Els sistemes aquàtics continental representen un dels ecosistemes més amenaçats a nivell mundial, com a conseqüència de l'ús intensiu quel'home en fa. La conca del Guadiana no està lliure d'aquestes pressions antròpiques. Les grans infraestructures hidràuliques i l'escorrentia provinent de l'agricultura són només exemples dels greus problemes que pateix la conca. Aquests problemes es fan especialment palesos en la zona alta de la conca, on l'escassetat d'aigua no fa més que agreujar el problema.Tot això ha generat la necessitat urgent d'avaluar l'estat de conservació d'aquests ecosistemes aquàtics continentals, poder determinar la mesura i la magnitud de les pertorbacions que els estan afectant i així proposar mesures de gestió destinades a restaurar-ne la integritat ecològica. El principal objectiu que presenta aquest és determinar els patrons de distribució de les comunitats de algals (amb una menció especial en el grup de les diatomees) i de les seves causes en la conca del Guadiana i associades, amb la finalitat d'establir i proposar eines que permetin avaluar l'estat de conservació de les masses d'aigua d'aquestes conques. / In many parts of the world freshwater ecosystems are intensely modified and degraded by human activities. Rivers have been altered since historical times, but such modifications have intensified in the last century. Water extraction and heavily regulated reservoirs are spread disturbances in the Guadiana network. Drainage from agricultural land in river systems results in large inputs of nitrates and phosphates that eutrophy river waters. Inputs of fertilizers and water extraction are especially remarkable in the North-East Guadiana watershed. The development of tools to assess ecological status of water bodies has been a key issue in the management of the freshwater ecosystems in the last decades in order to protect and restore the biological integrity of ecosystems. The present study aims to determine distributional patterns of benthic algae communities (with a special attention on diatom microflora) and their causes in Guadiana and associated watersheds in order to establish tools for assessing the conservation status of the water bodies in those watersheds.

Coeliac disease (CD) is a disorder resulting from interactions between diet, genome and immunity. This research seeks to further our understanding of the pathology of CD in regard to its secondary effects on the diversity of enteral microflora via changes in immune tolerance. It proposes that enteral mucosal pro-inflammatory change in CD is associated with a decrease in microbial diversity whilst remission from inflammation may result in an increase in enteral microbial diversity that could contribute to the restoration of tolerance. The first study analyses whether remission from active CD is associated with change in generic enteral microbial diversity by assessing people at diagnosis and following their response to gluten exclusion. A comparison is made to people without CD consuming a ’normal diet’. DGGE profiling of faecal microflora in subjects with CD at diagnosis (confirmed by serology and by duodenal biopsy) and over three consecutive months on a gluten-free diet (GFD) was performed and profiles were compared with those of age and gender matched control subjects taken at monthly intervals. Diversity of faecal microflora (measured as Simpsons Index ) was significantly lower in people with CD than in control subjects. It was possible to distinguish the profiles of coeliac subjects at diagnosis from those obtained after three months on a GFD but it was not possible to distinguish between the samples from control subjects taken at monthly intervals. The profiles of CD subjects after three months on a GFD were more dissimilar to those of the control subjects than those obtained prior to dietary treatment, chiefly on the basis of three bands that were not found in the faeces of any control subjects. The second study analyses dietary intake to determine if a lack of nutrients at diagnosis (before institution of a GFD) and at monthly intervals for three consecutive months post diagnosis (on a GFD) exists, as it is known that CD is associated with nutrient deficiencies resulting from malabsorption due to intestinal inflammation and damage. Subjects completed a customised food questionnaire at each sampling period. Dietary intake was analysed using Foodworks Professional 2007. Significant differences were identified in gluten, starch and carbohydrate intake but not in other macronutrients. Contrary to established literature, these analyses identified few significant differences in micronutrient intake within coeliac subjects over time, however, significant differences were found in iron and sodium.

The goals of the work are the evaluation of the pathogenic fungi occurence, detection of the occurence frequency, the strategy of losses estimates and the appreciation of the disease intensity. In the following I observe the infection with fungi pathogens from the tribe Fusarium including the interpretation of the proceeds items and the surface microflora on the seed of Avena nuda.

