Orientador: Márcia Ferreira da Rosa Sobreira / Coorientador: Gisele Fabrino Machado / Banca: Aureo Evangelista Santana / Banca: Maria Angélica Dias / Resumo: A cinomose canina é uma importante afecção contagiosa, com elevada taxa de letalidade e comprometimento multissistêmico, incluindo o sistema nervoso central. A leucoencefalite desmielinizante é a principal alteração neuropatológica observada, sendo sua patogênese pouco conhecida. A quebra da barreira hematoencefálica (BHE) com penetração viral e infiltração leucocitária são aspectos cruciais no desenvolvimento da encefalite desmielinizante. Em seres humanos e animais com alterações semelhantes, as metaloproteinases da matriz (MMPs), principalmente 2 e 9, vem sendo apontadas como os agentes desestruturantes da integridade da BHE. Assim, buscou-se estabelecer o envolvimento das MMPs 2 e 9 na quebra da BHE de cães com cinomose em fase neurológica. O presente estudo teve como escopo principal determinar e quantificar tais enzimas, pela técnica de zimografia, no soro e no liquor de cães hígidos (Gcon, n=10) e cães com cinomose na fase neurológica (Gcin, n=10). Além disso, foram determinados os parâmetros físico-químicos do liquor para a obtenção de maiores informações acerca da integridade da BHE. A análise estatística multivariada exploratória mostrou padrões distintos segundo as variáveis estudadas, sendo estas comparadas entre os grupos com o teste t / Abstract: Canine distemper is an important contagious disease with high mortality rate and multisystem involvement including the central nervous system. The demyelinating leukoencephalitis is the main neuropathological alteration observed and its pathogenesis is not well known. The rupture of the blood brain barrier (BBB) with viral penetration and leukocyte infiltration are crucial for the development of demyelinating encephalitis. In humans and animals with similar disorders, matrix metalloproteinases (MMPs) especially 2 and 9 have been pointed as the agents that destabilize the integrity of the BBB. Thus, we sought to establish the involvement of MMPs 2 and 9 in BBB rupture of dogs with distemper at the neurological phase. The present study aimed to determine and quantify these enzymes by the technique of zymography in serum and cerebrospinal fluid of healthy dogs (Gcon, n=10) and dogs with distemper at neurological stage (Gcin, n=10). Furthermore, we determined the physicochemical parameters of liquor to obtain more information about the integrity of the BBB. Exploratory multivariate analysis showed distinct patterns according to the variants studied, which were compared between groups with t test / Mestre

Orientador: Maria Leticia Cintra / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T19:11:09Z (GMT). No. of bitstreams: 1 Scarpelli_EdsonMartins_M.pdf: 3928334 bytes, checksum: 3ef78d4f7a3f4ec5c95969a5e584a0e9 (MD5) Previous issue date: 2008 / Resumo: A cinomose canina (CC) é uma doença viral sistêmica de carnívoros, cujo diagnóstico é embasado em sinais neurológicos e extra-neurais. A desmielinização que ocorre nesta doença assemelha-se à das doenças desmielinizantes humanas, o que a torna um modelo animal para estudo de doenças neurodegenerativas humanas. Lesões iniciais do SNC caracterizam-se por desmielinização sem considerável inflamação e lesões tardias por um processo inflamatório crônico. Vários métodos são empregados para o diagnóstico da CC, destacando-se a RT-PCR como precisa e sensível. Estudar, prospectivamente, os achados clínicos e histopatológicos do cérebro de 49 cães com cinomose diagnosticada clinicamente, e confrontá-los com os resultados da RT-PCR foi o nosso objetivo. A presença de sinais neurológicos foi significante para o diagnóstico da doença, especialmente a mioclonia e ataxia. O comprometimento inflamatório era significante no cerebelo, hipocampo e diencéfalo sendo que a reação inflamatória era maior no diencéfalo. Quanto maior o peso cerebral, menor era a desmielinização. Nos animais na fase clínica aguda, a freqüência de desmielinização cerebelar foi maior. Foi significante a localização linização, mas, também, havia forte comprometimento cortical, embasando os comemorativos clínicos. A pesquisa do vírus da CC pela RT-PCR foi negativa em 34% dos animais doentes; 88% deles encontravam-se na fase crônica, onde o vírus esta praticamente ausente. Em todos os cães sadios, a RT-PCR resultou positiva, provavelmente por vacinação ou doença prévia. Nossos achados ressaltam a importância da avaliação clínica para o diagnóstico da CC e dos achados anatomopatológicos respaldando a clínica / Abstract: Introduction: canine distemper (CD) is a carnivores viral systemic disease whose clinical diagnosis is based on neurological and extra neural signs. The CD demyelination resembles those of human