In the course of this work a novel strategy has been developed for linking the adjuvant Escherichia coli heat-labile enterotoxin subunit B (LTB) to Simian Immunodeficiency Virus (SIV) proteins via an antibody bridge and the systemic and mucosal immunogenicity of such SIV-MAb-LTB complexes have been investigated. A short peptide tag, termed Pk, was joined to the 3'-end of the gene coding for LTB and expression studies revealed that the gene product, LTB-Pk, could be efficiently synthesised and secreted from non-pathogenic Vibrio sp.60. Analysis of the functional properties of LTB-Pk demonstrated that LTB-Pk , like native LTB, was a heat-labile oligomer, that could bind to the glycolipid GM1-ganglioside and was immunogenic in vivo. In attempts to purify LTB-Pk for immunisation studies, both hydrophobic and ion-exchange chromatography schedules were analysed, the latter procedure being more efficient. Strategies were developed for joining LTB-Pk to one arm of an anti-Pk MAb, (MAb SV5-P- k) and Pk-linked SIV proteins to the other arm, and such SFV-MAb-LTB complexes bound to GM1 -ganglioside in vitro. Systemic immunisation studies suggested that SIV-MAb-LTB complexes, using recombinant p17 as the target antigen, promoted both humoral and cell- mediated immunity to the recombinant p17. In addition, it was later shown that conjugation of LTB-Pk to recombinant SIV proteins via an antibody bridge, resulted in a more efficent presentation of the recombinant SIV protein to the immune system, than co-administration of LTB-Pk with the recombinant SIV protein. However, intranasal administration of p17-MAb-LTB complexes did not induce immunity to recombinant p17. Subsequently it was shown that the recombinant p17 was highly susceptible to mucosal degradation, suggesting the poor mucosal immunogenicity of p17-MAb-LTB complexes may be related to the instability of recombinant p17 in the mucosal environment. Further investigations into the stability of other recombinant SIV proteins in the mucosa, revealed that recombinant p27 was more resilient to mucosal degradation. p27-MAb-LTB complexes were constructed and initial intranasal immunisation studies revealed that both systemic and cell-mediated immunity to recombinant p27, could be induced following intranasal administration. Furthermore, mucosal immunity to recombinant p27 was evident in the lungs of vaccinated mice, with anti-recombinant p27 IgG-secreting cells predominating.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-03-31Bitstream added on 2014-06-13T18:44:29Z : No. of bitstreams: 1 garcia_gr_dr_jabo.pdf: 313576 bytes, checksum: 3615e7690e51b47d22430c998944cfd2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho objetivou caracterizar um isolado de Bacillus subtilis para utilização como agente probiótico para bovinos. Foram determinadas “in vitro” a capacidade e o tipo de efeito inibitório do isolado de B. subtilis do produto comercial Biotop sobre Salmonella, Escherichia coli e Clostridium perfringens, além de sua estabilidade, viabilidade, resistência a antimicrobianos e potencial de ação em comparação com a Nisina e em combinação com EDTA. Foram avaliados os efeitos da adição do isolado na dieta de 32 bezerros da raça Holandesa, em quatro diferentes tratamentos (controle; 1 g/dia; 2 g/dia e 4 g/dia), sobre o consumo de matéria seca, perímetro torácico, ganho de peso, consistência fecal e incidência de doenças. Também foi realizado um desafio com E. coli e monitoramento do “score” de fezes, temperatura retal (oC) e parâmetros sanguíneos. O isolado de B. subtilis foi mais eficaz contra C. perfringens, principalmente quando associado com a Nisina e EDTA. A adição do probiótico na dieta de bezerros aumentou o consumo de matéria seca, ganho de peso e perímetro torácico. No desafio bacteriano não foram observadas diferenças significativas para presença de Bacillus spp. e E. coli nas fezes. A análise do “score” de fezes, temperatura retal e parâmetros sanguíneos não demonstraram diferenças significativas entre os tratamentos. O produto avaliado demonstrou resultados satisfatórios quanto aos parâmetros de produção animal, sendo recomendada sua utilização para bezerros lactantes, principalmente na dosagem de 4 g/animal/dia. / The present work aimed to characterize an isolated of Bacillus subtilis to be used as a probiotic for calves. Were determinate in vitro its inhibition capacity and the type of effect of the isolated, that is present in the commercial product Biotop against Salmonella, Escherichia coli and Clostridium perfringens. It also was tested its stability, viability, resistance to antibiotics and its potential action in comparison with Nisin and in combination with EDTA. They were appraised the effects of the addition of the isolated in the diet of 32 calves Holstein Frisian in four different treatments (control; 1 g/day; 2 g/day and 4 g/day), over the intake of dry matter, thoracic perimeter, gain of weight, faecal consistence and incidence of diarrhoea. Also a challenge was accomplished with E. coli and the score of faeces monitored, rectal temperature (oC) and sanguine parameters. The isolated of B. subtilis was more effective against C. perfringens, mainly when associated with Nisin and EDTA. The addition of the probiotic in the diet of calves increased the dry matter intake, weight enhance and thoracic perimeter. In the bacterial challenge significant differences were not observed in the counting of Bacillus spp. and E. coli in the faeces. The analysis of the score of faeces, rectal temperature and sanguine parameters didn't demonstrate significant differences between the treatments. The appraised product demonstrated satisfactory results as for the parameters of animal production, being recommended to be use for nursing calves, mainly in the dosage of 4 g/animal/day.

