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The Effect of External Shell Treatments on Salmonella Penetration of Chicken Eggs
1084
W. C. MORRIS AND S. L. BALLOUN of diet on xanthine dehydrogenase in chicken tissues. J. Biol. Chem. 193: 659-667. Scholz, R. W., and W. R. Featherston, 1968. Effect of alterations in protein intake on liver xanthine dehydrogenase in the chick. J. Nutrition, 95: 271— 277. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. 6th Ed. The Iowa State University Press, Ames, Iowa. Stripe, F., and E. D. Corte, 1965. Regulation of xanthine dehydrogenase in chick liver. Effect of starvation and of administration of purines and purine nucleotides. Biochem. J. 94: 309-314. Strittmatter, C. F., 1965. Studies on avian xanthine dehydrogenases. Properties and patterns of appearance during development. J. Biol. Chem. 240: 2557-2564. Summers, J. D., and J. Fisher, 1961. Net protein values for the growing chicken as determined by carcass analysis: Exploration of the method. J. Nutrition, 75: 435-442. Summers, J. D. and H. Fisher, 1962. Net protein values for the growing chicken from carcass analysis with special reference to animal protein sources. J. Sci. Food Agr. 13: 496-500.
The Effect of External Shell Treatments on Salmonella Penetration of Chicken Eggs J. E. WILLIAMS AND L. H. DILLARD Southeast Poultry Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, 934 College Station Rd., Athens, Georgia 30601 (Received for publication September 18, 1972) ABSTRACT The penetration patterns of salmonella organisms, pathogenic for poultry, through the outer egg structures can be markedly altered through external shell treatments before surface contamination. The influences of chemical disinfectants added to exposure cultures and applied to the shell after exposure were also evaluated using a new technique for detecting bacterial penetration through each of the outer egg structures. The shell surfaces of fresh eggs were exposed to formaldehyde fumigation, EDTA, hydrochloric acid, formalin solution, cetyl pyridinium chloride, and ethanol before salmonella contamination. Some treatments had a decided influence on established egg penetration patterns of salmonella organisms. Others had little or no effect. When disinfectant solutions in recommended concentrations were added to bacterial suspensions to which eggs were exposed, salmonella penetration was completely prevented. Using disinfectants to wash eggs after exposure to salmonella organisms, followed by 1J hours' incubation, did not significantly alter the penetration pattern. This finding indicated that organisms that have penetrated under the shell were not reached. POULTEY SCIENCE 52: 1084-1089,
INTRODUCTION
V
ARIOUS species of poultry, including chickens and turkeys, are often fecal shedders of salmonella serotypes
1973
which may infect the shell surface of eggs and, under proper conditions, rapidly penetrate the outer egg structures (Williams et at., 1968). When such infections
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of nitrogen balance and carcass analysis methods. J. Nutrition, 55:493-498. Gehrt, A. J., M. J. Caldwell and W. P. Elmslie, 1955. Chemical method for measuring relative digestibility of animal protein feedstuffs. J. Agric. Food Chem. 3: 159-162. Kakade, M. L., and I. E. Liener, 1969. Determination of available lysine in proteins. Anal. Biochem. 27: 273-280. Kazemi-Shirazi, R., 1973. Urea and diammonium citrate utilization by poultry. Poultry Sci. 52: 44-50. Moran, E. T., Jr., J. D. Summers and S. J. Slinger, 1966. Keratin as a source of protein for the growing chick. Poultry Sci. 45: 1257-1266. Morris, W. C , and S. L. Balloun, 1973. Effect of processing methods on utilization of feather meal by broiler chicks. Poultry Sci. 52: 858-866. Naber, E. C , and C. L. Morgan, 1956. Feather meal and poultry meat scrap in chick starting rations. Poultry Sci. 35: 888-895. National Academy of Sciences—National Research Council, 1966. Nutrient requirements of domestic animals: I. Nutrient requirements of poultry. Remy, C , and W. W. Westerfield, 1951. The effect
SHELL TREATMENTS AND SALMONELLA PENETRATION
face of market shell eggs subsequent to dipping in liquid cultures of the organisms. Quaternary ammonium compounds were considered the most effective compounds used for destroying organisms on the shell surface. These compounds had a residual germicidal effect when eggs were infected with salmonella organisms after dipping. Harry (1954) did not find that eggs exposed to fumigation with formaldehyde gas possessed a residual germicidal effect on their surface for salmonella organisms. This paper is a report of research on the effect of various shell surface treatments with specific chemicals and disinfectants on the penetration of salmonella organisms through the outer structures of chicken hatching eggs. MATERIALS AND METHODS
Eggs. Brown-shell, broiler type hatching eggs from White Rock and Athens Randombred (ARB) chickens maintained in isolation on the laboratory premises were employed. Eggs were collected from nests into clean baskets and held at room temperature for approximately 24 hours before use. Shell treatments. Eggs were exposed at room temperature to the following chemical treatments: 1) Soaked in a 10% solution of disodium ethylenediamine tetraacetate (EDTA) (pH 7.5) for 30 minutes. The cuticle was washed free under running tap water; 2) exposed to 0.1 N HCL for 20 seconds. Rinsed in running tap water; 3) left submerged in 95% ethanol for 18 hours. Eggs were not rinsed but allowed to dry at room temperature; 4) soaked in a 2% aqueous solution of formalin (40%) for 18 hours. Eggs were not rinsed but allowed to dry at room temperature; and 5) submerged in a 1-100 solution of cetyl pyridinium chloride for 5 minutes. Antonova (1966) reported that