demyelinating diseases; therefore it becomes an animal model for human neurodegenerative diseases study. Central nervous system (CNS) initial lesions are characterized by demyelination with no considerable inflammation and late lesions by a chronic inflammatory process. Several methods are used for the diagnosis of CD; and RT-PCR stands out for being precise and sensitive. Purpose: to study, prospectively, clinical and histopathologic signs of 49 dogs' brains with distemper clinically diagnosed, and compare them to RT-PCR results. Results and Conclusions: the presence of neurological signs was significant for the diagnosis of the disease, especially myoclonus and ataxia. The inflammatory reaction was significant in the cerebellum, the hippocampus and the diencephalons; the inflammatory reaction was larger in the diencephalon. The higher the cerebral weight the lowest the demyelination. In animals on the acute clinical form, the demyelination frequency in the cerebellum was higher. Cerebellum and diencephalons localization in the demyelination was significant, but there was also strong cortical compromising, embasing clinical observations. RT-PCR for CD virus tested negative in 34% of the sick animals; 88% of them were in the chronic phase, in which the virus is practically non-existent. In all healthy dogs, RT-PCR resulted positive, probably due to vaccination or previous disease. Our findings stress the importance of clinical evaluation for CD diagnosis and histopathological examination supporting clinical findings / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Memoria para optar al Título Profesional de Médico Veterinario / El Virus Distemper Canino (VDC) es un agente infeccioso de amplia distribución, responsable de la enfermedad denominada Distemper Canino (DC) que afecta a una amplia diversidad de carnívoros terrestres (reportándose incluso en primates) y marinos. Afecta agresivamente a perros domésticos (con una tasa de mortalidad aproximada del 50%). Por tanto, el impacto clínico del VDC es importante. El VDC es codificado por 6 genes, N, P/V/C, M, F, H y L. Casi todos ellos se han usado para identificación de linajes y diagnóstico de la enfermedad. En este trabajo y utilizando Reacción en Cadena de la Polimerasa asociada a Retrotranscripción (RT-PCR) se analizaron 22 fragmentos de Ácido Ribonucleico (RNA) obtenidos de sangre periférica de 22 perros diagnosticados con VDC clínicamente y positivos a VDC mediante RT-PCR (gen N) con el objetivo de lograr implementar la detección del gen de la proteína grande (L) como alternativa diagnóstica. En este sentido, se diseñó un par de partidores que generaron un fragmento de ácido desoxirribonucleico (DNA) de alrededor de 450 pares de bases (pb), el cual mediante análisis bioinformático confirmó su identidad (PIN>94%). Así, esta implementación abre la discusión sobre el mejor método alternativo, en conocimiento de que la detección del gen P mediante RT-PCR anidado lo es actualmente. Un análisis de sensibilidad pondría dilucidar esta incógnita prontamente. Se concluye por tanto que esta técnica molecular ofrece un efectivo método diagnóstico para la presencia de VDC en perros domésticos. / The Canine Distemper Virus (CDV) is a widespread responsible for the Canine Distemper Disease that affects a broad diversity of carnivore mammals (and some cases in primates have been reported) globally. Domestic dogs are extensively and aggressively (with a ~50% mortality rate) infected by the CDV. Therefore, the clinical impact of the CDV is remarkable. The CDV is encoded by six genes, N, P/V/C, M, F, H and L. Almost all of which have been previously studied for lineage identification and disease diagnosis. In this study we used Reverse Polymerase Chain Reaction tests (RT-PCR) to analyseobtain 22 fragments of Ribonucleic acid (RNA) from peripheral blood of 22 subject dogs clinically diagnosed with CDV and positive to CDV by RT-PCR (N gene). The aim of this study was to implement a protocol of detection of the Large Protein Gene (L) to be employed as an alternative diagnostic. To achieve this, a pair of primers that generated a fragment of deoxyribonucleic acid (DNA) of ~450 base pairs (bp) was designed, which, using a bioinformatic analysis, confirmed its identity (PIN > 94%). Therefore, the implementation of this protocol opens the discussion about the best alternative method, within the context that the detection of the P gene through Nested RT-PCR is currently the best available protocol. A sensitivity analysis may potentially elucidate this question. Consequently, the main conclusion is that the molecular technique implemented in this study offers an effective diagnostic method to optimize the detection of VDC in domestic dogs. / Financiamiento: Proyecto FIV 121014019102010.

