Orientador: Helio José Montassier / Banca: Samir Issa Samara / Banca: Alessandro de Mello Varani / Banca: João Pessoa de Araujo Junior / Banca: Ricardo Luiz Moro de Sousa / Resumo: O vírus da doença de Newcastle (VDN) é o agente causador de uma das mais importantes enfermidades em aves e representa uma ameaça para a indústria avícola. O VDN é membro da família Paramyxoviridae, subfamília Paramyxovirinae, gênero Avulavirus. São vírus envelopados, não-segmentados dotados de genoma RNA de fita simples sentido negativo, associado à doença do trato respiratório, digestivo e nervoso das aves. O controle da DN se baseia em biossegurança, uso de vacinas e detecção precoce de lotes infectados. No presente estudo foi feito o sequenciamento do genoma completo da estirpe vacinal lentogênica (LaSota) e de um isolado de campo brasileiro patogênico (APMV-1/Chicken/Brazil/SJM/75) do VDN, seguido de análise filogenética. Os resultados revelaram que o genoma das estirpes LaSota e APMV-1/Chicken/Brazil/SJM/75 estão constituídos respectivamente por um total de 15.186 e 15.192 nucleotídeos, seis genes na ordem 3'-NP-P-M-F-HN-L-5'. Para a estirpe LaSota o local de clivagem da proteína de fusão (F) apresenta a sequência de aminoácidos correspondentes às encontradas em estirpes não virulentas do VDN, oposto ao que acontece com a estirpe APMV-1/Chicken/Brazil/SJM/75, no qual o sítio de clivagem da proteína F apresenta a sequência de aminoácidos correspondente às encontradas nas estirpes virulentas do VDN. O genoma completo da estirpe LaSota não apresentou grandes diferenças genômicas, de forma que na análise filogenética ficou evidenciado que esta estirpe está classificada no genótipo II, já a estirpe APMV-1/Chicken/Brazil/SJM/75 apresentou grandes diferenças genômicas nas sequências deduzidas de aminoácidos de suas principais proteínas estruturais, de forma que na análise filogenética do genoma completo foi demonstrado que esse vírus está classificado no genótipo V. Os dados obtidos a partir do presente estudo podem contribuir consideravelmente para ... / Abstract: Newcastle disease virus (NDV) is the agent that causes one of the most important diseases in birds and represents a threat to industrial aviculture. NDV is a member of the Paramyxoviridae family, Paramyxovirinae subfamily, Avulavirus genus. Viruses consist of single-stranded, non-segmented, negative-sense, RNA, associated with diseases in respiratory tract, digestive and nervous in birds. The ND control is based on biosafety, use of vaccines and early detection of infected batches. Here we present the complete genome sequence and phylogenetic analysis of a lentogenic strain (LaSota) and a velogenic strain (APMV-1/Chicken/Brazil/SJM/75) of NDV. The results revealed that the genome of LaSota and APMV-1/Chicken/Brazil/SJM/75 are constituted respectively by a total of 15,186 and 15,192 nucleotides and six genes in the order 3'-NP-P-M-F-HN-L-5'. The cleavage site of fusion protein (F) in LaSota strain shows the amino acid sequence found in non-virulent NDV strains, as opposed to happen with APMV-1/Chicken/Brazil/SJM/75 strain, the cleavage site of the F protein has the amino acid sequence corresponding to that found in virulent strains of NDV. The complete genome of LaSota strain showed no genomic differences and the phylogenetic analysis evidenced that this strain is classified in genotype II. The APMV-1/Chicken/Brazil/SJM/75 strain there are large differences in the genomic sequences and in the deduced amino acid sequences of its major structural proteins, so the phylogenetic analysis of the complete genome was demonstrated that this virus is classified in genotype V. This study can contribute significantly to understand better the genomic evolution of NDV in Brazil and in Americas, such as these strains with genotypic and phenotypic characteristics were no longer isolated in Brazil after 1975, while continued to be isolated in North America / Mestre

Orientador: Antonio Carlos Paulillo / Banca: Elizabeth Moreira dos Santos Schmidt / Banca: Antonio Carlos Alessi / Resumo: As avaliações dos parâmetros clínicos, imunitários e epidemiológicos da vacinação contra a doença de Newcastle (DN) em agapornis (Agapornis roseicollis) foram investigadas em dois experimentos. Amostras vacinais Ulster 2C, B1 e LaSota do vírus da doença de Newcastle (VDN) foram utilizadas para imunização das aves. No experimento 1, utilizaram-se 48 agapornis, distribuídos em quatro tratamentos de 12 animais cada, num total de três repetições, submetidos a diferentes esquemas imunoprofiláticos. A resposta imune foi avaliada pelo teste de HI. Não foram observados sinais