Efectos tóxicos de la zearalenona y sus metabolitos en líneas celulares

La zearalenona (ZEA) y sus metabolitos α-zearalenol (α-ZOL) y β-zearalenol (β-ZOL), son metabolitos secundarios de hongos del genero Fusarium. La ZEA se absorbe y se metaboliza a α-ZOL y β-ZOL. Tanto la ZEA como sus metabolitos producen sus efectos tóxicos por la interacción con los receptores endocrinos aunque no es el único mecanismo por el cual pueden producir toxicidad. Se sabe que, además de disruptores endocrinos la ZEA y sus metabolitos son inmunotóxicos, hepatotóxicos y hematotóxicos causando alteraciones en el sistema reproductivo. La citotoxicidad de la ZEA y sus metabolitos ha sido determinada por distintos tiempos de exposición en diferentes células de mamíferos obteniéndose diferentes valores de IC50. Se investigó la conversión de ZEA y sus metabolitos en estos u otros derivados de ZEA por células CHO-K1 y HepG2 observando diferentes derivados de la ZEA y sus metabolitos. La exposición a estas micotoxinas causa estrés oxidativo observándose un aumento de las especies reactivas de oxigeno (ROS) que es proporcional al tiempo de exposición y la concentración de las micotoxinas. Se ha visto que este aumento de ROS conduce a una alteración de las vías metabólicas, disminución de los antioxidantes celulares y provoca que se oxiden estructuras macromoleculares, entre las que se encuentran las proteínas y lípidos de las membranas celulares y el ADN. Se evaluó la actividad estrogénica de la ZEA y sus metabolitos usando células de cáncer de mama humano (MCF-7) y se observó un aumento de esta actividad siendo el α-ZOL el que producía un mayor aumento de la actividad estrogénica. Se determinó si la ZEA y sus metabolitos activan los mecanismos de defensas antioxidantes en las células CHO-K1 y HepG2. Para ello, se determinó la actividad del glutatión (GSH) y de las enzimas catalasa (CAT), superóxido dismutasa (SOD) y glutatión peroxidasa (GPx) tras exponer las células durante 24 h en las células CHO-K1 y HepG2. Los resultados mostraron una disminución de los niveles de GSH y de la actividad de la catalasa. Mientras que se evidenció un aumento en la actividad de la SOD y GPx. Todos estos efectos tóxicos pueden conducir a un riesgo en la salud humana.Zearalenone (ZEA) and its metabolites α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL) are secondary metabolites of Fusarium fungy. ZEA is absorbed and metabolized to α-ZOL and β-ZOL. Boths ZEA and its metabolites produce their toxic effects through interactions with endocrine receptors, although it is not the only mechanism by which they can produce toxicity. It is known that in addition to endocrine disruptors ZEA and its metabolites are immunotoxic, hepatotoxic and hematotoxic causing alterations in the reproductive system. The cytotoxicity of ZEA and its metabolites has been determined by different exposure times in different mammalian cells obtaining different IC50 values. It was investigated the conversion of ZEA and its metabolites into these or other ZEA derivatives in CHO-K1 and HepG2 cells by observing different derivates of ZEA and its metabolites. The exposure to these mycotoxins causes oxidative stress observing an increase of the reactive oxygen species (ROS) that is proportional to the time of exposure and the concentration of the mycotoxins. It is seen that this growth of ROS leads to an alteration of metabolic pathways, a decrease in cellular antioxidants and causes oxidation of macromolecular structures, among which are the proteins and lipids of cell membranes and DNA. The estrogenic activity of ZEA and its metabolites was evaluated using human breast cancer cells (MCF-7) and an increase of this activity was observed. α-ZOL was the one that produced a greater increase of the estrogenic activity. It was determined if ZEA and its metabolites activate the mechanisms of antioxidant defenses in CHO-K1 and HepG2 cells. For this purpose, the activity of glutathione (GSH) and catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were determined after exposed the cells for 24 h in CHO-K1 and HepG2 cells. The results showed a decrease in GSH levels and a decrease in catalase activity. While an increase in the activity of SOD and GPx was evidenced. All these toxic effects can lead to a risk to human health

The moderate drift towards less tetracycline-susceptible isolates of contagious agalactia causative agents might result from different molecular mechanisms.

