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Disinfection of Eggs Artificially Inoculated with Salmonellae
Disinfection of Eggs Artificially Inoculated with Salmonellae 1. APPLICATION OF SEVERAL DISINFECTANTS 1 S. S. RIZK, J. C. AYRES AND A. A. KRAFT Department of Dairy and Food Industry, Iowa State University, Ames, Iowa (Received for publication December 20, 1965)
of shell eggs by orC ONTAMINATION ganisms of the genus Salmonella is one
1 Journal Paper No. J-5201 of the Iowa Agricultural and Home Economics Experiment Station, Ames. Project No. 1544; Center for Agricultural and Economic Development cooperating. A report of work done under contract with the TJ. S. Department of Agriculture and authorized by the Research and Marketing Act of 1946. The contract is being supervised by the Western Utilization Research and Development Division of the Agricultural Research Service. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
2 Nacconal NR—an anionic detergent-sanitizer containing 40% active organic sulfante and 60% sodium1 sulphate. National Aniline Division, Allied Chemical and Dye Corp., New York, N. Y. 3 Clorox—a liquid detergent sanitizer containing 5.25% sodium hypochlorite. Clorox Chemical Co., Oakland, Calif. 4 Vel—an alkaline detergent containing a sulfonated monoglyceride. Colgate-Palmolive Peet Co., Jersey City, N. J. "Kleneg—an alkaline detergent sanitizer containing 5% trimethyloctadecadienyl ammonium chlorides (Saneg), tetrasodium pyrophosphate, so-
764
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of the major problems of the egg-breaking industry. Although Salmonellae do not cause a rapid deterioration of the infected eggs, they are a public health hazard. A considerable proportion of the outbreaks of salmonellosis is due to poultry and poultry products. It has been shown that Salmonellae can penetrate hen's eggs (Schalm, 1937; Bigland and Papas, 1953; and Stokes et al., 1956). Consequently, washing eggs with various sanitizing agents is considered an expedient procedure for eliminating penetration by Salmonellae into the shell membrane. However, many investigators have shown that there is a higher percentage of bacterial spoilage in washed eggs than in unwashed eggs (Gillespie et al., 1950; Johns et at., 1946; Dawson and Davidson, 1951; Lorenz et al., 1962). On the other hand, a number of investigators have reported no harm from washing eggs (Bryant and Sharp, 1934; Miller et al., 1950; Pino, 1950; Rhodes, 1950; Williams
and Gable, 1950; Winter et al., 1952; Lorenz and Starr, 1952; and Forsythe et al., 1953). Problems arising from washed eggs are primarily the result of failure to follow proper practices. The day the eggs were gathered Pino (1950) washed clean and soiled eggs in an anionic detergent-sanitizer, Nacconal NR,2 and held them four weeks at 60°F. There was no spoilage among the washed eggs. Miller et al. (1950) washed soiled eggs in water containing black rot bacteria and dipped part of them in 1% Roccal and in 0.5% pentachlorophenol. A slight reduction in spoilage was obtained by use of sanitizers. According to Lorenz et al. (1952), the keeping quality of eggs from ranches was better after washing in trisodium phosphate, an anionic detergent, or Clorox3 than in water alone. Forsythe et al. (1953) removed 82% of the bacteria from shell eggs by washing in 0.5% Vel,4 93% by Roccal (200 p.p.m.) and 93% by 0.5% Kleneg.5 In 1953, Botwright reported that very few of the detergent-sanitizers on the market cleaned eggs satisfactorily un-
DISINFECTION OF EGGS
MATERIALS AND METHODS
The application of several disinfectants was evaluated under different conditions of inoculation with Salmonellae by dipping the eggs in disinfectants for 5 min. at room temperature. Disinfectants employed were dium carbonate, trisodium phosphate and polyethylene-glycol ester of oleic acid. Armour and Co., Chicago, 111.
