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Research to Understand and Control Salmonella enteritidis in Chickens and Eggs
Research to Understand and Control Salmonella enteritidls in Chickens and Eggs RICHARD K. GAST and C. W. BEARD USDA, Agricultural Research Service, Southeast Poultry Research Laboratory, Athens, Georgia 30605
1993 Poultry Science 72:1157-1163
Initial reactions to the evidence that associated human illness due to SE with A large increase in the incidence of the consumption of eggs included a subhuman Salmonella enteritidis (SE) outbreaks stantial amount of disbelief. After imin the United States during the 1980s plementation of the Egg Products Inspecfocused attention on possible sources of tion Act in 1970, uncracked and sanitized infection (Centers for Disease Control, eggs had a long history of being consid1992). Epidemiological investigations im- ered safe (even if consumed uncooked). plicated the consumption of eggs or foods However, when epidemiological investicontaining eggs as responsible for a sig- gations of human SE outbreaks implicated nificant proportion of SE outbreaks (St. Grade A eggs as vehicles of disease Louis et al., 1988). The SE outbreaks in the transmission, disbelief was replaced with United Kingdom and Western Europe alarm. A similar involvement of eggs in were also attributed to contaminated eggs human SE outbreaks in the United King(Cowden et al., 1989; Perales and Au- dom resulted in a precipitous drop in egg dicana, 1989). Nearly 82% of the SE consumption that had devastating ecooutbreaks in the United States between nomic consequences for the British egg 1985 and 1989 for which a food source was industry. To address the threat to public identified were associated with eggs health posed by contaminated eggs and to (Centers for Disease Control, 1990). In protect the economic viability of the egg several instances, eggs implicated during industry, research programs were rapidly investigations of SE outbreaks were traced initiated to investigate the many unanback to infected laying flocks (Telzak et al., swered questions about SE in eggs and 1990; Mishu et al, 1991). chickens. These research efforts have been concentrated in five broadly defined areas. The first such area involves issues relating Received for publication August 4, 1992. to the dynamics of production of contamiAccepted for publication January 25, 1993. INTRODUCTION
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ABSTRACT When it became evident that the association of human Salmonella enteritidis (SE) outbreaks with the consumption of contaminated Grade A eggs posed a threat to public health and to the economic viability of the egg industry, research programs were rapidly initiated to investigate the many unanswered questions about SE in eggs and chickens. Research efforts have focused on the dynamics of deposition, survival, and growth of SE in eggs, the pathogenesis of SE in chickens, strategies for detecting SE-infected flocks, opportunities for intervening to prevent infection, the sources of SE in laying flocks, options for effectively cleaning poultry houses, and the epidemiology of SE infections of humans and chickens. This research has provided a substantially better understanding of the SE problem in poultry, but many further questions about the basis for and the prevention of eggborne transmission of SE remain to be answered. (Key words: Salmonella enteritidis, chickens, eggs, food safety, research)
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Both naturally and experimentally infected hens have been observed to lay eggs with SE in the yolk or albumen. After infection of hens of three age groups with a large oral dose of a phage Type 13a SE strain, Gast and Beard (1990a) collected and sampled more than 800 eggs. Eggs with contaminated contents were produced at a relatively high frequency by both orally inoculated and horizontally contact-exposed hens, but only during a brief period after infection (the last contaminated egg was laid 23 days postinoculation). Most eggs with contaminated contents were produced between 7 and 11 days postinoculation. The frequency of recovery of SE from eggshells was considerably lower than from yolks or albumens, suggesting that penetration of SE through the shell was not likely to have been the principal source of contamination of egg contents. In another study, Shivaprasad et al. (1990) reported the isolation of SE from eggs laid by inoculated hens only during the first 14 days after infection. Timoney et al. (1989) found SE in the contents of as many as 15% of the eggs produced on particular days during the first 2 wk postinoculation. Eggs
with contaminated contents have been produced by hens infected with as few as 103 SE cells (Humphrey et al, 1991a). Naturally infected hens have generally been observed to produce contaminated eggs at very low frequencies (Mawer et al, 1989). Humphrey et al. (1991b) isolated SE from the contents of less than 1% of over 5,700 eggs from 15 infected British commercial laying flocks. These contaminated eggs, moreover, generally contained only very small numbers of SE cells (Humphrey et al, 1991b). Evidence from experimentally infected hens has similarly indicated that contaminated eggs do not often harbor large numbers of SE (Gast and Beard, 1992b). Eggs held at a temperature able to support bacterial multiplication, however, experienced a significant increase in the numbers of SE cells in their contents (Gast and Beard, 1992b). Salmonella enteritidis has been found to multiply rapidly in eggs at temperatures as low as 10 to 15 C (Humphrey, 1990a). The multiplication of SE in eggs held at warm temperatures has been identified as an important factor in many SE outbreaks, particularly in institutional settings. Lin et al. (1988) traced an SE outbreak back to a restaurant breakfast bar. Incompletely cooked scrambled eggs were allowed to continue cooking slowly at the bar's maintenance temperature. Telzak et al. (1990) reported an instance of improper handling of eggs in a kitchen that led to a hospital outbreak. After mayonnaise was made from raw eggs, it was added to a tuna-macaroni salad and left unrefrigerated for 5 h before being served and consumed. The ability of cooking to eliminate SE from eggs and egg products has also received considerable scrutiny. This issue is of particular consequence for providing egg users with dependable instructions about the safe preparation of eggcontaining foods. Although SE phage Type 4 has been reported to be more heatresistant than some other egg-associated Salmonella (Humphrey et al, 1990), Baker (1990) observed that SE was destroyed in eggs cooked using the same time and temperature standards effective against other serotypes. Humphrey et al. (1989) found that some SE strains could survive
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nated eggs by SE-infected hens and the factors that affect survival and growth of SE in eggs. Second, several researchers have examined various questions that relate to the pathogenesis of SE in chickens. Third, a number of strategies for detecting infected flocks have been evaluated. Options for intervening to prevent SE infections are also under investigation. A fourth area of research concerns the means by which SE is introduced into and spreads throughout poultry flocks. Fifth, the regional and national epidemiology of SE in poultry has recently received intensive analysis. The objective of the present paper was to briefly summarize some of the finished and ongoing research in each of these areas. The current paper was not intended as an exhaustive literature review of all pertinent work, but rather as an interpretive overview of current research themes regarding SE in eggs and chickens.
