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     

On the Ultrastructure of Mast Cells and Globule Leucocytes in the Common Bile Duct of Cattle Chronically Infected With Fasciola Hepatica

Mast cells and globule leucocytes in the walls of the common bile ducts of seven uninfected slaughtered cows and of five cows chronically infected with Fasciola hepatica were studied by electron microscopy. The mast cells displayed numerous small intracytoplasmic granules, the matrixes of which were either strongly electron-opaque and homogeneous or less dense with a fine-granular ultrastructure. Unattached ribosomes were abundant in the cytoplasm of the cells. Many mast cells in the walls of chronically infected bile ducts were normal-looking while the cells in the subepithelial tissues often showed striking irregularities in the ultrastructure of the granules. Globule leucocytes were most numerous in the surface epithelium and were not connected by desmosomes to the epithelial cells. The nuclei of the globule leucocytes were similar to those in the mast cells except that indentations caused by the globules were more usually seen. The globules were considerably larger than the mast cell granules but bounded by similar agranular capsules. The matrix of most globules was dense and homogeneous while the other globules showed dense reticulate or lamellate structures. The relationship between the mast cell and globule leucocyte in the liver is briefly discussed with reference to recent reports on these cells in the intestinal wall.     

Globule leukocyte and mast cell in bile ducts of cattle naturally infected with liver flukes

In n previous report dealing with the pathology of bovine fascio-liasis the author described an unknown cell type in the epithelium of bile ducts. The histological and histochemical investigations published in this paper suggest that the cell may be considered a globule leukocyte. Globule leukocytes are rare in uninfected livers but are occurring in abundance in main bile ducts of cattle with spontaneous fascioliasis and also in small perilobular ducts in dicrocoeliasis. Liver fluke infection causes an increase in the population of subepithelial mast cells. Mast cell and globule leukocyte present similarities In their cytochemical properties. However, at low pH toluldinc blue shows a stronger but Alcian blue a weaker affinity for mast cells than for globule leukocytes.     

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     

Failure to induce homologous immunity to Fasciola hepatica in sheep vaccinated with irradiated metacercariae

Vaccination of sheep with either 100 or 1000 γ-irradiated (2.5 krad) metacercariae of Fasciola hepatica, on two occasions six weeks apart, did not generate significant protection against intraruminal challenge with F. hepatica six weeks after the second vaccinating dose as measured by recovery of flukes from liver and bile ducts, twenty weeks after challenge. There was, however, a significant increase in the proportion of flukes retarded in the parenchyma of both vaccinated groups. The percentage of retarded flukes was positively correlated with the degree of liver damage and increased weight of the hepatic lymph nodes. It was not possible to determine if the retarded flukes were derived from the vaccine or challenge infections or both.Challenge infection of both vaccinated and unvaccinated sheep significantly increased the numbers of eosinophils and globule leucocytes in the parenchymal bile duct and the numbers of mast cells and globule leucocytes in the abdominal bile duct. In addition the numbers of eosinophils and globule leucocytes in the parenchymal bile duct were significantly correlated with the percentage of retarded flukes in both vaccinated groups. In the abdominal bile duct, only the numbers of eosinophils in the low level vaccination group were significantly correlated with fluke retardation.Vaccination did not protect against the pathogenic effects of challenge infection as measured by reduced packed cell volumes and weight gain.     

Liver fluke infestation in New World camelids. Parasitology, pathology, clinical findings and therapy]

In Llamas and Alpacas infestation with Fasciola hepatica or Dicrocoelium dendriticum can cause liver damage, sometimes even with lethal outcome. Once infected South American Camelids (SACs) react more sensitively to these parasites than other domestic ruminants. We report here on the pathology, parasitology, clinics and therapy of this disease. Concerning Dicrocoelium dendriticum we describe own clinical results and therapeutic outcome in addition to the pathological investigation. According to anatomic corrosion casts, the bile ducts of SACs show more similarity with the equine bile system than with the bile system of domestic ruminants.     

Relationship of gastrointestinal histology and mucus antiparasite activity with the development of resistance to trichostrongyle infections in sheep

Changes in the numbers of globule leucocytes, mast cells, eosinophils and goblet cells in the gastrointestinal mucosa were examined in relation to the development of resistance and elimination of nematodes in grazing sheep in their firsty year of life. Sheep immunised against Trichostrongylus colubriformis, and sheep treated with dexamethansone were also examined. A strong association between resistance to infection and the presence of globule leucocytes was found. In contrast, the numbers of mast cells or goblet cells were not correlated with resistance. Globule leucocyte and eosinophil numbers were also correlated with antiparasite activity in mucus.