as follows: Roccal, a mixture of C8-C18 alkyl dimethyl benzyl ammonium chlorides; Spartec, a mixture of C12, C14, C16, n-alkyl dimethyl benzyl ammonium chlorides; Kleen-Shel, Roccal 5%, sodium metasilicate pentaphosphate and sodium carbonate; Anti-bac, 1,3 dichloro 5,5 dimethyl hydantoin 22.2%; Steri-Chlor, dichloro (5) triozine trione 5.7%; Dowicide A, sodium o-phenylphenate 4 H 2 0 97%; and zinc sulphate. Nest clean, non-fertile, unwashed eggs were obtained from the Iowa State University Poultry Farm. These eggs were candled and those with obvious shell defects were excluded. Inoculation: The eggs were kept overnight at room temperature and inoculated by immersing in a chilled suspension of brightened S. anatum and S. lexington (Paton and Ayres, 1964) in 0.1% peptone at 4.5°C. for 5 min. The optical density of the suspension was determined in a Spectronic 20 colorimeter (Bausch and Lomb, Rochester, N.Y.) at 625 m\t.; 50% T corresponded to approximately 1 X 10s cells per ml. After inoculation, the eggs were kept at room temperature (about 25°C, R.H. 55%) for 30 min. The eggs were then treated with different disinfectant solutions at 28-29°C. for 5 min. Since it was found that the organisms on the shell surface died during the incubation period at 37°C. for 10 days, shell surface counts were taken before incubation. After incubation the eggs were analyzed for Salmonellae on the shell membrane system and in the egg contents. To determine if the organisms invade the eggs without being sucked inside, a second method of inoculation was used. This consisted of immersing the eggs for 5 minutes at room temperature in pooled nutrient broth cultures of S. senftenberg, S. typhimurium, S. bovis, S. newport, S. enteritidis,
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less they contained a quaternary ammonium compound combined with an alkaline detergent. Several investigations have been made on the effect of washing and infection of eggs by Salmonellae. Williams (1959) indicated that egg washing procedures are not practical or effective for the prevention of salmonellosis. Lancaster et al. (1952) reported that sodium hypochlorite solution and a solution of sodium para-toluene-sulphono chloramide, cetyltrimethyl ammonium bromide and chlorometaxylenol were each successful in removal of S. pullorum from the shells of artificially infected eggs. These workers also stated that S. typhimurium and 5. thompson were resistant to the disinfectants tested and that the practice was of no value in controlling egg "rot" during storage. In studies performed by Frank and Wright (1956), when eggs were artificially infected with S. typhimurium and dipped for five minutes in sodium hydroxide at concentrations up to 2%, penetration of the egg shell by the organisms was not prevented. Bierer et al. (1961) used 16 components for washing eggs as a practice to destroy artificially introduced 5. typhimurium. Later, Bierer and Barnett (1962) also tested quaternary ammonium compounds. sodium hypochlorite, zinc sulphate, and formaldehyde fumigation against 5. pullorum. They indicated that zinc sulphate was superior to all chemicals tested.
765
766
S. S. RIZK, J. C. AYRES AND A. A. KRAFT
sion of shell and of contents for enumeration of Salmonellae by an MPN technique (3 tubes per dilution). Also, brightened organisms were spotted under ultraviolet light, the membrane scraped in the areas where fluorescent spots were seen and swabs of such areas taken for culture on brilliant green agar. To determine if the disinfectants used have a residual effect, eggs were treated with the disinfectants and then kept at
Sample Lactose broth Selenite cystine B. G . Agar
4 T
urea media probably Salmonellae
Proteus
4^
+H2S
-H2$ Salmonellae,polyvalent antiserum I
Sal monellae polyvalent antiserum
- 1
+ Sal monellae
not Salmonel la
Dulcitol lysine, Iron sugar agar o r G i l l i e s tubes
branes and in egg contents, eggs were im mersed in 1:1000 Roccal solution for 5 min. and in 75% ethanol for 2 min. The blunt end of the shell of each egg was flamed and, after fracturing the shell with sterile forceps, contents of 5 eggs were poured into a sterile jar. Shells of these same eggs were placed in another sterile jar with 100 ml. of lactose broth and blended with an Osterizer for 2 min. (John Oster Mfg. Co., Milwaukee, Wisconsin). Serial dilutions were made of the suspen-
I
*
-
D u l c i t o l lysine, iron sugar agar o r G i l l i e s tubes
room temperature until dry (3 hrs). After this, the eggs were dipped momentarily in a suspension of Salmonellae and allowed to dry again. Eggs were examined for Salmonellae by the methods already described. RESULTS AND DISCUSSION
Recoveries of Salmonellae from egg surfaces treated by different disinfectants show a marked reduction in the number of organisms recovered compared with that of
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S. thompson, S. montevideo, S. pullorum, S. anatum, and S. lexington. For shell surface counts, the entire surface of each individual egg was swabbed with 0.1% peptone and the swab transferred to 10 ml. of 0.1% peptone. One ml. of the suspension was spread in 0.2 ml. quantities on brilliant green (B.G.) agar plates. Plates were incubated at 37°C. for 24 hrs. Counts were made and Salmonellae were confirmed by the method shown below: To determine Salmonellae on egg mem-
767
DISINFECTION OF EGGS TABLE 1.
-Salmonella counts after disinfection of eggs inoculated with a suspension at a temperature lower than that of the eggs1 Bacterial Counts
Treatment
6 4 6 6 4 6 6 6
B Shell membrane XI,000 per shell
Range
Median
Range
780-2,030 <0.01-0.27 <0.01-0.06 <0.01 <0.01-0.59 <0.01-0.02 <0.01 <0.01-0.02
1,200 0.02 <0.01 <0.01 0.03 <0.01 <0.01 <0.01
0.84-32.0 0.48-30.0 1.86-22.0 0.30-22.0 1.86-42.0 1.5-10.6 0.58-22.0 0.56- 4.8
Median 15.2 11.2 3.0 8.2 4.1 5.6 3.6 2.4
C Egg content No. ijer gram Range
Median
<0.03-0.091 <0.03-0.036 <0.03-0.036 <0.03-0.073 <0.03-0.061 <0.03-0.091 <0.03-0.036 <0.03
0.054 <0.03-0.03 3 <0.03 <0.03 <0.03-0.036 3 0.036 <0.03 <0.03
6
<0.01-0.01
<0.01
0.86- 9.2
3.6
<0.03-0.036
<0.03
4
<0.01-0.21
0.01
9.2 -30.0
22.0
<0.03-0.03
<0.03-0.03'
6 4 6 6
<0.01-0.02 <0.01-0.05 <-.01 <0.01-0.32
<0.01 <0.01 <0.01 0.11
1.86- 9.2 0.88-11.0 0.22-15.0 0.86-48.0
<0.03-0.036 <0.03-0.036 <0.03-0.073 <0.03-0.036
<0.03 <0.03 <0.03 <0.03-0.03 3
4.4 5.5 8.9 1.86
1 Inoculation was accomplished by dipping the eggs in a chilled brightened Salmonella suspension (Spectronic 20 colorimeter at 625 m/x: 50% T = 108 organisms per ml.) at 3-4°C. for 5 min. after the eggs were kept overnight at room temperature. 2 5 eggs per trial. 8 Median count lies between negative tubes at lowest dilution and positive tubes at next highest dilution.
