Frasca Salvatore

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Frasca
First Name
Salvatore
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  • Article
    Nuclear small-subunit ribosomal RNA gene-based characterization, molecular phylogeny and PCR detection of the Neoparamoeba from western Long Island Sound lobster
    (National Shellfisheries Association, 2005-10-01) Mullen, Thomas E. ; Nevis, Kathleen R. ; O'Kelly, Charles J. ; Gast, Rebecca J. ; Frasca, Salvatore
    Western Long Island Sound (LIS) lobsters collected by trawl surveys, lobstermen and coastal residents during 2000 to 2002 were identified histologically as infected with a parasome-containing amoeba. Primers to conserved SSU rRNA sequences of parasome-containing amoebae and their nonparasome-containing relatives were used to amplify overlapping SSU rRNA fragments of the presumptive parasite from gill, antenna, antennal gland and ventral nerve cord of infected lobsters. The consensus sequence constructed from these fragments had 98% or greater nucleotide sequence identity with SSU rRNA gene sequences of strains of Neoparamoeba pemaquidensis and associated with high confidence in distance- and parsimony-based phylogenetic analyses with strains of Neoparamoeba pemaquidensis and not members of the family Paramoebidae, e.g., Paramoeba eilhardi. Primers designed to SSU rRNA sequences of the lobster amoeba and other paramoebid/vexilliferid amoebae were used in a nested polymerase chain reaction (PCR) protocol to test DNA extracted from formalin-fixed paraffin-embedded tissues of lobsters collected during the 1999 die-off, when this amoeba initially was identified by light and electron microscopy and reported to be a paramoeba of the genera Paramoeba or Neoparamoeba (Mullen et al. 2004). All sequences amplified from 1999 lobsters, with the exception of one, had 98% to 99% identity to each other, and the 1999 PCR product consensus had 98% identity to Neoparamoeba pemaquidensis strains CCAP 1560/4 (AF371969.1) and 1560/5 (AF371970.1). Molecular characterization of the amoeba from western LIS lobsters by direct amplification circumvents a collective inability to culture the organism in vitro, provides insight into the molecular epidemiology of neoparamoebiasis in American lobster, and allows for PCR-based detection of infected lobsters for future research and diagnostics.
  • Article
    Development of a one-step duplex RT-qPCR for the quantification of phocine distemper virus
    (Wildlife Disease Association, 2015-04) Bogomolni, Andrea L. ; Frasca, Salvatore ; Matassa, Keith A. ; Nielsen, Ole ; Rogers, Kara ; De Guise, Sylvain
    Worldwide, stranded marine mammals and the network personnel who respond to marine mammal mortality have provided much of the information regarding marine morbillivirus infections. An assay to determine the amount of virus present in tissue samples would be useful to assist in routine surveying of animal health and for monitoring large-scale die-off events. False negatives from poor-quality samples prevent determination of the true extent of infection, while only small amounts of tissue samples or archived RNA may be available at the time of collection for future retrospective analysis. We developed a one-step duplex real-time reverse transcriptase-quantitative-PCR assay (RT-qPCR) based on Taqman probe technology to quantify phocine distemper virus (PDV) isolated from an outbreak in harbor (Phoca vitulina concolor) and gray seals (Halichoerus grypus) along the northeast US coast in 2006. The glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene was selected to assess RNA quality. This duplex assay is specific for PDV and sensitive through a range of 100 to 109 copies ds-plasmid DNA. For the GAPDH target, the reaction in duplex amplified 100 to 109 copies of ds-plasmid DNA and was detectable in multiple seal species. This assay reduced the likelihood of false negative results due to degradation of tissues and well-to-well variability while providing sensitive and specific detection of PDV, which would be applicable in molecular epidemiologic studies and pathogen detection in field and laboratory investigations involving a variety of seal species.