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Salmonella Infection of Cracked Eggs
Salmonella Infection of Cracked Eggs D. V. VADEHRA, R. C. BAKER AND H. B. NAYLOR Department of Poultry Science, Cornell University, Ithaca, New York 14850 (Received for publication October 31, 1968)
L
Eggs. One-day-old eggs produced by a single strain of White Leghorn hens were procured from the Cornell University farms and used throughout this investigation. For the purpose of this study a
Culture. Salmonella typhimurium LT2 was used in this study. The bacteriological medium used throughout the study was glucose yeast extract broth or agar. The inoculum was grown for 24 hours at 37°C.
This study was carried out to determine the relative susceptibility of cracked and normal shell eggs to invasion by Salmonella organisms. The effect of the presence of several chemicals in the wash water on the spoilage of cracked and normal shell eggs was also investigated.
954
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
MATERIALS AND METHODS
cracked egg is one in which only the shell has been damaged without any visual damage to the shell membrane. The eggs were candled to make sure that leakers (eggs with damaged shells and membranes) and extensively cracked eggs were not used in this experiment. Washing procedure. The cracked eggs were washed in an immersion type commercial egg washer at 42°C. for three minutes. The washing procedure was varied to study the effect of several chemicals under various commercial conditions. The following conditions were used: a) normal washing procedure using a sanitizer-detergent containing a quaternary ammonium compound; b) washing, using five times the normal amount of sanitizerdetergent as was used in "a"; c) the use of a commercial laundry detergent with no sanitizer; d) sanitizer-detergent containing chlorine as the sanitizer; e) 200 p.p.m. of active chlorine but no detergent; f) 0.5% sodium lauryl sulfate. Following washing the eggs were rinsed with water at 45°C. to remove the washing compound. Although this procedure is not usually recommended, it was considered necessary in these experiments to prevent a residual effect of the sanitizer-detergent on bacteria. In another experiment wash water containing; a) chicken manure (3/4 pound in eight gallons of water); b) sterile chicken manure (same amount as "a"); and c) 0.5% bile salts was used.
ITTLE work has been done on the susceptibility of cracked eggs to infection and spoilage by microorganisms, as compared to the extensive literature of spoilage of normal shell eggs. Cracked eggs constitute about 2-3% of total egg production and are of great economic importance to the poultry industry. With the recent reports of occurrence of Salmonella in egg and egg products, the safety of cracked eggs and the products produced from them for human consumption has been questioned by public health authorities and others. Previous work done by McNally (1953) indicated that temperature and humidity changes affect cracked eggs adversely as compared to normal shell eggs. Brown et al. (1966) working with Pseudomonas aeruginosa showed that bacterial numbers in the dip water were more important than a temperature differential for infection. They also reported that cracked eggs were no more susceptible to infection by P. aeruginosa than uncracked eggs as long as they were handled properly.
955
SALMONELLA I N F E C T I O N OF CRACKED E G G S
TABLE 1.—Degree of spoilage of cracked and normal eggs by Salmonella typhimurium under several conditions
Treatment
Cracked, washed Cracked, unwashed Cracked Control (unexposed) Normal washed Normal unwashed Normal Control (unexposed)
% showing infection after 30 days of incubation* 23° C.
10° C.
10.0 3.0 0 3.0 0 0
4.0 0 0 0 0 0
* Accumulative data of three experiments (total of 150 eggs exposed to infection).
TABLE 2.—The effect of normal and abnormal conditions during washing procedure on the amount of spoilage of cracked eggs when exposed to artificial infection
Treatment
% spoilage after 30 days at 23°C*
Quaternary Ammonium, detergent Quaternary Ammonium (5X), detergent Laundry detergent Chlorine, detergent Chlorine 200 p.p.m. Sodium lauryl sulfate (0.5%)
13 23 27 32 41 18
* Accumulative data of two experiments (total number of eggs exposed to infection was 100).
