Paul F. Basch

CULTIVATION OF SCHISTOSOMA MANSONI IN VITRO. I. ESTABLISHMENT OF CULTURES FROM CERCARIAE AND DEVELOPMENT UNTIL PAIRING

Procedures are described for mass cultivation of Schistosoma mansoni. Cercariae were pooled, concentrated, and axenized through a series of washes in base medium containing antibiotics. Cercariae were sheared through a double-Luer-ended needle connected to two syringes, and tails separated and discarded. Young schistosomules were grown in a medium based upon BME augmented with lactalbumin hydrolysate, glucose, hormones, and other amendments and supplemented with human serum. Human blood cells (Type O) were added to cultures. Intestinal pigment was seen on the 5th day and gut cecal fusion began on day 11. Schistosomules developed steadily to pairing, which was seen first during the 7th wk of culture. Pairing occurred somewhat later in relation to development in vivo.

Intermediate host specificity in Schistosoma mansoni.

Abstract Miracidia of Schistosoma mansoni penetrate into many kinds of snails, but development of normal sporocysts takes place only in certain species of Biomphalaria . Different populations of this snail vary greatly in laboratory infection rates with S. mansoni originating from diverse geographic localities. Cross-exposure experiments show that compatibility factors exist in both snails and parasites. Susceptibility of stocks of Biomphalaria to particular strains of S. mansoni is genetically determined and may be modified by selection in the laboratory. In a compatible snail, the sporocyst develops without host tissue reaction; in incompatible snails the early larvae are rapidly surrounded by amebocytes and fibroblasts, and destroyed. This reaction resembles the generalized host cellular response elicited by any foreign body. An individual snail exposed to many miracidia may have both developing and encapsulated sporocysts side by side within its tissues. The weight of current evidence suggests that elicitation or absence of this cellular response resides in the recognition or nonrecognition of the sporocyst as a foreign body. The sporocyst tegument surface, which forms within a few hours after miracidial penetration, may have a molecular conformation identical with that of the snail, or may be able to bind specific host molecules, so that detection and subsequent encapsulation by host cells are averted. Presuming genetic determination of the sporocyst surface structure and of the host cell detection capability, differing infection rates would result from the particular frequencies of relevant genes in the populations concerned.

Evidence that the reduced surface antigenicity of developing Schistosoma mansoni schistosomula is due to antigen shedding rather than host molecule acquisition

Summary Antibody and lectin binding characteristics of Schistosoma mansoni schistosomula maturing in vivo and in vitro were quantitatively assessed and compared in order to investigate the basis of the reduced surface antigenicity of host derived larval schistosomes. Quantitative indirect immunofluorescence assays showed that schistosomula recovered from mice at 24 h and 5–10 days post infection bound low or insignificant amounts of a variety of anti‐schistosome antibodies including those from chronically infected and radiation attenuated cercariae‐vaccinated mice, a vaccinated rabbit and rabbits hyper‐immunized with non‐living larval and adult schistosome antigen preparations. In contrast, parasites maturing in vitro continued to bind highly significant levels of each of these antibody preparations until at least 10 days post transformation. To investigate the basis of the decreased surface antigenicity of parasites maturing in vivo, 6‐day‐cultured parasites were injected intravenously into mice and recovered from the lungs at various times thereafter and examined for their ability to bind both anti‐parasite and anti‐host antibodies. After 30 min in vivo, cultured schistosomula exhibited a significantly decreased capacity to bind anti‐parasite antibodies and concanavalin A (Con A), and by 16 h had lost their binding sites for fucose binding protein (FBP) as well. That this reduction in antigenicity was due to shedding of surface antigens was suggested by the observation that the reduced ability of these parasites to bind anti‐parasite antibodies coincided closely with the loss of 125I‐labelled surface proteins. Furthermore unlike 6 day schistosomula which had developed wholly in vivo, 6‐day‐cultured parasites recovered after 30 min in vivo failed to bind anti‐host antibodies suggesting that in these organisms parasite antigens were not masked by host molecules. These data argue that surface antigen shedding may explain the reduced surface antigenicity of schistosomula developing in vivo. While this surface modulation apparently independently of host antigen uptake, it is dependent upon an as yet unindentified host factor.

