A erliquiose monocitotrópica canina é caracterizada como uma infecção persistente, podendo evoluir para doença fatal. Mecanismos imunopatogênicos estão implicados no desenvolvimento da doença, porém não é completamente compreendido o papel da resposta imune celular nas infecções causadas por Ehrlichia canis transmitida pelo carrapato Rhipicephalus sanguineus. A infecção do vetor ocorre somente durante o repasto em cães riquetsêmicos, porém não é reconhecido o nível de riquetsemia infectante durante o curso da infecção nos cães. O objetivo desse estudo foi verificar os níveis de riquetsemia relativa capazes de infectar o vetor (R. sanguineus) durante o curso da erliquiose canina; e avaliar a resposta imune celular induzida por exposição de cães a carrapatos infectados com Ehrlichia canis. Um cão foi utilizado para produção do inoculo e infecção de ninfas de R. sanguineus. Subseqüentemente, os carrapatos adultos infectados, oriundos das ninfas que se alimentaram nos cães infectados, foram utilizados para infecção dos cães dos grupos I (n=3) e II (n=3). Cães do grupo III (n=3) foram inoculados com sangue infectado com E. canis, por via intravenosa. Os grupos IV (n=3) e V (n=3) foram compostos por cães não infectados. Os grupos II e IV foram reinfestados periodicamente com ninfas não infectadas de R. sanguineus. Foram observadas, alterações clínicas como febre, apatia e disorexia nos cães infectados, durante a fase aguda da infecção, porém de forma mais intensa e precoce nos cães infectados por inoculação intravenosa (grupo III). As alterações hematológicas mais evidentes foram redução do número de plaquetas, leve redução na série vermelha e branca. Todos os animais infectados soroconverteram aos 14 dias após inoculação ou infestação com carrapatos infectados (dpi), mantendo títulos entre 10240 e 81920. Os níveis de riquetsemia foram variáveis durante o curso da infecção, persistindo até 364 dpi, porém mais constante na fase aguda da doença. Um pequeno número de carrapatos alimentados nos cães infectados apresentou amplificação de DNA de E. canis, porém foram demonstrados até 308 dpi. Foi observada uma redução na relação CD4:CD8 nos animais infectados, no 28° dpi, mantendo-se de forma mais branda até 252 dpi. Uma maior proporção de linfócitos T CD4+ produtores de IFN-γ e IL-4 foi observada no tempo zero de cães que não adquiriram infecção após infestação com carrapatos infectados. Os níveis séricos de citocinas mostraram de forma mais evidente a elevação de IL-10 e TNF-α em um cão que desenvolveu doença fatal, podendo indicar a participação de mecanismos imunes na apresentação clínica e na persistência do agente no organismo hospedeiro. / The canine monocytic ehrlichiosis is characterized as a persistent infection, which can develop fatal disease. Immunopathogenic mechanisms are implicated in the development of the disease, however it is not completely understood the role of cellular immune in the infection caused by Ehrlichia canis transmitted by the tick Rhipicephalus sanguineus. The vector becomes infected only while feeding on rickettsemic dogs, however the level of rickettsemia is not recognized during the course of the infection in the dogs. The objective of this study was to verify the level of relative rickettsemia capable to infect the vector (R. sanguineus) during the course of the canine ehrlichiosis; and to evaluate the cellular immune induced in dogs exposed to Ehrlichia canis-infected ticks. A dog was used for production of the inoculum and infection of nymphs of R. sanguineus. Infected adult ticks that had fed as nymphs on the infected dog were used to feed on and transmit the infection to dogs of groups I (n=3) and II (n=3). Group III dogs (n=3) were intravenously inoculated with E. canis-infected dogs. Group IV (n=3) and V (n=3) dogs were the control groups, never exposed to E. canis. Dogs of groups II and IV were reinfested periodically with uninfected R. sanguineus nymphs. Clinical alterations such as fever, apathy and dysorexia were observed in the infected dogs during the acute phase of the infection, however in a more intense and precocious form in the dogs infected by intravenous inoculation (group III). The more evident hematological alterations were reduction of the number of platelets, mild reduction in the red and white cells series. All infected animals soroconverted by 14 days after inoculation or infestation by infected ticks (dpi), showing titers between 10240 and 81920 during the study. The rickettsemia levels were variable during the course of the infection, persisting up to 364 dpi, however more constant in the acute phase of the disease. A small number of ticks that fed on infected dogs presented amplification of E. canis DNA, however they were demonstrated up to 308 dpi. A reduction in the relationship CD4:CD8 in the infected animals was observed in 28th dpi, keeping in a mild form up to 252 dpi. A larger proportion of lymphocytes T CD4+ producing IFN-γ and IL-4 it was observed in the zero time of dogs that did not acquire infection after infestation with infected ticks. The cytokine seric levels showed the elevation in a more evident form of IL-10 and TNF-α in a dog that developed fatal disease, what could indicate the participation of immune mechanisms in the clinical presentation and in the persistence of the agent in the organism host.

