Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). I. Identification of the aetiological agent and in vitro modulation of zoosporulation by temperature and salinity

Casas, Sandra M.; Villalba, António; Reece, Kimberly S.

doi:10.3354/dao050051

Cited by 127 publications

(136 citation statements)

References 28 publications

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“…P. atlanticus ITS sequences from this study grouped with other P. atlanticus sequences and the P. olseni sequences with 100% bootstrap support in a clade that was sister to ITS sequences from P. marinus. Many of the sequences from the isolate culture DNAs were identical to Perkinsus sequences previously obtained by specific amplification of Perkinsus sequences from P. atlanticus-infected host tissue DNA (Casas et al 2002). …”

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confidence: 67%

“…The primer pair designated 'D' was used to amplify an approximately 675 bp fragment of the ITS region from genomic DNA of the cultured P. atlanticus. PCR amplification, cloning and sequencing was done as previously described (Casas et al 2002).The resulting ITS region sequences were subjected to BLAST searches (Altschul et al 1990) Characterisation: enlargement of cultured cells in RFTM. The enlargement of cultured cells in RFTM was tested for 9 different isolates, 3 established from gill fragments, 3 established from haemolymph and 3 established from hypnospores.…”

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confidence: 99%

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

Casas¹,

Peyre²,

Reece³

et al. 2002

Dis. Aquat. Org.

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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...

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confidence: 67%

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confidence: 99%

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

Casas¹,

Peyre²,

Reece³

et al. 2002

Dis. Aquat. Org.

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“…(n = 54; Table 1), polymerase chain reaction (PCR) assay was prepared using a pair of Perkinsus genus-specific primers, PerkITS85 / PerkITS750 (CASAS et al, 2002). These primers specifically hybridize to the conserved regions of the ribosomal ribonucleic acid (RNA) gene, including internal transcribed spacer 1 (ITS), the 5.8S gene and ITS2 (ITS rDNA).…”

Section: Pcr Analysis To Confirm the Genus Perkinsusmentioning

confidence: 99%

Epizootiology of Perkinsus sp. inCrassostrea gasar oysters in polyculture with shrimps in northeastern Brazil

Silva

Costa

Araújo

et al. 2016

Rev. Bras. Parasitol. Vet.

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Bivalve culture is of considerable economic and social interest in northeastern (NE) Brazil. The polyculture is an alternative approach to traditional monoculture for reducing the environmental impact of shrimp farming and improving oyster culture. Perkinsus marinus and Perkinsus olseni were found infecting oysters in NE Brazil and can threaten oyster production. This study evaluated Perkinsus spp. occurrence in Crassostrea gasar during all production stages. Oyster spats were produced in a hatchery and grown in shrimp ponds in Rio Grande do Norte state. Perkinsus spp. were surveyed by Ray's fluid thioglycollate medium and confirmed by polymerase chain reaction. Prevalence and intensity of infection were determined in oysters until they reached 7 cm. Results showed that the broodstock was already infected by Perkinsus (60%), but the derived spats were Perkinsus-free. Oyster spats acquired Perkinsus infection when transferred to ponds. The prevalence gradually increased in the seven months following placement in ponds (73%), and then decreased to 17% by the tenth month. The infections were initially mild, but intensity increased at the final growth stage. In conclusion, it is possible to produce Perkinsus-free C. gasar oyster spats from infected broodstock, and their culture in shrimp ponds is feasible.Keywords: Crassostrea gasar, hatchery, oyster, polyculture, Perkinsus sp., sanitary control. ResumoO cultivo de bivalves é de grande interesse econômico e social no Nordeste (NE) do Brasil. O policultivo é uma alternativa ao monocultivo tradicional de camarões para reduzir o impacto ambiental e melhorar a produção de ostras. Perkinsus marinus e Perkinsus olseni foram identificados infectando ostras no Nordeste do Brasil e representam uma ameaça a produção de ostras. Este estudo avaliou a ocorrência de Perkinsus spp. em Crassostrea gasar durante todas as fases de produção. Sementes de ostras foram produzidas em laboratório e cultivadas em viveiros de camarão no Rio Grande do Norte. Perkinsus spp. foram diagnosticados com o uso do meio de tioglicolato fluido de Ray e confirmado por reação em cadeia da polimerase. A prevalência e intensidade de infecção foram determinadas em ostras até atingirem 7 cm. Os resultados mostraram que os reprodutores encontravam-se infectados por Perkinsus (60%), mas as sementes produzidas estavam livres de Perkinsus. As sementes adquiriram a infecção por Perkinsus quando transferidas para os viveiros. A prevalência aumentou gradualmente nos sete meses após a colocação nos viveiros (73%) e, em seguida, diminuiu para 17% até o décimo mês. As infecções foram inicialmente leves, mas aumentaram até a fase final do crescimento. Em conclusão, é possível produzir sementes de ostras C. gasar livres de Perkinsus a partir de reprodutores infectados e seu cultivo em viveiros de camarão é viável.Palavras-chave: Crassostrea gasar, unidade de reprodução, ostras, policultivo, Perkinsus sp., controle sanitário.

