Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. I. The histopathology of the liver and bile ducts

Comparative studies were made on the histopathology of the liver and bile ducts in dicrocoeliasis and fascioliasis of the goat. Investigations revealed certain similarities in the nature of the pathological phenomena produced by D. dendriticum and F. hepatica. However, a feature peculiar to fascioliasis was the migratory tracks of immature flukes in the liver parenchyma. Further, the lesions produced by fascioliasis were distinctly more pronounced than those present in dicrocoeliasis.In both parasitic diseases the bile-duct walls showed glandular hyperplasia, increased frequency of goblet cells and globule leucocytes, and extensive fibrosis in the outer layers. The desquamation produced by Dicrocoelium remained superficial whereas Fasciola was capable of causing deep erosions in the walls of the main bile ducts. The bile-duct walls did not become calcified. Increased fibrosis was present in the interlobular areas, too, but only fascioliasis produced extensive disorganisation of the hepatic architecture, due to the scars originating from repaired migratory tracks. The liver parenchymal cells were degenerated, showed decreased glycogen but were increasingly infiltrated by neutral fats.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts     

Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. 3. The histochemistry of mast cells and globule leucocytes

Comparative studies were made on the structure and histochemistry of mast cells and globule leucocytes in dicrocoeliasis and fascioliasis of the goat. Investigations showed that D. dendriticum and F. hepatica infections in the goat produce an increase in the number of hepatic mast cells and frequent occurrence of globule leucocytes in the epithelium of the bile ducts. The only difference between these infections was that globule leucocytes were more frequent in small bile ducts in dicrocoeliasis than in fascioliasis.Histochemical methods showed the presence of sulphomucins in the granules of mast cells, while both neutral mucins and carboxymucins were revealed in the globules of globule leucocytes. The acidity of the mucosubstances could not be shown to result from hyaluronic acid, chondroitin-4 and -6-sulphates, or sialic acid. The globules of globule leucocytes also contained histochemically identifiable highly basic proteins.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts, mast cell, globule leucocyte     

Studies on the pathology of dicrocoeliasis and fascioliasis in the goat. II. The histochemistry of bile-duct mucosubstances

Histochemical studies were made on the characteristics of carbohydrate-rich compounds in the bile-duct mucosa of goats infected either with Dicrocoelium dendriticum or with Fasciola hepatica. In a previous study the author showed that a chronic infection with these parasites produces glandular hyperplasia in the walls of bile ducts. This study revealed a definitely increased secretion of qualitatively normal mucosubstances by the hyperplastic epithelial cells.Epithelial mucosubstances in the bile-duct walls were characterized by the presence of periodate-reactive hydroxy groups and both carboxyl and sulphate radicals. The intensity of the PAS reaction was moderate or strong and not definitely inhibited by the acidic or acetyl radicals. Specific enzymatic digestions did not reveal periodate-reactivity produced by glycogen or basophilia due to hyaluronic acid or chondroitin-4 and -6-sulphates in the epithelial cells. On the other hand, sialidase digestion showed that the main part of the carboxymucins was sialidase-labile sialomucins. Their susceptibility towards sialidase was not definitely increased after deacetylation. The single and sequential histochemical methods showed a more intense staining reaction for sulphate radicals in the mucosubstances of the deep glandular and goblet cells than in those of the superficial glands, which were characterized by high concentration of carboxyl groups.Keyword: Dicrocoelium dendriticum, Fasciola hepatica, liver, bile ducts, mucosubstances     

Liver flukes promote cholelithiasis in sheep

The main objective of this study was to investigate whether cholelithiasis in sheep is related to parasitism or other commonly observed disorders such as liver abscesses. Additionally, the features of the observed biliary calculi are described. The livers of 254 randomly selected clinically healthy adult dairy sheep were used. All visible concretions in the bile were considered as stones. Based on the macroscopical examination, 60 livers were normal, 40 were parasitized with Fasciola hepatica, 42 were parasitized with Dicrocoelium dendriticum, 28 were parasitized with both D. dendriticum and F. hepatica, 40 livers had abscesses and 44 had hydatid cysts. Biliary calculi were detected in 40 livers. Twenty livers had pigment stones and 20 livers had cholesterol stones. The percentage of cholelithiasis was significantly higher in livers parasitized with flukes compared with the others (P<0.05) and the parasitoses with F. hepatica and D. dendriticum were proven to be significant risk factors (P<0.05) for the presence of cholelithiasis in sheep.     

