Publication: Advances in non-invasive biomarkers of health and welfare in swine
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Date
2025-12-19
Authors
Ortín Bustillo, Alba
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Escuela Internacional de Doctorado
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Martínez Subiela, Silvia ; Muñoz Prieto, Alberto ; Tvarijonaviciute, Asta
Publisher
Universidad de Murcia
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DOI
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info:eu-repo/semantics/doctoralThesis
Description
Tesis por compendio de publicaciones
Abstract
La importancia de la saliva como fuente de biomarcadores para evaluar la salud y el bienestar ha crecido en los últimos años. Los objetivos del doctorado fueron: (1) la validación de nuevos métodos para medir biomarcadores en saliva porcina; (2) la evaluación del efecto de factores pre-analíticos en biomarcadores salivales seleccionados; (3) la evaluación de la respuesta de biomarcadores salivales seleccionados a patologías comunes y su tratamiento.Para ello, este doctorado se dividió en dos partes principales:La primera parte se centra en el desarrollo y la validación de nuevos ensayos para la medición de zinc, cobre, hierro y ferritina utilizando kits comerciales en un analizador bioquímico automatizado (Artículo no1), ITIH4 (también conocido como Pig-Map) mediante un kit ELISA comercial (Artículo no9), y haptoglobina, proteína C reactiva y S100A12 mediante tecnología AlphaLISA (Artículos no7 y no8). Estos nuevos ensayos mostraron una adecuada precisión, exactitud y alta sensibilidad para sus mediciones en saliva porcina y se aplicaron en diferentes condiciones fisiológicas y patológicas. El zinc y la ferritina mostraron una reducción significativa en sus niveles en cerdos no suplementados con hierro (Artículo no1). Los niveles de S100A12 aumentaron en la saliva de lechones con diarrea por Escherichia coli (Artículo no7) y también aumentaron los niveles de ITIH4 en cerdos con mordedura de cola (Artículo no9). Además, la haptoglobina y la proteína C reactiva aumentaron en la saliva de lechones con meningitis por Streptococcus suis y disminuyeron en lechones con respuesta adecuada al tratamiento (Artículo no8). Otros biomarcadores salivales relacionados con el estrés, el estado redox, la inflamación y el sistema inmunitario mostraron cambios en estas situaciones.La segunda parte aborda la evaluación del efecto de factores pre-analíticos en la determinación de biomarcadores salivales seleccionados. En esta parte se estudiaron cuatro aspectos principales:Se estudió la influencia de la edad, el sexo y algunos parámetros productivos en cerdos de engorde; Se encontró que la mayoría de los analitos salivales mostraron cambios con la edad y algunos con el género (Artículos no2 y no5).El efecto del muestreo a diferentes horas del día en la medición de biomarcadores salivales. Muchos de los analitos mostraron cambios entre las tomas, lo que resalta la importancia de considerar el horario de muestreo en la recolección de muestras de saliva en estudios experimentales y para la monitorización del estado de salud (Artículos no3 y no5).Los efectos de la contaminación por heces de corral y pienso en los biomarcadores salivales, y si los tratamientos de centrifugación, filtración y clarificación podrían revertir los efectos de estos contaminantes en la evaluación de biomarcadores. En este punto, se observaron diversos resultados para cada analito y, en términos generales, la centrifugación fue el tratamiento con los mejores resultados para la eliminación de contaminantes (Artículos no4 y no5).El material utilizado para el muestreo de saliva. Para ello, se compararon diferentes materiales de recolección para estudiar si podían afectar de forma diferente los niveles de biomarcadores salivales. El material utilizado para la recolección de saliva puede influir en el volumen de saliva obtenido y podría influir en algunos biomarcadores; por lo tanto, sería deseable utilizar el mismo tipo de material de recolección durante el mismo estudio (Artículo no6).En general, la tesis proporciona información sobre nuevos biomarcadores y ensayos que pueden aplicarse en saliva, los factores pre-analíticos que pueden generar variaciones en los resultados y sus posibles aplicaciones como biomarcadores de enfermedades. Se espera que contribuya a una mejor monitorización de la salud y el bienestar de los cerdos y al conocimiento para futuras aplicaciones en este campo
The importance of the saliva as a source of biomarkers to evaluate health and welfare is growing in the last years. The objectives of the PhD were: (1) validation new methods to measure biomarkers in porcine saliva; (2) evaluation of the effect of pre-analytical factors on selected salivary biomarkers; (3) evaluation of the response of selected salivary biomarkers to naturally occurring pathologies and their treatment.For this purpose there were two main parts in this PhD:The first part is focused on the development and validation of new assays for the measurement of zinc, copper, iron, and ferritin using commercial kits on automated biochemistry analyser (Article no1), ITIH4 (also known as Pig-Map) by a commercial ELISA kit (Article no9), and haptoglobin, C reactive protein and S100A12 by AlphaLISA technology. (Articles no7 and no8).These new assays showed an adequate precision, accuracy, and high sensitivity for their measurements in porcine saliva and were also applied in different physiological and pathological conditions. Zinc and ferritin showed significant reduction in their levels in non-supplemented pigs with iron (Article no1). S100A12 levels were increased in saliva of piglets with diarrhea by Escherichia. coli (Article no7) and also ITIH4 in pigs that suffered tail biting (Article no9). Moreover, haptoglobin