Vaccine 24 (2006)

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1 Vaccine 24 (2006) Oral immunisation of laying hens with the live vaccine strains of TAD Salmonella vac E and TAD Salmonella vac T reduces internal egg contamination with Salmonella Enteritidis Inne Gantois, Richard Ducatelle, Leen Timbermont, Filip Boyen, Lotte Bohez, Freddy Haesebrouck, Frank Pasmans, Filip van Immerseel Department of Pathology, Bacteriology and Avian Diseases, Research Group Veterinary Public Health and Zoonoses, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium Received 22 February 2006; received in revised form 18 May 2006; accepted 23 May 2006 Available online 9 June 2006 Abstract Eggs are a major source of human infections with Salmonella. Therefore controlling egg contamination in laying hen flocks is one of the main targets for control programmes. A study was carried out to assess the effect of oral vaccination with TAD Salmonella vac E, TAD Salmonella vac T and with both vaccines TAD Salmonella vac E and TAD Salmonella vac T, on colonization of the reproductive tract and internal egg contamination of laying hens with Salmonella Enteritidis. Three groups of 30 laying hens were vaccinated at 1 day, 6 weeks and 16 weeks of age with either one of the vaccine strains, or a combination of both vaccine strains, while a fourth group was left unvaccinated. At 24 weeks of age, the birds were intravenously challenged with 0.5 ml containing cfu Salmonella Enteritidis PT4 S1400/94. The number of oviducts from which Salmonella was isolated, was significantly lower in the vaccinated than in the non-vaccinated hens at 3 weeks post-challenge. Significantly less egg contents were Salmonella positive in the birds vaccinated with TAD Salmonella vac E or TAD Salmonella vac T (12/105 batches of eggs in both groups) than in the unvaccinated birds (28/105 batches of eggs). Internal egg contamination in the hens vaccinated with both TAD Salmonella vac E and TAD Salmonella vac T was even more reduced, as over the whole experiment, only one batch of eggs was positive. In conclusion, these data indicate that vaccination of laying hens with these live vaccines could be considered as a valuable tool in controlling internal egg contamination Elsevier Ltd. All rights reserved. Keywords: Salmonella Enteritidis; Vaccination; Internal egg contamination; Intravenous challenge 1. Introduction Salmonella Enteritidis has become a worldwide problem, due to the unique ability of this serotype to contaminate eggs without causing discernible illness in the infected hen [1]. Eggs can be contaminated with Salmonella Enteritidis by penetration through the egg shell from contaminated faeces during or after oviposition [2,3] or by direct contamination of yolk, albumen, egg shell membranes or egg shells before oviposition, originating from the infection of reproductive organs with Salmonella Enteritidis [4,5]. The association Corresponding author. Tel.: ; fax: address: inne.gantois@ugent.be (I. Gantois). between chicken reproductive tract infection, egg contamination and human food poisoning was a strong incentive to develop effective control programs focussing on parent birds and laying hens. One of the most widely used control measures is vaccination of laying hens. In several countries (e.g. in the UK), the implementation of vaccination in laying hens in national control programs led to a dramatic decline in the number of recorded human cases of Salmonella Enteritidis infection [6]. Currently, two types of Salmonella vaccines are commercially available: the bacterins and the live vaccines. It is claimed that currently available vaccines reduce shedding and colonization of host tissues. Studies documenting protection against egg contamination by vaccination of laying X/$ see front matter 2006 Elsevier Ltd. All rights reserved. doi: /j.vaccine

2 I. Gantois et al. / Vaccine 24 (2006) hens are however limited. Evidence of protection against egg contamination was shown in an intravaginal challenge model in hens immunized with an oil-emulsion bacterin [7]. More recently, an iron-restricted Salmonella Enteritidis bacterin vaccine was efficient at reducing egg contamination after intravenous challenge with Salmonella Enteritidis [8]. Because of the intracellular parasitism of Salmonella, it is believed that Salmonella vaccines must have the potential to induce a cell-mediated immune response, which is more achievable with live vaccines [9]. The efficacy of live vaccines in poultry has been tested in experimental and field studies but to our knowledge, only a few studies have demonstrated a clear protective effect of immunization