Paul H. Silverman

In vitro Cultivation Procedures for Parasitic Helminths

Publisher Summary This chapter focuses on the in vitro cultivation procedures for parasitic helminths. In most in vitro cultures of parasitic helminths, cultivation is carried out under a protective umbrella of antibiotics, which maintains a bacteriostatic environment for the helminths under investigation. Because of the variable source of helminth material, there is considerable variation among individual cultures in the types of associated micro-organisms. The only meaningful environmental condition is the true axenic one, which, for successful rearing of some helminthes is an absolute prerequisite. A wide variety of criteria have been used for assessing success in in vitro cultivation procedures. These depend on the parasite and the stage with which one begins and include motility and reaction to stimulus and physiological criteria, such as the utilization of substrates, cytological evidence of an increase in mitotic division, organogeny, gametogenesis, and oviposition. In vitro cultivation procedures provide an insight into many of the essential physiological requirements of the parasite and when used intelligently in conjunction with carefully planned in vivo experiments, they help to elucidate some of the many aspects of the host–parasite relationship. Work on the metabolism and physico-chemical requirements of parasites, investigations of certain features of behavior in parasites, and studies of antigens and other materials secreted and excreted during growth and metamorphosis, can be implemented effectively by means of in vitro culture techniques.

In vitro cultivation procedures for parasitic helminths: recent advances.

Publisher Summary This chapter describes the in vitro cultivation of various stages of parasitic worms. It emphasizes the application of this technique for elucidation of problems concerning parasite physiology and development. The chapter considers axenic culture of free-living and insect or plant parasitic nematodes. It also reviews the effect of variety of environmental conditions that must be satisfied before successful adaptation of a parasitic helminth to an in vitro culture can be achieved. Environmental conditions considered include (1) host stimuli and trigger mechanisms, (2) precise conditions required during successive stages of development, (3) factors influencing development and organogenesis as distinct from maintenance or survival, (4) immunological inhibition, (5) the toxicity and elimination of metabolic waste products, and (6) pretreatment factors acting on the parasite before cultivation. Several techniques along with trigger mechanisms of various kinds and media and various conditions are presented. The chapter concludes with description of recent studies concerning trematodes, cestodes, nematodes, and applications of metazoan in vitro cultivation procedures.

Cultivation of Ascaris suum larvae in supplemented and unsupplemented chemically defined media.

Second stage Ascaris suum larvae were maintained in several defined or partially defined media. The best medium as judged by rate of development appeared to be Caenorhabditis briggsae medium (Sayre, Hansen, and Yarwood, 1963) supplemented with 16.7% pig serum. The highest survival rate was obtained in Eagles minimum essential medium constituted in Earles salt solution plus 16.6% to 22.2% pig serum. In both of these media, the larvae grew in length and diameter, exsheathed, and developed to the third stage. While Ascaris suum adults will survive for many days in various media and even in physiological salt solution (see review by Silverman, 1965 and Rogers, 1958), no one has yet succeeded in cultivating these nematodes from larvae to the adult stage. Pitts and Ball (1955) and Pitts (1962) obtained limited growth of the larvae from eggs. Cleeland and Laurence (1962) and Cleeland (1963) maintained viable A. suum second stage larvae as long as 110 days in Medium 199 containing 20% bovine serum. These larvae increased considerably both in length and diameter. The present report summarizes an attempt to ascertain and compare the ability of A. suum larvae to grow and survive in various defined and partially defined media. MATERIALS AND METHODS

AXENIC HELMINTH CULTURES AND THEIR USE FOR THE PRODUCTION OF ANTIPARASITIC VACCINES

Research on immunologic resistance of vertebrate animals to metazoan parasites has encompassed a wide variety of infections including cestode, trematode, and nematode parasites. In this presentation, special emphasis is placed on nematode parasites of the families Metastrongylidae and Trichostrongylidae. These helminths parasitize domestic animals and in some cases, man. In general, the parasitic nematodes go through a life cycle involving a free-living stage that takes place in the host’s fecal excrement. Helminth eggs or embryos are passed out in the feces, where they develop and grow, molting twice, into ensheathed third-stage larvae. If the infective third-stage larva is ingested by a suitable host, the larva exsheaths, casting off the extra cuticle retained from the second free-living ecdysis. Inside the host, the larva enters the parastic or histotropic stage and undergoes a further two molts in the host’s tissues or in intimate contact with the intestinal mucosa. After the fourth and final molt, the worm matures, copulates, and eggs are produced and excreted into the host’s intestinal lumen to passed out in the feces. Work of the past thirty years on the mechanism of immunity to infection with parasitic helminths indicates that the sources of antigen and the site of antibody inhibition occur during growth and development of the histotropic stages. More specifically, it has been shown that inhibition of parasite development in resistant hosts is evident at the first, and to a lesser extent, during the second parasitic ecdysis. The functional antigens, which have been incriminated as important stimulants of the host immune response, are apparently elaborated during certain transitory stages (Silverman & Patterson, 1960). The stages which so far have been identified as particularly involved in the immune stimulating process are the molting third to fourth stage and the molting fourth and early fifth (or young adult) stage (Silverman, 1965a). Antigens prepared from these stages obtained by in vivo (Silverman & Patterson, 1960) and in vitro (Silverman, Poynter & Podger, 1962; Silverman, 1965a) means have proved to be effective in inducing resistance to a number of different parasites in animals. Work is presently under way in several laboratories to translate these promising results into practical vaccination procedures in veterinary and human medicine. This paper proposes to deal with some of the problems involved in the large-scale axenic culture of the histotropic stage of helminths for the production of antigen to be used in antiparasitic vaccines.

