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Eggshell characteristics and penetration by Salmonella through the productive life of a broiler breeder flock
Eggshell Characteristics and Penetration by Salmonella Through the Productive Life of a Broiler Breeder Flock M. E. BERRANG,*,1 J. F. FRANK,† R. J. BUHR,* J. S. BAILEY,* N. A. COX,* and J. MAULDIN‡ *USDA-ARS, Russell Research Center, Athens, Georgia 30604-5677, †Department of Food Science and Technology and ‡Department of Poultry Science Extension, University of Georgia, Athens, Georgia 30602 weight loss/d per torr) showed any clear trend throughout the life of the flock despite the increase in egg weight. Conductance values were not correlated with specific gravity. The number of eggs positive for Salmonella penetration after 24 h incubation showed a general upward trend with flock age; however, penetration frequency and hen age were not found to be significantly correlated (P > 0.05). No relationship was found between egg specific gravity, conductance, or egg weight and the likelihood of Salmonella to penetrate the eggshell. Because shell characteristics did not change over time and the penetration patterns did vary, it is likely that factors other than specific gravity and conductance were involved in the penetration of eggshells by Salmonella.
(Key words: Salmonella, specific gravity, conductance, egg weight, hatching eggs) 1998 Poultry Science 77:1446–1450
INTRODUCTION Salmonella and other bacteria can penetrate the shell and associated membranes of the chicken egg (Wolk et al., 1950; Stokes et al., 1956; Williams et al., 1968; Vadehra et al., 1970; Neill et al., 1985; Padron, 1990). Salmonella, when present in an egg at hatch, can cross-contaminate many chicks in the hatching cabinet via the fan-driven air (Cason et al.,1994). Some published reports suggest a relationship between eggshell quality and bacterial penetration (Sauter and Petersen, 1974; Nascimento and Solomon, 1991). There are several objective measures of eggshell quality. Conductance is a measure of eggshell porosity indicating the ability of a shell to allow the passage of water vapor and other gasses. Conductance is important to the developing embryo because diffusive respiration and weight loss in the form of water vapor are essential for hatch. Shell strength and thickness are also measures of shell quality. Specific gravity is positively correlated with shell thickness (Peebles and
Brake, 1987). Sauter and Peterson (1974) found that eggs with low specific gravity were more likely to be penetrated by Salmonella. Another study showed that eggs judged by visual examination to have poor quality shells were more likely to allow bacterial penetration than those judged to have good quality shells (Nascimento and Solomon, 1991). There are various studies reporting the effect of hen age on the quality of eggshells. Some investigators have found that the quality of eggshells as measured by specific gravity tends to decline with age (McDaniel et al., 1979; Peebles and Brake, 1987; Bennett, 1992). Another study found that eggshell membranes from older hens were lighter than that from young hens (Britton, 1977). Another report indicates that percentage weight loss through incubation (related to shell conductance) is not affected by the age of the flock (Reinhart and Hurnik, 1984). Comparison of these studies is difficult because some used different groups of hens at different ages and some used experimental housing whereas others employed commercial broiler breeder farms.
Received for publication December 9, 1997. Accepted for publication April 22, 1998. 1To whom correspondence should be addressed.
Abbreviation Key: TTC = 2,3,5 triphenyl tetrazolium chloride.
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ABSTRACT Egg weight, specific gravity, conductance, and ability of Salmonella to penetrate the shell and membranes were determined for hatching eggs from a commercial broiler breeder flock. Thirty unsanitized eggs were sampled on Weeks 29, 34, 39, 42, 48, 52, and 56 of flock age for specific gravity and conductance. An additional 10 intact eggs were inoculated with Salmonella by a temperature differential immersion method for 1 min. Eggs were then emptied of contents and filled with a selective medium that allowed visualization of Salmonella growth on the inside of the shell and membrane complex. Over the 27-wk sampling period, egg weight increased from 56 to 66 g and was positively correlated with hen age (r = 0.96, P < 0.05). However, neither specific gravity (ranging from 1.077 to 1.082) nor eggshell conductance (ranging from 14.7 to 17.9 mg
EGGSHELL CHARACTERISTICS AND SALMONELLA PENETRATION
A clear understanding of the relation of hen age, shell quality, and bacterial penetration could be a valuable tool in planning intervention strategies to prevent the introduction of human pathogens to commercial hatcheries, chicks, growout farms, and processing plants. This study was designed to examine eggs from one commercial broiler breeder flock through the entire production period. Our objective was to determine the affect of flock age on eggshell quality and furthermore the ability of Salmonella typhimurium to penetrate the shell and membrane complex of intact eggs. Our goal was to determine the effect of hen age and shell quality on the likelihood of hatching eggs becoming contaminated with Salmonella postoviposition.
MATERIALS AND METHODS
Specific Gravity On each sample day, specific gravity was measured on 30 eggs. Specific gravity was calculated by the Archimedes method as described by Hempe et al. (1988). This method is based on comparing the weight of the egg to the weight of water the egg displaces. Briefly, the room temperature egg is weighed dry and then reweighed under water. Specific gravity is calculated using the following formula: Sg = ew/dww(tc), where Sg = specific gravity; ew = egg weight; dww = displaced water weight; and tc = water temperature correction found in Hempe et al. (1988).
