| Aeromonas hydrophila, which has caused a big loss in aquaculture in past years, is receiving an increasing attention as a primary pathogen both in humans and in aquatic and terrestrial animals. So far, the pathogenesis of the organism has been known to involve multiple factors. However, the precise pathogenesis mechanism is not known. As an effective tool for environmental toxicological study, Tetrahymena can be used to assess the presence of toxic bacterial metabolites. In this study, we investigated the relationships among the virulence factors of A. hydrophila, and evaluated the potential of Tetrahymena thermophila as the host model of A. hydrophila by the coculture of the ciliate and the bacterium.Three main extracellular virulence factors of A. hydrophila, including aerolysin, cytotoxic enterotoxin and extracellular serine protease, were investigated in84strains of A. hydrophila by PCR. On the basis of the detection results, all the strains were divided into seven exotoxin genotypes:61.9%(52/84) of strains were aer+alt+ahp+;14.3%(12/84) were aer+alt+ahp-;10.7%(9/84) were aer+alfahp+;4.8%(4/84) were aer alt+ahp+;3.6%(3/84)were aer-alfahp+;2A%(2/84)were aer+alt-ahp-and2.4%(2/84)were aer-alt-ahp-, It suggested that the dominant strains were those virulent strains carrying with all the three exotoxin genes. Twenty-six representative strains from the seven genotypes were randomly selected, and individually injected into zebrafish to calculate the LD5o. The results showed that virulence factors get synergistic reaction to each other and the bacterial strains with more virulence factors have lower LD5o and stronger pathogenicity to zebrafish. It is the first time to develop a PCR method for detecting aer-alt-ahp genes to predict the virulent strains of A. hydrophila.On the basis of zebrafish experiment, highly virulent strain BSK-10and lowly virulent strain NJ-4were selected to coculture with Tetrahymena, respectively. The OD450values were recorded and the growth curves of the bacteria outside the ciliate were drawn. By coculturing again the two strains of A. hydrophila with the hungry Tetrahymena respectively, the growth curves of Tetrahymena and the bacteria inside them were drawn. The results showed that the highly virulent strain BSK-10grew well, while the lowly virulent strain NJ-4had a serious growth inhibition and the biomass of bacteria declined rapidly at2h after coculture. The number of Tetrahymena cocultured with BSK-10got nearly0at48h, and the bacteria in them had a significant increasing. However, the number of Tetrahymena cocultured with NJ-4declined slowly, and the biomss of bacteria inside got smaller fluctuation. Tetrahymena were further mixed with the24h-culture supernatant of BSK-10and NJ-4respectively, and then the change in the number of Tetrahymena was recorded. It showed that Tetrahymena cultured in the supernatant of BSK-10gained a higher lethal rate than those in the NJ-4. All these indicated that Tetrahymena might be susceptible to the extracellular products of A. hydrophila, and showed the difference in survival when cocultured with highly and lowly virulent strains respectively. It suggested that Tetrahymena could be a potential host model for assessing A. hydrophila virulence.By coculturing A. hydrophila J-1labelled with green fluorescent protein (AhJ-1GFP) with hungry Tetrahymena, lots of phagocytic vacuoles containing the bacteria with green fluorescent could be observed under fluorescence microscope. The observation by transmission electron microscopy showed that phagocytic vacuoles containing lowly virulent strain were round, while those with highly virulent strain were not round and the clear border could not be seen. Most of the lowly virulent bacteria inside the parasites had the irregular shapes while the highlt virulent bacteria were regular and tended to break through the phagocytic vacuoles. The scanning electron microscopical observation showed that most of cilia on the surface of Tetrahymena cocultured with BSK-10were lost and something wrapped around the ciliate, while those cocultured with NJ-4got rich cilia and clear gyri. The reference strain AhJ-1was cocultured with Tetrahymena both in PBSS and SM, respectively, and then the virulent gene (aer and ahp) expressions of the bacteria inside and outside the ciliate were detected by RT-PCR at6h after coculture. The results showed that virulent genes of the bacteria inside both up-regulated in PBSS and SM media, while those of the bacteria outside got a little up-regulation in the former medium, and had no change in the latter. The results indicated that the virulent A. hydrophila might inhibit the phagocytosis of Tetrahymena and even kill the ciliate by up-regulating the virulent gene expression. |
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