Transmisibilidad e inocuidad de un candidato vacunal...
Bovine tuberculosis (BTB), distributed worldwide, represents a problem for the\nlivestock sector and a Public Health concern due to its zoonotic nature. The main\ncausative agent is Mycobacterium bovis (M. bovis). BTB has a negative impact on the\nproductivity and profitability of livestock farms, r...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2023
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| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7156 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7156.dir/7156.PDF |
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I28-R145-HWA_7156 |
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dspace |
| institution |
Universidad de Buenos Aires |
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I-28 |
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R-145 |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
| topic |
Tuberculosis Bovina Diagnostico Vacuna Bovine Tuberculosis Diagnosis Vaccine Tuberculosis Bovinos |
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Tuberculosis Bovina Diagnostico Vacuna Bovine Tuberculosis Diagnosis Vaccine Tuberculosis Bovinos Ferrara Muñiz, Ximena Transmisibilidad e inocuidad de un candidato vacunal... |
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Tuberculosis Bovina Diagnostico Vacuna Bovine Tuberculosis Diagnosis Vaccine Tuberculosis Bovinos |
| description |
Bovine tuberculosis (BTB), distributed worldwide, represents a problem for the\nlivestock sector and a Public Health concern due to its zoonotic nature. The main\ncausative agent is Mycobacterium bovis (M. bovis). BTB has a negative impact on the\nproductivity and profitability of livestock farms, reducing production, due to sending\ninfected animals to slaughter, confiscation in slaughterhouses and, because of the\nrestrictions imposed on the commercialization and export of meat and dairy products, and their derivatives. In Argentina, although the National Control and Eradication Program (Law 128/2012, Senasa) reduced its prevalence based on ante-mortem diagnosis in herds and post-mortem epidemiological surveillance in slaughterhouses, due to its complexity, the disease continues to be a challenge. Vaccination, as a\ncomplementary intervention strategy, could reduce the impact of M. bovis infection in cattle, its main host. At present, there are no commercial vaccines, which must be developed in parallel to a differential diagnosis system that allows to distinguish between the vaccinated animal and the naturally infected one, without interfering with the official diagnostic test. In Argentina, the vaccine candidate M. bovis?mce2-phoP, deleted in two independent regions of the genome, the phoP gene (Mb0780) and the mce2AB genes (Mb0604-Mb0605), was obtained. M. bovis?mce2-phoP induced protection against challenge with a virulent strain of M. bovis in the murine model.\nIn order to contribute to the characterization of the M. bovis?mce2-phoP\ncandidate, the general objective of this work was to evaluate the safety and\ntransmissibility of the M. bovis?mce2?phoP strain under experimental and natural infection conditions, and to develop a diagnostic system that allows to differentiate a\nvaccinated animal from an infected one.\nDifferent aspects of the M. bovis?mce2-phoP strain were studied when\ninoculated subcutaneously, such as its safety, its tropism, transmission, excretion, immunological aspects related to the diagnosis of BTB, such as its ability to generate an immune response in intradermal test reaction and gamma interferon release, and its dissemination to the environment. The experimental approach consisted in characterizing these attributes in different experimental animal models and in cattle. In the murine model, the airborne transmission capacity of M. bovis?mce2-phoP was evaluated in a prolonged coexistence assay of BALB/c mice inoculated with the experimental vaccine strain and sentinel mice. Residual virulence was studied in the\nguinea pig model, the laboratory specie most susceptible to infection by M. bovis and that best reproduces tuberculosis, considering the presence of granulomatous lesions in the inoculated animal, possible excretion in feces, and immunological reactivity by intradermal inoculation of tuberculin and recombinant antigens. Finally, in cattle, in a vaccination and infection trial under natural transmission conditions, the protection and safety of the strain was evaluated in inoculated and sentinel animals and for the\nenvironment, potential antigens for ante-mortem differential diagnosis of the vaccinated and infected animal, and post-mortem complementary molecular techniques.\nIn the murine model, the PCR technique demonstrated the presence of M. bovis DNA in tissues of the sentinel mice of the M. bovis?mce2-phoP vaccinated group, although its viability was not verified. Although it retains the ability to replicate in the\nspleen of mice and in lymph nodes of guinea pigs, its attenuation is confirmed by the low bacterial load, the absence of lesions and the survival of all the inoculated animals; compared to that observed with the parental strain. The development of a differential\nPCR allowed us to conclude that, in cattle, neither M. bovis?mce2-phoP DNA nor viability was detected, confirming the findings about its attenuation. In relation to transmissibility,\nno specific genomic sequences were detected in the DNA extracted from stool in guinea pigs and cattle, as well as in soil and drinking water surrounding the cattle under study.