Conductance After taking measurements for specific gravity, the same 30 eggs were used to determine conductance. Conductance was calculated from the weight loss meas-
2Difco, 3Sigma
Detroit, MI 48201. Chemical Co., St. Louis, MO 63178-9916.
ured over 2 d of egg incubation. Eggs were held in a 35 C incubator in which the temperature and humidity were recorded every 60 s. Egg weight was recorded 18 to 24 h after placement into the incubator (to allow moisture picked up during specific gravity measurement to dissipate) and again after two further consecutive 24-h periods of incubation (wgt1 and wgt2). These measurements were used to calculate conductance by the following formula: G = wgt1 – wgt2/SVP – (%RH × SVP)/ (#hours/24 h); where G = conductance; wgt1 and wgt2 = the first and second egg weights taken during incubation, respectively; SVP = saturated water vapor pressure at the average temperature at which the eggs were incubated; and %RH = average relative humidity at which the eggs were incubated (Tullet, 1981).
Penetration of Shell and Membranes by Salmonella The ability of S. typhimurium to penetrate the eggshell as well as outer and inner eggshell membranes was studied through the life of the flock. Ten eggs, separate from those used for shell analysis, were examined on each sample day. The method used was designed to measure bacterial penetration into the unbroken egg. Ten eggs were warmed to 42 C overnight and dipped into a cell suspension of a nalidixic acid resistant strain of S. typhimurium at a level of 104 cells per milliliter 1% peptone water (Berrang et al., 1991) at 25 C. The temperature differential cools the egg causing the contents to contract and the resultant negative pressure can draw bacteria into and through the shell. The eggs remained submerged in the cell suspension for 60 s were removed and allowed to remain at room temperature until dry. An agar molding technique similar to that described by Neill et al. (1985) was used to visualize S. typhimurium penetration. The narrow end of each egg was sanitized with 70% ethanol and a hole about 1 cm in diameter was aseptically cut in the shell and membranes with sterile scissors. The egg contents were drained through the hole and the egg was then filled with sterile molten 45 C Plate Count Agar2 containing 200 ppm sodium salt of nalidixic acid3 and 0.1% 2,3,5 triphenyl tetrazolium chloride (TTC).3 The addition of nalidixic acid assures that only resistant bacteria will be able to grow thus holding down competitors for the resistant S. typhimurium. The TTC is reduced by growing bacteria resulting in dark red colonies. The eggs were allowed to remain in the narrow end up position until the agar hardened, at that point the hole was sealed with molten paraffin. When the paraffin hardened, the eggs were turned over and incubated at 35 C for 24 h. Penetration was recorded as the number of red colonies visible on the molded agar when the agar filled eggs were candled after 24 and 48 h. If the egg showed no visible dots due to the reduction of TTC at 24 h, then the egg was allowed to incubate overnight again and reexamined. Any red colonies seen to be continuous with the hole were assumed to result from contamination during cutting, filling, or sealing and not counted as penetration. All agar
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All eggs were collected from a commercial broiler breeder house in Northeast Georgia stocked with Hubbard hens and Petersen roosters. Eggs were separated by the producer into hatchery quality and cull groups as they appeared on the automated collection lines. Eggs for analysis were gathered at random from the hatchery quality group before application of any sanitization procedures. Eggs from the midday collection were selected to eliminate the first or last eggs in a series (normally laid early in the morning or late in the afternoon, respectively). Thus, we focused on eggs with more uniform shell quality associated with the middle of a hen’s series (Berg, 1945). Eggs were sampled from early in the productive life through the last week of production on a roughly monthly schedule. Samples were drawn on Weeks 29, 34, 39, 42, 48, 52, and 56 of flock age. Eggs were transported to the laboratory and analyzed within 2 h.
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TABLE 1. Effect of hen age on eggshell characteristics and penetration by Salmonella typhimurium through 24 h of incubation
Hen age
Egg weight1
Specific gravity1
(wk) 29 34 39 42 48 52 56
(g) 56.1 60.2 61.9 62.2 64.4 66.0 66.2
1.082 1.077 1.080 1.078 1.080 1.079 1.078
± ± ± ± ± ± ±
1.3 1.6* 1.6 1.2 1.2 1.6 1.3
± ± ± ± ± ± ±
0.002 0.002* 0.003 0.004 0.002 0.003 0.002
Conductance
No. eggs penetrated by Salmonella
Total number of penetration spots on 10 eggs (variance)
(mg/d/torr) 14.7 ± 0.8 17.1 ± 1.0* 17.9 ± 1.1 16.5 ± 0.7 17.9 ± 0.9* 16.6 ± 0.7* 16.2 ± 1.1
(#/10 eggs) 4 6 6 8 6 10 7
8 19 9 114 50 256 38
(1.51) (4.98) (0.77) (187) (115) (220) (19.9)
1Plus/minus a 95% confidence interval. *This figure represents a significant change (P ≤ 0.05) from the same measurement taken in the previous months sampling (Kolmogrov-Smirnov test).
Egg Production and Hatchability The total number of cull, nest clean, and dirty eggs per sample day (at hen age 29, 34, 39, 42, 48, 52, and 56 wk) were calculated from the farm egg classification sheets. Hatchability records were obtained from the commercial hatchery personnel. Hatch is reported as average percentage hatch of all eggs set by the hatchery from the subject farm in the week that egg samples were drawn. All eggs from this farm were subjected to eggshell sanitization in a two stage spray machine and thus both nest clean and sanitized dirty eggs are used in calculation of the hatch percentages.