the controls (Table 1). The higher concentration of disinfectants used seemed to eliminate the organisms entirely from the egg surface. Roccal and Spartec were effective in a dilution of 1:2000 whereas Antibac was effective at a concentration of 200 p.p.m. of available chlorine. No Salmonellae could be recovered from egg surfaces treated by any of these disinfectants, in the concentration mentioned. The use of 200 p.p.m. available chlorine (Steri-Chlor) could not eliminate the Salmonellae entirely from the egg surfaces whereas the same concentraton of Antibac seemed to be sufficient for such purpose. The pH of the disinfectants might account for these results, since a dilution of Antibac containing 200 p.p.m. of free chlorine has a pH of 6.7 whereas the same concentrate of SteriChlor has a pH 8.2. Table 1, Column B presents the numbers of Salmonellae recovered from the egg
membrane system. No special pattern was shown. Also, the recoveries of the Salmonellae from the membrane system varied within the same treatment. The treatment of eggs with the various disinfectants failed to kill Salmonellae on the shell membrane system. These quantitative results confirmed the positive results obtained by scraping the membrane and swabbing within the areas where fluorescent spots were seen under untraviolet light. Examinations revealed the presence of viable organisms from all brightened colonies after application of any of the disinfectants. Table 1, Column C, indicates numbers of Salmonellae recovered per gram of egg contents. In most instances, the number of Salmonellae in egg contents of the control eggs was slightly higher than that of treated eggs. The number of organisms in general was usually very low. In order to determine if the organisms
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Control Roccal 1:5,000 Roccal 1:3,000 Roccal 1:2,000 Spartec 1:5,000 Spartec 1:3,000 Spartec 1:2,000 Kleen Shell: 250 Na o-phenyl phenate 1:500 Steri-Chlor 100 p.p.m. Steri-Chlor 200 p.p.m. Antibac 100 p.p.m. Antibac 200 p.p.m. Zinc sulphate 1%
No. of trials2
A Egg surface X I , 000 per shell
768
S. S. RIZK, J. C. AYRES AND A. A. KRAFT TABLE 2.—Salmonella counts after disinfection of eggs inoculated with a suspension at the same temperature as the eggs* Bacterial counts A Egg surface No. per shell
No. of trials b
Treatment
Range
Median
Range
Median
<10 c <10 <10 <10 <10 <10 <10-1,860 12,300-224,000
<10 <10 <10 <10 <10 <10 640 65,200
<0.03° <0.03 <0.03 <0.03-46 <0.03-420 28-93 <0.03 36-3,600
<0.03 <0.03 <0.03 <0.03-36 111 72 <0.03 621
a Eggs were inoculated by immersion for 5 min. in broth culture of ten different Salmonella spp. after the eggs were stored overnight at room temperature (average counts of Salmonellae in the nutrient broth 5.9X10 7 /per ml.). b 5 eggs per trial. 0 No. colonies on plates and no. positive MPN tubes at lowest dilution.
could invade the egg without being drawn inside by suction, the eggs were immersed for 5 min. in a nutrient broth culture which had the same temperature as that of the eggs. Table 2, Column 1 indicates that the disinfectants at the dilution used removed the Salmonellae from the treated egg surface, although ten species of Salmonellae suspended in nutrient broth had been used. These results were in agreement with those of Table 1. Table 2, Column B, shows it is possible for Salmonellae to invade the shell to the shell membrane without a temperature difference existing between the eggs and the bacterial suspension. However, the TABLE 3.—No. of organisms on shell eggs treated with disinfectants and dipped momentarily in in a Salmonella suspension No. of eggs fS per tnal
Na o-phenyl phenate 1:500 Anti-bac 200 p.p.m. Steri-Chlor 200 p.p.m. Zinc sulphate 1% Roccal 1:2,000 Spartec 1:2,000 Control
10 10 10 10 10 10 10
A
™ r *f
„
10.2 16.8 20.0 13.5 0.24 0.32 18.8
45.5 10.8 -6.3 27.8 87.1 82.9 0
n % No of organisms _,destructriak trials x l 0 0 0 t;on per shell
2 2 2 2 2 2 2
Number of Salmonellae in the suspension was 2.3X 10s organisms per ml. Eggs allowed to dry at room temperature (ca. 25"C.) for 3 hr. and then dipped rapidly in and out of Salmonella suspension for inoculation.