Immunising infections of T. colubriformis given to 10-month-old sheep, their duration limited by thiabendazole treatment, gave rise to considerable immunity to homologous challenge infections. Larvae that developed to the 4th stage were as effective at stimulating immunity as those that developed to the 5th stage. Dexamethasone treatment abrogated resistance to trickle challenge infection with T. colubriformis and reduced mucosal globule leucocyte and mast cell numbers. After cessation of drug treatment, the re-establishment of resistance and adult worm elimination were associated with repopulation of the mucosa with large numbers of globule leucocytes and high antiparasite activities in mucus.     

Morphological, Histochemical and Ultrastructural Features of the Globule Leukocyte of the Bovine Respiratory Tract

The histochemical and ultrastructural features of the globular leukocyte of the bovine respiratory tract is described and found to be similar to those localized in the abomasum and bile ducts of cattle. Histochemical analysis of these cells demonstrated low amounts of carboxylated mucin mixed with neutral mucin and large amounts of basic protein. Two types of globules were seen in the leucocytes; one with a homegeneous electron dense material and another with vesiculated, crastalloid containing bodies.     

Bovine nasal granuloma. Gross and microscopic lesions.

The anterior nasal mucosa of 21 cattle had small closely-packed polypoid nodules. These had a distribution pattern similar to that of inhaled particles. Acute inflammatory changes consisting of eosinophil infiltration, mast cell and globule leucocyte hyperplasia, and oedema were frequent in the epithelium, terminal gland ducts and adjacent lamina propria. Epithelium over nodules was metaplastic to non-keratinising stratified squamous, whilst epithelium of terminal gland ducts was metaplastic to mucus-secreting pseudostratified columnar. Focal accumulation of granulation tissue between gland ducts caused herniations of the superficial lamina propria to form the nodules.     

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.     

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.     

On the ultrastructure of epithelial cells in bile ducts of cattle chronically infected with fasciola hepatica

This paper describes briefly the normal ultrastructure of bile-duct epithelial cells in cattle and the main ultrastructural alterations produced in them by chronic infection with Fasciola hepatica. The studies showed only inconsiderable abnormalities in the ultrastructure of the epithelium in infections of a low degree as also during the recovery phase of the disease. On the other hand, in heavily infected livers pronounced ultrastructural alterations were seen in the surface epithelium of the main intrahepatic bile ducts invaded by the flukes. In such areas the intercellular spaces were filled with dense homogeneous material, and intact desmosomes and junctional complexes were not regularly seen, which indicates that foreign material may leak into the bile. The extracellular material was probably composed of albumin, and the significance of the present results regarding the etiology of hypoalbuminaemia during chronic fascioliasis is discussed.     

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.     

Light and scanning electron microscopic studies of the extrahepatic bile duct of sheep with experimentally induced Fasciola hepatica infection.

Changes in the common bile duct that contained adult Fasciola hepatica of sheep were evaluated by light (LM) and scanning electron microscopy (SEM). Nine ewes were inoculated with F hepatica metacercariae and necropsied 18 weeks after inoculation. The proximal portion of the common bile duct (CBD) that contained adult flukes was recovered and examined by LM and SEM. The CBD from 2 noninoculated ewes were used for control. On gross examination, CBD were markedly large because of the adult flukes, which were free in the lumen of the ducts. Extensive hemorrhage was not found either in intrahepatic or in extrahepatic bile ducts of any sheep. Histologic examination revealed changes, such as villous hyperplasia and hypertrophy of the epithelium; cell infiltration, predominantly with eosinophils or macrophages; and arterial intimal proliferation. By SEM, the epithelial surface of the CBD appeared intact. Villous hyperplasia and hypertrophy of the epithelium observed by LM was clearly seen by SEM. Epithelial damage, seen as small areas of denuded surface by LM and SEM, was confined to a few areas of the mucosa. Lack of extensive hemorrhage and confined epithelial damage were evaluated relative to the mode of feeding of adult flukes.     

Fasciola hepatica: comparison of flukes from Korea and the United States by isoelectric focusing banding patterns of whole-body protein.