©2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted version of a Published Work that appeared in final form in Veterinary Microbiology. To access the final edited and published work see https://doi.org/10.1016/j.vetmic.2018.05.001Contagious agalactia is a mycoplasmosis that affects small ruminants, is associated with loss of milk production and high morbidity rates, and is highly deleterious to dairy industries. The etiological agents are four mycoplasma (sub)species, of which the relative importance depends on the countries and the animal host. Tetracyclines are non 23 expensive, broad-spectrum antimicrobials and are often used to control mastitis in dairy herds. However, the in vitro efficiency of tetracyclines against each of the etiological agents of contagious agalactia has been poorly assessed. The aims of this study were i) to compare the tetracycline susceptibilities of various field isolates, belonging to different mycoplasma (sub)species and subtypes, collected over the years from different clinical contexts in France or Spain, and ii) to investigate the molecular mechanisms behind the decreased susceptibility of some isolates to tetracyclines. The Minimum Inhibitory Concentrations (MICs) of tetracyclines were determined in vitro on a set of 120 isolates. Statistical analyses were run to define the significance of any observed differences in MICs distribution. As mutations in the genes encoding the ttracycline targets (rrs loci) are most often associated with increased tetracycline MICs in animal mycoplasmas, these genes were sequenced. The loss of susceptibility to tetracyclines after year 2010 is not significant and recent MICs are higher in M. agalactiae, especially isolates from ovine mastitis cases, than in other etiological agents of contagious agalactia. The observed increases in MICs were not always associated with mutations in the rrs alleles which suggests the existence of other resistance mechanisms yet to be decipher

23S rRNA and L22 ribosomal protein are involved in the acquisition of macrolide and lincosamide resistance in Mycoplasma capricolum subsp. capricolum

© 2018. Elsevier. This document is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted version of a Published Work that appeared in final form in Veterinary Microbiology. To access the final edited and published work see https://doi.org/10.1016/j.vetmic.2018.02.014Mycoplasma capricolum subsp. capricolum (Mcc) is one of the causative agents of contagious agalactia, and antimicrobial treatment is the most commonly applied measure to treat outbreaks of this disease. Macrolides and lincosamides bind specifically to nucleotides at domains II and V of the 23S rRNA gene. Furthermore, rplD and rplV genes encode ribosomal proteins L4 and L22, which are also implicated in the macrolide binding site. The aim of this work was to study the relationship between these genes and the acquisition of macrolide and lincosamide resistance in Mcc. For this purpose, in vitro selected resistant mutants and field isolates were studied. This study demonstrates the appearance of DNA point mutations at the 23S rRNA encoding genes (A2058G, A2059G and A2062C) and rplV gene (Ala89Asp) in association to high minimum inhibitory concentration values. Hence, it proves the importance of 23S rRNA domain V and ribosomal protein L22 as molecular mechanisms responsible for the acquisition of macrolide and lincosamide resistance in both field isolates and in vitro selected mutants. Furthermore, these mutations enable us to provide an interpretative breakpoint of antimicrobial resistance for Mcc at MIC 0.8 µg/ml

Efflux Might Participate in Decreased Susceptibility to Oxytetracycline in Contagious Agalactia-Causative Mycoplasma spp.

Contagious agalactia is associated with mastitis, keratoconjunctivitis, arthritis, pneumonia, and septicemia in small ruminants in countries with large dairy industries worldwide. The causative agents belong to four (sub)species of the Mycoplasma genus that have remained essentially susceptible to antimicrobials, including to the widely-used tetracycline family. However, some clinical isolates have been detected that show increased minimum inhibitory concentrations of tetracyclines, although they do not harbor the mutation in the 16SrRNA gene usually associated with resistance. The present work aimed to assess whether efflux pumps, infrequently described in mycoplasmas, could participate in the observed moderate loss of susceptibility. General efflux mechanisms were measured (i) using the fluorescence property of ethidium bromide when accumulated intracellularly and intercalated in the mycoplasma genomes, its active extrusion resulting in a temperature-dependent decrease in fluorescence and (ii) monitoring the growth inhibition of mycoplasmas by subinhibitory concentrations of tetracycline with or without reserpine, a known inhibitor of efflux in other bacteria. Both methods revealed non-specific efflux phenomena in most of the isolates tested, although their efficacy was difficult to quantify. This property could contribute to the acquisition of mutations conferring resistance by maintaining intracellular concentrations of tetracyclines at subinhibitory levels

