineus are not only a vector but could be a reservoir of R. Conorii. A focus of spotted fever was investigated in France in May 2007. In addition to the originality of theses cases (ophthalmic involvements, the second reported case of R. Massiliae infection), we provide evidence that this cluster of cases was related to a warming-mediated increase in the aggressiveness of Rh. Sanguineus, leading to increased human attacks. The temperature seems to be essential on the ecology and the epidemiology of the tick borne rickettsioses. New studies are necessary to better understand the relation between the rickettsies and their vector - ticks

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Rickettsiosis is a human disease caused by bacteria of the genus Rickettsia, and its main vector ticks (Ixodidae), which are also considered reservoirs of the Spotted Fever Group rickettsiae in Brazil. Small mammals are frequent hosts for immature stages of rickettsial diseases vector ticks, and it is known that some species of small mammals are hosts amplifiers for Rickettsia sp..The aim of this study was to determine seropositivity against five species of Rickettsia and tick fauna of small mammals captured in periurban region of Uberlândia, MG, Brazil. For this purpose, 416 small mammals representing 13 species of wild rodents and marsupials were captured between the months of July 2011 to August 2012. Of these, 48 were infested with ticks of the genus Amblyomma and Ixodes, and 70 (16.8%) of the animals were seropositive for Rickettsia spp.. Our results indicate the circulation of four species of Rickettsia, R. rickettsii, R. parkeri, R. rhipicephali and R. bellii, in the periurban area of Uberlândia. The first two Rickettsia species are considered pathogenic to man and thus a more thorough investigation to confirm the species of bacteria and epidemiological aspects related to these Rickettsia spp. is the detection locations.
Riquetsiose é uma doença causada em humanos por bactérias do gênero Rickettsia, e tem como principais vetores os carrapatos, que são considerados também reservatórios de riquétsias do grupo da febre maculosa no Brasil. Frequentemente os estágios imaturos de carrapatos vetores de riquetsioses têm pequenos mamíferos como hospedeiros, e sabe-se que algumas espécies de pequenos mamíferos são hospedeiros amplificadores para Rickettsia sp.. O objetivo deste trabalho foi determinar a sororreatividade contra cinco espécies de riquétsias e a fauna de carrapatos de pequenos mamíferos na região periurbana de Uberlândia, MG, Brasil. Foram capturados, entre julho de 2011 a agosto de 2012, 416 animais representando 13 espécies de roedores silvestres e marsupiais. Destes, 48 estavam infestados com carrapatos dos gêneros Amblyomma e Ixodes, e 70 (16,8%) dos animais foram soropositivos para Rickettsia spp.. Nossos resultados indicam a circulação de quatro espécies de riquétsias, R. rickettsii, R. parkeri, R. rhipicephali e R. bellii, próximo à área urbana do município de Uberlândia. As duas primeiras espécies são consideradas patogênicas ao homem, evidenciando a necessidade de se realizar uma investigação mais minuciosa tanto para a confirmação das espécies de bactérias circulantes, quanto para a compreensão do ambiente que mantém as riquétsias nos locais de detecção.
Mestre em Imunologia e Parasitologia Aplicadas

Orientadores: Luiz Jacintho da Silva, Raquel Silveira Bello Stucchi
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Causada pela bactéria Rickettsia rickettsii e transmitida pelos carrapatos Amblyomma cajennense e Amblyomma aureolatum, a febre maculosa brasileira (FMB), após décadas de aparente silêncio epidemiológico, desde sua reemergência nos anos 80, vem figurando como importante problema de saúde pública no estado de São Paulo, sobretudo, em decorrência da aparente expansão das áreas de transmissão e da elevada letalidade a ela associada. O objetivo principal do presente estudo foi descrever características clínicas e epidemiológicas da FMB a partir da análise retrospectiva de casos confirmados da doença em áreas endêmicas no estado de São Paulo. Foi observado que manifestações inespecíficas como febre, cefaléia, mialgia e exantema, foram os sinais clínicos mais freqüentes e precoces. Embora variáveis, elevadas frequências de fenômenos hemorrágicos (22,9%-77,6%), icterícia (16,7%-52%), alterações neurológicas (27,2%-51,7%) e insuficiência respiratória (17,5%-62%) foram observadas. A taxa de letalidade média no estado de São Paulo no período de 2003 a 2008 foi de 29,6%(21,9%-40%). Trombocitopenia e elevação de transaminases hepáticas foram as alterações laboratoriais mais frequentes, ocorrendo em até 100% dos casos. Na faixa etária pediátrica, a FMB também se apresentou como doença severa, associando-se, embora em menor freqüência, às complicações acima mencionadas e à elevada letalidade (28,4%). Entretanto, em estudo comparativo entre o perfil clínico da FMB nos estados de São Paulo e Santa Catarina, observou-se no estado do Sul uma doença com evolução benigna, pequena frequência de sinais de gravidade e ausência de óbitos. Entre pacientes com FMB, as síndromes febris hemorrágica, icterohemorrágica e exantemática foram as mais comumente observadas (37,1%, 33,9%, 11,3%, respectivamente). Dentre os principais diagnósticos diferenciais da FMB, leptospirose, dengue e doença meningocócica foram as mais prevalentes (28,5%, 17,2%, 5,4%, respectivamente). Em 31% dos casos descartados para FMB não foi possível a identificação do diagnóstico etiológico. A doença foi mais incidente no gênero masculino e entre indivíduos da faixa etária de 20 a 49 anos. Em áreas em que o A. cajennense é o principal vetor foi possível observar maior incidência da doença entre o período de Junho a Setembro. Surtos de FMB se associaram a elevadas taxas de letalidade e a diferentes determinantes ecoepidemiológicos. Atividades de lazer e ocupacionais em áreas de mata, pastagem, próximas a coleções hídricas e/ou com presença de animais (cavalos, capivaras e, eventualmente, cães) foram consideradas importantes exposições de risco para infecção. O presente estudo permitiu observar que a infecção pela R. rickettsii no estado de São Paulo se associa à elevada morbimortalidade, sendo, aparentemente, mais severa que a febre das Montanhas Rochosas nos Estados Unidos. Exantema, icterícia e hemorragias são importantes marcadores clínicos que devem ser considerados na suspeição da doença e seus diagnósticos diferencias. O conhecimento das características epidemiológicas e dos fatores de risco para infecção deve fundamentar as ações de prevenção e controle da FMB. A maior severidade da infecção pela R. rickettsii no estado de São Paulo, a ocorrência de casos atípicos em Santa Catarina e a ausência de elucidação diagnóstica em casos descartados para FMB sugerem que cepas de R. rickettsii com distintos padrões de virulência, bem como outras espécies de riquétsias e, eventualmente, outros microorganismos transmitidos por carrapatos devam estar ocorrendo no Brasil
Abstract: Brazilian spotted fever (BSF) is caused by Rickettsia rickettsii and transmited by Amblyomma cajennense and Amblyomma aureolatum ticks. After decades of an apparent epidemiological silence, BSF reemerged as an important public health problem in São Paulo State in the 1980's, mostly because the possible expansion of its transmission areas and the high BSF related fatality-rate. The main objective of the present study was to describe clinical and epidemiological features of BSF through a retrospective analysis of BSF confirmed cases in endemic areas. Non-specific clinical signs like fever, myalgia, headache, and exanthema were the earliest and most frequent clinical signs. A high frequency of hemorrhagic manifestations (22.9%-77.6%), icterus (16.7%-52%), neurological signs (27.2%-51.7%), and respiratory distress (17.5%-62%) was also observed. Case-fatality ratio in São Paulo State between 2003 and 2008 was 29.6% (21.9%-40%). Thrombocytopenia and elevated liver enzymes were the most frequent laboratorial abnormalities, reaching 100% in some groups. In the pediatric age-group, BSF also presented as a severe disease with a slightly lower rate of clinical complications, but a similar high lethality rate (28.4%). Interestingly, when we compared the clinical profile of BSF cases between São Paulo state and Santa Catarina state, located in the southernmost part of Brazil, a milder disease, with a lower frequency of clinical signs of severity and no fatalities was observed in the latter. The most frequent clinical syndromes occurring in BSF patients were hemorrhagic, ictero-hemorrhagic, and exanthematic acute febrile syndromes (37.1%, 33.9% and 11.3% respectively). The most important differential diagnosis to BSF was leptospirosis, dengue fever, and meningococcal disease (28.5%, 17.2%, and 5.4%, respectively). In 31% of non-confirmed BSF cases, no etiological diagnosis was defined. A higher incidence of BSF was observed in males and in the 20-49 years age-group. In areas where A. cajennense is recognized as the most important vector, a higher BSF incidence was observed from June to September. Clusters of BSF were associated to elevated fatality rates and a wide number of ecoepidemiological determinants. Recreational and occupational activities in rural, periurban, and waterside areas, with presence of animals (mostly horses and capybaras, and eventually dogs) were considered the most important exposure risk factors to infection. The present study suggests a more severe pattern of R. rickettsii in São Paulo state when compared with Rocky Mountain spotted fever in United States. Exanthema, icterus, and hemorrhage are important clinical markers of BSF and should be considered in the suspicion of this disease and as a differential diagnosis. Knowledge of clinical, epidemiological, and risk factors for infection should be used to structure and improve the measures for control and prevention of BSF. Together, the higher severity of R. rickettsii infection in São Paulo state, the occurrence of atypical cases in Santa Catarina, and the unknown etiological diagnosis of a high percentage of post-tick exposure febrile patients suggest that more virulent R. rickettsii strains, other Rickttsiae species and, eventually, other tick-borne diseases could be occurring in Brazil
Doutorado
Clinica Medica
Doutor em Clínica Médica

