Eosinophil responses to Fasciola hepatica in rodents

Qualitative and quantitative cellular changes in the peripheral blood and bone marrow of resistant (rat) and susceptible (mouse) hosts of Fasciola hepatica have been examined. Eosinophil numbers in the peripheral blood and bone marrow of both hosts increased almost immediately following infection. Rats responded more rapidly than mice. Bone marrow colony formation in both rats and mice was greatly enhanced following F. hepatica infection. Injection of excretory/secretory (E/S) antigens of the fluke into rats and mice caused peripheral eosinophilia. Eosinophil levels in mice dropped by day 7 post-injection, but those in rats remained high. Eosinophil precursors in the bone marrow of injected animals also rose. Bone marrow colony formation in antigen-injected mice peaked sharply at day 7 but then fell rapidly. Rats injected with E/S antigens had about twice the level of bone marrow colonies as controls, 12 days post-injection. For most parameters measured, the magnitude of the responses of rats was greater than mice, which may be significant in the context of the rat's ability to acquire resistance to reinfection.     

Attempts to immunise rats and mice against infection with fasciola hepatica using antigens prepared from taenia hydatigena.

Attempts were made to immunise rats and mice against infection with F. hepatica by oral dosing with T. hydatigena eggs, or by vaccination with various T. hydatigena antigen preparations. These antigens included extracts from T. hydatigena cysticerci and cyst fluid, and antigens collected during short-term (48 h) and long-term (14 days) in vitro cultivation of larvae. Immunity was assessed by the numbers of F. hepatica recovered from the challenge infection in rats, and the mortality rates of infected mice. None of the immunisation regimes with T. hydatigena antigens induced consistent, significant immunity. This was in contrast to the high level of immunity shown by rats dosed orally with F. hepatica metacercariae four weeks prior to challenge infection.     

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.     

Glutathione S-transferases in Fasciola hepatica

Glutathione S-transferases (GST's) are widespread in the tissues of the liver fluke, Fasciola hepatica, and consist of multiple isozymes. Following purification to apparent homogeneity by affinity chromatography on glutathione agarose, fluke GST's were shown to comprise 2 components with molecular weights of about 25,000. Fluke GST's were immunogenic to rats, but when used as a vaccine conferred no protection on the animals against a challenge infection with F. hepatica metacercariae.     

A novel Fasciola hepatica saposinlike recombinant protein with immunoprophylactic potential

A member of the Fasciola hepatica saposinlike/NK-lysin protein family with lytic activity on human peripheral blood mononuclear cells and erythrocytes was recently described. The current study was designed to test the immunoprophylactic potential of this protein termed FhSAP-2 against infection with F. hepatica in rabbits. Two doses of 50 microg of recombinant FhSAP-2 (rFhSAP-2) emulsified in TiterMax were injected subcutaneously on the dorsal surface of 4 rabbits at 2-wk intervals. Four weeks after the second immunization, the rabbits were infected orally with 25 F. hepatica metacercariae. Four non-immunized-infected rabbits were used as controls. An enzyme-linked immunosorbent assay revealed high levels of antibodies to both rFhSAP-2 and F. hepatica excretory-secretory antigens by 2 wk after the first immunization, which were always significantly higher in immunized-infected rabbits than in control-infected rabbits. On the completion of the trial, vaccinated rabbits had 81.2% less flukes than controls. Moreover, F. hepatica egg counts in feces, as well as in bile collected from the gall bladders from vaccinated animals, were lower, 83.8 and 73%, respectively, compared with controls. The vaccinated rabbits also had significantly lower amounts of parasite antigen in stool and bile samples than controls. Last, evaluation of macroscopic liver lesions revealed that the rabbits vaccinated with rFhSAP-2 had milder lesions than the infected-control rabbits. These findings support the hypothesis that this novel rFhSAP-2 protein has immunoprophylactic potential against fascioliasis in rabbits including antifecundity and antipathology effects. This is the first report on experimental vaccination of rabbits against F. hepatica with a purified, defined, recombinant protein related to a member of the saposinlike protein family.     

The stimulation of resistance in sheep to Fasciola hepatica by infection with Cysticercus tenuicollis

Infection of sheep with Cysticerus tenuicollis for 12 weeks generated a high level of protection (> 95%) against intra-ruminal challenge with metacercariae of Fasciola hepatica as measured by recovery of flukes from liver and bile ducts and counts of fluke eggs in faeces. The animals were resistant to Fasciola whether challenge was superimposed upon the cestode infection or after removal of the cestode with mebendazole.Previous infection with C. tenuicollis also protected against the pathogenic effects of challenge infection with F. hepatica. Liver fibrosis was much less extensive in resistant sheep than controls and PCV's were not affected although these were reduced during fluke infection in the control animals.     

Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products.     

Age-dependent infectivity of orally transferred juvenile Fasciola hepatica.

Juvenile Fasciola hepatica is infective when administered orally. To determine whether the age of juveniles is a factor in infectivity by oral transfer, experimental mice were challenged orally with immature F. hepatica that had been grown in donor mice for 12, 14, 16, and 18 days. Experimental mice were examined for infections 12 16 days after the oral transfers. The infection success in experimental mice decreased with the age of juveniles. The worm recovery also decreased according to the age of juveniles. None of the juveniles was infective when grown for longer than 11 days. Once infected, orally transferred worms continued to grow. Juvenile age was a significant factor in determining the infectivity of orally transferred juvenile F. hepatica.     

Effect of BCG on the resistance of rats to infection with Fasciola hepatica

The effects of three different doses of BCG, given at various periods before infection, on the subsequent establishment of Fasciola hepatica metacercariae were studied. Although evidence was found to suggest that rats which received BCG had mounted a cell mediated immune response, there were no significant differences in worm recovery between BCG-treated rats and controls. The significance of these results in the light of current knowledge on immunity to F. hepatica in rats is discussed.     

Fasciola hepatica: an antigen fraction derived from newly excysted juveniles, containing an immunoreactive 32-kDa protein, induces strong protective immunity in rats

Crude antigens of adult Fasciola hepatica and of newly excysted juveniles (NEJ) and a low-molecular-weight fraction of antigen from NEJs were tested for inducing protective immunity in rats. Two routes of vaccination were applied. The results showed that intraperitoneal vaccination induced significantly better protection (P <0.05) than intramuscular vaccination. Intraperitoneal vaccination with antigens from NEJs induced more effective protection: after challenge infection, rats that were so vaccinated had 92.6% (+/-2.5% SEM) fewer parasites in their liver and 57.3% (+/-13.3% SEM) fewer parasites penetrating the gut wall than control rats. Rats that were vaccinated with a low-molecular-weight fraction of antigen from NEJs were also highly protected against a challenge. F. hepatica antigens that are immunoreactive were identified on immunoblots, using sera collected from highly protected rats that had been vaccinated with NEJ antigens and also sera from cattle and rats that were experimentally infected with F. hepatica. The low-molecular-weight fraction of antigen from NEJs contained an immunodominant 32-kDa protein that was recognized by serum antibodies of vaccinated rats and immune cattle. This 32-kDa protein was not detected in partially purified antigens from adult flukes. We conclude that antigens of NEJs of F. hepatica, when injected intraperitoneally in rats, are highly protective. In particular, the 32-kDa protein contained in these antigens may be highly valuable for the development of an effective vaccine against F. hepatica.     

Initial feasibility studies of the fast-ELISA for the immunodiagnosis of fascioliasis

The Falcon assay screening test enzyme-linked immunosorbent assay was adapted for the detection of antibodies to Fasciola hepatica excretion-secretion (FhES) antigens in various animal models. Pooled serum from 5 5-wk-old sheep infected with 400 F. hepatica metacercariae had high absorbance levels by 2 wk of infection and rose again at 8-10 wk. Pooled serum from 5 6-wk-old Holstein calves infected with 700 F. hepatica metacercariae had an increase in absorbance levels by 2 wk of infection, rising through 6 wk of infection. Rabbits with a primary F. hepatica infection (6-7 worms) developed antibodies to FhES by 3 wk of infection, peaking by 5 wk and remaining at high levels through the 16 wk tested. Mice with a primary F. hepatica infection developed antibodies to FhES rapidly, rising by 1 wk of infection and peaking 1-3 wk later. The sera from mice with a primary Schistosoma mansoni infection were also examined for the production of antibodies to both S. mansoni worm antigens (SmWWE) and to FhES. Antibodies to SmWWE rose by 5 wk of infection, peaking 1-3 wk later; the antibody levels to FhES rose at 6 wk with the absorbance values peaking 1 wk later and were always lower than those to SmWWE. This suggests that the anti-FhES antibodies in murine schistosomiasis mansoni may be due to cross-reactive antibodies to S. mansoni egg antigens.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Fasciola hepatica: Comparative studies on fascioliasis in rats and mice