the agar and tested for Salmonella on a Triple Sugar Iron medium. RESULTS AND DISCUSSION
Effect of washing procedure on infection. T h e d a t a are presented in Table 1. A batch of cracked and normal (uncracked) were not exposed to any artifical infection and these served as controls. T h e u n washed eggs were treated similarly except they were dipped directly after cracking and were not given any washing treatment. These data demonstrated t h a t cracked unwashed eggs had a lower percentage of spoilage as compared to cracked eggs which were washed in a sanitizer-detergent. Similar results were observed with normal shell eggs. However, the degree of spoilage was higher (10%) in cracked shell eggs as compared to uncracked eggs ( 3 % ) . As expected, the eggs which were not challenged by artificial infection did not show any spoilage, and the eggs stored at 10°C. showed a slower rate of spoilage. T h e effect of the addition of several chemicals in the water during the washing procedure on the spoilage of eggs is shown in Table 2. All the chemicals tested showed a higher degree of spoilage of eggs when compared to control washing pro-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
Exposure to infection. T h e eggs were dipped in a water b a t h containing approximately 100,000 S. typhimurium L T 2 per ml. T h e temperature of the eggs was 40°C. while the dip water had a temperature of 15°C. T h e dipping time was five minutes and the eggs after dipping were allowed to dry at room temperature. Storage of eggs. The eggs after exposure to infection were incubated a t 10°C. or 23° C. Two eggs were examined every third day and the experiment was terminated b y examining the remaining eggs after 30 days incubation. In another experiment, 100 eggs were exposed to infection, half of which were examined after 15 days of incubation and the remaining after 30 days of incubation. Bacteriological examination. T h e eggs for bacteriological examination were swabbed at the small end with 2 % iodine in 7 0 % alcohol, allowed to dry and then a circular cut was made through the shell, but without injuring the membranes, with a Dremel H o b b y Tool. T h e cut area was reswabbed and the small end of the egg was removed by sterile forceps. Bacteriological counts were made according to the Standard Methods Procedure (1960). The plates were incubated at 37°C. for 48 hours. Several colonies were picked from
956
D. V. VADEHRA, R. C. BAKER AND H. B. NAYLOR
TABLE 3.—The effect of different microbial loads and bile salts on the spoilage of cracked eggs incubated at 23°C. and 10°C* % spoilage after
Cracked—manure Cracked—sterile manure Cracked—0.5% bile salts Cracked—broth culture Cracked—normal wash
23°C.
10°C.
17.0 16.0 20.0 12.0 10.0
5.0 3.0 12.0 2.0 3.0
cedure which involved t h e use of quaternary ammonium compounds. Spoilage was greatest (41%) when 200 p.p.m. of chlorine (commercial bleach) was used. However, when sanitizer-detergent containing chlorine was used according to manufacturer's directions, the spoilage was somewhat lower ( 3 2 % ) . This study indicates t h a t washing of cracked shell eggs in a sanitizer-detergent containing quaternary ammonium compound is somew h a t better than a sanitizer-detergent containing chlorine. This study also points out t h a t t h e use of higher amounts of the washing compound than t h a t recommended b y the manufacturer has a deteriorative effect rather than a beneficial effect. I t also demonstrated t h a t t h e presence of sodium lauryl sulfate in t h e wash water h a d about t h e same effect as the presence of quaternary ammonium compounds on spoilage. T h e increased amount of spoilage by chlorine or chlorine containing sanitizer-detergent could be due to its strong oxidation properties in general and t h e action on sulfhydryl groups in particular. Since the membranes are exposed in cracked eggs, chlorine probably breaks t h e structure of the membranes thus making them more permeable and increasing spoilage. T h e action of laundry detergent and sodium lauryl sulfate m a y be due to their surface
TABLE 4.—Degree of spoilage of cracked eggs treated as shown after 30 days of incubation after infection* Incubation period Treatment
Quaternary Ammonium, detergent Chlorine, detergent Chlorine Manure Sterile manure
15 days % showing spoilage a t
30 days % showing spoilage a t
23°C.
10°C.
23°C.
10°C.