\nThese results reinforce those obtained in the murine model and suggest that the vaccine candidate is not excreted, with no potential for transmission to susceptible animals and the environment. Regarding ante-mortem diagnosis in guinea pigs and cattle, the\nrecombinant proteins, PhoP and Mce2B, were used for the first time. Although they\ninduced a certain degree of cell-mediated immune response, they would not be\npromising candidates for the development of a differential diagnosis between natural\ninfection and vaccination, since they induced a response in animals vaccinated with the\nM. bovis?mce2-phoP strain.\nIn conclusion, the attenuation of the M. bovis?mce2-phoP strain in cattle was confirmed, and it was shown that its subcutaneous inoculation does not represent a source of contamination for the environment since it would not be excreted, with no potential for transmission to other susceptible animals or to humans. Although a\ndifferential post-mortem detection tool was developed, more studies are required in relation to the generation of differential diagnosis tools for the live animal. The realization of this work, in addition to deepening the study of key aspects of vaccine candidates such as the protection and safety of a genetically modified organism, strengthened professional skills and multidisciplinary interaction, a strategic link in the optimization of this type of trial. The knowledge generated may be used in the future for purposes like those proposed in this research project. |
| author2 |
Cataldi, Ángel Adrian |
| author_facet |
Cataldi, Ángel Adrian Ferrara Muñiz, Ximena |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Ferrara Muñiz, Ximena |
| author_sort |
Ferrara Muñiz, Ximena |
| title |
Transmisibilidad e inocuidad de un candidato vacunal... |
| title_short |
Transmisibilidad e inocuidad de un candidato vacunal... |
| title_full |
Transmisibilidad e inocuidad de un candidato vacunal... |
| title_fullStr |
Transmisibilidad e inocuidad de un candidato vacunal... |
| title_full_unstemmed |
Transmisibilidad e inocuidad de un candidato vacunal... |
| title_sort |
transmisibilidad e inocuidad de un candidato vacunal... |
| publisher |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias |
| publishDate |
2023 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7156 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7156.dir/7156.PDF |
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AT ferraramunizximena transmisibilidadeinocuidaddeuncandidatovacunal AT ferraramunizximena transmisibilidadeinocuidaddeuncandidatovacunalcontralatuberculosisbovinayevaluaciondetecnicascomplementariasparaeldiagnosticodiferencialenlainfeccionnatural |
| _version_ |
1824356461092798464 |
| spelling |
I28-R145-HWA_71562024-10-23 Transmisibilidad e inocuidad de un candidato vacunal... Bovine tuberculosis (BTB), distributed worldwide, represents a problem for the\nlivestock sector and a Public Health concern due to its zoonotic nature. The main\ncausative agent is Mycobacterium bovis (M. bovis). BTB has a negative impact on the\nproductivity and profitability of livestock farms, reducing production, due to sending\ninfected animals to slaughter, confiscation in slaughterhouses and, because of the\nrestrictions imposed on the commercialization and export of meat and dairy products, and their derivatives. In Argentina, although the National Control and Eradication Program (Law 128/2012, Senasa) reduced its prevalence based on ante-mortem diagnosis in herds and post-mortem epidemiological surveillance in slaughterhouses, due to its complexity, the disease continues to be a challenge. Vaccination, as a\ncomplementary intervention strategy, could reduce the impact of M. bovis infection in cattle, its main host. At present, there are no commercial vaccines, which must be developed in parallel to a differential diagnosis system that allows to distinguish between the vaccinated animal and the naturally infected one, without interfering with the official diagnostic test. In Argentina, the vaccine candidate M. bovis?mce2-phoP, deleted in two independent regions of the genome, the phoP gene (Mb0780) and the mce2AB genes (Mb0604-Mb0605), was obtained. M. bovis?mce2-phoP induced protection against challenge with a virulent strain of M. bovis in the murine model.\nIn order to contribute to the characterization of the M. bovis?mce2-phoP\ncandidate, the general objective of this work was to evaluate the safety and\ntransmissibility of the M. bovis?mce2?phoP strain under experimental and natural infection conditions, and to develop a diagnostic system that allows to differentiate a\nvaccinated animal from an infected one.