Studies comparing canine distemper virus (CDV) strains from Africa with that of previously described lineages have been hampered due to a lack of field strains for in vitro experiments. There are no reports of CDV isolations in southern Africa, and although CDV is said to have geographically distinct lineages, molecular information of African strains has not yet been documented. Clinical specimens consisting of whole blood, spleen, lungs, brain and cerebrospinal fluid from dogs with clinical signs suggestive of distemper were obtained from private veterinary practices and diagnostic laboratories in Gauteng province, South Africa. One hundred and sixty two specimens from 124 dogs were inoculated into Vero cells expressing canine signalling lymphocyte activation molecule (Vero.DogSLAM). Cytopathic effects (CPE) in the form of syncytia formation and cell necrosis were observed in 33 (20.4%) specimens within 24 hours of inoculation and the presence of CDV was confirmed with the aid of the direct fluorescent antibody test and electronmicroscopy (EM). Viruses isolated in cell cultures were subjected to reverse transcription – polymerase chain reaction (RT-PCR), and the complete H gene was sequenced and phylogenetically analysed with other strains from GenBank. The animals all displayed the catarrhal, systemic, or nervous signs of the disease. Four of the dogs positive for CDV by viral isolation had a history of prior vaccinations to canine distemper. Seventy three percent of all positive dogs were less than 12 months old. There was no breed or sex predisposition to CDV infection in domestic dogs. Phylogenetic comparisons of the complete H gene of CDV isolates from different parts of the world (available in GenBank) with wild-type South African isolates revealed nine clades. All South African isolates form a separate African clade of their own and thus are clearly separated from the American, European, Asian, Arctic and vaccine virus clades. It is likely that only the ‘African lineage’ of CDV may be circulating in South Africa currently, and the viruses isolated from dogs vaccinated against CDV are not the result of reversion to virulence of vaccine strains, but infection with wild-type strains. Copyright / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Memoria para optar al Título Profesional de Médico Veterinario / El virus distemper canino (VDC) es el agente causal de una de las enfermedades infecciosas más relevantes en carnívoros a nivel mundial. En las últimas décadas se han reportado diversos brotes con alta mortalidad en especies silvestres, los que provocaron una gran disminución en las poblaciones afectadas, poniendo en riesgo la conservación de especies amenazadas. Con el objetivo de determinar la presencia de VDC en diferentes especies silvestres en Chile, se realizó un estudio longitudinal de detección del gen de la Fosfoproteína (gen P) mediante n-RT-PCR. Se analizaron 136 muestras de sangre entera de carnívoros clínicamente sanos provenientes de un zoológico de la Región Metropolitana y de zorros chilotes (Lycalopex fulvipes) de vida libre. Como resultado, no se detectó el gen P en ninguna de las muestras analizadas, siendo todas clasificadas como negativas. La evidencia encontrada, en conjunto con estudios previos, sugiere que el contacto de los zorros chilotes con perros infectados con VDC podría tener graves consecuencias en cuanto a su salud y conservación, causando disminución poblacional o extinciones locales a lo largo de su distribución. Se recomienda realizar estudios serológicos y moleculares de carnívoros silvestres y domésticos en zonas habitadas por zorro chilote que permitan comprender la epidemiología del virus en ambientes naturales para diseñar medidas preventivas adecuadas para proteger ésta especie. Finalmente, se recomienda continuar la vigilancia del virus mediante pruebas serológicas que confirmen la utilidad de la vacunación como medida preventiva en zoológicos y mantener protocolos sistemáticos de vacunación. / Canine distemper virus (CDV) is the causal agent of one of the most relevant infectious diseases in carnivores worldwide. In the last decades, several outbreaks with high mortality in wildlife species have been reported, which caused a large decline of the affected populations, putting at risk the conservation of threatened species. In order to determine the presence of CDV in different wild species in Chile, a longitudinal study of detection of the Phosphoprotein gene (P gene) was carried out by n-RT-PCR. A total of 136 whole blood samples were