Bacterial Penetration of the Eggshell and Shell Membranes of the Hatching Chicken Eggs_Berrang 1999

8/8/2019 Bacterial Penetration of the Eggshell and Shell Membranes of the Hatching Chicken Eggs_Berrang 1999

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01999Applied Poultry Science,Inc

BACTERIALENETRATION OF THEEGGSHELLND SHELL EMBRANESFTHE CHICKENATCHINGGG:A REVIEW

M. E. BEFUNGIUSDA,ARS, PMQR U, Russell Research Center, Athens, G A 3060 4567 7Phone: (706)546-3551FAX;. 706 ) 546-3633E-mail: m [email protected]

N . A. COXUSDA,ARS, PM SRU , Russell Research Center, Athen s, G A 30604-5677J. E FRANKUniversityof Georgia, Food Science and Technology,Athens, GA 30602R.J. BUHRUSDA,ARS, PPM QRU , Russell Research Center, Athen s, GA 306045677

Primary Audience: Researchers, Hatchery Managers

DESCRIPTIONF PROBLEMBacteria including the human pathogen

salmonellae can readily penetrate the shelland membranes of an intact hatching egg. Theresult of this penetration is contamination notonly of the embryo within but also of manyother chicks during hatch in the commercial

hatching cabinet. Such contamination can becarried onto the grow-out farm and pose asignificant food safety hazard. Understandingthe manner in which bacteria can penetratethe shell is important if producers hope tointervene. This information may be of use tohatchery managers, breeder managers, andproducers in their efforts to control the

1 Towhom correspondence should be addressed


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500 BACTERIAL PENETRATION OF EGGSSalmonella problem in the commercial hatch-ery and its product, the chick.

How BACTERIAPENETRATEGGSHELLSAND MEMBRANESBacteria can penetrate the outer struc-tures of an egg. Once within the egg, bacteria

can cause depression of hatchability and/orcontamination of the chick. Contamination ofthe chick with a human enteropathogen hasimportant food safety implications.MECHANISM FOR TRANS-SHELLPENETRATIONEggshells can be penetrated by bacteriawhen water or some other Liquid is present,especially if there is a temperature differentialbetween the egg and the liquid. When an eggis laid,it is warmer than the environment, sincethe body temperature of the hen is 42C. Thewarm egg cools to environmental tempera-ture, and this cooling causes the contents ofthe egg to contract. Such contraction tends toform a negative pressure within the egg.Bacteria present in the environment or on theegg surface can then be pulled into andthrough the eggshell and its membranes [l,21.This method of trans-shell contaminationhas been confirmed through years of research.Early research showed that application of amechanical vacuum to simulate a negativepressure could cause penetration of a partialeggshell [3]. Later experiments proved thatimmersion of an egg into a cool cell suspen-sion led to penetration of the intact egg [41.W-s et al. [5] found that eggs are pene-trated almost immediately when challengedwith moist Salmonella-contaminated chickenfeces. They [5 ] further noted that shell thick-ness did not have a significant effect on bac-terial penetration, but the presence of cuticleplugging the shell pores is more important.Also of interest was their observation thatbacterial motility is not related to ability topenetrate.AREA OF EGG MOST LIKELY TO BEPENETRATEDBy covering areas of the egg with moltenwax,Vadehra et al. [6] ested which area ismost likely to be penetrated by Pseudomonasaeruginosa, a common egg spoilage organism.

