- Email: [email protected]
Effect of Sweating on Shell Penetration of Salmonella enteritidis
Q1998 Applied PoulUy Science, Inc
EFFECT OF SWEATING ON SHELL PENETRATION OF S ENTERITIDIS
Primary Audience: Egg Processors, Egg producers, Egg Marketers, Poultry Specialists
into a warm, humid environment [l,21. SweatDESCRIPTION OF PROBLEMing has been reported to increase bacterial The USDA Food Safety Inspection Service is considering changes in table egg regulations that would lower the required storage temperature after processing and packaging. It is well known that eggs held at lower temperatures are more likelyto sweat when moved 1 To whom correspondence should be addressed
penetration of shell eggs [3]. Research has shown that prolonged storage (30 or 60 days) at high relative humidity (97%) results in greater salmonellae penetration into egg contents than does storage at 75% or 86% relative humidity (41. As a result of these reports, we
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
R. A. ERNST' and L.FUQUA Avian Sciences D e p m e n t , Universityof California -Davis, Davis, CA 95616 Phone: (916) 752-3513 F M : (916) 752-8960 H.€! RIEMANN and S. HIMATHONGKHAM Population, Health, and Reproduction, School of Veterinay Medicine, University of California - Davis, Davis G1 95616
EGG SWEATING AND SALMONELLA
82
TEST 2 Test 2 used cracked eggs that had been in storage for 17 days. One group of eggs was cultured on the day after sweating; a second group was cultured 8 days later to determine whether additional storage after sweating would impact SE penetration. The test was repeated to provide replicated data for statistical purposes.
were concerned that decreased egg storage temperature would increase the probability that eggs would sweat during shipment, distribution, and marketing and that this sweating would increase the probability of Salmonella enteritidis (SE) penetration. These studies were undertaken to determine whether sweating increases the probability that SE would move from the exterior of the shell into egg albumen.
EGG CONTAMINATION AND SWEAT-
MATERIALS AND METHODSING
CULTURING TECHNIQUES To determine the number of Salmonella in egg contents the eggs were dipped without agitation in 75% ethanol at the same temperature as the eggs (24°C) for 15 min. The eggs were then broken aseptically into a Whirlpak bag and most probable number estimation was made as described below. To determine the number of Salmonella on the shell after contamination, eggs were allowed to dry for 5 hr. The whole eggs were then placed in a
TEST 1 Test 1 used sound eggs that had been in storage for 32 days. One group of eggs was cultured on the day after sweating; additional groups were cultured 8 and 14 days later to determine whether additional storage after sweating would impact SE penetration. The test was repeated to provide replicated data for statistical purposes. TABLE 1. Experimental desianA8
1
2
3
4
Inoculated
No
No
Yes
Yes
Sweated
No
Yes
No
Yes
GROUP NUMBER
I
BFive or six eggs were used per replicate; two replicates per treatment combination.
I
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
A human isolate of Salmonella ententidis [5] was used to prepare a 24-hr (37°C) brainheart infusion culture. Eggs at 20-25°C were dipped into this culture without agitation for 5 sec and then dried at the same temperature for 7 hr. Eggs were then placed in sterile plastic bags and stored at 24°C overnight. The following morning the eggs to be sweated were removed from storage and immediatelyplaced in a small glass-topped, forced draft incubator at 32°C and about 95% relative humidity. In a few minutes the incubator returned to 32°C and was then turned off and left sealed for 3 hr. Under this protocol the eggs were observed to sweat continuously for 3 hr. The wet eggs were then removed from the incubator and allowed to dry at room temperature for 5 hr. When dry they were placed in sterile plastic bags and returned to 4°C storage. The unsweated egg samples remained in refrigerated storage during this time.
