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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