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J. Fd Hyg. Safety Vol. 27, No. 1, pp. 50 54 (2012) Available online at Bactericidal Efficacy of Vital- Oxide , Disinfectant Solution Against Salmonella Typhimurium and Brucella Ovis Chun-Nam Cha , Yeo-Eun Lee , In- Jin Kang , Chang-Yeul Yoo , Sunjeong An , Suk Kim, and Hu- Jang Lee* Research Institute of Live Sciences, College of Veterinary Medicine, Gyeongsang National University, Chinju 600-701, Korea Engineering Research Institute, Department of Industrial Systems Engineering, Gyeongsang National University, Chinju 600-701, Korea Department of Environmental Health, Graduate School of Public Health, Gyeongsang National University, Chinju 660-751, Korea Department of Computer Information, Gyeongnam Provincial Namhae College, Namhae 668-801, Korea Department of Occupational Therapy, Inje University, Gimhae 621-749, Korea (Received February 7, 2012/Revised February 24, 2012/Accepted March 12, 2012) ABSTRACT - Salmonella spp. and Brucella spp. have caused a considerable disease of farmed animals and eco- nomic loss in animal farming and food industry. In this study, the disinfection efficacy of Vital-Oxidel disinfectant, composed to chlorine dioxide, betaine hydrochloride, and propylene glycol was evaluated against S.
typhimurium and Brucella ovis. A bactericidal efficacy test by broth dilution method was used to determine the lowest effective dilution of the disinfectant following exposure to test bacteria for 30 min at 4 C. Vital-Oixdel teria were diluted with distilled water (DW), hard water (HW) or organic matter suspension (OM) according to treat- ment condition. On OM condition, the bactericidal activity of Vital-Oixdel against S. typhimurium and Brucella ovis was lowered compared to that on HW condition. As Vital-Oxidel possesses bactericidal efficacy against animal pathogenic bacteria such as S. typhimurium and Brucella ovis, this disinfectant solution can be used to control the spread of bacterial diseases.
Key words: Vital-Oxide , Salmonella typhimurium, Brucella ovis, Disinfectant efficacy Salmonella extensively causes self-limiting enteritis, fatal through water and poultry to humans infection in animals, food-borne infection, and typhoid fever Brucellae are Gram-negative, facultative, and intracellular . Salmonella infections are zoonotic disease and bacteria that can infect many species of animals and human.
can be transferred between humans and nonhuman animals.
Based on differences in pathogenicity and host preference, six Many infections are due to ingestion of contaminated food.
species are recognized within the genus Brucella.
The etiologic agents of salmonellosis are Salmonella spp.
B. melitensis and B. suis are responsible for bovine brucellosis, characterized by motile, Gram-negative, rod-shaped bacteria ovine and caprine brucellosis, and swine brucellosis, re- and facultative intracellular pathogens that can multiply within spectively. These three Brucella species may cause abortion in professional and nonprofessional phagocytes.
their hosts, which could result in huge economic losses. In survive for weeks outside a living body and are not destroyed addition, B. ovis is responsible for lamb epididymitis As Salmonella and Brucella infections are becoming harder Salmonella typhimurium (S. typhimurium) is one of the to control because of resistance to commonly used antibiotics, most frequently isolated serotypes from pig farms, slaughtered the effective cleaning and disinfection regimes are essential swine and human foodborne illness . Also, S. typhimurium for the prevention of infections and outbreaks can survive in different reservoirs and is easily transmitted cleaning and disinfectant regimes depend on the proper use of biocides, and there is the concern that the resulting increased use of biocides in farming, food production, and *Correspondence to: Hu-Jang Lee, College of Veterinary Medicine, hospital settings, and the home could contribute to the Gyeongsang National University, 900 Gajwa-dong, Chinju 660-701, selection of antibiotic-resistant strains as some mechanisms of biocide resistance also confer antibiotic resistance Tel: 82-55-772-2352, Fax: 82-55-772-2308 E-mail: Biocides are often composed of a mixture of ingredients that Antimicrobial efficacy of Citra-Kill , disinfectant solution act upon a wide range of cellular mechanisms and targets, Table 1. Experimental design for the determination of the bactericidal efficacy of Vital-Oxide which makes it difficult for bacteria to become resistant to Contents according to treatment condition** Salmonellosis and Brucellosis in livestock animals and human may cause enormous economic loss livestock animals caused by intensive farming practices, and the development of antibiotic-resistant bacteria are among the major reasons for the increased frequency of bacterial disease . Highly hygienic measures including the use of disinfectant are very effective for successful control of dis- *DW, distilled water; HW, standard hard water; OM, organic matter.
