Immature Fasciola Hepatica Research Articles

Assessment of the effective dose of an experimental intramuscular formulation against immature and adult Fasciola hepatica in sheep

The effective dose of an injectable prodrug, named compound alpha prodrug, against immature and adult Fasciola hepatica in experimentally infected sheep was determined. In a first experiment, 30 sheep were infected with Fasciola hepatica on day 0 and 50. After microscopic detection of faecal eggs on day 80, groups (n = 6) 1 to 3 were treated with 6, 8 and 10 mg/kg of the experimental water-soluble prodrug compound alpha intramuscularly, respectively. Group 4 was treated with closantel and group 5 remained untreated. Copromicroscopical examinations were made on day 0, 80 and 108. On day 110, trematodes were collected from the bile ducts. Fasciolicide efficacy was assessed as a percentage of fluke-egg and adult-fluke reduction. Fluke length was also recorded. In a second experiment aimed to assess the fasciolicide activity of compound alpha prodrug against four-week-old flukes, 12 sheep were infected on day 0 and allocated into two groups (n = 6). On day 50 post infection, group A was treated with the experimental water-soluble prodrug compound alpha at 6 mg/kg/IM and B remained untreated. Fasciolicide activity was assessed on day 80 after collection, microscopic observation and measurement of flukes present in the parenchyma for immature stages and on day 108 for adults. Egg output decreased 91.2, 96.0, 98.8 and 94.9% for groups 1, 2, 3 and 4, respectively. Compound alpha prodrug cleared 97.6%, 98.51% and 100% of adult stages in a dose-dependent manner. Closantel killed 81.95% flukes. Regarding the second experiment, 81.2% efficacy was achieved. Immature flukes were significantly smaller in the treated group. It is concluded that the intramuscular application of compound alpha prodrug exerted fasciolicide efficacy against adults of Fasciola hepatica.

Acute fasciolosis in cattle in southern Brazil

This study describes the epidemiological and pathological aspects of an outbreak of acute fasciolosis in cattle in southern Brazil. Fifteen out of 70 three-year-old pregnant cows lost weight in the 30-40 days prior to calving. Clinical signs included diarrhea, weakness, mild anemia and jaundice. Dark yellow fluid in the abdominal cavity was observed at necropsy. Fibrin and clotted blood were adhered to the pericardium and lung, primarily in the diaphragmatic lobes. The liver was enlarged, and the capsular surface was irregular with clear areas and petechiae. At the cut surface, the liver was irregular, firm and edematous, and several hemorrhagic channels could be observed. Areas of fibrosis through the parenchyma and whitish thrombi occluding the great vessels were also observed. The livers of 10 cows that not died were condemned at slaughter for lesions of fasciolosis similar to those observed at necropsy. Microscopically, the liver showed areas of coagulation necrosis, extensive hemorrhages in the streaks or foci and disruption of the parenchyma with neutrophil and eosinophil infiltration. Fibrosis and bile duct proliferation were also observed. Immature Fasciola hepatica flukes were observed in the parenchyma surrounded by degenerated hepatocytes, neutrophils, eosinophils, and hemorrhages. The outbreak occurred on a farm located in an area endemic for fasciolosis, although the acute form of the disease is not common in cattle in this region. It is likely that the cows were infected by F. hepatica metacercariae released in the late fall or early spring in the rice stubble where the herd was grazing prior to calving. Although mortality due to fasciolosis in cattle is infrequent, outbreaks can occur and treatments that are effective in both the immature and adult forms of the parasite should be administered to prevent economic losses.

Specificity of a coproantigen ELISA test for fasciolosis: lack of cross-reactivity with Paramphistomum cervi and Taenia hydatigena

A commercial coproantigen ELISA test for fasciolosis, based on the use of MM3 monoclonal antibody for antigen capture, was investigated for possible cross-reactivity with Paramphistomum cervi, a trematode that commonly...