Made available in DSpace on 2014-08-20T14:38:00Z (GMT). No. of bitstreams: 1 dissertacao_anelize_felix.pdf: 541679 bytes, checksum: e35d84480d181fc2e13d21300abf6a98 (MD5) Previous issue date: 2010-02-24 / Demodicosis is considered one of the most severe canine skin disease. It is caused by the excessive proliferation of the Demodex canis mite, normal member of the canine skin. Skin lesions caused by the parasite predispose the skin to secondary infections that will further aggravate the patients clinical aspects. The objective of this work was to evaluate the clinical a histopathological evolution of the disease, as well as to study the seric levels of interleukin 10 (IL10) in demodicosis patients. The first study was conducted using 20 animals, 10 demodicosis patients (GD), and 10 control dogs (GC). All these animals were clinically evaluated, and submitted to deep skin scraping in search of Demodex mites. The dogs in the GD group were treated with moxidectin and evaluated in days 0, 20, 40, 60, and 80. Five of these animals were healed and submitted to skin biopsies on days 0 and 80, for the observation of histopathological alterations. A second study used 26 animals, 17 on G1 (demodicosis patients) and 9 on G2 (healthy dogs). All G1 animals were positive for demodicosis on the skin scrape test, and were submitted to clinical evaluation. Blood was harvested from all the animals, with the interleukin 10 dosage being carried out with the comrcial kit Quantikine Canine IL-10 Immunoassay® (R&D Systems) . Results obtained in the first experiment showed considerable clinical and scrape test improvement in GD, but no evolution in the histopathological patern. The GC presented higid skin and negative skin scrape test. In the second experiment, dogs in the G1 group presented hi clinical scores, indicating severe desiase. G1 had a mean IL10 level of 184,38 (+258,9) pg/mL, while the mean for G2 was11,94 (+ 2,27) pg/mL, indicating that hi levels of IL10 may be related to the development of the disease. The results described in this work show that, even when clinically healed, and with negative skin scrape test, demodicosis carriers present persistence of the lesions and the mite in the histological structure of the skin. This work also shows that demodicosis patients tend to have higher IL10 levels than healthy animals. / A demodicose é considerada uma das mais graves dermatopatias que acomete cães. É causada pela proliferação excessiva do ácaro Demodex canis, comensal da pele canina. Devido à lesão causada pelo parasita, a pele torna-se predisposta à instalação de infecções secundárias que agravam o quadro clínico do paciente. Objetivou - se avaliar a evolução clínica e histopatológica, assim como estudar os níveis de interleucina -10 sérica em cães portadores de demodicose. Para a primeira etapa, foram estudados 20 cães, 10 apresentando demodicose (GD) e 10 animais controle (GC). Todos foram avaliados clinicamente e submetidos a raspado cutâneo profundo para pesquisa de ácaros. Os animais do GD foram tratados com moxidectina e avaliados nos dias 0, 20, 40, 60 e 80. Cinco destes animais obtiveram cura e foram submetidos à biópsia cutânea no dia 0 e no dia 80, para análise das alterações histopatológicas. Na segunda etapa, foram utilizados 26 animais, 17 no G1 (portadores de demodicose) e 9 no G2 (cães hígidos). Todos os animais do GD tiveram raspado positivo para Demodex canis e foram avaliados clinicamente. Foi feita coleta de sangue para obtenção de soro em todos os animais sendo realizada dosagem dos níveis de interleucina 10 através do kit comercial Quantikine Canine IL-10 Immunoassay® (R&D Systems). Os resultados obtidos na primeira etapa, demonstraram uma melhora clinica considerável e negativação do raspado no GD, porém em relação ao padrão histopatológico não houve evolução. Quando comparados os dois aspectos, não houve diferença significativa. O GC apresentou pele hígida e raspado cutâneo negativo. Já na segunda etapa, os cães do G1 apresentaram escores clínicos altos, indicando severidade da doença. Os níveis de interleucina 10 no G1 tiveram média de 184,38 pg/ml (+258,9 pg/ml) enquanto o G2 apresentou média igual a 11,94 pg/ml (+ 2,27 pg/ml), indicando que níveis altos de IL10 podem estar relacionados com o desenvolvimento da doença. Os resultados demonstraram que mesmo clinicamente curados e com raspado cutâneo negativo, os cães portadores de demodicose apresentam persistência das lesões e do ácaro na estrutura histológica da pele, e também que cães com demodicose apresentaram níveis de interleucina 10 elevados quando comparados com animais sadios.