They found that the blunt oraircell end ismostprone to penetration when challenged by atemperature differential immersion. Interest-ingly, this is the only area where the inner andouter shell membranes do not remain in closecontact, forming the air cell which may re-spond more rapidly to a change in tempera-ture than the rest of the egg contents.PRODUCTION STAGES WHERE ANEGG MAY BE PENETRATEDSince moisture and presence of bacteriaare needed to allow penetration, any stage ofproduction where these two factors may bepresent provides an opportunity for bacterialinvasion. When the factor of a positive temper-ature differential (egg warmer than the envi-ronment) is added, there is an increasedpossibility of bacterial attack. Padron [ foundthat when eggs were placed on Satmonella-contaminated nest box shavings for 10 min,the eggshell and membranes were penetratedby that Salmonella organism in 59% of thesamples.In a field survey, Smeltzer etal. foundthat eggs laid on the dirty chicken house floorwere more likely to exhibit internal bacterialcontamination than were eggs laid in a nestbox [8]. Eggs are most vulnerable to bacterialpenetration in the first 30 to 60 sec after laybefore the cuticle hardens and effectively capsthe pores.Other stages of egg production andhandling can be implicated in bacterial pene-tration. After collection, eggs are usuallyplaced in a cold room at the farm until hatch-ery personnel transport them to the hatchery.When cool eggs are removed from the farm ona warm day, they may "sweat"due to conden-sation of moisture from the air.Despite thelack of a positive temperature differential, thepresence of moisture on the egg surface mayprovide opportunity for contamination andcauses concern among many researchers asreviewed by Bruce and Drysdale [2].

METHODSSED TOMEASUREACTERIALPENETRATIONMany articles have been publishedon themeasurement of bacterial invasion of eggs.However, several stand ou t as real contribu-tions in the area of how best to assess thepenetration of intact eggs or membranes.


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Review ArticleBERRANG et al. 501EGGSHELL AND MEMBRANESWilliams and Whittemore [9] reported anexcellent test for bacterial invasion under sim-ulated fecal contamination conditions. Thismethod essentially involves gluing a shortaluminum tube to the area of interest andfllinp it with sterile chicken feces. The fecescan then be seeded with a known bacteria.Penetration is demonstrated by culturallyretrieving the microorganism from within theegg. The egg is emptied and the inside of theshell (or shell and membranes, depending onthe interest of the researcher) is sampled witha swab. This method is excellent for examina-tion of specific regions of the shell. However,if one is interested in the entire egg or largenumbers of samples, this method becomescumbersome.

Board and Board [ lo] developed amethod whereby the entire egg surface canbe quickIy and easily tested for bacteria1 pen-etration. The intact egg is first subjected to apositive temperature differential challenge byimmersion in a cool bacterial cell suspension.After drying, the egg is emptied of contentsand fdled with microbiological growth me-dium with the addition of tetrazolium. Uponreduction by bacterial growth, tetrazolium ischemically changed to formazon, which is adeep red color. After the growth media hashardened, the egg is sealed with paraffin andincubated to allow for bacterial growth. Theresulting red spots appear through the shellusing a standard portable candler. This samemethod has been adapted for use to show thatCampylobacter ejuni can penetrate the outerstructures of an egg [l l] .Another method developed to test in-tact eggs for penetration uses a luminescentstrain of Salmonella enleritkiis (121. In thismethod the egg is inoculated by immersion ina culture of luminescent S. enteritidis. Theegg is then sealed in a plastic bag with theluciferase substrate decanal. Luminescencecould then be detected through the shell withan imaging system. This method is exciting andshows promise because the egg does notneed to be opened or cracked during thepenetration test. However, it does requiresome specialized expertise in the manufac-ture, handling, and detection of luminescentcultures.AS an alternative to using bacterial cul-tures, Kim and Slavik [13] developed an indi-

