Comparative study of structural features and parasite–parasite interactions between MCPX, a novel pathogen identified from the Manila clam Ruditapes philippinarum, and Perkinsus olseni

Co-infections involving multiple pathogens are common and can have varied effects on the host, ranging from beneficial to detrimental or insignificant. However, the mechanisms driving these interactions remain poorly understood. This study aimed to investigate the parasite–parasite interactions regulating disease progression, focusing on two protist parasites: the novel parasite MCPX, identified from the Manila clam (Ruditapes philippinarum), and Perkinsus olseni. Structural variations between MCPX and P. olseni were evaluated using Ray's fluid thioglycollate medium and immunofluorescence assay. A large vacuole, a characteristic feature, was observed only in the enlarged prezoosporangia of P. olseni, while MCPX prezoosporangia contained a dense cytoplasm. Both parasites were stained with Lugol's iodine. However, only MCPX prezoosporangia were digested by 2 M NaOH. Immunofluorescence staining revealed that only P. olseni trophozoites reacted with the P. olseni-specific antibody, highlighting structural differences. To assess parasite–parasite interactions, growth kinetics were measured during co-culture and in the presence of extracellular products (ECPs) from each parasite. Flow cytometry results indicated a notable decline in MCPX cell numbers by day 10, while P. olseni grew normally. This finding was further confirmed by assessing the fluorescence intensity observed in the immunofluorescence assay. A significant reduction in MCPX propagation was observed in the presence of P. olseni ECPs, suggesting an adverse effect of P. olseni ECPs on MCPX growth. These findings reveal the distinct structural properties of MCPX, potential antagonistic effect of P. olseni against MCPX, and presence of proteases in P. olseni ECPs that may modulate MCPX propagation.