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Immunization with outer membrane protein A from Salmonella enterica serovar Enteritidis induces humoral immune response but no protection against homologous challenge in chickens
Immunization with outer membrane protein A from Salmonella entérica serovar Enteritidis induces humoral immune response but no protection against homologous challenge in chickens M. Okamura,*^ M. Ueda,* Y. Noda,* Y. Kuno,* T. Kashimoto,t K. Takehara,*^ and M. Nakamura*^ *Laboratory of Zoonoses, and fLaboratory of Veterinary Public Health, Kitasato University School of Veterinary Medicine, Towada, Aomori 034-8628, Japan ABSTRACT Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. We evaluated the efficacy of outer membrane protein A (OmpA) as a novel vaccine candidate against Salmonella in poultry. Native OmpA purified from Salmonella entérica serovar Enteritidis was mixed with adjuvant and administered intramuscularly to 41-d-old chicks. The vaccinated birds showed no decrease in cecal excretion and tissue colonization compared with the unvaccinated birds after oral challenge with 10^ cfu of the homologous strain at 28
d postimmunization. However, this vaccination induced an increased level of serum anti-OmpA IgC. Similar results were obtained in the replication experiments using a recombinant OmpA with single and double doses. For the development of more effective component vaccines for avian salmonellosis, the vaccine efñcacy of outer membrane proteins other than OmpA and route of immunization other than parenteral administration should be evaluated with regard to protection and immune responses, including mucosal Ig A.
Key words: Salmonella, outer membrane protein, outer membrane protein A, vaccine 2012 Poultry Science 91:2444-2449 http://dx.doi.org/10.3382/ps.2012-02303
INTRODUCTION Salmonella spp. has been recognized as a major cause of food-borne illness in humans worldwide. Although a decreased incidence has been reported recently (Cogan and Humphrey, 2003; Mumma et al., 2004), major outbreaks still occur in the United Kingdom (Little et al., 2007) and the United States (Kuehn, 2010), causing it to remain relevant to public health. Although many different foods have been implicated in salmonellosis outbreaks, most cases are usually caused by the consumption of poultry and eggs (Schlundt et al., 2004). Therefore, the production stage is the primary target for the control of Salmonella in the food chain. Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. Live and killed vaccines against Salmonella entérica serovars Enteritidis and Typhimurium for poultry are available worldwide. However, these vaccines are ©2012 Poultry Science Association Inc. Received March 14, 2012. Accepted June 24, 2012. ^Corresponding author: [email protected] ^Present address; Laboratory of Veterinary Hygiene, Tokyo University of Agriculture and Technology, Fnchu, Tokyo 183-8538, Japan. ^Present address: Research Institute for Animal Science in Biochemistry and Toxicology, 3-7-11 Hashimotodai, Midori-ku, Sagamihara, Kanagawa 252-0132, Japan.
not applicable to other serovars with different O and H antigens, such as S. entérica serovar Infantis, which has been isolated from broilers frequently and also linked to human cases in Japan to date (Noda et al., 2010). To comply with the increasing public demand for crossprotective vaccines against multiple Salmonella serovars, the development of component vaccines with highly conserved antigens has great value. Outer membrane proteins (OMPs) are considered effective antigens to stimulate immune responses because they are exposed on the bacterial surface and easily recognized by the host immune system. Among OMPs, outer membrane protein A (OmpA) is an approximately <40-kDa monomeric pore-forming protein inserted abundantly into the enterobacterial outer membrane at about 100,000 copies/cell (Koebnik et al., 2000). Although its relevance in pore formation is still controversial, OmpA is considered essential for the conservation of cell structure by physical linkage between the outer membrane and peptidoglycan (Sonntag et al., 1978). The OmpA is also reported to function in host-pathogen interactions, including the adhesion and invasion of epithelial cells, immune target and evasion, and biofllm formation (Smith et al., 2007). The roles of OmpA in Salmonella infection in chickens are not clear, but antibody against OmpA was produced specifically in hens naturally infected with S. entérica serovar
2444
SALMONELLA OUTER MEMBRANE PROTEIN A VACCINE
Enteritidis (Ochoa-Repáraz et al., 2004). In addition, based on bioinformatic analysis, OmpA is well conserved among Salmonella serovars. Therefore, OmpA seems to hold promise as a component vaccine against avian salmonellosis caused by different serovars. The present study evaluates the efficacy of OmpA as a vaccine candidate against Salmonella in poultry.
MATERIALS AND METHODS
Bacteria and Growth Conditions The S. Enteritidis phage type 4 strain HY-1 used in this study was made resistant to rifampicin through selective pressure for ease of recovery (Okamura et al., 2007). To prepare the bacterial suspension and inocula, the organisms were grown on a desoxycholate-hydrogen sulfate-lactose agar plate supplemented with 100 \ig/ mL of rifampicin (DHL-rif), and 1 or 2 isolated colonies were transferred to a heart infusion broth (HIB; Eiken Ghemical, Tokyo, Japan) and incubated at 37°G for 5 to 6 h with shaking until a density of 10^ cfu/mL was obtained, as determined by optical density values at 600 nm. The bacterial concentration was adjusted by serial dilution with PBS, and the actual inoculation dose was determined retrospectively by colony counts of serial 10-fold dilutions that were spread on DHL-rif plates.
Preparation of Native Antigens Grude OMPs were purified from the bacterial suspension according to Lobos and Mora (1991). To further purify OmpA, the crude OMPs were boiled in a sample buffer for 5 min and applied to SDS-PAGE (Laemmli, 1970). The samples were electrophoresed on a 10% polyacrylamide gel followed by Goomasie Brilliant Blue staining. The band of approximately 35 kDa was identified as OmpA (NGBI Reference Sequence: NP_460044.1) by orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry and tandem mass spectrometry in combination (QSTAR Pulsar i, Applied Biosystems, Foster Gity, GA) with trypsin digestion, followed by a peptide mass fingerprint search using Mascot (Matrix Science, Boston, MA). The band was then cut from the gel and homogenized separately in an elution buffer (20 mMTris-HGl with 1% SDS, pH 8.0), followed by removal of the gel using a microspin column (GE Healthcare, Buckinghamshire, UK). After centrifugal concentration, ice-cold acetone was added, placed at -80°G, and centrifuged at 20,000 x g for 15 min. The pellet was suspended in PBS. After adjustment at the appropriate concentration, the protein solution was used as native OmpA (nOmpA).