against egg contamination [7,8,10,11]. Only a few live vaccines have been registered and are commercially available for use in poultry. The commercially available live Salmonella Gallinarum vaccine strain SG9R, a rough strain, has been shown to give cross-protection against Salmonella Enteritidis, as immunization was able to reduce egg contamination [10]. In the USA, a live Salmonella Typhimurium vaccine strain, attenuated by mutating genes coding for adenylate cyclase (cya) and the camp receptor protein (crp) is now used in the control of Salmonella infections in chickens. It was shown that this vaccine can induce an excellent protection against reproductive tract colonization and egg contamination [11]. Other vaccine strains were developed on the basis of the principle of metabolic drift mutation [12,13], i.e. they carry negative mutations in essential enzymes and metabolic regulatory centres. TAD Salmonella vac E and TAD Salmonella vac T vaccine strains are produced by chemical mutagenesis and have an increased permeability of the cell membrane to antibiotics and detergents due to the mutations [14]. As a consequence these mutants have prolonged generation times and corresponding reductions in virulence [14]. Scientific data on the efficacy of these metabolic drift mutant vaccine strains in protection against egg contamination is lacking. Therefore, in the present study, the efficacy of oral vaccination with the two commercially available metabolic drift mutant vaccine strains, TAD Salmonella vac E and TAD Salmonella vac T, on internal organ colonization and internal egg contamination with Salmonella Enteritidis was evaluated in an intravenous challenge model. 2. Materials and methods 2.1. Live vaccine strains and challenge inoculum TAD Salmonella vac E and TAD Salmonella vac T are metabolic drift mutants of Salmonella Enteritidis and Salmonella Typhimurium, respectively, and are produced by chemical mutagenesis [12,13]. TAD Salmonella vac Eis resistant to streptomycin and rifampicin and TAD Salmonella vac T is resistant to nalidixic acid and rifampicin. The vaccine strains were prepared according to the manufacturers instructions. The challenge strain was Salmonella Enteritidis S1400/94, kindly provided by Prof. M.J. Woodward, V.L.A., Weybridge, UK. The characteristics of this strain have been described previously [15]. The strain was statically incubated overnight at 37 C in Luria Bertani (LB) medium (Sigma, St. Louis, MO, USA). Ten-fold dilutions were plated on brilliant green agar (BGA) (Oxford, Basingstoke, Hampshire, UK) to determine the titer. The bacterial suspension was diluted in phosphate buffered saline (PBS) to 10 8 colony forming units (cfu) per ml, and this inoculum was used to challenge the birds Animals One hundred and twenty SPF Lohmann White LSL hens (Lohmann Tierzucht GMBH, Cuxhaven, Germany) were housed in battery cages in two separate rooms. The nonvaccinated and the double vaccinated birds were kept in one room and the single vaccinated birds also (empty battery cages were in between the two groups so there was no contact between the two different groups of birds kept in one room). The animals received 12 h of light per day and had free access to food and water Experimental set-up Three different groups (n = 30) of animals were orally immunized at the day of birth, at 6 weeks of age and finally at 16 weeks of age through crop instillation of 0.5 ml containing 10 8 cfu of TAD Salmonella vac E (group 1), 10 8 cfu of TAD Salmonella vac T (group 2), or 10 8 cfu of each TAD Salmonella vac E and TAD Salmonella vac T (group 3). A fourth group of birds (n = 30) was kept as non-immunized but Salmonella challenged positive controls (group 4). At the age of 18 weeks, serum samples were taken from six hens of each group to test for anti-salmonella Enteritidis and anti-salmonella Typhimurium antibodies in a LPS-ELISA [16]. The LPS was purified of Salmonella Enteritidis PT4, strain NIDO 76Sa88 and Salmonella Typhimurium, strain 742Sa91. A 1/200 dilution of the sera (100 l) was added to the wells. As negative control, sera from a Salmonella free specific pathogen free (SPF) chicken and as positive control, sera from a chicken that had been infected experimentally with Salmonella Enteritidis PT4, strain NIDO 76Sa88, was used. The cut-off OD value was calculated as the mean obtained from the sera from the Salmonella free chickens (the non-vaccinated birds) plus five times the standard deviation. At the same time, cloacal swabs were taken from six animals in each group, and bacteriologically examined for the presence of the vaccine strains. The swabs were incubated overnight at 37 C in buffered peptone water (BPW) (Oxoid, Basingstoke, Hampshire, UK) and afterwards a loopful was plated on LB plates supplemented with 200 g/ml streptomycin (Sigma, St. Louis, MO, USA) and 200 l/mg rifampicin (Sigma, St. Louis, MO, USA) for detection of

