Utility of Schistosoma bovis adult worm antigens for diagnosis of human schistosomiasis by enzyme-linked immunosorbent assay and electroimmunotransfer blot techniques.

Immunodiagnostic methods based on the detection of antibodies continue to be the most effective and practical methods for the diagnosis of imported schistosomiasis. Schistosoma bovis is a species whose final natural hosts are bovines, ovines, caprines, and small wild ruminants. Different studies have demonstrated the analogies existing between S. bovis and other Schistosoma species which affect humans. The objective of this work was to evaluate the utility of S. bovis adult worm antigens (AWA) for the diagnosis of imported human schistosomiasis by enzyme-linked immunosorbent assay (ELISA) and electroimmunotransfer blotting (EITB) techniques. By detecting eggs, the ELISA for S. bovis AWA was able to definitively detect imported cases with a sensitivity of 94%. The specificity of the ELISA for S. bovis AWA was 97%. There were no differences between the results of the S. bovis AWA ELISA for patients infected with Schistosoma mansoni and those infected with Schistosoma haematobium. The EITB technique showed bands of 85, 37, and 20 kDa, which are characteristic of infections with Schistosoma spp. Specific bands to indicate infection by different species of Schistosoma have not been detected. The combined use of the ELISA for S. bovis AWA and EITB increased the global sensitivity of the study to 97%. Our findings suggest that the ELISA for S. bovis AWA is a useful test for the immunodiagnosis of imported schistosomiasis and that EITB for detecting S. bovis AWA permits the confirmation of diagnosis when the ELISA for S. bovis AWA is positive.

Vaccination of mice and sheep with Fh12 FABP from Fasciola hepatica using the new adjuvant/immunomodulator system ADAD.

We evaluate the ability of a Fasciola hepatica FABP native antigen (Fh12) with a new vaccination system called ADAD to protect mice and sheep against an experimental F. hepatica infection. The vaccination protocol consists of a set of two injections. The first injection contains a micelle in which two components are included, saponin from Quillaja saponaria (Qs) and/or Anapsos (A) a Polypodium leucotomos hydroalcoholic extract, both emulsified in a non-mineral oil (Montanide) in a water/oil emulsion (30/70). This is subcutaneously injected to achieve the "adaptation" of the immune system to subsequent stimuli. The second injection contains in addition the Fh12 antigen. Two different experiments were carried out using two mouse strains (BALB/c and CD-1). Mice vaccinated with Qs+A+Fh12 presented a survival rate of 40%, when compared with control groups. Furthermore, we evaluated the efficiency of the vaccination in sheep against an experimental F. hepatica challenge. The vaccinated sheep presented lower fluke recovery (24.5%), number of eggs in bile fluid (58.1%) and faeces (40.3%) than control groups. The recovered flukes were shorter (32.7%), immature (34.0%) and with lower body mass (31.6%) than non-complete vaccinated sheep. Thus, the new ADAD system could be a good alternative for future vaccination experiments against fasciolosis.

Immunoprophylaxis against Fasciola hepatica in rabbits using a recombinant Fh15 fatty acid-binding protein.

Previous studies of ours have demonstrated that a recombinant protein (Fh15) related to fatty acid-binding proteins did not induce significant protection in rabbits challenged 2 or 4 wk postimmunization over nonimmunized controls. In the current study, rabbits were immunized with Fh15 and challenged with Fasciola hepatica metacercariae 12 and 20 wk later. In the current study in which longer lag periods for challenge infection after the second immunization were used, worm burden reductions compared to adjuvant controls were a significant 43% and 76%, respectively. Importantly, rabbits immunized with Fh15 had significant numbers of immature flukes, 66% in the 12-wk period and 84% in the 20-wk lag period as compared to controls. In addition, liver lesions were clearly diminished in the vaccinated rabbits. Enzyme-linked immunosorbent assay absorbance values showed that immunized rabbits developed high antibody levels to Fh15 from 8 wk after the first immunization and did not increase after challenge. These results suggest that a recombinant F. hepatica molecule related to fatty acid-binding proteins induces protective (worm burden reductions), anti-fecundity (immature flukes), and anti-pathology (less liver lesions) effects in rabbits and may serve as a model for the immunoprophylaxis of fascioliasis.

Vaccination of sheep against Fasciola hepatica with homologous fatty acid binding proteins.

The current study was designed to test the immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in sheep subsequently infected with the fluke. Thirty lambs were divided into six groups according to various patterns of immunisation and times of infection and necropsy. The antigens were emulsified in Freund's adjuvant. Levels of specific anti-nFh12 and anti-rFh15 antibodies rose rapidly by 2 weeks after the first immunisation and were always significantly higher in immunised-infected sheep than in control-infected sheep. On completion of the trial there was no difference in fluke burden between groups vaccinated with either of the antigens and non-immunised controls. However, worm size and faecal egg counts were significantly diminished in the sheep vaccinated with either of the antigens, suggesting an anti-fecundity effect. This is the first report of experimental vaccination of sheep against F. hepatica with purified native and recombinant antigens related to fatty acid binding proteins.

Vaccination of mice against schistosoma bovis with a recombinant fatty acid binding protein from Fasciola hepatica.

