Paul P. Weinstein

Development in vitro of some parasitic nematodes of vertebrates.

Discussion of the cultivation of parasitic nematodes of vertebrates is more the expression of a state of mind than of reality. Not a single species has been reared through successive generations outside the host, and partial success has been obtained for relatively few of the vast numbers of such nematodes found in nature. The problems presented by parasitic helminths because of their complex life cycles are perhaps more challenging than those for many other organisms. I t may be postulated with a high degree of confidence that each new stage in the growth of these parasites may demand a different set of nutritional and physicochemical conditions, since a worm may proceed through a sequence of free-living larval stages, parasitic larval stages in intermediate hosts and, finally, through an involved organ and tissue migration in the definitive host. The variations in the patterns of development that exist between species will probably reveal even further differences in growth requirements. There is some comfort, however, in the fact that the free-living stages of a number of “strongyles ” (Weinstein and Jones, 1956a; Weinstein and Jones, 19573; Jones and Weinstein, 1957), which have the practice in common of feeding upon living bacteria, have been successfully reared under axenic conditions in essentially the same media. These media, however, have been very complex, usually containing tissue homogenate and other supplements which may well mask any specific differences in requirements that exist between species. Partial development in vitro of the parasitic stages of a strongyle was first achieved by Glaser and Stoll (1938) with Haemonchus contortus. Subsequently we reported the cultivation from the egg to sexually mature fifthstage worms of Nippostrongylus muris (Weinstein and Jones, 1 9 5 6 ~ ) and from the egg to immature fifth-stage worms of Nematospiroides dubius (Jones and Weinstein, 1957). The media used for the development of the parasitic stages a t first consisted of a combination of sodium caseinate, liver extract, fresh tissue homogenate, and serum. More recently we have investigated the role of homogenate-serum. In this paper we shall discuss the growth of N . muris in this basal medium alone and with various supplements added; also included are some preliminary observations on the development of Necator americanus, the hookworm found in humans.

In vitro nutritional requirements of Nippostrongylus brasiliensis—I. Effects of sterols, sterol derivatives and heme compounds on the free-living stages☆

Abstract 1. 1. Nippostrongylus brasiliensis free-living stages developed in culture on Escherichia coli K-12 supplemented with a sterol or sterol derivative. 2. 2. Excessive washing of living E. coli removed a “growth factor” which was replaced by a heme compound. 3. 3. Although supplemented living E. coli supported N. brasiliensis development, better growth occurred when formalin-killed E. coli was used as a culture medium base. 4. 4. Heat-killed E. coli did not support complete development of N. brasiliensis free-living stages even upon addition of sterol and heme. 5. 5. N. brasiliensis larvae in culture took up and incorporated 3H-7α cholesterol.

Ultrastructure of cuticle formation in the nematodes Nippostrongylus brasiliensis and Nematospiroides dubius

The ultrastructure of cuticle formation was studied in the third molt of Nippostrongylus brasiliensis , a parasite of rats. For comparative purposes, early events in the formation of adult cuticle during the fourth molt of Nematospiroides dubius , a parasite of mice, were also examined. In the former, the third cuticle became separated from the hypodermis plasma membrane, and a filamentous coat was deposited on the external surface of the membrane. The surface coat increased in size and the hypodermis plasma membrane folded regularly to form plicae which circumscribed the worm. An inner layer was deposited between the hypodermis plasma membrane and the filamentous outer layer, which in turn differentiated into the triple-layered limiting membrane of the fourth cuticle. Corresponding events in formation of adult cuticle in N. dubius were similar to those seen in N. brasiliensis . These observations indicated that the cuticle, in both organisms, formed external to the hypodermis plasma membrane.

Ultrastructure of the hypodermis during cuticle formation in the third molt of the nematode Nippostrongylus brasiliensis.

SummaryThe fine structure of the hypodermis of Nippostrongylus brasiliensis, a parasitic nematode of the rat, was studied in free-living third-stage filariform (infective) larvae, and in parasitic forms undergoing the third molt. In filariform larvae the hypodermis displayed a comparatively poorly developed RER, and few Golgi complexes, mitochondria, and vesicles. Nuclei contained large amounts of heterochromatin and no nucleoli. After worms reached the lungs and the third molt began, the RER, Golgi complexes, mitochondria, multivesicular bodies, and coated vesicles greatly increased in amounts. Nuclei displayed less heterochromatin and contained prominent nucleoli. These morphological changes were associated with transition from the free-living to the parasitic mode in life. The results were correlated with the fact that the hypodermis in N. brasiliensis is actively synthesizing culticular collagen during the third molt.

Experimental Schistosome Dermatitis in Rabbits.

that in the skin of unsensitized persons there was a slow dissolution of the parasites in the epidermis with the formation of parakeratotic plaques. On the other hand, Olivier (1949a, 1953) showed that following skin penetration of unsensitized mice, hamsters, guinea pigs, rabbits, and rhesus monkeys by avian schistosome cercariae, some of these worms migrated to the lungs of these animals. Penner (1941) showed that a schistosome of small mammals, Schistosomatium douthitti, could penetrate the skin of monkeys and migrate to the lungs. These observations demonstrated that although some of the schistosome cercariae that penetrate unsensitized laboratory animals may be destroyed in the skin, others leave the skin and reach the lungs. Furthermore, the observations on the migration of the schistosomes in monkeys suggest that migration of the same species to the lungs of man is a possibility. In order to learn more concerning the fate of avian schistosomes in both sensitized and unsensitized animals, a study was made in rabbits of the skin reactions to these parasites. Rabbits were sensitized to schistosomes; skin biopsies were then made from these and from control rabbits after a challenge exposure to the same species of parasite. MATERIALS AND METHODS

Effects of an azasteroid on growth, development and reproduction of the free-living nematodes Caenorhabditis briggsae and Panagrellus redivivus.