**+, presence; -, absence eases from bacteria, fungi and parasites in farmed animals hydrochloride and propylene glycol have been used for negative control) in Table 1. HW, an ingredient of HW decontamination of farmed animal and food borne dis- treatment condition, was made by adding anhydrous CaCl2 . However, there is not the efficacy test for the 0.305 g and MgCl ·6H O 0.139 g into one liter distilled water.
disinfectant composed of chlorine dioxide, betaine hydrochlo- Organic suspension, an ingredient of OM treatment condition, ride and propylene glycol against bacterial animal diseases.
is a solution of 5% (w/v) yeast extract in HW. The test Therefore, this study was carried out to examine bactericidal organisms were prepared by titration of each cultural broth efficacy of a disinfectant solution against S. typhimurium and into at least 10 CFU/ml viable organisms with the same kind of diluents of treatment condition. Materials and methods Experimental procedures For the efficacy test against S. typhimurium, Vital-Oxide Bacteria and culture was diluted 2.0, 2.25, 2.5, 2.75, and 3.0 times with DW and HW, and diluted 1.0, 1.1, 1.2, 1.3, and 1.4 times with OM, Brucella ovis (ATCC 25840)) were obtained from the Korean respectively. For the efficacy test against Brucella ovis, Vital- Veterinary Culture Collection (KVCC, Seoul, Korea). The Oxide was also diluted 2.4, 2.7, 3.0, 3.3, and 3.6 times with strains were maintained as frozen glycerol stock. S. typhimurium DW, and diluted 2.0, 2.25, 2.5, 2.75, and 3.0 times with HW, cells were cultured in Luria-Bertani (LB) broth containing and diluted 1.0, 1.1, 1.2, 1.3, and 1.4 times with OM, 1.5% agar. Brucella ovis were spread in Brucella broth containing 5% fetal bovine serum and incubated at 37 C To verify the lowest effective dilution of the disinfectant, under CO condition.
five serial dilutions of the disinfectant were prepared and placed at 4 C prior to test reaction. 2.5 ml of each disinfectant dilution was mixed with the same amount of test organism The active ingredients for Vital-Oxide , the tested dis- followed by contact time of 30 min at 4 C.
infectant solution, are chlorine dioxide (0.01% v/v), betaine During this period, the mixture was shaken at 10 min hydrochloride (0.50% v/v) and propylene glycol (0.30% v/ interval. At the end of 30 min contact period, one ml of the v). Vital-Oxide was provided by Dae Han New Pharm Co.
mixture was neutralized with 9 ml of Nutrient broth containing (Seoul, Korea). The disinfectant solution was stored in the 5% inactivated horse serum (Becton Dickinson & Co., MD, dark in room temperature and prepared for dilution on the USA) at 37 C. 0.1 ml of the neutralized reaction mixture was day of evaluation. Determination of the antimicrobial efficacy subcultured into 10 ml of recovery each cultural broth at of the disinfectant was based on Animal, Plant and Fisheries 37 C for 48 h in incubator. The valid dilution was determined Quarantine and Inspection Agency Regulation No. 2008-14, that the greatest dilution showing no growth in two or more in the five replicates was confirmed. The final dilution time was statistically determined by a median value among three Diluents and treatment condition valid dilution of the triplicate test, but each value of which Testing was based on bactericidal effects of disinfectant should be within 20% experimental error.
diluents in three treatment conditions (distilled water (DW) condition, standard hard water (HW) condition, and organic matter (OM) condition), pathogen control (disinfectant negative control) and DW control (both disinfectant and pathogen Table 2 shows the final valid dilution of Vital-Oxide Chun-Nam Cha, Yeo-Eun Lee, In-Jin Kang, Chang-Yeul Yoo, Sunjeong An, Suk Kim, and Hu-Jang Lee Table 2. Final valid dilution of Vital-Oxide against S. typhimurium and Brucella ovis Treatment condition Bacterial strains *DW, distilled water; HW, standard hard water; OM, organic matter; DT, dilution time.
