A Thesis for applying for the degree of Doctor of Philosophy in Veterinary Medicine and Food Science (Food Hygiene and Food Quality) ; Campylobacteriosis, caused by thermotolerant Campylobacter species, is continued to be one of the most commonly reported zoonosis in humans in the European Union, and Campylobacter is recognized as the most common causative agents of bacterial gastroenteritis in developed world. Listeriosis, the disease caused by Listeria monocytogenes, is a lifethreatening disease for individuals with compromised immune systems. Consumption of different foods, including poultry products, have been associated with campylobacteriosis and listeriosis. Healthy birds may shed Campylobacter spp. and L. monocytogenes in fecal material asymptomatically. Poultry meat may become contaminated with fecal matter during slaughter or through cross-contamination during processing. The main goals of the present thesis were 1) to determine the prevalence and counts of Campylobacter spp. and the prevalence of L. monocytogenes in Estonian and imported raw poultry meat, 2) to serotype and genotype using pulsed-field gel electrophoresis both Campylobacter and L. monocytogenes isolates, and 3) to determine the antimicrobial susceptibility of the Campylobacter isolates. The studies showed that the poultry products of Estonian origin and imported to Estonia were contaminated with Campylobacter spp. and L. monocytogenes. There was high sero- and genotype diversity among Campylobacter and L. monocytogenes isolates. High level of resistance to fluoroquinolones, tetracycline, ampicillin, and erythromycin among the Campylobacter isolates were detected. ; Termofiilsed kampülobakterid on paljudes maades ühed põhilised inimese bakteriaalse mao-peensoolepõletiku põhjustajad ja Euroopa Liidu riikides on kampülobakterioos jätkuvalt kõige sagedamini esinev zoonoos. Listerioos on nakkushaigus, mille kutsub esile bakter Listeria monocytogenes ja mis ohustab eelkõige immuunpuudulikkusega inimesi. Haigusjuhtude põhjuseks on kas bakteriga Campylobacter spp. või bakteriga L. monocytogenes saastunud toiduainete, sh linnuliha, tarbimine. Linnud võivad olla nii kampülobakterite kui ka listeeriate asümptomaatilised kandjad. Peamiselt saastub linnuliha fekaalse saastumise teel lindude algtöötlemisel tapamajas või ristsaastumise teel linnuliha edaspidise töötlemise käigus. Käesoleva väitekirja peamised eesmärgid olid 1) määrata bakteri Campylobacter spp. levimus ja arvukus ning bakteri L. monocytogenes levimus Eestis toodetud ja Eestisse imporditud toores linnulihas, 2) serotüpiseerida ja genotüpiseerida pulseeriva välja geelelektroforeesi meetodil toorest linnulihast isoleeritud patogeenitüved ja 3) määrata bakteri Campylobacter spp. isolaatide tundlikkus antibiootikumide suhtes. Tehtud uuringud näitasid, et Eestis toodetud ja Eestisse imporditud linnuliha osutus saastunuks bakteritega Campylobacter spp. ja L. monocytogenes. Uuritud patogeenide tüpiseerimine viitas sero- ja genotüübilisele mitmekesisusele. Tuvastati isoleeritud kampülobakterite tüvede suur resistentsus fluorokinoloonide, tetratsükliini, ampitsilliini ja erütromütsiini suhtes. ; The publication of this dissertation is granted by the Estonian University of Life Sciences
The taxonomic position of members of the Mycobacterium abscessus complex has been the subject of intensive investigation and, in some aspects confusion, in recent years as a result of varying approaches to genetic data interpretation. Currently, the former species Mycobacterium massiliense and Mycobacterium bolletii are grouped together as Mycobacterium abscessus subsp. bolletii. They differ greatly, however, as the former M. bolletii has a functional erm(41) gene that confers inducible resistance to macrolides, the primary therapeutic antimicrobials for M. abscessus, while in the former M. massiliense the erm(41) gene is non-functional. Furthermore, previous whole genome studies of the M. abscessus group support the separation of M. bolletii and M. massiliense. To shed further light on the population structure of Mycobacterium abscessus, 43 strains and three genomes retrieved from GenBank were subjected to pairwise comparisons using three computational approaches: verage ucleotide dentity, enome to enome istance and single nucleotide polymorphism analysis. The three methods produced overlapping results, each demonstrating three clusters of strains corresponding to the same number of taxonomic entities. The distances were insufficient to warrant distinction at the species level, but met the criteria for differentiation at the subspecies level. Based on prior erm(41)-related phenotypic data and current genomic data, we conclude that the species M. abscessus encompasses, in adjunct to the presently recognized subspecies M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a third subspecies for which we suggest the name M. abscessus subsp. massiliense comb. nov. (type strain CCUG 48898(T) =CIP 108297(T) =DSM 45103(T) = KCTC 19086(T)). ; research grants FFC from Fondazione Ricerca Fibrosi Cistica ; European Union PathoNgen-Trace project ; German Center for Infection Research (DZIF) ; Ist Sci San Raffaele, Emerging Bacterial Pathogens Unit, Milan, Italy ; Leibniz Zentrum Med & Biowissensch, Mol & Expt Mycobacteriol, Borstel, Germany ; Univ Texas Hlth Ctr Tyler, Dept Microbiol, Mycobact Nocardia Res Lab, Tyler, TX USA ; Univ Fed Sao Paulo, Escol Paulista Med, Dept Microbiol Imunol & Parasiotol, Sao Paulo, SP, Brazil ; Univ Madrid, Dept Prevent Med Publ Hlth & Microbiol, Madrid, Spain ; Diagnost Serv Manitoba, Winnipeg, MB, Canada ; Univ Texas Hlth Sci Ctr Tyler, Dept Pulm Med, Tyler, TX USA ; Meyer Univ Hosp, Reg Reference Ctr Cyst Fibrosis, Florence, Italy ; IRCCS Ca Granda, Cyst Fibrosis Microbiol Lab, Milan, Italy ; IRCCS Ca Granda, Cyst Fibrosis Ctr, Milan, Italy ; Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil ; research grants FFC from Fondazione Ricerca Fibrosi Cistica: 27/2014 ; European Union PathoNgen-Trace project: FP7-278864-2 ; Web of Science
It is well established that bacteria are the first organisms to adhere and colonize both abiotic and biotic surfaces. Their subsequent multiplication and production of exopolymeric substances (EPS) bring to biofilm formation which is believed to influence the settlement of following colonisers starting biofouling process on surfaces. Biofilm and fouling often have a deleterious effect on colonized surfaces and materials. They explicate a complex and various range of processes on abitic surfaces that cause physical damages, chemical alterations, loss of functionality and discolouration of surfaces, generating tremendous environmental and economical harm for human society. The control of the deleterious biofilm and the following fouling is today a great challenge. Currently, we have two choices: to remove biofilm by traditional methods or to research new effective antifouling approaches. Traditional techniques consist mainly in the application of biocides, however this practice has several disadvantages. In fact, traditional biocides are not generally specifically target against detrimental microorganisms and they are often potentially toxic both for humans and the environment. In addition, biological matter released by their use can offer a favourable substratum for subsequent colonisations. Others problems concern the development of resistance in target pest populations and the low biodegradability of these chemicals. Moreover, biocides are not always effective at low concentrations because life inside the biofilm leads to increased resistance to antimicrobial products up to 1000-fold compared to planktonic cells. Finally, current legislation in UE and USA regulates the use of biocides and lately several products have been withdrawn from the market. New antifouling strategies need to be effective, economic, safe for the public and to pose negligible risk to human health and the environment. In the last years new strategies able to control biofilm growth have been proposed as alternative to traditional active substances. However, several years will be required to set up and test satisfactory methods, so, these novel strategies can be only a long term solution. In the meantime, as short term solution, it is vital to study methods for a more sustainable use of traditional antimicrobial agents. Therefore, the aims of the PhD project here presented were: 1) to use biocides for the removal of deleterious biofilm from relevant abiotic surfaces in the most sustainable way; 2) to exploit the ability of a new promising inorganic compound, the photocatalytic titanium dioxide, as an innovative non-toxic antifouling system to control biofilm formation on abiotic surfaces. The study reported in the chapter 3 addresses the first aim. Generally, broad-range biocides are used to remove alterative biofilms from historical and artistic surfaces. In order to obtain a use as sustainable as possible of antimicrobial agents, the strategy has been to identify alterative microorganisms so as to use a suitable biocidal product which targets specifically the biodeteriogen agents. In this work, for the first time, an integrated biotechnological system that enables the cleaning of cultural heritage stone affected by both biological and chemical alteration process was used. The study was conducted on alterations found on two stone sculptures decorating the courtyard of the Buonconsiglio Castle in Trento (Italy). Stone, especially if exposed to the weather, is subject to chemical, physical and aesthetical deterioration. In this regard, pollution and environmental parameters play an important role since they are most responsible of deterioration process. Samples from altered and unaltered areas were characterized using stereomicroscope, cross-section observations and Fourier transform infrared (FTIR) analyses. Results reveled that stone was an oolitic limestone and changes were both discolorations ascribed to biological agents and chemical alteration represented by black crusts composed mainly by gypsum with a small amount of calcite, nitrate and silicates. Cultural and biomolecular methods were adopted to study microbial biofilm from powders samples. The cultural analyses proved that heterotrophic bacteria, fungi and prokaryotic and eukaryotic algae were present on surfaces and that in some samples the counts were quite high (up to 7 logCFU/g for bacteria and up to 6 logCFU/g for fungi). Denaturing gradient gel electrophoresis (DGGE) and sequencing from total DNA extracted allowed to identify taxa of microorganisms causing discolorations: they were Cyanobacteria, Chlorophyta green algae (Myrmecia and Friedmannia), Streptophyta green algae (Klebsormidium), microcolonial black fungi (Alternaria and Cladosporium) and other species of fungi able to deposit melanin in the cell wall (Verticillium). Fluorescent in situ hybridisation (FISH) highlighted that the Cyanobacteria generally were dominant (more than 60%) among the other prokaryotics belonging to the Bacteria domain. In this case, in order to remove the discolourations, despite the initial purpose to use a product that targeted only the small population of harmful microorganisms, we were forced to choose the biocide BIOTIN N (constituted by a mixture of tributyltin naphthenate (20% w/v) and didecyl dimethyl ammonium chloride (35% w/v)) with a broad spectrum of activity due to the taxonomical variety of the biodeteriogens. The same traditional and biomolecular methods were carried out on the samples collected after the cleaning to verify the removal of alterative microorganisms. Treatment with the biocide resulted in a decrease of the bacterial load (up to five orders of magnitude), and neither culturable fungi nor culturable prokaryotic and eukaryotic algae grew. Indeed the DGGE profiles showed far fewer bands than before treatment and proved that Cyanobacteria and most of the green algae and dematiaceous fungi had been efficiently removed. Chemical alterations were removed by a biocleaning treatment, an innovative, efficient and highly selective bioremediation technology, alternative to the use of chemicals, that uses viable cells of sulphate-reducing bacteria able to remove sulphates from stone ornamental surfaces. In this work for the first time this approach was applied on limestone. Sulphate-reducing bacteria have been Desulfovibrio vulgaris subsp. vulgaris (ATCC 29579). The treatment consisted in three 12-h applications for a total duration of 36 h. Comparison of the results of the chemical analyses performed before and after the biocleaning proved that gypsum, nitrates and silicates were almost completely removed. In addition, both optical evidence and FTIR analysis showed that the limestone substratum was preserved. In general, the cases of biofilms with large taxonomical variety (as the biofilms on the sculptures from Buonconsiglio Castle in Trento) are very frequent. Therefore, due to the side-effects of biocides, the development of alternative strategies for the prevention and control of deleterious biofilm becomes imperative. They must allow to protect materials from biodeterioration, and thus preserve their usefulness for as long as possible. In addition, new approaches must be safe for human beings and the environment. The study reported in the chapter 4 refers to this second aim of the project. The ability of photoactivated titanium dioxide (TiO2), a promising alternative to biocide for the biofilm control and prevention, was investigated. The biocidal activity of TiO2 against planktonic cells thanks to its strong photocatalytic properties has been reported since 1985. TiO2 per se is non-toxic, as the tests in rats prove, and has been approved by the American Food and Drug Administration (FDA) for use in human food, drugs, cosmetics and food contact materials. Moreover, it is considered an environmentally friendly photocatalyst, it is relatively inexpensive, chemically stable and effective under weak solar irradiation in ambient atmospheric environment. Few attention was dedicated to study the potential of photocatalytic TiO2 against the bacterial biofilm so far. The aim was to investigate the ability of photocatalytic TiO2 as a new non-toxic antifouling nanotechnology to deter and prevent the attachment and biofilm formation of selected bacteria on TiO2-coated surfaces. Aeroxide P25 (Degussa) was employed as source of TiO2. The effects of both photocatalyst nanopowder suspensions (3g/l concentration) and thin TiO2-film applied on glass coverslides by sol-gel method were valued. During the experiments TiO2 was photoactivated by a lamp emitting radiation over a UV-A wavelength range with light intensity similar to outdoor solar irradiation (between 3000 and 500 μW/cm2). An efficient protocol for the photoactivation of TiO2 was set up degrading the dye rhodamine B. Thus activity was investigated on Bacillus cereus-group sp. (Gram-positive) and P. stutzeri and P. aeruginosa (Gram-negative) planktonic cells. The results proved that photoactivated TiO2 provoked a significant decrease of CFU/ml. Biocidal activities of nanopowder suspension in demineralised water in Bacillus sp., P. stutzeri and P. aeruginosa were respectively 1-log reduction after 24 h, 2-log reduction after 30 min and 1-log reduction after 2 h compared to non-photoactivated TiO2. TiO2 thin film also produced a complete disinfection of P. aeruginosa planktonic cells in 24 h. Finally, the activity of photoactivated TiO2 was investigated on P. aeruginosa biofilm at various formation steps both at the solid-liquid and at the solid-air interface. It was proved that neither TiO2 nanopowder nor photocatalytic film showed any biocidal activity on P. aeruginosa biofilm at all the interfaces investigated. The experiments have demonstrated that the lack of cell inactivation by photocatalytic action on biofilm was not due to 1) the presence of phosphates that could block active sites on the catalyst surface, 2) scavenge oxidative radicals produced at the surface, nor to insufficient presence of O2 at the TiO2 surface, which maintains charge transfer in photocatalytic reactions, 3) biofilm growth that screens UV-A light, deactivating TiO2, 4) insufficient build-up of photocatalytically-generated reactive species necessary for cell inactivation. In addition, confocal laser scanning microscope analysis demonstrated that not even exopolysaccharides produced by biofilm cells were the cause of fail inhibition since almost absent in the very young tested biofilms. The only possible explanation for these findings was that the cells, when live in sessile form, invoke a genetic response that imparts them an increased resistance to oxidative stress generated by photoactivated TiO2. In conclusion, the studies reported here have demonstrated that: a) Biotechnologies could facilitate a more sustainable use of biocides addressing the choice toward a suitable product that targets only the biodeteriogen microorganisms. b) The biocleaning treatment is an effective technology, alternative to use of chemicals, to remove selectively sulphates from stone. c) The photocatalytic TiO2 is not a good candidate to develop an effective technology that is alternative to traditional biocides for the control of deleterious biofilm. Further studies with other promising environmentally-friendly compounds may provide new ways to move forward in the search and to pursue the goal of an efficient surface coating methods able to prevent biofilm formation or, at least, to interfere with their inconvenient increased resistance to biocides, respecting the human health and environment. The research for new non-toxic antifouling strategies continues.