rate of infection was extremely low compared with that found using the other method. The disinfectants might have destroyed organisms which had invaded the shell by capillary action before they actually penetrated to the shell membrane. Despite recoveries of Salmonellae from the shell membrane, no Salmonellae could be recovered from egg contents when eggs were inoculated by immersion in nutrient broth at the same temperature as that of the eggs. Results obtained (Table 3) indicate that quaternaries have more residual effect than other disinfectants. Lower counts were obtained from eggs treated with Roccal and Spartec than from eggs treated with any other disinfectants. Na o-phenyl phenate and zinc sulphate had an intermediate residual effect. CONCLUSIONS 1. None of the disinfectants applied could eliminate Salmonellae from the shell membrane system after the organisms had penetrated the shell. 2. The application of such disinfectants may be very useful to control the Sal-
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4 4 4 4 4 4 4 4
Roccal 1:2,000 Spartec 1:2,000 Kleen Shel 1:250 Na o-phenyl phenate 1:500 Steri-Chlor 200 p.p.m. Antibac 200 p.p.m. Zinc sulphate 1% Control
B No. per shell membrane
DISINFECTION OF EGGS
REFERENCES Bierer, B. W., H. D. Valentine, B. D. Barnett and W. H. Rhodes, 1961. Germicidal efficiency of egg washing compounds on egg artificially contaminated with Salmonella typhimurium. Poultry Sci. 40: 148-152. Bierer, B. W., and B. D. Barnett, 1962. Killing Salmonella on egg shells with disinfectants. Amer. Vet. Med. Assoc. J. 140: 159-161. Bigland, C. H., and G. Papas, 1953. Experiment in egg penetration by Salmonella. Can. J. Comp. Med. Vet. Sci. 17: 105-109. Botwright, W. E., 1953. Detergent-sanitizers for cleaning eggs. Amer. Egg Poultry Rev. 15: 3438. Bryant, R. L., and P. R. Sharp, 1934. Effect of washing on the keeping quality of hens eggs. J. Agr. Res. 48: 67-69. Dawson, L. E., and J. A. Davidson, 1951. Farm practices and egg quality. Part II. Production practices affecting decline in egg quality. Michigan Agri. Exp. Sta. Quart. Bull. 34, 89-104. Frank, J. F., and G. W. Wright, 1956. The disinfection of eggs contaminated with 5. typhimurium. Canad. J. Comp. Med. 20: 406-410. Forsythe, R. H., J. C. Ayres and J. L. Radio, 1953. Factors affecting the microbiological population of shell eggs. Food Tech. 7: 49-56.
Gillespie, J. M., W. J. Scott and F. R. Vickery, 1950. Studies on the preservation of shell eggs. III. The storage of machine washed eggs. Australian J. App. Sci. 1:313-329. Johns, C. K., and H. L. Berard, 1946. Effect of certain methods of handling upon the bacterial content of dirty eggs. Sci. Agr. 26, 11-15. Lancaster, J. E., R. F. Gordon and J. Tucker, 1952. The disinfection, prior to incubation of hens eggs contaminated with Salmonella pullorum. British Vet. J. 108: 418-431. Lorenz, F. W., F. X. Ogasawara and P. B. Starr. 1952. Spoilage of washed eggs. 3. A survey of ranch practices and results. Poultry Sci. 3 1 : 221-226. Lorenz, F. W., and P. B. Starr, 1952. Spoilage of washed eggs. 1. Effect of sprayed versus static water at various temperatures. Poultry Sci. 3 1 : 204-214. Miller, M. W., V. Taukausky and A. Kraght, 1950. Experiments relating to the spoilage of washed eggs. Poultry Sci. 29: 27-33. Paton, A. M., and J. C. Ayres, 1964. Use of a fluorescent brightener for tracing the passage of Salmonella in eggs. Nature, 204, 4960: 803-804. Pino, J. A., 1950. Effect of washing with a hot detergent solution on keeping quality and hatachability of eggs. Poultry Sci. 29: 888894. Rhodes, W. F., 1950. The effect of washing eggs on their hatchability and storage. M. S. thesis, Oklahoma A & M College. Schalm, O. W., 1937. Study of a paratyphoid infection in chicks. J. Infect. Dis. 6 1 : 208-216. Stokes, J. L., W. W. Osborne and H. G. Bayne, 1956. Penetration and growth of Salmonella in shell eggs. Food Res. 2 1 : 510-518. Williams, I. W., and J. W. Gable, 1950. Egg washing studies. Nebraska State Agr. Exp. Sta. 63rd An. R p t , p. 126. Williams, J. E., 1959. Paratyphoid and paracolon infection. Diseases of Poultry. Biester and Schwarte, Fourth Ed., Iowa State Univ. Press, Ames, Iowa, 218-228. Winter, A. R., B. Burkart and C. Wettling, 1952. Cleaning eggs for market. Ohio Agr. Exp. Sta. Res. Bui. 710.
AUGUST 6-7. WORLD'S TURKEY FEDERATION CONGRESS, LONDON, ENGLAND AUGUST 10-12. ANNUAL MEETING, ASSOCIATION OF AMERICAN FEED CONTROL OFFICIALS, RADISSON HOTEL, MINNEAPOLIS, MINN.
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monellae on the surface of the egg shell. In this way, the probability of organisms invading the eggs might be reduced. 3. The organisms could penetrate the shells to the shell membrane without a temperature difference existing between the eggs and the bacterial suspension. 4. Quaternary ammonium compounds might be the most effective compounds to be used, since they have a good germicidal action at alkaline pH values and at high dilution, they can be used at a high temperature, have a residual effect, and are less influenced by organic matter than other disinfectants tested.
