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Keywords:

  • Salmonella ;
  • vaccine;
  • egg contamination;
  • efficacy

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

To evaluate the efficacy of a novel attenuated Salmonella Enteritidis (△lon△cpxR) vaccine candidate (JOL919), chickens were immunized through oral and intramuscular routes to reduce egg contamination against S. Enteritidis challenge. Birds were orally immunized with JOL919 on the first day of life and were subsequently boosted in the 6th and 16th weeks through oral (group B) or intramuscular (group C) route, while control birds were unimmunized (group A). The chickens of all groups were challenged intravenously with the virulent S. Enteritidis strain in the 24th week. The immunized groups B and C showed significantly higher plasma IgG and intestinal secretory IgA levels as compared to those of the control group. The lymphocyte proliferation response and CD45+CD3+ T-cell number in the peripheral blood of the groups B and C were significantly increased. In addition, the egg contamination rates were significantly lower in the group B (0%, 10.7% and 0%) and the group C (3.6%, 14.3% and 3.6%) as compared to the group A (28.6%, 42.8% and 28.6%) in the 1st, 2nd and 3rd weeks post-challenge. All animals in the groups B and C showed lower organ lesion scores in the liver and spleen and lower bacterial counts in the liver, spleen and ovary at the 3rd week post-challenge. These results indicate that this vaccine candidate can be an efficient tool for prevention of Salmonella infections by inducing protective humoral and cellular immune responses. In addition, this vaccine did not prevent egg contamination, but did appear to reduce incidence. Booster immunizations, especially via oral administration route, showed an efficient protection against internal egg contamination with S. Enteritidis.

Impacts
  • The oral route of immunization was more effective than the intramuscular route for reducing internal organ colonization and egg contamination by Salmonella Enteritidis.
  • Immunization of poultry with attenuated live vaccines by the oral route supports efforts to reduce human salmonellosis resulting from contamination by Salmonella Enteritidis.
  • Assay-based evaluation for the cellular and mucosal sIgA immune responses indicated that the oral prime and booster immunization strategy is the optimal option.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) is a major cause of food-borne disease. Poultry-derived products, particularly meat and chicken eggs, are considered a major source of human infection with this pathogen (Rampling, 1993; Bäumler et al., 2000). Eggs can be contaminated with S. Enteritidis by penetration through the egg shell from contaminated faeces (Barrow and Lovell, 1991; Humphrey et al., 1991) or by direct contamination from the infection of reproductive organs (Shivaprasad et al., 1990; De Buck et al., 2004). It is believed that the most important route of egg contamination is via infected reproductive tissues (Gast and Beard, 1990; Keller et al., 1995). To control S. Enteritidis infection in chickens, different strategies have been used at poultry breeding and rearing farms. Among the strategies used, vaccination is a supportive approach for establishing immunity in birds for prevention of Salmonella infection in poultry farms (Bäumler et al., 2000). It has been suggested that vaccination of laying hens has contributed to a dramatic decline in the number of recorded human cases of S. Enteritidis infection (Cogan and Humphrey, 2003).

Although most commercially available vaccines are in the killed bacteria form (WHO, 2002), a few registered S. Enteritidis live vaccines are commercially available for poultry. These live vaccines are developed on the principle of either metabolic drift mutations or auxotrophic double-marker mutants obtained through chemical mutagenesis (EFSA, 2004). It has been suggested that live vaccines may be more effective at preventing Salmonella infection because they may elicit higher cellular immunity compared with killed vaccines (Babu et al., 2003). It has also been claimed that currently available Salmonella vaccines reduce colonization of bacteria in the host tissues (Selke et al., 2007; Jiang et al., 2010). However, the efficacy of live vaccines is variable and not always satisfactory because residual virulence may remain that requires host clearance (Matsui et al., 2003; Takaya et al., 2003; Singh, 2009). The efficacy of live vaccines in poultry has been examined in experimental and field studies, but to our knowledge, only a few studies have shown improvements in the protective effect of immunization against egg contamination (Nassar et al., 1994; Hassan and Curtiss, 1997; Gantois et al., 2008).

