Search

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 iflto 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°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 login 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. [ABSTRACT FROM AUTHOR]

Although Salmonella deposition inside yolks is uncommon in naturally contaminated eggs, migration through the vitelline membrane into the nutrient-rich yolk contents could enable rapid bacterial multiplication. Egg refrigeration restricts both penetration and growth, but a recently proposed national Salmonella Enteritidis control program would allow unrefrigerated ambient temperature storage of eggs on farms for up to 36 h. The present study used an in vitro egg contamination model to assess the ability of small numbers of 4 Salmonella Enteritidis strains and 4 Salmonella Heidelberg strains to penetrate the vitelline membrane and multiply inside yolks during 36 h of storage at either 20 or 30°C. After inoculation onto the exterior surface of the vitelline membrane, all 8 Salmonella strains penetrated to the yolk contents (at a mean frequency of 45.1%), and most strains grew to significantly higher levels (with a mean log10 bacterial concentration of 2.2 cfu/mL) during incubation at 30°C. Significant differences in penetration frequency and yolk multiplication were observed between individual strains and between serotypes (Salmonella Enteritidis > Salmonella Heidelberg for both parameters). Penetration and multiplication were significantly less frequent during incubation at 20°C. These results demonstrate that controlling ambient temperatures during prerefrigeration storage may be an important adjunct to prompt refrigeration for limiting Salmonella growth in eggs and thereby for preventing egg-transmitted human illness. [ABSTRACT FROM AUTHOR]

Eggs that harbor Salmonella in their edible contents pose a significant risk of transmitting disease to consumers. Although Salmonella deposition inside yolks does not usually occur at a high frequency in naturally contaminated eggs, bacterial penetration through the vitelline membrane could lead to rapid and extensive multiplication in the nutrient-rich yolk contents. The present study used an in vitro egg contamination model to assess the ability of Salmonella strains to penetrate the vitelline membrane and multiply inside yolks. An S. enteritidis strain and 2 Salmonella heidelberg strains, initially inoculated onto the outside of the vitelline membrane, were able to enter the yolk contents (at frequencies ranging from 10 to 25% of experimentally contaminated eggs) during 24 h of incubation at 30°C. Variants of these parent strains, obtained by in vivo passage into eggs laid by infected hens, penetrated the yolk membrane at significantly higher frequencies. These results demonstrate that pathogens such as S. enteritidis and S. heidelberg can penetrate into and begin to multiply inside the yolks of contaminated eggs during the first day of storage at warm temperatures. [ABSTRACT FROM AUTHOR]

An abstract of the article "Effect of passage through laying hens on organ invasiveness and phenotypic heterogeneity of Salmonella Enteritidis," by R.K. Gast and colleagues is presented.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

The environmental sampling of layer housing systems is essential to identifying potential pathogens that are of concern to human health. To identify the natural occurrence of pathogens (Listeria, Campylobacter, and Salmonella) at various locations in a cage-free aviary housing system, swabs were collected when hens were 22 to 39 wks of age. Duplicate environmental swabs were taken and inoculated with a low dose (10 1 cfu) Salmonella Enteritidis (SE) and examined for the recovery of SE from environmental samples. Detection of Listeria (P < 0.0001) and Campylobacter (P < 0.0001) varied between the environmental sample types taken: concrete dust, drag swabs, egg belt dust, manure belt scraper swabs, and wall dust. Detection of Listeria (P < 0.0001) was the highest (70.0%) at the beginning of the study (22 wk) and decreased over time. Detection of Campylobacter (P < 0.001) was also the highest at 22 wk, however the decrease over time was more gradual. Interestingly, detection of Campylobacter (P < 0.0001) was the greatest in concrete dust samples (96.25%), which can be attributed to the presence of rodent excreta in the samples. Drag swabs and manure belt scraper swabs were the best sampling types for high detection of Listeria and Campylobacter. It should be noted that Listeria recovered was not of human health concern. No naturally occurring Salmonella was identified in this study. The recovery of the SE inoculum increased over time, reaching the greatest recovery in drag (81.25%; P < 0.0001), egg belt dust (100.00%; P < 0.0001) and wall dust swabs (100.00%; P < 0.0001) by 39 wk. This high rate of SE recovery occurred just before US mandatory SE environmental monitoring at 40 to 45 wks of age. Based on this study, the use of drag and manure belt scraper swabs are effective in detecting Listeria and Campylobacter in cage-free aviary housing. Along with good pest management, the occurrence of pathogens could be monitored and reduced in laying hen flocks., (Published by Elsevier Inc.)