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occur in hatching eggs, they are important to the profitable production of poultry, and when present in market eggs, they pose a serious public health problem. Since the external surface of the chicken egg offers the first barrier to invasion of the egg contents by salmonella organisms, the influence on salmonella penetration patterns of alterations in its structure is important. Investigations in this field have been directed mostly to the influence of shell treatments on penetration characteristics of common egg spoilage organisms. Brown et al. (1966), employing a dye penetration technique, demonstrated that physical and chemical treatments designed to alter the shell surface generally increased the susceptibility of White Leghorn market eggs to spoilage by Pseudomonas aeruginosa. Studies of this nature have not been reported in which hatching eggs are exposed to salmonella organisms and examined for penetration by using more refined methods. Such methods would demonstrate the organisms moving through each of the outer egg structures. Treatment of the shell surface of eggs with disinfectants to destroy salmonella organisms has been studied using both hatching eggs (Wilson, 1948; Lancaster et al, 1952; Frank and Wright, 1956; Gordon et al., 1956; and Bierer et al., 1961) and market eggs (Rizk et al., 1966). Gordon et al. (1956) studied the germicidal effects of 9 chemical dipping solutions for destroying S. typhimurium and S. thompson organisms experimentally deposited on the surface of chicken eggs. Most of the compounds effectively sanitized the shell surfaces and did not affect the flavor of the egg contents nor the hatchability of dipped eggs. Rizk et. al. (1966) applied several disinfectants to destroy salmonella organisms on the sur-
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to the salmonella organisms in the same manner and incubated along with test groups. Disinfectant exposure. The eggs were exposed to two types of disinfectant treatments: 1) Sani-Squad. Room temperature eggs were dipped for 5 minutes in a room temperature 24-hour veal infusion broth culture of S. typhimurium diluted 1-5 using as a diluent a 1-100 solution of SaniSquad (Vineland Laboratories, Vineland, N. J.). A 1-100 dilution of Sani-Squad provides a use level solution containing 0.25% formaldehyde, 200 p.p.m. quaternary and 250 p.p.m. phenolic agent. This same exposure procedure was used with the addition of 5 gm. of fresh chicken feces to each liter of disinfectant-broth solution. After the above treatment, the eggs were allowed to dry and then incubated at 37°/30°C. for 24 hours before examining for penetration to the areas under the shell, between the membranes, and to the inner surface of the inner membrane. Control eggs were soaked for 5 minutes in a broth culture of S. typhimurium diluted 1-5 with sterile tap water. After being removed, these eggs were incubated at 37°/30°C. for 24 hours and sampled in the 3 areas mentioned above. In a second group of tests, room temperature eggs were also allowed to remain submerged in a 24-hour broth culture of S. typhimurium diluted 1-5 with sterile tap water for 5 minutes. They were then removed and left at room temperature for 1J hours at which time they were washed for 5 minutes in a 1-100 dilution of Sani-Squad. These eggs were incubated at 37 o /30°C. for 24 hours before sampling under the shell, between the membranes and on the inner surface of the inner membrane. Control eggs were soaked in the 1-5 dilution of a 24-hour
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this compound facilitated the penetration of erythromycin into eggs which were dipped for elimination of mycoplasma. After treatment, eggs were removed and brushed individually with a nylon brush as they were rinsed in running tap water. After the above shell treatments, eggs in all the groups were allowed to dry. They were then spot-exposed to approximately 5 X 106 viable cells of Salmonella typhimurium, strain M-24, in normal fresh chicken feces following procedures described in detail by Williams and Whittemore (1967) and Williams et al. (1968). The contaminated eggs were incubated at 37°C. dry bulb and 30°C. wet bulb (37°/30°C.) for 24 hours before sampling the areas under the shell, between the inner and outer membranes, and the inner surface of the inner membrane using methods described by Williams and Whittemore (1967). Control eggs with no shell treatments were also included with each treated group. Fumigation. Eggs were fumigated on plastic racks in a tight enclosure for 20 minutes with a high level of formaldehyde gas (1.2 ml. of formalin (40%) mixed with 0.6 gm. potassium permanganate (KMnO4)/0.0283 cu.m. (1 cu. ft.) of cabinet space). Tests were also conducted with eggs fumigated at twice the above level. At the end of the fumigation, the gas was exhausted to the exterior for 15 minutes before eggs were removed from the cabinet. Immediately after fumigation, eggs were spot-exposed, as above, to approximately 5 X 106 cells of S. typhimurium. The salmonella-exposed eggs were left in separate groups for 15 minutes and for 1 hour at 37°/30°C. before sampling for penetration to the area under the shell only (Area 1) using the method of Williams and Whittemore (1967). Nonfumigated control eggs were also exposed
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SHELL TREATMENTS AND SALMONELLA PENETRATION
broth culture of S. typhimurium, incubated for 1^ hours at room temperature and then washed in tap water rather than Sani-Squad for 5 minutes. 2) Zinc Sulfate. An experimental design similar to the above was followed using as the disinfectant a 1% solution of zinc sulfate (Bierer et al., 1961). The only difference in these tests was that feces was not added to the disinfectant-broth solutions before egg exposures.