The humoral immune response seems to be necessary for the protection against Salmonella infection (Mittrucker et al., 2000). In addition, cell-mediated immune mechanisms are presumed to be of special importance for resolution of Salmonella infections (Carvajal et al., 2008). We are not aware of any data published before the present study on the potentials of immune induction through different routes to reduce egg contamination against salmonellosis. We performed this study to evaluate the efficacy of a newly developed attenuated S. Enteritidis vaccine candidate using oral and intramuscular routes of inoculations to reduce internal egg contamination with Salmonella. To evaluate the vaccine efficacy, we used ELISA assays to measure plasma IgG and intestinal sIgA antibody titres to S. Enteritidis-specific antigen, a lymphocyte proliferation assay for induction of cellular immunity, flow cytometry to analyse the population of CD45+CD3+ T cells, observation of gross lesions and bacterial recovery from the internal organs. We also examined the egg production and internal egg contamination rates after virulent S. Enteritidis challenge.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Animals

One-day-old, female Brown Nick chickens were used in this study and given antibiotic-free food and water ad libitum. The animal experiments mentioned in this study were conducted with approval (CBU 2011-0017) from the Chonbuk National University Animal Ethics Committee, in accordance with the guidelines of the Korean Council on Animal Care.

Bacterial strains and growth conditions

The S. Enteritidis mutant strain JOL919 was constructed by deletion of the lon and cpxR genes of the wild-type S. Enteritidis JOL860 as described previously (Nandre et al., 2011). The wild-type S. Enteritidis isolate JOL1182 was used as a virulent challenge strain (Nandre et al., 2011). All strains were grown in Luria–Bertani (LB) broth (Difco, Sparks, MD, USA) at 37°C with shaking at 40 g, washed three times with phosphate-buffered saline (PBS, pH 7.4) and adjusted to appropriate concentrations based on the optical density at 600 nm for inoculations.

Immunization and challenge inoculations

Two different groups (= 20) of birds were orally immunized on the first day of life, and subsequent booster immunizations were performed in the 6th and 16th weeks through oral (group B) or intramuscular (group C) administration of 0.1 mL containing 1 × 107 colony-forming units (cfu) of live attenuated vaccine. Control birds (= 20) were orally inoculated on the first day of life and in the 6th and 16th weeks with 0.1 mL PBS as the unimmunized control group (group A). In the 24th week, all birds were intravenously challenged in the wing vein with 0.5 mL containing 1 × 10cfu of the S. Enteritidis virulent strain JOL1182.

Enzyme-linked immunosorbent assay (ELISA) for systemic IgG and mucosal IgA

Plasma and intestinal wash samples were collected from five birds of each group to determine systemic IgG and mucosal IgA, respectively, as described previously (Nandre et al., 2011). Indirect ELISA was performed with an outer membrane protein fraction (OMP) antigen, which was extracted from the wild-type S. Enteritidis JOL860 (Kang et al., 2002). Chicken IgG and IgA ELISA Quantitation Kits (Bethyl laboratories, USA) were used for determination of IgG and IgA concentrations, respectively. Microlon ELISA plate wells (Greiner Bio-One GmbH, Germany) were coated with OMP at a concentration of 0.2 mg/mL. The plasma samples were diluted 1 : 80 with PBS, and intestinal wash samples were diluted 1 : 4 with PBS. After dilution, these samples were added to the OMP-coated plates and incubated for 1 h, followed by reaction with goat anti-chicken IgG and IgA horseradish peroxidase (HRP) conjugates for 1 h. The bound HRP activity was determined using o-phenylenediamine dihydrochloride (Sigma-Aldrich, St. Louis, MO, USA). The OD492 was determined with an ELISA reader after adding 3m H2SO4 to stop the reaction.