Parasites of livestock in Asir Province, southwestern Saudi Arabia

Thirteen parasite species, three trematodes, five cestodes, one nematode, and four arthropods, have been reported in livestock in Asir, southwestern Saudi Arabia. The occurrence of Dicrocoelium dendriticum in imported sheep and Paramphistomum cervi in indigenous cattle is recorded for the first time in Saudi Arabia.     

Percutaneous ultrasound-guided cholecystocentesis in cows.

A method was developed for percutaneous ultrasound-guided cholecystocentesis in cattle. The procedure was performed on the right side in the 9th, 10th, or 11th intercostal space of 30 cows. Of the 30 cows, 20 were slaughtered 24 hours after cholecystocentesis and the remaining 10 cows were slaughtered after a 10-day observation period. Changes in the peritoneum and gallbladder wall, observed at slaughter, were minimal. During the 10-day observation period, general behavior, attitude, and appetite of the 10 cows were normal. A transient, slight increase in rectal temperature was observed in 6 cows at 4, 5, or 8 days after cholecystocentesis. Total and differential WBC counts and total protein and fibrinogen concentrations, determined daily, were all within normal ranges. Bile samples from 20 cows were examined microscopically and biochemically. Fasciola hepatica and Dicrocoelium dendriticum eggs were observed in bile from 7 and 12 cows, respectively. Fecal examination revealed F hepatica eggs in 4 cows; D dendriticum eggs were not identified in any of the fecal samples. In 1 cow, F hepatica eggs were observed in the feces, but not in the bile. Bile acids concentration in bile varied from 12.5 to 68.5 mmol/L (mean +/- SD, 45.3 +/- 3.05 mmol/L) and in serum from 3.8 to 281.0 mumol/L (41.6 +/- 17.24 mumol/L). Negative correlation was obtained between bile acids concentration in bile and that in serum (r = -0.60, P less than 0.01). It was concluded that percutaneous ultrasound-guided cholecystocentesis in cows is a safe procedure and that microscopic and biochemical examinations of obtained bile can be useful diagnostic aids.     

Pathophysiological studies on Dicrocoelium dendriticum infection in sheep

Red cell kinetics and plasma protein metabolism were investigated in two experiments using 20 adult sheep naturally infected with Dicrocoelium dendriticum, but free of other liver and gastrointestinal helminths. In the first experiment, where groups of animals with low to high Dicrocoelium burdens were injected with 51Cr-labelled red cells and 125I-labelled albumin, the results indicated that there were no significant differences in the turnover rate of labelled red cells or albumin between any of the groups. In the second experiment, two groups of sheep with low and high worm burdens were studied using the same radioisotope tracers; in addition, 59Fe-citrate was used to assess red cell iron incorporation rates in the two groups. Although the red cell half-lives of the infected sheep were just significantly longer, both were within normal limits and the difference was attributed to random variation within the two small groups of sheep. No significant differences were found in the other parameters. It was concluded that burdens of up to 4000 D. dendriticum do not cause significant blood or plasma protein loss in sheep.     

The influence of flotation solution, sample dilution and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal strongyles and Dicrocoelium dendriticum in sheep

