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.     

IL-4受体阻断对小鼠急性感染肝片吸虫后肝脏单核细胞悬液中细胞因子的影响

本研究旨在探索肝片吸虫感染小鼠急性期肝脏单核细胞悬液中细胞因子表达模式及其原因。试验将小鼠分为4组,分别对感染抗体处置组、感染非抗体处置组、抗体处置非感染组和非抗体处置非感染组的肝脏单核细胞培养上清液中IL-4、IL-5、IL-13及IFN-γ进行测定;同时也对肝脏中IL-4及IL-12P40 mRNA水平进行荧光定量PCR检测。结果显示,IL-4和IL-5的表达模式相似。在非感染状态下,无论是否进行抗体处置,两者都处于较低表达水平。在感染状态下,两者在非抗体处置小鼠中的表达显著高于抗体处置组(P<0.05)。IL-13与IL-4及IL-5的表达差异在于感染并用抗体处置后,小鼠肝脏单核细胞中的IL-13仍有较高水平表达。IFN-γ的表达量在感染后有所增加,但在有无抗体处理间无显著差异(P>0.05)。mRNA检测结果显示,在非感染状态下,抗体处置后的IL-4 mRNA水平显著低于非抗体处置组(P<0.05)。感染后非抗体处置组小鼠中IL-4 mRNA水平极显著高于抗体处置组(P<0.01),同时也极显著高于非感染非抗体处置组(P<0.01)。IL-12P40 mRNA的水平在感染状态下,抗体处置组的IL-12P40 mRNA虽较非抗体处置组低,但无显著差异(P>0.05)。但感染后非抗体处置组的IL-12P40 mRNA水平均极显著高于感染组抗体处置和非抗体处置组(P<0.01)。本试验结果表明,IL-4Rα对肝片吸虫感染急性期IL-5和IL-4的表达极其重要,但对IL-13及IFN-γ的影响较小。     

Covariate selection in multivariate spatial analysis of ovine parasitic infection

Gastrointestinal (GI) strongyle and fluke infections remain one of the main constraints on health and productivity in sheep dairy production. A cross-sectional survey was conducted in 2004-2005 on ovine farms in the Campania region of southern Italy in order to evaluate the prevalence of Haemonchus contortus, Fasciola hepatica, Dicrocoelium dendriticum and Calicophoron daubneyi from among other parasitic infections. In the present work, we focused on the role of the ecological characteristics of the pasture environment while accounting for the underlying long range geographical risk pattern. Bayesian multivariate spatial statistical analysis was used. A systematic grid (10 km×10 km) sampling approach was used. Laboratory procedures were based on the FLOTAC technique to detect and count eggs of helminths. A Geographical Information System (GIS) was constructed by using environmental data layers. Data on each of these layers were then extracted for pasturing areas that were previously digitalized aerial images of the ovine farms. Bayesian multivariate statistical analyses, including improper multivariate conditional autoregressive models, were used to select covariates on a multivariate spatially structured risk surface. Out of the 121 tested farms, 109 were positive for H. contortus, 81 for D. dendriticum, 17 for C. daubneyi and 15 for F. hepatica. The statistical analysis highlighted a north-south long range spatially structured pattern. This geographical pattern is treated here as a confounder, because the main interest was in the causal role of ecological covariates at the level of each pasturing area. A high percentage of pasture and impermeable soil were strong predictors of F. hepatica risk and a high percentage of wood was a strong predictor of C. daubneyi. A high percentage of wood, rocks and arable soil with sparse trees explained the spatial distribution of D. dendriticum. Sparse vegetation, river, mixed soil and permeable soil explained the spatial distribution of the H. contortus. Bayesian multivariate spatial analysis of parasitic infections with covariates from remote sensing at a very small geographical level allowed us to identify relevant risk predictors. All the covariates selected are consistent with the life cycles of the helminths investigated. This research showed the utility of appropriate GIS-driven surveillance systems. Moreover, spatial features can be used to tailor sampling design where the sampling fraction can be a function of remote sensing covariables.     

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%).     

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     

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.     

Experimentally induced Fasciola hepatica infection in white-tailed deer. II. Pathological features.

