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PubMed1995

Immunodiagnosis of fasciolosis in ruminants using a 28-kDa cysteine protease of Fasciola gigantica adult worms.

A homogeneous extract of a 28-kDa cysteine protease of Fasciola gigantica adult worms was used as the antigen for immunodiagnosis of fasciolosis in cattle, sheep and goats using the Falcon assay screening test enzyme-linked immunosorbent assay technique. This antiprotease assay technique was found to be very rapid and sensitive and may be useful as a supplementary method for the diagnosis of fasciolosis in ruminants.

Animals↗
PubMed1995

Distribution of fasciolosis in Kansas, with results of experimental snail susceptibility studies.

A total of 278 veterinarians throughout Kansas were sent mail-in survey forms asking specific questions relating to their experience with fasciolosis in their practice area. Replies were received from 178 (64%) veterinarians representing six practice types; one-third reported having seen cases of fasciolosis in their practice. The results of our survey indicate that the majority of the cattle diagnosed with liver fluke disease in Kansas are imported from other areas of the USA. However, in both central and southeastern regions of Kansas, some cattle that had never been out of the state were infected with Fasciola hepatica. Thus, these areas of Kansas should be considered endemic for liver fluke disease. Methods of diagnosis, types of operations, and improvements seen after treatment were also discussed. In order to ascertain the existence of one or more possible snail intermediate hosts within Kansas, five species of lymnaeid snails were collected from central and southeastern parts of the state and tested for their susceptibility to infection by Fasciola hepatica. The snails collected included Pseudosuccinea columella, Fossaria obrussa, Fossaria bulimoides, Fossaria parva and Fossaria dalli. Of these, Pseudosuccinea columella and Fossaria bulimoides proved susceptible to experimental infection by Fasciola hepatica. Metacercariae obtained from experimentally infected snails were used to infect both a weanling calf thereby completing the life cycle of the parasite. This report is the first to identify the existence of suitable snail intermediate hosts for Fasciola hepatica in Kansas.

Animals↗
PubMed1995

Development and application of a dot-ELISA test for the detection of serum antibodies to Fasciola hepatica antigens in llamas.

A microenzyme-linked immunosorbent assay (dot-ELISA) was developed to detect serum antibodies against Fasciola hepatica antigens in llamas. Sera from five F. hepatica-infected and 11 non-infected llamas were used in initial test development. Nitrocellulose filter disks containing F. hepatica excretory-secretory product were placed in 96-well microtiter plates, washed, blocked with Tween-20, then incubated with four-fold serial dilutions of llama sera. After incubation with rabbit anti-llama IgG followed by peroxidase-conjugated goat anti-rabbit IgG, addition of precipitable substrate resulted in purple dots on white background (positives) easily read by eye. The technique was further evaluated at titers of 1:512 using an additional six known positive and eight known negative llamas. Test results showed 6/6 known positive as positive and 8/8 known negative as negative. Sera were collected, at approximately weekly intervals, from three llamas experimentally infected with F. hepatica. The dot-ELISA detected antibodies to F. hepatica as early as the second week post-infection in all llamas. In a serologic survey of 256 llamas from an F. hepatica endemic area, the dot-ELISA detected antigen-specific serum antibodies to F. hepatica in 42 (16%) of the llamas. Although no difference was noted in antibody prevalence between sexes, prevalence increased in llamas over 6 months of age.

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PubMed1995

Detection of circulating antigen in sera of Fasciola gigantica-infected cattle with antibodies reactive with a Fasciola-specific 88-kDa antigen.

Antibodies against a specific 88-kDa antigen of Fasciola gigantica were used for the detection of circulating antigen in the sera of cattle with experimental and natural infections of F. gigantica by a double antibody ELISA. Circulating antigen was detectable as early as the second and third weeks after infection and positive absorbance values were obtained for the entire duration of infection. Absorbance values decreased below the cutoff point 3 weeks after chemotherapy with oxyclozanide. This immunoassay also greatly enhanced the specificity of immunodiagnosis of fasciolosis in naturally infected cattle. The test system has excellent potential for the accurate diagnosis of ruminant fasciolosis.

Animals↗
PubMed1995

Time-course analysis of antibody response by EITB and ELISA before and after chemotherapy in sheep infected with Fasciola gigantica.