TABLE 2.—Effect of high-level formaldehyde fumigation on the penetration patterns of S. typhimurium through the cuticle and shell of chicken eggs incubated at 37°C. % penetrated into Area If after incubation for: Level of
IX* Control eggs (unfumigated) 2X** Control eggs (unfumigated)
114 119
7.90% 10.08%
425 368
13.18% 12.23%
196 197
2.55% 4.06%
363 363
10.47% 10.74%
RESULTS
The various chemical treatments of the egg shell before contamination with 5. typhimurium greatly increased the number of eggs penetrated (Table 1). The most marked effect was noted in eggs exposed to 10% EDTA. A total of 68.92% of such eggs were penetrated by S. typhimurium to the inner surface of the inner membrane in contrast to only 4.80% of control eggs. Eggs soaked in ethanol and 2% formalin solution and not rinsed following such treatment had no viable organisms in the area just under the shell 24 hours following contamination. However, such eggs did show penetration into the deep outer egg structures revealing that organisms had earlier traversed the area TABLE 1.—Influence of various chemical treatments of the shell on the penetration patterns of S. typhimurium through, the shell and shell membranes of eggs incubated at 37°C. Chemical Treatment 10% EDTA 0.1NHC1 Ethanol (95%) Formalin (2%) Cetyl pyridinium chloride (1-1000) Control eggs (no treatment)
No. of eggs
% penetrated after 24 hr. into: . Area2" Ar«a 3f
Arealonly
%
%
%
74 77 30 29
1.35 14.28 0.00 0.00
16.22 10.39 10.00 27.58
68.92 24.68 20.00 20.69
170
5.29
8.82
14.70
125
7.20
3.20
4.80
* Area 1 only = Penetration through cuticle and shell. ** Area 2 =Penetration through cuticle, shell, and outer shell membrane. t Area 3 =Penetration through cuticle, shell, and both shell membranes.
just under the shell. High-level formaldehyde fumigation of eggs immediately before contamination of the shell surface with S. typhimurium did not appreciably affect the penetration of the organisms through the shell (Table 2). Effective residual concentrations of formaldehyde did not remain on the shell surface to protect the eggs from invasion by salmonellae. Even levels of the fumigant twice those conventionally used for on-the-farm preincubation fumigation practices offered no residual protective effect. When broth cultures of S. typhimurium were mixed with either Sani-Squad ( 1 100) or 1% aqueous zinc sulfate disinfectant solutions immediately before dipping, penetration of salmonella organisms into the eggs was entirely prevented (Table 3). In the tests with Sani-Squad, chicken feces was also introduced into the dip solutions, but the presence of such organic matter did not eliminate the efficacy of the disinfectant in killing the organisms or preventing their penetration of the egg shells. Disinfectant solutions had no appreciable effect in changing the penetration patterns of S. typhimurium through the outer egg structures if the organisms were allowed to gain entrance to the area under the shell (Table 4). Results were
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* 1 X = 1.2 ml. of formalin mixed with 0.6 gm. KMnOi/0.0283 cu.m. (1 cu.ft.). ** 2 X =Twice the 1X level. f Area 1 =Penetration through the cuticle and shell only.
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J. E. WILLIAMS AND L. H. DILLARD
TABLE 3.—The penetration of S. typhimurium through the outer structures of chicken eggs dipped into broth cultures diluted immediately before dipping with Sani-Squad and zinc sulfate disinfectant solutions.
The results obtained with the chemical
t Area 3 = Penetration through cuticle, shell, and both shell membranes.
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treatments reported here were apparently due to the partial or complete removal of the cuticular layer on the surface of the egg as well as other outer shell layers be% penetrated after 24 hr. low the cuticle. With these vital barNo. of at 37°C. into: riers damaged or removed, the salmonella Area 1 only* Area 2** Area 3t organisms were able to rapidly penetrate % % % Sani-Squad through the outer egg structures. Any 36 0.00 0.00 0.00 (Without feces) type of egg treatment that injures or Sani-Squad 36 0.00 0.00 0.00 (With feces) eliminates the outer coating on chicken Zinc sulfate (1%) 67 0.00 0.00 0.00 without feces eggs increases the potential of salmonella Control eggs 108 8.33 20.37 14.81 as well as other organisms to penetrate * Area 1 only = Penetration through cuticle and shell. ** Area 2 = Penetration through cuticle, shell, and outer shell into the egg. membrane. t Area 3 =Penetration through cuticle, shell, and both shell Formaldehyde fumigation at a high membranes. level has readily killed salmonella orgavery similar for both disinfectant-treated nisms on the shell surface (Wilson, 1951; and control eggs when samplings were Frank and Wright, 1955). The work remade in each of the 3 areas of the outer ported here shows clearly, however, that egg structures. The 1J hour penetration no residual protective effect is afforded to period before the eggs were exposed to the surface of the egg following fumigathe disinfectant solutions allowed the or- tion to prevent shell penetration by salganisms to reach subsurface areas of the monella organisms even when eggs are egg where they were adequately protected fumigated with twice the usual level of from the germicidal action of the disin- gas. fectant solutions. While the 2 disinfectants used in these studies were effective in preventing peneDISCUSSION tration of salmonella organisms into eggs, Using qualitative procedures to detect if they were mixed with the organisms at the presence of salmonella organisms the time of shell exposure, they had no efunder the shell, between the shell mem- fect on organisms that had been allowed branes, and on the inner surface of the to penetrate under the shell. These findinner membrane, it is possible to deter- ings suggest that if disinfectants are emmine the effect of a large variety of external shell treatments on the penetration properties of salmonella organisms TABLE 4.—S. typhimurium isolations from the outer structures of chicken eggs exposed to the organisms through the outer egg structures. These by dipping into broth cultures followed by 1\ hr. of incubation before washing in studies represent the first application of disinfectant solutions. these procedures for this purpose. Previous techniques have involved culture of % penetrated after 24 hr. Disinfectant No. of at 37°C. into: the egg contents after a set period of exSolution eggs Area 1 only * Area 2** Area 3f posure and very little attention has been % % % directed to the penetration process as it Sani-Squad (1-100) 70 28.57 7.14 7.14 Zinc sulfate (1%) 79 12.65 12.65 2.64 occurs in the outer structures of the egg Control eggs 15.80 76 7.90 2.63 following various treatments of the shell * Area 1 only=Penetration through cuticle and shell. ** Area 2 =Penetration through cuticle, shell, and outer shell surface. membrane.
