In vitro embryonation of Syphacia obvelata eggs

Inrernofionalfournalfor Printed in Grear Briroin

Porosirology

Vol. 23, No. 2, pp. 257-260,

1993 0

IN VITRO

EMBRYONATION OF SYPHACIA

002Cb7519/93 $6.00 + 0.00 Pergamon Press Lrd I993 Australian Sociery for Parmilology

OBVELATA

EGGS

R. L. GRICE and P. PROCIV* Department

of Parasitology,

The University

of Queensland,

St. Lucia, Brisbane,

Queensland

4072, Australia

(Received 27 August 1992; accepted 3 November 1992) Abstract-&ICE R. L. and PROW P. 1993.In vitro embryonation of Syphacia obvelata eggs. International Journal for Parasitology 23: 257-260. Mouse infections with the pinworm, Syphacia obvelata, were evaluated as a potential model of human enterobiasis. Eggs of S. obveluta were found to be much less resistant to adverse environmental factors than those of Enterobius vermiculuris, perishing rapidly when exposed to desiccation or to water. The average number of eggs produced by a female worm was 317 f 29 S.D.(range: 266347), which is about 2-3% of the fecundity of E. vermiculurir. Eggs expressed from gravid S. obvelata were incubated under various conditions, but the only reliable method of supporting complete embryonation was culture on a floating cellophane membrane. At 3o’C on this substrate, eggs were found to be infective between 6 and 42 h, inclusive. The pre-patent period in mice fed these eggs was 11-15 days. The more fastidious developmental and survival requirements of Syphacia eggs indicate that transmission of this species depends on much more intimate contact between hosts than is required by E. vermicularis. INDEX

KEY WORDS:

Syphaciu obvelufa: Enterobius vermicularis; pinworms;

nematode

eggs; mouse

parasites; embryonation.

INTRODUCTION of Syphaciu obveluta infection in mice as a model of human enterobiasis, fundamental differences between the two host-parasite systems became apparent. Conspicuous among these were the embryonation requirements of eggs from the two nematode species. While conclusions from most earlier studies of S. obvelutu are open to question because inappropriate methods were used to embryonate eggs, and/or underlying infections in the mice were not reliably excluded, it is not surprising that Enterobius vermiculurk and S. obvelutu should share important common features. Eggs of both species are deposited by worms onto the perianal skin of their hosts, where they then need to embryonate rapidly (Chan, 1952). However, published findings indicate that eggs of S. obvelutu may be more fastidious in their developmental re-

in water (Philpot, 1924; Cram, 1943; Hsu, 1951). Eggs from macerated gravid female S. obveluta were reported to infect mice by Lawler (1939), who did not specify the incubation period; his findings have not been replicated by others. Prince (1950) failed to embryonate S. obveluta eggs in vitro. Chan (1952) found that, in S. obvelutu eggs incubated in excessively moist, hypotonic conditions, the opercula opened prematurely. while dryness reduced egg viability. In fact, Chan (1952) seems to be alone in consistently being able to embryonate eggs, recovered from worms dissected from mice, by incubating on cellophane membranes at 37 or 29°C. Chan (Chan, K. F. 1956. Abstract in Journal of Parasitology 42 (Suppl.): 18) also succeeded in embryonating eggs, released spontaneously by worms when immersed in normal saline, which became infective after 3-5 h in saline at 37°C. The purpose of this study was to find a reliable means of producing embryonated S. obvelutu eggs for use in infection experiments.

EARLY in our appraisal

quirements. The failure of S. obvelutu eggs to develop in water was first reported by Philpot (1924) who could not sustain eggs expressed from gravid worms in either water or Ringer’s solution, at 22 or at 37°C except on one occasion. By contrast, E. vermicularis eggs embryonate readily and survive for relatively long periods

*To whom all correspondence

MATERIALS AND METHODS Source ofworms. Juvenile outbred Quackenbush mice were purchased from the Central Animal Breeding House of the University of Queensland and maintained under standard conditions, with free access to pelleted food and water. Mice found by perianal sticky-tape testing to be infected were each given an intragastric dose of 1 ml piperazine citrate solution,

should be addressed. 257

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R. L.

