Baseline Assessment of WOAH-Listed Protozoan Parasites in Wild Mediterranean Mussels Mytilus galloprovincialis and Pacific Oysters Crassostrea gigas from Port-Adjacent Coastal Waters of Korea in 2023

1. Introduction

In the international seafood trade, importing countries implement sanitary and phytosanitary measures in line with the World Organization for Animal Health (WOAH) guidelines to protect their aquatic ecosystems and aquaculture industries [1]. Scientific surveillance data demonstrating that exported products are free from WOAH-listed aquatic animal diseases are a key requirement for meeting import sanitary standards [2]. Bivalves, due to their sessile lifestyle and filter-feeding behavior, are widely used as sentinel species that reflect environmental conditions and the presence of pathogens in coastal ecosystems [3]. Evidence of pathogen dispersal through maritime transport has been suggested by detections of Perkinsus beihaiensis infections in Mediterranean mussels Mytilus galloprovincialis in Tokyo Bay, Japan, and in bivalve populations in Panama [4,5,6].

The Mediterranean mussel M. galloprovincialis is a well-recognized invasive species [7] that can be infected by Marteilia refringens, P. olseni, and P. beihaiensis, indicating its susceptibility to a range of protozoan parasites affecting bivalves [5,8,9,10,11]. The Pacific oyster Crassostrea gigas is susceptible to P. marinus and has also been suggested as a potential vector of Bonamia ostreae [12,13]. These protozoan parasites are of particular concern because they can cause significant mortality and productivity loss in wild and farmed bivalves and may be translocated between regions through the movement of seed, live shellfish, and shipping activities [14]. Along the Korean coast, P. olseni is known to be prevalent in Manila clams and blood cockles [15], and previous PCR-based monitoring reported no WOAH-listed protozoan parasites in C. gigas along parts of the southern coast [16]. More recently, however, P. marinus was detected in wild C. gigas on the west coast of Korea, and B. ostreae infection was reported in wild flat oysters Ostrea denselamellosa near Gunsan Port [12,17], raising concerns about the introduction and local establishment of these exotic protozoan parasites. Among the targeted protozoan parasites, P. marinus, P. olseni, Bonamia spp., and M. refringens are WOAH-listed, whereas P. beihaiensis is a non-listed but emerging Perkinsus species with documented occurrences in bivalves from Japan and Panama [4,5,6].

2. Materials and Methods

2.1. Sample Collection

To evaluate the occurrence of WOAH-listed protozoan parasites in port-associated bivalve populations, we collected wild M. galloprovincialis and C. gigas from small harbors located near ten major trading ports along the west and south coasts of Korea and screened them for P. marinus, P. olseni, B. ostreae, B. exitiosa, M. refringens, and P. beihaiensis using species-specific PCR assays. In May and September 2023, mussels and oysters were sampled from 18 sites around Taean, Boryeong, Janghang, and Gunsan on the west coast and Gwangyang, Yeosu, Tongyeong, Gohyeon, Okpo, and Busan on the south coast (Figure 1 and Figure 2), yielding a total of 1,080 mussels and 1,080 oysters for analysis. Shell height (for mussels) or shell length (for oysters) and tissue weight were measured for each individual, and gill and digestive gland tissues were excised, with aliquots stored at −70 °C for molecular analysis and remaining tissues preserved in Davidson’s solution for potential histology.

2.2. PCR Assay

For PCR screening, approximately 30 mg of pooled gill and digestive gland tissues from six individuals were combined to form a single sample, and genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following proteinase K digestion, resulting in pooled DNA samples that were used as templates for species-specific PCR assays.