Tipificación molecular y mecanismos de resistencia antimicrobiana en micoplasmas asociados a la agalaxia contagiosa

La presente tesis doctoral se ha realizado en formato de compendio de publicaciones y los artículos que la conforman tienen el objetivo común de evaluar distintas técnicas disponibles para la detección de M. agalactiae en muestras de leche de cabra, así como la tipificación molecular de las cepas circulantes y los mecanismos de resistencia antimicrobiana de las micoplasmas causantes de la agalaxia contagiosa. De esta forma, los objetivos específicos y la metodología seguida fueron: 1) Evaluar los límites de detección de las técnicas de cultivo microbiológico y PCR empleadas para la detección de M. agalactiae en muestras de leche de cabra inoculadas experimentalmente; 2) Determinar la concordancia entre las técnicas de cultivo microbiológico y PCR utilizadas para detectar M. agalactiae en muestras de mamitis procedentes de rebaños caprinos crónicamente infectados; 3) Evaluar la variabilidad genética de cepas de campo de M. mycoides subsp. capri y M. capricolum subsp. capricolum con diferentes características epidemiológicas procedentes de áreas endémicas de agalaxia contagiosa; 4) Evaluar la susceptibilidad in vitro a diferentes antimicrobianos de cepas de M. capricolum subsp. capricolum aisladas en España e Italia y 5) Estudiar la importancia de los genes de la región QRDR en el aumento de la resistencia a quinolonas así como determinar las mutaciones específicas que nos aportan información acerca de la susceptibilidad a quinolonas de las cepas de M. agalactiae y M. capricolum subsp. capricolum. Los resultados obtenidos permiten concluir que: 1) En muestras de leche de cabra, la técnica del cultivo microbiológico presenta un límite de detección de M. agalactiae inferior al obtenido por la PCR. Por lo tanto, en muestras de leche de tanque se recomienda la realización del diagnóstico mediante cultivo, mientras que en muestras de mamitis clínicas ambas técnicas presentan una buena concordancia; 2) Los esquemas de MLST son una herramienta útil para la detección de diferencias genéticas entre aislamientos de M. mycoides subsp. capri procedentes de distintos orígenes geográficos. Asimismo, esta técnica permite detectar diferencias entre cepas presentes en un mismo rebaño al mismo tiempo; 3) M. mycoides subsp. capri y M. capricolum subsp. capricolum presentan una gran variabilidad genética entre cepas aisladas en una misma área endémica de agalaxia contagiosa, mayor que la observada anteriormente en otras especies de micoplasmas como M. agalactiae; 4) No existe una relación directa entre la evolución filogenética de las cepas de M. capricolum subsp. capricolum y sus perfiles de susceptibilidad a distintos antimicrobianos; 5) La doxiciclina es el antimicrobiano más eficaz para inhibir el crecimiento de las cepas estudiadas de M. capricolum subsp. capricolum. Asimismo, algunos de los aislamientos analizados resultaron ser resistentes a quinolonas y macrólidos; 6) El aumento de la resistencia a quinolonas en M. capricolum subsp. capricolum y M. agalactiae se debe a la aparición de mutaciones en los genes que codifican la DNA girasa y la topoisomerasa IV bacterianas y 7) El gen parC es el que aporta mayor información acerca de la susceptibilidad a quinolonas de M. capricolum subsp. capricolum y M. agalactiae.This doctoral thesis is presented as a compendium of publications and the articles that form part of it have the common goal of evaluate different diagnostic techniques for the detection of M. agalactiae in goat milk samples and the characterization of field isolates of contagious agalactia causing mycoplasmas by molecular typing and the study of their antimicrobial resistance mechanisms. Thus, the specific objectives and methods were: 1) To determine the lower M. agalactiae detection limits of culture and PCR techniques in experimentally inoculated goat milk samples, 2) To establish the agreement between PCR and culture techniques for the detection of M. agalactiae using mastitic milk samples retrieved from chronically infected commercial dairy herds, 3) To assess the genetic variability of different M. mycoides subsp. capri and M. capricolum subsp. capricolum field isolates with different epidemiological characteristics isolated from contagious agalactia endemic areas, 4) To evaluate the in vitro antimicrobial susceptibility of M. capricolum subsp. capricolum field isolates retrieved from Spain and Italy and 5) To study the importance of the QRDR in the acquisition of quinolone resistance, determining the specific genes that provide useful information about quinolone resistance in M. agalactiae and M. capricolum subsp. capricolum isolates. The obtained results allowed us to conclude: 1) In goat milk samples, bacterial culture technique has a lower detection limit of M. agalactiae than PCR. Therefore, culture is recommended when bulk tank milk samples are analyzed, whereas both techniques have a good agreement when detecting this pathogen in mastitic milk samples; 2) MLST schemes are a useful tool to detect genetic differences between different M. mycoides subsp. capri field isolates coming from different geographical areas. Moreover, they also allow the detection of variations between isolates retrieved from the same herd at the same time; 3) M. mycoides subsp. capri and M. capricolum subsp. capricolum show great genetic variability between field isolates coming from the same contagious agalactia endemic area. These differences are greater than those reported in other mycoplasma species such as M. agalactiae; 4) There is no connection between phylogeny and antimicrobial susceptibility in the studied M. capricolum subsp. capricolum strains; 5) Doxycycline is the most effective antimicrobial inhibiting M. capricolum subsp. capricolum growth. Besides, some of the analyzed field isolates were resistant to quinolones and macrolides; 6) The acquisition of quinolone resistance in M. capricolum subsp. capricolum and M. agalactiae is due to mutations in the genes that encode enzymes DNA gyrase and topoisomerase IV and 7) parC is the gene providing most information about quinolone susceptibility in M. capricolum subsp. capricolum and M. agalactiae