Statistical Analyses Pearson correlations were done to examine the relationships between factors measured or between any one factor and hen age. F tests and analysis of variance were used to examine and compare variance values for egg penetration data. A randomized design model was used with the variation within group as the error term. The Kolmogrov Smirnov test was used to determine significance between measurement results on particular sample days. All 4Datamost
Corp., Salt Lake City, UT 84165.
statistical calculations were done using the Statmost statistical software package.4
RESULTS
Eggshell Characteristics Eggshell characteristic data are shown in Table 1. Egg weight increased 10 g as the flock aged 6 mo and egg weight was significantly correlated with hen age (r = 0.96, P = 0.0005). However, neither hen age nor egg weight was significantly correlated with specific gravity throughout flock life. Specific gravity showed no trend through the life of the flock despite an increase in egg weight. Likewise, eggshell conductance was not affected by hen age. However, a significant increase in egg weight and conductance and a significant decrease in specific gravity was noted between Weeks 29 and 34 of hen age; after that point, specific gravity remained fairly stable through the end of production. Conductance value showed another significant peak at 48 wk of flock age without a corresponding decrease in specific gravity or significant increase in egg weight. Overall, conductance did not correlate with specific gravity.
Eggshell Penetration Eggshell penetration results are presented in Table 1. The number of eggs out of 10 challenged found to show penetration in 24 h gave a general upward trend through the production period of the flock. The correlation between number of eggs penetrated in 24 h and age approaches significance (R = 0.68, P = 0.089); a larger sample size may have shown this relationship more clearly. The number of eggs showing penetration after an additional 24 h incubation (data not shown) had a lower probability of a significant correlation with hen age (P = 0.176). This result was likely a function of the fact that eggs found to be negative for penetration after the first 24 h of incubation and positive after an additional 24 h were really showing the ability of S. typhimurium located
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molded eggs showing red spots were aseptically opened; the inside of the inner membrane was swabbed at the red spots with a sterile cotton tipped applicator. The applicator was used to transfer the bacteria to a plate of BG Sulfa Agar2 with the addition of 200 ppm sodium salt of nalidixic acid3 for confirmation as nalidixic acid resistant Salmonella. Isolates from the inside of the inner membrane were further confirmed as Salmonella with Poly O serology.2 Thus penetration through the shell and associated membranes could be demonstrated. Upon completion of the 48 h incubation, any eggs still not found to show penetration with candling were aseptically opened and the agar and membranes were examined directly for signs of bacterial growth.
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EGGSHELL CHARACTERISTICS AND SALMONELLA PENETRATION TABLE 2. Egg production and hatchability across the production period of a commercial broiler breeder flock Number of eggs by category—Percentage hatch of set Week 29 34 39 42 48 52 56
Nest clean 4,482 5,022 4,968 4,536 4,428 4,050 3,456
1Percentage
Cull 512 400 332 320 310 213 350
(8.9%)1 (6.8%) (5.6%) (5.6%) (5.8%) (4.4%) (8.0%)
Dirty
Total
Percentage hatch
756 486 648 810 594 594 540
5,750 5,908 5,948 5,666 5,332 4,857 4,346
82.5 85.0 89.0 89.0 85.3 81.7 73.5
of total eggs classified as cull.
Egg Production Egg production data are presented in Table 2. The numbers of nest clean eggs suitable for the hatchery peaked at 34 wk of age (5,022) and suffered a noticeable decline through the rest of the production period. As dirty eggs were counted separately, most cull eggs in this study consisted of thin-shelled, cracked-shelled, or irregular shaped eggs. However, eggshell quality characteristics in this study were only determined on noncull eggs and as
such were not correlated with the number of cull eggs on each sample day. Like total egg numbers, the number of cull eggs was found to have a significant (P ≤ 0.05) downward trend over flock life. However, the percentage of total eggs made up by cull eggs did not decline. Except for the first and last week of production (8.9 and 8.0% respectively), the percentage of eggs characterized as cull remained fairly stable around an average of 5.6%. Percentage hatch of eggs set peaked for eggs from hens at age 39 to 42 wk and declined through later production. Although percentage hatch was not significantly correlated with hen age through the first 52 wk of flock life, it dropped steeply between 52 and 56 wk of age. A significant negative correlation (r = –0.95, P = 0.04) between hen age and percentage hatch was noted from 42 to 56 wk of age. Percentage hatch was calculated from the same class of eggs used to measure eggshell quality (noncull eggs). However, percentage hatch was not correlated with specific gravity, conductance, or egg weight.
DISCUSSION In this study, restriction-fed broiler breeders tended to lay heavier eggs as the flock aged. However, in contrast to conventional wisdom, the shells did not become significantly thinner through flock life. Instead, shell thickness and quality, as examined by specific gravity and conductance, did not correlate with hen age. In fact, the eggs tested had consistently good specific gravity at or near the industry standard of 1.080. However, it seems that a relationship does exist between egg weight, specific gravity, and conductance. At Week 34, the hens were at peak production (nest clean egg numbers) and these eggs were significantly heavier than those laid 1 mo earlier. Furthermore, these eggs had significantly thinner shells and higher conductance than eggs sampled 1 mo before. In studies in which feed was consumed ad libitum, we might expect these relationships to carry on as a trend through flock life. However, in the case of carefully managed restricted-fed broiler breeders, egg weight increase is apparently controlled. Therefore, a decline in eggshell quality is not necessarily observed through flock life.