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cooking in egg yolk by methods that were observed in mortality rates among allowed part of the yolk to remain liquid. chicks and in total egg production, Moreover, very large SE populations (such the frequency of production of SEas those in eggs stored at warm tempera- contaminated eggs, and the serum antitures for several days) were not com- body response among hens. pletely eliminated by any standard cookEfforts to explain the differences being method (Humphrey et ah, 1989). On tween SE strains (and thus the basis for SE the other hand, prior exposure to refriger- pathogenesis) have focused on several key ator temperatures increased the suscepti- issues. Lipopolysaccharide structural varibility of SE to cooking (Humphrey, 1990b). ation between SE isolates (in terms of Pasteurization under standard conditions quantitative expression of O-antigen) has (60 C for 3.5 min) can achieve the desired been found to correlate with the frequency seven-decimal reduction in SE numbers of colonization of the spleen of inoculated required for product safety (Baker, 1990). chicks (Petter, 1992). The presence of Humphrey et al. (1990) similarly con- plasmids has been the most intensively cluded that SE phage Type 4 should not investigated possible explanation for survive pasteurization. pathogenic behavior by SE strains. A 38 MDa plasmid was previously observed to be essential for the full expression of PATHOGENESIS OF SALMONELLA virulence of SE phage Type 4 in mice ENTERITIDIS IN CHICKENS (Chart et al, 1989). A plasmid of similar Although SE colonizes the intestinal size was recently identified in most SE tract of chickens, invades to reach internal isolates (of a variety of phage types) from tissues, provokes a specific antibody re- poultry and poultry nouses in Maine sponse, and is deposited in the contents of (Singer et al, 1992). eggs, no clear evidence of clinical disease An additional important issue in SE has been associated with SE infections in pathogenesis concerns possible variations the United States. Phage Type 4 infections in host susceptibility caused by exposure in the United Kingdom have been respon- to potentially immunocompromising treatsible for increased morbidity and mortal- ments. In one study, prior infection with ity in broiler chickens (Lister, 1988). The infectious bursal disease virus increased SE phage types found in the United States mortality, persistence of SE in the intestihave reduced total egg production by nal tract, and duration of SE organ experimentally infected hens (Gast and colonization in chicks subsequently inBeard, 1990a), but such an effect has not fected with SE (Opitz et al, 1990). In been commonly reported in infected com- another study, induced molting was associated with increased bowel inflammamercial flocks. Considerable variation, nonetheless, has tion and increased numbers of SE shed in been observed in the consequences of alimentary secretions after oral inoculation infections of chickens with different SE of hens (Holt and Porter, 1991). strains. Salmonella enteritidis isolates from chickens and humans have been found to DETECTION AND INTERVENTION colonize the ovaries and oviducts of Detection of SE-infected laying flocks inoculated hens more frequently than did isolates from horses or cows (C. E. Benson, has become a vital part of efforts to reduce University of Pennsylvania, Kennett the frequency of eggborne transmission of Square, PA 19348, personal communica- SE to human consumers. A testing protion, 1992). Barrow (1991) determined that gram, administered by the USDA-Animal SE phage Type 4 invaded to reach the and Plant Health Inspection Service spleen more often than did other phage (APHIS) SE Task Force, has been estabtypes. Gast and Beard (1992a) evaluated lished to evaluate the infection status of the effects of eight SE strains (representing flocks implicated by epidemiological inthree different phage types) in experimen- vestigations as the sources of eggs respontally infected chicks and laying hens. sible for human outbreaks (USDA, 1991). Significant differences between strains This program bases initial identification of
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a poultry house as infected on the isola- incidence of SE infections in chickens has tion of SE from environmental samples. been directed toward vaccination. The Culturing environmental samples col- protective efficacy of live attenuated lected by the drag-swab technique has Salmonella vaccines has been wellbeen shown to be a highly sensitive established (Barrow et al, 1991), but conindicator that a house is infected with SE cerns about safety are likely to prevent the (Opara et al, 1992). Final confirmation of use of such products in many countries infection status is provided when SE is (including the United States). Although isolated from internal organ samples. capable of achieving substantial reducSalmonella enteritidis has been observed to tions in relevant parameters, killed bacteinvade to internal organs at a high fre- rins generally provide only partial protecquency in experimentally infected hens tion against Salmonella infections. A killed (Gast and Beard, 1990c). oil-emulsion bacterin reduced (but did not A more controversial question regard- eliminate) the frequency of isolation of SE ing SE detection involves the usefulness of from internal organs and eggs of tests for specific antibodies. Detectable challenged hens (Gast et al, 1992). Addilevels of antibodies have been found at tional preliminary work has indicated that high frequencies in serum (Gast and bacterins can also reduce the numbers of Beard, 1990b) and egg yolks (Gast and SE shed in the feces of vaccinated hens Beard, 1991) of experimentally infected after oral challenge (R. K. Gast, unpubhens. Chart et al. (1990) similarly detected lished data). antibodies to SE lipopolysaccharide in a high percentage of serum samples from SOURCES AND CLEANUP OF two naturally infected flocks. In many SALMONELLA ENTERITIDIS IN cases, however, serological tests have not LAYING FLOCKS been effective for identifying infected commercial flocks, perhaps as a conseThe development of effective strategies quence of low exposure doses (Humphrey for reducing tike incidence of SE in laying et al., 1991a). Significant improvements in flocks requires