3 6252 I. Gantois et al. / Vaccine 24 (2006) TAD Salmonella vac E and on LB plates supplemented with 20 g/ml nalidixic acid (Sigma, St. Louis, MO, USA) and 200 g/ml rifampicin for detection of TAD Salmonella vac T. At 21 weeks of age, all the hens were in lay and eggs were collected daily during 3 weeks for bacteriological analysis for detection of the vaccine strain in the egg content. At 24 weeks of age, all the animals were intravenously inoculated in the wing vein with 0.5 ml containing cfu of the Salmonella Enteritidis strain S1400/94. This protocol was already used to produce high levels of internal egg contamination [17]. The eggs were collected daily during 3 weeks. Three weeks after challenge inoculation, all the animals were euthanized by intravenous embutramid injection (T61, Intervet, Belgium). A part of the liver and the whole caeca, spleen, oviduct and ovary were aseptically removed Bacteriological examination of the challenged birds Caeca, spleen, liver, oviduct and ovary were weighed. The organs were diluted 10 times in BPW and homogenized with a stomacher. The number of cfu of Salmonella bacteria/g of sample was determined by direct plating of 10-fold dilutions of the organs (in PBS) on BGA. Samples that tested negative after direct plating were pre-enriched overnight at 37 Cin BPW (1/10) and afterwards enriched in tetrathionate brilliant green broth by overnight incubation at 37 C. Then, a loopful of the tetrathionate brilliant green broth was plated on BGA Egg production and bacteriological examination of eggs Eggs were collected daily and the egg production was measured. Each day, eggs of six chickens per group were pooled in one batch so the number of eggs per batch varied between one and six. Upon collection, faeces on the surface of the eggs were removed and the eggs were decontaminated by dipping in ethanol (95%) for 1 min. The eggs were broken aseptically and the total content of the eggs was pooled and homogenized. A volume of forty ml of BPW per egg was added to the pooled egg content and incubated 48 h at 37 C. To detect the vaccine strains, a loopful of the enrichment broth was plated on LB plates supplemented with 200 g/ml streptomycin and 200 l/ml rifampicin or with 20 g/ml nalidixic acid and 200 g/ml rifampicin. For isolation of the challenge strain, further enrichment overnight at 37 C was done in tetrathionate brilliant green broth and after incubation, a loopful of broth culture was streaked onto BGA. Antibiotic resistance testing was then used to confirm that the colonies were challenge strain bacteria Statistical analysis The data were analysed with SPSS 12.0 software using binary regression to compare the numbers of positive tissue samples, the numbers of positive egg pools and the number of eggs that were laid after challenge. 3. Results 3.1. Serological analysis As appeared from the LPS-ELISA detecting anti- Salmonella Enteritidis antibodies, four hens in the group vaccinated with TAD Salmonella vac E were positive and five hens in the group vaccinated with both TAD Salmonella vac E and TAD Salmonella vac T had OD values higher than the cut-off value. Antibodies to Salmonella Typhimurium were measured with the Salmonella Typhimurium LPS-ELISA. Three hens vaccinated with TAD Salmonella vac T and four hens vaccinated with the combined TAD Salmonella vac E and TAD Salmonella vac T were positive. In both LPS-ELISA tests, the OD values of the sera from the animals, vaccinated with the combined TAD Salmonella vac E and TAD Salmonella vac T vaccines were higher than sera from hens, vaccinated with either TAD Salmonella vac E or TAD Salmonella vac T. Sera collected from the hens vaccinated with TAD Salmonella vac E were negative for Salmonella Typhimurium antibodies and sera obtained from hens vaccinated with TAD Salmonella vac T were negative for Salmonella Enteritidis antibodies, except once, where an OD value above the cut-off value was found (Table 1) Analysis of cloacal swabs and eggs for the presence of vaccine strains The vaccine strains were not isolated from any of the cloacal swabs and the egg content samples Clinical signs and egg production after challenge Over the whole experiment, one bird died in the unvaccinated group and two died in the single TAD Salmonella vac E vaccinated group. In the first week after challenge infection, the non-vaccinated birds and the birds vaccinated with the single vaccine strains were depressed and their water and feed intake was reduced. In contrast, the animals vaccinated with the combined TAD Salmonella vac E and TAD Salmonella vac T vaccine did not show any clinical signs and their feed and water intake was not reduced. The egg production rate after infection in the unvaccinated group was 51%, 39% and 60% in the first, second and third week post-infection (pi) (Fig. 1). The vaccinated hens recovered faster from the challenge infection compared with the unvaccinated hens and their egg production was already increasing again in the second week pi. In the group vaccinated with both vaccine strains, the egg pro-