Comparisons were made between the flukes from Chonnam, Korea and Oregon, USA by isoelectric focusing (IEF) of whole-body protein. Adult Fasciola hepatica were recovered from bile ducts of Korean native cattle. Whole-body protein of the flukes was subjected to IEF, and the banding patterns of the fluke protein were compared with those of North American F. hepatica recovered from experimentally infected calves. The overall banding pattern of F. hepatica from Korea was essentially identical to that of F. hepatica from the United States. These results provide further support for the usefulness of this technique in differentiating Fasciola species in other geographical areas.     

Cellular and humoral local immune responses in sheep experimentally infected with Oestrus ovis (Diptera: Oestridae)

Cellular and humoral local responses were investigated following repetitive artificial Oestrus ovis infections in lambs. The presence of larvae induced a huge local recruitment of either leucocytes (T and B lymphocytes, macrophages) or granulocytes (eosinophils, mast cells and globule leucocytes). This cellular response was more pronounced in the ethmoid and sinus (development sites of second and third instar larvae) than in the septum or turbinates where first instar larvae migrate. Infected lambs produced Oestrus ovis specific IgG and IgA antibodies in their mucus. This local humoral response was mainly directed against larval salivary gland antigens and not against larval digestive tract antigens. Compared to the control animals, the sinusal mucosa of infected animals was extremely thickened and the epithelium exhibited hyperplasia, metaplasia and eosinophilic exocytosis. The possible roles of these local immune responses in the regulation of O. ovis larvae populations in sheep are discussed.     

The abomasal histology of worm-free sheep given primary and challenge infections of Haemonchus contortus

The histological changes in the abomasal mucosa of pairs of worm-free ewes and lambs were compared at various stages after infection and reinfection with 350 Haemonchus contortus L3 per kg bodyweight. In terms of worm burdens the ewes were relatively resistant to reinfection compared with the lambs and the histological changes in the abomasum at most stages after infection were more marked in the adult animals. These included increases in the numbers of mast cells, globule leucocytes, eosinophils and IgA plasma cells in the mucosa.     

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.     

Impact of compound alpha treatment in vivo on egg production by the liver fluke, Fasciola hepatica

Sheep infected with the triclabendazole-susceptible Cullompton isolate of Fasciola hepatica were treated with compound alpha at a dosage of 15 mg/kg at 12 weeks post-infection. Adult flukes were recovered from the bile ducts at 24h, 48 h and 72 h post-treatment (pt). They were processed for whole mount analysis, histology and transmission electron microscopy of the female reproductive system: specifically, the uterus, Mehlis' gland, ovary and vitellaria. As judged by the appearance of the uterus, normal egg production ceased within 24h of treatment; this phenomenon preceded significant changes to the other reproductive organs. Over the 3-day pt period, there was a progressive decline in the number of oogonia in the ovary, together with an increase in the number of eosinophilic and apoptotic oocytes and vacuolation and shrinkage of the ovarian tubules. There was a shift in the cell population within the vitelline follicles at 48 h pt, with relatively greater numbers of mature vitelline cells and fewer immature cells. The follicles were vacuolated and the shell globule clusters in the mature cells were disorganised. Greater disruption was seen at 72 h pt, with a reduction in the size of the follicles and rupture of cells, releasing their content into the lumen of the follicles. These histological observations were confirmed and extended at the TEM level. Thus, examination of electron micrographs showed that disruption of the shell globule clusters was evident at 48 h pt, which coincided with the start of the breakdown of the mature cells and of the nurse cell network. These degenerative changes were more conspicuous at 72 h pt. In the Mehlis' gland, shrinkage and vacuolation of the cells and their cytoplasmic extensions became progressively greater from 48 h to 72 h pt, and secretory activity declined. The changes in the reproductive organs and inhibition of egg production are put in context of the overall time-course of drug action.     

Dimensional analysis of milk fat globules in sow milk: effects of the lactation stage and fat content and comparison with vaccine milk

To assess the differences in the granulometry of milk fat globules between swine and bovine species, milk samples from 30 lactating sows were analyzed for fat globule dimensions and compared with cow milk samples. Results showed differences between the fat globules: sow milk presents reduced globule diameters compared with cow milk (volume-weighted diameter 2.62 vs. 3.27 µm, p < 0.001) and reduced interglobular distance. A positive relationship was observed between milk fat content and globule diameter, while a slight, insignificant inverse trend was detected between the day of lactation and fat globule diameter. These complex interactions between milk lipids, globule membrane proteins, and globule dimensions provide a better understanding of digestion/absorption phenomena in the design of milk replacers.