Molecular biological methods are fast and efficient tool for the identification of microorganisms in real samples. The aim of this diploma thesis was analysis of microflora in control and contaminated brined cheeses. DNA isolated from analyzed samples was used to optimize the PCR course using primers with GC clamp on the distribution of amplicons using DGGE. DGGE products were reamplified after optimization and prepared for DNA sequencing. DNA isolated from analyzed samples was used in real-time PCR with high resolution melt analysis of the amplicons (HRMA). Samples of cheese and bacterial cultures isolated from cheeses were compared by DGGE and HRMA. Comparing the position of the amplicons was found that contaminants may be Bacillus licheniformis, Staphylococcus epidermidis, and Acinetobacter baumanii/ calcoaceticus. Sequence analysis of cheese and pickles amplicons, the presence of Bacillus sp. and other microorganisms spree in five genera were detected. Representatives of the tree genera were cultured. It is considered contamination Bacillus sp., or microorganisms which are not culturable methods used. The method is suitable for the analysis of complex microflora in cheese and pickles after further optimization.

The goal of presented dissertation has been complex analysis of changes in content of fatty acids, enzymatic and low-molecular antioxidants in apples and related evaluation of perception of originators of storage diseases for apples stored in various conditions. Main part of the work has been dedicated to study of the impact of storage of apples in modified atmosphere with reduced amount of oxygen and in reference “normal” atmosphere for six months. Next part of the work has been dedicated to study how apple storage in common, consumer affordable, conditions (storage in a cellar, in a refrigerator, and in room temperature) affects content of low-molecular antioxidants. For testing has been selected apple kinds Jonagored, Golden Delicious, Idared, Šampion, and Granny Smith. Within the scope of this work has been optimized the method for determination of fatty acids in plant material with higher content of wax. The measured values imply the apples are valuable source of many important nutrition substances like vitamins, provitamins and antioxidants. During the storage process these substances exhibit considerable protective function. Long-term storage, mainly in the atmosphere with reduced amount of oxygen (FAN), enables preservation of majority of these important nutrition substances depending on the kind of apple and conditions of storage. Freezing process is conservative to apples (mainly in the presence of protective substances), while in the process of drying the values of all monitored antioxidants decrease depending on temperature and conditions of drying. Upon the choice of the storage method one must consider nutrition, sensoric characteristic and consumer demands.

Die Besiedlung des kindlichen Darms beginnt unmittelbar mit der Geburt. Hier findet der erste Kontakt mit verschiedensten Bakterien statt. Deshalb war die zentrale Fragestellung unserer Untersuchung, wie sich die Bakterienarten bei den Geburtsmodi unterscheiden und wie sich die bakterielle Zusammensetzung im Stuhl von Neugeborenen im Laufe der Jahre verändert hat. Wir untersuchten die Stuhlproben von 42 Neugeborenen, wovon 22 vaginal und 20 per Sectio entbunden wurden, am ersten, zweiten und dritten Lebenstag auf Bakterienwachstum. Es wurden die aufgetretenen Bakteriengattungen mittels Kultur bestimmt und von den aufgetretenen Staphylokokken und Enterokokken die Arten und deren Resistenzen mittels des Phönix-Vollautomaten untersucht. Wir konnten zeigen, dass im Gegensatz zu früheren Untersuchungen heutzutage vermehrt Staphylokokken den Darm von Neugeborenen besiedeln. Außerdem fiel auf, dass per Sectio entbundene Kinder eine verminderte Heterogenität in ihrer Stuhlflora haben und erst später besiedelt werden als vaginal entbundene Kinder.