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of shell eggs by orC ONTAMINATION ganisms of the genus Salmonella is one
1 Journal Paper No. J-5201 of the Iowa Agricultural and Home Economics Experiment Station, Ames. Project No. 1544; Center for Agricultural and Economic Development cooperating. A report of work done under contract with the TJ. S. Department of Agriculture and authorized by the Research and Marketing Act of 1946. The contract is being supervised by the Western Utilization Research and Development Division of the Agricultural Research Service. Reference to a company or product name does not imply approval or recommendation of the product by the U. S. Department of Agriculture to the exclusion of others that may be suitable.
2 Nacconal NR—an anionic detergent-sanitizer containing 40% active organic sulfante and 60% sodium1 sulphate. National Aniline Division, Allied Chemical and Dye Corp., New York, N. Y. 3 Clorox—a liquid detergent sanitizer containing 5.25% sodium hypochlorite. Clorox Chemical Co., Oakland, Calif. 4 Vel—an alkaline detergent containing a sulfonated monoglyceride. Colgate-Palmolive Peet Co., Jersey City, N. J. "Kleneg—an alkaline detergent sanitizer containing 5% trimethyloctadecadienyl ammonium chlorides (Saneg), tetrasodium pyrophosphate, so-
764
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of the major problems of the egg-breaking industry. Although Salmonellae do not cause a rapid deterioration of the infected eggs, they are a public health hazard. A considerable proportion of the outbreaks of salmonellosis is due to poultry and poultry products. It has been shown that Salmonellae can penetrate hen's eggs (Schalm, 1937; Bigland and Papas, 1953; and Stokes et al., 1956). Consequently, washing eggs with various sanitizing agents is considered an expedient procedure for eliminating penetration by Salmonellae into the shell membrane. However, many investigators have shown that there is a higher percentage of bacterial spoilage in washed eggs than in unwashed eggs (Gillespie et al., 1950; Johns et at., 1946; Dawson and Davidson, 1951; Lorenz et al., 1962). On the other hand, a number of investigators have reported no harm from washing eggs (Bryant and Sharp, 1934; Miller et al., 1950; Pino, 1950; Rhodes, 1950; Williams
and Gable, 1950; Winter et al., 1952; Lorenz and Starr, 1952; and Forsythe et al., 1953). Problems arising from washed eggs are primarily the result of failure to follow proper practices. The day the eggs were gathered Pino (1950) washed clean and soiled eggs in an anionic detergent-sanitizer, Nacconal NR,2 and held them four weeks at 60°F. There was no spoilage among the washed eggs. Miller et al. (1950) washed soiled eggs in water containing black rot bacteria and dipped part of them in 1% Roccal and in 0.5% pentachlorophenol. A slight reduction in spoilage was obtained by use of sanitizers. According to Lorenz et al. (1952), the keeping quality of eggs from ranches was better after washing in trisodium phosphate, an anionic detergent, or Clorox3 than in water alone. Forsythe et al. (1953) removed 82% of the bacteria from shell eggs by washing in 0.5% Vel,4 93% by Roccal (200 p.p.m.) and 93% by 0.5% Kleneg.5 In 1953, Botwright reported that very few of the detergent-sanitizers on the market cleaned eggs satisfactorily un-
DISINFECTION OF EGGS
MATERIALS AND METHODS
The application of several disinfectants was evaluated under different conditions of inoculation with Salmonellae by dipping the eggs in disinfectants for 5 min. at room temperature. Disinfectants employed were dium carbonate, trisodium phosphate and polyethylene-glycol ester of oleic acid. Armour and Co., Chicago, 111.
as follows: Roccal, a mixture of C8-C18 alkyl dimethyl benzyl ammonium chlorides; Spartec, a mixture of C12, C14, C16, n-alkyl dimethyl benzyl ammonium chlorides; Kleen-Shel, Roccal 5%, sodium metasilicate pentaphosphate and sodium carbonate; Anti-bac, 1,3 dichloro 5,5 dimethyl hydantoin 22.2%; Steri-Chlor, dichloro (5) triozine trione 5.7%; Dowicide A, sodium o-phenylphenate 4 H 2 0 97%; and zinc sulphate. Nest clean, non-fertile, unwashed eggs were obtained from the Iowa State University Poultry Farm. These eggs were candled and those with obvious shell defects were excluded. Inoculation: The eggs were kept overnight at room temperature and inoculated by immersing in a chilled suspension of brightened S. anatum and S. lexington (Paton and Ayres, 1964) in 0.1% peptone at 4.5°C. for 5 min. The optical density of the suspension was determined in a Spectronic 20 colorimeter (Bausch and Lomb, Rochester, N.Y.) at 625 m\t.; 50% T corresponded to approximately 1 X 10s cells per ml. After inoculation, the eggs were kept at room temperature (about 25°C, R.H. 55%) for 30 min. The eggs were then treated with different disinfectant solutions at 28-29°C. for 5 min. Since it was found that the organisms on the shell surface died during the incubation period at 37°C. for 10 days, shell surface counts were taken before incubation. After incubation the eggs were analyzed for Salmonellae on the shell membrane system and in the egg contents. To determine if the organisms invade the eggs without being sucked inside, a second method of inoculation was used. This consisted of immersing the eggs for 5 minutes at room temperature in pooled nutrient broth cultures of S. senftenberg, S. typhimurium, S. bovis, S. newport, S. enteritidis,
Downloaded from http://ps.oxfordjournals.org/ at University of Queensland on June 22, 2015
less they contained a quaternary ammonium compound combined with an alkaline detergent. Several investigations have been made on the effect of washing and infection of eggs by Salmonellae. Williams (1959) indicated that egg washing procedures are not practical or effective for the prevention of salmonellosis. Lancaster et al. (1952) reported that sodium hypochlorite solution and a solution of sodium para-toluene-sulphono chloramide, cetyltrimethyl ammonium bromide and chlorometaxylenol were each successful in removal of S. pullorum from the shells of artificially infected eggs. These workers also stated that S. typhimurium and 5. thompson were resistant to the disinfectants tested and that the practice was of no value in controlling egg "rot" during storage. In studies performed by Frank and Wright (1956), when eggs were artificially infected with S. typhimurium and dipped for five minutes in sodium hydroxide at concentrations up to 2%, penetration of the egg shell by the organisms was not prevented. Bierer et al. (1961) used 16 components for washing eggs as a practice to destroy artificially introduced 5. typhimurium. Later, Bierer and Barnett (1962) also tested quaternary ammonium compounds. sodium hypochlorite, zinc sulphate, and formaldehyde fumigation against 5. pullorum. They indicated that zinc sulphate was superior to all chemicals tested.
765
766
S. S. RIZK, J. C. AYRES AND A. A. KRAFT
sion of shell and of contents for enumeration of Salmonellae by an MPN technique (3 tubes per dilution). Also, brightened organisms were spotted under ultraviolet light, the membrane scraped in the areas where fluorescent spots were seen and swabs of such areas taken for culture on brilliant green agar. To determine if the disinfectants used have a residual effect, eggs were treated with the disinfectants and then kept at
Sample Lactose broth Selenite cystine B. G . Agar
4 T
urea media probably Salmonellae
Proteus
4^
+H2S
-H2$ Salmonellae,polyvalent antiserum I
Sal monellae polyvalent antiserum
- 1
+ Sal monellae
not Salmonel la
Dulcitol lysine, Iron sugar agar o r G i l l i e s tubes
branes and in egg contents, eggs were im mersed in 1:1000 Roccal solution for 5 min. and in 75% ethanol for 2 min. The blunt end of the shell of each egg was flamed and, after fracturing the shell with sterile forceps, contents of 5 eggs were poured into a sterile jar. Shells of these same eggs were placed in another sterile jar with 100 ml. of lactose broth and blended with an Osterizer for 2 min. (John Oster Mfg. Co., Milwaukee, Wisconsin). Serial dilutions were made of the suspen-
I
*
-
D u l c i t o l lysine, iron sugar agar o r G i l l i e s tubes
room temperature until dry (3 hrs). After this, the eggs were dipped momentarily in a suspension of Salmonellae and allowed to dry again. Eggs were examined for Salmonellae by the methods already described. RESULTS AND DISCUSSION
Recoveries of Salmonellae from egg surfaces treated by different disinfectants show a marked reduction in the number of organisms recovered compared with that of
Downloaded from http://ps.oxfordjournals.org/ at University of Queensland on June 22, 2015
S. thompson, S. montevideo, S. pullorum, S. anatum, and S. lexington. For shell surface counts, the entire surface of each individual egg was swabbed with 0.1% peptone and the swab transferred to 10 ml. of 0.1% peptone. One ml. of the suspension was spread in 0.2 ml. quantities on brilliant green (B.G.) agar plates. Plates were incubated at 37°C. for 24 hrs. Counts were made and Salmonellae were confirmed by the method shown below: To determine Salmonellae on egg mem-
767
DISINFECTION OF EGGS TABLE 1.
-Salmonella counts after disinfection of eggs inoculated with a suspension at a temperature lower than that of the eggs1 Bacterial Counts
Treatment
6 4 6 6 4 6 6 6
B Shell membrane XI,000 per shell
Range
Median
Range
780-2,030 <0.01-0.27 <0.01-0.06 <0.01 <0.01-0.59 <0.01-0.02 <0.01 <0.01-0.02
1,200 0.02 <0.01 <0.01 0.03 <0.01 <0.01 <0.01
0.84-32.0 0.48-30.0 1.86-22.0 0.30-22.0 1.86-42.0 1.5-10.6 0.58-22.0 0.56- 4.8
Median 15.2 11.2 3.0 8.2 4.1 5.6 3.6 2.4
C Egg content No. ijer gram Range
Median
<0.03-0.091 <0.03-0.036 <0.03-0.036 <0.03-0.073 <0.03-0.061 <0.03-0.091 <0.03-0.036 <0.03
0.054 <0.03-0.03 3 <0.03 <0.03 <0.03-0.036 3 0.036 <0.03 <0.03
6
<0.01-0.01
<0.01
0.86- 9.2
3.6
<0.03-0.036
<0.03
4
<0.01-0.21
0.01
9.2 -30.0
22.0
<0.03-0.03
<0.03-0.03'
6 4 6 6
<0.01-0.02 <0.01-0.05 <-.01 <0.01-0.32
<0.01 <0.01 <0.01 0.11
1.86- 9.2 0.88-11.0 0.22-15.0 0.86-48.0
<0.03-0.036 <0.03-0.036 <0.03-0.073 <0.03-0.036
<0.03 <0.03 <0.03 <0.03-0.03 3
4.4 5.5 8.9 1.86
1 Inoculation was accomplished by dipping the eggs in a chilled brightened Salmonella suspension (Spectronic 20 colorimeter at 625 m/x: 50% T = 108 organisms per ml.) at 3-4°C. for 5 min. after the eggs were kept overnight at room temperature. 2 5 eggs per trial. 8 Median count lies between negative tubes at lowest dilution and positive tubes at next highest dilution.