Efficacy of an injectable combination anthelmintic (nitroxynil + clorsulon + ivermectin) against early immature Fasciola hepatica compared to triclabendazole combination flukicides given orally or topically to cattle

The objective was to compare the efficacy against artificially induced 2- and 4-week old early immature triclabendazole-susceptible liver flukes (Fasciola hepatica) of an injectable combination of nitroxynil, clorsulon and ivermectin with oral and pour-on combination formulations containing triclabendazole.Groups of yearling Angus or Angus cross cattle were confirmed fluke free before being artificially infected with 500 Sunny Corner strain triclabendazole-susceptible liver fluke metacercariae. Two or four weeks after infection, cattle were treated with the test combination Nitromec® (10.2mg/kg nitroxynil, 2.0mg/kg clorsulon, 0.2mg/kg ivermectin), or oral Flukazole® C+Se (triclabendazole/oxfendazole/Selenium), oral Fasimec® C (triclabendazole/ivermectin) or Genesis® Ultra Pour-On (triclabendazole/abamectin).At intervals cattle were weighed, faecal sampled for liver fluke egg counts and blood sampled for liver serum enzyme analysis. Cattle were slaughtered 14 weeks after infection for recovery of adult flukes; fluke egg counts and liver pathology assessment.All cattle increased in body weight by 0.4–0.8kg/day but there were no significant differences between control and treated groups or between the treatment groups.Geometric mean 14-week fluke egg counts and total fluke counts for all treatments, were significantly less (p<0.05) than the control group, except for the group treated with Genesis® Ultra Pour-On, 2 weeks after infection. Nitromec treatment of 2-week old flukes was 83% and 95% effective as assessed by 14-week egg and fluke counts, respectively, compared to Flukazole C; 96% and 99%, Fasimec C; 70% and 46%, and Genesis Pour-On, which was ineffective, with egg and fluke count reductions of 0% and 8%, respectively. Against 4-week old flukes, Nitromec treatment was 88% and 99% effective when assessed by 14-week egg and fluke counts, respectively, with Flukazole C; 98% and 99%, Genesis Pour-On; 98% and 82% and Fasimec C; 91% and 61% effective, respectively.Group mean levels of the bile duct-associated enzyme gamma glutamyl transpeptidase (GGT) and the parenchymal associated enzymes, aspartate amino-transferase (AST) and glutamate dehydrogenase (GLDH) increased above the normal range 8 and 11 weeks after infection in the untreated control animals and the group treated 2 weeks after infection with Genesis Pour-On. The groups treated with Fasimec at 2 or 4 weeks after infection, also had elevated enzyme levels. The use of liver-associated enzyme assay is supported as supplementary indicators of fluke-induced pathology.

Seasonal differences in the efficacy of pour-on formulations of triclabendazole and ivermectin or abamectin against late immature liver fluke ( Fasciola hepatica) in cattle

Three controlled studies were conducted to determine the efficacy against late immature (6 weeks) Fasciola hepatica of two currently available fasciolicides (Genesis ® Ultra and Coopers ® Sovereign ®) which are applied externally to cattle. Efficacy of the two products was assessed when application was made under winter, spring and summer conditions. Efficacies for winter, spring and summer respectively, based on arithmetic mean total fluke counts, were 78.9%, 91.7% and 99.6% for Coopers ® Sovereign ® and 73.4%, 89.7% and 99.6% for Genesis ® Ultra. Seasonal differences with treatment efficacy were indicated. The studies also confirmed previous observations that liver fluke egg counts overestimate the efficacy of fasciolicides and that total fluke counts is the most reliable method for assessing efficacy of such products.

Immature triclabendazole-resistant Fasciola hepatica: tegumental responses to in vitro treatment with the sulphoxide metabolite of the experimental fasciolicide compound alpha

