Characterization and localization of saposin-like protein-2 (SAP-2) in Fasciola gigantica

Abstract

Fasciola gigantica saposin-like protein-2 (FgSAP-2) belongs to a family of lipid-interacting proteins that are involved in the cytolysis of target cells. In this study, we have cloned and expressed FgSAP-2 and produced the antibody against this recombinant protein. Rabbit antiserum against rFgSAP-2 reacted with a similar native protein in the whole body extracts of the 4-week-old juvenile and adult stage, as well as a protein in their excretion-secretion, but not in the tegument. In situ hybridization and immunofluorescence detection revealed the presence of SAP-2 mRNA transcripts and proteins in the cecal epithelial cells of 4-week-old juvenile and adult parasites, but not in the metacercariae and newly excysted juveniles. Moreover, SAP-2 is present only in the cecal epithelial cells lining the distal part of the digestive tract, but not in the tegumental-type epithelium lining the proximal part of the digestive tract. The rFgSAP-2 reacted with antisera from rabbits infected with F. gigantica metacercariae collected at 5 weeks, but not at 2 weeks after infection. Anti-rFgSAP-2 did not exhibit any cross-reactivity with the other parasites' antigens, including Opisthorchis viverrini, Eurytrema pancreaticum, Cotylophoron cotylophorum, Fischoederius cobboldi, Gigantocotyle explanatum, Paramphistomum cervi, Setaria labiato-papillosa, and Haemonchus placei. This finding indicated that SAP-2 is a unique protein that is expressed only in late juvenile and adult F. gigantica, and it could be considered for immunodiagnostic and as a vaccine candidate for fasciolosis.

Differential expression and localization of saposin-like protein 2 of Fasciola hepatica

“…Indeed, we previously reported the presence of this protein in the excretion-secretion material of F. hepatica (Espino and Hillyer, 2003). Interestingly, native FhSAP2 was also localized toward the tegument surface of both NEJs and adult F. hepatica , as demonstrated by weak fluorescence staining, which is a localization that has not been reported for any other members of the parasite SAPLIP family (Don et al , 2008; Don et al , 2007; Grams et al , 2006; Kueakhai et al , 2011). The tegument of flukes is a surface syncytial layer covering the parasite and bounded externally by an outer membrane coated with a carbohydrate-rich glycocalix (Anuracpreeda et al , 2009; Dalton, 2004; Dangprasert et al , 2001).…”

Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica

“…Juvenile parasites were obtained from the livers of hamsters infected with metacercariae via a stomach tube at the second and the fourth weeks post infection. Adult F. gigantica, Cotylophoron cotylophorum, Setaria labiatopapillosa, Fischoederius cobboldi, Gigantocotyle explanatum, Gastrothylax crumenifer, and Eurytrema pancreaticum were collected from infected cattle killed at a local abattoir as described earlier (Kueakhai et al 2011). All parasites were subsequently washed with 0.85 % normal saline and used immediately.…”

“…Immunolocalization of CatL1H in developmental stages of F. gigantica F. gigantica metacercariae, NEJ, 2-and 4-week-old juveniles, and adult were fixed in 4 % paraformaldehyde (w/v) in PBS, pH 7.4, at 4°C for 3 h. The parasite samples were prepared for paraffin embedding as described in Kueakhai et al (2011). The 5-μm-thick tissue sections were deparaffinized and rehydrated through a serial concentrations of ethyl alcohol (100-70 %).…”

“…Whole body (WB) extracts of various stages of F. gigantica (metacercariae, NEJ, 2-and 4-week-old juveniles, and adult) and other adult parasites (C. cotylophorum, S. labiatopapillosa, F. cobboldi, G. explanatum, G. crumenifer, and E. pancreaticum) were prepared as previously described (Kueakhai et al 2011). Adult F. gigantica excretory-secretory (ES) and tegumental antigens (TA) were prepared as previously described (Chaithirayanon et al 2002).…”

Immunohistochemical localization of sex hormone receptors in two Raillietina tapeworms

“…The immunolocalization procedure was similar to that used previously (Brattig et al, 2010;Kueakhai et al, 2011), with some modifications. Deparaffinated section slides were placed in 0.01 M citrate buffer solution, pH 6.0, and incubated for 10 min in a microwave.…”