01996Applied Poultry sfienc+ Inr EVALUATION HATCHING EGGS OF SANITIZERS FOR SIMON M. SHANE' and ANN FAUST Department of Epidemiology and Community Health, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803 Phone: (504) 3463327 FAX: (504) 363295 Primary Audience: Production Managers, Quality Control Technicians, Avian Health Professionals DESCRIPTION OF PROBLEM The competitive environment of modem intensive broiler production dictates high efficiency through optimal hatchability, chick viability, and growout performance. Microbial contamination of egg shells inherent in housing broiler parent flocks on a combination of slats and deep litter may result in a depression in hatch to fertile ratios compared to genetic potential. Such contamination may also cause an increase in omphalitis in chicks, and egg contamination and shell penetration may result in subcutaneous cellulitis of the ab- dominal and inguinal areas. Most integrators specify that the contractor carry out a program to decontaminate the surface of hatchingquality nest eggs shortly after each collection. Phenolic [l] or quaternary ammonium compounds [2] are usually sprayed on eggs either stacked on collecting flats or trayed in setter racks. During the past 5 years, formaldehyde fumigation has been phased out of routine decontamination programs for hatching eggs despite its efficacy as a disinfectant [3J because of federal restrictions and concern over possible liability claims arising from exposure of farm workers or hatchery personnel 1 To whom correspondence should be addressed
Research Report SHANE and FAUST 135 to a toxic and potentially carcinogenic compound [4]. Hatchmg egg disinfectants can be evaluated on the basis of safety, spectrum of antimicrobial action, cost-effectiveness, and ability to support hatchability and chick viabdity. This paper documents the evaluation of the cost-effectiveness of four chemical disinfectants for broiler hatching eggs. MATERIALS AND METHODS Hatching-quality broiler parent eggs were obtained at a morning collection from a single 45-wk old flock of 9,000 hens operated by an integrator in west central Mississippi. The flock was housed in a conventional breeder house with deep litter and slats. Eggs were hand gathered from nests and were not subjected to any disinfection procedure. Within 24 hr of gathering, eggs were conveyed on new fiber trays to the laboratory, where they were stored at room temperature for two days before experimental procedures. Eggs were then candled to confirm that shells were free of hairline cracks or other obvious imperfections, and any with shell abnormalities, including hypo-pigmentation or atypical shape, were discarded. Eggs were individually weighed, then distributed among the eight treatments to achieve an approximately similar mean weight for each treatment group of five eggs. We selected Escherichia coli ATCC 25922 (Bactol@ Difco Laboratories) as the test organism. A single disk was placed in 10 ml brain-heart infusion broth (BHI, Difco) and incubated at 37 C for 10 hr. A 10 ml aliquot was transferred to 990 ml BHI and was incubated at 37 C for 3 hr, and the cell concentration was quantified using a hemocytometer. Procedures relating to infection of egg shells, manipulation of eggs, and enumeration of bacterial counts were standardized by performing a series of pilot trials. Each egg was individually wiped with sterile gauze saturated with 95% ethanol and then sprayed with 95% ethanol dispensed from a plastic squeeze bottle. Eggs were allowed to air dry in previously sterilized metal racks for 30 min at room temperature. Five dried eggs acting as a post-cleaning control were placed in individual sterile 500 ml Whirlpak@ bags (Nasco) containing 9 ml of 1% sterile peptone water. Bags were sealed and agitated gently for 5 min. The bags were then shaken vigorously and a 1 ml aliquot was obtained from each of the five bags and pooled in a sterile disposable plastic tube. After agitating this solution on a vortex apparatus, we used it to prepare three serial 10-fold dilutions in sterile peptone water. A 0.02 ml aliquot was transferred and spread across the surface of half of a 100 mm diameter petri dish containing McConkey s agar (Difco). The procedure, when repeated, produced three replicates of each of the three 10-fold dilutions (lo- to lo ). We incubated the inoculated plates at 37 C for 12 hr, then counted colonies using an illuminated magnifier. The remaining 35 eggs were placed in individual plastic Whirlpak bags containing 25 ml of the E. coli solution for 10 min