Juvenile triclabendazole-resistant liver flukes, Fasciola hepatica, were incubated in vitro with 10 microg/ml of the sulphoxide metabolite of the experimental fasciolicide, compound alpha [5-chloro-2-methylthio-6-(1-naphthyloxy)-1H-benzimidazole], for 6 and 18 h. Following treatment, the specimens were examined using scanning electron microscopy (SEM), transmission electron microscopy (TEM) and tubulin immunocytochemistry. The SEM results revealed a posterior-directed disruption comprised predominantly of swelling and blebbing of the tegument; these changes were more severe and extensive after the longer 18-h incubation. Along with swelling of the tegument and blebbing, the TEM results also revealed swelling of the mitochondria and basal infolds. A decrease in the number of both T1 and T2 secretory bodies was observed in the syncytium and cytoplasmic connections after the 18-h treatment. The circular muscle bundles were also disrupted, in that the organisation of the muscle fibres was irregular and the total number of muscle fibres was reduced. The immunocytochemical studies revealed no significant disruption to the distribution of tubulin immunoreactivity within the tegumental syncytium, the cytoplasmic connections or the associated tegumental cells. The results indicate that alpha.SO is capable of disrupting the tegument of 4-week-old triclabendazole-resistant liver flukes, though the morphological changes were not associated with any significant differences in tubulin immunostaining.

The effect of a parenteral ivermectin/closantel injection on the growth and reproductive development of early immature Fasciola hepatica in cattle

Sixteen calves approximately 6 months old were each infected with 500 metacercariae of Fasciola hepatica. Thirty-two days later they were weighed and divided into two groups, and on day 35 all calves in one of the groups were injected subcutaneously with an ivermectin/closantel combination. Both groups were sacrificed between days 70 and 72 to enable counting and examination of the flukes recovered from the bile ducts. Eggs released by the flukes were collected for incubation, hatching and estimation of egg viability. Flukes were counted, flat-fixed in 70% ethanol, stained with catechol and carmine and measured. The reproductive organs, namely testis, vitelline glands, ovary and uterus, were examined and scored on a 0–3 scale according to their state of development. This was visually assessed on the basis of size, distribution and staining density of their constituent tissues and the abundance of eggs in the uterus. A representative sample of flukes from each animal was fixed in formalin for histological sectioning to enable more detailed examination of the reproductive structures. Treatment of the immature flukes reduced the population in cattle by 42.6% as compared with the controls and as a result of the stunting effect due to the presence of closantel during early development the size of treated flukes was reduced by 43.9%. A bimodal pattern of size and reproductive score was also observed in flukes from treated cattle, suggesting that the stunting effect on individual flukes differed depending on whether or not they had gained access to the bile ducts or were still migrating in the hepatic parenchymal tissue at the time of drug exposure with the effect being greater once the fluke had gained access to the bile ducts. The mean reproductive score for untreated flukes was 8.76 and for treated flukes 5.64, a 35.6% reduction. This difference was highly significant ( p < 0.001). Egg shedding from treated flukes was significantly less than that from controls ( p < 0.05), but there were no differences in hatchability, suggesting that whilst drug treatment reduced the energy supply available for gametogenesis/oogenesis, it did not induce functional defects in the gonads or accessory reproductive organs. Histological examination confirmed that there was a reduction in development of testes, ovaries and vitellaria in treated flukes, with a consequent reduction in egg production. In the treated flukes, early spermatogonia and oogonia were the predominant cell types in the testes and ovary, whilst undifferentiated stem cells were abundant in the vitelline follicles. In untreated flukes, cells representing more advanced stages in gametogenesis and vitellogenesis predominated in the respective organs. It is likely that this inhibition of gametogenesis and vitellogenesis was caused by the effects of closantel treatment on intermediary metabolism in the flukes. Clearly these effects were evident even at a relatively early stage of fluke growth, and because of the impact on egg output may have epidemiological importance in addition to the reduction in fluke numbers.

The effect of the 5-chloro-2-methylthio-6-(1-naphtyloxy)-1H-benzimidazole on the tegument of immature Fasciola hepatica in their natural host

The damage to the tegument of 3-week-old Fasciola hepatica was evaluated by scanning electron microscopy (SEM) following treatment with the 5-chloro-2-methylthio-6-(1-naphtyloxy)-1H-benzimidazole (called compound alpha) in its natural host. For the present study, flukes were raised in pelibuey sheep infected orally with metacercariae of F. hepatica; the parasites were recovered from the liver of the sacrificed sheep after 6, 12 and 24 h of treatment with compound alpha. At 6 h of treatment, the flukes showed some lesions on the ventral surface of the anterior region, such as a swollen tegument and blebs. At 12 h after treatment, the specimens showed structural disorganization and spine loss in the ventral anterior region. The tegument of the flukes treated for 24 h was completely lost in some areas of the ventral surface, leaving an exposed basal lamina. The tegument of immature F. hepatica might be a target organ for compound alpha to exert its fasciolicide effect.

