VETERINARY MICROBIOLOGY

Author: LACOMBE ANTONELI ÁNGELA.
Year: 2003.
University: EXTREMADURA [www.unex.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Author: BAU PUIG MARTA.
Year: 2004.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE VETERINÀRIA.
Place of preparation: ESCUELA DE POSTGRADO.

Summary: In this report Doctoral Thesis has studied mycobiota of grapes ripening at different periods, with the aim of identifying the species ocratoxígenas present. This has been sampled 7 vineyards distinct, located mainly in the Spanish Mediterranean coast, during 2001.2002 and 2003. It has also analyzed the capacity to produce ochratoxin A potentially producing strains isolated in this sampling. On the other hand, have been characterized at the molecular level 226 strains of the genus Aspergillus section Nigri potentially ocratoxígenas: 53 strains belonging to the species A.carbonarius and 173 strains belonging to the aggregate A.niger. All strains of the study are molecular isolation of grapes from different geographical origins: Spain, France, Greece, Israel, Italy and Portugal. The mycobiota predomiante of grape vines Spaniards sampled belonged to the genus Alternaria, Cladosporium and Aspergillus, whereas gender Penicillim represented less than 3% of the isolates. The high incidence of Alternaria spp. And Cladosporium spp. In the first sampling times decreased with the maturation of the grapes, while the presence of Aspergiullus spp. Increased significantly. In all cases, significant differences in the incidence of these genera in each sampling period. During the three years of sampling the grapes were isolated from a total of 1812 strains of Aspergillus spp., Among which only 13 were identified as A.ochraceus, while 1423 were identified as belonging to aggregate A. Niger, and 226 as A.carbonarius. The only isolation identified as P.verrucosum among 214 strains of Penicillium spp.aisladas showed the ability to produce citrinina but not OA. The isolates from Aspergillus section Nigri represenarona about 90% of the strains isolated gender. Although presented in all periods of sampling, the presence of species added A.niger and A.caronarius was significantly higher in the harvest. Moreover, the number of aislamien cough in the middle DRBC higher than in MEA, although these differences were not significant. The pepetias grape analyzed no fungal contamination, therefore it is assumed that the fungus contamination producers of OA in the grape comes from the surface of the fruit. As to the results obtained in the study of OA production of the strains isolated from vineyards Spaniards, has shown the important contribution of A.carbonarius in pollution OA in grape and thus in the wine. This assertion stems not only from the high incidence introducing this species in the grapes, particularly in the harvest, but also the percentage of strains producing a 100% in this study. The range of OA in production of this species OA was 0,06-477 ug / g of culture medium. In terms of other species of Aspergillus Nigri isolated section, only 0.7% of the isolates of adding A.niger proved ocratoxígenos, in a range of 0,05-231 ug OA / g of culture medium, while none of the isolates produced uniseriate OA. It was also detected the production of OA by other species of Aspergillus non-section Nigri. So, 7 of 13 isolates A.ochraceus, and 3 of 6 from A.mellus produced OA. Also 3 isolates A.ostianus and 6 of A.aliaceus were producers. Some of these species produce OA in large quantities, but its low incidence in grapes makes the importance as a source of contamination is low. In connection 8 to stud 6fb io molecular A.carbonarius, has been characterized by RAPD and sequencing of the region ITS-5.8S of rDNA 53 strains isolated from grapes from different European countries. Forty-nine of the strains had a very similar pattern of RAPD and sequencing of the region ITS-5.8S virtually identical. All these strains OA produced in different amounts. The 4 strains showed a different pattern of RAPD and sequencing of the region ITS-5.8S different from the others, in addition to being the only non-producing strains of OA. These isolates could represent a new species under section Nigri. Moreover, the study molecular added A.niger have been characterized 173 strains from different geographical origins, using a technique of RFLP in the region ITS-5.8S of rDNA. All strains grouped in 2 patterns RFLP described previously: N-type (43% of the strains) and T (57% of the strains). It detected the production of OA in 20 of the 173 strains (11.6%). All strains ocratoxígenas showed the pattern type N.