some knowledge of the the sensitivity of serological methods may sources of introduction of SE into poultry be provided by recent research regarding houses. Much early speculation suggested ELISA antigens (Kim et al, 1991) and that contaminated feed was a likely culmonoclonal antibodies (Lin et al, 1992). prit. Several feed ingredients are recogAnother testing option involves cultur- nized as being frequent sources of various ing eggs. This approach has the advantage Salmonella, but no convincing evidence has of being a direct measure of the criterion emerged linking SE infections in laying of ultimate epidemiological significance, flocks to the consumption of contaminated whereas tests that measure flock infection feed. Another potential source of SE is parameters only indirectly assess the found in breeder flocks. By vertical transpotential of a flock to produce SE- mission and horizontal spread in the contaminated eggs. Small numbers of SE hatchery, Salmonella can be readily transcells, experimentally added to egg con- mitted to the progeny of an infected tents, can be efficiently recovered from breeder flock. The SE testing program pools of 10 to 30 eggs if the pools are requires the monitoring of breeder flocks incubated before culturing to allow mul- for evidence of SE infection (USDA, 1991). tiplication of SE to detectable levels (Gast, Vertical transmission (including hatchery 1993). Preliminary screening to select eggs contamination) has been identified as the with blood spots for culturing has been source of SE in only a small percentage of noted to improve the frequency of SE infected laying flocks, with a much larger isolation from eggs from implicated com- percentage accounted for by contaminamercial flocks (D. Kradel, Pennsylvania tion of the layer house environment State University, State College, PA 16803, (Opitz, 1992). personal communication, 1992). One of the major reservoirs for inThe greatest amount of interest in troduction of SE into poultry house enoptions for intervention to reduce the vironments may be infected mice. Highly
SYMPOSIUM: MICROBIAL SAFETY OF POULTRY PRODUCTS
EPIDEMIOLOGY OF SALMONELLA ENTERITIDIS IN CHICKENS The page types of SE isolates, based on lysis patterns obtained with a defined set of bacteriophages, have been evaluated as potential epidemiological markers. Although the significance of phage types to
the virulence of SE strains is not entirely clear, phage typing has been helpful for establishing clonal relationships among strains from different sources. In several investigations of human outbreaks, SE isolates from infected patients and implicated laying flocks have been found to be of the same phage type (Telzak et al, 1990; Mishu et al, 1991). In the United Kingdom, most egg-associated human SE outbreaks have involved phage Type 4 (Bartlett et al, 1989). In the United States, phage Type 8 isolates are the most prevalent from both human and poultry sources (Rodrigue et al, 1992). Singer et al. (1992) observed a consistent correlation between the phage types and plasmid profiles of SE isolates. Another important epidemiological issue concerns the regional and national distribution of SE infection among humans and laying flocks. The isolation rate of SE from humans has been high in the New England and Mid-Atlantic states since the early 1980s, although the percentage of SE outbreaks occurring in other regions has increased in recent years (Centers for Disease Control, 1992). Two surveys of SE in poultry flocks conducted by the USDA-APHIS SE Task Force have provided valuable information for understanding the regional patterns of human outbreaks. In a study that involved culturing pools of ceca collected when spent hens were slaughtered, 45% of the samples from laying houses in the Northern Region, 17% from the Central and Western Regions, and 3% from the Southeastern Region were SE-positive (Mason and Ebel, 1992). In a study of unpasteurized liquid egg material at egg-breaking plants, SE was isolated from 20% of samples from the Northern Region, 15% from the Central Region, 6% from the Southeastern Region, and 5% from the Western Region (Mason and Ebel, 1992). The overall magnitude of the eggassociated SE problem continues to provide an urgent challenge to researchers. Between January, 1990 and June, 1992, 28% of the 145 reported human SE outbreaks in the United States were attributed to the consumption of contaminated eggs (J. Mason, USDA-APHIS SE Task Force, Hyattsville, MD 20782, per-
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susceptible to SE infection, mice may serve as amplifiers by excreting very large numbers of SE in their feces. In a study of layer complexes, SE culture-positive environments could always be correlated with SE culture-positive mice (Eckroade et al, 1992). Mice infected with SE have been observed to persist through the cleaning and disinfection process in poultry houses and may be the most important factor in the spread of SE from house to house (Opitz, 1992). In a study that found SEinfected mice on all contaminated premises but not on SE-clean premises, the same phage types were isolated from mice and the environment (Henzler and Opitz, 1992). Some debate has occurred regarding the efficacy of cleaning and disinfection of poultry houses that contained SE-positive flocks. Apparent cleanup failures have been observed, in some instances possibly due to the use of pond or stream water (S. Davison, University of Pennsylvania, Kennett Square, PA 19348, personal communication, 1992). The effectiveness of cleaning and disinfection procedures has been observed to vary with the composition of the surfaces being treated. In one study, chemical and hot water disinfection effectively removed Salmonella from galvanized steel, but not from wood (El-Assaad et al, 1990). Opitz (1992) reports that an integrated program has successfully eliminated SE from previously contaminated Maine layer complexes in nearly all instances. Thorough cleaning and disinfection is applied and verified by negative culture results. A permanent rodent control system is implemented to achieve at least a 95% reduction in mice. Houses are restocked with tested SE-clean pullets from United States Sanitation Monitored parents. Replacement pullets to be housed in previously contaminated complexes are vaccinated with a killed bacterin.
