Tesis sobre el tema "Rickettsia infections"

Orientadores: Luiz Jacintho da Silva, Raquel Silveira Bello Stucchi<br>Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas<br>Made available in DSpace on 2018-08-18T15:04:22Z (GMT). No. of bitstreams: 1 Angerami_RodrigoNogueira_D.pdf: 7670692 bytes, checksum: f6634d52b90eaf383ed11a180b9179aa (MD5) Previous issue date: 2011<br>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<br>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<br>Doutorado<br>Clinica Medica<br>Doutor em Clínica Médica

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

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<br>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

The spotted fever rickettsia, Rickettsia helvetica, is an endemic tick-borne bacteria in Sweden. It causes infections in humans, manifested as aneruptive fever, headache, arthralgia and myalgia, and sometimes an inoculation eschar or a rash. There have also been two known cases of human infections with R. felis in Sweden. The present thesis starts by investigating dispersal of ticks and Rickettsia spp. by migrating birds flying from Africa to Europe. Almost 15,000 birds were searched and 734 ticks collected, mainly of the species Hyalomma marginatum complex. Almost half (48%) of the ticks were infected with Rickettsia spp., 96% of which was R. aeschlimannii, the remaining R. africae and undefined species. The next study focused on questing ticks over a large area in Sweden and determining the prevalence of Rickettsia spp., Anaplasma spp. and Coxiella burnetii. Rickettsia spp. was found in 9.5-9.6% of the ticks and A. phagocytophilum in 0.7%; no C. burnetii was found. The last three papers in the thesis focused on the clinical presentation of rickettsiosis, the symptoms associated with the infection in general and particularly in patients with neurological complications. A tick-exposed population in Sweden was investigated to gain a better understanding of symptoms due to rickettsioses, also in relation to co-infections with other tick-borne bacteria. Based on symptoms, it was not possible to distinguish what pathogen caused the infections. Most patients had erythema migrans, some had serological reactions to Rickettsia spp., Borrelia spp. or co-infections by Rickettsia spp., Borrelia spp. and/or Anaplasma spp. In the fourth and fifth papers, we found associations between antibodies against Rickettsia spp. and sudden deafness (in 10-24% of patients) and facial nerve paralysis (in 8.3-25% of patients). In three patients R. felis was detected in the cerebrospinal fluids.    Briefly, the thesis helps to clarify our knowledge about tick dispersal, shows a narrower prevalence estimate of Rickettsia spp. in Swedish ticks, and illuminates symptoms of rickettsioses and co-infections with other tick-borne infections. It also shows that presence of erythema migrans may be explained by more than Lyme disease and indicates a possible association between rickettsiosis and sudden deafness and facial nerve paralysis.

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.

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

Globally, vector borne diseases cause more than a million deaths each year and more than a billion infections in humans. Ticks are of big medicinal importance since they can transmit pathogens that can cause serious infections. Some recently discovered pathogens that can cause infections in humans are Anaplasma phagocytophilum (A. phagocytophilum) that can cause human granulocytic anaplasmosis (HGA) and Candidatus Neoehrlichia mikurensis (N. mikurensis) that can cause Neoehrlichiosis. It is still widely unknown how prevalent these pathogens are, if ticks can be infected with both of these pathogens and if these pathogens can be transovarially transmitted from adult female to egg and larvae. This study aims to screen for these pathogens in collected ticks from southern Sweden and to detect eventual co-infections and transovarial transmission. A real-time qPCR assay targeting the 16S rRNA gene of N. mikurensis and other Anaplasmataceae was applied on 1356 Ixodes ricinus (I. ricinus) ticks collected from 5 sites in southern Sweden. Positive samples were subjected to Sanger sequencing. A. phagocytophilum occurred in 4.64 % of the ticks, N. mikurensis occurred in 1.33 % of the ticks and also Rickettsia was found to occur in 6.27 % of the ticks. No co-infection was detected. Some samples of tick larvae showed positive results after qPCR, indicating transovarial transmission, but none of the sequences were readable.

Anaplasma phagocytophilum and A. marginale are the etiologic agents of bovine anaplasmosis and human granulocytic anaplasmosis, respectively. As obligate intracellular pathogens, binding and entry of host cells is a prerequisite for survival. The molecular events associated with these processes are poorly understood. Identifying the adhesins mediating binding, delineating their key functional domains, and determining the molecular determinants to which they bind not only benefits better understanding of Anaplasma spp. pathobiology, but could also benefit the development of novel approaches for protecting against infection. We previously demonstrated that A. phagocytophilum outer membrane protein A (ApOmpA) is critical for bacterial binding and entry host through recognition of α2,3-sialic acid and α1,3-fucose of its receptors, including 6-sulfo-sLex. In this study, we determined that two amino acids, G61 and K64, within its binding domain (ApOmpA59-74), are essential for ApOmpA function. We also confirmed the ability of ApOmpA to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. We next extended our studies to A. marginale as it also expresses OmpA (AmOmpA) and its role in infection has not been studied. Molecular models of ApOmpA and AmOmpA were nearly identical, especially in the ApOmpA binding domain and its counterpart in AmOmpA. Antisera raised against AmOmpA or its putative binding domain inhibit A. marginale infection. AmOmpA G55 and K58 are contributory and K59 is essential for AmOmpA to bind to host cells. AmOmpA binding is dependent on α2,3-sialic acid and α1,3-fucose. Coating inert beads with AmOmpA conferred the ability to bind to and be taken up by host cells, confirming that it acts as an adhesin and invasin. 6-sulfo-sLex is dispensable for AmOmpA binding and A. marginale infection. ApOmpA works cooperatively with Asp14 (14-kDa A. phagocytophilum surface protein) to promote optimal infection of host cells. We found that Asp14 is conserved across A. phagocytophilum strains and in A. marginale and confirmed the ability of Asp14 to act as an adhesin and invasin as it conferred adhesiveness and invasiveness to inert beads. Collectively, this work advances our understanding of A. phagocytophilum and A. marginale adhesion and invasion of host cells.

Les rickettsies sont de petites bactéries à Gram-négatif associées à différentes espèces d'arthropodes. Leur nature intracellulaire stricte a longtemps été un obstacle à la compréhension des mécanismes moléculaires responsables de leur pathogénicité qui restent mal connus. L'adhésion bactérienne, qui est une étape clef de l'invasion des tissus de l'hôte, met en jeu les protéines rOmpA et rOmpB (rickettsial outer membrane proteins), identifiées depuis longtemps comme des antigènes de surface majeurs des rickettsies. L'objectif de cette thèse a été de caractériser une autre adhésine potentielle de Rickettsia prowazekii récemment identifiée, soit Adr2. La stratégie mise en œuvre a été basée sur la production d'anticorps monoclonaux spécifiques de cette protéine, dont une forme recombinante a été exprimée. Cet outil a permis, non seulement de localiser Adr2 à la surface des rickettsies, mais aussi d'apporter la preuve de son rôle dans le phénomène invasif puisque les anticorps anti-Adr2 diminuent significativement la cytotoxicité des rickettsies sur les cellules épithéliales. Un autre aspect de la pathogénicité que nous avons abordé concerne la mobilité des rickettsies du groupe boutonneux, fonction attribuée à la protéine RickA lorsque ce travail a été initié. La résolution des images obtenues par immunofluorescence, ou par microscopie électronique après marquage immunogold, montrent que l'expression de RickA est non-polarisée et répartie sur la surface entière de Rickettsia conorii. Enfin, plusieurs protéines recombinantes ont été utilisées dans des tests de screening sérologiques avec des sérums de patients infectés par diverses rickettsies, avec des résultats encourageants. L'ensemble de ces résultats contribue à une meilleure connaissance de la pathogénicité des bactéries du genre Rickettsia.

