Serologic detection of experimental Salmonella enteritidis infections in laying hens by fluorescence polarization and enzyme immunoassay

Detection of infected poultry flocks is essential for controlling eggborne transmission of Salmonella enteritidis to humans. The present study evaluated the detection of antibodies in the sera of experimentally infected chickens by a fluorescence polarization assay with a tracer prepared from the O-polysaccharide of S. enteritidis and an enzyme-linked immunosorbent assay (ELISA) with an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with either 10(6) or 10(8) colony-forming units (CFU) of S. enteritidis (phage type 13a) or with 10(8) CFU of Salmonella typhimurium. Serum samples were collected before inoculation and at five subsequent weekly intervals. Both assays successfully detected the majority of hens infected with S. enteritidis at either dose level, but they also identified a substantial number of hens infected with S. typhimurium as seropositive. The fluorescence polarization test detected S. enteritidis infection significantly more often and cross-reacted with sera from hens infected with S. typhimurium significantly less often than the ELISA. The fluorescence polarization assay also offered advantages in terms of speed and methodologic simplicity.     

Airborne infection of laying hens with Salmonella enteritidis phage type 4.

Hens were exposed to small-particle aerosols containing different concentrations of Salmonella enteritidis phage type 4. They developed a systemic infection and some birds were still excreting the organism in the faeces when killed 28 days after infection. S enteritidis was present for a similar period in a wide range of alimentary tract issues and in the ovary and oviduct.     

Salmonella enteritidis and other Salmonella in laying hens and eggs from flocks with Salmonella in their environment.

Seven Canadian layer flocks with Salmonella enteritidis in their environment were investigated to determine the numbers of hens infected with S. enteritidis, the localization of S. enteritidis in organs of infected hens and the numbers of S. enteritidis-infected eggs produced by two affected flocks. By a microagglutination test (MAT) using S. pullorum antigens, these flocks had more seropositive hens (mean 51.9 +/- 16.9%) than two Salmonella-free flocks (mean 13.0 +/- 4.2%). Culture of tissues of 580 hens (433 seropositive) from the seven flocks detected 26 (4.5%) S. enteritidis-infected hens from two flocks. In one flock, 2/150 hens were infected with S. enteritidis phage type (PT) 8, which was confined to the ceca, and no Salmonella spp. were isolated from 2520 eggs (one day's lay). In the second flock, where 24/150 hens were infected with S. enteritidis PT13, extraintestinal infection was found in nine hens and involved the ovaries and/or oviduct in two hens. Salmonella enteritidis PT13 was isolated from one sample of egg contents and from one sample of cracked shells from among 14,040 eggs (one day's lay) from this flock. The overall prevalence of S. enteritidis-contaminated eggs from the two flocks with infected hens was less than 0.06%. Other Salmonella spp. isolated were S. heidelberg from 58 hens (10%), and S. hadar, S. mbandaka and S. typhimurium from one hen (0.2%) each. The MAT with antigens of S. pullorum had a sensitivity of 81% and a specificity of 24% for detecting S. enteritidis-infected hens.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infection of laying hens with Salmonella enteritidis PT4 by conjunctival challenge.

The direct administration of either 103 or 108 cells of Salmonella enteritidis phage type 4 on to the conjunctiva of laying hens resulted in systemic infection. The bacterium was isolated from a variety of tissues, including the ovary and oviduct, and it was excreted in faeces for at least 27 days after infection.     

Colonization of reproductive organs and internal contamination of eggs after experimental infection of laying hens with Salmonella heidelberg and Salmonella enteritidis

