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404 S. Wattanaphansak et al. / Veterinary Microbiology 136 (2009) 403–407 (Wattanaphansak et al., 2005). Both assays make it possible to evaluate the effectiveness of disinfectants against L. intracellularis using bacterial viability as an indicator. Stalosan 1 F (Stormollen, Tureby, Storstrom, Denmark), a powder disinfectant mainly composed of phosphate compounds (85%), copper sulfate (2.5%), ferrous sulfate (2.1%), active chlorine (0.25%), perica oil (0.05%), and al- silicate (10.1%), is indicated for use in livestock farms for reducing the number of microorganisms in the environ- ment, absorption of moisture, and reduction of ammonia production. The susceptibility of L. intracellularis to Stalosan F has not been reported. Therefore, the objective of this study was to evaluate both the modified tissue culture method and the direct count method for testing the efficacy of disinfectants against L. intracellularis and to determine the bacteriocidal activity of Stalosan F against L. intracellularis using both methods. 2. Materials and methods 2.1. Microorganism strains and preparations L. intracellularis strains VPB4 and PHE/MN-01, which were isolated from proliferative hemorrhagic enteropathy infected pigs in the United States in 1991 and 2000, respectively (Guedes and Gebhart, 2003a), were used throughout this study, prepared independently and tested twice. Both were grown, maintained, and harvested as described previously (Guedes and Gebhart, 2003b; Wat- tanaphansak et al., 2005). The final concentration of L. intracellularis was determined using a direct count staining procedure as described previously (Guedes and Gebhart, 2003b). 2.2. Disinfectants and test procedures Two forms of Stalosan F preparations, a powder disinfectant and an aqueous suspension, were used for testing. For use as a powder, Stalosan F was tested at three final concentrations which were 2 X , 1 X , and 0.5 X of recommended dosages ( X = dose recommended on label). Three hundred microliters of bacterial solution containing approximately 10 8 L. intracellularis /ml were added in duplicate and spread onto 10 cm  10 cm square sterile Petri dishes. Then, 1 g, 0.5 g, or 0.25 g of Stalosan F powder was applied evenly to cover the entire surface of the Petri dish. These yielded final concentrations of Stalosan F equivalent to 100 g/m 2 , 50 g/m 2 , and 25 g/m 2 , respectively. For testing as an aqueous suspension, Stalosan F was prepared to final concentrations of 1%, 4%, 8%, 16%, and 32% in Dulbecco’s modified Eagle medium (DMEM). The suspension was mixed and 8 ml of each concentration was aliquoted in duplicate, with the addition of 300 m l of 10 8 L. intracellularis /ml to each. In both applications, L. intracellularis was exposed to Stalosan F for 0.5 h, 1 h, 2 h, and 4 h at room temperature. The controls for each time point were live L. intracellularis in DMEM without exposure to Stalosan F and dead L. intracellularis in which the bacteria were exposed to isopropyl alcohol for 30 min. After incubation, the powder in the Petri dishes was washed with 8 ml DMEM and the suspension was immediately transferred to 15 ml tubes. The bacteria in both applications were separated from the powder by passing the suspension through 5 m m filters into microcentrifuge tubes and centrifuged at 10,000 rpm for 3 min. The pellet was washed twice with sterile distilled water and half was re-suspended with 2 ml sterile distilled water for enumeration by the direct count method. The other half was re-suspended with 2 ml of L. intracellularis culture media and used to infect 1-day-old McCoy cells in the modified tissue culture method. 2.3. Bacterial survival assay The percentage of L. intracellularis surviving after exposure to the disinfectant was assessed using both direct count and the modified tissue methods. The direct count method was conducted using Live/Dead 1 BacLight TM staining as described in a previous study (Wattanaphansak et al., 2005). In this study, only green fluorescent cells of live bacteria that stained with SYTO-9 were counted. The modified tissue culture method was performed as described previously (Wattanaphansak et al., 2009) to determine L. intracellularis viability. The effec- tiveness of Stalosan F was determined by evaluating the number of heavily infected cells (HICs), which were defined as the relative number of cells that were infected with the surviving L. intracellularis after exposure to the Stalosan F. These numbers were used as a live L. intracellularis indicator. 