Proc. Helminthol. Soc. Wash. 55(1), 1988, pp. 15-18

In Vitro Culture of procyonis and Initial Analysis of Larval Excretory-Secretory Antigens

W. M. BOYCE, B. A. BRANSTETTER, AND K. R. KAZACOS Department of Veterinary Microbiology, Pathology and Public Health, School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907

ABSTRACT: Larvae of hatched from embryonated eggs were cultured in vitro for 6 wk. Larvae molted once during culture, and increased in length from x = 0.278 to 0.513 mm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of excretory-secretory (ES) ma- terials collected from these cultures demonstrated a heterogeneous group of antigenic proteins ranging from 10 to 70 kd. Immunoblots using these ES antigens allowed for the differential diagnosis of B. procyonis from infections in rabbits, and they may prove useful in the immunodiagnosis of B. procyonis infections in other , including humans. KEY WORDS: , in vitro culture, Baylisascaris procyonis, Toxocara canis, excretory-secretory (ES) antigens, SDS-PAGE, immunoblotting, immunodiagnosis.

Baylisascaris procyonis, the common ascarid dium (DMEM, GIBCO), containing 100 units/ml pen- of (Procyon lotor}, has recently emerged icillin and 100 /ug/ml streptomycin, in 24-well poly- as an important cause of migrans in ani- styrene tissue culture plates (Corning Glass Works, Corning, New York). Each well contained 2 ml DMEM mals and humans (Kazacos, 1986). Currently, with a larval concentration of 10,000/ml. Larvae were definitive diagnosis of larva migrans due to B. maintained for 6 wk at 37°C in 5% CO2 and compressed procyonis requires microscopic identification of air and the media changed weekly. Larval mortality larvae either within or recovered from tis- was assessed on a weekly basis by examining 100 larvae stained with 1:10,000 new methylene blue. Dead larvae sues (Buffet al., 1984; Fox etal., 1985). A specific rapidly absorbed the dye along their cuticles while live immunoassay for B. procyonis infections is larvae remained largely unstained and were motile. therefore highly desirable. Excretory-secretory Samples of larvae were also collected at regular inter- (ES) materials released by parasitic vals and fixed in hot alcohol-formalin-acetic acid (AFA) during in vitro culture represent a potential source for morphometric and developmental analyses. Culture medium collected at weekly intervals was of useful diagnostic antigens. This report de- dialyzed against DPBS and concentrated by diafiltra- scribes an in vitro culture technique for larvae tion using spiral cartridge and stir cell apparatuses with of B. procyonis, and the initial analysis of ES molecular weight exclusion limits of 10,000 (Amicon antigens collected from this system. Corp., Danvers, Massachusetts). Protein concentration determinations, SDS-PAGE, and immunoblotting were Materials and Methods performed according to manufacturer's instructions, utilizing equipment and reagents obtained from Bio- Adult B. procyonis were obtained from the intestines Rad (Richmond, California). SDS-PAGE was per- of naturally infected raccoons, and eggs were collected formed under reducing conditions in a discontinuous and embryonated as described by Kazacos et al. (1981). system utilizing 4% stacking and either 12% or 15% Embryonated eggs were washed 3 times in 0.85% NaCl separating gels. Following completion of the SDS-PAGE and decoated for 30-90 min (without agitation) at 37°C run, gels were either silver stained or the proteins were in a 1:1 mixture of 5.25% sodium hypochlorite and 1% electrophoretically transferred from the gels onto ni- NaOH. Decoating was stopped when the outer layers trocellulose using a Bio-Rad transblot apparatus. SDS- of the egg shells had been removed and the remaining PAGE and silver staining were performed with be- vitelline membranes became distorted by the move- tween 3.5 and 4.5 ng of protein collected from each of ments of the enclosed larvae. Following decoating, eggs the 6 wk of culture. Immunoblotting was performed were washed 4 times in 0.85% NaCl and hatched in with pooled protein samples collected from the 1st 3 Earle's balanced salt solution (EBSS, Grand Island Bi- wk of culture, utilizing 200 /ug of protein per 16x11 ological Co. [GIBCO], Grand Island, New York) as cm gel. described for suum by Urban et al. (1981). The Following protein transfer, the portion of the nitro- resulting larval suspension was washed 2 times in EBSS cellulose containing molecular weight standards was and layered over sterile cotton gauze in a glass funnel cut off and stained with 0.1% amido black. Reactive and maintained overnight at 37°C. The following sites were blocked with 3% gelatin and the membranes morning, larvae were collected aseptically in 10 ml incubated for 3 hr in a 1:100 dilution of antisera. Anti- aliquots and washed 6 times in warm, sterile Dulbec- sera were obtained from New Zealand White (NZW) co's phosphate buffered saline (DPBS, GIBCO). Larvae rabbits orally inoculated with 10,000 embryonated eggs were dispensed into Dulbecco's modified Eagle's me- of B. procyonis or T. canis. Serum was collected from