The azasteroid, 25-azacoprostane (ASA-6), was evaluated for its effects on the growth, development and reproduction of the free-living nematodes, Caenorhabditis briggsae and Panagrellus redivivus. The axenic culture medium for either species of nematode consisted of Caenorhabditis briggsae Maintenance Medium (CbMM): formalin-killed Escherichia coli (1:1) with or without the addition of 5 micrograms cholesterol per ml and/or 25 micrograms ASA-6 per ml medium. All cultures also contained 50 micrograms Tween 80 per ml medium. After two generations of growth in sterol-deficient media, both species displayed a decrease in mean length, a decrease in the percent development to the adult stage and an inhibition of reproductive capability. These effects were more apparent in the sterol-deficient medium containing ASA-6. In the presence of cholesterol and ASA-6, growth and reproduction of C. briggsae, but not of P. redivivus, was inhibited after five generations. Morphologic abnormalities of azasteroid-inhibited worms were similar to those shown by worms cultured in sterol-deficient medium. These results suggest that different species of nematodes may exhibit different responses to azasteroid and that sterol utilization and metabolism may vary between nematode species. In addition, the similarities between the known effects of azasteroid inhibition in insects and those presented in this study on nematodes suggest a similar mechanism of action by the inhibitor in both groups of organisms.

Synthesis of cuticular protein during the third molt in the nematode Nippostrongylus brasiliensis

Abstract 1. 1. The uptake of 14 C-proline and its subsequent incorporation into the fourth-stage cuticle of Nippostrongylus brasiliensis was studied employing both in vitro and in vivo systems. 2. 2. Third-stage larvae absorbed and assimilated 14 C-proline under in vitro conditions. 3. 3. Isotopically labeled worms were injected into rats where they continued development to late fourth stage. These worms were recovered from rats, placed into an in vitro system and completed the fourth molt. 4. 4. Shed cuticles were isolated and shown to be radioactive. The majority of radioactivity in the cuticles was localized in proline and hydroxyproline indicating they are utilized for synthesis of cuticular protein.

Effects of azasteroids on growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius

25-Azasteroids were evaluated for their effects on the growth and development of the free-living stages of Nippostrongylus brasiliensis and Nematospiroides dubius. Increasing the concentration of 25-azasteroids in axenic cultures of either species resulted in a decrease in the percentage and mean length of larvae that developed to the third stage. Morphologic abnormalities of inhibited larvae were similar to those shown by larvae cultured in sterol-deficient medium. Addition of cholesterol to the culture medium reversed the inhibitive effects of azasteroid. Azasteroid completely inhibited growth and development of N. brasiliensis when the only sterol present in the culture medium was sitosterol. These results suggest similar pathways of sterol metabolism and similar mechanisms of action by azasteroids in the nematodes and insects that have been studied.

Spirometra mansonoides: lectin analysis of tegumental glycopeptides.

Abstract Teguments from spargana of Spirometra mansonoides were disrupted and removed using 0.2% Triton ×-100. Tegumental fractions were obtained by differential centrifugation and the proteins and glycoproteins of this surface layer were partially characterized in 9 to 20% linear gradient sodium dodecyl sulfate-polyacrylamide slab gels. Electrophoretic analysis of the microtriches (brush border) and vesicular fractions revealed nine polypeptides that were common to these tegumental fractions. The polypeptide composition of the microtriches and vesicular fractions differed qualitatively and with respect to the relative concentrations of certain polypeptides. Glycopeptides of the microtriches and vesicular fractions were identified by the direct application of the following fluorescein isothiocyanate-conjugated lectins to slab gels: concanavalin A, wheat germ agglutinin, Ricinus communis agglutinin-120, soybean agglutinin, and Ulex europaeus agglutinin-1. The major polypeptides associated with the tegument were found to be glycopeptides. U. europaeus agglutinin-1 failed to label any tegumental glycopeptides. Based on the different sugar specificities of the lectins tested, the oligosaccharide chains of tegumental glycoproteins of S. mansonoides may contain the following carbohydrates: d -mannose, d -glucose, N -acetyl- d -glucosamine, N -acetylneuraminic acid, d -galactose, and N -acetyl- d -galactosamine.

Ultrastructural analysis of pathologic lesions in sterol-deficient Nippostrongylus brasiliensis larvae

A variety of cellular lesions were manifested by the free-living larval stages of Nippostrongylus brasiliensis cultured axenically in medium lacking cholesterol. Pathologic changes developed rapidly and were most apparent in intestinal cells which displayed generalized degradation of membranous organelles. Mitochondria, endoplasmic reticulum, and Golgi complexes became disassociated and vacuolated. Autophagosomes appeared within intestinal cells and contained a wide variety of cellular components. By the 5th day gross vacuolization and degeneration of intestinal cells occurred and the hypodermis and lateral cords displayed lysed cytoplasmic regions. The latter structures are concerned with synthesis of cuticle and their degeneration correlates with the suppression of molting and the abnormal molts that occurred.