[Show abstract][Hide abstract] ABSTRACT: Haemonchus contortus is an economically important gastrointestinal parasite that infects primarily sheep and goats. In order to survive inside the host, the parasite must overcome the host immune response. In the present study, we have identified and characterized a complement C3 binding protein (H.c-C3BP) from this parasite employing biochemical and molecular biology tools. Initially, a truncated form of the protein was isolated from the excretory secretory products of the parasite using C3-Sepharose column that facilitated its identification by mass spectroscopy. Subsequently, the parent molecule was generated in E. coli and sequence analysis confirmed it as glyceraldehydes-3-phosphate dehydrogenase (GAPDH). GAPDH reacted with the antiserum raised against the truncated protein and the truncated protein reacted with anti-GAPDH antiserum. The protein inhibited complement function as measured by hemolytic assay and membrane attack complex (MAC) formation. Sera from H. contortus-infected animals reacted with GAPDH as well as the truncated form of the protein which further lend support to protein secretion. Thus, the C3 binding property of H. contortus GAPDH is a new function and it represents a new entity of complement binding protein. Identification and characterization of H.c-C3BP should facilitate development of new therapeutics considering a key role of this protein in immune modulation. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an immune response in mice, by delivering the gene coding for this protein with mannosylated-polyethylenimine (PEI) to peritoneal cells. Mice were immunized with 50 microg recombinant plasmid DNA (Group I) or DNA-PEI-mannose (a 22 kDa linear cationic polymer with mannose ligand) (Group II) via the intraperitoneal route. Antibody studies showed no significant humoral immune response evoked to this DNA immunization with either PEI-mannose-delivered or naked DNA. However, on protein boosting of these DNA-primed mice there was a significant enhancement of antibody titre. Flow cytometric bead array was used to measure quantities of interleukin (IL)-2, IL-4, IL-5, interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) cytokines. Overexpression of T-helper 1 (Th1) cytokines such as IFN-gamma and TNF, with a lower but significant expression of the T-helper 2 (Th2) cytokine IL-5 was detected. Gene delivery using polyethylenimine-mannose ligand showed significant expression of IFN-gamma and TNF (P < 0.05), but no significant difference in IL-2, IL-4 and IL-5 (P>0.05) cytokine expression was observed between naked-DNA- and mannosylated PEI-DNA-delivered mice. Naked- or PEI-delivered-DNA immunization produced insignificant levels of IL-2 and IL-4 (P>0.05) cytokines in both groups of mice.
Journal of Helminthology 09/2009; 84(2):149-55. · 1.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present communication reports on the kinetics of immunoglobulin isotype response in Fasciola gigantica infected bovine calves. Fifteen Holstein-Friesian cross-bred male calves were assigned to 3 groups (Gr) of 5 calves each and infected with 4-month (Gr-A) and 16-month-old (Gr-B) F. gigantica metacercariae (n=400), respectively, while Gr-C calves served as uninfected control. Infection was terminated by treating the animals with triclabendazole on 28 weeks post-infection (WPI). Sera were collected on 0, 4, 10 and 14 days post-infection (DPI) and subsequently at weekly interval up to 32 WPI. The immunoglobulin isotype response was analyzed by ELISA, using anion exchange purified antigen fraction. An IgG response against F. gigantica infection was evoked by 3 and 2 WPI in animals of Gr-A and Gr-B, respectively with peak antibody response at 13 WPI. Elicitation of an early IgG1 response by 10 and 14 DPI but a delayed IgG2 response at 6 and 4 WPI, in animals of Gr-A and Gr-B, respectively was recorded. An early IgM response was evoked by 10 and 14 DPI and the level peaked at 13 and 12 WPI, with no detectable level at 21 and 15 WPI in animals of Gr-A and Gr-B, respectively. IgA response was elicited at 4 WPI in both the groups and showed the highest titre at 25 and 27 WPI in animals of Gr-A and Gr-B, respectively. Present study indicated an early and predominant response of IgG1, with concurrent expression of delayed and weak IgG2 in calves experimentally infected with F. gigantica.
[Show abstract][Hide abstract] ABSTRACT: Haemonchus contortus is an economically important gastrointestinal parasite of domestic animals. The parasite secretes calreticulin (CalR), a Ca(++) binding protein which modulates the host immune response. One way by which this protein acts is by inhibiting the classical complement pathway by binding to complement C1q protein. Understanding CalR-C1q interaction is important to develop methods to enhance host immune response. In this study, we have mapped the regions in the N-domain of CalR that facilitates C1q binding by generating small recombinant fragments of the domain and using synthetic peptides. In addition to already identified C1q binding motifs in human CalR, two additional sites in the N-domain of H. contortus were revealed with the following sequences-GKYYDDAKRD and the AKFPKKFT. The significance of multiple C1q binding motifs in CalR is discussed in relation to host-parasite interactions.
