Dicrocoelium ova can block the induction phase of experimental autoimmune encephalomyelitis.

AIMS: This study aimed at investigating the impact of Dicrocoelium ova on experimental autoimmune encephalomyelitis (EAE) treatment in C57BL6 mice. METHODS AND RESULTS: Twenty-eight C57BL/6 mice were assigned into four groups as PBS, prophylaxis (P), treatment1 (T1) and treatment2 (T2). Prior to induction of EAE in prophylaxis group and on days 7 and 18 in T1 and T2 groups, respectively, Dicrocoelium eggs were injected intraperitoneally to each mouse. The clinical score, weight changes and incidence time of EAE were recorded. IFN-γ and IL-4 expression is quantified on spleen cells. Also, histopathological study by (H&E) and Toluidine-Blue (TB), and Luxol Fast Blue (LFB) were performed. The data were analysed using SPSS version 21. Mean disease scores were significantly lower in P and T1 groups than the PBS group (P = .01). IFN-γ was lower in P and T1 groups than the PBS group. The highest level of IL-4 was observed in T1 group. The total number of neuroglia cells of corpus callosum was similar in all groups, but the density increased in T1 group compared to the PBS group (P = .03). CONCLUSIONS: Dicrocoelium eggs have a great potential to stimulate immunomodulation towards treatment of EAE during the initial phase.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region.

Despite its extensive 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. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS-PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS-PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

On the presence and immunoregulatory functions of extracellular microRNAs in the trematode Fasciola hepatica.

Liver flukes represent a paraphyletic group of endoparasitic flatworms that significantly affect man either indirectly due to economic damage on livestock or directly as pathogens. A range of studies have focussed on how these macroscopic organisms can evade the immune system and live inside a hostile environment such as the mammalian liver and bile ducts. Recently, microRNAs, a class of short noncoding gene regulators, have been proposed as likely candidates to play roles in this scenario. MicroRNAs (miRNAs) are key players in development and pathogenicity and are highly conserved between metazoans: identical miRNAs can be found in flatworms and mammalians. Interestingly, miRNAs are enriched in extracellular vesicles (EVs) which are secreted by most cells. EVs constitute an important mode of parasite/host interaction, and recent data illustrate that miRNAs play a vital part. We have demonstrated the presence of miRNAs in the EVs of the trematode species Dicrocoelium dendriticum and Fasciola hepatica (Fhe) and identified potential immune-regulatory miRNAs with targets in the host. After our initial identification of miRNAs expressed by F. hepatica, an assembled genome and additional miRNA data became available. This has enabled us to update the known complement of miRNAs in EVs and speculate on potential immune-regulatory functions that we review here.

Analysis of an expressed sequence tag library from Dicrocoelium dentriticum.

Dicrocoeliosis caused by Dicrocoelium dendriticum is an important liver disease, which affects ruminants all around the world. Despite the significant economic losses caused by this trematode, molecular knowledge is very scarce. In fact, there is no information in the expressed sequence tag (EST) database about the parasite. Furthermore, the immunological diagnosis of dicrocoeliosis remains unsatisfactory, and there aren't available recombinant proteins that could be tested in the diagnosis. For this reason a cDNA library was constructed with mRNA extracted from D. dendriticum adults for first time. A random preliminary screening of 230 phage plaques from the library resulted in the identification of 173 new EST. The deduced proteins expressed by these genes have been described as possible vaccine targets in other trematodes, and/or as relevant diagnosis antigens. Then, our goal was to identify D. dentriticum diagnosis genes to be used as recombinant antigens in the specific immunological diagnosis of the trematodoses. A D. dendriticum cDNA encoding an 8-kDa recombinant protein has been cloned, expressed in Escherichia coli and evaluated in dicrocoeliosis diagnosis using both Western Blot and enzyme-linked immunosorbent assay (ELISA). The recombinant expression molecule has demonstrated its value as a diagnosis antigen of dicrocoeliosis, able to discriminate between positive and controls on day 30 post infection. This is the first research conducted for identification and characterization of D. dendriticum ESTs, which can serve as a starting point for future research on immunodiagnosis and immunoprofilaxis of dicrocoeliosis.

