Search

Paramphistomidosis has recently been identified as an emerging parasitosis in Europe. This study estimated the prevalence of rumen flukes, Fasciola hepatica and Dicrocoelium dendriticum, in small ruminants in Germany and identified occurring rumen fluke species and potential predictors for fluke infections. Pooled fecal samples from 223 sheep farms and 143 goat farms in northern and southern Germany were examined by the sedimentation technique, and molecular species identification was performed on rumen-fluke-positive samples. In sheep, a flock prevalence of 2.2% was detected for rumen flukes. Calicophoron daubneyi was identified on four of five positive farms, while species identification failed in one flock. No rumen fluke eggs were detected in the examined goat herds. F. hepatica eggs were detected in 2.7% of the sheep flocks, while the herd prevalence was 5.6% in goats. Higher prevalence values of 21.1% (sheep) and 7.0% (goats) were observed for D. dendriticum. Mixed grazing with other ruminants and previously identified infections with rumen flukes and/or F. hepatica were identified as predictors for paramphistomidosis. The distribution of the three trematode species followed a geographical pattern associated with conditions favoring the relevant intermediate hosts. C. daubneyi is an established parasite in German sheep at a currently low prevalence.

Aim: Fasciolosis and dicrocoeliosis are important parasitic diseases worldwide, causing significant financial losses due to decrease in production and viscera condemnation in animals. We performed the current research to assess the epidemiology of these infections and determine their significance from an economic perspective in Arak, Iran. Materials and Methods: In total, we evaluated 118,463 sheep, 207,652 goats, and 43,675 cattle through necropsic analysis at the slaughterhouses. The average weight of sheep, goat, and cattle liver was 1000, 900, and 5000 g, respectively. The average price of liver in the market was 8 USD/kg. Moreover, the elimination of fundamental nutrients and vitamins was evaluated in infected livers. The prevalence of fasciolosis and dicrocoeliosis was determined. Analysis of variance test was applied for the statistical analysis, and the significance level was

Background and Aim: Cystic echinococcosis, Echinococcus granulosus, and liver flukes, such as Fasciola spp. and Dicrocoelium dendriticum, are important parasitic zoonoses, where they able to cause significant veterinary, medical, and economic problems. The present study was carried out to obtain the updated knowledge on the frequency of hydatidosis, fasciolosis, and dicrocoeliosis in the slaughtered sheep and cattle. Materials and Methods: Information were collected from meat inspection records using systematically visual inspection, palpation, and incision of the visceral organs in the industrial abattoir in Tehran, the capital of Iran, between February 1, 2015, and January 31, 2018. For an analysis of the data, SPSS version 16 was applied. Results: The hydatidosis infection in sheep and cattle was 2.48% and 2.25%, respectively. With respect to liver flukes, 0.62% and 0.25% sheep and cattle were infected by Fasciola spp., respectively; furthermore, 2.86% sheep and 0.79% cattle were positive for D. dendriticum. Conclusion: The findings will provide considerable awareness for the future monitoring and control of these potentially important infections.

Background: Food-borne trematode infections and hydatidosis are endemic diseases caused by helminths in Iran that are responsible for great economic loss and getting public health at risk. Aim: Aim of this study was to determine the prevalence of fasciolosis, dicrocoeliasis, and hydatidosis infections in slaughtered sheep and goats in Nishapour, Khorasan Razavi province of Iran. Materials and Methods: A survey was implemented on 130,107 sheep and goats slaughtered at an abattoir in Nishapour (Neyshbur) city, north central Khorasan Razavi Province, Iran, to determine the prevalence of fascioliasis, dicrocoeliosis and presence of hydatidosis. Results: During a 1-year period of study, among 130,107 of sheep and goats slaughtered at Nishapour abattoir, 1064 and 7124 livers were condemned totally and partially, respectively. A total of 255 (0.19%), 181 (0.12 %), and 7751 (5.95%) of livers were condemned due to cysts of Echinococcus granulosus, flukes of Fasciola spp., and Dicrocoelium dendriticum, respectively. Totally, 1932 (1.48%) lungs were condemned due to hydatidosis. The significant seasonal pattern was seen for fasciolosis, dicrocoeliosis, and hydatidosis, statistically (p

Background This study investigated the epidemiological and molecular aspects of dicrocoeliosis in extensive sheep farms. Methods From 2013 to 2014, copromicroscopical analyses in 190 dairy sheep farms and anatomo-pathological inspections in six slaughterhouses were carried in Sardinia, Italy. Rectal faecal samples were analyzed using the FLOTAC® method, and anatomo-pathological examinations were based on detecting thickened terminal bile ducts (TTBDs). In addition, genetic analyses were conducted on representative DNA samples of adult Dicrocoelium spp. Results Ninety-seven (51.1%) out of 190 sheep farms were coprologically positive for Dicrocoelium spp. In the liver, on the surface and cut surface, TTBDs were reported in 40.1% (309/770) and 15.3% (118/770) of the animals examined, respectively, with an overall prevalence of 25.5% (196/770). No intraspecific genetic variation was observed among the Dicrocoelium dendriticum isolates. Conclusions Our survey reveals the widespread presence of D. dendriticum in Sardinia, although seasonal, geographical and climatic conditions might be key factors in modulating the infection prevalence. Examining typical lesions due to D. dendriticum in the liver in abattoirs can be used as a marker for tracking chronic dicrocoeliosis infection.

