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Salmonella enterica serovar Enteritidis is the leading cause of salmonellosis in people, and modeling of infections in chickens is used to identify intervention strategies. A review of 80 manuscripts encompassing 119 experiments indicated that the mean dose of infection was 10 8 CFU per bird. Experiments of less than 10 6 CFU were primarily conducted in immature birds. To address a lack of information on the impact of low dosages on the hen at lay, two experiments were conducted in triplicate. Experiment A addressed issues associated with vaccination; thus, hens were infected intramuscularly at 10 3 , 10 5 , and 10 7 CFU. For Experiment B, which was focused more on colonization and invasion, hens were infected orally with 5 × 10 3 CFU with 4 strains from different genomic clades. Samples from liver, spleen, ovarian pedicle, and paired ceca in both experiments were cultured 5, 6, 7, and 8 days postinfection. Eggshell microbiome taxa were assessed in Experiment B. Results indicated that dosages of 10 3 CFU in both experiments produced enough positive samples to be used within models. The intramuscular route resulted in approximately twice as many positive samples as the oral route. The kinetics of infection appeared to differ between low and high dosages suggestive of a J-curve response. These results could impact risk assessments if the hen at lay has a nonlinear response to infectious dose.

More than 90% of the commercial egg production in the United States is pledged to be in cage-free systems by 2025. Management practices like induced molting and litter area management have come under scrutiny because of the housing system change. The aim of this study was to determine the welfare and production implications of different litter substrates and also evaluate induced molting of hens in a cage-free system. Bovan White hens were housed in a multi-tier aviary system with daily access to open litter area of either Astroturf (AT), wood shavings (SH), or straw (ST) and bare concrete floor (CO) serving as control. At 68 wk of age, molt was induced in half of the hens whereas the other half continued without molting to 116 wk. Production and welfare parameters were measured periodically throughout first and second cycles. Litter substrate did not influence hen-day production and case-weight measurements. However, CO had the lowest total number of eggs produced during the first cycle (P < 0.05). Hen-day percentage was approximately 14% greater in molted hens during the second cycle with egg case weight being heavier in non-molt hens toward the end of second cycle (P < 0.05). The only welfare parameter influenced by litter substrate during the first cycle was a greater crop feather loss in AT than ST at mid-lay (P < 0.05). Keel deformations increased with age irrespective of the litter substrate with 91.5% of palpated hens having keel deformations at the end of first cycle (P < 0.05). Molting did not influence the keel palpation and footpad scores whereas frequency of moderate comb wound was greater in molt hens during molt (P < 0.05). Severe feather loss was seen in non-molt hens during the second cycle (P < 0.05). Litter substrate does not affect production and physical parameters of welfare of hens in a multi-tier aviary system. Additionally, induced molting can be successfully carried out in the multi-tier cage-free system.

In the United States, there is an increase in need for cage-free eggs in retail and food manufacturing sectors. Understanding the impact of cage-free systems and the corresponding management on egg quality is pertinent as the U.S. industry adapts existing housing and builds new cage-free housing structures. A study was conducted comparing 2 brown shell and 2 white shell hen strains housed in a cage-free aviary system. Each set of eggs were placed in cold storage and assessed at 0, 2, 4, 8, and 12 wk. Eggs were collected at 21, 31, 42, and 60 wk of hen age. A full profile of physical quality measurements was conducted on up to 18 intact eggs for each hen strain/egg storage/hen age combination. Egg weight increased approximately 10 g for brown shell and 14 g for white shell eggs as hens aged. Many of the properties monitored were significantly impacted by all 3 main effects (hen strain, egg storage, and hen age) resulting in 3-way interactions. A brown and a white shell strain had stronger shells (44 N; P < 0.0001) than the remaining brown and white shell strains (42 N and 39 N, respectively). The current study also determined volume of shell, total length, maximum width, and percent length at maximum width to more accurately indicate egg shape than shape index. One brown shell strain produced eggs with the most consistent shape characteristics over the hen ages monitored. White shell eggs from the cage-free aviary housing produced the highest whole-egg total solids between 31 to 60 wk of hen age, whereas brown shell eggs resulted in the most consistent level of whole-egg total solids (22-23.5%). The brown and white shell strains in the current study produce cage-free aviary eggs with distinctive physical quality attributes. The outcomes from this study can be utilized by the U.S. egg industry in planning management strategies and market placement of cage-free eggs.