2445
dorf, Germany) according to the manufacturer's instructions. The DNA sequence of S. Enteritidis ompA (NGBI Reference Sequence: YP_002243066.1) was obtained by PGR using the Expand High-Fidelity^^^^ PGR System (Roche Applied Science, Mannheim, Germany). The ompA DNA was amplified with the forward (5'-ÇAXAT:GGGTGGGAAAGATAAGAGGTGGTAG-3') and reverse primers (5'-GTGGAGAGGGTGGGGGTGAGTTAGGAGGTG-3') ; the product lacked the signal sequence of the first 63 bp (containing the start codon) and the stop codon of full-length ompA and instead contained the recognition sites of the restriction enzymes Ndel and Xhol, respectively (underlined), which allowed the directional cloning of the PGR fragment into the expression vector. The PGR product was cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and the plasmid was transformed into One Shot TOPIO competent Escherichia coli (Invitrogen, Garlsbad, GA). The transformed cells were recovered by blue-white selection. The plasmid DNA from the selected clones was sequenced. Digestion of the pGEM-T Easy plasmid with Ndel and Xhol resulted in restriction fragments of ompA, which were subcloned into the expression vector pET29b (Novagen, Milwaukee, WI). This inducible vector system allows the expression of recombinant proteins with a 6-histidine tag at the C-terminus under the presence of isopropyl ß-D-1-thiogalactopyranoside (IPTG). The plasmid was transformed into E. coli BL21 (DE3) competent cells and grown on LB agar containing 100 |xg/ mL of kanamycin. The recombinant OmpA (rOmpA) protein was expressed in the insoluble fraction under the presence of 0.4 mMIPTG and was purified by affinity chromatography on a Ni-NTA His-Bind Resin (Takara Bio Inc., Shiga, Japan) after solubilization with 8 M urea. The purified protein was pooled and dialyzed in Spectra/Por 4 Regenerated Gellulose Dialysis Membranes (Spectrum Laboratories Inc., Rancho Dominguez, GA). The protein concentration was determined by the DG Protein Assay (Bio-Rad) based on the Lowry method (Lowry et al, 1951). The purified proteins were confirmed to be rOmpA by Western blotting using anti-polyhistidine mouse monoclonal antibody (Sigma, St. Louis, MO) followed by horseradish peroxidase-conjugated anti-mouse IgG rabbit sera (BD Transduction Laboratories, Lexington, KY). The reaction was visualized by the peroxidase substrate (Sigma).
Preparation of Component Vaccines Both nOmpA and rOmpA were adjusted to 320 [xg/ mL with PBS and mixed well with the same volume of complete or incomplete Fi-eund adjuvant (Sigma) for primary and secondary immunizations, respectively. The vaccination dose was 80 \xg in 0.5 mL/bird.
DNA Cioning and Expression
Expérimentai Birds
The bacterial genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Düssel-
In the present study, 37- to 41-d-old non-SPF White Leghorn chicks (Lohman, Julia), none of which had
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OKAMURA ET AL.
been vaccinated against Salmonella, were obtained from a local commercial layer farm. The birds were reared in individual wire cages with a nonmedicated layer ration and water supphed ad libitum in an isolation building. The birds were confirmed to be Salmonella-iree bacteriologically. All of the animal experiments in this study followed the institutional guidelines for the care and use of laboratory animals and were approved by the Animal Research Gommittee, Kitasato University School of Veterinary Medicine, Japan.
Efficacy of Vaccination with nOmpA Twenty healthy chicks of 37-d-old were assigned to 2 groups: nOmpA-vaccinated and S. Enteritidis-challenged; unvaccinated and S. Enteritidis-challenged (n = 10/group). The chicks were immunized intramuscularly in their legs at 41 d of age and challenged orally with lO'^ cfu of S. Enteritidis at 26 d postimmunization. The cecal droppings were collected at 3, 7, and 14 d postchallenge, and the bacterial excretion was examined. Ghickens were necropsied at 18 d postchallenge, and the liver, spleen, and cecal contents were examined for Salmonella. Sera were also collected at 0, 7, 14, 21, 26, 33, and 40 d after vaccination for antibody measurement .
Efficacy of Vaccination with rOmpA Thirty healthy chicks of 41-d-old were assigned to 6 groups: vaccinated once, vaccinated twice, adjuvant administered once, adjuvant administered twice, PBS administered once, and PBS administered twice (n — 5/ group). The groups with one administration were vaccinated intramuscularly in their legs at 43 d of age and challenged orally with 10^ cfu of 5*. Enteritidis at 28 d postvaccination. The remaining 3 groups with 2 administrations were vaccinated intramuscularly in their legs at 43 and 73 d of age and challenged orally with 10^ cfu of S. Enteritidis at 26 d after the second vaccination (56 d after the first vaccination). The cecal droppings were collected at 3, 6, and 13 d postchallenge, and the bacterial excretion was examined. Ghickens were necropsied at 14 d postchallenge, and the hver, spleen, and cecal contents were examined for Salm,onella. Sera were also collected weekly after the first vaccination for antibody measurement.
Recovery of Bacteria from Cecal Droppings and Tissue Samples The cecal droppings after challenge and the liver, spleen, and cecal contents at necropsies were taken aseptically and used for the enumeration of Salmonella according to a previous study (Okamura et al., 2007). The samples were separately homogenized with a 9-fold volume of Hajna Tetrathionate broth (HTT; Eiken, Tokyo, Japan) for the cecal droppings and cecal contents, and HIB for the liver and spleen. The serial
10-fold dilutions were then spread on DHL-rif plates and incubated at 37°G for 24 h. The homogenates were also incubated at 4I.5°G (cecal contents) or 37°G for 24 h for enrichment. After enumeration of colonies, loopfuls of homogenates that were negative initially were then streaked on DHL-rif plates. After incubation at 37°G for 24 h, the samples with Salmonella colonies were recorded as positive and assumed as 100 cfu/g. For the samples negative for Salmonella in the primary enrichment, the delayed secondary enrichment (DSE) was performed as described previously (Waltman et al., 1991). Briefly, the negative samples were kept at room temperature for 5 to 7 d to avoid the effects of inhibitory factors on Salmonella growth, of which 0.5 mL was inoculated into 4.5 mL of a fresh HTT and incubated at 41.5°G for 24 h. Salmonella was detected as described above for the primary enrichment, and the positive samples by DSE were assumed to contain Salmonella at 10 cfu/g.