Editado por: Toledo, R.; Fried, B.--Digenetic Trematodes.-- Colección: Advances in Experimental Medicine and Biology 766

Dicrocoelium dendriticum is the most common liver fluke in domestic ruminants in Italy, reaching prevalence values of 67% on ovine farms in the southern regions. It causes economic losses and clinical problems. The present paper reviews the results of a controlled field trial conducted in southern Italy to evaluate the efficacy of a single or double administration of albendazole micronised against D. dendriticum in pastured sheep naturally infected by the small liver fluke. The trial was conducted in a sheep farm located in the Campania region, southern Italy. Sixty female crossbreed sheep were selected on the basis of the D. dendriticum positive faecal egg counts (FEC). Ewes were divided into 3 groups, of 20 sheep each, based on D. dendriticum FEC. One group (A-group) was treated once and the other group (B-group) treated twice at an interval of one week, whereas the control group (C-group) was untreated. The efficacy of treatment was evaluated by FEC and necropsy examination for D. dendriticum. Following the WAAVP guidelines for evaluating the efficacy of anthelmintics in ruminants, the double administration of albendazole micronized, showed a high efficacy (up to 99% based on FEC reduction), whereas the single treatment was less effective (up to 96% based on FEC reduction) against D. dendriticum. This difference was significant (P < 0–0.05). The results were also confirmed by necropsy examinations. At day 14 the mean D. dendriticum burdens of the slaughtered sheep of the A-group, B-group and C-group were 87, 70 and 1274 adults (P < 0.001), respectively following necroscopic examinations.

We report four cases of dicrocoeliosis in sheep of untraced origin at an abattoir in Wales in 2015. Liver presentation ranged from severe, with extensive disseminated fibrosis and small bile duct hyperplasia and ectasia, to mild, characterised by occasional small white lesions on the subcapsular surface. Incising the liver revealed black fluid containing Dicrocoelium dendriticum. In another case, from North West England, adult parasites were found in the gall bladder—there were no liver lesions—and eggs were present in faeces. The cases demonstrate that this condition may appear in sheep in UK outside the known endemic areas of western Scotland. Surveillance is therefore necessary but only achievable through accurate diagnosis. Investigators must be aware that lesions can appear similar in some cases to those caused by other parasites, especially Fasciola hepatica and Cysticercus tenuicollis, and that coprological methods to detect sheep nematode eggs are insensitive for this parasite.

Objective: The liver flukes, Fasciola spp. and Dicrocoelium dendriticum, infect ruminants and other mammalian extensively and cause major diseases of livestock that produce considerable economic losses. Methods: A survey of 2 391 sheep and goats slaughtered at an abattoir in Amol region, northern Iran was used to determine the prevalence of the liver flukes infection based on season, sex and specie of the animals. Results:The results revealed that the prevalence rate of Fasciola spp. and Dicrocoelium dendriticum was 6.6% and 4.3% respectively. Dicrocoeliosis was more dominant in female animals (7.1%) whereas there was no sex-related difference in the prevalence of Fasciola spp. in male and female animals. Furthermore, Fasciolosis was significantly more prevalent than dicrocoeliosis in both sheep and goats. The Seasonal prevalence of Fasciola spp. was highest (P

Fasciola spp. and Dicrocoelium dendriticum as liver flukes, contaminate ruminants and other mammalian extensively and cause major diseases of livestock that create considerable economic losses. This retrospective study has been done to evaluate contamination rate of slaughtered animals with fasciolosis and dicrocoeliosis at Lorestan abattoirs. In this survey, prevalence rate of fasciolosis and dicrocoeliosis in slaughtered animals in a 3-year period (2010–2013) has been analyzed. A total of 356,605 livestock including 265,692 sheep and 90,913 goats were slaughtered in the 3-year period and overall 39,613 (11.1 %) livers were condemned. Fascioliasis and dicrocoeliosis were responsible for 6.3 and 4.8 % of total liver condemnations in this period, respectively. Fasciola spp. and D. dendriticum infection in sheep (7.1 and 5.6 %, respectively) were considerably higher than goats (3.9 and 2.6 %, respectively). The annual prevalence rates showed a significant decline in the fasciolosis and dicrocoeliosis infection in goats (p

Dicrocoeliosis is mainly caused by Dicrocoelium dendriticum , which is also known as ‘lancet fluke' or ‘small liver fluke' as opposed to Fasciola spp., otherwise known as the ‘liver fluke'. This underestimated parasitic disease affects the liver of domestic and wild ruminants, which can lead to weight loss and reduced milk production, and is also common among grazing ruminants. Reports of dicrocoeliosis are increasing, mainly due to the expansion of dry desertified habitats and the increase in anthelmintic resistance. This article provides insights into the latest investigations on diagnosis and control of dicrocoeliosis, and discusses the feasibility of environmentally friendly management strategies.