Internal contamination of eggs laid by hens infected with Salmonella enteritidis has been a prominent international public health issue since the mid-1980s. Considerable resources have been committed to detecting and controlling S. enteritidis infections in commercial laying flocks. Recently, the Centers for Disease Control and Prevention also reported a significant association between eggs or egg-containing foods and S. heidelberg infections in humans. The present study sought to determine whether several S. heidelberg isolates obtained from egg-associated human disease outbreaks were able to colonize reproductive tissues and be deposited inside eggs laid by experimentally infected hens in a manner similar to the previously documented behavior of S. enteritidis. In two trials, groups of laying hens were orally inoculated with large doses of four S. heidelberg strains and an S. enteritidis strain that consistently caused egg contamination in previous studies. All five Salmonella strains (of both serotypes) colonized the intestinal tracts and invaded the livers, spleens, ovaries, and oviducts of inoculated hens, with no significant differences observed between the strains for any of these parameters. All four S. heidelberg strains were recovered from the interior liquid contents of eggs laid by infected hens, although at lower frequencies (between 1.1% and 4.5%) than the S. enteritidis strain (7.0%).     

Prevalence of Salmonella enteritidis in spent hens.

As part of a USDA/APHIS study on the prevalence of Salmonella enteritidis in spent laying hens, 3700 pooled cecal samples were cultured for Salmonella. Samples were received from a single processing plant and represented 81 commercial egg-type layer flocks from nine southern states. Salmonella were isolated from 2418 of the 3700 (65.4%) cecal pools, but only six isolates were serotype enteritidis. S. enteritidis was isolated from three flocks from two states but was detected in only six of 140 samples from those flocks. Various Salmonella isolation media and procedures were compared. Xylose-lysine-tergitol-4 plates detected 64% of the total Salmonella-positive cecal samples. Brilliant green agar with novobiocin detected 72% of the total Salmonella-positive samples. When used in combination, 82% of the positive samples were detected with these two plates. The remaining 425 Salmonella-positive samples were detected after delayed secondary enrichment.     

The effect of killed Salmonella enteritidis vaccine prior to induced molting on the shedding of s. enteritidis in laying hens

Effects of administering killed Salmonella enterica serovar enteritidis (SE) vaccines to laying hens prior to induced molting on egg production and on shedding of SE were investigated. Forty hens were vaccinated with one of two SE vaccines available commercially in the United States and Japan. Twenty-five days after vaccination, feed was withdrawn for 2 wk from 20 vaccinated plus 10 unvaccinated hens to induce molt. Four days after molt induction, all hens were challenged with a dose of 2.4 X 10(9) of SE. For the 25 days following administration of the SE bacterins, egg production in vaccinated hens showed approximately a 15% decrease. After molt induction, egg production in molted hens ceased and then returned to normal levels 8 or 9 wk postvaccination. Through the 3-mo experimental period, the decreases in numbers of eggs laid in the unvaccinated/molted group and two vaccinated/molted groups were 225 (26.2%), 245 (28.4%), and 274 (31.9%), respectively, compared with 860 in the unvaccinated/unmolted group. There was no significant difference in egg lay at the P < 0.05 level among the former three groups. Hens in the vaccinated/molted groups shed about two logs less SE than hens in the unvaccinated/molted group 3 14 days postchallenge (P < 0.05 or 0.01). These results indicate that vaccination prior to induced molting might be effective in preventing the exacerbation of SE problems within flocks in which the potential for SE contamination may exist.     

Environmental contamination and detection of Salmonella enterica serovar enteritidis in laying flocks.

Faecal, dust and other environmental samples were collected from the floors, droppings belts, egg-collection systems and other areas of 14 cage-layer flocks, 10 barn egg production flocks and seven free-range flocks, and cultured for Salmonella species. The distribution of the organism varied with its prevalence and with the vaccination status of the birds. No one sample type was found to be suitable for identifying all contaminated houses. Salmonella was also frequently found on egg-packing equipment and in samples from rodents and wild birds.     