4 I. Gantois et al. / Vaccine 24 (2006) Table 1 Anti-Salmonella Enteritidis- and anti-salmonella Typhimurium LPS-ELISA OD values in sera (measured at OD 492 nm ) of six hens 2 weeks after the final vaccination (16 weeks) in the group of animals vaccinated with TAD Salmonella vac E (SE), TAD Salmonella vac T (ST) and with both vaccine strains TAD Salmonella vac E/TAD Salmonella vac T (SE/ST) Chicken number Anti-Salmonella Enteritidis LPS-ELISA OD values Anti-Salmonella Typhimurium LPS-ELISA OD values Non-vaccinated SE ST SE/ST Non-vaccinated SE ST SE/ST The cut-off OD value, measured according to Desmidt et al. [16], was and for the anti-salmonella Enteritidis LPS-ELISA and the anti-salmonella Typhimurium LPS-ELISA, respectively Isolation of the challenge strain from the organs of the hens Fig. 1. The egg production rate in the unvaccinated and the vaccinated groups at 1 3 weeks after challenge with Salmonella Enteritidis. Vaccination was done with TAD Salmonella vac E (SE), TAD Salmonella vac T (ST) and with both vaccine strains TAD Salmonella vac E and TAD Salmonella vac T (SE/ST). duction rate was 57%, 66% and 82% in the first, second and third week after infection, respectively. Over the whole experiment, infection with the challenge strain resulted in a significantly larger reduction of egg production in the nonvaccinated birds (50%) than in the birds vaccinated with both vaccine strains (68%) (P = 0). There was no significant difference in number of eggs laid by the unvaccinated hens, the single vaccinated hens with TAD Salmonella vac E (51%) and the single vaccinated hens with TAD Salmonella vac T (57%). Some hens of the non-vaccinated group laid some malformed, thin-shelled eggs in the first week of infection. Results of the isolation of the Salmonella Enteritidis challenge strain from different organs at 1 3 weeks after challenge are summarized in Table 2. Three weeks after challenge, Salmonella Enteritidis was not isolated from any of the caeca from all groups. Spleen samples were only positive after enrichment, and the number of positive spleen samples in non-immunized birds (80%) was significantly higher than in TAD Salmonella vac E (43%), in TAD Salmonella vac T (50%) and in the combined TAD Salmonella vac E/TAD Salmonella vac T vaccinated birds (30%). Few liver samples were Salmonella positive after enrichment. Salmonella was isolated with a high frequency from the ovaries in the nonvaccinated group (80%). In contrast to the non-immunized hens, fewer ovaries were positive in both of the groups, vaccinated with either TAD Salmonella vac E (57%) or vac T (57%), and in the group of animals, vaccinated with both vaccine strains (37%). The number of Salmonella positive oviduct samples was significant higher in the unvaccinated group with 51% of the samples being positive after enrichment, compared to the single TAD Salmonella vac E (21%) and TAD Salmonella vac T (13%) vaccinated hens, and the hens vaccinated with both vaccine strains (13%) Isolation of the challenge strain from egg contents Table 3 presents the percentage of positive pools of eggs in the four different groups. During the first week, a total Table 2 Isolation of Salmonella Enteritidis from the organs of the unvaccinated control hens and the hens orally vaccinated with the live vaccine strains TAD Salmonella vac E, TAD Salmonella vac T or both TAD Salmonella vac E and TAD Salmonella vac T Group Spleen Liver Oviduct Ovary Control 23/29 a (0 b ) A 2/29 (0) A 15/29 (1) A 23/29 (14) A TAD Salmonella vac E 12/28 (0) B 1/28 (0) A 6/28 (1) B 16/28 (12) AB TAD Salmonella vac T 15/30 (0) B 6/30 (0) A 4/30 (0) B 17/30 (9) AB TAD Salmonella vac E/TAD Salmonella vac T 9/30 (0) B 1/30 (0) B 4/30 (0) B 11/30 (7) B Values with different letters are statistically significant different from each other within the same column (P < 0.05). a Number of positive samples after enrichment/total number of samples. b Number of positive samples after direct plating.