clínicos de reação pós-vacinal em qualquer grupo experimental. Os resultados dos títulos de anticorpos (HI) mostraram que a estirpe vacinal LaSota conferiu maior grau de imunidade aos agapornis, quando comparada às estirpes Ulster 2C e B1. Estas aves foram posteriormente desafiadas frente a uma estirpe patogênica do VDN (EID50=108,15/0,1mL), aos 12 meses de idade. Em todos os grupos, procedeu-se a pesquisa do RNA viral a partir de suabes cloacais, através da técnica de RT-PCR. Os agapornis de todos os grupos não demonstraram qualquer sinal clínico sugestivo da DN, mostrando-se refratários à doença clínica frente a este vírus. Entretanto, ficou caracterizado o estado de portador de VDN nesta espécie decorridos até 21 dias da infecção experimental com este patógeno. No experimento 2, foram utilizadas aves SPF ("Specific-Pathogen-Free") conviventes com agapornis inoculados com uma estirpe patogênica do VDN. Observou-se a transmissão do VDN dos agapornis para as aves SPF conviventes decorridos até 21 da infecção experimental com este patógeno, o que realça a importância do agapornis como fonte potencial de infecção do VDN para aves domésticas / Abstract: The evaluations of clinical, immunological and epidemiological parameters of vaccination against Newcastle disease (ND) in lovebirds (Agapornis roseicollis) were investigated in 2 experiments. Ulster 2C, B1 and LaSota vaccines strains of the Newcastle disease virus (NDV) were used to immunization of birds. In experiment 1, 48 lovebirds were distributed into 4 different treatments, with 12 birds in each, with a total of 3 repetitions, submitted to different vaccination programs. The immunological responses were measured by HI test. Lovebirds from all groups did not show any clinical signs of post vaccinal reaction. The antibody titers (HI) results showed that the immune vaccine programs adopted were different in stimulating protective levels of humoral immune response. These birds were also challenge with a pathogenic NDV strain (EID50=108.15/0.1mL) at 12 months of age. After the challenge in all groups, cloacal swabs were collected for RT-PCT to find the pathogenic viruses. Lovebirds from all groups did not demonstrate clinical signs of ND, being refractory to the clinical disease with the NDV. However, a NDV carrier state was demonstrated in this specie until 21 days after experimental infection. In experiment 2, SPF chicks were housed with lovebirds previously inoculated with a pathogenic NDV strain. The pathogenic virus (NDV) was transmitted from lovebirds to SPF chicks until 21 days after challenge, showing the importance of the lovebirds as source of dissemination of NDV to domestic birds / Mestre

For many paramyxoviruses, including Newcastle disease virus (NDV), syncytia formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (23, 73). Because a potential interaction in paramyxovirus infected cells has never been clearly demonstrated, such an interaction was explored in Chapter 2 using coimmunoprecipitation and crosslinking. Both HN and F proteins could be precipitated with heterologous antisera after a five minute radioactive pulse as well as after a two hour chase in non-radioactive media, but at low levels. Chemical crosslinking increased detection of complexes containing HN and F proteins at the cell surface. After crosslinking, intermediate as well as high molecular weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after crosslinking resulted in the detection of complexes which electrophoresed in the stacker region of the gel, from 160-300 kD, at 150 kD and at 74 kD. Precipitates obtained with anti-F after crosslinking contained species which migrated in the stacker region of the gel, between 160-300 kD, at 120 kD and at 66 kD. The 3-4 discrete complexes ranging in size from 160-300 kD contained both HN and F proteins when precipitated with either HN or F antisera. That crosslinking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating crosslinking at early time points post infection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically crosslinked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the RER. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a subsequent change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion. Chapter 3 explores the stalk region of the NDV HN protein, which has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (7, 51, 57). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled-coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha-helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic "a" position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.

