O alfaherpesvírus bubalino 1 (BuHV1) e os alfaherpesvírus bovinos 1 (BoHV1), 5 (BoHV5) e são membros da ordem Herpesvirales, família Herpesviridae, subfamília Alphaherpesvirinae, gênero Varicellovirus. O BoHV1 e o BoHV5 são subdivididos em subtipos (BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c). No Brasil circulam BoHV1 e BoHV5 de todos os subtipos até o presente reconhecidos. Em vista dessa variedade de alfaherpesvírus potencialmente infecciosos para bovinos e bubalinos, o conhecimento sobre estes agentes é essencial para a implementação de medidas de controle ou erradicação. Com o objetivo de permitir algumas comparações entre estes agentes e as respostas por eles induzidas em seus hospedeiros, primeiramente foi realizado o sequenciamento do genoma completo de uma amostra de BuHV1 (b6), isolada a partir de prepúcio de búfalos (Bubalus bubalis) em 1972, na Austrália. O objetivo foi disponibilizar a primeira sequência completa de um alfaherpesvírus bubalino e avaliar o grau de similaridade entre o genoma desse vírus e os alfaherpesvírus de bovinos. O genoma sequenciado compreende 137.452 pares de bases (pb), com um nível de similaridade nucleotídica de 92,2% com BoHV5 (SV507/99) e 76,7% com BoHV1 (NVSL). Estes resultados permitirão estudos futuros buscando reconstruir a história evolutiva destes vírus, a importância de infecções interespécies na geração de variantes destes agentes e possíveis associações entre tais infecções e patogenicidade. Na segunda etapa dos estudos, foram realizados diversos testes sorológicos buscando definir uma metodologia de diagnóstico adequada para a identificação de anticorpos contra BuHV1 e todos os diferentes subtipos de BoHV1 e BoHV5. Inicialmente, 600 amostras de soros de bovinos de campo foram testadas através do teste de soroneutralização (SN), realizada frente aos sete alfaherpesvírus em estudo. Os resultados da SN foram influenciados pela escolha do vírus desafio utilizado no ensaio. Para obter a sensibilidade máxima do teste (259/600), foi necessário combinar resultados positivos frente a pelo menos cinco diferentes amostras virais. Em seguida, foram preparados ensaios do tipo “ELISA” para detecção de anticorpos utilizando antígenos preparados com cada um dos diferentes tipos/subtipos desses vírus (BuHV1; BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c), individualmente (chamado “single ELISA” ou “sAgELISA”). Os resultados obtidos foram comparados com os resultados da soroneutralização (SN). A sensibilidade do sAgELISA variou significativamente conforme a amostra viral utilizada no preparo dos antígenos. Considerando os resultados frente a apenas um antígeno, a maior sensibilidade foi de 96,1% (249/259), obtida com apenas uma amostra de BoHV5c. Foram necessários pelo menos seis antígenos (BuHV1; BoHV1.2a, 1.2b; BoHV5a, b, c) para atingir a sensibilidade máxima do teste (263/259). Assim, foi desenvolvido um ELISA combinando vários antígenos em um mesmo teste (chamado “multiple ELISA” ou “mAgELISA”). O mAgELISA detectou 263 amostras positivas, resultando em uma concordância ótima entre sAgELISA e mAgELISA (κ=0,99). Frente a SN, o mAgELISA apresentou uma sensibilidade de 96,5% e especificidade de 96,1% (κ=0,93; VPP=95,0%; VPN=97,3%). Essas diferenças não foram estatisticamente significativas. Os resultados obtidos com a SN e mAgELISA foram comparados também a um ELISA comercial para a detecção de anticorpos contra BoHV1. O ELISA comercial utilizado nas comparações não apresentou resultados significativamente diferentes dos demais testes realizados, no entanto, revelou o maior número de resultados discrepantes em relação ao SN, considerado padrão-ouro. Estes resultados revelam que o mAgELISA aqui relatado é adequado para a detecção de anticorpos para BuHV1, BoHV1 e BoHV5, com sensibilidade e especificidade significativamente comparáveis às da SN. / Bubaline alphaherpesvirus 1 (BuHV1), Bovine alphaherpesviruses 1 (BoHV1) and 5 (BoHV5) are members of the order Herpesvirales family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. The BoHV1 and BoHV5 are divided into subtypes (BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c). In Brazil, circulates BoHV 1 and BoHV 5 from all subtypes known to the present. In view of this variety of potentially infectious alphaherpesviruses for bovines and buffaloes, knowledge about these agents is essential for the implementation of control or eradication measures. In order to gain a deeper understanding about these agents and the responses induced by them in their