analyzed, collected from different clinically healthy carnivores from a zoo in the Metropolitan Region and free-ranging Darwin’s foxes (Lycalopex fulvipes). As a result, the presence of P gene was not detected in any of the analyzed samples, so all were classified as negative. The evidence found, in conjunction with previous studies, suggests that the contact of Darwin’s foxes with CDV infected dogs could have severe consequences in their health and conservation status, causing population decline or local extinctions throughout its distribution. It is recommended to perform serological and molecular studies in wild and domestic carnivores in areas inhabited by Darwin’s foxes that allow understanding the epidemiology of the virus in natural environments, in order to design adequate preventive measures to protect this specie. Finally, it is recommended to continue monitoring the virus through serological tests that confirm the efficacy of vaccination as a preventive method in zoos and maintain systematic vaccination protocols. / Proyecto Buin Zoo para la conservación del zorro chilote

No description available.

The higher proximity among humans, domestic animals and wildlife favours disease spill-over both from wildlife to domestic animals and vice versa, which is a potential risk for the extinction of wildlife populations and could be influencing the emergence and/or re-emergence of zoonotic diseases. The domestic dog (Canis familiaris) is the most abundant and widely distributed carnivore worldwide and is known to be carrying many infectious diseases. Among these diseases, domestic dogs are known to be source of canine distemper virus (CDV), canine parvovirus (CPV) and Echinococcus granulosus to wild carnivores and human being. Populations of domestic dogs inhabiting urban areas can be the source of infection of directly transmitted pathogens, since in these areas a high density of domestic dogs can facilitate the maintenance of these infections to both domestic and wild carnivore populations. In addition, the knowledge of the diseases present in the domestic dog populations in close proximity to wildlife is essential for conservation planning and for control of both zoonotic diseases and diseases of conservation concern. This thesis explores the effect of urbanization on the epidemiology of CDV, CPV, and E granulosus in domestic dogs and wild carnivores of the Coquimbo region of Chile as for example, chilla (L. griseus) and culpeo (L. culpaeus) foxes and assess the risk factors that could be facilitate disease transmission between canid inhabiting urban and rural areas. The first of the chapters containing original data, Chapter 3, describe the demography of dogs in the study area, indicating that urban sites have a greater population and a higher density of domestic dogs, a high growth rate and therefore a high turnover of susceptible than rural areas, which can be of relevance for the differences in diseases transmission patterns between these sites. Chapter 4 describe the degree of interaction between wild and domestic carnivores and its effect on interespecific disease transmission; indicating that in the study area there are many opportunities for domestic/wild carnivores interactions, as for example livestock predation by carnivores, by approaching to peridomestic environments, facilitating in this scenario the transmission of CDV, CPV and also E. granulosus by predating on livestock contaminated with cyst echinococcosis. Chapter 5 indicate that urban areas hold domestic dog populations with higher CDV seroprevalence than rural sites and probably these areas are the source of infection to rural sites. In contrast, a more stable CPV seroprevalence was found between urban and rural areas, indicating that possibly this pathogen follow an endemic state across the study area. Chapter 6 describe the factors for E. granulosus prevalence in domestic dogs, livestock and human being, suggesting that more cases of E. granulosus in livestock and in humans are found in provinces of the Coquimbo region with higher percentage of rural population; however, and unexpectedly, more cases of E. granulosus in domestic dogs were found in urban areas, although analysis of risk factors indicated that those domestic dogs inhabiting in the borders of urban areas, were at greater risk of being infected with E. granulosus than those in the centre of these areas. The results of this study exemplify how three pathogens are found in urban areas which can be source of infection to domestic and wild carnivores in the study area.