Mycoplasma excretion in reproductive male and female goats

Three experiments were designed in order to study the excretion routes of mycoplasmas involved in caprine contagious agalactia in chronically infected goat populations. The results obtained for external auricular canal (EAC) swabs were compared to other samples: 1) nasal swabs in 95 bucks; 2) conjunctival swabs in 85 bucks; 3) milk and vaginal swabs in 23 goats. Serology to detect specific antibodies against Mycoplasma agalactiae (Ma) was also conducted. Out of 1061 samples, the presence of Ma, Mmc and Mcc was detected in 23, 18 and 8 samples, respectively, which belonged to 24 bucks and 7 goats. EAC analyses detected 21 bucks (87.5%) and 4 infected goats (57.1%). Additional sampling (conjunctival, nasal swabs and vaginal swabs in goats) allowed the detection of further infected animals, in comparison to the sole sampling of the EAC

Zoonosis y salud laboral en la profesión veterinaria

ABSTRACT The veterinary profession implies a greater risk of infection by zoonotic pathogens than the overall population. The aim of the present work was to evaluate the impact of zoonoses on the occupational health of veterinarians reviewing the published surveys addressing this subject. Following these inquiries, between 4% and 64.3% of the surveyed professionals acknowledge to have suffered at least one zoonotic disease, and dermatophytosis is the most frequently described zoonosis. In addition, from a qualitative point of view, it is necessary to highlight the occurrence of different diseases of important clinical seriousness and the existence of infections by antimicrobial-resistant pathogens. Due to the absence of updated studies in Spain, the present work reflects on the need to recognize the health determinants associated to the veterinary profession within the characteristics of our labour market. The available data suggest that the occurrence of zoonoses as occupational diseases is underestimated in Spain. Therefore, performing surveys on occupational health, documenting and publishing cases and reviewing the risks and the impact of zoonotic diseases on the veterinary profession would contribute to the description and notification of such diseases on behalf of the healthcare administration and would also become an essential tool in occupational risk prevention.RESUMEN La profesión veterinaria presenta mayor riesgo de infección frente a agentes patógenos de carácter zoonósico que la población general. Con el objetivo de valorar el impacto de las zoonosis, el presente trabajo revisa las encuestas publicadas sobre salud laboral en la profesión veterinaria. En dichas encuestas, el rango de profesionales que refieren haber padecido al menos una zoonosis oscila entre el 4% y el 64,3%, siendo la dermatofitosis la zoonosis comunicada con mayor frecuencia. Además, desde el punto de vista cualitativo, cabe destacar la comunicación de diferentes procesos de especial gravedad clínica, así como la existencia documentada de infecciones por agentes patógenos resistentes a los antimicrobianos. Dada la ausencia de estudios actualizados en España, se reflexiona sobre la necesidad de conocer los determinantes de salud asociados a las características del mercado laboral de la profesión en nuestro entorno. Los datos existentes sugieren que la consideración legal de enfermedad profesional para las zoonosis se encuentra infraestimada en nuestro país. Por todo ello, la realización de encuestas de salud laboral, la documentación y publicación de los casos, así como la revisión de los riesgos y el impacto de las zoonosis en la profesión veterinaria, pueden contribuir a la calificación y notificación de dichas enfermedades por parte de la administración sanitaria, al tiempo que suponen una herramienta fundamental en el ámbito de la prevención de riesgos laborales