hosts, initially, the complete genome sequence of BuHV1-b6, one of the first BuHV1 viruses isolated in Australia in 1972, is reported. The objective was to provide the first complete sequence of a buffalo alphaherpesvirus and to evaluate the degree of similarity between BuHV1 and bovine alphaherpesviruses genomes. The BuHV1-b6 genome is a linear double-stranded DNA molecule, 137,452 bp long, with overall sequence of the 92.2% similarity at the nucleotide level to the reference BoHV5 (SV507/99) strain and 76.7% with BoHV1 (NVSL) strain. These results are expected to be valuable for further studies involving evolutionary chain of these viruses, the interspecies infections importance in generation of variants and possible associations between such infections and pathogenicity. In the second stage of the study, several serological tests were performed in order to define a appropriate diagnostic methodology for the identification of antibodies against BuHV1 and all different subtypes of BoHV1 and BoHV5. Initially, 600 bovine field serum samples were screened in serum neutralization tests (SN) performed against all seven virus types/subtypes. The SN results were influenced by the choice of the challenge virus. The maximum number of positive sera (259/600) was detected by adding the positive results obtained with at least five different viruses. Seven enzyme linked immunoassays were prepared with each of the seven antigens (BuHV1; BoHV1.1, 1.2a, 1.2b; BoHV5a, b, c), individually (single antigen ELISAs; sAgELISAs). The results obtained were compared to the SN. The sensitivity of the sAgELISAs was also influenced by the choice of antigen. Considering the results against only one of them, the best sensitivity was 96.1% (249/259), obtained with BoHV5c. Maximum sAgELISA sensitivity (263/259) was achieved when positive results of at least six viruses (BuHV1; BoHV1.2a, 1.2b; BoHV5a, b, c) were combined. A multiple antigen ELISA (mAgELISA) was then prepared by combining of different viral antigens in the same test. The mAgELISA detected 263 positive samples, resulting an optimum concordance between sAgELISA and mAgELISA (κ=0.99). When compared to SN, the mAgELISA revealed 96.5% sensitivity and 96.1% specificity (κ=0.93; PPV=95.0%; NPV=97.3%). These differences were not statistically significant. The results of both SN and mAgELISA were compared to a commercially available (IBRgB) ELISA. The results of the commercial ELISA did not vary significantly in relation to others tests performed, however, revealed the highest number of discrepant results in relation to SN, taken as the gold standard. These results reveal that the mAgELISA reported here is suitable for the detection of antibodies to BuHV1, BoHV1 and BoHV5 with sensitivity and specificity significantly comparable to those of SN.

Orientador: José Maurício Sforcin / Banca: Anderson de Sá-Nunes / Banca: Sueli Aparecida Calvi / Resumo: Nosso grupo tem se dedicado à investigação das ações biológicas da própolis, alecrim-do-campo, capim-limão e cravo-da-Índia. A própolis tem despertado a atenção dos pesquisadores em virtude de suas inúmeras propriedades biológicas, e o alecrim-do-campo é uma das principais fontes deste apiterápico em nossa região. Já o capim-limão e o cravo-da-Índia têm sido pouco avaliados no tocante à sua ação imunomoduladora. O objetivo deste trabalho foi avaliar o efeito imunomodulador do extrato e respectivos compostos isolados da: própolis (ácidos cumárico e cinâmico), do alecrim-do campo (ácido cafeico), do cravo-da-Índia (eugenol) e do capim-limão (citral) sobre a produção de citocinas (IL-1, IL-6 e IL-10) por macrófagos peritoneais de camundongos BALB/c. Em protocolos com LPS, macrófagos foram incubados ora com os produtos naturais supracitados em diferentes concentrações e posteriormente desafiados com LPS; ora desafiados com LPS e posteriormente incubados com os produtos naturais. A dosagem das citocinas foi realizada através da técnica de ELISA. A própolis exerceu ação moduladora sobre a resposta imune e inflamatória, e os ácidos cinâmico e cumárico podem estar envolvidos em sua ação imunomoduladora. O alecrim-do-campo e o ácido cafeico também demonstraram efeito imunomodulador quanto à produção de citocinas. O capim-limão exerceu efeito inibitório sobre a produção de citocinas, sendo este efeito mais pronunciado em ensaios com o citral. Resultados semelhantes foram observados com o cravo-da-Índia e o eugenol. Nossos resultados sugerem que o potencial imunomodulador dos produtos naturais merece ser melhor explorado