Sweden has through its membership of the EU, an obligation to strive that the wolf population should be both long and short time viability. The signed Convention on Biological Diversity means that the wolf population must meet a favorable conservation status, which has required a great need for scientific facts about what a sustainable status is. The relationship between science and decision making can be difficult, especially in complex environmental problems with many uncertainties. The purpose of this study is to examine the various scientific uncertainties and find out how they influence decision-making within the Swedish wolf policy. The focus is on the impact of the scientific advice for decision-makers. The study is a case study in which the results are based on five interviews with experts in the field as well as a deeper text analysis of the reports and regulations used in the decision-making process. In conclusion, the study indicates that research and scientific findings have had a major role in the decision-makers in wolf management. To some extent, it has also complicated the issue because of the lack of consensus among researchers. The attitude around Sweden's wolves extends far back in time and the conflict today is a contentious question that seems to act much about values ​​and not just scientific contradictions. / Sverige har genom sitt medlemskap i EU en skyldighet att sträva efter att vargens population ska vara både kortsiktigt och långsiktigt livskraftig. Den undertecknade konventionen om biologisk mångfald medför att vargstammen måste uppfylla en gynnsam bevarandestatus, vilket har krävt ett stort behov av vetenskapliga fakta kring vad en hållbar status är. Sambandet mellan vetenskap och beslutsfattande kan vara svår, särskilt i komplicerade miljöproblem med många osäkerhetsfaktorer. Syftet med studien är att granska de olika vetenskapliga osäkerheterna och ta reda på hur de påverkar beslutsprocessen inom svensk vargpolitik. Fokus ligger på den inverkan de vetenskapliga råden har för beslutsfattarna. Studien är en fallstudie där resultatet bygger på fem intervjuer med experter inom området, samt en djupare textanalys av de rapporter och förordningar som använts i beslutsprocessen. Sammanfattningsvis tyder studiens resultat på att forskning och vetenskapliga resultat har haft en stor roll för beslutsfattarna inom vargförvaltningen. Till viss del har den också försvårat frågan på grund av avsaknaden av konsensus bland forskarna. Attityden kring Sveriges vargar sträcker sig långt bak i tiden och konflikten är idag en laddad fråga som inte bara handlar om vetenskapliga motsättningar, utan även om värderingar.

Long-distance vocalizations by canids play an important role in communication among individuals. I evaluated efficacy of broadcasted coyote (Canis latrans) group-yip calls and gray wolf (C. lupus) lone howls to elicit vocal responses from 18 GPS-collared coyotes on 144 occasions. I concluded that eliciting coyote vocalizations where wolves are present will not bias responses, and recommend eliciting coyote vocalizations using recorded coyote group-yip howls during July–September to estimate species’ presence or density. From foraging theory, generalist predators should increase consumption of prey if prey availability increases. I estimated densities for coyotes, adult deer, and fawns, and collected coyote scat to estimate occurrence and biomass of adult and fawn deer consumed by coyotes during 2 periods. I suggest that consumption rates of coyotes was associated positively with increases in fawn density, and fawn consumption by coyotes follows predictions of foraging theory during this pulsed resource event.