Orientador: Ruben pablo Schocken-Iturrino / Banca: Maria Luiza Poiatti / Banca: Ricardo de Albuquerque / Banca: Hélio José Montassier / Banca: Ricardo Andrade Reis / Resumo: O presente trabalho objetivou caracterizar um isolado de Bacillus subtilis para utilização como agente probiótico para bovinos. Foram determinadas "in vitro" a capacidade e o tipo de efeito inibitório do isolado de B. subtilis do produto comercial Biotop sobre Salmonella, Escherichia coli e Clostridium perfringens, além de sua estabilidade, viabilidade, resistência a antimicrobianos e potencial de ação em comparação com a Nisina e em combinação com EDTA. Foram avaliados os efeitos da adição do isolado na dieta de 32 bezerros da raça Holandesa, em quatro diferentes tratamentos (controle; 1 g/dia; 2 g/dia e 4 g/dia), sobre o consumo de matéria seca, perímetro torácico, ganho de peso, consistência fecal e incidência de doenças. Também foi realizado um desafio com E. coli e monitoramento do "score" de fezes, temperatura retal (oC) e parâmetros sanguíneos. O isolado de B. subtilis foi mais eficaz contra C. perfringens, principalmente quando associado com a Nisina e EDTA. A adição do probiótico na dieta de bezerros aumentou o consumo de matéria seca, ganho de peso e perímetro torácico. No desafio bacteriano não foram observadas diferenças significativas para presença de Bacillus spp. e E. coli nas fezes. A análise do "score" de fezes, temperatura retal e parâmetros sanguíneos não demonstraram diferenças significativas entre os tratamentos. O produto avaliado demonstrou resultados satisfatórios quanto aos parâmetros de produção animal, sendo recomendada sua utilização para bezerros lactantes, principalmente na dosagem de 4 g/animal/dia. / Abstract: The present work aimed to characterize an isolated of Bacillus subtilis to be used as a probiotic for calves. Were determinate "in vitro" its inhibition capacity and the type of effect of the isolated, that is present in the commercial product Biotop against Salmonella, Escherichia coli and Clostridium perfringens. It also was tested its stability, viability, resistance to antibiotics and its potential action in comparison with Nisin and in combination with EDTA. They were appraised the effects of the addition of the isolated in the diet of 32 calves "Holstein Frisian" in four different treatments (control; 1 g/day; 2 g/day and 4 g/day), over the intake of dry matter, thoracic perimeter, gain of weight, faecal consistence and incidence of diarrhoea. Also a challenge was accomplished with E. coli and the score of faeces monitored, rectal temperature (oC) and sanguine parameters. The isolated of B. subtilis was more effective against C. perfringens, mainly when associated with Nisin and EDTA. The addition of the probiotic in the diet of calves increased the dry matter intake, weight enhance and thoracic perimeter. In the bacterial challenge significant differences were not observed in the counting of Bacillus spp. and E. coli in the faeces. The analysis of the score of faeces, rectal temperature and sanguine parameters didn't demonstrate significant differences between the treatments. The appraised product demonstrated satisfactory results as for the parameters of animal production, being recommended to be use for nursing calves, mainly in the dosage of 4 g/animal/day. / Doutor

Os vírus da Influenza Equina (EIV) (H3N8 e H7N7) pertencem à família Orthomyxoviridae, gênero Influenza A. Apesar de existirem poucos relatos de infecção humana pelo EIV, é conhecido o risco zoonótico e infecção interespécies. Serviços de vigilância epidemiológica da OIE e WHO informam que o subtipo H3N8 é isolado de surtos que ocorrem mundialmente, enquanto o subtipo H7N7, menos patogênico, não é isolado desde 1980, sendo então considerado um vírus extinto. Embora o EIV seja endêmico em nosso meio, há poucos trabalhos nacionais que tenham versado sobre a avaliação atual de anticorpos (Ac) anti-EIV presentes nos equídeos do Estado de São Paulo, o que motivou a realização do presente estudo. Os objetivos do presente trabalho foram: 1) avaliar a ação de diferentes tratamentos de soro descritos pela OIE e WHO para a remoção de inibidores inespecíficos da hemaglutinação em soros de 10 equinos vacinados (H3N8 A/Equi/Kentucky/1/1997), sendo eles: a) TPH: tripsina, metaperiodato de potássio seguido de adsorção de hemácias; b) KH: kaolin 20% seguido de adsorção de hemácias; e c) RDEH: RDE seguido de adsorção de hemácias; 2) avaliar a presença de Ac contra os vírus H3N8 e H7N7, em 84 equídeos não vacinados do Estado de São Paulo; 3) comparar a frequência de Ac contra H3N8 entre equídeos amostrados do estado de SP e de um painel de soros de equídeos do município de Mossoró - RN, região onde não há estudos sobre a circulação do EIV. Constatou-se que não houve diferença estatística entre os tratamentos de soro para a remoção de inibidores inespecíficos da hemaglutinação (p>0,05; confiança de 95%), todavia o tratamento RDEH apresentou resultados mais consistentes, corroborando a recomendação da OIE e da WHO de utilizar preferencialmente este tratamento. O perfil sorológico dos animais amostrados de SP sugere que circule o subtipo H3N8 e que o subtipo H7N7 circule de forma subclínica nos equídeos, o que é sustentado por outros trabalhos realizados no Brasil. Há evidências no Brasil sobre a detecção de anticorpos em equinos contra o subtipo H7N7, mesmo não havendo o isolamento deste no mundo desde 1980. No painel