Márcia Bohrer MentzI; Carlos Graeff-TeixeiraII
ISetor de Parasitologia do Departamento de Microbiologia do Instituto de Ciências Básicas da Saúde da Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil IIInstituto de Pesquisas Biomédicas da Pontifícia Universidade Católica de Porto Alegre,RS, Brasil
ABSTRACT
Fifteen pairs (male/female) of Angiostrongylus costaricensis were kept in vitro in Waymouth medium for three days to evaluate the amount and duration of egg laying. At 24, 48 and 72 hours, the mean egg counts were 321, 24 and 4 eggs/10 microliters, respectively. Most of the eggs were eliminated within the first 24 hours, suggesting they are expelled under non-physiological conditions. These results indicate that in vitro conditions are not appropriate for drug trials of egg-laying inhibitors for treatment of abdominal angiostrongylosis.
Key-words: Angiostrongylus costaricensis. Abdominal angiostrongyliasis. Egg laying.
RESUMO
Quinze duplas (macho/fêmea) de Angiostrongylus costaricensis foram mantidas in vitro, em meio de Waymouth durante 3 dias, para observação da quantidade e duração da oviposição. Médias de 321, 24 e 4 ovos em 10 microlitros foram registradas em 24, 48 e 72 horas, respectivamente. A maioria dos ovos foi eliminada nas primeiras 24 horas, sugerindo terem sido expulsos em condições não fisiológicas. Estes resultados indicam que as condições in vitro não são adequadas para testes de drogas inibidoras da oviposição, para tratamento da angiostrongilíase abdominal.
Palavras-chaves: Angiostrongylus costaricensis. Angiostrongilíase abdominal. Oviposição.
Angiostrongylus costaricensis Morera & Cespedes, 1971, is a parasite nematode of the mesenteric arterial system in wild rodents5 16 19. The intermediate hosts are terrestrial mollusks, mostly of the Veronicellidae family8 18. Abdominal angiostrongylosis may result from accidental human infection, leading to severe ischemic and inflammatory intestinal lesions4 6 17. Since the first report in Costa Rica19, many human cases have been reported from Mexico14 15 to Brazil1 2 3 6 13 23 24 . Besides its medical importance, Angiostrongylus costaricensis has been selected as a model to study the diseases caused by tissue nematodes and the efficacy of anthelmintics20. The large scale production of larvae and worms, as well as the principle of minimizing laboratory animal use justifies the need for in vitro maintenance of parasites. None of the reports from trials of in vitrocultivation of A. costaricensis were concerned with the quantity and duration of egg laying10 11 12 20 21. However, these data are important to evaluate the usefulness of an in vitro model for drug therapy of abdominal angiostrongylosis. Eggs are considered one of the main causative factors for granulomatous inflammatory reaction in human disease. The objectives of this experiment were to evaluate the quantity and duration of in vitro egg laying by Angiostrongylus costaricensis. Male, 6-week-old, Mus domesticus, Swiss strain, were used as the definitive host and were inoculated (per os) with 10 infective third-stage larvae of A. costaricensis (Santa Rosa strain) each. These were obtained from experimentally-infected Biomphalaria glabrata snails, by artificial digestion using 0.03% pepsin (Sigma P 7125) solution in 0.7% HCL for 2 hours at 37ºC8 22. Following exposure to larvae, each group of five animals were caged and given food and water ad libitum. All surviving mice were sacrificed at 34 days post infection (dpi) through anesthetic depression. Adult worms were recovered from the mesenteric artery, transferred into Petri dishes and washed with PBS. After washing, a total of 15 male/female pairs were separated and transferred onto another plate, containing Waymouth medium (GIBCO) plus antibiotic and amphotericin and incubated at 37ºC and 5% CO2. The medium was changed at 1-day intervals. Eggs were examined under a stereomicroscope and counted, every 24 hours, for 72 hours. Most of the eggs were recovered in the first 24 hours, with a mean 321 eggs/female/10mL. After 48 hours, the number of eggs decreased dramatically, to 24 eggs/female/10mL. After 72 hours, only a few dark and deformed eggs were recovered (Table 1).
Chemically defined Waymouth medium, is reportedly the best culture medium for the development of L3 into young adult stage, with a 60% success rate10 11 12. Otherwise, when mature female worms removed from the definitive host are kept in the same culture medium, a very intense and extensive egg elimination takes place, suggestive of a stress response.
In conclusion, these data indicate that the in vitro conditions tested are not a good experimental model for testing drug therapy for abdominal angiostrongylosis.
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Correspondece to
Dra. Márcia Bohrer Mentz
R. Fernandes Vieira 634/801, Bairro Bom Fim
90035-090 Porto Alegre, RS
e-mail: mbmentz@uol.com.br
Recebido para publicação em 6/2/2004
Aceito em 16/12/2004
Financial support: CAPES-PROBAL, 055/97 AND PUC/RS