While studying packs of the eastern timber wolf in Algonquin Provincial Park in Ontario, DNA profiles at 8 microsatellite loci and the mitochondrial control region were found to be similar to those of the red wolf, C. rufus. Based on this it was suggested that both the red wolf and the eastern timber wolf have a common origin, evolving in North America, with the coyote diverging from them 150,000-300,000 years ago and with neither having any recent connection with the gray wolf that evolved in Eurasia. It was further proposed, that the eastern timber wolf retain its original species designation of C. lycaon instead of the present status of a subspecies of the gray wolf. Four "types" or "races" of wolves have been previously described in Ontario. Using DNA profiles, assignment tests identified four groups, which were typified by animals in Algonquin Provincial Park, Pukaskwa National Park, Frontenac Axis and those north of Lake Superior. The tests indicate that Frontenac animals are hybrids between the western coyote and C. lycaon and represent the eastern coyote. Pukaskwa maintains a small wolf population, which is genetically closer to the gray wolves of the Northwest Territories than the surrounding C. lycaon. These may represent an isolated remnant population of the original "Ontario type" (C. lupus). Animals north of Lake Superior were identified as C. lycaon, but represent products of hybridization between C. lycaon and C. lupus. Currently within Ontario, Algonquin Park contains the largest protected area of the eastern timber wolf. DNA profiles, including Y-linked microsatellite loci were used to establish maternity, paternity and kin relationships for 102 animals from 24 packs over a 12-year period. A complex pack structure was identified. A pack is not composed simply of an unrelated breeding pair and their offspring and subordinates appear to enter pack systems through adoption, pack splitting, dispersal and immigration. Relatively high genetic structuring was found between the Park animals and the "Tweed" wolves to the southeast suggesting introgression of coyote genetic material is not a present concern to the integrity of park animals. Evidence of gene flow with animals to the west, northeast and northwest coupled with the high genetic diversity, suggest that the Park animals are not an island population, but the southern part of a larger metapopulation of C. lycaon. Increased interest in the relationship of the red and eastern wolves led to the investigation of a gene in the major histocompatibility complex. Allelic variation in the exon 2 region of the DLA-DQA1 locus was analysed for gray wolves, red wolves, the eastern timber wolf and the western coyote. Twelve alleles were identified, seven of which were previously characterized in dogs. Non-synonomous nucleotide substitutions was 3.0 times higher than the synonomous changes, indicative of strong positive selection. These data provide baselines for the determination of allele frequencies and their distribution across the geographical range of the four species in North America. The results in this thesis have sparked numerous debates with respect to the protection of the wolves in Algonquin Provincial Park and reintroduction of wolves into Northeastern United States. The data support the idea that the C. lycaon population in Ontario is relatively large, numbering in the thousands rather than the hundreds. Concern for the conservation of wolves in Ontario should be directed at the declining numbers of gray wolves present in Ontario. / Thesis / Master of Science (MSc)

Babesia canis est un hémoparasite du phylum des Apicomplexes transmis par la morsure de tique et responsable de la piroplasmose canine en Europe. Dans la perspective de développer un vaccin recombinant, nous avons réalisé deux études visant à mieux comprendre les interactions hôte/parasite au cours du cycle érythrocytaire. Nous avons étudié d'une part le rôle des antigènes parasitaires solubles (APS) dans le déclenchement des signes cliniques et d'autre part celui de l'antigène Bc28.2 codé par la famille multigénique Bc28 dans les mécanismes d'échappement à l'hôte.Les APS de B. canis induisent une réponse protectrice anti-maladie chez les chiens vaccinés. Toutefois, leur rôle exact dans la pathogénèse reste à définir. Contrairement à ce qui est décrit dans la pathogenèse à B. bovis, nos analyses réfutent l'hypothèse qu'ils pourraient agir sur le système kallicréine-kinine plasmatique. Par contre elles suggèrent, pour la première fois chez le genre Babesia, un rôle direct des APS dans le déclenchement précoce de la réponse inflammatoire observée au cours de la pathogenèse.De part leur fonction essentielle dans la survie parasitaire, les antigènes localisés à la surface de l'érythrocyte (rôle dans l'agglutination des hématies parasitées) ou des mérozoïtes (rôle dans l'invasion) sont de bons candidats vaccins. Cependant et probablement dans une perspective d'échappement à l'hôte, ils sont codés par des familles multigéniques. Chez B. canis, la famille multigénique Bc28 contient le gène Bc28.1 qui code pour un antigène à ancrage GPI préalablement caractérisé à la surface du mérozoïte. Nous montrons qu'un autre gène, désigné Bc28.2 (plusieurs copies dans le génome) contient 2 cadres de lecture chevauchant et serait capable d'exprimer des antigènes polymorphes de 28 kDa et 50 kDa par un mécanisme de recodage traductionnel +1. De façon originale, ces protéines seraient localisées non pas à la surface des mérozoïtes mais à la surface des hématies parasitées. / Babesia canis is an apicomplexan haemoparasite transmitted by tick bite and responsible of canine babesiosis in Europe. Understanding host/parasite relationships during the erythrocytic cycle is crucial for further development of a recombinant vaccine. In that way, the role of soluble parasite antigens (SPA) in the onset of clinical signs and the role of Bc28.2 antigen (encoded by the B. canis Bc28 multigene family) in host evading process were investigated.B. canis-derived SPA induce a protective anti-disease immunity in vaccinated dogs but their precise role during the pathogenesis remains unknown. In contrast to B. bovis, our analysis disproved the hypothesis that B. canis SPA could act on the plasma kallikrein-kinin system. However they strongly suggest, for the first time in the genus Babesia, a direct role of these SPA in the onset of the inflammatory response which is early observed during pathogenesis. Because of their essential function in the parasite survival, antigens located on the surface of infected erythrocyte (role in agglutination of erythrocytes) or on the surface of merozoites (role in the invasion) are good vaccine candidates.However, and probably for host evading, they are encoded by multigene families. In B. canis, the Bc28 multigene family contains the Bc28.1 gene that encodes for a GPI-anchored antigen previously characterized on the merozoite surface. We demonstrated here, that another gene designated Bc28.2, is multicopy and composed of two overlapping open reading frames (Orf1 and Orf2). It allows, though +1 programmed ribosomal frameshift, expression of polymorphic antigens of 28 kDa and 50 kDa. Unexpectedly, these proteins seem localized on the surface of parasitized erythrocytes, suggesting they play a crucial function in evading host through agglutination process of infected erythrocytes.