A doença de Newcastle, causada por cepas patogênicas de paramixovírus aviário 1 (APMV-1), é uma doença de aves importante por causar altos índices de mortalidade e perdas econômicas. Em aves da ordem Columbiformes, vários surtos têm sido relatados ao longo de 30 anos, em diferentes partes do mundo, causados por uma cepa denominada pigeon paramyxovirus tipo 1 (PPMV-1). Este trabalho descreve um surto de mortalidade em pombos domésticos (Columba livia), provenientes de uma praça pública, no município de Porto Alegre, no Sul do Brasil, ocorrido no mês de novembro de 2014. Aves moribundas e mortas, no intervalo de cinco semanas, foram submetidas ao exame de necropsia, exame histopatológico, imuno-histoquímico anti- Newcastle, de transcrição reversa seguida da reação em cadeia da polimerase (RTPCR), exame de sequenciamento e analise filogenética. Foram acometidas aves adultas, de ambos os sexos e a mortalidade foi estimada em 80%, os sinais neurológicos apresentados foram tremores da cabeça, torcicolo, dificuldade em manter-se em estação, dificuldade de locomoção, paresia, paralisia, asas caídas e vômito. As lesões encontradas no exame macroscópico eram inespecíficas e no exame histológico do sistema nervoso central eram caracterizadas por encefalite e encefalomielite não supurativas. No rim, fígado e pâncreas foi observado infiltrado inflamatório mononuclear, que por vezes era associado à necrose. No baço, além de necrose, foi observado depleção linfoide e infiltrado de macrófagos. Das 24 aves testadas para a RTPCR, seis foram positivas para a proteína da matriz (M) e através do sequenciamento destas amostras, pode-se identificar que todas as aves foram acometidas pela mesma cepa viral. Para confirmação de que a cepa encontrada tratava-se de uma cepa virulenta, foi feita a análise por sequenciamento do sítio de clivagem da proteína F, comparando a sequência de aminoácidos encontrada, 112RRQKRF117, com outras cepas já conhecidas. Observou-se que as amostras analisadas apresentaram aminoácidos na região do sítio de clivagem da proteína F, compatíveis com cepas virulentas. De acordo com a análise filogenética, o virus foi classificado como pertencente à classe II e ao genótipo VI. Ao exame imuno-histoquimico, a marcação foi observada no cérebro, no citoplasma de astrócitos e no núcleo de neurônios; no fígado, associada ao infiltrado inflamatório no interior de macrófagos; em células epiteliais do pâncreas exócrino e no citoplasma de células epiteliais do rim. / The Newcastle disease caused by avian paramyxovirus 1 strains (APMV-1) is an important avian disease involved into high rates of mortality and economic losses. Over the last 30 years several outbreaks have been reported in the order Columbiformes in many parts of the world caused by a strain, known as pigeon paramyxovirus type 1 (PPMV-1). This paper describes a mortality outbreak in free-living pigeons (Columba livia) from a public square in a city in Southern Brazil, occurred in November 2014. Moribund or freshly dead pigeons, within five weeks interval, were submitted to necropsy, histopathological, immunohistochemical (anti-Newcastle), Reverse transcription polymerase chain reaction (RT-PCR), sequencing analisys and phylogenetic analysis. It was affected only adult free-living pigeons of both sexes, and the mortality was estimated at 80%. Neurological signs presented by the pigeons were head tremors, stiff neck, lack of balance, incoordination, paresis, paralysis, drooped wings, and vomit. Gross findings were nonspecific. Histological findings in the central nervous system were characterized by encephalitis and encephalomyelitis nonsuppurative. In the kidney, liver and pancreas was observed mononuclear inflammatory infiltrate, which sometimes was associated with necrosis. In the spleen was observed necrosis, lymphoid depletion and macrophage infiltration. Out of 24 pigeons examined by RT-PCR, 6 pigeons had positive signal for the presence of matrix (M) protein gene and by sequencing analysis it appears that the sequences were identical to each other. The complete genome sequence and the complete coding sequence of the fusion (F) gene according to the unified NDV classification system showed that isolate had cleavage site 112RRQKRF117 which is characteristic of velogenic strains. Phylogenetic analysis showed that this strain could be classified into class II and genotype VI. Immunohistochemical analysis showed that the virus antigens were detected in astrocytes and in neurons in the brain, in liver macrophages, in exocrine pancreas epithelial cells, and in kidney epithelial cells.

