Microbiological Investigations of Bacteria Infecting Yolks of Broiler and Broiler Breeder Embryos and Hatchlings

Abstract

Enterococcus cecorum has been identified as causing early sepsis in commercial broilers. We hypothesized that vertical transmission may contribute to the spread of the pathogen. Sampling of 360 commercial broiler eggs from a previously infected flock detected Enterococcus gallinarum and Enterococcus faecalis in yolks of 18-day incubated eggs. Enterococcus avium was recovered by enrichment from 1 of 31 unincubated yolks. Direct sampling without enrichment of 297 egg yolks from a previously infected broiler breeder flock detected E. faecalis, Escherichia coli, Globicatella sanguinis, and E. gallinarum, often in high numbers, or in coinfections. Microbiological sampling of day-of-hatch chicks suggested only the yolk sac was likely to yield bacterial growth, and that yolks could be sampled directly using swabs streaked onto growth medium. The most common isolate from sampling 30 day-of-hatch broiler chicks was E. faecalis from 16 different chicks. Further, E. coli was recovered as a mixed infection with E. faecalis in 3 of those 16 residual yolk sacs. From swab-sampling of residual yolk sacs from 40 day-of-hatch broiler breeder chicks, E. faecalis was recovered from 7 chicks. Thus, yolk infections by Enterococcus species were the primary isolates cultured from commercial broilers and broiler breeders in Arkansas. Identification of these species at high numbers in yolks is likely contributing to reduced hatchability, early chick death, and transmission of bacteria to subsequent flocks. Therefore, the broiler industry needs to pursue methods to identify infected laying hens and management strategies to mitigate vertical transmission. Importance: We were unable to identify E. cecorum being spread from flock to flock via vertical transmission. We did identify specific bacterial species infecting and reproducing within the yolks of developing embryos and day-old chicks of commercial, meat-type chickens. These particular species were detected at low and high levels, indicating growth within the yolk. The implication is that these species colonized the yolk within the hen’s reproductive tract and were vertically transmitted to the chicks and subsequent flocks. Some infections represent only a single species, while others involve two species that may represent cooperative infections. With the removal of the use of antibiotic growth promoters during early development, these species may be reducing chicken reproduction through embryo death or early chick mortality.

1. Introduction

The ever-increasing demand for chicken meat and eggs has necessitated modern poultry farming to meet the growing needs of consumers. While chicken breeding has undoubtedly enhanced chicken production, it also presents challenges in terms of animal health and welfare [1]. One of these challenges lies in the incidence of embryonic mortality during the incubation period, which can result in significant economic losses [2]. This early-stage mortality can be attributed to a range of infectious and non-infectious agents [2,3,4,5,6,7] that reduce hatchability. In 2023, the USDA National Agricultural Statistics Service reported a recent 3% decline in the hatchability of broiler chicks in the United States [8], highlighting hatchability as an urgent concern.

