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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

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

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.

Dicrocoelium dendriticum is a helminth which lives in the bile ducts and gall bladder of mammalian definitive hosts. Dicrocoeliosis is thought to be endemic in some countries and it has an increasing prevalence in Northern Iraq, potentially due to increased importation of infected animals, particularly sheep, in high numbers from neighboring countries. The parasite's ability to infect rodents, wild animals, livestock and humans means this parasite is of significant interest in veterinary and human medicine. While D. dendriticum causes relatively mild clinical disease in animals, infection leads to liver condemnation at slaughter and subsequent economic losses to farmers. In this study, the livers of 91,486 sheep slaughtered at Sulaymaniyah New Slaughterhouse (Northern Iraq) were visually inspected for D. dendriticum infection between November 2013 and March 2014, with 1269 livers rejected due to D. dendriticum in this period, representing 29.4% of all condemned livers. The highest rate of rejection due to D. dendriticum infection was seen in December, possibly linked to increased numbers of intermediate host snails during the wet season. Routine inspection of condemned livers revealed adult flukes of D. dendriticum and eggs were seen in the faeces of imported sheep, indicating these animals may be a vector for introduction of D. dendriticum to this region. Due to the complex life cycle and wide range of animals which it infects, we cannot establish the exact route of introduction into Iraq; however, our study suggests that slaughterhouse workers, farmers and local health authorities should be aware of the presence of D. dendriticum, and the potential risks it represents to both human and animal health. Our data also suggest that some level of quarantine or border checks may be useful to prevent further introduction of D. dendriticum or other pathogens into Iraq, although this may prove difficult until accurate diagnostic assays are developed.

The concentration of chemical ements (Pb, Cd, Cu, As, Zn, Hg, Fe, Co, Mn) in the liver of healthy cattle and those affected by Fasciola hepatica and Dicrocoelium lanceatum in Poltava region (central part of Ukraine) was determined. The research was carried out by the method of atomic and absorption spectrometry carried out at the Regional State Laboratory of Veterinary Medicine in Poltava region. The liver samples (n = 30) from healthy cattle black-and-white breed and those affected by F. hepatica and D. lanceatum were taken at the meat processing plant. The ages of the cattle ranged from 6 to 8 years. The samples were immediately cooled, transported to the laboratory and stored at –20 °C for further analysis. The results of the research determined the average indicators of concentration of some toxic elements in the livers of healthy cattle and those infected by the trematodes. The content of chemical elements in the liver of healthy animals and those affected by fasciola can be represented in the form of a decreasing rank number: Zn > Fe > Cu, and for dicrocoeliosis, respectively, Fe > Zn > Cu. It has been established that Cu and Zn are involved in the metabolic processes of the body of trematodes, which is confirmed by our research. The presence of F. hepatica and D. lanceatum in the body of cattle significantly reduces the level of copper and zinc, with a high inverse correlation dependence on the intensity of infection, thus indicating the possibility of their accumulation by helminths. Concentration of Cu and Zn in the liver of cattle with fasciolosis was 6.82 ± 0.29 and 35.77 ± 1.93 mg/kg, while for animals with dicrocoeliosis it was 3.90 ± 0.25 and 41.91 ± 2.22 mg/kg. The content of cobalt and manganese in the liver of healthy animals was, respectively, 0.05 ± 0.01 and 1.95 ± 0.06 mg/kg. In the case of Fasciola parasitising in the liver tissue, the level of cobalt (0.10 ± 0.02) and manganese (2.55 ± 0.16) significantly increased, positively correlating with the intensity of the infection, indicating no effect on the exchange and bioaccumulation of these elements by helminths.

Dicrocoeliosis is a helminthosis caused by the small liver fluke Dicrocoelium spp. (Trematoda, Dicrocoeliidae) parasitizing in the bile ducts and gall bladder of ruminants as well as many other animal species including humans. In the biological life cycle of Dicrocoelium, land snails are first intermediate hosts and ants are second intermediate hosts. Sporocysts and cercaria, which are larval stages, live in the hepatopancreas of land snails and metacercaria, which is also the larval stage, lives in the abdomen and brain of ants. Land snails, which are the first intermediate host of this parasite in Turkey, include Helicopsis derbentina, Helicopsis protea, Helicopsis krynickii, Cernuella virgata, Trochoidea pyramidata, Cochicella acuta, Monacha carthusiana, Helicella candicans, Helix aspersa, Helix lucorum, and Chondrus tournefortianus. Dicrocoeliosis is widespread in ruminants and affects their liver, which can lead to weight loss and reduced milk production. The number of reports on dicrocoeliosis is increasing due to the expansion of dry habitats and parasites becoming resistant to antihelminthic drugs. This study provides information on the epidemiology and control methods of Dicrocoelium.

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

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.