En Europe, la présence de grands carnivores dans des paysages anthropisés entraîne une forte compétition avec l'homme et alimente d'importantes polémiques concernant leur protection légale. La perception antagoniste de ces espèces à la fois emblématiques pour certains et sources de conflits pour d'autres, rend la gestion de leurs populations très délicate. Depuis la recolonisation spontanée du loup (Canis lupus) dans les Alpes françaises au début des années 1990, la population s'est accrue numériquement et spatialement. Parallèlement, les dégâts occasionnés par le loup sur la filière élevage ont suivi la même tendance. L'Etat met en place aujourd'hui un contrôle raisonné de la population, sous réserve que les objectifs de conservation, exigés par la Directive Habitat, soient respectés. En s'inspirant du cas d'étude du loup en France, nous proposons dans cette thèse un cadre de prise de décision structurée adapté pour la gestion et la conservation d'espèces rares et difficilement observables, protégées par des accords législatifs mais qui, dans un contexte social conflictuel, peuvent être régulées. La modélisation séquentielle du processus décisionnel s'est déroulée dans un contexte de forte incertitude selon plusieurs étapes : 1) appréhender les objectifs de conservation et/ou contrôle du loup en France pour les formaliser sous forme mathématique via une fonction d'utilité, 2) suivre la population par une méthode non-invasive pour définir des indicateurs de gestion fiables et évaluer le statut de conservation de la population, 3) coupler les mesures létales adoptées actuellement à un modèle démographique décrivant la dynamique du loup et intégrant sa structure sociale, 4) et déterminer la décision. Cette dernière étape est réalisée à l'aide d'une méthode d'optimisation qui calcule la stratégie optimale de gestion en fonction de la structure sociale de la population et des différentes sources d'incertitude accumulées à chaque étape du processus décisionnel. Nous avons choisi comme indicateur de gestion le taux de croissance, à partir duquel nous avons défini l'utilité. Cet indicateur était robuste à l'incertitude d'échantillonnage émergeant de la détection partielle et hétérogène des individus. Des analyses de sensibilité de la décision ont montré une forte influence de la fonction d'utilité sur la stratégie optimale, soulignant ainsi l'importance de définir correctement les objectifs. Nous avons également montré que la stratégie optimale était sensible aux variations des paramètres démographiques, montrant ainsi l'intérêt des méthodes de capture-marquage-recapture pour les estimer correctement. Nous discutons enfin de l'extension de notre approche à un cadre décisionnel de gestion adaptative pour traiter des problèmes de conservation dans un contexte conflictuel. / Large carnivore management in Europe is controversial because of conflictive objectives arising from the legal protection of threatened species vs. the possible necessity of culling individuals to prevent severe damages on human activities. Since the wolf recovery in the French Alps in the early 90's, the population has been numerically and spatially increasing. In parallel, livestock depredations have been following the same trend. As an EU member state, France is bound to the European Habitat Directive, which provides full protection of wolf populations and their habitat. Nevertheless, derogatory killings are allowed for individuals causing problems on livestock and some lethal control is now incorporated into the national management plan, as long as the population growth and its distribution range are not being threatened. Illustrating with the case study of the wolf in France, my dissertation proposes a structured decision making framework for the management and the conservation of elusive species that are legally protected but, in a conflictive context, are subject to population control. The sequential modeling of our decision process occurred in the following steps: 1) define the multiple objectives and formulate them in terms of a utility function, 2) monitor the population through a non-invasive approach in order to define the population conservation status, 3) build a demographic model to predict the consequences of harvesting on population dynamics and social structure, 4) obtain optimal state-dependent decisions. The last step is done with stochastic dynamic programming (SDP), acknowledged to be one of the most useful optimization methods in decision making. We provide an optimal solution for wolf management that gives the highest chance of meeting objectives, defined on population growth rate. This demographic indicator was found to be robust to sampling uncertainty arising from partial and heterogeneous detection of individuals. We ran decision sensibility analyses and found a strong effect of the utility function on the optimal strategy, highlighting the importance of defining explicit objectives. We also found that the optimal strategy was sensitive to demographic parameters, which demonstrate the general need of using solid statistical approaches to estimate them properly. This structured decision making framework can further be extended to adaptive management, acknowledged as being a convenient framework for wildlife management.