5 6254 I. Gantois et al. / Vaccine 24 (2006) Table 3 Results of bacteriological examination of batches of eggs (comprising one to six eggs and containing the egg contents) from the vaccinated and the unvaccinated groups at 1 3 weeks after challenge with Salmonella Enteritidis Group Week 1 Week 2 Week 3 Control 15/35 a A 7/35 A 6/35 A TAD Salmonella vac E 9/35A * 2/35 * AB 1/35 * AB TAD Salmonella vac T 9/35 * A 2/35 * AB 1/35 * AB TAD Salmonella vac E/TAD Salmonella vac T 1/35 B 0/35 B 0/35 B Values with different letters are statistically significant different from each other within the same column (P < 0.05). a Number of positive batches/total number of batches. * Statistically different from the control animals at P < 0.1. of 34 batches were positive for Salmonella but from 1 week post-infection on, the number declined rapidly, resulting in 11 and 8 positive batches of eggs in the second and third week, respectively. The unvaccinated hens laid more positive eggs than vaccinated ones during the whole 3-week period. In the group of animals immunized with the combined vaccine, only one batch of eggs was positive over the whole experiment. When analysed collectively for all groups, 27% of the batches of the control group were culture-positive, whereas only 11% of the batches of the group of animals, vaccinated with the single TAD Salmonella vac Eorvac T strains was positive for Salmonella. Moreover, for the group vaccinated with both vaccine strains, only in 1% of the batches Salmonella was detected. 4. Discussion TAD Salmonella vac E and TAD Salmonella vac T have been used for years in different countries to control Salmonella infections in poultry flocks [14]. Active immunization with these live vaccines protects the birds from mortality and significantly reduces faecal excretion by Salmonella Enteritidis and Salmonella Typhimurium [14].In the present study, it was shown that immunisation with these attenuated strains also reduces reproductive tract colonization and internal egg contamination in hens intravenously challenged with Salmonella Enteritidis. The vaccine strains TAD Salmonella vac E and TAD Salmonella vac T were not detected in a total of 1575 eggs from the vaccinated groups. Immunization with the single live vaccine strains of TAD Salmonella vac E and TAD Salmonella vac T and with both vaccine strains reduced organ colonization and internal egg contamination by Salmonella Enteritidis. Indeed, we observed a significant reduction of Salmonella Enteritidis positive oviduct samples in the vaccinated animals, and this was reflected in lower egg contamination rates. The difference in egg contamination between the vaccinated and non-vaccinated groups is even higher, when it is taken into account that the batches of eggs from unvaccinated animals contained generally less eggs due to the decrease in egg production. Interestingly, the TAD Salmonella vac T, containing a Salmonella Typhimurium strain, was almost equally effective as TAD Salmonella vac E, containing a Salmonella Enteritidis strain, in the induction of a protective response against challenge with Salmonella Enteritidis. Previous experiments with TAD Salmonella vac T also demonstrated cross-protection against intestinal colonization with Salmonella Enteritidis, although the protection was only partial when compared with the homologous protection [14]. Other scientific reports also mention cross-protection between serogroups. A genetically modified cya crp Salmonella Typhimurium vaccine strain was able to confer protection against Salmonella Enteritidis and other homologous and heterologous Salmonella serotypes. Vaccination induced cross-protection against intestinal colonization and colonization of the spleen, bursa of fabricius and ovary [18]. Another study demonstrated that vaccination with the cya crp Salmonella Typhimurium strain prevented transmission of Salmonella Enteritidis into eggs [11]. In contrast, Parker et al. [19] found no significant protection against egg contamination or reproductive tract colonization in laying hens orally vaccinated with an aroa-mutant of Salmonella Typhimurium after challenge with Salmonella Enteritidis. Vaccination with TAD Salmonella vac E and with both TAD Salmonella vac E and TAD Salmonella vac T induced Salmonella Enteritidis specific antibodies in serum. Although vaccination with TAD Salmonella vac T was effective against challenge with Salmonella