Lymphocyte proliferation assay

The peripheral lymphocytes were separated from the blood of five randomly selected chickens per group using the gentle swirl technique (Gogal et al., 1997). After trypan blue dye exclusion testing, 100 μL of viable mononuclear cell suspensions at 1 × 105 cfu/mL in RPMI-1640 medium was incubated in triplicate in 96-well tissue culture plates with 50 μL of medium alone or medium containing 4 μg/mL of soluble antigen at 40°C in a humidified 5% CO2 atmosphere for 72 h. The proliferation of stimulated lymphocytes was measured using adenosine triphosphate (ATP) bioluminescence as a marker of cell viability with a ViaLight® Plus Kit (Lonza Rockland Inc., Rockland, ME, USA) to provide an estimation of mitochondrial activity (Crouch et al., 1993) according to the product information. The emitted light intensity was measured using a luminometer (TriStarLB941, Berthold Technologies GmbH & Co., Bad Wildbad, Germany) with an integrated program for duration of 1 s. The blastogenic response against the specific antigen was expressed as the mean stimulation index (SI), as previously reported (Rana and Kulshreshtha, 2006).

Flow cytometric analysis

For analysis of changes in T-cell composition upon Salmonella vaccination, heparinized blood from the wing vein of each individual animal (n = 5) was aseptically collected at the fourth week after prime and booster immunizations. The collected blood samples were mixed with 3% hetastarch (Sigma Immuno Chemicals, USA) at a ratio of 1 : 2 and centrifuged at 65 × g for 10 min to allow erythrocytes to sediment. The cells of the supernatant were used for flow cytometric analysis of blood lymphocytes, as described previously (Crouch et al., 1993). Then, 100 μL of isolated leucocytes was incubated with the APC-conjugated monoclonal antibody CD45 and with FITC-labelled monoclonal antibody CD3 (Beckman Coulter, USA) for 20 min in the dark. After washing the cells, aliquots of 2 × 104 cells per sample were analysed using a FACSCalibur (BD Bioscience, Heidelberg, Germany) equipped with a 15 mW, 488 nm (FITC) and 635 nm (APC) argon ion laser. The percentages of positively stained cells were calculated with the CellQuestPro 4.0.2 software (BD Bioscience, Germany).

Egg contamination bacteriology to isolate the vaccine strain

At 21st week of age, all hens were in lay. Eggs were collected daily for 3 weeks before virulent S. Enteritidis (JOL1182) challenge to hens. Surfaces of the eggs were cleaned to remove droppings, and the eggs were dipped in ethanol for 1 min. Eggs were broken aseptically and contents were pooled. A volume of 40 mL of buffered peptone water (BPW, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) per egg was added to the pooled egg content and homogenized. A loop full of the homogenized content was plated on BGA plates. For enrichment, the egg content samples were further incubated overnight at 37°C in BPW, transferred to Rapport Vassiliadis R10 (RV, Becton, Dickinson and Company, USA) broth and kept at 42°C for 48 h. A loop-full was then plated on Brilliant Green Agar (BGA, Becton, Dickinson and Company, USA).

Assessment of egg production

To assess the influence of vaccination on egg production, egg production rates were observed for 3 weeks after challenge. Eggs laid by the immunized and control birds were collected daily for 3 weeks. The egg production rate was calculated as follows: egg production rate per week = (the total number of eggs laid)/ (the number of hens × 7 days) × 100.

Isolation of challenge strain from eggs

Each day, the eggs from five chickens per group were pooled in one batch, so the number of eggs per batch varied between one and five. For each group, total batches were 28 for 20 birds in a week (four batches in a day). Eggs were processed as described above. A volume of 40 mL of BPW per egg was added to the pooled egg content and homogenized. To detect the challenge strain, a loop full of the broth was plated on BGA plates. Negative samples after direct plating were kept for further enrichment in RV broth, as described previously. All positive samples were confirmed by PCR, as described previously (Matsuda et al., 2010). The egg contamination rates were calculated as follows: egg contamination rate per week = (the total number of infected batches)/ (the total number batches × 7 days) × 100.