Measurement of Antibody Levels in Sera Serum IgG levels against OmpA were measured by ELISA according to a previous study (Okamura et al., 2007). Each well of 96-well microtiter plates (Sumitomo Bakelite Go., Ltd., Tokyo, Japan) was coated overnight at 4°G with 50 ^iL of 10 |j,g/mL of rOmpA in a carbonate-bicarbonate buffer. Sera were diluted 10-fold with 0.5% BSA in 0.05% Tween20-PBS and applied to the plates at 50 |aL/well. Rabbit anti-chicken IgG antibody conjugated with horseradish peroxidase (Sigma), peroxidase substrate (Sigma), and 2 A/'H2SO4 (50 |a,L/well) was used for the reaction, and the optical density values were measured at 450 nm. The assay was performed in duplicate. The sera from experimentally infected hens (Okamura et al., 2007) and naive chicks collected in an earlier study (unpublished) were used as positive and negative controls, respectively. The reactivity of these sera to S. Enteritidis heat-inactivated and formalininactivated antigens were confirmed by Western blot before this study (data not shown).
Statistical Analyses Data of cfu enumeration and antibody levels were compared between groups by the unpaired Student's i-test. P-values of less than 0.05 were considered significant.
RESULTS
Efficacy of Vaccination with nOmpA In all the groups challenged with the homologous strain of S. Enteritidis, the levels of cecal excretion and tissue colonization were less than 10^ cfu/g, and the vaccinated birds showed no decrease in cecal excretion (Figure lA) and tissue colonization (Figure IB) compared with the unvaccinated birds throughout the ex-
SALMONELLA OUTER MEMBRANE PROTEIN A VACCINE
périment. However, vaccination with nOmpA successfully induced an increased level of serum anti-OmpA IgC (Figure lC). In the unvaccinated control birds, the specific antibody slightly increased postchallenge (Figure lC). Although the initial optical density value was relatively high (approximately 0.6), it was similar to that of the negative control sera. Together with the Western blot analyses (Supplemental Figure 1; available online at http://ps.fass.org/), this indicates that such a high background signal was not due to the possibility that the chickens had 5a¿mone/Za-specific or antiOmpA antibody before study initiation.
2447
who suggested the potential limitation of the interaction between an antibody and OmpA of live bacteria by 0-chain of LPS. Tarkka et al. (1989) also pointed out that it is difficult for the anti-Omp antibody to reach the Omp protein on live Neisseria meningitidis. Meenakshi et al. (1999) referred to an irrelevance of the increased antibody level as an indicator of a protective effect against Salmonella.
Efficacy of Vaccination with rOmpA The level of S. Enteritidis detected in cecal droppings was 10^ to 10^ cfu/g at 3 d postchallenge, and gradually decreased to 10^ to 10^ cfu/g at 13 d postchallenge. There were no significant differences between groups administered with rOmpA, adjuvant, and PBS (Figure 2A). Tissue colonization was less than 10^ cfu/g in all samples taken at necropsy without significant differences between groups (Figure 2B). As seen in the experiment using nOmpA, rOmpA-vaccinated birds showed a significant increase in anti-OmpA IgC in sera, and adjuvant- and PBS-administered birds showed increasing levels of anti-OmpA IgC after the challenge (Figure 2C). Similar results were obtained in both single- and double-dose administrations. Second immunization with rOmpA did not add any booster effects on cecal excretion, tissue colonization, and antibody levels (data not shown).
— nOmpA - Control
2
4
6
8
10
12
14
16
Days postchallenge
B
DISCUSSION Various proteins of gram-negative bacteria including Salmonella have been tested for their immunological or protective effects as vaccines. Among them, OMPs or porins have been reported to induce the host humoral responses and in turn inhibit postchallenge bacterial colonization. Meenakshi et al. (1999) showed that the vaccine composed of Salmonella OMPs and oil adjuvant inhibited the fecal shedding of Salmonella. The use of a different evaluation system with S. Callinarum, which is more lethal than S. Enteritidis, could provide more promising results. Cómez-Verduzco et al. (2010) demonstrated that 53 to 70% of the progeny chicks from breeding hens immunized with S. Callinarum porins were protected from death when challenged with 20 to 500 LD50 of S. Callinarum. Our study demonstrated that OmpA has a strong immunogenicity to induce the humoral response. Nevertheless, the bacterial shedding after challenge was not reduced by vaccination with OmpA. A potential reason is that the anti-OmpA antibody did not reach or recognize the OmpA on the outer membrane of live Salmonella due to the presence of other properties, such as LPS, pili, flagella, and other porin proteins, which could have masked the OmpA. This possibility is supported by Singh et al. (2003),
Liver
10
Spleen
20
Cecum
30
40
Days postimmunization
Figure 1. (A) Salmonella excretion in the cecal droppings collected at 3, 7, and 14 d postchallenge. The data represent the mean bacterial count and the SEM (n = 10 for each group). (B) Viable counts of Salmonella in the liver, spleen, and cecal contents collected at 18 d postchallenge. The data represent the mean bacterial count and the SEM (n = 10 for each group). (C) Anti-native outer membrane protein A (nOmpA) antibody response induced by vaccination determined by ELISA. Sera were collected from 10 birds/group at 0, 7, 14, 21, 26, 33, and 40 d after vaccination. Arrow indicates the date of challenge (26 d postimmunization). The data represent the mean absorbance and the SEM (n = 10 for each group), and asterisks indicate significant differences between groups (P < 0.01). OD = optical density.
OKAMURA ET AL.