In 1914, Rettger described yolk infections in embryos from flocks that had experienced outbreaks of bacillary white diarrhea (a.k.a. Pullorum) disease caused by Bacterium pullorum (since renamed Salmonella serovar Gallinarum biovar Pullorum) [4]. Further, he identified the same bacterium in the ovaries of hens from flocks that had outbreaks. Thus, he demonstrated vertical transmission from hen to chicks through infected ovary, to infected yolk, to infected chick, and thus the spread of bacterial infections from one flock to another through contaminated eggs. In 1992, surveys of Salmonella sero-positive layer hens detected only 2 out of 300 hens with Salmonella in their ovaries or oviduct and 0.06% Salmonella-positive eggs [9]. A different survey from a human infection outbreak found 0.03–0.9% Salmonella-contaminated layer eggs [10]. However, further work has suggested that the primary vehicle for vertical transmission of Salmonella appears to be through the albumen [11,12,13,14,15,16,17]. Campylobacter has been cultured from the reproductive tract of >60 week-old hens from flocks that are fecal-positive for Campylobacter [18]. However, Campylobacter has been primarily cultured from egg shells and hatchery waste (reviewed in [19]), not yolk. Studies have reported contamination of eggs by Salmonella serovar Enteriditis during egg formation in the reproductive tract [11,13,20]. Further studies have suggested that Salmonella present in the oviduct could contaminate the albumen, inner and outer shell membranes, or the eggshell [13,14,15], implicating the albumen as the most probable site of contamination. Others found the yolk to be the most frequently contaminated, suggesting that the ovary is the first site of bacterial colonization [4,11,13,16,17,21,22]. A wide range of aerobic bacterial genera have been isolated from non-viable chicken embryos, including Escherichia, Micrococcus, Salmonella and Staphylococcus [3,23]. Avian pathogenic Escherichia coli presents a range of infections, including yolk sac infections, omphalitis, respiratory infections, swollen head syndrome, pericarditis, airsacculitis, perihepatitis, arthritis, osteomyelitis, septicemia, and cellulitis [24]. Farm surveys of omphalitis and residual yolk sac infections in Bangladesh, Egypt and Ethiopia identified E. coli, Salmonella spp., and Staphylococcus aureus as the most frequently recovered bacteria contributing to a concern for early chick death [25,26,27,28]. Enterococcus species, which are known commensals in the gastrointestinal tracts of chickens, have also been associated with a range of pathological issues in broiler chickens [29,30,31,32,33,34,35]. Notably, Enterococcus cecorum has been linked to recently emerging and economically significant infectious diseases in the poultry industry, including sepsis in young chicks as well as osteomyelitis and spondylitis in older chickens [31,34,36,37,38,39,40]. Given the recent emergence of E. cecorum sepsis outbreaks in young chicks, this new disease manifestation is likely an outcome of the removal of routine feed additions of antibiotic growth promoters (AGPs) during early growth of broiler chicks, with E. cecorum adapting through mutation to colonize a newly available niche [39].

Therefore, the objective of this study was to identify the specific bacteria associated with yolk infections in embryos and newly hatched chicks of commercial broilers and broiler breeders. Our initial goal was to determine whether E. cecorum was being passed from flock to flock through vertical transmission through the egg. Although we did not find E. cecorum in eggs from flocks that had experienced previous E. cecorum sepsis outbreaks, we did identify other Enterococcus species infecting yolks, often at very high levels. Our results, therefore, represent a new survey of fertile broiler breeder and broiler eggs since the discontinuation of AGP use during early growth. Although our survey was limited to the area around Northwest Arkansas, the results are cautionary for other broiler commercial operations and may relate to recent declines in hatchability [8].

2. Materials and Methods

2.1. Media

Media and supplements included CHROMagar Orientation (DRG International, Springfield Township, NJ, USA), tryptic soy broth and bacteriological agar (Difco, Becton, Dickinson and Company, Franklin Lakes, NJ, USA), and chicken serum (Life Technologies, Gaithersburg, MD, USA). Media abbreviations: CO—CHROMagar Orientation; TSA—tryptic soy agar; TSB—tryptic soy broth; TSBcs—TSB + 2% chicken serum; TSAcs—TSA + 2% chicken serum. Note: We have found some Enterococcus isolates that require chicken serum for continuous passage and better growth performance.

2.2. Bacterial Cultures

Bacterial isolates were archived in 40% glycerol at −80 °C. Working stocks were passaged monthly on TSA or TSAcs slants stored at 4 °C.

2.3. Hatchery and Breeder Flock Specifics

Broiler and broiler breeder fertile eggs or day-of-hatch chicks were provided by commercial broiler or breeder companies operating in the Northwest Arkansas region. Company names, specific hatcheries, and details on parent flocks were withheld, subject to Non-Disclosure Agreements.