Proteomic analysis of the tegument and excretory-secretory products of Dicrocoelium dendriticum (Digenea) adult worms.

Dicrocoeliosis, caused by Dicrocoelium dendriticum, is an important hepatic parasitosis in ruminants, whose immunological diagnosis and control remain unsatisfactory. There are very few studies on the antigens of this trematode and molecular knowledge about it is practically nil. Therefore the aim of this study was to identify the major antigenic proteins in the tegument (TG) and excretory-secretory (ES) antigenic extracts of D. dendriticum. The separation conditions of the protein extracts were optimized using 2-D PAGE; the gels were stained with colloidal Coomassie or transferred to carry out immunodetection with anti-Dicrocoelium dendriticum sera. The proteins of interest excised from the gels were identified by mass spectrometry (MALDI). The proteomic maps of the TG and ES extracts of D. dendriticum were defined first, detecting 332 spots in the TG and 284 in the ES, with a similar distribution in both. 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 dicroceliosis and help to produce possible vaccines to control it.

Detection of antibodies in wild ruminants to evaluate exposure to liver trematodes.

Wild ruminants sharing pastures with domestic livestock are at risk of infection by liver trematodes. Detection of antibodies provides a very useful tool to gain more knowledge about the distribution of these parasites. Non-lethal methods are strongly encouraged for the analysis of the risk of infection among wild ruminants. A seroepidemiological survey was conducted to analyze exposure to hepatic trematodes ( Fasciola hepatica and Dicrocoelium dendriticum ) in wild ruminants from southern Spain. Blood samples were collected from 69 bovids (Mouflon + Spanish ibex) and 143 cervids (red deer + fallow deer) from Sierra de Cazorla, Segura and Las Villas Natural Park. The samples were analyzed using the excretory/secretory antigens of each trematode to determine the IgG response. All the animals were examined at necropsy for the presence of flukes, and the species, age, and gender of the animals were recorded. Fasciola hepatica were only observed in cervids (3%; 95% confidence interval [CI]  =  2-8), while D. dendriticum specimens were recorded in 1% (0-8) of bovids and 4% (CI  =  2-9) of the cervids. The IgG-seroprevalence against F. hepatica was significantly higher in the cervids. Statistical differences according to gender were observed. The bovids exhibited the greatest percentages of positive cases to D. dendriticum antigens, and the DdES-seroprevalence was related to age of the animals. When considering all the factors, the FhES-seroprevalence was initially distributed according to the type of ruminant (cervids), gender (male), and age (>2 yr).

Experimental ELISA for diagnosis of ovine dicrocoeliosis and application in a field survey.

Dicrocoeliosis is a parasitic disease caused by trematodes belonging to the genus Dicrocoelium. It usually produces mild symptoms, and for this reason, dicrocoeliosis often remains undetected. Its diagnosis is mostly based on postmortem examination of the liver or on coprological assays for in vivo diagnosis. However, the latter method has scant sensitivity and because of the long pre-patency of Dicrocoelium spp. only permits late diagnosis. In the present study, an enzyme-linked immunosorbent assay (ELISA) was developed based on excretory/secretory antigen of Dicrocoelium dendriticum. The ELISA detected antibodies (IgG) in experimental infected sheep starting from day 30 post-infection (d.p.i.), whereas coprological samples were positive from 58 d.p.i. The ELISA was used in a field study in order to assess dicrocoeliosis seroprevalence in ovine flocks from the province of Trento (northeastern Italy), and this was the first sero-epidemiological study of ovine dicrocoeliosis in northern Italy. Altogether, 842 sheep sera were tested. In accordance with previous surveys carried out in other regions of Italy, a high prevalence of 80% to 100% was found.

Immunohistochemical study of the local immune response in lambs experimentally infected with Dicrocoelium dendriticum (Digenea).