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<br>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

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&lt;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<br>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&lt;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

Many insects form symbioses with maternally inherited, intracellular bacteria, which can have major effects on the ecology and evolution of the insect host. Here I investigated the interaction between a global agricultural pest, Bemisia tabaci (the sweetpotato whitefly), and a Rickettsia bacterial symbiont. Rickettsia had previously been tracked sweeping through field populations of B. tabaci in the southwestern USA and had been shown to dramatically increase whitefly fitness under laboratory conditions. In contrast, the Rickettsia present in whiteflies in Israel has few observable fitness effects and is declining in frequency in field populations. I explored the population dynamics of Rickettsia in B. tabaci field populations in the USA and Israel, and assessed the genetic diversity of the Rickettsia in these populations. In laboratory experiments, there was no observable effect of Rickettsia on the heat shock or constant temperature tolerance of USA B. tabaci. Instead, whitefly genetic background appears to influence the effects of Rickettsia. Lastly, analysis of the genome sequence of Rickettsia provided insights into the mechanism of the fitness benefit and evolutionary history of the bacterium. Taken together, these integrated ecological, physiological and genomic studies provide some explanation for the contrasting and wide-ranging phenotypes associated with whitefly Rickettsia, and provide support for the hypothesis that the fitness benefit provided by Rickettsia is context dependent. The Rickettsia symbiosis exhibits geographically distinct population dynamics, is affected by whitefly genotype, and may involve manipulation of host plants and/or defense against pathogens rather than nutritional supplementation. Overall, these results highlight the important role that microbial symbionts may play in the adaptation of invasive species to changing environments.

Rickettsia africae is a gram-negative bacterium which causes African tick bite fever (ATBF) in humans. ATBF is a febrile disease mainly affecting travellers to Southern Africa. This bacterium is known to be transmitted by Amblyomma hebraeum and Amblyomma variegatum ticks. In Southern Africa, the principal vector is A. hebraeum. This project was performed in a rural community in Mpumalanga province and aimed at addressing knowledge gaps of R. africae infection in A. hebraeum ticks. Infection rates in adult ticks and larvae as well as transovarial transmission efficiency of R. africae from the tick to its offspring were determined. To accomplish this, 106 adult A. hebraeum ticks were collected from cattle from Utah A and 106 from Welverdiend A. Larvae (n=1060) were collected by dragging at each of the two targeted dip tanks. Engorged female A. hebraeum ticks (n=53) were also collected from cattle from each of the two dip tanks and they were incubated in a humidity tank to oviposit and egg masses were collected from each tick. DNA was extracted from the engorged ticks and the egg masses as well as from the adult ticks and the larvae. After DNA quantification, a real-time quantitative PCR targeting Rickettsia gltA gene was performed to screen all samples for Rickettsia DNA. The gltA gene is common in all Rickettsia species. Samples positive for the gltA gene were subjected to conventional PCR targeting the ompA gene, which is specific for the Spotted Fever Group to which R. africae belongs. The samples positive for amplicons of ompA gene were sequenced and all the sequenced samples were found to be 99.98% identical to R. africae sequences from GenBank. From the sampled adult ticks, 13.43% tested positive for R. africae and 13.20% of the larvae also tested positive. The infection rate for larvae and adult ticks collected from Utah A was 15.09% and those from Welverdiend A was 11.79%. From these results, there were no notable differences in the infection rates of ticks at different stages of development. R. africae infection rates for the two study sites were found to be similar which can be an indication of even distribution of R. africae in this area. Transovarial transmission was found to be 100% in engorged female ticks collected from Utah and 71.43% in ticks from Welverdiend, which indicates a high transmission rate of the pathogen to the offspring of the vector. The presence of R. africae in A. hebraeum ticks from this area is a cause of concern since there are chances of people getting ATBF after bites by these ticks.<br>Dissertation (MSc)--University of Pretoria, 2019.<br>Veterinary Tropical Diseases<br>MSc<br>Unrestricted