Fasciola hepatica:Stage-Specific Expression of Novel Gene Sequences as Identified by Differential Display

Reed, M. B., Spithill, T. W., Strugnell, R. A., and Panaccio, M. 1998.Fasciola hepatica:Stage-specific expression of novel gene sequences as identified by differential display.Experimental Parasitology89, 169–179. Differences in gene expression between adult and immatureFasciola hepatica(liver fluke) parasites isolated from the mammalian host were investigated using the technique of differential display. For any given primer combination used to produce these displays there were, on average, 22% apparently adult-specific and 14% apparently immature-specific cDNA products able to be identified, consistent with a high degree of differential gene expression between these two parasite developmental stages. Several cDNA fragments specific to immature parasite RNA were isolated and cloned. An abundant 400- to 500-bp RNA species was identified on a Northern blot by hybridization to the cloned DD2 cDNA fragment and was determined to be expressed at levels at least 10-fold higher in immature parasites relative to adult parasites. mRNA transcripts corresponding to the remaining cDNA fragments (DD14, DD16, DISP10, and DISP2) were apparently expressed at levels below the sensitivity limits of Northern analysis, although differential expression of these transcripts was confirmed by reverse transcriptase PCR (RT-PCR). The identities or functional significance of each of the five differentially expressed cDNAs identified in this study is still unclear due to the lack of any significant sequence similarity to the entries currently held within sequence databases.

The efficacy of triclabendazole (fasinex ®) against immature and adult fasciola hepatica in experimentally infected cattle

Eighteen Chinese cattle were experimentally infected with metacercariae of Fasciola hepatica and randomly assigned to 6 groups. Five groups of cattle were treated with a single oral dose of triclabendazole at a dose rate of 12 mg kg −1. At necropsy, the reduction in fluke burden compared with the untreated group was 85, 99.6, 99.8, 100 and 100% for cattle treated 2, 6, 8, 12 and 16 weeks after infection, respectively. Data are also presented on body weight changes during the experimental period and on serum γ-GT activity in cattle from selected groups. Triclabendazole is considered to be safer and more efficacious than currently available fasciolicides in China.

Thiol proteases released in vitro by Fasciola hepatica

Immature Fasciola hepatica release 11 distinct proteases when cultured in vitro for 16 h as revealed by gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six of these proteases are active in the pH range 4.5 to 8.0. Five are acid proteases, being most active in the pH range 3.0 to 4.5. The majority of proteases released in vitro by immature flukes are also released by mature flukes; however, a 40-kDa protease released by immature flukes is a very minor protease released by mature flukes. The activity of all proteases is inhibited by leupeptin, l- trans-epoxysuccinyl-leucylamido(4-guanidino)butane, phenylmethylsulfonyl fluoride and iodoacetamide and enhanced or stabilized by the reducing agents cysteine and dithiothreitol. Therefore, all F. hepatica in vitro-released proteases identified by gelatin-substrate SDS-PAGE are thiol proteases.

Flukicidal action of closantel against immature and mature Fasciola hepatica in experimentally infected rats and sheep

The relative importance of peak level- and residual level-related flukicidal activity of closantel against immature and mature Fasciola hepatica was evaluated in a comparative efficacy trial using two animal species with a different plasma elimination pattern, that is, the rat and the sheep with an elimination half-life of less than one week and of two to three weeks, respectively. The rats were dosed orally with closantel at 20 mg kg-1 at two, four, six, eight and 10 weeks; the sheep at 10 mg kg-1 at eight, 10 and 12 weeks after artificial infection. Necropsy was performed either one week after treatment or 12 weeks after infection. Efficacy rates and the length of the recovered flukes were evaluated. It was demonstrated that the flukicidal effect of closantel is directly related to its peak plasma levels and less to its residual plasma concentrations. In the rat, a high efficacy (P less than 0.001) could be demonstrated against immature stages of four weeks or older. The two-week immature stages were less markedly affected. No significant differences in efficacy and size of the flukes were noted between the animals autopsied one week after treatment and those autopsied 12 weeks after infection. In the sheep, the efficacy against six-week and eight-week-old immature stages varied between 70.3 and 76.8 per cent and between 92.8 and 96.5 per cent, respectively. As in the rats, no marked differences in efficacy were noted between the animals autopsied one week after treatment and those autopsied 12 weeks after infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Fasciola hepatica: Action in vitro of triclabendazole on immature and adult stages