Tesis doctoral de la Universidad de León y el Instituto de Ganadería de Montaña (CSIC). Leída el 23-10-2013., [EN] The main goal of this PhD thesis was to improve Dicrocoelium dendriticum diagnosis in its definitive and intermediate hosts, using immunological and molecular techniques, for which we have carried out the following procedures. A PCR technique has been developed for the accurate identification of D. dendriticum in molluscs and ants, the first and second intermediate hosts, and their early detection. Also, proteomics techniques have been used to identify the major antigenic proteins in the tegument (TG) and excretory–secretory (ES) antigenic extracts of D. dendriticum. Finally, a random preliminary screening of a D. dendriticum adult cDNA library has been used for the first time to study a collection of EST (Expressed Sequence Tag), and three recombinant proteins have been obtained and assessed for the diagnosis of dicrocoeliosis in the definitive host. Detection of D. dendriticum in molluscs and ants The main aim of this PhD thesis was to develop, perfect and validate an analytical method based on PCR (polymerase chain reaction) techniques which would, on the one hand, allow precise identification of D. dendriticum in mollusc and ant intermediate hosts and, on the other, allow early detection of their infection in order to avoid false negatives. First, we collected specimens of molluscs and ants of different species, both naturally infected and uninfected, D. dendriticum adult parasites and other species of flukes for specificity tests. Furthermore, experimental infections of a batch of 80 mollusc species of C. (X.) cespitum arigonis and C. (C.) virgata were carried out with D. dendriticum eggs. Faced with the lack of D. dendriticum sequences, five pairs of degenerate oligonucleotide were designed and tested by aligning mitochondrial sequences of phylogenetically close parasites available in databases. A primer pair that amplified a 1034 bp of mitochondrial DNA fragment was chosen, which was submitted to the GenBank database with Accession No. JF690758. From this sequence, a second pair of specific primers was designed, which amplified a 169 bp fragment. The first primers permitted the detection even 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 D. dendriticum to be detected in the molluscs, they did not discriminate Brachylaimidae metacercariae found in the same mollusc. A new pair of primers was designed to amplify a 93 bp fragment of the nuclear region ITS-2., The PCR designed is D. dendriticum specific as it did not amplify D. chinensis, Brachylaimidae, F. hepatica, C. daubneyi, Plagiorchiidae or Notocotylidae. Besides, this technique is very sensitive since it permitted D. dendriticum to be detected in the molluscs from the first day post-infection as well as the brainworm in the head of the ants and only 1 D. dendriticum metacercaria from the abdomen of the ants. Natural infection by D. dendriticum was confirmed for the first time in 10 species of naturally infected molluscs. Proteomic analysis of the TG and ES antigens The second purpose was to study and identify the major antigen proteins in the adult D. dendriticum tegument and excretion-secretion extracts using bidimensional electrophoresis (2-DE) and mass spectrometry (MS) techniques. First the extraction process of the TG and ES antigen fractions of D. dendriticum was optimized, after which the best conditions of separation by 2D electrophoresis and immunodetection of antigenic proteins were determined. The best results in terms of spot resolution and reproducibility were obtained using the following parameters: treating the samples with the "ReadyPrep 2D clean-up" kit; 3-10 IPG non-linear strips, colloidal Coomassie staining. Under those conditions the maps of the protein ES and TG extracts of D. dendriticum were obtained. In the TG 332 spots were detected and 284 in the ES, both extracts showing a similar distribution in the number of spots as in the ranges of molecular weights and isoelectric points. Also, an immunodetection of antigenic proteins onto nitrocellulose membranes was carried out with sera from experimentally infected sheep. Majority and antigenic proteins were subsequently excised from colloidal Coomassie stained gels and identified by Mass Spectrometry. A quantity of 29 proteins in the excretion-secretion products and 43 in the teguments were identified first in D. dendriticum, 23 of them antigenic, involved in various processes such as: metabolism, detoxification, chaperone, transport or structural molecules. These results could help us to understand the complex parasite-host relationships, improve the diagnosis of dicrocoeliosis and help to produce possible vaccines to control it. Construction of an expression library and EST analysis The next objective was to construct a cDNA library of D. dendriticum to identify specific genes to be used as recombinant antigens in the specific immunological diagnosis of dicrocoeliosis and extend molecular information about it. For this purpose, mRNA from 400 adult parasites obtained from naturally infected sheep was extracted and purified. A double-strand cDNA was synthesized from mRNA and ligated with Uni-ZAP XR vector, then E. coli XL1-Blue MRF’ cells were transformed with the ligation mixture and used for library propagation and maintenance., After construction and titration, the cDNA library was amplified for greater stability. The amplified cDNA library had 2 x 107 plaque forming units (pfu)/ ml, the cDNA insertion rate was 90% and the length of the cDNAs ranged from 120 bp to 1300 pb. We can establish that the library was of good quality, because less than 20% useless sequences were found and 80% of the total sequenced genes were different ones. A random screening of 230 phage plaques was carried out by plaque-PCR and then cloned in the pGEM-T easy vector. After edition and analysis sequences, the trimmed ESTs were assembled into 158 clusters, the most abundant of which was identified as mitochondrial DNA. All other ESTs were registered in GenBank under accession numbers JZ330400- JZ330572. Those EST sequences that displayed significant similarities with known sequences were categorized by their Biological Process, Molecular Function and Cellular Component according to information obtained from the Gene Ontology database. Many of the molecules described in this work could carry out important functions such as: penetration and migration in host tissues, immunoevasion, digestion, redox homeostasis or cellular stress response that could serve as a starting point for further research in disease control. Obtaining and evaluation of recombinant protein in