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A Taenia crassiceps cDNA sequence encoding a putative immunodiagnostic antigen for bovine cysticercosis.

A cDNA expression library was constructed in lambda gt11 using poly A mRNA from the metacestode stage of Taenia crassiceps. The library was screened with rabbit antiserum to a previously defined protein fraction from Taenia hydatigena immunodiagnostic for bovine cysticercosis and with sera from cattle with experimentally induced cysticercosis. One clone (lambda TcA2) containing a 279-bp cDNA insert, reacted strongly with both antisera. A second clone (lambda TcA5.5) revealed the full-length cDNA sequence to be 361 bp. Data from Southern blots and enzymatically amplified genomic DNA segments were consistent with multiple copies or a gene family within the genome. The lambda TcA2 cDNA insert was subcloned into the plasmid pPR987 which generated a 47-kDa maltose-binding fusion protein (TcA2-MBP). Affinity-purified TcA2-MBP antigen reacted positively by ELISA with sera from cattle with experimentally induced T. saginata infections but not with sera from cattle with Fasciola hepatica or common gastrointestinal parasite infections. Rabbit polyclonal, monospecific antisera to TcA2-MBP recognized a 10-kDa protein in the cyst fluid, body wall and excretory/secretory products of the metacestode stage of T. crassiceps and immonolocalized this protein to organelles within the matrix of the cyst wall.

Animals↗

Precipitating antibodies against excretory/secretory antigens of Fasciola hepatica in sheep serum.

Gel diffusion techniques were used to study antigen-antibody reactions in precipitates forming around juvenile, semi-mature and adult Fasciola hepatica cultured in serum from infected sheep. The number of reactions was analysed in both primary and challenge infections. The number of antigens shared by the various developmental stages of the fluke was also examined. At least 2 antigens were involved in precipitate formation around juvenile flukes. These antigens were also produced by the later stages of development. Two additional antigens were produced by semi-mature flukes and these, together with two others, produced by adult flukes. Challenge infections had little effect on the number of antigens or antibodies in the precipitate.

Animals↗

Fasciola hepatica: attempts to immunise rats and mice with metabolic and somatic antigens derived from juvenile flukes.

Attempts were made to vaccinate rats and mice against Fasciola hepatica using either somatic or metabolic antigens derived from juvenile flukes between 10 and 16 days old. None of the regimes tried induced a good resistance to subsequent infection, though metabolic antigens derived from 13-14-day-old flukes when injected subcutaneously into rats with adjuvant did produce some protection to challenge.

Animals↗

Antibodies to Fasciola hepatica antigens during experimental infections in cattle measured by ELISA.

Chromatography of a soluble PBS extract of adult Fasciola hepatica on Sephadex G200 produced 7 fractions, F1-F7. The antigenicity of these fractions varied with F1 being the most antigenic. Antibody responses to these fractions showed different time courses. Antibodies to F1, F2 and F3 appeared early on in infection and, for the most part, persisted throughout infection, whereas antibodies to F4, and F5 increased gradually during infection. A standardised assay was developed to measure anti-F1 antibodies. The assay was reproducible and able to detect antibodies by 3 weeks post-infection in experimentally-infected calves.

Animals↗

Productivity in sheep treated with diamphenethide at different times after infection with Fasciola hepatica.

Sheep with potentially fatal Fasciola hepatica burdens were treated with diamphenethide 4, 8 or 12 weeks after infection. Specific effects of fluke on growth rate and wool growth over and above any anorectic effects were measured by including a pair fed group to each infected group. After drenching, feed intakes returned to that of the uninfected control group. With the exception of sheep drenched in Week 4 after infection, all sheep had significantly lower production after drenching than the control sheep. No compensatory growth was observed and productivity remained depressed for the duration of the trial (26 weeks).

Acetanilides↗

A review of the antigens of Fasciola hepatica.