GRICE

450 g I-’ (Pharmacia-Chem. Co., Salisbury, Queensland), then placed in wire-bottomed cages over water for 24 h. Voided live worms were collected from the water with a fine mounted needle, and teased apart with a 26G needle to release eggs. Fecundity of S. obvelata. Eight gravid female worms were teased apart on a glass slide, and the number of eggs in each was counted under a compound microscope. Embryonation studies. Eggs were incubated, in triplicate, under the following conditions: (i) smeared onto glass slides kept in humidified Petri dishes at room temperature (2026’C); (ii) spread over the surfaces of glycerine jelly, nonnutrient agar, agar containing antibiotics and anti-fungal agents, and petroleum jelly, which were then kept in covered Petri dishes at room temperature; (iii) mixed into moist, activated charcoal in covered 5 cm Petri dishes, maintained at room temperature and at 37°C; (iv) in 0.85% saline (NaCl solution) under similar conditions; (v) in 1.7% saline at 3o’C; (vi) in 10% formalin at 30°C; (vii) on a 20 pm thick, waterabsorbent (wettable) cellophane membrane (Austpap, Melbourne, Australia), as described by Chan (1952) at 30°C. Circles of cellophane were cut to a size slightly larger than a 10 cm Petri dish. With fine, mounted pins, eggs were pressed from female worms and smeared over the cellophane pieces, which were then floated on distilled water 1cm deep in 3 cm deep Petri dishes and incubated, uncovered. Open jars of water were kept in the incubator to maintain humidity. From cellophane cultures, the eggs were examined microscopically for development only at 5 and 20 h. From the other substrates, small numbers of eggs were removed at 5 h intervals over the first 25 h of incubation and examined. Evidence of viability included intact opercula, progressing development or visible larval movement. Confirmation of infectivity. Apparently viable eggs from cellophane membrane culture were transferred onto tips of orange sticks smeared thinly with petroleum jelly, then into the mouths ofjuvenile parasite-free mice. These comprised a group of 20 4-week old Quackenbush mice which had been confirmed free of S. obvelata infection by daily perianal tape tests for 16 days. Each mouse received approximately 100 eggs embryonated for 20 h, except for 10 mice kept as a

control group. Onset and duration of egg infectivity. Adult worms were dissected from the terminal colons of freshly killed mice, and the eggs expressed were cultured on cellophane membranes. They were fed to 4 week-old SPF mice at 6 h intervals from 0 to 54 h after incubation, then each 12 h until day 4, then on day 8. The mice were swabbed daily for 15 days following exposure. RESULTS

Fecundity of S. obvelata The number of eggs in gravid female S. obvelata was determined as 3 17 * 29 s D (range: 266347). Embryonation Eggs kept on slides appeared first to develop slowly but then dried out, to collapse before embryonation was complete. Eggs kept on glycerine jelly, agar and

and P. PROCIV

and petroleum jelly did not appear to develop beyond the morula stage, although it was difficult to observe embryonation because of the opacity and surface markings of the shells. Most larvae began to degenerate within 25 h without ever developing to an apparently infective form. Eggs kept in water or 0.85% saline, or those which became frankly wet, such as those in charcoal, perished because their prominent opercula burst open. The shells of eggs incubated in 1.7% saline remained transparent with intact opercula, and early embryonation proceeded rapidly, although only in one instance was larval motility observed. Most larvae began to degenerate within 24 h, not having attained an apparently infective form. Similarly, eggs in formalin remained intact, but neither larval development nor motility were observed. Most of the eggs cultured for 20 h on cellophane membranes contained motile larvae. Conjirmation of infectivity The floating cellophane membrane was used for this component of the study, because it was the only substrate on which the eggs developed to apparently infective stages (at 20 h). These eggs were fed to known parasite-free mice, which remained negative by tape test for the first 10 days. On day 11, perianal eggs were found in six of 20 experimentally infected mice and, on days 12 and 13, a further eight were found to be infected. On day 14, all inoculated mice had positive tests and were killed; S. obvelata was found in the large intestine of each, without any other parasites. All control mice were free of S. obvelata by tape tests and necropsy. Onset and duration of egg infectivity Eggs incubated on cellophane at 30°C were already infective at 6 h and were infective at all intervals at 42 h, but not beyond this. Infections in mice fed these eggs became patent at 12-l 5 days after inoculation. DISCUSSION The mean egg production of 319 per female S. obveluta found here is comparable with the 350 reported by Chan (1952). The larger gravid females of E. vermiculuris are much more prolific, each producing an average 10,780 eggs, with individual output ranging from 4672 to 16,888 (Reardon, 1938). The unreliability of reproducing the natural conditions under which S. obvelata eggs embryonate was first recognized by Prince (1950) who criticized previously published methods. Earlier findings most likely were misinterpreted, as the infections detected were probably established in the mice before experimental exposure to supposedly infective eggs. The available anthelminthics could not eradicate entire