Observations of gross lesions and bacterial recovery from infected organs

Hens were euthanized 3 weeks after challenge. Gross lesions of enlarged and necrotic foci in the liver and spleen were given scores of 0, 1, 2 or 3. A score of 0 indicated no lesion, a score of 1 was assigned to necrotic foci, a score of 2 indicated enlarged and necrotic organs, and a score of 3 was associated with more debilitated, necrotic and distorted organs. Pieces of the liver, spleen, caecum and ovary were aseptically removed. Organs were weighed and then suspended in 2 mL BPW and homogenized. The cfu of Salmonella/g for each sample was determined by direct plating of 100 μL of the organ suspensions on BGA. The bacterial cell number was counted and expressed as mean ± standard deviation (SD) log10 cfu/g, as previously described (Betancor et al., 2005). Samples that tested negative after direct plating were pre-enriched overnight at 37°C in BPW and enriched in RV broth for 48 h at 42°C. Then, a loop full of the RV broth was plated on BGA. Colonies were confirmed by PCR using the lon/cpxR gene-specific primer sets (Matsuda et al., 2010).

Statistical analysis

The statistical analyses were performed with the spss 16.0 program (SPSS Inc., Chicago, IL, USA). For the immunological analysis, the mean optical density (OD) values, SI values and flow cytometric data obtained from this experiment were compared between the immunized and control birds by Mann–Whitney U-test. In addition, the Mann–Whitney U-test was used for the comparison of gross lesion scores and bacterial count between the immunized and control groups. For differences in frequency of egg contamination between the immunized and control hens, Fisher's exact probability test was applied. The probability values, which are considered to be significant at < 0.05, are not significant at < 0.01.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Systemic and mucosal immune responses measured by ELISA

Antibody responses against S. Enteritidis-specific antigen in the plasma and intestinal wash samples of the immunized and non-immunized groups were monitored for 24 weeks post-prime immunization (PPI). At the 4th week of age, the plasma IgG levels were 2.0 and 2.2 times higher in groups B and C, respectively, compared with that of the control group. The IgG response was significantly elevated after booster immunization. The plasma IgG levels were increased approximately 2.5 and 2.8 times in groups B and C, respectively, compared with the control group at the 10th week PPI (Fig. 1). In addition, the plasma IgG levels were approximately 2.6 and 2.8 times higher in groups B and C, respectively, than those in the control group at the 20th week PPI. The immunized birds showed a significant secretory IgA (sIgA) response at the 2nd week PPI. The sIgA levels were 2.0 and 1.8 times higher in groups B and C, respectively, compared with the control group A at the 2nd week PPI (Fig. 2). The sIgA levels were also significantly higher in groups B and C after booster immunization. The sIgA levels were approximately 2.5 and 2.2 times higher in groups B and C, respectively, than those in the control group at the 10th week PPI (Fig. 2), and sIgA levels were 2.8 and 2.4 times higher in groups B and C, respectively, than those in the control group at the 20th week PPI. The sIgA response was slightly higher in the group boosted with oral immunizations (group B) compared with the group boosted with intramuscular immunizations (group C) (Fig. 2).

image

Figure 1. Plasma IgG immune responses against the Salmonella Enteritidis -specific antigen in chickens immunized with the Salmonella vaccine candidate by different routes. The arrow indicates secondary immunization at the 6th and 16th weeks of age. Group A chickens were unimmunized control, group B chickens were orally primed and boosted with oral immunizations, and group C chickens were orally primed and intramuscularly boosted. Values are the mean values in each group, and error bars demonstrate standard deviations (SD). Error bars refer to individual differences within a single experiment. Lower case letters indicate a significant difference (< 0.05) between the immunized and control groups as follows: (b), group B compared with the group A; (c), group C compared with the group A.

