S.C. Gupta

Interferon-gamma and interleukin-4 expression during Fasciola gigantica primary infection in crossbred bovine calves as determined by real -time PCR

Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) expression in crossbred (Bos taurusxBos indicus) bovine calves during primary infection with Fasciola gigantica was measured. Ten crossbred calves of 1-year age were divided into two groups of five calves each, group I uninfected control and group II calves orally infected with a dose of 1000 metacercariae of F. gigantica. The two cytokines were measured 10, 30 and 75 days post-infection (PI) by real-time polymerase chain reaction (RT-PCR) with the double stranded DNA-binding dye SYBR Green. IL-4 was present in detectable levels in peripheral blood mononuclear cells (PBMCs) of infected animals at 10, 30 and 75 days PI but no IFN-gamma was detected in PBMCs of infected animals at 10 and 30 days PI. However, at 75 days PI, IFN-gamma in two infected animals was present in detectable level. Eosinophil count increased from 2nd fortnight after infection and the increased level persisted till the termination of experiment. Present study indicated that T-cell response during F. gigantica infection was Th2-type during earlier phase of infection, which may be polarized in chronic infection to that of a Th0-type.

Efficacy and pharmacokinetics of triclabendazole in buffalo with induced fasciolosis.

A study was conducted to understand pharmacokinetics and flukicidal activity of intraruminal administration of triclabendazole (TCBZ) at 12.0, 24.0 and 36.0 mg kg-1 body weight in experimentally Fasciola gigantica-infected buffaloes on Week 2 and 10 post-infection. No fluke eggs in faeces and no flukes could be recovered from the liver of buffaloes following intraruminal administration of triclabendazole at 24.0 and 36.0 mg kg-1 body weight both on Weeks 2 and 10 post-infection, while the recommended therapeutic dose at 12.0 mg kg-1 body weight was 19-23% effective. Pharmacokinetic analysis of the data revealed a significantly higher (P < 0.05) concentration maximum of both the metabolites and area under concentration-time curve of TCBZ-SO2 in animals treated at 12.0 mg kg-1 body weight on Week 10 post-infection, whereas a significantly higher area under the concentration-curve and elimination half-life of both the metabolites and significantly higher concentration maximum and area under the concentration-time curve of both the metabolites were observed in animals treated on Week 10 post-infection at the dose rates of 24.0 and 36.0 mg kg-1 body weight, respectively. Bioavailability of triclabendazole was more in buffaloes with mature flukes than with immature flukes.

Early detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay and dot enzyme-linked immunosorbent assay

In an attempt to develop a suitable serological test for early detection of Fasciola gigantica infection in buffaloes, a group of proteins were isolated from the somatic antigen of the parasite by immunoaffinity chromatography. The process of isolation of the proteins has been standardized and significant level of repeatability was achieved. To test the diagnostic potentiality of the antigens, two serological tests, viz., enzyme-linked immunosorbent assay (ELISA) and dot enzyme-linked immunosorbent assay, were standardized using the sera from experimentally noninfected (group A) and infected (group B) animals. Further, the sensitivity and the specificity of the tests were evaluated employing the field sera from animals of different parasitic load viz., F. gigantica positive (group C), F. gigantica and Gastrothylax crumenifer positive (group D), F. gigantica and Gigantocotyle explanatum positive (group E), a group of sera without F. gigantica but other trematode infection (group F), only G. crumenifer positive (group G), only G. explanatum positive (group H), G. crumenifer and G. explanatum positive (group I), and PM negative (group J) collected from slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). In plate ELISA, the sensitivity of the antigen and the test was 75.75% while the specificity was 97%, 95%, and 98%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. In the case of dot ELISA the sensitivity was 86.5% and specificity was 92.3%, 94.7%, and 90%, respectively, against G. crumenifer, G. explanatum, and mixed infection of G. crumenifer and G. explanatum, respectively. The potentiality of the antigen in the diagnosis of field infection is discussed.

Vaccination of buffaloes with Fasciola gigantica recombinant glutathione S-transferase and fatty acid binding protein

Fasciola gigantica, causative agent of tropical fasciolosis, inflicts substantial economic losses on the livestock industry, affecting severely buffalo productivity in the tropical countries. Very few vaccination trials with different target antigens against F. gigantica infection have been conducted in this host. Present study describes a vaccination trial in buffaloes with F. gigantica recombinant glutathione S-transferase and fatty acid binding protein. The two recombinant proteins were expressed in Escherichia coli and evaluated for their immunoprophylactic potential in buffalo calves, using montanide 70 M-VG, a mineral oil-based adjuvant, for delivering the antigens. Buffalo calves were distributed in three groups, with group I, II and III calves immunized with recombinant glutathione S-transferase, fatty acid binding protein and a cocktail of these two antigens, respectively. Immunization of the calves evoked a mixed IgG1 and IgG2 antibody response. Present vaccination trial in these animals achieved a maximum protection level of 35%, when the two antigens were used in combination. Eosinophils were measured in both immunized and non-immunized challenge control animals, which showed a steady increase in their count in response to immunization with both the antigens and infection with F. gigantica, respectively.