em futuras investigações, avaliando sua eficiência em doenças inflamatórias / Abstract: Our group has been investigating the biological action of propolis, "alecrim-do-campo", lemongrass and clove. Propolis has attracted the researchers' attention due to its several biological properties, and "alecrim-do-campo" is its main vegetal source in our region. However, little is known concerning lemongrass and clove immunomodulatory action. The goal of this work was to evaluate the immunomodulatory effect of the following extracts and isolated compounds: propolis (coumaric and cinnamic acids), "alecrim-do-campo" (caffeic acid), clove (eugenol) and lemongrass (citral) on cytokines production (IL-1, IL-6 and IL-10) by peritoneal macrophages of BALB/c mice. In LPS protocols, macrophages were incubated either with natural products in different concentrations and then challenged with LPS; or with LPS and then incubated with the natural products. Cytokine concentrations were measured by ELISA. Propolis exerted a modulatory action on the immune and inflammatory response and cinnamic and coumaric acids may be involved in its immunomodulatory action. "Alecrim-do-campo" and caffeic acid also exerted an immunomodulatory action on cytokines production. Lemongrass showed an inhibitory action on cytokines production, mainly in the assays with citral. Similar results were found using clove and eugenol. Our results suggest that the immunomodulatory potential of the natural products should be investigated in further studies, evaluating their efficacy in inflammatory diseases / Mestre

Made available in DSpace on 2016-04-28T20:15:25Z (GMT). No. of bitstreams: 1 2007-Fabiola do Nascimento Correa.pdf: 1170487 bytes, checksum: 12941bc2fa78c4d327c2059d03bf1c5c (MD5) Previous issue date: 2007-02-27 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Rearing buffaloes in Brazil has been increasing notoriously and leaving of being a simple activity to justify the use of poor fertility lands. Right now the rearing buffalo is considered a promissory lucrative activity, but the information about sanitary aspects to improve buffaloes health are not consistent. Borreliosis is a systemic infectious disease caused by many Borrelia species, which have a cosmopolitan distribution, and affects various species of domestic and wild animals, including human beings. Borrelia theileri is the specie more commonly reported in ruminants. However, these animals can also be infected by B. burgdorferi sensu lato and B. coriaceae, which cause Lyme borreliosis and abortion epizootic bovine, respectively. Meanwhile, there are no seroepidemiologic studies of borreliosis in buffaloes. The aim of this study was to know the frequency of anti- B. burgdorferi homologous antibodies in buffaloes serum samples proceeding from Castanhal, Santa Isabel, Nova Timboteua and Santar?m Novo, in the continental part of Par? state and Cachoeira do Arari in Maraj? Island were collected. Serums of 491 buffaloes were analyzed by indirect ELISA test. The serologic analysis of the samples showed that 412 serums (83.91%) were positive, without different statistical significance between positive animals from Maraj? Island 81.469% (2362/284) and from the continental part 86.96% (180/207). Was observed that buffaloes of these five municipalities studied have high antibodies frequency against spirochetes B. burgdorferi. The Correspondence Analysis test showed the formation of three different municipalities groups of according with seropositives animals number. The first group was formed by Cachoeira do Arari and Castanhal, the second by Nova Timboteua and Santar?m Novo. The third group was constituted by Santa Isabel only, which presented statisticment loss antibodies frequency than others municipalities. The high frequency of positives animals found can be explained by presence of tick Boophilus microplus and by the existence of report on Borrelia sp. infecting buffalo in the studied region. These facts suggest cross-reactivity between strain g39/40 of B. burgdorferi used as antigenic substratum and Borrelia species that infect buffaloes in Par? state. Despite of low specificity of indirect ELISA test used in this study, it is a good method to select e screen infected animals in studies about Borrelia sp. in buffaloes. / A cria??o de b?falos tem expandido no Brasil deixando de ser um simples elemento de ocupa??o de terras pouco f?rteis para tornar-se sin?nimo de produ??o pecu?ria rent?vel. No entanto, os pecuaristas e profissionais de sa?de carecem de