The housing of laying hens is important for social, industrial, and regulatory aspects. Many studies have compared hen housing systems on the research farm, but few have fully examined commercial housing systems and management strategies. The current study compared hens housed in commercial cage-free aviary, conventional cage, and enriched colony cage systems. Environmental and eggshell pool samples were collected from selected cages/segments of the housing systems throughout the production cycle and monitored for Salmonella and Campylobacter prevalence. At 77 wk of age, 120 hens per housing system were examined for Salmonella and Campylobacter colonization in the: adrenal glands, spleen, ceca, follicles, and upper reproductive tract. All isolates detected from environmental swabs, eggshell pools, and tissues were identified for serotype. Two predominant Salmonella were detected in all samples:S.Braenderup andS.Kentucky.Campylobacter coli and C. jejuni were the only Campylobacter detected in the flocks. Across all housing systems, approximately 7% of hens were colonized with Salmonella, whereas >90% were colonized with Campylobacter Salmonella Braenderup was the isolate most frequently detected in environmental swabs (P<0.0001) and housing system impacted Salmonella spp. shedding (P<0.0001).Campylobacter jejuni was the isolate most frequently found in environmental swabs (P<0.01), while housing system impacted the prevalence of C. coli and jejuniin ceca (P<0.0001). The results of this study provide a greater understanding of the impact of hen housing systems on hen health and product safety. Additionally, producers and academia can utilize the findings to make informed decisions on hen housing and management strategies to enhance hen health and food safety., (Published by Oxford University Press on behalf of Poultry Science Association 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Hen housing for commercial egg production continues to be a societal and regulatory concern. Controlled studies have examined various aspects of egg safety, but a comprehensive assessment of commercial hen housing systems in the US has not been conducted. The current study is part of a holistic, multidisciplinary comparison of the diverse aspects of commercial conventional cage, enriched colony cage, and cage-free aviary housing systems and focuses on environmental and egg microbiology. Environmental swabs and eggshell pools were collected from all housing systems during 4 production periods. Total aerobes and coliforms were enumerated, and the prevalence of Salmonella and Campylobacter spp. was determined. Environmental aerobic and coliform counts were highest for aviary drag swabs (7.5 and 4.0 log cfu/mL, respectively) and enriched colony cage scratch pad swabs (6.8 and 3.8 log cfu/mL, respectively). Aviary floor and system wire shell pools had the greatest levels of aerobic contamination for all eggshell pools (4.9 and 4.1 log cfu/mL, respectively). Hens from all housing systems were shedding Salmonella spp. (89-100% of manure belt scraper blade swabs). The dry belt litter removal processes for all housing systems appear to affect Campylobacter spp. detection (0-41% of manure belt scraper blade swabs) considering detection of Campylobacter spp. was much higher for other environmental samples. Aviary forage area drag swabs were 100% contaminated with Campylobacter spp., whereas enriched colony cage scratch pads had a 93% positive rate. There were no differences in pathogen detection in the shell pools from the 3 housing systems. Results indicate egg safety is enhanced when hens in alternative housing systems use nest boxes. Additionally, current outcomes indicate the use of scratch pads in hen housing systems needs to be more thoroughly investigated for effects on hen health and egg safety., (© The Author 2015. Published by Oxford University Press on behalf of Poultry Science Association.)

A move from conventional cages to either an enriched cage or a noncage system may affect the safety or quality, or both, of the eggs laid by hens raised in this new environment. The safety of the eggs may be altered either microbiologically through contamination of internal contents with Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) or other pathogens, or both, or chemically due to contamination of internal contents with dioxins, pesticides, or heavy metals. Quality may be affected through changes in the integrity of the shell, yolk, or albumen along with changes in function, composition, or nutrition. Season, hen breed, flock age, and flock disease-vaccination status also interact to affect egg safety and quality and must be taken into account. An understanding of these different effects is prudent before any large-scale move to an alternative housing system is undertaken.

Although deposition of Salmonella Enteritidis inside yolks is less common than deposition in albumen or on the vitelline (yolk) membrane in naturally contaminated eggs laid by infected hens, bacterial migration into the yolk to reach its nutrient-rich contents could lead to extensive multiplication. The present study used an in vitro egg contamination model to assess the ability of small initial numbers of Salmonella Enteritidis to penetrate the vitelline membrane and multiply inside yolks of eggs laid by 6 genetically distinct commercial lines of hens during 24 h of storage at 30 degrees C. Eggs from each line were tested at 4 different hen ages by inoculation of approximately 100 cfu of Salmonella Enteritidis onto the outside of the vitelline membranes of intact yolks in plastic centrifuge tubes and then adding back the albumen into each tube before incubation. Overall, the frequency of penetration of Salmonella Enteritidis into the yolk contents of eggs from individual lines of hens ranged from 30 to 58% and the mean concentration of Salmonella Enteritidis in yolk contents after incubation ranged from 0.8 to 2.0 log(10) cfu/mL. For both of these parameters, values for one hen line were significantly higher than for 2 other lines, but no other differences were observed. Hen age did not have a significant effect on egg yolk penetration by Salmonella Enteritidis. These results indicate that opportunities for the migration and growth of small initial numbers of Salmonella Enteritidis to attain more dangerous levels inside contaminated eggs during storage at warm temperatures can sometimes vary between different lines of laying hens.

