Universidad de Buenos Aires Facultad de Ciencias Veterinarias ...
Trichinellosis is a zoonotic disease transmitted by the consumption of foods infected with Trichinella spp. In Argentina the disease is endemic and mainly transmitted by pigs, reemerging in the period 1990/2005. The diagnosis can be made through direct and indirect methods. The artificial digestion...
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Universidad de Buenos Aires. Facultad de Ciencias Veterinarias
2024
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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_7380 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7380.dir/7380.PDF |
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I28-R145-HWA_7380 |
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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) |
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Español |
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Trincchinella spp Diagnostico PCR en tiempo real Tolerancia a la congelación Serologia Trichinella spp Diagnosis Real time PCR Freezing tolerance Serology Trinchinella Jabalies Ciencias Veterinarias |
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Trincchinella spp Diagnostico PCR en tiempo real Tolerancia a la congelación Serologia Trichinella spp Diagnosis Real time PCR Freezing tolerance Serology Trinchinella Jabalies Ciencias Veterinarias Bessi, Clara Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... |
| topic_facet |
Trincchinella spp Diagnostico PCR en tiempo real Tolerancia a la congelación Serologia Trichinella spp Diagnosis Real time PCR Freezing tolerance Serology Trinchinella Jabalies Ciencias Veterinarias |
| description |
Trichinellosis is a zoonotic disease transmitted by the consumption of foods infected with Trichinella spp. In Argentina the disease is endemic and mainly transmitted by pigs, reemerging in the period 1990/2005. The diagnosis can be made through direct and indirect methods. The artificial digestion and molecular techniques are the method of \nchoice for the direct observation of the parasite. While the indirect ones detect the host's \nresponse to the parasite, particularly in pigs, the ELISA technique is used and it is confirmed by means of a Western Blot. These methodologies are carried out either to develop prevalence studies or direct diagnoses of this disease. The aim of this work was to study the acute and chronic infection of the disease, the species susceptibility and the \nimmune response of wild boars experimentally infected with T. patagoniensis and T.\npseudospiralis. As well as to evaluate the detection capacity of newborn larvae during the window period in sera from wild boars and pigs infected with T. patagoniensis, T. \npseudospiralis and T. spiralis by means of the real-time PCR technique. For this study, \n17 wild boars (Sus scrofa) and 6 pigs (Sus scrofa domestica) were used. The animals were inoculated with 20,000 larvae of T. patagoniensis (5 wild boars and 1 pig), with \n20,000 larvae of T. pseudospiralis (5 wild boars and 2 pigs), and with 20,000 larvae of T. spiralis (4 wild boars and 2 pigs). These animals were divided into groups based on \ntheir animal species and the Trichinella species inoculated. The rest of the animals were kept as control (3 wild boars and 1 pig). These animals were checked periodically, and during the acute phase of the disease they were weighed weekly. Blood samples were taken weekly throughout the study. These samples were used for various purposes. On one hand, this was used to evaluate the relative eosinophil count in wild boars infected with T. patagoniensis and T. pseudospiralis, by means of blood smears. Moreover, the \nsera obtained from the blood samples of wild boars infected with T. patagoniensis and \nT. pseudospiralis was also used to determine the antibody titer using the ELISA technique, applying a commercial kit named PrioCHECK Trichinella Ab. On the other hand, the sera of all infected animals was used to evaluate the presence of newborn larvae (NBL) using the real-time PCR technique. This was measured in wild boars serum at 7- and 14-days post infection (d.p.i.), in pigs infected with T. spiralis