Six white-tailed deer (Odocoileus virginianus) and six sheep were inoculated with metacercariae of Fasciola hepatica. Two animals of each species were given 100, 500 or 2500 metacercariae. One animal in each inocluated group was killed and examined at six weeks postinoculation and the remainder at 15 weeks postinoculation. At six weeks postinoculation the parietal surface of the livers from inoculated deer was covered with gray fibrous plaques and rust colored patches. Fibroplasia with mononuclear cell infiltration characterized Glisson's capsule on the parietal surface. Granulomas were found in the hepatic parenchyma and on the dorsal surface of the lung. Fresh and healing tracks were occasionally found in the liver. In the sheep fibrinous exudate and numerous subcapsular tracks were found on both surfaces of the liver. Inflammatory changes in portal areas and numerous fresh and healing tracks in the hepatic parenchyma were prominent features. At 15 weeks postinoculation inflammatory changes in Glisson's capsule of inoculated deer were less marked than at six weeks but portal fibrosis and hyperplasia of bile duct epithelium were more advanced. A zone of hemorrhage surrounded ducts that contained mature F. hepatica in one deer. The livers from the sheep were rough, pitted and covered with fibrous tags and adhesions to the diaphragm and greater omentum were common. Hemorrhagic tracks were common in the sheep given 500 and 2500 metacercariae. Portal fibrosis and hyperplasia of bile duct epithelium were seen in the sheep (100 metacercariae) that harbored mature F. hepatica.     

Host responses during experimental infection with Fasciola gigantica or Fasciola hepatica in Merino sheep I. Comparative immunological and plasma biochemical changes during early infection

This study reports the early biochemical changes in plasma, comparative host-immune responses and parasite recovery data in Merino sheep during the first 10 weeks of infection with Fasciola gigantica and Fasciola hepatica. One group of sheep were uninfected, four groups of sheep received incremental challenge doses of F. gigantica metacercariae (50, 125, 225 and 400, respectively) and the sixth group was challenged with 250 F. hepatica metacercariae. At 10 weeks post infection (wpi), sheep challenged with F. hepatica showed the greatest fluke recovery (mean 119, range 84-166); a significantly higher biomass of parasites recovered (2.5-fold greater than the highest dose of F. gigantica); and a greater mean % parasite recovery (39.3%, range 27-55%) than any group challenged with F. gigantica. Within the groups dosed with F. gigantica a strong dose-dependent response was observed in both fluke recovery and fluke biomass with increasing dose of metacercariae. The mean % parasite recovery of F. gigantica infected groups 1-5 were 26, 23, 26 and 25%, respectively, suggesting a uniform viability of parasite establishment independent of infection dose. At 6 wpi, elevated levels of plasma GLDH were observed in the F. gigantica infected groups compared to the uninfected sheep (p<0.005) whereas the F. hepatica challenged group had four-fold higher levels of GLDH compared to the F. gigantica infected group (p<0.001). Elevated levels of GGT as an indicator of epithelial damage in the bile duct was only seen in the group challenged with F. hepatica at 10 wpi when it rose from below 100 IU/l to approximately 250 IU/l (p<0.0001) whereas no detectable increase in GGT was observed in any of the groups challenged with F. gigantica. The white blood cell response to F. hepatica infection was biphasic with the initial peak at 4 wpi and a second peak at 9 wpi, corresponding to the period of migration of juvenile fluke in the liver and the time when adult flukes are migrating into the bile duct, respectively. This biphasic response was also evident in the changes in the eosinophil counts and serum haemoglobin levels. There was a trend toward higher parasite-specific IgG2 titres in sheep infected with lower worm burdens, suggesting that higher F. gigantica or F. hepatica burdens suppress IgG2 responses. The findings of this study suggest that, in early infection in a permissive host, F. hepatica appears to be more pathogenic than F. gigantica because of its rapid increase in size and the speed of its progression through the migratory phases of its life cycle.     