SHELL TREATMENTS AND SALMONELLA PENETRATION
ployed to dip, wash, or spray eggs, they should be applied as soon as possible after the eggs are laid. ACKNOWLEDGMENT The authors t h a n k Mr. Steven Benson for technical assistance in conducting this work.
REFERENCES
Gordon, R. F., E. G. Harry and J. F. Tucker, 1956. The use of germicidal dips in the control of bacterial contamination of the shells of hatching eggs. Vet. Record, 68: 33-38. Harry, E. G., 1954. The influence of certain chemico-physical characteristics of formaldehyde on its use as a disinfectant. Proc. 10th World's Poultry Cong.: 217-221. Lancaster, J. E., R. F. Gordon and J. Tucker, 1952. The disinfection prior to incubation of hen eggs contaminated with Salmonella pullorum. Brit. Vet. J. 108:418-431. Rizk, S. S., J. C. Ayres and A. A. Kraft, 1966. Disinfection of eggs artificially inoculated with salmonellae. Poultry Sci. 45: 764-769. Williams, J. E., and A. D. Whittemore, 1967. A method for studying microbial penetration through the outer structures of the avian egg. Avian Dis. 11:467-490. Williams, J. E., L. H. Dillard and G. O. Hall, 1968. The penetration patterns of Salmonella typhimurium through the outer structures of chicken eggs. Avian Dis. 12: 445-466. Wilson, J. E., 1948. Avian salmonellosis. Vet. Record, 60: 615-624. Wilson, J. E., 1951. The control of salmonellosis in poultry with special reference to fumigation of incubators. Vet. Record, 63: 501-503.
NEWS AND NOTES {Continued from page 1074) C.D.A. NOTES
ALBERTA NOTES R. J. Chernos has been appointed Poultry Specialist serving the industry in the southern part of Alberta. His office is in the Science Building, Lethbridge Community College. Born in Oliver, British Columbia, he received a B.Sc. degree at the University of Alberta in 1970, majoring in poultry science. He worked with Master Feeds Limited, Sunrise Poultry Farm, and the Poultry Section, Animal Research Institute, Canada Department of Agriculture, Ottawa. QUAKER NOTES Dr. R. E. Smith, Research Branch, Quaker Oats Co., Barrington, Illinois, has been promoted to Assistant Director of Research and DevelopmentNutrition. He has also been appointed to the Technical Advisory Group, Committee on Nutrition, American Academy of Pediatrics.
Dr. I. R. Sibbald, formerly on the staff of John Labatt Ltd., London, Ontario, joined the Animal Research Institute, Central Experimental Farm, Canada Department of Agriculture, Ottawa, Ontario, on December 1. He will be working in the area of poultry nutrition.
4TH EUROPEAN POULTRY CONGRESS The complete Proceedings of the 4th European Poultry Conference, organized by the United Kingdom Branch of the World's Poultry Science Association, and held in London, September 3-8, 1972, will be published by British Poultry Science Ltd. and will be obtainable from Longmans Group Ltd., Journal Division, 33 Montgomery Street, Edinburgh EH7 5JX, Scotland. Date of publication and price have not been an-
(Continued on page 1123)
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Antonova, M. E., 1966. Disinfection of eggs against Mycoplasma infection. Vestnik selskokhozyaistvennoi Nauki (Moskva), No. 8: 97-100. Bierer, B. W., B. D. Barnett and H. D. Valentine, 1961. Experimentally killing Salmonella typhimurium on egg shells by washing. Poultry Sci. 40: 1009-1014. Brown, W. E., R. C. Baker and H. B. Naylor, 1966. Shell treatments as affecting microbial egg spoilage. Poultry Sci. 45: 276-279. Frank, J. F., and G. W.Wright, 1955. Susceptibility of salmonella organisms to formaldehyde fumigation. Canad. J. Comp. Med. Vet. Sci. 19: 71-75. Frank, J. F., and G. W. Wright, 1956. The disinfection of eggs contaminated with Salmonella typhimurium. Canad. J. Comp. Med. Vet. Sci. 20: 406-410.