and C reactive protein were increased in saliva of piglets suffering from meningitis by Streptococcus suis infection and decrease in piglets responded adequately to the treatment (Article no8). In addition, other salivary biomarkers related stress, redox status, inflammation and immune system showed changes in these situations.The second part deals with the assessment of the effect of pre-analytical factors on selected salivary biomarker determinations. In this part four main aspects were studied:The influence of age, gender and some productive parameters were studied in fattening pigs; where it was found that most of the salivary analytes showed changes with age and some of them with gender (Articles no2 and no5).The effect of sampling at different times of the day in salivary biomarker measurements. Many of the analytes showed changes between sampling times raising the importance of consider the sample time in the saliva sample collection in experimental studies and for health monitoring (Articles no3 and no5).The effects of pen faeces and feed contamination in salivary biomarkers, and if treatments of centrifugation, filtration and clarification could revert the effects of those contaminants in biomarker evaluation. At this point, diverse results were observed in each analyte and in general terms centrifugation was the treatment with the best results obtained in order to remove contaminants (Articles no4 and no5).The material used for sampling saliva. For this purpose, different collection materials were compared in order to study if they could differently affect the levels of salivary biomarkers. The material used for saliva collection can influence the volume of saliva obtained and could have influence in some biomarkers; hence the use of the same collection material type would be desirable during the same study (Article no6).Overall, the thesis provides information about new biomarkers and assays that can be applied in saliva, the pre-analytical factors that can provide variations in the results and potential applications as biomarkers of diseases. It is expected that will contribute to a better monitoring of health and welfare in swine and to knowledge for further applications in this field.
The importance of the saliva as a source of biomarkers to evaluate health and welfare is growing in the last years. The objectives of the PhD were: (1) validation new methods to measure biomarkers in porcine saliva; (2) evaluation of the effect of pre-analytical factors on selected salivary biomarkers; (3) evaluation of the response of selected salivary biomarkers to naturally occurring pathologies and their treatment.For this purpose there were two main parts in this PhD:The first part is focused on the development and validation of new assays for the measurement of zinc, copper, iron, and ferritin using commercial kits on automated biochemistry analyser (Article no1), ITIH4 (also known as Pig-Map) by a commercial ELISA kit (Article no9), and haptoglobin, C reactive protein and S100A12 by AlphaLISA technology. (Articles no7 and no8).These new assays showed an adequate precision, accuracy, and high sensitivity for their measurements in porcine saliva and were also applied in different physiological and pathological conditions. Zinc and ferritin showed significant reduction in their levels in non-supplemented pigs with iron (Article no1). S100A12 levels were increased in saliva of piglets with diarrhea by Escherichia. coli (Article no7) and also ITIH4 in pigs that suffered tail biting (Article no9). Moreover, haptoglobin and C reactive protein were increased in saliva of piglets suffering from meningitis by Streptococcus suis infection and decrease in piglets responded adequately to the treatment (Article no8). In addition, other salivary biomarkers related stress, redox status, inflammation and immune system showed changes in these situations.The second part deals with the assessment of the effect of pre-analytical factors on selected salivary biomarker determinations. In this part four main aspects were studied:The influence of age, gender and some productive parameters were studied in fattening pigs; where it was found that most of the salivary analytes showed changes with age and some of them with gender (Articles no2 and no5).The effect of sampling at different times of the day in salivary biomarker measurements. Many of the analytes showed changes between sampling times raising the importance of consider the sample time in the saliva sample collection in experimental studies and for health monitoring (Articles no3 and no5).The effects of pen faeces and feed contamination in salivary biomarkers, and if treatments of centrifugation, filtration and clarification could revert the effects of those contaminants in biomarker evaluation. At this point, diverse results were observed in each analyte and in general terms centrifugation was the treatment with the best results obtained in order to remove contaminants (Articles no4 and no5).The material used for sampling saliva. For this purpose, different collection materials were compared in order to study if they could differently affect the levels of salivary biomarkers. The material used for saliva collection can influence the volume of saliva obtained and could have influence in some biomarkers; hence the use of the same collection material type would be desirable during the same study (Article no6).Overall, the thesis provides information about new biomarkers and assays that can be applied in saliva, the pre-analytical factors that can provide variations in the results and potential applications as biomarkers of diseases. It is expected that will contribute to a better monitoring of health and welfare in swine and to knowledge for further applications in this field.
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