Author: MARTÍNEZ PULGARÍN SUSANA.
Year: 2004.
University: COMPLUTENSE DE MADRID [www.ucm.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: S. Aureus is a ubiquitous microorganism causing various diseases in humans as in pets. In veterinary medicine stresses primarily for his involvement in intramammary infections, which cause important economic losses in the production of cattle, sheep and goats. In recent years it has become clear that S. Aureus is capable of intemalizarse, survive and sometimes multiplied in different cell types, including cells fagociticas, this fact may contribute to the persistence of the infection in the body to protect the bacteria from the activity of the immune system and treatment antibiotics and facilitate its spread. This intracellular survival can be detellllinada by the ability of S. Aureus to cope with the bactericidal action resulting from the oxidative metabolism of eukaryotic cells. In bacteria such as M. Tuberculosis, C. Jejuni and H. Pylori has been shown that the catalase, an enzyme responsible for the degradation of hydrogen peroxide produced during the metabolism and molecular oxygen in water, is essential for their survival in elinterior of various cell types, however, in S. Aureus has not been determined accurately so far, the possible role of this enzyme in its pathogenesis. Similarly, beta-toxina, an enzyme that acts deteriorated esfingomielina present in cell membranes, could be important during the life cycle of S. intracellular Aureus, participating, as well as other esfingomielinasas bacteria in the breakdown of the vacuola fagocitica and subsequent release of the bacteria to the cytoplasm of the infected cell and inducing cell apoptosis via the release of ceramide. However, the possible contribution of beta-toxina the pathogenesis of S. Aureus has not been conclusively demonstrated so far. Based on these facts, the main objective which was raised for this Doctoral Thesis was detellllinar the possible role of the catalase and the beta-toxina, as well as the combined effect of both, in the pathogenesis of S. Aureus. This simple mutants were constructed by delección of genes katA (catalase negative) and hlb (beta-toxina negative) and a double mutant by delección of both genes from a strain of S. Aureus, 2386, which had been isolated from a clinical case of mamitis in cattle, and compared the behavior of mutants with that of the parent strain using various biological models. Specifically, they studied their ability to intracellular survival on the line macrophagic murine J774A.1 and the linca epitclialmamaria bovine MAC-Ty its virulence in a mouse model of experimental infection, by the estimate of DLSO. In trials of intracellular survival in the online cellular J774A, he found no statistically significant differences attributable to the catalase or beta-toxina. In line cellular MAC- T, however, the absence of catalase activity affected fom1a significantly to the spread of bacteria in the early hours postinfección. This result suggests that the catalase promotes proliferation intracellular S. Aureus in bovine mammary epithelial cells, contributing to the defense of bacteria during the early hours postinfección, but did not significantly influence the survival of S. Aureus in the interior of cells macrofágicas murine, revealing the existence of differences in the relative weight of the system cytotoxic oxigeno-dependiente the bactericidal activity of these two cell types. The cell line MAC-T were observed also significant differences in favor of mutant beta-toxina negative of the persistence of intracellular bacteria but not their proliferation. The beta-toxina not therefore plays an important role in the proliferation of intracellular S. Aureus in bovine mammary epithelial cells, introducing, however, a slight effect on the persistence in these cells which 8 were not p 6d8 roduce in cells macrofugicas murine, which could be related to the different composition in esfingomielina of these two cell types . Finally, in studies conducted with the double mutant was found statistically significant differences in favor of this in the two cell lines used. These studies show that the catalase and beta-toxina act in a synergistic manner during the life cycle of S. intracellular Aureus, so that the simultaneous absence of both enzymes resulting in a delay in the proliferation of intracellular S. Aureus in bovine mammary epithelial cells and in a remarkable increase in their persistence in the two cell types studied, the latter as a result, possibly from a lower pathogenicity to the cell. Tests vi1111enciaen the mouse model (DLSO) indicate that the catalase and beta-toxina, by themselves. Not JUégan a decisive role in the virulence of S. Aureus, however, the combined effect of both enzimasjuega a role detellninante in the virulence of the bacteria in this model. The greatest permissiveness of intracellular replication and persistence, as well as the lower pathogenicity, shown by the double mutant, make him a good candidate for possible use as attenuated vaccine against the processes caused by S, aureus.

