Inflammation in meninges and/or brain is regularly noticed in red foxes and other wild carnivores during rabies control programs. Despite negative rabies virus (RABV) results, the etiologies of these cases remain unknown. Thus, the aim of this study was to provide an overview of the occurrence of pathogens that may cause diseases in the brains of wild carnivores and pose a risk to humans and other animals. In addition to RABV and canine distemper virus (CDV), a variety of pathogens, including members of Flaviviridae, Bornaviridae, Herpesviridae, Circoviridae, as well as bacteria and parasites can also cause brain lesions. In 2016 and 2017, brain samples of 1,124 wild carnivores were examined by direct fluorescent antibody test for RABV as well as (reverse-transcriptase) quantitative polymerase chain reaction (PCR) for the presence of CDV as part of a monitoring program in Saxony-Anhalt, Germany. Here, we applied similar methods to specifically detect suid herpesvirus 1 (SuHV-1), West Nile virus (WNV), Borna disease virus 1 (BoDV-1), canid alphaherpesvirus 1 (CaHV-1), canine parvovirus type 2 (CPV-2), fox circovirus (FoxCV), and Neospora caninum (N. caninum). Further, bacteriogical examination for the existence of Listeria monocytogenes (L. monocytogenes) and immunohistochemistry of selected cases to detect Toxoplasma gondii (T. gondii) antigen were performed. Of all pathogens studied, CDV was found most frequently (31.05%), followed by FoxCV (6.80%), CPV-2 (6.41%), T. gondii (4/15; 26.67%), nematode larvae (1.51%), L. monocytogenes (0.3%), and various other bacterial pathogens (1.42%). In 68 of these cases (6.05%), multiple pathogen combinations were present simultaneously. However, RABV, WNV, BoDV-1, SuHV-1, CaHV-1, and N. caninum were not detected. The majority of the histopathological changes in 440 animals were inflammation (320/440; 72.73%), predominantly non-suppurative in character (280/320; 87.50%), and in many cases in combination with gliosis, satellitosis, neuronophagia, neuronal necrosis, and/or vacuolization/demyelination, or in single cases with malacia. Thus, it could be shown that wild carnivores in Saxony-Anhalt are carriers mainly for CDV and sometimes also for other, partly zoonotic pathogens. Therefore, the existing monitoring program should be expanded to assess the spill-over risk from wild carnivores to humans and other animals and to demonstrate the role of wild carnivores in the epidemiology of these zoonotic pathogens.

(1) Background: Canine distemper virus (CDV)-induced demyelinating leukoencephalitis (CDV-DL) in dogs and Theiler’s murine encephalomyelitis (TME) virus (TMEV)-induced demyelinating leukomyelitis (TMEV-DL) are virus-induced demyelinating conditions mimicking Multiple Sclerosis (MS). Reactive oxygen species (ROS) can induce the degradation of lipids and nucleic acids to characteristic metabolites such as oxidized lipids, malondialdehyde, and 8-hydroxyguanosine. The hypothesis of this study is that ROS are key eector molecules in the pathogenesis of myelin membrane breakdown in CDV-DL and TMEV-DL. (2) Methods: ROS metabolites and antioxidative enzymes were assessed using immunofluorescence in cerebellar lesions of naturally CDV-infected dogs and spinal cord tissue of TMEV-infected mice. The transcription of selected genes involved in ROS generation and detoxification was analyzed using gene-expression microarrays in CDV-DL and TMEV-DL. (3) Results: Immunofluorescence revealed increased amounts of oxidized lipids, malondialdehyde, and 8-hydroxyguanosine in CDV-DL while TMEV-infected mice did not reveal marked changes. In contrast, microarray-analysis showed an upregulated gene expression associated with ROS generation in both diseases. (4) Conclusion: In summary, the present study demonstrates a similar upregulation of gene-expression of ROS generation in CDV-DL and TMEV-DL. However, immunofluorescence revealed increased accumulation of ROS metabolites exclusively in CDV-DL. These results suggest dierences in the pathogenesis of demyelination in these two animal models.