Purification and binding properties of hemagglutinin from Biomphalaria glabrata

Abstract A hemagglutinin has been purified from Biomphalaria glabrata (PR-B) hemolymph, albumin glands, and egg masses using affinity chromatography with Sephadex gels. The purified material from any of the sources above demonstrated identical immunological properties during immunoelectrophoresis or immunodiffusion, and similar serological specificity for human A1 erythrocytes and to a lesser extent A2 erythrocytes. Hemagglutinin was able to bind in vitro to the tegumental surface of cultured Schistosoma mansoni sporocysts, cercariae, and miracidia. Sporocysts dissected from infected snails and shed cercariae were already found to have hemagglutinin on their tegumental surface as demonstrated by immunofluorescence. It is postulated that hemagglutinin binding to the surface of larval helminths may “mask” them from being recognized by the snail hosts cellular defense system.

The miracidium-sporocyst transition in Schistosoma mansoni: surface changes in vitro with ultrastructural correlation.

Miracidia of Schistosoma mansoni, hatched aseptically, readily shed their ciliated epidermal cells in a culture medium. Electron microscopy shows that within 200 min in culture a new sporocyst tegument has formed, presumably from material mobilized from the subepidermal region. Granules resembling the alpha and beta particles of glycogen are involved in this process, passing up into the new tegument where they appear to disintegrate. The surface at this time bears simple microvilli, which become abundant and branched by 20 hr incubation. Mother sporocysts cultured for 6, 8, or 10 days were injected into Biomphalaria glabrata snails in which some at each age continued development leading to production of cercariae. Within the first 24 to 48 hr after miracidial penetration, focal tissue responses in the snail, Biomphalaria glabrata, act to destroy certain individual sporocysts of Schistosoma mansoni while others proceed to normal development (Newton, 1954; Pan, 1963). Elicitation or absence of this host foreign body response is probably associated with surface features of very young mother sporocysts (Wright, 1971). Information about the nature and changes in the parasite surface during the critical period of transformation from miracidium to sporocyst is therefore basic to comprehension of specificity between host and parasite. It is difficult to follow these events in vivo because the precise moment of miracidial penetration usually cannot be determined, and the tiny organisms are hard to find within the snail tissue. Induction of the same changes in vitro, with viability of sporocysts demonstrated by subsequent normal development upon implantation into snails, offers a means whereby these phenomena may be readily observed. We present here a simple culture method together with a description of surface changes between miracidium and sporocyst of S. mansoni in vitro. MATERIALS AND METHODS Golden hamsters, Mesocricetus auratus, were infected with 150 to 200 cercariae of S. mansoni. After about 8 weeks an animal was killed by rapid cervical dislocation, rinsed with 82% alcohol, its liver removed aseptically and dropped into a Received for publication 8 July 1974. * Supported by Grant AI-10271 from the NIAID, NIH, Bethesda, Maryland. sterile, ice-cold stainless steel semi-micro blender container (Eberbach Corporation) along with 100 ml of sterile ice-cold distilled water containing 200 units of Penicillin G per ml. All subsequent procedures were done aseptically using sterile glassware and reagents. The liver was minced for about 10 sec at high speed and the container placed in the refrigerator for 20 to 30 min. The top 80 ml of fluid was then carefully decanted, and 100 ml of fresh water with antibiotics added. The container was again refrigerated for 20 to 30 min. Meanwhile 2 X 105 units of Penicillin G were added to each of 2 1-liter volumetric flasks which had earlier been filled to the base of the neck with filtered aquarium water, capped (aluminum 35-mm film cans were used), and autoclaved. After the second sedimentation of the comminuted liver, 10 ml of sediment was transferred by pipette to each volumetric flask. The body of each flask was covered with aluminum foil, sterile water added up to the liter mark, and the upper neck illuminated with a small desk lamp. When miracidia were abundant in the top few cm of the water column, 5-ml aliquots, usually containing several hundred organisms, were withdrawn at intervals and transferred to 15-ml screwcapped conical centrifuge tubes, which were then placed into shaved ice in an insulated bucket. After miracidia had concentrated at the bottom of the tubes, as much water as possible was removed with a drawn-out Pasteur pipette, while observing through a binocular dissecting microscope. Two to 3 ml of sporocyst culture medium (DiConza and Basch, 1974) was added and the tubes incubated at room temperature (25 ?+ 1 C). Medium was made up with horse or fetal calf serum (Grand Island Biological Co.) or human serum from our laboratory staff. All sera were inactivated (56 C, 30 min) before use. Some cultures were transferred to 10by 75-mm disposable glass culture tubes which were tightly sealed with 00 silicone stoppers. Medium usually was not changed during the culture period. Obser-