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outside the membranes to grow through the membranes and not truly penetrating at the time of challenge. There was a general upward trend in the number of spots on the eggs penetrated by S. typhimurium as the hens aged. However, on the last week of sampling this relationship broke down; overall, there was no significant correlation (P > 0.05) between hen age and number of spots on the shells and membranes that were penetrated. Variance values for number of spots penetrated per 10 eggs are shown in Table 1. The variances were similar for these data collected in Weeks 29, 34, 39, and 56 of hen age; variance values for the same data from eggs collected in Weeks 42, 48, and 52 were higher. An analysis of variance showed that the differences in the number of spots penetrated per 10 eggs can be thought of as due to random effects in Weeks 29, 34, 39, and 56 (P > 0.05). However, the higher variance values noted in eggs from Weeks 42, 48, and 52 suggest that the number of spots penetrated in eggs from these sample days were different than numbers from the other group and that this difference is due to something other than chance. Thus these data indicate that two distinct groups of eggs were produced in the hen’s productive life relative to Salmonella penetration. However, the conductance and specific gravity did not show a noticeable change in Weeks 42, 48, and 52. Further analysis by Pearson correlation did not detect a relationship between specific gravity, conductance, or egg weight and the likelihood of S. typhimurium to penetrate the outer structures of an egg over the entire production period. Although the number of eggs penetrated (4 of 10 to 10 of 10) and the number of spots on penetrated eggs (8 to 256) varied through flock life, other than the peak at Week 34, the specific gravity and conductance did not change with any practical significance.
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REFERENCES Bennett, C. D., 1992. The influence of shell thickness on hatchability in commercial broiler breeder flocks. J. Appl. Poult. Res. 1:61–65. Berg, L. R., 1945. The relationship of clutch position and time interval between eggs to eggshell quality. Poultry Sci. 24: 555–563. Berrang, M. E., N. A. Cox, J. S. Bailey, and L. C. Blankenship, 1991. Methods for inoculation and recovery of Salmonella from chicken eggs. Poultry Sci. 70:2267–2270. Berrang, M. E., J. F. Frank, R. J. Buhr, J. S. Bailey, N. A. Cox, and J. M. Mauldin, 1997. Microbiology of sanitized broiler hatching eggs through the egg production period. J. Appl. Poult. Res. 6:298–305. Board, R. G., 1965. Bacterial growth on and penetration of the shell membranes of the hen’s egg. J. Appl. Bacteriol. 28: 197–205. Britton, W. M.. 1977. Shell membranes of eggs differing in shell quality from young and old hens. Poultry Sci. 56:647–653. Cason, J. A., N. A. Cox, and J. S. Bailey, 1994. Transmission of Salmonella typhimurium during hatching of broiler chicks. Avian Dis. 38:583–588.
Fajardo, T. A., R. C. Anantheswaran, V. M. Puri, and S. J. Knabel, 1995. Penetration of Salmonella enteritidis into eggs subjected to rapid cooling. J. Food Prot. 58:473–477. Garibaldi, J. A., and J. L. Stokes, 1958. Protective role of shell membranes in bacterial spoilage of eggs. Food Res. 23: 283–290. Hempe, J. M., R. C. Lauxen, and J. E. Savage, 1988. Rapid determination of egg weight and specific gravity using a computerized data collection system. Poultry Sci. 67: 902–907. Lifshitz, A., R. C. Baker, and H. B. Naylor, 1964. The relative importance of chicken egg exterior structures in resisting bacterial penetration. J. Food Sci. 29:94–99. McDaniel, G. R., D. A. Roland, and M. A. Coleman, 1979. The effect of egg shell quality on hatchability and embryonic mortality. Poultry Sci. 58:10–13. Nascimento, V. P., and S. E. Solomon, 1991. The transfer of bacteria (Salmonella enteritidis) across the eggshell wall of eggs classified as poor quality. Anim. Tech. 42:157–165. Neill, S. D., J. N. Campbell, and J. J. O’Brien, 1985. Egg penetration by Campylobacter jejuni. Avian Pathol. 14: 313–320. Padron, M. N., 1990. Salmonella typhimurium penetration through the eggshell of hatching eggs. Avian Dis. 34: 463–465. Peebles, E. D., and J. Brake, 1987. Eggshell quality and hatchability in broiler breeder eggs. Poultry Sci. 66: 596–604. Reinhart, B. S., and G. I. Hurnik, 1984. Traits affecting the hatching performance of commercial chicken broiler eggs. Poultry Sci. 63:240–245. Sauter, E. A., and C. F. Petersen, 1974. The effect of egg shell quality on penetration by various salmonellae. Poultry Sci. 53:2159–2162. Stokes, J .L., W. W. Osborne, and H. G. Bayne, 1956. Penetration and growth of Salmonella in shell eggs. Food Res. 21:510–518. Tullet, S. G., 1981. Theoretical and practical aspects of eggshell porosity. Turkeys 29:24–28. Vadehra, D. V., R. C. Baker, and H. B. Naylor, 1970. Infection routes of bacteria into chicken eggs. J. Food Sci. 35:61–62. 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. Wolk, J., E. H. McNally, and N. H. Spicknall, 1950. The effect of temperature on the bacterial infection of shell eggs. Food Technol. 14:316–318.