dicrocoeliosis diagnosis In order to identify those antigenic molecules of D. dendriticum that can be used in the serological diagnosis of dicrocoeliosis, 3 clones were selected to be expressed in recombinant form and evaluated in disease diagnosis. Proteins were selected based on the literature and the solubility and antigenicity results obtained through "in silico" sequence studies. Thus, the clones selected were: a / Clone No. 179 myoglobin, b / Clone No. 182 8kDa protein c / Clone No. 151 cystatin. Bioinformatic analysis of the three proteins was performed to ensure that they had a complete ORF, the presence of a possible signal peptide and to predict their secondary structure. Three cDNAs were subcloned into both the Glutation-S-Transferase (GST) pGEX6P vector (Health Care) and the His6-tag pRSET vector (Life Technologies). A PCR assay was carried out using specific primers, including the restriction enzyme sites. Subsequently, the cDNAs were cloned into both vectors and different IPTG expression conditions were tested in the transformed E. coli. The best results were obtained with pGEX-6P vector. In the case of the pRSET-A vector it was only possible to induce cystatin expression in the insoluble fraction and this hampers the subsequent purification process. After evaluating the best conditions, a large-scale purification was carried out by affinity chromatography using glutathione sepharose columns. Finally, a preliminary assessment of the three recombinant proteins was conducted to evaluate diagnosis potential by Western blotting with sera from experimentally infected lambs., The most satisfactory results were obtained with the 8-kDa protein, which was performed with a preliminary indirect ELISA test using polyclonal sera obtained throughout the post-infection period (up to 180 days pi). The results obtained from this trial demonstrated the diagnostic value of the 8-kDa protein, which was able to discriminate between positive and control animals from day 30 pi, with maximum values obtained from day 60 pi. Also, specificity of the test was evaluated using heterologous sera collected from monospecific animals and those experimentally infected with other flukes (C. daubneyi, F. hepatica and S. bovis) and nematodes (T. circumcinta, H. contortus and C. colubriformis). Cross-reactions were only observed in animals infected with S. bovis., [ES] 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 definitivos. Detección de D. dendriticum en moluscos y hormigas El primer objetivo de la presente Tesis fue desarrollar y validar un método analítico basado en la técnica de PCR, que permitiera identificar, con precisión y de forma precoz, D. dendriticum en moluscos y hormigas, primeros y segundos hospedadores intermediarios, respectivamente. En primer lugar, se recolectaron ejemplares de moluscos y hormigas de distintas especies, infectados de forma natural y sin infectar, así como parásitos adultos de D. dendriticum y de otras especies de trematodos para los ensayos de especificidad. Asimismo, se realizaron infecciones experimentales con huevos de D. dendriticum, de un lote de 80 moluscos de las especies C. (X.) cespitum arigonis y C. (C.) virgata. Ante la falta de secuencias nucleotídicas disponibles del parásito, se diseñaron y probaron cinco parejas de oligonucleótidos degenerados, mediante el alineamiento de las secuencias mitocondriales disponibles en las bases de datos de parásitos próximos filogenéticamente. Se seleccionó una pareja de cebadores que amplificó un fragmento de ADN mitocondrial de 1034 pb, que fue enviado a la base de datos GenBank con el nº de acceso JF690758. A partir de esta secuencia se diseñó una segunda pareja de cebadores específicos del parásito, que amplificó un fragmento de 169 pb. Mediante esta técnica fue posible la detección de una única metacercaria de D. dendriticum extraída del abdomen de hormigas de las especies F. rufibarbis y F. pratensis, así como la detección de la larva cerebral en la cabeza de hormigas recogidas en fase de tetania. Fue también posible detectar la infección en moluscos infectados experimentalmente con D. dendriticum., No se observaron reacciones cruzadas con adultos de D. chinensis, F. hepatica y C. daubneyi, ni con fases larvarias de otros digenea hallados en el molusco acuático G. truncatula. Sin embargo, no fue posible discriminar entre el ADN de D. dendriticum y las larvas de Brachylaimidae sp. encontradas en las mismas especies de moluscos terrestres. Debido a esta falta de especificidad se diseñó una nueva pareja de cebadores basada en la región nuclear ITS-2, con la que se amplificó un fragmento de 93 pb. La PCR diseñada a partir de esta región demostró ser específica de D. dendriticum, por lo que no se observaron reacciones cruzadas con el ADN de D. chinensis, Brachylaimidae, F. hepatica, C. daubneyi, Plagiorchiidae o Notocotylidae. Además, con esta técnica fue posible detectar fases larvarias de D. dendriticum en moluscos infectados experimentalmente desde el primer día p.i. También se pudo confirmar, por primera vez, la infección natural por D. dendriticum en 10 especies de moluscos infectados naturalmente. La PCR demostró, además, tener una elevada sensibilidad, ya que permitió la detección de la larva cerebral de las hormigas que actúan como segundos hospedadores intermediarios, así como hasta una única metacercaria de D. dendriticum obtenida del abdomen de hormigas infectadas. Análisis proteómico de los antígenos TG y ES El segundo objetivo era la identificación de las proteínas mayoritarias y antigénicas de los extractos de TG y ES de adultos de D. dendriticum, mediante el uso de técnicas de de electroforesis 2D y espectrometría de masas. Para ello se optimizaron los procesos de obtención de las fracciones antigénicas TG y ES de D. dendriticum, tras lo cual se establecieron las condiciones óptimas de separación mediante electroforesis 2D e inmunodetección de las proteínas antigénicas. Los mejores resultados en cuanto a resolución de spots y reproducibilidad fueron obtenidos al utilizar los siguientes parámetros: tratamiento de las muestras con el kit "ReadyPrep 2D clean-up”; tiras IPG 3-10 no lineal y tinción Coomassie coloidal. De esta manera se han obtenido, por primera vez, los mapas proteicos de los extractos TG y ES de D. dendriticum. Se han detectado 332 spots en el TG y 284 en el ES, con una distribución similar tanto en el número de spots como en los rangos de pesos moleculares y puntos isoeléctricos. Asimismo, se llevó a cabo la inmunodetección de las proteínas antigénicas en membranas de nitrocelulosa con sueros de corderos infectados experimentalmente. Las proteínas mayoritarias y antigénicas fueron posteriormente