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.

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.

Efficient detection of Salmonella enteritidis inside eggs is critical for confirming that individual commercial laying flocks present a risk to public health. In most standard bacteriological culturing protocols, an initial incubation step is necessary to allow the typically very small population of S. enteritidis cells in pools of egg contents to multiply to more easily detectable levels. In the present study, two rapid methods were evaluated as alternatives to plating on selective media for detecting S. enteritidis in incubated egg pools. By using either fluorescence polarization or lateral flow immunodiffusion assays, S. enteritidis could be consistently detected in egg pools at 10(8) cfu/mL (and in most pools at 10(7) cfu/mL). Although the rapid assays were significantly less sensitive than culturing, they both were consistently able to detect contamination when pools of 10 eggs were inoculated with approximately 10 cfu of S. enteritidis and incubated for 72 h at 25 degrees C.

The crop (ingluvies), an organ for food storage in most avian species when the proventriculus is full, is located at the base of the esophagus. Little is known about any immunological capacity in the crop, and the current study was conducted to determine whether any antibodies to SE could be found in crop flushes taken from White Leghorn hens following infection with this organism. Surprisingly, an exceptionally strong IgA anti-SE response could be detected in the crops of hens 17 d postchallenge, and a comparison at Day 22 of crop vs. intestinal IgA anti-SE responses showed a good correlation between anti-SE antibody levels in the two regions. Histologic examination of crop tissues revealed development of lymphoid aggregates in the crop walls following challenge with SE. These results indicate that the crop may serve a role in immune protection in addition to its capacity as a food storage organ.

Periods of inflammation due to infection, injury, or malignancy are marked by increases in serum constituents known as acute phase proteins (APP), and these proteins have been used as markers for early stages of disease. Four experiments were performed to examine whether levels in chickens of one such APP, alpha1 acid glycoprotein (AGP), would be affected by an infection with Salmonella enteritidis (SE) and if the added stress of induced molting via 14-d feed withdrawal would increase these effects. In all experiments but Experiment 1, hens were divided into four equal groups: molted infected, nonmolted infected, molted noninfected, nonmolted non-infected (Experiment 1 lacked this last group). Blood and intestinal samples were collected at various times from the hens and assayed for AGP and SE levels, respectively. Infection with SE significantly elevated serum AGP levels above those found in the noninfected groups of hens in two of four experiments, whereas in molted infected hens, serum AGP levels were significantly higher than those found in the noninfected counterparts in all four experiments. Comparison of AGP titer between the infected groups of hens revealed that significantly higher SE levels generally did not guarantee significantly higher AGP levels, although when individual values were plotted, a trend was observed toward increasing serum levels concomitant with increasing SE counts. Serum AGP levels show promise as a method to detect infection problems in hens, especially when the severity of the infection is exacerbated by stress situations. However, more work is needed to determine what other factors may elevate serum AGP levels and potentially confound the picture.

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.