12.0 18.0 11.0 8.0 9.0
0 0 0 0 0
24 37 41 37 35
0 2 0 1 0
* 100 eggs used per treatment.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
* Accumulative data of two experiments (total of 100 eggs exposed to infection).
active properties which affect t h e interface of the membranes. Brown et al. (1966) reported t h a t t h e microbial load played an i m p o r t a n t role in spoilage of cracked eggs. Their primary source of microorganisms in wash water was chicken fecal material. Results presented in Table 3, however, indicate t h a t increased spoilage of eggs containing chicken manure m a y not necessarily be due to increased microbial load because when similar quantity of sterile manure was added to wash water, the percentage of spoilage did not decrease. Also, when a similar load of (10,000/ml.) broth culture of S. typhimurium was added to wash water, the spoilage of eggs did not increase. These data indicate t h a t there m a y be some other active material in chicken fecal matter which affects t h e cracked eggs in such a way t h a t they become more prone to infection. I t is known t h a t chickens excrete bile acids in their droppings, which could affect the membranes and thus increase spoilage. Bile salts are known to behave like anionic detergents in which the fat soluble portion has a steroid structure. Table 3 gives t h e results of washing eggs in 0 . 5 % bile salts, the spoilage figure almost doubled when compared to normal washing procedure. I t would appear t h a t the increased spoilage b y fecal m a t t e r m a y be due to t h e presence of bile salts, and the action of
SALMONELLA INFECTION OF CRACKED EGGS
957
3. Unwashed cracked eggs are less prone to spoilage when compared to washed, cracked eggs when exposed to infection. 4. Cracked eggs when washed under adverse conditions such as excessive sanitizer-detergent, chicken manure, etc. show much more spoilage, when challenged. 5. The temperature of incubation of eggs following infection had a profound effect on the spoilage. It was observed that cracked eggs washed even under adverse conditions did not show a great deal of spoilage at 10°C.
SUMMARY
This work was sponsored by Public Health Service Grant No. 5 R01 UI00146.
1. Cracked eggs which were challenged[ to artificial infection by Salmonella organisms did not show any spoilage under conditions of this experiment. 2. Cracked eggs washed in the normalI procedures and exposed to artificial infection are more prone to infection when1 compared to normal eggs treated similarly.
ACKNOWLEDGEMENT
REFERENCES Brown, W. E., R. C. Baker and H. B. Naylor, 1966. The microbiology of cracked eggs. Poultry Sci. 45: 284-287. McNally, E. H., 1953. Some factors related to the incidence of bacterial infection of shell eggs. Poultry Sci. 32: 915.
Gonadotropic Hormones in Hens Treated with Ethylene Dibromide* EUGENIA ALTJMOT (OLOMUCKI) AND E. MANDEL Division of Animal Nutrition, Volcani Institute for Agricultural Research, Rehovot, Israel. (Received for publication November 4, 1968)
INTRODUCTION
E
THYLENE dibromide (EDB), used as a grain fumigant, caused a decrease in egg size when administered to laying hens (Bondi el al., 1955). This decrease was found to be due to impaired ovarian follicle growth. Olomucki (1957) * Contribution from the National and University Institute of Agriculture, Rehovot, 1967 Series; N 1180-E. Financed partly by a grant from the United States Dept. of Agriculture, under P.L. 480.
suggested that the latter might be caused by depressed activity of the follicle stimulating hormone. Either the formation or the release of the hormone could be affected. To test the first assumption, hormone levels were measured in the pituitary of hens treated with EDB. In another series of experiments, hormone preparations were injected into hens laying small eggs following treatment with EDB, to counteract the possible impaired release of the hormone.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
bile salts is probably due to its detergentt as well as the surface active properties3 which weaken the membrane and make; them more vulnerable to attack by bacteria. In another experiment the eggs were2 examined after incubation for 15 and 30) days instead of sampling the eggs everyj third day. The results are shown in Table; 4. Such a procedure has the advantage of ai definite incubation time for the whole; group, and gives spoilage at the end off incubation period rather than accummulative spoilage. As was expected the degree of spoilage increased but the effect of various chemicals showed the same trend.