Este trabalho teve como objetivo geral avaliar os fatores de risco de disseminação da Doença de Newcastle relacionados ao trânsito de veículos entre empresas integradoras e unidades de produção de frangos de corte. Uma área de grande produção avícola do estado do Rio Grande do Sul foi escolhida para ser objeto de estudo da presente pesquisa, sendo simuladas a partir de uma granja índex, as zonas de proteção e vigilância, como determina o Plano de Contingência para a Influenza Aviária e Doença de Newcastle. A metodologia foi fundamentada em análises espaciais e probabilísticas, associadas a situações reais que fazem parte do processo de logística das empresas. Foi realizada a análise espacial da região, através de técnicas de geoprocessamento, a extração da taxa reprodutiva básica, estudo exploratório da proporção de alojamento de frangos de corte e desenvolvimento do modelo epidêmico clássico de Reed-Frost, avaliando assim, as possibilidades de disseminação da doença na área de estudo. Os resultados obtidos demonstraram que somente o trânsito referente a visitas técnicas e caminhões de ração nas unidades de produção são fatores que podem desencadear um surto da doença. Quanto mais rápido for obtido o diagnóstico definitivo e tomadas medidas de contenção, menor é a probabilidade de disseminação da doença. / This thesis had as general objective to evaluate the factors of risk of spread of the Newcastle Disease related to the traffic of vehicles between companies and poultry farms. An area of great poultry production of the state of Rio Grande do Sul, Brazil, was chosen to be object of study of the present researches, being simulated starting from a farm index, the protection areas and surveillance, as it determines the Plan of Contingency for Avian Influenza and Newcastle Disease. The methodology was based in space analyses and probability, associated to real situations that are part of the process of logistics of the companies. The space analysis of the area was accomplished, through the use of geographical information systems, the extraction of the basic reproductive rate, study exploratory of the proportion of lodging of cut chickens and development of the model epidemic classic of Reed-Frost, evaluating like this, the possibilities of spread of the disease in the study area. The obtained results demonstrated that only the traffic regarding technical visits and ration trucks in the units of production is factors that can unchain an outbreak of the disease. The more fast it be obtained the definitive diagnosis and sockets contention measures, minor is the probability of spread of the disease.

O uso de antibióticos promotores de crescimento de forma indiscriminada vem causando enorme preocupação a cada ano por causa de uma possível resistência cruzada. Essa é uma das razões pela quais pesquisas vêm sendo desenvolvidas para substituição destes agentes por produtos naturais e/ou alternativos. Desde 1973 o Conceito de Exclusão Competitiva (CE) vem sendo estudado e usado na avicultura mundial. Com esse objetivo, inúmeras pesquisas relacionadas com produtos de Exclusão Competitiva vêm demonstrando, ao passar dos anos, não só a eficiência desse método no controle de patógenos indesejáveis como Salmonella ssp, como também sua eficácia em desempenho zootécnico. Dessa forma, o presente estudo teve como objetivo verificar a influência de um produto de Exclusão Competitiva sobre os índices zootécnicos e parâmetros de imunidade de frangos de corte, tendo um antibiótico e um controle para comparação. A revisão bibliográfica também apresenta pesquisas sobre o controle de Salmonella spp com o uso da CE. Foram utilizados 420 pintos de corte, criados até 42 dias de idade em gaiolas. O delineamento utilizado foi o inteiramente casualizado, com 3 tratamentos: Dieta basal (DB) com Antibiótico; DB com produto de Exclusão Competitiva, e DB sem Antibiótico (controle); sendo 20 repetições/tratamento. Quanto ao desempenho zootécnico e, considerando-se o período total de criação, o produto de Exclusão Competitiva testado demonstrou efeito benéfico para os valores de ganho médio de peso (GMP) e peso final (PF), que foram melhores ou similares comparados ao antibiótico principalmente até a fase final de criação. Já os resultados de conversão alimentar e mortalidade mostraram-se melhores com o uso da CE quando foram comprados com os demais tratamentos. Com relação à imunidade, a Exclusão Competitiva não apresentou efeito sobre o parâmetro avaliado neste trabalho. Diante dos resultados aqui obtidos, conclui-se que os ensaios com aditivos alternativos devem continuar, já que houve evidências de seus possíveis efeitos benéficos na produção animal. / The use of growth-promoting antibiotics inappropriately has been addressing concerns every year for possible cross-resistance. This is one of the reasons why studies have been carried through to replace such agents by natural and/or alternative products. Since 1973, the concept of Competitive Exclusion (CE) has been studied and used in poultry raising worldwide. Aiming at reaching the said objective, several studies on Competitive Exclusion products have been showing, over the years, not only how effective the method is in controlling undesirable pathogens, such as Salmonella ssp, but also its efficacy for animal performance. Thus, the present paper aims