Under in vitro conditions in a balanced salt solution, triclabendazole was found to accumulate in significant amounts in both immature (3 week old) and adult Fasciola hepatica. A viable parasite was needed to concentrate the drug, but a high percentage of the compound was also bound by the dead worm. The drug could penetrate into liver flukes even when the oral route had been closed off by ligation, indicating that the drug can be taken up by transtegumentary absorption. A 24 hr exposure to triclabendazole, at 10–25 μ M concentrations, was found to result in a strong inhibition of the parasite's motility. This effect was paralleled by dramatic changes in the worm's resting tegumental membrane potential. The onset of these actions was found to develop very slowly, and high drug levels had to accumulate within the parasite to initiate its immobilization. In addition to drug concentration and incubation time, physiological alterations observed were also dependent on other culture conditions, such as the presence or absence of serum albumin and the drug tissue/medium ratio. Biochemical examinations showed that triclabendazole significantly stimulated glucose derived acetate and propionate formation by adult liver flukes. Adenosine triphosphate levels were not changed even in the presence of high triclabendazole concentrations (25 μ M). Likewise, the activities of various membrane associated adenosine triphosphatases were not altered by the drug. However, the ability of the drug to inhibit colchicine binding to microtubular protein purified from adult liver flukes suggested an interference of the drug with microtubular structure and function. This interpretation of the drug's action was supported by the finding that triclabendazole strongly inhibited the release of proteolytic enzymes from immature and adult parasites, a process which is likely to be dependent on microtubular function. Similar physiological and biochemical effects as those described for triclabendazole were also found with its major metabolite, triclabendazole sulfoxide. Although triclabendazole was found to initiate a variety of potent physiological and metabolic effects on immature and adult liver flukes, the precise molecular mode of action of the fasciolicidal drug remains to be elucidated.

Chemoprophylaxis of fascioliasis with triclabendazole

In a field experiment, moderate to heavy natural concomitant infections with immature and mature Fasciola hepatica were treated with triclahendazole at a dose rate of 10 mg/kg and an efficiency of 99.8% was achieved. Subsequent treatments of all susceptible farm animals at the same dose rate at intervals of eight to eleven weeks were carried out for 14 months; no patent infections could be detected in sheep and cattle during the whole period. Evidence is presented that pasture contamination with liver fluke was reduced to a negligible level for a further 12 months after the final treatment. It is suggested that, if regular treatments with triclabendazole are given within the pre-patent period of Fasciola hepatica infection for the whole season, the infection can be eradicated or reduced to such a low level that control of the disease could be maintained with less frequent strategic drenching for a considerable period.

Identification and characterization of a major Schistosoma mansoni glycoprotein antigen cross-reactive with Fasciola hepatica.

A major surface antigen of Schistosoma mansoni has been identified and characterized as a glycoprotein of 66,000 molecular weight (Mr) and isoelectric point of 6.2-6.1 (SM66-GP) by use of a monoclonal antibody. The antigen was expressed by schistosome eggs, cercariae, larvae, and adults, and was recognized by sera of schistosome infected hosts. Direct immunofluorescence microscopy showed the antigen was distributed in a uniform pattern on the entire worm surface. Indirect immunofluorescence microscopy revealed that it was present in the parenchymal tissue of immature and mature Fasciola hepatica, in the gut of the mature fluke, and in embryonated fasciola eggs. The cross-reactive F. hepatica epitope recognized was expressed on a polypeptide of Mr 220,000.