The present study was aimed to evaluate the influence of flotation solution, sample dilution, and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal (GI) strongyles and Dicrocoelium dendriticum in a composite sample of faeces from naturally infected sheep. Fourteen flotation solutions having densities between 1.200 and 1.450, and six sample dilutions, 1:10, 1:15, 1:20, 1:30, 1:40 and 1:50 were used. Each of the six dilutions was divided into 70 aliquots in order to have five replicates of each of the 14 flotation solutions at each of the six dilutions. For each McMaster slide, the GI strongyle and D. dendriticum egg counts were performed under one grid (McM 0.15 ml), two grids (McM 0.3 ml), one chamber (McM 0.5 ml), and both chambers (McM 1.0 ml). Mean eggs per gram (EPG) of faeces of GI strongyles and D. dendriticum were calculated and statistical analyses were performed on the resulting data. The type of flotation solution used significantly influenced the EPG in the GI strongyles and in the D. dendriticum egg counts. All the sucrose-based solutions at density between 1.200 and 1.350 floated more GI strongyle eggs than the others. With respect to D. dendriticum, only six solutions were capable of floating eggs and the potassium iodomercurate solution (density 1.440) floated more eggs than the others. The reliability of the McMaster technique regarding sample dilution was high for both GI strongyle and D. dendriticum EPG at 1:10 and 1:15, and then progressively decreased with increasing dilution. The reliability of the McMaster technique regarding the choice of the McMaster slide area (volume) was high for both GI strongyle and D. dendriticum EPG at the McMaster slide area (volume) of 1.0 ml, i.e. the total area of the McMaster slide. The EPG counts resulting from choosing any of the other three McMaster slide areas (volumes), i.e. McM 0.15 ml, McM 0.3 ml, or McM 0.5 ml, produced unreliable over-estimates. The findings of the present study show that the highest reliability of the McMaster technique for estimating GI strongyle and D. dendriticum egg counts in faeces from pastured sheep is obtained when using flotation solutions based on sucrose for GI strongyles, and potassium iodomercurate for D. dendriticum, dilutions which do not exceed 1:15, and the McMaster slide area (volume) of 1.0 ml.     

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.     

A cross-sectional coprological survey of liver flukes in cattle and sheep from an area of the southern Italian Apennines

A cross-sectional coprological survey of liver flukes (Fasciola hepatica and Dicrocoelium dendriticum) was conducted on 81 bovine farms and 197 ovine farms with animals pasturing in an area (3971 km(2)) of the southern Italian Apennines. The farms were selected to be uniformly distributed throughout the study area using geographical information system (GIS) software. Between June 1999 and March 2000, faecal samples were collected from 975 cattle and 3940 sheep and examined using a modified McMaster technique. The results were subjected to statistical analysis and point distribution maps (PDMs) were drawn by GIS.Cattle of 9 of the 81 (11.1%) farms were positive for F. hepatica and of 43 (53.1%) for D. dendriticum. Sheep of 8 of the 197 (4.1%) farms were positive for F. hepatica and of 133 (67.5%) for D. dendriticum. Co-infection was found in cattle of 2 (2.5%) farms, and in sheep of 8 (4.1%) farms.The findings of the present survey show that D. dendriticum was the predominant liver fluke found in cattle and sheep with respect to egg count numbers for both farms and animals. In addition, the general trends of the PDMs show that D. dendriticum was widely and homogeneously spread throughout the study area, whereas F. hepatica was present only in a few concentrated zones of the study area that had both positive bovine and positive ovine farms.     

Effectiveness of cambendazole (Bonlam paste) in sheep invaded by tramatodes (Dicrocoelium dendriticum)]

Tests were performed to examine the effectiveness of a new form of cambendazole (Bonlam paste, produced by MSD, USA) in sheep, naturally invaded by the trematodes Dicrocoelium dendriticum. Single administration of 25 mg per 1 kg live weight induced a rapid decrease in the number of the excreted eggs of D. dendriticum, as counted during ovoscopic examination seven, ten, and fifteen days after administration of the chemical. The intenseffectiveness of the preparation, determined post mortem, was 95.1%. Despite this, several dicrocoelia with normal motility and with no changes on the cuticle or internal organs were found in all the animals dissected. The animals tolerated the treatment with no signs of side-effects.     

An evaluation of the efficacy of netobimin against Dicrocoelium dendriticum in sheep

A trial was carried out to assess the efficacy of a nitrophenylguanidine compound, netobimin against Dicrocoelium dendriticum in naturally infected sheep. At a dose rate of 20 mg/kg bodyweight administered orally the drug was highly effective, producing a mean reduction of 98.9 per cent in the fluke burdens of treated animals compared with untreated controls. No side effects were observed in the treated sheep.     

