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ARS Home » Pacific West Area » Hilo, Hawaii » Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center » Tropical Crop and Commodity Protection Research » Research » Publications at this Location » Publication #252550
Title: Improved molecular detection of Angiostrongylus cantonensis in mollusks and other environmental samples with a species-specific ITS1-based TaqMan assay

Author
item QVARNSTROM, YVONNE - US Department Of Health And Human Services (HHS)
item DA SILVA, ANA CRISTINA AR - Pontifical Catholic University
item TEEM, JOHN - Florida Department Of Agriculture And Consumer Services
item Hollingsworth, Robert
item BISHOP, HENRY - Centers For Disease Control And Prevention (CDC) - United States
item GRAEFF-TEIXEIRA, CARLOS - Pontifical Catholic University
item DA SILVA, ALEXANDRE - US Department Of Health And Human Services (HHS)

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2010
Publication Date: 8/1/2010
Citation: Qvarnstrom, Y., Da Silva, A.A., Teem, J.L., Hollingsworth, R.G., Bishop, H., Graeff-Teixeira, C., Da Silva, A.J. Improved molecular detection of Angiostrongylus cantonensis in mollusks and other environmental samples with a species-specific ITS1-based TaqMan assay. Applied and Environmental Microbiology; 76: 5287-5289.

Interpretive Summary: The nematode Angiostrongylus cantonensis (also called the rat lungworm) is the most common cause of human eosinophilic meningitis [meningitis with a high percentage of eosinophils (a type of white blood cell) in the cerebrospinal fluid]. Humans can become infected by ingesting food items contaminated with the third-stage infectious nematode larvae released from infected mollusks as well as by ingesting mollusks or paratenic hosts carrying the infectious stage of the parasite. Paratenic hosts are intermediate hosts in which the parasite can survive but not develop. In this study we report the development and application of an improved PCR (polymerase chain reaction) test which can be used to identify the presence of Angiostrongylus cantonensis in the tissue of mollusks (slugs and snails). DNA was extracted from third-stage larvae obtained from naturally and experimentally infected mollusks to establish the sequence variability of a specific genetic sequence found in A. cantonensis, A. vasorum and A. costaricensis. Based on these sequences, a real-time PCR assay was designed to specifically detect A. cantonensis. The assay was validated on 233 mollusk samples from U.S. regions and 18 samples containing other nematode species. Compared to a previously developed method based on PCR amplification of the 18S rRNA gene followed by sequencing analysis, this new assay was more specific, had superior detection limit and was less prone to producing false negative results due to the presence of PCR inhibitors in the samples. This real-time PCR assay will be a useful tool for environmental surveys of local wildlife to determine the geographic distribution of this reemerging human parasite.

Technical Abstract: Angiostrongylus cantonensis is the most common cause of human eosinophilic meningitis. Humans can become infected by ingesting food items contaminated with the third-stage infectious larvae released from infected mollusks as well as by ingesting mollusks or paratenic hosts carrying the infectious stage of the parasite. In this study we report the development and application of a real-time PCR TaqMan assay targeting the Internal Transcribed Spacer 1 (ITS1) for the identification of A. cantonensis in mollusk tissue. DNA was extracted from third-stage larvae obtained from naturally and experimentally infected mollusks and the ITS1 was sequenced to establish the sequence variability of the PCR target in A. cantonensis, A. vasorum and A. costaricensis. Based on these sequences, a real-time PCR assay was designed to specifically detect A. cantonensis. The assay was validated on 233 mollusk samples from U.S. regions and 18 samples containing other nematode species. Compared to a previously developed method based on PCR amplification of the 18S rRNA gene followed by sequencing analysis, this new assay was more specific, had superior detection limit and was less prone to producing false negative results due to the presence of PCR inhibitors in the samples. This real-time PCR assay will be a useful tool for environmental surveys of local wildlife to determine the geographic distribution of this reemerging human parasite.