The crop immune response against Salmonella Enteritidis (SE) challenge in eight commercial egg-layer strains (five white-egg layer and three brown-egg layer) and specific-pathogen-free (SPF) White Leghorn (WL) hens was investigated. Pre- and post-SE challenge mucosal immune responses within the crops were evaluated. Commercial layers and SPF WL hens were orally challenged with 10(8) CFU/ml SE PT13a and SE nalR PT13, respectively. Crop lavage samples were collected at weekly intervals from day 0 (pre-challenge) to day 25-27 postinfection (PI), and bacteriological examination was performed to monitor progression of SE infection. Crop lavage samples were analyzed for SE-lipopolysaccharide (LPS)-specific IgA using enzyme-linked immunosorbent assay (ELISA). H&E-stained slides of crop sections from day 34 PI and uninfected controls were assessed for lymphoid tissue via light microscopy. Lymphoid areas were graded based on morphology, size, and cellularity using a score 0 to 5 scale. The 0 to 5 (low to high) numerical values represented progressive increases in size and cellular density of lymphoid tissue. Bacterial culture results showed the highest percentage of SE-positive crop lavage samples from all hen groups at day 5-6 PI and day 11-12 PI. A progressive decline in percentage of SE-positive crop lavage samples did occur as time PI lengthened; however, at day 25-27 PI SE persisted in crop lavage samples from SPF WL hens and three commercial white-egg layer strains. A marked increase in SE-LPS-specific IgA was measured in crop lavage samples between day 0 and day 11-12 PI for all hen groups. Crop SE-LPS-specific IgA response remained elevated above day 0 baseline for the duration of the experiment. Well-defined score 3 to 5 lymphoid tissue aggregates were observed in crop tissue sections harvested at day 34 PI. Comparison of crop sections determined a 1.2-4.0 times increase in ratio of lymphoid tissue in day 34 PI SE-challenged hens vs. uninfected control hens.

The ileal Peyer's patches (Pp), secondary gut-associated lymphoid tissue of the mucosal immune system, may serve as an important site for monitoring inflammatory and immunologic responses of the host against enteric pathogens. Chicken Pp are often difficult to observe grossly, and a simple technique to enhance visualization of the Pp is lacking. Therefore, we designed a novel staining method that is quick, easy, and accurate to aid in gross identification and recovery of the chicken Pp from fresh tissue specimens. Lower alimentary tracts were harvested from White Leghorn hens and commercial broilers. The ileocecocolic region was excised intact, flushed with deionized water to remove ingesta, and a dilute eosin-Y solution was infused. After 1 min, the eosin-Y was gently extruded. Modified-crystal violet (mCV) was then injected into the gastrointestinal segment, where on the lymphoid tissue area became apparent at the serosal surface. The distal ileal Pp was visible as a pale whitish pink ovoid-focalized area with surrounding gut tissue stained light purple. The exact Pp site could be delineated at the serosal and mucosal surface by gross assessment. Light microscopy evaluation of hematoxylin and eosin-stained tissue slides prepared from the excised Pp site revealed lymphoid tissue aggregations with multiple follicular units indicative of Pp. The novel eosin-Y + mCV staining technique promotes rapid identification and accurate recovery of chicken Pp lymphoid tissue from fresh tissue specimens.

Detection of Salmonella enteritidis in the environment of commercial laying hens is critical for reducing the production of contaminated eggs by infected flocks. In the present study, an inexpensive and portable electrostatic air sampling device was used to collect S. enteritidis in rooms containing experimentally infected laying hens. After hens were orally inoculated with a phage type 13a S. enteritidis strain and housed in individual cages, air samples were collected 3 times each week with electrostatic devices onto plates of 6 types of culture media (brilliant green agar, modified lysine iron agar, modified semisolid Rappaport-Vassiliadis agar, Rambach agar, XLD agar, and XLT4 agar). Air sampling plates were incubated at 37 degrees C, examined visually for presumptive identification of typical S. enteritidis colonies and then subjected to confirmatory enrichment culturing. Air samples (collected using all 6 culture media) were positive for S. enteritidis for 3 wk postinoculation. Because visual determination of the presence or absence of typical S. enteritidis colonies on air sampling plates was not consistently confirmed by enrichment culturing, the postenrichment results were used for comparing sampling strategies. The frequency of positive air sampling results using brilliant green agar (66.7% overall) was significantly greater than was obtained using most other media. A combination of several plating media (brilliant green agar, modified lysine iron agar, and XLT4 agar) allowed detection of airborne S. enteritidis at an overall frequency of 83.3% over the 3 wk of sampling. When used with appropriate culture media, electrostatic collection of airborne S. enteritidis can provide a sensitive alternative to traditional methods for detecting this pathogen in the environment of laying flocks.

