Nuevos enfoques para el diagnóstico y el control de la dicrocoeliosis, importante parasitosis hepática de los rumiantes

259 p.El objetivo principal de la presente Tesis Doctoral fue mejorar el diagnóstico de la dicrocoeliosis en sus hospedadores intermediarios y definitivos, mediante técnicas inmunológicas y moleculares, para lo cual se han llevado a cabo los procedimientos que se resumen a continuación. Se ha desarrollado una técnica de PCR para la detección precoz de D. dendriticum en moluscos y hormigas que actúan como hospedadores intermediarios. Asimismo, se han utilizado técnicas de proteómica para conocer las proteínas mayoritarias y antigénicas que se expresan en los antígenos de tegumento y de excreción/secreción del parásito adulto. Por último, mediante la construcción de una genoteca de expresión se ha abordado el estudio, por primera vez, de una colección de EST (Expressed Sequenece Tag), y se han obtenido y evaluado tres proteínas recombinantes para el diagnóstico de la dicrocoeliosis en los hospedadores definitivo

Zona de transición de una línea ferroviaria situada entre una vía en balasto y una vía en placa de hormigón

Zona de transición de una línea ferroviaria situada entre una vía en balasto y una vía en placa de hormigón, que comprende: - un conjunto de traviesas de hormigón de longitud superior a las traviesas de las vías en placa de hormigón y en balasto, agrupadas en secciones de traviesas de la misma longitud, con longitud creciente desde la vía en balasto a la vía en placa de hormigón, - una lámina de amortiguación de la vibración situada en sentido transversal a la vía en placa de hormigón que se extiende al menos entre la capa de balasto de la zona de transición y la capa de hormigón de la vía en placa de hormigón, - almohadillas de apoyo bajo carril con una rigidez igual o creciente desde la vía en placa de hormigón hacia la vía en balasto, - sendos carriles adicionales internos y - sendos carriles adicionales externos.Solicitud: 201730552 (31.03.2017)Nº Pub. de Solicitud: ES2684429A1 (02.10.2018

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Detection of Dicrocoelium dendriticum larval stages in mollusc and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences

8 páginas, 6 figuras, 1 tabla.The aim of this study was to develop, perfect and validate the PCR (polymerase chain reaction) technique using mitochondrial (mt) and ribosomal (ITS-2) DNA for the accurate identification of Dicrocoelium dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. The first primers that were designed amplified a 169 pb mtDNA fragment of D. dendriticum permitted the detection of a single D. dendriticum metacercaria from the Formica rufibarbis and Formica pratensis abdomen, as well as the detection of the brainworm in the head of the ants collected in tetania. Although these primers did not amplify Dicrocoelium chinensis DNA and permitted detected D. dendriticum in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. The PCR that was designed to amplify a 93 bp fragment of the ITS-2 is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae and other trematodes. This technique is very sensitive since it permitted the detection of D. dendriticum in the molluscs from the first day post-infection, the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Both techniques are important, mainly the latter.This study was supported by the Spanish CICYT (Project AGL2007–62824). Martínez-Ibeas is supported by the ‘Castile and León’ Autonomy (Spain) and the European Social Funds (Contract for young researchers). Martínez- Valladares is supported by the Spanish National Research Council (CSIC) (JAE Doc Contract).Peer reviewe

PCR diagnosis of dicrocoelium dendriticum infection in mollusc and ant intermediate hosts

1 página.-- Trabajo presentado al XII International Congress of Parasitology (Melbourne, Victoria, Australia, 15-20 August, 2010)Dicrocoeliosis, caused by Dicrocoelium dendriticum, is an important hepatic trematodosis which affects a wide range of mammals, mainly ruminants, in Spain and many other countries. To apply sueeessful control programmes against dicroceliosis, prior study of its epidemiology is needed. This requires specific and early diagnosis in mammals, as well as in molluscs and ants, the first and second intermediate hosts, respectively. The aim of this study was to develop an analytical method based on PCR (Polymerase chain reaction) to detect the infection by D. dendriticum in their intermediate hosts. A PCR based on the diversity of nucleotide sequences in mitochondrial DNA among species was carried out. Since no mitochondrial sequenee of D. dendriticum was available, firstly we identified conserved regions of different parasite species with phylogenetic similarity to design general primers which anneal with D. dendriticum DNA. So ten primers,and several combinations, which flanked the large and small rRNA subunits , the NADH dehydrogenase gen and different cytochrome C oxidase subunits were tested. The pair of general primers, which partly flanked the cytochrome C oxidase I and the large rRNA subunits, amplified a band from adult D. dendriticum samples. Once this band was sequenced, internal specific primers were designed fo detect the infection in the intermediate hosts. Using PCR the primers amplified DNA obtained from: D. dendriticum adults, different species of land molluscs experimentally and naturally infected with D. dendriticum and infected ants collected in tetania (in León province, Spain). The PCR products, observed in agarose gel, permitted the detection of D. dentricum sporocysts in mollusc hepatopancreas as well as metacercariae and brainworm in ant abdomen and head, respectively. The specificity of the PCR was also established after testing the primers with other different parasites such as Fasciola hepatica, Calicophoron daubneyi and Brachylaimidae.Study supported by Spanish CICYT, Project AGL2007-62824Peer reviewe