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Copyright © 2011, The Helminthological Society of Washington 16 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY

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123 4 56 Figure 1. Silver-stained SDS-PAGE gel (15%) of B. procyonis ES proteins collected at weekly intervals (1-6). Molecular weight standards (in kilodaltons) are 14 in far left lane. these rabbits at 14 and 56 days postinfection, respec- A B C D E tively, as well as from uninfected control rabbits. Se- rum was also collected from 2 pet rabbits naturally Figure 2. I mmunohlots of B. procyonis ES antigens infected with B. procyonis following accidental expo- transferred from 12% SDS-PAGE gels. Lane A = Nor- sure to B. procyonis eggs from a previously mal rabbit sera. Lane B = Toxocara canis antisera, housed in the same cage. The size of the infective dose rabbit, 56 days PI. Lane C = Baylisascaris procyonis and the duration of infection could not be positively antisera, rabbit, 14 days PI. Lanes D and E = Sera determined for these rabbits. However, based on pre- from 2 rabbits naturally infected with Baylisascaris pro- vious observations they had probably ingested large cyonis. Approximate molecular weights (in kilodaltons) numbers of eggs within the previous 3-6 wk (Kazacos, are shown on the right. 1986). Following incubation in rabbit antisera, nitro- cellulose membranes were incubated for 1 hr in a 1:3,000 dilution of goat anti-rabbit IgG (H and L chains) conjugated with horseradish-peroxidase. Color devel- eggs, or recovered following hatching and Baer- opment was visualized after the addition of 4-chloro- mannization, were loosely enclosed in the cuticle 1-naphthol. of the previous stage. Within 24 hr 8% of the larvae had shed this cuticle, and by 72 hr > 90% Results and Discussion of larvae had completed this molt. Larvae con- Initially, decoating of embryonated eggs of B. tinued to grow throughout the culture period, but procyonis required periods of time varying from no further molts were detected. lar- 30 to 90 min. However, subsequent trials re- vae have also been shown to grow and develop vealed that decoating could routinely be accom- when cultured in DMEM (Urban and Tromba, plished in 30 min when 10 ml of eggs were gently 1982), whereas T. canis larvae did not molt in agitated with 2 changes of 5.25% sodium hy- DMEM when maintained for periods up to 18 pochlorite (without 1 % NaOH) on a shaker plat- mo (de Savigny, 1975). form. Larvae either expressed from embryonated In the present study it is unclear whether the