de soros do RN, onde a espécie Equus asinus era maioria, verificou-se a igualdade estatística entre as frequências de Equus caballus e Equus asinus positivos no teste de HI para o subtipo H3N8 (p>0,05; confiança de 95%), dado inédito em nosso meio. A frequência dos equídeos positivos no teste de HI para o subtipo H3N8 foi estatisticamente maior (p<0,05; confiança de 95%) em SP do que em RN. / The Equine influenza Virus (EIV) (H3N8 and H7N7) belong to Orthomyxoviridae family and Influenza A genus. Although there are few reports of human infection with EIV zoonotic and interspecies infection risk is known. OIE and WHO services on epidemiological surveillance report that H3N8 subtypes are isolated and characterized from worldwide outbreaks while H7N7 subtype less pathogenic has not being isolated since 1980 considered an extinct virus. Although the EIV is endemic in our country there are few national studies that had versed on the current evaluation of horses antibodies (Ab) from São Paulo State which motivated the present study. The study objectives were: 1) to evaluate the effects of different serum treatments described by OIE and WHO for the removal of nonspecific inhibitors of hemagglutination in 10 vaccinated (H3N8 - A/Equi/Kentucky/1/1997) horses sera being: a) TPH: trypsin, potassium metaperiodate followed by adsorption of erythrocytes b) KH: kaolin 20% followed by adsorption of erythrocytes and c) RDEH: RDE followed by adsorption of erythrocytes; 2) investigate the presence of antibodies against H3N8 and H7N7 viruses in 84 unvaccinated equines in São Paulo State; 3) compare the frequency of antibodies against H3N8 sampled between São Paulo State and a panel of equines sera from Mossoró - RN where there are no studies on EIV circulation. There was no statistical difference between the treatments for serum nonspecific inhibitors of hemagglutination removal (p>0.05; 95% confidence) however RDEH treatment showed results more consistent confirming OIE and WHO recommendation to use this treatment with priority. The serological profile of SP samples suggests H3N8 subtype circulates in those animals and H7N7 subtype might circulate in a subclinical form in equines, which is supported by other studies conducted in Brazil. There is evidence of antibodies detection against equine H7N7 subtype in Brazil, even without since 1980 isolation in the world. In animals from RN State which had Equus asinus representing a major fraction there was statistical equal frequencies of Equus caballus and Equus asinus positivity in HI test against H3N8 subtype (p>0.05; 95% of confidence), as unprecedented in the world. The frequency of positive equine against H3N8 subtype on HI test in SP was statistically higher (p<0.05; 95% of confidence) than in RN.

O controle da eficiência de bacterinas antileptospirose de uso animal é o teste de potência com desafio em hamsters, contudo, na atualidade, tem sido estimulada a busca de alternativas que dispensem o uso de animais de laboratório. O teste de inibição de crescimento de leptospiras in vitro (ICLIV) tem sido proposto como possível alternativa. O presente trabalho empregou os testes de ICLIV, soroaglutinação microscópica (SAM) e ELISA anti IgG para avaliar a intensidade e a duração da imunidade passiva em leitões em aleitamento e ativa em matrizes suínas e leitões desmamados imunizados com bacterina experimental antileptospirose aprovada no teste de potência em hamster. Foi produzida uma bacterina experimental antileptospirose com estirpe patogênica de Leptospira interrogans, sorovar Kennewicki, estirpe Pomona Fromm (LPF), padronizada para conter 109 leptospiras por mL e associada ao adjuvante de hidróxido de alumínio na proporção de 10% do volume da dose final. Para a avaliação da eficácia da concentração mínima de leptospiras a ser utilizada na bacterina, diluições seriadas de razão dez de cultivo de leptospiras variando de 105 a 109 leptospiras/mL, foram submetidas ao teste de potência com desafio em hamsters (Experimento A), apenas a bacterina produzida na concentração de 109 leptospiras/mL foi capaz de proteger os hamsters contra a infecção induzida pela estirpe LPF, quando a vacina foi testada na diluição de 1:800, critério internacional de aprovação. A bacterina na concentração de 109 leptospiras/mL foi submetida ao teste de potência em hamsters (Experimento B), imunizados com a vacina pura e em diluições seriadas de razão dois (200 a 25600). A bacterina foi aprovada no teste de desafio em hamster até a diluição de 1:6400. O controle do inóculo de desafio foi constituído por 100 DL50. Fêmeas suínas que nunca haviam sido vacinadas contra a leptospirose e que foram não reagentes no teste de soroaglutinação microscópica aplicado a leptospirose efetuado com 24 estirpes de referência e no teste ICLIV com a estirpe LPF (Experimento 1), receberam duas aplicações intervaladas de 30 dias e um reforço aos 210 da primeira dose da bacterina pura e em diluições seriadas de razão dois (400 a 3200). Estes animais foram 16 monitorados com colheitas de sangue efetuadas a cada 30 dias. Os picos máximos de anticorpos avaliados pelos testes de SAM e de ICLIV foram observados aos 30 dias da segunda aplicação da vacina, com maior magnitude para a vacina pura, contudo aos 120 dias da segunda aplicação da vacina houve um declínio acentuado nos níveis de anticorpos. Para encontrar um melhor intervalo entre as imunizações (Experimento 2), fêmeas suínas receberam duas aplicações