Cercarial Transformation and <em>in vitro</em> Cultivation of <em>Schistosoma mansoni</em> Schistosomules

Abstract

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ranks second to malaria among parasitic diseases in terms of public health and socio-economic impact (1-4). Schistosome parasites are trematode worms with a complex life cycle interchanging between a parasitic life in molluscan and mammalian hosts with intervening freeswimming stages. Briefly, free-swimming cercariae infect a mammalian host by penetrating the skin with the aid of secreted proteases, during which time the cercariae lose their tails, transforming into schistosomules. The schistosomules must now evade the host immune system, develop a gut for digestion of red blood cells, and migrate though the lungs and portal circulation en route to their final destination in the hepatic portal system and eventually the mesenteric veins (for S. mansoni) where male and female worms pair and mate, producing hundreds of eggs daily. Some of the eggs are excreted from the body into fresh water, where the eggs hatch into free-swimming miracidia (5-10). The miracidia infect specific snail species and transform into mother and daughter sporocysts, which in turn, produce infective cercariae, completing the life cycle. Unfortunately, the entire schistosome life cycle cannot be cultured in vitro, but infective cercariae can be transformed into schistosomules, and the schistosomules can be cultured for weeks for the analysis of schistosome development in vitro or microarray analysis. In this protocol, we provide a visual description of cercarial transformation and in vitro culturing of schistosomules. We shed infectious cercariae from the snail host Biomphalaria glabrata and manually transform them into schistosomules by detaching their tails using an emulsifying double-ended needle. The in vitro cercarial transformation and schistosomules culture techniques described avoid the use of a mammalian host, which simplifies visualization of schistosomes and facilitates the collection of the parasite for experimental analysis. in vitro transformation and culturing techniques of schistosomes have been done for years (11, 12), but no visual protocols have been developed that are available to the entire community. Video LinkThe video component of this article can be found at https://www.jove.com/video/3191/ Protocol Safety measures:Schistosome cercariae can directly penetrate the skin without the needs of a cut or pores, and cause the chronic disease schistosomiasis. S. mansoni is a biosafety level 2 organism, therefore proper personal protective equipment and proper safety measures must be followed when working with infective cercaria. Most of the equipment necessary to grow and transform schistosomes is standard, with a few key exceptions noted in the table at the end of the protocol. All cercarial work should be done in an isolated room. Schistosomules should be cultured in a sterile, approved biosafety hood. Two layers of nitrile or latex gloves should be worn, as should a...

Comparative Study of Transcriptome Profiles of Mechanical- and Skin-Transformed Schistosoma mansoni Schistosomula

“…The presence of certain skin lipids, yet is not essential [17], also plays a role in the process of cercariae transformation and penetration [18], [19] probably by triggering the release of acetabular glands contents [20]. The most popular method for obtaining artificially transformed schistosomula uses a mechanical transformation (MT) protocol that includes some sort of shear force (centrifugation [21]–[23], passages through an emulsifying needle [24], or shaking [15]) applied to freshly shed cercariae followed by separation of cercariae heads from tails (usually by centrifugation in a density gradient) and posterior incubation of the cercariae heads/schistosomula in culture media at 37°C. Parasites obtained using this protocol show no major morphological or biochemical differences with those recovered from natural infections [10], [15]; making the MT the method of choice for obtaining large quantities of schistosomula.…”

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

“…Mechanical transformation of cercariae to schistosomules, and their subsequent in vitro cultivation was performed as described (Milligan and Jolly, 2011). Schistosome eggs were isolated from livers of infected mice by digestion with collagenase B (Dalton et al, 1997).…”

In Vitro Cercariae Transformation: Comparison of Mechanical and Nonmechanical Methods and Observation of Morphological Changes of Detached Cercariae Tails

“…Three mechanical methods have been employed for the acquisition of schistosomules, including centrifugation, vortexing, or pressure shearing, such as using the 21-gauge–needle method (Colley and Wikel, 1974; Ramalho-Pinto et al, 1974). Recently, the double-ended–needle method that presumably was developed from the normal needle process has been the most widely used for cercariae transformation in vitro (Colley and Wikel, 1974; Mann et al, 2010; Milligan and Jolly, 2011). For example, double-ended–needle transformed schistosomula have been recently employed in microarray studies (Parker-Manuel et al, 2011), investigations on gene function (Bhardwaj et al, 2011), inquiries that utilize RNA interference (Stefanić et al, 2010), and for drug discovery (Manneck et al, 2011).…”

“…The supernatant (containing the cercariae) was transferred to a new 50-ml conical tube for further debris removal. The cercariae were then placed on ice and left to settle in the dark for 45 min (Milligan and Jolly, 2011). The ASW was removed and the cercariae were washed twice with enriched RPMI (eRPMI) medium, RPMI 1640 (containing L-glutamine; Invitrogen, New York, New York) with antibiotics (150 units/ml penicillin, 100 μg/ml streptomycin; GIBCO, New York, New York), and 5% heat inactivated fetal bovine serum (GIBCO).…”

“…Mechanical transformation of cercariae into schistosomula was performed according to Milligan and Jolly (2011) by passing the parasites between 2 10-ml syringes joined by a 22-gauge double-ended, luer lok emulsifying needle (Scientific Commodities Incorporated, Lake Havasu, Arizona) 20 times. Nonmechanical transformation of cercariae into schistosomules was accomplished by leaving the cercariae undisturbed in eRPMI.…”