Author: DE LA TORRE MARTÍNEZ MARIA EUGÈNIA.
Year: 2005.
University: AUTÓNOMA DE BARCELONA [www.uab.es].
Place of defense: FACULTAT DE VETERINARIA, UAB.
Place of preparation: FACULTAT DE VETERINARIA, UNIVERSITAT AUTÒNOMA DE BARCELONA.

Summary: The salmonellosis toxiinfección is transmitted through food products and animal by-products more important in developed countries. To this must be added the emergence of strains of Salmonella multidrug mainly due to misuse of antimicrobials in animal farms. These facts justifying the need for epidemiological surveillance and study of Salmonella worldwide. This requires identification and characterization methods appropriate, which may be useful to establish relations of similarity between the strains isolated and detect, the source of disease outbreaks, allowing track comparable globally. When in the year 1997 was isolated in Spain for the first time the strain of Salmonella monophasic formula antigenic (4,5,12: :-), were carried out various epidemiological studies in order to establish their possible origin. This "new serotype" often presented a profile of multidrug R-ACSSuT-GSxT with fagotipo U302 more than 55% of cases. Using different methods of characterization (determining the profile restricted by pulsed field electrophoresis (PFGE), the plasmid profile, the antibiorresistencia and fagotipo) determine the possible genetic relationship of 48 strains of Salmonella enterica subsp. Enterica samples from pork in Catalonia, isolated in our laboratory during the period between 1998 and 2000. Twenty-three of these strains belonging to S. Enterica (4,5,12: :-), 24 S. Enterica serotype Typhimurium (4,5,12: i: 1.2) and a strain to S. Enterica (4,5,12 :-:-). After combining PFGE profiles obtained with the use of two different restriction enzymes (XbaI and BlnI), it was observed that the 48 strains were grouped into 7 patterns restriction combined (XB profile). The 23 isolates serotype (4,5,12: :-) and 10 strains of serotype Typhimurium had the same pattern. Three of these 10 strains with fagotipo U302 isolated during the early years of the study, in turn shared the plasmid profile and pattern of multidrug resistance characteristic of the strain monophasic. From the analysis of the relationships between strains of the study, we concluded that the new serotype phase was actually a variety monophasic serotype Typhimurium U302. One factor to be taken into account in epidemiological studies and disease outbreaks, is the time needed to establish relationships between the strains isolated from clinical samples and determine the possible origin of the same. A tool established in a preliminary way and with a degree of reliability such relations would be helpful. The classical biochemical profile for the characterization and identification of strains of Salmonella, has only in mind the positive or negative results compared with specific substrates, used to identify the various subspecies of the genus. With our second study we determine relationships between 135 isolates of Salmonella belonging to multiple serotypes, including strains of the variety monophasic, using data from biochemical kinetics of tests used to identify subspecies of Salmonella. The biotipazo yielded six major groups that include the 94% of the strains (n = 127). The percentage of similarity within the same group was greater than or equal to 95%. Some of the groups were observed not only strains belonging to the same serotype varieties but also phenotypic them. The remaining isolates (n = 8), formed groups with a single isolation. This sug 8 iere that 3a0 data from the kinetics of the reactions of metabolism used to identify and biotipar the subspecies of Salmonella enterica, can be used to determine a preliminary relationship between isolates in a short period of time.