Phenotypic expression of inflammatory cells in liver and hepatic lymph nodes (HLN) has been examined in lambs experimentally infected with Dicrocoelium dendriticum using immunohistochemical techniques. Thirty-two lambs, 12 infected with 1,000 D. dendriticum metacercariae, 12 with 3,000, and 8 controls were used. Half the lambs in each group were slaughtered on days 60 and 180 post-infection (p.i.), respectively. Primary antibodies (Abs) against T cell epitopes (CD3+, CD4+, CD8+ and WC1+ gammadelta), B cell epitopes (CD79alphacy+, CD45R+), immunoglobulin (IgG)-bearing plasma cells, macrophages (CD14+, VPM32+) and major histocompatibility complex (MHC) class IIbeta antigen were used. T lymphocytes (CD3+, CD4+ and CD8+) and B lymphocytes (CD79alphacy+ and CD45R+) with diffuse pattern or forming lymphoid aggregates and follicles surrounded the septal bile ducts (SBD) and inter-lobular bile ducts, whereas the WC1 gammadelta T cells were scattered. Numerous IgG+ plasma cells were observed around SBD. CD14 and VPM32+ macrophages intermingled with lymphocytes were immunostained by the anti-MHC class IIbeta. This Ab also reacted with lymphoid cells. Likewise, increased positive immunostaining for all Abs used was observed in the HLN of infected lambs. There was no qualitative difference regarding the phenotype expression of inflammatory cells between the lambs infected with D. dendriticum. The humoral and cell-mediated local immune responses observed were similar in the two groups of lambs infected with different doses.

Preliminary protective capacity study of a Dicrocoelium dendriticum antigenic protein in hamsters.

The purpose of this study was to evaluate the protective capacity of 130 kDa Dicrocoelium dendriticum protein in hamsters experimentally infected with this parasite. Forty hamsters divided into four groups of ten animals each were used: G1 (control), G2 (infected), G3 (immunized with Freund's adjuvant and infected), G4 (130 kDa protein vaccinated + adjuvant and infected). Infection with 40 metacercariae/hamster was carried out 4 weeks after the last immunization. Parasitological studies [number of eggs per gram (epg) and worm burden] and biochemical parameters (total proteins, albumin, and total bilirubin), hepatic enzymes [aspartate aminotransferase (AST) and alanine aminotransferase (ALT)], and total IgG levels were determined. A reduction in epg in G3 and G4 was observed 16 weeks postinfection with the higher reduction percentage in the latter (25.2%). No statistically significant differences were detected in the number of recovered worms among groups, although the mean was slightly less in G4 (12.2 +/- 2.08, mean +/- SE) than in G2 (15.4 +/- 2.90). In G4, global protection was 20.9% and an increase in AST and ALT levels was observed. Total IgG levels were similar in the three infected groups. The protection obtained was inadequate, so the antigen dose, immunization-infection period, adjuvants, and immunization route must be optimized.

Field and experimental studies on Dicrocoelium dendriticum and dicrocoeliasis in northern Spain.

The transmission, control and the relationship between Dicrocoelium dendriticum and its definitive (sheep and cattle) and intermediate (molluscs and ants) hosts under natural and experimental conditions are described. Eleven species of molluscs and four of ants were found infected with larval D. dendriticum in León province, north-west Spain. Infected ants were observed between April and November and in tetania at 7.5-26.9 degrees C. The highest shedding of eggs by sheep and cattle was detected in winter. Two treatments applied in November and January were the most effective. In experimentally infected molluscs, the parasite was not visible under the stereomicroscope, at least until 50 days post-infection (p.i.). The prepatent period in experimentally infected lambs was 49-79 days p.i. The number of eggs per gram increased with the days p.i. and the parasite burden. The aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, leukocyte and neutrophil values of infected lambs increased, but those of lymphocytes decreased. Using the enzyme-linked immunosorbert assay technique, the IgG antibody response to excretory-secretory and somatic antigens of D. dendriticum was positive from day 30 p.i., although the maximum antibody levels were observed on day 60 p.i. The number of worms per lamb ranged between 30 and 2063. Cholangitis and cholangiectasia of the septal bile and hepatic ducts were observed. The best enzymatic systems for adult and larval D. dendriticum characterization were lactate dehydrogenase, glucose phosphate isomerase and phosphoglucomutase. Genetic variability of adult D. dendriticum was high using the random amplified polymorphic DNA technique.

Serum antibodies to Dicrocoelium dendriticum in sheep from Sardinia (Italy).

