In the presence of T. thermophila, the virulent strain grew as well in the absence of Tetrahymena (Fig. 1a), indicating that the A. hydrophila J-1 could overcome predation by T. thermophila. Conversely, in the presence of T. thermophila, A. hydrophila NJ-4

was cleared from the culture after 6 h (Fig. 1b). Our findings revealed that the virulent strain is less efficiently predated by Tetrahymena than the avirulent strain. It suggested that A. hydrophila resistance to T. thermophila-mediated phagocytosis was associated with bacterial virulence. The fact that J-1 is virulent and NJ-4 is avirulent in zebrafish (unpublished data) suggested that the Tetrahymena–Aeromonas model provides a relevant measure of the virulence of A. hydrophila towards fish. We measured the growth of T. thermophila learn more when these cells were co-cultured MAPK inhibitor with two bacterial strains. In this study, T. thermophila was suspended in PBSS. Under the culture conditions, the bacteria served as the only food source for T. thermophila. Co-culture in the presence

of A. hydrophila J-1 reduced T. thermophila growth significantly. The protozoan biomass was severely affected during the 48-h incubation period. By 36-h postculture, most of the T. thermophila grown in the presence of A. hydrophila J-1 were nonviable and undetectable by 48-h postculture (Fig. 1c). Hence, A. hydrophila J-1 does not support T. thermophila growth; instead, this bacterium causes T. thermophila death. Conversely, in the presence of A. hydrophila NJ-4, the number of T. thermophila cells was increased within 12-h postculture, and then slightly decreased and maintained

a steady concentration throughout the 48-h examination period (Fig. 1c). The data showed that A. hydrophila J-1 could kill Amino acid all T. thermophila in 2 days, but A. hydrophila NJ-4 had no negative effects on T. thermophila and actually served as a food source during the co-culture. Because A. hydrophila can be phagocytosed by T. thermophila, we examined the intracellular growth of both A. hydrophila J-1 and NJ-4 (Fig. 1d). Both bacteria were observed to proliferate inside T. thermophila, although their growth rates and profiles were different. Aeromonas hydrophila J-1 began to grow steadily 6 h postphagocytosis and declined 36 h later. This decline coincided with the death of T. thermophila observed in Fig. 1c at this same time point. This suggested that A. hydrophila J-1 phagocytosed by T. thermophila was not consumed by the ciliate. Conversely, A. hydrophila NJ-4 grew steadily and maintained the high growth rate throughout the 42-h incubation period. This increased the growth rate and higher A. hydrophila NJ-4 numbers can be explained as a result of feeding and dividing T. thermophila that phagocytosed more A. hydrophila NJ-4 cells, resulting in increased intracellular growth (Fig. 1d). The T. thermophila biomass was assessed in the presence of supernatants from either A. hydrophila J-1 or NJ-4 (Fig. 2). In the presence of A.