This study aimed to evaluate the ability of nymphs of Amblyomma ovale naturally infected with Rickettsia parkeri (Atlantic Forrest strain) in transmitting it to Cavia porcellus (guinea pigs), and analyze the infection in these animals. A total of 26 guinea pigs divided into three groups were used: G1 - 10 guinea pigs infested with nymphs of uninfected A. ovale; G2 - 10 guinea pigs infested with nymphs of A. ovale naturally infected with R. parkeri (Atlantic Forrest strain) and G3 - 6 uninfected guinea pigs. A tick infestation chamber was fixated on the animals, where 25 nymphs of A. ovale, either infected or not, were placed. In the first study, the vector competence of A. ovale nymphs in the transmission of R. parkeri (Atlantic Forrest strain) for C. porcellus (animals of G1 and G2) was evaluated. After the period of parasitism, engorged nymphs were collected and stored in a B.O.D incubator. To assess the anti-Rickettsia spp antibodies, blood was collected at 7, 14, 21 and 28 days post infestation (DPI) being evaluated by indirect immunofluorescence assay (IFA). To identify the multiplication of Rickettsia in the tissue from guinea pigs a polymerase chain reaction was carried out at 7, 10, 14 and 28 DPI. To verify the vector competence of nymphs, parasite periods, the percentage of molting, transstadial survival and IFA were analyzed. The average period of parasitism in G1 was 6.6 days and 6 days in G2. The average percentage of molting (nymph to adult) was 95%. In G2, the survival of transstadial Rickettsia was confirmed in 100% (PCR) and 80% (hemolymph test) of adults. In serological analysis, 100% of G1 animals were seronegative and 80% were seropositive in G2. No riquetsial DNA was detected in the tissues of animals. In the second study, the profile of experimental infection caused by rickettsia in guinea pigs (animals from G1, G2 and G3) were analyzed, seeking to identify the clinical, histopathological and hematological profile. Blood samples for hematological analyzes were performed in the same periods of the previous study and the collection of tissue for histopathological analyzes, occurred at 10 and 28 DPI. In serology, animals from G1 and G3 were negative and 80% of the G2 positive. The observed hematological results were: G1 - leukopenia at 7 DPI, increased total plasma proteins (TPP) and decreased platelets at 7, 14 and 21 DPI, G2: leukocytosis, neutrophilia and monocytosis at 7 DPI, increased platelets at 14 DPI and decreased PPT at 21 DPI. Histopathology observed: G1 - diffuse splenic hemosiderosis at 28 DPI (20%), G2 - diffuse splenic hemosiderosis at 10 DPI (10%), diffuse pulmonary congestion at 10 and 28 DPI (30%) and multifocal splenic follicular hyperplasia at 28 DPI (20%); G3 - diffuse pulmonary congestion at 10 DPI (33%). It was concluded that nymphs of A. ovale presented vector competence in the transmission of R. parkeri (Atlantic Forrest strain) to guinea pigs, being possible to determine an acute infection of subclinical character in these hosts.<br>Este estudo teve como objetivos avaliar a capacidade de ninfas de Amblyomma ovale naturalmente infectadas com Rickettsia parkeri (cepa Mata Atlântica) em transmiti-la para Cavia porcellus (cobaios) e, analisar a infecção nestes animais. Utilizou-se 26 cobaios divididos em três grupos: G1 - 10 cobaios infestados com ninfas de A. ovale não infectadas, G2 - 10 cobaios infestados com ninfas de A. ovale naturalmente infectadas com R. parkeri (cepa Mata Atlântica) e G3 - 6 cobaios não infestados. Foi fixada uma câmara de infestação de carrapatos nos animais onde foram colocadas 25 ninfas de A. ovale infectadas ou não, de acordo com o grupo. No primeiro estudo foi avaliado a competência vetorial de ninfas de A. ovale na transmissão de R. parkeri (cepa Mata Atlântica) para C. porcellus (animais do G1 e G2). Após o período do parasitismo, as ninfas ingurgitadas foram coletadas e armazenadas em estufa B.O.D. Para a pesquisa de anticorpos anti-Rickettsia spp., coletou-se soros sanguíneos aos 7, 14, 21 e 28 dias pós infestação (DPI) sendo avaliados por meio de reação de imunofluorescência indireta (RIFI). Para a identificação da multiplicação desta riquétsia nos tecidos dos cobaios foi realizada a reação em cadeia da polimerase aos 7, 10, 14 e 28 DPI. Para verificar a competência vetorial das ninfas, analisaram-se os períodos parasitários, o percentual de ecdise, a sobrevivência transestadial e a RIFI. O período médio de parasitismo no G1 foi de 6,6 dias e, de 6 dias no G2. O percentual médio de ecdise (ninfa para adulto) foi de 95%. No G2, a sobrevivência desta riquétsia de forma transestadial foi confirmada em 100% (PCR) e 80% (teste de hemolinfa) dos adultos. Na análise sorológica, 100% dos animais do G1 foram soronegativos e 80% do G2 soropositivos. Não foi detectado o DNA riquetsial nos tecidos dos animais. No segundo estudo, avaliou-se o perfil da infecção experimental causada por esta riquétsia nos cobaios (animais do G1, G2 e G3), buscando identificar as alterações clínicas, perfil hematológico e histopatológico. As coletas sanguíneas para análises hematológicas foram realizadas nos mesmos períodos do estudo anterior e, as coletas de tecidos para análises histopatológicas, ocorreram aos 10 e 28 DPI. Na sorologia, os animais do G1 e G3 fora negativos e, 80% dos animais do G2 positivos. Os resultados hematológicos observados foram: G1 - leucopenia aos 7 DPI, aumento de proteínas plasmáticas totais (PPT) e diminuição de plaquetas aos 7, 14 e 21 DPI; G2: leucocitose, neutrofilia e monocitose aos 7 DPI, aumento de plaquetas aos 14 DPI e diminuição de PPT aos 21 DPI. Na análise histopatológica observou-se: G1 - hemossiderose difusa esplênica aos 28 DPI (20%); G2 - hemossiderose difusa esplênica aos 10 DPI (10%), congestão difusa pulmonar aos 10 e 28 DPI (30%) e hiperplasia folicular multifocal esplênica aos 28 DPI (20%); G3 - congestão difusa pulmonar aos 10 DPI (33%). Concluiu-se que ninfas de A. ovale apresentaram competência vetorial na transmissão de R. parkeri (cepa Mata Atlântica) para cobaios, sendo possível determinar uma infecção aguda de caráter subclínico nestes hospedeiros.

Ixodid ticks are second only to mosquitos in their notorious role as vectors of pathogens to both animals and humans. Rickettsioses are among the most important tick-borne diseases in Europe, Mediterranean spotted fever in particular. To date, many studies have been performed in order to uncover the underlying mechanisms of this disease, in terms of the interactions among its constituents, i.e., the pathogen, Rickettsia conorii, its vector, Rhipicephalus sanguineus tick, and a vertebrate host. However, important gaps remain in this knowledge, among them studies of the relationship of its vector and pathogen, and also the role of tick saliva in vector-host interactions. Thus, in order to approach these limitations, in the first part of this dissertation three studies were performed in order to analyze the vector-pathogen-host interface in natural settings (chapter 2). As a result of the first of these studies, co-infections of Borrelia lusitaniae with R. helvetica and R. slovaca were found in ticks collected from a natural safari park in the south of Portugal; new host-pathogen associations were found in the second study described, performed in Madeira Island, namely lizards infected with R. monacensis, as well as detection of R. helvetica in ticks, which was a first occurrence in this island; and in the last study a new species of Rickettsia was isolated from soft ticks collected from pig pens from Alentejo, Portugal. In chapter 3 experimental studies on the vector were performed through analysis of R. massiliae in Rh. sanguineus tick organs. We performed a quantitative analysis of R. massiliae in the salivary glands of feeding Rh. sanguineus ticks, and observed a statistically significant increase in bacterial load during the first two days, followed by a plateau up to day 6 of feeding. An ultrastructural study was also performed on the salivary glands, ovaries and midgut of R. massiliae infected-Rh. sanguineus, where we observed the reactivation phenomenon of Rickettsia in the salivary glands of fed ticks as a result of tick feeding. In the final part of this dissertation the role of tick saliva was ascertained in terms of bacterial burden and immune responses in a murine susceptible host, using uninfected Rh. sanguineus ticks and C3H/HeJ mice. No statistically significant differences in bacterial load were observed between the two groups of R. conorii-infected animals, one of which infested with ticks. However, host cytokine analysis of both groups of animals revealed statistically significant differences, suggesting an inhibitory effect of tick saliva on host pro-inflammatory responses (chapter 4); RESUMO: Estudo das interacções imunes do hospedeiro e vector resultantes da infecção por Rickettsia spp. Os ixodídeos desempenham um papel fundamental como vectores de agentes patogénicos tanto em animais como em humanos. As rickettsioses, com destaque para a febre escaronodular, encontram-se entre as doenças transmitidas por carraças mais importantes na Europa. Até à data muitos estudos foram efectuados de modo a descortinar os mecanismos subjacentes a esta doença, em termos das interacções entre os seus constituintes, i.e., agente patogénico, Rickettsia conorii , o seu vector, o ixodídeo Rhipicephalus sanguineus, e um hospedeiro vertebrado. Todavia, existem ainda lacunas neste conhecimento a nível da relação vector-agente patogénico, para além do papel da saliva do vector nas interacções agente patogénico-hospedeiro. Assim, numa tentativa de colmatar estas limitações, na primeira parte desta dissertação foram realizados três estudos para análise dos fenómenos que ocorrem naturalmente na interface vector-agente patogénico-hospedeiro ocorrentes na natureza (capítulo 2). Como resultado do primeiro destes estudos, foram encontradas co-infecções de Borrelia lusitaniae com R. helvetica e R. slovaca em ixodídeos capturados num parque safari no sul de Portugal. No segundo estudo descrito, efectuado na ilha da Madeira, foram encontradas novas associações hospedeiro-agente patogénico, nomeadamente lagartixas infectadas com R. monacensis, detectou-se R. helvetica em carraças, pela primeira vez nesta ilha, e foi possível isolar uma nova espécie de Rickettsia a partir de carraças de corpo mole capturados em pocilgas no Alentejo, Portugal. O capítulo 3 aborda os resultados de estudos experimentais efetuados no vector, através da análise de R. massiliae em orgãos de Rh. sanguineus. Realizámos uma análise quantitativa da R. massiliae em glândulas salivares de Rh. sanguineus durante a sua refeição sanguínea, tendo-se observado um aumento estatisticamente significativo da carga bacteriana nos dois primeiros dias de alimentação, seguido de um patamar até ao dia 6 da alimentação sanguínea. Foi também efectuado um estudo ultraestrutural nas glândulas salivares, ovários e intestino médio de Rh. sanguineus infectados com R. massiliae, onde observámos o fenómeno de reactivação da Rickettsia nas glândulas salivares dos ixodídeos alimentados, resultante do processo de alimentação. Na parte final desta dissertação analisámos o papel da saliva de ixodídeos em termos da carga bacteriana e respostas imunes num hospedeiro murino susceptível, usando Rh. sanguineus não infectados e ratinhos C3H/HeJ. Não verificámos diferenças estatisticamente significativas entre as cargas bacterianas de dois grupos de animais infectados com R. conorii, em que apenas um dos grupos estava infestado com carraças. Todavia, a análise de citoquinas no hospedeiro em ambos os grupos experimentais revelou diferenças estatisticamente significativas, sugerindo um efeito inibitório da saliva dos ixodídeos nas respostas pro-inflamatórias do hospedeiro (capítulo 4).

