Thesis (M.Sc.( Medical Sciences)) --University of the North, 1983 / Refer to the document

Foi investigado o valor diagnóstico da resposta alérgica cutânea à tuberculina e do ELISA indireto, com antígeno recombinante MPB 70, em leitões experimentalmente sensibilizados, pela via intramuscular, com suspensões oleosas de M. bovis ou M. avium inativados. Foram utilizados 91 animais divididos em quatro grupos. Os grupos A e B, cada um com 25 indivíduos, grupos C e D com 21 e 20 indivíduos respectivamente, balanceando-se as características de raça, linhagem, faixa etária e sexo. Aos 21 dias de idade, todos os animais foram submetidos a uma triagem com a aplicação de tuberculina PPD de M. bovis, pela via intradérmica na base da orelha e não houve qualquer tipo de reação. Decorridos 60 dias do teste tuberculínico de triagem, o grupo A, recebeu injeção intramuscular de 0,5 mL de uma suspensão oleosa de M. avium estirpe D4; o grupo B, recebeu 0,5 mL de uma suspensão oleosa de M. bovis estirpe AN5; o grupo C (controle I), recebeu 0,5 mL do adjuvante oleoso e o grupo D (controle II), recebeu 0,5 mL de solução fisiológica. Foi realizado o exame histopatológico de biopsias das reações cutâneas e a colheita de sangue para o teste de ELISA de captura. Após 30 dias da sensibilização, foi efetuada a prova de tuberculinização comparativa com reação medida pela variação da espessura da pele com paquímetro às 0h, 24h, 48h e 72h, após a aplicação das tuberculinas. No teste comparativo, lido às 72 horas, a reação foi considerada negativa quando a diferença das reações entre o PPD bovino e o PPD aviário foi menor que 6,7 mm; suspeito ou inconclusivo quando a diferença se situou na faixa de 6,7 a 7,5 mm; e positiva para o tipo de PPD, considerando-se tuberculose para PPD M. bovis e micobacteriose para PPD M. avium, quando a diferença da reação foi superior a 7,5 mm. Nos exames histopatológicos, foi observado intenso infiltrado inflamatório linfocitário no local das reações intradérmicas dos animais testados com o PPD homologo ao tipo de micobactéria utilizada na suspensão oleosa sensibilizante. O ensaio de ELISA com antígeno, MPB 70 recombinante, foi capaz de revelar a presença de anticorpos contra o M. bovis, porém não revelou anticorpos para M. avium. / The diagnostic value of the cutaneous allergic response to tuberculin and Indirect ELISA test was investigated using MPB 70 recombinant antigen, in piglets experimentally sensitized intramuscularly with the oily suspensions of inactivated M. bovis or M. avium. The ninety-one animals used were divided into four groups. The groups A and B were formed each with 25 individuals, and groups C and D, with 21 and 20 individuals, respectively, balancing the characteristics of race, ancestry, age and sex. At the age of 21 days, all the animals were submitted to the screening test with the use of M. bovis PPD, by the intradermal route at the base of the ear and no reaction was detected. Sixty days after the screening tuberculin test, animals of the group A were injected intramuscularly with 0.5 mL of oily suspension of M. avium D4 strain; animals of group B received 0.5 mL of an oily suspension of M. bovis, AN5 strain; and the members of group C (control I) received 0.5 mL of an oily adjuvant and the individuals of group D (control II) received 0.5 mL of saline solution. Histological examinations of biopsies of skin reactions were carried out and blood collections made for capture ELISA. Following 30 days of sensitization, comparative skin reactions were measured by the variation in skin thickness with a caliper at 0h, 24h, 48h an 72h after applications of tuberculins. In the comparative test measured at 72h, the reaction was considered negative when the difference of the reactions between bovine PPD and avian PPD was less than 6.7 mm; suspected or inconclusive, when the difference stood in the range of 6.7 to 7.5 mm; and positive according to the type of PPD, considering tuberculosis the M. bovis PPD and mycobacteriosis the M. avium PPD, when the difference of the reaction was greater than 7.5 mm. In histopathological examinations, intense lymphocytic inflammatory infiltrate were observed at the site of intradermal reactions of the animals tested with PPD homologous to the type of mycobacteria used in sensitizing oil suspension. The ELISA assay with MPB 70 recombinant antigen was able to reveal the presence of antibodies against M. bovis, but did not reveal antibodies to M. avium.