2.4. Embryo Microbial Sampling

Fertilized broiler eggs (n = 360) were procured from a commercial hatchery in Arkansas in July 2022, while fertilized broiler breeder eggs (n = 360) were procured from a primary breeder in September 2022. The eggs were washed in a dilute dish soap 2% bleach mixture and then thoroughly rinsed with tap water. Eggs were stored for up to one week at 18°C at 60% humidity.

For egg yolk enrichment cultures, 15 mL of TSBcs was added to a sterile 100 mL wide-mouth media bottle. Unincubated eggs were cleaned, their exteriors rinsed with 95% ethanol, and they were air-dried. Using ethanol-rinsed, gloved hands, eggs were gently cracked, and as much of the egg albumen as possible was discarded. The yolk was then transferred to the media bottle, vigorously mixed to break the yolk, and incubated overnight at 37 °C with continuous shaking. The next day, a sterile swab was used to streak the yolk mixture onto a CO plate, which was incubated overnight at 37 °C.

For embryo sampling, eggs were set in groups of 90 eggs on successive days. Incubation was carried out in an auto-rotated incubator (NartureForm Hatchery Technologies LLC, Jacksonville, FL, USA) maintained at 37.2 °C and 60% relative humidity. On the 18th day of incubation, the eggs were candled, their surfaces sterilized with 95% ethanol, and they were allowed to air-dry in a laminar flow hood. Using ethanol-rinsed, gloved hands, each egg was then broken into a sterile (rinsed with 70% ethanol and oven-dried) plastic weigh boat. Embryos were scored as dead/sick-looking embryos, live embryos, or non-fertile/early-dead. The embryos were euthanized by cervical dislocation with scissors. Using sterile forceps and scissors, the abdominal cavity of each embryo was opened. For liver sampling, a portion of the liver was excised and rubbed over the surface of a CO plate. For yolk sac sampling, the membrane was cut and the contents sampled by insertion of a sterile cotton swab, which was then streaked over the surface of a CO plate. Plates were incubated overnight at 37 °C under 5% CO₂.

2.5. Microbial Sampling of Day-of-Hatch Chicks

Day-of-hatch broiler chicks (n = 30) were acquired from a local commercial hatchery in September 2024. Day-of-hatch broiler breeder chicks (n = 40) were acquired from a commercial hatchery in January 2025. Scissors were used for cervical dislocation and removal of the head, wings and legs, then the dermis was peeled off. Carcasses were either sampled immediately or placed individually in plastic zip-lock bags and frozen at −80°C until thawed for microbiological evaluation. For sampling, the carcass was sprayed with 95% ethanol, then placed in a sterile weigh boat in a laminar flow hood to dry. Using sterilized forceps and scissors, the abdomen was opened. Sampling was performed either through the collection of tissue samples with sterile scissors and forceps or with sterile cotton swabs. Tissue samples were transferred into 5 mL TSB, incubated with shaking overnight, and then streaked with a loop onto CO plates for individual colonies. Swabs of internal surfaces or residual yolk sac contents were spread directly onto CO plates that were incubated at 37 °C overnight for colony identification.

2.6. Evaluation of Microbial Sampling

In all cases, CO plates were evaluated after incubation overnight. Representative colonies were purified for single colony isolation by restreaking on CO plates for verification of uniform colony color and morphology.