In piglets, weaning is a critical event associated with acute social, environmental and dietary stress engendering fairly often post-weaning diarrhea and enterotoxigenic Escherichia coli infections. Current strategies imply the systematic use of antimicrobials now raising public health concerns due to the massive increase of antibiotic resistance genes among bacteria. Non-pharmacological preventive and curative strategies are necessary to fight against post-weaning diarrhea in piglets. In compliance with the European Union directive aiming to reduce, refine and replace the use of animals in research, the principal aim of this PhD project was to develop a new in vitro fermentation model of the weaning piglet colon implemented with mucin beads to simulate both luminal and mucosal microbiota. Following its development and validation towards in vivo data, the presently designed in vitro model was used to evaluate the impact of two of the major stress encountered at weaning: a dietary change and transient anorexia as well as an enterotoxigenic Escherichia coli strain responsible for post-weaning diarrhea on the composition and activity of the microbiota. Finally, the effects of a Saccharomyces live yeast strain to fight against microbiota dysbiosis and ETEC infections in weaning piglets was evaluated using the newly developed in vitro model and cell culture experiments. The data collected throughout this PhD thesis participated to a better understanding of microbiota dysbiosis associated with weaning and gave interesting insights about the interest of a probiotic strategy in the critical period of weaning in piglets. ; Chez les porcelets, le sevrage est une étape cruciale associée avec d'importants stress d'ordres sociaux, environnementaux et alimentaires qui engendrent fréquemment des diarrhées post-sevrage majoritairement d'origine colibacillaire. Actuellement, ces infections sont traitées de manière systématique à l'aide d'antibiotiques. Toutefois, le nombre de bactéries multi-résistantes aux antibiotiques augmente de façon alarmante et le besoin de stratégies alternatives aux antibiotiques pour lutter contre ces diarrhées post-sevrage devient urgent. En accord avec la directive européenne visant à réduire, remplacer et raffiner le nombre d'animaux utilisés à des fins de recherche, le principal objectif de ce projet de thèse était de développer un modèle in vitro reproduisant les paramètres physicochimiques et microbiens de l'environnement colique du porcelet au sevrage, le MPigut-IVM. Ce système permet, de manière unique, de distinguer les microenvironnements coliques luminaux et mucosaux et leurs microbiotes associés. Une fois validé comparativement à des données in vivo chez le porcelet, ce modèle innovant a été utilisé pour étudier l'effet des principaux stress rencontrés au sevrage, un jeûne alimentaire, suivi d'un changement d'alimentation et d'une infection par une souche d'Escherichia coli entérotoxigénique responsable de diarrhées sur la composition et l'activité du microbiote. Enfin, l'intérêt d'un traitement préventif à base de levures Saccharomyces a été évalué in vitro en utilisant une approche originale combinant le modèle colique développé et des cellules épithéliales intestinales en culture. Les données de cette thèse ont permis à la fois de mieux comprendre l'étiologie de la dysbiose du microbiote observé au sevrage chez les porcelets et d'évaluer in vitro l'intérêt d'une stratégie probiotique lors de cette phase critique de la vie de l'animal.
El uso inadecuado de los antibióticos en las unidades de cuidados intensivos pediátricos aumenta la resistencia bacteriana, la estancia hospitalaria y los costos en los servicios de salud. El objetivo de esta investigación fue describir los criterios para la prescripción y uso de antibióticos en menores de cinco años, hospitalizados con Enfermedad Respiratoria Aguda en una Unidad de Cuidados Intensivos Pediátrico (UCIP) de un hospital público en Bogotá, durante al año 2011. Se realizó un estudio tipo observacional, descriptivo, transversal, a partir de los datos de ingreso, diagnóstico, tratamiento y egreso según las historias clínicas de la UCIP en niños menores de cinco años, de enero a diciembre del 2011. De 62 pacientes, 60 recibieron tratamiento con antibióticos, 23 cumplieron al menos 7 días de medicación, 14 fueron dados de alta antes del séptimo día, 1 falleció y en 9 casos se les suspendió el tratamiento inicial. Se prescribieron 12 principios activos, los dos antibióticos con mayor prevalencia son ampicilina sulbactam 44,6% (n = 45) y ceftriazona 13,9% (n = 14). El 96,7% (n = 60) de pacientes medicados inicialmente presentó aislamiento microbiológico (n = 20) y aislamiento viral (n = 19). De estos 20, el 75% (n = 15) continuaron con la medicación empírica inicial según resultados microbiológicos. Las indicaciones de antibióticos para las cuales se formularon de manera inadecuada fueron en las bronquiolitis (n = 7) sin complicaciones bacterianas sobreagregadas, sin sepsis ni signos radiológicos de neumonía y con hemocultivos negativos. Conclusiones: en este estudio se identificaron los siguientes criterios para la prescripción inicial de antibióticos: presencia de fiebre, cuadro hemático, PCR y estudio radiológico, en los cuales la prevalencia de antibióticos prescritos fue del 96% y la tasa de prescripción de antibióticos en infecciones de etiología viral fue del 37%. Se evidencia que para la selección del antibiótico empírico en los niños con enfermedades respiratorias agudas, puede ser difícil ante la imposibilidad de diferenciar entre sí si es de origen bacteriano o viral, ya que no se cuenta con la identificación inicial del patógeno. La variabilidad del uso de antibióticos está influenciada por factores culturales, epidemiológicos, nivel de complejidad, entre otros; es prioritario que cada institución establezca una política para el uso racional de antibióticos de acuerdo con sus necesidades, teniendo en cuenta los antibióticos de amplio espectro o de primera línea para sospecha de infección o tratamiento empírico y el adecuado abordaje de infeccionesAbstractThe inadequate use of the antibiotics in the intensive care units pediatric, they increase the bacterial resistance, the hospitable stay and the costs in the services of health. The objective of this investigation was to describe the criteria for the prescription and use of antibiotics in five-year-old minors hospitalized with Respiratory Sharp Illness in an Intensive care unit Pediatric (UCIP) of a public hospital in Bogota during a year 2011. A study was conducted observational, descriptive, cross from the input data, diagnosis, treatment and egress as medical records the (UCIP) in children under five years, from January to December 2011. Of 62 patients, 60 were treated with antibiotics, 23 completed at least 7 days of medication, 14 were discharged before the seventh day, 1 died and in 9 cases initial treatment stopped. 12 were prescribed active, the two most prevalent are antibiotics ampicillin sulbactam 44.6% (n = 45) and ceftriaxone 13.9% (n = 14). 96.7% (n = 60) of patients originally medicated microbiological isolation presented (n = 20) and virus isolation (n = 19). Of these 20, 75% (n = 15) continued the initial empiric medication as microbiological results. The indications for antibiotics to which were improperly formulated in bronchiolitis (n = 7) without a superimposed bacterial complications, without sepsis or radiological signs of pneumonia and negative blood cultures. Conclusions: In this study identified the following criteria for the initial prescription of antibiotics: presence of fever, blood picture, PCR and radiological study. Where the prevalence of prescribed antibiotics was 96% and the rate of prescription of antibiotics in infections of viral etiology was 37%. It is evident that the selection of empiric antibiotics in children with acute respiratory diseases, it can be difficult to impossible to differentiate between whether it is bacterial or viral origin, since there is no initial identification of the pathogen. The variability of the use of antibiotics is influenced by cultural, epidemiologic factors, level of complexity, between others; it is priority that every Institution establishes a politics for the rational use of antibiotics in accordance with his needs, taking into account the broad spectrum antibiotics or frontline for suspected infection or empiric treatment and adequate management of viral infections. Keywords: drug use, antimicrobial, intensive care, prescription, microbial drug resistance.