informa??es consistentes sobre sanidade desses animais. A borreliose ? uma enfermidade sist?mica, infecciosa e cosmopolita, causada por microrganismos do g?nero Borrelia, que acometem diversas esp?cies de animais dom?sticos e silvestres, al?m do homem. A esp?cie de Borrelia mais comumente reportada em ruminantes ? B. theileri. Estes animais tamb?m podem ser infectados por B. burgdorferi sensu lato, agente da borreliose de Lyme e B. coriaceae que causa o aborto epizo?tico bovino. No entanto, n?o h? estudos soroepidemiol?gicos sobre borreliose em bubalinos. Com o objetivo de conhecer a freq??ncia de anticorpos hom?logos anti-B. burgdorferi de b?falos provenientes dos munic?pios Castanhal, Santa Isabel, Nova Timboteua e Santar?m Novo, na parte continental do estado do Par? e Cachoeira do Arari na Ilha de Maraj? foram coletados soros de 491 b?falos, os quais foram analisados por meio do teste ELISA indireto. A an?lise sorol?gica das amostras revelou que 412 soros (83,91%) foram positivos, n?o ocorrendo diferen?a estat?stica significativa entre os amimais positivos provenientes da Ilha de Maraj? 81,69% (232/284) e da por??o continental do estado 86,96% (180/207). Quanto ? freq??ncia de soropositivos por munic?pio estudado, a an?lise de correspond?ncia, indicou a forma??o de tr?s grupos distintos, o primeiro formado pelos munic?pios de Cachoeira do Arari e Castanhal, o segundo formado pelos munic?pios de Nova Timboteua e Santar?m Novo e o terceiro formado apenas pelo munic?pio de Santa Isabel. Este ?ltimo apresentou estatisticamente menor freq??ncia de anticorpos em rela??o aos outros quatro munic?pios. No entanto, foi observado que b?falos dos cinco munic?pios estudados possuem alta freq??ncia de anticorpos hom?logos contra espiroquetas B. burgdorferi. A alta freq??ncia de animais soropositivos encontrada pode ser explicada pela presen?a do carrapato Boophilus microplus e pela exist?ncia de relato sobre Borrelia sp. infectando b?falo na regi?o estudada. Estes fatos sugerem rea??o cruzada entre a cepa americana g39/40 de B. burgdorferi utilizada como substrato antig?nico e a esp?cie de Borrelia que infecta os b?falos no estado do Par?. Apesar da baixa especificidade do ELISA indireto usado neste estudo, este teste constitui-se em um bom exame para triagem e monitoramento de indiv?duos infectados por microrganismos do g?nero Borrelia.

Eastern Equine Encephalitis virus has a human mortality rate of 30% of those cases diagnosed, while 30% of those surviving infection remain with neurological sequelae for life (CDC.gov, 2007). The use of sentinel chickens for surveillance of arboviruses that are known to use birds as a reservoir host, such as St. Louis Encephalitis (SLE), West Nile (WN) virus, Eastern Equine Encephalitis (EEE) and Highlands J (HJ) virus, in Florida began with the Sentinel Chicken Arboviral Surveillance Network in 1978 (Day and Stark, 1996). This network enables the activation of an early warning system for citizens, as well as, county epidemiologists and those in mosquito control, allowing for a coordinated effort of disease prevention. Methods currently used at the Florida Department of Health, Tampa Branch Laboratory include screening of submitted sera for antibodies to these arboviruses of epidemiologic importance by way of the hemagglutination inhibition test (HAI), and confirmation by the IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and Plaque Reduction Neutralization test if the MAC-ELISA proves to be negative. While these tests combined are providing the results needed, the time to result can be a week or greater depending on the initial screening result in the HAI tests. The Microsphere Assay Technology provides an accurate, more rapid (a day or two instead of a week or more) detection method including both a screening and confirmation protocol specifically designed to test for antibody to EEE in sentinel chicken sera. Two sera out of the thousands tested that were tested by HAI shown to be negative in standard testing, resulted as positive by the MIA method and therefore indicated a missed positive. The sensitivity and specificity, positive and negative predictive values of this new protocol as compared with MAC-ELISA as a reference standard indicated that both tests were remarkably similar; Providing sensitivity near 80%, specificity and PPV at 99%, and negative predictive values at 90% for MAC-ELISA and 94% for the MIA. Finally it was determined that Highlands J virus will not have any impact on the testing protocol and results of this test.