The transport of albendazole and albendazole sulphoxide in the lancet fluke (Dicrocoelium dendriticum)

Albendazole (ABZ) is one of the most important benzimidazole compounds possessing high activity against the lancet fluke, Dicrocoelium dendriticum. ABZ sulphoxide (ABZ.SO) is the main molecule present in the bloodstream of an ABZ-treated host. The aim of this study was to characterise the pattern of ex vivo uptake of ABZ and ABZ.SO by lancet flukes and the export of both anthelmintics from these parasites. Transport of these anthelmintics in both living and dead flukes was compared. The adult flukes were collected from mouflons (Ovis musimon) which had been infected naturally. Results showed that more lipophilic ABZ was imported to a higher extent than ABZ.SO, and that significantly higher concentrations of ABZ were detected within living flukes as compared to dead ones. The same pattern was revealed in the study of ABZ and ABZ.SO export from the flukes' bodies. In addition to passive diffusion, active ABZ uptake and active efflux of ABZ and ABZ.SO in D. dendriticum could be assumed.     

Prevalence of Fasciola hepatica and Dicrocoelium dendriticum in the cow: inspection in an east Switzerland abattoir

On the basis of meat inspection in an abattoir in the eastern part of Switzerland, the incidence of liverflukes in cattle was recorded weekly during one year. The prevalence was compared with surveys from the seventies and nineties. The average incidence of Dicrocoelium dendriticum infection did not change. Although the prevalence of Fasciola hepatica apparently slightly decreased, an actual decrease of liverflukes could not be demonstrated.     

Hepatogenous photosensitisation in Scottish sheep casued by Dicrocoelium dendriticum

Dicrocoeliosis was identified as the probable predisposing cause of weight loss and hepatogenous photosensitisation affecting half of a group of 14month-old ewe lambs on a farm on the Inner Hebridean island of Coll. Oral dosing of the ewe lambs with 15mg/kg albendazole only achieved a 79.2% reduction in Dicrocoelium dendriticum egg count, 21day 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.     

Partial characterization and isolation of 130 kDa antigenic protein of Dicrocoelium dendriticum adults

The study focused on characterizing and isolating Dicrocoelium dendriticum antigens or their fractions that could be used for the immunological diagnosis of dicrocoeliosis. Somatic (SoAg) and excretory-secretory antigens (ESAg) were analyzed by SDS-PAGE and their specificity was evaluated by Western blot with homologous and heterologous sera. The antigens were partially purified by chromatographic techniques of gel-filtration (Sephacryl S-300) and ion exchange (Hitrap-DEAE-Sepharose). Western blot analysis using sera of ovine infected with D. dendriticum revealed eight main antigenic polypeptides ranging from 24 to 205 kDa for SoAg and seven for ESAg with apparent molecular mass in the range of 26-205 kDa. We detected a specific parasite protein with an approximate molecular weight of 130 kDa in SDS-PAGE gels, arranged as a 450 kDa tetramer in native conditions. It also showed strong immunoreactivity by Western blot against ovine sera experimentally infected with D. dendriticum. Gel filtration chromatography (Sephacryl S-300) also showed other specific proteins, one of about 24 kDa in SoAg and another of about 42 kDa in ESAg. The elution conditions of 450 kDa protein (130 kDa monomer) by DEAE chromatography were similar to those from the somatic antigen (pH 7.2, 0.1M NaCl, in 29-34 ml fractions) and from the excretion-secretion antigen (pH 8.0, 0.1M NaCl, in 29-35 ml fractions). The suitability of 130 kDa polypeptide for D. dendriticum infection diagnosis was confirmed by Western blot using a pool of sera as well as individual serum samples from experimentally infected sheep. The sequence of amino termini of 130 kDa polypeptide from both fractions was the same and identical to that reported for a peptide from D. dendriticum described as a globin. This sequence also revealed an appreciable similarity with the amino end of globins from some phylogenetically related worms.     