2448 7
f ^
6
•
5 4 3 • 2
•
— Control — Adjuvant
— lOmpA 2
4
6
8
10
12
14
Days postchallenge
B
3 1
^2.5
D Control • Adjuvant • rOmpA
"s 2
1•
> 0.5 0 + Cecum
Spleen
Liver 1.4 1
with crude OMPs was not significant (Okamura et al., 2003). Therefore, the lack of protection in the challenge experiments may imply that the induction of cellular immunity failed. Different OMPs also need to be considered because the Omps vaccine with successful result (Meenakshi et al., 1999) should contain many other OMPs. Furthermore, variable results were obtained by using different types of OMPs in Salmonella Typhimurium (Hamid and Jain, 2008) and other bacterial agents such as Pasteurella multocida (Vasfi Marandi and Mittal, 1997). In addition, parenteral administration of antigen is expected to induce little or no secretory IgA response, which may be the most important part of a mucosal infection, such as salmonellosis. Therefore, the results obtained in this study do not preclude efficacy of mucosal administration of OmpA with a robust specific secretory IgA response. In conclusion, parenteral immunization with adjuvanted OmpA induced the anti-OmpA IgG effectively but resulted in no protection against Salmonella in chickens. To develop more effective component vaccines for avian salmonellosis, the vaccine efficacy of OMPs other than OmpA and route of immunization should be evaluated in regard to protection and immune responses, including mucosal IgA.
ACKNOWLEDGMENTS
1.2
The authors thank the students of the Laboratory of Zoonoses, Kitasato University School of Veterinary Medicine for their technical assistance for the animal experiments.
1
g' 0.6 ^ 0.4
- û - - Control - • • - • Adjuvant —•— rOmp.'V
0.2 0 10
20 30 Days postimmuni:zation
40
Figure 2. (A) Salmonella excretion in the cecal droppings collected at 3, 6, and 13 d postchallenge after single vaccination. The data represent the mean bacterial count and the SEM (n = 5 for each group). (B) Viable counts of Salmonella in the liver, spleen, and cecal contents collected at 14 d postchallenge. The data represent the mean bacterial count and the SEM (n = 5 for each group). (C) Anti-recombinant outer membrane protein A (rOmpA) antibody response induced by vaccination determined by ELISA. Sera were collected from 5 birds/group at 0, 7, 14, 21, 28, 35, and 42 d after vaccination. Arrow indicates the date of challenge (26 d postimmunization). The data represent the mean absorbance and the SEM (n = 5 for each group), and asterisks indicate significant differences between the rOmpA-immunized group and other groups [P < 0.01). OD = optical density.
On the other hand, protection against salmonellosis requires the host immunity of both cellular and humoral arms (Babu et al, 2003; Mittrücker and Kaufmann, 2000). In mice, immunization with porin can induce these responses (Singh et al., 1999). However, although the cellular responses were not evaluated in the current study, the proliferative response of splenocytes upon ex vivo stimulation with crude porin after immunization
REFERENCES Babu, U., M. Scott, M. J. Myers, M. Okamura, D. Gaines, H. F. Yancy, H. Lillehoj, R. A. Heckert, and R. B. Raybourne. 2003. Effects of live attenuated and killed Salmonella vaccine on Tlymphocyte mediated immunity in laying hens. Vet. Immunol. Immunopathol. 91:39-44. Cogan, T. A., and T. J. Humphrey. 2003. The rise and fall of Salm,onella Enteritidis in the UK. J. Appl. Microbiol. 94(Suppl.):114S119S. Gómez-Verduzco, G., G. Tellez, A. L. Quintana, A. Isibasi, and V. Ortiz-Navarrete. 2010. Humoral immune response in breeding hens and protective immunity provided by administration of purified Salmonella Gallinarum porins. Poult. Sei. 89:495-500. Hamid, N., and S. K. Jain. 2008. Characterization of an outer membrane protein of Salmonella entérica serovar typhimurium that confers protection against typhoid. Glin. Vaccine Immunol. 15:1461-1471. Koebnik, R., K. P. Locher, and P. Van Gelder. 2000. Structure and function of bacterial outer membrane proteins: Barrels in a nutshell. Mol. Microbiol. 37:239-253. Kuehn, B. M. 2010. Salmonella cases traced to egg producers: Findings trigger recall of more than 500 million eggs. JAMA 304:1316. Laemmli, U. K. 1970. Gleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Little, G. L., S. Surman-Lee, M. Greenwood, E. J. Bolton, R. Elson, R. T. Mitchell, G. N. Nichols, S. K. Sagoo, E. J. Threlfall, L. R. Ward, I. A. Gillespie, and S. O'Brien. 2007. Pubhc health investigations of Salmonella Enteritidis in catering raw shell eggs, 2002-2004. Lett. Appl. Microbiol. 44:595-601.