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image

Figure 2. Intestinal secretory IgA immune responses against an Salmonella Enteritidis -specific antigen (OMP) in chickens immunized with the Salmonella vaccine candidate by different routes. The arrow indicates secondary immunization at the 6th and 16th weeks of age. Groups A to C refer to Fig. 1. Values are the mean values in each group, and error bars demonstrate standard deviations (SD). Error bars refer to individual differences within a single experiment. Lower case letters indicate a significant difference (< 0.05) between the immunized and control groups as follows: (b), group B compared with the group A; (c), group C compared with the group A.

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Cellular immune responses by lymphocyte proliferation assay and flow cytometry

Cellular immune responses in the chickens of both immunized groups were examined by the peripheral mononuclear cell proliferation assay using the S. Enteritidis-soluble antigen. The proliferative responses are presented in Fig. 3. After immunization, both immunized groups showed significantly elevated SI values compared with the control group. Sequential monitoring of lymphocyte responses revealed a progressive increase in the stimulation indices (SIs) in the immunized chickens after booster immunization. The stimulation indices for the immunized groups B and C were 1.9 and 1.6, respectively, at the 4th week PPI. After the first booster immunization, the stimulation indices in groups B and C increased to 2.4 and 1.7 times higher, respectively, than those of the control group at the 10th week PPI. After the second booster immunization, the stimulation indices were 2.7 and 2.3 times higher in groups B and C, respectively, compared with that of the control group at the 20th week PPI (Fig. 3).

image

Figure 3. Stimulation index of chicken lymphocyte samples from the peripheral lymphocyte proliferation assay using soluble antigen at the 4th week post-prime and post-booster immunizations. Groups A to C refer to Fig. 1. Asterisks indicate significant differences between the values of the immunized groups B and C and those of the control group (*< 0.05). Error bars refer to individual differences within a single experiment. image_n/zph12042-gra-0001.png, Group A;image_n/zph12042-gra-0002.png, Group B;image_n/zph12042-gra-0003.png, Group C.

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To quantify changes in T-cell composition of the control and immunized groups, we used flow cytometry to analyse the occurrence of relevant cells, especially CD45+CD3+ T cells, in peripheral blood. At the 4th week PPI, post-booster immunization (PBI) and post-secondary booster immunization (PSBI), the CD45+CD3+ T-cell populations in both immunized groups were higher than those in the control group (Fig. 4). After prime and first booster immunization, there were increases in the CD45+CD3+ T-cell populations, while after second booster immunization, the increase in the CD45+CD3+ T-cell population was even more pronounced in groups B and C.

image

Figure 4. Flow cytometry analysis of CD45+CD3+ cell populations in the control and immunized chickens. Numbers in each quadrant show percentages of CD45CD3+ (Upper Left), CD45+CD3+ (Upper Right), CD45+CD3 (Lower Right) and CD45CD3 (Lower Left) at the 4th week post-prime and post-booster immunizations, stained with APC-conjugated monoclonal antibody against CD45 and FITC-labelled monoclonal antibody against CD3. Groups A to C refer to Fig. 1.

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Assessment of egg production after challenge

In this experiment, the egg production rates in the control group were 74%, 61.6% and 60.9% in the first, second and third weeks post-challenge (PC), respectively. The infection with the challenge strain resulted in a larger reduction in egg production in the second and third weeks PC in the control birds. Several hens in the control group also laid malformed or thin-shelled eggs during the second week PC. The egg production rates in the first, second and third weeks PC were 79.7%, 65.4% and 81.2%, respectively, in the immunized group B; and 75.7%, 65.7% and 77.1%, respectively, in the immunized group C. Both groups of immunized hens recovered faster from the challenge, and their egg production increased at the third week PC. Overall, the egg production rates for the control group A, immunized group B and group C were 65.5%, 75.4% and 72.8%, respectively, for 3 weeks after the challenge.

