Many species of bacteria pathogenic to humans, such as Legionella, are thought to have evolved in association with amoebal hosts. Several novel unculturable bacteria related to Legionella have also been found in amoebae, a few of which have been thought to be causes of nosocomial infections in humans. Because amoebae can be found in cooling towers, we wanted to know whether cooling tower environments might enhance the association between amoebae and bacterial pathogens of amoebae in order to identify potential "hot spots" for emerging human pathogens. To compare occurrence of infected amoebae in natural environments with those in cooling towers, 40 natural aquatic environments and 40 cooling tower samples were examined. Logistic regression analysis determined variables that were significant predictors of the occurrence of infected amoebae, which were found in 22 of 40 cooling tower samples but in only 3 of the 40 natural samples. An odds ratio showed that it is over 16 times more likely to encounter infected amoebae in cooling towers than in natural environments. Environmental data from cooling towers and natural habitats combined revealed dissolved organic carbon (DOC) and pH were predictors of the occurrence of the pathogens, however, when cooling tower data alone were analyzed, no variables accounted for the occurrence. Several bacteria have novel rRNA sequences, and most strains were not culturable outside of amoebae. Such pathogens of amoebae may spread to the environment via aerosols from cooling towers. Studies of emerging infectious diseases should strongly consider cooling towers as a source of amoeba-associated pathogens.

Several factors were examined with respect to their influence on the attraction of protozoa towards yeast extract and on the inhibitory effect of cadmium on chemoattraction. These included: density of protozoa (numbers per volume), age of cultures, axenic and bacterized cultures, and starvation period before testing. A marine species, Miamiensis avidus, and a strain of Tetrahymena isolated from a local river, were tested separately. Two-fold increases in density of Miamiensis resulted in a three-fold increase in numbers attracted, whereas Tetrahymena resulted in a two-fold decrease in numbers attracted. However, inhibition by cadmium was not altered by using different densities. At 250 and 500 protozoa/25 μL, Tetrahymena was inhibited 87.8 ± 5.2 (average of four replicates ± 1 SD) and 89.0 ± 2.6%, respectively; at 117 and < 1 protozoa/25 μL, Miamiensis was inhibited 76.0 ± 4.4 and 67.5 ± 10.0%, respectively. Age of culture had an effect on cadmium sensitivity of Miamiensis but not of Tetrahymena. Older cultures of Miamiensis were more inhibited by cadmium (77.0 ± 2.0%) than younger cultures (57.3 ± 8.4%). Protozoa from axenic and bacterized cultures of Tetrahymena were equally affected by cadmium. This aspect was not tested for Miamiensis, since we did not have axenic cultures of this species. For both species, the starvation period clearly affected the numbers of protozoa attracted to yeast extract. Longer starvation periods resulted in greater attraction, yet their sensitivity to the metal did not change. After 1 and 72 h of starvation Tetrahymena was inhibited 79.0 ± 7.4 and 80.7 ± 10.8%, respectively, and Miamiensis was inhibited 78.5 ± 10.0 and 78.5 ± 5.0%, respectively. These results should provide suggestions for better standardization of behavioral tests using protozoa.