Molecular and Biochemical Parasitology 08/2009; 166(1):42-6. · 2.73 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calreticulin (CalR), a Ca(2+) binding multifunctional protein, is secreted by the parasitic nematode Haemonchus contortus. We have earlier observed binding of this protein to a 24-kDa polypeptide (p24) present in an enriched preparation of prothrombin. In the present study, the identity of p24 was established as a C-reactive protein (CRP) by several criteria. CalR binding to CRP is an elegant strategy devised by the parasite to survive in the host. The secreted CalR may achieve this either by limiting the free concentration of CRP, which has antiparasite activity or inhibit the activation of the classical complement pathway triggered on binding of CRP to C1q protein. CalR binding to CRP would also ensure a check on the procoagulant activity of the CRP enabling parasite to feed on the host blood. Thus, targeting CalR could be a novel strategy to tackle this parasite, which has developed resistance to many anthelmintics.
[Show abstract][Hide abstract] ABSTRACT: Metacercarial antigen of Fasciola gigantica was evaluated for early immunodiagnosis of experimental bovine fasciolosis using ELISA and Western blot. In ELISA, the experimental F. gigantica infection was detected as early as 2 weeks post-infection (WPI). The gradual increasing trend of antibody level was observed from 2 to 7 WPI, followed by a plateau, which was maintained up to 14 WPI. In Western blot, sera from experimentally infected calves recognized one distinct polypeptide of 21 kDa in fractionated metacercarial antigen as early as 10th day post infection. From 2 WPI, more polypeptide bands were reacting. Recognition of these protein bands persisted till the end of the experiment (14 WPI). Cattle sera collected from the field showed 34.5% seroprevalence of fasciolosis by ELISA using MAg. Comparative immunoblot studies of metacercarial antigen with anti-Gigantocotyle explanatum and anti-Paramphistomum epiclitum sera revealed that 21 and 25 kDa polypeptides of metacercarial antigen did not cross-react with any of these sera and appear to be unique to F. gigantica and having the desirable qualities of early and specific immunodiagnosis.
Indian journal of experimental biology 10/2006; 44(9):749-53. · 1.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A 66 kDa adult Haemonchus contortus excretory/secretory (E/S) antigen was identified in Western blot by reaction with sera from the infected goats. The protein was purified from the adult worm extract and E/S products by anion exchange and ConA-Sepharose chromatography. The purified protein inhibited monocyte function in vitro as judged by decreased production of hydrogen peroxide and nitric oxide in the culture medium. The protein also caused proliferation of peripheral blood mononuclear cells. The absence of protein in the free living L3 larvae suggests that the expression of this protein coincides with the adaptation to the parasitic life. A correlation of antibody titre and worm burden was observed in the infected goats with higher antibody levels in high worm burdened animals. Anti-protein antibody caused loss of adult worm motility in vitro resulting in the death of the parasite. The fact that the protein is recognized by the host together with in vitro killing of adult parasites by antibodies makes this protein a promising candidate for vaccination trial.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.
Indian journal of experimental biology 07/2005; 43(6):536-41. · 1.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An indirect enzyme-linked immunosorbent assay (ELISA) using 27 kDa glycoprotein of Fasciola gigantica has been evaluated for its potential use in the diagnosis of bovine fasciolosis. Following experimental infection of rabbits, F. gigantica infection-induced antibodies were isolated and later used as ligands in affinity chromatography for isolation of infection-induced antibody-specific proteins. Among the five infection-specific proteins isolated, a glycoprotein of 27 kDa was later isolated by second-step purification using concanavalin A matrix. In crossbred cattle receiving different doses of infection (100, 200 and 400 metacercariae), the anti-27 kDa antibodies were detected as early as the 2nd week post infection. No direct correlation between initial dose, antibody response and fluke establishment was recorded. No cross-reaction was noted with the sera of goats experimentally infected with Paramphistomum epiclitum. ELISA with the 27 kDa glycoprotein could be a feasible diagnostic tool for the early detection of bovine fasciolosis.
Veterinary Research Communications 03/2005; 29(2):123-35. · 1.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polymerase chain reaction (PCR) was used to detect Fasciola gigantica infection in the snail intermediate host. Fasciola specific primers amplified a 124 bp fragment in PCR when the genomic DNA isolated from F. gigantica infected Lymnaea auricularia snails was used as template. In addition to the 124 bp amplicon, a ladder of DNA fragments representing amplification of the 124 bp repetitive sequences was observed. Genomic DNA of the parasite was used as a positive control, which also gave an amplification of the 124 bp fragment. DNA isolated from non-infected snails was used as a negative control and no amplification of this sequence was observed. This technique is highly specific and sensitive and possesses fairly good prospects of its utility as an epidemiological tool for ascertaining the infectivity status in ubiquitous snail populations.
[Show abstract][Hide abstract] ABSTRACT: The antibody response and circulating antigen levels in bovine calves, infected experimentally with Fasciola gigantica, were monitored using enzyme-linked immunoelectrotransfer blot (EITB) and sandwich ELISA, respectively. By EITB, the infected calves' sera recognized the polypeptides in the range of 54-58 kDa as early as 2 weeks post-infection. By 12th week post-infection, the lower two polypeptides of 12 and 8 kDa had disappeared. In sandwich ELISA, the circulating 54 kDa and whole worm antigen of F. gigantica were detected in the sera samples of infected calves as early as 2 weeks post-infection and persisted until the end of experiment (26th week PI). The 54 kDa antigen of F. gigantica appears to be specific and possesses promising immunodiagnostic potential for early prepatent diagnosis of bovine fasciolosis.