First record of Perkinsus marinus infecting Crassostrea sp. in Rio Grande do Norte, Brazil, using real-time PCR

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus , particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus , the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus , P. olseni , and P. chesapeaki , revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-024-76822-y.

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Perkinsus marinus in bioreactor: growth and a cost-reduced growth medium

Murphy,

Fernández Robledo,

van Walsum

2023

Journal of Industrial Microbiology and Biotechnology

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Perkinsus marinus (Perkinsea) is an osmotrophic facultative intracellular marine protozoan responsible for ‘Dermo’ disease in the eastern oyster, Crassostrea virginica. In 1993, three laboratories developed the in vitro culture of P. marinus in the absence of host cells. Two of the culture media and conditions went through several optimization reiterations, with one becoming the standard for the small-scale growth of Perkinsus species. Compared to most intracellular protozoan parasites, the availability of P. marinus to grow in the absence of host cells has provided the basis to explore its use as a heterologous expression system, which could potentially be used for therapeutic applications, such as vaccines. As the genetic toolbox is becoming available, there is also the need for larger-scale cultivation and lower-cost media formulations. Here, we took an industrial approach to scaled-up growth from a small culture flask to bioreactors which required developing new cultivation parameters, including aeration, mixing, pH, temperature control, and media formulation. Our approach also enabled more real-time data collection on growth. The bioreactor cultivation method showed similar or accelerated growth rates of P. marinus compared to culture in T-flasks. Redox measurements indicated sufficient oxygen availability throughout the cultivation. Replacing fetal bovine serum with chicken serum showed no differences in the growth rate and a 60% reduction in the medium cost. This study opens the door to furthering P. marinus as a valid heterologous expression system by showing the ability to grow in bioreactors.

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First record of Perkinsus marinus infecting Crassostrea sp. in Rio Grande do Norte, Brazil, using real-time PCR

Cited by 3 publications

References 29 publications

Perkinsus spp. Occurrence in South America: A review

Perkinsus spp. Occurrence in South America: A review

A highly sensitive and specific real-time quantitative polymerase chain reaction assay for Perkinsus marinus detection in oysters

Perkinsus marinus in bioreactor: growth and a cost-reduced growth medium

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