Memoria para optar al Título Profesional de Médico Veterinario / El crecimiento y expansión de la población humana ha ocasionado un mayor contacto entre los humanos, sus animales domésticos y la fauna silvestre. La interacción física entre estas especies ha favorecido la diseminación de enfermedades infecciosas entre ellas y tienen consecuencias para la salud y la conservación. En asociación con lo anterior, la presencia del perro doméstico (Canis familiaris) en áreas protegidas, puede afectar la conservación de carnívoros silvestres debido, principalmente, a la transmisión de enfermedades mediante “salto taxonómico”, como del virus distemper canino (VDC), que ha sido reportado en todas las familias de carnívoros terrestres y algunos marinos. Esta enfermedad representa una amenaza para estos depredadores y los perros pueden actuar como reservorio del agente infeccioso, manteniendo el virus en sus poblaciones y diseminándolo hacia otros hospederos. El objetivo presente estudio es determinar los factores de riesgo asociados a la presencia de VDC en perros y carnívoros silvestres de la Reserva de la Biósfera de Janos. Se realizó el diagnóstico de VDC mediante serología y se corroboró que el agente se encuentra circulando tanto en poblaciones de perros como de carnívoros silvestres. Los resultados muestran que existe una interacción física entre especies domésticas y silvestres, sin embargo, no sugieren la existencia de un riesgo de infección interespecífico entre los individuos seropositivos. / Human population growth and expansion have caused a habitat overlap between humans, their domestic animals and wildlife population. Physical interactions between these species has enabled the spread of infectious diseases and has had implications for public health and conservation. In addition, the presence of domestic dogs (Canis familiaris) in protected areas may impact wild carnivore’s conservation due to, mainly, disease transmission caused by spillover infection. A good example of this is canine distemper virus (CDV), that has been reported in every family of terrestrial and some marine carnivores. The disease poses a threat for these predators and dogs are the principal reservoir of the infectious agent, keeping the virus among their population and spreading it towards other hosts. The objective of the present study is to determine the risk factors related to the presence of CDV in dogs and wild carnivores of the Janos Biosphere Reserve. The virus diagnosis was made by serological testing and it was found that the virus is circulating in both dogs and wild carnivore population. The results show that there is a physical interaction between domestic and wild species, however, they suggest that there is no risk of interspecies infection between positive individuals. / Financiamiento: Proyecto Conacyt CB-179482 (Consejo Nacional de Ciencia y Tecnología de México)

Diferentes amostras biológicas foram avaliadas (zaragatoa ocular, genital, urina e células mononucleares do sangue periférico) pela RT-PCR em 50 cães com sintomas neurológicos compatíveis como cinomose, na presença ou não de outros sintomas sistêmicos. O gene da nucleoproteína do vírus da cinomose foi detectado em 43 das 50 amostras avaliadas. Considerando apenas os animais com resultado positivos pela hemi-nested - PCR, os sintomas neurológicos observados com freqüência maior que 50%, foram mioclonia e alteração locomotora (maioria dos cães evoluiu a tetraparesia); convulsão e vocalização foram observados em 32% dos casos Outros sintomas sistêmicos, sintomas oculares, respiratórios ou digestivos, prévios ou associados aos sintomas neurológicos, foram observados em 82% dos cães. Também observou-se hiperqueratose nasal ou de coxins em 44% dos casos. Somente 20% dos animais positivos haviam sido vacinados contra o vírus da cinomose. Zaragatoas genitais forneceram maior número de resultados positivos (40), seguidos por zaragatoas oculares e urina (37) e células mononucleares do sangue periférico (34). A coleta associada de duas amostras biológicas (zaragatoa genital e urina) por animal aumentou a possibilidade de detecção de animais positivos, principalmente nos casos de cães suspeitos que não apresentaram sintomas respiratórios, gastrintestinais e oculares, assim como nos animais vacinados e cronicamente infectados ou convalescentes. / Using Reverse transcription-PCR assay, different biological samples were avaliabled (conjunctival and genital swabs, urine and peripheral blood mononuclear cells) from dogs with or with no extraneural signs prior to or accompanying the neurologic signs. Canine distemper nucleoprotein were detected in 43 from 50 dogs availabled by hemi-nested-PCR. Considering just dogs with distemper confirmed by hemi-nested PCR, gait abnormalities (most commonly tetraparesis) and myoclonus were the neurological signs observed in over 50%; vocalizing and seizures occurred in 32%. Other systemic signs (respiratory, gastroenteric or ocular signs). preceding or associated to neurological signs, occurred in 82% of dogs Hyperkeratosis of the footpads or nose were observed in 44 % of the cases. Only (20%) were vaccinated against canine distemper. A greater number of positive results were obtained from genital swabs (40), followed by conjuctival swabs and urine (37) and PBMCs (33). Sensitivity of detection positive results were increased by using two clinical samples association (genital swab and urine), specially in dogs that had not shown extra neural signs, vaccinate dogs or during the convalescent and late stage of canine distemper.