da bacterina pura intervaladas de 30 dias e o reforço aos 150 da primeira dose. A redução do intervalo de revacinação após as duas doses iniciais determinou a persistência dos títulos de aglutininas e de anticorpos neutralizantes com níveis sempre superiores a 0,4 log. Nos leitões em aleitamento filhos das matrizes imunizadas com bacterina na concentração de 109 leptospiras/mL foi constatada a transferência de imunidade passiva, confirmada pelos títulos de anticorpos aglutinantes detectáveis no quinto dia de vida e de neutralizantes no quinto e décimo dia de vida. Nos ensaios realizados em leitões desmamados imunizados com uma série de diluições de razão dez variando de a 109 leptospiras/mL os picos máximos de anticorpos foram observados aos 30 dias da segunda imunização, com maior magnitude para a bacterina testada na concentração de 109 leptospiras/mL. Os parâmetros finalmente obtidos foram que matrizes suínas e leitões desmamados, primovacinados com duas aplicações intervaladas de 30 dias da bacterina aprovada no teste de potência com desafio em hamster, apresentaram no teste de ICLIV efetuado aos 60 dias da primo-vacinação, intervalos de títulos de anticorpos (95%) expressos em log variando, respectivamente de (0,87 a 1,35) e de (1,22 a 1,58). Os valores máximos (12.800) para o teste de ELISA anti IgG das matrizes suínas foram obtidos após o reforço efetuado aos 210 dias. / The potency of antileptospirosis bacterins is controlled by in vivo experimental assay performed in hamsters; however, nowadays the search for in vitro methodologies that could replace the use of laboratory animals has been stimulated. For this purpose, the In vitro leptospiral growth inhibition test (IVLGIT) has been proposed as an alternative test. In this work, the intensity and duration of the passive immunity in suckling piglets and active immunity in weaned piglets and adult sows were evaluated, after vaccination with an experimental leptospirosis bacterin, which had been approved previously on the potency test in hamsters. The in vitro tests applied to swine sera were the growth inhibition test (IVLGIT), microscopic agglutination (MAT) and anti-IgG ELISA. The bacterin was produced with a pathogenic Leptospira interrogans serovar Kennewicki strain identified as Pomona Fromm (LPF), and added with aluminum hydroxide as adjuvant at a concentration of 10% of the final volume dose. In order to evaluate the efficacy of the minimum concentration of the leptospires to be used in the bacterin, ten-fold serial dilutions of a culture of leptospira varying from 105 to 109 leptospires/mL were submitted to potency-challenge test in hamsters (Trial A). Only the bacterin produced at the concentration of 109 leptospires/mL protected the hamsters against the infection by LPF strain, when the vaccine was tested at the dilution of 1:800, which is the international criterion for the approval. The bacterin at the concentration of 109 leptospires/mL was submitted to the potency test in hamsters (Trial B), which had been immunized with an undiluted and with two-fold serially diluted bacterin (from 1:200 to 1:25,600). In the challenge test in hamsters, the bacterin has passed till the dilution of 1:6,400. The control of the challenge inoculum presented a titer of 100 LD50. Adult sows, never been vaccinated against leptospirosis, and that showed negative MAT results using 24 reference serovars and with negative IVLGIT with the LPF strain (Trial 1) had administered two doses of bacterin with 30 day interval and a booster dose at 210th day after the first application of the undiluted bacterin and the two-fold diluted ones (1:400 to :3,200). The animals were 18 monitored with bleeding performed each 30 days. The levels of antibodies evaluated by MAT and IVLGIT showed the maximum peak at the 30th day after the second vaccination of the bacterin, with high magnitude found with the undiluted bacterin, however, at the 120th day after the second vaccination there were found a marked decline in antibodies levels. To find a better immunization scheme (Trial 2), the sows received two doses of undiluted bacterin with 30 day interval and the booster dose at the 150th day after the first dose. The reduction on the interval of revaccination after the two initial doses determined the persistence of a higher levels agglutinins and neutralizing antibodies with titers 0.4 log. In suckling piglets born from sows immunized with the bacterin at the concentration of 109 leptospires/mL, the passive transference of antibodies was confirmed by MAT titers detected on the fifth day, and by IVLGIT titers, at the fifth and tenth days after birth. In the study of weaned piglets immunized with the bacterin at the concentrations of 106, 107, 108 and 109 leptospires/mL (Trial 3), the highest antibodies levels were observed at the 30th day after the second immunization, with greater magnitude for the bacterin tested at the concentration of 109 leptospires/mL. The final results indicate that sows and weaned piglets, primo-vaccinated and revaccinated with 30 day interval with bacterin that had passed in the potency-challenge test in hamsters, presented the IVLGIT results varying (95% CI) from 0.87 log to 1.35 log for sows and 1.22 to 1.58 log for weaned piglets, at the 60th day after the first vaccination. In adult sows the highest anti-IgG ELISA titer reached 12,800, obtained after the booster vaccine dose performed at 210th day from the first vaccination.