The time-course analysis of the antibody response to Fascicola gigantica infection in sheep was studied by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunoelectrotransfer blot (EITB). Sera from sheep experimentally infected with F. gigantica were reacted with excretory-secretory antigens of the worm before and after chemotherapy with oxyclozanide. In ELISA, there was a significant increase in anti-Fasciola antibody by 2 weeks after infection and there was a sharp decrease in antibody titer by 4 weeks after treatment. By EITB, the infected sheep sera recognised four polypeptides in the range of 43-75 kDa as early as 2 weeks after infection, with more polypeptides being recognised as the infection progressed. Recognition of an 87 kDa antigen was lost by 2 weeks after treatment and is therefore a good marker for treatment efficacy. Comparative immunoblotting with sheep anti-Paramphistomum, anti-Dicrocoelium and anti-Fasciola sera revealed that the 17 kDa, 21 kDa, 57 kDa and 69 kDa proteins are specific to fasciolosis and are good antigens for early and specific immunodiagnosis of F. gigantica infection in sheep.

Animals↗
PubMed1995

Enzyme-linked immunoelectrotransfer blot analysis of excretory-secretory proteins of Fascioloides magna and Fasciola hepatica.

Fasciola hepatica is a parasite of cattle (Bos taurus), but not of white-tailed deer (Odocoileus virginianus), while Fascioloides magna is a parasite of white-tailed deer which also infects cattle as dead-end host. Adult parasites were collected from naturally infected white-tailed deer or cattle. Excretory-secretory proteins (ESP) were obtained from each parasite. Protein banding patterns were analysed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and probed using sera from experimentally infected deer of cattle using enzyme-linked immunoelectrotransfer blot (EITB, also known as Western blot) analysis. Protein banding patterns of the two species were different. EITB analysis of Fascioloides magna ESP using sera from Fascioloides magna infected deer or cattle identified three bands of approximately 17, 22 and 27 kDa of which the 27 kDa antigen cross-reacted with sera from Fasciola hepatica infected cattle. EITB analysis of Fasciola hepatica ESP probed with sera from Fasciola hepatica infected cattle identified three bands of approximately 15, 26 and 46 kDa. The 46 and 26 kDa ESP cross-reacted with sera from Fascioloides magna infected cattle, but not with sera from Fascioloides magna infected deer. The band at 15 kDa which reacted specifically for Fasciola hepatica infected cattle sera consisted of two protein bands close to each other as seen on the SDS-PAGE gel. The EITB reaction at approximately 17 kDa and 22 kDa of Fascioloides magna ESP, and at approximately 15 kDa of Fasciola hepatica ESP can be used for species specific diagnosis.

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PubMed1994

Early immunodiagnosis of caprine fasciolosis using the specific f2 antigen in a passive hemagglutination test.

An improved hemagglutination (HA) test using the purified specific f2 antigen of Fasciola hepatica has been evaluated with respect to its potential use in the diagnosis of caprine fasciolosis. Following experimental infection of 1-year-old goats with a single heavy infection of 300 metacercariae, f2-specific antibodies were detected 2-3 weeks after infection and increased steadily to reach a maximum titer 9 weeks after infection, after which the antibody level declined. In animals receiving multiple infections of a lower dose of 50 metacercariae given at weekly intervals for 6 weeks, f2-specific antibodies were detected 3 weeks after infection and increased to reach a plateau 11 weeks after infection which was maintained until the end of the experiment (15 weeks after infection). Depending on animals and groups, eggs appeared in the feces between 7 and 9 weeks after infection. The HA test may provide valuable information about the early detection of caprine fasciolosis, particularly during the prepatent period.

Animals↗
PubMed1994

Appetite depression in sheep experimentally infected with Fasciola hepatica L.

The purpose of this study was to determine the effects of experimental fasciolosis at various stages of development on the daily food intake of sheep. Five male Churra sheep, 4 months of age, were infected orally with 300 Fasciola hepatica metacercariae over a 30 day period. There was a significant increase in serum glutamate dehydrogenase (GLDH) activity from 40 days post-infection and in aspartate aminotransferase (AST) activity from 60 days post-infection. Both enzyme activities reached maximum levels in the serum of infected animals at 80 days and then progressively decreased. Serum gamma-glutamyltransferase (GGT) activity was significantly increased from 80 to 120 days post-infection. Glycaemia was significantly decreased from 60 days post-infection. The average daily food intake was shown to steadily decrease until approximately 100 days. The coincidence of decreased food intake with the period of significant increase, both in AST and GLDH activities, indicated that damage caused around the time of migration of immature flukes through the liver parenchyma may be involved in appetite depression.

Animals↗
PubMed1994

The overwintering of eggs, intramolluscal stages and metacercariae of Fasciola hepatica under the temperatures of a Mediterranean area (Madrid, Spain).

