Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR

Anna Cutarelli; Francesca Carella; Francesca De Falco; Bianca Cuccaro; Fabio Di Nocera; Donatella Nava; Gionata De Vico; Sante Roperto

doi:10.3390/pathogens12080994

Detection and Quantification of Nocardia crassostreae, an Emerging Pathogen, in Mytilus galloprovincialis in the Mediterranean Sea Using Droplet Digital PCR

Pathogens. 2023 Jul 28;12(8):994. doi: 10.3390/pathogens12080994.

Authors

Anna Cutarelli¹, Francesca Carella², Francesca De Falco³, Bianca Cuccaro³, Fabio Di Nocera¹, Donatella Nava¹, Gionata De Vico², Sante Roperto³

Affiliations

¹ Istituto Zooprofilattico Sperimentale del Mezzogiorno, 80055 Portici, Italy.
² Dipartimento di Biologia, Università degli Studi di Napoli Federico II, 80126 Napoli, Italy.
³ Dipartimento di Medicina Veterinaria e Produzioni Animali, Università degli Studi di Napoli Federico II, 80137 Napoli, Italy.

Abstract

Nocardia crassostreae is a novel pathogen responsible for infections in oysters (Crassostrea gigas) and mussels (Mytilus galloprovincialis). N. crassostreae is also responsible for nocardiosis both in immunocompetent and immunocompromised patients. We investigated N. crassostreae DNA in mussels grown in marine sites of the Mediterranean Sea in the Campania Region. We examined 185 mussel pooled samples by droplet digital PCR (ddPCR) and real-time quantitative PCR (qPCR), each pool composed of 10 mussels and 149 individual mussels. ddPCR detected N. crassostreae DNA in 48 mussel pooled samples and in 23 individual mussel samples. qPCR detected N. crassostreae DNA in six pooled samples and six individual mussel samples. The two molecular assays for the detection of N. crassostreae DNA showed significant differences both in the pooled and in individual samples. Our study demonstrated that ddPCR outperformed real-time qPCR for N. crassostreae DNA detection, thus confirming that ddPCR technology can identify the pathogens in many infectious diseases with high sensitivity and specificity. Furthermore, in individual mussels showing histological lesions due to N. crassostreae, the lowest copy number/microliter detected by ddPCR of this pathogen was 0.3, which suggests that this dose could be enough to cause infections of N. crassostreae in mussels.

Keywords: Mediterranean Sea; Mytilus galloprovincialis; Nocardia crassostreae; droplet digital PCR (ddPCR); mussel; real-time qPCR.

Grants and funding

This study was in part supported by Istituto Zooprofilattico Sperimentale del Mezzogiorno. The funding bodies had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to publish.