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.     

Galectin secretion and binding to adult Fasciola hepatica during chronic liver fluke infection of sheep

Galectins are increasingly recognised as important mediators of immune homeostasis and disease regulation, but comparatively little is known about their role in parasite infection. This study investigates the interaction between two ovine galectins, galectin-11 and galectin-14, and the parasitic liver fluke, F. hepatica. Galectin-14 was found in eosinophils infiltrating the tissue surrounding infected bile ducts and secreted in the connective tissue, while galectin-11 was specifically induced in epithelial cells of bile ducts from infected sheep. Strong nuclear staining was observed for galectin-11. Both galectins were found to be secreted into the bile fluid of parasite infected sheep, and were also detected in the excretory/secretory products of adult flukes, following their removal from the ovine host. Recombinant galectin-14, but not recombinant galectin-11, was found to bind specifically to the surface tegument of adult flukes in a carbohydrate dependent manner. This study shows for the first time that both galectin-14 and galectin-11 are produced in liver tissue after chronic liver fluke infection and that they can directly interact with the parasite in the bile ducts. Galectin-11 may also be involved in epithelial cell turnover and cancerogenesis.     

Response of sheep to challenge infection with Fasciola hepatica

Sheep were infected with 100 metacercariae of Fasciola hepatica and reinfected 16 weeks later with a further 100 metacercariae. Serum samples were taken weekly for 36 weeks after primary infection. Serum was assayed for the presence of the enzymes glutamate dehydrogenase (GLDH) and gamma-glutamyl transferase (gamma-GT), as indicators of liver and bile duct damage respectively, and for levels of precipitating antibody. Antibody and GLDH levels rose following the primary infection but fell after patency had been reached . A peak in gamma-GT activity was associated with the onset of patency. After the challenge infection levels of both enzymes rose substantially and there were persistent fluctuations in activity. Antibody levels did not rise markedly following challenge but fluctuated at low levels until autopsy, 20 weeks after challenge. There was no resistance to challenge judged by worm recoveries at autopsy. It is suggested that the presence of adult flukes in the bile ducts suppresses the antibody response to challenge infection. Tissue damage, which is shown by fluctuations in GLDH and gamma-GT levels after adult flukes have become established in the bile ducts, is considered to be due to the feeding activity of adult flukes and the deposition of immune complexes in the liver parenchyma.     

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.     

The pharmacodynamics of the flukicidal salicylanilides, rafoxanide, closantel and oxyclosanide

The pharmacokinetics of oxyclosanide, rafoxanide and closantel were investigated in sheep (n = 5). All three drugs were extensively (greater than 99%) bound to plasma proteins and the plasma concentration/time curve was best described by a tri-exponential equation. Closantel and rafoxanide had long terminal half-lives (mean 14.5 and 16.6 days, respectively) compared with oxyclosanide (mean 6.4 days). In a study of the efficacy of rafoxanide against Fasciola hepatica, a dose rate of 7.5 mg kg-1 against 6-week-old flukes appeared to be similarly effective to a dose rate of 2.5 mg kg-1 against 10-week-old flukes (86% and 88% efficacy, respectively), as assessed at autopsies carried out on all sheep when the flukes were 14 weeks old. Part of this putative efficacy against immature flukes may be due to rafoxanide persisting in the plasma and affecting the mature flukes when they reach the bile ducts.     

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.     

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     

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.     

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.     

Uptake of albendazole and albendazole sulphoxide by Haemonchus contortus and Fasciola hepatica in sheep

The pattern of in vivo uptake of albendazole (ABZ) and its major metabolite, ABZ-sulphoxide (ABZSO), by Haemonchus contortus and Fasciola hepatica recovered from ABZ-treated sheep, was investigated. Concentration profiles of both compounds were simultaneously measured in target tissues/fluids from the same infected sheep. In addition, the proportion of the (+) and (-) ABZSO enantiomers was determined in plasma, bile and F. hepatica recovered from treated sheep. Sheep naturally infected with H. contortus were intraruminally (i.r.) treated with ABZ (micronized suspension, 7. 5mg/kg) and the plasma concentrations of ABZSO and ABZ-sulphone (ABZSO(2)) determined in addition to the concentration of ABZ and ABZSO in H. contortus, abomasal mucosa and fluid content samples. In addition, F. hepatica artificially infected sheep were treated i.r. with the same ABZ suspension (7.5mg/kg), and samples of blood, bile, liver tissue and adult flukes were collected and analysed by HPLC to determine the concentrations of ABZ and both enantiomers of ABZSO. ABZSO and ABZSO(2) were the analytes recovered in plasma with ABZ and ABZSO present in H. contortus. ABZ was the analyte recovered at the highest concentration in H. contortus and abomasal mucosa, whereas higher concentrations of ABZSO were measured in abomasal fluid content. Only low concentrations of ABZ were detected in F. hepatica and bile, but markedly higher concentrations of ABZ were measured in liver tissue. ABZSO was the main molecule recovered in F. hepatica, plasma and bile samples collected from ABZ-treated sheep. The (+) enantiomer of ABZSO was recovered at a higher proportion in plasma (75%), bile (78%) and F. hepatica (74%) after ABZ administration to infected sheep.     