and gently agitated to ensure complete saturation of the shell surface. The eggs were removed and air dried on sterilized metal racks for 30 min. Surface contamination on the five eggs representing the group inoculated with E. coli was calculated following immersion in 1% peptone water and subsequent plating of serial 10-fold dilutions on McConkey s agar as previously described. We distributed the remaining 30 eggs among six sanitizing treatments: a commercial phenolic egg sanitizer, 0.4% Tek-Trol@ (Bio- Tek Industries); an experimental oxyhalogen sanitizer, at 1500 dilution; 1.5% hydrogen peroxide; 250 ppm chlorine solution prepared from commercial 5.25% sodium hypochlorite; distilled water; and phosphate buffer saline (PBS). Each treatment was applied by a handheld sprayer under conditions simulating farm use to achieve thorough saturation of the shell surface of an egg previously contaminated with E. coli. Eggs were then allowed to air dry for 30 min at room temperature, standing on previously sterilized metal racks. We determined the residual level ofe. coli on the shell surface by immersion in 1% peptone water followed by calculation as previously described. The number of E. coli per ml solution was calculated from the mean number of colonies at the highest countable dilution multiplied by the dilution factor and multiplied by 50 representing the volume plated. The % destruction rate was calculated from the formula: - mean residual E coli colony count after treatment mean E coli colony count of non -treated controls x loo
136 SANITIZERS FOR HATCHING EGGS Each of the five eggs of the six treatments and the negative and positive E. coli controls were then placed in a Kuhl Model AT600 setter and maintained at 48 C and 40% relative humidity. Viability and the gross appearance of embryos was reviewed at 16 day break-out. A Culturette@ swab specimen obtained from the yolk sac was plated on McConkey's agar and incubated for 24 hr at 37 C prior to examination for bacterial growth. We calculated the relative cost of each treatment based on the cost of the solution as applied to eggs, using purchase price per unit of volume and the appropriate dilution rate. RESULTS AND DISCUSSION The mean weight of eggs in the six treatment groups and two control groups ranged from 61.02 g to 61.13 g, indicating similar mass and relatively uniform surface area. The concentration of E. coli in the inoculum was determined to be 1.25 x 108 CFU/mL,. The mean concentration of E. coli in the wash solution of eggs after treatment and the corresponding percent destruction is shown in Table 1. The commercially available compounds (0.4% Tek-Trol, 1.5% hydrogen peroxide, and 250 ppm chlorine) and the experimental oxyhalogen compound all demonstrated acceptable activity against E. coli on the surface of broiler hatching eggs. Hydrogen peroxide achieved complete disinfection of the shell surface, in contrast to distilled water and phosphate buffer which only removed 83% and 48% of thee. coli contamination, respectively. Levels below 95% are considered unacceptable when extrapolated to commercial conditions. All eggs were fertile and, with the I -- TREATMENT TABLE 1. Relative efficiencv of ma sanitizers Post ethanolwash (negative control) Post E. &contamination (positive control) 15% hydrogen peroxide solution 0.2% oxyhalogen solution @ 0.4% Tek-Trol solution 250 ppm chlorine from 5.25% hypochlorite solution Distilled water wash exception of the oxyhalogen treatment viable embryos, free of gross malformations at 16-day break-out examination. Three of five embryos in the oxyhalogen treatment group had died at approximately 10-12 days of incubation, suggesting that this compound is toxic at the recommended application rate. Attempts to culture E. coli from the yolk sac contents of eggs at 16 days of incubation yielded growth from all eggs in all infected treatments except for three of the five eggs treated with 1.5% hydrogen peroxide. This observation suggests that although shell infection can be inactivated by spraying the surface of the egg with a disinfectant, penetration of the shell by motile E. coli may occur prior to application of an antibacterial solution. Organisms may also be protected by pores during decontamination and may be responsible for subsequent infection of the interior of the egg. The presence of a potentially pathogenic organism within the yolk sac of 16-day incubated embryos confirms penetration of the shell in addition to the inner and outer shell membranes. The presence of pathogens within the egg may be