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W. C. MORRIS AND S. L. BALLOUN of diet on xanthine dehydrogenase in chicken tissues. J. Biol. Chem. 193: 659-667. Scholz, R. W., and W. R. Featherston, 1968. Effect of alterations in protein intake on liver xanthine dehydrogenase in the chick. J. Nutrition, 95: 271— 277. Snedecor, G. W., and W. G. Cochran, 1967. Statistical Methods. 6th Ed. The Iowa State University Press, Ames, Iowa. Stripe, F., and E. D. Corte, 1965. Regulation of xanthine dehydrogenase in chick liver. Effect of starvation and of administration of purines and purine nucleotides. Biochem. J. 94: 309-314. Strittmatter, C. F., 1965. Studies on avian xanthine dehydrogenases. Properties and patterns of appearance during development. J. Biol. Chem. 240: 2557-2564. Summers, J. D., and J. Fisher, 1961. Net protein values for the growing chicken as determined by carcass analysis: Exploration of the method. J. Nutrition, 75: 435-442. Summers, J. D. and H. Fisher, 1962. Net protein values for the growing chicken from carcass analysis with special reference to animal protein sources. J. Sci. Food Agr. 13: 496-500.
The Effect of External Shell Treatments on Salmonella Penetration of Chicken Eggs J. E. WILLIAMS AND L. H. DILLARD Southeast Poultry Research Laboratory, Agricultural Research Service, U. S. Department of Agriculture, 934 College Station Rd., Athens, Georgia 30601 (Received for publication September 18, 1972) ABSTRACT The penetration patterns of salmonella organisms, pathogenic for poultry, through the outer egg structures can be markedly altered through external shell treatments before surface contamination. The influences of chemical disinfectants added to exposure cultures and applied to the shell after exposure were also evaluated using a new technique for detecting bacterial penetration through each of the outer egg structures. The shell surfaces of fresh eggs were exposed to formaldehyde fumigation, EDTA, hydrochloric acid, formalin solution, cetyl pyridinium chloride, and ethanol before salmonella contamination. Some treatments had a decided influence on established egg penetration patterns of salmonella organisms. Others had little or no effect. When disinfectant solutions in recommended concentrations were added to bacterial suspensions to which eggs were exposed, salmonella penetration was completely prevented. Using disinfectants to wash eggs after exposure to salmonella organisms, followed by 1J hours' incubation, did not significantly alter the penetration pattern. This finding indicated that organisms that have penetrated under the shell were not reached. POULTEY SCIENCE 52: 1084-1089,
INTRODUCTION
V
ARIOUS species of poultry, including chickens and turkeys, are often fecal shedders of salmonella serotypes
1973
which may infect the shell surface of eggs and, under proper conditions, rapidly penetrate the outer egg structures (Williams et at., 1968). When such infections
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on May 14, 2015
of nitrogen balance and carcass analysis methods. J. Nutrition, 55:493-498. Gehrt, A. J., M. J. Caldwell and W. P. Elmslie, 1955. Chemical method for measuring relative digestibility of animal protein feedstuffs. J. Agric. Food Chem. 3: 159-162. Kakade, M. L., and I. E. Liener, 1969. Determination of available lysine in proteins. Anal. Biochem. 27: 273-280. Kazemi-Shirazi, R., 1973. Urea and diammonium citrate utilization by poultry. Poultry Sci. 52: 44-50. Moran, E. T., Jr., J. D. Summers and S. J. Slinger, 1966. Keratin as a source of protein for the growing chick. Poultry Sci. 45: 1257-1266. Morris, W. C , and S. L. Balloun, 1973. Effect of processing methods on utilization of feather meal by broiler chicks. Poultry Sci. 52: 858-866. Naber, E. C , and C. L. Morgan, 1956. Feather meal and poultry meat scrap in chick starting rations. Poultry Sci. 35: 888-895. National Academy of Sciences—National Research Council, 1966. Nutrient requirements of domestic animals: I. Nutrient requirements of poultry. Remy, C , and W. W. Westerfield, 1951. The effect
SHELL TREATMENTS AND SALMONELLA PENETRATION
face of market shell eggs subsequent to dipping in liquid cultures of the organisms. Quaternary ammonium compounds were considered the most effective compounds used for destroying organisms on the shell surface. These compounds had a residual germicidal effect when eggs were infected with salmonella organisms after dipping. Harry (1954) did not find that eggs exposed to fumigation with formaldehyde gas possessed a residual germicidal effect on their surface for salmonella organisms. This paper is a report of research on the effect of various shell surface treatments with specific chemicals and disinfectants on the penetration of salmonella organisms through the outer structures of chicken hatching eggs. MATERIALS AND METHODS
Eggs. Brown-shell, broiler type hatching eggs from White Rock and Athens Randombred (ARB) chickens maintained in isolation on the laboratory premises were employed. Eggs were collected from nests into clean baskets and held at room temperature for approximately 24 hours before use. Shell treatments. Eggs were exposed at room temperature to the following chemical treatments: 1) Soaked in a 10% solution of disodium ethylenediamine tetraacetate (EDTA) (pH 7.5) for 30 minutes. The cuticle was washed free under running tap water; 2) exposed to 0.1 N HCL for 20 seconds. Rinsed in running tap water; 3) left submerged in 95% ethanol for 18 hours. Eggs were not rinsed but allowed to dry at room temperature; 4) soaked in a 2% aqueous solution of formalin (40%) for 18 hours. Eggs were not rinsed but allowed to dry at room temperature; and 5) submerged in a 1-100 solution of cetyl pyridinium chloride for 5 minutes. Antonova (1966) reported that
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis S on May 14, 2015