An indirect enzyme-linked immunosorbent assay (ELISA) with Dicrocoelium dendriticum excretory/secretory antigens was used to evaluate the presence of serum antibodies against the trematode in 738 sheep randomly chosen in Sardinia (Italy). Coprological sedimentation was used to discover egg-output. Seropositivity was detected in 86.2% tested sheep, whereas faecal prevalence was 6.7%; all that were faecal-positive also were ELISA-positive.

Morphologic and biochemical changes caused by experimentally induced dicroceliosis in hamsters (Mesocricetus auratus).

BACKGROUND AND PURPOSE: The aim of the study reported here was to investigate the pathomorphologic changes caused by experimentally induced dicroceliosis and their correlation with hepatobiliary function. METHODS: Studies were carried out at days 80 and 120 after oral inoculation of hamsters with 40 metacercariae of Dicrocoelium dendriticum. RESULTS: The parasite-induced pathologic changes were assessed by presence of fluke eggs in feces, increased plasma alanine transaminase and aspartate transaminase activities and morphologic alterations. Dicroceliosis was characterized by bile ductular proliferation and enlargement of the bile duct surface area caused by hyperplastic cholangitis in septal bile ducts. The liver from infected animals contained portal tracts infiltrated with small to moderate numbers of lymphocytes, macrophages, and eosinophils. Simultaneously, there was an increase in portal tract collagen that extended to the interlobular septa and caused pressure atrophy of the hepatic parenchyma. The concentration of thiobarbituric acid-reactive substances and the ratio of oxidized to reduced glutathione, measured as markers of oxidative stress, were significantly increased. CONCLUSIONS: The presence of oxidative alterations could be related to the morphologic evidence of chronic inflammatory response as well as to liver cellular injury indicated by cellular swelling, and increased presence of peroxisomes and lysosomes.

Time-course analysis of antibody response by EITB and ELISA before and after chemotherapy in sheep infected with Fasciola gigantica.

The time-course analysis of the antibody response to Fascicola gigantica infection in sheep was studied by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Sera from sheep experimentally infected with F. gigantica were reacted with excretory-secretory antigens of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in anti-Fasciola antibody by 2 weeks after infection and there was a sharp decrease in antibody titer by 4 weeks after treatment. By EITB, the infected sheep sera recognised four polypeptides in the range of 43-75 kDa as early as 2 weeks after infection, with more polypeptides being recognised as the infection progressed. Recognition of an 87 kDa antigen was lost by 2 weeks after treatment and is therefore a good marker for treatment efficacy. Comparative immunoblotting with sheep anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 17 kDa, 21 kDa, 57 kDa and 69 kDa proteins are specific to fasciolosis and are good antigens for early and specific immunodiagnosis of F. gigantica infection in sheep.

Somatic antigens of adult Dicrocoelium dendriticum recognised by bile antibodies of naturally infected cattle.

Bile samples, from slaughtered cattle harbouring between 120 and 280 adult lancet flukes, were used to investigate the range of somatic proteins inducing local antibody responses in naturally infected animals. Lancet fluke infections induced local (bile) antibody responses against Tris-buffered saline (TBS) soluble, sodium dodecyl sulphate (SDS) soluble and 2-mercaptoethanol (2-Me) soluble somatic proteins of adult Dicrocoelium dendriticum. IgA antibody isotypes predominated in the response against buffer-soluble somatic antigens, whereas SDS-soluble and 2-Me-soluble proteins induced similar level of both IgA and IgG1 antibodies. Analysis of the antigens recognised by particular isotype-specific bile antibodies suggests that different antigens preferentially induce isotype restricted antibody responses. The bile antibody response was highly species specific, only one antigen from somatic protein extracts of Fasciola hepatica being precipitated by bile samples showing the highest reactivity against D. dendriticum.

Common antigens of Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis and their relevance to serology.