Identifying infected laying flocks is a critical component in efforts to prevent eggborne transmission of Salmonella enteritidis to humans. In the present study, egg yolk samples from experimentally infected chickens were tested for specific antibodies with a very rapid fluorescence polarization assay using tracers prepared from the O-polysaccharides of S. enteritidis and S. typhimurium and a conventional ELISA using an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with 106 or 10(8) cfu of S. enteritidis (phage type 13a) or with 10(8) cfu of S. typhimurium. Eggs were collected during five weekly postinoculation intervals. Both fluorescence polarization and ELISA detected the majority of hens infected with S. enteritidis at either dose level, although they also frequently cross-reacted with samples from hens infected with S. typhimurium. Fluorescence polarization with an S. typhimurium tracer was likewise able to consistently detect S. typhimurium infection but also tended to cross-react with samples from hens infected with S. enteritidis. Fluorescence polarization appears to offer a simple and rapid alternative to conventional serological methodology, although concerns about specificity may limit the usefulness of antibody testing data.

Molting is an important economic management tool for the layer industry as a means of maximizing the effective laying life of a flock. Previous work has shown that molting birds through feed removal (FM) increased the severity of a Salmonella Enteritidis (SE) infection. The current study was conducted to follow the progression of an SE infection in unmolted hens versus hens molted via 14-day FM or ad libitum feeding of wheat middlings (WM), in the presence or absence of 2.5% lactose administered in the drinking water. In two trials of the experiment, all hens were infected with approximately 1 x 10(7) SE at day 4 of molt and sampled for SE shedding on days 4, 10, 17, and 24 postinfection (PI). Organ levels of SE were determined on day 7 PI. All molt procedures caused cessation of egg lay within 3 to 7 days. In trials 1 and 2, birds subjected to total FM shed 3 to 5 logs more SE than either the control birds (unmolted) or the birds fed WM on days 4 and 10 PI. Liver and spleen, ovary, and cecum counts were also significantly (P < 0.05) higher in the fasted birds in one trial and liver and spleen and cecum counts in the second. No differences in any of the SE counts were observed in unmolted versus WM-fed birds. Lactose supplementation in drinking water did not provide any advantage in reducing SE infection in either trial. These results indicate that there are alternative methods to long-term FM that can be used to molt birds and not increase the risk for SE problems. How these alternative methods compare with FM with regard to second-cycle egg production and the mechanisms involved in the reduced SE shedding remain to be investigated.

Detecting infected laying flocks is a vital part of many efforts to control egg-associated transmission of Salmonella enteritidis to humans. The relationship between the development of a specific antibody response in infected hens and the deposition of S. enteritidis in eggs is important for establishing the epidemiologic relevance of serologic testing methods. In two trials, laying hens were infected with large oral doses of phage types 13a and 14b isolates of S. enteritidis. Approximately 38% of all infected hens produced at least one contaminated egg, at an overall incidence of 5.2%, between 3 and 23 days postinoculation. As determined by enzyme-linked immunosorbent assay with an S. enteritidis flagellar antigen, 91.7% of inoculated hens produced specific serum antibodies. Although hens with very high antibody titers were associated with a significantly elevated frequency of egg contamination, a consistently direct relationship was not evident between the magnitude of the antibody responses of individual hens and the frequency at which they laid contaminated eggs. Accordingly, although serologic tests can be valuable screening tools for preliminary detection of S. enteritidis infections in poultry, the magnitude of the antibody responses detected in individual hens may not predict the overall risk of egg contamination associated with particular laying flocks.

The effect of normal avian gut flora (NAGF) and enrofloxacin administration on the early infection of young chicks by Salmonella enteritidis (SE) was determined using day-old White Leghorn chicks. Day- old chicks were divided into two groups, untreated control and NAGF-treated, and then infected with 10(6) cfu of SE per chick by oral gavage. The untreated, infected chicks were further divided into two groups and were either left untreated or medicated with a regimen of 10 mg/kg of enrofloxacin in drinking water daily for 10 d, followed by two doses of NAGF beginning at 10 and 8 wk of age in Trial 1 and Trial 2, respectively. Liver, spleen, and cecum samples were tested for the presence of SE, and immunological responsiveness was investigated up to 12 wk of age. Compared with the untreated group, the cecal colonization of SE was significantly (P < 0.05) decreased in the NAGF-treated group in Trials 1 and 2. No significant differences in organ infection were observed in the NAGF-treated vs. untreated birds. Although a significant effect of the combined treatment of enrofloxacin treatment and NAGF on the early infection was not shown in Trial 1, compared with enrofloxacin only or the untreated group, a significant reduction (P < 0.05) in the number of infected chickens and in the number of SE in the cecal contents was observed at 10 wk of age in Trial 2. The enrofloxacin treatment did not increase opportunistic colonization by SE due to the use of the antibiotic in either trial. The plasma and intestinal immunological responses were not significant at the early age (up to 12 wk) of the birds. The use of enrofloxacin, followed by NAGF, could aid the elimination of SE from young chicks persistently infected at an early age. The combined treatment, compared with enrofloxacin alone, protected chickens from reinfection by 40%.