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“…DNA extraction was achieved with a DNAzol  kit (Invitrogen) following the manufacturerʼs directions, while PCR was performed with the primer set Perk ITS 85/750 (Casas et al, 2002) which specifically hybridizes with conserved regions of the internal transcribed spacers (ITS) from the rRNA gene complex, unique to members of the genus Perkinsus (except for Perkinsus qugwadi incertae sedis). DNA extracted from cells of Perkinsus beihaiensis was used as positive control.…”

Section: Detection Of Perkinsus By Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Perkinsus sp. infecting the oyster Crassostrea rhizophorae from estuaries of the septentrional Northeast, Brazil

Dantas-Neto

Sabry²,

Ferreira

et al. 2015

Braz. J. Biol.

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The mangrove oyster Crassostrea rhizophorae is an estuarine resource exploited by riverside communities in Northeast Brazil. Despite its socioeconomic importance, studies on the health status of this bivalve are scanty in this region. The purpose of the present study was to investigate the presence of the protozoan Perkinsus sp. in C. rhizophorae collected in August and September 2011 in three estuaries of the septentrional Northeast, Brazil: Jaguaribe (Ceará), Camurupim (Piauí) and Carnaubeiras (Maranhão) (n= 150 specimens/site). The samples were submitted to Ray's fluid thioglycollate medium (RFTM), PCR and histology assays. The RFTM assay revealed spherical, blue or bluish-black hypnospores of the genus Perkinsus in 50 specimens (Jaguaribe= 17.3%, Camurupim= 5.3%, Carnaubeiras= 10.6%). The intensity of the infection ranged from very light (1-10 cells per slide) to severe (more than 40 cells in each of 10 fields of the slide) for Jaguaribe; very light for Camurupim and very light to moderate (at least 40 cells observed in each of 10 fields of the slide) for Carnaubeiras. When submitted to confirmatory PCR analysis, 6 cases were confirmed (Jaguaribe=3, Camurupim=1, Carnaubeiras=2). The histology confirmed 21 cases of infection in specimens from the three estuaries. Although local collectors have reported no mortality in oyster populations that might be attributed to infection by Perkinsus, health surveillance of oyster populations in the septentrional region of Northeast Brazil is advisable.Keywords: Perkinsus, Crassostrea rhizophorae, RFTM, PCR. Perkinsus sp. infectando a ostra Crassostrea rhizophorae de estuários do Nordeste setentrional, Brasil ResumoA ostra-do-mangue Crassostrea rhizophorae é um recurso estuarino explorado por comunidades ribeirinhas do Nordeste do Brasil. Apesar de sua importância socioeconômica, estudos sobre o estado de saúde deste bivalve são escassos na região. O objetivo deste estudo foi investigar a presença do protozoário Perkinsus sp. em C. rhizophorae coletada em agosto e setembro de 2011, em três estuários da região setentrional do Nordeste brasileiro: Jaguaribe (Ceará), Camurupim (Piauí) e Carnaubeiras (Maranhão) (n = 150 espécimes/local). As amostras foram submetidas ao meio líquido de tioglicolato de Ray (RFTM), PCR e ensaios histológicos. A análise em RFTM revelou hipnósporos esféricos azuis ou preto-azulados do gênero Perkinsus em 50 espécimes (Jaguaribe= 17,3%, Camurupim= 5,3%, Carnaubeiras= 10,6%). A intensidade de infecção variou de muito leve (1-10 células por lâmina) a severa (mais de 40 células em cada um dos 10 campos da lâmina) para o Rio Jaguaribe; muito leve para o Rio Camurupim e muito leve a moderada (pelo menos 40 células observadas, em cada um dos 10 campos da lâmina) para o Rio Carnaubeiras. Quando submetidos à análise confirmatória por PCR, foram confirmados 6 casos (Jaguaribe= 3, Camurupim= 1, Carnaubeiras= 2). A histologia confirmou 21 casos de infecção em espécimes dos três estuários. Embora os coletores locais não tenham relatado nenhuma mortalida...

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Study of perkinsosis in the carpet shell clam Tapes decussatus in Galicia (NW Spain). I. Identification of the aetiological agent and in vitro modulation of zoosporulation by temperature and salinity

Cited by 127 publications

References 28 publications

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

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

Epizootiology of Perkinsus sp. inCrassostrea gasar oysters in polyculture with shrimps in northeastern Brazil

Perkinsus sp. infecting the oyster Crassostrea rhizophorae from estuaries of the septentrional Northeast, Brazil

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