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Gast, R. K., 1993. Recovery of Salmonella enteritidis from inoculated pools of egg contents. J. Food Prot. 56:21-24. Gast, R. K., and C. W. Beard, 1990a. Production of Salmonella enteritidis-contaminated eggs by experimentally infected hens. Avian Dis. 34: 438-446. Gast, R. K., and C. W. Beard, 1990b. Serological detection of experimental Salmonella enteritidis infections in laying hens. Avian Dis. 34:721-728. Gast, R. K., and C. W. Beard, 1990c. Isolation of Salmonella enteritidis from internal organs of experimentally infected hens. Avian Dis. 34: 991-993. Gast, R. K., and C. W. Beard, 1991. Detection of Salmonella serogroup D-specific antibodies in the REFERENCES yolks of eggs laid by hens infected with Salmonella enteritidis. Poultry Sci. 70:1273-1276. Baker, R. C, 1990. Survival of Salmonella enteritidis on and in shelled eggs, liquid eggs, and cooked egg Gast, R. K., and C. W. Beard, 1992a. Evaluation of a chick mortality model for predicting the conseproducts. Dairy Food Environ. Sanit. 10: quences of Salmonella enteritidis infection in 273-275. laying hens. Poultry Sci. 71:281-287. Barrow, P. A., 1991. Experimental infection of chickens with Salmonella enteritidis. Avian Gast, R. K., and C. W. Beard, 1992b. Detection and enumeration of Salmonella enteritidis in fresh and Pathol. 20:145-153. stored eggs laid by experimentally infected Barrow, P. A., M. A. Lovell, and A. Berchieri, 1991. hens. J. Food Prot. 55:152-156. The use of two live attenuated vaccines to immunize egg-laying hens against Salmonella Gast, R. K., H. D. Stone, P. S. Holt, and C. W. Beard, 1992. Evaluation of the efficacy of an oilenteritidis phage type 4. Avian Pathol. 20: emulsion bacterin for protecting chickens 681-692. against Salmonella enteritidis. Avian Dis. 36: Bartlett, C.L.R., J. R. Davies, R. J. Gilbert, C. Roberts, 992-999. B. Rowe, and J.W.G. Smith, 1989. Memorandum of evidence to the agriculture committee inquiry Henzler, D. J., and H. M. Opitz, 1992. The role of mice in the epizootiology of Salmonella enterition Salmonella in eggs. Public Health Laboratory dis infection on chicken layer farms. Avian Dis. Service Microbiol. Dig. 6:1-9. 36:625-631. Centers for Disease Control, 1990. Update: Salmonella enteritidis infections and shell eggs—United Holt, P. S., and R. E. Porter, 1991. Effects of induced molting on immunocompetence and susceptibilStates, 1990. Morbid. Mortal. Weekly Rep. 39: ity to Salmonella enteritidis (SE) infection in 909-912. laying hens. Poultry Sci. 70(Suppl. l):53.(Abstr.) Centers for Disease Control, 1992. Outbreak of Salmonella enteritidis infection associated with Humphrey, T. J., 1990a. Growth of salmonellas in intact shell eggs: influence of storage temperaconsumption of raw shell eggs, 1991. Morbid. ture. Vet. Rec. 126:292. Mortal. Weekly Rep. 41:369-372. Chart, H., B. Rowe, A BaskerviUe, and T. J. Humphrey, T. J., 1990b. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage Humphrey, 1990. Serological response of chicktemperatures before heating. J. Appl. Bacteriol. ens to Salmonella enteritidis infection. Epidemiol. 69:493-497. Infect. 104:63-71. Chart, H., E. J. Threlfall, and B. Rowe, 1989. Humphrey, T. J., A. BaskerviUe, H. Chart, B. Rowe, and A. Whitehead, 1991a. Salmonella enteritidis Virulence of Salmonella enteritidis phage type 4 is PT4 infection in specific pathogen free hens: related to the possession of a 38 MDa plasmid. influence of infecting dose. Vet. Rec. 129: Fed. Eur. Microbiol. Soc. Microbiol. Lett. 58: 482-485. 299-304. Humphrey, T. J., P. A. Chapman, B. Rowe, and R. J. Cowden, J. M., D. Lynch, C. A. Joseph, M. O'MaGilbert, 1990. A comparative study of the heat hony, S. L. Mawser, G. E. Spain, L. Ward, and B. resistance of salmonellas in homogenized whole Rowe, 1989. Case-control study of infections egg, egg yolk or albumen. Epidemiol. Infect. with Salmonella enteritidis phage type 4 in 104:237-241. England. Br. Med. J. 299:771-773. Humphrey, T. J., M. Greenwood, R. J. Gilbert, B. Eckroade, R. J., S. Davison, C. E. Benson, and E. S. Rowe, and P. A. Chapman, 1989. The survival of Bryant, 1992. Environmental contamination of salmonellas in shell eggs cooked under simupullet and layer houses with Salmonella enteritilated domestic conditions. Epidemiol. Infect. dis. Pages 14-20 in: Proceedings of the Sympo103:35-45. sium on the Diagnosis and Control of Humphrey, T. J., A. Whitehead, A.H.L. Gawler, A. Salmonella, United States Animal Health AsHenley, and B. Rowe, 1991b. Numbers of sociation, San Diego, CA. Salmonella enteritidis in the contents of naturally El-Assaad, F. G., L. E. Stewart, and L. E. Carr, 1990. contaminated hens' eggs. Epidemiol. Infect. 106: Disinfection of Poultry Transport Cages. Ameri489-496. can Society of Agricultural Engineers Annual Kim, C. J., K. V. Nagaraja, and B. S. Pomeroy, 1991. Meeting Paper No. 90-6015. Columbus, OH. Enzyme-linked immunosorbent assay for the
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sonal communication, 1992). During that period, the USDA-APHIS Task Force identified 19 SE-infected laying flocks during epidemiological traceback investigations, resulting in restrictions being imposed on the sale of 1.1 billion eggs (J. Mason, personal communication, 1992). The severe economic and public health consequences of this problem mandate further research efforts to reduce the frequency of SE contamination of eggs.
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detection of Salmonella enteritidis infection in Petter, J. G., 1992. Identification of variable LPS forms chickens. Am. J. Vet. Res. 52:1069-1074. and replication rates in Salmonella enteritidis Lin, A. W.-C, R. A. Goldsby, and G. H. Snoeyenbos, avirulent and virulent field isolates. Page 55 in: 1992. Salmonella enteritidis-specific monoclonal Proceedings of the American Society for antibodies. Avian Dis. 36:455-458. Microbiology Annual Meeting, New Orleans, Lin, F.-Y. C, J. G. Morris, Jr., D. Trump, D. Tilghman, LA.(Abstr.) P. K. Wood, N. Jackman, E. Israel, and J. P. Rodrigue, D. C, D. N. Cameron, N. D. Puhr, F. W. Libonati, 1988. Investigation of an outbreak of Brenner, M. E. St. Louis, I. K. Wachsmuth, and Salmonella enteritidis gastroenteritis associated R. V. Tauxe, 1992. Comparison of plasmid with consumption of eggs in a restaurant chain profiles, phage types, and antimicrobial resisin Maryland. Am. J. Epidemiol. 128:839-844. tance patterns of Salmonella enteritidis isolates in Lister, S. A., 1988. Salmonella enteritidis infection in the United States. J. Clin. Microbiol. 30:854-857. broilers and broiler breeders. Vet. Rec. 123:350. Mason, J., and E. Ebel, 1992. APHIS Salmonella Shivaprasad, H. L., J. F. Timoney, S. Morales, B. Lucio, and R. C. Baker, 1990. Pathogenesis of enteritidis control program. Pages 78-109 in: Salmonella enteritidis infection in laying chickProceedings of the Symposium on the Diagnosis ens. I. Studies on egg transmission, clinical and Control of Salmonella. United States Anisigns, fecal shedding, and serologic responses. mal Health Association, San Diego, CA. Avian Dis. 34:548-557. Mawer, S. L., G. E. Spain, and B. Rowe, 1989. Salmonella enteritidis phage type 4 and hens' Singer, J. T., H. M. Opitz, M. Gershman, M. M. Hall, I. G. Muniz, and S. V. Rao, 1992. Molecular eggs. Lancet 1:280-281. characterization of Salmonella enteritidis isoMishu, B., P. M. Griffin, R. V. Tauxe, D. N. Cameron, lates from Maine poultry and poultry farm R. H. Hutcheson, and W. Schaffner, 1991. Salmonella enteritidis gastroenteritis transmitted environments. Avian Dis. 36:324-333. by intact chicken eggs. Ann. Intern. Med. 115: St. Louis, M. E., D. L. Morse, M. E. Potter, T. M. 190-194. DeMelfi, J. J. Guzewich, R. V. Tauxe, and P. A. Opara, O. O., E. T. Mallinson, C. R. Tate, L. E. Carr, Blake, 1988. The emergence of grade A eggs as a R. G. Miller, L. Stewart, C. Kelleher, R. W. major source of Salmonella enteritidis infecJohnson, and S. W. Joseph, 1992. The effect of tions. New implications for the control of exposure, storage times, and types of holding salmonellosis. J. Am. Med. Assoc. 