Background: Almost all foals develop transient diarrhoea within the first weeks of life. Studies indicated different viral, bacterial, and parasitic causes, such as rotavirus, Clostridium perfringens, Escherichia coli, and Cryptosporidium are discussed. But little is known about the development of intestinal microflora in foals. The present study investigated whether the supplementation with Bacillus cereus var. toyoi would modify the developing intestinal microflora and consequently reduce diarrhoea in foals. From birth, the foals were randomly assigned to three treatment groups: placebo (10 mL isotonic NaCl, n = 8), low dosage (LD; 5 × 108 cfu B. cereus var. toyoi, n = 7) and high dosage (HD; 2 × 109 cfu B. cereus var. toyoi, n = 10). Treatment groups were supplemented orally once a day for 58 days. Faeces scoring and sampling were performed within the first 24 h after birth and on day 9, 16, 23, 30, 44, 58 of the foal’s life and also on the first day of diarrhoea. Culture-plate methods were used to analyse the bacterial microflora. Results: Eighty-eight per cent of the foals developed diarrhoea (placebo 7/8, LD 5/7, HD 10/10) during the first 58 days of life. Bacillus cereus var. toyoi supplementation had no effect on bacterial microflora. Clostridium perfringens and enterobacteria were equally prevalent in foals with diarrhoea and those who were not afflicted. Conclusions: We conclude that the supplementation of B. cereus var. toyoi had no effect on the occurrence of diarrhoea and health status in the foals.

Clinical and experimental studies have shown that estradiol (E2) can enhance protection against sexually transmitted infections such as HSV-2 and HIV-1. Antigen presenting cells (APCs) such as Dendritic cells (DCs) are critical for generating immune responses against these infections, and it is unclear whether unique factors present in the genital mucosa can influence immune responses by directly modulating the phenotype and function of local APCs. To address this, I hypothesized that sex hormones, such as E2 and innate factors in the local microenvironment can regulate the phenotype and function of vaginal APCs. The work summarized in this thesis addressed this central hypothesis. In the first section of the thesis, I examined whether vaginal APCs were distinct in their phenotype and function compared to those in other mucosal tissues or spleen. The results show that the vagina was enriched in CD11c+ CD11b+ MHCII− DCs. Functionally, vaginal tissue cells (TC) and CD11c+ DCs were more potent inducers of Th17 responses in co-cultures with CD4+ T cells, compared to lung, small intestine or spleen APCs. E2 was critical for the conditioning of vaginal DCs to prime these Th17 responses through an IL-1-dependent pathway, indicating that sex hormones such as E2 can directly influence the function of vaginal APCs. In the next section, I determined whether other co-factors in the genital microenvironment such as microflora and innate lymphocytes could also influence vaginal APC functions. We found that while microflora was not essential, IL-17 produced by innate lymphocytes was critical for the induction of IL-1 from DCs, and consequently for potentiating Th17 responses. Finally, I attempted to develop an in vivo mouse model where the effect of E2 on vaginal APCs could be examined in the context of genital HSV-2 infection. I tested a 7-day injectable E2 and a 21-day E2 pellet delivery model, and found that both regimes had limitations for examining E2-effects on anti-viral responses. Yet, subsequent to the work done in this thesis, we were able to confirm our observations of E2-conditioned Th17 responses in vivo in an intranasal immunization model utilizing E2 pellet delivery, and thereby addressed the mechanism underlying enhanced anti-viral protection following E2-treatment. In conclusion, this is the first study to show the effect of E2 on genital tract APCs and their ability to prime Th17 responses. It provides future avenues to examine whether modulation of this microenvironment can help optimize vaccine-induced immune responses against STIs. On a more fundamental level, it highlights the need to consider the inherent distinctions in APC populations among different mucosal tissues. / Dissertation / Doctor of Philosophy (PhD)

