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In human tuberculosis (Mycobacterium tuberculosis), molecular epidemiology has accurately indicated the risk factors involved in active transmission of the disease, by comparing individuals whose isolates belong to a cluster with patients whose strains are considered unique. Nevertheless, this application has not been used in bovine tuberculosis (Mycobacterium bovis). Our study describes the integration of epidemiological data into molecular classification data on M. bovis isolates. These were isolated from wild ungulates in Extremadura (western Spain) with the objective of detecting the risk factors linked to the association of strains in clades, which are indicators of the active spread of the disease. The molecular markers used were spoligotyping + VNTR typing (loci: VNTR 2165, VNTR 2461, VNTR 0577, VNTR 0580, VNTR 3192 VNTR 2163a and VNTR 2163b) on a population of 59 M. bovis strains isolated from deer (Cervus elaphus), 112 from wild boar (Sus scrofa), six from bovines, 28 from pigs and 2 from goats (n=207). Epidemiological variables included the animal species from which the strain was isolated, pathological condition of the host (incipient lesion, early and late generalisation), date of sampling (during or after the reproductive period) and hunting season. Bivariant analysis was used to establish the risk factors connected to the association of strains and later, the variables were evaluated by means of logistic regression. Molecular typing grouped a total of 131 strains (64.21%) in 28 clusters and 76 isolates shows unique profiles. The association of strains was connected to the appearance of macroscopic lesions during the reproductive period (O.R. 4.80; 95% CI 1.09-22.99, P

This study aimed to evaluate molecular typing methods useful for standardization of strains in experimental work on dermatophilosis. Fifty Dermatophilus congolensis isolates, collected from sheep, cattle, horse and a deer, were analyzed by randomly amplified polymorphic DNA (RAPD) method using twenty-one different primers, and the results were compared with those obtained by typing with a pulsed field gel electrophoresis (PFGE) method using the restriction digest enzyme Sse8387I. The typeability, reproducibility and discriminatory power of RAPD and Sse8387I-PFGE typing were calculated. Both typing methods were highly reproducible. Of the two techniques, Sse8387I-PFGE was the least discriminating (Dice Index (DI), 0.663) and could not distinguish between epidemiologically related isolates, whereas RAPD showed an excellent discriminatory power (DI, 0.7694–0.9722). Overall, the degree of correlation between RAPD and PFGE typing was significantly high (r, 0.8822). We conclude that the DNA profiles generated by either RAPD or PFGE can be used to differentiate epidemiologically unrelated isolates. The results of this study strongly suggest that at least two independent primers are used for RAPD typing in order to improve its discriminatory power, and that PFGE is used for confirmation of RAPD results.

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases.

Problem addressed : Shiga toxin-producing Escherichia coli (STEC), have emerged as food poisoning pathogens which can cause severe diseases in humans. Objective : The aim of this study was to determinate the serotypes and virulence genes of STEC strains isolated from sheep in Spain, with the purpose of determining whether sheep represent a potential source of STEC pathogenic for humans. Methods and approach : Faecal swabs obtained from 697 healthy lambs on 35 flocks in Spain during the years 2000 and 2001 were examined for STEC using phenotypic (Vero cells) and genotypic (PCR) methods. Results : STEC O157:H7 strains were isolated from seven (1%) animals in six flocks, whereas non-O157 STEC strains were isolated from 246 (35%) lambs in 33 flocks. A total of 253 ovine STEC strains were identified in this study. PCR showed that 110 (43%) strains carried stx 1 genes, 10 (4%) possessed stx 2 genes and 133 (53%) both stx 1 and stx 2 . Enterohaemolysin ( ehxA ) and intimin ( eae ) virulence genes were detected in 120 (47%) and in 9 (4%) of the STEC strains. STEC strains belonged to 22 O serogroups and 44 O:H serotypes. However, 70% were of one of these six serogroups (O6, O91, O117, O128, O146, O166) and 71% belonged to only nine serotypes (O6:H10, O76:H19, O91:H–, O117:H–, O128:H–, O128:H2, O146:H21, O157:H7, O166:H28). A total of 10 new O:H serotypes not previously reported in STEC strains were found in this study. Seven strains of serotype O157:H7 possessed intimin type γ1, and two strains of serotype O156:H– had the new intimin ζ. STEC O157:H7 strains were phage types 54 (four strains), 34 (two strains) and 14 (one strain). Conclusions : This study confirms that healthy sheep are a major reservoir of STEC pathogenic for humans. However, because the eae gene is present only in a very small proportion of ovine non-O157 STEC, most ovine strains may be less pathogenic.

belong to Institutes of Consecrated Life. This article is a commentary on a concrete case of a person belonging to an Institute of Consecrated Life who was declared incapable and for whom the claim was later made: the family claimed that guardianship was the responsibility of her sister or niece; on the other hand, the Institute claimed that it fell to the Director of the residence which belonged to the Institute. The decision of the First Instance Court gave awarded guardianship to the director; this was appealed by the family but the higher court turned down the appeal and handed down the same decision. Both edicts were based on the possibility of a civil judge altering the order established by the Civil Code and designating someone else, taking into account the welfare of the incapacitated person. The benefit to the person was explained by the better care and attention which the incapacitated woman had always received from her companions and by the contractual link which she had acquired on taking perpetual vows. In the commentary we state that canon law is not binding on a civil judge, except in those matters which have been formally agreed with the state. In this case however, we note the respect which the civil resolutions accorded to the contractual situation which a member of an Institute acquires; however, things do not always work out this way, and we propose a solution by means of a Concordat.