[spa] El uso indiscriminado y/o inadecuado de medicamentos veterinarios es la causa de posibles efectos adversos para la salud por el riesgo que entren en la cadena alimentaria y la aparición de sus residuos en los productos alimenticios de origen animal. Además, en el caso de los antibióticos, este hecho ha sido identificado como una de las causas de la aparición de mecanismos de resistencia a los antimicrobianos en las bacterias que causan enfermedades humanas, lo cual es causa de una gran preocupación en las autoridades sanitarias, distintos organismos gubernamentales y de la sociedad en general. Actualmente, existe una creciente necesidad de garantizar la seguridad y calidad de los productos alimenticios y los consumidores también han comenzado a ser más exigentes obligando a la industria a introducir sus preocupaciones en sus procedimientos, en términos de conseguir los productos más naturales, ecológicos y saludables con un origen de los ingredientes rastreable. Hoy en día, el control de alimentos se realiza en laboratorios centralizados que en la mayoría de casos utilizan procedimientos muy fiables, pero necesitan equipos caros, personal especializado y elevados tiempos de tratamiento de muestra. Con el fin de mejorar drásticamente esta situación, la Comisión Europea (CE) y los organismos de los Estados Miembros apoyan firmemente actividades de investigación orientadas a aumentar la eficiencia de los métodos de análisis actuales. Una estrategia es tomar ventaja de los últimos avances en las biomicro-nanotecnologías y de las capacidades complementarias de los equipos de investigación multidisciplinarios para desarrollar metodologías más rápidas, sensibles y específicas capaces de detectar una amplia variedad de sustancias químicas, biológicas o cualquier otro riesgo para la salud asociado a la industria agroalimentaria, a lo largo de toda la cadena alimentaria. En esta tesis se describe la investigación llevada a cabo en relación a la posibilidad de desarrollar alternativas más adecuadas para el análisis de residuos de alimentos basados en la combinación de receptores selectivos y las nuevas micro/nanotecnologías. En particular, el objetivo final de esta tesis fue dirigido hacia el desarrollo de un dispositivo sensor multiplexado para detectar prácticas agrícolas inadecuadas y/o la contaminación de alimentos, principalmente la leche, por residuos de antibióticos. A este respecto, la producción de receptores selectivos con un amplio reconocimiento de las familias de antibióticos más importantes que se utilizan en el sector veterinario ha sido uno de los objetivos más importantes de este trabajo de investigación. Así, lo constata la investigación realizada en relación con el desarrollo de receptores sintéticos para antibióticos de sulfonamida (SA), polímeros de huella molecular (MIPs), y su evaluación como enfoque racional para otros contaminantes. Además, la producción de anticuerpos genéricos (o selectivos de clase) para SAs y antibióticos de tetraciclina (TC) también ha sido abordada mediante el diseño racional y síntesis de haptenos. La evaluación de las características de los anticuerpos producidos se ha logrado mediante el desarrollo de ELISAs (Enzyme-Linked ImmunoSorbent Assay - ensayo por inmunoabsorción ligado a enzimas) en microplacas. Los resultados muestran que a pesar de que ha sido posible obtener anticuerpos selectivos de clase para las SA (hasta 11 congéneres pueden ser detectados), el enfoque adoptado para el caso de los anticuerpos TCs ofrece una alta selectividad frente a TC que poseen un grupo alquilo/alqueno en la posición 6 del anillo C, pero que carecen del grupo hidroxilo en esta posición. Los protocolos necesarios para aplicar estos procedimientos inmunoquímicos para el análisis de muestras de leche y el cabello han sido establecidos mostrando que fue posible la determinación de estos antibióticos en leche de acuerdo con la normativa de la CE. Para el caso del cabello, no existen regulaciones por el momento. Sin embargo, esta matriz tiene un gran valor en cuanto a su uso potencial para seguir tratamientos inadecuados a lo largo de la vida de los animales de granja. Los inmunoreactivos y procedimientos inmunoquímicos establecidos se han implementado en un dispositivo sensor óptico desarrollado por el Centre Suisse de Electrónica y Microelectrónica Inc. (CSEM, Neuchâtel - Suiza). Este dispositivo se basa en el principio de onda evanescente usando un diseño tecnológico particular basado en acopladores de rejilla de guía de ondas que es muy sensible a los cambios en el índice de refracción producida en la superficie del transductor. Además, el chip desarrollado por CSEM tiene 24 zonas de detección que permiten múltiples mediciones con el mismo transductor. Como consecuencia de esta colaboración ha sido posible desarrollar un dispositivo biosensor capaz de detectar residuos de SA en muestras de leche en el cumplimiento de la normativa CE. Investigaciones posteriores, han llevado al desarrollo de un dispositivo biosensor multiplexado que combina inmunoreactivos (antibióticos SAS y fluoroquinolonas (FQs)) producidos en el grupo de Receptores Moleculares Aplicados (AMRg) del CSIC, con bioreceptores (ß-lactámicos (BLs) y TCs) proporcionados por Unisensor SA (Lieja - Bélgica). Los resultados obtenidos fueron muy buenos. Alrededor de 34 antibióticos de cuatro familias diferentes de antibióticos pueden ser detectados en muestras de leche siguiendo todas las normativas de la CE con el dispositivo desarrollado. Anteriormente, se desarrolló un ELISA multianalito (en microplacas) mediante la combinación de los mismos bioreactivos para evaluar el sus características y establecer los procedimientos inmunoquímicos más adecuados. Los métodos inmunoquímicos desarrollados en esta tesis, incluyendo los inmunoensayos e inmunosensores, han sido evaluados en colaboración con el Centro de Investigación de Nestlé (NRC), en Lausana, en Suiza, con el fin de demostrar su rendimiento en las muestras de leche real. ; [eng]The indiscriminate and/or improper use of veterinary drugs is the cause of potential adverse health effects due to the risk of entering into the food chain and the appearance of residues in food products of animal origin. Moreover, in the case of antibiotics, this fact has been identified as one of the causes for the appearance of antimicrobial resistance mechanisms in bacteria causing human diseases, which is the cause of a big concern within the health authorities, distinct governmental agencies and the society in general. There is an increased need to ensure safety and quality of the foodstuff and customers have also started to become more exigent forcing the industry to introduce consumer worries in their daily procedures, in terms of getting more natural, ecological and healthy products with a clear traceable origin of the ingredients. Nowadays, food control is performed on centralized laboratories that in most cases use very reliable procedures, but involving expensive equipment, specialized personnel and time consuming sample treatment procedures. In order to drastically improve this situation, the European Commission (EC) and the member state agencies are strongly supporting research activities aimed to increase the efficiency of the actual analytical methods. An strategy is to take advantage of the latest bio-micro-nanotechnological advances and of the complementary skills of multidisciplinary research teams to develop more rapid, sensitive and specific methodologies capable of detecting a wide variety of chemical, biological or any other health risk associated to the agrofood industry and along to the whole food chain. This thesis describes the research performed in respect to the possibility to develop improved alternatives for food residue analysis based on the combination of selective receptors and novel micro/nanotechnologies. Particularly, the final objective of this thesis was addressed to the development of a multiplexed sensor device to detect inappropriate farm practices and or the contamination of food products by antibiotic residues, mainly milk. In this respect, production of selective receptors with a broad recognition of the most important antibiotic families used in the veterinary field has been one of the most important aims of this research work. Thus, we report here the investigation made regarding development of synthetic receptors for sulfonamide antibiotics (SAs), particularly molecular imprinted polymers (MIPs), and their evaluation of a rational approach. Moreover, production of generic (or class-selective) antibodies for SAs and tetracycline antibiotics (TCs) has also been approached through the rational design and synthesis of appropriate haptens. Evaluation of the features of the antibodies produced has been accomplished through the development of microplate-based ELISAs (enzymelinked immunosorbent assays). The results show that although it has been possible to obtain class-selective antibodies for SAs (up to 11 congeners can be detected), the approach followed for the case of the TCs afforded antibodies with a high selectivity versus TCs possessing an alkyl/alkene group at position 6 of the C-ring, but lacking the hydroxyl group at this position. The necessary protocols to apply these immunochemical procedures to the analysis of milk and hair samples have been established showing that determination of these antibiotics according to the EC regulations was possible for the case of milk. For the case of hair, no regulations do exist at the moment. However this matrix holds great value regarding their potential use to trace inappropriate treatments through the life of the farm animals. The immunoreagents and immunochemical procedures established have been implemented on an optical sensor device developed by the Centre Suisse for Electronics and Microelectronics Inc. (CSEM, Neuchâtel - Switzerland). This device is based on the evanescent wave principle using a particular technological design based on waveguide grating couplers and it is very sensitive to the changes in the refractive index produced at the surface of the transducer. Moreover, the chip developed by CSEM has 24 sensing pads which allow multiple measurements with the same transducer. As a consequence of this collaboration it has been possible to develop a biosensor device able to detect SA residues in milk samples in compliance with the EC regulations. Further investigation, has led to the development of a multiplexed biosensor device by combining immunoreagents (SAs and fluoroquinolone antibiotics (FQs)) produced at the Applied Molecular Receptors group (AMRg) of the CSIC, with bioreceptors (ß lactam antibiotics (BLs) and TCs) provided by UNISENSOR S.A. (Liege - Belgium). The results obtained were very good. About 34 antibiotics from four different antibiotic families can be detected in milk samples following all the EC regulations with the device developed. Before, a multianalyte microplate-based ELISA had been developed combining the same bioreagents to evaluate performance and to establish the most appropriate immunochemical procedures. The immunochemical methods developed within this thesis, including immunoassays and immunosensors, have been preliminarily evaluated in collaboration with the Nestlé Research Centre (NRC), at Lausanne in Switzerland, in order to demonstrate performance in real milk samples.
La presenza di residui chimici negli alimenti, costituiti sia da composti farmaceutici che da contaminati ambientali, è un argomento di crescente interesse e preoccupazione per la sanità pubblica. L'Unione Europea periodicamente sancisce leggi e rapporti aggiornati, con lo scopo di condurre piani di monitoraggio e linee guida sull' impiego di tali composti a livello industriale, agricolo e terapeutico e livelli massimi residuali (LMR e ML) negli alimenti, al fine di prevenire il rischio per il consumatore. Sulla base di queste considerazioni, il presente elaborato ha lo scopo di studiare la presenza di residui chimici in diversi alimenti di origine animale, al fine di caratterizzare il rischio per il consumatore. In primo luogo, ci siamo focalizzati sullo studio di alimenti provenienti dal settore ittico, che è un'ottima fonte di nutrienti, con importanti benefici per la salute umana. Ci siamo concentrati su cozze e vongole, animali filtratori e bioindicatori adatti per le loro caratteristiche a bioaccumulare un'ampia gamma di inquinanti ambientali. Lo scopo del primo elaborato è stato quello di valutare il rischio per il consumatore dei principali metalli (Hg, Cd, Pb, Ni, Cre As), attraverso il consumo di molluschi sulla base dei limiti massimi dichiarati dall'Unione Europea o, ove disponibili, sulla base dei valori soglia dichiarati dall'EFSA. Dal primo lavoro è emerso che vi è un basso rischio per il consumatore medio; tuttavia, i consumatori ai percentili superiori, possono essere soggetti a lesioni cutane e/o neoplasie polmonari, cutanee e vescicali per l'elevata assunzione di As. Soggetti Ni sensibili, possono invece essere soggetti a dermatiti allergiche. Il secondo lavoro di ricerca si è invece concentrato sulla ricerca dei medesimi metalli nel tonno, il cui consumo è in aumento secondo i dati della commissione europea, per le sue capacità di bioaccumulo. Sono stati, così, analizzati 131 campioni provenienti da diverse zone FAO. Dai risultati, è emerso che solo un tonno rosso, proveniente dal mare Adriatico e 11 tonni gialli hanno superato i livelli massimi residuali di Pb; tre tonni rossi provenienti da diverse sottozone del 7 Mediterraneo hanno superato i livelli massimi consentiti per il mercurio. La valutazione degli effetti tossicologici cumulativi ha indicato un rischio trascurabile sia per i medi che alti consumatori. L'obiettivo del terzo studio è stato quello di studiare la presenza di inquinanti organici persistenti e di antimicrobici nei salmoni selvatici e di allevamento di diverse aree geografiche. I salmoni d'allevamento hanno mostrato una presenza di contaminanti ambientali superiore a quelli selvatici, probabilmente a causa di un maggiore impatto demografico. Il rischio legato ai composti organofosforati, agli idrocarburi policiclici aromatici, ai policlorobifenili