The survival of embryonated and unembryonated eggs, of snails with mature or immature infection and of metacercariae over the winter of a Mediterranean area was analyzed. Embryonated eggs were more resistant than unembryonated eggs to cold weather, leaving a residual contamination which was responsible for earlier spring infection of the snail. Overwintering was similar in snail populations with both mature and immature infections although the lifespan was shorter in the former. Both snail populations survived in the mildest winter but not in the coldest. Only metacercariae from mid autumn were able to overwinter in an significant proportion (45%) but they were non-viable by mid spring. Our results suggest that in very cold winters no risk for grazing animals should be expected in spring.

Animals↗
PubMed1994

Complement depletion in rats infected with Fasciola hepatica: in vivo and in vitro studies.

The action of Fasciola hepatica on rat complement was evaluated by measuring the haemolytic activity. Extracts from metacercariae and eggs and the excretory and secretory products of adult flukes reduced the classical pathway of complement-mediated lysis by normal rat sera in a protein dose-dependent manner. Whole metacercariae produced the same effect. In vitro incubation of newly excysted juveniles and intact eggs with normal rat serum had no detectable effect on serum complement activity by either the alternative or classical pathway. During the course of rat fasciolosis a depletion in the classical pathway of complement-mediated lysis was demonstrated particularly 6-9 weeks post-infection; the alternative pathway of complement-mediated lysis was not affected.

Animals↗
PubMed1994

Rapid growth of Lymnaea viridis, the intermediate host of Fasciola hepatica, under laboratory conditions.

Reported in this communication is a unusually rapid growth of Lymnaea viridis, the intermediate host of Fasciola hepatica, maintained in the laboratory for experimental purposes. When the snails were fed on blue-green algae at about 20 degrees C, they attained a shell length of 12 mm in 37 days from the date of hatching. The snails began laying eggs from 18 to 24 days following hatching.

Animals↗
PubMed1996

Involvement of excretion-secretion products from Fasciola hepatica inducing suppression of the cellular immune responses.

Normal rats i.p. injected with Fasciola hepatica excretor-secretor antigen (ESA) induced a population of spleen mononuclear (SpM) cells, which suppressed the delayed type hypersensitivity (DTH) response to parasite antigens as well as to non-related antigens (human serum albumin) by adoptive transfer. A similar effect was observed when the cell transfer was performed with SpM cells non-adherent to nylon wool. The DTH was not modified by cells transfer adherent to nylon wool in syngeneic receptor animals. The observed suppression depended on the concentration and inoculation moment of the antigen; 1.8 mg of protein ESA being enough to suppress the DTH response at the different days studied, before and after immunization with whole F. hepatica antigens. A marked suppression was observed when ESA was injected on day 7 pre-immunization. On the other hand, inoculation of ESA treated with 0.01 M sodium periodate (carbohydrate oxidant) diminished the suppressor effect found after the native ESA inoculation, indicating participation of ESA glucidic components in induced suppression. Inoculation of ESA fractions obtained from polyacrylamide gel elution with different MW range, showed that components between 12 and 23 kDa actively induced suppression to the DTH response to parasite antigens.

Animals↗
PubMed1996

Herd evaluation by enzyme-linked immunosorbent assay for the determination of Fasciola hepatica infection in sheep and cattle from the Altiplano of Bolivia.

A study was designed to determine by ELISA the seroprevalence of fasciolosis both in sheep (29 herds totaling 184 sheep), in samples collected in 1988, and in cattle (41 herds totaling 299 animals, samples collected in 1988; 34 herds totaling 147 animals, samples collected in 1989) in the same area of Corapata in which a seroprevalence survey had been done in humans. The results show high seropositivity in sheep (89%) and lower seropositivity in cattle (58% in 1988, and 57% in 1989). The seroprevalence in cattle in 1988 was essentially identical to that detected in 1989. Faecal examinations were also done in the 1988 sheep and 1989 cattle. Results of the study showed that of the 184 sheep examined, 22 were positive for F. hepatica eggs, while 163 were positive by serology. All of the 22 sheep which were positive parasitologically were also positive serologically for a sensitivity of 100%. On the other hand, of 147 cattle tested, 38 were positive parasitologically while 84 were positive serologically. Of the 38 positives for F. hepatica eggs, 31 were positive by serology (sensitivity 82%).

Animals↗
PubMed1996

Experimental ovine fasciolosis: antipyrine clearance as indicator of liver damage.

Antipyrine clearance was used to assess microsomal oxidative function in eight female Churra breed sheep at 20, 30, 40, 60, 80 and 100 days after infection by an oral administration of 150 metacercariae of Fasciola hepatica. Experimental infection was ascertained by an ELISA test and by faecal analysis. A significant increase in plasma glutamate dehydrogenase (GLDH) activity from 20 days post-infection and in gamma-glutamyltransferase (GGT) activity from 40 days post-infection was found. Both enzyme activities reached maximum levels in plasma of infected sheep at 80 days post-infection, progressively decreasing thereafter. A significant reduction in the total plasma clearance of antipyrine occurred from 60 to 100 days post-infection and a significant increase in mean residence time occurred by 80 days post-infection. The decrease of antipyrine metabolism coincided with the entrance of parasites in bile ducts and the highest liver damage caused by migrating juvenile flukes.