2.7. Paramagnetic Bead DNA Extraction for PCR

DNA from cultures were extracted by lysis with NaOH and DNA capture with magnetic beads based on published procedures [41], using either: (1) a sterile toothpick to sample individual colonies into 90 µL of sterile deionized water in a 1.5 mL microfuge tube, or (2) 20 µL of an overnight culture in TSB pelleted in a 1.5 mL microfuge tube and resuspended in 90 µL of sterile deionized water. Cells were lysed by the addition of 10 µL of 1 M NaOH, and the mixture was incubated at room temperature for 10 min. Silica-coated paramagnetic beads (5 µL; PureSil-Silica beads, BioChain Institute Inc., Newark, CA, USA) were added, along with 100 µL of bead-binding buffer. The resulting mixture was vortexed, then incubated for 10 min at room temperature. The paramagnetic beads were subsequently captured using a magnetic rack. The supernatant was carefully pipetted off and discarded. Following this, the captured beads were released from the rack and washed with 150 µL of 70% ethanol, followed by magnetic capture and solution discarded—this rinse step was performed twice. Microfuge tubes were opened, and the beads were allowed to air-dry for 6 min. Subsequently, 50 µL of Te (10 mM TrisCl, 0.1 mM EDTA, pH 7.5) was added to the beads and incubated at room temperature for 5 min. The tubes were returned to the magnetic rack to capture the beads, and the eluate was collected into a new 1.5 mL microfuge tube for subsequent qPCR analysis.

2.8. Bacterial Isolate Species Identification

The Illumina 16S metagenome primers, which amplify the V3-V4 region of the 16S rDNA gene, F: 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACCGGGNGGCWGCAG; R: 5′-;GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC, were synthesized by Integrated DNA Technologies (Coralville, IA, USA). PCR (20 µL) included 2 µL of 10× Taq buffer (500 mM TrisCl pH 8.3, 10 mM MgCl₂, 3 mg/mL bovine serum albumin), 0.2 µL of 20 mM dNTPs, 0.2 µL of 50 µM primers, 1 µL of 20× EvaGreen^® Dye (Biotium Inc., Fremont, CA, USA), and 0.1 µL of 40 U/µL Taq Polymerase. Triplicate reactions were performed in 96-well plates and analyzed using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.,Hercules, CA, USA). Cycling parameters were an initial denaturation step at 90 °C for 45 s, followed by 5 cycles of denaturation at 90 °C for 15 s, annealing at 71.5 °C for 15 s, and extension at 72 °C for 60 s. Afterward, 35 cycles were performed with identical parameters, with plate reads following each cycle. Subsequently, a High-Resolution Melt (HRM) analysis was carried out, which entailed incubation at 72 °C for 120 s, 90 °C for 60 s, and 65 °C for 120 s. The melt gradient was from 70 °C to 90 °C in increments of 0.1 °C for 5 s with a plate read.

The amplified PCR products were purified with RapidTip^® (Chiral Technologies, West Chester, PA, USA) and shipped to Eurofins Genomics LLC (Louisville, KY, USA) for capillary sequencing. Sequence data were inspected and edited using SeqBuilder (LaserGene v 17.3, DNAStar, Madison, WI, USA) and subsequently used for BLASTn (v 2.17.0) searches at NCBI of the bacterial 16S rDNA database.

3. Results

3.1. Egg Microbiology

This project was initiated to screen for vertical transmission of E. cecorum, which has recently been reported as causing early sepsis onset in broiler breeders and broilers [39,40,42]. We obtained fertile eggs from two specific flocks that had recovered from E. cecorum outbreaks in 2022. The first evaluation was for fertile broiler breeder eggs (from a broiler breeder parent flock). The second evaluation was for fertile broiler eggs (from a broiler breeder flock).

We first investigated microbial contamination of unincubated eggs through direct enrichment culture of whole yolks. As a positive control we spiked an additional yolk with circa 10³ CFU of E. cecorum 1415 [39,43], which, after the overnight enrichment and streaking onto a CO plate, produced a uniform population representative of E. cecorum (i.e., very small white colonies with green halo on CO plates). For the 31 yolks examined by culture enrichment, 30 were negative for growth, while one yolk produced a uniform culture identified by partial 16S sequencing as Enterococcus avium.