Chapman C. B. and Mitchell G. F. 1982. Fasciola hepatica: comparative studies on fascioliasis in rats and mice. International Journal for Parasitology12: 81–91. Certain characteristics of infection differ between rats and mice exposed to metacercariae of the trematode parasite, Fasciola hepatica. Rats develop a degree of age-related resistance (and infected older females contain fewer parasites than older males), resistance to reinfection in infected rats is demonstrated readily though is partial, and a comparable degree of resistance can be obtained in recipients of infected rat serum provided the serum is given at about the time of challenge. None of these features of F. hepatica infection is seen in mice. Rats also differ from mice in that they can be vaccinated against infection (although again, resistance is incomplete) using larval antigen mixtures in adjuvants. Mice do respond to infection by production of antilarval antibodies and a slight IgG₁ hypergammaglobulinaemia and larvae will sensitize mice for delayed hypersensitivity. The results of this study indicate that sera from infected rats versus infected mice will be useful in pinpointing antigens of F. hepatica larvae which are involved in expression of partial host protection.     

Eicosanoid production by adult Fasciola hepatica and plasma eicosanoid patterns during fasciolosis in sheep.

Fasciola hepatica infection in sheep is known to cause anaemia, fever and elevated levels of liver enzymes. It was hypothesised that eicosanoids play a role in these pathophysiological changes, so the pattern of plasma eicosanoids during the course of acute and chronic fasciolosis was studied in sheep infected with a single dose of 800 F. hepatica metacercariae. Blood plasma was collected weekly until week 17 p.i. from infected sheep, and from uninfected controls. Adult F. hepatica were then recovered from bile ducts and incubated for production of ES products. Eicosanoids were determined by enzyme immuno-assay in blood plasma, fluke homogenates and ES products after chromatographic purification of the samples. Fever and anaemia were seen from 3 to 12 weeks p.i. and from 8 to 17 weeks p.i., respectively. Onset of fever was accompanied by elevated liver enzyme activities (aspartate amino transferase and gamma glutamyl transferase) in the plasma. In general, the plasma levels of prostaglandin E2 (PGE2), prostaglandin I2 (PGI2) and leukotriene B4 (LTB4) were reduced during the acute and chronic stages of the infection, whereas thromboxane B2 (TXB2) was reduced only at 8 weeks p.i. The TXB2/PGI2 ratio was increased in favour of TXB2 at 3 and 11 weeks p.i. Additionally, TXB2, PGI2, PGE2 and LTB4 were detected both in ES products and in homogenates of F. hepatica. It was concluded that eicosanoid depletion in the plasma is caused by parasite-induced liver damage. The changes in eicosanoid levels are highly correlated to the clinical signs of the disease. Changes in the pattern of host plasma eicosanoids during fasciolosis, as well as parasite-derived eicosanoids, may reflect or contribute to the pathology of the disease.     

Experimentally induced Fasciola hepatica infections in black-tailed deer.

The susceptibility of black-tailed deer (Odocoileus hemionus columbianus) to the common liver fluke (F. hepatica) was studied. Two deer and one sheep comprised each of three experimental groups. Animals in each group were inoculated individually with 250, 500, or 1000 F. hepatica metacercariae. One deer and one sheep given 1000 metacercariae died with lesions consistent with black disease 7 weeks after inoculation. At necropsy 6 or 15 weeks postinoculation, the mean percentage recovery of the inoculum was 38.9% from the deer and 51.9% from the sheep. Fluke eggs recovered from the deer were viable and metacercariae cultured from the eggs were fully infective for sheep. Pathologic changes associated with F. hepatica infection were more severe in the infected deer; consequently, the deer were less resistant to the lethal effects of the parasite than sheep. Considering the experimental results and the fact that naturally acquired common liver fluke infection has been reported infrequently from black-tailed deer, it was concluded that black-tailed deer do not constitute a significant reservoir for F. hepatica in domestic livestock.     

Strongyloides ratti: formation of protection in rats by excretory/secretory products of adult worms

Formation of a marked protective immunity against the challenge infection was found in the rats immunized with excretory/secretory (ES) products of Strongyloides ratti adult worms. Immunization by intraduodenal injection of ES products reduced both the fecal egg counts and the adult worm burden by subcutaneous inoculation of infective larvae and by an intraduodenal implantation. The duration of parasitism in the immunized rats, however, was not shortened compared with that of control rats. The normal migration of subcutaneously challenged larvae was not affected by ES product immunization. Intestinal mastocytosis occurred according to the appearance of adult worms in the small intestine of the immunized rats earlier than it did in controls. This result suggests that mastocytosis is involved in the induction of protection by ES products of S. ratti adult worms.     