Animals↗
PubMed1996

Immune responses of chronically infected adult cattle to Fasciola hepatica.

Eight adult cows, with an existing chronic Fasciola hepatica infection, were experimentally infected with 1300 metacercariae of F. hepatica, given as trickle infections, over two separate 10-day periods. Two fluke-naive heifers were similarly treated. Analysis of parasite-specific immunoglobulin isotypes IgM, IgG1, IgG2 and IgA showed IgG1 to be the dominant isotype in both chronically infected and previously naive animals. Lymphocyte proliferation assays demonstrated (a) an association between lymphocyte response and mature fluke burden in the chronically infected cattle and (b) no association between lymphocyte response and mature or immature fluke burden in naive heifers. There was no production of gamma-interferon (IFN gamma) by lymphocytes responding to adult fluke antigen. At post-mortem examination the burden of immature flukes in chronically infected and previously naive heifers was similar. This suggests that chronically infected animals may be tolerant to a moderate superinfection and that the prevailing immune mechanism in operation may be a non-protective response generated by the Th2 lymphocyte subset.

Animals↗
PubMed1996

Distribution of potential intermediate hosts for Fasciola hepatica and Fascioloides magna in Montana, USA.

A collection of lymnaeid snails in Montana was made over a 3 year period, in conjunction with a state-wide survey of the distribution of liver flukes in Montana. Collection areas were selected based on reports of infected cattle, sheep or wildlife, and with the intent of covering all geographic regions of the state. Snails were found at all 97 of the locations chosen for collections, with lymnaeids collected at 71 of the locations. The 97 sites were located in 28 of Montana's 56 counties. Nine lymnaeid species were collected, five of which have been reported either as natural or experimental intermediate hosts for Fasciola hepatica or Fascioloides magna. The two snail species most widely distributed over the areas enzootic for the flukes were Lymnaea modicella and Lymanaea caperata.

Animals↗
PubMed1996

Detection of antibodies of Fasciola hepatica excretory--secretory antigens in experimentally infected goats by enzyme immunosorbent assay.

An ELISA with excretory-secretory (ES) antigens has been evaluated as a technique for the early detection of specific antibodies in Fasciola hepatica infections in goats. Goats were experimentally infected with 100 or 200 metacercariae of bovine origin and serum samples were taken periodically over 365 days. The ELISA test was performed with ES antigens (10 micrograms mL-1), a single dilution of sera (1:800) and anti-goat IgG conjugate (1:1000). ES specific antigens were detected in all infected goats between 15 and 30 days postinfection (PI) and maximum antibody levels were reached at 90 days PI. Positive antibody levels (significantly different from those of controls) were still found at 365 days PI. No significant differences were observed between goats infected with 100 or 200 metacercariae. In all infected goats, eggs appeared in faeces between 60 and 90 days PI. ELISA with ES antigens could be a feasible method for the early diagnosis of goat fasciolosis.

Animals↗
PubMed1996

Efficacy and pharmacokinetics of triclabendazole in buffalo with induced fasciolosis.

A study was conducted to understand pharmacokinetics and flukicidal activity of intraruminal administration of triclabendazole (TCBZ) at 12.0, 24.0 and 36.0 mg kg-1 body weight in experimentally Fasciola gigantica-infected buffaloes on Week 2 and 10 post-infection. No fluke eggs in faeces and no flukes could be recovered from the liver of buffaloes following intraruminal administration of triclabendazole at 24.0 and 36.0 mg kg-1 body weight both on Weeks 2 and 10 post-infection, while the recommended therapeutic dose at 12.0 mg kg-1 body weight was 19-23% effective. Pharmacokinetic analysis of the data revealed a significantly higher (P < 0.05) concentration maximum of both the metabolites and area under concentration-time curve of TCBZ-SO2 in animals treated at 12.0 mg kg-1 body weight on Week 10 post-infection, whereas a significantly higher area under the concentration-curve and elimination half-life of both the metabolites and significantly higher concentration maximum and area under the concentration-time curve of both the metabolites were observed in animals treated on Week 10 post-infection at the dose rates of 24.0 and 36.0 mg kg-1 body weight, respectively. Bioavailability of triclabendazole was more in buffaloes with mature flukes than with immature flukes.

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