Detection of Fasciola gigantica infection in buffaloes by enzyme-linked immunosorbent assay

The process of isolation of the 27-kDa glycoprotein from the somatic antigen of Fasciola gigantica was standardized and the diagnostic potentiality was evaluated for the detection of bubaline fasciolosis by indirect enzyme-linked immunosorbent assay. Initially, the test was standardized using the sera from experimentally noninfected(n = 20) and infected (n = 5)animals. Further, the sensitivity and the specificity of the test were evaluated through the sera of buffaloes with different natural infections, i.e., F. gigantica (n = 8 animals), F. gigantica and Gastrothylax crumenifer(n = 15), F. gigantica and Gigantocotyle explanatum (n = 6), trematode infections other than F. gigantica (n = 9), only G. crumenifer (n = 36), only G. explanatum (n = 18), G. crumenifer and G. explanatum positive (n = 39), and PM negative (n = 102). All animals came from the slaughterhouses of Bareilly (Uttar Pradesh, India) and Patna (Bihar, India). The level of sensitivity observed in the present study was 81.0%, while 97–98% specificity against G. crumenifer, G. explanatum, or a mixed infection with both parasites was noted. The study showed F. gigantica prevalence rate of 18–20% in the buffaloes of the study area. Enzyme-linked immunosorbent assay with a 27-kDa glycoprotein could be a feasible diagnostic method for the early detection of bovine fasciolosis.

On the viability of Fasciola gigantica metacercariae ingested by Lymnaea auricularia.

The effect of ingestion by Lymnaea auricularia on the viability and infectivity of Fasciola gigantica metacercariae was studied. The cyst wall was unaffected by the snails digestive processes. Two rabbits, each infected with 50 ingested metacercariae, died at 83 and 87 days post-infection. Eight and 10 immature flukes were recovered from the livers, indicating that the metacercariae had remained infective after passing through the intestine of the snail.

Pharmacokinetics and efficacy of long-term low-level administration of triclabendazole in urea molasses blocks against induced bovine and bubaline fasciolosis

The pharmacokinetics and flukicidal efficacy of triclabendazole delivered in low doses on a daily basis through urea molasses blocks were studied in cattle and buffaloes experimentally infected with Fasciola gigantica. The observations were compared with single therapeutic doses at 12.0 and 24.0 mg/kg body weight in cattle and buffaloes, respectively, prior to becoming experimentally infected. After receipt of triclabendazole by cattle and buffaloes at 0.83 and 3.0 mg/kg body weight, respectively, on a daily basis, both the animal species exhibited equilibrium between its absorption and disposition of its metabolites in plasma on day 4 and remained almost unchanged thereafter. The continuous subtherapeutic dosing of triclabendazole through urea molasses block at 0.83 mg/kg body weight in cattle and 3.0 mg/kg body weight in buffalo proved to be efficacious against mature liver flukes.

Molecular cloning and characterization of a glutathione S-transferase in the tropical liver fluke, Fasciola gigantica.

Glutathione S-transferase from an Indian isolate of Fasciola gigantica of buffalo origin was isolated and characterized. Total RNA was transcribed to cDNA by reverse transcription and an amplicon of 657 bp glutathione S-transferase gene was obtained by polymerase chain reaction (PCR). The present isolate showed 99.1% sequence homology with the published sequence of the F. gigantica GST gene of cattle origin, with six nucleotide changes causing an overall change of four amino acids. Glutathione S-transferase protein was expressed in Escherichia coli using a prokaryotic expression vector pPROEXHTb. The recombinant protein was purified under non-denaturing and denaturing conditions by nickel nitrilotriacetic acid (Ni-NTA) affinity chromatography. Recombinant GST protein detected F. gigantica infection in naturally infected buffaloes by dot-ELISA.

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.

Haematological and biochemical changes in Fasciola gigantica infected buffaloes fed on diet containing deoiled mahua (Bassia latifolia) seed cake

A study was conducted to evaluate the effect of condensed tannins and saponins from deoiled mahua seed cake (DMSC) on the development of fasciolosis. Fifteen male buffaloes were randomly divided into three groups of five each and were orally infected with Fasciola gigantica metacercaria at 800 per animal. DMSC was included in the concentrate mixture of group 2 (M5) and group 3 animals (M10) at 5 and 10%, respectively and group 1 remained as the non-supplemented control (C) group. Hypo-albuminemia, hyper-globulinemia and decrease in Albumin:Globulin (A:G) ratio were the significant features in C and M5 group. The group M10 was characterised by higher haemoglobin and packed cell volume and lower plasma total protein concentration, antibody response and faecal egg count than C and M5 group. This combined with higher plasma albumin, creatinine and lower globulin concentration in group 3 animals were conclusive of lower intensity infection as compared to other groups. It is concluded that condensed tannin and saponins of DMSC (10% of DMSC in concentrate mixture) possess remarkable anti-fasciolic activity.