2.4. Scanning electron microscopy (SEM) For SEM observations, L. intracellularis was exposed to 0.5 g of Stalosan F powder and 16% of Stalosan F suspension for 30 min. The bacteria were then filtered through a 5 m m filter and washed with phosphate buffer saline (PBS) twice. The samples were fixed with 2.5% glutaraldehyde in PBS for 1 h at room temperature. After three washes with PBS, the bacterial cells were fixed with 1% osmium tetroxide in PBS and washed three more times with PBS. The bacterial cells were dehydrated with increasing concentrations of etha- nol (25%, 50%, 75%, 95%, and 100%) and dried in a Balzer Critical Point Dryer 010 1 unit. The fixed bacteria were coated with a thin film of gold–palladium and examined using a VPSEM-Hitachi S-3500N scanning electron micro- scope. 2.5. Data analysis The number of HIC and the number of green fluorescent bacteria in each treatment of Stalosan F were expressed as percentages as compared to the controls. The correlation between the modified tissue culture and direct count methods was estimated with Spearman’s coefficient of rank correlation using the MedCalc 1 version 9.1.0.1 software. 3. Results The results (Figs. 1 and 2) show that both strains of L. intracellularis were similar in their susceptibilities to both
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406 S. Wattanaphansak et al. / Veterinary Microbiology 136 (2009) 403–407 Fig. 2. L. intracellularis exposed to Stalosan F powder at 0.25 g, 0.5 g, and 1 g for 0.5 h. The viability of L. intracellularis was measured with the direct count method (A) and the modified tissue culture method (B). Scanning electron micrographs of normal L. intracellularis and after exposure to 0.5 g of Stalosan F (C). wall of the bacterium seemed cloudy and intact. After exposure to Stalosan F powder at 0.5 g for 30 min, the bacterial cell wall became more translucent, indicating damage to the cell wall. The similar appearance of bacteria is found after exposure to aqueous suspension at 4% and 16% of Stalosan F (picture not shown). 4. Discussion The use of chemical disinfectants in swine facilities is a first line of defense against virus, bacteria, and parasite infection. However, the effect of disinfectants on L. intracellularis is very difficult to measure in vitro . Unlike other bacteria, L. intracellularis is an organism that propagates itself only inside enterocytes. Cell-free culture methods have not been successfully established and so there are no standard in vitro assays for assessing the efficacy of disinfectants against L. intracellularis . In this study, we compared two systems, a modified tissue culture method and a direct count method, for evaluating the efficacy of Stalosan F in killing L. intracellularis . The results from both methods showed that either the powder or suspension forms of Stalosan F could be used for L. intracellularis inactivation. The reduction of L. intracellu- laris viability depended on dose and exposure time of Stalosan F. According to the manufacturer, the compound is to be applied directly on the floor at a concentration of 50 g/m 2 , which equaled to 0.5 g/cm 2 in this study, for reducing the number of viable organisms in the environ- ment. This concentration would be able to kill 100% or > 99% of L. intracellularis after exposure for 30 min, according to the tissue culture and direct count assay, respectively. Although the mode of action of Stalosan F is not fully understood, it has been shown that the viabilities of pathogenic bacteria and viruses were significantly reduced when those organisms were exposed to Stalosan F (Methling et al., 1997). After exposure to the powder and suspension forms of Stalosan F, a few viable L. intracellularis ( < 1%) were detected in the direct count method, while none of the viable bacteria were found when measured with the modified tissue culture method. In this situation it is possible that the green labeled L. intracellularis in the direct count method (which were believed to be live) might have been in the viable but nonculturable state. The bacteria in this state generally remain viable and capable of revival under favorable conditions, but the standard culture methods cannot detect the bacteria. The state of viable but nonculturable has been reported in many Gram- negative bacteria (Xu et al., 1982; Roszak et al., 1984; Gupte et al., 2003; Tholozan et al., 1999). However, this state of viable but nonculturable for L. intracellularis has not been described. In addition, Millard and Roth (1997) found that in dead bacteria with minimal or partial membrane damage, propidium iodine showed limited penetration and accumulation in the cytoplasm of the bacteria. Therefore, it is plausible that the green fluor- escent bacteria are dead L. intracellularis with minimal membrane damage causing a greater accumulation of SYTO-9 than propidium iodine. Clearly, the concentrations of bacteria dramatically decreased after exposure to both forms of Stalosan F. Although the mechanism of action of