A la suite de mortalites dans la population de coquilles st-jacques, pecten maximus, de la baie de st-brieuc, un procaryote intracellulaire a ete mis en evidence dans les branchies et apparente a l'ordre des rickettsiales sur la base de criteres ultrastructuraux. Un protocole de purification, reposant sur des centrifugations differentielles et isopycniques, a ete elabore. Les proteines constitutives majeures et plusieurs activites enzymatiques ont ete caracterisees. Des anticorps polyclonaux et monoclonaux ont ete prepares et appliques a des immunodiagnostics de la rickettsie. Des fragments de restriction de l'adn du procaryote ont ete clones et analyses en tant que sondes. Plusieurs fragments ont ete sequences et des oligonucleotides specifiques ont ete synthetises et utilises comme amorces selon la technique de pcr. L'application de plusieurs de ces methodes et reactifs de caracterisation a ete validee pour d'autres rickettsies de pectinides. L'epidemiologie de la rickettsiose a ete etudiee d'un point de vue spatio-temporel. A l'echelle europeenne, les taux et les degres d'infection se sont averes tres differents. En baie de st-brieuc, la disparition des animaux les plus infectes est apparue concomitante avec les mortalites a la fin de l'hiver. La transmission horizontale du procaryote a ete etablie dans le milieu et au laboratoire. L'impact de la rickettsiose sur la physiologie de l'hote se traduit par un bilan energetique negatif chez des animaux fortement infectes maintenus a base temperature. Il a ete demontre que les hemocytes de p. Maximus sont capables de produire des radicaux toxiques de l'oxygene lors de la phagocytose de particules, mais la rickettsie interfere avec le processus microbicide. Ces resultats sont en faveur chez p. Maximus de l'hypothese d'un role pathogene de la rickettsiose

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CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
A Febre Maculosa é uma doença infecciosa aguda, causada por bactérias do gênero Rickettsia e transmitida pela picada de carrapatos infectados. Não transmitida de pessoa a pessoa, sendo humanos hospedeiros acidentais dentro da cadeia ecoepidemiológica. A evolução da doença pode ocorrer de forma assintomática, sintomatologia discreta até formas graves com elevada taxa de letalidade. O presente trabalho objetiva elucidar a ecoepidemiologia da doença no município de Divinópolis, Minas Gerais, Brasil, que possui ocorrência da doença, inclusive com óbito. Foram analisadas áreas predispostas (presença de humanos, hospedeiros vertebrados e carrapatos positivos) e áreas de transmissão (casos de FM confirmados). Quatro áreas foram escolhidas para coleta, duas de transmissão e duas predispostas, todas pertencentes à mancha de transição entre bioma Cerrado e Mata Atlântica. Foram coletados 5.706 carrapatos com padrão sazonal bem definido, tendo maior incidência de larvas no outono e inverno, ninfas na primavera e adultos no verão. Os espécimes foram identificados através de chaves dicotômicas específicas e através de biologia molecular, sendo todos da espécie Amblyomma sculptum. Alíquotas foram destinadas a ensaios moleculares para detecção de Rickettsia spp. resultando em uma fêmea positiva para Rickettsia rickettsii para os genes gltA (gênero Rickettsia) e ompA (Grupo da Febre Maculosa), fato este demonstrado pela primeira vez no referido município. Reforçando a importância de mais estudos e atenção da vigilância epidemiológica nessas áreas.
Spotted Fever is an acute infectious disease caused by bacteria of the genus Rickettsia and transmitted by the bite of infected ticks. Not transmitted from person to person, being accidental human hosts within the ecoepidemiological chain. The evolution of the disease can occur asymptomatically, discrete symptomatology to severe forms with a high lethality rate. The present study aims to elucidate the epidemiology of the disease in the municipality of Divinópolis, Minas Gerais, Brazil, which has an occurrence of the disease, including death. Predisposing areas (presence of humans, vertebrate hosts and positive ticks) and transmission areas (confirmed SF cases) were analyzed. Four areas were selected for collect, two for transmission and two predisposed, all belonging to the transition spot between Cerrado and Atlantic Forest. A total of 5.706 ticks were collected with a well defined seasonal pattern, with a higher incidence of larvae in autumn and winter, nymphs in spring and adults in summer. Specimens were identified through specific dichotomous keys and through molecular biology, all of them being Amblyomma sculptum. Aliquots were assigned to molecular assays for the detection of Rickettsia spp. resulting in a female positive for Rickettsia rickettsii for the genes gltA (genus Rickettsia) and ompA (Spotted Fever Group), a fact that was demonstrated for the first time in said municipality. Reinforcing the importance of further epidemiological surveillance studies and attention in these areas.