259: media on the drag-swab monitoring technique 2103-2107. for Salmonella. Avian Dis. 36:63-68. Telzak, E. E., L. D. Budnick, M.S.Z. Greenberg, S. Opitz, H. M., 1992. Progress being made in Blum, M. Shayegani, C. E. Benson, and S. Salmonella enteritidis reduction on the farm. Schultz, 1990. A nosocomial outbreak of Poult. Dig. (Mar.):16-22. Salmonella enteritidis infection due to the conOpitz, H. M., R. A. Phillips, and D. Beane, 1990. sumption of raw eggs. N. Engl. J. Med. 323: Persistence and transovarian transmission of 394-397. phage type 8 Salmonella enteritidis in normal and IBDV-infected chickens. Page 108 in: Proceed- Timoney, J. F., H. L. Shavaprasad, R. C. Baker, and B. Rowe, 1989. Egg transmission after infection of ings of the American Veterinary Medical Ashens with Salmonella enteritidis phage type 4. sociation Annual Meeting, San Antonio, Vet. Rec. 125:600-601. TX.(Abstr.) Perales, I., and A. Audicana, 1989. The role of hens' United States Department of Agriculture, 1991. Rules and regulations: chickens affected by Salmonella eggs in outbreaks of salmonellosis in north enteritidis. Fed. Reg. 56:3730-3743. Spain. Int. J. Food Microbiol. 8:175-180.
1993 Poultry Science 72:1157-1163
Initial reactions to the evidence that associated human illness due to SE with A large increase in the incidence of the consumption of eggs included a subhuman Salmonella enteritidis (SE) outbreaks stantial amount of disbelief. After imin the United States during the 1980s plementation of the Egg Products Inspecfocused attention on possible sources of tion Act in 1970, uncracked and sanitized infection (Centers for Disease Control, eggs had a long history of being consid1992). Epidemiological investigations im- ered safe (even if consumed uncooked). plicated the consumption of eggs or foods However, when epidemiological investicontaining eggs as responsible for a sig- gations of human SE outbreaks implicated nificant proportion of SE outbreaks (St. Grade A eggs as vehicles of disease Louis et al., 1988). The SE outbreaks in the transmission, disbelief was replaced with United Kingdom and Western Europe alarm. A similar involvement of eggs in were also attributed to contaminated eggs human SE outbreaks in the United King(Cowden et al., 1989; Perales and Au- dom resulted in a precipitous drop in egg dicana, 1989). Nearly 82% of the SE consumption that had devastating ecooutbreaks in the United States between nomic consequences for the British egg 1985 and 1989 for which a food source was industry. To address the threat to public identified were associated with eggs health posed by contaminated eggs and to (Centers for Disease Control, 1990). In protect the economic viability of the egg several instances, eggs implicated during industry, research programs were rapidly investigations of SE outbreaks were traced initiated to investigate the many unanback to infected laying flocks (Telzak et al., swered questions about SE in eggs and 1990; Mishu et al, 1991). chickens. These research efforts have been concentrated in five broadly defined areas. The first such area involves issues relating Received for publication August 4, 1992. to the dynamics of production of contamiAccepted for publication January 25, 1993. INTRODUCTION
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ABSTRACT When it became evident that the association of human Salmonella enteritidis (SE) outbreaks with the consumption of contaminated Grade A eggs posed a threat to public health and to the economic viability of the egg industry, research programs were rapidly initiated to investigate the many unanswered questions about SE in eggs and chickens. Research efforts have focused on the dynamics of deposition, survival, and growth of SE in eggs, the pathogenesis of SE in chickens, strategies for detecting SE-infected flocks, opportunities for intervening to prevent infection, the sources of SE in laying flocks, options for effectively cleaning poultry houses, and the epidemiology of SE infections of humans and chickens. This research has provided a substantially better understanding of the SE problem in poultry, but many further questions about the basis for and the prevention of eggborne transmission of SE remain to be answered. (Key words: Salmonella enteritidis, chickens, eggs, food safety, research)
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Both naturally and experimentally infected hens have been observed to lay eggs with SE in the yolk or albumen. After infection of hens of three age groups with a large oral dose of a phage Type 13a SE strain, Gast and Beard (1990a) collected and sampled more than 800 eggs. Eggs with contaminated contents were produced at a relatively high frequency by both orally inoculated and horizontally contact-exposed hens, but only during a brief period after infection (the last contaminated egg was laid 23 days postinoculation). Most eggs with contaminated contents were produced between 7 and 11 days postinoculation. The frequency of recovery of SE from eggshells was considerably lower than from yolks or albumens, suggesting that penetration of SE through the shell was not likely to have been the principal source of contamination of egg contents. In another study, Shivaprasad et al. (1990) reported the isolation of SE from eggs laid by inoculated hens only during the first 14 days after infection. Timoney et al. (1989) found SE in the contents of as many as 15% of the eggs produced on particular days during the first 2 wk postinoculation. Eggs
with contaminated contents have been produced by hens infected with as few as 103 SE cells (Humphrey et al, 1991a). Naturally infected hens have generally been observed to produce contaminated eggs at very low frequencies (Mawer et al, 1989). Humphrey et al. (1991b) isolated SE from the contents of less than 1% of over 5,700 eggs from 15 infected British commercial laying flocks. These contaminated eggs, moreover, generally contained only very small numbers of SE cells (Humphrey et al, 1991b). Evidence from experimentally infected hens has similarly indicated that contaminated eggs do not often harbor large numbers of SE (Gast and Beard, 1992b). Eggs held at a temperature able to support bacterial multiplication, however, experienced a significant increase in the numbers of SE cells in their contents (Gast and Beard, 1992b). Salmonella enteritidis has been found to multiply rapidly in eggs at temperatures as low as 10 to 15 C (Humphrey, 1990a). The multiplication of SE in eggs held at warm temperatures has been identified as an important factor in many SE outbreaks, particularly in institutional settings. Lin et al. (1988) traced an SE outbreak back to a restaurant breakfast bar. Incompletely cooked scrambled eggs were allowed to continue cooking slowly at the bar's maintenance temperature. Telzak et al. (1990) reported an instance of improper handling of eggs in a kitchen that led to a hospital outbreak. After mayonnaise was made from raw eggs, it was added to a tuna-macaroni salad and left unrefrigerated for 5 h before being served and consumed. The ability of cooking to eliminate SE from eggs and egg products has also received considerable scrutiny. This issue is of particular consequence for providing egg users with dependable instructions about the safe preparation of eggcontaining foods. Although SE phage Type 4 has been reported to be more heatresistant than some other egg-associated Salmonella (Humphrey et al, 1990), Baker (1990) observed that SE was destroyed in eggs cooked using the same time and temperature standards effective against other serotypes. Humphrey et al. (1989) found that some SE strains could survive