at verifying the influence of a Competitive Exclusion product on zoological properties and immunity parameters for broilers, having one antibiotic group and one control group for comparison. In addition, the bibliography includes studies on the control of Salmonella ssp when CE is used. A total of 420 broilers, reared in cages for maximum 42 days of age, have been used. The study design consisted in a randomized study, with three treatment groups: Baseline diet (DB) and antibiotic; DB and Competitive Exclusion, DB without Antibiotic (control); with 20 repetitions/treatment. With regard to animal performance and, considering total breeding period, the investigational Competitive Exclusion product has showed beneficial effect on average weight gain (GMP) and final weight (PF) values, which have been found to be improved or similar to the antibiotic group, specially when used until the final breeding stage. In turn, the results for feed conversion and mortality have showed to be improved when CE was used in comparison to the other two treatments. In relation to immunity, Competitive Exclusion has not affected the immunity parameter assessed in this paper. According to the results found in this study, it can be concluded that the use of alternative additives should be further studied, as evidences of their possible beneficial effects on animal raising have been found.

In the modern poultry industry, intense genetic selection for meat production has negatively influenced the reproductive performance of commercial birds. Parthenogenesis, embryonic development in unfertilized eggs without any sperm-egg interactions, is known to hinder the normal fertilization process and could be one of the reasons for this reduced reproductive performance in the poultry industry. Therefore, the overall objective of this research was to gain a better understanding of the process of parthenogenesis using Chinese painted quail as the model. Studies on Chinese painted quail reproduction revealed that they are very inefficient in sustained sperm storage and that number of sperm penetrating the egg and subsequent embryonic development potentially alter egg transit time through the oviduct. This poor sperm storage capacity and high sperm-egg interaction requirement might be responsible for the occurrence of parthenogenesis in this species; and in fact, this makes Chinese painted quail an excellent choice for parthenogenesis research. Further, dams selected for parthenogenesis as well as embryonic development, including parthenogen size, alter egg components by possibly delaying the transit time of the egg through the oviduct. Also, both dams and sires selected for the parthenogenesis trait appear to influence their progenies performance, including 1st wk mortality and occurrence of parthenogenesis. Additionally, vaccination of virgin hens with live pigeon pox virus increases parthenogenesis as well as parthenogen size and livability by the direct action of the virus on the embryo. Moreover, live Newcastle disease virus under in vitro conditions was found to have similar effects on the embryo. Because parthenogenesis exists in the modern poultry industry, even the accidental selection of the trait in either males or females could have a negative impact on overall chick production and performance. Also, as vaccination is a routine practice in the industry, it is possible that vaccination of birds that carry the trait will reduce fertility and hatchability due to enhanced parthenogenesis. Overall, currently it appears that, parthenogenesis is adversely affecting the poultry industry; and therefore, additional research on the accurate determination of losses in the poultry industry due to parthenogenesis could further benefit the industry.

Newcastle disease virus (NDV) is one of the paramyxovirus that has been studied at length since this virus infects the birds of all species. NDV is highly virulent in chickens and results in a high mortality rate because the V protein of NDV is found to inhibit the avian immune response system. No drugs are available for treating NDV therefore, determining the structure of V protein would help in developing a drug that can inactivate the V protein, thereby increasing the host immune response against viral infection. The research here is focused on purification and initial structural analysis of the V protein of NDV. The V protein was purified by gel filtration chromatography and the structure was studied using fluorescence and CD spectroscopy, and NMR. The results suggested that the V protein is unstructured. The research also involved purification and structural analysis of the flowering locus T (FT) protein, which is found to play a major role in theninitiation of flowering in plants. Structural analysis of the FT protein may help in finding the possible domains of the FT protein that interacts with other plant proteins, leading ton flowering. The FT protein was purified by ion exchange chromatography and the structure was studied by fluorescence and CD spectroscopy. The fluorescence data suggested that the FT protein may be folded, where as the CD data was inconclusive. More accurate secondary structure information about the protein could be obtained using NMR, but since the concentration of the FT protein was too low (0.007 mM), proper NMR study was not possible.