cator test whereby blue lake dye was appliedto eggs. Penetration was assessed by detectionof blue spots on the membranes below thesurface of the shell. The dye penetrationmethod correlated well with eggshell penetra-tion by Salmonella.MEMBRANE ALONEThe literature does not contain manyreferences to measurement of bacterialpenetration of membranes alone. Lifshitzetal.[14] were able to test penetration of thesestructures by emptying the egg, removing theshell from the wide end of the egg, and leavingthe membrane intact. The egg was then setinto a bacterial cell suspension and the inte-rior was filled with sterile broth. Periodicculture of the broth from inside the shellwas used to determine the extent of bacterialpenetration.A more practical and repeatable methodwas originally developed to test the membraneas a barrier to radioactive amino acids. Themembrane can be placed across the openingin a ball and socket ground glass connection.The radioactive amino acid is placed in theupper tube with the ball part of the connectionand assayed in the lower socket section [15].This type of method was modified by Berranget ul. [16]. An apparatus was fashioned withPVC pipe and a union, whereby eggshellmembrane was used to cover the only openingbetween an upper and lower chamber. Whena cell suspension of Salmonella typhimuriumwas placed in the upper chamber, penetrationcould be demonstrated by using a syringeand needle to aseptically draw samples fromthe bottom chamber through a sealed rubberseptum.Wong et ul. [17] developed a method toexamine eggshell membrane microscopicallyfollowing positive temperature differentialimmersion of an egg in a Salmonella cell sus-pension. A confocal laser scanning micro-scope was used to visualize Salmonella withinthe meshwork of the outer membrane. With aSalmonella-specific antibody conjugated to acolor-producing chemical, this method couldallow detection of penetration without theneed for culturing.


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502 BACTERIAL, PENETRATION OF EGGS

THEEGGS EFENSEAGAINSTACTERIALINVASIONDespite the fact that bacteria can pene-

trate the shell and membrane, the egg is notwithout defense against these invaders.PHYSICAL DEFENSESThe outermost physical defense that abacterial cell encounters on an egg is the cuti-cle. The cuticle is a very effective barrier towater and carbon black particles[MI.espitethe fact that the cuticle allows gas passage, itseems to effectively fill the pores of the egg-shell [Z]. However, this defense is not perfect.A small percentage of eggs are laid withoutcuticle; these eggs may easily be contaminatedby water and carbon black [MI. Even whencuticle is present, for the first few minutesafter lay it is an ineffective barrier to bacterialinvasion until it hardens [19]. Nevertheless,the hardened cuticle is the major barrier toliquid and therefore is of utmost importanceinbacterial exclusion [19].However, the eggshell is an ineffectivebarrier to bacterial penetration. The poresare wide enough to allow entry. Eggshellscharacterized to be of excellent quality (fromeggs with specificgravityabove 1.090 and pre-sumably thicker shell than poor quality eggs)were found to be more resistant to penetrationby salmonellae [20], possibly because of thelonger time needed for bacteria to movethrough a thick shell.Eggshell membranes do not have an in-herent anti-bacterial property and can bepenetrated by bacteria. However, the mem-branes do add some protective value to theshell alone, improving the ability to hold outbacteria over the short term [21]. The timeneeded for bacteria to penetrate the combinedinner and outer eggshell membranes is notclearly related to the amount of open spacebetween fibers in the outer surface of the outermembrane [16]. When comparing the shell,inner, and outer membranes for ability to pre-vent bacterial entry, the inner membrane is themost effective [14] because of the tightermeshwork of the inner membrane relative tothe outer membrane.

CHEMICAL DEFENSESThe eggs albumen is a substance unin-viting to most bacteria. The pH of egg albumenis about 7.6 when laid. This pH rises to about9.5 during storage, unfavorable for microbialgrowth [22,23]. Conalbumin, an iron-bindingagent is also present in the albumen. Unlessthis chemical can be quenched with iron, therewill not be enough free iron available to allowmicrobial growth in the egg [22]. Furtherprotection is afforded the egg by the actionof lysozyme which can lead to the ruptureof procaryotic cell walls [22]. The typical in-cubator temperature also seems to increasethe antibacterial effect of egg albumen [24].Nevertheless, in an in-vitro test Pseudomonasputida cells could move through the albumen;those reaching the surface of the yolk wereable to reproduce quickly, leading to a gener-alized infection of the egg [l].While bacterial cells that contact the al-bumen early may be killed, the area withinthe shell and membranes can provide a safeniche. Bacteria can remain in the membranes,which have been shown to select for gramnegative over gram positive bacteria [19].When the chick hatches, these bacteria areingested or can cause cross contamination.Thus, in the hatching egg industry the chemicaldefenses of the egg are not totally effective.Also, in a fertile egg, the chemistry changes atabout the 7th day of embryonic developmentand is no longer as hostile to bacteria such asSalmonella [25].