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nated eggs by SE-infected hens and the factors that affect survival and growth of SE in eggs. Second, several researchers have examined various questions that relate to the pathogenesis of SE in chickens. Third, a number of strategies for detecting infected flocks have been evaluated. Options for intervening to prevent SE infections are also under investigation. A fourth area of research concerns the means by which SE is introduced into and spreads throughout poultry flocks. Fifth, the regional and national epidemiology of SE in poultry has recently received intensive analysis. The objective of the present paper was to briefly summarize some of the finished and ongoing research in each of these areas. The current paper was not intended as an exhaustive literature review of all pertinent work, but rather as an interpretive overview of current research themes regarding SE in eggs and chickens.
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cooking in egg yolk by methods that were observed in mortality rates among allowed part of the yolk to remain liquid. chicks and in total egg production, Moreover, very large SE populations (such the frequency of production of SEas those in eggs stored at warm tempera- contaminated eggs, and the serum antitures for several days) were not com- body response among hens. pletely eliminated by any standard cookEfforts to explain the differences being method (Humphrey et ah, 1989). On tween SE strains (and thus the basis for SE the other hand, prior exposure to refriger- pathogenesis) have focused on several key ator temperatures increased the suscepti- issues. Lipopolysaccharide structural varibility of SE to cooking (Humphrey, 1990b). ation between SE isolates (in terms of Pasteurization under standard conditions quantitative expression of O-antigen) has (60 C for 3.5 min) can achieve the desired been found to correlate with the frequency seven-decimal reduction in SE numbers of colonization of the spleen of inoculated required for product safety (Baker, 1990). chicks (Petter, 1992). The presence of Humphrey et al. (1990) similarly con- plasmids has been the most intensively cluded that SE phage Type 4 should not investigated possible explanation for survive pasteurization. pathogenic behavior by SE strains. A 38 MDa plasmid was previously observed to be essential for the full expression of PATHOGENESIS OF SALMONELLA virulence of SE phage Type 4 in mice ENTERITIDIS IN CHICKENS (Chart et al, 1989). A plasmid of similar Although SE colonizes the intestinal size was recently identified in most SE tract of chickens, invades to reach internal isolates (of a variety of phage types) from tissues, provokes a specific antibody re- poultry and poultry nouses in Maine sponse, and is deposited in the contents of (Singer et al, 1992). eggs, no clear evidence of clinical disease An additional important issue in SE has been associated with SE infections in pathogenesis concerns possible variations the United States. Phage Type 4 infections in host susceptibility caused by exposure in the United Kingdom have been respon- to potentially immunocompromising treatsible for increased morbidity and mortal- ments. In one study, prior infection with ity in broiler chickens (Lister, 1988). The infectious bursal disease virus increased SE phage types found in the United States mortality, persistence of SE in the intestihave reduced total egg production by nal tract, and duration of SE organ experimentally infected hens (Gast and colonization in chicks subsequently inBeard, 1990a), but such an effect has not fected with SE (Opitz et al, 1990). In been commonly reported in infected com- another study, induced molting was associated with increased bowel inflammamercial flocks. Considerable variation, nonetheless, has tion and increased numbers of SE shed in been observed in the consequences of alimentary secretions after oral inoculation infections of chickens with different SE of hens (Holt and Porter, 1991). strains. Salmonella enteritidis isolates from chickens and humans have been found to DETECTION AND INTERVENTION colonize the ovaries and oviducts of Detection of SE-infected laying flocks inoculated hens more frequently than did isolates from horses or cows (C. E. Benson, has become a vital part of efforts to reduce University of Pennsylvania, Kennett the frequency of eggborne transmission of Square, PA 19348, personal communica- SE to human consumers. A testing protion, 1992). Barrow (1991) determined that gram, administered by the USDA-Animal SE phage Type 4 invaded to reach the and Plant Health Inspection Service spleen more often than did other phage (APHIS) SE Task Force, has been estabtypes. Gast and Beard (1992a) evaluated lished to evaluate the infection status of the effects of eight SE strains (representing flocks implicated by epidemiological inthree different phage types) in experimen- vestigations as the sources of eggs respontally infected chicks and laying hens. sible for human outbreaks (USDA, 1991). Significant differences between strains This program bases initial identification of
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a poultry house as infected on the isola- incidence of SE infections in chickens has tion of SE from environmental samples. been directed toward vaccination. The Culturing environmental samples col- protective efficacy of live attenuated lected by the drag-swab technique has Salmonella vaccines has been wellbeen shown to be a highly sensitive established (Barrow et al, 1991), but conindicator that a house is infected with SE cerns about safety are likely to prevent the (Opara et al, 1992). Final confirmation of use of such products in many countries infection status is provided when SE is (including the United States). Although isolated from internal organ samples. capable of achieving substantial reducSalmonella enteritidis has been observed to tions in relevant parameters, killed bacteinvade to internal organs at a high fre- rins generally provide only partial protecquency in experimentally infected hens tion against Salmonella infections. A killed (Gast and Beard, 1990c). oil-emulsion bacterin reduced (but did not A more controversial question regard- eliminate) the frequency of isolation of SE ing SE detection