A serosurvey was conducted to determine rickettsial infection rates in dogs from two distinct areas in Indiana. Sera were collected from dogs and tested for the presence of antibodies to R. rickettsii, R. montana, R. rhipicephali, and R. bellii using the micro-immunofluoresence test. Results from this study indicate an association between canine and human rickettsial infections. Dogs in southwestern Indiana were found to have significantly higher rickettsial infection rather than those in east central Indiana. Human RMSF cases have also been reported more frequently from southern Indiana.All rickettsial species were detected at some level, with many dogs reacting to more than one antigen Evidence suggests that R. montana is the predominant rickettsial species in Indiana. In addition, indicative of a more suitable tick habitat, dogs sampled from rural areas were seropositive more frequently than the urban/suburban dogs. This study suggests that dogs are exposed to the same tick population as humans and can serve as indicators of the presence of rickettsial agents. Indiana residents should be aware of the potential for RMSF transmission throughout the state.<br>Department of Physiology and Health Science

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.

Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-05-19T13:08:10Z No. of bitstreams: 1 Maria Fernanda Melo Monteiro.pdf: 639501 bytes, checksum: 8ac4c3c948754bb276d69058aaf30f8b (MD5)<br>Made available in DSpace on 2016-05-19T13:08:10Z (GMT). No. of bitstreams: 1 Maria Fernanda Melo Monteiro.pdf: 639501 bytes, checksum: 8ac4c3c948754bb276d69058aaf30f8b (MD5) Previous issue date: 2016-02-19<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES<br>Rickettsial diseases are zoonotic infections caused by gram-negative bacteria of the family Rickettsiaceae which are transmitted by several ectoparasites such as ticks belonging to the genus Amblymomma, however, the species Rhipicephalus sanguineus sensu lato (s.l.) and Ctenocephalides felis felis have been reported as potential vectors.The aim of this study was to detect Rickettsia spp. DNA in R. sanguineus s.l. and C. felis felis through molecular examination (Polymerase Chain Reaction PCR). A total of 728 ectoparasites were collected of 155 dogs from the states of Pernambuco (municipalities of Recife and Bezerros) and Alagoas (municipalities of Viçosa and Arapiraca) were used in this study. All specimens were morphologically identified (R. sanguineus s.l. and C. felis felis), separated in pools (n = 3) and analyzed molecularly through PCR. Out of the 136 pools of R. sanguineus s.l. analyzed, 20.58% (28/136) and 80 pools of C. felis felis analyzed 31.20% (39/80) it was observed amplification fragment of 401 bp compatible with Rickettsia spp. In relation to the positivity of ectoparasites in the municipalities studied, it was observed that R. sanguineus s.l. was positive in the Metropolitan Area of Recife (20.49%) and Bezerros (22.22%), however in the state of Alagoas was not observed the positivity for Viçosa municipality, but, Arapiraca got 25% positive. With regard to C. felis felis it was observed that in Pernambuco 54.54% of samples were positive being derived from Metropolitan Area of Recife, while Bezerros municipality was not observed amplification of the fragment compatible with Rickettsia spp. In Alagoas, it was observed 12.50% of positivity for Arapiraca and 50.00% for Viçosa. This study reports, for the first time, the detection of Rickettsia spp. in R. sanguineus s.l. parasitizing dogs from the state of Pernambuco and R. sanguineus s.l. and C. felis felis parasitizing dogs from the state of Alagoas. Although there are no reports of Brazilian Spotted Fever (BSF) in the areas studied, the presence of infected ticks and fleas suggests the circulation of the pathogen between vertebrate hosts and vectors.<br>As riquetsioses são doenças zoonóticas causadas por bactérias gram-negativas da família Rickettsiaceae sendo transmitidas por diversos ectoparasitos, destacando-se os carrapatos do gênero Amblymomma, entretanto, as espécies Rhipicephalus sanguineus sensu lato (s.l.) e Ctenocephalides felis felis vêm sendo relatadas como vetores em potencial. Desta forma, o objetivo deste trabalho foi diagnosticar a infecção por Rickettsia spp. em R. sanguineus s.l. e C. felis felis através da Reação em Cadeia da Polimerase (PCR). Para tanto foram coletados 728 ectoparasitos de 155 cães de dois municípios do estado de Pernambuco (Recife e Bezerros) e de dois municípios do estado de Alagoas (Viçosa e Arapiraca). Todos os espécimes foram identificados morfologicamente (R. sanguineus s.l. e C. felis felis), sendo separados em pools (n = 3) e analisados através da PCR. Dos 136 pools de R. sanguineus s.l. analisados 20,58% (28/136) e dos 80 pools de C. felis felis analisadas 31,20% (39/80) foi observado amplificação de fragmento de 401 pb compatíveis com Rickettsia spp. Em relação à positividade dos ectoparasitos nos municípios estudados, observou-se que R. sanguineus s.l. foi positivo na Região Metropolitana do Recife (20,49%) e em Bezerros (22,22%), entretanto, no estado de Alagoas não foi observada positividade para o município de Viçosa, porém, Arapiraca obteve 25% de positividade. Em relação à C. felis felis observou-se que em Pernambuco 54,54% das amostras foram positivas sendo elas procedentes da Região Metropolitana do Recife, enquanto que no município de Bezerros não foi observada amplificação do fragmento compatível com Rickettsia spp. Já em Alagoas observou-se positividade de 12,50% para Arapiraca e 50,00% para Viçosa. Este estudo reporta pela primeira vez a detecção de Rickettsia spp. em R. sanguineus s.l. parasitando cães provenientes do estado de Pernambuco e R. sanguineus s.l. e C. felis felis parasitando cães provenientes do estado de Alagoas. Apesar de não existirem relatos de Febre Maculosa Brasileira (FMB) nas regiões estudadas, a presença de carrapatos e pulgas infectados sugere a circulação do patógeno entre hospedeiros vertebrados e vetores.