RESULTFBACTERIALNVASIONFHATCHINGGGSIn the past much of the concern regardingbacterial infection of hatching eggs resultedfrom the possibility of decreased hatch orrotting eggs in the incubators. Current con-cern has shifted to the food safety aspects ofbacterial invasion. Human enteropathogen

invasion of hatching eggs is a real problem.Salmonellae have been isolated from hatchingeggs in the field [26,27, 28, 291. The trans-mission of Salmonella through an integratedpoultry company has been documented.Bainset al. [30]details the chain of Salmonella frombreeder feed to the nest box, the hatchery,onto the grow-out farm, and to dressed car-casses. Broiler hatcheries have been shown to


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Review irticleBERRANG et al.be reservoirs forSalmonella. In one study71%of eggshell fragment samples were contami-nated [27]. In another study involving sixcommercial breeder hatcheries, 15.2% ofthe eggshell fragments were contaminatedwith salmonellae[a].almonella contamina-tion of eggs can lead to extensive cross con-tamination of chicks in the hatching cabinet[31, 32, 331. In a 1996 study [34] Salmonellawere introduced into the day-of-hatch chickthrough an assortment of body openings(mouth, cloaca, eye, nasal passage, and navel).All of these routes produced birds contami-nated with Salmonella. These data emphasizethe need to control salmonellae in breeder

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flocks, hatching cabinets, hatchery environ-ments, and broiler houses to minimize theproduction of seeder chicks. Salmonella iso-lates from the hatchery have been found tooccasionally make their way not only to thegrow-out farm but also to the processing plantand the final product [35].In order to produce chicken and chickenproducts free of human bacterial pathogens,the source of the pathogens needs to be inter-rupted. The hatching egg and the hatchery arevery important control points for introductionof some human pathogens such as Salmonellainto the growing chicken and ultimately ontothe final product [36,37,38].

CONCLUSIONSNDAPPLICATIONS1. Bacteria, including salmonellae, can penetrate eggshells and associated membranes.Penetration can be detected and measured and may lead to cross contamination of manychicks in the hatchery. This situation can cause a food safety problem when the contami-nation is carried onto the farm and the processing plant.2. The natural defenses of the egg are not entirely adequate to prevent penetration andsuMval of salmonellae.3. Likelihood of penetration can be lessened by avoiding contact between the egg andcontaminated surfacesor substances such as feces or dirty nest pads. Also, excess moisture(other than properly applied disinfectant solutions) on the eggs must be avoided, especiallyat times of positive temperature differential (egg warmer than the environment).

REFERENCESNDNOTES1. Lock, J.L,J. Dolman, and R.G. Board, 1992. Ob-senations on the mode of bacterial infection of henseggs. FEMS Microbiol. Letters 100:71-74.2. Bruce, J. and E M . Drysdale, 1994. Trans-shelltransmission. Pages 63-91 in: Microbiology of the AvianEgg. 1st Edition. RG. Board and R.Fuller, ed. Chapmanand Hall, London, England.3. Haines, R B . a n d T. Moran, 1940. Porosity of, andbacterial invasion through, the shell of the hens egg.J. Hyg. 4 0 4 5 H 6 1 .4. Stokes, J.L,W.W.Osborne, an dH.G. Bayne, 1956.Penetration and growthof Salmonella in shell eggs. FoodRes. 1:510-518.5 .WUliams, J.E,LH . Dillard, and G.O. Hall, 1968.The penetration patterns of Salmonella the ou ter structures of chicken e6. Vadehra, D.V., RC . Baker, and H.B. Naylor, 1970.Infection routes of bacteria into chicken eggs.J. Food Sci.3561-62.7. Padron, M.N., 1990.--en-etration through the eggshell of hatching eggs. Avian Dis.34A63465.8. Smeltzer, T.I., K. Orange, B. Peel, and 6 . Runge,1979. Bacterial penetration in floor and nest box eggsfrom meat and layer birds. Au t . Vet.J. 55592-593.