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The present study reports a serologic survey of the main viral infections of dogs in Santa Maria, RS, Brazil, and the production of cell lines of canine, swine and leporine origin resistant to bovine viral diarrhea virus (BVDV). Canine distemper virus (CDV), parvovirus (CPV), adenovirus (CAV) and coronavirus (CCoV) infections have been associated with significant morbidity and mortality among dogs worldwide. The aim of this study was to determine the prevalence of antibodies against these viruses in the canine population of Santa Maria. To this purpose, 817 blood samples were collected from non-vaccinated dogs of 14 neighborhoods and tested by virus neutralization (CDV, CAV and CCoV) and by hemagglutining inhibition (CPV). Specific antibodies to CDV were detected in 27.3% (223/817) of the samples, to CPV in 68.7% (561/817), to CAV in 43% (353/817) and to CCoV in 50.4% (412/817) of the dogs. These results indicate that CDV, CPV, CAV and CCoV infections are spread among dogs in Santa Maria. However, a significant part of the population is seronegative and therefore unprotected against these viruses. This indicates a need for extending the vaccination programs against these viruses. During the standardization of serologic tests and expansion of cell cultures for virus amplification, the canine MDCK cell line was found to be contaminated with BVDV, the main viral contaminant of cultured cells. The inadverted contamination of cultured cells with BVDV may represent a serious problem for diagnostic virology, research and production of biologicals. The second part of this dissertation reports the production and characterization of three cell lines resistant BVDV, obtained out of each parental cell line (canine MDCK, porcine PK-15 and leporine RK-13) that were contaminated with BVDV. Initially, the cells were submitted to four rounds of infection with a highly cytolytic BVDV strain. The cells surviving infection were then cloned out, expanded and assayed for their susceptibility to BVDV. The resistance to BVDV was investigated by search for viral proteins by immunofluorescence and by cocultivation with susceptible cells following inoculation of BVDV at high titers. All three cell lines were resistant to three standard BVDV strains (Singer, NADL e Oregon) and 10 field isolates. Inoculation of these cells with BVDV at a multiplicity of infection of 10 TCID50/cell resulted in frequencies of infection of <10-5 for MDCK-R and PK-15R cells and of 3,3x10-4 for RK- 13R. Compared to the parental ones, the resistant cells were >10.000 (MDCK-R), >20.000 (PK-15R) and 600 (RK-13R) times less susceptible to BVDV. The inoculation of virus in the resistant cells in the presence of polyethylene-glicol (PEG) resulted in an increase in susceptibility in the order of >437 (MDCK-R), >346 (PK-15R) and 87 (RK-13R) times. These results indicate that the resistance of these cell lines is probably due to a block in viral entry which can be overcome by addition of PEG. On the other hand, each resistant cell line retained the susceptibility to other three viruses of interest which replicate in the parental cells. Thus, these cells may be useful for virology diagnostic, virus propagation and for vaccine production, without the risk of being inadvertedly contaminated with BVDV. / O presente trabalho relata um inquérito sorológico das principais infecções víricas de cães em Santa Maria, RS, Brasil e a obtenção de linhagens celulares de origem canina, suína e leporina resistentes ao vírus da Diarréia Viral Bovina (BVDV). As infecções pelo vírus da cinomose (CDV), parvovírus (CPV), adenovírus (CAV) e coronavírus (CCoV) são importantes causas de morbidade e de mortalidade em cães em todo o mundo. Com o objetivo de determinar a prevalência de anticorpos contra esses vírus na população canina da cidade de Santa Maria, coletou-se amostras de sangue de 817 cães não-vacinados, em 14 bairros. Estas foram testadas pela técnica de soroneutralização (CDV, CAV e CCoV) ou inibição da hemaglutinação (CPV). Anticorpos específicos contra o CDV foram detectados em 27,3% (223/817) das amostras, contra o CPV em 68,7% (561/817), contra o CAV em 43% (353/817) e contra o CCoV em 50,4% (412/817) dos cães. Esses resultados demonstram que esses vírus estão difundidos na população canina dos bairros da cidade. Por outro lado, demonstram também que uma parte considerável da população é soronegativa e, portanto está desprotegida contra esses agentes, indicando a necessidade de se ampliar os programas de vacinação para essas infecções. Durante a padronização das técnicas sorológicas e expansão dos cultivos celulares para amplificação dos vírus, detectou-se a contaminação da linhagem de células caninas MDCK com o BVDV, o principal vírus contaminante de cultivos celulares. A contaminação inadvertida de cultivos celulares com o BVDV pode representar um sério problema para o diagnóstico virológico, pesquisa e produção de imunobiológicos. A segunda parte dessa dissertação descreve a produção e caracterização de três linhagens celulares resistentes ao BVDV, obtidas a partir das células parentais de origem canina (MDCK), suína (PK-15) e leporina (RK-13) que estavam contaminadas com o BVDV. Essas células foram submetidas a quatro ciclos de infecção com uma cepa citolítica de BVDV. As células que sobreviveram a infecção lítica foram clonadas, expandidas e