Prompt refrigeration to temperatures capable of restricting microbial growth has been recommended as an approach to reducing the likelihood that contaminated eggs will transmit Salmonella enteritidis to humans. By using experimentally contaminated egg components, the present study determined the extent to which small numbers of S. enteritidis could grow to more dangerous levels at different temperatures over a period up to 3 d. This model was intended to simulate the potential opportunities for S. enteritidis multiplication following oviposition and prior to the achievement of internal temperatures able to prevent further microbial growth in eggs. At a relatively warmer incubation temperature (25 C) and with higher inoculum doses (150 cells), rapid and substantial S. enteritidis multiplication often occurred, especially when the bacteria had an opportunity for access to yolk nutrients and when contaminated eggs were incubated for 2 or 3 d before sampling. Extensive multiplication of S. enteritidis was less frequently observed at lower inoculum doses (15 cells), shorter storage times (1 d), and lower temperatures (10 to 17.5 C) and when contaminants were introduced into the albumen.

Salmonella Enteritidis is an important pathogen for the layer industry, primarily because of its ability to infect hens and ultimately contaminate egg contents. Studies have shown that stress situations, such as flock recycling (induced molting), can increase Salmonella Enteritidis problems in the flock. The present study examined the effect of antibiotic treatment and competitive exclusion (CE) on Salmonella Enteritidis shedding in the period following molt and 14-day feed withdrawal. In two separate trials, 48 birds after molt and feed withdrawal were divided into one group that was treated for 10 days with enrofloxacin in water followed by administration of CE culture and a group that was left untreated. Salmonella Enteritidis shedding was significantly reduced in the antibiotic-CE group. The Salmonella Enteritidis shedding rate was 33 and 25% in untreated birds versus 4 and 0% in the enrofloxacin-CE group on the two test days. These results indicate that treatment of Salmonella Enteritidis-positive laying hens after molting with enrofloxacin and CE culture can substantially reduce Salmonella Enteritidis problems due to molting and would be a possible alternative to diverting eggs for pasteurization or slaughtering the infected flock. Possible development of bacterial resistance in conjunction with antibiotic use is also discussed.

Newly hatched chicks lack immunological maturity, which could compromise their ability to respond to infection by pathogens such as Salmonella enterica serovar enteritidis (S. enteritidis; SE). A study was conducted in which chicks were infected with a sublethal dose of SE at 1 d posthatch, and the systemic and intestinal immune responses to the challenge were followed over time. Birds infected at this age experienced difficulty in clearing the infection, and 50% of the individual birds remained persistently infected until 23 wk of age. These birds exhibited only a marginal systemic and mucosal humoral immune response to the infection. No response or little response was observed 1 wk postchallenge; responses increased somewhat over time. On many of the sampling times, 50% or more of the culture-positive birds lacked a detectable plasma or intestinal response. Levels of 10(3) to 10(5) SE/g of feces could be found in the intestines of birds eliciting a good IgA response, indicating that, when these birds did respond mucosally, the IgA produced was incapable of clearing the organism once the infection was established. Birds infected during this time also experienced reduced ability to respond to vaccination. Compared with uninfected controls, depressed responsiveness to an S. enteritidis bacterin was observed in infected birds 1 and 2 wk after administration, whereas those individuals receiving an inactivated Newcastle disease vaccine (NDV) experienced a reduced response 4 and 6 wk postvaccination, indicating that the persistent infection affected the ability of the immune system to respond to homologous and heterologous antigens. These results demonstrate that exposure of chickens to SE early in life interferes with the ability of these individuals to respond humorally to the infection and to other antigenic stimuli; such effects can be observed for at least 23 wk.

Electrostatic space chargers were used to impart a negative charge to airborne dust particles and thereby cause them to be attracted to grounded surfaces. To determine whether negative air ionization could affect the airborne transmission of Salmonella enteritidis, chicks were housed in four controlled-environment isolation cabinets in which airflow was directed across an unoccupied central area from one ("upstream") group of birds to another ("downstream") group. Negative air ionizers were installed in two of these cabinets. In three replicate trials, groups of chicks were placed in the upstream ends of the transmission cabinets and orally inoculated with S. enteritidis at 1 wk of age. On the following day, 1-d-old chicks were placed in the downstream ends of the cabinets. When chicks were sampled at 3 and 8 d postinoculation, S. enteritidis was found on the surface of 89.6% of the downstream chicks from cabinets without negative air ionizers, but on only 39.6% of the downstream chicks in the presence of the ionizers. Similarly, S. enteritidis was recovered from the ceca of 53.1% of sampled downstream chicks in cabinets without ionizers, but from only 1.0% of the ceca of chicks in cabinets in which ionizers were installed. The presence of the ionizers was also associated with reduced levels of circulating airborne dust particles. Reducing airborne dust levels may thus offer an opportunity to limit the spread of S. enteritidis infections throughout poultry flocks.

