Schistosoma mansoni: Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll

Lazdins, Janis; Stein, Marsha J.; David, John R.; Sher, Alan

doi:10.1016/0014-4894(82)90090-x

Cited by 123 publications

(75 citation statements)

References 11 publications

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“…Cercariae-derived schistosomula were prepared as described by Lazdins et al (1982), and cultured at 37 o C under 5% CO 2 for 4-6 days in 10-90% FCS in RPMI medium, or medium 169 (Harrop and Wilson 1993) supplemented with 300 U/ml penicillin, 300 µg/ml streptomycin, and 160 µg/ml gentamicin (all from BioWhittaker). The viability of schistosomula was assessed by determining the percentages which excluded Trypan blue, and appeared healthy, transparent, motile, and contractile.…”

Section: Methodsmentioning

confidence: 99%

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

Tallima¹,

Ridi²

2008

FOLIA PARASIT

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Abstract. 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% CO 2 (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.

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Section: Methodsmentioning

confidence: 99%

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

Tallima¹,

Ridi²

2008

FOLIA PARASIT

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“…Briefly, cercariae were concentrated by centrifugation (2000 x rpm/ 10 min) and washed once with somule wash medium (RPMI 1640 supplemented with 200 U/ml Penicillin G sulfate, 200 μg/ml streptomycin sulfate, 500 ng/ml amphotericin B, 10 mM HEPES). Cercarial tails were sheared off by 20 passes through 22G emulsifying needles after which schistosomule bodies were isolated free from tails by Percoll gradient centrifugation [19]. Schistosomula were washed 3 times in wash medium and cultured at 37°C under 5% CO 2 in air in modified Basch's medium [16] supplemented with small quantities of washed human erythrocytes.…”

Section: Parasitesmentioning

confidence: 99%

RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade

Morales

Rinaldi

Gobert

et al. 2008

Molecular and Biochemical Parasitology

115

111

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The aspartic protease cathepsin D (Clan AA, Family A1) is expressed in the schistosome gut where it plays an apical role in the digestion of hemoglobin released from ingested erythrocytes. In this report, RNA interference approaches were employed to investigate the effects of knockdown of schistosome cathepsin D. Cultured schistosomules of Schistosoma mansoni were exposed by square wave electroporation to double stranded RNA (dsRNA) specific for cDNA encoding S. mansoni cathepsin D. RNAi mediated reductions in transcript levels led to phenotypic changes including significant growth retardation in vitro and suppression of aspartic protease enzyme activity. In addition, black-pigmented heme, the end point by-product of normal hemoglobin proteolysis that accumulates in the schistosome gut, was not apparent within the guts of the treated schistosomules. Rather, their guts appeared to be red in color, rather than black, apparently indicating the presence of intact rather than digested host hemoglobin. These phenotypic effects were apparent when either of two forms of dsRNA, a long form spanning the entire target transcript or a short form specific for the 3'-region were employed. Off-target effects were not apparent in transcript levels of the gut-localized cysteine protease cathepsin B1. Finally, cathepsin D may be an essential enzyme in the mammal-parasitic stages of schistosomes because schistosomules treated ex vivo with dsRNA did not survive to maturity after transfer into Balb/c mice. These and earlier findings suggest that, given its essential function in parasite nutrition, schistosome cathepsin D could be developed as a target for novel anti-schistosomal interventions.

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“…Since then, preparation of schistosomules has been done using either the shearing strategy with a needle and syringe as described in this protocol, or using a vortexing approach and separated with gradients of percoll (14,15) or swirling as demonstrated in this paper. These two techniques for preparing schistosomules are beautifully described by Fred Lewis (16) and more information for further culturing of schistosomules for long-term growth can be found at the NIAID Schistosome Resource Center (www.schisto-resource.org).…”

Section: Discussionmentioning

confidence: 99%

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

Milligan

Jolly

2011

JoVE

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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...

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Schistosoma mansoni: Rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll

Cited by 123 publications

References 11 publications

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade

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

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