testadas para a sua susceptibilidade ao BVDV e outros vírus de interesse. A resistência ao BVDV foi investigada pela pesquisa de antígenos virais por imunofluorescência indireta e por cocultivo com células susceptíveis após a inoculação do vírus em altos títulos. As três linhagens celulares demonstraram ser resistentes a três cepas-padrão (Singer, NADL e Oregon) e a 10 isolados de campo do BVDV. A inoculação do BVDV nessas células com uma multiplicidade de infecção de 10 DICC50/célula resultou em freqüências de infecção de <10-5 para as células MDCK-R e PK-15R; e de 3,3x10-4 para as células RK-13R. Comparando-se com as células parentais, verificou-se que as linhagens resistentes são >10.000 (MDCK-R), >20.000 (PK-15R) e 600 (RK-13R) vezes menos susceptíveis ao BVDV. A inoculação do vírus nas células resistentes na presença de polietilenoglicol (PEG) resultou em um aumento na susceptibilidade dessas células na ordem de >437 (MDCK-R), >346 (PK-15R) e 87 vezes (RK-13R). Esses resultados indicam que a resistência dessas linhagens ao BVDV reside em um bloqueio na penetração do vírus, que pode ser parcialmente revertido pela adição do PEG. Por outro lado, cada linhagem resistente conservou a susceptibilidade a outros três vírus que replicam nas células parentais. Essas características tornam essas linhagens celulares potencialmente úteis para o diagnóstico, amplificação de vírus e produção de vacinas, sem o risco de contaminação acidental com o BVDV.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The respiratory viruses of dogs are associated with a disease called canine infectious respiratory disease (CIRD). The main etiological agents of CIRD are canine distemper virus (CDV), canine parainfluenza virus (cPIV), canine adenovirus type 2 and canid herpesvirus type 1 (CaHV-1), which may cause single or mixed infections. CIRD occurs most frequently in places with high animal density and constant movement. CDV, cPIV, CAdV-2 and CaHV-1 infections have been described worldwide, however, few reports of molecular identification of these viruses are available in Brazil. Thus, the objective of this study was to investigate the occurrence of respiratory viruses in dogs in Santa Maria, RS, and in dog shelters in RS, trying to correlate their occurrence with the environmental conditions. Nasal secretions were collected from dogs with respiratory signs submitted to veterinary clinics in Santa Maria; and from dogs of three shelters of RS (Cachoeira do Sul [shelters #1 and #2] and Passo Fundo [shelter #3]). Viral detection/identification was performed by polymerase chain reaction (PCR) for CDV, cPIV, CAdV-2 and CaHV-1. Positive samples were sequenced and, for some viruses, phylogenetic analysis was performed, comparing with sequences deposited in GenBank. Samples of shelters #1 and #3 were obtained during the cold season. Shelter #1 presented poor sanitary and nutrition conditions, high animal density and constant direct contact among dogs. In this shelter 78% (58/74) of the respiratory samples were positive for at least one virus. The single infections were caused by cPIV in 30% (22/74) of the samples and CAdV-2 in 5% (4/74). Coinfections represented 23% (cPIV and CAdV-2); 13% cPIV, CDV and CAdV-2; 4% cPIV-2 and CDV; and 3% CDV and CAdV-2. Shelters #2 and #3 presented satisfactory sanitary and nutrition conditions, with large outdoors exercise areas (#2) and animal separation by groups (#3). In shelter #2, 8% (5/35) of the samples were positive to cPIV and 6% to CaHV-1; in shelter #3, 8% (7/77) of the samples were positive to CAdV-2 and 1% to CDV. Of samples obtained in Santa Maria, 40% (10/25) were positive for virus, being 28% (7/25) for cPIV, and 4% (1/25) to each of the other viruses. Thus, the results obtained demonstrate that infections and coinfections by respiratory viruses are common in shelter dogs in RS, and their occurrence is related to population density, health and nutritional conditions and season. These viruses are also circulating in domestic dogs in Santa Maria, associated with respiratory disease. This study reinforces the importance of preventive measures such as vaccination and good environmental conditions to prevent/reduce infections caused by respiratory viruses in dogs. / Os vírus respiratórios de cães estão associados com uma enfermidade denominada doença respiratória infecciosa canina (canine infectious respiratory disease - CIRD). Os principais agentes da CIRD são o vírus da cinomose (canine distemper virus - CDV), vírus da parainfluenza canina tipo 2 (canine parainfluenza virus - cPIV), adenovírus canino tipo 2 (canine adenovirus type 2 - CAdV-2) e herpesvírus canino tipo 1 (canid herpesvirus 1 - CaHV-1), que podem causar infecções simples ou mistas. A CIRD ocorre