A conservação dos animais selvagens de vida livre é ameaçada pela fragmentação de habitat, caça, diminuição de presas e, em menor escala pela ocorrência de doenças infecciosas. Reconhecendo a importância das doenças para a conservação, e considerando que o crescimento da população humana no entorno de áreas protegidas propicia o aumento do contato de animais domésticos e selvagens, o presente estudo teve como objetivo pesquisar a exposição de carnívoros selvagens de vida livre e cães domésticos da região do Parque Nacional das Emas (PNE), Goiás à cinomose, parvovirose, brucelose e brucelose canina. Entre as espécies de carnívoros selvagens amostradas estão o lobo-guará, cachorro-do-mato, raposinha-do-campo, onça-parda, jaguatirica, gato-palheiro, gatomourisco, jaratataca e quati. Foram realizados os testes de soroneutralização, inibição de hemaglutinação, imunodifusão em gel ágar e PCR, para cinomose, parvovirose, brucelose canina e brucelose, respectivamente. Lobos-guará (12,7%, n=9), cachorros-do-mato (11,6%, n=7), jaguatiricas (18,2%, n=2) e cães domésticos (71,4%, n=25) foram expostos à cinomose. Todas as espécies de carnívoros selvagens amostradas, com exceção do quati, sendo 40,4% (n=65) dos indivíduos, e 37,1% (n=13) dos cães domésticos foram expostos à parvovirose. Apenas o lobo-guará (1,67%, n=2) foi exposto à brucelose canina e a raposinha-do-campo à Brucella spp. (1,47%, n=1). Este é o primeiro relato da exposição de gato-palheiro, gatomourisco e jaratataca ao parvovírus, e de lobo-guará à Brucella canis. A cinomose e a parvovirose merecem atenção no PNE pela possibilidade de envolvimento de cães domésticos na sua transmissão, embora não possa ser comprovada no presente estudo. A brucelose, no momento, parece não ser uma ameaça sanitária para as populações de carnívoros do PNE. / The conservation of free ranging wildlife is threatened by habitat fragmentation, hunting, decrease of prey and, to a lesser extent by the occurrence of infectious diseases. Recognizing the diseases importance for conservation, and considering that the increase of human population around protected areas provides increased contact with domestic and wild animals, the present study aimed to investigate the exposure of free ranging wild carnivores and domestic dogs in the region of Emas National Park (ENP), Goiás, Brazil for canine distemper virus (CDV), parvovirus, brucellosis and canine brucellosis. Among the species of wild carnivores sampled are the maned wolf, crab-eating fox, hoary fox, puma, ocelot, pampas cat, jaguarondi, skunk and coati. The tests performed for CDV, parvovirus, canine brucellosis and brucellosis tests were the serum neutralization test, hemagglutination inhibition, agar gel immunodiffusion and PCR, respectively. Maned wolves (12.7%, n = 9), crab-eating foxes (11.6%, n = 7), ocelots (18.2%, n = 2) and domestic dogs (71.4%, n = 25) were exposed to CDV. All species of wild carnivores sampled, except coatis, being 40.4% (n = 65) of individuals, and 37.1% (n = 13) of domestic dogs were exposed to parvovirus. Only maned wolves (1.67%, n = 2) were exposed to canine brucellosis and hoary fox to Brucella spp. (1.47%, n = 1). This is the first report of pampas cat, jaguarondi and skunk exposure to parvovirus, and of maned wolf to Brucella canis. CDV and parvovirus deserve attention in ENP by the possibility of involvement of domestic dogs in its transmission, although it can not be proven in this study. At the moment, brucellosis doesn\'t seem to be a sanitary threat for carnivores population at ENP.