escindidas de los geles teñidos con Coomassie coloidal e identificadas mediante Espectrometría de Masas., Se han identificado, por primera vez, 43 proteínas en el TG, 12 de ellas antigénicas, mientras que en ES se han identificado 29, 11 de las cuales también resultaron antigénicas. Las proteínas identificadas en este trabajo fueron clasificadas por su función molecular en 16 categorías, algunas con funciones tan importantes como: detoxificación, chaperona, transporte, estructural o metabolismo. Además, varias de las moléculas descritas en este trabajo han sido propuestas como posibles candidatos vacunales y/o antígenos relevantes para el diagnóstico de otras parasitosis. Construcción de una genoteca de expresión y análisis EST El siguiente objetivo fue la construcción de una genoteca de ADNc de D. dendriticum, con la que poder obtener antígenos recombinantes que nos permitan mejorar el diagnóstico inmunológico de la dicrocoeliosis, así como ampliar la información molecular del mismo. Para ello, se extrajo y se purificó el ARNm de 400 parásitos adultos vivos obtenidos del hígado de ovejas infectadas de forma natural. A partir del ARNm se sintetizó la cadena de ADNc que se ligó en el vector de clonación Uni-Zap XR, con el que se infectaron las células E. coli XL1-Blue MRF’, utilizadas para la propagación y mantenimiento de la genoteca. Tras la construcción y titulación de la misma se amplificó para conseguir una mayor estabilidad. El título de la genoteca amplificada fue de 3 x 108, el porcentaje obtenido de recombinantes del 90%, y el tamaño de los insertos osciló entre 120-1300 pb. Esto demuestra la elevada calidad de la genoteca de expresión por la alta proporción de insertos mayores de 500 pb y la elevada proporción de nuevos genes secuenciados. Se realizó un cribado al azar de la misma y se aislaron 230 fagos que se secuenciaron y posteriormente clonaron en el vector pGEM-T easy. Tras la edición y análisis de las secuencias se obtuvieron 200 EST que se ensamblaron en 158 clústeres, de los cuales el más abundante —formado por 27 secuencias— se corresponde con ADN mitocondrial. Esta es la primera colección obtenida de EST de D. dendriticum que se ha depositado en la base de datos del GenBank bajo los nos. de acceso: JZ330400- JZ330572. Tras el análisis de los EST se clasificaron según su función molecular, proceso biológico y componente celular, de acuerdo a la base de datos GeneOntology. Muchas de las moléculas aquí descritas tienen importantes funciones, tales como: penetración y migración en los tejidos del hospedador, inmunoevasión, digestión, homeostasis redox o respuesta al estrés celular, y aportan una valiosa información para futuras investigaciones del parásito., Obtención y evaluación de proteínas recombinantes en el diagnóstico de la dicrocoeliosis Con el fin de identificar aquellas moléculas antigénicas de D. dendriticum que puedan ser empleadas en el diagnóstico serológico de la dicrocoeliosis, se seleccionaron 3 clones para ser expresados en su forma recombinante y ser evaluados en el diagnóstico de la enfermedad. Las proteínas se seleccionaron en base a la bibliografía y a los resultados de aparente solubilidad y antigenicidad, obtenidos mediante los estudios “in silico” de las secuencias. De esta manera, se seleccionaron los siguientes clones: a/ Clon Nº179 mioglobina, b/ Clon Nº 182 proteína de 8kDa y c/ Clon Nº 151 cistatina. Se realizo el análisis bioinformático de las tres moléculas para comprobar que poseían un ORF completo, la presencia de un posible péptido señal y predecir su estructura secundaria. Se llevó a cabo la subclonación de los tres ADNc por duplicado en los vectores pGEX-6P-2 (Health Care) y pRSET-A (Life Technologies), que expresan la proteína recombinante fusionada a GST y a una cola de 6-His, respectivamente. Para ello, se amplificaron los fragmentos de interés mediante PCR, utilizando los cebadores específicos en los que se incluyeron los sitios de corte de las enzimas de restricción. Posteriormente, se clonaron en ambos vectores y se probaron las condiciones de expresión en E. coli mediante su inducción con IPTG. Se obtuvieron los mejores resultados con el vector pGEX-6P. En el caso del vector pRSET-A tan solo fue posible inducir la expresión de la cistatina, que se encontraba en la fracción insoluble, lo que dificulta el posterior proceso de purificación. Tras evaluar las mejores condiciones, se realizó el cultivo a gran escala de las correspondientes bacterias y se indujo la expresión de las proteínas recombinantes, que se purificaron por cromatografía de afinidad mediante columnas de glutation sepharosa. Finalmente, se realizó una evaluación preliminar para el diagnóstico mediante Western Blot con sueros de corderos infectados de forma experimental de las tres proteínas recombinantes obtenidas en este trabajo. Los resultados más satisfactorios se obtuvieron con la proteína de 8-kDa, con la que se realizó un ensayo preliminar de ELISA indirecto utilizando sueros policlonales obtenidos a lo largo de todo período post-infección (hasta los 180 días p.i.). Los resultados obtenidos mediante este ensayo demostraron el valor diagnóstico de la proteína e 8-kDa, que fue capaz de discriminar entre animales positivos y controles desde el día 30 p.i., obteniendo los máximos valores a partir del día 60 p.i. Asimismo se evaluó la especificidad del test, empleando sueros heterólogos de animales monoespecífica y experimentalmente infectados con otros trematodos (C. daubneyi, F. hepatica y S. bovis) y nematodos (T. circumcinta, H. contortus y C. colubriformis). Únicamente se observaron reacciones cruzadas con animales infectados con S. bovis., Junta de Castilla y León, Ministerio de Ciencia y Educación, Fondo Social Europeo, MARTÍNEZ-IBEAS, A. M., MARTÍNEZ-VALLADARES, M., GONZÁLEZ-LANZA, C., MIÑAMBRES B. & MANGA-GONZÁLEZ M.Y.(2011). Detection of Dicrocoelium dendriticum larval stages in mollusc and ant intermediate hosts by PCR, using mitochondrial and ribosomal internal transcribed spacer (ITS-2) sequences. Parasitology, 138: 1916-1923. Cambridge University Press. http://dx.doi.org/10.1017/S003118211001375, MARTÍNEZ-IBEAS, A. M., GONZÁLEZ-LANZA C. & MANGA-GONZÁLEZ, M.Y.(2013). Proteomic analysis of the tegument and excretory-secretory products of Dicrocoelium dendriticum (Digenea) adult worms. Experimental Parasitology, 133: 411-420. Elsevier. http://dx.doi.org/10.1016/j.exppara.2013.01.010, MARTÍNEZ-IBEAS, A. M., PERTEGUER, M.J., GONZÁLEZ-LANZA C., GARATE, T. & MANGA-GONZÁLEZ, M.Y.(2013). Analysis of an expressed sequence tag library from of Dicrocoelium dendriticum. Experimental Parasitology, 135: 287-296. Elsevier. http://dx.doi.org/10.1016/j.exppara.2013.07.005