Foi investigado o valor diagnóstico da resposta alérgica cutânea à tuberculina e do ELISA indireto, com antígeno recombinante MPB 70, em leitões experimentalmente sensibilizados, pela via intramuscular, com suspensões oleosas de M. bovis ou M. avium inativados. Foram utilizados 91 animais divididos em quatro grupos. Os grupos A e B, cada um com 25 indivíduos, grupos C e D com 21 e 20 indivíduos respectivamente, balanceando-se as características de raça, linhagem, faixa etária e sexo. Aos 21 dias de idade, todos os animais foram submetidos a uma triagem com a aplicação de tuberculina PPD de M. bovis, pela via intradérmica na base da orelha e não houve qualquer tipo de reação. Decorridos 60 dias do teste tuberculínico de triagem, o grupo A, recebeu injeção intramuscular de 0,5 mL de uma suspensão oleosa de M. avium estirpe D4; o grupo B, recebeu 0,5 mL de uma suspensão oleosa de M. bovis estirpe AN5; o grupo C (controle I), recebeu 0,5 mL do adjuvante oleoso e o grupo D (controle II), recebeu 0,5 mL de solução fisiológica. Foi realizado o exame histopatológico de biopsias das reações cutâneas e a colheita de sangue para o teste de ELISA de captura. Após 30 dias da sensibilização, foi efetuada a prova de tuberculinização comparativa com reação medida pela variação da espessura da pele com paquímetro às 0h, 24h, 48h e 72h, após a aplicação das tuberculinas. No teste comparativo, lido às 72 horas, a reação foi considerada negativa quando a diferença das reações entre o PPD bovino e o PPD aviário foi menor que 6,7 mm; suspeito ou inconclusivo quando a diferença se situou na faixa de 6,7 a 7,5 mm; e positiva para o tipo de PPD, considerando-se tuberculose para PPD M. bovis e micobacteriose para PPD M. avium, quando a diferença da reação foi superior a 7,5 mm. Nos exames histopatológicos, foi observado intenso infiltrado inflamatório linfocitário no local das reações intradérmicas dos animais testados com o PPD homologo ao tipo de micobactéria utilizada na suspensão oleosa sensibilizante. O ensaio de ELISA com antígeno, MPB 70 recombinante, foi capaz de revelar a presença de anticorpos contra o M. bovis, porém não revelou anticorpos para M. avium. / The diagnostic value of the cutaneous allergic response to tuberculin and Indirect ELISA test was investigated using MPB 70 recombinant antigen, in piglets experimentally sensitized intramuscularly with the oily suspensions of inactivated M. bovis or M. avium. The ninety-one animals used were divided into four groups. The groups A and B were formed each with 25 individuals, and groups C and D, with 21 and 20 individuals, respectively, balancing the characteristics of race, ancestry, age and sex. At the age of 21 days, all the animals were submitted to the screening test with the use of M. bovis PPD, by the intradermal route at the base of the ear and no reaction was detected. Sixty days after the screening tuberculin test, animals of the group A were injected intramuscularly with 0.5 mL of oily suspension of M. avium D4 strain; animals of group B received 0.5 mL of an oily suspension of M. bovis, AN5 strain; and the members of group C (control I) received 0.5 mL of an oily adjuvant and the individuals of group D (control II) received 0.5 mL of saline solution. Histological examinations of biopsies of skin reactions were carried out and blood collections made for capture ELISA. Following 30 days of sensitization, comparative skin reactions were measured by the variation in skin thickness with a caliper at 0h, 24h, 48h an 72h after applications of tuberculins. In the comparative test measured at 72h, the reaction was considered negative when the difference of the reactions between bovine PPD and avian PPD was less than 6.7 mm; suspected or inconclusive, when the difference stood in the range of 6.7 to 7.5 mm; and positive according to the type of PPD, considering tuberculosis the M. bovis PPD and mycobacteriosis the M. avium PPD, when the difference of the reaction was greater than 7.5 mm. In histopathological examinations, intense lymphocytic inflammatory infiltrate were observed at the site of intradermal reactions of the animals tested with PPD homologous to the type of mycobacteria used in sensitizing oil suspension. The ELISA assay with MPB 70 recombinant antigen was able to reveal the presence of antibodies against M. bovis, but did not reveal antibodies to M. avium.