In each of two replicate trials, 1-d-old chicks were inoculated orally with a phage type 13 Salmonella enteritidis isolate (resistant to nalidixic acid). Although S. enteritidis was found in the livers, spleens, and ceca of all sampled chicks at 1 wk postinoculation, colonization generally persisted beyond 4 wk postinoculation only in the ceca. Nearly half of the remaining hens were still shedding S. enteritidis in their feces at 24 wk of age, but only 1 of 62 hens laid eggs that were internally contaminated with S. enteritidis during the initial 4 to 6 wk of egg production. Chickens exposed to S. enteritidis shortly after hatching can apparently remain infected until maturity, at which time they might produce contaminated eggs or spread the infection to other susceptible, previously unexposed hens.

Although direct contact with infected birds and indirect contact with contaminated environmental surfaces are known to be important factors in the dissemination of Salmonella enteritidis in poultry flocks, the potential role of airborne transmission is less clearly defined. This study considered the mechanism by which S. enteritidis might spread between groups of chicks housed in controlled-environment disease transmission cabinets, separated by an unoccupied space that prevented any direct or indirect contact. Airflow in these cabinets was directed across the unoccupied area from one ("upstream") group of chicks to the other ("downstream") group. In each of four replicate trials, two groups of 25 chicks were placed in the upstream ends of transmission cabinets and orally inoculated with S. enteritidis at 1 week of age. One day later, 25 1-day-old chicks were placed in the downstream end of each cabinet. When chicks were removed and sampled at 3 and 7 days postinoculation, S. enteritidis was found on the feathers of 77% of the downstream chicks. Moreover, 33% of the downstream chicks became infected with S. interitidis. The comparative frequencies of recovery of S. enteritidis from various downstream sampling sites suggested that infection was apparently transmitted principally by oral ingestion, perhaps from environmental surfaces contaminated by airborne movement of the pathogen. Reducing the airborne movement of S. enteritidis in poultry houses should thus help limit the spread of infection within flocks and thereby diminish the incidence of production of contaminated eggs.

The identification of infected commercial poultry flocks has become a pivotal component of efforts to reduce the incidence of egg-associated transmission of Salmonella enteritidis to humans. To assess the sensitivity with which testing for specific antibodies in egg yolks can be applied to detect S. enteritidis infection in laying chickens, groups of hens were orally inoculated with either 10(3), 10(5), or 10(7) cfu of a phage type 13a strain of S. enteritidis. Eggs from these hens were collected for 4 wk after inoculation and yolk samples were tested for antibodies to S. enteritidis flagella by ELISA. All hens that were inoculated with 10(7) cfu of S. enteritidis were detected as infected by the egg yolk ELISA when eggs were tested individually, as were up to 66 and 35% of hens inoculated with 10(5) or 10(3) cfu, respectively. Even when yolks from infected hens were diluted 1:10 in yolk from uninfected hens, specific antibodies could still be found in eggs from 31% of hens given 10(7) cfu of S. enteritidis and 13% of hens given 10(3) cfu. These results demonstrate that egg yolk antibody testing can provide a highly sensitive indication of prior exposure to S. enteritidis, and should accordingly be useful for verifying the effectiveness of programs designed to reduce the incidence of S. enteritidis infection in poultry.

Phage type 4 Salmonella enteritidis strains have only recently been isolated from poultry and humans in the United States, although this phage type predominates in many other countries. The present study assessed the ability of S. enteritidis isolates of various phage types found in the United States (including phage type 4) to colonize the intestinal tract and invade to reach internal organs in experimentally infected chicks. Groups of 5-day-old single-comb white leghorn chicks were inoculated with a range of oral doses of three phage type 4 isolates and three isolates of other phage types. Although some significant differences were observed between individual S. enteritidis isolates in the frequencies at which they colonized the intestinal tracts and invaded to reach the spleens of inoculated chicks, no consistent overall pattern differentiated phage type 4 isolates from isolates of other phage types.

Bacterial culturing of eggs for Salmonella enteritidis has become an important tool in efforts to identify laying flocks that potentially threaten public health. As pools of egg contents are generally incubated before culturing to allow S. enteritidis numbers to multiply to easily detectable levels, any differences in the multiplication of S. enteritidis strains in egg pools could result in similar differences in the likelihood of detection. To assess whether 12 S. enteritidis strains would multiply to reach different final levels in pools of egg contents, 100-mL samples of liquid whole egg were experimentally contaminated with < 10 cfu of the various strains. After incubation for 24 h at 37 C, the number of colony-forming units of S. enteritidis in each pool was determined. Significant differences were observed between strains in the extent of expansion of the S. enteritidis population during incubation (some strains grew to levels more than a thousand times greater than others). Iron supplementation of the pools during incubation significantly increased S. enteritidis growth and reduced the extent of variation between strains.