Brazil has 58 species of chigger mites parasitizing different animal groups. Of these, only 6 species were reported for birds, being 1 of Apolonia, 2 of Eutrombicula, 1 of Neoschoengastia and 2 of Parasecia. The larvae of chiggers cause deep lesions and cutaneous reactions in the host, and are often cited as vectors of pathogens. In the United States, public health departments have come across the need for cataloging and knowledge of the biology of these mites as potential vectors of Rickettsia spp. In Brazil, the first cases of Brazilian Macular Fever (BMF) diagnosed in São Paulo were associated with these mites because they were found in outbreaks of the disease. However, its role in the epidemiology of rickettsial disease has not been confirmed. Another concern is dermatitis caused by the bite of these mites, popularly known as trombiculiasis. As it is well known, thrombiculid mites are not specific and several cases of bites in humans have been reported. Thus, the purpose of the present study is to know the current situation of chigger mites parasitizing birds, to clarify aspects of its taxonomic complexity, as well as to provide information about its participation in the epidemiology of Rickettsia in some localities in the southeastern region of the country. From the material examined in the present study were identified 8 species: Blankaartia shatrovi n. sp., Blankaartia sinnamaryi, Microtrombicula n. sp., Eutrombicula tinami, Eutrombicula goeldii, Parasecia n. sp.1, Parasecia n. sp.2, Parasecia n. sp.3. The mites were kept in absolute alcohol, were submitted to DNA extraction and investigation of the presence of Rickettsia. One of the samples showed 100% for Rickettsia felis. Therefore, for Brazil now, we have 13 species described for the entire national territory, with the exception of the Midwest region, and, in addition, the first record of the presence of Rickettsia in chigger mites on South America.
O Brasil possui 58 espécies de ácaros trombiculídeos parasitando diferentes grupos animais. E destas, somente 6 espécies foram reportadas para aves, sendo 1 de Apolonia, 2 de Eutrombicula, 1 de Neoschoengastia e 2 de Parasecia. As larvas de trombiculídeos provocam lesões profundas e reações cutâneas no hospedeiro, sendo por muitas vezes citados como vetores de agentes patogênicos. Nos Estados Unidos, os departamentos de saúde pública se depararam com a necessidade de catalogação e conhecimento da biologia desses ácaros por serem considerados potenciais vetores de Rickettsia spp. No Brasil, os primeiros casos de Febre Maculosa Brasileira (FMB) diagnosticados em São Paulo, foram associados a esses ácaros porque terem sido encontrados em focos da doença. No entanto, o seu papel na epidemiologia da riquetsiose não foi confirmado. Outra preocupação é a dermatite causada pela mordida desses ácaros, popularmente conhecida como trombiculíase. Como é bem conhecido, trombiculídeos não são específicos e vários casos de mordidas em humanos já foram relatados. Dessa forma, a proposta do presente estudo é conhecer a situação atual dos trombiculídeos de aves, esclarecer os aspectos de sua complexidade taxonômica, bem como fornecer informações sobre sua participação na epidemiologia de riquétsias em algumas localidades da região sudeste do país. Do material examinado no presente estudo foram identificadas 8 espécies: Blankaartia shatrovi sp. n., Blankaartia sinnamaryi, Microtrombicula sp. n., Eutrombicula tinami, Eutrombicula goeldii, Parasecia sp. n.1, Parasecia sp. n.2, Parasecia sp. n.3. Os ácaros mantidos em álcool absoluto foram submetidos à extração de DNA e investigação da presença de Rickettsia. Uma das amostras aprentou 100% para Rickettsia felis. Portanto, para o Brazil agora, temos 13 espécies descritas para todo o território nacional, com excessão da região Centro-Oeste, e, além disso, o primeiro registro da presença de Rickettsia em ácaros trombiculídeos da América do Sul.

Le genre Rickettsia, sont des petites bactéries Gram-négatives et symbiotes intracellulaires obligatoires des eucaryotes. Les Rickettsia sont surtout connus pour leur pathogénicité et pour provoquer des maladies graves chez l'homme et les autres animaux. À ce jour, 26 espèces valides de Rickettsies ont été identifiées dans le monde entier, dont 20 sont des agents pathogènes éprouvées. Toutes les espèces de Rickettsies validées sont associées à des arthropodes. Les phylogénies basées sur divers marqueurs moléculaires ont présenté des topologies discordantes, avec seulement R. bellii et R. canadensis qui ne sont classées ni parmi la fièvre boutonneuse groupe rickettsies, ni parmi le typhus groupe rickettsies. En utilisant les méthodes avancées de séquençage de génomes entiers, nous avons obtenu et analysé quatre séquences génomiques de Rickettsies : R. helvetica, R. honei, R. australis et R. japonica. Via la phylogénomique qui constitue une nouvelle stratégie permettant de mieux comprendre leur évolution, l'on remarque que ces micro-organismes ont subi une évolution génomique réduite au cours de spécialisation en intracellulaire. Plusieurs caractéristiques évolutives, comme le réarrangement des gènes, la réduction génomique, le transfert horizontal de gènes et l'acquisition d'ADN égoïste, ont formé les génomes Rickettsia d'aujourd'hui. Ces processus peuvent jouer un rôle important pour équilibrer la taille du génome afin de l'adapter au mode de vie intracellulaire. En outre, la pathogénicité des rickettsies peut être associée à la réduction génomique
The Rickettsia genus is composed of small, Gram-negative, bacteria that are obligate intracellular eukaryotic symbionts. Members of the genus Rickettsia are best known for infecting and causing severe diseases in humans and other animals. To date, 26 valid Rickettsia species have been identified worldwide, including 20 that are proven pathogens. All validated Rickettsia species are associated to arthropods that act as vectors and/or reservoirs. The phylogenies based on various molecular markers have resulted in discrepant topologies, with R. bellii and R. canadensis being classified neither among spotted fever nor typhus group rickettsiae. In this thesis, using the advanced whole genomic sequencing methods, we have and analyzed the genomic sequences from four Rickettsia species, including R. helvetica, R. honei, R. australis and R. japonica. Phylogenomics constitute a new strategy to better understand their evolution. These microorganisms underwent a reductive genomic evolution during their specialization to their intracellular lifestyle. Several evolutive characteristics, such as gene rearrangement, reduction, horizontal gene transfer and aquisition of selfish DNA, have shaped Rickettsia genomes. These processes may play an important role in free-living bacteria for balancing the size of genome in order to adapt the intracellular life style. In addition, in contrast with the concept of bacteria becoming pathogens by acquisition of virulence factors, rickettsial pathogenecity may be linked to genomic reduction of metabolism and regulation pathways