the controls (Table 1). The higher concentration of disinfectants used seemed to eliminate the organisms entirely from the egg surface. Roccal and Spartec were effective in a dilution of 1:2000 whereas Antibac was effective at a concentration of 200 p.p.m. of available chlorine. No Salmonellae could be recovered from egg surfaces treated by any of these disinfectants, in the concentration mentioned. The use of 200 p.p.m. available chlorine (Steri-Chlor) could not eliminate the Salmonellae entirely from the egg surfaces whereas the same concentraton of Antibac seemed to be sufficient for such purpose. The pH of the disinfectants might account for these results, since a dilution of Antibac containing 200 p.p.m. of free chlorine has a pH of 6.7 whereas the same concentrate of SteriChlor has a pH 8.2. Table 1, Column B presents the numbers of Salmonellae recovered from the egg
membrane system. No special pattern was shown. Also, the recoveries of the Salmonellae from the membrane system varied within the same treatment. The treatment of eggs with the various disinfectants failed to kill Salmonellae on the shell membrane system. These quantitative results confirmed the positive results obtained by scraping the membrane and swabbing within the areas where fluorescent spots were seen under untraviolet light. Examinations revealed the presence of viable organisms from all brightened colonies after application of any of the disinfectants. Table 1, Column C, indicates numbers of Salmonellae recovered per gram of egg contents. In most instances, the number of Salmonellae in egg contents of the control eggs was slightly higher than that of treated eggs. The number of organisms in general was usually very low. In order to determine if the organisms
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Control Roccal 1:5,000 Roccal 1:3,000 Roccal 1:2,000 Spartec 1:5,000 Spartec 1:3,000 Spartec 1:2,000 Kleen Shell: 250 Na o-phenyl phenate 1:500 Steri-Chlor 100 p.p.m. Steri-Chlor 200 p.p.m. Antibac 100 p.p.m. Antibac 200 p.p.m. Zinc sulphate 1%
No. of trials2
A Egg surface X I , 000 per shell
768
S. S. RIZK, J. C. AYRES AND A. A. KRAFT TABLE 2.—Salmonella counts after disinfection of eggs inoculated with a suspension at the same temperature as the eggs* Bacterial counts A Egg surface No. per shell
No. of trials b
Treatment
Range
Median
Range
Median
<10 c <10 <10 <10 <10 <10 <10-1,860 12,300-224,000
<10 <10 <10 <10 <10 <10 640 65,200
<0.03° <0.03 <0.03 <0.03-46 <0.03-420 28-93 <0.03 36-3,600
<0.03 <0.03 <0.03 <0.03-36 111 72 <0.03 621
a Eggs were inoculated by immersion for 5 min. in broth culture of ten different Salmonella spp. after the eggs were stored overnight at room temperature (average counts of Salmonellae in the nutrient broth 5.9X10 7 /per ml.). b 5 eggs per trial. 0 No. colonies on plates and no. positive MPN tubes at lowest dilution.
could invade the egg without being drawn inside by suction, the eggs were immersed for 5 min. in a nutrient broth culture which had the same temperature as that of the eggs. Table 2, Column 1 indicates that the disinfectants at the dilution used removed the Salmonellae from the treated egg surface, although ten species of Salmonellae suspended in nutrient broth had been used. These results were in agreement with those of Table 1. Table 2, Column B, shows it is possible for Salmonellae to invade the shell to the shell membrane without a temperature difference existing between the eggs and the bacterial suspension. However, the TABLE 3.—No. of organisms on shell eggs treated with disinfectants and dipped momentarily in in a Salmonella suspension No. of eggs fS per tnal
Na o-phenyl phenate 1:500 Anti-bac 200 p.p.m. Steri-Chlor 200 p.p.m. Zinc sulphate 1% Roccal 1:2,000 Spartec 1:2,000 Control
10 10 10 10 10 10 10
A
™ r *f
„
10.2 16.8 20.0 13.5 0.24 0.32 18.8
45.5 10.8 -6.3 27.8 87.1 82.9 0
n % No of organisms _,destructriak trials x l 0 0 0 t;on per shell
2 2 2 2 2 2 2
Number of Salmonellae in the suspension was 2.3X 10s organisms per ml. Eggs allowed to dry at room temperature (ca. 25"C.) for 3 hr. and then dipped rapidly in and out of Salmonella suspension for inoculation.