The serum glucose and beta-hydroxybutyrate levels in sheep with experimental Fasciola hepatica and Fasciola gigantica infection

The influence of Fasciola hepatica and Fasciola gigantica infection on serum glucose and beta-hydroxybutyrate (beta-HOB) in sheep was evaluated. This was done by setting up two groups of sheep. The first group (n=13) was split in two sub-groups, one experimentally infected with F. hepatica (n=9) and the other (n=4) as uninfected control. A second group consisting of a sub-group experimentally infected with F. gigantica (n=9) the other sub-group (n=6) left as uninfected control was also set up. The results of weight gain, parasitological and serum liver enzymes activity (glutamate dehydrogenase [GLDH] and gamma glutamyltransferase [gamma-GT]) used in monitoring the infection showed that all infected animals developed fasciolosis. It was observed that a reduction in serum glucose levels was significantly lower (p<0.05) in F. hepatica infected sheep than in uninfected control sheep starting from 5 weeks post-infection (wpi) to the end of the experiment. Similar reduction was recorded in F. gigantica infected sheep between 8 and 19 wpi. In contrast, serum beta-HOB levels were elevated in F. hepatica infected sheep between 6 and 16 wpi and in F. gigantica infected sheep between 7 and 15 wpi. It would appear from these serum glucose and beta-HOB levels that fasciolosis does lead to energy deficiency (low glucose) and ketosis (increased beta-HOB). The decrease in serum glucose and increase in serum beta-HOB levels in infected sheep may help in understanding the interaction between fasciolosis and nutritional status of infected ruminants especially in young growing animals.     

Circulating oxidative stress status in desert sheep naturally infected with Fasciola hepatica

Oxidative stress is a general mechanism whereby free radicals induce oxidative damages and reduce the antioxidant defences of the biological systems. The aim of the present study was to determine plasma malondialdehyde levels as a biomarker of lipid peroxidation and its relation to the antioxidants status (plasma ascorbate and blood glutathione concentrations), liver function tests and anaemia in spontaneous ovine fascioliasis. For this purpose, jugular blood samples and livers of 27 infected ewes with Fasciola hepatica along with blood samples of 20 healthy (control) ewes were collected from animals slaughtered in a F. hepatica endemic area (Kharga oasis, Egypt). An increase (P<0.001) in plasma malondialdehyde (141.1%) accompanied by decreased levels (P<0.001) of albumin (29.3%) and ascorbate (36.2%) in plasma and glutathione in blood (31.6%) of infected sheep was noticed when compared with control values. In the infected group, malondialdehyde values were positively correlated with liver fluke burden (r=0.57, P=0.002) and the activity of plasma aspartate aminotransferase (r=0.39, P=0.0.046) and gamma-glutamyltransferase (r=0.64, P=0.0003) and negatively correlated with the concentrations of albumin (r=-0.53, P=0.004), ascorbate (r=-0.46, P=0.0.17) and glutathione (r=-0.41, P=0.034). In conclusion, oxidative stress is a significant feature of chronic F. hepatica infection in grazing sheep.     

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     

Progress in the development of Fasciola hepatica vaccine using recombinant fatty acid binding protein with the adjuvant adaptation system ADAD

Fatty acid binding proteins (FABP) have been designed as a potential vaccine against fasciolosis. In this work, the immunoprophylaxis of the recombinant Fh15 FABP from F. hepatica (Fh15) in adjuvant/immunomodulator ADAD system was evaluated using mice and sheep challenged with F. hepatica. The ADAD system combines the Fh15 antigen with an immunomodulator (hydroalcoholic extract of Polypodium leucotomos; PAL) and/or an adjuvant (saponins of Quillaja saponaria; Qs) in a water/oil emulsion (30/70) with a non-mineral oil (Montanide). All the infected control mice died by 41-48 days post-infection. The mice vaccinated with ADAD only with PAL+Fh15 present a survival rate of 40-50% and those vaccinated with ADAD containing PAL+Qs+Fh15 had a survival rate of 50-62.5%. IgG1 antibodies were lower in surviving mice in comparison with non-surviving mice. The sheep vaccinated with ADAD PAL+Qs+Fh15 showed lower fluke recovery (43%), less hepatic lesions and higher post-infection daily weight gain than F. hepatica infected control animals. Thus, the ADAD system using recombinant fatty acid binding proteins from F. hepatica could be a good option to develop vaccines against F. hepatica.     

