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.     

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.     

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.     

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.     

Serological detection of experimental Salmonella enteritidis infections in laying hens

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.     

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%).     

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.     

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.     

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.     

The relationship between the duration of fecal shedding and the production of contaminated eggs by laying hens infected with strains of Salmonella enteritidis and Salmonella Heidelberg

Egg contamination by Salmonella Enteritidis has remained a significant public health problem for nearly two decades, and Salmonella Heidelberg has also been recently implicated in egg-transmitted human illness. Colonization of the intestinal tract is a necessary precursor to the invasion of reproductive organs and subsequent deposition inside eggs laid by infected hens, but the relationship between the persistence of Salmonella in the intestinal tract and the likelihood of egg contamination has been uncertain. In this study, groups of laying hens were inoculated with large oral doses of strains of Salmonella Enteritidis and Salmonella Heidelberg, including variants of the original parent strains that had been reisolated from eggs laid by infected hens in a prior study. The shedding of Salmonella in voided feces was monitored for 6 wk postinoculation, and all eggs laid by infected hens between 5 and 22 days postinoculation were cultured for Salmonella in their contents. The mean duration of fecal shedding was significantly longer for the previously passaged Salmonella strains (26.7 days) than for the original parent strains (17.5 days), and the passaged strains caused a significantly higher frequency of egg contamination (6.4%) than did the parent strains (3.3%). However, the duration of fecal shedding and the frequency of egg contamination were not correlated for any of the Salmonella Enteritidis or Salmonella Heidelberg strains.     

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.     

Salmonella enteritidis in chicken eggs

After an outbreak caused by salmonella enteritidis (SE) of which home-made mayonnaise and remoulade sauce were found to be the cause, SE was detected in ten of the remaining eggs. Subsequently 409 eggs of the next shipment from the same outlet were examined bacteriologically. Of 70 of the eggs examined, SE was found in 5 egg yolk samples and in 3 egg white samples as well as in 2 of 7 pooled shell samples. Five out of 35 pooled whole egg samples comprising a total of 349 eggs were likewise positive for SE. The isolates belonged to the phage types 4 and 7 carried the virulence plasmid pRQ29.     

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.     

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.     

Longitudinal monitoring of two commercial layer flocks and their environments for Salmonella enterica serovar enteritidis and other Salmonellae

Between August 20, 2001, and September 17, 2002, 1429 samples including drag swabs, egg belt or egg rollout swabs, fan-blade swabs, rodent organ and intestinal pools, beetle (Alphitobius diaperinus) pools, housefly (Musca domestica) pools, chicken organ and intestinal pools, and egg pools were obtained for Salmonella culture from two flocks from two different commercial layer ranches. The two ranches were purposefully selected for the study based on their previous status of Salmonella Enteritidis isolation using environmental drag swabs in cooperation with practicing veterinarians. Salmonella sp. was isolated from 337 out of 979 (34.42%) non-egg samples. No Salmonella was isolated from 450 egg pools collected from either ranch. S. enteritidis was isolated from samples obtained from ranch 1 from manure drag swabs, 4/284 (1.4%); rodent organs, 1/24 (4.2%); and housefly pool cultures 1/21 (4.8%). Salmonella Enteritidis was isolated from ranch 2 from mouse organ and intestinal pool samples, 1/24 (4.2%). Salmonella group B was isolated from all sample types except the insects. There was a statistically significant difference in isolation rates among seven serogroups of Salmonella: groups B, C1, C2, D, E, K, and untypeable (Pearson chi-square 18.96, P = 0.002). Overall, statistically significant differences were observed with respect to Salmonella isolation among the types of samples taken (Pearson chi-square 118.54, P < 0.0001). Intensive monitoring for Salmonella Enteritidis can be used to optimize a Salmonella reduction program for an individual poultry biosecurity unit.     

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.     

Serological and bacteriological investigations of chickens from flocks naturally infected with Salmonella enteritidis

Groups of 10 birds were obtained from four flocks which had shown evidence of natural salmonella infection. S enteritidis had been isolated from three flocks and S typhimurium from the fourth. Each bird was housed in a separate cage and blood samples and cloacal swabs were taken weekly to follow the course of natural infection. After four weeks the birds were killed and examined post mortem. The isolation of Salmonella species could not be related to the serological results. In individual birds the rapid slide test and tube agglutination test could not be relied upon to detect infection; the microantiglobulin test and the enzyme-linked immunosorbent assay (ELISA) were more sensitive than the other tests and detected some infected birds that were negative by the rapid slide and tube agglutination tests, and also showed high titres in some birds from which Salmonella species could not be isolated post mortem. Sera obtained from two flocks which had a history of natural S enteritidis infection were evaluated by all the tests; evidence of infection was found with the microantiglobulin and ELISA tests but not with the other tests.     

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.     

The relationship between the magnitude of the specific antibody response to experimental salmonella enteritidis infection in laying hens and their production of contaminated eggs.

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.