The site of deposition of Salmonella enteritidis in eggs could influence the extent to which this pathogen multiplies before refrigeration achieves growth-inhibiting internal temperatures. The first part of this study sought to determine whether S. enteritidis inoculated onto the exterior (vitelline) membrane surface of egg yolks was able to penetrate into and multiply within the yolk contents. When 10(2) cfu of S. enteritidis was inoculated onto the exterior surface of intact egg yolks, multiplication within the interior yolk contents occurred in 10% of samples after 6 h of incubation and in 75% of samples after 24 h at 25 C (reaching mean levels of about 10(4) cfu/mL) but in only 20% of samples incubated for 72 h at 15 C. The second part of this study applied an oral infection model in laying hens to establish the relative proportions of contaminated eggs in which S. enteritidis deposition was associated with the yolk membrane or was found inside the yolk contents. Although approximately 4.3% of egg yolks were positive for S. enteritidis when both yolk contents and membranes were sampled, only about 0.5% of samples of yolk contents (without membranes) were positive. Although deposition of S. enteritidis within egg yolks appears to occur infrequently, rapid refrigeration of eggs is necessary to prevent the penetration of S. enteritidis into and multiplication within egg yolks.

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%.

Serological surveillance can be an important component for egg quality assurance programs geared toward controlling problems with Salmonella enterica serovar Enteritidis (S. enteritidis) within a flock. Serum is the primary sample source for the procedures, although egg yolk antibody assays have become popularity in recent years. However, these assays tend to be labor intensive, requiring procedures for extracting antibodies from the yolk followed by assaying the samples. We describe an adaptation of the agar gel precipitin (AGP) test for use in detecting antibodies to S. enteritidis deposited in egg yolks of infected hens. Yolk or sera from infected birds were administered to wells cut into seven-well clusters in an agar gel plate, and detection antigen was added to the center well. The agar gels were incubated for 24 h and then examined for the presence of precipitin lines formed by the interaction of antibody with antigen. Three different antigens were tested: S. enteritidis flagella, SEF14 (a 14-kDa fimbrial protein produced ostensibly by S. enteritidis), and a sodium deoxycholate extract of whole S. enteritidis organism. Flagella and the organism extract detected antibodies to S. enteritidis in the yolk and sera, whereas SEF14 was not reactive. Positive reactions were observed in serum 1 wk postchallenge, whereas in yolks, this was further delayed by 1 wk. The sensitivity of the test was slightly less than the standard microagglutination assay, although specificity was slightly higher, as indicated by results from sera and yolks from birds infected with Salmonella enterica serovar Typhimurium. Simplicity and low labor requirements of the assay would allow for the potential testing of several hundred egg samples within a day, which would make up for test shortcomings due to sensitivity. The AGP test could be an important tool for individuals using serological testing to monitor the S. enteritidis situation within their flocks or as a rapid screen for vaccine responses. The assay could also be used in tandem with other AGP tests to screen for the presence of multiple avian pathogens.

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.

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.

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.

Despite extensive testing for Salmonella pullorum infections in commercial poultry, occasional outbreaks of pullorum disease still occur. In some recent instances, questions have been raised about whether standard serological assays, which employ antigen strains selected several decades ago, are still able to detect currently prevalent S. pullorum strains. The present study evaluated the ability of standard rapid whole-blood plate agglutination test and serum tube agglutination test antigens to detect infection in Single Comb White Leghorn hens inoculated with large oral doses of six recent S. pullorum isolates. Two commercially available plate test antigens were obtained and three tube test antigens were prepared. All five antigens identified most inoculated hens as seropositive, although some differences in sensitivity were evident between the two assay types and between the two plate test antigens. The antigenic composition of the tube test antigens did not affect their ability to detect infections with the various inoculum strains. Regardless of the antigen used, hens infected with antigenically intermediate or variant S. pullorum strains were detected as seropositive less often than were hens infected with antigenically standard strains. Positive culturing results for S. pullorum in the livers and ovaries of infected hens were nearly always predicted by positive serological test results.

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.