! , growth; ×, growth inhibition composed to chlorine dioxide, betaine hydrochloride and low concentrations) and then penetrating into the cell and dis- propylene glycol. On DW condition, S. typhimurium and B.
rupting the protein synthesis . At the various concentration ovis were completely inactivated with 2.5 and 3.0 fold dilu- of chlorine dioxide, the method of rapid bacterial kill appears tions of the disinfectant, respectively. When the bactericidal to be the softening and destroying of the cell wall or viral effect on HW condition was evaluated, the antibacterial envelope. However, human cells do not have cell walls and activity of the disinfectant showed on 2.5 fold dilutions against are apparently unaffected. Human skin and bodies are likely both S. typhimurium and B. ovis. With the investigation of the protected from the general oxidative effects of chlorine dioxide bactericidal effect of the disinfectant on OM condition, both by the many reducing agents in human cells and blood S. typhimurium and B. ovis were inactivated on 1.1 fold previously reported that in fish exposed to the chlorine dilutions. Because organic material interferes with efficacy dioxide-treated wastewater effluents, there was no induction by either inactivating the disinfectant or blocking it from of plasma vitellogenin or reduction in the weight of the surface contact, the bactericidal activity of the disinfectant fatpad, a secondary sex character in males. Daniel et al.
on the OM condition was lowered against animal pathogenic exposed groups of 10 male and 10 female Sprague-Dawley bacteria compared with DM or HW conditions.
rats to chlorine dioxide in drinking water for 90 days at Comparing the results of the disinfectant against two concentrations of 0, 25, 50, 100, or 200 mg/l. No exposure- pathogenic bacteria in the present study, the bactericidal effect of Vital-Oxide against B. ovis was higher than that against parameters were reported. But, exposure to over than 50 mg/ S. typhimurium on the DM condition and was same on the l resulted in significant reductions in terminal body weights HW and OM condition. and water consumption. In the short-term toxicological studies of chlorine dioxide , a group of 10 healthy male adults drank 1,000 ml of a 0 or 24 mg/l chlorine dioxide solution.
Neither study found any physiologically relevant alterations Vital-Oxide is a potential antibacterial disinfectant which in general health, vital signs, serum clinical chemistry and was composed of chlorine dioxide, betaine hydrochloride and hematologic parameters. Chang and Schneider propylene glycol. Chlorine dioxide has been used for food that the treatment for 15 sec with chlorine dioxide-based dis- sanitization as an alternative of chlorine-based sanitizer.
infectant at the dose of 5 µg/ml reached a 3.0 logCFU/ml Aqueous chlorine dioxide has much higher oxidation capacity reduction on the surface of tomatoes contaminated with than chlorine and doses not generate undesirable chemicals Salmonella. Trinetta et al.
investigated on the effectiveness like trihalomethanes . The main action of chlorine dioxide of chlorine dioxide, ozone gas and e-beam irradiation treat- consists in the oxidation of cellular constituents. Chlorine ments for inactivation of pathogens inoculated onto tomato, dioxide has a direct action on cell membranes, either altering cantaloupe and lettuce seeds. The result suggested that (at high concentrations) or disrupting their permeability (at chlorine dioxide, ozone gas and e-beam irradiation treatments Antimicrobial efficacy of Citra-Kill , disinfectant solution were 5.3, 4.4 and 4.0 log CFU/g reduction against Salmonella Salmonella typhimurium on contaminated tomato seeds, respectively. And Shams et L] !ma= >d },K !ma9 ‰Š5… u‹ carried out the disinfectant efficacy test for the chlorine ŒQ)Ž L) 3‚ƒy, / OX? !m 3 T dioxide solution against Brucella species. At the concentration U, _/ ‘l, ’ “VW ”.•G of 0.25 mg/l chlorine dioxide in portable water, Brucella species Salmonella typhimurium were inactivated by at least 3.0 logCFU/ml within 10 min. % –= *+1—T 01y, _ !m 9v ˜E F‚ With the consideration of previous studies, Vital-Oxide is ™š, L OX?) pmT 01 ›72 ?œ5)Ž L a more effective and safe disinfectant than chlorine and other l d F2 “VW ž•G treatments like ozone and beam irradiation against pathogenic In this study, disinfectant efficacy of Vital-Oxide has limitation that the results are based on in vitro test. Organic 1. Cleaveland, S., Laurenson, M.K. and Taylor. L.H.: Diseases material in suspension (OM condition) could not represent all of humans and their domestic mammals: pathogen character- possible parameters of Salmonella and Brucella contaminated istics, host range and the risk of emergence. B. Biol. Sci. 356, farm and food-industry environments.
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Die wahrscheinlichkeit, dass katzen mit husten an einer atemwegserkrankung leiden, ist bei weitem größer als die wahrscheinlic

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