occur in hatching eggs, they are important to the profitable production of poultry, and when present in market eggs, they pose a serious public health problem. Since the external surface of the chicken egg offers the first barrier to invasion of the egg contents by salmonella organisms, the influence on salmonella penetration patterns of alterations in its structure is important. Investigations in this field have been directed mostly to the influence of shell treatments on penetration characteristics of common egg spoilage organisms. Brown et al. (1966), employing a dye penetration technique, demonstrated that physical and chemical treatments designed to alter the shell surface generally increased the susceptibility of White Leghorn market eggs to spoilage by Pseudomonas aeruginosa. Studies of this nature have not been reported in which hatching eggs are exposed to salmonella organisms and examined for penetration by using more refined methods. Such methods would demonstrate the organisms moving through each of the outer egg structures. Treatment of the shell surface of eggs with disinfectants to destroy salmonella organisms has been studied using both hatching eggs (Wilson, 1948; Lancaster et al, 1952; Frank and Wright, 1956; Gordon et al., 1956; and Bierer et al., 1961) and market eggs (Rizk et al., 1966). Gordon et al. (1956) studied the germicidal effects of 9 chemical dipping solutions for destroying S. typhimurium and S. thompson organisms experimentally deposited on the surface of chicken eggs. Most of the compounds effectively sanitized the shell surfaces and did not affect the flavor of the egg contents nor the hatchability of dipped eggs. Rizk et. al. (1966) applied several disinfectants to destroy salmonella organisms on the sur-
1085
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to the salmonella organisms in the same manner and incubated along with test groups. Disinfectant exposure. The eggs were exposed to two types of disinfectant treatments: 1) Sani-Squad. Room temperature eggs were dipped for 5 minutes in a room temperature 24-hour veal infusion broth culture of S. typhimurium diluted 1-5 using as a diluent a 1-100 solution of SaniSquad (Vineland Laboratories, Vineland, N. J.). A 1-100 dilution of Sani-Squad provides a use level solution containing 0.25% formaldehyde, 200 p.p.m. quaternary and 250 p.p.m. phenolic agent. This same exposure procedure was used with the addition of 5 gm. of fresh chicken feces to each liter of disinfectant-broth solution. After the above treatment, the eggs were allowed to dry and then incubated at 37°/30°C. for 24 hours before examining for penetration to the areas under the shell, between the membranes, and to the inner surface of the inner membrane. Control eggs were soaked for 5 minutes in a broth culture of S. typhimurium diluted 1-5 with sterile tap water. After being removed, these eggs were incubated at 37°/30°C. for 24 hours and sampled in the 3 areas mentioned above. In a second group of tests, room temperature eggs were also allowed to remain submerged in a 24-hour broth culture of S. typhimurium diluted 1-5 with sterile tap water for 5 minutes. They were then removed and left at room temperature for 1J hours at which time they were washed for 5 minutes in a 1-100 dilution of Sani-Squad. These eggs were incubated at 37 o /30°C. for 24 hours before sampling under the shell, between the membranes and on the inner surface of the inner membrane. Control eggs were soaked in the 1-5 dilution of a 24-hour
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this compound facilitated the penetration of erythromycin into eggs which were dipped for elimination of mycoplasma. After treatment, eggs were removed and brushed individually with a nylon brush as they were rinsed in running tap water. After the above shell treatments, eggs in all the groups were allowed to dry. They were then spot-exposed to approximately 5 X 106 viable cells of Salmonella typhimurium, strain M-24, in normal fresh chicken feces following procedures described in detail by Williams and Whittemore (1967) and Williams et al. (1968). The contaminated eggs were incubated at 37°C. dry bulb and 30°C. wet bulb (37°/30°C.) for 24 hours before sampling the areas under the shell, between the inner and outer membranes, and the inner surface of the inner membrane using methods described by Williams and Whittemore (1967). Control eggs with no shell treatments were also included with each treated group. Fumigation. Eggs were fumigated on plastic racks in a tight enclosure for 20 minutes with a high level of formaldehyde gas (1.2 ml. of formalin (40%) mixed with 0.6 gm. potassium permanganate (KMnO4)/0.0283 cu.m. (1 cu. ft.) of cabinet space). Tests were also conducted with eggs fumigated at twice the above level. At the end of the fumigation, the gas was exhausted to the exterior for 15 minutes before eggs were removed from the cabinet. Immediately after fumigation, eggs were spot-exposed, as above, to approximately 5 X 106 cells of S. typhimurium. The salmonella-exposed eggs were left in separate groups for 15 minutes and for 1 hour at 37°/30°C. before sampling for penetration to the area under the shell only (Area 1) using the method of Williams and Whittemore (1967). Nonfumigated control eggs were also exposed
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SHELL TREATMENTS AND SALMONELLA PENETRATION
broth culture of S. typhimurium, incubated for 1^ hours at room temperature and then washed in tap water rather than Sani-Squad for 5 minutes. 2) Zinc Sulfate. An experimental design similar to the above was followed using as the disinfectant a 1% solution of zinc sulfate (Bierer et al., 1961). The only difference in these tests was that feces was not added to the disinfectant-broth solutions before egg exposures.