The interaction between the antigens of Fasciola hepatica and the host immune response are reviewed. This paper evaluates not only more recent work, but the older literature as well. Antigens from each stage in the life cycle are considered with the idea of identifying those antigens with a potential for use in an effective vaccine. Antigens which cross-react with other parasite species are detailed as well as those that cross-react between different stages in the life cycle of F. hepatica. The objective of the review is to demonstrate for other investigators that vaccination against F. hepatica is a distinct possibility. We hope to encourage more investigators to initiate work on this aspect of an economically-important cosmopolitan parasite.

Animals↗

Effect of inoculum on the size and location of Fasciola hepatica subsequently recovered from the livers of rats.

Two outbred strains of Sprague-Dawley rats were given, by intraperitoneal injection, 5, 10, 20, 30 or 50 newly excysted juvenile (NEJ) Fasciola hepatica. Ninety days post-infection all rats were killed and their livers teased apart under 10 X magnification for quantitation of the flukes present. There was no significant difference in worm numbers between the rat strains. However, several significant differences (P less than 0.05) between the various infection groups were observed. As the size of the infective dose was increased from 5 to 50 NEJ, the percentage of the infective dose recovered from the livers of the infected rats decreased from 36.7 to 12.1%. With the 5 and 10 NEJ infective doses, the worms recovered were large mature flukes (1.52 and 1.68 cm, respectively) and were found in the common bile ducts. In the rats receiving infections of 30 and 50 NEJ, the flukes were smaller (0.88 and 0.55 cm, respectively), immature, and were primarily located in the liver parenchyma. These findings are important in light of previous studies on the development of resistance in the rat to challenge infections in which immunity was based on both the size and the location of the flukes recovered. The results from our study indicate that in a primary infection of 20 or greater NEJ, many small immature flukes remain in the liver parenchyma, even after several months. When testing for resistance to F. hepatica in rats, these flukes may erroneously be thought to comprise a portion of the challenge infection.

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Suppression of Babesia microti infection in mice concurrently infected with Fasciola hepatica.

Blood cell parasitaemia of Babesia microti and the associated haematological changes were examined in mice harbouring patent Fasciola hepatica infections and in fluke-free control mice. B. microti parasitaemia was markedly suppressed in mice harbouring primary 7-week F. hepatica infections, as reflected in a reduction in the percentage of erythrocytes parasitised and in the incidence of multiple B. microti infections in the red cells. This suppression was accompanied by an annulment of B. microti induced reductions in haemoglobin and haematocrit levels in F. hepatica infected mice. In naive recipient mice, inoculated with blood from mice concurrently infected with F. hepatica and B. microti, the course of B. microti infection was characterised by a prolonged pre-parasitaemia period, a reduced peak parasitaemia and a delayed fall in haematocrit levels as compared to those inoculated with blood from mice infected with B. microti only. This feature may presumably be dose-related. The present study does not reveal the actual mechanism(s) involved in the suppression of the blood protozoan by F. hepatica. However, since B. microti has a preference for mature erythrocytes, the suppression may be a result of the altered erythrocyte kinetic state induced by the removal of erythrocytes by the blood-sucking fluke resulting in high levels of reticulocytes.

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Worm recovery, haemagglutinating antibodies and IgE-levels after immunisation against Fasciola hepatica in rats.

Female inbred Hooded Lister (HL) rats were each infected with 20 metacercariae (Mc) of Fasciola hepatica. Remarkable variations between the number of flukes established in the bile ducts suggest the presence of individual, perhaps genetically controlled, differences in immune responsiveness of HL rats to F. hepatica. Serum (4 ml) from HL rats infected with 20 Mc 6 weeks prior to transfer partially protected rats against a F. hepatica challenge infection. However, 1 X 10(6) lymphoid cells originating from rats of the same age and stage of infection did not show the same protective qualities. Furthermore, attempts to immunise HL rats i.p. with either juvenile or adult excretory/secretory (ES) products, or somatic tissue antigens and AlOH3-gel as adjuvant failed. When compared to other investigations, the present results further suggest that both the adjuvant and the route of administration are crucial for the stimulation of a protective immunity to F. hepatica. Low titers and low anamnestic responses of haemagglutinating antibodies after prior immunisation with juvenile ES antigens or both juvenile ES and somatic tissue antigen suggest the occurrence of an immunosuppressive effect caused by juvenile ES products. The total serum IgE-levels in immunised groups were generally lower when compared to the challenge control group, whereas the F. hepatica ES-specific IgE-levels rose after challenge, but immediately decreased again when compared to challenge controls. These findings support the hypothesis of an immunomodulatory effect caused by the vaccination scheme.