<p>U radu je ispitana zastupljenost influenca A virusa, njihova antigenska i genetička svojstva i osetljivost na antivirotik oseltamivir.</p><p>Ispitivanje je sprovedeno u toku četiri uzastopne sezone, od 2010/2011 do 2013/2014 &nbsp;i obuhvatilo je 887 briseva nosa i grla pacijenata sa simptomima gripa, sa teritorije Južnobačkog okruga. Svi uzorci su&nbsp;testirani na prisustvo influenca A(H1N1)pdm09, A(H3N2), A(H1N1), A(H5) i A(H7) i influenca B virusa, real-time RT PCR testom. Pozitivni uzorci iz sezona 2012/2013 i 2013/2014, podvrgnuti su izolaciji na MDCK ćelijskim kulturama, a zatim je izvr&scaron;eno ispitivanje sposobnosti dobijenih izolata da aglutiniraju eritrocite koko&scaron;ke, čoveka i zamorca u reakciji virusne hemaglutinacije. Antigenska svojstva izolata sa hemaglutinacionim titrom &ge;40, ispitana su reakcijom inhibicije hemaglutinacije. Genetičkoj karakterizaciji, sekvenciranjem hemaglutinin i neuraminidaza gena, podvrgnuti su reprezentativni izolati iz sezona 2012/2013 i 2013/2014. Za ispitivanje osetljivosti odabranih izolata virusa na oseltamivir upotrebljen je hemiluminiscentni test inhibicije aktivnosti neuraminidaze.</p><p>Ukupno 46,3% (411/887) uzoraka bilo je influenca pozitivno, od čega je 73% (300/411) bilo influenca A pozitivno, a 27% (111/411)influenca B pozitivno (p&lt;0,0001). &nbsp;Influenca A(H1N1)pdm09 podtip je detektovan u 48% (144/300), a A(H3N2) podtip u&nbsp;52% (156/300) influenca Apozitivnih uzoraka. Najveći procenat influencaA pozitivnih zabeležen je u uzrastnoj grupi 5-14 godina (48,2%, 77/160) i kod pacijenata sa lak&scaron;im kliničkim manifestacijama gripa (43,7%, 153/350).</p><p>Influenca A(H1N1)pdm09 podtip preovladavao je u uzrastnoj grupi 15-29 godina (66%, 31/47, p=0,0400) i 30-64 godina (55,9%,71/127, p=0,0215), kao i kod pacijenata sa te&scaron;kom akutnom respiratornom bole&scaron;ću (63,5%, 80/126, p&lt;0,0001), fatalnih slučajeva (100%,9/9, p=0,0039) i pacijenata sa hroničnim bolestima i stanjima (68,8%, 84/122, p&lt;0,0001).&nbsp;</p><p>Influenca A(H3N2) podtip dominirao je kod dece uzrasta do 4 godine (72,2%,13/18, p=0,0381) i 5-14 godina (75,3%, 58/77, p&lt;0,0001), kod pacijenata sa lak&scaron;im oblikom bolesti (69,3%,106/153, p&lt;0,0001) i bez hroničnih bolesti ili stanja (66,3%, 118/178,&nbsp;p&lt;0,0001).</p><p>Najznačajniji predikcioni faktori komplikacija influence bili su: prisustvo hroničnih bolesti ili stanja i uzrast &ge;15 godina. Prisustvo hroničnih bolesti ili stanja nosilo je 34 puta, a uzrast &ge;15 godina 10 puta veći rizik od nastanka te&scaron;kih oblika bolesti.</p><p>Izolacija influenca virusa na MDCK ćelijskim kulturama, bila je uspe&scaron;na u 34,3% (70/204) slučajeva, pri čemu je u grupi uzoraka sa real-time RT-PCR Ct vrednostima &lt;30 ona iznosila 80,5% (62/77), kod uzoraka sa Ct vrednostima 30-34 svega 8,7% (8/92), a izolacija iz uzoraka sa Ct vrednostima &gt;34 nije bila moguća. U reakciji hemaglutinacije, najbolji rezultati su postignuti sa eritrocitima zamorca, koje je u titru &ge;40 aglutiniralo 56% (14/25) A(H1N1)pdm09 virusa i 62,5% (15/24) A(H3N2) virusa. Sa humanim eritrocitima dobar titar dalo je 16% (4/25) influenca A(H1N1)pdm09 i 8,3% (2/24) A(H3N2) virusa, a sa koko&scaron;ijim eritrocitima 8% (2/25) A(H1N1)pdm09 virusa i nijedan virus A(H3N2) podtipa.</p><p>Rezultati antigenske karakterizacije pokazali su da je svih 23 influenca virusa A(H1N1)pdm09 podtipa, iz sezona 2012/2013 i 2013/2014, antigenski bilo slično referentnom, vakcinalnom virusu A/California/7/2009. Nasuprot tome, samo 1 od 7 ispitanih A(H3N2) virusa iz sezone 2012/2013, antigenski je bio sličan vakcinalnom virusu A/Victoria/361/2011, a samo 2 od 20 iz sezone 2013/2014 antigenski je bilo slično vakcinalnom A/Texas /50/2012 virusu.</p><p>Filogenetska analiza hemaglutinin gena influenca A(H1N1)pdm09 virusa iz sezone 2012/2013, pokazala je da su u na&scaron;oj sredini, bili prisutni virusi iz dve različite genogrupe, 6C i 7, dok su naredne sezone svi analizirani virusi pripadali genogrupi 6B. Virusi iz na&scaron;e sredine bili su filogenetski srodni A(H1N1)pdm09 virusima iz drugih evropskih zemalja. Svi ispitani A(H3N2) virusi iz sezone 2012/2013 i2013/2014, pripadali su genetičkoj grupi&nbsp; 3C.3.Filogenetski su bili srodni sa virusima iz drugih gografskih regiona Evrope.</p><p>Svih 20 izolata influenca A(H1N1)pdm09 podtipa i 23 A(H3N2) podtipa pokazali su normalnu inhibiciju aktivnosti neuraminidaze pod dejstvom oseltamivira.ekvenciranje neuraminidaza gena jednog A(H3N2) virusa, koji je imao 8 puta redukovanu inhibiciju aktivnosti neuraminidaze oseltamivirom, ukazalo jena prisustvo retke mutacije Q391H, povezane sa rezistencijom na inhibitore neuraminidaze.</p><p>Rezultati ovog rada ukazali su na značaj influenca A virusa kao etiolo&scaron;kih uzročnika akutnih respiratornih obolenja u na&scaron;oj sredini, naročito za osobe sa hroničnim bolestima koje su pod povećanim rizikom od razvoja te&scaron;kih oblika gripa. U ovom istraživanju stečena su i saznanja koja imaju praktičnu primenu u postupku antigenske karakterizacije influenca A virusa, koja je jedna od ključnih faza u procesu pripreme vakcine protiv gripa. Značajna antigenska razlika A(H3N2) virusa koji su cirkulisali u sezonama 2012/2013 i 2013/2014 u odnosu na viruse koji su bili u sastavu vakcina u datim sezonama, ukazala je na neophodnost unapređenja proizvodnje vakcine protiv gripa. Dobijeni su i prvipodaci orezistenciji na antivirotik oseltamivir, kao i o filogenetskim odnosima i genetičkim grupama virusa koji su&nbsp; cirkulisali u na&scaron;oj sredini.