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Because only eggs with good quality shells were used in the study, no direct conclusions can be drawn about the effect of eggshell quality changes on S. typhimurium penetration. However, because shell quality measures did not change greatly over time and the S. typhimurium penetration patterns did vary, it is likely that factors other than just shell quality are involved in bacterial penetration of eggshells. Age of the hen was not directly correlated with bacterial penetration through the eggshell and membrane complex. When coupled with the fact that eggs from older hens do not necessarily have higher bacterial populations (Berrang et al., 1997), this finding would indicate that the eggs from older hens are not more likely to be contaminated postoviposition with human enteropathogens. It may be that eggshell membranes play a pivotal role in exclusion of bacteria from the inside of an egg, as suggested by some researchers (Garibaldi and Stokes, 1958; Lifshitz et al., 1964; Board, 1965; Williams, et al., 1968; Fajardo et al. 1995). Further research into the effect of age on eggshell membranes and on penetration specifically through the membranes may shed some light on the process of bacterial invasion of the intact egg.
(Key words: Salmonella, specific gravity, conductance, egg weight, hatching eggs) 1998 Poultry Science 77:1446–1450
INTRODUCTION Salmonella and other bacteria can penetrate the shell and associated membranes of the chicken egg (Wolk et al., 1950; Stokes et al., 1956; Williams et al., 1968; Vadehra et al., 1970; Neill et al., 1985; Padron, 1990). Salmonella, when present in an egg at hatch, can cross-contaminate many chicks in the hatching cabinet via the fan-driven air (Cason et al.,1994). Some published reports suggest a relationship between eggshell quality and bacterial penetration (Sauter and Petersen, 1974; Nascimento and Solomon, 1991). There are several objective measures of eggshell quality. Conductance is a measure of eggshell porosity indicating the ability of a shell to allow the passage of water vapor and other gasses. Conductance is important to the developing embryo because diffusive respiration and weight loss in the form of water vapor are essential for hatch. Shell strength and thickness are also measures of shell quality. Specific gravity is positively correlated with shell thickness (Peebles and
Brake, 1987). Sauter and Peterson (1974) found that eggs with low specific gravity were more likely to be penetrated by Salmonella. Another study showed that eggs judged by visual examination to have poor quality shells were more likely to allow bacterial penetration than those judged to have good quality shells (Nascimento and Solomon, 1991). There are various studies reporting the effect of hen age on the quality of eggshells. Some investigators have found that the quality of eggshells as measured by specific gravity tends to decline with age (McDaniel et al., 1979; Peebles and Brake, 1987; Bennett, 1992). Another study found that eggshell membranes from older hens were lighter than that from young hens (Britton, 1977). Another report indicates that percentage weight loss through incubation (related to shell conductance) is not affected by the age of the flock (Reinhart and Hurnik, 1984). Comparison of these studies is difficult because some used different groups of hens at different ages and some used experimental housing whereas others employed commercial broiler breeder farms.
Received for publication December 9, 1997. Accepted for publication April 22, 1998. 1To whom correspondence should be addressed.
Abbreviation Key: TTC = 2,3,5 triphenyl tetrazolium chloride.
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ABSTRACT Egg weight, specific gravity, conductance, and ability of Salmonella to penetrate the shell and membranes were determined for hatching eggs from a commercial broiler breeder flock. Thirty unsanitized eggs were sampled on Weeks 29, 34, 39, 42, 48, 52, and 56 of flock age for specific gravity and conductance. An additional 10 intact eggs were inoculated with Salmonella by a temperature differential immersion method for 1 min. Eggs were then emptied of contents and filled with a selective medium that allowed visualization of Salmonella growth on the inside of the shell and membrane complex. Over the 27-wk sampling period, egg weight increased from 56 to 66 g and was positively correlated with hen age (r = 0.96, P < 0.05). However, neither specific gravity (ranging from 1.077 to 1.082) nor eggshell conductance (ranging from 14.7 to 17.9 mg
EGGSHELL CHARACTERISTICS AND SALMONELLA PENETRATION
A clear understanding of the relation of hen age, shell quality, and bacterial penetration could be a valuable tool in planning intervention strategies to prevent the introduction of human pathogens to commercial hatcheries, chicks, growout farms, and processing plants. This study was designed to examine eggs from one commercial broiler breeder flock through the entire production period. Our objective was to determine the affect of flock age on eggshell quality and furthermore the ability of Salmonella typhimurium to penetrate the shell and membrane complex of intact eggs. Our goal was to determine the effect of hen age and shell quality on the likelihood of hatching eggs becoming contaminated with Salmonella postoviposition.
MATERIALS AND METHODS
Specific Gravity On each sample day, specific gravity was measured on 30 eggs. Specific gravity was calculated by the Archimedes method as described by Hempe et al. (1988). This method is based on comparing the weight of the egg to the weight of water the egg displaces. Briefly, the room temperature egg is weighed dry and then reweighed under water. Specific gravity is calculated using the following formula: Sg = ew/dww(tc), where Sg = specific gravity; ew = egg weight; dww = displaced water weight; and tc = water temperature correction found in Hempe et al. (1988).
Conductance After taking measurements for specific gravity, the same 30 eggs were used to determine conductance. Conductance was calculated from the weight loss meas-
2Difco, 3Sigma
Detroit, MI 48201. Chemical Co., St. Louis, MO 63178-9916.
ured over 2 d of egg incubation. Eggs were held in a 35 C incubator in which the temperature and humidity were recorded every 60 s. Egg weight was recorded 18 to 24 h after placement into the incubator (to allow moisture picked up during specific gravity measurement to dissipate) and again after two further consecutive 24-h periods of incubation (wgt1 and wgt2). These measurements were used to calculate conductance by the following formula: G = wgt1 – wgt2/SVP – (%RH × SVP)/ (#hours/24 h); where G = conductance; wgt1 and wgt2 = the first and second egg weights taken during incubation, respectively; SVP = saturated water vapor pressure at the average temperature at which the eggs were incubated; and %RH = average relative humidity at which the eggs were incubated (Tullet, 1981).