Dissertação de Mestrado Integrado em Medicina Veterinária
As doenças transmitidas por vectores são causadas por parasitas, bactérias ou vírus e são veiculadas pela picada de artrópodes (principalmente ixodídeos e mosquitos). Estas doenças afectam cães a nível mundial e a maioria delas têm potencial zoonótico. Nestas estão incluídas as doenças sob estudo: Erliquiose, Babesiose, Riquetsiose, Anaplasmose e Leishmaniose. A patogénese das doenças sob estudo, baseia-se fundamentalmente na resposta humoral exagerada e não protectora desenvolvida pelo hospedeiro, que provoca sintomas semelhantes aos de uma doença auto-imune. Isto é, ao contrário do que se podia pensar, não é o parasita em si que é responsável por todos os sintomas da doença, mas sim a resposta imunológica do hospedeiro à sua presença. No que respeita ao diagnóstico, os médicos veterinários podem ter a sua tarefa complicada devido à ausência de sintomas específicos e à presença de co-infecções (infecção por mais do que um parasita). A compreensão por parte do clínico, da patogenia e imunologia das doenças transmitidas por vectores, é uma ferramenta essencial para o diagnóstico rápido e preciso. Sob o ponto de vista zoonótico, se por um lado os canídeos actuam como sentinelas de doenças, por outro, são reservatórios de parasitas e importantes hospedeiros de transporte de vectores. Devido a isto um dos pontos fulcrais da prevenção destas hemoparasitoses é o controlo de vectores. Neste estudo foi realizado o rastreio de cinco doenças transmitidas por vectores, causadas pelos agentes: Ehrlichia canis, Anaplasma phagocytophilum, Rickettsia conorii, Babesia canis e Leishmania infantum. Este estudo incidiu sobre uma população de um canil de Setúbal, no qual foram testados 80 cães (29 machos e 51 fêmeas) escolhidos aleatoriamente. Os testes utilizados foram testes comerciais de imunofluorescência indirecta (IFI). Dos animais testados 58,75% dos animais encontravam-se infectados (20% co-infectados e 38,75% mono-infectados). As prevalências de anticorpos contra os 5 agentes foram: Rickettsia conorii (23,75%), Babesia canis (20%), Ehrlichia canis (16,25%), Leishmania infantum (16,25%) e Anaplasma phagocytophilum (12,5%).
ABSTRACT - Vector-borne diseases in kennel dogs, a study - Vector-borne diseases are caused by parasites, virus and bacteria, and transmitted through the bite of arthropods. These diseases have a great zoonotic potential and affect dogs worldwide. The five diseases studied (Ehrlichiosis, Babesiosis, Rickettsiosis, Anaplasmosis and Leishmaniasis) are included in this category of illness. The pathogenesis, it‘s based mainly on the humoral response developed by the host, that being exaggerated and not protective, causes symptoms similar to an autoimmune disease. Meaning, on the contrary to what might be thought, is not the parasite itself that is responsible for all the symptoms, but the host‘s immune response to their presence. Concerning the diagnosis, veterinarians can have their task complicated by the absence of specific symptoms and the presence of co-infection (infection for more than a parasite).The understanding of the clinical, the pathogenesis and immunology of vector-borne diseases, is an essential tool for rapid and accurate diagnosis. From the zoonotic point of view, dogs act as sentinels of disease, and are important reservoirs of parasites and carry vectors. Due to this, one of the key points for prevention of vector-borne diseases in animals and humans is vector control. In this study was performed the screening of five vector-borne diseases caused by the agents: Ehrlichia canis, Anaplasma phagocytophilum, Rickettsia conorii, Babesia canis and Leishmania infantum. This study focused on a population of a kennel of Setúbal, in which 80 dogs (29 males and 51 females), randomly chosen, were tested. The tests used were indirect fluorescence antibody (IFA) commercial. 58.75% of the animals tested animals were infected (20% co-infected and 38.75% single infections). The point prevalence for different pathogens was: Rickettsia conorii (23.75%), Babesia canis (20%), Ehrlichia canis (16.25%), Leishmania infantum (16.25%) and Anaplasma phagocytophilum (12.5%).