L
Eggs. One-day-old eggs produced by a single strain of White Leghorn hens were procured from the Cornell University farms and used throughout this investigation. For the purpose of this study a
Culture. Salmonella typhimurium LT2 was used in this study. The bacteriological medium used throughout the study was glucose yeast extract broth or agar. The inoculum was grown for 24 hours at 37°C.
This study was carried out to determine the relative susceptibility of cracked and normal shell eggs to invasion by Salmonella organisms. The effect of the presence of several chemicals in the wash water on the spoilage of cracked and normal shell eggs was also investigated.
954
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
MATERIALS AND METHODS
cracked egg is one in which only the shell has been damaged without any visual damage to the shell membrane. The eggs were candled to make sure that leakers (eggs with damaged shells and membranes) and extensively cracked eggs were not used in this experiment. Washing procedure. The cracked eggs were washed in an immersion type commercial egg washer at 42°C. for three minutes. The washing procedure was varied to study the effect of several chemicals under various commercial conditions. The following conditions were used: a) normal washing procedure using a sanitizer-detergent containing a quaternary ammonium compound; b) washing, using five times the normal amount of sanitizerdetergent as was used in "a"; c) the use of a commercial laundry detergent with no sanitizer; d) sanitizer-detergent containing chlorine as the sanitizer; e) 200 p.p.m. of active chlorine but no detergent; f) 0.5% sodium lauryl sulfate. Following washing the eggs were rinsed with water at 45°C. to remove the washing compound. Although this procedure is not usually recommended, it was considered necessary in these experiments to prevent a residual effect of the sanitizer-detergent on bacteria. In another experiment wash water containing; a) chicken manure (3/4 pound in eight gallons of water); b) sterile chicken manure (same amount as "a"); and c) 0.5% bile salts was used.
ITTLE work has been done on the susceptibility of cracked eggs to infection and spoilage by microorganisms, as compared to the extensive literature of spoilage of normal shell eggs. Cracked eggs constitute about 2-3% of total egg production and are of great economic importance to the poultry industry. With the recent reports of occurrence of Salmonella in egg and egg products, the safety of cracked eggs and the products produced from them for human consumption has been questioned by public health authorities and others. Previous work done by McNally (1953) indicated that temperature and humidity changes affect cracked eggs adversely as compared to normal shell eggs. Brown et al. (1966) working with Pseudomonas aeruginosa showed that bacterial numbers in the dip water were more important than a temperature differential for infection. They also reported that cracked eggs were no more susceptible to infection by P. aeruginosa than uncracked eggs as long as they were handled properly.
955
SALMONELLA I N F E C T I O N OF CRACKED E G G S
TABLE 1.—Degree of spoilage of cracked and normal eggs by Salmonella typhimurium under several conditions
Treatment
Cracked, washed Cracked, unwashed Cracked Control (unexposed) Normal washed Normal unwashed Normal Control (unexposed)
% showing infection after 30 days of incubation* 23° C.
10° C.
10.0 3.0 0 3.0 0 0
4.0 0 0 0 0 0
* Accumulative data of three experiments (total of 150 eggs exposed to infection).
TABLE 2.—The effect of normal and abnormal conditions during washing procedure on the amount of spoilage of cracked eggs when exposed to artificial infection
Treatment
% spoilage after 30 days at 23°C*
Quaternary Ammonium, detergent Quaternary Ammonium (5X), detergent Laundry detergent Chlorine, detergent Chlorine 200 p.p.m. Sodium lauryl sulfate (0.5%)
13 23 27 32 41 18
* Accumulative data of two experiments (total number of eggs exposed to infection was 100).