Cross-resistance between Schistosoma mansoni and Fasciola hepatica in sheep

Five sheep were exposed to 5,000 S. mansoni cercariae percutaneously and the stools examined for 20 wk to determine patency. The sheep were found to be partially susceptible to a primary infection and showed great individual variations in their pathophysiological responses. All of the sheep acquired a patent infection with S. mansoni and eggs were first seen in feces 9 wk postexposure with no eggs detected after 14 wk. At necropsy 20 wk postexposure only dead S. mansoni worms were found. KOH digests revealed that tissue egg counts were low, ranging from 0 to 133 in the liver, and 0 to 257 in the intestine. Primary infection of sheep with S. mansoni followed by oral infection with F. hepatica metacercariae 10 wk later resulted in a reduction of 51% in F. hepatica worms recovered over controls infected with F. hepatica for 10 wk. All 5 of the S. mansoni-infected/F. hepatica-challenged sheep developed 71 or less F. hepatica worms. In contrast, 3 of the 5 F. hepatica-infected sheep developed 113-197 worms. However, although the experimental mean worm burden was lower than the control group, the variability in the control group was too great to obtain significance between the groups. There was a clear tendency toward normocytic normochromic anemia following a primary infection with S. mansoni; however, blood values were more reduced in the F. hepatica challenge controls than in the animals that received primary infection with S. mansoni.     

Fasciola hepatica in rats: transfer of immunity by serum and cells from infected to F. hepatica naive animals

Immune, hyperimmune, and nonimmune serum samples were collected from inbred rats following 10 to 15 weeks of one [5 metacercariae (mc)/rat], two (5 mc followed by 30 mc/rat) or no (uninfected) exposure to Fasciola hepatica. Lymphoid cells also were collected from these donors. Inbred, naive rats in groups receiving immune serum, hyperimmune serum, nonimmune serum (serum control), immune cells, hyperimmune cells, and nonimmune cells (cell control) received intraperitoneally either a total of 20 ml of serum or a total of 3 x 10(8) viable lymphoid cells. A challenge infection of 30 mc/rat was administered orally at about the time of serum or cell transfer. The transfer of immunity was evaluated by examining recipient rats for parasites 4 and 8 weeks after challenge. Some hematological parameters and the precipitating antibody response of the recipients were monitored also. Hyperimmune serum, unlike immune serum, consistently provided a significant degree of protection in recipient rats. The precipitating antibody titre of this serum was higher than that obtained from the immune donor group. The importance of a second sensitization to obtain sufficiently potent serum was demonstrated. Lymphoid cells from infected donors did not consistently confer protection on recipients. Thus, the expression of protective immunity against F. hepatica seemed to be more dependent on the presence of antibodies than on cells. The hematological parameters of the recipients, in general, supported this observation. The precipitating-antibody response of protected rats was lower than that of unprotected animals following challenge, presumably because the development of fewer worms in the former provided less antigenic stimulation.     

Experimental fascioliasis in the rat-like hamster,Tscherskia triton,and other rodent hosts

Experimental infection with Fasciola hepatica and parthenogenetic Fasciola sp. in laboratory animals have been conducted in rats and rabbits. Inoculation of less than 5 metacercariae into rat-like hamsters, Tscherskia triton, is sufficient to establish Fasciola infections. The prepatent period of F. hepatica and the parthenogenetic Fasciola sp. in T. triton was shorter than that in rats and rabbits, suggesting that T. triton is a suitable experimental model for these flukes. In contrast, F. gigantica infection in T. triton did not yield adult flukes; T. triton, is therefore, considered to be an unsuitable host for F. gigantica. The cotton rat, Sigmodon hispidus, was an unsuitable host for the parthenogenetic Fasciola sp.     

Aspects of the maintenance of the life cycle of Fasciola hepatica in Lymnaea columella in Minas Gerais,Brazil

Fascioliasis is a parasitic disease of domestic ruminants that occurs worldwide. The lymnaeid intermediate hosts of Fasciola hepatica include Lymnaea columella, which is widely distributed in Brazil. A colony of L. columella from Belo Horizonte, MG, was reared in our laboratory to be used in studies of the F. hepatica life cycle, the intermediate host-parasite relationship and development of an anti-helminthic vaccine. In the first experiment 1,180 snails were exposed to miracidia of F. hepatica eggs removed from the biliary tracts of cattle from the State of Rio Grande do Sul. In the second and third experiments the snails were exposed to miracidia that had emerged from F. hepatica eggs from Uruguay, maintained in rabbits. The rates of infection in the first, second and third experiments were 0, 42.1 and 0% respectively. Over 15,806 metacercariae were obtained and stored at 4 degrees C. Four rabbits weighing 1.5 kg each were infected with 32-44 metacercariae and two with 200. Three rabbits begin to eliminate eggs of the parasite in the feces from 84 days after infection onwards. The biological cycle of F. hepatica in L. columella and the rabbit was completed within 124 days.