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United States Department of Agriculture

Agricultural Research Service

Research Project: POSTHARVEST TREATMENT OF TROPICAL COMMODITIES FOR QUARANTINE SECURITY, QUALITY MAINTENANCE, AND VALUE ENHANCEMENT

Location: Tropical Crop and Commodity Protection Research

Title: PCR-based detection of Angiostrongylus cantonensis in tissue and mucus secretions from molluscan hosts

Authors
item Qvarnstrom, Yvonne - CDC
item Sullivan, James - CDC
item Bishop, Henry - CDC
item Hollingsworth, Robert
item Da Silva, Alexandre - CDC

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 19, 2006
Publication Date: March 31, 2007
Citation: Qvarnstrom, Y., J.J. Sullivan, H.S. Bishop, R.G. Hollingsworth, A.J. da Silva. 2007. PCR-based detection of Angiostrongylus cantonensis in tissue and mucus secretions from molluscan hosts. Applied and Environmental Microbiology. 73:1415-1419.

Interpretive Summary: Angiostrongylus cantonensis is a nematode which causes a type of meningitis, or swelling of the brain and the surrounding tissues. Recent outbreaks of this disease made it necessary to determine the distribution of this nematode in the environment, in order to prevent the disease from occurring. The body structure of the nematode A. cantonensis is generally identified in the molluscan intermediate host (a slug or a snail) by microscopic examination, a labor-intensive method. The aim of this study was to develop a PCR-based method (polymerase chain reaction) to detect A. cantonensis directly from molluscan tissue. Thirty-four Parmarion cf. martensi (Simroth) semi-slugs, of which 25 were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces were digested with pepsin-HCl to recover third stage larvae for microscopic exam, or were used for DNA extraction. PCR primers were designed to amplify 1134 base pairs from the A. cantonensis 18S ribosomal RNA gene and amplicons produced were sequenced for confirmation of the species. Both microscopy and PCR with DNA sequencing identified the same 25 semi-slugs as positive for A. cantonensis, indicating that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas PCR can also be performed on frozen tissue. The PCR with DNA sequencing was further evaluated on tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. This is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.

Technical Abstract: Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection in endemic regions have prompted a need to determine the distribution of this nematode in the environment to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. Thirty-four Parmarion cf. martensi (Simroth) semi-slugs, of which 25 were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces of approximately 25 mg were digested with pepsin-HCl to recover third stage larvae for morphologic identification, or used for DNA extraction. PCR primers were designed to amplify 1134 base pairs from the A. cantonensis 18S ribosomal RNA gene and amplicons produced were sequenced for confirmation of the species. Both microscopy and PCR with DNA sequencing identified the same 25 semi-slugs as positive for A. cantonensis, indicating that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas PCR can also be performed on frozen tissue. The PCR with DNA sequencing was further evaluated on tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.

Last Modified: 9/29/2014
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