A significant proportion of human Salmonella enteritidis (SE) outbreaks in recent years has been traced to the consumption of contaminated eggs produced by infected laying flocks. Experimental SE infections in chickens have been used to acquire a considerable amount of basic information about the interaction between SE and the avian host. After oral or parenteral inoculation of chickens, SE can colonize the intestinal tract, invade and disseminate to reach numerous internal organ sites, and elicit the production of specific antibodies in serum and egg yolks. Experimental infection of laying hens can result in the deposition of SE in the contents of eggs before oviposition, although generally in rather small numbers and at a relatively low frequency. The consequences of experimental SE infection have been shown to vary significantly with the strain and dose of the inoculum. Some SE isolates have been shown to produce various clinical effects, including decreased egg production by hens. The information provided by experimental SE infections in chickens has played an important role in the formulation of appropriate strategies for reducing the incidence of SE in commercial laying flocks and thereby also reducing the incidence of transmission of SE to consumers via contaminated eggs.

Two replicate experiments were conducted to test the efficacy of two different Salmonella enteritidis oil-emulsion bacterins (an experimentally prepared acetone-killed vaccine and a commercially available vaccine) for protecting laying hens against intestinal colonization following oral exposure to S. enteritidis. Each vaccine was administered twice (4 weeks apart), and all hens were challenged with 10(8) cells of a nalidixic-acid-resistant S. enteritidis strain 2 weeks after the second vaccination. Fecal samples from vaccinated and unvaccinated control hens were cultured at three weekly intervals post-challenge to determine the incidence of intestinal colonization and the numbers of S. enteritidis shed into the environment. Both vaccines significantly reduced the incidence of intestinal colonization (P < 0.05) and the mean number of S. enteritidis cells shed in the feces (P < 0.01) at 1 week post-challenge. However, the degree of protection afforded by vaccination was only partial, as more than half of the vaccinated hens still shed substantial numbers of S. enteritidis. If used in conjunction with other flock sanitation and infection-monitoring strategies, vaccination with bacterins could potentially reduce the overall level of environmental contamination and thereby also reduce the horizontal transmission of S. enteritidis within and between laying flocks.

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.

Two experiments were conducted to determine whether Salmonella enteritidis could be detected efficiently in pooled samples of the contents of eggs laid by experimentally infected hens. In Experiment 1, groups of laying hens were orally inoculated with either 10(4) or 10(6) S. enteritidis cells. In Experiment 2, one-third of the hens in each group were orally inoculated with 10(6) S. enteritidis cells. At 2 wk postinoculation, culturing egg pools yielded positive results at frequencies similar to those obtained by culturing fecal samples, culturing internal organs, or testing for specific serum antibodies. Culturing fecal samples detected a higher percentage of exposed hens than egg pool culturing at 1 and 3 wk, and serum antibody tests were superior at 3 and 4 wk. In Experiment 1, more than 46% of the hens inoculated with 10(6) S. enteritidis cells laid at least one contaminated egg during the 2nd wk postinoculation. In Experiment 2, over 34% of the hens produced at least one contaminated egg during the 2nd wk postinoculation, but very few contaminated egg pools were identified during the 3rd and 4th wk. Bacteriological culturing of pooled egg contents for S. enteritidis, therefore, can apparently provide an effective method for assessing the potential public health threat posed by a flock.

To assess the potential protective efficacy of a Salmonella enteritidis bacterin, an acetone-killed oil-emulsion vaccine was prepared from a phage type 13a S. enteritidis strain and administered subcutaneously to hens in two experiments. Hens were housed individually, and every other hen was vaccinated (at 23 weeks of age in one experiment and at 45 weeks in the other). A second (booster) bacterin injection was administered 6 weeks later in both experiments. Three weeks after the second vaccination, all hens were challenged with an oral dose of approximately 10(9) cells of a heterologous (phage type 14b) S. enteritidis strain. In both trials, S. enteritidis was isolated from fewer internal organs (spleens, ovaries, and oviducts) and pools of egg contents from vaccinated hens than from unvaccinated control hens. Vaccination did not, however, affect the percentage of hens that shed S. enteritidis in feces in either experiment.

The effects of four domestic Salmonella enteritidis (SE) isolates were compared in experimentally infected chicks and laying hens. The pathogenicity of each strain for 1-day-old chicks was determined by recording postinoculation mortality. The effects of the SE strains on adult hens were measured in terms of changes in total egg production, the frequency of production of SE-contaminated eggs, the dissemination of SE to internal organs, and the elicitation of a specific antibody response. Significant differences in the consequences of infection with different SE strains were observed in mortality rates among chicks and in total egg production, the frequency of production of contaminated eggs, and the serum antibody response among laying hens. The usefulness of a chick mortality model for predicting the probable frequency of production of contaminated eggs by laying hens infected with particular SE strains was then further evaluated by infecting chicks and laying hens with four other field isolates of SE. Although significant differences between SE strains were observed in both chick mortality and the frequency of production of contaminated eggs by hens, a strong correlation between these two parameters was not evident.