SALMONELLA OUTER MEMBRANE PROTEIN A VACCINE Lobos, S. R., and G. C. Mora. 1991. Alteration in the electrophoretic mobility of OmpC due to variations in the ammonium persulfate concentration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis 12:448-450. Lowry, O. H., N. Rosenbrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Meenakshi, M., C. S. Bakshi, G. Bntchaiah, M. P. Bansal, M. Z. Siddiqui, and V. P. Singh. 1999. Adjuvanted outer membrane protein vaccine protects poultry against infection with Salmonella enteritidis. Vet. Res. Commun. 23:81-90. Mittriicker, H. W., and S. H. Kaufmann. 2000. Immune response to infection with Salmonella typhimurium in mice. J. Leukoc Biol 67:457-463. Mnmma, G. A., P. M. Griffin, M. I. Meltzer, C. R. Braden, and R. V. Tauxe. 2004. Egg quality assurance programs and egg-associated Salmonella enteritidis infections. United States. Emerg. Infect. Dis. 10:1782-1789. Noda, T., K. Murakami, Y. Ishignro, and T. Asai. 2010. Chicken meat is an infection source of Salmonella serovar Infantis for humans in Japan. Foodborne Pathog. Dis. 7:727-735. Ochoa-Repáraz, J., B. Sesma, M. Alvarez, M. Jesus Renedo, J. M. Irache, and C. Gamazo. 2004. Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens. Vet. Res. 35:291-298. Okamura, M., H. S. Lillehoj, R. B. Raybourne, U. Babu, and R. Heckert. 2003. Antigen-specific lymphocyte proliferation and interleukin production in chickens immunized with killed Salmonella enteritidis vaccine or experimental subunit vaccines. Avian Dis. 47:1331-1338. Okamura, M., H. Tachizaki, T. Kubo, S. Kikuchi, A. Suzuki, K. Takehara, and M. Nakamura. 2007. Comparative evaluation of a
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bivalent killed Salmonella vaccine to prevent egg contamination with Salmonella entérica serovars Enteritidis, Typhimurium, and Gallinarum biovar PuUorum, using 4 different challenge models. Vaccine 25:4837-4844. Schlundt, J., H. Toyofuku, J. Jansen, and S. A. Herbst. 2004. Emerging food-borne zoonoses. Rev. Sei. Tech. 23:513-533. Singh, M., H. Vohra, L. Kumar, and N. K. Ganguly. 1999. Induction of systemic and mucosal immune response in mice immunised with porins of Salmonella typhi. J. Med. Microbiol. 48:79-88. Singh, S. P., Y. U. Williams, S. Miller, and H. Nikaido. 2003. The C-terminal domain of Salmonella entérica serovar typhimurium OmpA is an immunodominant antigen in mice but appears to be only partially exposed on the bacterial cell surface. Infect. Immun. 71:3937-3946. Smith, S. G., V. Mahon, M. A. Lambert, and R. P. Fagan. 2007. A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol. Lett. 273:1-11. Sonntag, L, H. Schwarz, Y. Hirota, and U. Henning. 1978. Cell envelope and shape of Escherichia coli: Multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. J. Bacteriol. 136:280-285. Tarkka, E., A. Muotiala, M. Karvonen, K. Saukkonen-Laitinen, and M. Sarvas. 1989. Antibody production to a meningococcal outer membrane protein cloned into liv Salmonella typhimurium aroA vaccine strain. Microb. Pathog. 6:327-335. Vasfi Marandi, M., and K. R. Mittal. 1997. Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida. Infect. Immun. 65:4502-4508. Waltman, W. D., A. M. Home, C. Pirkle, and T. G. Dickson. 1991. Use of delayed secondary enrichment for the isolation of Salmonella in poultry and poultry environments. Avian Dis. 35:88-92.
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d postimmunization. However, this vaccination induced an increased level of serum anti-OmpA IgC. Similar results were obtained in the replication experiments using a recombinant OmpA with single and double doses. For the development of more effective component vaccines for avian salmonellosis, the vaccine efñcacy of outer membrane proteins other than OmpA and route of immunization other than parenteral administration should be evaluated with regard to protection and immune responses, including mucosal Ig A.
Key words: Salmonella, outer membrane protein, outer membrane protein A, vaccine 2012 Poultry Science 91:2444-2449 http://dx.doi.org/10.3382/ps.2012-02303
INTRODUCTION Salmonella spp. has been recognized as a major cause of food-borne illness in humans worldwide. Although a decreased incidence has been reported recently (Cogan and Humphrey, 2003; Mumma et al., 2004), major outbreaks still occur in the United Kingdom (Little et al., 2007) and the United States (Kuehn, 2010), causing it to remain relevant to public health. Although many different foods have been implicated in salmonellosis outbreaks, most cases are usually caused by the consumption of poultry and eggs (Schlundt et al., 2004). Therefore, the production stage is the primary target for the control of Salmonella in the food chain. Vaccination of poultry is one promising strategy to mitigate Salmonella infection in poultry and, in turn, humans as well. Live and killed vaccines against Salmonella entérica serovars Enteritidis and Typhimurium for poultry are available worldwide. However, these vaccines are ©2012 Poultry Science Association Inc. Received March 14, 2012. Accepted June 24, 2012. ^Corresponding author: [email protected] ^Present address; Laboratory of Veterinary Hygiene, Tokyo University of Agriculture and Technology, Fnchu, Tokyo 183-8538, Japan. ^Present address: Research Institute for Animal Science in Biochemistry and Toxicology, 3-7-11 Hashimotodai, Midori-ku, Sagamihara, Kanagawa 252-0132, Japan.
not applicable to other serovars with different O and H antigens, such as S. entérica serovar Infantis, which has been isolated from broilers frequently and also linked to human cases in Japan to date (Noda et al., 2010). To comply with the increasing public demand for crossprotective vaccines against multiple Salmonella serovars, the development of component vaccines with highly conserved antigens has great value. Outer membrane proteins (OMPs) are considered effective antigens to stimulate immune responses because they are exposed on the bacterial surface and easily recognized by the host immune system. Among OMPs, outer membrane protein A (OmpA) is an approximately <40-kDa monomeric pore-forming protein inserted abundantly into the enterobacterial outer membrane at about 100,000 copies/cell (Koebnik et al., 2000). Although its relevance in pore formation is still controversial, OmpA is considered essential for the conservation of cell structure by physical linkage between the outer membrane and peptidoglycan (Sonntag et al., 1978). The OmpA is also reported to function in host-pathogen interactions, including the adhesion and invasion of epithelial cells, immune target and evasion, and biofllm formation (Smith et al., 2007). The roles of OmpA in Salmonella infection in chickens are not clear, but antibody against OmpA was produced specifically in hens naturally infected with S. entérica serovar
2444
SALMONELLA OUTER MEMBRANE PROTEIN A VACCINE
Enteritidis (Ochoa-Repáraz et al., 2004). In addition, based on bioinformatic analysis, OmpA is well conserved among Salmonella serovars. Therefore, OmpA seems to hold promise as a component vaccine against avian salmonellosis caused by different serovars. The present study evaluates the efficacy of OmpA as a vaccine candidate against Salmonella in poultry.
MATERIALS AND METHODS
Bacteria and Growth Conditions The S. Enteritidis phage type 4 strain HY-1 used in this study was made resistant to rifampicin through selective pressure for ease of recovery (Okamura et al., 2007). To prepare the bacterial suspension and inocula, the organisms were grown on a desoxycholate-hydrogen sulfate-lactose agar plate supplemented with 100 \ig/ mL of rifampicin (DHL-rif), and 1 or 2 isolated colonies were transferred to a heart infusion broth (HIB; Eiken Ghemical, Tokyo, Japan) and incubated at 37°G for 5 to 6 h with shaking until a density of 10^ cfu/mL was obtained, as determined by optical density values at 600 nm. The bacterial concentration was adjusted by serial dilution with PBS, and the actual inoculation dose was determined retrospectively by colony counts of serial 10-fold dilutions that were spread on DHL-rif plates.
