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A "universal non-metazoan" polymerase chain reaction (UNonMet-PCR) that selectively amplifies a segment of nonmetazoan Small Subunit (SSU) rDNA gene was validated. The primers used were: 18S-EUK581-F (5'-GTGCCAGCAGCCGCG-3') and 18S-EUK1134-R (5'-TTTAAGTTTCAGCCTTGCG-3') with specificity provided by the 19-base reverse primer. Its target site is highly conserved across the Archaea, Bacteria, and eukaryotes (including fungi), but not most Metazoa (except Porifera, Ctenophora, and Myxozoa) which have mismatches at bases 14 and 19 resulting in poor or failed amplification. During validation, UNonMet-PCR amplified SSU rDNA gene fragments from all assayed protists (n = 16 from 7 higher taxa, including two species of marine phytoplankton) and Fungi (n = 3) but amplified very poorly or not at all most assayed Metazoa (n = 13 from 8 higher taxa). When a nonmetazoan parasite was present in a metazoan host, the parasite DNA was preferentially amplified. For example, DNA from the parasite Trypanosoma danilewskyi was preferentially amplified in mixtures containing up to 1,000 x more goldfish Carassius auratus (host) DNA. Also, the weak amplification of uninfected host (Chionoecetes tanneri) SSU rDNA did not occur in the presence of a natural infection with a parasite (Hematodinium sp.). Only Hematodinium sp. SSU rDNA was amplified in samples from infected C. tanneri. This UNonMet-PCR is a powerful tool for amplifying SSU rDNA from non-metazoan pathogens or symbionts that have not been isolated from metazoan hosts.

The protistan parasite Mikrocytos mackini, the causative agent of Denman Island disease in the oyster Crassostrea gigas in British Columbia, Canada, is of wide concern because it can infect other oyster species and because its life cycle, mode of transmission, and origins are unknown. PCR and fluorescent in situ hybridization (FISH) assays were developed for M. mackini, the PCR assay was validated against standard histopathological diagnosis, and a preliminary phylogenetic analysis of the M. mackini small-subunit ribosomal RNA gene (SSU rDNA) was undertaken. A PCR designed specifically not to amplify host DNA generated a 544 bp SSU rDNA fragment from M. mackini-infected oysters and enriched M. mackini cell isolates, but not from uninfected control oysters. This fragment was confirmed by FISH to be M. mackini SSU rDNA. A M. mackini-specific PCR was then designed which detected 3 to 4 × more M. mackini infections in 1056 wild oysters from Denman Island, British Columbia, than standard histopathology. Mikrocytos mackini prevalence estimates based on both PCR and histopathology increased (PCR from 4.4 to 7.4%, histopathology from 1.2 to 2.1%) when gross lesions were processed in addition to standard samples (i.e. transverse sections for histopathology, left outer palp DNA for PCR). The use of histopathology and tissue imprints plus PCR, and standard samples plus observed gross lesions, represented a 'total evidence' approach that provided the most realistic estimates of the true prevalence of M. mackini. Maximum parsimony and evolutionary distance phylogenetic analyses suggested that M. mackini may be a basal eukaryote, although it is not closely related to other known protistan taxa. KEY WORDS: Denman Island disease · Mikrocytos mackini · PCR validation · Fluorescent in situ hybridization Resale or republication not permitted without written consent of the publisherDis Aquat Org 54: [219][220][221][222][223][224][225][226][227] 2003 (OIE 2000) because it is pathogenic to several oyster species and because its life cycle, mode of transmission, and origins are unknown.Mikrocytos mackini might be understood more thoroughly and managed more effectively if specific molecular diagnostic assays existed to complement conventional histopathological techniques. However, M. mackini small-subunit ribosomal DNA (SSU rDNA) eluded identification for a decade, presumably because 'universal' eukaryotic PCRs preferentially amplified the oyster DNA predominating in host-parasite mixtures, and because pure isolates of M. mackini are not available. We describe here: successful amplification of M. mackini 's SSU rDNA gene using a primer pair designed specifically not to amplify host DNA; proof of the identity of this gene using fluorescent in situ hybridization (FISH); design and validation of a PCR assay specific for M. mackini SSU rDNA; and a preliminary SSU rDNA phylogenetic examination of M. mackini 's affinities. MATERIALS AND METHODSAmplification of presumptive Mikrocytos mackini SSU rDNA. A rapid PCR cloning approa...

The pathogenic protozoan that has caused high mortalities among cultured Japanese scallops, Patinopecten yessoensis (introduced into British Columbia in 1983), is described as a new species, Perkinsus qugwadi. As in other species in the genus Perkinsus, vegetative multiplication occurred in the intercellular spaces and haemolymph sinuses of all organs of the host. Although the morphology of all developmental stages was similar to that of other Perkinsus spp., differences were observed in the ultrastructural morphology of some organelles in the biflagellate zoospore of P. qugwadi. Also, unlike all other Perkinsus spp., P. qugwadi multiplied and was pathogenic at low temperatures (8-15°C), the development of zoospores was found to occur only within the interstitial spaces of living hosts (and only in some heavily infected juvenile scallops that were less than 50 mm in valve height), and the thioglycollate culture technique that is a diagnostic test for all previously described Perkinsus spp. was negative for P. qugwadi.

Patients with neurologic disease make up a substantial minority of medical inpatients in Ethiopia. Noninfectious neurologic disease is at least as common as infectious neurologic disease. Reaching a well-defined final diagnosis occurs in only a minority of cases. Areas for improving the mortality rate include improving the barriers to diagnostic certainty and increasing treatment options for Ethiopian patients.