Desinfektionsmittel sind ein elementarer Bestandteil der Tierseuchenbekämpfung und damit auch der Lebensmittelsicherheit. Die Prüfung chemischer Desinfektionsmittel ist Voraussetzung für deren zuverlässige Wirksamkeit und zielgerichteten Einsatz. In Deutschland geschieht dies nach den Richtlinien der Deutschen Veterinärmedizinischen Gesellschaft e.V. (DVG). Seit der ersten Fassung sind die Richtlinien einem ständigen Anpassungsprozess unterworfen. Im Zuge der europäischen Harmonisierung gilt es nun, sich gesamteuropäischen Richtlinien, verfasst durch das europäische Komitee für Normung (Comité Européen de Normalisation) (CEN) anzupassen. Das Thema dieser Arbeit entwickelte sich im Kontext der derzeitigen Diskussion über Verbesserungsvorschläge zu den bestehenden Richtlinien und deren Anpassung an die europäischen Normen. Es wurden je zwei Testviren für die Bereiche Tierhaltung und tierärztliche Praxis ausgewählt, um sie auf Eignung für die Viruzidieprüfung zu testen und gegebenenfalls zu etablieren. Des Weiteren wurde in einem zweiten Teil, in Anlehnung an die Forderungen der europäischen Normen die Prüfung zu vereinfachen, ein alternatives Zellkulturnachweissystem für das Newcastle-Disease-Virus (NDV) geprüft. Die Prüfung der viruziden Wirksamkeit erfolgte mit fünf verschiedenen Grundsubstanzen, gewählt um ein möglichst breites Spektrum an Desinfektionsmittelwirkstoffen abzudecken. Es wurden Glutaraldehyd, Ethanol, Natronlauge, Natriumhypochlorit und Peressigsäure verwendet. Die Versuche wurden mit einer niedrigen Eiweißbelastung und bei einer Temperatur von 20°C durchgeführt. Um eine praxisnahe Situation zu simulieren wurde auf, bereits in den DVG-Richtlinien, verankerten Stahl- und Holzkeimträgertests zurückgegriffen. Als mögliche Prüfviren für die Tierhaltung wurden das Equine Arteritis-Virus (EAV) und das Bovine Virus Diarrhoe Virus verwendet. Bei beiden Viren handelt es sich um weit verbreitete Tierseuchenerreger mit einer großen epidemiologischen Bedeutung. Die Untersuchung von fünf verschiedenen Desinfektionsmitteln erfolgte im Keimträgertest auf Holz. Sowohl EAV als auch BVD stellen ein weniger geeignetes Prüfvirus dar, da beide Viren enorme Titerverluste im Trocknungsvorgang der Holzkeimträger zeigten. Die Viren ließen sich zwar leicht vermehren, aber die erzielten Ausgangstiter reichten nicht aus um die Trocknungsverluste zu kompensieren und aussagekräftige Ergebnisse zu produzieren. Für den Bereich tierärztliche Praxis wurden das Feline Coronavirus (FCoV) und das Murine Parvovirus (MPV) genutzt. FCoV ist ein weltweit in Hauskatzenpopulationen vorkommendes Virus mit einer hohen Seroprävalenz und wurde daher ausgewählt. MPV wurde als Stellvertreter für die, in der Praxis häufig vorkommenden Parvovirusinfektionen gewählt. Es schien ein ideales Modellvirus aufgrund seiner weiten Verbreitung in der Forschung zu sein. Bei beiden Viren erfolgte die Prüfung auf Stahlkeimträgern. Unter Laborbedingungen konnte FCoV ohne Probleme zu hohen Titern vermehrt werden. Es gab keine nennenswerten Trocknungsverluste. FCoV erwies sich als geeignetes Prüfvirus. MPV hingegen ist bedingt durch die langen Versuchszeiten und schwierig auszuwertenden Zellkulturen, sowie wegen der niedrigen Ausgangstiter weniger geeignet als Modellvirus für die Desinfektionsmittelprüfung. Die Anzucht von NDV in Allantoisflüssigkeit von SPF Hühnereiern erschien sehr aufwendig und mit hohem Eiweißfehler belastet. In den Versuchen konnte ein deutlich höherer Eiweißgehalt als in den vergleichend geprüften, in Zellkultur angezogenen Viren nachgewiesen werden. Infolge der Probleme mit der Kultivierung der LMH-Zelllinie und den damit verbundenen langen Wartezeiten bis zur eigentlichen Versuchsdurchführung kann nur eine teilweise Empfehlung, von auf Zellkultur vermehrtem NDV (NDV (ZK)) gegeben werden. Nach Behebung dieser Probleme ist durchaus eine Ablösung, von in Allantoisflüssigkeit angezüchtetem NDV durch NDV (ZK) zu empfehlen. Die Verfälschung der Ergebnisse durch die höheren Eiweißgehalte bei Desinfektionsmitteln mit deutlichem Eiweißfehler könnten so vermieden werden.