Ascaris Suum: Immune reactions in mice: I. Larval metabolic and somatic antigens☆

Abstract The immunologie activity of somatic and metabolic antigens derived from second and third stage Ascaris suum larvae was studied in mice. The results demonstrate that metabolic antigens harvested from third stage A. suum larvae in vitro were capable of inducing resistance to reinfection almost comparable to the immunity stimulated by a combined primary and rechallenge infection. The nonviable metabolic antigen(s) did not appear to be adversely affected by lyophilization. Third stage larvae, recovered from the lungs of infected mice, were cultured for 5 days in Eagles Minimum Essential Medium, and supplementation with protein did not seem to improve the yield of metabolic antigen(s).

Ascaris suum: immune reactions in mice. II. Metabolic and somatic antigens from in vitro cultured larvae.

Abstract The immunologic activity of somatic and metabolic antigens derived solely from in vitro cultured second- and early third-stage Ascaris suum larvae was studied in mice. Eagles Minimum Essential Medium and Caenorhabditis briggsae medium 75 were used. The results demonstrate for the first time that metabolic and somatic antigens harvested after 12 days from in vitro cultures were capable of inducing resistance to reinfection. The effect on immunity to A. suum in mice of somatic and metabolic antigens from: (1) second- and third-stage in vitro cultured A. suum larvae; (2) metabolic and somatic antigens from third- and fourth-stage A. suum ; (3) C. briggsae metabolic antigens; and (4) desalted Haemonchus contortus third-stage larvae exsheathing fluid are described.

Development of Haemonchus contortus in media designed for studies on Caenorhabditis briggsae.

Techniques and media designed for nutritional studies of the free-living nematode Caenorhabditis briggsae were used in culture studies of Haemonchus contortus. A chemically defined medium supplemented with a proteinous growth factor was found to support partial development of Haemonchus contortus to the beginning of the fourth molt. Studies of stability of media at the higher incubation temperature required by the parasite are reported. Previous work has shown that the free-living nematode Caenorhabditis briggsae can be maintained in vitro in serial subculture when a proteinous growth factor in an activated form is added to a chemically defined basal medium. In the defined medium without this activated factor, C. briggsae in any of the four larval stages undergoes only one additional molt before development stops (Hansen, Buecher, and Yarwood, 1965). Activation of this factor was originally produced by freezing (Hansen, Sayre, and Yarwood, 1961) in the defined medium. Several additional and more useful methods have now been developed. Activation produced at the temperature of homeothermic animals is of particular interest (Hansen et al., 1964). One can now postulate that active growth factor is available to parasitic nematodes in vertebrate tissues and also that such an activated growth factor might be of use in cultivation of the parasitic stages in vitro. This paper describes activation and stabilization of growth factor by several different methods. C. briggsae was used as the test organism; the results were then applied to culture of Haemonchus contortus. We found that development of H. contortus into the fourth molt was supported by mixtures of defined medium and activated growth factor (Silverman, Hansen, and Buecher, 1965). MATERIALS AND METHODS