Validation of a spatial liver fluke model under field conditions in Ireland

Fasciola hepatica is the causative agent of fasciolosis, a global disease of a wide range of mammals, particularly sheep and cattle. Liver fluke infection causes annual losses estimated at around €2.5 billion to livestock and food industries worldwide. Various models have been developed to define risk factors and predict exposure to this liver fluke in ruminants in European countries, most of them based exclusively on data from dairy herds. The aim of this study was to validate a published theoretical baseline risk map of liver fluke exposure and cluster maps in Ireland, by including further explanatory variables and additional herd types that are spatially more widespread. Three approaches were employed: i) comparison of predicted and actual exposure; ii) comparison of cluster distribution of hotspots and coldspots; and iii) development of a new model to compare predicted spatial distribution and risk factors. Based on new survey data, the published baseline predictive map was found to have a sensitivity of 94.7%, a specificity of 5%, a positive predictive value of 60% and a negative predictive value of 38.2%. In agreement with the original model, our validation highlighted temperature and rainfall among the main risk factors. In addition, we identified vegetation indices as important risk factors. Both the previously published and our new model predict that exposure to Fasciola is higher in the western parts of Ireland. However, foci of high probability do not match completely, nor do the location of clusters of hotspots and coldspots

Development and validation of a mtDNA multiplex PCR for identification and discrimination of Calicophoron daubneyi and Fasciola hepatica in the Galba truncatula snail

8 páginas, 8 figuras, 1 tabla.Paramphistomosis and Fasciolosis caused by Calicophoron daubneyi and Fasciola hepatica, respectively, are frequent and important trematodoses in ruminant livestock worldwide. Both parasites use the same snail, Galba truncatula, as intermediate host. The aim of this study was to develop and validate an analytical method based on a mitochondrial DNA (mtDNA) multiplex PCR technique which would allow the early and specific identification, in one step, of C. daubneyi and F. hepatica infection in G. truncatula. First of all, a 1035 bp fragment of mtDNA from adult C. daubneyi worms was obtained. Then two pairs of specific mtDNA primers, which amplified a DNA fragment of 885 pb in the case of C. daubneyi, and of 425 pb in that of F. hepatica, were designed. By means of the multiplex PCR technique developed, there was always a specific amplification in samples from adult F. hepatica and C. daubneyi, but not from Calicophoron calicophorum, Cotylophoron cotylophorum, Cotylophoron batycotyle or Dicrocoelium dendriticum. Likewise, specific amplifications of the expected DNA fragments happened in all samples from snails harbouring larval stages of C daubneyi or F. hepatica, previously detected by microscopy. However, amplifications were not seen when DNA from snails harbouring other Digenea (Plagiorchiidae, Notocotylidae and furcocercous cercariae) was analysed. Moreover, DNA from G. truncatula molluscs free from infection was not amplified. The multiplex PCR assay permitted infection in the snails experimentally infected with 4 miracidia to be detected as early as day 1 p.i. in the case of F. hepatica and with only 2 miracidia from day 2 p.i. in both, C daubneyi and F. hepatica. Nevertheless it was necessary to wait until days 29 and 33 p.i. to see C. daubneyi and F. hepatica immature redia, respectively, using microscope techniques. The detection limit of the PCR technique was very low: 0.1 ng of DNA from C daubneyi and 0.001 ng of DNA from F. hepatica. This allowed infection by either F. hepatica or C daubneyi to be detected even when pools made up with only 1 mu l (60 ng of DNA) from infected snail plus 99 mu l from non-infected ones were analyzed. Moreover, simultaneous detection of both parasites was experimentally possible in pools made up with uninfected (98 mu l), C. daubneyi infected (1 mu l) and F. hepatica infected (1 mu l) snails. The most precise and early diagnosis of the infections using the multiplex PCR technique designed will allow more realistic epidemiological models of both infections to be established and consequently a better strategic control.This study was financially supported by the Spanish INIA, Project FAU2006-00021-C03-00 and by the European Social Funds, as well as by the Castile and León Autonomy (Spain) Project LE023A10-2.A.M. Martínez-Ibeas is supported by the ‘Castilla y León’ Autonomy (Spain) and the European Social Funds (Contract for young researchers). M. Martínez- Valladares is supported by the Spanish National Research Council (CSIC) and the European Social Funds (JAE Doc Contract). B. Mi˜nambres was supported by ‘Ramón y Cajal’ contract.Peer reviewe

