Immune responses to polyethylenimine delivered plasmid DNA encoding a Fasciola gigantica fatty acid binding protein in mice and rabbits.

Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an effective immune response in mice and rabbits, when delivered as a DNA vaccine in muscle cells. Polyethylenimine (PEI), 25 kDa, branched cationic polymer was used as a delivery vehicle for this DNA in the muscle cells of mice and rabbits. Naked DNA evoked mixed Th1 and Th2 responses in mice. PEI condensed DNA, at amine nitrogen over DNA phosphate (N/P) ratios of 4, 6 and 8 and with various DNA concentrations, failed to evoke a significantly higher antibody response compared to naked DNA in mice. Similarly, the humoral immune response to naked DNA administration in rabbit thigh muscles was poor and no boosting of this antibody response on administration of DNA complexed to PEI was observed. On metacercarial challenge, rabbits failed to show any significant protective immune response in both the naked DNA and PEI-DNA immunized groups. Administration of PEI alone (12.5 mug) in mouse thigh muscles caused significant muscle cytotoxicity but condensation of DNA with PEI had less of a toxic effect on muscle cells, which was inversely related to the N/P ratio. Delivery of plasmid DNA encoding F. gigantica FABP by high molecular weight polyethylenimine (branched, 25 kDa) did not boost the effective immune response in both the animal species, which could either be attributed to cytotoxicity associated with this cationic polymer or muscle cells being unsuitable target cells for PEI condensed DNA delivery.

Immuno-diagnosis of bubaline fasciolosis with Fasciola gigantica cathepsin-L and recombinant cathepsin L 1-D proteases.

Fasciola gigantica cathepsin-L cysteine proteinase and recombinant cathepsin L 1-D were assessed for their potential in the immuno-diagnosis of F. gigantica infection in buffaloes. A diagnostic ELISA, based on these two antigens, was developed to detect antibodies against F. gigantica in water buffaloes. Sensitivity of the ELISA was assessed using sera from buffaloes experimentally or naturally infected with F. gigantica from F. gigantica endemic areas and its specificity by probing the sera of the host from F. gigantica non-endemic area. Our earlier studies under experimental setting showed 100% sensitivity of cathepsin-L ELISA in the diagnosis of fasciolosis in buffaloes, with the earliest detection of infection at 4 weeks post-infection. However, under field situation of natural F. gigantica infection, this sensitivity declined to 97.1% but specificity of the test remained 100%. Cross-reactivity of the antigen was checked with Schistosoma indicum, S. spindale, Paramphistomum epiclitum, Gastrothylax spp., Gigantocotyle explanatum, hydatid and Strongyloides papilossus in the bubaline host, naturally infected with these helminths. F. gigantica cathepsin-L and the recombinant cathepsin L-1D does not cross-react with these helminth parasites in natural mono or mixed infection of the host. The present ELISA contributes a relatively sensitive and reliable tool for the early serodiagnosis of bubaline fasciolosis.

Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection.

Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants.

Fasciola gigantica cathepsin-L cysteine proteinase in the detection of early experimental fasciolosis in ruminants.

Cathepsin-L cysteine proteinase was purified from Fasciola gigantica regurgitant by two-step alcoholic fractionation, followed by ion-exchange chromatography. The purification strategy was evolved to eliminate other contaminating proteins co-precipitating with the purified proteinase during alcoholic fractionation. The enzyme was stable on long-term storage at -20 degrees C rendering it more suitable for field diagnostic use. The purified cathepsin-L cysteine proteinase was assayed for detection of F. gigantica experimental infection in sheep and buffaloes and could detect infection, as early as 4 weeks post-infection by ELISA, Western blotting and Dipstick ELISA. The 28-kDa cathepsin-L cysteine proteinase seems a promising antigen for the diagnosis of tropical fasciolosis in domestic animals.

Vaccination of buffaloes with Fasciola gigantica recombinant fatty acid binding protein.

Recombinant fatty acid binding protein (rFABP) of Fasciola gigantica was expressed in Escherichia coli and used as vaccine in Freund's adjuvant to evaluate the level of protection induced in buffalo (Bubalus bubalis) calves. Fifteen buffalo calves were distributed to three groups of five calves each. An antigen dose of 400 mug for each of the three immunizations at 3-week intervals, and a challenge dose of 600 metacercariae was administered per calf. Levels of anti-FABP antibodies increased rapidly by 2 weeks after the first immunization and were always significantly higher in the immunized-challenged group than in the infected control group. Immunization with FABP induced both humoral and cell-mediated immune response in these animals. Vaccination showed a moderate level of protection in terms of reduced fluke burden (35.8%) and liver damage as assayed by aspartate aminotransferase and sulfhydryl group levels as well as anti-fecundity effect of the vaccine.

Humoral immune response against Fasciola gigantica fatty acid binding protein.

Recombinant fatty acid binding protein of Fasciola gigantica was expressed in Escherichia coli and purified by nickel chelating affinity chromatography. The recombinant protein along with native fatty acid binding protein (FABP) isolated from the parasite were evaluated for their potential in the diagnosis of F. gigantica infection in sheep, cattle and buffaloes, both by ELISA and western blotting. Results of this study indicate that there is no humoral immune response generated against this protein in the experimental infection of these ruminants with F. gigantica, thereby limiting the usefulness of this antigen in the early diagnosis of fasciolosis in these animals. Also, the paper discusses the probable reasons for the failure of this protein in detecting humoral response in these animals by ELISA and immunoblotting.