and T. \npseudospiralis at 2, 5, 9, 12 and 15 d.p.i., and in pigs infected with T. patagoniensis at \n2, 6, 10, 12 and 15 d.p.i. In order to evaluate the distribution pattern of the muscle larvae, \nsamples of muscles of parasitological and commercial interest were taken during the \nnecropsy of wild boars infected with T. patagoniensis and T. pseudospiralis. These \nsamples were: tongue, diaphragm, masseter, intercostals, muscular portion of the esophagus, upper forelegs, upper hind limbs, pork shoulders and sirloin. These were analyzed using the artificial digestion technique. At the same time, samples of pork shoulders, upper foreleg and upper hind limbs were also taken from wild boars infected with T. pseudospiralis to evaluate the tolerance of muscle larvae (ML) to freezing. These muscles were subjected to temperatures of -18ºC for 14 days. The sampling dates were 2, 4, 7, 9, 11 and 14 days, and on each date a sample of each muscle was digested, by artificial digestion, in order to recover the ML there, and then inoculate them in BALB/c mice to determine the Reproductive Capacity Index (ICR). No animal presented clinical signs related to this disease. During the acute phase, no differences in weight gain were evident between infected wild boars and controls. An increase in the relative eosinophil count was seen immediately after infection in wild boars infected with T. pseudospiralis (1 week p.i.) and at week 2 p.i. in wild boars infected with T. patagoniensis. Regarding antibody dynamics, wild boars inoculated with T. patagoniensis sero-converted between \n2 4 weeks p.i., while wild boars inoculated with T. pseudospiralis at 2 weeks p.i. All animals, except controls, remained above the cut-off value throughout the experience (19 weeks). Statistically significant differences were observed in optical density (OD) values between wild boars inoculated with T. pseudospiralis and those inoculated with T. patagoniensis at 2 weeks p.i. (P<0.050). The early detection of NBL showed variable results depending on the animal species and the Trichinella species under study. \nSamples taken at 7 d.p.i. of wild boars infected with T. spiralis were positive when \nanalyzed with the Rep primer. These positive samples presented a Ct range of 29.26 ± \n0.16 for one sample and up to 33.37 ± 0.45 for another. and the average Ct (of the \npositive samples) was 31.91 ± 1.70. At 14 d.p.i. NBL DNA was not detected in the \nanalyzed sera. Regarding pigs infected with T. spiralis, the Rep primer failed to detect circulating DNA in serum on any sampled date. The 18S primer failed to detect NBL DNA in wild boars infected with T. pseudospiralis or T. patagoniensis on the sampled dates (7 \nand 14 d.p.i.). With this primer, DNA was only detected in a sample at 7 d.p.i. in a single \nwild boar infected with T. spiralis when 1 l of DNA was used, when increasing to 5 l of DNA the other 3 samples were also detected as positive. The sample made with 1 l of DNA presented an average Ct value of 37.32 ± 0.45, while the samples analyzed with 5 l presented a Ct range of 35.13 ± 1.02 to 36.80 ± 0 .29, with an average Ct of 36.21 ± 1.02. The muscle distribution of the larvae of T. pseudospiralis and T. patagoniensis gave the following results. T. patagoniensis had a larval density considerably lower than T. pseudospiralis. Both Trichinella species showed a predilection mainly for the tongue and diaphragm. Followed by the upper forelegs for T. patagoniensis, and the sirloins and \npork shoulders for T. pseudospiralis. The highest larval loads were: for T. pseudospiralis\n134.3 larvae per gram (lpg) (in the tongue and intercostal muscles) and for T. \npatagoniensis 0.087 lpg (in the tongue). The T. pseudospiralis larvae recovered from the \nmuscles subjected to freezing treatment (-18 ºC) remained viable until 48 hours after being placed in the proposed medium. From day 4 onwards, the RCI value was equal to 0 in all muscle groups studied. These results allow us to evaluate how T. patagoniensis behaves when it infects wild boars. This study allows us to understand how these species develop when they affect wild boars, as well as allowing us to explore their biological capabilities. Using various diagnostic methods not only allowed us to delve into how this disease develops, but also allowed us to begin to build new diagnostic strategies to reach its early diagnosis and treatment or even be able to prevent this disease |