Foram coletados cinqüenta e oito (58) amostras de cachorro-do-mato (Cerdocyon thous) de vida livre, provenientes do Estado do Espírito Santo, Brasil. Os animais eram mortos por atropelamento ao longo da rodovia estadual ES-060 que liga os municípios de Vila Velha à Guarapari, passando por duas reservas florestais de Mata Atlântica. Todos os animais eram encaminhados para a Universidade de Vila Velha UVV pela concessionária RodoSol, onde passavam por detalhado exame necroscópico com coleta de tecidos e ectoparasitas para pesquisa de patógenos pela técnica de Reação em Cadeia de Polimerase (PCR). Todas as amostras obtidas foram testadas quanto à presença de agentes pertencentes à família Anaplasmataceae e membros dos gêneros Rickettsia, Borrelia, Babesia, Coxiella, Leishmania, Hepatozoon e Ehrlichia. No total foram colhidos 25 espécimes de carrapatos, todos em estágio ninfal e pertencentes à espécie Amblyomma cajennense. Na pesquisa de patógenos pela PCR, não foi encontrada nenhuma amostra de animal ou carrapato positiva para Leishmania spp, Rickettsia spp, Borrelia spp, Babesia spp e Coxiella spp. Das 58 amostras de tecidos, 29 (50%) foram positivas para Hepatozoon spp no gene 18S rRNA, tendo sido identificado dois genótipos, um denominado Hepatozoon sp. ex Cerdocyon thous, presente em 96,55% dos animais testados, com máxima similaridade de 98,67% com a espécie Hepatozoon sp. curupira 2 (AY461377). O outro genótipo foi encontrado em somente um animal (3,44%), denominado Hepatozoon sp. P20 Cerdocyon thous, que apresentou máxima similaridade de 97,5% com Hepatozoon sp. 744C (EU430234). Na pesquisa para Ehrlichia spp, seis amostras foram positivas (10,34%). As seis amostras foram caracterizadas como uma possível nova espécie de Ehrlichia, denominada Ehrlichia sp. ex Cerdocyon thous, com máxima similaridade de 97,57% com a espécie Ehrlichia ruminantium (DQ482915) para o gene 16S e 82,51% similar com E. ruminantium str. Gardel (CR925677) para o gene dsb. Todas as espécies de carrapatos foram negativas. Hepatozoon spp e Ehrlichia spp são agentes infecciosos transmitidos por carrapatos, com potencial zoonótico, de potencial impacto para saúde animal e humana.<br>We collected samples from fifty-eight (58) free-living crab-eating fox (Cerdocyon thous) from the State of Espírito Santo, Brazil. All animals were hit by vehicles passing through the highway ES-060, which crosses an Atlantic rainforest reserve, located between the cities of Guarapari and Vila Velha. The animals were collected by the Rodosol Company, and taken to the University of Vila Velha - UVV, where they underwent post-mortem examination, and collection of tissue samples and ectoparasites for research through the polymerase chain reaction (PCR) technique. All samples were tested for the presence of agents belonging to the family Anaplasmataceae and members of the genera Rickettsia, Borrelia, Babesia, Coxiella, Leishmania, Hepatozoon, and Ehrlichia. We collected 25 specimens of nymphal ticks, which were all identified as Amblyomma cajennense. Search of pathogens by PCR did not find any positive animal or tick sample for Leishmania spp, Rickettsia spp, Borrelia spp, Babesia spp and Coxiella spp. From the 58 tissue samples, 29 (50%) were positive for Hepatozoon sp by the PCR targeting the 18S rRNA gene. Amplicons generated two different genotypes, one named Hepatozoon sp. ex Cerdocyon thous, present in 96.55% of the animals, with a maximum of 98.67% similarity with the sequence of Hepatozoon sp. curupira 2 (AY461377); the second genotype was found in only one animal (3.44%), named Hepatozoon sp. P20 Cerdocyon thous, which showed maximum similarity (97.5%) with Hepatozoon sp. 744C (EU430234). The survey for Ehrlichia spp resulted in six positive samples (10.34%). The six samples were characterized as a possible new species of Ehrlichia, named Ehrlichia sp. ex Cerdocyon thous, with maximum similarity (97.57%) with the species Ehrlichia ruminantium (DQ482915) for the 16S and 82.51% similar to E. ruminantium str. Gardel (CR925677) for the dsb gene. All species of ticks were negative for all pathogens searched. Hepatozoon spp and Ehrlichia spp are tick-borne infectious agents with zoonotic potential; the present findings are of potential impact for both animal and human health.

Dissertação de Mestrado Integrado em Medicina Veterinária<br>A Leishmaniose Canina é uma doença sistémica grave, crónica, debilitante e potencialmente fatal, provocada por um protozoário difásico, a Leishmania infantum. È endémica em grande parte do território de Portugal Continental e nos últimos anos têm-se assistido a um aumento da sua prevalência nos cães. É transmitida ao hospedeiro vertebrado através da picada do insecto vector, Phlebotomus perniciosus. As doenças transmitidas por vectores constituem um grupo de doenças infecciosas caracterizadas, muitas vezes, por sintomatologia inespecífica e semelhante entre as diferentes doenças e o seu diagnóstico pode ser um desafio. As co-infecções podem explicar, em parte, os casos de sintomatologia atípica, agravamento do quadro clínico e casos de insucesso à terapêutica instituída. Com esta dissertação pretendeu-se avaliar se os sinais clínicos, as alterações do hemograma e do proteinograma apresentam semelhanças entre canídeos mono-infectados com Leishmania infantum e canídeos co-infectados com Leishmania infantum e com pelo menos um dos seguintes agentes patogénicos: Babesia canis, Ehrlichia canis e/ou Rickettsia conorii. Foi realizado um estudo retrospectivo observacional, em 35 cães com diagnóstico clínico e serológico de leishmaniose e/ou hemoparasitas. Os animais foram divididos em dois grupos de acordo com a positividade serológica: Grupo A, constituído por 17 animais monoinfectados com Leishmania infantum e, Grupo B, constituído por 18 animais co-infectados a Leishmania infantum e pelo menos um dos seguintes agentes patógenicos: Babesia canis, Ehrlichia canis e Rickettsia conorii. Este estudo revelou que as principais alterações clínicas e alterações laboratoriais, a nível do hemograma e do proteinograma, são semelhantes em canídeos mono-infectados com leishmaniose e nos canídeos co-infectados com Leishmania e hemoparasitas. A identificação de infecções concomitantes nos canídeos com leishmaniose é essencial na medida em que o seu reconhecimento e posterior tratamento poderão ter repercussões importantes quer na qualidade de vida dos animais infectados quer a nível do prognóstico e evolução clínica da leishmaniose.<br>ABSTRACT - Canine Leishmaniasis is a severe systemic, chronic, debilitating and potentially fatal disease caused by a diphasic protozoan, Leishmania infantum. It is endemic in much of Portugal mainland showing in recent years an increase in its prevalence in dogs. It is transmitted to the vertebrate host through the bite of the insect vector, Phlebotomus perniciosus. The vector-borne diseases are a group of infectious diseases characterized often by nonspecific symptoms and similar between the different diseases and its diagnosis can be a challenging task. The co-infections may explain, in part, cases of atypical symptoms, aggravation of the clinical cases and the failure of therapy. The aim of this thesis is to assess whether the clinical signs, changes in the protein profile and blood count show similarities between canine mono-infected with Leishmania infantum and canine co-infected with Leishamania infantum and at least one of the following pathogens: Babesia canis, Ehrlichia canis and/or Rickettsia conorii. A retrospective observational study was conducted in 35 dogs with clinical and serological diagnosis of Leishmaniasis and/or Hemoparasites. The animals were divided into two groups according its sero-positivity: Group A, consisting of 17 animals mono-infected by Leishmania infantum, and Group B consisting of 18 animals co-infected with Leishmania infantum and at least one of the following pathogens: Babesia canis, Ehrlichia canis and Rickettsia conorii. This study revealed that the clinical and laboratory changes at the CBC and protein profile are similar in dogs mono-infected with canine Leishmaniasis and those co-infected with Leishmaniasis and Hemoparasites. The identification of concomitant infections in canine Leishmaniasis is essential to the extent that their recognition and subsequent treatment may have important implications both in quality of life of infected animals at the prognosis and clinical evolution of Leishmaniasis.

