Susceptibility of Snails to Infection with Schistosomes is influenced by Temperature and Expression of Heat Shock Proteins.

The freshwater snail, Biomphalaria glabrata is the obligate intermediate host for the transmission of the parasitic trematode, Schistosoma mansoni the causative agent of the chronic debilitating neglected tropical disease, schistosomiasis. We showed previously that in juvenile snails, early and significant induction of stress manifested by the expression of stress proteins, Hsp 70, Hsp 90 and reverse transcriptase (RT) of the non- LTR retrotransposon, nimbus, is a characteristic feature of juvenile susceptible NMRI but not resistant BS-90 snails. These latter, however, could be rendered susceptible after mild heat shock at 32°C, revealing that resistance in the BS-90 resistant snail to schistosomes is a temperature dependent trait. Here we tested the hypothesis that maintenance of BS-90 resistant snails at the permissive temperature for several generations affects the resistance phenotype displayed at the non-permissive temperature of 25°C. The progeny of BS-90 snails bred and maintained through several generations (F1 to F4) at 32°C were susceptible to the schistosome infection when returned to room temperature, shedding cercariae at four weeks post-infection. Moreover, the study of expression levels of the heat shock protein (Hsp) 70 protein by ELISA and western blot analysis, showed that this protein is also differentially expressed between susceptible and resistant snails, with susceptible snails expressing more protein than their resistant counterparts after early exposure to wild-type but not to radiation-attenuated miracidia. These data suggested that in the face of global warming, the ability to sustain a reduction in schistosomiasis by using refractory snails as a strategy to block transmission of the disease might prove challenging since non-lethal elevation in temperature, affects snail susceptibility to S. mansoni.

Trichinella spiralis-specific monoclonal antibodies and affinity-purified antigen-based diagnosis.

Hybridomas secreting monoclonal antibodies (MAbs) to Trichinella spiralis were produced. Myeloma cells were fused with splenocytes of a mouse immunized with excretory-secretory (E-S) antigen of infective larvae. A large percentage of growing hybrids secreted antibodies cross-reactive to many of 23 heterologous parasites tested. Only 6 monoclones (designated 3F2, 5D1, 10F6, 11E4, 13D6 and 14D11) secreted MAbs specific to the E-S antigen and/or a crude extract (CE) of T. spiralis infective larvae. The 6 monoclones secreted IgM, IgG3, IgM, IgG3, IgG3 and IgG3, respectively. Clone 5D1 was selected to mass produce MAbs which were then coupled to CNBr-activated Sepharose CL-4B to prepare an affinity-purified antigen. Dot-blot ELISA with either purified antigen or CE was evaluated. There were 17 patients with acute trichinellosis and 76 individuals convalescing from T. spiralis infection (group 1). Controls were 170 patients with parasitic infections other than trichinellosis (group 2) and 35 healthy parasite-free controls (group 3). CE-ELISA was positive in all group 1 patients. However, sera from many group 2 patients also were reactive (opisthorchiasis-44.2%, schistosomiasis-44%, gnathostomiasis-30%, paragonimiasis-28.6%, taeniasis-27.3%, strongyloidiasis-23.1% and hookworm infections-20%). Affinity-purified antigen was 100% specific, all sera from group 2 and group 3 individuals tested negative. Although 74 of 76 patients (97.4%) with convalescing trichinellosis tested positive, sera from only 3 of 17 patients (17.6%) with acute T. spiralis were reactive. Thus, CE antigen is appropriate when sensitivity is needed, while purified antigen should be used when specificity is required. Dot-blot ELISA is easier to perform, more rapid and less expensive than indirect ELISA. Many samples can be assayed simultaneously, special equipment is not required, and results can be preserved for retrospective analysis. Dot-blot ELISA is therefore the method of choice for the rapid diagnosis of trichinellosis, particularly when more complex laboratory tests are unavailable.

Differential diagnosis of schistosomiasis mekongi and trichinellosis in human.

An indirect (plate) ELISA and, a more convenient version, a dot-blot (membrane) ELISA have been developed using haemocyanin of a mollusk, Megathura crenulata, i.e. keyhole limpet haemocyanin (KLH) and purified, specific antigen of Trichinella spiralis (APTsAg) obtained from a monoclonal antibody-affinity column chromatography, for differential diagnosis of schistosomiasis mekongi and trichinellosis. Serum samples of patients with parasitologically confirmed trichinellosis were reactive to both antigens in both versions of ELISA while sera of patients with schistosomiasis mekongi were positive only to the KLH. Both ELISA were negative when used to test sera of normal controls and patients with gnathostomiasis, paragonimiasis and opisthorchiasis.