We incubated 297 broiler breeder eggs for 18 days. Candling revealed 21 as nonfertile or early-dead, so these early-deads were sampled by swab-inoculation for yolk sac only. One swab-inoculation produced a lawn of very small green colonies evaluated as Globicatella sanguinis. One swab-inoculation produced a single colony of Enterococcus faecalis. One swab-inoculation produced a green lawn of Enterococcus gallinarum. For the developed eggs, 37 embryos were evaluated as dead or stressed and were sampled for liver and yolk sac microbiology. One yolk swab-inoculation produced a lawn of E. gallinarum and one a lawn of E. faecalis. There were no colonies from any of the liver tissue samples, even for the two embryos with lawns of E. gallinarum or E. faecalis from yolk swabs. Therefore, the bacterial growth in the yolk had apparently not translocated into the blood system and organs. For 239 normal embryos, all were sampled for yolk sac microbiology, with 175 sampled for liver tissue microbiology. One liver sample produced a single colony determined to be Streptococcus salivarius. Two yolk sac samples produced single colonies determined to be Lactobacillus cremoris, and a Bacillus species (licheniformis or safensis). These single colony samples were presumed to likely represent random contaminations during the isolation/sampling procedures. However, one yolk swab-inoculation produced 10 colonies determined to be E. gallinarum.

For the second evaluation of broiler eggs, we incubated 360 eggs for 18 days, and only yolk sac microbiology was investigated using swab-inoculations. We scored 22 eggs as nonfertile or early-dead, with one producing a mixed lawn of green colonies of E. faecalis and pink colonies of Escherichia coli. The 36 yolks swab-inoculations from stressed or possibly dead embryos produced no colonies. For the 302 live embryos’ yolk swab-inoculations, one gave a green lawn of E. faecalis, one gave a single colony of Streptococcus gordonii, and one gave four colonies of Staphylococcus pasteuri.

3.2. Day-of-Hatch Microbiology

In September 2024, broiler chicks were obtained from a commercial hatchery on day-of-hatch, with no provision of water or food. First, we sampled eight for intestine (circa 1 cM of small intestine) and residual yolk sac (circa 1 cM² of yolk sac membrane with adherent yolk material), which were pooled for enrichment through overnight growth in TSB (see Section 2). Single colony streaking of the overnight broth culture determined that there was limited microbiological diversity. One chick produced a uniform culture of Staphylococcus warneri (white colonies), three produced uniform cultures of E. faecalis (green colonies), two produced mixed cultures of S. warneri and E. faecalis, one produced a mixed culture of E. faecalis and E. coli (purple colonies), and one a mixed culture of E. coli (both white colonies and purple colonies) and E. faecalis (green colonies). The second round of sampling was for four carcasses using direct swab streaking on CO plates for peritoneal fluid, and residual yolk swabs, each onto duplicate plates with one incubated at ambient CO₂ and one at 5% CO₂. Additionally, small intestine tissue and residual yolk sac were sampled into TSB for broth overnight enrichment. Three of four small intestine TSB enrichments were turbid; all four residual yolk sac TSB enrichments were turbid. The enrichment cultures were not further evaluated, as the direct swabs were determined to be more reliable for detecting significant bacterial presence. Peritoneal swabs for two of four embryos produced single green colonies, which were not evaluated further. Residual yolk sac direct swabs onto CO plates were the same whether incubated at ambient or 5% CO_2, where one chick gave no colonies, one gave a single green colony, one gave seven green colonies determined to be E. faecalis and one gave a mixed lawn of white colonies of E. coli and green colonies of E. faecalis. We then evaluated the 18 remaining carcasses only by residual yolk sac swabs directly spread on CO plates that were incubated at ambient CO₂. Eight of the 18 gave only green colonies diagnosed as E. faecalis: three produced single colonies, one produced two colonies, one produced three colonies, one produced five colonies, one produced sixteen colonies, and one produced a lawn.