Characteristics of Salmonella enteritidis contamination in eggs after oral,aerosol, and intravenous inoculation of laying hens

Experimental infection models are useful tools for understanding how Salmonella enteritidis is deposited in eggs and for testing potential strategies to control eggborne transmission of disease to humans. Oral inoculation of laying hens is presumed to provide the closest simulation of naturally occurring infections, but alternatives such as intravenous or aerosol inoculation have sometimes been recommended as options to induce higher incidences of egg contamination. The present study compared the frequency, level, and location of S. enteritidis deposition in egg contents after experimental inoculation by three different routes. In two replicate trials, specific-pathogen-free laying hens were infected with an S. enteritidis culture mixture prepared to optimize invasive behavior. Groups of hens received either an oral dose of 10(9) S. enteritidis, an aerosol dose of 10(9) S. enteritidis, or an intravenous dose of 10(5)-10(7) S. enteritidis. Oral inoculation led to the highest incidence of fecal shedding of S. enteritidis, whereas intravenous inoculation produced the highest specific antibody titers. Eggs laid during the first 21 days postinoculation were cultured to detect and enumerate S. enteritidis in the yolk and albumen. No significant differences were observed among the three inoculation routes in the frequencies of isolation of S. enteritidis from either yolk or albumen. For all three routes of administration, S. enteritidis was recovered more often from yolk (at frequencies ranging from 4% to 7%) than from albumen (0 to 2%). Over 73% of contaminated eggs harbored fewer than 1 colony-forming unit (CFU) of S. enteritidis per milliliter, and only 3% of such eggs contained more than 100 CFUs/ml. Significantly higher levels of S. enteritidis contaminants were associated with intravenous inoculation than with the other routes. No advantage of using aerosol or intravenous administration of S. enteritidis as an alternative to oral inoculation for inducing the production of contaminated eggs was evident in this study.     

Detection of airborne Salmonella enteritidis in the environment of experimentally infected laying hens by an electrostatic sampling device

Bacteriologic culturing of environmental samples taken from sources such as manure pits and egg belts has been the principal screening tool in programs for identifying commercial laying flocks that have been exposed to Salmonella enteritidis and are thus at risk to produce contaminated eggs. Because airborne dust and aerosols can carry bacteria, air sampling offers a potentially efficient and inexpensive alternative for detecting S. enteritidis in poultry house environments. In the present study, an electrostatic air sampling device was applied to detect S. enteritidis in a room containing experimentally infected, caged laying hens. After oral inoculation of hens with a phage type 13a S. enteritidis strain, air samples were collected onto agar plates with the electrostatic sampling device, an impaction air sampler, and by passive exposure to the settling of aerosols and dust. Even though the floor of the room was cleaned once per week (removing most manure, dust, and feathers), air samples were positive for S. enteritidis for up to 4 wk postinoculation. On the basis of both the number of S. enteritidis colonies observed on incubated agar plates and the frequency of positive results, the efficiency of the electrostatic device was significantly greater than that of the passive exposure plates (especially at short collection intervals) and was similar to that of the far more expensive impaction sampler. The electrostatic device, used for a 3-hr sampling interval, detected airborne S. enteritidis on 75% of agar plates over the 4 wk of the study.     

Colonization of a marker and field strain of Salmonella enteritidis and a marker strain of Salmonella typhimurium in vancomycin-pretreated and nonpretreated laying hens

This study was conducted to evaluate the influence of a vancomycin pretreatment on the ability of marker (nalidixic-acid resistant) Salmonella Enteritidis (SE(M)), field Salmonella Enteritidis (SE(E)), and marker Salmonella Typhimurium (ST(M)) strains to colonize within the intestinal and reproductive tracts and translocate to other organs of leghorn laying hens. In each of three trials, caged laying hens (76, 26, and 33 wk ofage) were divided into six groups designated to receive SE(M), SE(F), or ST(M), and half were pretreated with vancomycin (n = 11-12 hens). Vancomycin-treated hens received 10 mg vancomycin in saline/kilogram body weight orally for 5 days to inhibit Gram-positive bacteria within the intestines. On Day 6, all hens were concurrently challenged by oral, intravaginal, and intracolonal routes with Salmonella and placed into separate floor chambers by Salmonella strain. Two weeks postinoculation, all hens were euthanatized and the ceca, spleen, liver/gall bladder (LGB), upper (URT), and lower (LRT) reproductive tracts, and ovarian follicles were aseptically collected, and analyzed for Salmonella. Results did not differ for the three hen's ages and were therefore combined. The vancomycin pretreatment also had no significant effect on the colonization ability of SE(M), SE(F) or ST(M), and therefore results were combined within Salmonella strain. The marker strain of Salmonella Enteritidis was recovered from 21% of ceca, 4% of LGB, 9% of LRT, and 17% of the fecal samples. The field strain of Salmonella Enteritidis was recovered from 88% of ceca, 96% of spleen, 92% of LGB, 30% of LRT, 4% of URT, 13% of follicle, and 42% of the fecal samples. The marker strain of Salmonella Typhimurium was recovered from 100% of ceca, 74% of spleen, 91% of LGB, 30% of LRT, 9% of URT, 9% of follicle, and 100% of the fecal samples. Among ceca, spleen, LGB, and fecal samples, SE(F) and ST(M) colonization was significantly greater than SE(M) colonization. Overall prevalence of Salmonella in the reproductive tracts of challenged hens was relatively low, ranging from 4% to 30%.     