e ai pesticidi organoclorurati derivanti dall'assunzione di salmone si è rilevato molto basso, mentre la presenza di polobromodifenilietere congenere 99 (PBDE99) e acido perfluoroottanoico (PFOA) suscita maggiore preoccupazione. Gli antibiotici sono stati riscontrati con bassa frequenza solo nel salmone allevato. Da questi lavori, sulla base dei dati ottenuti, possiamo confermare che vi è basso rischio per il consumatore medio Il consumo regolare di carne e prodotti a base di carne fornisce un significativo apporto di proteine e micronutrienti essenziali. La carne suina, ad esempio, è impiegata in molti paesi per produrre prodotti derivati (prosciutti e salumi) ad alto valore qualitativo. Anche il consumo di carne di selvaggina, pur essendo un prodotto di nicchia, è in costante aumento e i cacciatori, le loro famiglie e le persone a loro strettamente legate possono essere considerati una sottopopolazione ad alto consumo. Nel quarto lavoro abbiamo cosi studiato la presenza di polibromodifenilietere e sostanze perfluoroalchiliche provenienti da otto Stati membri dell'UE (Austria, Danimarca, Francia, Germania, Olanda, Italia, Polonia e Spagna). La commissione Europea non ha definito limiti massimi per tali composti e dai nostri risultati non sono state rilevate sostanze perfluoroalchiliche ad eccezione dell'acido perfluoroottanoico in un solo campione austriaco. I polibromodifenilietere sono stati rilevati solo in 3 dei 77 campioni investigati. I risultati mostrano che i campioni analizzati non rappresentano un rischio per il consumatore. Recentemente una successiva relazione EFSA ha richiesto di porre maggiore attenzione sulla presenza dei perfluoroalchilici, i cui valori soglia sono stati ridotti drasticamente per il loro rischio tossicologico. Nel quinto lavoro ci siamo focalizzati su quattro diverse specie selvatiche (camoscio, capriolo, capriolo, cervo e cinghiale) con abitudini alimentari differenti. Gli animali selvatici sono considerati 8 specie sentinelle e quindi ottimi indicatori ambientali. Campioni muscolari di settantanove animali sono stati raccolti durante la stagione venatoria in una zona montana dell'Italia settentrionale. Nei campioni non sono stati trovati polibromodifenilieteri. Al contrario i pesticidi organoclorurati e organofosforati e i policlorobifenili sono stati rilevati in quasi tutti i campioni a diversi intervalli di concentrazione, mostrando una frequenza maggiore nelle specie di ungulati rispetto al cinghiale. I PFA ,invece, sono stati riscontrati solo nei cinghiali. Tra gli idrocarburi, antracene e benzopirene, sono stati trovati solo nel camoscio a basse concentrazioni. Possiamo nuovamente concludere che per il frequente ritrovamento a basse concentrazioni dei contaminati, ad eccezione di singoli composti riscontrati ad alte concentrazioni, e del basso consumo di carne di selvaggina rapportata ad altre tipologie di carne, vi è un basso rischio per il consumatore italiano. Un ulteriore tema di crescente interesse per la sanità pubblica è stato lo studio dei contaminati ambientali persistenti nel miele, in particolare il miele biologico. Infatti, nonostante l'apicoltura biologica escluda (o consenta in modo restrittivo) l'impiego di farmaci o pesticidi, molti inquinanti possono contaminare api, miele e polline. Pertanto, l'attenzione si è concentrata sullo studio di un ampio spettro di analiti quali, pesticidi, inquinanti organici persistenti e antibiotici in mieli organici raccolti in diverse aree produttive con diverso impatto agricolo, zootecnico o antropico per verificare il potenziale trasferimento di xenobiotici nella catena di approvvigionamento da fonti diverse rispetto alle pratiche apistiche. È stata confermata la presenza di diversi composti, come policlorobifenili, i polibromodifenilietere e gli idrocarburi policiclici aromatici non solo nelle arnie in prossimità di centri altamente urbanizzati, dove le concentrazioni erano più elevate, ma in tutti i contesti ambientali, confermando la possibilità di trasferimento da fonti ambientali e l'ubiquità di tali composti. Il mancato ritrovamento di antibiotici nei campioni analizzati esclude la possibilità di trasferimento accidentale delle molecole dall'ambiente in cui sono posizionate le arnie. Per ottenere una così ampia e diversificata ricerca, ogni lavoro è stato approciato in modo differente per il pretrattamento dei campioni, l'ottimizzazione del metodo analitico, l'estrazione degli analiti e il loro successivo clean up prima dell'analisi con cromatografia liquida in spettrometria di massa tandem (LC-MS/MS) o gas spettrometria (GC-MS/MS). L'approccio di natura analitico-strumentale ha richiesto per ogni ricerca un'accurata e ampia ricerca per ottenere l'ottimizzazione delle prestazioni strumentali e delle fasi di pretrattamento dei campioni, al fine di raggiungere buoni livelli di sensibilità, specificità e robustezza dei metodi analitici impiegati per poi fare 9 considerazioni di natura qualitativa, quantitativa e statistica. La pianificazione delle prove, l'ottimizzazione e la convalida dei metodi sono state eseguite secondo la Commissione SANTE/10553/2018 (SANTE 2018). I risultati di questo lavoro suggeriscono che il rischio per la salute media dei consumatori è basso. Le concentrazioni ambientali dei composti organoclorurati persistenti sembrano diminuite negli ultimi due decenni, probabilmente grazie ai progressi nella rilevazione analitica e al miglioramento dei controlli europei. I PCB sono ancora presenti nell'ambiente a causa del loro ampio impiego a livello industriale nel secolo scorso e delle loro peculiarità chimico fisiche, anche se il loro uso, oggi, è stato vietato in molte applicazioni. Per quanto riguarda i composti emergenti, i PFAs destano preoccupazione a causa del loro ampio uso e del loro possibile ruolo tossicologico. Recentemente la Commissione Europea ha infatti drasticamente diminuito i livelli soglia per queste classi per salvaguardare la salute umana. Gli antibiotici sono ancora motivo di preoccupazione e necessitano di uno stretto controllo per garantire la sicurezza umana e ridurre le resistenze, tema tuttora più che attuale. ; The presence of xenobiotic residues, both drugs and environmental contaminants, in food is a cause for concern and therefore the European Authorities issue reports or laws in order to propose monitoring plans, Health-based Guidance Values (HBGV) and maximum residue levels or maximum levels (MRLs and MLs). Based on these considerations, this doctoral thesis studies the presence of residues in different foods of animal origin, aimed at a characterization of the risk for the consumer. Firstly, we studied seafood, which is an excellent source of nutrients, with important human health benefits. We focused on mussels and clams, filter feeders animals, suitable bio indicator organisms due to their bioaccumulation ability of a wide range of environmental pollutants. In the first research study, we evaluated the Italian consumer risk related to metal exposition through molluscs, on the basis on the MLs stated by the European Union, where available, or, otherwise, based on the HBGV stated by EFSA. About our results, regarding the human metal exposure, we conclude that there is a low risk for the average consumer; however, high percentile consumers, may be subjected to skin lesions, and lung, skin and bladder cancer due to high intake of As, while Ni sensitive individuals can undergo allergic dermatitis due to constant Ni presence in the studied molluscs. Subsequently, we focused on most consumed fish like salmon, tuna which consumption has consistently risen. In the second study about salmon, the aim was to investigate the presence of persistent organic pollutants (POPs) and antimicrobials in wild and farmed salmons from different geographic areas. Farmed salmons showed slightly higher presence of environmental contaminants than wild ones, likely due to the decreased possibility of a constant exposition. Antibiotics were seldom found only in farmed salmon. Risk related to organophosphate compounds (Ops), polycyclic aromatic hydrocarbons (PAHs), polychlorobiphenyls (PCBs) and organochlorine pesticides (OCPs) deriving from salmon intake were of is very low concern, while the presence of polybromodiphenyl ether (PBDE99) and perfluorooctanoic acid (PFOA), is a cause for a bit higher concern. The substantial lack of data about the detected antibiotics in salmon did not allow an extrapolation from MRLs of terrestrial animals and a risk characterization In the third work on tuna, a long-living fish with high biomagnification ability, we studied the presence of metals with high toxicological importance for public health (Hg, Pb, Cd, As, Cr, Ni). One hundred thirty-one samples were analysed. One red tuna from the Adriatic Sea and 11 yellow tunas exceeded Pb maximum levels (MLs); three red tunas from different Mediterranean sub-areas exceeded Hg MLs. The evaluation of cumulative effects indicated that only a negligible health hazard could derive from the ingestion of tuna, for both average and high consumers. The risk of carcinogenicity from Cr is still under debate at the concentrations detectable in food. In these two works, we confirm a low risk, related to the studied compounds, for average consumer health due to fish consumption. The regular consumption of meat and meat products provides a significant intake of proteins and essential micronutrients. Pork meat, for example, is used in many countries to produce derivative products (hams and cured meats) with high qualitative value. Also, game animal meat consumption, though being a niche product, is constantly increasing and hunters, their families and persons closely associated with them can be regarded as a high consumption subpopulation. Furthermore, game animals are a suitable indicator about environmental pollutant such as PCBs, PBDEs, PAHs and brominated flame retardants (BFRs). In the fourth work we studied the occurrence of PBDEs and perfluoroalkyl substances (PFASs) from eight EU Member States (Austria, Denmark, French, Germany, Holland, Italy, Poland and Spain). The European Commission has not stated maximum limits (MLs) for some environmental pollutants such as polybrominated diphenyl ether PBDEs and PFASs; no perfluoroalkyl substances were detected, except PFOA, in only one Austrian sample. PBDEs were detected in three out of 77 samples: the one coming from Germany showed the presence of all congeners analyzed the ones from Netherland and Italy, respectively PBDE 153 and PBDE 100. The results show that the analyzed samples do not pose a risk for human beings about PFASs and PBDEs. A following report from EFSA, requires a new attention on PFAS, with HBGV being drastically reduced. In the fifth work we studied four different animal species (chamois, roe deer, red deer and wild boar) that have different nutrition habits. Game animals are a suitable sentinel species to have a picture of the environment. Muscle samples from seventy-nine animals were collected during the hunting season in a Northern Italy mountain area. No PBDEs were found in the samples. OCPs, OPs and PCBs were detected in almost all samples at different concentration ranges, showing higher frequency in ungulate species than in wild boar. PFAs were found only in wild boar. Anthracene and benzopyrene, among PAHs, were found only in chamois at low concentrations. A low risk for consumers can be indicated due to the frequent detection of contaminants at trace levels, to the scarce prevalence of high concentrations of some contaminants and to the low consumption of game animal meat. An important topic in the researches carried out in my doctorate was the investigation of POPs in organic honey. However, even if organic beekeeping excludes (or restrictively allows) the use drugs or pesticides many pollutants may contaminate bee matrices, comprising bee, honey and pollen. Therefore, the focus was the investigation of a broad spectrum of analytes, pesticides, persistent organic pollutants and antibiotics in organic honeys collected in different productive areas with different agricultural, zootechnical or anthropic impact to verify the potential transfer of xenobiotics into supply chain from different sources than beekeeping practices. The presence of several compounds, such as PCBs, PBDE and PAHs was confirmed, not only in proximity to highly urbanised centres, where the concentrations were higher, but in all environment contexts, confirming the theory that these are ubiquitous contaminants. No antibiotics were found in samples analysed suggesting that presence of antibiotics is from beekeeping practices. The analytes in the different matrices required different approaches for sample pretreatment, extraction, clean up and fractionation before the analysis with liquid chromatography–tandem mass spectrometry (LC-MS/MS) or – gas mass spectrometry (GC-MS/MS). The approach of analytical-instrumental nature has provided for the optimisation of instrumental performances as well as of the steps of sample pretreatment, in order to achieve good levels of sensitivity, specificity and robustness of the method to then make considerations of qualitative, quantitative and statistical nature. The trials planning, optimisation and validation of the methods were performed according to Commission SANTE/10553/2018 (SANTE 2018). The results of this manuscript suggest that there is a low risk for the average consumer health. Environmental concentrations of persistent organochlorine compounds have been decreasing over the past two decades, and this correlates with remarkable advances in the detection of exceedingly low levels of these compounds in human populations and the improvement of European control. PCBs still are present in environment due to their industrial source even if their use was banned in many industries application. Regarding emerging compounds, PFAs still need to be concern due to their wide use and their possible toxicological role. Recently European commission decreased the HBGVs for these classes to safeguard human health. Antibiotics still are a matter of concern and need a close control to ensure human safety and decrease antimicrobial resistance.