ANTECEDENTS. Les rickettsiosis, conegudes des de fa dècades, han passat a ser considerades malalties emergents en els últims anys. HIPÒTESIS. L’espècie de Rickettsia Bar 29, aïllada prèviament de paparres en la nostra àrea, causa infecció subclínica en humans. Aquesta espècie és un agent etiològic de la febre botonosa mediterrània (FBM) en la nostra àrea. El cultiu shell vial i la tècnica de la PCR són tècniques útils per a la detecció de rickèttsies. OBJECTIUS. Realitzar un estudi epidemiològic de la presència de la infecció per R. massiliae Bar 29 en humans, reservoris i vectors a Catalunya, i avaluar l’eficàcia de les tècniques diagnòstiques. METODOLOGIA. Estudis de seroprevalença en humans enfront de Bar 29 per immunofluorescència indirecta (MIF). Detecció de Bar 29 per MIF i Western inmunoblot, cultiu shell vial i PCR en pacients amb sospita de FBM i estudis per MIF en reservoris animals. Estudi en vectors per PCR i per la tècnica Tick-borne Bacteria Flow Chip (Master Diagnóstica). RESULTATS. S’ha trobat una prevalença del 8,5% enfront de Bar 29. L’associació entre Bar 29 i el contacte amb gossos ha estat estadísticament significativa (p= 0,018, OR 3,59 1,29-10,05). S’ha trobat reaccions creuades amb R. conorii en 8 dels sèrums i 10 d’ells han presentat tan sols anticossos enfront de Bar 29. En un primer estudi, de 15 sèrums estudiats enfront de R. conorii i Bar 29, 5 sèrums han presentat títols enfront de Bar 29 superiors a R. conorii, i l’especificitat de les reaccions s’ha confirmat per inmunoblot en dos d’ells. En un estudi posterior amb 136 sèrums, s’han trobat 53 sèrums positius almenys a un dels dos microorganismes, 12 sèrums amb títols superiors a Bar29 i dos casos només amb anticossos enfront de Bar 29. Durant el període 1994-2003, s’ha cultivat la sang de 59 pacients per shell vial. S’han obtingut 6 cultius positius, i s’han confirmat les soques per PCR-RFLP com a R. conorii. En l’estudi de reservoris animals del cicle silvestre (guineus) i del domèstic (ovelles, cabres i bòvids), s’han recollit mostres de sang de 135 guineus, 138 vaques braves, 137 ovelles i 98 cabres. En guineus s’ha trobat un 45,9% de les mostres amb anticossos enfront de R. massiliae Bar29 i un 25,1% a R. conorii. En les vaques se n’ha trobat un 39,8% amb anticossos enfront de R. massiliae Bar 29 i un 26% de R. conorii. En les ovelles, un 20,4% han estat positives a Rickettsia Bar 29 i un 24,8% a R. conorii. Finalment, de les cabres, un 26,5% han estat positives a Rickettsia Bar 29 i un 39,7% a R. conorii. De les guineus s’han recollit 137 paparres del complex Rhipicephalus sanguineus, que s’han analitzat per la tècnica Tick-Borne Bacteria Flow Chip. El 83,2 % han resultat positives a Rickettsia. Un cop seqüenciades aquestes rickèttsies, se n’han identificat 107 com a R. massiliae Bar 29, 3 com a R. aeschlimannii, una com a R. slovaca i en les altres tres sols s’ha arribat a Rickettsia sp.. S’han detectat, a més, 21 coinfeccions amb Bartonella sp., 12 amb Borrelia sp. i tres amb les dues a la vegada. S’han recollit, a més, 117 paparres en fase lliure que s’han identificat dins del grup R. sanguineus. S’han analitzat per la tècnica de Tick-Borne Bacteria Flow Chip. El 94% d’elles han resultat positives a Rickettsia del grup de les febres tacades. Se n’han pogut seqüenciar 85, 71 com a R. massiliae Bar 29 i 14 com a Rickettsia sp., i s’ha detectat en 23 paparres coinfecció amb Borrelia sp. CONCLUSIONS 1. La població humana sana de la nostra àrea presenta una seroprevalença enfront de R. massiliae Bar 29 del 8,5%. 2. Dels resultats dels dos estudis en pacients amb sospita de FBM es pot deduir que un nombre de casos de FBM dels nostres pacients podrien estar causats per R. massiliae Bar 29 . 3. Mitjançant el cultiu shell vial podem arribar a través de PCR i seqüenciació a identificar les diferents espècies de rickèttsies de les soques aïllades. 4. La nova tècnica Tick-Borne Bacteria Flow Chip ens augmenta notablement la sensibilitat respecte a la PCR ompA per a la detecció de rickèttsies en els vectors.
HYPOTHESIS: The Rickettsia species Bar 29 previously isolated from ticks in our area causes subclinical infection in humans. This species is an etiological agent of Mediterranean spotted fever (MSF) in our area. The centrifugation shell-vial culture and PCR are useful techniques for the detection of rickettsiae. GOALS: To perform an epidemiological study of the presence of the infection by R. massiliae Bar 29 in humans, reservoirs and vectors in Catalonia, and to assess the effectiveness of the diagnostic techniques. METHODOLOGY: Seroprevalence studies in humans and animal reservoirs against Bar 29 through indirect immunofluorescence (MIF). Detection of Bar 29 through MIF and Western immunoblot, shell-vial culture and PCR in patients suspected of MSF. Study in vectors through PCR and the Tick-Borne Bacteria Flow Chip technique (Master Diagnóstica). RESULTS: An 8.5% prevalence against Bar 29 was found. Cross reactions with R. conorii were found in 8 of them and 10 sera showed antibodies only against Bar 29. Out of 15 sera from patients with MSF against R. conorii and Bar 29, 5 sera showed higher titers against Bar 29 than against R. conorii, and 2 of them were confirmed by immunoblot. From a later study with 136 sera, 53 sera were found to be positive at least against one of the microorganisms, 12 sera with higher titers against Bar 29 and 2 cases with antibodies only against Bar 29. During the 1994-2003 period, blood from 59 patients was cultured by shell vial. 6 cultures were found to be positive, and the strains were confirmed by PCR-RFLP as R. conorii. In the study of animal reservoirs, blood samples from 135 foxes, 138 cows, 137 sheep and 98 goat were collected. From those samples, there was found a 45.9%, 39.8%, 20.4% and 26.5% of antibodies against R. massiliae Bar 29 and a 25.1%, 26%, 24.8% and 39.7% against R. conorii in foxes, cows, sheep and goats respectively. From the foxes, 137 ticks of the Rhipicephalus sanguineus complex were collected. They were analyzed through the Tick-Borne Bacteria Flow Chip technique. 83.2% of them were found to be positive for Rickettsia, 107 of which were identified as R. massiliae Bar 29. Besides, 21 coinfections with Bartonella spp., 12 with Borrelia ssp. and 3 with both of them there were detected. Furthermore, 117 ticks on the ground were collected, which were identified within the R. sanguineus group. 94% of them were found to be positive for Rickettsia from the spotted fever group, 71 as R. massiliae Bar 29, and coinfection with Borrelia sp. was detected in 23 ticks. CONCLUSIONS: 1. The healthy human population of our area shows an 8.5% seroprevalence against R. massiliae Bar 29. 2. From the results of the two studies in patients suspected of MSF, it can be deduced that a number of MSF cases could be caused by R. massiliae Bar 29 . 3. Using shell-vial culture, the various species of rickettsiae can be identified from the isolated strains through PCR and sequencing. 4. The new Tick-Borne Bacteria Flow Chip technique notably increases the sensitivity with respect to ompA PCR for the detection of rickettsiae in vectors.

Le genre Rickettsia est composé de bactéries Gram-négatives, intracellulaires obligatoires qui causent un éventail de maladies humaines à travers le monde. Des nouvelles techniques ont permis de progresser dans l'identification et la classification des Rickettsia, y compris l'introduction de méthodes moléculaires comme la comparaison de séquences de gènes (ARNr 16S, ompA, ompB, gltA, sca4 …) et la création du statut de sous-espèce. La génomique et les techniques de séquençage de nouvelle génération ont permis d’accéder à une nouvelle façon d’en apprendre davantage sur la pathogenèse et l'évolution de Rickettsia. La première partie de cette thèse est une revue sur les avantages et les limites de la génomique en taxonomie des procaryotes, tandis que la seconde partie est constituée des analyses génomiques de cinq sous-espèces de Rickettsia et une nouvelle espèce de Rickettsia. En utilisant des méthodes de séquençage à haut débit, nous avons obtenu les génomes de R. sibirica sibirica, R. sibirica mongolitimonae, R. conorii indica, R. conorii caspia, R. conorii israelensis et R. gravesii. Ce travail constitue la base d’autres études qui permettront de mieux comprendre les mécanismes physiopathologiques, l’évolution, et la taxonomie des rickettsies
The Rickettsia genus is composed of Gram-negative, obligate intracellular bacteria that cause a range of human diseases around the world. New techniques have led to progress in the identification and classification of Rickettsia, including the introduction of molecular methods like sequence comparison (16S rRNA, ompA, ompB, gltA, sca4 …) and the creation of the subspecies status. Genomics and next-generation sequencing have opened a new way to learn more about the pathogenesis and evolution of Rickettsia. The first part of this thesis is a review on the advantages and limitations of genomics in prokaryotic taxonomy, while the second part consists of the genomic analyses of five Rickettsia subspecies and a new Rickettsia species. Using high-throughput sequencing methods, we obtained the draft genomes of R. sibirica sibirica, R. sibirica mongolitimonae, R. conorii indica, R. conorii caspia, R. conorii israelensis, and R. gravesii. This work can be a basis of further studies to increase the understanding on the disease-causing mechanisms, evolutionary relationships, and taxonomy of rickettsiae