In order to study eggs that had been washed and handled in the same way as eggs in commercial channels, we obtained samples from cartoned eggs in a commercial processing plant immediately after processing. The eggs were candled to obtain samples of sound eggs and cracked eggs. The cracked eggs were those with small line checks that candlersoften miss. Cracked eggs were included in the study because a small percentage of them are allowed in USDA Grade A eggs. These egg samples were placed in flats and transported to the laboratory, where they were placed in storage at 4°C. Eggs were intentionally stored before use in the study because previous research [3] had indicated that older eggs are more likely to be contaminated through the shell when allowed to sweat. Five or six eggs were used in each experimentalgroup because48 eggs were the maximum number that could be cultured during one week.
Research Report 83
ERNSTetal.
Means for percent eggs positive were analyzed by factorial ANOVA using a completely randomized design [q.
RESULTS AND DISCUSSION Culture of whole inoculated eggs demonstrated successful inoculation of approximately lo6 SE per egg. None of the uninoculated eggs (36 sound eggs and 28 cracked eggs) were found to be positive for SE regardless of sweating treatment. When sound table eggs were SE inoculated (Table 2), Salmonella were as likely to be isolated from unsweated eggs (1/36) as sweated eggs (2/35). Table 3 shows that similar results were obtained with unsweated (23/28) vs. sweated (19/28) cracked eggs. The range in number of SE found per egg was similar in sweated and unsweated groups (Tables 2 and 3). In a previous study [3], bacteria were more likely to be present in the albumen or yolk of eggs allowed to sweat for 1,3,or 5 hr. The procedures in that study, however, differed from those we used. For example, eggs in the aforementioned study were returned to storage after sweating while still wet, while eggs in our study were thoroughly dried before return to storage. In our study cracked eggs were frequently culture positive, and we recovered relatively large numbers of SE from these eggs (Table 3).
TABLE 2. Effect of sweating on penetration of SE into sound table eggs inoculated with 6 million CFU/egg vest I)* % POSITIVE
1
1/12
8.3
0- c 0.7
1
0/12
0
8
0/11
0
0-0 0-0
POSTCONTAMINATION STORAGE (Days)
Yes
No YeS
EGGS
RANGE OF SE/EGG (LOG)
NUMBER EGGS POSITIVEPIUMBER EGGS TESIED
SWEATED
No
8
1/12
8.3
0-1.8
YeS
14
1/12
8.3
0-0.7
No
14
0/12
0
0-0
Yes
1-14
2/35
5.T
0-0.7
1-14
1/36
2.ga
0-1.8
No
aMeans were not significantly different (P = 59).
I
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
Whirlpak bag, crushed with 90 mL lactose broth, and cultured as described below. The primary sample (egg contents or egg plus shell) was transferred to a sterile 18-oz Whirlpak bag and mixed in the bag with 50 mL lactose broth. From this mixture ten-fold dilutions were made by serially transferring 10 mL of the mixture to bags with 90 mL lactose broth. Four ten-fold dilutions were made with two bags of lactose broth at each dilution level. These pre-enrichment cultures were incubated 16-20 hr at 37T, after which 10pL of the cultures were transferred to 250pL of tetrathionate broth in microtiter plate wells for selective enrichment. After 24 hr at 37°C the tetrathionate cultures were streaked on brilliant green novobiocin agar and XLT 4 agar. Suspect colonies on these media were transferred to triple sugar iron agar for presumptive confirmation. Before incubation of the pre-enrichment cultures, 100-pL quantities were spread directly on the agar media to permit quantification if large numbers of salmonella were present. The calculation of most probable numbers of Salmonella was made based on the number of positive cultures, following the recommendations of Fisher and Yates [6]. The detection level for SE with this procedure was 1-4 Salmonella bacteria per egg.