Calibration and diagnostic accuracy of simple flotation, McMaster and FLOTAC for parasite egg counts in sheep

The present study was aimed at carrying out a calibration and a comparison of diagnostic accuracy of three faecal egg counts (FEC) techniques, simple flotation, McMaster and FLOTAC, in order to find the best flotation solution (FS) for Dicrocoelium dendriticum, Moniezia expansa and gastrointestinal (GI) strongyle eggs, and to evaluate the influence of faecal preservation methods combined with FS on egg counts. Simple flotation failed to give satisfactory results with any samples. Overall, FLOTAC resulted in similar or higher eggs per gram of faeces (EPG) and lower coefficient of variation (CV) than McMaster. The "gold standard" for D. dendriticum was obtained with FLOTAC when using FS7 (EPG=219, CV=3.9%) and FS8 (EPG=226, CV=5.2%) on fresh faeces. The "gold standard" for M. expansa was obtained with FLOTAC, using FS3 (EPG=122, CV=4.1%) on fresh faeces. The "gold standard" for GI strongyles was obtained with FLOTAC when using FS5 (EPG=320, CV=4%) and FS2 (EPG=298, CV=5%). As regard to faecal preservation methods, formalin 5% and 10% or freezing showed performance similar to fresh faeces for eggs of D. dendriticum and M. expansa. However, these methods of preservation were not as successful with GI strongyle eggs. Vacuum packing with storage at +4°C permitted storage of GI strongyle eggs for up to 21 days prior to counting. Where accurate egg counts are required in ovine samples the optimum method of counting is the use of FLOTAC. In addition, we suggest the use of two solutions that are easy and cheap to purchase and prepare, saturated sodium chloride (FS2) for nematoda and cestoda eggs and saturated zinc sulphate (FS7) for trematoda eggs and nematoda larvae.     

Assessment of climatic and orographic conditions on the infection by Calicophoron daubneyi and Dicrocoelium dendriticum in grazing beef cattle (NW Spain)

From February 2003 to March 2004, 1148 faecal samples from autochthonous Rubia Gallega cattle breed were collected in 170 farms from Lugo (NW Spain), in order to find out the prevalence and intensity of egg-excretion by Calicophoron daubneyi and Dicrocoelium dendriticum, and the possible influence of external factors as annual mean temperature and rainfall, altitude and mean slope. Twenty-six percent (95% CI 19%, 33%) and the 18% (12, 24) of the farms were infected with rumen and lancet flukes. C. daubneyi egg-output (Me=24) was found in 13% (11, 15) of the individual samples. Six percent (5, 7) of the animals in the study had low D. dendriticum eggs (Me=8). By estimating the odds ratio values it was shown that the highest probability of infection by C. daubneyi was in pastures with a mean slope of less than 13% (OR=1.9) and situated under 600 m (OR=1.6). Annual mean rainfall and temperature were not identified as risk factors for the infection with rumen flukes. Mountainous pastures with a mean slope of higher than 25% (OR=5.8) and situated over 600 m (OR=24.6) where precipitation was high (>1000 mm; OR=7) and temperature low (<11 degrees C; 2.8) had involved the highest risk of infection by D. dendriticum. Because of the prevalences found in this study, employment of suitable management practices with strategic treatments with efficacious anthelmintics are needed to reduce the presence of both trematodes and to increase the health status of grazing beef cattle.     

Abattoir condemnation due to parasitic infections and its economic implications in the region of Trikala,Greece

The prevalence of parasitic infections responsible for the condemnation of carcasses and viscera during meat inspection, and their economic implication, was estimated in a year long abattoir survey of 10 277 slaughtered farm animals in the region of Trikala, Greece. The organs examined for the presence of parasitic lesions during meat inspection were: liver and lungs of all animals, rumen of cattle, small intestine of lambs and kids, and muscles of cattle and swine. The parasitic lesions observed in the lungs of cattle, sheep and goats were caused only by hydatid cysts. No hydatid cysts were observed in the lungs of swine. The parasitic lesions observed in the liver of cattle, sheep and goats were as a result of hydatid cysts and flukes of Fasciola hepatica and Dicrocoelium dendriticum, while those of swine were due to milk spots only. Moniezia sp. proglottids were found in the small intestine of lambs only. The prevalence of parasites responsible for the condemnation of marketable organs was low (0.26%). Parasites were responsible for 22% of the total of condemned organs, and their annual cost was 99, 00 GDR (approximately 292 Euros). The parasites most contributing to marketable organ condemnation were hydatid cysts (26%) and D. dendriticum flukes (26%).