Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne
Bovine tuberculosis, a disease caused by Mycobacterium bovis (M. bovis), is endemic\nin Argentine. Its presence in cattle represents a risk to human health, due to its\nzoonotic nature and can also affect various mammals. In human beings, M. bovis\ncauses disease with pulmonary or extrapulmonary pre...
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| Formato: | Tesis doctoral acceptedVersion |
| Lenguaje: | Español |
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Facultad de Ciencias Veterinarias
2019
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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_3133 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_3133.dir/3133.PDF |
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I28-R145-HWA_3133 |
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dspace |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-145 |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
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spa |
| topic |
Mycobacterium bovis Spoligotyping MIRU-VNTR Resistencia a los antibióticos PCR Bovinos Tuberculosis Mycobacterium bovis Cepas Micobacterias Transmisión Comercialización Microbiología |
| spellingShingle |
Mycobacterium bovis Spoligotyping MIRU-VNTR Resistencia a los antibióticos PCR Bovinos Tuberculosis Mycobacterium bovis Cepas Micobacterias Transmisión Comercialización Microbiología Marfil, María Jimena Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| topic_facet |
Mycobacterium bovis Spoligotyping MIRU-VNTR Resistencia a los antibióticos PCR Bovinos Tuberculosis Mycobacterium bovis Cepas Micobacterias Transmisión Comercialización Microbiología |
| description |
Bovine tuberculosis, a disease caused by Mycobacterium bovis (M. bovis), is endemic\nin Argentine. Its presence in cattle represents a risk to human health, due to its\nzoonotic nature and can also affect various mammals. In human beings, M. bovis\ncauses disease with pulmonary or extrapulmonary presentation, being the clinical manifestation undistinguishable from that produced by M. tuberculosis. The most exposed are children who consume unpasteurized milk or its products, adults who\nworks with livestock and also hunters. To avoid the arrival of the agent to the\nconsumer, one of the main barriers of protection are the carcasses post-mortem\ninspection at the slaughterhouse, to detect and confiscate organs with lesions compatible with tuberculosis or even the hole animal. Inspection is performed visually\nand by palpation of the bovine organ, but due to the pathogenesis of the\nMycobacterium, only the chronic lesions can be evidenced in this inspection. The Mycobacterium might be present even in tissues without apparent lesion, being the lymph nodes and the lungs, the most affected organs: Other parenchymal organs such as the liver could also be affected. This can lead to escapes of the surveillance system\nallowing the Mycobacteria to reach the stores and hence, arrive to the consumer.\nIn this study, 210 lungs along with the corresponding lymph nodes and 6 bovine livers were collected, in one slaughterhouse and 6 butchers´ shops in the city of Buenos Aires and the great Buenos Aires area. The lungs collected in the slaughterhouse were\ninspected in situ and small portions of them were taken. Complete lungs and livers that were bought in butchers´ shops and were then inspected in the laboratory.\nA total of 5 isolates of M. bovis were obtained in Stonebrink medium and its identity confirmed by PCR IS-6110 (isolates number 134, 719, 179, 182 and 184). Additionally\n12 non-tuberculous mycobacteria were isolated and confirmed by the sequencing of the hsp65, rpoB and 16S rRNA genes. The isolates of M. bovis were molecularly\ncharacterized by Spoligotyping and MIRU-VNTR. We could differentiate 3\nSpoligotyping patterns that corresponded with the 3 patterns of MIRU-VNTR obtained. Three isolates shared both patterns and the other 2 were unique. Additionally, drug\nresistance to human treatment commonly used drugs (rifampicin, isoniazid,\nethionamide, levofloxacin and streptomycin) was investigated in all M. bovis isolates, and it was found that isolate number 184 was resistant to isoniazid and isolate number 134 was multi-resistant (showing resistance to the 4 antibiotics tested and considered "border" for levofloxacin). One non-tuberculous mycobacteria isolates from which good\nquality DNA was obtained was completely sequenced (whole genome equencing).\nThe genome was annotated and its characteristics were investigated as well as the \npresence of single nucleotide polymorphism (SNPs) in virulence genes and genes\nrelated to resistance to antibiotics and antigens used for diagnosis.