Penetration of Shell and Membranes by Salmonella The ability of S. typhimurium to penetrate the eggshell as well as outer and inner eggshell membranes was studied through the life of the flock. Ten eggs, separate from those used for shell analysis, were examined on each sample day. The method used was designed to measure bacterial penetration into the unbroken egg. Ten eggs were warmed to 42 C overnight and dipped into a cell suspension of a nalidixic acid resistant strain of S. typhimurium at a level of 104 cells per milliliter 1% peptone water (Berrang et al., 1991) at 25 C. The temperature differential cools the egg causing the contents to contract and the resultant negative pressure can draw bacteria into and through the shell. The eggs remained submerged in the cell suspension for 60 s were removed and allowed to remain at room temperature until dry. An agar molding technique similar to that described by Neill et al. (1985) was used to visualize S. typhimurium penetration. The narrow end of each egg was sanitized with 70% ethanol and a hole about 1 cm in diameter was aseptically cut in the shell and membranes with sterile scissors. The egg contents were drained through the hole and the egg was then filled with sterile molten 45 C Plate Count Agar2 containing 200 ppm sodium salt of nalidixic acid3 and 0.1% 2,3,5 triphenyl tetrazolium chloride (TTC).3 The addition of nalidixic acid assures that only resistant bacteria will be able to grow thus holding down competitors for the resistant S. typhimurium. The TTC is reduced by growing bacteria resulting in dark red colonies. The eggs were allowed to remain in the narrow end up position until the agar hardened, at that point the hole was sealed with molten paraffin. When the paraffin hardened, the eggs were turned over and incubated at 35 C for 24 h. Penetration was recorded as the number of red colonies visible on the molded agar when the agar filled eggs were candled after 24 and 48 h. If the egg showed no visible dots due to the reduction of TTC at 24 h, then the egg was allowed to incubate overnight again and reexamined. Any red colonies seen to be continuous with the hole were assumed to result from contamination during cutting, filling, or sealing and not counted as penetration. All agar
Downloaded from http://ps.oxfordjournals.org/ at Yale University on July 3, 2015
All eggs were collected from a commercial broiler breeder house in Northeast Georgia stocked with Hubbard hens and Petersen roosters. Eggs were separated by the producer into hatchery quality and cull groups as they appeared on the automated collection lines. Eggs for analysis were gathered at random from the hatchery quality group before application of any sanitization procedures. Eggs from the midday collection were selected to eliminate the first or last eggs in a series (normally laid early in the morning or late in the afternoon, respectively). Thus, we focused on eggs with more uniform shell quality associated with the middle of a hen’s series (Berg, 1945). Eggs were sampled from early in the productive life through the last week of production on a roughly monthly schedule. Samples were drawn on Weeks 29, 34, 39, 42, 48, 52, and 56 of flock age. Eggs were transported to the laboratory and analyzed within 2 h.
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TABLE 1. Effect of hen age on eggshell characteristics and penetration by Salmonella typhimurium through 24 h of incubation
Hen age
Egg weight1
Specific gravity1
(wk) 29 34 39 42 48 52 56
(g) 56.1 60.2 61.9 62.2 64.4 66.0 66.2
1.082 1.077 1.080 1.078 1.080 1.079 1.078
± ± ± ± ± ± ±
1.3 1.6* 1.6 1.2 1.2 1.6 1.3
± ± ± ± ± ± ±
0.002 0.002* 0.003 0.004 0.002 0.003 0.002
Conductance
No. eggs penetrated by Salmonella
Total number of penetration spots on 10 eggs (variance)
(mg/d/torr) 14.7 ± 0.8 17.1 ± 1.0* 17.9 ± 1.1 16.5 ± 0.7 17.9 ± 0.9* 16.6 ± 0.7* 16.2 ± 1.1
(#/10 eggs) 4 6 6 8 6 10 7
8 19 9 114 50 256 38
(1.51) (4.98) (0.77) (187) (115) (220) (19.9)
1Plus/minus a 95% confidence interval. *This figure represents a significant change (P ≤ 0.05) from the same measurement taken in the previous months sampling (Kolmogrov-Smirnov test).
Egg Production and Hatchability The total number of cull, nest clean, and dirty eggs per sample day (at hen age 29, 34, 39, 42, 48, 52, and 56 wk) were calculated from the farm egg classification sheets. Hatchability records were obtained from the commercial hatchery personnel. Hatch is reported as average percentage hatch of all eggs set by the hatchery from the subject farm in the week that egg samples were drawn. All eggs from this farm were subjected to eggshell sanitization in a two stage spray machine and thus both nest clean and sanitized dirty eggs are used in calculation of the hatch percentages.
Statistical Analyses Pearson correlations were done to examine the relationships between factors measured or between any one factor and hen age. F tests and analysis of variance were used to examine and compare variance values for egg penetration data. A randomized design model was used with the variation within group as the error term. The Kolmogrov Smirnov test was used to determine significance between measurement results on particular sample days. All 4Datamost
Corp., Salt Lake City, UT 84165.
statistical calculations were done using the Statmost statistical software package.4
RESULTS
Eggshell Characteristics Eggshell characteristic data are shown in Table 1. Egg weight increased 10 g as the flock aged 6 mo and egg weight was significantly correlated with hen age (r = 0.96, P = 0.0005). However, neither hen age nor egg weight was significantly correlated with specific gravity throughout flock life. Specific gravity showed no trend through the life of the flock despite an increase in egg weight. Likewise, eggshell conductance was not affected by hen age. However, a significant increase in egg weight and conductance and a significant decrease in specific gravity was noted between Weeks 29 and 34 of hen age; after that point, specific gravity remained fairly stable through the end of production. Conductance value showed another significant peak at 48 wk of flock age without a corresponding decrease in specific gravity or significant increase in egg weight. Overall, conductance did not correlate with specific gravity.