involves the usefulness of from internal organs and eggs of tests for specific antibodies. Detectable challenged hens (Gast et al, 1992). Addilevels of antibodies have been found at tional preliminary work has indicated that high frequencies in serum (Gast and bacterins can also reduce the numbers of Beard, 1990b) and egg yolks (Gast and SE shed in the feces of vaccinated hens Beard, 1991) of experimentally infected after oral challenge (R. K. Gast, unpubhens. Chart et al. (1990) similarly detected lished data). antibodies to SE lipopolysaccharide in a high percentage of serum samples from SOURCES AND CLEANUP OF two naturally infected flocks. In many SALMONELLA ENTERITIDIS IN cases, however, serological tests have not LAYING FLOCKS been effective for identifying infected commercial flocks, perhaps as a conseThe development of effective strategies quence of low exposure doses (Humphrey for reducing tike incidence of SE in laying et al., 1991a). Significant improvements in flocks requires some knowledge of the the sensitivity of serological methods may sources of introduction of SE into poultry be provided by recent research regarding houses. Much early speculation suggested ELISA antigens (Kim et al, 1991) and that contaminated feed was a likely culmonoclonal antibodies (Lin et al, 1992). prit. Several feed ingredients are recogAnother testing option involves cultur- nized as being frequent sources of various ing eggs. This approach has the advantage Salmonella, but no convincing evidence has of being a direct measure of the criterion emerged linking SE infections in laying of ultimate epidemiological significance, flocks to the consumption of contaminated whereas tests that measure flock infection feed. Another potential source of SE is parameters only indirectly assess the found in breeder flocks. By vertical transpotential of a flock to produce SE- mission and horizontal spread in the contaminated eggs. Small numbers of SE hatchery, Salmonella can be readily transcells, experimentally added to egg con- mitted to the progeny of an infected tents, can be efficiently recovered from breeder flock. The SE testing program pools of 10 to 30 eggs if the pools are requires the monitoring of breeder flocks incubated before culturing to allow mul- for evidence of SE infection (USDA, 1991). tiplication of SE to detectable levels (Gast, Vertical transmission (including hatchery 1993). Preliminary screening to select eggs contamination) has been identified as the with blood spots for culturing has been source of SE in only a small percentage of noted to improve the frequency of SE infected laying flocks, with a much larger isolation from eggs from implicated com- percentage accounted for by contaminamercial flocks (D. Kradel, Pennsylvania tion of the layer house environment State University, State College, PA 16803, (Opitz, 1992). personal communication, 1992). One of the major reservoirs for inThe greatest amount of interest in troduction of SE into poultry house enoptions for intervention to reduce the vironments may be infected mice. Highly
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EPIDEMIOLOGY OF SALMONELLA ENTERITIDIS IN CHICKENS The page types of SE isolates, based on lysis patterns obtained with a defined set of bacteriophages, have been evaluated as potential epidemiological markers. Although the significance of phage types to
the virulence of SE strains is not entirely clear, phage typing has been helpful for establishing clonal relationships among strains from different sources. In several investigations of human outbreaks, SE isolates from infected patients and implicated laying flocks have been found to be of the same phage type (Telzak et al, 1990; Mishu et al, 1991). In the United Kingdom, most egg-associated human SE outbreaks have involved phage Type 4 (Bartlett et al, 1989). In the United States, phage Type 8 isolates are the most prevalent from both human and poultry sources (Rodrigue et al, 1992). Singer et al. (1992) observed a consistent correlation between the phage types and plasmid profiles of SE isolates. Another important epidemiological issue concerns the regional and national distribution of SE infection among humans and laying flocks. The isolation rate of SE from humans has been high in the New England and Mid-Atlantic states since the early 1980s, although the percentage of SE outbreaks occurring in other regions has increased in recent years (Centers for Disease Control, 1992). Two surveys of SE in poultry flocks conducted by the USDA-APHIS SE Task Force have provided valuable information for understanding the regional patterns of human outbreaks. In a study that involved culturing pools of ceca collected when spent hens were slaughtered, 45% of the samples from laying houses in the Northern Region, 17% from the Central and Western Regions, and 3% from the Southeastern Region were SE-positive (Mason and Ebel, 1992). In a study of unpasteurized liquid egg material at egg-breaking plants, SE was isolated from 20% of samples from the Northern Region, 15% from the Central Region, 6% from the Southeastern Region, and 5% from the Western Region (Mason and Ebel, 1992). The overall magnitude of the eggassociated SE problem continues to provide an urgent challenge to researchers. Between January, 1990 and June, 1992, 28% of the 145 reported human SE outbreaks in the United States were attributed to the consumption of contaminated eggs (J. Mason, USDA-APHIS SE Task Force, Hyattsville, MD 20782, per-
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susceptible to SE infection, mice may serve as amplifiers by excreting very large numbers of SE in their feces. In a study of layer complexes, SE culture-positive environments could always be correlated with SE culture-positive mice (Eckroade et al, 1992). Mice infected with SE have been observed to persist through the cleaning and disinfection process in poultry houses and may be the most important factor in the spread of SE from house to house (Opitz, 1992). In a study that found SEinfected mice on all contaminated premises but not on SE-clean premises, the same phage types were isolated from mice and the environment (Henzler and Opitz, 1992). Some debate has occurred regarding the efficacy of cleaning and disinfection of poultry houses that contained SE-positive flocks. Apparent cleanup failures have been observed, in some instances possibly due to the use of pond or stream water (S. Davison, University of Pennsylvania, Kennett Square, PA 19348, personal communication, 1992). The effectiveness of cleaning and disinfection procedures has been observed to vary with the composition of the surfaces being treated. In one study, chemical and hot water disinfection effectively removed Salmonella from galvanized steel, but not from wood (El-Assaad et al, 1990). Opitz (1992) reports that an integrated program has successfully eliminated SE from previously contaminated Maine layer complexes in nearly all instances. Thorough cleaning and disinfection is applied and verified by negative culture results. A permanent rodent control system is implemented to achieve at least a 95% reduction in mice. Houses are restocked with tested SE-clean pullets from United States Sanitation Monitored parents. Replacement pullets to be housed in previously contaminated complexes are vaccinated with a killed bacterin.