JAPR EGG SWEATING AND SALMONELLA
84
EGGS SWEATED
POSTCONTAMINATION STORAGE (Days)
Yes
1
NUMBER EGGS POS-MBER EGGS TESI'ED
%POSITIVE
RANGEOF SEPOSITIVE EGG (LOG)
6111
54.5
0->3.85
No
1
8111
72.7
0->3.85
Yes
8
8111
72.7
e>3.85
No
8
9111
81.8
0-4.53
Yes
1-8
14/22
63.6a
0->3.85
No
1-8
17/22
77.3'
0-1.53
1. Sweating cracked eggs previously stored for 17 days or sound eggs previously stored for 32 days did not appear to enhance the penetration of SE into the eggs. 2. Shells of eggs with small line cracks dipped into SE culture were more likely to be penetrated than sound eggs similarly treated. 3. Egg shell integrity appears to be a more important factor than sweating in SE penetration of table eggs. 4. These results indicate that additional research is needed to determine the relationship between egg sweating and bacterial penetration of the shell.
REFERENCES AND NOTES 1. Stadelman, WJ., 1995. Egg roduction practices. Pages 34-35 in: Egg Science and fechnology. Haworth Food Products Press, New York, NY. 2. Sicer, J.W.,undated. Poultry for Profit: EggSweating Problem P-87, Animal Sciences Department, Purdue University, Lafayette, IN. 3. Fromm, D. and P.H. Margolf, 1958. The influence of sweatingandwashin on weight loss, bacterial contamination, and interior pfjsical quality of 12day-old shell eggs. Poultry Sci. 37:1273-1278. 4. SLmmons, ER, J.C. Ayns, and kk Kralt, 1970. Effect of moisture and temperature on ability of Salmonellae to infect eggs. Poultry Si.49761-768.
5. A human isolate of SE obtained from Dr. Bryan Walsh, University of California - Davis. 6. Fisher, RA and F. Yaks, 1963. Statistical Tables for Biological, Agricultural, and Medical Research. 6th Edition. Oliver & Boyd, Edinbourgh, England. 7. MSTAT Development Team, 1989. User's Guide to MSTAT-C. Michigan State University, East Lansing, MI.
ACKNOWLEDGEMENT We would like to thank the California Egg Commission for their encouragement to pursue this research and for significant financial support.
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
CONCLUSIONS AND APPLICATIONS
EFFECT OF SWEATING ON SHELL PENETRATION OF S ENTERITIDIS
Primary Audience: Egg Processors, Egg producers, Egg Marketers, Poultry Specialists
into a warm, humid environment [l,21. SweatDESCRIPTION OF PROBLEMing has been reported to increase bacterial The USDA Food Safety Inspection Service is considering changes in table egg regulations that would lower the required storage temperature after processing and packaging. It is well known that eggs held at lower temperatures are more likelyto sweat when moved 1 To whom correspondence should be addressed
penetration of shell eggs [3]. Research has shown that prolonged storage (30 or 60 days) at high relative humidity (97%) results in greater salmonellae penetration into egg contents than does storage at 75% or 86% relative humidity (41. As a result of these reports, we
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
R. A. ERNST' and L.FUQUA Avian Sciences D e p m e n t , Universityof California -Davis, Davis, CA 95616 Phone: (916) 752-3513 F M : (916) 752-8960 H.€! RIEMANN and S. HIMATHONGKHAM Population, Health, and Reproduction, School of Veterinay Medicine, University of California - Davis, Davis G1 95616
EGG SWEATING AND SALMONELLA
82
TEST 2 Test 2 used cracked eggs that had been in storage for 17 days. One group of eggs was cultured on the day after sweating; a second group was cultured 8 days later to determine whether additional storage after sweating would impact SE penetration. The test was repeated to provide replicated data for statistical purposes.
were concerned that decreased egg storage temperature would increase the probability that eggs would sweat during shipment, distribution, and marketing and that this sweating would increase the probability of Salmonella enteritidis (SE) penetration. These studies were undertaken to determine whether sweating increases the probability that SE would move from the exterior of the shell into egg albumen.