The effectiveness of direct plating of incubated egg contents pools onto selective agar media for detecting contamination by Salmonella enteritidis (SE) was evaluated in comparison to several broth enrichment strategies. Pools of mixed liquid whole egg were inoculated with very small number of SE cells, incubated for 4 days at 25 C, and sampled by direct plating or both enrichment culture. Methods involving broth nonselective (pre-enrichment) and selective broth culture steps detected a significantly higher percentage of contaminated egg pools than did direct plating, but required 48 h longer to provide results. Methods involving direct selective broth enrichment, without prior pre-enrichment, did not perform significantly better than direct plating. Direct plating appears to provide a relatively rapid and inexpensive method for detecting SE in egg pools, although greater sensitivity is attainable with more intensive methods.

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.

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.

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.

Fifty-eight cultures of Salmonella arizonae isolated in 1983 from poultry sources and 21 cultures isolated in 1972 to 1974 were screened for resistance to nalidixic acid, chloramphenicol, triple sulfa, ampicillin, tetracycline, streptomycin, and kanamycin by the standardized disc susceptibility method. Sensitivity to all seven drugs was observed in 51% of the cultures tested (76% of the older cultures and 41% of the recent isolates). Resistance to two or more drugs was observed in 36% of the cultures tested (14% of the older cultures and 43% of the newer ones). The highest incidence of resistance was to streptomycin. Salmonella arizonae cultures sensitive to all seven drugs were tested for the ability conjugatively to acquire kanamycin, streptomycin, tetracycline, and ampicillin resistance determinants from a multiply resistant strain of Escherichia coli K12NA. Complete or intermediate resistance to all antibiotics was transferred to 93% of the sensitive cultures at 41 C and 85% at 28 C. Kanamycin resistance was transferred most frequently, and ampicillin resistance least frequently.

Two trials were conducted to determine the transferability of antibiotic resistance in vivo between two strains of enteric bacteria. Newly hatched turkey poults were inoculated per os with a strain of Escherichia coli resistant to kanamycin, tetracycline, and ampicillin, a strain of Salmonella arizonae resistant to nalidixic acid and streptomycin, or both strains. Kanamycin was added to the drinking water of some poults. To test for S. arizonae cells which had received transferable resistance determinants in vivo from the E. coli strain, samples were collected by swabbing the recta of the poults and by removing segments of the intestines and livers after the birds were sacrificed. Nalidixic acid was added to the isolation media to prevent in vitro transfer from occurring after the samples were collected. Salmonella arizonae resistant to nalidixic acid, streptomycin, kanamycin, tetracycline, and ampicillin were isolated from 20% of the rectal samples taken from poults that had received both bacterial strains. S. arizonae cells which had received resistance determinants in vivo were also isolated from 73% of the intestinal samples and 8% of the liver samples taken from birds inoculated with both donor E. coli and recipient S. arizonae. Salmonella arizonae demonstrating resistance to all five antibiotics were recovered from all intestinal samples taken from birds given kanamycin in their drinking water immediately after the last S. arizonae inoculation but from only 43% of such samples taken from birds given no kanamycin.

Three experiments were conducted to assess the relationship between antibiotic administration to poultry and the transmission of drug-resistant Salmonella through a simulated food chain. Poults were inoculated per os with either multiply drug-resistant Escherichia coli and drug-sensitive S. typhimurium or with multiply drug-resistant S. typhimurium. One-half of the poults inoculated with drug-sensitive S. typhimurium and all poults given drug-resistant S. typhimurium received kanamycin in their drinking water. Liver tissue from these poults was incorporated into diets fed to rats, half of which were treated with kanamycin. Antibiotic administration to the poults was associated with a significantly (P less than .05) higher frequency of transmission of drug-resistant S. typhimurium to the rats through the simulated food chain. S. typhimurium was isolated only from rats treated with kanamycin. The highest frequency of isolation of drug-resistant S. typhimurium from rats (40%) was observed in kanamycin-treated rats fed a diet containing liver from kanamycin-treated poults. Results of this experiment indicate that antibiotic administration can increase the frequency of transmission of drug-resistant Salmonella through the food chain.

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.