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Gast, R. K., 1993. Recovery of Salmonella enteritidis from inoculated pools of egg contents. J. Food Prot. 56:21-24. Gast, R. K., and C. W. Beard, 1990a. Production of Salmonella enteritidis-contaminated eggs by experimentally infected hens. Avian Dis. 34: 438-446. Gast, R. K., and C. W. Beard, 1990b. Serological detection of experimental Salmonella enteritidis infections in laying hens. Avian Dis. 34:721-728. Gast, R. K., and C. W. Beard, 1990c. Isolation of Salmonella enteritidis from internal organs of experimentally infected hens. Avian Dis. 34: 991-993. Gast, R. K., and C. W. Beard, 1991. Detection of Salmonella serogroup D-specific antibodies in the REFERENCES yolks of eggs laid by hens infected with Salmonella enteritidis. Poultry Sci. 70:1273-1276. Baker, R. C, 1990. Survival of Salmonella enteritidis on and in shelled eggs, liquid eggs, and cooked egg Gast, R. K., and C. W. Beard, 1992a. Evaluation of a chick mortality model for predicting the conseproducts. Dairy Food Environ. Sanit. 10: quences of Salmonella enteritidis infection in 273-275. laying hens. Poultry Sci. 71:281-287. Barrow, P. A., 1991. Experimental infection of chickens with Salmonella enteritidis. Avian Gast, R. K., and C. W. Beard, 1992b. Detection and enumeration of Salmonella enteritidis in fresh and Pathol. 20:145-153. stored eggs laid by experimentally infected Barrow, P. A., M. A. Lovell, and A. Berchieri, 1991. hens. J. Food Prot. 55:152-156. The use of two live attenuated vaccines to immunize egg-laying hens against Salmonella Gast, R. K., H. D. Stone, P. S. Holt, and C. W. Beard, 1992. Evaluation of the efficacy of an oilenteritidis phage type 4. Avian Pathol. 20: emulsion bacterin for protecting chickens 681-692. against Salmonella enteritidis. Avian Dis. 36: Bartlett, C.L.R., J. R. Davies, R. J. Gilbert, C. Roberts, 992-999. B. Rowe, and J.W.G. Smith, 1989. Memorandum of evidence to the agriculture committee inquiry Henzler, D. J., and H. M. Opitz, 1992. The role of mice in the epizootiology of Salmonella enterition Salmonella in eggs. Public Health Laboratory dis infection on chicken layer farms. Avian Dis. Service Microbiol. Dig. 6:1-9. 36:625-631. Centers for Disease Control, 1990. Update: Salmonella enteritidis infections and shell eggs—United Holt, P. S., and R. E. Porter, 1991. Effects of induced molting on immunocompetence and susceptibilStates, 1990. Morbid. Mortal. Weekly Rep. 39: ity to Salmonella enteritidis (SE) infection in 909-912. laying hens. Poultry Sci. 70(Suppl. l):53.(Abstr.) Centers for Disease Control, 1992. Outbreak of Salmonella enteritidis infection associated with Humphrey, T. J., 1990a. Growth of salmonellas in intact shell eggs: influence of storage temperaconsumption of raw shell eggs, 1991. Morbid. ture. Vet. Rec. 126:292. Mortal. Weekly Rep. 41:369-372. Chart, H., B. Rowe, A BaskerviUe, and T. J. Humphrey, T. J., 1990b. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage Humphrey, 1990. Serological response of chicktemperatures before heating. J. Appl. Bacteriol. ens to Salmonella enteritidis infection. Epidemiol. 69:493-497. Infect. 104:63-71. Chart, H., E. J. Threlfall, and B. Rowe, 1989. Humphrey, T. J., A. BaskerviUe, H. Chart, B. Rowe, and A. Whitehead, 1991a. Salmonella enteritidis Virulence of Salmonella enteritidis phage type 4 is PT4 infection in specific pathogen free hens: related to the possession of a 38 MDa plasmid. influence of infecting dose. Vet. Rec. 129: Fed. Eur. Microbiol. Soc. Microbiol. Lett. 58: 482-485. 299-304. Humphrey, T. J., P. A. Chapman, B. Rowe, and R. J. Cowden, J. M., D. Lynch, C. A. Joseph, M. O'MaGilbert, 1990. A comparative study of the heat hony, S. L. Mawser, G. E. Spain, L. Ward, and B. resistance of salmonellas in homogenized whole Rowe, 1989. Case-control study of infections egg, egg yolk or albumen. Epidemiol. Infect. with Salmonella enteritidis phage type 4 in 104:237-241. England. Br. Med. J. 299:771-773. Humphrey, T. J., M. Greenwood, R. J. Gilbert, B. Eckroade, R. J., S. Davison, C. E. Benson, and E. S. Rowe, and P. A. Chapman, 1989. The survival of Bryant, 1992. Environmental contamination of salmonellas in shell eggs cooked under simupullet and layer houses with Salmonella enteritilated domestic conditions. Epidemiol. Infect. dis. Pages 14-20 in: Proceedings of the Sympo103:35-45. sium on the Diagnosis and Control of Humphrey, T. J., A. Whitehead, A.H.L. Gawler, A. Salmonella, United States Animal Health AsHenley, and B. Rowe, 1991b. Numbers of sociation, San Diego, CA. Salmonella enteritidis in the contents of naturally El-Assaad, F. G., L. E. Stewart, and L. E. Carr, 1990. contaminated hens' eggs. Epidemiol. Infect. 106: Disinfection of Poultry Transport Cages. Ameri489-496. can Society of Agricultural Engineers Annual Kim, C. J., K. V. Nagaraja, and B. S. Pomeroy, 1991. Meeting Paper No. 90-6015. Columbus, OH. Enzyme-linked immunosorbent assay for the
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sonal communication, 1992). During that period, the USDA-APHIS Task Force identified 19 SE-infected laying flocks during epidemiological traceback investigations, resulting in restrictions being imposed on the sale of 1.1 billion eggs (J. Mason, personal communication, 1992). The severe economic and public health consequences of this problem mandate further research efforts to reduce the frequency of SE contamination of eggs.
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