A reliable assessment of the viability of schistosome eggs trapped in host tissues is difficult. The use of a coupling azo dye method for the detection of alkaline phosphatase (A1P) in Schistosoma mansoni ova was found to be a specific and sensitive method for differentiating between viable and dead eggs, and can be used in both immature and mature eggs. In fully developed miracidia within an egg, A1P activity was demonstrated in germ cells and in the sensory endings of the neural cells. The embryonating miracidia displayed A1P activity on the body surface and in von Lichienberg’s envelope. The alkaline phosphatase test for egg viability shows increased sensitivity when compared with the more conventional Oogram and Hatching tests.
We have previously reported that Schistosoma mansoni larvae emerging from host lung at pH 7.5-7.8 and then fixed with diluted formaldehyde (HCHO) readily bind radiation-attenuated cercariae (RA) vaccine serum antibodies, as assessed by indirect membrane immunofluorescence (IF). Here we show that S. mansoni schistosomula emerging from lung pieces under 5% CO2 (pH <=7.3) readily bind RA vaccine serum antibodies, provided they have been incubated for 12 h at pH 7.5-7.8 in foetal calf serum-free RPMI medium, and fixed with diluted HCHO. Ex vivo larvae exposed during incubation to GW4869, a specific inhibitor of tegument-bound, neutral sphingomyelinase (nSMase) displayed significantly diminished binding of RA vaccine serum antibodies, thus suggesting that nSMase activity at pH >=7.5 leads to exposure of lung-stage larvae surface membrane antigens to specific antibody detection. More importantly, ex vivo larvae readily bound antibodies directed to dipeptidic multiple antigen peptide constructs, based on S. mansoni-specific sequences in S. mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). Lung-stage schistosomula IF reactivity was diminished following antiserum absorption with recombinant SG3PDH. The data together indicate that intact ex vivo, as well as, 5-day-old in vitro-grown larvae express SG3PDH on their surface membrane. The findings are discussed in relation to the importance of surface membrane proteins as candidate vaccine antigens.