Bactericidal Effect of Several Chemicals on Hatching Eggs Inoculated with Salmonella serovar Typhimurium

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1 2007 Poultry Science Association, Inc. Bactericidal Effect of Several Chemicals on Hatching Eggs Inoculated with Salmonella serovar Typhimurium N. A. Cox,* 1 L. J. Richardson,* R. J. Buhr,* M. T. Musgrove, M. E. Berrang, and W. Bright *USDA, Agricultural Research Service, Poultry Microbiological Safety Research Unit, Russell Research Center, Athens GA 30605; USDA, Agricultural Research Service, Egg Quality and Safety Research Unit, Russell Research Center, Athens GA 30605; USDA, Agricultural Research Service, Bacterial Epidemiology and Antibiotic Resistance Research Unit, Russell Research Center, Athens GA 30605; and South Carolina State University, Orangeburg, Primary Audience: Researchers, Hatchery Managers, Quality Control Personnel, Flock Supervisors SUMMARY Breeder flocks and commercial hatcheries represent an early contamination point for Salmonella entry into commercial integrated poultry operations. Utilizing effective antimicrobial treatments for hatching eggs is a critical part of reducing the incidence of Salmonella-colonized chicks on the farm. The objective of this study was to evaluate the bactericidal effect of several chemicals on Salmonella-contaminated hatching eggs. Four replications (n = 10/treatment per replicate) were conducted to determine the efficacy of 7 commercially available compounds. The compounds tested were as follows: 1) hydrogen peroxide, 2) water-oil emulsion droplets stabilized by detergent, 3) peroxyacetic acid, 4) 4 quaternary ammonium compounds attached to a polymer, 5) 2 quaternary ammonium compounds, 1 biguanide compound and bronopol attached to a polymer, 6) N-alkyl dimethyl benzyl ammonium chloride and stabilized urea, and 7) polyhexamethylenebiguanide hydrochloride. A naladixic acid-resistant Salmonella serovar Typhimurium was inoculated (10 3 cfu/ml) onto fertile hatching eggs by drip-inoculation. Controls included a positive control (no spray application) and a water control (spray containing water to take into account rinsing effects). Compounds 5 and 7 had a 100% reduction, and both of these chemicals included a biguanide. Compounds 4 and 3 were also effective with a 95 and 93.5% reduction, respectively. Compounds 6 and 2 were the least effective of all chemicals, with a reduction of 47.5 and 40%, respectively. Hydrogen peroxide (compound 1), which has been used by the poultry industry, had a 70% reduction, and the water control produced a 10% reduction due to the rinsing effect. Several antimicrobials tested were more effective than hydrogen peroxide. More detailed studies will be required to adequately evaluate these antimicrobials. Key words: hatchings egg, Salmonella, bactericide 2007 J. Appl. Poult. Res. 16: doi: /japr Corresponding author: ncox@saa.ars.usda.gov

2 624 DESCRIPTION OF PROBLEM JAPR: Research Report Salmonella is well known for its ability to cause disease in humans. When associated with foodborne outbreaks, Salmonella infection usually leads to gastroenteritis [1]. To produce chicken and chicken products free of human bacterial pathogens, the source of the pathogens needs to be interrupted [2, 3]. All sources of Salmonella are potentially important; however, in the integrated poultry industry, the hatchery may be the most important source. A freshly laid egg is wet, warm, and susceptible to rapid penetration by organisms such as Salmonella, usually from the feces. Once Salmonella passes through the shell and into the membranes of hatching eggs, it is difficult to prevent further invasion of the egg contents or developing embryo [4]. As the egg cools, the contents contract, and the resulting negative pressure pulls shell surface bacteria into and through the shell and membrane [5, 6]. Salmonella on or within a hatching egg are spread in the hatching cabinet when the chick pips a contaminated egg [7]. This contamination can then be carried by many of the hatchmates to grow-out facilities. In fact, Salmonella isolated from the hatchery have been found to make their way not only to the grow-out farm but also to the processing plant and the final product [8]. Therefore, eggshell decontamination is a critically important control point for preventing the entry of Salmonella and other human enteropathogens into poultry production and subsequent processing. The need to disinfect broiler hatching eggs was recognized at least as early as 1908, when Pernot reported on the use of formaldehyde gas to control microbial populations [9]. Since the Occupational Safety and Health Administration published its list of concerns in 1991 on the effects of repeated or prolonged exposure to formaldehyde, there have been numerous studies evaluating various chemical sprays or dips as potential replacements for formaldehyde [10].Therefore, effective bactericidal treatment of hatching eggs is critical to reduce the possibilities of broiler flock contamination due to these routes of transmission [4, 11]. Numerous studies have been conducted to determine the best antimicrobial to use in fertile hatching egg sanitation programs. In our laboratory at Russell Research Center, we have evaluated over 20 different commercial hatching egg sanitizers in the past 15 yr. This present study is a continuation of our efforts to identify the most effective chemicals for use in the poultry industry. The objective of this study was to evaluate the bactericidal effect of several untested chemicals on Salmonella contaminated hatching eggs along with reevaluation of hydrogen peroxide. MATERIALS AND METHODS Four repetitions were conducted to determine the bactericidal effects of 7 commercially available chemical products on elimination of inoculated naladixic acid-resistant Salmonella serovar Typhimurium on hatching eggs (Table 1). In addition, a positive control (no spray application) and a water control (spray containing water to take into account rinsing effects) were also utilized. For each repetition, eggs (n = 90) were inoculated with a yielding concentration of 10 3 naladixic acid-resistant Salmonella serovar Typhimurium by the method described by Berrang et al. [2], in which 10 L of cell suspension from a 10 4 dilution in saline was placed on the side of an egg that had been tempered to 42 C and spread with a sterile bent-glass rod. The eggs were then randomly divided into groups of 10, and each of the above 7 chemicals and 2 controls were assigned to 1 of the groups of 10 eggs. The inoculated eggs were allowed to dry for a period of 1 h before spray application of the chemicals. For spray application, each chemical was placed into a calibrated 750- ml sterile spray bottle (each spray bottle was calibrated to administer 10 ml of chemical per egg). Each chemical was spray-applied to each egg, and the eggs were allowed to sit at room temperature for 1 h. Each egg was aseptically opened, and the internal contents were discarded. A modified crush-and-rub method was used, each eggshell was placed into a 50-mL tube, and 30 ml of sterile 1% buffered peptone was added. For compounds 5 and 7, one milliliter of 10% powdered milk was added to deactivate the effects of the chemicals; preliminary experiments found that these were the only chemicals that needed deactivation to exclude effects of residual chemicals in the enrichment. The content inside each 50-mL tube was then