THE ROLE OF SCHISTOSOMA MANSONI MALES IN FEEDING AND DEVELOPMENT OF FEMALE WORMS

Female Schistosoma mansoni from unisexual infections have scant pharyngeal musculature, thin intestinal cecal walls, pale and scanty intestinal contents, and lack acidic thiol proteinase digestive enzyme as determined by indirect immunofluorescence using a monoclonal antibody. Their intake of host erythrocytes, measured by 51Cr labeling, is about one-fourth that of paired adult females, and they appear to be starved. In contrast, paired adult females have heavier pharyngeal musculature and intestinal cecal walls and abundant digestive enzyme in the anterior third of their intestinal tract. Females in worm pairs surgically transplanted into uninfected mice continued to feed, but separated females were carried into the liver and deteriorated. Adult female S. mansoni, newly separated from their male partners and incubated in vitro with labeled erythrocytes, ingested marginally fewer cells than did still-paired females, indicating their ability to continue feeding almost normally at least for a period after separation. Paired and ex-paired adult females declined similarly in feeding rate with increased time in vitro. In Schistosomatium douthitti, females grow and mature without males, the pharyngeal musculature and cecal walls are well developed, the gut is full of ingested blood, and the acidic thiol proteinase is present in both unisexual and paired female worms. There are different stimulatory pathways for growth and for reproductive maturation in S. mansoni, although both processes require physical contact with the male. We believe that the growth-stimulating function results from the muscular action of the clasping male, which helps the immature female to pump blood into her intestine, thereby overcoming a state of relative starvation.

An interpretation of snail-trematode infection rates: Specificity based on concordance of compatible phenotypes

Abstract A formula is proposed that bases host-parasite specificity upon genetically determined characters for compatibility in both snail and trematode. Individual snails are thereby considered to be susceptible or resistant to individual miracidia and infections rates are controlled by the relative frequencies of relevant genes in the particular populations concerned. The biological evidence for this hypothesis stems from published geographic, genetic, and histological studies in the Biomphalaria-Schistosoma host-parasite complex. The specific mechanism of incompatibility probably resides in the host cellular reaction to parasites recognized as foreign. The principles governing snail-trematode specificity are considered applicable to all combinations and underlie regional and evolutionary adaptations in these groups. The hypothesis of Donges that snail populations are homogeneous with regard to susceptibility is discussed.

Intergeneric reproductive stimulation and parthenogenesis in Schistosoma mansoni

When mice were infected with female cercariae of Schistosoma mansoni and male cercariae of Schistosomatium douthitti, many mixed pairs formed. The paired females were approximately the same size as those in unisexual infections, far smaller than females paired with S. mansoni males. Although the Sch. douthitti males possessed well-developed testes, sperm were not found in their female partners, which developed scanty vitelline glands and produced laterally spined eggs typical of S. mansoni. Such eggs yielded swimming miracidia infective to the snail host of S. mansoni, Biomphalaria glabrata, but not to the lymnaeid snail host of Sch. douthitti. Sporocysts arising from these miracidia were haploid and produced cercariae infective to mice. Parthenogenetically derived female cercariae in mice co-infected with either parthenogenetically derived male or normal diploid male S. mansoni developed to large adults of normal appearance, whose eggs yielded diploid miracidia and subsequent generations of normal diploid schistosomes. Parthenogenetically derived females co-infected with Sch. douthitti males also paired and produced some eggs containing viable miracidia, which gave rise once again to haploid sporocysts. These observations confirm previous suggestions that the stimulus for maturation in female S. mansoni is distinct from that for growth, and is independent of insemination and fertilization. It is concluded that both diploid and haploid S. mansoni females are capable either of parthenogenesis or of bisexual reproduction when appropriately stimulated.

CULTIVATION OF SCHISTOSOMA MANSONI IN VITRO. II. PRODUCTION OF INFERTILE EGGS BY WORM PAIRS CULTURED FROM CERCARIAE

Schistosoma mansoni grown from the cercarial stage in vitro produced infertile eggs in about 10% of the pairs studied. Cultured eggs were about half normal size, had a small and blunt lateral spine, and appeared to lack a germinal disc. The shell opposite the spine was typically thin and poorly formed. Eggs were often found in dyads. Vitelline conglomerates in long strands were sometimes deposited. Vitelline glands, ovaries, and testes were relatively poorly developed. Alterations of gas phase or addition of reducing agents, antioxidants, steroid hormones, specific adsorbents, or adult worm extracts did not improve the worms or augment egg production.

In vitro development of Schistosoma mansoni cercariae.

Early cercarial embryos of Schistosoma mansoni developed to swimming cercariae within seven days in vitro, via the same sequence of stages as reported in vivo. The presence of living Biomphalaria glabrata cells (Bge line) was indispensable for proper development. Cultured cercariae were water-sensitive, gave no cercarienhüllen reaction, and appeared to lack the surface glycocalyx. Further development was not detected in mice or hamsters.