\nDifferent aspects of the M. bovis?mce2-phoP strain were studied when\ninoculated subcutaneously, such as its safety, its tropism, transmission, excretion, immunological aspects related to the diagnosis of BTB, such as its ability to generate an immune response in intradermal test reaction and gamma interferon release, and its dissemination to the environment. The experimental approach consisted in characterizing these attributes in different experimental animal models and in cattle. In the murine model, the airborne transmission capacity of M. bovis?mce2-phoP was evaluated in a prolonged coexistence assay of BALB/c mice inoculated with the experimental vaccine strain and sentinel mice. Residual virulence was studied in the\nguinea pig model, the laboratory specie most susceptible to infection by M. bovis and that best reproduces tuberculosis, considering the presence of granulomatous lesions in the inoculated animal, possible excretion in feces, and immunological reactivity by intradermal inoculation of tuberculin and recombinant antigens. Finally, in cattle, in a vaccination and infection trial under natural transmission conditions, the protection and safety of the strain was evaluated in inoculated and sentinel animals and for the\nenvironment, potential antigens for ante-mortem differential diagnosis of the vaccinated and infected animal, and post-mortem complementary molecular techniques.\nIn the murine model, the PCR technique demonstrated the presence of M. bovis DNA in tissues of the sentinel mice of the M. bovis?mce2-phoP vaccinated group, although its viability was not verified. Although it retains the ability to replicate in the\nspleen of mice and in lymph nodes of guinea pigs, its attenuation is confirmed by the low bacterial load, the absence of lesions and the survival of all the inoculated animals; compared to that observed with the parental strain. The development of a differential\nPCR allowed us to conclude that, in cattle, neither M. bovis?mce2-phoP DNA nor viability was detected, confirming the findings about its attenuation. In relation to transmissibility,\nno specific genomic sequences were detected in the DNA extracted from stool in guinea pigs and cattle, as well as in soil and drinking water surrounding the cattle under study.\nThese results reinforce those obtained in the murine model and suggest that the vaccine candidate is not excreted, with no potential for transmission to susceptible animals and the environment. Regarding ante-mortem diagnosis in guinea pigs and cattle, the\nrecombinant proteins, PhoP and Mce2B, were used for the first time. Although they\ninduced a certain degree of cell-mediated immune response, they would not be\npromising candidates for the development of a differential diagnosis between natural\ninfection and vaccination, since they induced a response in animals vaccinated with the\nM. bovis?mce2-phoP strain.\nIn conclusion, the attenuation of the M. bovis?mce2-phoP strain in cattle was confirmed, and it was shown that its subcutaneous inoculation does not represent a source of contamination for the environment since it would not be excreted, with no potential for transmission to other susceptible animals or to humans. Although a\ndifferential post-mortem detection tool was developed, more studies are required in relation to the generation of differential diagnosis tools for the live animal. The realization of this work, in addition to deepening the study of key aspects of vaccine candidates such as the protection and safety of a genetically modified organism, strengthened professional skills and multidisciplinary interaction, a strategic link in the optimization of this type of trial. The knowledge generated may be used in the future for purposes like those proposed in this research project. Fil: Ferrara Muñiz, Ximena. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, Argentina Cataldi, Ángel Adrian Eirin, Maria Emilia Ferrara Muñiz, Ximena 2023-05-31 La tuberculosis bovina (TBB), distribuida mundialmente, constituye un problema para el sector pecuario y la Salud Pública por su naturaleza zoonótica. Su principal agente causal es Mycobacterium bovis (M. bovis). La TBB influye negativamente en la\nproductividad y rentabilidad de las explotaciones ganaderas, disminuyendo la\nproducción, por envío a faena de animales infectados, decomiso en frigorífico y por\nrestricciones en la comercialización y exportación de productos cárnicos, lecheros y\nderivados. En Argentina, si bien el Plan Nacional de Control y Erradicación (Resol.128/2012, Senasa) redujo su prevalencia basándose en el diagnóstico ante-mortem en los rodeos y en la vigilancia epidemiológica post-mortem en frigoríficos, por su complejidad, la enfermedad continúa siendo un desafío. La vacunación, como estrategia complementaria de intervención, podría reducir el impacto de la infección por M. bovis en el ganado bovino, su principal hospedador. Al presente, no existen vacunas comerciales, las cuales deben desarrollarse en paralelo a un sistema de diagnóstico diferencial que permita distinguir entre el animal vacunado del naturalmente infectado, sin interferir con la prueba diagnóstica oficial. En Argentina, se obtuvo el candidato vacunal M. bovis?mce2-phoP, delecionado en dos regiones independientes del\ngenoma, el gen phoP (Mb0780) y los genes mce2AB (Mb0604- Mb0605). M. bovis\n?mce2-phoP indujo protección frente al desafío con una cepa virulenta de M. bovis en el modelo murino.