com maior frequência em locais com alta densidade populacional e constante fluxo de animais. Infecções pelo CDV, cPIV, CAdV-2 e CaHV-1 tem sido descritas em vários países, contudo, são escassos os relatos da identificação molecular desses agentes no Brasil. Além disso, há falta de estudos relacionados aos fatores que favorecem a ocorrência e disseminação desses agentes em cães de abrigos. Desta forma, o objetivo do presente estudo foi investigar a ocorrência de vírus respiratórios em cães do município de Santa Maria, Rio Grande do Sul, e em cães de abrigos, buscando-se associar a ocorrência das infecções com as condições ambientais. Para isso, foram coletadas secreções nasais de cães com sinais respiratórios em clínicas veterinárias de Santa Maria; e de cães de três abrigos do estado do RS (dois em Cachoeira do Sul [#1 e #2] e um em Passo Fundo [#3]). A identificação viral foi realizada por reação em cadeia de polimerase (PCR) para o CDV, cPIV, CAdV-2 e CaHV-1. As amostras positivas foram sequenciadas e, para alguns vírus, foi realizada a análise filogenética, comparando-se com sequências depositadas no GenBank. As amostras dos abrigos #1 e #3 foram obtidas durante épocas de baixas temperaturas. O abrigo #1 apresentava condições sanitárias e nutricionais precárias, além de alta densidade populacional e constante contato entre os cães. Neste abrigo, 78% (58/74) das amostras foram positivas para, pelo menos, um dos vírus investigados. As infecções simples foram causadas pelo cPIV em 30% (22/74) das amostras e CAdV-2 em 5% (4/74). As coinfecções totalizaram 23% (17/74) para o cPIV e CAdV-2; 13% (10/74) para o cPIV, CDV e CAdV-2; 4% (3/74) para o cPIV e CDV; e 3% (2/74) para o CDV e CAdV-2. Os abrigos #2 e #3 eram higienizados corretamente e os cães recebiam alimentação adequada, sendo que no abrigo #2 os animais possuíam amplo espaço para se exercitarem, e no abrigo #3 os animais eram separados em grupos e alojados em gaiolas. No abrigo #2 foram detectadas 8% de amostras positivas para o cPIV e 6% para o CaHV-1; e no abrigo #3, 8% de amostras positivas para o CAdV-2 e 1% para o CDV. Das amostras obtidas em clínicas de Santa Maria, 40% (10/25) foram positivas para um dos vírus pesquisados, sendo 28% (7/25) para o cPIV, e 4% (1/25) para cada um dos outros vírus. Assim, os resultados obtidos demonstram que infecções e coinfecções por vírus respiratórios são comuns em cães de abrigos no estado do RS, estando relacionadas com a densidade populacional, condições sanitárias e nutricionais e estação do ano. Estes vírus também circulam em cães domésticos em Santa Maria, estando associados com doença respiratória. Este estudo reforça a importância de medidas de prevenção, tendo em vista que a vacinação e boas condições ambientais podem reduzir e/ou prevenir as infecções causadas por vírus respiratórios em cães.

Le travail présenté dans ce manuscrit traite des mécanismes d'émergence des maladies infectieuses. Une première partie du texte est consacrée à la réflexion menée sur ces processus, et à leurs conséquences pour la gestion des maladies. Dans une deuxième partie nous exposons les travaux de modélisation réalisés ; l’objectif est de tester si des changements dans l'expression d'une infection peuvent être expliqués par la dynamique de circulation de l'agent infectieux dans la population hôte, à travers la protection de groupe. Nous montrons que l'apparition de la maladie de Carré chez les lions du Serengeti peut être due à une perte d’immunité de groupe suite à l’extinction du virus. Nous étudions ensuite les conséquences sur le patron épidémique d'une protection partiellement croisée entre souches. Enfin, nous intégrons à cette problématique la structure spatiale de la population hôte, pour expliquer des observations faites sur les systèmes lagomorphes/lagovirus en France. / The study presented here aims to suggest and to test several mechanisms to explain how infectious diseases impact could increase, in relation to microparasite circulation and loss of herd protection. We first introduce the main characteristics of host/microparasite interaction and display some knowledge about emerging infectious diseases, and their consequences for diseases control. Stochastic modelling is then used to study epidemic patterns under several hypotheses and for different host/parasite systems: i) we explain canine distemper emergence in Serengeti lions through a break in virus circulation and the resulting loss of herd immunity; ii) we study the consequences of partial crossprotection induced in hosts by different strains in a small homogeneous host population; and iii) we extend this study to spatially structured host populations to explain data observed in lagomorphs/lagoviruses in France.