Ctenocephalides felis est une espèce de puce cosmopolite parasitant majoritairement les carnivores domestiques. Elle est vectrice de nombreux agents pathogènes zoonotiques dont les bactéries du genre Bartonella, bactéries intracellulaires facultatives. La compétence vectorielle de cette puce a été investiguée pour B. henselae, B. quintana, B. clarridgeiae, B. tribocorum et B. birtlesii. Dans ces conditions expérimentales, utilisant un système de gorgement sur membrane, ces espèces ont persisté pendant les trois jours d'une première étude, et pour B. henselae durant les 13 jours de survie des puces, dans une seconde étude. Les cinq espèces de bartonelles ont été retouvées dans les fèces. Pour ces cinq espèces, nos résultats montrent une absence de transmission verticale transovarienne chez la puce et suggèrent une possibilité de contamination horizontale. Nous proposons enfin un protocole original d'évaluation de l'efficacité d'un traitement antiparasitaire chez le chat, pour prévenir sa contamination par Bartonella spp. / Ctenocephalides felis is a cosmopolitan flea species mainly parasitizing pets, transmitting several pathogens of veterinary and zoonotic importance including the facultative intracellular bacteria of the genus Bartonella. The vector competence of this flea was investigated for B. henselae, B. quintana, B. clarridgeiae, B. tribocorum and B. birtlesii, using an artificial feeding system. In these experimental conditions, these bartonellae proved to persist for three days, in a first study, while B. henselae persisted for the 13 days of its life span, in a second study. All five bartonellae were excreted in the flea's faeces. On the whole, these five species were not transmitted transovarially in the fleas, though horizontal transmission was suggested. Furthermore, we propose an original protocol allowing the evaluation of the efficacy of ectoparasiticidal products against Bartonella spp. infection in cats.

A toxocaríase é considerada uma das cinco parasitoses negligenciadas pelo Centers for Disease Control and Prevention e recebe ainda pouca atenção. As metodologias diagnósticas conhecidas são bem estabelecidas, apresentando, porém, limitações caracterizadas, sobretudo, pela ocorrência de reações-cruzadas. A biologia molecular mostra grandes avanços para o diagnóstico eficaz de diversas parasitoses, mas ainda carece de estudos em amostras de fácil obtenção para o diagnóstico da toxocaríase. Para aprimorar o conhecimento sobre a importância da técnica da Reação em Cadeia da Polimerase Convencional (PCR) e sua relação com técnicas diagnósticas já conhecidas, foram utilizados 42 camundongos BALB/c, machos, entre 6 a 8 semanas de vida, divididos em três grupos, inoculados com 5, 50 ou 500 ovos larvados e sangrados pelo plexo orbital aos 15, 30, 60 e 90 dias pós infecção. Ainda, do total, 24 camundongos foram sangrados aos 120 dias pós infecção. Ao final do experimento, foi realizada a recuperação de larvas e a PCR de tecido hepático, cérebro e carcaça de camundongos dos grupos infectados. As amostras de soro foram processadas pelas técnicas de ELISA, Western-blotting e PCR. O ELISA e o Western-blotting mostraram resultados reagentes em todas as datas para a maioria dos inóculos de ovos, com relação diretamente proporcional entre a detecção de anticorpos e a carga parasitária. Durante o período da infecção, a detecção de IgG foi mais intensa próxima aos 60 dias pós-infecção para a maioria dos inóculos de ovos, por ambos os métodos imunológicos. Apesar de identificar DNA de larvas e vermes adultos, a PCR não foi capaz de detectar DNA do parasito em amostras de soro em todos os grupos e datas pós-infecção. Em contrapartida, foi detectado DNA do parasito em todos os órgãos com ao menos um dos primers utilizados. Foram recuperadas larvas na maioria dos órgãos com maior porcentagem de recuperação relatada nos animais inoculados com 50 ovos larvados. O diagnóstico molecular, utilizando sangue do paciente, ainda não pode ser considerado uma ferramenta para o diagnóstico dessa infecção / Toxocariasis is considered by the Centers for Disease Control and Prevention one of the five neglected diseases and still receives little attention. The diagnostic methods are well established, presenting, however, limitations characterized mainly by the occurrence of cross-reactions. Molecular