The prevalence of dicrocoeliosis was estimated by the examination of liver of slaughtered goats (n = 228) brought from two major goat rearing regions (Kandi and R.S. Pura) of Jammu province of India. Dicrocoelium dendriticum was found in 18.9 % of the goats, with mean fluke count (±SEM) of 24.9 ± 4.4 (ranged from 0 to 478). A significant seasonal variation (p

Dicrocoeliosis was identified as the probable predisposing cause of weight loss and hepatogenous photosensitisation affecting half of a group of 14 month-old ewe lambs on a farm on the Inner Hebridean island of Coll. Oral dosing of the ewe lambs with 15 mg/kg albendazole only achieved a 79.2% reduction in Dicrocoelium dendriticum egg count, 21 day post treatment. Thus, this report of dicrocoeliosis differs from the disease that occurs elsewhere, both in the severity and nature of its clinical signs, and in the response of the parasite to anthelmintic drug treatment. These differences could indicate the existence of a genetically divergent D. dendriticum population, which may have evolved within the unique and isolated biotope afforded by the machair on the island of Coll. Better understanding of these factors is a prerequisite for effective and sustainable disease control.

Human dicrocoeliosis is reported sporadically in various parts of the world. We report a case in a 21-year-old male, who had right upper abdominal pain, weight loss, and chronic relapsing watery diarrhea three to four times daily for four weeks. The patient had abdominal tenderness to palpation in the right upper quadrant. Alkaline phosphatase, alanine aminotransferase, and serum immunoglobulin E levels were slightly elevated; all other biochemical and hematological findings were in their normal ranges. The duodenal biopsy samples were normal and an abdominal ultrasonography showed no biliary or hepatic abnormality. Stool microscopy revealed numerous eggs of Dicrocoelium dendriticum. As pseudoparasitosis can result from eating raw, infected animal liver, the patient was given a liver-free diet for three days, to rule out that possibility. Subsequent stool examinations showed eggs in each of the samples indicating that the infection was genuine. The patient was treated with triclabendazole 10 mg/kg in a single dose. Four weeks later, no parasite eggs were detected in the microscopic examination of the stool samples. The patient got better gradually and the symptoms disappeared. Physicians should keep in mind parasitic diseases such as the rarely encountered dicrocoeliosis.