Detection of the larval stages of Fasciola hepatica and Calicophoron daubneyi in the Galba truncatula mollusc by multiplex PCR

1 página.-- Trabajo presentado al XII Congreso Ibérico de Parasitología (Zaragoza, 5 al 8 de julio del 2011)The aim of this study was to develop and validate a multiplex PCR technique using mitochondrial DNA (mtDNA) for the accurate and early detection of F. hepatica and C. daubneyi in G. truncatula, the mollusc intermediate host of both parasites...Study supported by Spanish INIA, Project FAU2006-00021-C03-OO. Martínez-Ibeas and Martínez-Valladares were supported by CSIC and Castilla y León, respectively.Peer reviewe

Study on the intraorganic location of paramphistomidae flukes infecting adult cattle in Galicia (Spain) and the species identification

1 página.-- Trabajo presentado al XII International Congress of Parasitology (Melbourne, Victoria, Australia, 15-20 August, 2010)The forestomachs of 722 cows, over 2 years oid, from 628 farms in the 4 provinces of Galicia (Coruña, Lugo, Ourense and Pontevedra), NW Spain, were colleeted from slaughterhouses. All the rumens and reticula were examined to detect and count the Paramphistomidae. To determine the parasite intraorganic distribution, 31 animals were chosen randomly and the parasites found in the different anatomical areas of the rumen (ruminal atrium, rumenoreticular sulcus, ventral sae, dorsal sac) and in the reticulum were counted and measured. Fifty parasites were coliected from each infected animal. The specific identification was done morphologically and by ITS2 (Internal Transcribed Spacer 2) sequencing. The infection prevalence was 17.3% and the parasite loads varied between 1- 21200 ( Mean = 1293 ± 2780 SD). Significant differences were observed between the infection prevalences in the four provinces, the highest being in Pontevedra (27.6%). Most parasites were located in the rumen (94.3%), with the percentages found in the atrium (58.2%) and in the rumenoreticular sulcus (26.5%) significantly higher than those found in the dorsal and ventral sacs. Statistically significant differences were also recorded in parasite size. In order to study the genetic variability of Calicophoron daubneyi a 401 bp fragment of the ribosomal RNA internal transcribed spacer 2 (ITS2) was sequenced. Firstly, we analysed the intraindividual variability after sequencing a total of 10 C. daubneyi recovered from two cows. No difference was shown in the ITS2 sequence between the adults taken from different parts of the rumen (ruminal atrium, rumenoreticular sulcus, ventral sac, dorsal sac) and reticulum , in the same eow. Secondly, the interindividual variability was also tested in 21 samples obtained in the four provinces of Galicia (Spain). All sequences were also similar between C. daubneyi adults from different origins.Stydy supported by Spanish INIA, Project FAU2006-00021-C03-00Peer reviewe

Preliminary study of the genetic variability of Calicophoron daubneyi (paramphistomidae) in cattle in north-west Spain

1 página.-- Trabajo presentado al XII Congreso Ibérico de Parasitología (Zaragoza, 5 al 8 de julio de 2011)The objective of this work was to study the genetic variability of Calicophoron daubneyi natural populations in cattle from Galicia and Castile and León by means of the analysis of parasite mitochondrial DNA (mtDNA) sequences. The species was molecularly identified by sequencing the ITS-2 (Internal Transcribed Spacer 2) intergene zone, using two primers designed from the C. daubneyi sequence published in GenBankTM (Access No AY790883). Our sequence homology was 100%...Study supported by Spanish INIA (Project FAU2006-000021-C03-00) and Castile and León (Project LEO23A1O-2).Peer reviewe