Development of S. obveIata eggs

worm burdens, and the only reliable means of diagnosing S. obvelata infection in mice would have been by necropsy; eggs on per&al skin signal only a terminated infection by an individual worm. The uncertainties of eliminating pinwo~ infections from laboratory mice have been well documented (Chan, 1956, abstract cited above; Owen & Turton, 1979). Humidity was not measured in our studies, but it is clear that the state of hydration was critical to the development and survival of S. obvelata eggs. They collapsed if allowed to dry, yet excessive moisture caused rupture and/or premature opening of opercula, killing the embryo. Eggs incubated in hypertonic saline retained their opercula and appeared to embryonate initially, but were not fed to mice because larvae appeared to degenerate after 24 h and did not exhibit motility. Chan (1956, abstract cited above) successfully cultured infective eggs in hypertonic saline, but they were from mature worms, recovered from the lower colon, which ovipositcd spontaneously into the medium. While saline is a convenient medium, our inability to duplicate Chan’s results may reflect the use of eggs dissected from worms expelled by an anthelminthic. They may have been not quite mature, or perhaps ‘natural’ spontaneous oviposition somehow protects the eggs, e.g. by coating with secretions during their release, which would then enable development under less hospitable conditions. Nevertheless, we successfully and repeatedly produced infective eggs by incubating on cellophane membranes at 30°C. This temperature was assumed to approximate that of the mouse perianal region, and the conditions seemed to afford a compromise between excessive moisture and dryness. Temperature influences both the rate of development and the longevity of S. obvefata eggs. While we found that eggs, recovered from worms dissected from freshly killed mice, become infective within 6 h at 30°C according to Chan (1952), eggs incubated at 37°C on cellophane became infective in 5 h, on average, but required 20-24 h at 29°C. We cannot explain this apparent discrepancy, which may reflect difficulties in timing the onset of embryonation and other less obvious experimental factors., Akagi (1973) briefly described the conditions required by mouse pinworm eggs for successful development to infectivity, but it is obvious that he was referring mainly to Aspiculuris tetraptera; he mentioned, in passing, that S. obvelata eggs were much more di~cult to emb~onate. By all accounts, the eggs of E. vermicu~ar~sseem capable of surviving a wider range of conditions than S. obveelata, especially with regard to temperature and humidity {Cram, 1943; Akagi, 1973). In suitable humidity, they become infective in 20 h at 20X, 6-7 h

259

at 33°C and 3-4 h at 37°C; they will not develop below 15°C and die rapidly at temperatures above 40°C (Akagi, 1973). E. vermicularis eggs survive longer in water than in dry air at the same temperature; at any temperature, survival increases with humidity and, at any humidity, survival increases at lower temperatures; even at temperatures of - 8 to - 10°C they can survive for up to 70 h, although with reduced viability (Cram, 1943). E. vermicularis eggs embryonate readily in water, by 5 hat 37°C and 68 h at 22°C (Philpot, 1924; Hsu, 1951). Embryonation has also been observed in horse serum at 37°C (Hsu, 1951), as well as in normal saline, saturated copper sulphate solution and formalin (Zawadowsky, M. M. & Schalimov, L. G. 1930. Abstract in Tropical Diseases Bdletin 27: 982-983). Motile larvae have been expressed from E. vermicularis eggs which were passed in urine, preserved in 5% formalin and examined 3 weeks later, after crossing Australia by mail (unpublished observation). The eggs have been shown to survive exposure to paradichlorobenzene, naphthalene and hydrocyanic acid gas (Cram, 1943). The maximum reported survival time for E. vermicularis eggs in vitro is 19 weeks (Akagi, 1973), whereas we found S. obve~ataeggs to be viable on cellophane for only 42 h. Chan (1956, abstract cited above) reported that S. obvebta eggs, embryonated in normal saline at 37°C would then survive storage at l--&C for up to 14 days. Clearly, eggs of E. vermicular~~ tolerate greater environmental variations, and survive for longer periods, than those of S. obvelata. The fastidious developmental requirements of S, obvelata are reflected by our finding that floating cellophane membrane culture seems to be the most reliable means of obtaining infective eggs. This suggests that its transmission may depend on more intimate contact between individual hosts than is required by E. vermic~lar~s.Findings from one species, therefore, must not be extrapolated to the other, at least in relation to the embryonation and environmental dispersal of their eggs. ~c~~~~~e~~e~e~?-This work was funded by the Australian National Health and MedicaI Research Council.

REFERENCES AKAGIK. 1973. Enterobius vermicularis and enterobiasis. In: Progress of Medical Parasitology in Japan, Vol. 5 (Edited by MORISHITA K., KOMIYAY. & MA~UEAYASHI H.), pp. 233-279. Meguro Parasitological Museum, Tokyo. CHAN K. F. 1952. Life cycle studies on the nematode Syphacia obvetata. American Journal of Hygiene 56: 14-20.

CRAME. B. 1943. Studies on oxyuriasis. XXVIII. Summary and conclusions. American Journal of the Diseases qf children 65: 46-59.

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R. L. GRICEand P. PROW

Hsu K. C. 1951. Experimental studies on egg development, hatching and retrofection in Aspiculuris tetraptera. Journal ofHelminthology 25: 131-160. LAWLER H. J. 1939. Demonstration of the life history of the nematode Syphacia obvelata. Journal of Parasitology 25: 442. OWEN D.

& TURTONJ. A. 1979. Eradication of the pinwonn Syphacia obvelata from the animal unit by anthelminthic therapy. Laboratory Animals 13: 115-l 18.

PHILFQT F.

1924. Notes on the eggs and early development of some species of Oxyuridae. Journal of Helminthology 11: 239-252.

PRINCEM. J. R. 1950. Studies on the life cycle of Syphacia obvelata, a common nematode parasite of rats. Science 111: 6667. REARLWNL.

1938. Studies on oxyuriasis. XVI. The number of eggs produced by the pinworm, Enterobius vermicularis,and its bearing on infection. Public Health Reports 53: 978-984.