</p> / <p>In this study we investigated the representation, antigenic and genetic properties, and sensitivity to antiviral drug oseltamivir of influenza A viruses. The study was conducted&nbsp; during 4 consecutiveseasons 2010/2011 - 2013/2014, and included 887 nasal and throat swabs taken from patients with influenza-like symptoms from South&nbsp; Backa district. All samples were tested for influenza A(H1N1)pdm09, A(H3N2), A(H1N1), A(H5), A(H7) and influenza B viruses, by real-time RT-PCR. Isolation on MDCK cell culture was performed with positive samples from seasons 2012/2013 and 2013/2014, and virus isolates were tested for ability&nbsp; to agglutinate guinea pig, chicken and human red blood cells in reaction of virus hemagglutination. Antigenic properties of isolates with hemagglutination titre &ge;40, were investigated using reaction&nbsp; of hemagglutination inhibition. Genetic characterization was performed by sequencing of neuraminidase and hemagglutination genes of representative isolates from seasons 2012/2013 and 2013/2014. Testing for sensitivity to oseltamivir was done with chemiluminescent neuraminidase inhibition assay.</p><p>Total of 46,3% (411/887) of samples were influenza positive, out of which 73% (300/411) were influenza A positive and 27% (111/4111, p&lt;0,0001) were influenza&nbsp; B positive. Influenza A(H1N1)pdm09 subtype was detected in 48% (144/300), and A(H3N2) subtype in 52% (156/300) of influenza A positive samples. The highest proportion of influenza A positive samples wasfound in age group 5-14&nbsp; years (48,2%,&nbsp; 77/160) and among patients with uncomplicated influenza (43,7%, 153/350).</p><p>Influenza A(H1N1)pdm09 subtype predominated in age group 15-29 years (66%, 31/47, p=0,0400) and 30-64 years (55,9%,71/127, p=0,0215), in patients with severe acute respiratory illness (63,5%, 80/126, p&lt;0,0001), in fatal cases (100%, 9/9, p=0,0039), and among patients with underlying chronic diseases and conditions (68,8%,84/122, p&lt;0,0001).</p><p>Influenza A(H3N2) subtype predominated in age group &le;4 years (72,2%, 13/18, p=0,0381) and 5-14 years (75,3%,58/77, p&lt;0,0001), in patients with mild form of influenza (69,3%,106/153, p&lt;0,0001), and in group of patients without chronic diseases and conditions (66,3%,60/478, p&lt;0,0001).</p><p>The most significant risk factors for severe influenza were: the presence of underlying diseases and conditions and age &ge;15 years. Patients with chronic illnesses and conditions had 34 times higher and patients &ge;15 years of age 10 times higher risk from severe influenza.</p><p>Isolation rate of influenza A viruses in MDCK cell cultures was 34,3% (70/204). For samples with real time RT-PCR Ct values &lt;30 isolation rate was 80,5% (62/77), for samples with Ct values 30-34 it was 8,7% (8/92), while isolation of viruses from samples with Ct values &gt;34 was not successful. In the reaction of virus hemagglutination, the best results were achieved with guinea pig red blood cells which agglutinated in titre &ge;40, 56% (14/25) of influenza A(H1N1)pdm09 viruses and&nbsp; 62,5% (15/24) of A(H3N2) viruses. With human erythrocytes, good titre gave 16% (4/25) of influenza A(H1N1)pdm09 and 8,3% (2/24)of A(H3N2) viruses and with chicken erythrocytes 8% (2/25) A(H1N1)pdm09 viruses and none of the A(H3N2) viruses.</p><p>Results of the antigenic characterization of 23 influenza A(H1N1)pdm09 viruses, showed that they were antigenically similarto referent, vaccine virus A/California/7/2009. On the contrary, only 1 out of 7 influenza A(H3N2) viruses from season 2012/2013,was antigenically similar to A/Victoria/361/2011 vaccine virus, and only 2 out of 20 from season 2013/2014 were antigenically similar to A/Texas/50/2012&nbsp; vaccine virus.</p><p>Filogenetic analysis of hemagglutinin genes indicated co-circulation of 2 distinct genetic groups, 6C and 7, of A(H1N1)pdm09 viruses during the season 2012/2013, while during the season 2013/2014 all tested viruses were from genetic group 6B. Influenza A(H1N1)pdm09 viruses from our region, were closely related to viruses from other European countries. All influenza A(H3N2) viruses from season 2012/2013 and 2013/2014 belonged to genetic clade 3C.3 and were closely related to viruses from different European countries.</p><p>Total of 20 A(H1N1)pdm09 isolates and 23 A(H3N2) isolates were tested for sensitivity to oseltamivir, and all of them showed normal inhibition of neuraminidase activity with oseltamivir. Sequencing of&nbsp; neuraminidase gene of one A(H3N2) virus with 8-fold reduced inhibition by oseltamivir, revealed rare mutation Q391H associated with antiviral resistance.</p><p>Results of this study indicate the significance of influenza A viruses as etiological factors of acute respiratory diseases in our area, especially for persons with chronic medical conditions who are at higher risk for severe influenza. Data gathered during&nbsp;the process of virus isolation and investigation of hemagglutination abilities of&nbsp; isolated viruses, have practical application in antigenic testing of influenza A viruses which is one of the key points of process of anti-flu vaccine production. Significant &nbsp;antigenic difference between influenza A(H3N2) viruses from seasons 2012/2013 and&nbsp; 2013/2014 and vaccine viruses, emphasis the importance of vaccine production improvement. During this study, the first data about antiviral resistance, filogenetic relationships and genetic groups of influenza viruses from our region, were obtained.</p>

