Fasciola gigantica: production and characterization of a monoclonal antibody against recombinant cathepsin B3.

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55-75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG(1) and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.

Fasciola gigantica: histology of the digestive tract and the expression of cathepsin L.

The digestive tract of Fasciola gigantica is composed of the oral sucker, buccal tube, pharynx, esophagus, and caecum. The tegumental-type epithelium lines the first four parts of the digestive tract while the caecal-type epithelium lines the remaining parts from the caecal bifurcation. The caecal-epithelial cells are classified into 3 types according to their staining properties and ultrastructural characteristics, as related to the amount of food contents in the caecal lumen. All caecal-type epithelial cells synthesize and secrete cathepsin L, a major group of enzymes in the digestive tract, as detected by in situ hybridization and immunolocalization. Moreover, the secreted cathepsin L is also adsorbed on the outer surface of the tegument and the glycocalyx coating of the surface of the tegument, whereas the tegumental cells and tegumental syncytium covering the parasite's body and lining the proximal part of the digestive tract exhibit no in situ hybridization signal and immunostaining for cathepsin L.

Identification, expression and immunolocalization of cathepsin B3, a stage-specific antigen expressed by juvenile Fasciola gigantica.

To identify antigens that could potentially be developed as vaccines against Fasciola gigantica, somatic antigens were analyzed by immunoprecipitation using pooled sera from rats infected with F. gigantica metacercariae. A prominent antigen of the newly excysted juveniles (NEJ), cathepsin B3 protease (FgCatB3), was identified by N-terminal sequencing and PCR screening of a cDNA library. Recombinant FgCatB3 (rFgCatB3) was expressed in Pichia pastoris, and shown to catalyse the digestion of gelatin, the fluorometric substrate Z-Phe-Arg-AMC and native fibronectin, suggesting that this enzyme may be involved in digesting host connective tissues during the fluke's penetration and migration in the host. Rabbit polyclonal sera against rFgCatB3 was produced and used to determine the distribution of the native cathepsin B3 protease in various developmental stages of F. gigantica. By Western blotting, cathepsin B3 was detected in the whole body (WB) extract of metacercariae and NEJ but not in 4-week-old juveniles or adult parasites which confirmed the stage-specific characteristics of cathepsin B3. Immunolocalization of cathepsin B3 protease in each parasite stage showed that high levels of FgCatB3 were present in the caecal epithelium of the metacercariae and NEJ. The differential distribution of FgCatB3 in the different life cycle stages suggests that this protease is functionally important for the juvenile stage of F. gigantica.

Prevalence of accessory head of flexor pollicis longus muscle and its relation to anterior interosseous nerve in Thai population.

A detailed description of the accessory head of flexor pollicis longus muscle (AHFPL) in the Thai population has not been reported. Because it is one of the causes of anterior interosseous nerve syndrome (AINS), a study was carried out on 120 Thai cadavers (70 embalmed, 50 fresh; 78 male, 42 female) to elucidate the prevalence of AHFPL, its morphology and relationship with the anterior interosseous nerve (AIN). The prevalence of AHFPL was 62.1% (149/240) with 74.5% (111/149) of its origin on medial epicondyle, 23.5% (35/149) on coronoid process and 2% (3/149) on flexor digitorum superficialis muscle. One hundred percent of its insertion was on the ulnar border of flexor pollicis longus tendon, and it was 98% (146/149) fusiform-shaped and 2% (3/149) slender shaped, with a diameter between 0.8-16.0 mm (average 6.7 mm), averaging 6.5 mm on the right and 4.2 mm on the left. The right was significantly statistically larger than the left (P < 0.05). The average distance from the mid-point of the distal wrist crease to the insertion point of AHFPL was 12.8 cm. Four patterns of relationship with AIN were noted including: 1) I AIN passed anterior to AHFPL, 13.4% (20/149); 2) AIN passed lateral to AHFPL, 65.8% (98/149); 3) AIN passed posterior to AHFPL, 8.1% (12/149); and 4) AIN passed both lateral and posterior to AHFPL, 12.8% (19/149). We believe that the latter two patterns (3 and 4) with AIN passing posteriorly would be more likely to be associated with AINS due to anatomic considerations.