Abstract
The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO2 atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.
Highlights
Angiostrongylus cantonensis (Nematode: Metastrongiloidea), a nematode parasite, lives in the rat pulmonary artery where it develops to sexual maturity [1], [2]
Minimum Essential Medium Eagle (MEM), amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA), fetal calf serum (FCS) and pepsin were obtained from Sigma-Aldrich Chemical Co
With 10%, 15% and 20% fetal calf serum (FCS), (Fig. 1A and 1B), AcL3 were significantly longer on day 36 than on day 0
Summary
Angiostrongylus cantonensis (Nematode: Metastrongiloidea), a nematode parasite, lives in the rat pulmonary artery where it develops to sexual maturity [1], [2]. A. cantonensis is thought to be the primary causative pathogen of human eosinophilic meningitis or meningoencephalitis in Taiwan, Japan, Southeast Asia and the Pacific Islands [3,4,5] This parasite has spread from its traditional endemic regions of the Pacific islands and Southeast Asia to the South Pacific, Africa, India, the Caribbean, and most recently, to Australia and North America, including the USA, Caribbean islands and Brazil [6]. The females lay eggs that hatch, producing first-stage larvae of A. cantonensis (AcL1) in the terminal branches of the pulmonary arteries. These AcL1 migrate to the pharynx, are swallowed, and are passed in the feces.
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