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Effect of "in vitro"cultivation time on the infectivity of Toxocara canis eggs

Toxocara canis; "in vitro" cultivation; Infectivity; Immune response

BRIEF COMMUNICATION

EFFECT OF "IN VITRO"CULTIVATION TIME ON THE INFECTIVITY OF Toxocara canis EGGS

Susana Zevallos LESCANO (1 (1 ) Instituto de Medicina Tropical de São Paulo, LIM 06, São Paulo, SP, Brasil. ), Maria Cristina NAKHLE (1 (1 ) Instituto de Medicina Tropical de São Paulo, LIM 06, São Paulo, SP, Brasil. ) & Pedro Paulo CHIEFFI (1 (1 ) Instituto de Medicina Tropical de São Paulo, LIM 06, São Paulo, SP, Brasil. )

KEYWORDS:Toxocara canis; "in vitro" cultivation; Infectivity; Immune response.

Human Toxocariasis is a serious epidemiological problem in many countries2. It is elicited by larvae of nematodes of the genus Toxocara; T. canis a parasite of canines is the major agent of human toxocariasis, whereas T.cati, infecting felines, causes this disease less frequently6. An important role in maintaining toxocariasis of carnivores may be played by small mammals-paratenic hosts of Toxocara spp. The occurrence of toxocaral antibodies in small mammals strongly suggests environmental contamination with infective eggs of Toxocara spp. The antibody level may be an indicator of the number of eggs ingested by the paratenic hosts, as a correlation has been ascertained between the infective dose and the antibody level1,8.

The eggs of T.canis are extremely resistant to chemical and climatic agents and it is suspected that they may survive in an appropriate external environment for about 6 years like the related nematode Ascaris lumbricoides9. Since they are quite sensitive to dessiccation and temperatures above 370C, we do not know how many eggs passed by dogs with patent infections remain infective in the soil.

The first references about the "in vitro" development of ascarids eggs are from STEWART14 who obtained the partial segmentation of Ascaris eggs submitting them to a saturated atmosphere by adding water to an hermetic system. PITTS11 got the total development of A.suum eggs maintained in saline solution at 370C during four weeks. Since then ,different substances were assayed looking for an optimal medium for experimental development of these eggs till the using of formalin solution. GONZALEZ7 did not find any significative difference among saline solution, formalin at 1%, Ringer-Lactate solution and Phosphate-buffered saline when used as media to incubate T.canis eggs.

Toxocara eggs are unembryonated and not infectious when passed in the feces of dogs and cats into the environment. Within a period of between 3 to 6 weeks to several months, depending on soil type and climatic conditions, such as temperature and humidity, eggs develop to an infectious stage that it is supposed to survive under optimal circumstances for at least 1 year10. Several studies from all over the world demonstrated high rates of soil contamination with Toxocara eggs in parks, playgrounds, sandpits and other public places with positivity values ranging from 1.3% found in Switzerland12 up to 87.5% found in Japan13. Many factors interfere on longevity of these eggs; it is known that they develop better in clay soil, with poor sun light exposure and the rain intensity is considered important to concentrate them. Here in Brasil CASEIRO3 made a survey on soil contamination with Toxocara spp.eggs in public places in Santos, State of São Paulo, between September 1995 and September 1996 and had found higher frequencies of viable eggs between September and November 1995 and between May and June 1996. A possible explanation for this fact would be the occurrence of lower rain index, and the lower temperature when compared with summer months, facilitating eggs survival in soil and justifying the presence of higher number of living eggs in these periods of time. Another explanation would be that proposed by CHIEFFI & MÜLLER4 who had found the same kind of anual variation in the soil of public places in Londrina,State of Paraná., ascribing it to the fact of being more frequent the birth of puppies in these periods, as a consequence of normal periodical variations in the ocurrence of bitches rut.

The present study deals on the effect of cultivation time on the infectivity of T.canis eggs and the level of specific antibodies generated by its inoculation in the murine model. We used 2% formalin to cultivate T.canis eggs at 280 C; larval development and viability were confirmed microscopically prior to inoculation. Inbred male BALB/c mice aged 6 weeks were orally infected with three doses of 100 eggs each in a week period. Mice were divided randomly into 3 groups: GI, 12 animals inoculated with 1 month cultivated eggs; GII, 10 animals inoculated with 11 months cultivated eggs, and 6 mice were maintained without infection as control group.The animals were bled from retroorbital plexus on days 23, 38 and 70 post infection. Antibodies were determined in mice sera using an enzyme-linked immunosorbent assay (ELISA) in microtitre plates using excretory-secretory (ES) antigen, obtained by in vitro cultivation of infective T.canis larvae5.