Preparation of Native Antigens Grude OMPs were purified from the bacterial suspension according to Lobos and Mora (1991). To further purify OmpA, the crude OMPs were boiled in a sample buffer for 5 min and applied to SDS-PAGE (Laemmli, 1970). The samples were electrophoresed on a 10% polyacrylamide gel followed by Goomasie Brilliant Blue staining. The band of approximately 35 kDa was identified as OmpA (NGBI Reference Sequence: NP_460044.1) by orthogonal matrix-assisted laser desorption/ionization hybrid quadrupole-quadrupole time-of-flight mass spectrometry and tandem mass spectrometry in combination (QSTAR Pulsar i, Applied Biosystems, Foster Gity, GA) with trypsin digestion, followed by a peptide mass fingerprint search using Mascot (Matrix Science, Boston, MA). The band was then cut from the gel and homogenized separately in an elution buffer (20 mMTris-HGl with 1% SDS, pH 8.0), followed by removal of the gel using a microspin column (GE Healthcare, Buckinghamshire, UK). After centrifugal concentration, ice-cold acetone was added, placed at -80°G, and centrifuged at 20,000 x g for 15 min. The pellet was suspended in PBS. After adjustment at the appropriate concentration, the protein solution was used as native OmpA (nOmpA).
2445
dorf, Germany) according to the manufacturer's instructions. The DNA sequence of S. Enteritidis ompA (NGBI Reference Sequence: YP_002243066.1) was obtained by PGR using the Expand High-Fidelity^^^^ PGR System (Roche Applied Science, Mannheim, Germany). The ompA DNA was amplified with the forward (5'-ÇAXAT:GGGTGGGAAAGATAAGAGGTGGTAG-3') and reverse primers (5'-GTGGAGAGGGTGGGGGTGAGTTAGGAGGTG-3') ; the product lacked the signal sequence of the first 63 bp (containing the start codon) and the stop codon of full-length ompA and instead contained the recognition sites of the restriction enzymes Ndel and Xhol, respectively (underlined), which allowed the directional cloning of the PGR fragment into the expression vector. The PGR product was cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and the plasmid was transformed into One Shot TOPIO competent Escherichia coli (Invitrogen, Garlsbad, GA). The transformed cells were recovered by blue-white selection. The plasmid DNA from the selected clones was sequenced. Digestion of the pGEM-T Easy plasmid with Ndel and Xhol resulted in restriction fragments of ompA, which were subcloned into the expression vector pET29b (Novagen, Milwaukee, WI). This inducible vector system allows the expression of recombinant proteins with a 6-histidine tag at the C-terminus under the presence of isopropyl ß-D-1-thiogalactopyranoside (IPTG). The plasmid was transformed into E. coli BL21 (DE3) competent cells and grown on LB agar containing 100 |xg/ mL of kanamycin. The recombinant OmpA (rOmpA) protein was expressed in the insoluble fraction under the presence of 0.4 mMIPTG and was purified by affinity chromatography on a Ni-NTA His-Bind Resin (Takara Bio Inc., Shiga, Japan) after solubilization with 8 M urea. The purified protein was pooled and dialyzed in Spectra/Por 4 Regenerated Gellulose Dialysis Membranes (Spectrum Laboratories Inc., Rancho Dominguez, GA). The protein concentration was determined by the DG Protein Assay (Bio-Rad) based on the Lowry method (Lowry et al, 1951). The purified proteins were confirmed to be rOmpA by Western blotting using anti-polyhistidine mouse monoclonal antibody (Sigma, St. Louis, MO) followed by horseradish peroxidase-conjugated anti-mouse IgG rabbit sera (BD Transduction Laboratories, Lexington, KY). The reaction was visualized by the peroxidase substrate (Sigma).
Preparation of Component Vaccines Both nOmpA and rOmpA were adjusted to 320 [xg/ mL with PBS and mixed well with the same volume of complete or incomplete Fi-eund adjuvant (Sigma) for primary and secondary immunizations, respectively. The vaccination dose was 80 \xg in 0.5 mL/bird.
DNA Cioning and Expression
Expérimentai Birds
The bacterial genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Düssel-
In the present study, 37- to 41-d-old non-SPF White Leghorn chicks (Lohman, Julia), none of which had
2446
OKAMURA ET AL.
been vaccinated against Salmonella, were obtained from a local commercial layer farm. The birds were reared in individual wire cages with a nonmedicated layer ration and water supphed ad libitum in an isolation building. The birds were confirmed to be Salmonella-iree bacteriologically. All of the animal experiments in this study followed the institutional guidelines for the care and use of laboratory animals and were approved by the Animal Research Gommittee, Kitasato University School of Veterinary Medicine, Japan.
Efficacy of Vaccination with nOmpA Twenty healthy chicks of 37-d-old were assigned to 2 groups: nOmpA-vaccinated and S. Enteritidis-challenged; unvaccinated and S. Enteritidis-challenged (n = 10/group). The chicks were immunized intramuscularly in their legs at 41 d of age and challenged orally with lO'^ cfu of S. Enteritidis at 26 d postimmunization. The cecal droppings were collected at 3, 7, and 14 d postchallenge, and the bacterial excretion was examined. Ghickens were necropsied at 18 d postchallenge, and the liver, spleen, and cecal contents were examined for Salmonella. Sera were also collected at 0, 7, 14, 21, 26, 33, and 40 d after vaccination for antibody measurement .