In January of 2025, surplus male chicks from a broiler breeder female line were similarly obtained on day-of-hatch, with no provision of food or water. Microbiology sampling was assessed as before, but without carcass freezing and only direct swab sampling of the residual yolk sac streaked on CO plates. Seven of 40 dissected chicks produced colonies. Six chicks produced only green colonies with counts of 1, 3, 6, 8, 15, and 120, where representative colonies were diagnosed as E. faecalis based on 16S sequencing. The seventh chick yielded 45 green colonies and 7 purple colonies diagnosed as E. faecalis and E. coli, respectively.

4. Discussion

Concerns in evaluating microbiological sampling of eggs, embryos and chicks include random contaminants from the instruments or investigators, especially when using broth enrichment methods. Our initial methods (direct yolk cultures) were designed to screen for E. cecorum, where we had verified that the yolk culture conditions were suitable for recovery of E. cecorum. Out of 31 yolks, we failed to find any E. cecorum but did recover E. avium from one yolk. We then sampled an additional 297 eggs from the same set of eggs after 18 days of incubation by direct sampling onto CO plates. From 21 infertile or early-dead yolks, we recovered three species: G. sanguinis, E. faecalis, and E. gallinarum. Although the E. faecalis was only a single colony and thus could be a contaminant, the other two were lawns, indicating a high level of growth for these two species, likely contributing to the failure of the embryo to develop. From 37 developed embryos evaluated as stressed or recently dead, we recovered a lawn of E. gallinarum from one embryo and a lawn of E. faecalis from another yolk sample, but no colonies from liver tissue samples. From 239 live embryos, we sampled 175 livers and got only a single colony of S. salivarius from one liver, which we suspect is a random contaminant. From the 239 live embryos, only one yolk sample produced colonies, which were 10 colonies of E. gallinarum. Thus, we conclude that there is sufficient evidence for yolk infections by E. gallinarum and E. faecalis. Clearly, one yolk was infected with G. sanguinis, since it produced a lawn from direct swab sampling without enrichment. Finding this species is surprising since it has been primarily associated with rare human infections [44,45] and has not been previously associated with chickens. Based on 16S rDNA, this species is closely related to Streptococcus and Enterococcus [46,47] but is distinguishable by specific phenotypes and at the genome sequence level. Full genome sequencing is necessary to verify the species identification for this isolate and determine its relationship to human isolates.

Microbiological surveys of yolk sacs from 360 18-day broiler embryos produced To Numerous to Count (TNTC) growth in a mixed infection of E. faecalis and E. coli in one nonfertile or early-dead egg. There was also TNTC growth of E. faecalis in the yolk of one live embryo. Therefore, our investigations of embryos from fertile eggs determined that severe yolk infections may affect 1 to 2% of eggs, and some of these infections may affect embryos that would be included in hatching baskets or that might even produce live chicks at hatch. Thus, there is substantial evidence that yolks can be the vector for vertical transmission of pathogenic bacteria, and thus the source for early colonization of other hatched chicks.

Our microbiological sampling of multiple tissues from 12 day-of-hatch broiler chicks from each colony revealed a low diversity of bacterial species. We suspect that the isolates of S. warneri could be contaminants in the enrichment growth. However, E. faecalis and E. coli were identified at significant levels without enrichment. We also identified a co-infection by E. faecalis and E. coli in a non-fertile or early-dead broiler yolk from an 18-day embryo. Mixed infections of embryos have been previously reported. In a Canadian study of early-dead embryos, E. faecalis predominated, with E. coli being the second most prevalent, but more than half the E. coli cases were co-infected with E. faecalis [37]. E. faecalis and E. coli have been previously associated with high levels of embryo mortality in chickens [48]. Further, E. faecalis is commonly co-isolated from chicken colibacillosis mortalities [35]. E. faecalis has been reported to be one of the first to colonize the gastrointestinal tract of poultry [49]. Therefore, the identification of E. faecalis in the majority of infections is in line with past findings, and we also see a probable synergism between E. faecalis and E. coli in infections of embryos, yolks, and day-old chicks’ residual yolk sacs.