Six primer sets were used to target WOAH-listed and non-listed protozoan parasites, specifically P. marinus, P. olseni, P. beihaiensis, B. ostreae, B. exitiosa, and M. refringens, as previously described for each species (Table 1). Each PCR reaction was performed in a 25 μL volume containing 100 ng of template DNA, buffer with MgCl₂, dNTPs, primers, and Ex Taq DNA polymerase (Takara, Japan), using thermal cycling conditions based on the original primer descriptions with minor modifications. For P. marinus and P. olseni, DNA extracted from in vitro cultures was used as a positive control, whereas plasmid DNA of B. ostreae, B. exitiosa, and M. refringens from the European Union Reference Laboratory for Mollusk Diseases and plasmid DNA of P. beihaiensis provided by the University of Tokyo served as positive controls for these targets. PCR products were separated on 1.5% agarose gels along with a 100 bp DNA ladder and visualized under UV illumination to detect specific amplicons corresponding to each protozoan parasite.

3. Results

3.1. PCR Screening Results

The mussels analyzed in this study had shell heights ranging from 21.2 to 96.1 mm and tissue weights from 0.2 to 22.1 g, whereas oysters had shell lengths ranging from 15.9 to 134.2 mm and tissue weights from 0.4 to 36.3 g, indicating that multiple size classes were represented across the 18 sampling sites (Table 2 and Table 3). Species-specific PCR screening revealed no evidence of infection by WOAH-listed protozoan parasites or by P. beihaiensis in any of the pooled mussel or oyster samples from port-adjacent waters on the west and south coasts of Korea: no specific amplicons were detected for P. marinus, P. olseni, B. ostreae, B. exitiosa, M. refringens, or P. beihaiensis in any field samples (Table 2 and Table 3, Figure 3). In contrast, all positive control reactions produced clear bands of the expected sizes, whereas negative controls remained free of bands, confirming that the assays were functional and that non-detection in field samples reflected a true lack of detectable infections rather than technical failure.

4. Discussion

These results demonstrate a complete absence of detectable infections by WOAH-listed protozoan parasites and P. beihaiensis in wild M. galloprovincialis and C. gigas inhabiting port-adjacent environments on the west and south coasts of Korea during the 2023 survey period. This finding is notable given recent detections of P. marinus in wild C. gigas from Hongseong [12] and B. ostreae in O. denselamellosa near Gunsan Port [17], both on the west coast of Korea, and the geographical proximity of some of our sampling sites, such as Namdang and Suryong, to these previously reported locations. The absence of P. marinus and Bonamia spp. in M. galloprovincialis and C. gigas from these areas suggests that infections with these protozoan parasites may currently be focal and limited in spatial extent and may also reflect strong host specificity, as Bonamia spp. are known to primarily infect Ostrea species, with substantially lower susceptibility reported for Crassostrea and Mytilus species [18].

Environmental factors, particularly temperature and salinity, strongly influence the prevalence and infection intensity of P. marinus [19], and differences in local or seasonal conditions between our survey and previous reports [12] may partly explain the discrepancies in detection. Although we did not measure environmental parameters concurrently, the complete absence of PCR-positive samples in our survey, combined with earlier negative findings from southern coastal C. gigas populations in 2020 [16], suggests that WOAH-listed protozoan parasites have not yet become widely established in port-associated mussel and oyster communities along the surveyed coasts. At the same time, the recent emergence of P. marinus and B. ostreae in Korea indicates that the risk landscape is changing, and continued surveillance of multiple host species, integrated with environmental monitoring, will be crucial for early detection and risk assessment of protozoan parasite incursions [12,17].

5. Conclusions

This Brief Communication reports that species-specific PCR assays of 1,080 M. galloprovincialis and 1,080 C. gigas collected from 18 port-adjacent sites along the west and south coasts of Korea in 2023 detected no infections by P. marinus, P. olseni, B. ostreae, B. exitiosa, M. refringens, or P. beihaiensis. These data provide important baseline information on the current absence of WOAH-listed protozoan parasites in wild mussel and oyster populations inhabiting high-risk harbor environments in Korea and will support future risk assessments, disease-free certifications in shellfish trade, and the design of environment-coupled surveillance programs for transboundary shellfish diseases.