TABLE 2.—Effect of high-level formaldehyde fumigation on the penetration patterns of S. typhimurium through the cuticle and shell of chicken eggs incubated at 37°C. % penetrated into Area If after incubation for: Level of
IX* Control eggs (unfumigated) 2X** Control eggs (unfumigated)
114 119
7.90% 10.08%
425 368
13.18% 12.23%
196 197
2.55% 4.06%
363 363
10.47% 10.74%
RESULTS
The various chemical treatments of the egg shell before contamination with 5. typhimurium greatly increased the number of eggs penetrated (Table 1). The most marked effect was noted in eggs exposed to 10% EDTA. A total of 68.92% of such eggs were penetrated by S. typhimurium to the inner surface of the inner membrane in contrast to only 4.80% of control eggs. Eggs soaked in ethanol and 2% formalin solution and not rinsed following such treatment had no viable organisms in the area just under the shell 24 hours following contamination. However, such eggs did show penetration into the deep outer egg structures revealing that organisms had earlier traversed the area TABLE 1.—Influence of various chemical treatments of the shell on the penetration patterns of S. typhimurium through, the shell and shell membranes of eggs incubated at 37°C. Chemical Treatment 10% EDTA 0.1NHC1 Ethanol (95%) Formalin (2%) Cetyl pyridinium chloride (1-1000) Control eggs (no treatment)
No. of eggs
% penetrated after 24 hr. into: . Area2" Ar«a 3f
Arealonly
%
%
%
74 77 30 29
1.35 14.28 0.00 0.00
16.22 10.39 10.00 27.58
68.92 24.68 20.00 20.69
170
5.29
8.82
14.70
125
7.20
3.20
4.80
* Area 1 only = Penetration through cuticle and shell. ** Area 2 =Penetration through cuticle, shell, and outer shell membrane. t Area 3 =Penetration through cuticle, shell, and both shell membranes.
just under the shell. High-level formaldehyde fumigation of eggs immediately before contamination of the shell surface with S. typhimurium did not appreciably affect the penetration of the organisms through the shell (Table 2). Effective residual concentrations of formaldehyde did not remain on the shell surface to protect the eggs from invasion by salmonellae. Even levels of the fumigant twice those conventionally used for on-the-farm preincubation fumigation practices offered no residual protective effect. When broth cultures of S. typhimurium were mixed with either Sani-Squad ( 1 100) or 1% aqueous zinc sulfate disinfectant solutions immediately before dipping, penetration of salmonella organisms into the eggs was entirely prevented (Table 3). In the tests with Sani-Squad, chicken feces was also introduced into the dip solutions, but the presence of such organic matter did not eliminate the efficacy of the disinfectant in killing the organisms or preventing their penetration of the egg shells. Disinfectant solutions had no appreciable effect in changing the penetration patterns of S. typhimurium through the outer egg structures if the organisms were allowed to gain entrance to the area under the shell (Table 4). Results were
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* 1 X = 1.2 ml. of formalin mixed with 0.6 gm. KMnOi/0.0283 cu.m. (1 cu.ft.). ** 2 X =Twice the 1X level. f Area 1 =Penetration through the cuticle and shell only.
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J. E. WILLIAMS AND L. H. DILLARD
TABLE 3.—The penetration of S. typhimurium through the outer structures of chicken eggs dipped into broth cultures diluted immediately before dipping with Sani-Squad and zinc sulfate disinfectant solutions.
The results obtained with the chemical
t Area 3 = Penetration through cuticle, shell, and both shell membranes.
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treatments reported here were apparently due to the partial or complete removal of the cuticular layer on the surface of the egg as well as other outer shell layers be% penetrated after 24 hr. low the cuticle. With these vital barNo. of at 37°C. into: riers damaged or removed, the salmonella Area 1 only* Area 2** Area 3t organisms were able to rapidly penetrate % % % Sani-Squad through the outer egg structures. Any 36 0.00 0.00 0.00 (Without feces) type of egg treatment that injures or Sani-Squad 36 0.00 0.00 0.00 (With feces) eliminates the outer coating on chicken Zinc sulfate (1%) 67 0.00 0.00 0.00 without feces eggs increases the potential of salmonella Control eggs 108 8.33 20.37 14.81 as well as other organisms to penetrate * Area 1 only = Penetration through cuticle and shell. ** Area 2 = Penetration through cuticle, shell, and outer shell into the egg. membrane. t Area 3 =Penetration through cuticle, shell, and both shell Formaldehyde fumigation at a high membranes. level has readily killed salmonella orgavery similar for both disinfectant-treated nisms on the shell surface (Wilson, 1951; and control eggs when samplings were Frank and Wright, 1955). The work remade in each of the 3 areas of the outer ported here shows clearly, however, that egg structures. The 1J hour penetration no residual protective effect is afforded to period before the eggs were exposed to the surface of the egg following fumigathe disinfectant solutions allowed the or- tion to prevent shell penetration by salganisms to reach subsurface areas of the monella organisms even when eggs are egg where they were adequately protected fumigated with twice the usual level of from the germicidal action of the disin- gas. fectant solutions. While the 2 disinfectants used in these studies were effective in preventing peneDISCUSSION tration of salmonella organisms into eggs, Using qualitative procedures to detect if they were mixed with the organisms at the presence of salmonella organisms the time of shell exposure, they had no efunder the shell, between the shell mem- fect on organisms that had been allowed branes, and on the inner surface of the to penetrate under the shell. These findinner membrane, it is possible to deter- ings suggest that if disinfectants are emmine the effect of a large variety of external shell treatments on the penetration properties of salmonella organisms TABLE 4.—S. typhimurium isolations from the outer structures of chicken eggs exposed to the organisms through the outer egg structures. These by dipping into broth cultures followed by 1\ hr. of incubation before washing in studies represent the first application of disinfectant solutions. these procedures for this purpose. Previous techniques have involved culture of % penetrated after 24 hr. Disinfectant No. of at 37°C. into: the egg contents after a set period of exSolution eggs Area 1 only * Area 2** Area 3f posure and very little attention has been % % % directed to the penetration process as it Sani-Squad (1-100) 70 28.57 7.14 7.14 Zinc sulfate (1%) 79 12.65 12.65 2.64 occurs in the outer structures of the egg Control eggs 15.80 76 7.90 2.63 following various treatments of the shell * Area 1 only=Penetration through cuticle and shell. ** Area 2 =Penetration through cuticle, shell, and outer shell surface. membrane.