the agar and tested for Salmonella on a Triple Sugar Iron medium. RESULTS AND DISCUSSION
Effect of washing procedure on infection. T h e d a t a are presented in Table 1. A batch of cracked and normal (uncracked) were not exposed to any artifical infection and these served as controls. T h e u n washed eggs were treated similarly except they were dipped directly after cracking and were not given any washing treatment. These data demonstrated t h a t cracked unwashed eggs had a lower percentage of spoilage as compared to cracked eggs which were washed in a sanitizer-detergent. Similar results were observed with normal shell eggs. However, the degree of spoilage was higher (10%) in cracked shell eggs as compared to uncracked eggs ( 3 % ) . As expected, the eggs which were not challenged by artificial infection did not show any spoilage, and the eggs stored at 10°C. showed a slower rate of spoilage. T h e effect of the addition of several chemicals in the water during the washing procedure on the spoilage of eggs is shown in Table 2. All the chemicals tested showed a higher degree of spoilage of eggs when compared to control washing pro-
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
Exposure to infection. T h e eggs were dipped in a water b a t h containing approximately 100,000 S. typhimurium L T 2 per ml. T h e temperature of the eggs was 40°C. while the dip water had a temperature of 15°C. T h e dipping time was five minutes and the eggs after dipping were allowed to dry at room temperature. Storage of eggs. The eggs after exposure to infection were incubated a t 10°C. or 23° C. Two eggs were examined every third day and the experiment was terminated b y examining the remaining eggs after 30 days incubation. In another experiment, 100 eggs were exposed to infection, half of which were examined after 15 days of incubation and the remaining after 30 days of incubation. Bacteriological examination. T h e eggs for bacteriological examination were swabbed at the small end with 2 % iodine in 7 0 % alcohol, allowed to dry and then a circular cut was made through the shell, but without injuring the membranes, with a Dremel H o b b y Tool. T h e cut area was reswabbed and the small end of the egg was removed by sterile forceps. Bacteriological counts were made according to the Standard Methods Procedure (1960). The plates were incubated at 37°C. for 48 hours. Several colonies were picked from
956
D. V. VADEHRA, R. C. BAKER AND H. B. NAYLOR
TABLE 3.—The effect of different microbial loads and bile salts on the spoilage of cracked eggs incubated at 23°C. and 10°C* % spoilage after
Cracked—manure Cracked—sterile manure Cracked—0.5% bile salts Cracked—broth culture Cracked—normal wash
23°C.
10°C.
17.0 16.0 20.0 12.0 10.0
5.0 3.0 12.0 2.0 3.0
cedure which involved t h e use of quaternary ammonium compounds. Spoilage was greatest (41%) when 200 p.p.m. of chlorine (commercial bleach) was used. However, when sanitizer-detergent containing chlorine was used according to manufacturer's directions, the spoilage was somewhat lower ( 3 2 % ) . This study indicates t h a t washing of cracked shell eggs in a sanitizer-detergent containing quaternary ammonium compound is somew h a t better than a sanitizer-detergent containing chlorine. This study also points out t h a t t h e use of higher amounts of the washing compound than t h a t recommended b y the manufacturer has a deteriorative effect rather than a beneficial effect. I t also demonstrated t h a t t h e presence of sodium lauryl sulfate in t h e wash water h a d about t h e same effect as the presence of quaternary ammonium compounds on spoilage. T h e increased amount of spoilage by chlorine or chlorine containing sanitizer-detergent could be due to its strong oxidation properties in general and t h e action on sulfhydryl groups in particular. Since the membranes are exposed in cracked eggs, chlorine probably breaks t h e structure of the membranes thus making them more permeable and increasing spoilage. T h e action of laundry detergent and sodium lauryl sulfate m a y be due to their surface
TABLE 4.—Degree of spoilage of cracked eggs treated as shown after 30 days of incubation after infection* Incubation period Treatment
Quaternary Ammonium, detergent Chlorine, detergent Chlorine Manure Sterile manure
15 days % showing spoilage a t
30 days % showing spoilage a t
23°C.
10°C.
23°C.
10°C.