The possession of common antigens by three trematode parasites which commonly occur together in ruminants in the tropics, Fasciola gigantica, Dicrocoelium hospes and Schistosoma bovis was studied in relation to the reliability of serodiagnosis of infection with these helminths. The crude antigenic extracts of the three trematodes were subjected to Sephacryl S-300 column chromatography and F. gigantica was fractionated into six peaks, S. bovis into nine peaks and D. hospes into seven peaks. Common antigens were found in these three trematodes in both the crude whole worm antigenic extracts and in the semi-purified fractions obtained by Sephacryl S-300 column chromatography. The implications of this finding and the limitation it imposes on the usefulness of serodiagnostic tests in routine use as regards their specificity are discussed in relation to previous studies.

[Antigenic structure of Dicrocoelium dendriticum (Rudolphi, 1819)]. / Badania struktury antygenowej Dicrocoelium dendriticum (Rudolphi, 1819).

The purpose of this paper was to study the antigenic structure of Dicrocoelium dendriticum with a particular consideration of its antigenic relationship between F. hepatica and Paramphistomum sp. Studies of the antigenic composition of somatic extract of D. dendriticum and its four fractions obtained by gel separation chromatography on Sephadex G 75 were carried out by using double diffusion reaction with homologous (SDd) and heterogeneous (SFh, SPs) rabbit sera as well as sera of sheep naturally infected with liver fluke. The highest seroantigenic activity was observed for the somatic extract and its fraction I. A distinct antigenic relationship was found between the fluke species studied, a closer relationship between D. dendriticum and F. hepatica than between D. dendriticum and Paramphistomum sp. The studied sera of 7 sheep naturally infected with liver fluke were inactive in double diffusion reaction with D. dendriticum extract and its fractions.

[Antigenic similarity between the spermatozoa of mammals and some endoparasites]. / Antigenno podobie mezhdu spermatozoidi na bozaintsi i niakoi endoparaziti.

Rabbit antisera to bull, ram, boar, and man spermatozoa were used in passive hemagglutination, passive skin anaphylaxis after Ovary, and immunoelectrophoresis tests to study water-salt extracts from spermatozoa and small and large liver fluke. the immunoelectrophoretic analysis revealed the presence of similar antigens in F. hepatica and D. lanceatum and in bull spermatozoa. A cross reaction was also observed (passive skin anaphylaxis in a guinea pig) between antisera to bull, ram, and man spermatozoa and water-soluble antigens of the two species of fluke. A similar reaction was noted also between sperm-agglutinating sera of infertile cows and the extracts from parasites. Discussed is the problem as to whether the cross reaction is due to common or similar antigenic determinants in the parasites and in some specific spermatozoid antigens. Stated is the clinical importance of these data and the ensuing link between liver fluke infection and the immunologically-mediated infertility of cows.

Experimental dicrocoeliasis: the humoral immune response of golden hamsters and rabbits to primary infection with Dicrocoelium dendriticum.

The results presented deal with the humoral immune response of golden hamsters to primary experimental infection with D. dendriticum. The development of serum antibodies has been comparatively investigated with three hamster groups (n = 43) harbouring different burdens of adult flukes. The mean numbers of parasites were 11, 30, or 130 per animal. Serum antibody response was studied during an observation period of at least 331 and up to 496 days postinfection. For antibody detection the sensitivities of precipitation test (PTs) (double diffusion test, immuno- and counterimmunoelectrophoresis), of the indirect haemagglutination test (IHAT), the complement fixation test (CFT), and the enzyme linked immuno sorbent assay (ELISA) were compared using aqueous crude fluke antigen and crude egg antigen. CFT and ELISA were most sensitive for the early detection of initial response. Thereafter all the tests employed revealed increasing antibody titres, which in general remained at constant levels and persisted until the end of the observation period with the exception of CF-antibodies. In general fluke antigen was found to be more sensitive than egg antigen. However, in CFT this antigen occasionally has been associated with unspecific inhibition of haemolysis. Comparison of the results shows that ELISA using crude fluke antigen gave the most realistic picture of the actual fluke burden. Also preliminary results on the precipitin response of rabbits (n = 3) after primary experimental exposure to different numbers of metacercariae (500, 1,000, and 3,000 per animal respectively) are reported. Employing the above mentioned PTs a persisting antibody response could be demonstrated only after exposure to at least 3,000 infective larvae. The initial response was found on day 63, the observation period was 550 days.