Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of Fasciola gigantica.

The digenetic trematode Fasciola gigantica is a parasite of great agricultural and economic importance. Along with Fasciola hepatica, F. gigantica incurs huge economic losses to the agricultural sector. Because of unavailability of an effective and commercial vaccine, the earliest diagnosis of the disease is the only way to control the disease. The conventional coprological techniques are able to detect the disease only after the parasites get matured and starts releasing their eggs with the faeces of host, therefore prepatent infection remain undiagnosed. The alternative method is by serological tests that uses circulatory antigens. Despite high sensitivity, their reliability is quite low because of the common antigens shared between different helminth parasites. To overcome this, investigation was shifted to identify the copro-antigens which could be more sensitive and reliable. In the present study, we tried to identify some of the immunodominant proteins from the Excretory Secretory (ES) product of F. gigantica which can be further characterized and used for early detection of infection and also as drug and vaccine candidates. The ES products of F. gigantica were collected and used for raising the polyclonal antibody in rabbit. The polypeptide profile was generated as well as immunogenic polypeptides were identified. The Source of ES antigen was immunolocalized using confocal microscopy and dot blot assay was performed to diagnose field infection. The polypeptide profile of ES products revealed a total of 24 polypeptides out of which 12 immunogenic polypeptides were identified by western blotting. Confocal micrographs showed the immunolocalization of antigens in the intestinal caecae, vitalline glands, gonads as well as in the tegument of the worm. The dot blot assay confirmed the utility of ES products for the detection of field infection. Subsequently, cross reactivity was found negative with Gigantocotyle explanatum; an amphitome parasite of same habitat. However, the cross reactivity with other helminths needs to be worked out

Immunolocalization and immunodetection of the excretory/secretory (ES) antigens of <i>Fasciola gigantica</i> - Fig 4

<p>Confocal micrograph of <i>Fasciola gigantica</i> in the liver of infected rabbit showing quite restricted immunolocalization of excretory secretory antigens as revealed by using FITC conjugated secondary antibody (A), counter staining using phalloidin TRITC (B) was done for actin localization. (C) Showing the merged A and B images, where green fluorescence is representing exclusive ES antigen distribution. [V vitelline gland, Tg tegument, Ht host tissue, Vs ventral sucker and HPi host-parasite interface].</p

Molecular weight of separated ES products polypeptides of <i>Fasciola gigantica</i> by SDS-PAGE.

<p>Molecular weight of separated ES products polypeptides of <i>Fasciola gigantica</i> by SDS-PAGE.</p

Western blot analysis of ES antigens of <i>Fasciola gigantica</i> using anti-FgES antibody.

<p>Western blot analysis of ES antigens of <i>Fasciola gigantica</i> using anti-FgES antibody.</p

SDS-polyacrylamide gel electrophoresis of excretory secretory products collected from <i>Fasciola gigantica</i> after 1h, 2h and 3h of incubation in Hanks’ medium (HBSS) Std: Standard molecular weight marker.

<p>SDS-polyacrylamide gel electrophoresis of excretory secretory products collected from <i>Fasciola gigantica</i> after 1h, 2h and 3h of incubation in Hanks’ medium (HBSS) Std: Standard molecular weight marker.</p

Differential immunolocalization of ES antigens in Fasciola gigantica as revealed by confocal microscopy, using anti-Fg ES hyperimmune sera and the FITC conjugated secondary antibody.

<p>Differential immunolocalization of ES antigens in Fasciola gigantica as revealed by confocal microscopy, using anti-Fg ES hyperimmune sera and the FITC conjugated secondary antibody.</p

Dot blot analysis of faecal samples collected from field, showing three positive <i>Fasciola gigantica</i> infected samples (within the red circle) detected by polyclonal antibodies raised against <i>Fasciola gigantica</i> ES products.

<p>Dot blot analysis of faecal samples collected from field, showing three positive <i>Fasciola gigantica</i> infected samples (within the red circle) detected by polyclonal antibodies raised against <i>Fasciola gigantica</i> ES products.</p