First report of a Mikrocytos-like parasite in European oysters Ostrea edulis from Canada after transport and quarantine in France

Abstract

As part of a disease resistance experiment, 112 apparently healthy European flat oysters Ostrea edulis L. were exported from Canada (Nova Scotia) into France to test their susceptibility to Bonamia ostreae infection. Twelve oysters died in transit and 17 others died within 2 wk of laboratory quarantine acclimation. All oysters were examined histologically, and the 17 that died during quarantine were assayed for microcells (Bonamia sp. and Mikrocytos mackini) using molecular techniques. A microcell parasite was detected in the connective tissue of 5 of the 112 oysters. Morphological appearance, tissue affinity and molecular characterization through PCR, in situ hybridization (ISH), fluorescence in situ hybridization (FISH) and sequencing revealed a protist related to M. mackini. This is the first report of a parasite of the genus Mikrocytos in a species belonging to the genus Ostrea from the Atlantic Ocean. KEY WORDS: Mikrocytos · Ostrea edulis · In situ hybridization · PCR · Histology Resale or republication not permitted without written consent of the publisherDis Aquat Org 80: [27][28][29][30][31][32][33][34][35] 2008 MATERIALS AND METHODSOysters. The oysters from Nova Scotia were produced in a hatchery and transferred to open water after 4 mo. They were held in suspended culture for the duration of grow out and were ca. 3 yr of age when shipped to France (A. Mallet, pers. comm.). In April 2001, 60 adult Ostrea edulis were collected from this population and histological examination of tissue cross-sections and heart smears to check for the absence of lesions and/or pathogens was done at the Shellfish Health Laboratory of the Fisheries and Oceans Canada, Gulf Fisheries Center (DFO-GFC), Moncton, NB, Canada. Subsequently, 112 cohorts from the same site were shipped in June 2001 and were received after 6 d of transit and maintained in quarantine at the IFREMER laboratory, La Tremblade, France. The quarantine system used chlorine treatment for effluent water, but the incoming water was not treated. Temperature of the water was 14°C.Following the detection of a presumptive Mikrocytos sp. in the oysters at IFREMER, 135 American oysters Crassostrea virginica and 275 Ostrea edulis were collected from several beds in Nova Scotia in late 2001 and in 2002. Of these, 3 samples totaling 189 O. edulis (46%) came from the initial site of sampling (Table 1). A total of 104 O. edulis from this site were examined by histology for pathogens and all of the oysters were examined by PCR for the presence of Mikrocytos sp. and Bonamia ostreae at DFO-GFC.Histology. Oysters were opened and a standard section through the digestive gland, gills, labial palps and mantle was fixed in Davidson's fixative (Moore et al. 1953), paraffin embedded and sections (5 µm thick) stained with haematoxylin and eosin (H&E). A complete examination of the cross-section was performed at 100 × magnification with further examination of any unusual pathology and specifically for the presence of microcells at 1000 × magnification (OIE 2003).Heart ...

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location

“…Although our ability to characterize this parasite was severely limited by our lack of success in visualizing it using histology or in situ hybridization, the fact that we were able to sequence a much larger portion of the rDNA cistron (1903 bp from the 18S to 28S gene) -compared to previous findings of Mikrocytos sp. organisms (486 bp of 18S; Gagné et al 2008, Wang et al 2010) -allows some preliminary investigation into its evolutionary relationship to M. mackini. The use of 18S rDNA sequence data for phylogenetic studies of eukaryotic taxa has been prolific since the 1980s (cf.…”

“…These fragments were sequenced as an early check for any divergent forms, as this PCR amplified Mikrocytos sp. parasites in earlier studies: herein referred to as 'Mikrocytos sp.-Atl', to reflect its origin in Ostrea edulis from the Atlantic Ocean (Gagné et al 2008), and 'Mikrocytos sp.-YS', to reflect its origin in C. gigas from the Yellow Sea, China (Wang et al 2010).…”

“…However, scant infections by a Mikrocytos sp. have been detected in European flat oysters Ostrea edulis collected in the Atlantic Ocean on the east coast of Canada (Gagné et al 2008), and in Pacific oysters from the Yellow Sea, China (Wang et al 2010). These 2 geographically disparate Mikrocytos sp.…”

“…The first, called Mikrocytos sp.-Atl here, was reported by Gagné et al (2008), and the second, called Mikrocytos sp.-YS here, was reported by Wang et al (2010). For our newly discovered Mikrocytos sp.-BC sample, we sequenced a total of 1903 bp (GenBank accession no.…”

“…mentioned above crossreacts with the PCR reaction originally designed by Carnegie et al (2003) to be specific to M. mackini (Gagné et al 2008, Wang et al 2010. Although M. mackini was recently de-listed by the World Organisation for Animal Health's (OIE) Aquatic Animal Health Code, it continues to be subject to regulatory testing in Canada.…”