Exsheathment Phenomenon in the Infective-Stage Larvae of Haemonchus contortus

The nature of exsheathment of 3rd-stage larvae of Haemonchus contortus was investigated further using a new simplified technique for the study of cuticular ring formation. The presence of a second ring on the 2nd-stage larval cuticle, 8 to 9 u from the anterior, is described. Exsheathing activity was observed to occur in 4 consecutive steps on isolated sheaths of H. contortus. Metabolic fluid and somatic extract of Ascaris suum and Caenorhabditis briggsae were investigated for their ability to stimulate exsheathment in H. contortus. Scanning electron microscopy revealed some aspects of the fine structure of cuticle. Exsheathing factor(s) was found to be thermostable, to require no metal ion activation, and to be active over a broad range of pH. Temperature appeared to be an important factor for the exsheathment activity. Refractile ring formation at 19 to 20 tL from the anterior end of larval Haemonchus contortus cuticles, as a criterion for exsheathing activity, was first reported by Lapage (1935). Empty sheaths with their caps still attached were also described by Veglia (1916). Sommerville (1957) described the presence of exsheathing fluid and its action on cuticles. Some properties of exsheathing fluid and its relation to the exsheathment phenomenon were investigated by Rogers and Sommerville (1957, 1960). The presence of leucine aminopeptidase in exsheathing fluid was first reported by Rogers (1963, 1965), and its role as an exsheathing enzyme was postulated by Rogers and Sommerville (1968). However, data of Ozerol and Silverman (1969) indicated that leucine aminopeptidase is not the exsheathing enzyme, and they pointed out the possible involvement of other factors in this phenomenon. The possible function of leucine aminopeptidase in exsheathing fluid was reemphasized by Rogers (1970); however, Slocombe and Whitlock (1971a, b) have recently presented additional data in support of Ozerol and Silvermans (1969) conclusions. The present studies were undertaken to investigate further the nature of the exsheathment phenomenon in infective larvae of Haemonchus contortus. MATERIALS AND METHODS Infective juvenile of H. contortus (L3) were used for all the experiments. Larvae, which were Received for publication 27 July 1971. * This work supported by Grant AI 05910, NIH, U. S. Public Health Service. kept f r several weeks at 10 C 1, were used for the isolation of the sheaths and preparation of the metabolic fluids (XL3F, XL4,5F) and somatic extracts (L3Fp, XLsFp, XL4.sFp) (Ozerol and Silverman, 1969). Preparation of metabolic fluid Metabolic fluid from third-stage (XLsF) and from fourth-fifth stage larvae (XL,.sF) were prepared in accordance with methods described by Silverman, Alger, and Hansen (1966), and Alger (1968). The fluid was separated by sedimentation of the larvae, withdrawn by syringe, and filtered through Whatman No. 1 paper containing some glass wool. Metabolic fluids from both stages of larvae were concentrated by lyophilization and subsequently fractionated as described by Ozerol and Silverman (1969, 1970). Concentration of the metabolic fluid In addition to lyophilization, the metabolic fluids were also concentrated by the following techniques in order to prevent the possible loss of biological and enzymic activities. (a) Lyphogel: Lyphogel, a polyacrylamide hydrogel in pellet form (Gelman Instrument Co., Ann Arbor, Mich.), was used for the concentration of fresh metabolic fluid from the third larval stage. One gram of lyphogel generally absorbs 5.4 ml of water. Approximately 10 ml fresh dialyzed metabolic fluid was placed in a beaker containing 1.7 g of lyphogel pellets. Concentration was carried out at 5 C + 1 for several hours. (b) Amicon membrane filter (Centriflo): Centriflo membrane ultrafilter cones (obtained from Amicon Corp., Lexington, Mass.) retains molecules above 5,000 MW. They are laminated to a tough, inert substrate which improves the handling qualities. Fresh, dialyzed metabolic fluid of third-stage larvae was filtered under gravitational force and the concentrate was recovered by pipette without damaging the membrane.

Enzymatic studies on the exsheathment of Haemonchus contortus infective larvae: The role of leucine aminopeptidase

Abstract 1. 1. Metabolic fluid of infective larvae of Haemonchus contortus and its role in the exsheathment phenomenon was investigated. Metabolic fluid and somatic extract of two different nematodes were tested for both enzymatic and exsheathing activiteis on H. contortus second stage sheaths. The data clearly indicate that leucine amonopeptidase is not involved in the exsheathment process as reported by other workers. 2. 2. Activators and inhibitors of leucine aminopeptidase were found to have little effect, if any, on the exsheathing activity of metabolic fluids. Exsheathing factor(s) was found to require no metal ion activation. 3. 3. The factor(s) which is responsible for exsheathment in H. contortus is thermostable and active over a broad pH range. 4. 4. The evidence for and against the view that leucine aminopeptidase is involved in this phenomenon is reviewed.

Active immunization against Ascaris suum minimum lethal dose in mice

SummaryAscaris suum metabolic and somatic antigens were obtained from second and third stage larvae cultured in vitro in supplemented Eagles minimum essential medium. Antigens were harvested after 12 and 16 days in in vitro culture.These four different antigens were injected intraperitoneally into mice with 4% sodium alginate. Metabolic antigens harvested from cultures after 12 and 16 days protected mice against a minimum lethal dose of 15000 A. suum embryonated eggs.