| author2 |
Pasqualetti, Mariana I. |
| author_facet |
Pasqualetti, Mariana I. Bessi, Clara |
| format |
Tesis doctoral Tesis doctoral acceptedVersion |
| author |
Bessi, Clara |
| author_sort |
Bessi, Clara |
| title |
Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... |
| title_short |
Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... |
| title_full |
Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... |
| title_fullStr |
Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... |
| title_full_unstemmed |
Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... |
| title_sort |
universidad de buenos aires facultad de ciencias veterinarias ... |
| publisher |
Universidad de Buenos Aires. Facultad de Ciencias Veterinarias |
| publishDate |
2024 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=avaposgra&cl=CL1&d=HWA_7380 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7380.dir/7380.PDF |
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AT bessiclara universidaddebuenosairesfacultaddecienciasveterinarias AT bessiclara caracterizaciondelainfeccionporespeciessilvestresdetrichinellaenjabaliiessusscrofa |
| _version_ |
1824356464344432640 |
| spelling |
I28-R145-HWA_73802024-08-27 Universidad de Buenos Aires Facultad de Ciencias Veterinarias ... Trichinellosis is a zoonotic disease transmitted by the consumption of foods infected with Trichinella spp. In Argentina the disease is endemic and mainly transmitted by pigs, reemerging in the period 1990/2005. The diagnosis can be made through direct and indirect methods. The artificial digestion and molecular techniques are the method of \nchoice for the direct observation of the parasite. While the indirect ones detect the host's \nresponse to the parasite, particularly in pigs, the ELISA technique is used and it is confirmed by means of a Western Blot. These methodologies are carried out either to develop prevalence studies or direct diagnoses of this disease. The aim of this work was to study the acute and chronic infection of the disease, the species susceptibility and the \nimmune response of wild boars experimentally infected with T. patagoniensis and T.\npseudospiralis. As well as to evaluate the detection capacity of newborn larvae during the window period in sera from wild boars and pigs infected with T. patagoniensis, T. \npseudospiralis and T. spiralis by means of the real-time PCR technique. For this study, \n17 wild boars (Sus scrofa) and 6 pigs (Sus scrofa domestica) were used. The animals were inoculated with 20,000 larvae of T. patagoniensis (5 wild boars and 1 pig), with \n20,000 larvae of T. pseudospiralis (5 wild boars and 2 pigs), and with 20,000 larvae of T. spiralis (4 wild boars and 2 pigs). These animals were divided into groups based on \ntheir animal species and the Trichinella species inoculated. The rest of the animals were kept as control (3 wild boars and 1 pig). These animals were checked periodically, and during the acute phase of the disease they were weighed weekly. Blood samples were taken weekly throughout the study. These samples were used for various purposes. On one hand, this was used to evaluate the relative eosinophil count in wild boars infected with T. patagoniensis and T. pseudospiralis, by means of blood smears. Moreover, the \nsera obtained from the blood samples of wild boars infected with T. patagoniensis and \nT. pseudospiralis was also used to determine the antibody titer using the ELISA technique, applying a commercial kit named PrioCHECK Trichinella Ab. On the other hand, the sera of all infected animals was used to evaluate the presence of newborn larvae (NBL) using the real-time PCR technique. This was measured in wild boars serum at 7- and 14-days post infection (d.p.i.), in pigs infected with T. spiralis and T. \npseudospiralis at 2, 5, 9, 12 and 15 d.p.i., and in pigs infected with T. patagoniensis at \n2, 6, 10, 12 and 15 d.p.i. In order to evaluate the distribution pattern of the muscle larvae, \nsamples