<p>U radu je ispitan i razvijen novi pristup tretmanu kože goveda prirodnom smolom &scaron;elak, koja je dozvoljena za kori&scaron;ćenje u hrani, u cilju redukcije unakrsne mikrobiolo&scaron;ke kontaminacije sa kože na goveđe meso. Mehanizam ovog tretmana je baziran na imobilizaciji mikroorganizama na dlaci tretirane kože i prevenciji njihovog fizičkog prenosa sa dlake na meso trupova tokom procesa obrade zaklanih goveda.<br />U in vitro uslovima, tretman uzoraka vizuelno čiste i suve kože 23% rastvorom &scaron;elaka u etanolu je redukovao prenos sa kože na sunđere kojima je koža uzorkovana brisevima: ukupne mikroflore (TVC) za 6,6 log (&gt;1000 puta vi&scaron;e u odnosu na 2,9 log redukcije kod tretmana samo etanolom), generičke Escherichia coli za najmanje 2,9 i Enterobacteriaceae za najmanje 4,8 log. Ove redukcije sve tri grupe mikroorganizama su bile značajno vi&scaron;e u odnosu na redukcije postignute tretmanom kože kombinacijom ispiranja sanitajzerom i vakumiranja. Značajno vi&scaron;e redukcije prenosa TVC sa kože na sunđerske briseve su postignute kori&scaron;ćenjem vi&scaron;ih koncentracija &scaron;elaka (23% i 30%) u odnosu na niže (4,8-16,7%) i u slučajevima kada je temperatura rastvora &scaron;elaka bila 20, 30 ili 40oC u odnosu na 50oC i 60oC. Takođe, tretman kože &scaron;elakom je značajno (3,7 puta) redukovao prevalencu E. coli O157 na prirodno kontaminiranoj, neinokulisanoj koži, kao i broj E. coli O157 na ve&scaron;tački inokulisanim kožama (redukcija od 2,1 log), u odnosu na odgovarajuće netretirane kontrole.<br />U uslovima laboratorijskog modela direktnog kontakta kože i mesa, tretman kože (različitih kategorija čistoće) 23% rastvorom &scaron;elaka je značajno smanjio prenos mikroorganizama sa tretirane kože na sterilno goveđe meso: do 3,6 log cfu/cm2 redukcije ukupnog broja bakterija (TVC), do 2,5 log cfu/cm2 Enterobacteriaceae (EC) i do 1,7 log cfu/cm2 generičke E. coli (GEC). Redukcija prenosa TVC je bila značajno vi&scaron;a, a redukcije EC i GEC slične, u odnosu na redukcije nakon tretiranja kože kombinacijom ispiranja-vakumiranja sanitajzerom.<br />U uslovima male komercijalne klanice sa nezadovoljavajućom procesnom praksom (klanje prljavih goveda i neadekvatna higijena procesa klanja i obrade), tretman koža zaklanih goveda 23% rastvorom &scaron;elaka je rezultirao značajnom mikrobnom redukcijom na mesu trupova goveda nakon skidanja kože: 1,7 log cfu/cm2 TVC, 1,4 log cfu/cm2 EC i 1,3 log cfu/cm2 GEC. Redukcija TVC na mesu trupova je bila značajno vi&scaron;a, a redukcije EC i GEC slične, u odnosu na redukcije nakon tretiranja kože ispiranjem-vakumiranjem sanitajzerom.<br />Ova istraživanja su po prvi put pružila naučne dokaze da se tretman kože goveda u cilju imobilizacije mikroflore na dlaci može uspe&scaron;no koristiti u cilju smanjenja kontaminacije mesa trupova tokom procesa skidanja kože, unapređenja finalnog mikrobiolo&scaron;kog statusa mesa i bezbednosti goveđeg mesa uop&scaron;te. Da bi se ostvario puni potencijal ovog novog tretmana u praksi, neophodna su dalja istraživanja u cilju njegove tehničke optimizacije u uslovima industrije mesa.</p> / <p> In this research, a new approach to cattle hide treatments, based on using a natural, food-grade resin, Shellac, to reduce microbial cross-contamination from the hides onto carcass meat, was developed and evaluated. The basis of this treatment is immobilisation of microorganisms on cattle hide&rsquo;s hair and subsequent reduction of their transmissibility from the hair onto carcass meat during dressing of slaughtered cattle.<br /> Under in vitro conditions, treatment of samples of visually clean and dry hides with 23% Shellac-in-ethanol solution reduced sponge-swabbing recoveries of general microflora (TVC) by a factor of 6.6 logs (&gt;1000-fold greater than the 2.9 log reduction observed by ethanol alone), and of generic E. coli (GEC) and Enterobacteriaceae (EC) by factors of at least 2.9 and 4.8 logs, respectively. The reductions of these three groups of microorganisms were superior to those achieved by a sanitizer rinse-vacuum hide treatment. Significantly greater reductions of TVC recoveries from hides were achieved when using higher Shellac concentrations (23.0% and 30.0% rather than 4.8-16.7%) and when Shellac solution temperatures were 20-40&deg;C rather than 50-60&deg;C. Furthermore, the Shellac-based treatment also markedly reduced the E. coli O157 prevalence (3.7-fold reduction) on natural, uninoculated hides, as well as the counts of E. coli O157 on artificially inoculated hides (2.1 log reduction) when compared to corresponding untreated controls.