New insights into the reaction of Schistosoma mansoni cercaria to the human complement system

Abstract

Schistosomes are parasitic worms that have a complex life cycle. The larval stage cercaria, infectious to mammals, is described as highly susceptible to the complement system, largely due to the glycocalyx that covers the cercarial membrane. In an attempt to have a more complete understanding of cercaria reaction to the complement system, three different approaches were used. Live cercariae exposed to normal human serum (NHS) as source of complement factors were assessed for: i) Membrane Attack Complex (MAC) deposition on the parasite surface; ii) cercaria survival rate by Hoechst staining of parasite DNA; and iii) transformation into schistosomula by detection of the glucose transporter protein 4 (SGTP4), a marker for new tegument formation. We found that 82–95% of cercariae directly exposed to NHS for 18 h were viable and retained their ability to shed the glycocalyx, suggesting minimal tegument damage. In contrast, inhibition of glycocalyx shedding using eserine caused significant MAC binding and parasite death. Culturing complement-exposed cercariae to measure long term survival showed that more parasites died over time, reaching a survival rate of 18–31% by day 6 in culture. The reason for this slow death is unknown, but the surviving parasites were able to form a new tegument as shown by detection of SGTP4 on the parasite surface. Furthermore, we found that complement activation significantly damaged the acetabular gland ducts and lysed secretory vesicles released by transforming cercariae. These findings should contribute for future in vivo studies of the effects of the complement system in skin migrating cercariae.

A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing

“…Such drugs would probably otherwise be missed. Taken together, we show that in contrast to cercariae that are susceptible to the complement system present in serum [44], schistosomulae not only tolerate the presence of HSe, but actually require it to progress to late-stage larval stages within their definite host, the human.…”

Determining the viability of Schistosoma mansoni cercariae using fluorescence assays: An application for water treatment

“…Stock solutions of FDA (catalog no F7378, 0.025 mg/ml in acetone), PI (catalog no P4170, 0.02 mg/ml in 1 X phosphate buffered saline (PBS)) and Hoechst (catalog no 94403, 5 mg/ml in 1 X PBS) were stored in the dark at 4˚C for up to 3 months. Stock solutions were added to cercaria samples to obtain final dye concentrations of 0.075, 0.5 and 2 μg/ml for Hoechst, FDA, and PI, respectively, based on previous literature [6,20,21]. All dyes were purchased from Sigma-Aldrich.…”

“…Here we adapt two fluorescence assays from previous schistosome viability studies [20,21] to identify if they can accurately determine cercarial viability. The two assays use a combination of vital and non-vital dyes that are widely available, have short incubation periods, and separate emission wavelengths: Hoechst 33258 (Hoechst), Propidium Iodide (PI) and Fluorescein Diacetate (FDA).…”

“…Hoechst is a cell-permeable dye that fluoresces blue when bound to DNA and is used as an indicator of membrane integrity, as fluorescence increases if cell membrane integrity is impaired [21]. PI is a non-vital, red fluorescent DNA stain, which is unable to cross intact cells and can only enter if there is a breach in the membrane [22,23].…”

“…However, other researchers have struggled to replicate these results [6,16], and limitations of the assay have been found in other fields of research [24]. We therefore also test the FDA-H dual stain, since Hoechst has been used to determine cercarial viability [25]. Using a combination of vital and non-vital dyes, instead of using a single dye, may help increase the accuracy and validates that differences in dye uptake and fluorescence result from differences in viability [20].…”