biology shows great advance for the effective diagnosis of several parasitic infections, but still lacks studies using samples that are easily obtained for the diagnosis of toxocariasis. In order to refine the knowledge about the importance of Conventional Polymerase Chain Reaction (PCR) and its relation with known techniques, 42 BALB/c male mice, between 6-8 weeks of age were inoculated with 5, 50 and 500 embryonated eggs respectively and bled by the orbital plexus at 15, 30, 60 and 90 days post infection. Also, 24 of 42 animals were bled the same way at 120 days post-infection. At the end of the experiment, larval recovery and conventional PCR were performed in liver, brain and carcass of mice of the infected groups. Serum samples were processed by ELISA, Western-blotting and PCR. The ELISA and Western-blotting techniques showed positive results in all days post infection for most eggs inocula and showed a directly proportional dependence between the infective dose and the level of antibodies. During the course of the infection, IgG detection was most intense near 60 days post infection for most eggs inocula, for both diagnostic methods. Despite positive DNA identification in larvae and adult worms, PCR wasn\'t able to detect parasite DNA in serum samples in all infected groups and days post infection. In contrast, parasite DNA was detected in all organs with at least one of the primers. Larvae were recovered from most organs, and animals inoculated with 50 embryonated eggs showed the highest percentage of larval recovery. Molecular diagnosis using patient\'s blood is not the best tool for toxocariasis diagnosis so far

INTRODUÇÃO: Toxocaríase é uma infecção parasitária de distribuição global, causada pela fase larval de Toxocara spp. Os hospedeiros naturais são cães e gatos, nos quais o parasita completa o ciclo chegando a fase adulta. Outros hospedeiros podem ser infectados pela fase larval do parasita, após ingestão de ovos embrionados do solo, mãos contaminadas, fomites, ou ingestão de carne ou vísceras de animais infectados. Em hospedeiros paratênicos o parasita não completa o ciclo, invadindo em estágio larval vísceras ou outros tecidos, onde podem sobreviver e induzir a patologia. O presente estudo teve como objetivo caracterizar o hamster (Mesocricetus auratus), como modelo experimental de toxocaríase, inicialmente através do estudo das lesões histopatológicas em fígado, pulmão e rim. A caracterização da resposta imunológica do modelo, foi feita através do estudo de citocinas envolvidas nas respostas Th1 e Th2, e foi sugerida uma correlação entre alterações glomerulares e depósitos de complexos antígenos-anticorpo pré-formados na circulação. MÉTODOS: Hamsters foram inoculados com ovos embrionados de Toxocara canis, e mantidos no biotério do Instituto de Medicina Tropical de São Paulo. O estudo histopatológico foi desenvolvido utilizando-se cortes parafinados corados por hematoxilina e eosina. Para detecção de antígenos nos tecidos foram realizadas reações imunohistoquímicas, utilizando-se anticorpo monoclonal e policlonal anti- Toxocara canis. Utilizando-se o soro dos animais infectados e animais controle, foi realizada pesquisa de antígeno e anticorpo por ELISA. Para pesquisa de imunoglobulinas IgG e IgM e complemento, foram utilizados cortes congelados de rins para realização de reação de Imunofluorescência. Fragmentos de rins foram incluídos para utilização em microscopia eletrônica, para detecção de antígenos de toxocara e de imune complexos. Para caracterização de resposta imunológica foram estudadas citocinas envolvidas na resposta Th1 e Th2 por técnica de RT-PCR. RESULTADOS: Os achados histopatológicos demonstraram desde o início da infecção, presença de larvas em maior número no fígado, seguido de pulmão e raramente rins. Em fígado remanescentes larvares foram visualizados cercados por reação inflamatória granulomatosa. Logo no início da infecção foi encontrado pneumonite intersticial e intraalveolar focal, e lesão renal com glomérulo apresentando hiperplasia focal de células mesangiais (glomerulite mesangio-proliferativa). Houve marcação de antígenos em todos os grupos de animais infectados, tanto pelo anticorpo monoclonal, como pelo policlonal. Depósitos de imunoglobulinas e complemento foram marcados em glomérulo por imunofluorescência A análise