Isolation of the vaccine and challenge strains from egg contents

The vaccine strain was not isolated from any of the egg content samples collected from any of the groups for the 3 weeks before the challenge. Table 1 presents the data on positive pools of eggs batches in the three different groups after the challenge. The egg contamination rates in the control group were 28.6%, 42.8% and 28.6% for the first, second and third weeks post-challenge, respectively, which are significantly higher than those of immunized groups B and C, which were 0%, 10.7% and 0%; and 3.6%, 14.3% and 3.6% respectively, in the stipulated period (< 0.05). Over the whole experiment, 33.3% of the batches in the control group were culture-positive, whereas 7.1% of the batches in immunized group C were positive for the challenge strain, and the strain was detected only in 3.6% of the batches in immunized group B after analysis of all three groups.

Table 1. Results of bacteriological examination of eggs batches from all three groups for the 3 weeks after the challenge
GroupaWeek 1Week 2Week 3
  1. a

    Each group contained 20 hens. Each batch contained five birds. The groups were designated as group A, non-immunized; group B, oral prime immunization and subsequent oral booster immunizations; and group C, oral prime immunization and subsequent intramuscular booster immunizations.

  2. b

    Total number of positive batches relative to the total number of batches after observation of bacterial recovery (egg contamination rate in percentage).

  3. All values were considered to be significant as compared to the control group if *< 0.05 or **< 0.01.

A8/28 (28.6)b12/28 (42.8)8/28 (28.6)
B0/28 (0)**3/28(10.7)*0/28 (0)**
C1/28 (3.6)*4/28 (14.3)*1/28 (3.6)*

Observations of gross lesions and bacterial recovery from the infected organs

Chickens from each group were killed for the examination of gross lesions and bacterial recovery from the infected organs (Table 2). As demonstrated in Table 2, the immunized groups showed significantly lower lesion scores than those of the control group. Persistence of the challenge strain in the tissues of liver, spleen, caeca and ovary was also examined. The challenge strain isolated from the positive samples was confirmed by PCR. Based on the colony count method in the infected organ tissues, the bacterial count was significantly lower in the immunized groups compared with the control group.

Table 2. Observation of gross lesions and bacterial recovery from the internal organs of chickens after challenge
GroupaNo. of challenged chickensNo. of Enteritidis- positive chickensGross lesionbBacterial recoveryd
LiverSpleenLiverSpleenCaecaOvary
  1. a

    Each group contained 20 hens. The groups were designated as group A, non-immunized; group B, oral prime immunization and subsequent oral booster immunizations; and group C, oral prime immunization and subsequent intramuscular booster immunizations.

  2. b

    Gross lesions were observed in livers and spleens of birds in each group at the third week post-challenge (Mean ± SD).

  3. c

    Significantly lower gross lesion score of the immunized groups than that in the control group at < 0.05.

  4. d

    Bacterial recovery was observed by direct and enrichment culture in liver, spleen and caeca of birds at the third week post-challenge (Mean ± SD log10 cfu/g).

  5. e

    Significantly lower bacterial count of the immunized groups than that in the control group at < 0.05.

  6. f

    Significantly lower number of Salmonella Enteritidis -positive birds from the immunized group compared with the control group after direct plating on BGA at the third week post-challenge (< 0.05).

A20121.1 ± 0.91.8 ± 0.90.5 ± 0.91.6 ± 1.40.3 ± 0.91.1 ± 1.8
B204f0.1 ± 0.3c0.4 ± 0.5c  0 ± 0e0.6 ± 1.2e  0 ± 0e0.2 ± 0.8e
C205f0.2 ± 0.4c0.5 ± 0.7c  0 ± 0e0.7 ± 1.3e0.1 ± 0.30.3 ± 0.9e

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

Worldwide, Salmonella Enteritidis has emerged as a major egg-borne zoonotic infection in the late 20th century (Singh, 2009). Vaccination is an effective tool for the prevention of Salmonella infections (Mastroeni et al., 2001). Experience with live vaccines suggests they contribute to protecting birds from infection with S. Enteritidis (Ilka, 2000). The ideal route of immunization for the live vaccines to poultry is orally via drinking water or food or by spray (Zhang-Barber et al., 1999). An oral immunization via drinking water or food eliminates animal stress and reduces efforts, costs and time for the personnel working on the farm compared to using numerous individual mass vaccinations. In this study, we evaluated the efficacy of a new live S. Enteritidis vaccine candidate constructed by deletion of lon and cpxR virulent genes, by analysing the effects of administering the vaccine via oral and intramuscular routes.

