Marcia Mika-Grieve

Active and passive immunization of mice against larval Dirofilaria immitis.

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.

IN VITRO CULTURE OF DIROFILARIA IMMITIS THIRD- AND FOURTH-STAGE LARVAE UNDER DEFINED CONDITIONS

The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.

Dirofilaria immitis: Surface properties of third- and fourth-stage larvae

The objective of this study was to analyze surface properties of larval Dirofilaria immitis with potential relevance to protective immunity. Comparisons were made between third (L3)- and fourth-stage larvae (L4) based on their net surface charge, surface carbohydrate and antigen composition, ability to nonspecifically absorb host proteins, complement activation, and nonspecific cellular adherence. It was determined that L3 had a net negative surface charge, whereas L4 had either a neutral or weakly positive surface charge. The lectin Con A, but not any of the other lectins tested, bound only to the surface of L4, and not to that of L3. Monoclonal antibodies were prepared which reacted with the surface of L3 or with the surface of L4, but never both. L4 were found to nonspecifically adsorb host protein to their surfaces, whereas L3 did not. Both L3 and L4 were found to activate complement through the alternate pathway. Finally, nonspecific cellular adherence was found on L3 both in vitro and in vivo but not on L4. The surfaces of L3 and L4 were thus shown to be significantly different and, potentially, in ways which would have great impact in the generation and effectiveness of a protective immune response.

Toxocara canis: monoclonal antibodies to larval excretory-secretory antigens that bind with genus and species specificity to the cuticular surface of infective larvae.

When maintained in culture, the infective-stage larvae of Toxocara canis produce a group of excretory-secretory antigens. Monoclonal antibodies to these antigens have been produced and partially characterized. Hybridomas were made using spleens from mice that had been given 250 embryonated eggs of T. canis followed by immunization with excretory-secretory antigens. Monoclonal antibodies were first screened against excretory-secretory antigens using an indirect enzyme-linked immunosorbent assay. Those antibodies positive in this assay were then screened against the surfaces of formalin-fixed, infective-stage larvae using an indirect fluorescent antibody assay. The two monoclonal antibodies showing fluorescence were also tested against the surfaces of infective-stage larvae of Toxocara cati, Baylisascaris procyonis, Toxascaris leonina, Ascaris suum, a Porrocaecum sp., and Dirofilaria immitis. One of these two antibodies bound to the surface of T. canis and T. cati while the other bound only to the surface of T. canis; neither were reactive with the other ascaridoid larvae or the larvae of D. immitis. Enzyme-linked immunoelectrotransfer blotting techniques were used to demonstrate that the cross-reactive antibody recognized antigens with molecular weights of about 200 kDa while the more specific monoclonal antibody recognized antigens with approximate molecular weights of 80 kDa. The specificity of these two antibodies for T. canis and T. cati should prove helpful in the development of more specific assays for the diagnosis of visceral and ocular larva migrans.

Development and dynamics of regional specialization within the syncytial epidermis of the rat tapeworm,Hymenolepis diminuta

SummaryThe epidermis of the tapewormHymenolepis diminuta is a highly organized syncytium, composed of an outer layer of continuous cytoplasm, or ectocytoplasm, and an inner layer of nucleated cell bodies, or perikarya. The perikarya are in direct cytoplasmic continuity with the ectocytoplasm via narrow plasmalemma-bound bridges called internuncial process. Although distinct structural and functional differences are apparent between ectocytoplasm and perikarya, all of the perikarya along the body of the cestode are morphologically similar, as are all regions of ectocytoplasm. However, immunocytochemically distinct subpopulations of perikarya and regionally defined areas of ectocytoplasm were identified along the tapeworm strobila by the use of monoclonal antibodies raised against a preparation of isolated tegument. The different types of perikarya and the regionally specialized areas of ectocytoplasm were organized in a topographically precise manner along the body of the parasite. Examination of labeling patterns after colchicine treatment suggests that different types of perikarya are specialized for biosynthesis of specific tegumental molecules and for turnover or recycling of tegumental material. Furthermore, it appears that a 52 kDa polypeptide synthesized by one population of perikarya is transported through the syncytium and ultimately resorbed by a different population of tegumental perikarya. These data suggest that the syncytial epidermis of parasitic platyhelminthes exhibits a more complex organization of function than previously appreciated.

Survival and viability of Dirofilaria immitis microfilariae in defined and undefined culture media.

In vitro cultivation of Dirofilaria immitis microfilariae has been utilized to observe worm development (Devaney, 1981, Acta Tropica 38: 251-260) as well as to perform immunological (El-Sadr et al., 1983, Journal of Immunology 130: 428-434; Rzepczyk and Bishop, 1984, Parasite Immunology 6: 443-457), pharmacological (Devaney and Howells, 1984, Annals of Tropical Medicine and Parasitology 73: 139-144), and physiological (Jaffe and Doremus, 1970, Journal of Parasitology 56: 254-260; Ando et al., 1980, American Journal of Tropical Medicine and Hygiene 29: 213-216) studies. The objective of the present study was to test various culture conditions for long-term maintenance of D. immitis microfilariae. Culture conditions that permitted extended microfilarial survival were further evaluated by testing the ability of cultured microfilariae to develop to infective larvae in mosquitoes. Microfilariae, recovered from infected canine blood by a saponin lysis technique (Grieve et al., 1984, Acta Tropica 41: 271-278), were placed in culture in 1 of the following 4 media: L-15, RPMI1640, F12 and F12K (Gibco, Grand Island, New York). Gentamicin (100 Ag/ml) was added to the various media, and culture fluid consisted of media alone or with heat-inactivated fetal calf serum (FCS) (Flow Laboratories, McLean, Virginia) at concentrations of 40%, 20%, 10%, or 5%. Microfilariae were cultured in 2 ml of media in 110 x 16-mm flat-sided tubes (Nunc, Kamstrup, Denmark) at a concentration of 15,000 microfilariae per ml. Cultures were maintained at 37 C in 5% CO2 in air, and microfilarial motility was assessed weekly. All of the media selected for evaluation in this study were equally proficient at maintaining microfilariae if they were supplemented with 40% FCS. At lower FCS concentrations, microfilariae survived for abbreviated time periods in L-15, RPMI and F12. Reducing the concentration of FCS in F12K to as low as 5% did not increase microfilarial mortality. Over 50% of microfilariae cultured in F12K without FCS survived for 4 wk; addition of 5% FCS allowed 50% survival for 6 wk (Fig. 1). Changing media every 8 days did not enhance microfilarial survival. Microfilariae, recovered after 2 wk in culture in F-12K or F-12K with 5% FCS, were inoculated into female Aedes aegypti mosquitoes using the fluid enema technique described by Grieve et al. (1984, Acta Tropica 41: 271-278) based on Klowden (1981, Transactions of the Royal Society of Tropical Medicine and Hygiene 75: 354358). Mosquitoes were also inoculated with microfilariae recovered directly from canine blood. A dose of 30 microfilariae was administered to each mosquito. Nineteen days after inoculation, the mosquitoes were dissected, and numbers and stages of larval D. immitis were recorded. It was determined that after 2 wk in culture in either F12K or F12K with 5% FCS, microfilariae were still capable of normal development in mosquitoes (Table I).