\nCon el fin de contribuir con la caracterización del candidato M. bovis ?mce2-\nphoP, el objetivo general del presente trabajo de tesis fue evaluar la inocuidad y\ntransmisibilidad de la cepa M. bovis ?mce2-phoP en condiciones experimentales y naturales de infección, y desarrollar un sistema de diagnóstico que permita diferenciar un animal vacunado de un animal infectado.\nSe estudiaron diferentes aspectos de la cepa M. bovis ?mce2-phoP al ser\ninoculada por vía subcutánea, como su seguridad, su tropismo, transmisión, excreción,\naspectos inmunológicos relacionados al diagnóstico de la TBB, como su capacidad de despertar respuesta inmune en pruebas de intradermorreacción y de liberación de interferón gamma; y su diseminación al medio ambiente. El abordaje experimental consistió en caracterizar estos atributos en diferentes modelos animales de\nexperimentación y en el bovino. En el modelo murino, se evaluó la capacidad de transmisión aerógena de M. bovis ?mce2-phoP en un ensayo de coexistencia\nprolongada de ratones BALB/c inoculados con la cepa vacunal experimental y ratones centinelas. En el modelo de cobayo, especie de laboratorio más susceptible a la infección por M. bovis y que mejor reproduce la tuberculosis, se estudió la virulencia residual ponderando la presencia de lesiones granulomatosas en el animal inoculado, posible excreción en heces y reactividad inmunológica por inoculación intradérmica de tuberculina y antígenos recombinantes. Finalmente, en el bovino, en un ensayo de vacunación e infección a campo en condiciones naturales de transmisión, se evaluó la inocuidad y seguridad de la cepa para el animal inoculado, animales centinelas y para\nel ambiente, potenciales antígenos para el diagnóstico diferencial ante-mortem del animal vacunado e infectado y técnicas moleculares complementarias post-mortem.\nEn el modelo murino, por la técnica de PCR se demostró la presencia de ADN\nde M. bovis en tejidos de los ratones centinela del grupo vacunado con el candidato M. bovis?mce2-phoP, aunque no se comprobó su viabilidad. Si bien retiene la capacidad de replicar en el bazo de ratones, y en linfonódulos de cobayos, se confirma su\natenuación por la baja carga bacteriana, la ausencia de lesiones y la supervivencia de la totalidad de los animales inoculados; en comparación con lo observado con la cepa parental. La puesta a punto de una PCR diferencial permitió concluir que, en el bovino, no se detectó ADN de M. bovis ?mce2-phoP ni viabilidad, confirmando los hallazgos acerca de su atenuación. En relación a la transmisibilidad, no se detectaron secuencias\ngenómicas específicas en ADN extraído de materia fecal en cobayos y bovinos, al igual que en suelo y agua de bebedero circundante a los bovinos en estudio. Estos resultados refuerzan los obtenidos en el modelo murino y sugieren que el candidato vacunal no se excreta teniendo potencial nulo de transmisión a animales susceptibles y al ambiente.\nEn cuanto al diagnóstico ante-mortem en cobayos y bovinos, se emplearon por primera vez las proteínas recombinantes PhoP y Mce2B. Si bien indujeron cierto grado de respuesta inmune mediada por células, no serían candidatos prometedores para desarrollo de un diagnóstico diferencial entre la infección natural y la vacunación, ya que indujeron respuesta en animales vacunados con la cepa M. bovis ?mce2-phoP.\nEn conclusión, se confirmó la atenuación de la cepa M. bovis ?mce2-phoP en el\nbovino, y se demuestra que su inoculación por vía subcutánea no representa una fuente de contaminación para el medioambiente ya que no sería excretada, con potencial nulo de transmisión a otros animales susceptibles o al ser humano. Si bien se desarrolló una herramienta diferencial de detección post-mortem, se requieren más estudios en\nrelación a la generación de herramientas de diagnóstico diferencial para el animal en\npie. La concreción de este proyecto de tesis doctoral, además de profundizar en\naspectos claves en el estudio de candidatos vacunales como la inocuidad y seguridad\nde un organismo genéticamente modificado, fortaleció las capacidades profesionales y la interacción multidisciplinaria, eslabón estratégico en la optimización de este tipo de\nensayos. Los conocimientos generados podrán ser empleados a futuro con fines similares al planteado en este proyecto de investigación application/pdf Tuberculosis Bovina Diagnostico Vacuna Bovine Tuberculosis Diagnosis Vaccine spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-ncnd/2.5/ar/ Tuberculosis Bovinos Doctora de Universidad de Buenos Aires en Ciencias Veterinarias Transmisibilidad e inocuidad de un candidato vacunal contra la tuberculosis bovina y evaluación de técnicas complementarias para el diagnóstico diferencial en la infección natural info:eu-repo/semantics/doctoralThesis info:ar-repo/semantics/tesis doctoral info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7156 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7156.dir/7156.PDF |