Ornithodoros brasiliensis Aragão é um carrapato endêmico do Brasil, restrito às regiões serranas e frias do estado do Rio Grande do Sul. É uma espécie bastante agressiva aos humanos, causando febre, muita dor e intensa resposta inflamatória no local da picada. O ciclo biológico de O. brasiliensis (ovo a adulto, única geração) realizado nos anos 50, utilizando ratos brancos como hospedeiros, levou um total de 103 e 184 dias de amplitude, com máximo de 7 ínstares ninfais observados e com emergência de adultos a partir de N4, estando os machos em maior proporção. Os hospedeiros apresentaram febre e uma bactéria espiroqueta denominada Borrelia brasiliensis foi descrita de esfregaços sanguíneos desses animais. Porém as borrélias não puderam ser cultivadas por muito tempo, e acabaram desaparecendo. Houve um silêncio de mais de 50 anos sobre relatos de ocorrência de O. brasiliensis, deixando inclusive dúvidas se a espécie estava extinta de fato. No entanto, há cerca de quatro anos, alguns espécimes de carrapatos da mesma localidade foram coletados e identificados como O. brasiliensis. O presente trabalho teve por objetivos estudar a morfologia e a biologia de O. brasiliensis, em condições de laboratório, bem como investigar a presença de bactérias do gênero Rickettsia em espécimes coletados. Ninfas e adultos foram alimentados em cobaias de laboratório e as fêmeas acasaladas realizaram posturas que resultaram nas gerações sucessivas. A larva e os adultos foram descritos e redescritos, e comparados com as espécies próximas, O. rostratus e O. turicata. Análises moleculares de uma porção do gene 16S rDNA mitocondrial foram comparadas com as sequências depositadas no GenBank e apresentaram valores de máximo bootstrap de 100% para ambas, O. rostratus e O. turicata. O ciclo biológico completo (ovo a ovo) de O. brasiliensis foi obtido entre 215,4 e 195 dias, em média, para duas gerações consecutivas, respectivamente. Os períodos médios de pré-oviposição, oviposição e incubação dos ovos (em dias), para a geração F1 foram 76,7 ± 27,01 (31-117); 15,55 ± 5,36 (10-24); 10,6 ± 4,5 (3-19) e para geração F2 foram 54,1 ± 8,7 (40-65), 15,8 ± 8,9 (7-36); 15,4. Os períodos médios de pré-ecdise larval foram de 12,5 ± 1,8 (10-15), considerando as duas gerações. A larva não se alimenta, enquanto que há um repasto sanguíneo para cada instar ninfal, antes de cada muda. Um total de 5 instares ninfais foi observado, sendo que os períodos médios de préecdise para os instares N1, N2, N3, N4 e N5, respectivamente para as duas gerações foram de 33,3 ± 4 (32-45), 38,6 ± 2,5 (36-47), 31,1 ± 3,4 (28-42), 34,8 ± 3,3 (31-43), 37,2 ± 2,4 (35-41) e 32,5 ± 3,3 (29-40), 37,5 ± 2,5 (34-44), 34,6 ± 3,2 (31- 37), 36,5 ± 2,5 (34-42), 32,5 ± 0,7 (32-33). Os períodos médios de alimentação para os ínstares ninfais, foram semelhantes nas duas gerações, sendo de 28,62 ± 3,67 (25,2 35,1) minutos. Adultos alimentam-se repetidas vezes, e as posturas sempre são precedidas de repasto sanguíneo, embora somente dois ciclos gonotróficos foram observados. O primeiro ciclo gonotrófico apresentou maior número de ovos depositados, com 139 ± 13,8 (53 197) em relação ao segundo 73,8 ± 10,6 (53 123). A investigação da presença de riquétsia foi avaliada em 107 espécimes recentemente coletados, evidenciando um total de 12 exemplares positivos. O produto amplificado pela PCR foi sequenciado e as análises moleculares apresentaram homologia de 98% com sequências disponibilizadas no Genbank, correspondentes a uma espécie asiática denominada RDa420 (AF497584) e outra brasileira, Rickettsia bellii (DQ865204) .<br>Ornithodoros brasiliensis Aragão is an endemic tick to Brazil, restricted to the highlands and cold regions of the state of Rio Grande do Sul. This species is very aggressive to humans, causing fever, great pain and intense inflammatory response at the site of the bite. The life cycle of O. brasiliensis (egg to adult, single generation) previously performed (studied during 50\') using white mice as host, spent a total of 103 and 184 days of amplitude, with maximum 7 nymphal instars observed, with adult emerging from N4, and males in greater proportion. The hosts had fever and spirochete bacteria called Borrelia brasiliensis was described from blood of the animals used. Nevertheless borrelias could not be cultivated for a long time, and they eventually disappeared. A silence of more than 50 years on reports of occurrence of O. brasiliensis, left doubts about whether the species was extinct. However, about four years ago, some specimens of ticks were collected in the same locality and identified as O. brasiliensis. This work aimed to study the morphology and biology of O. brasiliensis, under laboratory conditions, and investigate the presence of bacteria of the genus Rickettsia in the collected ticks. Nymphs and adults were fed on laboratory guinea pigs, and mated females laid eggs that resulted in the successive generations. The larvae and adults have been described and redescribed, respectively, and compared with the closely related species, O. rostratus and O. turicata. Molecular analyzes of a portion of the mitochondrial 16S rDNA gene was compared with the sequences deposited in GenBank, showing maximum bootstrap values out of 100% for both O. rostratus and O. turicata. In this study, the complete life cycle (egg to egg) of O. brasiliensis was obtained between 215,4 and 195 days, on average, for the two generations studied, respectively. The average periods of preoviposition, oviposition and incubation of the eggs to the F1 generation were 76,7 ± 27,01 (31-117), 15,55 ± 5,36 (10-24), and to F2 were 54,1 ± 8,7 (40-65), 15,8 ± 8,9 (7-36). The averaging periods to the preecdysis of the larvae were 12,5 ± 1,8 (10-15), considering the two generations. The larva does not feed, while for the nymphs there is a blood meal for each instar, before each molt. A total of five nymphal instars was observed, and the average periods of pre-molt instars for N1, N2, N3, N4 and N5, for two generations, respectively, were 33,3 ± 4 (32-45), 38,6 ± 2,5 (36-47), 31,1 ± 3,4 (28-42), 34,8 ± 3,3 (31-43), 37,2 ± 2,4 (35-41) and 32,5 ± 3,3 (29-40), 37,5 ± 2,5 (34- 44), 34,6 ± 3,2 (31-37), 36,5 ± 2,5 (34-42), 32,5 ± 0,7 (32-33). The average feeding time of nymphal instars, were similar in the two generations, being 28,62 ± 3,67 (25,2 35,1) minutes. Adults fed repeatedly, and the postures were always preceded by blood meal, although only two gonotrophic cycles have been observed. The first gonotrophic cycle presented higher average number of eggs deposited 139 ± 13,8 (53-197) that the second 73,8 ± 10,6 (53 123). The investigation to the presence of rickettsia was performed in 109 recently collected ticks, showing a total of 12 positive specimens. The amplified PCR product was sequenced and molecular analyzes showed 98% homology with sequences available in Genbank, corresponding to an Asian species named RDa420 (AF497584) e another Brazilian strain named Rickettsia bellii (DQ865204).