Dissertação de Mestrado Integrado em Medicina Veterinária
O otohematoma é uma entidade clínica frequente, que pode atingir vários animais domésticos, entre eles o cão e o gato. Caracteriza-se por ser uma tumefacção flutuante na orelha, cujo tamanho pode variar, e embora possa ocorrer em ambas as superfícies, geralmente está presente na sua superfície côncava. Foi realizado um estudo retrospectivo de 6 anos, em que foram recolhidos os dados das fichas clínicas de 55 animais com otohematoma. Neste estudo, os cães foram os maisafectados com 98,2% dos casos. Destes, a raça Indeterminada foi a que obteve maior prevalência de otohematomas (33,3%), o sexo masculino foi o mais atingido (79,6%), e a faixa etária mais representada foi a dos 9 aos 12 anos (38,9%). De referir que 41,9% dos cães apresentava um peso entre os 30 e os 40 kg, e 82,8% possuía orelha do tipo pendente. O único gato neste estudo era um macho da raça Europeu Comum, com 4 anos, 4,25 kg e orelha erecta. Os resultados obtidos permitem sugerir que os cães, e nestes, o sexo masculino, a idade adulta, o porte elevado e orelha do tipo pendente sejam factores de risco do otohematoma. Observou-se que 21,8% dos animais apresentavam história prévia de otite externa, enquanto que 43,6% possuíam otite externa no momento do diagnóstico do otohematoma. A riquetsiose foi a doença associada à otite externa mais prevalente, tanto anterior (28,6%), como concomitantemente (87,5%) ao otohematoma. Além disso todos os animais que possuíam esta doença quando o otohematoma foi diagnosticado, tiveram recidiva, independentemente da terapêutica utilizada. Quanto à terapêutica, a drenagem com agulha e injecção local de corticosteróides revelou ser uma boa opção no caso dos animais possuírem outras doenças, pois com esta técnica, o otohematoma não recidivou em 42,8% dos animais. A técnica que registou melhor eficácia nos animais sem outras doenças, foi a incisão em “S”,com drenagem e pontos, com 50% de eficácia, no entanto esta só foi aplicada em 2 animais. A drenagem com agulha revelou ser também uma boa técnica em animais sem outras doenças, pois registou uma eficácia de 45,4%, além de ter sido aplicada em 11 animais.
ABSTRACT - The aural hematoma is a common clinical entity, which can affect several domestic animals, including dog and cat. It is characterized by a fluctuating swelling in pinna, whose size can vary, and although it can occur on both surfaces, is usually present on its concave surface. A retrospective study of six years was performed, in which data were collected from clinical records of 55 animals with aural hematoma. In this study, dogs were the most affected with 98,2% of cases. Of these, Crossbreed dogs had the highest prevalence of aural hematomas (33,3%), male sex was the most affected (79,6%), and dogs between 9 to 12 years were the most represented (38,9%). Its recalled that 41,9% of dogs had a weight between 30 to 40 kg, and 82,8% had pendular pinna. The only cat in this study, was a European Shorthair male, with 4 years, 4,25 kg and erect pinna. The results may suggest that dogs, and in these, male sex , adult age, large size and pendular pinna seems to be risk factors of aural hematoma. It was observed that 21,8% of animals had a previous history of otitis externa, while 43,6% had otitis externa at diagnosis of aural hematoma. Rickettsiosis was the most prevalent disease associated with otitis externa, previous (28,6%) and concomitantly (87,5%) to aural hematoma. In addition, all animals that had this disease when the aural hematoma was diagnosed, relapsed, regardless of therapy used. As for therapy, the needle drainage andlocal injection of corticosteroids has proved to be a good choice for animals with other diseases, because with this technique, aural hematoma did not relapse in 42,8% animals. The most efficient technique in animals without other diseases, was the S-shaped incision, with drainage and sutures, with 50% of efficiency, however this was applied only in 2 animals. The needle drainage also proved to be a good technique in animals without other diseases, because had an efficiency of 45,4%, besides being applied em 11 animals.

La lutte anti-vectorielle est l’un des volets le plus important de l’entomologie médicale et nécessite une identification précise des vecteurs. Cette dernière décennie, la technique du MALDI-TOF MS a prouvé son potentiel comme outil rapide et efficace pour l'identification des arthropodes hématophages adultes. Dès lorsNous nous sommes intéressés à la mise au point d’un protocole d’identification des stades aquatiques de moustique par MALDI-TOF MS d’une part. D’autre part la détection d’un pathogène dans un arthropode n’implique pas forcement sa capacité à transmettre. L’incrimination d’un arthropode comme vecteur respecte certaines règles allant de la suspicion à la démonstration de sa compétence vectorielle au laboratoire. Afin de mieux comprendre l’épidémiologie de R. felis nous avons d’abord participé à une investigation conduite à l’Ile de la Réunion, en testant des puces, les seuls vecteurs biologiques connus jusqu’à présent. Ensuite Nous avons démontré le rôle potentiel des moustiques en particulier d’Anopheles gambiae à transmettre Rickettsia felis. Enfin nous avons utilisé le MALDI-TOF MS pour la détermination du statut infectieux d’Anopheles gambiae à R. felis. Nous proposons également un cycle probable de transmission de R. felis à l’homme incluant les psoques et les moustiques
Vector control is one of the most important aspects of medical entomology and requires accurate identification of vectors. Within the past decade, the MALDI-TOF MS technique has proven its potential as a fast and effective tool for identification of adult blood-sucking arthropods. From then on we were interested in the development of an identification protocol of aquatic stages of mosquitoes by MALDI-TOF MS. On the other hand, the detection of a pathogen in an arthropod does not necessarily mean its ability to transmit. Incrimination of an arthropod as vector follows certain rules ranging from suspicion to demonstrate its vector competence in the laboratory. To better understand the epidemiology of R. felis we first participated in an investigation conducted in Reunion, testing fleas, the only biological vectors known to date. We demonstrated the potential role of the mosquito particularly Anopheles gambiae, in the transmission of R. felis. Finally, we used the MALDI-TOF MS for the determination of the Anopheles gambiae infection status to R. felis. We also offer a probable transmission cycle of R. felis to man including psocids and mosquitoes

Fästingburna infektioner är ett ökande problem, och därmed även infektioner orsakade av Rickettsia spp. Syftet med studien var att undersöka risken att drabbas av en infektion efter bett av rickettsiainfekterad fästing. Specificitet och sensitivitet av Fluidigm-PCR jämfördes mot real time polymerase chain reaction (realtids-PCR) vid detektion av Rickettsia spp. i fästingar som bitit människor. Vidare undersöktes om det finns någon korrelation mellan fästingens blodsugningstid och serokonversion mot Rickettsia spp. 753 fästingar lämnades in av 104 deltagare i Sverige och på Åland. Fästingarna analyserades med realtids-PCR för att detektera gltA-genen som är specifik för Rickettsia spp. 3,5 % av fästingarna var positiva för Rickettsia spp. med realtids-PCR. Vid analysering med Fluidigm-PCR av samma material blev 1,3 % av proverna positiva. Beräkningar som gjordes med realtids-PCR som referens visade att Fluidigm-PCR har sämre specificitet och sensitivitet jämfört med realtids-PCR. Deltagare som serokonverterade (n=5) lämnade endast in rickettsianegativa fästingar som därför inte kunde kopplas till infektionen. Därmed kunde inga slutsatser dras om risken att drabbas av en infektion efter bett av rickettsiainfekterad fästing eller om det föreligger någon korrelation mellan fästingens blodsugningstid och serokonversion.
Tick-borne infections are increasing, including infections caused by Rickettsia spp. The aim of this study was to examine the risk of developing an infection after being bitten by a tick infected with Rickettsia spp. Specificity and sensitivity of a Fluidigm-PCR assay were compared to real time polymerase chain reaction (real-time PCR) assay when detecting Rickettsia spp. in ticks that had bitten humans. Possible correlation between the tick's feeding time and seroconversion against Rickettsia spp. was also investigated. A total of 753 ticks from 104 participants in Sweden and the Åland Islands (Finland) were analyzed with real-time PCR to detect the gltA gene specific for Rickettsia spp. 3.5 % of the samples were positive for Rickettsia spp. with real-time PCR, while only 1.3 % of the samples were positive with Fluidigm-PCR. Calculations showed that Fluidigm-PCR assay has lower specificity and sensitivity than the real-time PCR assay. Unfortunately, no conclusions could be drawn considering correlation between the tick's feeding time and seroconversion of the bitten humans since no participants who had seroconverted had also submitted ticks containing Rickettsia spp. Therefore, no conclusions could be drawn considering the risk of developing an infection after being bitten by a tick infected with Rickettsia spp.
STING-studien

Este estudo objetivou estudar os carrapatos e a infecção por patógenos, e sua correlação com os vertebrados desta região entre 10/2007 e 09/2008 foram realizadas mensalmente doze visitas a uma área de floresta ombrófila densa da Amazônica na Estação Ecológica de Samuel. Foram coletados, 60 aves, 36 mamíferos, 14 répteis e 11 anfíbios. Em vida livre foram realizadas 80 coletas de carrapatos em um total de 39.600m em cerca de 2.640min (44h) e coletados 265 carrapatos adultos de quatro espécies, sendo elas: A. scalpturatum (81), A. latepunctatum (84), A. oblongogutattum (18), A. naponense (69) e 597 ninfas de Amblyomma sp. Dos 104 adultos (11,8% do total de carrapatos) foram testados para Anaplasmataceae, obtive-se 27 (25,9%) positivos e mais 36 adultos (total de 140) e 11 carrapatos foram positivos (1) A. scalpturatum, (4) A. latepunctatum, (3) A. naponense. É notável o impacto da hidrelétrica sobre a fauna de pequenos mamíferos.
This study was aimed to analyze the ticks and the pathogenic infections, and their correlation with the vertebrates of such a region from October 2007 to September 2008, 12 surveys were monthly carried out at the Samuel Ecological Station in an Amazonian dense ombrophilous forest. They were collected from 60 birds, 36 mammals, 14 reptiles, and 11 amphibians. Eighty(80) collections of free-living ticks were carried out within an area of about 39.000 meters, during 2.640 minutes (44 hours), and 265 adult ticks or four species were picked up as following: 81 A.scalpturatum, 84 A. latepunctatum, 18 A. oblongogutattum, 69 A. naponense , and 597 Amblyomma sp. nymphs. 104 the adults, which accounted for 11.8% of total of ticks collected, were tested for Anaplasmataceae, and 27 (25.9%) were positive. In the Rickettsia spp. sample, additionally to the above mentioned 104 ticks tested, 36 adult ticks (out of a total of 140) were positive. It is noteworthy the impact caused by the hydropower plant on the small mammal fauna.

博士
高雄醫學大學
醫學研究所博士班
103
Background. In Taiwan, Q fever, rickettsioses (scrub typhus and murine typhus), and leptospirosis are common rickettsioses and zoonoses. Material and methods. The clinical characteristics of Q fever and rickettsioses were investigated by a multi-center and retrospective clinical cases study. The retrospective serological studies included the infective markers (C-reactive protein [CRP] and procalcitonin [PCT]), cytokines, and antibodies of atypical pneumonia pathogens and spotted fever group rickettsiosis (SFGR). The confirmed Q fever cases were followed up for study of possible complications and chronic Q fever. Q fever and community-acquired pneumonia (CAP) was investigated by a prospective study. The epidemiology studies of human Q fever and animals involved nationwide databases of reported Q fever and husbandry, and the geographic information software (GIS) was applied. Animal specimen was collected for detection of Q fever antibody and Coxiella burnetii gene. Finally, the multilocus variable number of tandem repeat analysis (MLVA) was used for molecular epidemiology study of human and animal Q fever. Results. In this study, human SFGR, particular infection of Rickettsia felis, were identified. Fever with hepatitis was the major presentation of Q fever. The percentage of animal contact history was low in Q fever and the days from disease onset to hospital visit and diagnostic examinations ordered by clinicians were different between Kaohsiung and Pingtung region. Comparison between Q fever/rickettsioses and leptospirosis, elevated liver enzymes, residence in or traveling to mountain area, and hepatomegaly or splenomegaly were the characteristics for the former, but the pigs contact history, sweating, diarrhea, and impaired renal function were those for later. The values of CRP and PCT increased in Q fever/rickettsioses and were correlated to clinical response to treatment. The changes of cytokines were different between each rickettsiosis, and the percentage of increased IL-2 and IL-10 in Q fever were significantly higher than scrub typhus. The seropositive rate of Mycoplasma pneumoniae IgM in Q fever (57.8%) was higher than scrub typhus (7.7%) and murine typhus (9.0%) (p<0.001). In the prospective study of adult CAP, 7.2% cases were seropositive for Q fever antibodies, but all were past-infection. Although 73.5% followed Q fever patients were found to have abnormality of cardiac valve, no chronic Q fever was identified. By analysis of Q fever database applied from Taiwan CDC, the confirmed Q fever mainly distributed in Kaohsiung and Pingtung regions, and cases have increased since 2004, but decreased in 2008 and 2011. The distribution of Q fever cases was similar with cattle and goat, however, the trend of case number was correlated with the number of goat rather than cattle. In the study of clinical and animal specimen, the seropositive rate and PCR positive rate of vaginal secretion were higher in goat (22.2% and 40.0%) than in cattle (13.8% and 27.2%). By MLVA analysis, the major strains of C. burnetii were different between goat and cattle, and the strains of human were more closed to those of goat rather than cattle. The identified strains were different from those of other countries. Conclusions. This study not only illustrated the characteristics and epidemiology of Q fever and rickettsioses in Taiwan, but also identified the closed association between human Q fever and goat by molecular investigation. Further study in combination of clinical and veterinary medicine is necessary to illustrate the epidemiology of this zoonosis.

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2021
As rickettsioses são zoonoses causadas por bactérias pertencentes ao género Rickettsia, e transmitidas por artrópodes infetados. São cocobacilos gram-negativo intracelulares obrigatórios, dividindo-se em dois grupos, o Grupo das Febres Exantemáticas e o Grupo do Tifo. A FEN, pertencente ao grupo das Febres Exantemáticas, é a zoonose mais prevalente na Europa, principalmente, nas áreas rurais. O seu agente etiológico é Rickettsia conorii, tendo-se identificado em Portugal, Rickettsia conorii subsp. conorii e Rickettsia conorii subsp. israelensis. A doença causada por esta infeção, na maior parte das vezes benigna, apresenta-se geralmente com um quadro inespecífico de febre, cefaleias, mialgias, náuseas e vómitos, surgindo um exantema maculo-papular com atingimento palmo-plantar, geralmente 3 a 5 dias depois do início dos sintomas. A presença de escara é característica, mas não é patognomónica nem obrigatória para o diagnóstico. Nos casos mais graves, pode associar-se a choque séptico, falência multiorgânica e morte. O seu diagnóstico é essencialmente clínico, confirmando-se com serologia e, para diagnóstico definitivo da subespécie, isolamento do agente por cultura ou deteção do DNA por técnica de Polimerase Chain Reaction (PCR). O início precoce de doxiciclina é essencial para o prognóstico do doente. Rickettsia conorii subsp. israelensis é considerada a subespécie mais virulenta, associada a uma doença mais grave, com maior número de internamentos em unidades de medicina intensiva e uma taxa de mortalidade mais elevada. A discussão de um caso clínico de FEN causada por Rickettsia conorii subsp. israelensis tem o intuito de reportar uma apresentação rara desta infeção, a purpura fulminans, assim como uma sequela, não descrita anteriormente na literatura, de neuropatia periférica sensitivo-motora. Por fim, realçar a importância da elevada suspeição clínica e início precoce da terapêutica antibiótica, principalmente tendo em conta a frequente ausência de tache noir e/ou exantema na apresentação inicial da infeção por Rickettsia conorii subsp. israelensis.
Rickettsiosis are zoonoses caused by bacteria belonging to the genus Rickettsia, transmitted by infected arthropods. Rickettsia is an obligately intracellular gram-negative coccobacillus, divided into the Spotted Fever group and the Typhus group. The Mediterranean Spotted Fever (MSF), belonging to the Spotted Fever group, is the most prevalent in Europe, mainly in rural areas. Its etiological agent is Rickettsia conorii and in Portugal has Rickettsia conorii subsp. conorii and Rickettsia conorii subsp. israelensis. This infection, mostly benign, presents with an initial nonspecific fever associated with headache, myalgia, nausea, and vomiting, appearing a maculopapular rash with palmoplantar affection, 3 to 5 days later. The presence of eschar is characteristic, but not pathognomonic or obligatory for diagnosis. In the most severe cases, it can be associated with septic shock, multiorgan failure, and death. Its diagnosis is essentially clinical, confirmed with serology, and for definitive diagnosis of the subspecies, isolation of the agent by culture or detection of DNA by PCR. The early onset of doxycycline is essential for the patient’s outcome. Rickettsia conorii subsp. israelensis is the most virulent subspecies, associated with a more serious disease, with a higher number of hospitalizations in intensive care and a higher mortality rate. The discussion of the clinical case of MSF caused by Rickettsia conorii subsp. israelensis has the purpose of reporting a rare presentation of this infection, the purpura fulminans, as well as an outcome of peripheral sensory-motor neuropathy, not previously described in literature. Finally, emphasize the importance of high clinical suspicion and early onset of therapy, especially considering the frequent absence of tache noir or rash in the initial presentation of infection by Rickettsia conorii subsp. Israelensis.

Orientação : Pedro Faísca ; co-orientação : Odete Almeida ; responsabilidade externa : Liliana Fernandes
As doenças transmitidas por vectores ocorrem pela picada, nomeadamente de mosquitos, flebótomos, ixodídeos e pulgas podem ser causadas por parasitas, bactérias ou vírus. Para além de algumas apresentarem prevalências elevadas e/ou serem endémicas, têm importância acrescida por muitas serem zoonoses. Nos últimos anos, tem havido uma emergência ou reemergência de algumas doenças, e muitas associadas a alterações epidemiológicas, sobretudo na sua expansão geográfica. Vários factores afectaram este fenómeno, especialmente o aumento da temperatura, que leva à alteração do microclima, biologia e comportamentos alimentares dos vectores, bem como as viagens de animais para zonas endémicas e a sua importação destas zonas. O objectivo do presente estudo foi conhecer e caracterizar a existência de doenças transmitidas por vectores no cão, numa clínica veterinária em Cascais, onde foram recolhidos dados retrospectivamente de 1 de Janeiro de 2010 e 29 de Fevereiro de 2012, e relacionou-se o seu diagnóstico com possíveis factores de risco, como a idade, a época do ano, sexo e raça. A amostra foi obtida através da base de dados da clínica, tendo sido analisados 42 cães com suspeita de Babesiose, 43 de Ehrlichiose, 46 de Rickettsiose e 190 de Leishmaniose. A Leishmaniose Canina foi a infecção com maior número de casos positivos, 53 em 190 (28%), seguida da Rickettsiose Canina com 40 em 46 (87%). A Babesiose apresentou apenas 8 casos em 42 (19%) e Ehrlichiose com 4 em 43 (9%). Foram também verificadas co-infecções entre Rickettsia conorii e Ehrlichia canis (n=4), Rickettsia conorii e Babesia canis (n=8) e Leishmania infantum e Rickettsia conorii (n=2). Os factores analisados não revelaram diferenças estatisticamente significativas no diagnóstico destes agentes.
Vector borne diseases, caused by parasites, bacterias or viruses can be transmitted by mosquitoes, sandflies, fleas and tick bites. The prevalence of these diseases is increasing, and some of them have a zoonotic character, which strengthen their importance. The aim of this study was to determine and characterize vector borne diseases in dogs, at a Veterinary Clinic located in Cascais (Portugal). A retrospective study was conducted, comparing vector borne diseases diagnosis with possible risk factors, such as age, time of year, sex and breed, in that particular geographic location, during the period of 1 January 2010 to 29 February 2012. There were a total of 207 patients of dogs who presented signs compatible with vector borne diseases: 42 suspected of Babesiosis, 43 suspected of Ehrlichiosis, 46 canine Ricketsiosis and 190 suspected of Leishmaniosis. It was concluded that in the area covered by this study, the elected factors didn’t have any statistic correlation with the positive diagnosis. It was also determined that Leishmaniasis had a bigger onset of positive cases, 53 out of 190 (28%), Ricketsiosis had 40 out 46 (87%), Babesiosis, 8 out of 42, (19%) and Ehrlichiosis, 4 out of 43, 9%.