<br /> Under the conditions of a hide-to-meat direct contact laboratory-based model, treatment of hides (of varying visual cleanliness) with the 23% Shellac solution produced significant reductions of microbial transfer from treated hide onto sterile beef: up to 3.6 log10 CFU/cm2 of TVC, up to 2.5 log10 CFU/cm2 of EC and up to 1.7 log10 CFU/cm2 of GEC. TVC reductions of microbial transfer from treated hide onto beef achieved by the Shellac hide treatment were superior to those achieved by the comparative sanitizer rinse-vacuum hide treatment, but reductions of EC and GEC did not differ between the two hide treatments.<br /> In a small commercial abattoir with unsatisfactory process practices (slaughtering dirty cattle, inadequate process hygiene), treatment of hides with Shellac produced significant microbial reductions on skinned beef carcasses: 1.7 log10 CFU/cm2, 1.4 log10 CFU/cm2 and 1.3 log10 CFU/cm2 of TVC, EC and GEC, respectively. TVC reductions on skinned beef carcasses achieved by the Shellac hide treatment were superior to those achieved by the comparative sanitizer rinse-vacuum hide treatment, but reductions of EC and GEC did not differ significantly between the two hide treatments.<br /> These investigations produced the first scientific evidence that treatment of cattle hides with aim of immobilising microflora on the hair can be very successfully used to reduce carcass meat contamination during the skinning operation, thus improving the microbiological status of the final beef carcasses as well as the beef safety in general. To achieve the full potential of this new treatment in practice, further research aimed at its further technical optimization under real-life meat industry conditions is necessary.</p>

Ce travail de thèse concerne l'étude de l'écologie microbienne d'un atelier de découpe de viande bovine, dans le but de mieux comprendre la persistance bactérienne, c'est-à-dire, la présence répétée d'un même clone bactérien pendant une longue période malgré l'application bien conduite et régulière du nettoyage et de la désinfection (N-D). Des prélèvements par « chiffonnages » multiples de surfaces d'équipements ont été réalisés lors de trois campagnes de prélèvement espacées les unes des autres d'au moins six mois. Les prélèvements ont été réalisés sur un tapis convoyeur en polychlorure de vinyle (PVC) et sur des machines éplucheuses en acier inoxydable avant et après N-D. Nous avons quantifié les cellules totales (les cellules vivantes et les cellules mortes) par PCR quantitative en temps réel (qPCR), les cellules viables par EMA-qPCR, et les UFC (provenant de cellules cultivables) par dénombrement après incubation à 25°C sur gélose tryptone soja. Les résultats montrent qu'avant N-D, les cellules totales (en moyenne 5,6 – exprimé en log10 cellules/cm2 – sur PVC et 4,7 sur acier inoxydable) sont plus nombreuses que les cellules viables (4,5 sur PVC et 4,4 sur acier inoxydable) lesquelles sont plus nombreuses que les UFC (3,8 sur PVC et 2,9 sur acier inoxydable). Le N-D entraîne moins d'une réduction décimale (RD) des populations à l'exception des UFC sur acier inoxydable qui subissent 1,5 RD en moyenne. Ce dernier chiffre s'explique par des forces d'adhésion faibles. L'étude de la diversité des bactéries cultivables montre que sur un total de 51 genres identifiés, 13 seulement sont retrouvés lors des trois campagnes de prélèvements. Les isolats de ces 13 genres représentent 75, 72 et 62% des isolats des campagnes1, 2 et 3 respectivement. Parmi ces isolats, les plus fréquents sont (par ordre décroissant du nombre d'isolats) : Pseudomonas, Staphylococcus, Microbacterium, Acinetobacter, Chryseobacterium, Psychrobacter et Kocuria. Le génotypage d'isolats de 3 genres majoritaires (Staphylococcus, Pseudomonas et Acinetobacter) montre qu'une seule souche, Staphylococcus equorum, est sans aucun doute persistante. L'ensemble de ces observations montrent que l'écosystème varie d'une campagne à une autre. Ces modifications de la