\nThe virulence and transmissibility of isolates number 134 and 182 were evaluated together with a reference strain M. bovis-AN5 (considered of intermediate virulence), in\nan experimental assay in a mouse model. A total of 99 BALB/c female mice aged 5\nweeks, were separated in 3 groups of 33 animals each. In turn, each group was divided into 6 cages, the first cage with 3 animals and the remaining cages with 6 animals. All animals in cage number 1 of each group and 3 from each of the remaining cages, were intratracheally inoculated under sedation with isoflurane, with 1x105 CFU of each of the strains to be evaluated, suspended in 100 ?L of PBS, keeping the relationship between\ninoculated animal and healthy contact 1:1. Virulence was evaluated in those animals that were inoculated, while in the healthy contacts that were living with the infected\nanimals the transmissibility of the strains was investigated. Euthanasia of 5 infected animals and 5 healthy contact animals of each group were performed at times 30, 60 and 90 days post-inoculation. The lungs and spleens of all infected animals and healthy contacts were cultured. The cultures were grown at 37°C for up to 2 months, with weekly evaluation of their growth. Spleens were further processed to measure the\ncytokines IL-4, TNF? and INF? with the use of a commercial Cytometric bead array kit. Strain number 134 showed to be virulent because it produced symptoms incompatible\nwith life on day 16 after inoculation, producing the death of 9 animals, the remaining 6 were euthanatized and processed. Strain number 182 generated spleen enlargement in the infected mice, but it did not generate the illness or death of any of them. The transmissibility of strain 182 could be evidenced, due to the observation of the growth\nof colonies from a spleen of a contact animal in the time of euthanasia 90 days after\ninoculation It was possible to detect the presence of both, zoonotic and environmental\nmycobacteria, in commercialized viscera. The virulence, transmissibility and drugresistance of the M. bovis strains was variable, requiring future studies for a better understanding of the dynamics of the disease. |
| author2 |
Zumárraga, Martín José |
| author_facet |
Zumárraga, Martín José Marfil, María Jimena |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Marfil, María Jimena |
| author_sort |
Marfil, María Jimena |
| title |
Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| title_short |
Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| title_full |
Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| title_fullStr |
Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| title_full_unstemmed |
Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| title_sort |
tuberculosis bovina : transmisibilidad de cepas de mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne |
| publisher |
Facultad de Ciencias Veterinarias |
| publishDate |
2019 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_3133 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_3133.dir/3133.PDF |
| work_keys_str_mv |
AT marfilmariajimena tuberculosisbovinatransmisibilidaddecepasdemycobacteriumbovisydetecciondemicobacteriasenmenudenciascomercializadasenbocasdeexpendiodecarne |
| _version_ |
1766017527077404672 |
| spelling |
I28-R145-HWA_31332020-02-21 Bovine tuberculosis, a disease caused by Mycobacterium bovis (M. bovis), is endemic\nin Argentine. Its presence in cattle represents a risk to human health, due to its\nzoonotic nature and can also affect various mammals. In human beings, M. bovis\ncauses disease with pulmonary or extrapulmonary presentation, being the clinical manifestation undistinguishable from that produced by M. tuberculosis. The most exposed are children who consume unpasteurized milk or its products, adults who\nworks with livestock and also hunters. To avoid the arrival of the agent to the\nconsumer, one of the main barriers of protection are the carcasses post-mortem\ninspection at the slaughterhouse, to detect and confiscate organs with lesions compatible with tuberculosis or even the hole animal. Inspection is performed visually\nand by palpation of the bovine organ, but due to the pathogenesis of the\nMycobacterium, only the chronic lesions can be evidenced in this inspection. The Mycobacterium might be present even in tissues without apparent lesion, being the lymph nodes and the lungs, the most affected organs: Other parenchymal organs such as the liver could also be affected. This can lead to escapes of the surveillance system\nallowing the Mycobacteria to reach the stores and hence, arrive to the consumer.\nIn this study, 210 lungs along with the corresponding lymph nodes and 6 bovine livers were collected, in one slaughterhouse and 6 butchers´ shops in the city of Buenos Aires and the great Buenos Aires area. The lungs collected in the slaughterhouse were\ninspected in situ and small portions of them were taken. Complete lungs and livers that were bought in butchers´ shops and were then inspected in the laboratory.