[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.
Full-text · Article · Aug 2013 · Parasite Immunology
[Show abstract][Hide abstract] ABSTRACT: An in vitro excystment protocol for Fasciola gigantica metacercariae is described in this communication. A hatching percentage of ~90% of the freshly harvested F. gigantica metacercariae was observed following this excystment protocol, which was found inversely related to the age of the metacercariae. In vitro excystment of the metacercariae is essential for determining their viability prior to their use in vaccination or primary infection studies in animals and for studies on immunological, chemotherapeutic or gene expression aspects of the parasite.
[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.
Full-text · Article · Sep 2009 · Journal of Helminthology
[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.
No preview · Article · Aug 2009 · Veterinary Parasitology
[Show abstract][Hide abstract] ABSTRACT: Protoscolices of buffalo origin were treated with 0.2% sodium dèoxycholate and 1% Triton X - 100 in order to isolate the membrane associated protein antigens. Both the surface antigens fractions had similar antibody response, suggesting that surface antigens of protoscolices can be used for immunodiagnosis.
No preview · Article · Aug 2009 · The Indian veterinary journal
[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.
No preview · Article · Aug 2009 · Molecular and Biochemical Parasitology
[Show abstract][Hide abstract] ABSTRACT: The partially purified infection specific polypeptide of Fasciola gigantica somatic antigen was used in ELISA to detect antibodies in groups A, B and C bovine calves experimentally infected with 100, 200 and 400 metacercariae of F. gigantica, respectively. All the calves showed detectable antibodies as early as 2 wpi with peak antibody titre on 11th wpi by group C and on 12th wpi by rest 2 groups' animals, thereafter, declining gradually up to 26th wpi. However, no statistical difference was observed in antibody response shown by the 3 groups of animals. The antigen did not show cross reaction to rabbit anti G. explanatum and S. spindale homogenate sera indicating greater opportunities for developing popular test for field use.
No preview · Article · Jun 2009 · The Indian journal of animal sciences
[Show abstract][Hide abstract] ABSTRACT: Swiss albino mice were experimentally infected with protoscolices of Echinococcus granulosus of buffalo origin through intraperitoneal route. The sera samples of experimentally infected mice were collected at an interval of 15 days and the antibody responses were tested in enzyme linked immunosorbent assay (ELISA) against different antigenic fractions of protoscolieces. The antibody response in mice against soluble proteins of protoscolieces (SP-P) was significant throughout the observation period of 180 days w.e.f. day 15-post infection (p.i.). The peak response was observed on day 120 p.i. although substantial ELISA values were recorded during 75 to 150 days p.i. With the excretory-secretory antigen of protoscolices (ES-P), a significant antibody response was recorded from day 15 p.i. and higher ELISA values were recorded during day 105 to 135 p.i. and the peak response was observed on day 120 p.i. The steady increase in antibody response up to around 120 days p.i. and thereafter declining trends of antibody response against the infection were evident. However, a significant response throughout the experiment was observed. The trend of anti-procoscolieces IgG level may be coinciding with cyst development in the present experimental infection.
[Show abstract][Hide abstract] ABSTRACT: A coprological survey to assess the incidence of fasciolosis in cattle and buffaloes of comparatively unexplored districts regions viz., Agra, Allahabad, Bareilly, Gorakhpur, Lucknow and Varanasi of Uttar Pradesh, India, was carried out. Faecal samples of the animals at the door steps of farmers in different villages of Agra (n=550) and Gorakhpur (n=564) districts while necropsy data (n=285) and faecal samples (n=1409) of buffaloes brought to the slaughterhouses of Allahabad (n=62+63), Bareilly (n=0+1207), Lucknow (n=80+20) and Varanasi (n=143+119) were collected. Faecal samples were examined microscopically and besides Fasciola gigantica eggs, other fluke eggs viz., species of amphistome spp. and schistosomes encountered during the examination were recorded and presented in the paper. In Gorakhpur region, 15.8% of 292 buffaloes and 7.7% of 272 cattle showed F. gigantica infection. Amphistome infection was recorded in 49.3% of buffaloes and 24.6% of cattle. Amongst the infected animals, 10.3% of buffaloes and 4.8% of cattle showed mixed infection. One buffalo and 2 cattle were also infected with Schistosoma spindale. In contrast, in Agra region, F. gigantica eggs were encountered only in 4.6% of 452 buffaloes while eggs of different species of amphistome were detected in 23.2% of buffaloes and 17.3% of 98 cattle faeces with 2.4% buffaloes showing mixed infection. Necropsy examination of buffaloes slaughtered at Varanasi, Allahabad and Lucknow showed fasciolosis in 9.1%, 8.1% and 37.5%, whereas amphistomosis was found in 53.8%, 32.3% and 52.5% of animals, respectively. Coproscopically F. gigantica eggs were detected in 1.7%, 23.8% and 35.0% of buffaloes while amphistome eggs were found in 21.8, 31.7 and 50.0% of buffalo faeces collected from Varanasi, Allahabad and Lucknow, respectively. At Bareilly, of 1207 buffalo faecal samples examined, 32.4%, 85.5% and 1.4% showed eggs of F. gigantica, species of amphistome and schistosome, respectively. Amongst schistosomosis, three species viz., Orientobilharzia duttai, S. spindale and S. indicum eggs were encountered with mixed or solitary infection. One of the animal's faeces showed eggs of all the three schistosome species along with F. gigantica and amphistome eggs. The study indicated a moderate to high prevalence of trematode infection amongst buffaloes and cattle in Uttar Pradesh.
[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.
Preview · Article · May 2008 · Parasite Immunology
[Show abstract][Hide abstract] ABSTRACT: Swiss albino mice were experimentally infected with protoscolices of Echinococcus granulosus of buffalo origin through intraperitoneal route. From 3-month post infection (p.i), small spots of echinococcal foci (lt;1 mm) were recorded on the surface of liver, lungs, spleen and kidney. At six-month infection, maximum number of cysts (gt; 1 mm) were recovered. Cysts or cystic foci were recorded from 66.66% of infected mice. Splenomegaly and hepatomegaly were the predominant gross changes in experimental infection. Histopathological changes in the organs of mice due to secondary hydatidosis have been discussed in the paper.
No preview · Article · Apr 2008 · The Indian journal of animal sciences
[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.
Full-text · Article · Oct 2006 · Indian journal of experimental biology
[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.
No preview · Article · Jul 2006 · Veterinary Parasitology
[Show abstract][Hide abstract] ABSTRACT: Anion exchange chromatography was attempted to isolate immunodiagnostic protein moieties from crude extracts of Fasciola gigantica. The somatic antigen was fractionated into 100 fractions of which 17 fractions collected by 0.1M, 0.15M and 0.2M NaCl gave strong reaction in dot ELISA using anti F. gigantica cattle sera raised experimentally. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of intensely reacted fractions resolved 13 polypeptides in the range of 86.3-11.8 kDA in fraction 4th, 10 polypeptides of 11.8 to 86.3 KDa in fraction 14 and 12 polypeptides of 97.4 to 11.8 kDa in fraction 21st. Of the different polypeptides, one protein band of 86.3 kDa was strongly reactive in all the fractions when tested by EITB. The purified antigens showed promising results for the early diagnosis of fasciolosis.
No preview · Article · Apr 2006 · The Indian journal of animal sciences
[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.
No preview · Article · Feb 2006 · Veterinary Parasitology
[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.
Full-text · Article · Jul 2005 · Indian journal of experimental biology
[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.
Full-text · Article · Mar 2005 · Veterinary Research Communications