The ability of diamphenethide to control immature Fasciola hepatica in sheep at a lower than standard dose level

Groups of eight Welsh Mountain sheep were dosed with diamphenethide at the rate of 70 mg/kg bodyweight at either one, four, six or eight weeks after artificial infection with approximately 300 Fasciola hepatica metacercariae. Comparisons were made with similarly infected but undosed sheep and with sheep which were neither infected nor dosed. The good clearance of flukes up to six weeks of age (above 97 per cent on pooled data) was reflected in the plasma concentrations of the accepted liver damage marker enzymes glutamate dehydrogenase and gamma-glutamyl transpeptidase. Highly significant correlations were demonstrated between the numbers of flukes recovered, the plasma levels of these enzymes and haemoglobin and plasma albumin values. At 70 mg/kg, diamphenethide was shown to be able to control F hepatica populations of up to six weeks of age. The systematic use of diamphenethide at this dose level at intervals of up to six weeks during the period of metacercarial challenge should prevent ovine fascioliasis.

Early migration of immature Fasciola hepatica and associated liver pathology in cattle

Immature Fasciola hepatica were successfully recovered from the body cavity of cattle from two to 21 days after oral infection. Flukes had penetrated the liver in large numbers by day 7 but there did not appear to be a corresponding reduction in the numbers recovered from the body cavity. Liver pathology showed a typical progressive tissue disruption with a neutrophil and lymphocyte infiltration by day 7 and a marked eosinophil response from day 14. The mesothelium of the liver capsule underwent metaplasia, becoming ultimately highly active and columnar.

Treatment of immature and mature Fasciola hepatica infections in sheep with triclabendazole

A new benzimidazole anthelmintic, triclabendazole (CGA-89317) was found to be highly efficient against mature and early immature Fasciola hepatica infections in sheep. At the dose rate of 2.5 mg/kg the efficiency was 90 and 98 per cent against flukes aged eight and 12 weeks respectively. At 5 mg/kg the drug was 92 and 98 per cent efficient against flukes aged four and eight weeks respectively and 100 per cent against 12-week-old flukes. An efficiency of 93 and 98 per cent was achieved against one-week-old flukes and 99 to 100 per cent against flukes aged two to four weeks at 10 mg/kg. At this dose rate the drug had 100 per cent efficiency against six-week-old flukes. If the dose was increased to 15 mg/kg, 98 per cent efficiency was achieved one day after infection. Triclabendazole was equally efficient when administered orally or by intraruminal or intra-abomasal injection. The maximum tolerated dose of 200 mg/kg was established.

Fasciola hepatica: Effects of diamfenetide free amine on in vitro physiology, biochemistry, and morphology

Short-term (1–3 hr) incubations in vitro of immature and adult Fasciola hepatica with 10 −4 to 10 −5 M free amine of diamfenetide (DPT-FA) demonstrated a time/dose-dependent, irreversible paralysis that involved an increase in muscular tension and decrease in contraction amplitude. The following events occurred preceding or concomitant with the paralysis: influx of Na +, decrease in surface membrane potential, increase in wet weight, swellings on the ventral surface, and inhibition of 3-O-methyl glucose transport. These events were all consistent with a disturbance in surface membrane functions. The effects of DPT-FA were more severe in immature flukes (3–5 weeks postinfection) than adults which agrees with observed in vivo efficacy.

Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica

Immature Fasciola hepatica release a papain or cathepsin B-like proteolytic enzyme which cleaves immunoglobulins (Ig) of mouse, rat, rabbit and sheep in vitro. Mouse IgG and IgM molecules are both susceptible to cleavage as is hemoglobin. Whether single or multiple proteases are responsible for Ig cleavage is unknown. The proteolytic activity of secreted enzyme(s) is optimal at pH 3.5–4.5, but activity is also present at pH 7. Proteolysis is enhanced in the presence of 5 mM dithiothreitol or 100 mM cysteine. Based on studies with protease inhibitors, the F. hepatica enzyme activity has been identified as a thiol protease. It is destroyed by heating at 56°C for 1 h, but retains activity after storage at −20°C for 7 days. Whether inhibition of the proteolytic activity increases the susceptibility of F. hepatica immature worm to any extant immune effector mechanisms in hosts remains to be determined.