Eggshell Penetration Eggshell penetration results are presented in Table 1. The number of eggs out of 10 challenged found to show penetration in 24 h gave a general upward trend through the production period of the flock. The correlation between number of eggs penetrated in 24 h and age approaches significance (R = 0.68, P = 0.089); a larger sample size may have shown this relationship more clearly. The number of eggs showing penetration after an additional 24 h incubation (data not shown) had a lower probability of a significant correlation with hen age (P = 0.176). This result was likely a function of the fact that eggs found to be negative for penetration after the first 24 h of incubation and positive after an additional 24 h were really showing the ability of S. typhimurium located
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molded eggs showing red spots were aseptically opened; the inside of the inner membrane was swabbed at the red spots with a sterile cotton tipped applicator. The applicator was used to transfer the bacteria to a plate of BG Sulfa Agar2 with the addition of 200 ppm sodium salt of nalidixic acid3 for confirmation as nalidixic acid resistant Salmonella. Isolates from the inside of the inner membrane were further confirmed as Salmonella with Poly O serology.2 Thus penetration through the shell and associated membranes could be demonstrated. Upon completion of the 48 h incubation, any eggs still not found to show penetration with candling were aseptically opened and the agar and membranes were examined directly for signs of bacterial growth.
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EGGSHELL CHARACTERISTICS AND SALMONELLA PENETRATION TABLE 2. Egg production and hatchability across the production period of a commercial broiler breeder flock Number of eggs by category—Percentage hatch of set Week 29 34 39 42 48 52 56
Nest clean 4,482 5,022 4,968 4,536 4,428 4,050 3,456
1Percentage
Cull 512 400 332 320 310 213 350
(8.9%)1 (6.8%) (5.6%) (5.6%) (5.8%) (4.4%) (8.0%)
Dirty
Total
Percentage hatch
756 486 648 810 594 594 540
5,750 5,908 5,948 5,666 5,332 4,857 4,346
82.5 85.0 89.0 89.0 85.3 81.7 73.5
of total eggs classified as cull.
Egg Production Egg production data are presented in Table 2. The numbers of nest clean eggs suitable for the hatchery peaked at 34 wk of age (5,022) and suffered a noticeable decline through the rest of the production period. As dirty eggs were counted separately, most cull eggs in this study consisted of thin-shelled, cracked-shelled, or irregular shaped eggs. However, eggshell quality characteristics in this study were only determined on noncull eggs and as
such were not correlated with the number of cull eggs on each sample day. Like total egg numbers, the number of cull eggs was found to have a significant (P ≤ 0.05) downward trend over flock life. However, the percentage of total eggs made up by cull eggs did not decline. Except for the first and last week of production (8.9 and 8.0% respectively), the percentage of eggs characterized as cull remained fairly stable around an average of 5.6%. Percentage hatch of eggs set peaked for eggs from hens at age 39 to 42 wk and declined through later production. Although percentage hatch was not significantly correlated with hen age through the first 52 wk of flock life, it dropped steeply between 52 and 56 wk of age. A significant negative correlation (r = –0.95, P = 0.04) between hen age and percentage hatch was noted from 42 to 56 wk of age. Percentage hatch was calculated from the same class of eggs used to measure eggshell quality (noncull eggs). However, percentage hatch was not correlated with specific gravity, conductance, or egg weight.
DISCUSSION In this study, restriction-fed broiler breeders tended to lay heavier eggs as the flock aged. However, in contrast to conventional wisdom, the shells did not become significantly thinner through flock life. Instead, shell thickness and quality, as examined by specific gravity and conductance, did not correlate with hen age. In fact, the eggs tested had consistently good specific gravity at or near the industry standard of 1.080. However, it seems that a relationship does exist between egg weight, specific gravity, and conductance. At Week 34, the hens were at peak production (nest clean egg numbers) and these eggs were significantly heavier than those laid 1 mo earlier. Furthermore, these eggs had significantly thinner shells and higher conductance than eggs sampled 1 mo before. In studies in which feed was consumed ad libitum, we might expect these relationships to carry on as a trend through flock life. However, in the case of carefully managed restricted-fed broiler breeders, egg weight increase is apparently controlled. Therefore, a decline in eggshell quality is not necessarily observed through flock life.
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outside the membranes to grow through the membranes and not truly penetrating at the time of challenge. There was a general upward trend in the number of spots on the eggs penetrated by S. typhimurium as the hens aged. However, on the last week of sampling this relationship broke down; overall, there was no significant correlation (P > 0.05) between hen age and number of spots on the shells and membranes that were penetrated. Variance values for number of spots penetrated per 10 eggs are shown in Table 1. The variances were similar for these data collected in Weeks 29, 34, 39, and 56 of hen age; variance values for the same data from eggs collected in Weeks 42, 48, and 52 were higher. An analysis of variance showed that the differences in the number of spots penetrated per 10 eggs can be thought of as due to random effects in Weeks 29, 34, 39, and 56 (P > 0.05). However, the higher variance values noted in eggs from Weeks 42, 48, and 52 suggest that the number of spots penetrated in eggs from these sample days were different than numbers from the other group and that this difference is due to something other than chance. Thus these data indicate that two distinct groups of eggs were produced in the hen’s productive life relative to Salmonella penetration. However, the conductance and specific gravity did not show a noticeable change in Weeks 42, 48, and 52. Further analysis by Pearson correlation did not detect a relationship between specific gravity, conductance, or egg weight and the likelihood of S. typhimurium to penetrate the outer structures of an egg over the entire production period. Although the number of eggs penetrated (4 of 10 to 10 of 10) and the number of spots on penetrated eggs (8 to 256) varied through flock life, other than the peak at Week 34, the specific gravity and conductance did not change with any practical significance.
