Aeromonas salmonicida subsp. salmonicida Early Infection and Immune Response of Atlantic Cod (Gadus morhua L.) Primary Macrophages

Abstract

In contrast to other teleosts, Atlantic cod ( Gadus morhua ) has an expanded repertoire of MHC-I and TLR components, but lacks the MHC-II, the invariant chain/CD74, and CD4 + T cell response, essential for production of antibodies and prevention of bacterial infectious diseases. The mechanisms by which G. morhua fight bacterial infections are not well understood. Aeromonas salmonicida subsp. salmonicida is a recurrent pathogen in cultured and wild fish, and has been reported in Atlantic cod. Macrophages are some of the first responders to bacterial infection and the link between innate and adaptive immune response. Here, we evaluated the viability, reactive oxygen species (ROS) production, cell morphology, and gene expression of cod primary macrophages in response to A. salmonicida infection. We found that A. salmonicida infects cod primary macrophages without killing the cod cells. Likewise, infected Atlantic cod macrophages up-regulated key genes involved in the inflammatory response (e.g., IL-1 β and IL-8 ) and bacterial recognition (e.g., BPI/LBP ). Nevertheless, our results showed a down-regulation of genes related to antimicrobial peptide and ROS production, suggesting that A. salmonicida utilizes its virulence mechanisms to control and prevent macrophage anti-bacterial activity. Our results also indicate that Atlantic cod has a basal ROS production in non-infected cells, and this was not increased after contact with A. salmonicida . Transmission electron microscopy results showed that A. salmonicida was able to infect the macrophages in a high number, and release outer membrane vesicles (OMV) during intracellular infection. These results suggest that Atlantic cod macrophage innate immunity is able to detect A. salmonicida and trigger an anti-inflammatory response, however A. salmonicida controls the cell immune response to prevent bacterial clearance, during early infection.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes

“…In summary, we found that Salmo salar erythrocytes have phagocytic capabilities (Figures ), but lower than primary macrophages (Soto‐Davila et al, ). Although the S. salar erythrocytes have the ability to phagocyte bacteria and particles (Figures ), A. salmonicida remains in a viable cultivable state in erythrocytes and blood of infected animals (Figure b).…”

“…Here, using in vivo and in vitro approaches, we present evidence that A. salmonicida invades S. salar erythrocytes, which is a novel feature for furunculosis that could benefit the bacterial dispersion throughout the host tissues. Additionally, we observed that S. salar erythrocytes have a minor phagocytic activity compared to primary macrophages of Atlantic salmon and Atlantic cod (Soto‐Davila et al, ). These results are coincident with previous studies that showed that O. mykiss RBC have phagocytic activity (Passantino et al, ).…”

“…In vitro tests indicate that the initial infection percentages in RBC are around 42% (Figure b; attachment), and similar values were founded in non‐phagocytic cells, (Valderrama et al, ). However, the attachment in phagocytic cells, like macrophages of Atlantic salmon and Atlantic cod, is around 10% (Soto‐Dávila et al, ). The number of intraerythrocytic A. salmonicida at 2 hr (0.047%; 5.90 × 10 3 CFU) and 3 hr (0.032%; 2.10 × 10 3 CFU) post‐infection was significantly lower than the intracellular A. salmonicida founded in different type of cells at the same infection times.…”

“…For instance, the number of intracellular A. salmonicida founded at 2 hr and 4 hr post‐infection in CHSE‐214 cells was 0.47% and 0.29%, respectively (Valderrama et al, ). The number of intracellular A. salmonicida founded at 2 hr and 3 hr post‐infection in primary Atlantic salmon cells was 0.42% and 0.26%, respectively, and in Atlantic cod primary macrophages, the number of intracellular A. salmonicida founded at 2 hr and 3 hr post‐infection was 0.42% and 0.37%, respectively (Soto‐Dávila et al, ). The results observed in RBC infected with A. salmonicida were verified by the FACS results (Figure ).…”

“…A. salmonicida has the ability to infect non-phagocytic cells (Valderrama et al, 2017) and phagocytic cells (Soto-Dávila, Hossain, Chakraborty, Rise, & Santander, 2019). Using the gentamicin exclusion assay, we evaluated whether A. salmonicida has the ability to infect and survive in primary S. salar RBC.…”