\nA total of 5 isolates of M. bovis were obtained in Stonebrink medium and its identity confirmed by PCR IS-6110 (isolates number 134, 719, 179, 182 and 184). Additionally\n12 non-tuberculous mycobacteria were isolated and confirmed by the sequencing of the hsp65, rpoB and 16S rRNA genes. The isolates of M. bovis were molecularly\ncharacterized by Spoligotyping and MIRU-VNTR. We could differentiate 3\nSpoligotyping patterns that corresponded with the 3 patterns of MIRU-VNTR obtained. Three isolates shared both patterns and the other 2 were unique. Additionally, drug\nresistance to human treatment commonly used drugs (rifampicin, isoniazid,\nethionamide, levofloxacin and streptomycin) was investigated in all M. bovis isolates, and it was found that isolate number 184 was resistant to isoniazid and isolate number 134 was multi-resistant (showing resistance to the 4 antibiotics tested and considered "border" for levofloxacin). One non-tuberculous mycobacteria isolates from which good\nquality DNA was obtained was completely sequenced (whole genome equencing).\nThe genome was annotated and its characteristics were investigated as well as the \npresence of single nucleotide polymorphism (SNPs) in virulence genes and genes\nrelated to resistance to antibiotics and antigens used for diagnosis.\nThe virulence and transmissibility of isolates number 134 and 182 were evaluated together with a reference strain M. bovis-AN5 (considered of intermediate virulence), in\nan experimental assay in a mouse model. A total of 99 BALB/c female mice aged 5\nweeks, were separated in 3 groups of 33 animals each. In turn, each group was divided into 6 cages, the first cage with 3 animals and the remaining cages with 6 animals. All animals in cage number 1 of each group and 3 from each of the remaining cages, were intratracheally inoculated under sedation with isoflurane, with 1x105 CFU of each of the strains to be evaluated, suspended in 100 ?L of PBS, keeping the relationship between\ninoculated animal and healthy contact 1:1. Virulence was evaluated in those animals that were inoculated, while in the healthy contacts that were living with the infected\nanimals the transmissibility of the strains was investigated. Euthanasia of 5 infected animals and 5 healthy contact animals of each group were performed at times 30, 60 and 90 days post-inoculation. The lungs and spleens of all infected animals and healthy contacts were cultured. The cultures were grown at 37°C for up to 2 months, with weekly evaluation of their growth. Spleens were further processed to measure the\ncytokines IL-4, TNF? and INF? with the use of a commercial Cytometric bead array kit. Strain number 134 showed to be virulent because it produced symptoms incompatible\nwith life on day 16 after inoculation, producing the death of 9 animals, the remaining 6 were euthanatized and processed. Strain number 182 generated spleen enlargement in the infected mice, but it did not generate the illness or death of any of them. The transmissibility of strain 182 could be evidenced, due to the observation of the growth\nof colonies from a spleen of a contact animal in the time of euthanasia 90 days after\ninoculation It was possible to detect the presence of both, zoonotic and environmental\nmycobacteria, in commercialized viscera. The virulence, transmissibility and drugresistance of the M. bovis strains was variable, requiring future studies for a better understanding of the dynamics of the disease. Fil: Marfil, María Jimena. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, Argentina Facultad de Ciencias Veterinarias Zumárraga, Martín José Marfil, María Jimena 2019-03-29 La tuberculosis bovina, causada por Mycobacterium bovis (M. bovis), es endémica en la Argentina. Su presencia en el ganado bovino representa un riesgo para la salud humana debido a su carácter zoonótico, pudiendo además afectar a diversos\nmamíferos. En el hombre, M. bovis causa enfermedad con presentación pulmonar o extrapulmonar siendo la clínica indistinguible de la producida por Mycobacterium\ntuberculosis. Los más expuestos son los niños que consumen leche o sus derivados sin pasteurizar, los adultos relacionados con la actividad laboral pecuaria y también los cazadores. Para evitar la llegada del agente al consumidor, una de las últimas barreras\nde protección es la inspección bromatológica en el frigorífico para poder detectar lesiones compatibles con tuberculosis y decomisar los órganos afectados y si está\ngeneralizado la res completa. Se realiza visualmente y por palpación, pero debido a la\npatogenia de la tuberculosis, sólo las lesiones crónicas pueden ser evidenciadas