Enteritidis, there was no cross reaction between anti-salmonella Typhimurium antibodies and the Salmonella Enteritidis LPS, except in one animal. Cross reactions between the Salmonella Typhimurium LPS antigen and the Salmonella Enteritidis antiserum and between the Salmonella Enteritidis LPS antigen and the Salmonella Typhimurium antiserum were not detected. This is rather unexpected because it is described that LPS from group B and D organisms, contain a common O antigens, which are responsible for cross reactions [20]. Hens vaccinated with both vaccine strains displayed higher ELISA OD values than the single vaccinated ones and this is probably correlated with a double dose of immunisation. Withanage et al. [21] demonstrated that the dynamics of the antibody levels in the oviduct are similar to those in the serum and that IgM and IgG levels reach a peak by 14 days post inoculation and remain elevated until the end of their experiment, which was 5 weeks. The higher ELISA OD values in the animals, vaccinated with the combined TAD Salmonella vac E and TAD Salmonella vac T vaccine could correlate with a lower recovery of challenge strain bacteria from the reproductive organs compared to the single TAD Salmonella vac E vaccinated hens. However, in two experimental trials, where laying hens were infected with Salmonella Enteritidis, there was no consistent relationship between the magnitude of the antibody responses of individual hens and the frequency at which they laid contaminated eggs [22]. It is proposed that cell-mediated immunity is more important than humoral responses for tissue clearance of virulent strains in poultry

6 I. Gantois et al. / Vaccine 24 (2006) [23]. As the induced protective effect in the birds vaccinated with TAD Salmonella vac T was not most likely due to the humoral antibody response, it could be correlated with a high cell-mediated response. This has however not been measured. In conclusion, the challenge study demonstrates a remarkable reduction of Salmonella colonization in the reproductive tract and internal egg contamination in laying hens vaccinated with live vaccines TAD Salmonella vac E and TAD Salmonella vac T in comparison to non-vaccinated control groups. The experimental results underline the high value of routine vaccination practices to complement other control measures to combat Salmonella in laying hen flocks and to aid in preventing transmission of Salmonella to eggs. Acknowledgements The authors would like to express their appreciation to Gunter Massaer, Venessa Eeckhaut, Paul De Groot, Maja Marien, Hilde Moyaert and Sofie Debruykere for their assistance during the bacteriological analysis. Furthermore, we would like to thank Dr. I. Schröder and Dr. D. Rebeski (Lohmann Animal Health GmbH & Co., KG, Cuxhaven, Germany) for helpful discussions. Dr. Filip van Immerseel is supported by a post-doctoral research grant of the Ghent University (BOF). References [1] Hedberg CW, David MJ, White KE, MacDonald KL, Osterholm MT. Role of egg consumption in sporadic Salmonella Enteritidis and Salmonella Typhimurium infections in Minnesota. J Vet Res 1993;59: [2] Barrow PA, Lovell MA. Experimental infection of egg-laying hens with Salmonella Enteritidis phage type 4. Avian Pathol 1991;20: [3] Humphrey TJ, Chart H, Baskerville A, Rowe B. The influence of age on the response of SPF hens to infection with Salmonella Enteritidis PT4. 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Egg contamination by Salmonella serovar Enteritidis following vaccination with -aroa Salmonella Typhimurium. FEMS Microbiol Lett 2001;195:73 8. [20] Chart H, Rowe B, Baskerville A, Humphrey TJ. Serological response of chickens to Salmonella Enteritidis infection. Epidemiol Infect 1990;104: [21] Withanage GSK, Sasai K, Fukata T, Miyamoto T, Baba E. Secretion of Salmonella-specific antibodies in the oviducts of hens experimentally infected with Salmonella Enteritidis. Vet Immunol Immunopathol 1999;67: [22] Gast RK, Holt PS. The relationship between the magnitude of the specific antibody response to experimental Salmonella Enteritidis infection in laying hens and their production of contaminated eggs. Avian Dis 2001;45: [23] Nagaraja KV, Rajashekara G. Vaccination against Salmonella enterica serovar Enteritidis infection: dilemma and realities. In: Saeed AM, editor. Salmonella enterica serovar Enteritidis in humans and animals. Ames, USA: Iowa State University Press; p