dos soros por ELISA, demonstrou na pesquisa de anticorpos aumento gradativo no decorrer da infecção, acompanhado de diminuição de antígenos. Depósitos de antígenos em glomérulos foram detectados por microscopia imonoeletrônica. No RT-PCR foi detectado aumento significativo do nível de IL-4, com tendência de elevação de IL-10 e IFN-?. CONCLUSÃO: O hamster demonstrou ser um modelo experimental eficiente para toxocaríase. Entretanto este modelo é mais adequado para infecções de curto prazo. A resposta imunológica avaliada por RT-PCR, com elevado nível da expressão de IL-4, sugere uma resposta Th2, mas a tendência de aumento de IL-10 e IFN-? poderia sinalizar uma resposta mista Th1 e Th2. Achados de depósitos de imunoglobulinas no glomérulo sugerem a possibilidade de que as manifestações renais com síndrome nefrótica em humanos possa vir a ter como base a toxocaríase / INTRODUCTION: Toxocariasis is a parasitic infection of global distribution, caused by the larval stage of Toxocara spp. The natural hosts are dogs and cats, in which the parasite completes the cycle reaching adulthood. Other hosts can be infected with the larval stage of the parasite, after ingestion of embryonated eggs from the soil, contaminated hands, fomites, or ingestion of meat or viscera of infected animals. In paratenics hosts the parasite not complete the cycle, encroaching on larval stage in viscera or other tissues where they can survive and induce pathology. The present study aimed to characterize the hamster, Mesocricetus auratus, as experimental model of toxocariasis, initially through the study of histopathological lesions in the liver, lung and kidney. The characterization of immune response model, was made through the study of cytokines Th1 and Th2 responses involved, and a correlation was suggested between glomerular changes and antibody-antigen complexes deposits preformed in the circulation. METHODS: Hamsters were inoculated with embryonated eggs of Toxocara canis, and kept in the bioterium of the Institute of Tropical Medicine of the São Paulo. The histopathologic study was developed using paraffin slides stained by hematoxylin and eosin. For detection of antigens in tissues immunohistochemistry reactions were performed using monoclonal and polyclonal anti-Toxocara canis sera. Using the serum of infected and control animals, search has been carried out of antigen and antibody by ELISA. For the search of immunoglobulins IgG, IgM and complement, were used slides prepared from frozen fragments of kidneys and a immunofluorescence reaction. Fragments of kidneys were included for electron microscopy to detect antigens of Toxocara and immune complexes. For characterization of Th1 and Th2 response cytokines involved were detected by RT-PCR technique. RESULTS: Histopathological findings demonstrated since the beginning of the infection the presence of larvae in greater numbers in the liver, followed by lung and rarely kidneys. In the liver larval remnants were surrounded by a granulomatous inflammatory reaction. Early in the infection was found interstitial pneumonitis with intraalveolar focal inflammatory infiltrate and renal injury with glomerulus showing mesangial cell focal hyperplasia (mesangioproliferative glomerulonephritis). There were the presence of antigens in all groups of animals infected detected by both the monoclonal and polyclonal antibodies. Deposits of immunoglobulin and complement were present in glomerulus by immunofluorescence analysis. ELISA, showed that the presence of antibodies increased gradually in the course of infection, accompanied by progressive diminution of antigens. Clusters of antigen/s were detected by immunoelectron microscopy. RT-PCR showed a significant increase of IL-4, with a tendency of increase of IL- 10 and IFN-?. CONCLUSION: The hamster has proved to be an efficient experimental model for toxocariasis. However this model is best suited for short-term infections. The immune response evaluated by RT-PCR, with high level of expression of IL-4, suggests a Th2 response, but the trend of increase of IL-10 and IFN-? might suggest a Th1 and Th2 mixed response. Findings of immunoglobulin deposits in glomeruli suggests the possibility that the renal manifestations with nephrotic syndrome in humans might have, in certain circunstances, as a basis the toxocariasis