Dermatophilus congolensis is the pathogenic actinomycete that causes dermatophilosis in cattle, lumpy wool in sheep and rain scald in horses. Phenotypic variation between isolates has previously been described, but its genetic basis, extent and importance have not been investigated. Standard DNA extraction methods are not always successful for D. congolensis due to its complex life cycle, one stage of which is encapsulated. Here we describe the development of rapid and reliable DNA extraction and random amplified polymorphic DNA (RAPD) methods that can be used for genotyping D. congolensis field isolates. Our results suggest that genotypic variation between isolates correlates with host species. Several DNA extraction methods and RAPD protocols were compared. An extraction method based on incubation of the bacterium in lysozyme, sodium dodecyl sulphate (SDS) and proteinase K treatments and phenolic extraction yielded high-quality DNA, which was used to optimize RAPD-polymerase chain reaction (PCR) protocols for two random primers. An alternative rapid, non-phenolic extraction method based on proteinase K treatment and thermal shock was selected for routine RAPD typing of isolates. DNA extracted from reference strains from cattle, sheep and horse using either method gave reproducible banding patterns with different DNA batches and different thermal cyclers. The rapid DNA extraction method and RAPD-PCR were applied to 38 D. congolensis field isolates. The band patterns of the field and type isolates correlated with host species but not with geographical location.

It is possible to study the in force right not knowing his origin, that is to say, ignoring his history. Of there the result of his investigation. To study the historical right demands to know the method – also – historically applied in his sources. Across him we will know the area reached in his juridical dogmatist with his successes and limitations in this moment. With this aim I get in the judicial procedure method, almost exclusively of our discipline, destined to investigate the procedural law in The XIXth and XXth Century. This work serves me to remember not such a distant period of our procedural history and especially the method used by his authors in his works when they were investigating our right to apply it to the society of the epoch.

La sucesión temporal de normas tras el inicio del proceso plantea cuestiones complejas, muy prolijas y en ocasiones de difícil solución. A dichas cuestiones trataron de dar respuesta las disposiciones transitorias de las distintas leyes procesales hasta el presente con mejor o peor fortuna y otras se siguen silenciando. La doctrina procesal española y extranjera no es ajena a la materia de ahí la razón de ser de este trabajo. Ello se debe a que en el proceso se aplican desde su inicio a fin las normas procesales y estas son de distinta especie pudiendo hablarse de las formales o de mero trámite y de las sustanciales. A su vez el Juez, en la sentencia, tiene que aplicar las normas materiales al caso concreto y en ocasiones dentro de éstas existen preceptos procesales que no pueden escindirse de lo material o sustancial. Se añade que el proceso pasa por diversos periodos, fases e instancias. Existe una diversidad de procesos sean generales o especiales según su objeto; y diversidad por el orden jurisdiccional ante el que se siguen y la jurisdicción ante la que se ventilan. Determinar el momento decisivo a partir del cual se puede hablar de irretroactividad, retroactividad o ultractividad es complejo y de base doctrinal. Sin olvidar que la opción que dé a estos sistemas el legislador tiene hoy relevancia y proyección constitucional.The temporal sequence of rules after the beginning of the process raises complex, very prolix questions and in occasions of difficult solutions. To these questions they tried to answer the transitory provisions of the various procedural laws to the present with better or worse fortune and they continue hushing up others. The procedural doctrine so much Spanish and foreign is not foreign to the matter of there the raison of this work. This is because the apply from the beginning to the procedural rules and these are of different species may speak of formal or purely procedural and substantial. In turn the Judge, in the judgment, you must apply the substantive rules to the concrete case and in occasions inside these there exist procedural rules that cannot split of the material or substantial thing. It adds that the process goes through diverse periods, phases and instances. There are a diversity of processes by general or special order according to his object; and diversity for the jurisdictional order before which they follow and the jurisdiction before which they ventilate. To determine the decisive moment from which it is possible to speak of non-retroactivity, retroactivity or ultractivity is complex and of doctrinal base. Without forgetting that the option that the legislator gives to these systems has today relevancy and constitutional projection.

Methods such as real time (RT)-PCR have not been developed for the rapid detection and diagnosis of Dermatophilus (D.) congolensis infection. In the present study, a D. congolensis-specific SYBR Green RT-PCR assay was evaluated. The detection limit of the RT-PCR assay was 1 pg of DNA per PCR reaction. No cross-reaction with nucleic acids extracted from Pseudomonas aeruginosa, Mycobacterium tuberculosis, Staphylococcus aureus, or Austwickia chelonae was observed. Finally, the RT-PCR assay was used to evaluate clinical samples collected from naturally infected animals with D. congolensis. The results showed that this assay is a fast and reliable method for diagnosing dermatophilosis.

An indirect fluorescent antibody (IFA) technique has been developed for detection of anti-Dermatophilus antibodies in sheep. Sera from 25 bacteriologically confirmed clinically affected sheep and from 10 negative non affected lambs were used. Whole cell antigen from brain heart infusion cultures of D. congolensis was used and all sera were tested in the same way for cross-reactivity against antigens obtained from cultures of Actinomyces viscosus, Micrococcus luteus, Nocardia asteroides, and Corynebacterium pseudotuberculosis. Sera from Dermatophilus-infected sheep gave positive results with D. congolensis antigen and negative results with the antigens from other bacteria. The whole cell antigens employed were simple to prepare and easy to recognise by microscopy. Cross-reactivity was further tested using the D. congolensis culture whole cell antigen and 3 sera from sheep with bacteriologically confirmed natural infections due to Corynebacterium pseudotuberculosis, Actinomyces pyogenes and Nocardia asteroides. None of these sera showed positive reactions. The authors recommend this technique for serological surveys and research on dermatophilosis.

API ZYM kit was used to test enzymatic activities on eighteen strains of Dermatophilus congolensis. All strains produced lipase and acid phosphatase, which act on lipids, and leucine arylamidase which act on proteins. Another 10 exoenzymes were present in at least one of the strains.