Dogs play an important role in the lives of humans, and because of this close contact they share with humans, they can serve as sentinels for infections to humans. Rickettsiae are small, obligate intracellular bacteria that are vector-borne and can cause mild to fatal diseases in humans worldwide. There is little information on the zoonotic rickettsial pathogens that may be harboured by dogs from rural localities in South Africa. Thus the primary aim of this study was to identify and characterise rickettsial pathogens infecting dogs using molecular techniques by screening blood samples and ectoparasites collected from domestic dogs in the Bushbuckridge area, Mpumalanga province of South Africa. A total of 141 blood samples, 103 ticks and 43 fleas were collected from owned, free-roaming dogs in the Mnisi community area in Bushbuckridge Local Municipality, Mpumalanga Province, South Africa under the project “Health and Demographic Surveillance System (HDSS) in dogs” (protocol number: V033/11). Samples were collected from October 2011 through May 2012. Ectoparasites identified are as follows: the tick species include Haemaphysalis elliptica, Amblyomma hebraeum, Rhipicephalus sanguineus, Rhipicephalus simus, and an unspeciated Ixodes spp. Fleas includes Ctenocephalides felis strongylus and Echidnophaga gallinacea. DNA was extracted from blood and ectoparasites and screened for the presence of Ehrlichia, Anaplasma, Theileria and Babesia species infections using the reverse line blot (RLB) hybridization assay. Almost half of the blood samples reacted with the genus-specific probes for Ehrlichia/Anaplasma (49%), and 16% of the samples investigated were positive for Ehrlichia canis DNA. Babesia rossi (9%) and Babesia vogeli (4%) were detected among the 14% of samples that were positive for the genus-specific probe 1 of Babesia. Furthermore, 14% of the samples were positive for the genus-specific probes of Theileria/Babesia, 2% were positive for the genus-specific probe of Theileria, and 1% for the genus-specific probe 2 of Babesia. Haemoparasite DNA could not be detected in 36% of the samples investigated. Results of the RLB assay for ectoparasites showed that 30% of tick pools were positive for the genus-specific probes of Ehrlichia/Anaplasma species, Babesia 1 and 2 (30%) and Theileria (10%) while haemoparasite DNA could not be detected in 30% of the tick pools. Haemoparasite species DNA detected from tick pools includes Neoehrlichia spp., Ehrlichia ruminantium, and Babesia microti. Flea pools were positive for the genus-specific probes of Babesia 1 and 2 (50%). Specific haemoparasite species were not detected from the flea pools. Rickettsia genus and species-specific qPCR assays were performed to detect zoonotic rickettsiae from DNA obtained from blood and ectoparasite samples. Rickettsiae were not detected in blood samples but in 70% of tick pools analyzed using a genus-specific qPCR assay, based on the 17-kD antigen gene. Rickettsial infections were detected in tick pools, namely Haemaphysalis elliptica, Amblyomma hebraeum, Rhipicephalus sanguineus, Rhipicephalus simus, and Ixodes, and in flea pools Ctenocephalides felis strongylus and Echidnophaga gallinacea. Using the species-specific assays, three pools of ticks (two Amblyomma hebraeum pools and a Haemaphysalis elliptica pool) tested positive on a R. africae genotype-specific qPCR assay, while flea pools (Ctenocephalides felis strongylus and Echidnophaga gallinacea) were positive on the R. felis qPCR assay. Blast analysis of nucleotide sequences of the V1 hypervariable region of the 16S rRNA gene of five selected Ehrlichia/Anaplasma positive samples on the RLB hybridization assay revealed an Anaplasma sp. South Africa dog strain 98% related to Anaplasma phagocytophilum in four of the five samples and an Anaplasma phagocytophilum strain in one sample. Furthermore, Rickettsia massiliae and Orientia tsutsugamushi sequences were also identified in two additional samples. Analysis of nucleotide sequences obtained from tick and flea pool DNA samples positive on R. africae and R. felis qPCR assays revealed Rickettsia africae and Candidatus Rickettsia asemboensis a new Rickettsia felis-like organism respectively. Analysis of nucleotide sequences of the 18S rRNA gene of three selected B. rossi positive samples on the RLB hybridization assay confirmed detection of B. rossi in all selected samples. The detection of zoonotic rickettsiae in canine blood and their ectoparasites suggests that dogs may play an important role in the lifecycle of these organisms; these data also provide preliminary information about the diversity of rickettsiae and other vector-borne pathogens circulating in domestic dogs and their ectoparasites in Bushbuckridge, South Africa. In conclusion the detection of zoonotic rickettsiales from blood samples of domestic dogs and their ectoparasites in a rural community in South Africa highlights the potential risk of human infection that may occur from these pathogens.<br>Dissertation (MSc)--University of Pretoria, 2014.<br>tm2015<br>Veterinary Tropical Diseases<br>MSc<br>Unrestricted

The present thesis examines questions related to the temporal origin of the Flavivirus genus and the ecology of tick-borne pathogens. In the first study, we date the origin and divergence time of the Flavivirus genus. It has been argued that the first flaviviruses originated after the last glacial maximum. This has been contradicted by recent analyses estimating that the tick-borne flaviviruses emerged at least before 16,000 years ago. It has also been argued that the Powassan virus was introduced into North America at the time between the opening and splitting of the Beringian land bridge. Supported by tip date and biogeographical calibration, our results suggest that this genus originated circa 120,000 (156,100–322,700) years ago if the Tamana bat virus is included in the genus, or circa 85,000 (63,700–109,600) years ago excluding the Tamana bat virus. In the second study we estimate the prevalence of tick-borne encephalitis virus (TBEV) in host-seeking Ixodes ricinus from 29 localities in Sweden and compare our data with those of neighbouring countries. Nymphs and adult ticks were screened for TBEV using a real-time PCR assay. The mean TBEV prevalence for all tick stages combined was 0.26% for Sweden and 0.28% for all Scandinavian countries, excluding Iceland. The low prevalence of TBEV in nature may partly be explained by the fact that TBEV occurs in spatially small foci and that the inclusion of ticks from non-infected foci will reduce the prevalence estimate. In the third and fourth study, we conducted the first large-scale investigations to estimate the prevalence and geographical distribution of Anaplasma spp. and Rickettsia spp. in host-seeking larvae, nymphs and adults of I. ricinus ticks in Sweden. Ticks were collected from several localities in central and southern Sweden and were subsequently screened for the presence of Anaplasma spp. and Rickettsia spp. using a real-time PCR assay. For all active tick stages combined, the mean prevalence of Anaplasma spp. and Rickettsia spp. in I. ricinus in Sweden was estimated to 1.1% and 4.8%, respectively. It was also shown that A. phagocytophilum and R. helvetica are the main Anaplasma and Rickettsia species occurring in Sweden.