Innate and adaptive resistance of Indonesian Thin Tail sheep to liver fluke: a comparative analysis of Fasciola gigantica and Fasciola hepatica infection

In the current study, three independent trials directly compared Fasciola gigantica and Fasciola hepatica infection of ITT sheep. In all trials, F. hepatica infection resulted in higher worm burden recoveries and greater physiological damage to ITT sheep. Developmental differences of the two Fasciola species were also observed during the first twelve weeks of a primary infection, where the migration and growth of F. hepatica was more rapid than F. gigantica. Various immunological blood parameters were measured and indicated similar kinetics in the humoral and cellular responses during the time course of infection with each Fasciola species. In contrast to F. hepatica infection, we demonstrate an innate and adaptive comparative ability of ITT sheep to resist the early stages of infection with F. gigantica infection. Unraveling the mechanisms leading to this differential resistance may potentially lead to new methods for the control of fasciolosis and other human liver flukes.     

The effect of Taenia hydatigena infection on existing and concurrent infections of Fasciola hepatica in sheep

Sheep given a primary infection of Fasciola hepatica were challenged 18 weeks later with Taenia hydatigena or F hepatica, or both parasites together, or were not challenged. At the same time, control sheep were infected with T hydatigena and/or F hepatica separately or concurrently. All sheep were killed seven weeks after challenge and the number of cysts and flukes counted. Challenge infection with T hydatigena did not affect the numbers of flukes recovered from either primary or challenge F hepatica infections. On the other hand, the numbers of cysticerci were reduced in sheep previously infected with F hepatica but not in those given T hydatigena and F hepatica concurrently.     

Effect of parasite burden on the detection of Fasciola hepatica antigens in sera and feces of experimentally infected sheep

The effect of Fasciola hepatica parasite burden on the detection of excretory/secretory (E/S) antigens in sera and feces of experimentally infected sheep was evaluated using a double antibody-based capture enzyme-linked immunosorbent assay (ELISA). Four groups of five sheep each were used. The first three groups were infected with 50, 100 and 200 metacercariae of F. hepatica, and the fourth group remained as non-infected control. On the day of infection and weekly thereafter, serum and fecal samples were taken. ELISA detected F. hepatica E/S antigen levels in serum from the first week post-infection (wpi) and in fecal supernatant from the fourth wpi, which were significantly (p<0.05) higher than controls. F. hepatica eggs were not detected until after the eighth wpi. The correlation between absorbance of E/S antigens in serum with the fluke burden was 0.77 (p<0.0001) and in feces 0.76 (p<0.0001) at 12th wpi. The sensitivity of the assay to detect E/S antigens in serum was 86.6% and in feces 93.3%. It is concluded that the ELISA technique used in this study offers a diagnostic alternative for detecting early infections of F. hepatica in sheep.     

Responses of Fasciola hepatica infected sheep to various infection levels

The response to Fasciola hepatica was studied in sheep infected with 5, 30, 150 metacercariae. The animals were necropsied 12 weeks post-infection (p-i) for counting and measuring flukes. Cellular and humoral responses were detected by peripheral eosinophil count, peripheral blood lymphocyte proliferation with excretory-secretory products (FhESP) and ELISA. All sheep were infected at necropsy except one sheep which was infected with 5 metacercariae. Mean parasitic intensities were 40%, 44% and 27% of the infection dose in sheep infected with 5, 30, 150 metacercariae respectively. FhESP-specific lymphocyte responses of the 3 infected groups were significantly enhanced in weeks 3 and 4 p-i (p < 0.05). The kinetics of the specific humoral response were similar for the 3 infected groups but the antibody level was significantly lower in animals infected with 5 metacercariae than in the 2 other infected groups from week 5 p-i to week 12 p-i (p < 0.05). Peripheral eosinophil count was significantly enhanced (p < 0.05) in infected groups. The numbers of peripheral eosinophils were significantly different between the 3 infected groups in week 3, 4 and 6 p-i and were related to infection level. These results confirm that sheep are highly susceptible to F. hepatica infection, even when infection pressure is very low. Peripheral eosinophilia was dependent of the infection level. The immune response was similar in sheep infected with various numbers of flukes.     