Cellular proliferation of bile ductules and gamma-glutamyl transpeptidase in livers and sera of young cattle following a single infection with Fasciola hepatica

During the course of experimental single infection with Fasciola hepatica in calves, the origin of the increased concentration of gamma-glutamyl transpeptidase (gamma-GT) found to be present in the serum during the biliary stage of the disease, was studied. The enzyme originates from the liver after flukes have caused intermittent and selective cholestasis in the bile-duct system. The enzyme which normally is present in the bile ducts can under these circumstances enter the surrounding interstitial tissues through broken-up junctions of the bile drainage system or be added to these from proliferating bile-ductule epithelium. The multiplication of bile-ductule cells can be looked upon as a neogenesis of cholangioles, which becomes essential when newly formed perilobular connective tissues prevent restoration of the anatomical connections of the bile capillaries with the biliary tree. The connective tissue growth is induced by bile fluid. An initially regional perilobular fibrosis is eventually distributed diffusely through the liver by migration of flukes. The continued and extending fibrosis is due to the constant recurrence of intermittent and selective cholestasis. The transient occlusions of branches of the bile ducts are very likely to be attributable to persistent cholangitis. The growth of connective tissues has a considerable effect on the gain in weight of the liver.     

Pathological changes of tracheal mucosa in chickens infected with infectious laryngotracheitis virus

Six-week-old chickens were inoculated via the posterior thoracic air sac with infectious laryngotracheitis virus. Chickens were sacrificed on various days through day 16 postinoculation (PI), and the trachea was examined by scanning electron microscopy (SEM) and light microscopy (LM). The pathological changes observed on day 1 PI were hypertrophy and hyperplasia of goblet cells. From day 3 PI, the epithelial cells protruded collectively and fused to form syncytia, which contained many intranuclear inclusion bodies. Subsequently, epithelial syncytia desquamated, one after another, and connective tissues were exposed in places. Serofibrinous exudate and detritus were abundant on the surface of the exposed connective tissues and seemed to form a pseudomembrane. On day 5 PI, the remaining epithelial cells began to repair the devastated mucosa just under the pseudomembrane. On day 6 PI, microvillus-rich regenerating epithelial cells were arranged like paving stones. On day 8 PI, the epithelial cells proliferated extensively and formed folds with cyst-like structures. By day 16 PI, the tracheal epithelium was covered with cilia and regained its normal histologic appearance.     

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.     

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     

Hepatic trematodiasis in a Ganges River dolphin

Hepatic trematodiasis caused by Cyclorchis campula was diagnosed in a juvenile Ganges River dolphin that had been in captivity at an aquarium for approximately 1 year. Histopathologic findings were severe chronic suppurative cholangitis, hyperplasia of the bile duct epithelium, and periductal fibrosis associated with fluke infection of the large bile ducts.     

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.     

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.     

Fasciola hepatica: comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study, F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response.     

肝片吸虫感染致斑纹角马死亡案例调查

首次报道了国内圈养斑纹角马肝片吸虫病确诊病例。诊断依据：（1）提示性临床症状：贫、消瘦、营养不良;（2）尸体剖检特征性病理变化：胆管显著扩张和胆管内挤排出大量肝片吸虫成虫的虫体;（3）肝片吸虫感染强度测定：运用麦克马斯特法测定了病死角马肝片吸虫感染强度为1 600个/g;（4）中间宿主调查：角马的饲养区内发现大量椎实螺,椎实螺体内含有大量的肝片吸虫尾蚴;（5）组织病理学检查：细支气管和肺泡腔内见有童虫的虫体结构。首次证实了角马肝片吸虫感染时,童虫在移行过程中可误移至肺,导致肺的病理变化。该病的发生与环境潮湿多水、环境中存在大量椎实螺而未被重视有关。肝片吸虫宿主范围广,应重视易感动物的生活环境,定期粪检确定感染率和感染强度,以及选择有效的药物防治,这是科学预防该病的关键。     

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