Author: ALVAREZ GONZALEZ BEATRIZ.
Year: 2005.
University: OVIEDO [www.uniovi.es].
Place of defense: FACULTAD DE BIOLOGIA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The pathogen Flavobacterium psychrophilum is salmonids causing sickness cold water. This disorder causes significant economic losses in farms, and there is a system of effective prophylaxis. Despite the great importance of this disease, little is known about the mechanisms of pathogenicity of F. Psychrophilum. The present work has designed a new culture medium to improve solid recovery of viable cells from the culture fluid, and have developed a number of methods such as conjugation, processing and expression vectors, which allow the genetic manipulation of F. Psychrophilum. Using technology developed have yielded a number of mutant characters described as involved in the virulence of F. Psychrophilum (motility, extracellular proteolytic activity and limiting growth in the absence of iron). Among these mutants, in the report was studied known as FP523, which was introduced simultaneously defects in the three phenotypic traits above and a decrease in virulence. In addition, this mutant also showed alterations in growth and an increase in the sensitivity and DCT, unlike the parental strain, was able to form biofilm. The gene interrupted in this mutant, called tlpB, codified tioldisulfuro oxidoreductase involved in the reduction of disulfide bridges in protein periplásmicas.

Author: VIDAL GALLEGO ANA BELEN.
Year: 2005.
University: LEÓN [www.unileon.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The main objective of this thesis was to determine the frequency of isolation of Salmonella in swine, evaluate the available tools for diagnosis, identification and characterization, as well as clarifying aspects of its epidemiology, necessary to establish effective strategies surveillance and control of Salmonella in swine production systems. A first part of the study was conducted in pigs in the slaughterhouse. It monitored different batches of pigs in the finals of bait at two different times, on the farm before his departure to the slaughterhouse and after slaughter. The study consisted of a bacteriological analysis of different types of samples, as well as a serological analysis. We evaluated the usefulness of a Mix-ELISA for the detection of antibodies to Salmonella and was determined to what extent the serological results reflect the presence of Salmonella in batches studied. Finally, we identified the factors and stages of production associated with the presence of salmonella in pigs after slaughter. The second part of this thesis was carried out on pigs sick in order to determine the frequency of detection, as well as serotypes and fagotipos involved. A third part of the work was determined the genetic variability of isolates from different fagotipos S. Typhimurkium and S. 4, 5, 12: i:-for which two molecular techniques were used: determining the profile of plasmids and pulsed field electrophoresis (PFGE) DNA previously digested with enzymes XbaI and BlnI. Finally, in the fourth and last part of the doctoral thesis, we studied different antimicrobial resistance in isolates of different serotypes and fagotipos salmonella from pigs healthy and the sick. The results obtained in this thesis illustrates the importance of the pig as a reservoir of salmonella serotypes frequently resistant to antimicrobial drugs. These resistant isolates were able to cause salmonellosis in humans. For this and other reasons, it would be important to carry out effective control measures that could reduce the prevalence of salmonella infection and the development of bacterial resistance in pig farms. These measures contribute to a decline in the entry of pork products contaminated with Salmonella in the food chain

Author: MENENDEZ GONZALEZ AURORA.
Year: 2006.
University: OVIEDO [www.uniovi.es].
Place of defense: UNIVERSIDAD DE OVIEDO.
Place of preparation: UNIVERSIDAD DE OVIEDO.

Summary: The mechanisms of pathogenicity of Lactococcus garvieae, causative agent of the lactococosis in various fish species of freshwater, brackish and saltwater, are still poorly known. To address their study, the present work has been used technique called mutagenesis marking (Signature tagged mutageneiss, STM). To that end, it was necessary to develop a whole set of genetic engineering in this microorganism as transformaicón, interrupting gene mutagenesis through transposition, ect. Then, and through the application of STM has been able to identify a set of mutant whose attenuated in its virulence genes have been identified and disrupted including genes associated with metabolism, genes involved in the transport and regulatory genes. Some of these mutants hans been studied in more depth both in vitro and in the host and the results obtenidios have enabled some of the specific mechanisms of pathogenicity of L.garvieae.