Continuous in vitro culture of the carpet shell clam Tapes decussatus protozoan parasite Perkinsus atlanticus

Abstract

Continuous in vitro cultures of the clam Tapes decussatus parasite Perkinsus atlanticuswere established from infected gill fragments, infected haemolymph and parasite hypnospores isolated from infected gill fragments following incubation in Ray's fluid thioglycollate medium (RFTM). No continuous cultures could be initiated from P. atlanticus zoospores. Cultures initiated from hypnospores yielded the highest percentage of continuous cultures (100%, 6/6), followed by cultures initiated from gill fragments (93%, 43/46) and from haemolymph (30%, 3/10). Failures to establish continuous cultures were due to microbial contamination. The source of parasite influenced the success rate, the time taken to establish cultures and the size of cultured cells. In vitro proliferation of parasite cells was mainly by vegetative multiplication. Zoosporulation, yielding motile biflagellated zoospores, was observed at a low frequency (<1% of dividing cells) in every culture. Morphology of cultured cells examined with light and transmission electron microscopy corresponded to that of P. atlanticus found in clam tissues. Cultured cells enlarged in RFTM and stained blue-black with Lugol's solution, which are characteristics of the Perkinsus species cells. DNA sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA gene complex matched those of P. atlanticus. All cultures were established in a medium designated JL-ODRP-2A that was similar in composition to the culture medium JL-ODRP-1 originally used to propagate Perkinsus marinus in vitro. Proliferation of P. atlanticus in vitro could be supported by the commercial culture medium (1:2 v/v) DME:Ham's F-12 with fetuin. KEY WORDS: Perkinsus atlanticus · Tapes decussatus · In vitro culture · Clam parasite · Ribosomal RNA gene complex · ITS · UltrastructureResale or republication not permitted without written consent of the publisher Dis Aquat Org 52: [217][218][219][220][221][222][223][224][225][226][227][228][229][230][231] 2002 spacer (ITS) region of the rRNA gene complex (Goggin 1994, de la Herrán et al. 2000, Robledo et al. 2000, Casas et al. 2002. Recently, Ordás & Figueras (1998) reported the in vitro culture of P. atlanticus from the haemolymph of an infected carpet shell clam. Identification of the cultured cells was based solely on their morphology (light and electron microscopy). Sequence analysis of the SSU small subunit rRNA gene, however, showed that those cultured cells did not correspond to a Perkinsus organism (Figueras et al. 2000). Continuous cultures of P. atlanticus, therefore, still need to be established.The development of continuous cultures of Perkinsus species can lead to a better understanding of this group of parasites . Cultures of the eastern oyster pathogen Perkinsus marinus, for example, have been used in a wide range of studies to address the parasite's environmental tolerance (Burreson et al. 1994, Dungan & Hamilton 1995, virulence , Bushek & Allen 1996a, Volety & Chu 1997, genetic composition (Reece et al. 1997, Reece et...

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists

“…12) have a limited or absent disease pathology, and/or (iii) disease is caused only in association with other infectious pathogens such as Ranavirus (43) or other forms of host stress (44). Other parasitic Perkinsea, such as Perkinsus sensu stricto (parasites of bivalves), are also widely geographically distributed, but infection and catastrophic host population MMEs are localized, with pathogenicity related in part to abiotic factors (33,(45)(46)(47).…”

Perkinsus chesapeaki in stout razor clams Tagelus plebeius from Delaware Bay

“…infection intensities were not associated with mortality (e.g. Figueras et al 1992, Ragone & Burreson 1993, Casas et al 2002, and this may also be true of Tagelus plebeius. Other factors that may be responsible for the routine mortality events observed over the years at this site include freezing during winter when snow and ice accumulate on the flats, shifting sediments, age-dependent mortality (Holland & Dean 1977b) or DN disease .…”

“…Histology sections (n = 1 per individual) were examined with standard light microscopy for evidence of parasites or pathological conditions, including disseminated neoplasia (DN disease) as detected in Tagelus plebeius from Chesapeake Bay . A genus-specific PCR assay (Casas et al 2002) was performed to identify individuals infected with Perkinsus spp. using 50 ng of template DNA extracted from 20 to 25 mg of labial palp tissue (Dungan et al 2002).…”

Discrete stochastic marine metapopulation disease model

“…The environment to host transmission occurs via pathogens released from infected animals that can survive in the marine environment for a certain amount of time from days to months (Casas et al, 2002) until they can invade host individuals finding suitable conditions for reproduction within the host. This is the case of the susceptible animal contacting with or filtering infective particles from the environment once are released by living or dead infected individuals; that is, the case of black-band disease (Richardson, 2004;Zvuloni et al, 2009) and Aspergillosis (Jolles et al, 2002) in corals, whithering syndrome (WS) in abalone (Moore et al, 2001(Moore et al, , 2002 and transmission of trematode cercariae (De Montaudouin et al, 1998), shrimps with White-Spot disease (Rudolf and Antonovics, 2007) shedding particles during decay and scavenging processes, OsHV1virus in pacific oysters (Schikorski et al, 2011), MSX (Haskin et al, 1966) and Dermo (Mackin et al, 1950) diseases in oysters; Perkinsosis in clams (Paillard, 2004;Dang et al, 2010).…”