9. Williams, J.E. and AD . Whittemore, 1967. Amethod for studying microbial penetration thrououter structu res of the avian egg. AvianDis. l l :46f%%10. Board, P A an d R.G. Board, 1967. A method ofstudying bacterial penetration of the shell of the hens egg.Lab. Prac. 16:472473,482.11.Neill, S.D.,J.N.Campbe bandJJ. Brien, 1985.Egg penetration by m. vian Path.14:313-320.12. Chen , J. , R.C. Clarke, and M .W. Grimths, 1996.

monito r contamination and survival in eggs. J. FoodProt. 59:915-921.13. Kim, J.W. and M.F. Slavik, 1996. Use of blue lakeasa n indicatorof bacterial penetration into eggs.J. RapidMethods Automat. Microbiol.4 1 8 S 1 9 0 .14. Lifshitz A, R.C. Baker, and H .B. Naylor, 1964.The relative importance of chickene exteriorstructuresin resisting bacterial penetration. J.Food Sci.29:94-99.15. Wedral, EM., D.V. Vadehra, and RC. Baker,1971. Mechanism of bacterial penetration through theeggsof -&. 2. Effect of penetration andgrowthon permeability of inner shell membrane. J. Food Sci.36520-522.16. Berrang, M E , J.F. Frank, RJ.Buhr, J.S. Bailey,an d N . h Cox, 1999. Eggshell membrane structure and

. . .Use of luminescent strains of Salmonella ententldls to


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ship, and RL Wilson, 1991. Research note: Extent ofsalmonellae contaminationin breeder hatcheries. PoultrjSci. 7041641 8.

29. Blankenship. LC., J.S. Bailey, N.A. Cox, N.J.Stern, R. Brewer, and 0.Williams, 1993. Two-step mu-cosal competitive exclusion flora treatment to diminishsalmonellae in commercial broiler chickens. Poultry Sci.72: 1667-1672.30. Bains, B.S. an dM.A. MacKenzie, 1974. Transmis-sion of Salmonella hrough an integrated poultry organi-zation. Poultry Sci. 53:1114-1118.31. Bailey, J.S., N.A. Cox, and M.E Berrang, 1994.Hatchery acquired salmonellae in broiler chicks. PoultrySci. 73:1153-1157.32. Cason,J.A., N . k C0q.andJ.S. Bailey, 1994.Trans-during hatching ofission of Salnlpnellabroiler chicks. Avlan D%%??%33. Bailey, J.S., RJ. Buhr, N.A. Cox, and M.EBerrang, 1996. Effect of hatcher cabinet sanitizationtreatments on -cross contamination and hatch-ability of broiler eggs.Poultry Sci. 75191-196.34. Cox, N.A., J.S. Bailey, and M . E Berrang, 1996.Alternative routes forSalmonella ntestinal tract coloni-zation of chick. J. Appl. Poultry Res. 5:282-288.35. Lahellec, C. and P. Colin, 1985. Relationship be-tween serotypes of salmonellae from hatcheries and rear-ing farms and those from processed poultry carcases.Br. Poultry Sci. 26:179-186.36. Cox, N.A., J.S. Bailey, and M.E Berrang, 1994.Attempts to break the salmonellae in oultchemically treating the freshly laid egg. Ag e 3 7 6 % k 284th General Meeting, American Society of Microbiol-ogy, Las Vegas, NV.37.Cox, SA. nd J.S. Bailey, 1995. Reducin bacteriafrom farm through processing. Pages 28-31 in: #roc.30thNatl. Meeting on Poultly Health and Processing,OceanCity. MD.38. Cox, N.A., J.S. Bailey, and NJ. Stern, 1995. Re-search efforts to intervene in the Salmonellacolonization

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Bacterial Penetration of the Eggshell and Shell Membranes of the Hatching Chicken Eggs_Berrang 1999

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