Copyright © 2011, The Helminthological Society of Washington OF WASHINGTON, VOLUME 55, NUMBER 1, JANUARY 1988 17 cuticle shed during the first few days of culture in T. canis ES components from different culture represents the 1 st or 2nd molt. Sprent et al. (1973) batches (Speiser and Gottstein, 1984; Badley et stated that the 2nd molt took place 1-2 wk after al., 1987). infection of mice with eggs of either B. devosi or Equal amounts of ES proteins were pooled from B. tasmaniensis, and Douvres et al. (1969) stated the 1 st 3 wk of culture for use in immunoblotting, that the 2nd molt of A. suum occurred in the since larval mortality was > 10% after 3 wk. The liver within 1 wk. In contrast, Maung (1978) major ES proteins identified by silver staining of reported that the 1st 2 molts of A. suum occur polyacrylamide gels were recognized by immu- within the egg, and that 3rd-stage larvae may noblot analysis with sera from rabbits infected hatch from the egg still enclosed within the 2nd- with B. procyonis (Fig. 2). Serum from the T. stage cuticle. Further work is needed to deter- canis infected rabbit cross-reacted with several mine which larval stage of B. procyonis, as well B. procyonis ES antigens, with the notable ex- as other ascarids, hatches from the embryonated ceptions of those having apparent molecular egg. weights of 18, 39 (doublet), and 43 kd. These After hatching and Baermannization, B. pro- results have been substantiated by immunoblot cyonis larvae measured an average of 0.278 mm analysis using sera from several other T. cams- in length (N = 25). The larvae continued to grow infected rabbits, and immunoblotting using ES averaging 0.333 mm at 1 wk, 0.409 mm at 2 wk, antigens has consistently allowed us to differ- 0.451 mm at 3 wk, and 0.513 mm at 6 wk (TV = entiate B. procyonis infections from T. canis in- 25 at each wk). Larval mortality progressively fections in rabbits. increased from 5% (wk 1), to 9% (wk 3), and Toxocara canis larval ES antigens are of prov- finally to near 50% by 6 wk when the cultures en value in the serodiagnosis of toxocaral visceral were terminated. During the 1st 3 wk of culture, larva migrans (Glickman and Schantz, 1981). each larva produced about 1-3 ng of protein per However, cross-reactions have been detected be- day. Protein production was not determined be- tween T. canis ES antigens and antisera against yond 3 wk because of the large number of dead various other ascarid nematodes (Nicholas et al., and dying larvae which were present in the cul- 1984). In the present study, cross-reactions were tures at this time. detected between T. canis antisera and B. pro- SDS-PAGE analysis of ES proteins collected cyonis ES antigens, but several ES antigens ap- at each week of culture revealed a heterogeneous peared to be specific for B. procyonis. Further mixture with molecular weights ranging from 10 studies are indicated to determine the usefulness to 70 kd (Fig. 1). Quantitative and qualitative of these ES antigens in differentiating B. pro- variations in ES components occurred between cyonis from other ascarid infections in a variety weeks as indicated by the relative intensity of of host species, including humans. staining and the presence or absence of certain bands. These variations tended to correlate with Literature Cited the age of larvae as well as the degree of larval mortality. The relative intensity of staining of Badley, J. E., R. B. Grieve, D. D. Bowman, L. T. Glick- man, and J. H. Hockey. 1987. Analysis of Tox- major components at 35 and 43 kd decreased ocara canis larval excretory-secretory antigens: with time, and by 5 and 6 wk these bands were physiochemical characterization and antibody barely discernible. A more diffuse banding pat- recognition. Journal of Parasitology 73:593-600. tern was detected after wk 4 with the appearance de Savigny, D. H. 1975. In vitro maintenance of Tox- ocara canis larvae and a simple method for the of new bands. This diffuse pattern is also seen production of Toxocara ES antigen for use in ser- with silver-stained gels of homogenized larvae odiagnostic tests for . Jour- (data not shown), and the presence of new bands nal of Parasitology 61:781-782. after wk 4 may be due to the release of proteins Douvres, F. W., F. G. Tromba, and G. M. Malakatis. from dead and dying larvae. Other ES compo- 1969. Morphogenesis and migration of Ascaris suum larvae developing to fourth stage in swine. nents (doublet at 39 kd and band at 60 kd) were Journal of Parasitology 55:689-712. only apparent after wk 1. However, SDS-PAGE Fox, A. S., K. R. Kazacos, N. S. Gould, P. T. Heyde- analyses of numerous other cultures have shown mann, C. Thomas, and K. M. Boyer. 1985. Fatal that these components are sometimes present in eosinophilic meningoencephalitis and visceral lar- va migrans caused by the raccoon ascarid Baylis- material collected from the 1 st week of culture, ascaris procyonis. New England Journal of Med- and they are always present by the 2nd week. icine 312:1619-1623. Qualitative differences have also been detected Glickman, L. T., and P. M. Schantz. 1981. Epide-

Copyright © 2011, The Helminthological Society of Washington 18

miology and pathogenesis of zoonotic toxocaria- tion from by immunization with ES sis. Epidemiologic Reviews 3:230-250. antigens. Australian Journal of Biology and Med- Huff, D. S., R. C. Neafie, M. J. Binder, G. A. De Leon, ical Science 62:619-626. L. W. Brown, and K. R. Kazacos. 1984. The first Speiser, F., and B. Gottstein. 1984. A collaborative fatal Baylisascaris infection in humans: an infant study on larval excretory-secretory antigens of with eosinophilic meningoencephalitis. Pediatric Toxocara canis for the immunodiagnosis of hu- Pathology 2:345-352. man toxocariasis with ELISA. Acta Tropica 41: Kazacos, K. R. 1986. Raccoon ascarids as a cause of 361-372. larva migrans. Parasitology Today 2:253-257. Sprent, J. F. A., J. Lamina, and A. McKeown. 1973. , W. L. Wirtz, P. P. Burger, and C. S. Christmas. Observations on migratory behavior and devel- 1981. Raccoon ascarid larvae as a cause of fatal opment of Baylisascaris tasmaniensis. Parasitol- central nervous system disease in subhuman pri- ogy 67:67-83. mates. Journal of the American Veterinary Med- Urban, J. F., Jr., F. W. Douvres, and F. G. Tromba. ical Association 179:1089-1094. 1981. A rapid method for hatching Ascaris suum Maung, M. 1978. The occurrence of the second molt eggs in vitro. Proceedings of the Helminthological of and Ascaris suum. Inter- Society of Washington 48:241-243. national Journal of Parasitology 8:371-378. , and F. G. Tromba. 1982. Development of Nicholas, W. L., A. C. Stewart, and G. F. Mitchell. immune responsiveness to Ascaris suum antigens 1984. Antibody responses to Toxocara canis us- in vaccinated with attenuated eggs. Veterinary ing sera from parasite-infected mice and protec- Immunology and Immunopathology 3:399-409.

Beltsville Symposia in Agricultural Research

XIII. "Biotic Diversity and Germplasm Preservation: Global Imperatives' 9-11 May 1988 Beltsville Agricultural Research Center Beltsville, Maryland Contact: Mrs. J. Weirman ARS/USDA Room 127, Building 001, Beltsville Agricultural Research Center-West Beltsville, Maryland 20705

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