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REFERENCES Bennett, C. D., 1992. The influence of shell thickness on hatchability in commercial broiler breeder flocks. J. Appl. Poult. Res. 1:61–65. Berg, L. R., 1945. The relationship of clutch position and time interval between eggs to eggshell quality. Poultry Sci. 24: 555–563. Berrang, M. E., N. A. Cox, J. S. Bailey, and L. C. Blankenship, 1991. Methods for inoculation and recovery of Salmonella from chicken eggs. Poultry Sci. 70:2267–2270. Berrang, M. E., J. F. Frank, R. J. Buhr, J. S. Bailey, N. A. Cox, and J. M. Mauldin, 1997. Microbiology of sanitized broiler hatching eggs through the egg production period. J. Appl. Poult. Res. 6:298–305. Board, R. G., 1965. Bacterial growth on and penetration of the shell membranes of the hen’s egg. J. Appl. Bacteriol. 28: 197–205. Britton, W. M.. 1977. Shell membranes of eggs differing in shell quality from young and old hens. Poultry Sci. 56:647–653. Cason, J. A., N. A. Cox, and J. S. Bailey, 1994. Transmission of Salmonella typhimurium during hatching of broiler chicks. Avian Dis. 38:583–588.
Fajardo, T. A., R. C. Anantheswaran, V. M. Puri, and S. J. Knabel, 1995. Penetration of Salmonella enteritidis into eggs subjected to rapid cooling. J. Food Prot. 58:473–477. Garibaldi, J. A., and J. L. Stokes, 1958. Protective role of shell membranes in bacterial spoilage of eggs. Food Res. 23: 283–290. Hempe, J. M., R. C. Lauxen, and J. E. Savage, 1988. Rapid determination of egg weight and specific gravity using a computerized data collection system. Poultry Sci. 67: 902–907. Lifshitz, A., R. C. Baker, and H. B. Naylor, 1964. The relative importance of chicken egg exterior structures in resisting bacterial penetration. J. Food Sci. 29:94–99. McDaniel, G. R., D. A. Roland, and M. A. Coleman, 1979. The effect of egg shell quality on hatchability and embryonic mortality. Poultry Sci. 58:10–13. Nascimento, V. P., and S. E. Solomon, 1991. The transfer of bacteria (Salmonella enteritidis) across the eggshell wall of eggs classified as poor quality. Anim. Tech. 42:157–165. Neill, S. D., J. N. Campbell, and J. J. O’Brien, 1985. Egg penetration by Campylobacter jejuni. Avian Pathol. 14: 313–320. Padron, M. N., 1990. Salmonella typhimurium penetration through the eggshell of hatching eggs. Avian Dis. 34: 463–465. Peebles, E. D., and J. Brake, 1987. Eggshell quality and hatchability in broiler breeder eggs. Poultry Sci. 66: 596–604. Reinhart, B. S., and G. I. Hurnik, 1984. Traits affecting the hatching performance of commercial chicken broiler eggs. Poultry Sci. 63:240–245. Sauter, E. A., and C. F. Petersen, 1974. The effect of egg shell quality on penetration by various salmonellae. Poultry Sci. 53:2159–2162. Stokes, J .L., W. W. Osborne, and H. G. Bayne, 1956. Penetration and growth of Salmonella in shell eggs. Food Res. 21:510–518. Tullet, S. G., 1981. Theoretical and practical aspects of eggshell porosity. Turkeys 29:24–28. Vadehra, D. V., R. C. Baker, and H. B. Naylor, 1970. Infection routes of bacteria into chicken eggs. J. Food Sci. 35:61–62. 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. Wolk, J., E. H. McNally, and N. H. Spicknall, 1950. The effect of temperature on the bacterial infection of shell eggs. Food Technol. 14:316–318.
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Because only eggs with good quality shells were used in the study, no direct conclusions can be drawn about the effect of eggshell quality changes on S. typhimurium penetration. However, because shell quality measures did not change greatly over time and the S. typhimurium penetration patterns did vary, it is likely that factors other than just shell quality are involved in bacterial penetration of eggshells. Age of the hen was not directly correlated with bacterial penetration through the eggshell and membrane complex. When coupled with the fact that eggs from older hens do not necessarily have higher bacterial populations (Berrang et al., 1997), this finding would indicate that the eggs from older hens are not more likely to be contaminated postoviposition with human enteropathogens. It may be that eggshell membranes play a pivotal role in exclusion of bacteria from the inside of an egg, as suggested by some researchers (Garibaldi and Stokes, 1958; Lifshitz et al., 1964; Board, 1965; Williams, et al., 1968; Fajardo et al. 1995). Further research into the effect of age on eggshell membranes and on penetration specifically through the membranes may shed some light on the process of bacterial invasion of the intact egg.