Abnormal T lymphocyte reactions in both autism and Alzheimer's disease (AD) have been reported. This research investigated the possibility that these abnormalities may involve circulating antithymic antibodies. Plasma samples from autistic patients, AD patients, and normal-matched controls were tested for reactivity against murine thymocytes. In the first of 3 studies results of the enzyme-linked immunosorbent assay (ELISA) were statistically significant for binding (P < 0.001) between antithymic antibodies in plasmas of AD patients and murine thymocytes. Binding (P < 0.05) in low dilutions (1/2.5 and 1/5} of autistic patient plasmas was also observed. In the second study, plasmas of neither autistic nor AD patients significantly inhibited DNA synthesis of thymic cells in the presence of interleukin-1 (IL-l} and phytohemagglutinin (PHA). In the third study, no significant increases (P > 0.05) in cytotoxic activities were detected using AD patient plasmas and both untreated and heat-treated autistic patient plasmas. After further testing, these heat-treated plasmas diluted 1/64 and 1/128 had increased cytotoxicities (P Therefore, circulating antithymic antibodies may be involved in abnormal T lymphocyte reactions in autism and AD. Since they probably do not act alone, future research should study these complex abnormalities using human thymocytes.

Tesis para optar al Grado de Magíster en Ciencias Animales y Veterinarias. / Las aflatoxinas son micotoxinas consideradas altamente carcinogénicas, siendo la aflatoxina B1, carcinógeno del Grupo 1, de acuerdo al IARC. Estas micotoxinas llegan a tener impactos en la salud y económicos significativos, lo que las convierte en objetivos importantes para la detección y cuantificación. La evidencia científica ha demostrado que el clima influye en su presencia, por lo cual, las regiones tropicales y subtropicales como la provincia de Huambo-Angola, son considerados propensas a su desarrollo, asociándose principalmente a los cultivos de maíz, maní y otras semillas. Para identificar y cuantificar la presencia de esta toxina en los granos, se implementó un método analítico de screening a través de la técnica inmunoenzimatica (ELISA), mediante la cual se analizó un total de 36 muestras (32 muestras de piensos y 4 de maíz en grano para alimento animal), utilizando un valor de corte de 5 ng/g como valor referencial analíticamente aceptable, siendo todos sus resultados no detectados a un nivel de control de 5 ng/g. Posteriormente, se reanalizaron 10 de estas muestras, utilizando cromatografía líquida de alta resolución acoplada a un detector de fluorescencia (HPLC-FL), técnica mediante la cual no se evidenció señal en los tiempos de retención, lo que fue concordante con los resultados obtenidos por el métodos ELISA. Por otra parte, se implementó un diseño de programa para monitorear aflatoxinas B1 en alimentos destinado a la producción avícola, en la provincia de Huambo–Angola, contribuyendo a la entrega de información para implementar acciones de control y mitigación en la calidad e inocuidad alimentaria en la industria avícola / Aflatoxins are mycotoxins considered to be highly carcinogenic, aflatoxin B1 being a carcinogen of group 1, according to IARC. These mycotoxins have significant health and economic impacts, which makes them important targets for detection and quantification. Scientific evidence has shown that climate influences their presence, therefore, tropical and subtropical regions such as Huambo - Angola province are considered prone to their development, associating mainly to the crops of corn, peanuts and other seeds. In order to identify and quantify the presence of this toxin in the grains, an analytical method of screening was implemented through the immunoenzymatic technique (ELISA), through which a total of 36 samples were analyzed (32 samples of feed and 4 of corn for animal food), using a cut-off value of 5 ng / g as an analytically acceptable referential value, all of which results were not detected at a control level of 5 ng / g. Subsequently, 10 of these samples, using high-performance liquid chromatography coupled to a fluorescence detector (HPLC-FL), a technique whereby no signal was observed in the retention times, which was consistent with the results obtained by the ELISA methods. On the other hand, a program design was implemented to monitor aflatoxins in food destined to poultry production, in the province of Huambo-Angola, contributing to the delivery of information to implement control and mitigation actions in the quality and food safety in the poultry industry

Bovine reproductive performance is one of the most important factors influencing dairy farm profitability. Present-day techniques for oestrus- and pregnancy-detection are unreliable and labour-intensive. Although measuring milk-progesterone at regular intervals allows the fertility status of a cow to be determined reliably, the labour cost of collecting and analysing samples is prohibitive. This project aimed to develop a progesterone sensing system that could be automated and integrated with the milking unit, thus minimising labour costs. The proposed system involved mixing the milk sample with an enzyme-antibody conjugate and then passing the sample through a column containing immobilised progesterone. Any progesterone in the milk would inhibit conjugate binding to the column. An enzyme substrate would then flow through the column and bound conjugate would be detected as a colour change at the column's outlet. Periodate-coupling was used to attach horseradish peroxidase enzyme to anti-progesterone antibody, and progesterone-3-carboxymethyloxime was immobilised on the polystyrene bead surface using amine-coupling. Both techniques are widely used. Initial experiments attempted to verify the success of these two reactions simultaneously, whereas later experiments focused on the bead-coating. Beads were suspended in a specially-constructed syringe and the antibody activity of the eluted solution was measured by SPR. However, a combination of non-specific binding and antibody stability and activity issues meant neither reaction was conclusively verified. Many trials were done to investigate how to overcome the problems encountered but a suitable, workable procedure was not developed. Despite poor progress, the problems encountered did not undermine the project's potential. There remains optimism of developing an on-line method if research were to continue.