Durch genetischen Fortschritt und eine Verbesserung des Managements werden in der modernen Milchviehhaltung immer höhere Milchleistungen erzielt, während der Metabolismus der einzelnen Kühe so vermehrt vor Herausforderungen gestellt wird. Die abnehmende Futteraufnahme rund um die Abkalbung führt zusammen mit dem Bedarf des Fetus für das Wachstum und den Bedarf der einsetzenden Laktation zu einer negativen Energiebilanz der Kuh. Es ist nicht überraschend, dass in dieser frühen Phase der Laktation die höchste Inzidenz an Produktionserkrankungen vorliegt. Daher konzentriert sich das Gebiet der Rinderernährung seit langer Zeit auf die Erforschung von Maßnahmen, welche die Futteraufnahme, den Energiestatus, den Metabolismus und die Gesundheit der Milchkuh in dieser kritischen Phase unterstützen. Solche Eigenschaften wurden für das ionophore Antibiotikum Monensin nachgewiesen, da gezeigt wurde, dass es die mikrobielle Pansenflora hin zu Gram-negativen Bakterien verschiebt und so zu einer gesteigerten Propionatbildung und einer verminderten Methanbildung führt. Propionat stellt den Hauptvorläufer der hepatischen Glukoneogenese dar und könnte daher die Glukoseverfügbarkeit rund um die Abkalbung verbessern und so dem postpartalen Energiemangel entgegenwirken. Nachdem Antibiotika zur Leistungsförderung in der Europäischen Union verboten wurden, hat sich die Suche nach natürlichen Alternativen zu Monensin stark vermehrt und insbesondere die ätherischen Öle sind in den Fokus gerückt. Denn für diese sekundären Pflanzenmetaboliten konnte ebenfalls eine Beeinflussung der Pansenflora nachgewiesen werden, obwohl die Ergebnisse hinsichtlich des Fermentationsprofiles der kurzkettigen Fettsäuren nicht immer konstant waren sondern abhängig von der verwendeten Komponente und der Dosis. Vor Kurzem wurde Monensin als intraruminaler Bolus wieder in der Europäischen Union für hoch konditionierte Kühe als Tierarzneimittel kurz vor der Abkalbung zugelassen. Doch im Angesicht des öffentlichen Interesses für mögliche Rückstände von Antibiotika in Nahrungsmittel tierischen Ursprunges und der Entwicklung von bakteriellen Resistenzen, besteht nach wie vor Bedarf nach natürlichen Alternativen zu Monensin. Den Blutmetaboliten nicht-veresterte Fettsäuren (NEFA) und beta-Hydroxybutyrat (BHB), welche besonders in Energiemangelsituation stark ansteigen, wird eine zentrale Rolle in der verminderten Immunfunktion rund um die Abkalbung nachgesagt. Daher stellt sich ebenso die Frage, ob Monensin und ätherische Öle auch einen Einfluss auf die Immunfunktion haben, zum einen indirekt, über eine Verbesserung des Energiehaushaltes der Kuh, oder aber direkt und so den Gesundheitsstatus in der frühen Laktation verbessern können. Hierfür wurde eine Studie mit 60 multiparen Deutsch Holsteinkühen (mittlere Laktation 2,3 ± 1,4 (Standardabweichung)) durchgeführt, welche auf einem Tiermodell zur Untersuchung der subklinischen Ketose basierte. Die Kühe wurden 6 Wochen vor der Abkalbung entsprechend ihrer Körperkondition (BCS) in eine Gruppe geringerer (LC, n = 14) und höherer Kondition unterteilt. Letztere wurden in der Trockensteherphase überfüttert und in der frühen Laktation verzögert mit Energie versorgt, um eine negative Energiebilanz mit einhergehender Körperfettmobilisierung und Ketonkörperbildung hervorzurufen. Diese hoch konditionierten Tiere wurden entweder einer Kontrollgruppe zugeordnet (HC, n = 13) oder sie erhielten einen Monensin-freisetzenden intraruminalen Bolus 3 Wochen vor der Abkalbung (HC/MO, n = 14) oder ein Gemisch von ätherischen Ölen von Tag -21 bis Tag +56 bezogen auf die Abkalbung (HC/EO, n = 15). Es wurden Futtermittelproben genommen und Leistungsparameter dokumentiert und des Weiteren Blut-, Pansensaft-, Milchproben und Leberbiopsien entnommen. Hierdurch wurden die Leistung und der Energiehaushalt der Kühe erfasst und das ruminale Fermentationsprofil, biochemische und hämatologische Variablen, Antikörper gegen das Bovine Virale Diarrhö Virus (BVBV) und die Populationen und Funktionen der Leukozyten ermittelt. Die Ergebnisse deuten darauf hin, dass die Ziele des Tiermodells erfolgreich erreicht wurden: Die Kühe konnten durch die Fütterung zur Abkalbung weiter zunehmen und haben nach dieser enorm in der Frühlaktation an Körpermaße verloren, wie man an den BCS und NEFA Werten erkennen kann. Die Prävalenz der subklinischen und klinischen Ketose (BHB > 1.2 mmol/L) lag in der HC Gruppe 36% höher als in der LC Gruppe. Im ruminalen Fermentationsprofil konnte eine gesteigerte Propionatbildung in der Monensingruppe, aber nicht in der ätherischen Öle Gruppe, nachgewiesen werden, welche in keiner der beiden Gruppen mit einer gesteigerten Blutglukosekonzentration einherging. Betrachtet man die Leistung der Kühe, konnten die Futteraufnahme (DMI) in der Laktation und die Milchmenge nicht durch die Supplemente verändert werden, aber Monensin führte zu einer gesteigerten Futtereffizienz (DMI/Energie-korrigierte Milch). Biochemische Parameter deuten darauf hin, dass in den hoch konditionierten Tieren eine verminderte Leberfunktion und -integrität vorlag, da die Konzentration im Blut von Bilirubin in allen hoch konditionieren Tieren und von Aspartat-Aminotransferase (AST), gamma-Glutamyltransferase (GGT) und Glutamat Dehydrogenase (GLDH) in der HC und HC/EO Gruppe erhöht waren. Monensin konnte die Konzentration von BHB im Blut senken, obwohl die NEFA Freisetzung unverändert war. Es führte zu einer geringeren Konzentration von Blutmetaboliten, die auf eine verminderte Hepatozytenintegrität hinweisen könnten. Die Immunparameter zeigten eine deutliche Beeinflussung durch die Abkalbung, wie es besonders an der Produktion von reaktiven Sauerstoffspezies (ROS) in den neutrophilen Granulozyten und den BVDV Antikörpertitern sichtbar wurde. Die Antikörperreaktion zeigte sich in der Monensin Gruppe besonders hoch in Vergleich zu der ätherischen Öle Gruppe. Milchkühe, die vor der Abkalbung eine hohe Körperkondition aufweisen, scheinen von einem Monensin-freisetzenden Bolus 3 Wochen vor der Abkalbung zu profitieren, da eine erhöhte Bereitstellung von ruminalem Propionat für die hepatische Glukoneogenese die Energieverfügbarkeit der Kuh zu verbessern scheint und so vermutlich einen antiketogene Wirkung ausübt. Des Weiteren konnte so die Futtereffizienz in der Frühlaktation gesteigert werden. Außerdem lassen die Ergebnisse darauf schließen, dass Monensin direkte oder indirekte Effekte auf die Leber und das Immunsystem ausübt, welche noch weiterer Abklärung bedürfen. Für die supplementierte Dosis des Gemisches von ätherischen Ölen konnte kein Einfluss auf die ruminale Propionatbildung, den Energiehaushalt, die Leistung und die Gesundheit der Milchkühe nachgewiesen werden. Andere Komponenten der ätherischen Öle, andere Gemische und andere Dosen könnten möglicherweise bessere Ergebnisse erzielen. ; Genetic and management progress in modern dairy farming allow continuing increasing milk performance of herds whereas metabolism of individual cows often lags behind. The decreasing feed intake at calving together with growth of the fetus and the initiated lactation leads to a negative energy balance of the cow. It is not surprising that the highest incidence of production diseases can be found in early lactation. Therefore, ruminant nutrition has concentrated for a long time on measures to increase and improve feed intake, energy status, metabolism and health in these critical weeks around calving. One approach is the modulation of fermentation processes in the rumen to increase efficiency to convert feed energy. The ionophore antimicrobial drug monensin was successfully applied for this intention as it leads to a shift of the ruminal microflora towards Gram-negative bacteria increasing propionate and decreasing methane production. Propionate is the main precursor of hepatic gluconeogenesis and may therefore improve glucose availability at calving and so antagonize the postpartal energy deficit. After the ban on antibiotics as feed additives in the European Union, the research in natural alternatives to ionophores was greatly enhanced while essential oils caught great interest. These secondary metabolites obtained from plant material also modulate the rumen microflora although changes in short-chain fatty acid profile are not consistent but dependent on used essential oils component and dose. Recently, monensin was relaunched in the European Union as a ruminal controlled-release capsule (CRC) indicated for high condition dairy cows administered just before calving. However, in times of public attention for possible antibiotic residues in milk and meat and the development of bacterial antibiotic resistances, the quest for alternatives to monensin still proceeds. The blood metabolites non-esterified fatty acids (NEFA) and beta-hydroxybutyrate (BHB) that are high during negative energy balance were attributed an etiological involvement in an impaired immune cell function at calving. This raised the question if monensin and essential oils are able to influence immune function, either indirectly via an improved energy status or even directly, and so to contribute to an improved health status in early lactation. Therefore, a study was conducted with 60 multiparous German Holstein cows (parity 2.3 ± 1.4 (SD)) based on an animal model to investigate subclinical ketosis. They were allocated 6 weeks before calving according to their body condition score (BCS) to high and low condition group (LC, n = 14) while an overfeeding in the dry period and an decelerated energy supple in the high condition group in early lactation was intended to provoke a negative energy balance with subsequent body fat mobilization and ketogenesis. High condition cows formed a control group (HC, n = 13), received a monensin CRC 3 weeks before calving (HC/MO, n = 14) or a blend of essential oils from day -21 until day +56 relative to calving (HC/EO, n = 15). Feedstuff samples and performance parameters together with samples of blood, rumen fluid and milk as well as liver biopsies were taken over the trial to assess performance, energy status, ruminal fermentation pattern, biochemical and hematological parameters, Bovine Viral Diarrhea virus (BVDV) antibody titer, leukocyte subsets and function. Results imply that the purposes of the animal model were successfully achieved as all high condition cows further increased their body condition until calving and showed a massive loss of condition in early lactation as seen in BCS and also serum NEFA concentration. The prevalence of either subclinical or clinical ketosis (BHB > 1.2 mmol/L) was 36% higher in HC than in LC group. Ruminal fermentation pattern showed an increased propionate production in HC/MO but not in HC/EO cows which was not accompanied by a higher serum glucose concentration in both groups. Concerning performance, dry matter intake (DMI) and milk yield were not different after calving whereas monensin increased feed efficiency (DMI/energy-corrected milk). Biochemical parameters give evidence for an impaired liver function and integrity as displayed in increased bilirubin concentrations in all high condition cows and increased levels of aspartate-aminotransferase (AST), gamma-glutamyltransferase (GGT) or glutamine dehydrogenase (GLDH) after calving in HC and HC/EO group. Monensin reduced serum BHB concentration although NEFA mobilization was unaltered and led to a reduced concentration of blood metabolites that may indicate in impaired hepatocyte integrity. Immune parameters were greatly influenced by the event of calving as seen in short-term effects on parameters like reactive oxygen species (ROS) production in polymorphonuclear cells and the BVDV antibody titer. From the immune parameters investigated, the BVDV antibody response was more pronounced in HC/MO compared to HC/EO. Dairy cows with a high condition around calving may profit from a monensin CRC administered 3 weeks before calving, as the energy status was improved likely via an increased supply of ruminal propionate to hepatic gluconeogenesis exerting an antiketogenic effect. Furthermore, the feed efficiency was increased in early lactation. Results suggest direct or indirect monensin effects on the liver and the immune system while nature of these effects needs to be clarified further. The supplemented dose of a blend of essential oils showed no effect on ruminal propionate production, energy status, performance and health of transition dairy cows under the current conditions whereas other solitary components and combinations or doses may achieve better results.