Despite its widespread presence among grazing ruminants, dicrocoeliosis, also known as "small liver fluke" disease, is poorly known and often underestimated by researchers and practitioners in many countries. This is primarily due to the multiple parasitic infections which affect ruminant livestock and mask the pathology of dicrocoeliosis, to the difficulties in diagnosing it with coprological techniques and, finally, to the few effective drugs found. Furthermore, the biological cycle of Dicrocoelium, which requires a snail and an ant as intermediate hosts, and the high number of ecological and epidemiological variables affecting the disease make it difficult to set up experimental designs to study dicrocoeliosis. In the past 50 years, many aspects of this disease have been broadly investigated (aetiology, life cycle, diffusion, epidemiology, pathogenesis and immunology) but its diagnosis and treatment still remain moot issues. Dicrocoeliosis often remains clinically undetected and its diagnosis is mostly based on adult dicrocoelia recovered in the liver post mortem or on egg detected at coprological examination. The prophylaxis of the small liver fluke has been difficult and unsatisfactory to date due to the complexity of its biological life cycle and epidemiology. Many anti-helminthic drugs are practically ineffective against dicrocoeliosis if used at the dosage recommended against other gastrointestinal helminths and lungworms. The most important aspects of the aetiology, biological cycle, spread, epidemiology and pathogenesis of dicrocoeliosis are reviewed and the recent advances in the diagnosis and treatment are focused on.

6 pages, 2 tables, 2 figures.-- PMID: 8983169 [PubMed].--Available online 25 Jan 2005., The purpose of this investigation was to determine the effects of experimental dicrocoeliosis on the hepatic oxidative drug-metabolizing system in hamsters. Studies were carried out 80 and 120 days after infestation with an oral dose of 40 metacercariae of Dicrocoelium dendriticum. The parasitic pathology was ascertained by detection of the fluke eggs in faeces, increased serum alanine aminotransferase and aspartate aminotransferase activities, and postmortem and histological findings. Cytochrome P-450 concentration, aniline hydroxylase activity and ethoxycoumarin O-deethylase activity were significantly decreased in both groups of infected animals. Aminopyrine N-demethylase activity and erythromycin N-demethylase activity were only reduced 120 days after infection. Effects on drug metabolizing enzymes were unrelated to changes in the physical state of the microsomal membrane, as assessed by measurement of fluorescence polarization. The results of this study indicate that the capacity of the liver for handling drugs and xenobiotics may be impaired as a consequence of dicrocoeliosis., This study was supported by the Junta de Castilla y León (Project LE16/94)and the CICYT (Project AGF92-0588) Spain.

A survey incorporating field and abattoir studies was carried out on the prevalence of Dicrocoelium dendriticum in sheep and goats in the Kangra valley of Himachal Pradesh (India). Coprological studies on these migratory animals revealed that 8.1% of sheep and 4.1% of goats were positive for dicrocoeliosis, with a mean number of eggs per gram of faeces (EPG) of 702.1 for sheep and 566.7 for goats. The necropsy liver examination of slaughtered animals revealed that 24.1% of sheep and 12.3% of goats were infected with D. dendriticum and they had mean fluke burdens of 247.2 (sheep) and 298.1 (goats). No significant differences in fluke burden, liver lesion score and EPG count were observed between sheep and goats. The seasonal distribution of dicrocoeliosis indicated a higher percentage of infection in autumn and winter as compared with spring and summer. The potential role of migratory sheep and goats in the epizootiology of dicrocoeliosis is discussed.

The sensitivity and specificity of the agar gel precipitation test (AGPT), counter immunoelectrophoresis (CIEP) and passive haemagglutination test (PHT) were evaluated for the diagnosis of Dicrocoelium dendriticum infection in naturally infected sheep and goats. Two hundred and forty five sheep and goat sera samples were tested using phosphate buffered saline, pH 7.2 extracted adult fluke antigen. CIEP detected 69.8% of the infected animals and was found to be the most sensitive, followed by PHT which detected 50.0% of the infected sheep and goats. AGPT was found to be the least sensitive, detecting only 23.8% of the infected animals. The specificity of AGPT, CIEP and PHT were 93.3%, 84.0% and 93.3%, respectively. CIEP was found to be the most sensitive, specific and rapid test for the seroepidemiological survey of dicrocoeliosis in sheep and goats.