of muscles of parasitological and commercial interest were taken during the \nnecropsy of wild boars infected with T. patagoniensis and T. pseudospiralis. These \nsamples were: tongue, diaphragm, masseter, intercostals, muscular portion of the esophagus, upper forelegs, upper hind limbs, pork shoulders and sirloin. These were analyzed using the artificial digestion technique. At the same time, samples of pork shoulders, upper foreleg and upper hind limbs were also taken from wild boars infected with T. pseudospiralis to evaluate the tolerance of muscle larvae (ML) to freezing. These muscles were subjected to temperatures of -18ºC for 14 days. The sampling dates were 2, 4, 7, 9, 11 and 14 days, and on each date a sample of each muscle was digested, by artificial digestion, in order to recover the ML there, and then inoculate them in BALB/c mice to determine the Reproductive Capacity Index (ICR). No animal presented clinical signs related to this disease. During the acute phase, no differences in weight gain were evident between infected wild boars and controls. An increase in the relative eosinophil count was seen immediately after infection in wild boars infected with T. pseudospiralis (1 week p.i.) and at week 2 p.i. in wild boars infected with T. patagoniensis. Regarding antibody dynamics, wild boars inoculated with T. patagoniensis sero-converted between \n2 4 weeks p.i., while wild boars inoculated with T. pseudospiralis at 2 weeks p.i. All animals, except controls, remained above the cut-off value throughout the experience (19 weeks). Statistically significant differences were observed in optical density (OD) values between wild boars inoculated with T. pseudospiralis and those inoculated with T. patagoniensis at 2 weeks p.i. (P<0.050). The early detection of NBL showed variable results depending on the animal species and the Trichinella species under study. \nSamples taken at 7 d.p.i. of wild boars infected with T. spiralis were positive when \nanalyzed with the Rep primer. These positive samples presented a Ct range of 29.26 ± \n0.16 for one sample and up to 33.37 ± 0.45 for another. and the average Ct (of the \npositive samples) was 31.91 ± 1.70. At 14 d.p.i. NBL DNA was not detected in the \nanalyzed sera. Regarding pigs infected with T. spiralis, the Rep primer failed to detect circulating DNA in serum on any sampled date. The 18S primer failed to detect NBL DNA in wild boars infected with T. pseudospiralis or T. patagoniensis on the sampled dates (7 \nand 14 d.p.i.). With this primer, DNA was only detected in a sample at 7 d.p.i. in a single \nwild boar infected with T. spiralis when 1 l of DNA was used, when increasing to 5 l of DNA the other 3 samples were also detected as positive. The sample made with 1 l of DNA presented an average Ct value of 37.32 ± 0.45, while the samples analyzed with 5 l presented a Ct range of 35.13 ± 1.02 to 36.80 ± 0 .29, with an average Ct of 36.21 ± 1.02. The muscle distribution of the larvae of T. pseudospiralis and T. patagoniensis gave the following results. T. patagoniensis had a larval density considerably lower than T. pseudospiralis. Both Trichinella species showed a predilection mainly for the tongue and diaphragm. Followed by the upper forelegs for T. patagoniensis, and the sirloins and \npork shoulders for T. pseudospiralis. The highest larval loads were: for T. pseudospiralis\n134.3 larvae per gram (lpg) (in the tongue and intercostal muscles) and for T. \npatagoniensis 0.087 lpg (in the tongue). The T. pseudospiralis larvae recovered from the \nmuscles subjected to freezing treatment (-18 ºC) remained viable until 48 hours after being placed in the proposed medium. From day 4 onwards, the RCI value was equal to 0 in all muscle groups studied. These results allow us to evaluate how T. patagoniensis behaves when it infects wild boars. This study allows us to understand how these species develop when they affect wild boars, as well as allowing us to explore their biological capabilities. Using various diagnostic methods not only allowed us to delve into how this disease develops, but also allowed us to begin to build new diagnostic strategies to reach its early diagnosis and treatment or even be able to prevent this disease Fil: Bessi, Clara. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Buenos Aires, Argentina Pasqualetti, Mariana I. Bessi, Clara 2024-02-28 La trichinellosis es una enfermedad zoonótica transmitida por el consumo de alimentos infectados con Trichinella spp. En Argentina la enfermedad es endémica y \nprincipalmente transmitida por cerdos, reemergente en el período 1990/2005. El \ndiagnóstico de esta parasitosis se puede realizar por medio de métodos directos e indirectos. Dentro de los métodos de observación directa del parásito están la técnica de digestión artificial y técnicas moleculares. Mientras que los indirectos detectan la respuesta del hospedador al parásito, particularmente en cerdos se utiliza la técnica de ELISA y la misma se confirma por medio de un Western Blot. Estas metodologías se \nrealizan ya sea para realizar estudios de prevalencias como de métodos de detección directos de esta enfermedad. El objetivo de este trabajo fue estudiar la infección aguda y crónica de la enfermedad, la susceptibilidad de especie y la respuesta inmune de \njabalíes infectados experimentalmente con T. patagoniensis y T. pseudospiralis. Como \ntambién evaluar la capacidad de detección de larvas recién nacidas durante el período \nventana en sueros de jabalíes y cerdos infectados con T. patagoniensis, T. \npseudospiralis y T. spiralis por medio de la técnica de PCR en tiempo real. Para este estudio se emplearon 17 jabalíes (Sus scrofa) y 6 cerdos (Sus scrofa domestica). Los \nanimales fueron inoculados con 20000 larvas de T. patagoniensis (5 jabalíes y 1 cerdo), con 20000 larvas de T. pseudospiralis (5 jabalíes y 2 cerdos), y con 20000 larvas de T. \nspiralis (4 jabalíes y 2 cerdos), estos animales fueron divididos en grupos en función a su especie animal y a la especie de Trichinella inoculada. Se mantuvieron como control 3 jabalíes y 1 cerdo. Estos animales fueron revisados periódicamente y durante la fase aguda de la enfermedad fueron pesados semanalmente. Se realizaron extracciones de \nsangre cada 7 días durante todo el estudio. Estas muestras de sangre se utilizaron para varios estudios paralelos. Por un lado, esto se utilizó para evaluar el recuento relativo de eosinófilos en los jabalíes infectados con T. patagoniensis y T. pseudospiralis, por \nmedio de frotis sanguíneos. Además, se utilizó el suero obtenido de las muestras de \nsangre de los jabalíes infectados con T. patagoniensis y T. pseudospiralis, para \ndeterminar el título de anticuerpos mediante la técnica de ELISA, utilizándose un kit \ncomercial ¨PrioCHECK Trichinella Ab¨, esto se evaluó hasta el fin de la experiencia. Por otra parte, el suero de todos los animales infectados también fue utilizado para evaluar la presencia de larvas recién nacidas (LRN) a partir de la técnica de PCR en tiempo real. \nEsto se midió en los jabalíes a los 7 y 14 días post infección (d.p.i.), en los cerdos infectados con T. spiralis y T. pseudospiralis a los 2, 5, 9, 12 y 15 d.p.i, y en los cerdos \ninfectados con T. patagoniensis a los 2, 6, 10, 12 y 15 d.p.i. Con el fin de evaluar el patrón de distribución de las larvas musculares se tomaron muestras de músculos de interés parasitológico y comercial durante la necropsia de los jabalíes infectados con T. \npatagoniensis y T. pseudospiralis. Los mismos fueron: lengua, diafragma, maseteros, intercostales, porción muscular del esófago, paletas, jamones, bondiola y solomillo. \nEstos se analizaron por medio de la técnica de digestión artificial. A su vez, también se tomaron muestras de bondiola, jamón y paleta de los jabalíes infectados con T. \npseudospiralis para evaluar la tolerancia de las larvas musculares (LM) a la congelación. \nEstos músculos se sometieron a temperaturas de -18 ºC durante 14 días. Las fechas de muestreo fueron 2, 4, 7, 9, 11 y 14 días, y en cada fecha se digirió una muestra de cada músculo, por medio de la digestión artificial, con el fin de recuperar las larvas allí \npresentes y así luego inocularlas en ratones BALB/c para determinar el Índice de Capacidad Reproductiva (ICR). Ningún animal presentó signos clínicos relacionados con esta parasitosis. Durante la fase aguda de la enfermedad, no se evidenciaron diferencias