Particularidades etiológicas y epidemiológicas de la agalaxia contagiosa ovina: similitudes y diferencias con el caprino

Contagious agalactia (CA) is an infectious syndrome caused by several species of Mycoplasma spp. which affects small ruminants. The aim of this review is to describe the main differences noted between the disease in sheep and goats, especially with regard to its etiologic and epidemiological peculiarities. Thus, the presence of asymptomatic auricular carriers of CA-causing mycoplasmas and the stud’s role in the transmission of the disease are well known in goats, but scarcely evaluated in sheep. Further studies are needed to determine if these differences are real or if they are due to the shortage of scientific work in this matter.Este trabajo describe las diferencias existentes en la presentación de la agalaxia contagiosa, síndrome infectocontagioso causado por varias especies del genero Mycoplasma spp., en el ganado ovino y caprino. Las particularidades etiológicas y epidemiológicas de la infección crónica en la cabra, y la ausencia de datos similares en el ovino, evidencian la necesidad de realizar nuevos trabajos que determinen si las diferencias observadas en referencia a la presencia de portadores auriculares en los rebaños o la participación de los sementales en la difusión de la enfermedad son fruto de la escasez de trabajos científicos o por el contrario, establecen verdaderas diferencias sobre las que desarrollar estrategias dirigidas de control en función de la especie de rumiante afectada

Implicaciones sanitarias del manejo del calostro en el ganado caprino

Colostrum represents the first source of immunity for the ruminants, and thus determines its resistance to disease during the first hours of life. However, colostrum intake could be itself the way of transmition of several diseases, as paratuberculosis, caprine arthritis-encephalytis, or micoplasmosis like contagious agalactia. This risk could be avoided by means of an artificial rearing program which should include correct management practices for colostrum. Between them, the treatment of colostrum represents a critical point. In this sense, thermic treatments have been used to higienitize colostrum, showing different results about microorganism viability. Nevertheless, it should be considered the negative effect of these treatments over nutritional components of colostrum, particularly the loss of immunoglobulines. As an alternative to thermic treatments, there have been experimentally assayed other methods as the addition of sodium dodecyl sulphate, which inactivates AIDS virus in breast milk, and others as liophilization or high pression methods. In this works, apart from the effect of the treatment should be also taken into account its economical viability and on-farm feasibility.El calostro supone la primera fuente de inmunidad para los rumiantes y por tanto determina su resistencia a enfermedades durante las primeras horas de vida. No obstante, la ingesta de calostro puede suponer en sí misma una vía de transmisión de diversas enfermedades, como la paratuberculosis, la artritis-encefalitis caprina o la agalaxia contagiosa. Este riesgo puede evitarse siguiendo un régimen de lactancia artificial con unas adecuadas pautas de manejo del calostro. Entre dichas pautas, el tratamiento del calostro supone un punto crítico. En este sentido, se han empleado los tratamientos térmicos para higienizar el calostro, observándose resultados diversos en la viabilidad de distintos microorganismos. Al mismo tiempo, se debe considerar el efecto negativo del calor sobre la composición nutricional del calostro, principalmente la pérdida de inmunoglobulinas. Como alternativa a los tratamientos térmicos, a nivel experimental, se han empleado métodos como la adición de dodecil sulfato de sodio, capaz de inactivar el virus del síndrome de inmunodeficiencia humana en leche, y otros procesos como la liofilización o el uso de altas presiones. Previamente a la aplicación práctica de las diferentes opciones de tratamiento del calostro se deberá considerar su viabilidad económica y su factibilidad en la explotación