Bacteriologic and pathologic studies of hepatic lesions in sheep

At an abattoir, lesion specimens from 140 condemned sheep livers were collected for bacteriologic culture and for pathologic examination. Grossly, 23 lesions were abscesses; from 9 of which, Fusobacterium necrophorum biovar A (3 in pure culture and 6 in mixed culture) was isolated and from 14 of which, biovar B (6 in pure culture and 8 in mixed culture) was isolated. Escherichia coli was the predominant facultative anaerobic bacterium and Clostridium perfringens was the predominant obligate anaerobic bacterium isolated from the 14 lesions with mixed bacterial infection. Histologically, these lesions had a core of coagulation necrosis, encircled by a zone of necrotic phagocytic cells and bacteria with cellular characteristics of F necrophorum biovars A or B, and a connective tissue capsule. Of the 117 lesions without F necrophorum, 49 were culture-positive (for other organisms) and 69 were culture-negative. These 117 lesions were fibrous and were smaller than the 23 abscesses. A variety of gram-positive and gram-negative facultative anaerobic and obligate anaerobic bacteria was isolated from the culture-positive lesions, but always in low numbers. Eleven culture-negative and 18 culture-positive lesions were examined and had histologic characteristics of parasite-induced granulomas, with numerous eosinophils and epithelioid giant cells. Results of the study indicated that the histologic appearance of ovine hepatic lesions with F necrophorum was similar to bovine liver abscesses caused by F necrophorum, but unlike bovine liver abscesses, F necrophorum biovar B was isolated more frequently than was biovar A and often in pure culture. Most of the lesions in the condemned livers were parasite-induced granulomas.     

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 antibody isotype responses of Fasciola-infected sheep and cattle to excretory and secretory products of Fasciola species

This study investigated the immunoglobulin isotype responses of sheep and cattle chronically infected with Fasciola hepatica and Fasciola gigantica to adult F. hepatica excretory/secretory products (Fh-ES) or F. gigantica excretory/secretory products (Fg-ES), respectively. An antibody enzyme-linked immunosorbent assay (Ab-ELISA) was used to determine serum antibody (total Ig, IgG(1), IgM, IgG(2) and IgA) responses. At necropsy, the mean number of flukes recovered was lower in cattle than in sheep. All F. hepatica and F. gigantica infected sheep and cattle showed an increased total Ig levels from 3 to 4 weeks post-infection (wpi). Among isotypes IgG(1) was most dominant while IgM was the earliest (2 wpi) to be detected in both sheep and cattle infected with both F. hepatica and F. gigantica animals. IgG(2) response was early (2 wpi) in sheep infected by F. hepatica but there was no response in sheep infected with F. gigantica. There was a late and strong IgG(2) response in cattle infected with both flukes. The IgA isotype showed an early and a clear biphasic response in sheep with F. hepatica but was less pronounced in F. gigantica infected sheep. While IgA response to Fh-ES was noticed 5 wpi in F. hepatica infected cattle, it appeared much later (21 wpi) in those infected with F. gigantica. The dominance of IgG(1) isotype in infected sheep and cattle suggest an associated Th2 response. This early response to adult Fasciola spp. ES antigen suggests an early exposure to the antigen presumably through the cross-reacting ES products of juvenile flukes. There is clearly difference in IgG(2) isotype response in cattle (resistant) compared to sheep (susceptible). The late IgG(2) response in cattle may suggest late Th1 involvement in bovine cellular responses to adult Fh-ES/Fg-ES.     

Monoclonal antibody sandwich immunoassay detection of coproantigen to evaluate the efficacy of treatment in natural ovine fasciolosis

The monoclonal antibody-based sandwich immunoassay (mAb Sandwich ELISA) was used to evaluate the effectiveness of triclabendazole treatment by the detection of coproantigens in Fasciola hepatica naturally infected sheep. Twelve sheep (2 to 5 years of age) were separated into two groups. The first group (three sheep) remained untreated; the other group (nine sheep) was treated with a single dose of 5% triclabendazole at 10 mg kg-1 of body weight. All but one of the treated group had negative optical density values (OD492) after two weeks of treatment, while seven sheep intermittently shed eggs during the course of the study. In all but one of the treated sheep, no F. hepatica infection; the one positive ELISA in 5th week after treatment according to the mAb Sandwich, had one fluke in the liver. The results of the parasitological examinations, as well as OD492 values obtained by the mAb Sandwich ELISA for the detection of coproantigens are described. The findings at necropsy, of the treated group in comparison to the untreated group are shown. The mAb Sandwich ELISA could be a useful and accurate method with which to monitor the efficacy of flukicides in F. hepatica natural infections.