A Perkinsus organism with 96-98% homology in the ITS and IGR ribosomal gene regions with P. olseni was identified in a moribund chronically-infected ornamental reef clam, Tridacna crocea, imported from Vietnam and housed alone in a home aquarium for nearly two years.5 This is consistent with a shipment of Vietnamese T. crocea imported into the USA to a research setting discovered to be heavily infected with large 7-20 µm Perkinsus sp. organisms by alternative Ray's thioglycollate incubation (ARFTM) and histopathology for trophozoites and schizonts. Polymerase chain reaction (PCR) analysis was strongly positive with Perkinsus genus-specific primers and weakly positive with P. olseni-specific primers6 reflecting a dual infection with a previously uncharacterized P. olseni-like organism and a minor component of P. olseni. Although these findings confirmed the first known incursion of the exotic and OIE-reportable P. olseni and a closely related subspecies or species into the USA,3 the sources, extent of importation, and extent of dissemination of infected ornamental clams within the USA was highly speculative pending further investigation. The current study surveyed T. crocea (3-5 per group) randomly purchased from five popular internet aquarium store sites based in California (2), Pennsylvania, Connecticut, and Florida selling directly to the public and offering shipment throughout the USA. No information was available regarding possible co-housing with animals from other origins or shipments following collection. Gill and mantle of all animals were screened by ARFTM, histopathology and PCR. PCR products for the ITS region were generated using the Perkinsus genus-specific primer set PerkITS-85/750,1 the P. olseni-specific primer set Patl-ITS 140F/600R,4 and the Vietnamese Perkinsus primer set TcPITS (unpublished), and in the IGR nontranscribed spacer (NTS) region using the P. olseni-specific PK1/PK2 primer set.2 Individual purified ITS PCR products were sequenced and compared using ClustalW and NCBI Blastn search to a consensus sequence (Genebank EU871715.1) generated for the new Perkinsus sp. Vietnam, isolated from two infected Vietnamese T. crocea shipments, encompassing the ITS1 (partial), 5.8S ribosomal RNA gene, and ITS2 (partial) and all known Perkinsus sp. The NTS sequences were cloned (Invitrogen TOPO XL PCR cloning kit) and individually sequenced identifying a common nucleotide sequence similar but not identical to P. olseni. All T. crocea from all five vendors were heavily infected as indicated by every method and a few arrived clinically sick or dead; the sandbed clams were negative. Efforts to create a rapid hand-held on-site Perkinsus sp. sensor, generate primary replicating cultures, and ultrastructural characterization are underway. Final designation as a new species or P. olseni subspecies is imminent and will determine the reportable status of the organism which is likely to negatively impact domestic ecosystems and shellfish industries given the wide host range of the aggressively pathogenic P. olseni.7
The authors wish to thank Mr. George Papadi for his technical assistance.
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