Os vírus da Influenza Equina (EIV) (H3N8 e H7N7) pertencem à família Orthomyxoviridae, gênero Influenza A. Apesar de existirem poucos relatos de infecção humana pelo EIV, é conhecido o risco zoonótico e infecção interespécies. Serviços de vigilância epidemiológica da OIE e WHO informam que o subtipo H3N8 é isolado de surtos que ocorrem mundialmente, enquanto o subtipo H7N7, menos patogênico, não é isolado desde 1980, sendo então considerado um vírus extinto. Embora o EIV seja endêmico em nosso meio, há poucos trabalhos nacionais que tenham versado sobre a avaliação atual de anticorpos (Ac) anti-EIV presentes nos equídeos do Estado de São Paulo, o que motivou a realização do presente estudo. Os objetivos do presente trabalho foram: 1) avaliar a ação de diferentes tratamentos de soro descritos pela OIE e WHO para a remoção de inibidores inespecíficos da hemaglutinação em soros de 10 equinos vacinados (H3N8 A/Equi/Kentucky/1/1997), sendo eles: a) TPH: tripsina, metaperiodato de potássio seguido de adsorção de hemácias; b) KH: kaolin 20% seguido de adsorção de hemácias; e c) RDEH: RDE seguido de adsorção de hemácias; 2) avaliar a presença de Ac contra os vírus H3N8 e H7N7, em 84 equídeos não vacinados do Estado de São Paulo; 3) comparar a frequência de Ac contra H3N8 entre equídeos amostrados do estado de SP e de um painel de soros de equídeos do município de Mossoró - RN, região onde não há estudos sobre a circulação do EIV. Constatou-se que não houve diferença estatística entre os tratamentos de soro para a remoção de inibidores inespecíficos da hemaglutinação (p>0,05; confiança de 95%), todavia o tratamento RDEH apresentou resultados mais consistentes, corroborando a recomendação da OIE e da WHO de utilizar preferencialmente este tratamento. O perfil sorológico dos animais amostrados de SP sugere que circule o subtipo H3N8 e que o subtipo H7N7 circule de forma subclínica nos equídeos, o que é sustentado por outros trabalhos realizados no Brasil. Há evidências no Brasil sobre a detecção de anticorpos em equinos contra o subtipo H7N7, mesmo não havendo o isolamento deste no mundo desde 1980. No painel de soros do RN, onde a espécie Equus asinus era maioria, verificou-se a igualdade estatística entre as frequências de Equus caballus e Equus asinus positivos no teste de HI para o subtipo H3N8 (p>0,05; confiança de 95%), dado inédito em nosso meio. A frequência dos equídeos positivos no teste de HI para o subtipo H3N8 foi estatisticamente maior (p<0,05; confiança de 95%) em SP do que em RN. / The Equine influenza Virus (EIV) (H3N8 and H7N7) belong to Orthomyxoviridae family and Influenza A genus. Although there are few reports of human infection with EIV zoonotic and interspecies infection risk is known. OIE and WHO services on epidemiological surveillance report that H3N8 subtypes are isolated and characterized from worldwide outbreaks while H7N7 subtype less pathogenic has not being isolated since 1980 considered an extinct virus. Although the EIV is endemic in our country there are few national studies that had versed on the current evaluation of horses antibodies (Ab) from São Paulo State which motivated the present study. The study objectives were: 1) to evaluate the effects of different serum treatments described by OIE and WHO for the removal of nonspecific inhibitors of hemagglutination in 10 vaccinated (H3N8 - A/Equi/Kentucky/1/1997) horses sera being: a) TPH: trypsin, potassium metaperiodate followed by adsorption of erythrocytes b) KH: kaolin 20% followed by adsorption of erythrocytes and c) RDEH: RDE followed by adsorption of erythrocytes; 2) investigate the presence of antibodies against H3N8 and H7N7 viruses in 84 unvaccinated equines in São Paulo State; 3) compare the frequency of antibodies against H3N8 sampled between São Paulo State and a panel of equines sera from Mossoró - RN where there are no studies on EIV circulation. There was no statistical difference between the treatments for serum nonspecific inhibitors of hemagglutination removal (p>0.05; 95% confidence) however RDEH treatment showed results more consistent confirming OIE and WHO recommendation to use this treatment with priority. The serological profile of SP samples suggests H3N8 subtype circulates in those animals and H7N7 subtype might circulate in a subclinical form in equines, which is supported by other studies conducted in Brazil. There is evidence of antibodies detection against equine H7N7 subtype in Brazil, even without since 1980 isolation in the world. In animals from RN State which had Equus asinus representing a major fraction there was statistical equal frequencies of Equus caballus and Equus asinus positivity in HI test against H3N8 subtype (p>0.05; 95% of confidence), as unprecedented in the world. The frequency of positive equine against H3N8 subtype on HI test in SP was statistically higher (p<0.05; 95% of confidence) than in RN.