en la ganancia de peso, entre los jabalíes infectados y los controles. Se evidenció un aumento en el recuento relativo de eosinófilos la primera semana p.i. en \nlos jabalíes infectados con T. pseudospiralis y en la semana 2 p.i. en los jabalíes \ninfectados con T. patagoniensis. Con respecto a la dinámica de anticuerpos, los jabalíes \ninoculados con T. patagoniensis seroconvirtieron entre las 2 4 semanas p.i., mientras \nque los jabalíes inoculados con T. pseudospiralis a las 2 semanas p.i. Todos los \nanimales, excepto los controles, se mantuvieron por encima del valor de corte durante toda la experiencia (19 semanas). Diferencias estadísticamente significativas se \nobservaron en los valores de densidad óptica (OD) entre los jabalíes inoculados con T. \npseudospiralis y los inoculados con T. patagoniensis a las 2 semanas p.i. (P <0.050). La \ndetección temprana de LRN mostró resultados variables en función a la especie animal y la especie de Trichinella bajo estudio. Las muestras tomadas a los 7 d.p.i. de los \njabalíes infectados con T. spiralis fueron positivas al ser analizadas con el cebador Rep. \nEstas muestras positivas presentaron un rango de Ct de 29,26 ± 0,16 para una muestra y de hasta 33,37 ± 0,45 para otra, y el Ct promedio (de las muestras positivas) fue de 31,91 ± 1,70. A los 14 d.p.i. no se detectó ADN de LRN en los sueros analizados. Con respecto a los cerdos infectados con T. spiralis, el cebador Rep no logró detectar ADN circulante en suero en ninguna fecha muestreada. El cebador 18S, no logró detectar ADN de LRN en los jabalíes infectados con T. pseudospiralis, ni T. patagoniensis en las \nfechas muestreadas (7 y 14 d.p.i.). Con este cebador sólo se logró detectar ADN en una muestra a los 7 d.p.i. en un solo jabalí infectado con T. spiralis cuando se utilizó 1 l de ADN, al aumentar a 5 l de ADN se lograron detectar las otras 3 muestras también como positivas. La muestra realizada con 1 l de ADN presentó un valor de Ct promedio de 37,32 ± 0,45, mientras que las muestras analizadas con 5 l presentaron un rango de \nCt de 35,13 ± 1,02 a 36,80 ± 0,29, con un Ct promedio de 36,21 ± 1,02. La distribución \nmuscular de las larvas de T. pseudospiralis y T. patagoniensis arrojaron los siguientes resultados. T. patagoniensis presentó una densidad larvaria considerablemente inferior a T. pseudospiralis. Ambas especies de Trichinella mostraron predilección \nprincipalmente por la lengua y el diafragma. Seguidamente, los músculos más predilectos fueron para T. patagoniensis las paletas, mientras que para T. pseudospiralis los solomillos y bondiolas. Las cargas larvarias más altas fueron: para T. pseudospiralis\n134,3 larvas por gramo (lpg) (en lengua y músculos intercostales) y para T. \npatagoniensis de 0,087 lpg (en la lengua). Las larvas de T. pseudospiralis recuperadas de los músculos sometidos al tratamiento de congelación (-18 ºC) se mantuvieron viables hasta las 48 h posteriores de ser colocadas en el medio propuesto. A partir del día 4 el valor del RCI fue igual a 0 en todos los grupos musculares estudiados. Este \nestudio nos permite comprender cómo se desarrollan estas especies cuando afectan a \njabalíes, como también nos permitieron ahondar en sus capacidades biológicas. Utilizar \ndiversos métodos diagnósticos no sólo nos permitió evaluar cómo se desarrolla esta \nenfermedad, sino también nos permitió comenzar a construir nuevas estrategias \ndiagnósticas para tratar de prevenir esta parasitosis o incluso poder llegar a su \ndiagnóstico y tratamiento precoz. application/pdf Trincchinella spp Diagnostico PCR en tiempo real Tolerancia a la congelación Serologia Trichinella spp Diagnosis Real time PCR Freezing tolerance Serology spa Universidad de Buenos Aires. Facultad de Ciencias Veterinarias info:eu-repo/semantics/openAccess htpps://creativecommons.org/licenses/by-nend/2.5/ar/ Trinchinella Jabalies Ciencias Veterinarias Doctora de Universidad de Buenos Aires en Ciencias Veterinarias Caracterización de la infección por especies silvestres de Trichinella en jabaliíes (Sus scrofa) 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_7380 https://repositoriouba.sisbi.uba.ar/gsdl/collect/avaposgra/index/assoc/HWA_7380.dir/7380.PDF |