diversité bactérienne reflèteraient les modifications de flores des viandes traitées dans l'atelier, qui ont des origines multiples. En outre, il apparaît que, contrairement à ce qui est généralement admis, les bactéries à coloration de Gram négative cultivables sont plus facilement inactivées par le N-D que les bactéries à coloration de Gram positive. L'étude de l'écosystème par PCR-DGGE a permis d'identifier sept genres bactériens et montre que les espèces dominantes sont toutes sous forme vivante, autrement dit, aucune des espèces dominantes n'a été détectée uniquement sous forme de cellules mortes. Sur les sept genres identifiés six sont des Gram – dont majoritairement les genres Acinetobacter, Pseudomonas et Psychrobacter. Cette dominance montre que le N-D permet une forte perte de cultivabilité des bactéries Gram – mais qu'une grande partie n'est pas détachée. La dominance des bactéries Gram – observée par PCR-DGGE masque les staphylocoques qui ne sont pas détectés alors qu'ils sont majoritaires parmi la flore cultivable. Seul un genre bactérien, Propionibacterium, est identifié par PCR-DGGE uniquement mais il n'est trouvé qu'à une seule campagne et uniquement sur l'acier inoxydable avant N-D. En conclusion, l'avancée majeure de ce travail est la mise en évidence qu'une proportion importante de bactéries survit après les opérations très poussées de N-D mais pour une période transitoire. / The aim of this work is to acquire a better knowledge of the microbial ecology of a beef processing plant to understand bacterial persistence, e.g. the presence of a clone isolated several times on several visits in the same processing plant despite regular Cleaning and disinfection (C&D) procedures. Successive swabbing were performed on a PVC conveyor belt and skinning machines made of stainless steel before and after C&D during three surveys in minimal 6 month-intervals. Total cells (live and dead cells) were quantified using real-time quantitative PCR (qPCR). Viable cells e.g. cells with intact membrane, were assessed using Ethidium Monoazide combined with qPCR. Culturable cells (CFU) were determined from plate counts on Tryptone Soy Agar. Before C&D, total cells (5.6 log cells/cm2 and 4.7 log10 cells/cm2 on PVC and stainless steel respectively) were greater than viable cells (4.5 and 4.4 log10 cells/cm2) and CFUs (3.8 and 2.9 log10 CFU/cm2). C&D lead to less than 1 log10 reduction in bacterial populations except for CFU counts on stainless steel where a 1.5 log reduction is observed. This result is highlighted by the weak attachment strengths observed on stainless steel for CFUs. Identification of the culturable microbiota revealed that out of 51 genera identified, 13 were found at all the visits. These genera represented 75, 72 and 62% of the total isolates. The most frequently identified bacteria were Pseudomonas, Staphylococcus, Microbacterium, Acinetobacter, Chryseobacterium, Psychrobacter and Kocuria. Molecular typing of three dominant genera (Staphylococcus, Pseudomonas and Acinetobacter) showed that only one strain, Staphylococcus equorum, was persistent in the premises. Our results show that the microbial ecosystem is different from one survey to another, which reflect the various geographical origins of meat products. Contrary to widespread belief, Gram negative strains were more easily eliminated by C&D than Gram positive strains. Furthermore, the microbial diversity assessed by PCR-DGGE allowed the identification of 7 genera. This molecular approach showed that dominant species are all in a viable state: none of these species was solely detected in a dead state. Of the 7 genera identified, 6 were Gram negative, Acinetobacter, Pseudomonas and Psychrobacter being predominant. This result highlights that C&D induced the lost of culturability of Gram negative bacteria although a high proportion was not detached from the surface. The predominance of Gram negative microflora, didn’t allow the detection of staphylococcal isolates which were numerous in the culturable microflora. One genus, Propionibacterium, isolated in one survey on stainless steel before C&D was only identified by PCR-DGGE. In conclusion, the present study has demonstrated that a large proportion of bacteria can survive drastic cleaning and disinfection for a transient period.