<p></p><p>The antifouling paint Fabi 3959 is painted on the hulls of vessels to avoid fouling caused by marine organisms attached to surfaces. The paint is registered for use on pleasure boats and other vessels weighing over 200 kg which are mainly running on the Swedish west coast (www.kemi.se).</p><p>Fabi 3959 contains copper as its active component, which is highly toxic to marine organisms and thus classified as a biocide.</p><p>Fabi antifouling paint was tested under laboratory conditions on the red macro alga <em>Ceramium tenuicorne</em>, in natural brackish water taken from the Baltic Sea. The <em>Ceramium</em> growth inhibition-test was performed using cloned algae exposed to leakage water with and without sediment. The samples containing only water held concentrations in the range of 0.11% of volume-18% of volume per liter, while the samples using sediment held doses measuring between 0.11% of volume-36% of volume leakage water per liter.</p><p>The study showed a growth inhibiting effect on the <em>Ceramium</em> in both water and sediment samples down to the lowest concentration used in the test. There was a difference between the water series and the sediment series in the EC₅₀ values of the leakage water. The mean EC₅₀value was almost 10 times lower within the sediment series compared to the water series (0.114±0.10 and 1.024±0.75, respectively). This indicates that the sediment series are more toxic to <em>Ceramium</em> than the water series. However, if the mean values of EC₅₀ are expressed as copper-concentration, there is no clear difference between the two series (0.59 ± 0.13µg/l for the sediment series and 0.62 ± 0.12 µg/l for the water series). Apparently, the test did not indicate that the sediment was absorbing the copper. Instead it cannot be excluded that another substance involved could have a growth inhibiting impact on <em>Ceramium</em>.</p><p> </p> / <p> </p><p>Båtbottenfärgen Fabi 3959 målas på fartygsskrov för att undvika påväxt av marina organismer. Färgen är registrerad att användas på fritidsbåtar och andra fartyg med en egenvikt på över 200 kg och med huvudsaklig fart på Västkusten (www.kemi.se). Den aktiva komponenten i Fabi 3959 är koppar, vilket är mycket giftigt för marina organismer och därför klassificeras den som en biocid.</p><p>Fabi båtbottenfärg testades i laborativ miljö, på den röda makroalgen <em>Ceramium tenuicorne</em> i naturligt brackvatten från Östersjön. Ett tillväxthämningstest utfördes på <em>Ceramium</em>-kloner vilka exponerades för lakvatten i bägare med och utan sediment. Proverna endast innehållande vatten bestod av koncentrationer i intervallen 0,11-18 volym% per liter medan proverna med sedimentvatten hade koncentrationer på 0,11-36 volym% per liter.</p><p>Studien visade på en signifikant tillväxthämningseffekt på <em>Ceramium</em> i både vatten och sediment, ner till den lägsta använda koncentrationen. Det förelåg en tydlig skillnad mellan vattenserierna och sedimentserierna med avseende på resultaten av EC₅₀-värdena på lakvattnet; EC₅₀-värdena av den toxiska nivån för lakvattnet visade sig ligga tio gånger lägre i sedimentserierna än i vattenserierna (0,114 ± 0,10 i sediment och 1,024 ± 0,75 i vatten). Medelvärdena av EC₅₀för koppar visar inte någon påtaglig skillnad mellan serierna (0,59 ± 0,13µg/l för sediment 0,62 ± 0,12 µg/l för vatten). Testet indikerade därmed inte att sedimentet absorberade koppar. Istället föreligger en möjlighet att en annan substans orsakade tillväxthämning på<em> Ceramium.</em></p><p> </p>

<p>Antifouling paints are applied on vessels to prevent growth of fouling organisms such hasbarnacles. Presently, there are a number of different paints available on the Swedish marketwith different strategies and active substances. The paints might work by either continuouslyreleasing biocides or physically by peeling off or provide an easily cleansed surface whereorganisms cannot attach. The physically working paints do not need to register an activesubstance since its purpose is not to affect living organisms by a chemical or biological modeof action. In this study, two commercially available paints, the copper-based Fabi 3959(International Paint Ltd) and physically eroding, biocide-free labelled Mille Light (HempelFärg AB) were compared to Hard Racing superior, containing copper and the forbiddensubstance Tributyltin. Fabi International is only allowed to be used on the Swedish west coastdue to 6% added as active substance while the biocide-free Mille Light is eligible for eastcoast usage. The toxic effect from respective paint was investigated by assembling a growthinhibition test with the red macro alga Ceramium tenuicorne. The results show that all thestudied paints had a negative effect on growth and therefore leaked substances inconcentrations high enough to be harmful to the alga. The toxic response differed with theeffect on growth being in the following order, Hard racing superior>Fabi >Mille Light.Implications regarding the current legalization involving biocide-free labelled antifoulingpaints are discussed.</p>

Made available in DSpace on 2014-08-26T17:15:06Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 91.pdf: 7442873 bytes, checksum: 27b69519fc283b4bdd1b15559627fb79 (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / O presente estudo é uma aplicação prática do conceito dos Três Rs (3Rs) de Russell e Burch (1959) nos ensaios de controle da qualidade de imunobiológicos para Raiva preconizados pela Farmacopéia Brasileira através de: uma análise retrospectiva de dados para a Redução do nº de animais no Ensaio de Potencia NIH para vacina contra raiva de uso humano (vaccinum rabiei ad usum humanum); mudanças no método de avaliação da inativação viral destas vacinas utilizando animais e células e validação de um ensaio in vitro para substituir o ensaio in vivo no teste de potência de imunoglobulinas anti-rábicas (Immunosera rabicum ex animali ad usum humanum e immunoglobulinum humanum rabicum). Todos os três protocolos de ensaio testados no estudo demonstraram viabilidade de utilização. / The present study is a practical application of the Russell and Burch 3Rs concept (1959) in the in vivo tests described in the Farmacopéia Brasileira for quality control of rabies biologicals by testing: A reduction in the number of mice in the NIH potency test for rabies vaccine for human use (vaccinum rabiei ad usum humanum); changes in the evaluation of virus inactivation method using suckling mice and cells as an alternative to the current Brazilian Pharmacopoeia official test (Reduction, Refinement and Replacement) and the validation of an in vitro assay to replace the in vivo assay for the potency test of Rabies Immunoglobulins (Immunosera rabicum ex animali ad usum humanum e immunoglobulinum humanum rabicum) (Replacement). All the three assay protocols have shown viability of use.