La rage est une zoonose mortelle, susceptible d’atteindre autant les mammifères sauvages et domestiques que l’Homme. Elle est à l’origine d’environ 70 000 décès humain déclarés par an, majoritairement des enfants dans les pays en développement. Le chien, réservoir majeur de l’espèce RABV, est à l’origine de 98-99% de ces décès. Quatorze espèces de Lyssavirus, circulant majoritairement chez les chiroptères sont actuellement reconnues. La vaccination, associée à des mesures sanitaires, reste le meilleur outil de prévention et de maîtrise de la maladie. A l’heure actuelle, seule la sérologie permet de contrôler l’efficacité de la vaccination antirabique, le développement des anticorps neutralisants étant le premier témoin d’une immunité protectrice. Les travaux s’appuyant sur la séroneutralisation virale, et notamment ceux auxquels j’ai participé, ont mis en évidence l’influence de différents facteurs dont certains ont conduit à préconiser des modifications de protocoles vaccinaux. Ils ont également permis d’assurer le suivi de l’efficacité de la vaccination individuelle ou de groupe sur le terrain et de contribuer à son amélioration. Les tests de séroneutralisation sont également utilisés dans le cadre de l’épidémiosurveillance de populations animales non vaccinées. La mise en œuvre de ces tests chez les chiroptères en France, après leur adaptation au Lyssavirus d’intérêt que j’ai menée à bien, a permis d’obtenir des informations sur la circulation des espèces virales EBLV-1 et EBLV-2, sur une base uniquement sérologique pour ce dernier. D’autre part, elle a permis de mettre en évidence au sein d’une même colonie des phénomènes de transition sérologique au cours du temps, dont l’étude mériterait d’être approfondie. Les tests de séroneutralisation sont cependant difficilement transférables aux pays où la rage est très présente, du fait de ressources limitées. Mes travaux, proposant l’utilisation d’un test ELISA comme méthode alternative, ont contribué à remettre en cause le dogme du recours nécessaire à la séroneutralisation. Ce test, couplé à un système de collecte d’échantillons sanguins adapté au terrain, devrait améliorer le suivi de l’efficacité des campagnes de vaccination de la faune sauvage comme des animaux domestiques, y compris dans les pays d’enzootie où la qualité des prélèvements de sang ne peut être assurée. Ainsi, les outils d’évaluation de la réponse immunitaire humorale sont des outils très précieux au service de la lutte et de la surveillance de la rage animale dans le monde. Mes travaux, complémentaires à ceux réalisés par d’autres équipes, ont contribué à rendre envisageable l’objectif prioritaire des organisations internationales : l’éradication de la rage canine dans le monde à l’horizon 2030. Il est cependant nécessaire de les poursuivre pour améliorer les outils disponibles et d’en proposer de plus adaptés, afin d’atteindre l’ensemble des objectifs d’éradication, de la rage canine comme de la rage selvatique / Rabies is a deadly zoonosis that can affect wild and domestic mammals as much as humans. About 70,000 human deaths are reported each year, mostly in children from developing countries. Dogs, which are the major reservoir and source of the RABV species, account for 98-99% of these deaths. Currently, fourteen species of Lyssavirus, mainly circulating in chiroptera, are officially recognized. Vaccination, combined with sanitary measures, remains the best tool for preventing and controlling the disease. To date, only serology has allowed to control the effectiveness of rabies vaccination, as the production of neutralizing antibodies is the first evidence of protective immunity. Studies based on viral seroneutralisation, including my own studies, have highlighted the influence of various factors. Some of them have led to recommend modifications of vaccine protocols. They also contributed to monitor the effectiveness of individual or group vaccination field programmes and to improve these programmes. Seroneutralisation tests are also used in the context of the epidemiological surveillance of unvaccinated animal populations. I first successfully adapted these tests to lyssaviruses of interest in France. In a second step, their implementation in chiropters in France provided information on the circulation of EBLV-1 and EBLV-2 species, (only on a serological basis for the latter). This survey also allowed to highlight, within a specific colony, a phenomenon of serological transition over time, which should deserve to be studied further. However, seroneutralisation tests are difficult to be implemented in countries where rabies is very prevalent, mainly because of limited resources. My work, which recommends the use of an ELISA test as an alternative method, contributed to questioning the dogma of the necessary use of seroneutralisation tests. This test, coupled with a blood sampling system adapted to the field, should improve the monitoring of the effectiveness of vaccination campaigns for both wildlife and domestic animals, including in enzootic countries, where the quality of the blood samples cannot be guaranteed. Humoral immune response assessment tools are very valuable tools for the control and surveillance of animal rabies all around the world. My work, complementary to those carried out by other teams, has helped to make the priority objective of international organizations possible, i.e. the eradication of canine rabies in the world by 2030. However, further works are needed to improve the available tools and to propose more adapted ones, in order to achieve all the goals of eradication, for both canine and sylvatic rabies