rate of infection was extremely low compared with that found using the other method. The disinfectants might have destroyed organisms which had invaded the shell by capillary action before they actually penetrated to the shell membrane. Despite recoveries of Salmonellae from the shell membrane, no Salmonellae could be recovered from egg contents when eggs were inoculated by immersion in nutrient broth at the same temperature as that of the eggs. Results obtained (Table 3) indicate that quaternaries have more residual effect than other disinfectants. Lower counts were obtained from eggs treated with Roccal and Spartec than from eggs treated with any other disinfectants. Na o-phenyl phenate and zinc sulphate had an intermediate residual effect. CONCLUSIONS 1. None of the disinfectants applied could eliminate Salmonellae from the shell membrane system after the organisms had penetrated the shell. 2. The application of such disinfectants may be very useful to control the Sal-
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4 4 4 4 4 4 4 4
Roccal 1:2,000 Spartec 1:2,000 Kleen Shel 1:250 Na o-phenyl phenate 1:500 Steri-Chlor 200 p.p.m. Antibac 200 p.p.m. Zinc sulphate 1% Control
B No. per shell membrane
DISINFECTION OF EGGS
REFERENCES Bierer, B. W., H. D. Valentine, B. D. Barnett and W. H. Rhodes, 1961. Germicidal efficiency of egg washing compounds on egg artificially contaminated with Salmonella typhimurium. Poultry Sci. 40: 148-152. Bierer, B. W., and B. D. Barnett, 1962. Killing Salmonella on egg shells with disinfectants. Amer. Vet. Med. Assoc. J. 140: 159-161. Bigland, C. H., and G. Papas, 1953. Experiment in egg penetration by Salmonella. Can. J. Comp. Med. Vet. Sci. 17: 105-109. Botwright, W. E., 1953. Detergent-sanitizers for cleaning eggs. Amer. Egg Poultry Rev. 15: 3438. Bryant, R. L., and P. R. Sharp, 1934. Effect of washing on the keeping quality of hens eggs. J. Agr. Res. 48: 67-69. Dawson, L. E., and J. A. Davidson, 1951. Farm practices and egg quality. Part II. Production practices affecting decline in egg quality. Michigan Agri. Exp. Sta. Quart. Bull. 34, 89-104. Frank, J. F., and G. W. Wright, 1956. The disinfection of eggs contaminated with 5. typhimurium. Canad. J. Comp. Med. 20: 406-410. Forsythe, R. H., J. C. Ayres and J. L. Radio, 1953. Factors affecting the microbiological population of shell eggs. Food Tech. 7: 49-56.
Gillespie, J. M., W. J. Scott and F. R. Vickery, 1950. Studies on the preservation of shell eggs. III. The storage of machine washed eggs. Australian J. App. Sci. 1:313-329. Johns, C. K., and H. L. Berard, 1946. Effect of certain methods of handling upon the bacterial content of dirty eggs. Sci. Agr. 26, 11-15. Lancaster, J. E., R. F. Gordon and J. Tucker, 1952. The disinfection, prior to incubation of hens eggs contaminated with Salmonella pullorum. British Vet. J. 108: 418-431. Lorenz, F. W., F. X. Ogasawara and P. B. Starr. 1952. Spoilage of washed eggs. 3. A survey of ranch practices and results. Poultry Sci. 3 1 : 221-226. Lorenz, F. W., and P. B. Starr, 1952. Spoilage of washed eggs. 1. Effect of sprayed versus static water at various temperatures. Poultry Sci. 3 1 : 204-214. Miller, M. W., V. Taukausky and A. Kraght, 1950. Experiments relating to the spoilage of washed eggs. Poultry Sci. 29: 27-33. Paton, A. M., and J. C. Ayres, 1964. Use of a fluorescent brightener for tracing the passage of Salmonella in eggs. Nature, 204, 4960: 803-804. Pino, J. A., 1950. Effect of washing with a hot detergent solution on keeping quality and hatachability of eggs. Poultry Sci. 29: 888894. Rhodes, W. F., 1950. The effect of washing eggs on their hatchability and storage. M. S. thesis, Oklahoma A & M College. Schalm, O. W., 1937. Study of a paratyphoid infection in chicks. J. Infect. Dis. 6 1 : 208-216. Stokes, J. L., W. W. Osborne and H. G. Bayne, 1956. Penetration and growth of Salmonella in shell eggs. Food Res. 2 1 : 510-518. Williams, I. W., and J. W. Gable, 1950. Egg washing studies. Nebraska State Agr. Exp. Sta. 63rd An. R p t , p. 126. Williams, J. E., 1959. Paratyphoid and paracolon infection. Diseases of Poultry. Biester and Schwarte, Fourth Ed., Iowa State Univ. Press, Ames, Iowa, 218-228. Winter, A. R., B. Burkart and C. Wettling, 1952. Cleaning eggs for market. Ohio Agr. Exp. Sta. Res. Bui. 710.
AUGUST 6-7. WORLD'S TURKEY FEDERATION CONGRESS, LONDON, ENGLAND AUGUST 10-12. ANNUAL MEETING, ASSOCIATION OF AMERICAN FEED CONTROL OFFICIALS, RADISSON HOTEL, MINNEAPOLIS, MINN.
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monellae on the surface of the egg shell. In this way, the probability of organisms invading the eggs might be reduced. 3. The organisms could penetrate the shells to the shell membrane without a temperature difference existing between the eggs and the bacterial suspension. 4. Quaternary ammonium compounds might be the most effective compounds to be used, since they have a good germicidal action at alkaline pH values and at high dilution, they can be used at a high temperature, have a residual effect, and are less influenced by organic matter than other disinfectants tested.
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