Detection of Paragonimus metacercariae in the Japanese freshwater crab, Geothelphusa dehaani, bought at retail fish markets in Japan.

Metacercariae, the encysted larval stage of flukes capable of infecting the final and/or paratenic hosts, of Paragonimus miyazakii and of both diploid and triploid forms of P. westermani are found in the Japanese freshwater crab, Geothelphusa dehaani, which acts as the second intermediate host in Japan. This crab is known as Sawagani in Japanese and is widely distributed in Japan, from Hokkaido to Kyushu islands, including Yakushima Island. Both Paragonimus spp. are known to be medically important causes of human infection, although the respiratory symptoms that develop in patients vary according to the form and species of the causative lung fluke. Chronic cough with rusty-colored sputum is the most common symptom of patients infected with the triploid form of P. westermani, while infection with P. miyazakii and the diploid form of P. westermani usually causes pleural effusion without remarkable lesions in the lung parenchyma (1). In Japan, the incidence of Paragonimus infection has increased among long-term foreign residents (2,3). It is postulated that long-term residents from Asian countries such as China, Korea and Thailand maintain their dietary habits in Japan and, thus, ingest uncooked Sawagani in their ethnic dishes. Infection of people outside of these groups who eat these dishes has also been reported. There is a need for caution regarding paragonimiasis associated with these eating habits. In some cases, the causative foodstuff included in these dishes was identified as Sawagani sold at local retail fish markets. In the present study, we purchased Sawagani originating from three prefectures (Shizuoka Prefecture in the Tokai district, and Miyazaki and Nagasaki prefectures in the Kyushu district) at retail fish markets in the Tokyo metropolitan area between April 2004 and February 2008 and examined these crabs for the prevalence of Paragonimus metacercariae (Table 1). Lung fluke metacercariae were detected in 44 (17%) of 266 examined crabs. The positive crabs harbored a total of 169 metacercariae, with the average numbers of metacercariae being 3.8 and 0.64 per positive crab and per crab of the total number of crabs examined, respectively. The maximum number of metacercariae in a single crab was 23 in a crab originating in Miyazaki Prefecture that was purchased in February 2008. Individual metacercariae isolated from the crabs were identified to the species (P. westermani or P. miyazakii) and, further, to the form (diploid or triploid) for P. westermani. The metacercariae of P. miyazakii could be morphologically discriminated from those of P. westermani by the presence of a membranous substance, as well as by the absence of a stylet (1). Of a total of 169 isolated metacercariae, both of these characteristics were confirmed in only 20 metacercariae, which were identified as P. miyazakii. The remaining metacercariae were subjected to molecular identification by PCR-restriction fragment length polymorphism (RFLP) analysis and sequencing. First, the total genomic DNA was prepared from individual metacercariae following our previously described method (4). The ITS2 region of the nuclear ribosomal DNA (rDNA) and a portion of the 16S mitochondrial rDNA were amplified by PCR using primer pairs 3S (forward: 5 -́GGTA CCGGTGGATCACTCGGCTCGTG-3 ́) with A28 (reverse: 5 -́GGGATCCTGGTTAGTTTCTTTTCCTCCGC-3 ́) (5) and T7-1 (forward: 5 -́ATTTACATCAGTGGGCCGTC-3 ́) with SP6-1 (reverse: 5 -́GATCCAAAAGCATGTGAAAC-3 ́) (6), respectively. The amplified products were treated with restriction enzymes and separated by electrophoresis on agarose gel (RFLP analysis). For the RFLP analyses, we selected restriction enzymes SnaBI and BssSI to digest the ITS2 PCR products from P. westermani and P. miyazakii (4). We selected enzymes SnaBI and BsrDI based on the theoretical restriction maps generated from the 16S mitochondrial rDNA sequences of diploid and triploid forms of P. westermani (6,7). Undigested amplicons were sequenced using the corresponding primers to verify the identification made by RFLP analyTable 1. Prevalence, number and species of Paragonimus metacercariae in Japanese freshwater crabs, Geothelphusa dehaani, sold at retail fish markets in the Tokyo metropolitan area, Japan