3 COX ET AL.: EFFECT OF CHEMICALS ON HATCHING EGGS 625 Table 1. The active ingredients of the 7 chemicals tested on reducing Salmonella in fertile hatching eggs Compound Concentration 1 tested Company Active ingredient (ppm) 1 J. T. Baker Inc., Hydrogen peroxide 14,000 Stone Mountain, GA 2 Nanobio Inc., Water-oil emulsion droplets (200 to 400 nm) 100,000 Ann Arbor, MI stabilized by detergent 3 FMC Inc., Peroxyacetic acid and hydrogen peroxide 35,000 Princeton, NJ 4 Bytrol Inc., 4 quaternary ammoniums attached to a 40,000 Spartanburg, SC polymer 5 Bytrol Inc. 2 quaternary ammoniums, a biguanide and 111,000 bronopol attached to a polymer 6 UPI, N-Alkyl dimethyl benzyl ammonium 800 Atlanta, GA chloride and stabilized urea 7 Arch Chemicals Inc., Polyhexamethylenebiguanide hydrochloride 200 Norwalk, CT 1 The concentration was determined by the recommendation of the manufacturer. crushed using a 2.2-mL sterile pipette and was incubated at 37 C overnight. From the tubes containing buffered peptone and sample, 20 L was then streaked for isolation onto brilliant green sulfa agar plates containing 200 ppm naladixic acid and incubated overnight at 37 C. Plates were then observed for growth of the inoculated Salmonella Typhimurium, confirmed by latex agglutination and results recorded. For interpretation of the data, the chisquared test for independence was utilized. RESULTS AND DISCUSSION In this study, compounds 5 and 7 were the only compounds tested that produced a 100% reduction. None of the 40 eggs inoculated with Salmonella were found to contain the marker organism after treatment with these 2 chemicals (Table 2). Both of these chemicals contain a biguanide. Other studies, 1 conducted over 2 decades ago and another more recent study, also found that compounds containing a biguanide were very effective even in very dilute solutions as an eggshell-sanitizing agent regardless of whether it was applied as an immersion or a spray [12, 13]. Therefore, this study further substantiated the effectiveness of biguanides to be used as a fertile egg sanitizer. In regards to compound 5, which attaches several chemicals by utilizing a polymer, it also contained 2 quaternary ammoniums and a bronopol. The combined benefit of these chemicals in the compound could not be determined; however, the combination of biocides adds to its broad spectrum of efficiency against both gram-positive and -negative bacteria, viruses, and other microorganisms. It has been shown that quaternary ammoniums are less effective than biguanides as a fertile egg sanitizer [3]. However, further investigations are needed to determine whether the attachment of these chemicals to form a compound increases the effectiveness that could be achieved compared with compounds that only contain a biguanide. Compounds 4 and 3 were also effective, resulting in 95 and 93.5% reductions, respectively. There was no significant difference between these compounds or between compounds 5 and 7. Compound 3 was a mixture of peroxyacetic acid and hydrogen peroxide, which forms peracetic acid, whereas compound 4 was 4 quaternary ammonium chemicals attached together by a polymer. Compounds 2 and 6 were relatively ineffective, because approximately half of the inoculated eggs remained positive after treatment and significantly less effective than hydrogen peroxide. Compound 1, which was hydrogen peroxide, resulted in removal of Salmonella from 70% of the inoculated eggs. This was somewhat similar to previous studies with hydrogen peroxide [4, 11]. A significant difference (P < 0.05) was seen between compound