Pathologic and bacteriologic findings in 27-week-old commercial laying hens experimentally infected with Salmonella enteritidis, phage type 4

Two strains of 27-wk-old commercial laying chickens (strain A, brown-egg-laying type and strain B, white-egg-laying type) were inoculated either orally (PO) or intravenously (IV) with a field isolate of Salmonella enteritidis phage type 4. Chickens were sequentially necropsied at regular intervals throughout the 17-wk observation period. Gross and microscopic lesions were most evident between 1 and 14 days postinoculation (DPI). Gross lesions consisted of enlarged livers with white foci, enlarged and mottled white spleens, fibrinous exudate in the peritoneum, and atretic, misshapen ovarian follicles. Microscopic lesions included multifocal coagulative necrosis of hepatocytes and inflammation, fibrinous exudation in vascular sinuses of the spleen, and fibrinosuppurative inflammation of the peritoneum and ovarian follicles. The proportion of reproductive organ infections (ovary and oviduct) in the IV group, 83% (20/24, P = 0.007; 50% and 33% for strains A and strain B birds, respectively), was higher than that of the PO group, 46% (11/24; 29% and 17% for strains A and B, respectively), for the first 16 days of observation postinoculation. The proportion of fecal shedding for the IV group of birds was significantly (P = 0.009) lower, 29% (7/24; 33% and 25% respectively for strain A and strain B birds, respectively), than the PO group, 67% (16/24; 75% and 58% for strain A and strain B birds, respectively). Three (2.6%) of 234 egg pools were culture-positive for group D Salmonella from strain A chickens (1 of 119 pools from the IV group and 2 of 115 pools from the PO group of birds). Chickens infected with the field strain of S. enteritidis phage type 4 harbored the organism in tissues only for a brief time, most clearing within 8 DPI and nearly all within 16 DPI. Overall the percentage of culture-positive birds did not differ significantly (P > 0.05) between birds with and without lesions, but isolation of S. enteritidis tended to be more frequent when lesions were evident. This experiment also demonstrated that brown-egg-laying-type chickens were more susceptible than white-egg-laying-type chickens to S. enteritidis phage type 4 isolated from California based on gross and microscopic lesions and bacteriologic findings.     

Effect of prior serial in vivo passage on the frequency of Salmonella enteritidis contamination in eggs from experimentally infected laying hens

Experimental infection models are valuable tools for understanding and preventing the deposition of Salmonella enteritidis inside eggs. Oral inoculation is believed to closely simulate naturally occurring S. enteritidis infections of chickens, but oral infection studies have often generated relatively low frequencies of egg contamination. The present study assessed whether repeated in vivo passage of an S. enteritidis strain could affect its ability to cause egg contamination in experimentally infected hens. The incidence of egg contamination was determined in groups of hens inoculated orally with either a phage type 13a S. enteritidis strain or derivatives of this parent strain that were obtained by three successive rounds of passage and reisolation from tissues of infected hens. Passaged S. enteritidis isolates recovered from ovaries and oviducts induced a significantly higher incidence of egg contamination (16.97%) than was attributed to the parent strain (8.27%). However, passaged S. enteritidis isolates recovered from livers and spleens were not associated with a significantly increased frequency of deposition in eggs. By either inducing or selecting for the expression of relevant microbial properties, passage of S. enteritidis through reproductive tissues of chickens may be useful for improving the efficiency at which experimental infection models produce egg contamination.     