associated with depressed hatchability, occurrence of omphalitis, or reduced viability. In one study, the application of a quaternary ammonium sanitizer reduced levels of aerobic bacteria on shells by 99% and raised hatchability by 6% compared to levels in untreated eggs [2]. Hydrogen peroxide achieved total decontamination of the shell surface under controlled experimental conditions. This result may be significant for use in commercial hatcheries where proliferation and lateral transmission of bacterial pathogens occur in both setters and hatchers. BACIEFUA PER ML IMMERSION SOLUTION % DFSIRUCIION 0 Not applicable 6.067 lo5 Not applicable 0 100.0 2.83 lo3 9953 1.07 x Id 99.82 1.91 lo4 98.65 1.02 los 83.19
SHANE and FAUST Research Report 137 Table 2 shows the relative cost of the four hatching egg sanitizers. The effectiveness of the sodium hypochlorite solution in relation to cost is noteworthy. Chlorine at 250 ppm is almost as efficient as the more sophisticated (and presumably more costly) oxyhalogen compound. Sodium hypochlorite is obviously an acceptable egg sanitizer in applications where cost is a significant criterion and would be suitable for non-industrialized countries. The commercial phenolic offers a compromise between cost and efficiency. At the recommended dilution rate (0.4%), which yields a 2c cost per liter of working solution, we estimate that eggs can be sanitized at a value equivalent to 6c/loOO chicks hatched, or 0.05% of the current production cost of a broiler chick. If hatchability or first week livability is improved by only OS%, a 1O:l benefit to cost ratio would result. The cost of hydrogen peroxide solution is significantly higher than either hypochlorite or the commercial orthophenol, so use of this compound can be justified only if egg-borne infection resists other sanitizers. Hydrogen peroxide may also be an appropriate agent to decontaminate eggs from high-value breeding stock where cost is not a consideration. It should be noted that this trial involved eggs with clean shells, free of imperfections, that had been disinfected with alcohol prior to contamination under controlled conditions. Inactivation of E. coli and embryo viability were the measures of efficiency. Under commercial conditions, sanitizers are required to inactivate a broad range of bacterial and fungal pathogens. This predicates a phenolic or quaternary ammonium compound compatible with a manual or mechanical delivery system. In addition to short-term efficacy, sanitizing compounds should demonstrate residual bacteriostasis and fungicidal activity, be innocuous to embryonic development, and not affect shell permeability. Compounds also should not damage egg handling equipment. Chlorine will corrode metal components of buggies when applied consistently under farm conditions. TABLE 2. Relative cost of hatchina eaa sanitizers COMMERCIAL PRODUCT WORKING SOLUTION Cost/Liter Concentration CostLiter Concentration Hydrogen peroxide Tek-Trol@ $20.00 $5.00 30% 12% OPPA me 2e 1.5% 0.4% Oxyhalogen compound Sodium hypochlorite Undetermined $0.25-5.25% - o.1e 0.2% 0.25% CONCLUSIONS AND APPLICATIONS 1. The orthophenol-based sanitizer Tek-Trol and 1.5% hydrogen peroxide are both effective in reducing levels of E. coli on the shell surface of broiler hatching eggs, when applied according to a standard laboratory-contamination protocol. 2. We found the experimental oxyhalogen compound unacceptable due to its effect on embryo mortality at the concentration applied, despite its apparent effectiveness as a surface disinfectant. 3. Sodium hypochlorite (250 ppm chlorine) offers acceptable reduction in shell E. coli at a lower cost than commercial compounds. 4. Cost-effectiveness and ultimately benefit:cost ratio should determine choice of hatching egg sanitizers.
138 JAPR SANITIZERS FOR HATCHING EGGS 1. Scott, T.A. and C. Swelnam, 1993. Screening sanitizing agents and methods of application for hatching eggs. 11. Effectiveness against microorganisms on the egg shell. J. Appl. Poultry Res. 2:7-11. 2. Brake, J. and B.W. Sheldon, 1990. Effect of a quaternary ammonium sanitizer for hatching eggs on their contamination, ermeability, water loss, and hatchability. Poultry Sci. 69:&7-525. REFERENCES AND NOTES 3. Williams,J.E, 1969. Effect of high-level formaldehyde fumigation on bacterial PO ulations on the surface of chicken hatching eggs. Avian his. 14:38&391. 4. Scott, T.A. and C. Swctnam, 1993. Screening sanitizin agents and methods of ap lication for hatching eggs. I. knvironmental and user kendliness. J. Appl. Poultry Res. 21-6.