EGG CONTAMINATION AND SWEAT-
MATERIALS AND METHODSING
CULTURING TECHNIQUES To determine the number of Salmonella in egg contents the eggs were dipped without agitation in 75% ethanol at the same temperature as the eggs (24°C) for 15 min. The eggs were then broken aseptically into a Whirlpak bag and most probable number estimation was made as described below. To determine the number of Salmonella on the shell after contamination, eggs were allowed to dry for 5 hr. The whole eggs were then placed in a
TEST 1 Test 1 used sound eggs that had been in storage for 32 days. One group of eggs was cultured on the day after sweating; additional groups were cultured 8 and 14 days later to determine whether additional storage after sweating would impact SE penetration. The test was repeated to provide replicated data for statistical purposes. TABLE 1. Experimental desianA8
1
2
3
4
Inoculated
No
No
Yes
Yes
Sweated
No
Yes
No
Yes
GROUP NUMBER
I
BFive or six eggs were used per replicate; two replicates per treatment combination.
I
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
A human isolate of Salmonella ententidis [5] was used to prepare a 24-hr (37°C) brainheart infusion culture. Eggs at 20-25°C were dipped into this culture without agitation for 5 sec and then dried at the same temperature for 7 hr. Eggs were then placed in sterile plastic bags and stored at 24°C overnight. The following morning the eggs to be sweated were removed from storage and immediatelyplaced in a small glass-topped, forced draft incubator at 32°C and about 95% relative humidity. In a few minutes the incubator returned to 32°C and was then turned off and left sealed for 3 hr. Under this protocol the eggs were observed to sweat continuously for 3 hr. The wet eggs were then removed from the incubator and allowed to dry at room temperature for 5 hr. When dry they were placed in sterile plastic bags and returned to 4°C storage. The unsweated egg samples remained in refrigerated storage during this time.
In order to study eggs that had been washed and handled in the same way as eggs in commercial channels, we obtained samples from cartoned eggs in a commercial processing plant immediately after processing. The eggs were candled to obtain samples of sound eggs and cracked eggs. The cracked eggs were those with small line checks that candlersoften miss. Cracked eggs were included in the study because a small percentage of them are allowed in USDA Grade A eggs. These egg samples were placed in flats and transported to the laboratory, where they were placed in storage at 4°C. Eggs were intentionally stored before use in the study because previous research [3] had indicated that older eggs are more likely to be contaminated through the shell when allowed to sweat. Five or six eggs were used in each experimentalgroup because48 eggs were the maximum number that could be cultured during one week.
Research Report 83
ERNSTetal.
Means for percent eggs positive were analyzed by factorial ANOVA using a completely randomized design [q.
RESULTS AND DISCUSSION Culture of whole inoculated eggs demonstrated successful inoculation of approximately lo6 SE per egg. None of the uninoculated eggs (36 sound eggs and 28 cracked eggs) were found to be positive for SE regardless of sweating treatment. When sound table eggs were SE inoculated (Table 2), Salmonella were as likely to be isolated from unsweated eggs (1/36) as sweated eggs (2/35). Table 3 shows that similar results were obtained with unsweated (23/28) vs. sweated (19/28) cracked eggs. The range in number of SE found per egg was similar in sweated and unsweated groups (Tables 2 and 3). In a previous study [3], bacteria were more likely to be present in the albumen or yolk of eggs allowed to sweat for 1,3,or 5 hr. The procedures in that study, however, differed from those we used. For example, eggs in the aforementioned study were returned to storage after sweating while still wet, while eggs in our study were thoroughly dried before return to storage. In our study cracked eggs were frequently culture positive, and we recovered relatively large numbers of SE from these eggs (Table 3).
TABLE 2. Effect of sweating on penetration of SE into sound table eggs inoculated with 6 million CFU/egg vest I)* % POSITIVE
1
1/12
8.3
0- c 0.7
1
0/12
0
8
0/11
0
0-0 0-0
POSTCONTAMINATION STORAGE (Days)
Yes
No YeS
EGGS
RANGE OF SE/EGG (LOG)
NUMBER EGGS POSITIVEPIUMBER EGGS TESIED
SWEATED
No
8
1/12
8.3
0-1.8
YeS
14
1/12
8.3
0-0.7
No
14
0/12
0
0-0
Yes
1-14
2/35
5.T
0-0.7
1-14
1/36
2.ga
0-1.8
No
aMeans were not significantly different (P = 59).