LEISHMANIA INFANTUM INFECTION IN STRAY CATS IN A NON-ENDEMIC AREA IN NORTHERN ITALY E. Spada, DVM, PhD, Researcher 1, A. Della Pepa, DVM 1, A. Migliazzo, DVM, PhD 2, G. Bagnagatti De Giorgi, DVM 1, R. Perego, DVM, PhD 1, D. Proverbio, DVM, PhD, Professor 1 1Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare, Universita degli Studi di Milano, Milan, Italy 2Centro di Referenza Nazionale per le Leishmaniosi,Istituto Zooprofilattico Sperimentale della Sicilia, Palermo, Italy Tipologia: Ricerca Originale Area di interesse: Medicina interna Purpose of the work. To assess the prevalence of leishmaniosis in a large representative sample of stray cats from this non-endemic area, and to analyze the results according to clinical, laboratory and infectious data 2. MOLECULAR STUDY ON VECTOR-BORNE INFECTIONS IN URBAN STRAY COLONY CAT IN NORTHENRN ITALY Eva Spada§, DVM, PhD, Researcher Daniela Proverbio§, DVM, PhD, Professor Alessandra Della Pepa§, DVM Paola Galluzzo*, Biologist Roberta Perego§, DVM, PhD Giada Bagnagatti De Giorgi§, DVM Abstract Feline vector-borne diseases are caused by a wide range of pathogens, which are transmitted by arthropods. Many of these infections have zoonotic implications and feral cats may potentially act as sentinels of human and pet health. The present study investigated the prevalence of vector-borne infections in feral colony cats in the city of Milan in northern Italy. Blood samples from 260 feral cats were evaluated, with conventional PCR, for the presence of DNA associated with hemoplasmas (Mycoplasma haemofelis and Mycoplasma haemominutum), Rickettsia spp., Anaplasma phagocytophilum, Ehrlichia spp. and Babesia microti. Odd ratios (OR) were calculated to identify risk factors for infection with vector-borne pathogens. Positive PCR was found in 156 out of 260 subjects (60%), with a prevalence of 33.1% for hemoplasmas, 31.9% for Rickettsia spp., 17.7% for A. phagocytophilum , 6.7% for Ehrlichia spp. (out of 30 samples), and 1.2% for B. microti spp (out of 168 samples). Statistical analysis revealed a correlation between infections with Rickettsia spp. and hemoplasmas (OR=1.95, P=0.02). Additionally, Rickettsia spp. infection was associated with ocular infection (OR=2.21, P=0.02). We conclude that vector-borne infections, including zoonotic diseases, are present in feral cats of Milan. Thus, domestic cats exposed to the outdoors should be routinely monitored and treated for ectoparasites to minimize disease onset and potential transmission of zoonotic agents to humans. Moreover, as these vector-borne infections are transmitted through blood, feline blood donors from this area should be screened by PCR.

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

Research Doctorate - Doctor of Philosophy (PhD)<br>At the commencement of this project in 2014, some data were available on scrub typhus (ST) in Bhutan but none on the Spotted Fever Group (SFG) and Typhus Group (TG) Rickettsia and Q fever (QF). Following an ST outbreak in 2009, heightened awareness increased case notifications from 91(no deaths) in 2010 to 753 (3 deaths) in 2017. Another outbreak in 2014 saw two deaths from meningoencephalitis. These events inspired this doctoral thesis to explore the epidemiology of rickettsioses in Bhutan. Clinico-demographic information, human and animal samples were collected from Bhutan and analyzed at the Australian Rickettsial Reference Laboratory. Findings suggests that approximately 15% (159/1044) of patients with acute undifferentiated fevers attending 14 Bhutanese hospitals were due to rickettsioses, ST being the commonest (6.7%). About 49% of 864-healthy Bhutanese showed evidence of past exposure to ST (22.6%), SFG (15.7%), QF (6.9%) and TG (3.5%). ST and SFG exposure significantly increased with age and farming activities. Trongsa district residents had the highest exposure to ST but residents at altitudes >2000 meters were relatively protected. In animals, an overall seropositivity of 46% (106/294) with SFG (36%), ST (21%), TG (15%), and QF (4%) was determined. Seropositivity differences between animal species appeared to have been significant and warrant confirmation. A few inferences should be interpreted with caution, but findings in general constitute baseline data for Bhutan and serve to prompt further research. Rickettsial outbreaks and the high incidence and prevalence rates established the endemicity of rickettsioses in Bhutan. Health authorities should ensure that services are equipped to manage these infections by developing diagnostic and clinical guidelines. Increased human-livestock sector collaborations in research, diagnostics and therapeutics through a ‘One Health’ approach is recommended. Future studies should consider vector profiles, geospatial, biosocial and environmental risk assessment for strategic prevention and control of rickettsioses.

碩士<br>國立屏東科技大學<br>獸醫學系<br>88<br>Since October 1992, the first outbreak of the disease caused by rickettsia-like organism（RLO）in cultured tilapias in Taiwan, there has been a great number of mortalities approximately 30 ~ 90% on the most cultured tilapias farms each year. In consequence of the major difficulty in the isolation and cultivation of the RLO, the research of the disease has made a little progress in the past. Therefore, the aim of this study was to establish the culturing methods and to develop the polymerase chain reaction（PCR）test for the detection of RLO. The RLO of tilapias was successfully to be cultivated in CHSE-214 cell line system, but was unable to be cultured in ten different synthetic media. The RLO was a gram-negative, intracellular parasite and frequently been observed within intracytoplasmic vacuoles of the host cell. The RLO was reisolated from the inoculated tilapia using the RLO infected CHSE-214 cells as an inoculum. According, the RLO isolated from the natually infected tilapia was confirmed by Koch's postulates. The bacterial 16S rDNA sequence was amplified directly from tissues of related fishes with RLO infection by polymerase chain reaction, first with broad-range primers（PTS-1 ＆ PTS-2）and then with specific primers（PTS-f ＆ PTS-r）. A unique 1304-base bacterial 16S rDNA sequence was amplified from spleen and kidney of all infected fishes. We then detected the sequence in tissue from all RLO infected fishes. The specificity of PCR was assessed with a panel of 17 bacterial strains（ATCC） and 9 strains of fish bacterial pathogens isolated and identified in our fish disease laboratory. Products derived from amplification were observed only from RLO, indicating that PCR technique can serve as a diagnostic tool for the detection of the RLO. Base on our results, we confirmed the fact that（1）this organism is existed in the environment, and（2）the RLO isolated and identified is a new species of gamma subdivision of Eubacteria infection in tilapias.

Copies of author's previously published articles inserted<br>Includes bibliographical references<br>[ix], 261, [101] leaves, [4] leaves of plates : ill. (some col.) ; 30 cm.<br>Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1992