Pathogenicity of Citrobacter freundii Causing Mass Mortalities of Macrobrachium rosenbergii and Its Induced Host Immune Response

<i>Citrobacter freundii</i> is an opportunistic pathogen of freshwater aquatic animals, which severely restricts the sustainable development of the aquaculture industry. In this study, a dominant strain, named FSNM-1, was isolated from the hepatopancreas of diseased <i>Macrobrachium rosenbergii</i>. This strain was identified as <i>C. freundii</i> based on a comprehensive analysis of its morphological, physiological, and biochemical features and molecular identification. Challenge experiments were conducted to assess the pathogenicity of <i>C. freundii</i> to <i>M. rosenbergii</i>. The results showed that the FSNM-1 strain had high virulence to <i>M. rosenbergii</i> with a median lethal dose (LD₅₀) of 1.1 × 10⁶ CFU/mL. Histopathological analysis revealed that <i>C. freundii</i> infection caused different degrees of inflammation in the hepatopancreas, gills, and intestines of <i>M. rosenbergii</i>. The detection of virulence-related genes revealed that the FSNM-1 strain carried colonization factor antigen (<i>cfa1</i>, <i>cfa2</i>), ureases (<i>ureG</i>, <i>ureF</i>, <i>ureD</i>, <i>ureE</i>), and outer membrane protein (<i>ompX</i>), and virulence factor detection showed that the FSNM-1 strain had lecithinase, amylase, lipase, gelatinase, and hemolysin activities but did not produce protease and DNase activities. To investigate the immune response of <i>M. rosenbergii</i> to <i>C. freundii</i>, the expression levels of <i>ALF3</i>, <i>MyD88</i>, <i>SOD</i>, <i>proPO</i>, <i>TRAF6</i>, and <i>TNF</i> immune-related genes were monitored at different points of time in the hepatopancreas, gills, intestines, and hemocytes of <i>M. rosenbergii</i> after infection. The results demonstrated a significant upregulation in the expression levels of the <i>ALF3</i>, <i>MyD88</i>, <i>SOD</i>, <i>proPO</i>, <i>TRAF6</i>, and <i>TNF</i> genes in <i>M. rosenbergii</i> at the early stage of <i>C. freundii</i> infection. This study highlights <i>C. freundii</i> as a major pathogen causing mass mortality in <i>M. rosenbergii</i> and provides valuable insights into its virulence mechanisms and the host's immune response.