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The effect of pre-exposure to Fasciola hepatica or Schistosoma mansoni on challenge infection with Fasciola hepatica.

Two groups of 12 and 6 rats were inoculated with Fasciola hepatica and Schistosoma mansoni, respectively. The Schistosoma-inoculated group, as well as 6 Fasciola-inoculated rats and 6 uninfected rats were challenged 8 weeks later with F. hepatica. A control group of 6 rats was left unexposed. Eight weeks after the challenge exposure all rats were necropsied and subjected to post-mortem examination. The number of Fasciola recovered after challenge was lower in both groups of rats primarily infected with F. hepatica or S. mansoni. F. hepatica-induced pathological changes were observed in all infected rats, but were pronounced in the group which was first exposed at the time of challenge of the primarily infected groups. No Schistosoma eggs or adults were detected in Schistosoma-inoculated rats. The results demonstrated that rats primarily infected with F. hepatica acquired resistance against a challenge exposure to the homologous parasite. Also S. mansoni, even without patency, can provide partial protection against F. hepatica infection.

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Interaction of bovine complement with Fasciola hepatica.

The host-parasite relationship of Fasciola hepatica in cattle was characterized by determining the effects of the parasite on the bovine complement system. Phosphosaline extracts of F. hepatica adults inhibited both the classical and alternative pathways of complement activation in normal bovine and human sera in a protein dose-dependent manner. The in vitro reaction of viable newly excysted juveniles (NEJ) with bovine serum (NBS) and with bovine serum containing antibodies specific to F. hepatica (IBS) resulted in no detectable changes in serum hemolytic complement activity for either pathway. This lack of complement consumption occurred even though these same flukes incubated in IBS for at least 24 h developed a precipitate that adhered to the parasite tegument, a feature that may reflect antibody--antigen reactions.

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The susceptibility of the goat to Fasciola hepatica infections.

Mixed breed goats were infected with metacercariae of Fasciola hepatica and the resulting worm burdens were quantitated after primary and secondary exposure of the goats to the parasite. Mean length and width of the parasite recovered after all primary exposures were 1.91 +/- 0.2 cm and 0.91 +/- 0.2 cm, respectively. A mean of 71.8 +/- 5.9% of the flukes were recovered from all of the primary infections. In the secondary infections, the mean length and width of the flukes from the physically smaller population was 0.88 +/- 0.27 cm and 0.53 +/- 0.19 cm, respectively. A mean of 67 +/- 6.7% of the flukes were recovered from this secondary infection. It appears that the goat is susceptible to challenge infections with F. hepatica and that its response to this infection is much like that of sheep.

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Early migration of radiolabeled larvae of Fasciola hepatica in the mouse.

Metacercariae of Fasciola hepatica were labeled with 75Se by exposure of infected Lymnaea columella to 75Se-M, 20 muCi/snail, in 5 ml of water. Seventy-two mice were inoculated with 50 75Se-labeled metacercariae. At intervals after infection, the distribution of metacercariae in gut, body cavities and liver was studied by compressed organ autoradiography, and a variant of this technique adapted to gut content and body cavity washes. Ag degree focus counts from autoradiograms expressed as percentage of actual exposure level were subjected to probit analysis. Estimates of the average departure time of radio-labeled maritas from gastrointestinal tracts and average arrival time into the abdominal cavities were 12.2 and 12.5 h, respectively. The peak accumulation of maritas in abdominal cavities occurred at 24 h post-inoculation. A 7.7-h discrepancy between estimated average departure time from abdominal cavities (92 h) and average arrival time at livers (99.7 h) was attributed to experimental error.

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Peripheral androgen levels in peripuberal rams infected with Fasciola hepatica.