12.0 18.0 11.0 8.0 9.0
0 0 0 0 0
24 37 41 37 35
0 2 0 1 0
* 100 eggs used per treatment.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
* Accumulative data of two experiments (total of 100 eggs exposed to infection).
active properties which affect t h e interface of the membranes. Brown et al. (1966) reported t h a t t h e microbial load played an i m p o r t a n t role in spoilage of cracked eggs. Their primary source of microorganisms in wash water was chicken fecal material. Results presented in Table 3, however, indicate t h a t increased spoilage of eggs containing chicken manure m a y not necessarily be due to increased microbial load because when similar quantity of sterile manure was added to wash water, the percentage of spoilage did not decrease. Also, when a similar load of (10,000/ml.) broth culture of S. typhimurium was added to wash water, the spoilage of eggs did not increase. These data indicate t h a t there m a y be some other active material in chicken fecal matter which affects t h e cracked eggs in such a way t h a t they become more prone to infection. I t is known t h a t chickens excrete bile acids in their droppings, which could affect the membranes and thus increase spoilage. Bile salts are known to behave like anionic detergents in which the fat soluble portion has a steroid structure. Table 3 gives t h e results of washing eggs in 0 . 5 % bile salts, the spoilage figure almost doubled when compared to normal washing procedure. I t would appear t h a t the increased spoilage b y fecal m a t t e r m a y be due to t h e presence of bile salts, and the action of
SALMONELLA INFECTION OF CRACKED EGGS
957
3. Unwashed cracked eggs are less prone to spoilage when compared to washed, cracked eggs when exposed to infection. 4. Cracked eggs when washed under adverse conditions such as excessive sanitizer-detergent, chicken manure, etc. show much more spoilage, when challenged. 5. The temperature of incubation of eggs following infection had a profound effect on the spoilage. It was observed that cracked eggs washed even under adverse conditions did not show a great deal of spoilage at 10°C.
SUMMARY
This work was sponsored by Public Health Service Grant No. 5 R01 UI00146.
1. Cracked eggs which were challenged[ to artificial infection by Salmonella organisms did not show any spoilage under conditions of this experiment. 2. Cracked eggs washed in the normalI procedures and exposed to artificial infection are more prone to infection when1 compared to normal eggs treated similarly.
ACKNOWLEDGEMENT
REFERENCES Brown, W. E., R. C. Baker and H. B. Naylor, 1966. The microbiology of cracked eggs. Poultry Sci. 45: 284-287. McNally, E. H., 1953. Some factors related to the incidence of bacterial infection of shell eggs. Poultry Sci. 32: 915.
Gonadotropic Hormones in Hens Treated with Ethylene Dibromide* EUGENIA ALTJMOT (OLOMUCKI) AND E. MANDEL Division of Animal Nutrition, Volcani Institute for Agricultural Research, Rehovot, Israel. (Received for publication November 4, 1968)
INTRODUCTION
E
THYLENE dibromide (EDB), used as a grain fumigant, caused a decrease in egg size when administered to laying hens (Bondi el al., 1955). This decrease was found to be due to impaired ovarian follicle growth. Olomucki (1957) * Contribution from the National and University Institute of Agriculture, Rehovot, 1967 Series; N 1180-E. Financed partly by a grant from the United States Dept. of Agriculture, under P.L. 480.
suggested that the latter might be caused by depressed activity of the follicle stimulating hormone. Either the formation or the release of the hormone could be affected. To test the first assumption, hormone levels were measured in the pituitary of hens treated with EDB. In another series of experiments, hormone preparations were injected into hens laying small eggs following treatment with EDB, to counteract the possible impaired release of the hormone.
Downloaded from http://ps.oxfordjournals.org/ at Florida International University on June 8, 2015
bile salts is probably due to its detergentt as well as the surface active properties3 which weaken the membrane and make; them more vulnerable to attack by bacteria. In another experiment the eggs were2 examined after incubation for 15 and 30) days instead of sampling the eggs everyj third day. The results are shown in Table; 4. Such a procedure has the advantage of ai definite incubation time for the whole; group, and gives spoilage at the end off incubation period rather than accummulative spoilage. As was expected the degree of spoilage increased but the effect of various chemicals showed the same trend.