Tesis de la Universidad de León. Facultad de Biología. Leída el 3 de julio de 1995. Este trabajo se llevó a cabo gracias a la financiación recibida de la Junta de Castilla y León, a través del Proyecto de Investigación número 0701/89, y la Beca de Formación de Personal Investigador concedida en virtud del Convenio de Cooperación CSIC-Caja de Ahorros y Monte de Piedad de León., [EN] Entre 1987 y 1991 se ha realizado en el valle de Redipollos, situado en la cuenca alta del río Porma (León, Noroeste de España), un estudio epidemiológico sobre la dicroceliosis, parasitosis producida por Dicrocoelium dendriticum (Rudolphi, 1819) Looss, 1899. Esto ha exigido investigaciones tanto en el ganado ovino (hospedador definitivo) como en los moluscos y hormigas (primeros y segundos hospedadores intermediarios, respectivamente). Como resultado de los muestreos efectuados, para el estudio de diversos aspectos biológicos y ecológicos de los moluscos presentes en el área de estudio, se identificaron 29 especies, todas ellas de la clase Gastropoda, subclase Pulmonata, orden Stylommatophora, excepto una de ellas. Sin embargo, en el estudio helmintológico de dichos moluscos, solamente Helicella itala (Linnaeus, 1758) y Helicella corderoi Gittenberger & Manga, 1977 se hallaron parasitadas por D. dendriticum. Moluscos de dichas especies que albergaban esporocistos con cercarias maduras se encontraron en todos los meses del año, aunque predominaron en primavera y otoño. En lo que respecta a los segundos hospedadores intermediarios, se identificaron 21 especies de hormigas de la Familia Formicidae, de las cuales únicamente albergaban metacercarias del parásito Formica cunicularia Latreille, 1789, Formica rufibarbis Frabricius, 1794, Formica sanguinea Latreille, 1789 y Formica nigricans Emery, 1909 (=Formica pratensis Retzius, según Seifert 1992). Ejemplares de hormigas parasitadas de esta última especie fueron hallados en fase de tetania en primavera y verano. Cuando se consideró la totalidad de los animales y los dos años de muestreo, se detectó eliminación de huevos del parásito por el ganado ovino en todos los meses del año, aunque el porcentaje más elevado de animales parasitados se observó en enero-febrero y el más bajo en julio. Por otra parte, el nº medio de huevos por gramo se incrementó en otoño y alcanzó su valor máximo a finales del invierno., [ES] An epidemiological study of dicrocoeliosis, a parasitosis produced by Dicrocoelium dendriticum (Rudolphi, 1819) Looss, 1899, was carried out in the upper basin of the River Porma (León, northwest Spain) between 1987 and 1991. This required research into sheep (the definitive host) as weII as in molluscs and ants (first and second intermediate hosts, respectively). A total of 29 species were identified as a result of the sampling carried out for the study of diverse biological and ecological aspects of the molluscs present in the study area. All, except one, belonged to the Gastropoda class, subclass Pulmonata, order Stylommatophora. However, only Helicella itala (Linnaeus, 1758) and Helicella corderoi Gittenberger & Manga, 1977, were recorded as parasitized by D. dendriticum in the helminthological study of these molluscs. Those hosting sporocysts with mature cercariae were found in every month of the year although they predominated in spring and autumn. As far as the second intermediate hosts are concerned, 21 ant species of the Formicidae family were identified. OnIy Formica cunicularia Latreille, 1789, Formica rufibarbis Fabricius 1794, Formica sanguinea Latreille, 1789 and Formica nigricans Emery, 1909 (=Formica pratensis Retzius, according to Seifert 1992) hosted metacercariae of the parasite. Infected specimens of the Iast named species were found at the “tetania” stage in spring and summer. On taking into consideration all the animals and the two years of sampling, parasite egg shedding by sheep was recorded in every month of the year, although the highest percentage of parasitized animals was found in January-February and the lowest in JuIy. On the other hand, the mean number of eggs per gram increases in autumn and was at its highest and the end of winter.

Duchaaek L., J. Lamka: Dicrocoeliosis ‐ the Present State of Knowledge with Respect to Wildlife Species. Acta Vet. Brno 2003, 72: 613-626. The present paper summarizes contemporary knowledge concerning dicrocoeliosis (chronic disease of the liver caused by the lancet fluke Dicrocoelium dendriticum) as to its spraed, etiology, life cycle, epidemiology, pathogenesis, immunogenesis, clinical symptoms, diagnostics, therapy, prevention of dicrocoeliosis, and human infection. The major part of published data is connected with farm animal husbandry, dicrocoeliosis of wildlife species is examined to a limited extent only. Although dicrocoeliosis is intensively studied, numerous aspects of this trematodosis have to be verified. Control of dicrocoeliosis in domesticated and wildlife species is not fully effective at present. Dicrocoelium dendriticum, lancet fluke Dicrocoeliosis is a helmithosis caused by the lancet fluke Dicrocoelium dendriticum Rudolphi, 1819 (D. dendriticum, syn. D. lanceolatum, Dicrocoelidae, Platyhelminthes), parasitizing in the liver of ruminants as well as many other animals species including man. This parasitosis belongs to the six principal and economically most important pasture helminthoses of sheep and cattle‐trichostrongylidosis, dictyocaulosis, protostrongylidosis, moniesiosis, fasciolosis, and dicrocoeliosis (Hiepe 1994). Parasitosis accompanies man and farm animals breeding from time immemorial. The eggs of D. dendriticum were found in the coprolites and paleofaeces of the prehistoric human population in Central Europe. Nevertheless, it is not certain whether the lancet fluke D. dendriticum was a real parasite, or whether the found eggs indicate pseudoparasitism after consummation of animal liver (Aspock et al. 1999).