Efficacy of Vaccination with rOmpA Thirty healthy chicks of 41-d-old were assigned to 6 groups: vaccinated once, vaccinated twice, adjuvant administered once, adjuvant administered twice, PBS administered once, and PBS administered twice (n — 5/ group). The groups with one administration were vaccinated intramuscularly in their legs at 43 d of age and challenged orally with 10^ cfu of 5*. Enteritidis at 28 d postvaccination. The remaining 3 groups with 2 administrations were vaccinated intramuscularly in their legs at 43 and 73 d of age and challenged orally with 10^ cfu of S. Enteritidis at 26 d after the second vaccination (56 d after the first vaccination). The cecal droppings were collected at 3, 6, and 13 d postchallenge, and the bacterial excretion was examined. Ghickens were necropsied at 14 d postchallenge, and the hver, spleen, and cecal contents were examined for Salm,onella. Sera were also collected weekly after the first vaccination for antibody measurement.
Recovery of Bacteria from Cecal Droppings and Tissue Samples The cecal droppings after challenge and the liver, spleen, and cecal contents at necropsies were taken aseptically and used for the enumeration of Salmonella according to a previous study (Okamura et al., 2007). The samples were separately homogenized with a 9-fold volume of Hajna Tetrathionate broth (HTT; Eiken, Tokyo, Japan) for the cecal droppings and cecal contents, and HIB for the liver and spleen. The serial
10-fold dilutions were then spread on DHL-rif plates and incubated at 37°G for 24 h. The homogenates were also incubated at 4I.5°G (cecal contents) or 37°G for 24 h for enrichment. After enumeration of colonies, loopfuls of homogenates that were negative initially were then streaked on DHL-rif plates. After incubation at 37°G for 24 h, the samples with Salmonella colonies were recorded as positive and assumed as 100 cfu/g. For the samples negative for Salmonella in the primary enrichment, the delayed secondary enrichment (DSE) was performed as described previously (Waltman et al., 1991). Briefly, the negative samples were kept at room temperature for 5 to 7 d to avoid the effects of inhibitory factors on Salmonella growth, of which 0.5 mL was inoculated into 4.5 mL of a fresh HTT and incubated at 41.5°G for 24 h. Salmonella was detected as described above for the primary enrichment, and the positive samples by DSE were assumed to contain Salmonella at 10 cfu/g.
Measurement of Antibody Levels in Sera Serum IgG levels against OmpA were measured by ELISA according to a previous study (Okamura et al., 2007). Each well of 96-well microtiter plates (Sumitomo Bakelite Go., Ltd., Tokyo, Japan) was coated overnight at 4°G with 50 ^iL of 10 |j,g/mL of rOmpA in a carbonate-bicarbonate buffer. Sera were diluted 10-fold with 0.5% BSA in 0.05% Tween20-PBS and applied to the plates at 50 |aL/well. Rabbit anti-chicken IgG antibody conjugated with horseradish peroxidase (Sigma), peroxidase substrate (Sigma), and 2 A/'H2SO4 (50 |a,L/well) was used for the reaction, and the optical density values were measured at 450 nm. The assay was performed in duplicate. The sera from experimentally infected hens (Okamura et al., 2007) and naive chicks collected in an earlier study (unpublished) were used as positive and negative controls, respectively. The reactivity of these sera to S. Enteritidis heat-inactivated and formalininactivated antigens were confirmed by Western blot before this study (data not shown).
Statistical Analyses Data of cfu enumeration and antibody levels were compared between groups by the unpaired Student's i-test. P-values of less than 0.05 were considered significant.
RESULTS
Efficacy of Vaccination with nOmpA In all the groups challenged with the homologous strain of S. Enteritidis, the levels of cecal excretion and tissue colonization were less than 10^ cfu/g, and the vaccinated birds showed no decrease in cecal excretion (Figure lA) and tissue colonization (Figure IB) compared with the unvaccinated birds throughout the ex-
SALMONELLA OUTER MEMBRANE PROTEIN A VACCINE
périment. However, vaccination with nOmpA successfully induced an increased level of serum anti-OmpA IgC (Figure lC). In the unvaccinated control birds, the specific antibody slightly increased postchallenge (Figure lC). Although the initial optical density value was relatively high (approximately 0.6), it was similar to that of the negative control sera. Together with the Western blot analyses (Supplemental Figure 1; available online at http://ps.fass.org/), this indicates that such a high background signal was not due to the possibility that the chickens had 5a¿mone/Za-specific or antiOmpA antibody before study initiation.
2447
who suggested the potential limitation of the interaction between an antibody and OmpA of live bacteria by 0-chain of LPS. Tarkka et al. (1989) also pointed out that it is difficult for the anti-Omp antibody to reach the Omp protein on live Neisseria meningitidis. Meenakshi et al. (1999) referred to an irrelevance of the increased antibody level as an indicator of a protective effect against Salmonella.
Efficacy of Vaccination with rOmpA The level of S. Enteritidis detected in cecal droppings was 10^ to 10^ cfu/g at 3 d postchallenge, and gradually decreased to 10^ to 10^ cfu/g at 13 d postchallenge. There were no significant differences between groups administered with rOmpA, adjuvant, and PBS (Figure 2A). Tissue colonization was less than 10^ cfu/g in all samples taken at necropsy without significant differences between groups (Figure 2B). As seen in the experiment using nOmpA, rOmpA-vaccinated birds showed a significant increase in anti-OmpA IgC in sera, and adjuvant- and PBS-administered birds showed increasing levels of anti-OmpA IgC after the challenge (Figure 2C). Similar results were obtained in both single- and double-dose administrations. Second immunization with rOmpA did not add any booster effects on cecal excretion, tissue colonization, and antibody levels (data not shown).