Three-month-old, Polled Dorset rams infected with Fasciola hepatica and uninfected control rams were monitored throughout the peripuberal phase for peripheral concentrations of testosterone and for body weight. No differences between groups occurred in these measurements from early spring to mid-autumn. However, the infected postpuberal rams demonstrated a reduced ability to remove exogenous testosterone from the circulation. Liver damage induced by moderate infections of flukes apparently impairs hepatic catabolism of this steroid but compensatory mechanisms appear to maintain circulating levels comparable to those of uninfected rams.

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Separation of parasite antigens by molecular exclusion, anion exchange, and chromatofocusing utilizing FPLC protein fractionation systems.

Excretory-secretory products (ESP) were harvested from balanced salt solutions in which adult Fasciola hepatica had been incubated for 4-6 h at 37 degrees C. The ESP was fractionated by standard low pressure molecular exclusion chromatography and FPLC (fast protein liquid chromatography) using the principles of molecular exclusion, anion exchange, and chromatofocusing. The dot-enzyme-linked immunosorbent assay (Dot-ELISA) was used to demonstrate the immunoreactivity of eluted fractions. Compared to Sephacryl S-200, separation by Superose-6 (FPLC) was faster and resolved more peaks (four with Sephacryl S-200 and nine with Superose-6). Peaks from Sephacryl S-200 were resolved by the first anion exchange (Mono Q) separation into seven peaks; when these peaks were subjected to a second anion exchange, 15 peaks were resolved. Thirty-eight peaks were resolved by chromatofocusing (Mono P) in the pH range 7-4. Immunoreactive fractions from narrow-range (single pH unit) chromatofocusing were identified by the Dot-ELISA. The FPLC system proved to be a means of rapid and high resolution separation of F. hepatica antigens.

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Heterologous resistance to Fasciola hepatica conferred upon rats by passive transfer of serum from different breeds of sheep.

Two experiments compared the protection against oral challenge with 20 metacercariae of Fasciola hepatica conferred on rats by intraperitoneal injection of serum from three breeds of sheep infected with F. hepatica (Barbados Blackbelly, St. Croix, Florida Native). Experiment 1 used serum from sheep 5-6 months of age following two infections of 250 metacercariae each, while Experiment 2 utilized serum collected from the same sheep at 10-11 months of age following either a primary (first exposure) or challenge (after two previous exposures of 250 metacercariae each) infection with 500 metacercariae. Similar numbers of flukes were recovered from rats given either immune or nonimmune (control) serum from each breed of sheep in Experiment 1. In Experiment 2, rats given serum from infected St. Croix sheep had significantly fewer flukes than rats given either control or immune serum from Barbados Blackbelly or Florida Native sheep. There was no significant correlation of fluke counts between individual serum donors (sheep) and serum recipients (rats).

Analysis of Variance↗

Seasonal variations in egg passage of Fasciola hepatica in dairy cows in Quebec.

Seasonal changes in Fasciola hepatica egg passage were documented in 17 dairy herds in Portneuf County, Quebec (Canada). Egg passage was low or absent in spring and summer but rose in autumn to reach a peak in winter, then subsequently returned to low levels. This study suggests that there are two periods of transmission to the bovine host during the grazing season: a minor period in the spring, due to overwintered snails, and a major period in August and September due to snails that acquired the infection in May or June.

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Epidemiology of Fasciola hepatica infection in the Paraíba River Valley, São Paulo, Brasil.

The study revealed that although Lymnaea columella and Stenophysa marmorata were found in the same habitat, only L. columella harbored intramolluscan stages of F. hepatica. The population density of L. columella cyclically decreased between September and February, and increased from March to September. The drought that occurred between June and November 1981 dramatically reduced the number of snails collected. Larger snails were collected in March-July and November-December, while the smaller snails were collected in January-February and August-October, suggesting that there may be two generations of L. columella per year. The highest prevalence levels of infection in L. columella were: first year, June 8.82%, September 9.09% and October 10.52%; second year, March 6.25% and April 6.89%; third year, July 7.69%, August 10.25%, April 17.91% and May 13.91%. Results obtained with tracer sheep showed that the largest numbers of metacercariae were on pasture between June and October, and between March and April, but the infection was present during most of the year.

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