Orientador: Helio Jose Montassier / Banca: Ricardo Luiz Moro de Sousa / Banca: Manoel Victor Franco Lemos / Banca: Camillo Del Cistia Andrade / Banca: Everlon Cid Rigobelo / Resumo: Foi realizada a clonagem e expressão do gene da glicoproteína HN da estirpe La Sota do vírus da doença de Newcastle (VDN), como proteína recombinante de fusão, contendo uma cauda de poli-histidina no sistema hospedeiro constituído por leveduras da espécie Saccharomyces cerevisiae, que apesar de tentativas de otimização, não evidenciou a expressão da proteína recombinante. Após isso, foram produzidas as porções N-terminal e C-terminal da glicoproteína HN como proteínas recombinantes de fusão contendo uma cauda de poli-histidina e o peptídeo SUMO no sistema hospedeiro constituído pela bactéria Escherichia coli. Contatou-se que o sistema procarioto de expressão foi mais eficiente e permitiu a geração de dois peptídeos, que depois de devidamente caracterizados e purificados foram utilizados como antígenos para a realização de testes de imunodiagnóstico em sustituição aos kits comerciais utilizados atualmente. Foram desenvolvidos dois ensaios de ELISA baseados na adsorção de um antígeno formado pela expressão da porção N-terminal da glicoproteína HN (ELISA HN N-terminal) e na porção C-terminal da mesma glicoproteína (ELISA HN C-terminal), recuperados a partir da purificação da fração solúvel de culturas de E. coli. O ELISA C-terminal mostrou os melhores coeficientes de correlação com o teste padrão HI e com o teste S-ELISA-ConA, além de melhores índices de sensibilidade, especificidade e acurácia. Com isso, o ELISA baseado em um antígeno da porção C-terminal da glicoproteína HN e uma única diluição do soro desenvolvido neste estudo pode ser aplicado no diagnóstico e monitoramento pós-vacinal do VDN / Abstract: Was carried out the cloning and expression of the glycoprotein gene of the strain La Sota HN of the Newcastle disease virus (NDV), as a recombinant fusion protein containing a poly-histidine tail at the host system consisting of the yeast species Saccharomyces cerevisiae, which despite attempts at optimizing, showed no expression of recombinant protein. After that, the portions N-terminal and C-terminal from glycoprotein HN were produced as recombinant proteins containing a fusion tail and the poly-histidine peptide SUMO at the host system consisting of Escherichia coli. It was noted that the prokaryotic expression system was more efficient and allowed the generation of two peptides, which once characterized and purified were used as antigens for immunodiagnostic tests replacement in the commercial kits currently used. Were developed two ELISA assays based on the adsorption of the antigen formed by expression of the N-terminal portion of the HN glycoprotein (HN ELISA N-terminal) and the C-terminal portion of the same glycoprotein (HN ELISA C-terminus), recovered from purification of the soluble fraction of cultures of E. coli. The C-terminal ELISA showed the best correlation coefficients with the standard HI test and ELISA test S-ConA-, and higher sensitivity, specificity and accuracy. Therefore, the ELISA of the antigen based on a C-terminal portion of the glycoprotein HN and a single serum dilution developed in this study can be applied in the diagnosis and monitoring of post-vaccination VDN / Doutor