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Poly lactic-co-glycolic acid (PLGA) particles safely and effectively deliver pharmaceutical ingredients, with many applications approved for clinical use in humans. In fishes, PLGA particles are being considered as carriers of therapeutic drugs and vaccine antigens. However, existing studies focus mainly on vaccine antigens, the endpoint immune responses to these (e.g., improved antibody titres), without deeper understanding of whether fishes react to the carrier. To test whether or not PLGA are recognized by or interact at all with the immune system of a teleost fish, we prepared, characterized and injected PLGA microparticles intraperitoneally into common carp. The influx, phenotype of inflammatory leukocytes, and their capacity to produce reactive oxygen species and phagocytose PLGA microparticles were tested by flow cytometry, qPCR, and microscopy. PLGA microparticles were indeed recognized. However, they induced only transient recruitment of inflammatory leukocytes that was resolved 4 days later whereas only the smallest µm-sized particles were phagocytosed. The overall response resembled that described in mammals against foreign materials. Given the similarities between our findings and those described in mammals, PLGA particles can be adapted to play a dual role as both antigen and drug carriers in fishes, depending on the administered dose and their design.

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99–100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans.

In poikilothermic vertebrates, seasonality influences different immunological parameters such as leukocyte numbers, phagocytic activity, and antibody titers. This phenomenon has been described in different teleost species, with immunological parameters peaking during warmer months and decreased levels during winter. In this study, the cellular immune responses of rainbow trout (Oncorhynchus mykiss) kept under constant photoperiod and water temperature against intraperitoneally injected Aeromonas salmonicida during the summer and winter were investigated. The kinetics of different leukocyte subpopulations from peritoneal cavity, spleen, and head kidney in response to the bacteria was measured by flow cytometry. Furthermore, the kinetics of induced A. salmonicida-specific antibodies was evaluated by ELISA. Despite maintaining the photoperiod and water temperature as constant, different cell baselines were detected in all organs analyzed. During the winter months, B- and T-cell responses were decreased, contrary to what was observed during summer months. However, the specific antibody titers were similar between the two seasons. Natural antibodies, however, were greatly increased 12 h post-injection only during the wintertime. Altogether, our results suggest a bias toward innate immune responses and potential lymphoid immunosuppression in the wintertime in trout. These seasonal differences, despite photoperiod and water temperature being kept constant, suggest an internal inter-seasonal or circannual clock controlling the immune system and physiology of this teleost fish.

The daily change of light and dark periods influences different physiological processes including feeding, resting and locomotor activity. Previously, several studies on mammalian models revealed a strong link between day-night rhythms and key immunological parameters. Since teleost fishes possess innate and adaptive immune responses like those observed in higher vertebrates, we aimed to elucidate how changes in light-dark cycles shape the immune system of fish. Using the rainbow trout laboratory model, we investigated the link between diurnal rhythms and immune competence of fish. Initially, the cell composition and phagocytic activity of leukocytes was analyzed in the circulation as well as in the head kidney, the functional ortholog of mammalian bone marrow. Once the baseline was established, we evaluated the ability of fish to respond to a bacterial stimulus, as well as the changes in antimicrobial activity of the serum. Our results suggest increased immune competence during the day, manifested by the higher presence of myeloid cells in the circulation; increased overall phagocytic activity; and higher capacity of the sera to inhibit the growth of Aeromonas salmonicida. Notably, our flow cytometric analysis identified the myeloid cells as the major population influenced by the time of day, whereas IgM+ B cells and thrombocytes did not vary in a significant manner. Interestingly, the presence of myeloid cells in blood and head kidney followed complementary trends. Thus, while we observed the highest number of myeloid cells in the blood during early morning, we witnessed a reverse trend in the head kidney, suggesting a homing of myeloid cells to reservoir niches with the onset of the dark phase. Further, the presence of myeloid cells was mirrored in the expression of the proinflammatory marker tnfa as well as in the number of leukocytes recruited to the peritoneal cavity in the peritonitis model of inflammation. Overall, the data suggest a connection between diurnal rhythms and the immune response of rainbow trout and highlight the relevance of rhythmicity and its influence on experimental work in the field of fish chronoimmunology.