Figure 1 shows the Optical Density (OD) values from sera at a 1:160 dilution in different days post infection. It can be seen that in mice infected with 1 month cultivated eggs the level of specific antibodies increases continuosly and all the sera were positive with the maximum response on day 70 post infection. GII mice exhibited a weaker overall immune response and it can be seen that at day 23 p.i. all the sera were still negative.On day 38p.i. 70% of sera showed positive results but the OD values were lower than that observed in GI; the same happened with the OD values on day 70 p.i.. Even though the eggs of the two groups harboured infective larvae and the viability was confirmed microscopically the infective degree observed was quite different.

Fig. 1
- The dynamics of ELISA estimated circulating T. canis antibodies in BALB/c mice infected with 1 (

Analyzing our results we can suppose that the degree of infectivity of T. canis eggs maintained in optimal "in vitro"conditions may be variable, decreasing sharply after, at least, 11 months of laboratory maintainance. On the other hand, we can presume that in field conditions, usually more agressive than those found in our experiment, the survival time of T.canis eggs could be shorter. New studies should be done to confirm this hypothesis.

Correspondence to: Susana Zevallos Lescano, Instituto de Medicina Tropical de São Paulo, Lab. Imunopatologia da Esquistossomose, Av. Dr. Enéas C. Aguiar 470, 05403-000 São Paulo, SP, Brasil.

Received 24 April 1998

Accepted: 08 June 1998

  • 1. ALJEBOORI, T.I. & IVEY, M.H.- Toxocara canis infections in baboons. I. Antibody, white-cell and serum-protein responses following infections. Amer. J. trop. Med. Hyg.,19: 249-254, 1970.
  • 2. BARRIGA, O.O.- A critical look at the importance, prevalence and control of toxocariasis and the possibilities of immunological control. Vet. Parasit., 29:195-234, 1988.
  • 3. CASEIRO, M.M.- Síndrome de Larva Migrans Visceral causada por larvas de Toxocara canis (Werner, 1782 e Stiles,1905), no Município de Santos, Săo Paulo, 1994-1996 Săo Paulo, 1996. (Dissertaçăo de Mestrado-Faculdade de Medicina da Universidade de Săo Paulo).
  • 4. CHIEFFI , P.P. & MÜLLER, E.E.- Prevalęncia de parasitismo por Toxocara canis em căes e presença de ovos de Toxocara sp no solo de localidades públicas da zona urbana do Município de Londrina, Estado do Paraná, Brasil. Rev.Saúde públ. (S.Paulo), 10: 367-372, 1976.
  • 5. DE SAVIGNY, D.H.- In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigen for use in serodiagnostic tests for Visceral Larva Migrans. J. Parasit., 61: 781-782, 1975.
  • 6. GILLESPIE, S.H.- The epidemiology of Toxocara canis Parasit. today, 4: 180-182, 1988.
  • 7. GONZÁLEZ, S.- Estudio experimental de Toxocara canis en fase larvaria. Madrid, 1983. (Tesina de licenciatura, Facultad de Farmacia de Madrid).
  • 8. KAYES, S.G.; OMHOLT, P.E. & GRIEVE,RB.- Immune responses of CBA/J mice to graded infections with Toxocara canisInfect.Immun. 48: 697-703, 1985.
  • 9. MUELLER, G.- Untersuchungen uber die Lebensdauer von Askarideiern in Gertenerde. Zentalbl.Bakt.I.Orig., 159: 377-379,1953.
  • 10. OVERGAAUW, P. A. M. - Aspects of Toxocara epidemiology: toxocarosis in dogs and cats. Crit. Rev. Microbiol., 23: 233-251, 1997.
  • 11. PITTS, T.D.- Experimental hatching of the eggs of Ascaris suum Proc. Soc. exp. Biol.,69: 348-351,1948.
  • 12. SALM, C.- Helminthen des Hundes-potentielle Gefahrdung der menschlichen Gesundheit? Eine Untersuchung zur umwelthygienischen Bedeutung des Hundekots am Beispiel des Kantons Basel-Stadt. Schweiz.Arch.Tierheilkd., 128:53, 1986.
  • 13. SHIMIZU, T.- Prevalence of Toxocara eggs in sandpits in Tokushima city and its outskirts. J. vet. med. Sci. 55: 807-811, 1993.
  • 14. STEWART, F.H.- A method of incubating Ascaris eggs.J. med. Res., 22:1, 1934.
  • (1
    ) Instituto de Medicina Tropical de São Paulo, LIM 06, São Paulo, SP, Brasil.
  • Publication Dates

    • Publication in this collection
      19 Jan 1999
    • Date of issue
      May 1998

    History

    • Received
      24 Apr 1998
    • Accepted
      08 June 1998
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