Every year, through mass drug administration (MDA), hundreds of millions of the world's poorest people receive a single annual dose of one or more drugs to eliminate certain parasitic worm or bacterial infections. Some of these infections, mostly prevalent in tropical areas, have traditionally been neglected from the public health and research point of view. These conditions, collectively known as the neglected tropical diseases (NTDs), still cause, at the cusp of the second decade of the 21st century, a significant amount of morbidity and mortality. The existing control measures for NTD have an enormous potential, although there are still some challenges that require further investigation. For some diseases, alternative strategies may be needed, including longer duration of MDA programmes or modified drug regimens. For other diseases, such as yaws, the work must start almost from scratch, since little has been achieved in terms of control of this disease in the past 50 years. Although eight NTDs affect the region, two diseases pose a major public health problem in the South Pacific Islands, namely yaws and lymphatic filariasis and are the basis for his thesis. These two infections were selected for a number of reasons. First, they affect the South Pacific region disproportionately. Secondly, little research has been conducted in the past years. And third, but more importantly, several epidemiological, technological and historical factors make these two diseases amenable to elimination. Safe and effective tools and interventions to achieve these targets are available and concerted efforts to scale them up are likely to lead to success. Yaws is one of the most neglected of the NTDs. Yaws was one of the first diseases to be targeted for eradication on a global scale, efforts which almost led to the disease disappearance as a result of a massive treatment program started in the 1950s. After the successful eradication campaigns the primary health care systems were supposed to give the last push towards eradication of yaws. However a combination of various factors including poor political commitment and limited funding resulted in a progressive abandonment of efforts and the resurgence of the disease. Every new case of yaws was the disappointing confirmation that the public health world had missed a great opportunity. Today yaws has resurged in many tropical areas and presents new challenges including its unknown epidemiological situation, the attenuated clinical forms of the disease, a poor awareness and knowledge among health care workers, the lack of knowledge about the effectiveness of classic treatment with penicillin and, an obvious need for research into simplified administration schemes or new antibiotic treatments, particularly oral ones. There is an enormous knowledge gap regarding current reliable epidemiological information about the disease. Certainly we know little about the burden in the three Melanesian countries where the disease is highly endemic, Papua New Guinea, Solomon Islands and, Vanuatu. In Solomon Islands and Vanuatu there are indications that Yaws is widespread and prevalent, but we know that the diagnosis is unreliable. This takes us to the next point, what does a diagnosis of yaws mean? Overall the natural history of the disease in this era, where it is often subject to inadequate antibiotic pressure, is very unclear. Some authors have suggested that yaws appears to be attenuated in both Solomon Islands and Vanuatu. They state that bone involvement in yaws is now rare and implies that yaws is a mild disease not requiring efforts for elimination. However, the first paper of this thesis describes the epidemiology of yaws in Lihir Island (Papua New Guinea, PNG) and shows a high rate of classical primary ulcers (almost 60%) and significant bone and periosteal involvement (more than 15%), suggesting that "attenuation" is not an important issue. When we look at the diagnostic criteria for yaws, signs and symptoms alone are still used often in many areas to diagnose the disease. This reliance on clinical findings was the result of the difficulty of performing serological tests in remote areas. Today, available rapid serological tests are simple, rapid, inexpensive and useful for guiding confirmation of cases, making them adequate tools for the diagnosis and monitoring of the disease. The clinical diagnosis of yaws is complicated because its clinical manifestations may be unspecific. Thus, it is possible that a significant proportion of yaws cases may in fact have been falsely diagnosed. We show, in the first article, that in our experience only 60% of the cases with a clinical suspicion of yaws were finally confirmed by serologic tests. Therefore, a proper diagnosis of yaws requires the interpretation of clinical findings with reference to laboratory results and the epidemiologic history of the patient. Serological testing in yaws is not only important for diagnostic accuracy, but also is very helpful in defining the disease's evolution and eventual cure after treatment. Rapid plasma regain (RPR) titres should decline within 6-12 months, becoming negative in less than 2 years. The second article of this thesis combines a clinical and serological approach to assess the response after treatment with benzathine benzylpenicillin, and it identifies an overall 20% treatment failure. This could be related to resistance to the antimicrobial drug used or to re-infection caused. The distinction between re-infection and true resistance to antibiotic treatment is difficult to make but these failures are worrisome. This article also proposes a multivariate model performed to identify independent determinants of failure that affected the outcome after treatment. The risk for reinfection caused by repeated contact with infected children seems to be a pivotal predictor of failure. Low baseline titters (<1:32) of RPR are also an important and independent predictor of failure, possibly as a result of the greater difficulty in resolving chronic infections which are usually accompanied by low titters. With yaws re-emerging, the development of new strategies against this infection aimed at simplifying its treatment and potentially re-focussing strategies towards its eradication seems essential. Injectable penicillin is still effective but management with an oral drug that can be easily administered on a large scale should be the preferred method for treatment. To date, there had been no studies that directly compared the efficacy of penicillin with any of the potentially alternative agents shown to work in the treatment of the non-venereal treponematoses. The fourth paper in this thesis has shown that a single-dose of oral azithromycin is non-inferior to benzathine benzylpenicillin for the treatment of yaws in children in PNG. In an open-label randomised trial, at 6-month follow-up, 96% of patients treated with azithromycin were cured, as were 93% in the benzathine benzylpenicillin group. The prospects of eliminating and eventually eradicating yaws may now be enhanced by the use of a single-dose of oral azithromycin in mass drug administration campaigns. Community based mass administration of azithromycin has been widely used in many locations for the control of trachoma, which, like yaws, is a disease of poor rural communities in developing countries, and has been used in a more limited way to control granuloma inguinale and outbreaks of venereal syphilis. Elimination of yaws and lymphatic filariasis in the South-Pacific Islands is now considered biologically feasible and programmatically attainable. The Global Programme to Eliminate Lymphatic Filariasis (GPELF) has expanded quickly to reach the target of elimination by 2020. On the other hand the strategy to eliminate yaws is again at the centre of discussions and given that infected humans are the only source of disease, its eradication could be achieved within a very relatively short time. The fifth article of the thesis comprehensively reviews antimicrobial treatments and elimination strategies against yaws. In order to control yaws and push it towards elimination, we propose to move away from penicillin to azithromycin and use mass treatment campaigns of the entire population in endemic communities irrespective of the prevalence. Also, to make sure all cases are tracked down and treated, strict follow-up measures and selective mass treatment will be required until zero case prevalence is reached. Importantly, we suggest testing the principle of interrupting transmission in pilot implementation studies, including prevalence surveys to assess the impact of the intervention and macrolide resistance monitoring which in our opinion will be essential evaluation tools to guide us towards a sustainable elimination. Lymphatic filariasis (LF), caused by the mosquito-borne nematode Wuchereria Bancrofti, is a major public-health problem in the Melanesian countries. Annual MDA over five years is currently the WHO's recommended strategy to eliminate lymphatic filariasis. This approach aims to suppress microfilaraemia in infected individuals and bring the infection below a threshold that leads to interruption of transmission. However theoretical work and clinical field experience has highlighted how the ecological diversity between different endemic regions can result in elimination thresholds that vary between local communities. This means that the duration required might be different for different areas. Other variables have also been previously identified as potentially having an influence on the outcome of the program, including baseline prevalence of infection, vector density or the treatment coverage. The last article of this thesis provides data about the impact of a five-year filariasis control program in Papua New Guinea. The findings reported support this strategy for areas with low-to-moderate rates of transmission in regions where anopheline mosquitoes transmit this infectious disease. Additional measures or longer periods of treatment may be necessary in areas with a high rate of transmission. The experience acquired on Lihir Island in MDA programs during the campaigns for the elimination of filariasis, will be very valuable when implementing a pilot strategy for yaws control. Also, in the near future it might be important to link yaws mass treatment with other mass programmes to increase efficiency. The plan for elimination of lymphatic filariasis in PNG was approved as a pilot project in 2005 but the program still needs to be extended to the total of 20 provinces in the country where filariasis is endemic. In this context, an integrated approach to NTD control could represent an important global public health solution in PNG and other South Pacific Islands. Little has been achieved in the past decade in NTDs. We are now in a good position to translate into policies the results of our research projects. A new elimination policy for yaws around the azithromycin pillar has been sketched a WHO consultation meeting held in Morges, Switzerland last March. In the intentions of the organization, a last global mass campaign to tackle yaws should permit to reach zero cases in 2017, and the subsequent certification of worldwide interruption of transmission by 2020. ; Cada año, a través de la administración masiva de medicamentos (MDA), cientos de millones de personas, las más pobres del mundo, reciben una dosis única de uno o más medicamentos para eliminar ciertas infecciones, parasitarias o bacterianas. Algunas de estas infecciones, frecuentes sobre todo en las zonas tropicales, han sido tradicionalmente desatendidas desde el punto de vista de salud pública e investigación. Estas enfermedades, conocidas comúnmente como las enfermedades tropicales desatendidas (ETD), aún causan, en el inicio de la segunda década del siglo 21, una cantidad significativa de morbilidad y mortalidad. Las medidas de control actuales para ETDs tienen un enorme potencial, pero todavía existen algunas cuestiones que requieren investigación. Para algunas de estas infecciones, son necesarias estrategias alternativas, incluyendo una mayor duración de los programas de MDA o regímenes modificados de medicamentos. Para otras enfermedades, como la enfermedad de pián, el trabajo debe comenzar casi desde cero, ya que poco se ha logrado, en términos de control de esta enfermedad, en los últimos 50 años. Aunque ocho ETDs afectan a la región, dos enfermedades constituyen un problema importante de salud pública en las Islas del Pacífico Sur, a saber: el pián y la filariasis linfática y son la base de esta tesis. Estas dos infecciones fueron elegidas por muchas razones. En primer lugar, afectan a la región del Pacífico Sur de forma desproporcionada. En segundo lugar, pocas investigaciones se han llevado a cabo en los últimos años. Y en tercer lugar, pero lo más importante, varios factores epidemiológicos, tecnológicos e históricos hacen que estas dos enfermedades sean susceptibles de eliminación. Existen armas terapéuticas seguras y eficaces para lograr este objetivo, y esfuerzos coordinados para ejecutar los programas de control pueden conducir al éxito. El pián es una de las más olvidadas de las ETDs. Ésta fue una de las primeras enfermedades en ser objetivo de erradicación a escala global. Los esfuerzos de un programa de tratamiento masivo, que se inició en la década de 1950, casi llevaron a la desaparición de la enfermedad. Después de las exitosas campañas de erradicación, los sistemas de salud de atención primaria debían dar el último empujón hacia la erradicación del pián. Sin embargo, una combinación de varios factores, incluyendo un pobre compromiso político y una financiación limitada, dieron como resultado el abandono progresivo de los esfuerzos y el resurgimiento de la enfermedad. Cada nuevo caso de pián era la decepcionante confirmación de que el mundo de la salud pública había perdido una gran oportunidad. Hoy la enfermedad de pián ha resurgido en muchas áreas tropicales con nuevos desafíos: una situación epidemiológica desconocida, formas clínicas atípicas o atenuadas, poco conocimiento de la enfermedad entre el personal sanitario, la falta de datos acerca de la eficacia del tratamiento clásico con penicilina inyectable y la necesidad de desarrollar esquemas terapéuticos simplificados o investigar en nuevos tratamientos antibióticos, en especial de administración oral. Actualmente hay una enorme brecha de conocimiento entorno a la información epidemiológica fiable sobre la enfermedad. Ciertamente, sabemos poco acerca de la incidencia en los tres países melanesios, donde la enfermedad es altamente endémica, Papúa Nueva Guinea (PNG), Islas Salomón y Vanuatu. En las Islas Salomón y Vanuatu, las cifras de incidencia son muy altas lo que demuestra que el pián es una enfermedad frecuente