Beak and Feather Disease Virus (BFDV) is a circovirus which causes ill-thrift, feather loss and immunosuppression leading to secondary infections and eventually death in psittacine birds. The development of standardised reagents for the detection and characterisation of BFDV infections and for the production of protective vaccines has been difficult as no cell culture system has yet been found to grow the virus successfully in vitro. However, the development of consistent and effective diagnostic tests and vaccines is now more practical through the application of nucleic acid-based detection methods and recombinant technology. A quantitative real-time PCR assay for the detection of BFDV DNA was developed, using primers designed to amplify a conserved 81 bp fragment of ORFV1 and SYTO9, a fluorescent intercalating dye, with assays run on a Corbett RotorGene 3000. A synthetic oligonucleotide was used to establish standard curves for the quantitation of viral load in both blood and feather preparations. The assay was very sensitive, with a detection limit of 50 copies/ìL. The assay was developed using BFDV-positive DNA extracts from the feathers of 10 different species of birds and validated with blood and feather samples from corellas vaccinated with an experimental BFDV vaccine, then challenged with live virus. Viral DNA was reliably detected in the blood of all control (non-vaccinated) birds and in some vaccinated birds. Contamination of the environment with the feather dander of BFDV-infected birds meant that HA feather preparations were unreliable for the detection and quantitation of viral excretion. Nonetheless, the assay should prove to be a useful and sensitive test for the detection of viral DNA in a range of samples in future investigations. A recombinant BFDV capsid protein was also produced and a specific monoclonal antibody developed against it. The behaviour of the protein in haemagglutination (HA) assays and the behaviour of the monoclonal antibody in western blotting, immunohistochemistry (IHC), ELISA and haemagglutination-inhibition (HI) assays were characterised. The protein had the ability to agglutinate galah erythrocytes as per the wild-type virus and this agglutination was successfully inhibited by antibodies to wild-type BFDV from naturally immune psittacine birds. Furthermore, the protein self-assembled into virus-like particles as determined by electron microscopy. The antibody was specific for both the recombinant BFDV capsid protein and the whole virus and had similar optimal titres when used in western blotting and IHC. The antibody also had HI activity and detected BFDV virus from 3 genera of psittacine birds, including the recently described cockatiel BFDV isolate. A novel “blocking” (or “competitive”) ELISA (bELISA) for the detection of anti- BFDV antibodies in psittacine sera (Ab-bELISA) was also developed and validated with 166 samples from eastern long-billed corellas vaccinated with the recombinant capsid protein and challenged with live virus. The bELISA was found to be both sensitive and specific and correlated strongly with the HI test, thus it should have wide application for the serodiagnosis of BFDV. A survey of cockatiels (n=88) housed at commercial aviaries was conducted to investigate whether BFDV infection occurs in cockatiels. All birds were diagnosed as being virus-free by PCR and HA and had no detectable antibody titre by HI assay. In addition to this, the genomes of two BFDV isolates obtained from diseased cockatiel feathers were sequenced and cross-reactivity assays performed using virus eluted from these feathers and sera from naturally immune psittacine birds. Serological cross-reactivity results and phylogenetic analysis of the nucleotide sequences indicated that the cockatiel virus isolates were serologically and genetically different to other BFDV isolates. This is the first report of an antigenically distinct BFDV in psittacine birds. Since the Ab-bELISA has a lower limit of detection than the HI assay, it was used to repeat the cockatiel sero-survey. No antibodies were detectable in any of the cockatiels tested and thus questions about the real prevalence of BFDV infection in cockatiels and the possible existence of a novel BFDV serotype adapted to cockatiels remain unanswered. The successful control of PBFD in both pet and wild birds depends on the development of vaccines that incite a strong specific immune response and can be efficiently produced in large quantities. Recombinant BFDV capsid proteins have recently been considered as