SHELL TREATMENTS AND SALMONELLA PENETRATION
ployed to dip, wash, or spray eggs, they should be applied as soon as possible after the eggs are laid. ACKNOWLEDGMENT The authors t h a n k Mr. Steven Benson for technical assistance in conducting this work.
REFERENCES
Gordon, R. F., E. G. Harry and J. F. Tucker, 1956. The use of germicidal dips in the control of bacterial contamination of the shells of hatching eggs. Vet. Record, 68: 33-38. Harry, E. G., 1954. The influence of certain chemico-physical characteristics of formaldehyde on its use as a disinfectant. Proc. 10th World's Poultry Cong.: 217-221. Lancaster, J. E., R. F. Gordon and J. Tucker, 1952. The disinfection prior to incubation of hen eggs contaminated with Salmonella pullorum. Brit. Vet. J. 108:418-431. Rizk, S. S., J. C. Ayres and A. A. Kraft, 1966. Disinfection of eggs artificially inoculated with salmonellae. Poultry Sci. 45: 764-769. Williams, J. E., and A. D. Whittemore, 1967. A method for studying microbial penetration through the outer structures of the avian egg. Avian Dis. 11:467-490. Williams, J. E., L. H. Dillard and G. O. Hall, 1968. The penetration patterns of Salmonella typhimurium through the outer structures of chicken eggs. Avian Dis. 12: 445-466. Wilson, J. E., 1948. Avian salmonellosis. Vet. Record, 60: 615-624. Wilson, J. E., 1951. The control of salmonellosis in poultry with special reference to fumigation of incubators. Vet. Record, 63: 501-503.
NEWS AND NOTES {Continued from page 1074) C.D.A. NOTES
ALBERTA NOTES R. J. Chernos has been appointed Poultry Specialist serving the industry in the southern part of Alberta. His office is in the Science Building, Lethbridge Community College. Born in Oliver, British Columbia, he received a B.Sc. degree at the University of Alberta in 1970, majoring in poultry science. He worked with Master Feeds Limited, Sunrise Poultry Farm, and the Poultry Section, Animal Research Institute, Canada Department of Agriculture, Ottawa. QUAKER NOTES Dr. R. E. Smith, Research Branch, Quaker Oats Co., Barrington, Illinois, has been promoted to Assistant Director of Research and DevelopmentNutrition. He has also been appointed to the Technical Advisory Group, Committee on Nutrition, American Academy of Pediatrics.
Dr. I. R. Sibbald, formerly on the staff of John Labatt Ltd., London, Ontario, joined the Animal Research Institute, Central Experimental Farm, Canada Department of Agriculture, Ottawa, Ontario, on December 1. He will be working in the area of poultry nutrition.
4TH EUROPEAN POULTRY CONGRESS The complete Proceedings of the 4th European Poultry Conference, organized by the United Kingdom Branch of the World's Poultry Science Association, and held in London, September 3-8, 1972, will be published by British Poultry Science Ltd. and will be obtainable from Longmans Group Ltd., Journal Division, 33 Montgomery Street, Edinburgh EH7 5JX, Scotland. Date of publication and price have not been an-
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Antonova, M. E., 1966. Disinfection of eggs against Mycoplasma infection. Vestnik selskokhozyaistvennoi Nauki (Moskva), No. 8: 97-100. Bierer, B. W., B. D. Barnett and H. D. Valentine, 1961. Experimentally killing Salmonella typhimurium on egg shells by washing. Poultry Sci. 40: 1009-1014. Brown, W. E., R. C. Baker and H. B. Naylor, 1966. Shell treatments as affecting microbial egg spoilage. Poultry Sci. 45: 276-279. Frank, J. F., and G. W.Wright, 1955. Susceptibility of salmonella organisms to formaldehyde fumigation. Canad. J. Comp. Med. Vet. Sci. 19: 71-75. Frank, J. F., and G. W. Wright, 1956. The disinfection of eggs contaminated with Salmonella typhimurium. Canad. J. Comp. Med. Vet. Sci. 20: 406-410.
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