y ampliamente extendida, pero sabemos que el diagnóstico no es muy fiable. Esto nos lleva al siguiente punto: ¿Cuáles son los criterios diagnósticos del pián? En general, la historia natural de la enfermedad en la época actual, donde la bacteria es objeto de presión antibiótica inadecuada, no es muy clara. Algunos autores han escrito que el pián parece presentar manifestaciones "atenuadas" en las Islas Salomón y Vanuatu. Afirman que la afectación ósea en el pián es poco frecuente, lo que implica que el pián es una enfermedad leve que no requeriría esfuerzos para su eliminación. Sin embargo, el primer trabajo de esta tesis describe la epidemiología del pián en la Isla de Lihir (Papúa Nueva Guinea) y muestra una alta tasa de úlceras primarias clásicas (casi el 60% de casos) y una afectación significativa del hueso y periostio (más del 15%) que sugiere que la "atenuación" no es un tema importante. Cuando nos fijamos en los criterios diagnósticos, únicamente signos y síntomas todavía se utilizan en muchas áreas para el diagnóstico de la enfermedad. Esta confianza en los hallazgos clínicos fue el resultado de la dificultad de realizar pruebas serológicas en las zonas remotas. Hoy en día, las pruebas serológicas rápidas son simples, rápidas, económicas y útiles para orientar la confirmación de los casos. El diagnóstico clínico del pián es complicado debido a que sus manifestaciones pueden ser inespecíficas. Así, es posible, que una proporción significativa de los casos de pián puedan haber sido falsamente diagnosticados. En el primer artículo, presentamos que, en nuestra experiencia, sólo el 60% de los casos con sospecha clínica de pián fueron finalmente confirmados por pruebas serológicas. Por lo tanto, un diagnóstico adecuado del pián requiere la interpretación de los hallazgos clínicos con referencia a los resultados de laboratorio y la historia epidemiológica de los pacientes. Las pruebas serológicas en el pián no sólo son importantes para el diagnóstico de la enfermedad, también son muy útiles en la definición de curación después del tratamiento. En la prueba de la Reagina plasmática rápida (RPR) los títulos deben descender a los 6-12 meses, llegando a ser negativa en menos de 2 años. El segundo artículo de esta tesis combina un enfoque clínico / serológico para evaluar la respuesta a bencilpenicilina benzatina, e identifica una tasa de fracaso terapéutico del 20% a los 12 meses del tratamiento. Esto podría estar relacionado con resistencia al fármaco antimicrobiano, o bien indicar una re-infección por re-exposición. La distinción entre la re-infección y la resistencia verdadera al tratamiento es difícil, pero estos fracasos terapéuticos son preocupantes. En este artículo se describe un modelo multivariante realizado para identificar los factores determinantes del fracaso terapéutico. El riesgo de re-infección causado por el contacto repetido con otros niños infectados parece ser un predictor fundamental de fracaso. También es un factor de riesgo, los títulos basales bajos (< 1:32) de RPR. Este último factor podría estar relacionado con la mayor dificultad para resolver infecciones crónicas (en estadio secundario), habitualmente acompañadas de títulos bajos. Con la enfermedad de pián re-emergiendo, el desarrollo de nuevas estrategias contra la infección para hacer más fácil los esfuerzos de erradicación es esencial. La penicilina inyectable sigue siendo eficaz, pero el tratamiento con un fármaco por vía oral que pueda ser fácilmente administrado a gran escala es el método preferido para el tratamiento, prevención y finalmente eliminación en todas las regiones endémicas del mundo. Hasta la fecha, no ha habido estudios que comparen directamente la eficacia de la penicilina con cualquiera de los agentes alternativos en el tratamiento de las treponematosis no venéreas. El cuarto artículo de esta tesis ha demostrado que una dosis única de azitromicina por vía oral no es inferior a la bencilpenicilina benzatina intramuscular, para el tratamiento del pián en niños en Papúa Nueva Guinea. En un ensayo abierto, aleatorio, el 96% de los pacientes tratados con azitromicina estaban curados a los 6 meses de seguimiento, al igual que el 93% en el grupo de bencilpenicilina benzatina. Las perspectivas de finalmente erradicar el pián son ahora mayores, mediante el uso de una dosis única de azitromicina oral en campañas masivas de tratamiento. El tratamiento masivo con azitromicina ha sido ampliamente utilizado para el control del tracoma, que, al igual que el pián es una enfermedad de comunidades rurales pobres de países en desarrollo. También se ha utilizado de una manera más limitada para controlar el granuloma inguinal y brotes de sífilis venérea. En general, el uso de azitromicina ha demostrado ser seguro, y de hecho ha habido beneficios inesperados de salud en algunos programas. La eliminación del pián y la filariasis linfática en las Islas del Pacífico Sur se considera ahora biológicamente factible y operacionalmente alcanzable. El Programa Global para Eliminar la Filariasis Linfática (GPELF) se ha expandido rápidamente para alcanzar la meta de eliminación en el año 2020. Por otro lado la estrategia para eliminar el pián es nuevamente centro de atención. Además, dado que los seres humanos infectados son la única fuente de la enfermedad, su eliminación podría lograrse en un plazo relativamente corto. El quinto artículo de la tesis revisa de forma integral el tratamiento con antimicrobianos y las estrategias de eliminación contra el pián. Con el fin de controlar el pián hasta la erradicación, se propone pasar de la penicilina a la azitromicina, y el uso de campañas de tratamiento masivo de toda la población en todas las comunidades endémicas. Además, para asegurar que todos los casos son encontrados y tratados, serán necesarias medidas estrictas de seguimiento y tratamiento masivo selectivo hasta llegar al objetivo de cero casos clínicos. Es importante destacar que el principio de interrupción de la transmisión se debe probar en estudios piloto, incluyendo estudios de prevalencia, para monitorizar el impacto de la intervención, y también la valoración de resistencia a macrolidos, que en nuestra opinión, serán herramientas fundamentales que nos guíen en el camino hacia una eliminación sostenible La filariasis linfática (FL), causada por el nematodo Wuchereria bancrofti, es otro de los grandes problemas de salud pública en los países de la Melanesia. Un curso de MDA anual, durante cinco años, es la estrategia que la OMS recomienda para eliminar la FL. Este enfoque tiene como objetivo suprimir la microfilaremia en los individuos infectados y disminuir los niveles de infección por debajo de un umbral que conduzca a la interrupción de la transmisión. Sin embargo, trabajo teórico y experiencia práctica clínica han puesto de relieve cómo la diversidad ecológica, entre diferentes regiones endémicas, puede resultar en que los umbrales de eliminación varíen en diferentes comunidades. Esto significa que la duración requerida podría ser diferente para diferentes áreas. Algunas variables que han sido previamente identificadas como potenciales determinantes en el resultado de un Programa para la eliminación de FL (PELF) son la prevalencia basal de infección por filariasis, la densidad de vectores (mosquitos) o la cobertura del tratamiento en la población. El último artículo de esta tesis, proporciona datos sobre el impacto de un PELF de cinco años en PNG. Los resultados obtenidos apoyan la estrategia descrita para las zonas con baja a moderada tasas de transmisión en regiones donde mosquitos anofelinos transmiten la infección (pe. Melanesia, África). Medidas adicionales o períodos más largos de tratamiento pueden ser necesarios en áreas con una alta tasa de transmisión. La experiencia adquirida en la Isla de Lihir en los programas de tratamiento masivo durante las campañas para la eliminación de la filariasis, será muy valiosa en la aplicación de una estrategia piloto para el control del pián. Además, en un futuro próximo podría ser importante vincular los programas para el control del pián con otros programas de tratamiento masivo (por ejemplo, filariasis) para aumentar la eficiencia y reducir los costos. El plan para la eliminación de la filariasis linfática en PNG fue aprobado como proyecto piloto en 2005 en la provincia de Milne Bay. El programa todavía tiene que ser extendido a un total de 20 provincias en el país, donde la filariasis es endémica. En este contexto, un enfoque integrado para el control de enfermedades tropicales olvidadas podría representar una importante solución global de salud pública en PNG. Poco se ha logrado en la última década en enfermedades tropicales desatendidas. Ahora estamos en una buena posición para traducir los frutos de nuestra investigación en políticas de salud. Durante una consulta celebrada en la sede de la OMS en Ginebra el pasado mes de marzo, ya se ha esbozado una nueva política de eliminación para el pián que toma como pilar el tratamiento con azitromicina. La intención de la OMS es que una última campaña global debe permitir llegar a cero casos de pián en 2017, y la posterior certificación de la interrupción de la transmisión en todo el mundo en el año 2020.
Esta tesis doctoral se ha realizado dentro del marco de un acuerdo de co-tutela entre la Universidad de Zaragoza (Universidad de origen), la Universidad de Calabria (Universidad anfitriona) y la Facultad de Ciencias y Tecnología de la Universidad NOVA de Lisboa (FCT NOVA) (Universidad anfitriona). El trabajo de investigación se ha llevado a cabo dentro del programa de Doctorado en Ingeniería de Membranas Erasmus Mundus (EUDIME), (FPA 2011-0014), financiado por la Unión Europea. La tesis se centró principalmente en el uso de la técnica de electrohilado para producir diferentes tipos de membranas que puedan ser utilizadas en distintas aplicaciones biomédicas. Se sintetizaron y produjeron nanopartículas orgánicas e inorgánicas para ser utilizadas como rellenos o como portadores (sistema de administración de fármacos), así como membranas nanofibrosas electrohiladas. Este trabajo se llevó a cabo en el Instituto de Nanociencia de Aragón (INA), específicamente en el grupo de Nanostructured Films and Particles (NFP) bajo la supervisión de la profesora Silvia Irusta y la Dra. Gracia Mendoza. Una parte importante de la caracterización físico-química se realizó en el INA. En la Universidad de Calabria se trabajó bajo la supervisión de la Dra. Loredana de Bartolo en el Instituto de Tecnología de Membranas (ITM). Allí se utilizaron técnicas específicas tanto para la caracterización como para estudiar diferentes señales biológicas producidas por las membranas sintetizadas, bajo la supervisión. Por otro lado, la movilidad llevada a cabo en la Facultad de Ciencias y Tecnología (FCT NOVA) de la Universidade NOVA (FCT NOVA) bajo la supervisión de la profesora Ana Isabel Aguiar-Ricardo, permitió realizar una caracterización completa de dos membranas asimétricas siguiendo diferentes Normas Internacionales que establecen diferentes ensayos a realizar en apósitos primarios utilizados en heridas. El desarrollo de nuevos scaffolds cargados con proteínas morfogenéticas o antibióticos es de gran interés en el campo de la ingeniería de tejidos óseos. Scaffolds electrohilados con una microporosidad mejorada puede ser beneficioso para mejorar la viabilidad celular debido a que una alta porosidad junto a la presencia de microporos puede proporcionar un entorno tridimensional (3D) que no solamente facilita la siembra y difusión celular sino también proporciona una mejor difusión de los nutrientes y residuos a través del scaffolds. La adición de cerámica de fosfato de calcio ha sido ampliamente investigada para fabricar scaffolds altamente porosos para la ingeniería de tejidos óseos debido a que presentan una composición muy similar al hueso, incluyendo excelentes propiedades de biocompatibilidad, osteoinductivas y osteoconductoras. Partículas cargadas con proteínas morfogenéticas de hueso distribuidas homogéneamente en el scaffolds podrían asegurar una liberación continua del factor de crecimiento proporcionando de esta forma las señales bioquímicas necesarias para la reparación y regeneración ósea. Los scaffolds cargados con antibióticos pueden proporcionar una liberación sostenida del fármaco en el sitio de interés, así como el mantenimiento de propiedades osteogénicas mejoradas para la regeneración exitosa del hueso. Evitando de esta forma que se alcancen niveles de toxicidad o niveles ineficaces en la zona de interés, así como la aparición de efectos secundarios indeseados en los pacientes que provocan un rechazo a los tratamientos prolongados de fármacos por vía sistemática (vía oral e intravenosa). Otra aplicación biomédica interesante de las membranas electrohiladas es la fabricación de apósitos inteligentes eficientes para el tratamiento de heridas. Para lograr una curación rápida de la herida es necesario desarrollar membranas apropiadas con poros interconectados capaces de prevenir la deshidratación rápida y la penetración de bacterias. Para mantener un ambiente húmedo en el lecho de la herida se necesita una alta capacidad de absorción y una adecuada transmisión de vapor de agua. Además, si la membrana electrohilada presenta propiedades bactericidas facilitará el proceso de curación. El objetivo principal de esta tesis fue el desarrollo mediante electrohilado de membranas fibrosas con las características apropiadas para ser utilizadas en la ingeniería de tejidos óseos o como apósito para heridas. En los Capítulos II al V se plantean una serie de objetivos específicos con el fin de cumplir el objetivo principal. Este documento de tesis se dividió en las siguientes secciones: CAPÍTULO I, corresponde a la introducción general donde se describen los conceptos de biomateriales, scaffolds, ingeniería de tejidos y el objetivo principal de los sistemas de liberación de fármacos. Así como, la clasificación de los biomateriales y la ingeniería de tejidos según el origen de los materiales. Además se ponen de manifiesto todos los factores que deben tenerse en cuenta para desarrollar y aplicar adecuadamente los apósitos para heridas. Se mencionaron las diferentes técnicas utilizadas en la literatura haciendo énfasis en el uso de electrohilado y electropulverización para producir scaffolds o membranas para su uso en la ingeniería del tejido óseo y como apósitos para heridas. CAPÍTULO II, se enfoca en el desarrollo y mejora de andamios 3D capaces de promover una eficiente regeneración ósea junto con la liberación de antibióticos dirigidos para prevenir la colonización de bacterias. El objetivo de este trabajo fue sintetizar y caracterizar un sistema de liberación de fármacos que consiste en nanofibras electrohiladas de policaprolactona (PCL) decoradas con