candidate vaccines against BFDV and recombinant techniques allow the development of other candidate vaccines, including DNA vaccines. In order to examine the potential of DNA vaccination as a strategy for the prevention and control of BFDV, two DNA vaccines, based on the nucleotide sequence encoding the capsid protein of BFDV, were developed using the mammalian expression vector pVAX1. The vaccine constructs encoding both the full length and NLS-truncated capsid protein resulted in protein expression both in vitro and in vivo. Protein was detected in COS-7 cells transfected with the constructs with an indirect immunocytochemistry assay using the monoclonal antibody described in Chapter 5. Protein was present in the nucleus of cells transfected with the vaccine encoding the full-length nucleotide sequence and in the cytoplasm of cells transfected with the vaccine encoding the NLS-truncated sequence as expected. Both DNA vaccine constructs induced detectable levels of anti-BFDV antibodies in vaccinated birds, determined using the Ab-bELISA described in Chapter 5. Thus, DNA vaccines similar to those presented here may have application in the prevention and control of BFDV and some options for the further development of these vaccines into effective methods for the control of BFDV are discussed.

Background: Despite a declining trend in the incidence of gastric cancer (GC), it is still a major global public health concern of the 21st century. It afflicts one million people and kills 750,000 annually. It is believed that both genetic and environmental factors contribute to the gastric carcinogenesis. However geographic variation and immigrant studies highlight the role of environmental factors. Objective: To evaluate the association of GC with the environmental factors of diet, helicobacter pylori (H. pylori) infection, lifestyle and occupation as well as family history in Iran. Methodology: A population based case-control study was conducted in the Northwest of Iran where one of the highest incidence rates of the world has been reported. Two hundred and seventeen cases of GC and 394 age and gender matched controls were recruited. Participants were interviewed using a structured questionnaire which elicited information on demographic characteristics, socioeconomic status, family and medical history, lifestyle (smoking, alcohol drinking and substance abuse) and occupation. Ten milliliters of each subject???s blood was collected for blood grouping and to investigate presence of IgG antibodies against H. pylori using an ELISA kit which had been locally validated for this study. Results: Diet and H. pylori infection were found to be the most important determinants of GC in this study. High intake of allium vegetables and fruit, especially citrus fruit, appears to play a protective role. In addition to the consumption of fruit and vegetables, consumption of fresh fish was also inversely associated with GC. On the other, hand consumption of red meat and dairy products were positively associated with the risk of GC. Other dietary practices were also found to be important factors in the etiology of GC. People who had a preference for higher salt intake and drinking strong and hot tea were at higher risk. Finally, H. pylori infection was found to increase the risk of GC. Conclusion: This study has provided important and original information about the etiology of gastric cancer particularly in the Iranian context. These findings could be used in planning preventive strategies for this malignancy, which is a major health problem in Iran.

<p>Staphylococcus aureus is an important human and animal pathogen that causes a wide range of infections. These infections can be very serious and sometimes hard to get rid of, because of the many virulence factors the bacteria produce during infections.</p><p>This project was a research of the extracellular fibrinogen-binding protein, Efb, which is a 15.9 kDa protein that has been shown to be an important virulence factor during S. aureus infections.</p><p>The purpose with the project was to find out if the protein has more than one binding site to fibrinogen and if people produce antibodies against Efb.</p><p>This was performed with methods such as affinity chromatography, ELISA, coagulation test and western blot. It was shown that Efb has two binding sites to fibrinogen. One is placed on the C-terminal part of Efb and the other on the N-terminal. It was also shown that the production of antibodies against Efb rises significantly in people during an ongoing infection.</p>