por este monitoreo. La micobacteria puede estar presente incluso en tejidos sin lesión aparente, siendo los principales órganos afectados los linfonódulos y los pulmones, pudiéndose encontrar en otros órganos parenquimatosos como el hígado. Esto puede\nconducir a escapes del sistema de vigilancia permitiendo que las micobacterias lleguen a los comercios y posteriormente al consumidor. En este trabajo fueron recolectados 210 pulmones junto con los linfonódulos orrespondientes y 6 hígados bovinos, provenientes de un frigorífico y de 6 carnicerías\nde la Ciudad Autónoma de Buenos Aires y de la provincia de Buenos Aires. Los pulmones recolectados en el frigorífico fueron inspeccionados in situ y se tomaron muestras del órgano para el trabajo en el laboratorio. Los pulmones e hígados que fueron comprados completos en carnicerías y fueron inspeccionados en el laboratorio. Se obtuvieron un total de 5 aislamientos de M. bovis en medio Stonebrink confirmados\npor PCR IS-6110 (aislamientos N° 134, 719, 179, 182 y 184) y además 12 aislamientos de micobacterias no tuberculosas confirmadas por la secuenciación de\nlos genes hsp65, rpoB, 16S ARNr y secuenciación genómica de alto rendimiento para\nuno de ellos. Los aislamientos de M. bovis fueron caracterizados molecularmente por\nSpoligotyping y MIRU-VNTR. Se pudieron distinguir 3 patrones de Spoligotyping que\nse correspondieron con 3 patrones de MIRU-VNTR, 3 aislamientos compartieron\nambos patrones y los otros 2 fueron únicos. Adicionalmente, se investigó en 3 de los\naislamientos su capacidad de resistencia a drogas utilizadas en el tratamiento de la tuberculosis en humanos (rifampicina, isoniazida, etionamida, levofloxacina y\nestreptomicina), encontrándose que el aislamiento 184 era resistente a isoniazida y el\naislamiento 134 era multirresistente (presentando resistencia a los 4 antibióticos probados y considerado ?border? para levofloxacina). Se realizó la secuenciación completa del genoma de uno de los aislamientos identificado como M. kansasii\nempleando una plataforma Illumina mySeq. El genoma fue anotado y se describió sus características así como también se evaluó la presencia de single nucleotide polymorphism (SNPs) en genes de virulencia y genes relacionados a la resistencia a antibióticos y codificantes de antígenos empleados para el diagnóstico de tuberculosis. Se evaluó la virulencia y transmisibilidad de los aislamientos N° 134 y 182 junto con una cepa de referencia M. bovis AN5 (considerada de virulencia intermedia), en un\nmodelo murino. Los animales fueron inoculados intratraquealmente, con 1x105 UFC de cada una de las cepas a evaluar, y dichos animales se pusieron en contacto con ratones sin inocular en una relación 1:1. Se evaluó la virulencia en los animales inoculados, mientras que en los contactos sanos, se evaluó la transmisibilidad de las\ncepas. Se realizó la eutanasia de 5 animales infectados y 5 animales contactos sanos\nde cada grupo, en los tiempos 30, 60 y 90 días post-inoculación. Se cultivaron los pulmones y bazos de todos los animales infectados y los contactos sanos en los medios apropiados, evaluando su crecimiento semanalmente. Adicionalmente, los\nbazos fueron procesados para cuantificar las citoquinas IL-4, TNF ? e INF? por citometría de flujo mediante el kit comercial Cytometric bead array. El aislamiento N° 134 demostró ser virulento por producir sintomatología incompatible con la vida al día 16 posterior a la inoculación, produciendo la muerte de 9 animales, los 6 restantes fueron eutanasiados. El aislamiento N° 182 generó esplenomegalia en los ratones infectados, pero no se observó el desmejoramiento ni la muerte de los animales.\nAdemás, se pudo describir la transmisibilidad del aislamiento 182, mediante el\naislamiento de la micobacteria a partir de bazo de uno de los animales ?contacto?. A partir del presente trabajo se pudo detectar la presencia de micobacterias tanto\nzoonóticas como ambientales en vísceras comercializadas. La virulencia,\ntransmisibilidad y la resistencia a antibióticos de cada una de ellas fue variable, requiriéndose de estudios futuros para una mejor comprensión del impacto de estas\nobservaciones en la dinámica de la enfermedad tanto en el ámbito veterinario como en\nSalud pública.\n application/pdf Mycobacterium bovis Spoligotyping MIRU-VNTR Resistencia a los antibióticos PCR spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-ncnd/2.5/ar/ Bovinos Tuberculosis Mycobacterium bovis Cepas Micobacterias Transmisión Comercialización Microbiología Tuberculosis bovina : transmisibilidad de cepas de Mycobacterium bovis y detección de micobacterias en menudencias comercializadas en bocas de expendio de carne 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_3133 http://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_3133.dir/3133.PDF |