partículas de poli (ácido láctico-coglicólico) (PLGA) cargadas con rifampicina (RFP). Este material debe promover la reparación ósea evitando el deterioro del scaffolds provocado por una infección. Se realizó la evaluación in vitro de la capacidad bactericida del material electrohilado sintetizado contra bacterias Gram positivas (Staphylococcus aureus) y Gram negativas (Escherichia coli), así como su citocompatibilidad en cultivos 3D con osteoblastos humanos. Estos resultados se enviaron a la Revista de farmacia "International Journal of Pharmaceuitics" para su publicación en formato de artículo y está bajo revisión. CAPÍTULO III, se describe la síntesis y caracterización de membranas con estructura de núcleo-envoltura de PCL y acetato de polivinilo (PVAc) obtenidas por electrohilado. Las fibras se cargaron con nanopartículas de hidroxiapatita sintética (HAn) para aumentar la bioactividad de los materiales. Los scaffolds desarrollados se trataron con ablación láser para crear características topográficas deseadas a nivel micrométrico con el objetivo de favorecer la adhesión y crecimiento celular. Todas las membranas obtenidas presentaron una estructura de poros tridimensionalmente interconectados y el tratamiento con láser provocó un aumento en la viabilidad y densidad celular. Además, el aumento en la biocompatibilidad de los scaffolds sugiere que los microporos pequeños favorecen la adhesión y proliferación celular. Estos resultados fueron publicados en el artículo titulado "Laser-treated electrospun fibers loaded with nano-hydroxyapatite for bone tissue engineering". Javier Aragon, Nuria Navascues, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics 525,112–122, 2017. DOI:10.1016/j.ijpharm.2017.04.022. CAPÍTULO IV, se refiere al desarrollo de un scaffold electrohilado compuesto por fibras con estructura de núcleo-cubierta de PCL o PCL/PVAc cargado con HAn sintética. Estas fibras se decoraron con partículas de PLGA cargadas con proteína morfogenética ósea 2 (BMP2) mediante el uso simultaneo de electrohilado coaxial y electropulverización. El objetivo de este trabajo fue evaluar las propiedades estructurales y físico-químicas así como el proceso de biodegradación de los nuevos scaffolds desarrollados y su capacidad para abordar las características arquitectónicas, bioquímicas y funcionales del tejido óseo. Para esto, se probó la bioactividad del scaffold mediante el cultivo de osteoblastos humanos sobre ellos y se monitoreo de la viabilidad celular durante 4 semanas. Se evaluó la actividad osteogénica in vitro de las células sembradas sobre los scaffolds determinando la actividad de la fosfatasa alcalina (ALP) y la expresión de osteocalcina (OCN) y osteopontina (OPN) como proteínas osteogénicas. Estos resultados fueron publicados en el artículo titulado "Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering". Javier Aragón, Simona Salerno, Loredana De Bartolo, Silvia Irusta and Gracia Mendoza. Journal of Colloid and Interface Science, 531 (2018) 126–137. DOI:10.1016/j.jcis.2018.07.029. El CAPÍTULO V, describe la síntesis de un apósito antimicrobiano para heridas, con una resistencia mecánica adecuada que es capaz de absorber exudados y evitar la deshidratación rápida de una herida. Se prepararon membranas asimétricas de PCL/PVAc cargadas con carvacrol (CRV) mediante el uso simultáneo de electrohilado y electropulverización. Las membranas constan de dos capas; la primera es una capa de PCL electrohilado; la segunda, una lámina de PVAc que estaría en contacto con la piel liberando a su vez el compuesto antimicrobiano. Se demostró que el uso de diferentes disolventes pueden dar lugar a la obtención de diferentes morfologías de la capa PVAc-CRV. Los valores obtenidos de elongación máxima de las membranas antes de romperse son adecuados para ser utilizados como apósitos para heridas ya que están en el mismo rango reportado de elongaciones en la piel humana. Las membranas presentan una tasa óptima de Transmisión de vapor de agua (WVTR) con valores que se encuentran en el rango requerido para mantener un buen balance entre humedad y pérdida de agua en la herida. En la primera semana, se liberó más del 60 % del CRV cargado, mientras que después de tres semanas, las membranas liberaron entre el 85 y el 100 % del CRV cargado mediante la contribución de un proceso de difusión de tipo Fickiano y la relajación delas cadenas poliméricas. Las membranas sintetizadas son candidatas potenciales para ser utilizadas como apósitos para heridas. El manuscrito que resume estos resultados se envió a la revista "Materials Science and Engineering C" y está bajo revisión (MSEC_2018_3013). CAPÍTULO VI, resume las conclusiones generales del trabajo de tesis. APÉNDICE 1, describe las principales técnicas de caracterización y los métodos para evaluar diferentes propiedades en función de las posibles aplicaciones. APÉNDICE 2, resume los artículos publicados y la participación en foros científicos durante el período de tesis. 1 The current Doctoral Thesis work has been performed under a co-supervision agreement between University of Zaragoza (Home University), University of Calabria (Host University) and Faculty of Sciences and Technology of the NOVA University of Lisbon (FCT NOVA) (Host University). This research has been carried out inside the Erasmus Mundus Doctorate in Membrane Engineering program (EUDIME), (FPA 2011-0014), funded by the European Union. This thesis focused mainly on the use of the electrospinning technique to produce different kind of membranes for biomedical applications. In particular, it described the synthesis and production of inorganic and organic nanoparticles to be used as fillers or as carriers (drug delivery system) as well as the production of electrospun nanofibrous membranes. This work was carried out within the Institute of Nanoscience of Aragon (INA), specifically in the Nanostructured Films and Particles (NFP) group under the supervision of the Professor Silvia Irusta and Dr Gracia Mendoza. Also an important part of the physico-chemical characterization was done at INA. The study of different biological signals and the use of specific techniques for membrane characterization were acquired at the University of Calabria under the supervision of Dr. Loredana De Bartolo in the Institute on Membrane Technology of the National Research Council of Italy (ITM-CNR). On the other hand, the mobility carried out at the Faculty of Sciences and Technology (FCT NOVA) of Universidade NOVA (FCT NOVA) under the supervision of Professor Ana Isabel Aguiar-Ricardo, allowed a total characterization of two asymmetric membranes following different International Standards to accomplish testing for primary wound dressing. The development of novel membranes loaded with morphogenetic proteins or antibiotic are of great interest in the field of bone tissue engineering. To promote the cellular viability and extracellular matrix production, electrospun membranes with enhanced porosity and micro-scale pores could be beneficial since increased porosity and pore size can provide a three-dimensional (3D) environment that not only facilitates cell seeding/diffusion but also provides better diffusion of nutrients and waste throughout the membranes. The addition of calcium phosphate ceramics has been extensively investigated to fabricate highly porous membranes to bone tissue engineering due to their close similar composition of bone, including excellent biocompatibility, osteoinductive and osteoconductive properties. A homogeneous distribution of the bone morphogenetic protein-loaded particles along the entire membrane could be ensuring a continuous release of the growth factor to provide the necessary biochemical cues for bone repair and regeneration. Antibiotic-loaded membranes may provide drug targeted and sustained release, avoiding the long-term oral and intravenous systematic multidrug administration, which implies toxic side effects, low delivery to the target site and low patient adherence to the treatment. Therefore, membranes loaded with antibiotics can overcome the drawbacks of the traditional therapy sustaining enhanced osteogenic properties for the successful regeneration of the bone. Another interesting biomedical application of electrospun membranes is the fabrication of efficient smart dressings for the treatment of wounds. A rapid wound healing requires developing appropriate membranes with interconnected pores that allow the oxygen diffusion and transport of metabolic waste, as well as an adequate pore size to prevent rapid dehydration and bacteria penetration. A high absorption capacity and adequate water vapor transmission will be necessary to keep a moist environment in the wound bed. Besides, if the electrospun membrane has some bactericidal properties will be better for the healing process. The main goal of this thesis was the development of fibrous membranes by electrospinning with the appropriate characteristics to be used in bone tissue engineering or as wound dressing materials. To achieve this target, several specific objectives were defined, which are described in Chapters II to V. The thesis was divided in the following sections: CHAPTER I, is an introduction where the concepts of biomaterials, scaffolds and tissue engineering and the main target of drug delivery systems are described. The chapter includes the classification of biomaterials according to the origin of the materials and tissue engineering is also described as well as all the factors that must be taken into account to develop and properly apply a wound dressing are discussed. Different kind of techniques used in the literature to produce scaffolds or membranes for bone tissue engineering and wound dressings are mentioned, focusing on the use of electrospinning and electrospray to produce them. CHAPTER II, focuses on the development of enhanced 3D membranes able to promote efficient bone regeneration together with targeted antibiotic release to prevent bacteria colonization. The aim of this work was to synthesize and characterize a drug delivery system consisting of polycaprolactone (PCL) electrospun nanofibers decorated with rifampicin (RFP) loaded into poly(lactic-coglicolic acid) (PLGA) particles. This material would promote bone repair avoiding the impairment of the membrane mediated by infection. The bactericidal ability of the synthesized electrospun material was assessed In vitro against gram positive (Staphylococcus aureus) and gram negative (Escherichia coli) bacteria, as well as its cytocompatibility in human osteoblasts 3D cultures. These results are included in the accepted article entitled "Composite scaffold obtained by electro-hydrodynamic technique for infection prevention and treatment in bone repair". Javier Aragon, Sergio Feoli, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics. CHAPTER III, describes the synthesis and characterization of core-shell membranes of PCL and polyvinyl acetate (PVAc) obtained by electrospinning. The fibers were loaded with synthetic hydroxyapatite nanoparticles (HAn) to increase the bioactivity of the materials. The prepared membranes were then treated by laser ablation to create desired microscale topographical features in order to favor cell adhesion and growth. All prepared membranes exhibited a three-dimensional network structure with interconnected pores; the laser treatment has modified the structural characteristics of the membrane causing an increase the cell viability and cell density. The materials biocompatibility is affected by the structural properties of the membranes, indeed smaller micropore sizes favor cell adhesion and proliferation. These results are published in the article entitled "Laser-treated electrospun fibers loaded with nano-hydroxyapatite for bone tissue engineering". Javier Aragon, Nuria Navascues, Gracia Mendoza, Silvia Irusta. International Journal of Pharmaceutics 525,112–122, 2017. DOI:10.1016/j.ijpharm.2017.04.022. CHAPTER IV, refers to the development of a composite electrospun membrane of PCL or PCL/PVAc core–shell fibers loaded with synthetic HAn. These fibers were decorated with bone morphogenetic protein 2 (BMP2) loaded in/into PLGA particles via simultaneous electrospraying and coaxial electrospinning. The aim of this study was to evaluate the structural and physico-chemical properties and biodegradation processes of the newly developed membranes assessing their ability to address the architectural, biochemical, and functional features of bone tissue. For this purpose, the membrane bioactivity was tested by culturing human osteoblasts on the membranes and by monitoring cell viability up to 4 weeks. The In vitro osteogenic activity of cells seeded onto the membranes was evaluated by assessing alkaline phosphatase (ALP) activity and the expression of osteogenic proteins osteocalcin (OCN) and osteopontin (OPN). These results are published in the article "Polymeric electrospun scaffolds for bone morphogenetic protein 2 delivery in bone tissue engineering". Javier Aragón, Simona Salerno, Loredana De Bartolo, Silvia Irusta and Gracia Mendoza. Journal of Colloid and Interface Science, 531 (2018) 126–137. DOI:10.1016/j.jcis.2018.07.029. CHAPTER V, describes the synthesis of an antimicrobial wound dressing material, with appropriate mechanical resistance avoiding rapid dehydration and absorbing exudates. PCL/PVAc asymmetric membranes loaded with carvacrol (CRV) were prepared by electrospinning and electrospraying simultaneously. The membranes consist of two layers: the first is an electrospun PCL sheet, the second a PVAc sheet that would be in contact with the skin releasing the antimicrobial compound. The use of different solvents results in different morphologies for the PVAc-CRV layer. The membranes exhibit mechanical properties with strain to failure values that are in the range of human skin, being adequate to be deposited over a wound surface. The samples present Water Vapor Transmission (WVTR) values in the required range to keep good moisture balance with water loss from the wound at the optimal rate. In the first week, more than 60 % of the loaded CRV was released while after three weeks membranes released between 85 to 100 % of the loaded CRV through a Fickian diffusion and diffusion due to polymer relaxation. The synthesized membranes are potential candidates to be used for wound dressing applications. The manuscript summing up these results has been submitted to a scientific journal and is currently under review. GENERAL CONCLUSIONS, summarizes the conclusions of the thesis work. APPENDIX 1, describes the main characterization techniques and the methods to evaluate different properties according to the possible applications. APPENDIX 2, summarizes the articles published and the participation in scientific forums during the thesis period.