— nOmpA - Control
2
4
6
8
10
12
14
16
Days postchallenge
B
DISCUSSION Various proteins of gram-negative bacteria including Salmonella have been tested for their immunological or protective effects as vaccines. Among them, OMPs or porins have been reported to induce the host humoral responses and in turn inhibit postchallenge bacterial colonization. Meenakshi et al. (1999) showed that the vaccine composed of Salmonella OMPs and oil adjuvant inhibited the fecal shedding of Salmonella. The use of a different evaluation system with S. Callinarum, which is more lethal than S. Enteritidis, could provide more promising results. Cómez-Verduzco et al. (2010) demonstrated that 53 to 70% of the progeny chicks from breeding hens immunized with S. Callinarum porins were protected from death when challenged with 20 to 500 LD50 of S. Callinarum. Our study demonstrated that OmpA has a strong immunogenicity to induce the humoral response. Nevertheless, the bacterial shedding after challenge was not reduced by vaccination with OmpA. A potential reason is that the anti-OmpA antibody did not reach or recognize the OmpA on the outer membrane of live Salmonella due to the presence of other properties, such as LPS, pili, flagella, and other porin proteins, which could have masked the OmpA. This possibility is supported by Singh et al. (2003),
Liver
10
Spleen
20
Cecum
30
40
Days postimmunization
Figure 1. (A) Salmonella excretion in the cecal droppings collected at 3, 7, and 14 d postchallenge. The data represent the mean bacterial count and the SEM (n = 10 for each group). (B) Viable counts of Salmonella in the liver, spleen, and cecal contents collected at 18 d postchallenge. The data represent the mean bacterial count and the SEM (n = 10 for each group). (C) Anti-native outer membrane protein A (nOmpA) antibody response induced by vaccination determined by ELISA. Sera were collected from 10 birds/group at 0, 7, 14, 21, 26, 33, and 40 d after vaccination. Arrow indicates the date of challenge (26 d postimmunization). The data represent the mean absorbance and the SEM (n = 10 for each group), and asterisks indicate significant differences between groups (P < 0.01). OD = optical density.
OKAMURA ET AL.
2448 7
f ^
6
•
5 4 3 • 2
•
— Control — Adjuvant
— lOmpA 2
4
6
8
10
12
14
Days postchallenge
B
3 1
^2.5
D Control • Adjuvant • rOmpA
"s 2
1•
> 0.5 0 + Cecum
Spleen
Liver 1.4 1
with crude OMPs was not significant (Okamura et al., 2003). Therefore, the lack of protection in the challenge experiments may imply that the induction of cellular immunity failed. Different OMPs also need to be considered because the Omps vaccine with successful result (Meenakshi et al., 1999) should contain many other OMPs. Furthermore, variable results were obtained by using different types of OMPs in Salmonella Typhimurium (Hamid and Jain, 2008) and other bacterial agents such as Pasteurella multocida (Vasfi Marandi and Mittal, 1997). In addition, parenteral administration of antigen is expected to induce little or no secretory IgA response, which may be the most important part of a mucosal infection, such as salmonellosis. Therefore, the results obtained in this study do not preclude efficacy of mucosal administration of OmpA with a robust specific secretory IgA response. In conclusion, parenteral immunization with adjuvanted OmpA induced the anti-OmpA IgG effectively but resulted in no protection against Salmonella in chickens. To develop more effective component vaccines for avian salmonellosis, the vaccine efficacy of OMPs other than OmpA and route of immunization should be evaluated in regard to protection and immune responses, including mucosal IgA.
ACKNOWLEDGMENTS
1.2
The authors thank the students of the Laboratory of Zoonoses, Kitasato University School of Veterinary Medicine for their technical assistance for the animal experiments.
1
g' 0.6 ^ 0.4
- û - - Control - • • - • Adjuvant —•— rOmp.'V
0.2 0 10
20 30 Days postimmuni:zation
40
Figure 2. (A) Salmonella excretion in the cecal droppings collected at 3, 6, and 13 d postchallenge after single vaccination. The data represent the mean bacterial count and the SEM (n = 5 for each group). (B) Viable counts of Salmonella in the liver, spleen, and cecal contents collected at 14 d postchallenge. The data represent the mean bacterial count and the SEM (n = 5 for each group). (C) Anti-recombinant outer membrane protein A (rOmpA) antibody response induced by vaccination determined by ELISA. Sera were collected from 5 birds/group at 0, 7, 14, 21, 28, 35, and 42 d after vaccination. Arrow indicates the date of challenge (26 d postimmunization). The data represent the mean absorbance and the SEM (n = 5 for each group), and asterisks indicate significant differences between the rOmpA-immunized group and other groups [P < 0.01). OD = optical density.
On the other hand, protection against salmonellosis requires the host immunity of both cellular and humoral arms (Babu et al, 2003; Mittrücker and Kaufmann, 2000). In mice, immunization with porin can induce these responses (Singh et al., 1999). However, although the cellular responses were not evaluated in the current study, the proliferative response of splenocytes upon ex vivo stimulation with crude porin after immunization
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bivalent killed Salmonella vaccine to prevent egg contamination with Salmonella entérica serovars Enteritidis, Typhimurium, and Gallinarum biovar PuUorum, using 4 different challenge models. Vaccine 25:4837-4844. Schlundt, J., H. Toyofuku, J. Jansen, and S. A. Herbst. 2004. Emerging food-borne zoonoses. Rev. Sei. Tech. 23:513-533. Singh, M., H. Vohra, L. Kumar, and N. K. Ganguly. 1999. Induction of systemic and mucosal immune response in mice immunised with porins of Salmonella typhi. J. Med. Microbiol. 48:79-88. Singh, S. P., Y. U. Williams, S. Miller, and H. Nikaido. 2003. The C-terminal domain of Salmonella entérica serovar typhimurium OmpA is an immunodominant antigen in mice but appears to be only partially exposed on the bacterial cell surface. Infect. Immun. 71:3937-3946. Smith, S. G., V. Mahon, M. A. Lambert, and R. P. Fagan. 2007. A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol. Lett. 273:1-11. Sonntag, L, H. Schwarz, Y. Hirota, and U. Henning. 1978. Cell envelope and shape of Escherichia coli: Multiple mutants missing the outer membrane lipoprotein and other major outer membrane proteins. J. Bacteriol. 136:280-285. Tarkka, E., A. Muotiala, M. Karvonen, K. Saukkonen-Laitinen, and M. Sarvas. 1989. Antibody production to a meningococcal outer membrane protein cloned into liv Salmonella typhimurium aroA vaccine strain. Microb. Pathog. 6:327-335. Vasfi Marandi, M., and K. R. Mittal. 1997. Role of outer membrane protein H (OmpH)- and OmpA-specific monoclonal antibodies from hybridoma tumors in protection of mice against Pasteurella multocida. Infect. Immun. 65:4502-4508. Waltman, W. D., A. M. Home, C. Pirkle, and T. G. Dickson. 1991. Use of delayed secondary enrichment for the isolation of Salmonella in poultry and poultry environments. Avian Dis. 35:88-92.
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