Pathologico-anatomic, bacteriologic and serologic findings in laying hens from small neighborhood flocks with Salmonella enteritidis phage type 4 infection

Two small free range flocks with 32 hens, whose Salmonella enteritidis contaminated eggs caused salmonellosis in man, were investigated for localisation of the agent. Salmonella enteritidis phage type 4 was isolated from 12 of 32 hens (37.5%). Eight hens (25%) harbored the bacterium in the ovary and/or the oviduct. The rapid slide agglutination test with Salmonella pullorum-antigen revealed 21 positive hens (66%). All eight hens with Salmonella enteritidis phage type 4 in the reproductive tract were seropositive. The significance of these findings for the control of Salmonella enteritidis phage type 4-infections in poultry is discussed.     

Isolation of Salmonella enteritidis from internal organs of experimentally infected hens

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.     

Growth of Salmonella typhimurium and Salmonella enteritidis in egg yolks from highly immunized hens.

This experiment was conducted to ascertain whether the growth of Salmonella Enteritidis (SE) or Salmonella Typhimurium (ST) would be suppressed in the presence of antibodies contained in egg yolks. Specific pathogen-free chickens (102 days of age) were subcutaneously immunized with oil-adjuvanted bacterin of SE or ST, twice within a four-week interval. During 160 to 170 days of age, eggs were collected, the yolks were removed and mixed with an equal volume of physiological buffered saline, inoculated with ten colony forming units (CFU) of SE or ST, and incubated at 37 degrees C or 20 degrees C for 23 hr. The growth of organisms in each yolk solution was examined. The egg yolk derived from non-immunized hens was examined in the same manner as the controls. There was no difference in the growth titer between the antibody-positive yolk and the negative yolk. The result suggests that the antibodies in the yolk do not influence the growth of each organism, even if the hens are highly immunized.     

Prevention of salmonella infections in laying hens by vaccination]

After multiple infections with S. enteritidis in humans and demonstration of S. enteritidis in egg products, S. enteritidis (lysotype 6, plasmid profile 40) could be isolated from organs of hens, anal swab tests of chicken, liquid manure, egg shells and non pasteurized egg contents. Because of the largeness of the hen farm prophylactic vaccinations seemed to be advisable additionally to improvement of the management and hygiene. A vaccine registered in 1987 for use in pigeons and water fowl "Zoosal oral Dessau" was used. As a test three were vaccinated three times and artificially infected. Cross immunity against S. enteritidis could be demonstrated. In October 1987 all chicken of the farm were vaccinated three times at the age of 4, 6 and 7 weeks; random samples were tested for immunity and cross immunity. Until the end of the laying period immunity against S. typhimurium could be proven in 98% of the hens, against S. enteritidis in 95%.     

Salmonella in Belgian laying hens: an identification of risk factors

Since the 1980s, the prevalence of Salmonella in Belgian poultry layers and broilers has greatly fluctuated with a rise observed in 2003 and a significant decrease in 2005. In order to alleviate the risk at egg consumer level, it is crucial to understand the factors which influence the contamination and the spread of Salmonella in laying hens. To study such determinants we explored the Belgian data from the 2005 baseline study on the prevalence of Salmonella in laying flocks of Gallus gallus in the European Union. The response variables corresponded to presence or absence of Salmonella from dust and faecal samples taken from the environment of a Belgian layer flock. The explanatory variables included: region of Belgium, sampling time (month the flock was sampled), production type (cage or barn and free range), Salmonella vaccination status, flock age and flock size. Analyses of these data were performed using a bivariate logistic regression model assuming independence between the two responses and bivariate generalized estimating equations model, which incorporates the correlation between the two responses on the same flock. The main risk factor that was identified was rearing flocks in cages compared to barns and free-range systems. The results also showed a significant higher risk for Salmonella for a 1 week increase in flocks’ age as well as with a unit increase in the size of the flock.