Staphylococcus, Enterococcus, Enterobacter, and Escherichia have all been previously cultured from embryos and day-old chicks [3,6,50,51,52]. First Week Mortality (FWM) of broiler chicks on Dutch farms differed significantly between chicks originating from eggs of different breeder flocks and correlated with breeder age, egg storage length, season, and feed company for the breeders [2]. All of these could affect breeder hen colonization by opportunistic bacterial pathogens. E. coli has been commonly associated with yolk sac infections and subclinical or acute septicemia, contributing to increased FWM in broilers [53]. Others have reported Enterococcal infections in non-viable chicken embryos [37]. Different lines of research have suggested Salmonella colonization of eggs could occur through the egg shell post-ovulation or in the ovary in the reproductive tract (reviewed in [11]). Our data are most consistent with bacterial contamination of the yolk, most likely from the breeder hens. Depending on the level of the initial infection, this could lead to early death of the embryo, FWM, or even persistent infection of some chicks after the first week. Although we initiated these experiments to pursue the vertical transmission of E. cecorum from breeders to broilers, the fact that we recovered three other species of Enterococci suggests that this same vertical transmission could vector E. cecorum, albeit at lower levels than the numbers of eggs examined in our surveys. With the removal of prophylactic antibiotics during early chick growth as well as in breeder flocks, opportunistic bacterial infections are not surprising. The levels of contamination that we detect could result in a 2% reduction in hatchability and increased FWM. This is based on 8 yolks with significant or heavy bacterial growth out of 688 embryos, see summary in

Table 1. Summary of bacterial species recovery from different egg or chick sources. Eggs or chicks (n = count) from different sources were sampled or cultured using different methods (see Section 2), with indicated evidence for significant growth yield for the indicated species.

Source	n	Sampling Method	Significant Growth
Broiler breeder eggs, no incubation	31	Yolk enrichment	1 each lawn E. avium
Broiler breeder eggs, 18 days of incubation	297	Yolk swab	1 each lawn G. sanguinis 2 each lawn E. gallinarum 1 each lawn E. faecalis 1 each 10 CFU E. gallinarum
Broiler eggs, 18 days of incubation	360	Yolk swab	1 each lawn of E. coli and E. faecalis 1 each lawn of E. faecalis
Broiler chicks, day of hatch	4	Residual yolk sac swab	1 each 7 CFU E. faecalis 1 each lawn of E. coli and E. faecalis
	18	Residual yolk sac swab	3 each 1 CFU E. faecalis 1 each 2 CFU E. faecalis 1 each 3 CFU E. faecalis 1 each 5 CFU E. faecalis 1 each 16 CFU E. faecalis 1 each lawn E. faecalis
Broiler breeder chicks, day of hatch	40	Residual yolk sac swab	1 each 1 CFU E. faecalis 1 each 3 CFU E. faecalis 1 each 6 CFU E. faecalis 1 each 8 CFU E. faecalis 1 each 15 CFU E. faecalis 1 each 120 CFU E. faecalis

. Additionally, vertical transmission from the breeder hens to the new flock could “seed” that flock for subsequent bacterial outbreaks, including sepsis, colibacillosis, or osteomyelitis (lameness). This is based on the recovery of bacteria from residual yolk sacs of day-old chicks (16 out of 62 chicks; Table 1)

5. Conclusions

Further pursuits should be aimed at repeated evaluation of eggs and tracking eggs with significant bacterial loads back to breeder hens to determine whether specific hens are responsible for the infected egg yolks in a Typhoid Mary scenario. If specific hens are repeatedly producing eggs with infected yolks, then the genetics of these particular hens could be examined for whether they have a genetic predisposition or if infection is stochastic. If environmental factors are more critical for which hens are producing infected eggs, then probiotic administration should reduce colonization of the hen. Alternatively, vaccines for the most common egg contaminant species might be an effective strategy, with eBeam vaccines as a suitable candidate [54].