Our results showed that the plasma IgG and intestinal sIgA antibody levels were significantly elevated after subsequent booster immunizations by oral or intramuscular administration. In addition, the sIgA response was slightly higher in the group boosted with oral immunizations as compared to the group boosted with intramuscular immunizations. Primary immunization, whether oral or parenteral, seeds memory cells into the gut, also called gut-localized memory (Kantele et al., 2005). The oral route of re-immunization targets the immune response more broadly than the parenteral route and simultaneously strengthens the intestinal immune response (Kantele et al., 2005).

Immunization with live vaccines is considered to confer better protection against intracellular pathogens compared with killed vaccines due to the ability of live vaccines to induce not only humoral immune responses, but also cell-mediated immune responses (Chong et al., 1996; Mastroeni et al., 2001). In the present study, the assay for lymphocyte proliferation and for CD45+CD3+ T cells detected significant responses after primary and booster immunizations in both immunized groups. The group B showed slightly higher cellular immune responses as compared to the group C after booster immunizations (Fig. 3 and Fig. 4). It is generally accepted that oral administration of live vaccine induces a more significant cellular immune response (Norimatsu et al., 2004; Meeusen et al., 2007; Boyen et al., 2008).

In the vaccinated chickens, a significant induced immune response did not necessarily prevent organ invasion or egg contamination but did contribute to the reduction in S. Enteritidis (Tables 1 and 2). In the present study, no significant difference in immune response between two vaccination regimens was observed. However, slightly higher sIgA and cellular immune responses were evident in the oral re-immunization as compared to the intramuscular re-immunization. This higher immune response may be responsible for the lower bacteria counts that were shown in the results for the caecal samples of group B. In addition, the number of contaminated egg batches and the bacterial counts in all examined organs were more effectively reduced in the group B. This result shows that differences in the immune responses between two vaccination regimens may be indicative of a higher protection efficacy against salmonellosis for the oral booster. Intravenous S. Enteritidis challenge provided a stringent test of the vaccine. However, the repetition of this result after an oral challenge with the virulent S. Enteritidis would be necessary to adequately test the potency of the vaccine candidate. In the safety examination, we evaluated whether any Salmonella recovered from eggs in this study was from vaccination, and determined that no example of this was found. However, this study is limited in scope and numbers, and transfer of a vaccination programme to a farm setting would require further evaluation for an event that may occur infrequently.

In conclusion, the present study showed that the live vaccine candidate S. Enteritidis JOL919 induced plasma IgG and intestinal sIgA, proliferation of lymphocytes and augmentation of the CD45+CD3+ T-cell population in peripheral blood under the conditions tested. These immune responses may be correlated with the reduction in Salmonella in the internal organs of vaccinated chickens and the eggs they produced. Because this study is the first attempt to evaluate the efficacy of a new live attenuated S. Enteritidis vaccine candidate to reduce internal egg contamination, further comparative evaluation experiments are needed to prove the efficiency of this vaccine candidate over the currently available live and/or inactivated vaccines.

Double oral immunizations may be required to achieve the best results from flocks in production. In addition, periodic booster vaccinations may be advised. However, the strategy of using live attenuated vaccines that can be delivered through feed or drinking water is an advantage over strategies requiring handling of individual birds for injections.

Acknowledgement

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References

This work was supported by Mid-career Researcher Program through NRF grant funded by the MEST (No. 2012R1A2A4A01002318) in Republic of Korea.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgement
  8. References
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