4 626 JAPR: Research Report Table 2. Bactericidal effect of several chemicals on Salmonella-inoculated hatching eggs Positive Positive Reduction Reduction Treatment (n) (%) (%) to water (%) Positive control 40/40 a 100 Water control 36/40 a /40 c /40 b /40 d /40 d /40 d /40 b /40 d a d Means in a column with no common superscript are significantly different (P < 0.05). 1 and compounds 3, 4, 5, and 7, in which the aforementioned compounds significantly (P < 0.05) reduced the number of positive hatching eggs compared with hydrogen peroxide. The rinsing effect of water was shown to eliminate the inoculated Salmonella from 10% of the inoculated eggs; however, it was not significant from the untreated control. Salmonella were recovered from 100% of the untreated controls. Because 4 of these compounds were found to be significantly (P < 0.05) more effective than hydrogen peroxide, future research is needed in the following areas. Studies need to CONCLUSIONS AND APPLICATIONS 1. The 2 compounds that both contain a biguanide performed the best with a 100% reduction. 2. Four compounds performed better than hydrogen peroxide. 3. Before commercial recommendation of these 4 chemicals, further studies need to be conducted to determine the effect of these antimicrobials on hatchability. 4. Studies also need to be conducted to determine that the inoculated Salmonella is not present in the young hatchling. 1. Cox, N. A., M. E. Berrang, and J. A. Cason Salmonella penetration of egg shells and proliferation in broiler hatching eggs. Poult. Sci. 79: Berrang, M. E., N. A. Cox, J. S. Bailey, and L. C. Blankerenship Methods for inoculation and recovery of Salmonella from chicken eggs. Poult. Sci. 70: Cox, N. A., J. S. Bailey, and M. E. Berrang Bactericidal treatment of hatching eggs. I. Chemical immersion treatments and Salmonella. J. Appl. Poult. Res. 7: Cox, N. A., J. S. Bailey, J. M. Mauldin, and L. C. Blankenship Presence and impact of Salmonella contamination in commercial broiler hatcheries. Poult. Sci. 69: REFERENCES AND NOTES be performed to determine the effects of these chemicals on hatchability of the fertile eggs. In addition, acid tests need to be conducted in which these chemicals are applied to inoculated fertile eggs, and then the eggs are incubated and hatched. The hatched chicks then need to be put in a sterile isolator for 7 d, and the intestinal tract of the bird needs to be examined for the marker organism that was applied to the eggs. Then and only then can the chemicals be recommended to the poultry industry concerning its effectiveness and suitability for commercial use. 5. Loch, J. L., L. J. Dolman, and R. G. Board Observations on the mode of bacterial infection of hen s eggs. FEMS Microbiol. Lett. 100: Bruce, J., and E. M. Drysdale Trans-shell transmission. Pages in Microbiology of the Avian Egg. 1st ed. R. G. Board and R. Fuller, ed. Chapman and Hall, London, UK. 7. Cason, J. A., N. A. Cox, J. S. Bailey, and L. C. Blankenship Transmission of Salmonella Typhimurium during hatching of broiler chicks. Avian Dis. 38: Lahellec, C., and P. Colin Relationship between serotypes of salmonellae from hatcheries and rearing farms and those from processed poultry carcasses. Br. Poult. Sci. 26:

5 COX ET AL.: EFFECT OF CHEMICALS ON HATCHING EGGS Pernot, E. F An investigation of the mortality of incubator chicks. Oregon Agric. Exp. Bull Schwab Brothers, Portland, OR. 10. Occupational Safety and Health Administration Occupational exposure to formaldehyde. Response to court remand. Fed. Regist. 55: Cox, N. A., J. S. Bailey, J. M. Mauldin, L. C. Blankenship, and R. L. Wilson Extent of salmonellae contamination in breeder hatcheries. Poult. Sci. 70: Cox, N. A., J. S. Bailey, J. E. Thomson, G. H. Snoeyenbos, and S. A. Vezey The use of polyhexamethylenediquanide hydrochloride to eliminate Salmonella and other microorganisms on hatching eggs. Poult. Sci. 61: Cox, N. A., M. E. Berrang, R. J. Buhr, and J. S. Bailey Bactericidal treatment of hatching eggs. II. Use of chemical disinfectants with vacuum to reduce Salmonella. J. Appl. Poult. Res. 8: