Identification of circulating antibodies in fasciolosis and localization of 66 kDa antigenic target using monoclonal antibodies.

We identified three specific circulating antibodies in serum of cattle naturally infected with Fasciola gigantica. Two of the antibodies were found to react specifically to 97 and 66 kDa antigenic molecules of adult worm tegumental membrane extract. The third antibody was identified by the reaction with 26-28 kDa molecule of the excretory/secretory antigens. Monoclonal antibody against 66 kDa protein was developed and used for localization of its antigenic target in adult worm frozen sections. The experiment demonstrated that 66 kDa protein is a component on the outer surface membrane and on the membrane lining of the caecal epithelial of adult worm. The 66 kDa antigen was considered as a promising candidate for immunodiagnosis and vaccine.

Fasciola gigantica: studies of the tegument as a basis for the developments of immunodiagnosis and vaccine.

The tegument of bile-dwelling Fasciola gigantica is the interfacing layer that helps the parasite to maintain its homeostasis, and evade the hostile environment, including the host's immune attacks. The tegument is a syncytial layer about 10 mm thick, that is formed by the fusion of cytoplasmic processes of tegument cells, whose soma lie underneath the two muscle layers. The surface of the tegument is highly folded and invaginated into numerous ridges, pits and spines, which help to increase the surface area of the tegument for the absorption and exchanging of molecules, as well as for attachment. The outer membrane covering the tegument is a trilaminate sheet about 12 nm thick, and coated with a carbohydrate-rich glycocalyx layer that also bears high negative charges. Some host molecules may also be adsorbed onto this layer. These unique characteristics enable the parasite to evade the antibody-dependent cell-mediated cytotoxicity (ADCC) reaction exerted by the host. The outer membrane and glycocalyx is continuously replaced by the reserved membrane synthesized and stored in secretory granules of tegument cells, that are transported via cell processes towards the tegument by microtubules. The basal membrane of the tegument is trilaminate and invaginated to form membrane infoldings with closely aligned mitochondria. The tegument cytoskeleton is composed of a highly cross-linked network of 4-6 nm knobby microtrabecular fibers, bundles of intermediate filaments, microtubules that splay out from the tegument cells' processes. Major proteins of the cytoskeleton are actin, paramyosin and tubulin. The flukes' antigens that can elicit strong immunological responses in animal hosts are synthesized and released mainly from the tegument and the cecum. The majority of antigens derived from the surface membrane and the tegument are of MW 97, 66, 58, 54, 47 and 14 kDa, while those released from the cecum are cysteine proteases of MW 27, 26 kDa. Monoclonal antibodies have been raised against some of these antigens, and have been employed in immunodiagnosis of the infection. From the protection conferred to animal models and the in vitro killing assays of young parasites by specific antibodies, candidate vaccines could be selected from these antigens, such as, an antioxidant enzyme, glutathione-S-transferase, the digestive enzyme cysteine proteases, the surface-tegument proteins, such as fatty acid binding protein (14 kDa), membrane proteins (at 66 kDa), as well as muscle protein paramyosin, and hemoprotein. Ongoing research have been directed at deciphering the genetic codes and the syntheses of some of these antigens by recombinant DNA technology.

Diagnosis of cattle fasciolosis by the detection of a circulating antigen using a monoclonal antibody.

A monoclonal antibody (MoAb) 1C12 that reacts with a 66 kDa surface tegumental (ST) antigen of adult worms of Fasciola gigantica was used to detect circulating antigen in sera of experimentally and naturally infected cattle. A combination of rabbit anti ST-antigens and MoAb 1C12 were used to capture and detect the circulating antigen in sandwich ELISA. The dilutions of 1:1,000 of rabbit anti ST-antigens and 1:100 for MoAb 1C12 were used to reduce cross-reactivity with other trematodes' antigens. The circulating antigen of F. gigantica was demonstrated in sera of all experimentally infected animals as early as the first week after the infection, and it remained detectable until the experiment was terminated at week 32 after the infection. Of the 97 serum samples from naturally infected cattle, the sensitivity of 86.6% was observed when the cut-off point was calculated from 32 serum specimens from uninfected control calves. The sensitivity increased to 100% when the commercial fetal calf and trematode-free baby calves sera were used for calculation of the control cut-off point. Based on these results, the combination of rabbit anti ST-antigens and MoAb 1C12 sandwich ELISA appeared to be sensitive, specific, and applicable in the immunodiagnosis of fasciolosis in cattle for epidemiological study and monitoring of chemotherapeutic efficacy.

Development and characterization of monoclonal antibodies against excretory-secretory antigens of Fasciola gigantica.

Monoclonal antibodies (MAbs) directed against Fasciola gigantica excretory-secretory (ES) antigens were developed from BALB/c mice. Four were selected for further study, from the panel of hybridomas. The antigen specificities of these MAbs were characterized and localized by enzyme-linked immunoeletrotransfer blot (EITB) and immunoperoxidase technique. The target epitopes of these MAbs are 66 kDa protein (MAb 2D10), 66 and 27-26 kDa proteins (MAbs 5D10 and 4F5) and 27-26 kDa protein (MAb 2D9). MAb 2D9 reacted to the antigenic components of the luminal content and epithelial cell lining the cecum, whereas MAb 2D10 reacted specifically to the antigens of the tegument and surface membrane. It was found that all MAbs cross-reacted to various degrees with the antigens extracted from Schistosoma mansoni, S. mekongi, S. spindale and Paramphistomum spp. However, when MAbs were diluted to 1:100 or 1:400 significant reduction of their cross-reactivities was observed.