Two strains of mice (NMRI and C57/BL) were each immunized with a 15kDa recombinant Fasciola hepatica fatty acid binding protein (FABP) (Fh15) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh15 had significant reductions in S. bovis worm burden recoveries (72% reductions over controls). When using NMRI mice, Fh15 in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice, only antibodies to the IgG2a isotype increased after the second immunization and remained high through 8 weeks of S. bovis infection. This is the first time that a heterologous recombinant molecule from F. hepatica has been used in vaccination against S. bovis, obtaining a significant reduction in the number of worms in C57/BL mice.

IgG antibody response to ES or somatic antigens of Dicrocoelium dendriticum (Trematoda) in experimentally infected sheep.

The ELISA technique was used to study the kinetics of IgG antibodies against excretory-secretory (ES) and somatic (So) antigens of Dicrocoelium dendriticum in the sera of 32 lambs: 12 experimentally infected with 1,000 metacercariae, 12 with 3,000 and 8 controls. Both antigen types were used at a 2 microg/ml concentration. Dilutions of sera and the anti-sheep IgG peroxidase conjugate were: 1:200 and 1:6,000, respectively. Optical density values for each type of antigen in the two infected groups were higher than the cut-off point from day 30 post infection (p.i.), showing positive infection. Maximum antibody levels were observed 60 days p.i. and remained high until the experiment ended 180 days p.i. This pattern was similar for both ES and So antigens, although with slightly lower figures in the latter. Antibody kinetics were very similar for each of the two doses, except on day 30 p.i. No correlation between the antibody level and parasite burden could be established.

Histopathological and immunohistochemical study of lambs experimentally infected with Fasciola hepatica and Schistosoma bovis.

The aim of this study was to investigate the cross-resistance between Fasciola hepatica and Schistosoma bovis in lambs assessing parasitologic, gross pathologic, histopathologic and immunohistochemical changes in liver and small intestine. Thirty Castellana breed lambs were divided into five comparable groups and exposed to F. hepatical S. bovis (group F/S), S. bovis/F. hepatica (group S/F), S. bovis (group S) or F. hepatica (group F) and six unexposed lambs were used as non-infected controls (group C). Primary patent infection with F. hepatica induced a lower number of schistosome eggs and a higher number of lymphocytes in intestinal and liver schistosome egg-induced granulomas in group F/S than in the groups S/F and S, liver damage being mainly attributed to F. hepatica. S. bovis infection followed by challenge with F. hepatica particularly increased the severity of the most significant liver alterations (cholangiohepatitis by F. hepatica and mesoendophlebitis by S. bovis) and F. hepatica seemed not to have an influence on established S. bovis infection. In addition, immunohistochemical results suggested that the predominant local immune response in both double-infected groups was different, being mainly a cell-mediated immune response in group F/S and a mucosal response in group S/F.

A fatty acid binding protein from Fasciola hepatica induced protection in C57/BL mice from challenge infection with Schistosoma bovis.

Three strains of mice (NMRI, C57/BL, BALB/c) were each immunized with a 12 kDa purified, native Fasciola hepatica fatty acid binding protein (Fh12) and challenged percutaneously with Schistosoma bovis cercariae. C57/BL mice immunized with Fh12 had significant reductions in S. bovis worm burden recoveries (96 and 87% reductions over controls in two separate experiments). When using NMRI or BALB/c mice, Fh12 alone or in Freund's adjuvant failed to induce significant protection against S. bovis. In C57/BL mice vaccinated against Fh 12, antibodies to the IgG2a isotype, but not to the IgG1 isotype, increased by 2 weeks after the second immunization and remained high through 8 weeks of S. bovis infection. Antibodies to S. bovis increased after 4 weeks of infection. Regarding cytokine production by spleen mononuclear cells, C57/BL mice vaccinated with Fh12 in adjuvant, and having the highest protective response against challenge infection with S. bovis, had an increase of IFN-gamma production with Concanavalin A but no increase of IL-4 in similarly stimulated cells. These results suggest that the protection obtained in this group of mice is mediated by a Th1 immune response.

Fasciola hepatica: vaccination of rabbits with native and recombinant antigens related to fatty acid binding proteins.

The current study was designed to compare the immunogenic and immunoprophylactic properties of native (nFh12) and recombinant (rFh15) antigens from Fasciola hepatica in rabbits infected with the fluke. Levels of specific anti-nFh12 and anti-rFh15 antibodies were significantly higher in the rabbits vaccinated twice compared with non-vaccinated infection controls. A reduction of 40% in worm burdens was found in rabbits immunized with nFh12 and infected 4 weeks after the second immunization. The recombinant vaccine induced lesser levels of protection than the native one, suggesting that both molecules may have slight differences either in immunogenicity or in their configuration. Further biochemical studies are required to define these differences. The mean length of flukes recovered was always smaller in all vaccinated rabbits. In addition, infected control rabbits had higher gamma glutamil transferase (GGT) levels than immunized rabbits. Lastly, gross anatomic observation always showed fewer liver lesions in all vaccinated rabbits than in controls. This finding clearly supports the possibility of vaccination regimes in fasciolosis.