Eggs laid by hens experimentally infected with Salmonella enteritidis were assayed for the presence of Serogroup D-specific yolk antibodies. Yolk antibodies were detected with S. enteritidis and Salmonella pullorum antigens in the microantiglobulin test as early as 9 days after inoculation of hens with S. enteritidis. Yolk antibody titers reached peak levels at 3 to 5 wk postinoculation and remained at detectable levels for at least 7 wk postinoculation in eggs from both orally inoculated and horizontally contact-exposed hens. Eggs laid by hens from commercial flocks implicated in epidemiological investigations of human S. enteritidis outbreaks were also tested. Serogroup D-specific yolk antibodies were detected in 5 to 22% of eggs from hens in houses identified as infected by bacteriological culturing of internal organs of hens for S. enteritidis.

Tissues from experimentally infected hens were examined for the presence of Salmonella enteritidis (SE). SE was recovered from internal organs of both orally inoculated hens and hens infected by horizontal contact transmission. SE was isolated from 58% of the ceca, 51% of the livers, 47% of the spleens, 17% of the ovaries, and 17% of the oviducts of hens sampled during the first 5 weeks after exposure. SE was recovered at a low frequency from all internal organs sampled for as long as 22 weeks after exposure.

The antibody response of laying hens to experimental Salmonella enteritidis infection was evaluated in microagglutination, tube agglutination, and rapid whole-blood plate agglutination assays. Hens of three different ages were infected by either oral inoculation or horizontal contact transmission. Blood was collected at weekly intervals, and the presence of specific antibodies was assessed by reaction with antigens prepared from strains of S. enteritidis and S. pullorum. The sensitivity of detection of infected hens did not vary significantly between the assays, as all three tests effectively identified most exposed hens as seropositive. Within each test, however, variation was observed in the detection sensitivity when different antigens were used. The microagglutination titers of serum samples were determined by serial dilution. Antibody titers peaked at 1 to 2 weeks postinoculation and declined steadily, although most birds were still identified as seropositive at 10 weeks postinoculation. The mean microtest titers obtained with S. enteritidis antigens were higher than with an S. pullorum antigen, indicating greater test sensitivity. However, use of the S. pullorum antigen resulted in fewer false positives when sera from uninfected control hens were tested. The titers of contact-exposed hens peaked later and at lower values than did those of inoculated hens, but these two groups of hens had similar antibody titers after the third week postinoculation.

Laying hens of three different ages were experimentally infected with a strain of Salmonella enteritidis by either oral inoculation or contact transmission. Total egg production was depressed in exposed hens of all three age groups. Persistent intestinal shedding was observed in a small number of hens. Eggs with contents contaminated by S. enteritidis were produced by exposed hens at a high frequency, but only during a fairly short period of time that extended through approximately 1 week postinoculation for older hens and through 2 weeks for younger hens. S. enteritidis was recovered from whole yolks and albumen of these eggs at similar frequencies, but not from the content of yolks. Eggs with contaminated shells were also produced, but at a lower frequency. Contaminated eggs were produced by orally inoculated and contact-exposed hens at similar frequencies. S. enteritidis was not isolated from the contents of eggs laid by hens infected with other S. enteritidis strains.

Four experiments were conducted to examine the relationship between antibiotic administration to poultry and the in vivo proliferation of Salmonellae. The frequency of isolation of drug-resistant transconjugant S. arizonae from the livers of chicks inoculated per os with multiply drug-resistant Escherichia coli and drug-sensitive S. arizonae was directly related to the concentration of kanamycin administered to the chicks in their drinking water. Kanamycin administration was also associated with a significant (P less than .05) increase in the frequency of isolation of drug-resistant transconjugant S. typhimurium from the intestines and livers of poults inoculated with drug-sensitive S. typhimurium and multiply drug-resistant E. coli. Kanamycin administration significantly reduced the spread of drug-sensitive S. typhimurium to the livers of poults inoculated only with that strain. These experiments demonstrate that antibiotic administration to poultry can enhance the proliferation of drug-resistant Salmonella.

Effects of age at exposure on the persistence of Salmonella in various tissues of chicks were assessed in two experiments. Broiler chicks, housed on wire floors in isolation cabinets, were orally inoculated with S. typhimurium at various ages (1 to 8 days after hatching). The postinoculation mortality of chicks declined significantly (P less than .05) as the age at inoculation increased. One experiment investigated the effect of age at inoculation on the persistence of S. typhimurium in the cecum. Salmonella persisted for 7 wk after inoculation in 81.3% of the chicks inoculated at 1 day of age and in 62.5% of the chicks inoculated at 8 days of age. The mean number of cecal Salmonella at 7 wk postinoculation was also greater for chicks inoculated on Day 1 than for those inoculated on Day 8. The second experiment examined the effect of age at inoculation on the adherence of S. typhimurium to and penetration through the cecal epithelium. The ceca of chicks inoculated at 1 day of age were colonized by significantly more adhering Salmonella at 2 days postinoculation (1.4 x 10(8)/g) than were those of chicks inoculated at 3, 5, or 7 days of age (8.0 x 16(6)/g or less), but age did not affect the recovery of S. typhimurium from livers or spleens.