I
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
Whirlpak bag, crushed with 90 mL lactose broth, and cultured as described below. The primary sample (egg contents or egg plus shell) was transferred to a sterile 18-oz Whirlpak bag and mixed in the bag with 50 mL lactose broth. From this mixture ten-fold dilutions were made by serially transferring 10 mL of the mixture to bags with 90 mL lactose broth. Four ten-fold dilutions were made with two bags of lactose broth at each dilution level. These pre-enrichment cultures were incubated 16-20 hr at 37T, after which 10pL of the cultures were transferred to 250pL of tetrathionate broth in microtiter plate wells for selective enrichment. After 24 hr at 37°C the tetrathionate cultures were streaked on brilliant green novobiocin agar and XLT 4 agar. Suspect colonies on these media were transferred to triple sugar iron agar for presumptive confirmation. Before incubation of the pre-enrichment cultures, 100-pL quantities were spread directly on the agar media to permit quantification if large numbers of salmonella were present. The calculation of most probable numbers of Salmonella was made based on the number of positive cultures, following the recommendations of Fisher and Yates [6]. The detection level for SE with this procedure was 1-4 Salmonella bacteria per egg.
JAPR EGG SWEATING AND SALMONELLA
84
EGGS SWEATED
POSTCONTAMINATION STORAGE (Days)
Yes
1
NUMBER EGGS POS-MBER EGGS TESI'ED
%POSITIVE
RANGEOF SEPOSITIVE EGG (LOG)
6111
54.5
0->3.85
No
1
8111
72.7
0->3.85
Yes
8
8111
72.7
e>3.85
No
8
9111
81.8
0-4.53
Yes
1-8
14/22
63.6a
0->3.85
No
1-8
17/22
77.3'
0-1.53
1. Sweating cracked eggs previously stored for 17 days or sound eggs previously stored for 32 days did not appear to enhance the penetration of SE into the eggs. 2. Shells of eggs with small line cracks dipped into SE culture were more likely to be penetrated than sound eggs similarly treated. 3. Egg shell integrity appears to be a more important factor than sweating in SE penetration of table eggs. 4. These results indicate that additional research is needed to determine the relationship between egg sweating and bacterial penetration of the shell.
REFERENCES AND NOTES 1. Stadelman, WJ., 1995. Egg roduction practices. Pages 34-35 in: Egg Science and fechnology. Haworth Food Products Press, New York, NY. 2. Sicer, J.W.,undated. Poultry for Profit: EggSweating Problem P-87, Animal Sciences Department, Purdue University, Lafayette, IN. 3. Fromm, D. and P.H. Margolf, 1958. The influence of sweatingandwashin on weight loss, bacterial contamination, and interior pfjsical quality of 12day-old shell eggs. Poultry Sci. 37:1273-1278. 4. SLmmons, ER, J.C. Ayns, and kk Kralt, 1970. Effect of moisture and temperature on ability of Salmonellae to infect eggs. Poultry Si.49761-768.
5. A human isolate of SE obtained from Dr. Bryan Walsh, University of California - Davis. 6. Fisher, RA and F. Yaks, 1963. Statistical Tables for Biological, Agricultural, and Medical Research. 6th Edition. Oliver & Boyd, Edinbourgh, England. 7. MSTAT Development Team, 1989. User's Guide to MSTAT-C. Michigan State University, East Lansing, MI.
ACKNOWLEDGEMENT We would like to thank the California Egg Commission for their encouragement to pursue this research and for significant financial support.
Downloaded from http://japr.oxfordjournals.org/ by guest on March 11, 2015
CONCLUSIONS AND APPLICATIONS