Research Notes
Humoral immune response in breeding hens and protective immunity
provided by administration of purified Salmonella Gallinarum porins
G. Gómez-Verduzco,* G. Téllez,†1 A. L. Quintana,* A. Isibasi,‡ and V. Ortiz-Navarrete§1
*Departamento de Producción Animal: Aves, Facultad de Medicina Veterinaria y Zootecnia,
Universidad Nacional Autonoma de Mexico, Mexico City, Mexico, D.F. 04510; †Department of Poultry Science,
University of Arkansas, Fayetteville 72701; ‡Unidad de Investigación Médica en Inmunoquímica del Hospital
de Especialidades del Centro Medico Nacional del Instituto Mexicano del Seguro Social, Mexico City, Mexico,
D.F. 03020; and §Departamento de Biomedicina Molecular del Centro de Investigación y de Estudios
Avanzados del Instituto Politécnico Nacional (CINVESTAV), Mexico City, Mexico, D.F. 07360
porin antibodies were detected in broiler breeder serum
and in fertile eggs (P < 0.05). Consequently, chickens
from immunized broiler breeder hens were protected
between 53 to 70% against challenges of 20 to 500 halfmaximal lethal dose of Salmonella Gallinarum (P <
0.001) when compared with control hens that were injected with PBS. These results suggest that Salmonella
Gallinarum porins, as those of other Salmonella species,
participate in the induction of the passive protective
immunity, and the humoral immune response may be
one of the mechanisms involved in the establishment of
this protection.
Key words: Salmonella Gallinarum, porin, immunity, chicken
2010 Poultry Science 89:495–500
doi:10.3382/ps.2009-00448
INTRODUCTION
Galdiero et al., 2003; Vega et al., 2003; Diaz-Quiñonez
et al., 2004; Secundino et al., 2006; Cervantes-Barragan
et al., 2009). In the present study, the porins of Salmonella Gallinarum were isolated, purified, and characterized to evaluate its capacity to induce humoral
response in broiler breeder hens and to transfer protective immunity in their progeny.
Among the total outer membrane proteins of the
gram-negative bacteria, porins are expressed more abundantly. Their structure, location, and large number on
bacterial surfaces assume them multiple functions (Achouak et al., 2001). Several studies have demonstrated
the efficacy of porins in the induction of active immunity against Salmonella Typhi in mice (Isibasi et al.,
1992), as well as Salmonella Gallinarum (Bouzoubaa et
al., 1987, 1989) and Salmonella Enteritidis (Meenakshi
et al., 1999) in chickens. Recent studies have indicated
that both cellular and humoral mechanisms of immunity are involved in protection achieved in mice (Blanco
et al., 1993; Isibasi et al., 1994; González et al., 1995;
MATERIALS AND METHODS
Bacterial Strain and Growth Conditions
A primary poultry isolate of Salmonella Gallinarum
was kindly donated by Mario Padron (Aviagen Inc.,
Mexico City, Mexico) for use in our laboratory. This
isolate was selected for resistance to nalidixic acid
(NA) and novobiocin (NO). An isolate of Salmonella
Typhi was originally isolated from a patient with typhoid fever and has been maintained in culture in our
laboratory since 1979. To isolate porins, cultures of Salmonella Gallinarum and Salmonella Typhi were grown
in minimum salts medium (medium A) containing 5%
©2010 Poultry Science Association Inc.
Received September 9, 2009.
Accepted November 22, 2009.
1
Corresponding authors: [email protected] and vortiz@cinvestav.
mx
495
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 17, 2016
ABSTRACT The current studies were undertaken to
assess the ability of humoral immune response in breeding hens to provide protective maternal antibody in the
progeny. A highly purified outer membrane protein, 34
kDa, was isolated from a virulent strain of Salmonella
Gallinarum. Cross-reactivity was observed between this
protein and Salmonella Typhi porins; thus we consider
this outer membrane protein as a Salmonella Gallinarum
porin. To evaluate passive immunity against Salmonella
Gallinarum, 200 broiler breeder hens were immunized
with either 10 μg of Salmonella Gallinarum porins, 30
μg of Salmonella Gallinarum porins, or PBS without
porins as a control group. Anti-Salmonella Gallinarum
496
Gómez-Verduzco et al.
Salmonella Gallinarum and Salmonella
Typhi Porin Isolation and Purification
Salmonella Gallinarum and Salmonella Typhi porins
were obtained according to the method of Nikaido
(1983) and modified by Isibasi et al. (1992). Briefly, the
supernatant from the sonicated culture was centrifuged
at 100,000 × g for 45 min and the pellet containing
the cell walls was resuspended in 2% SDS in 10 mM
Tris-HCl (pH 7.7). This suspension was kept at 32°C
for 30 min and then centrifuged at 100,000 × g for 30
min at 20°C. This procedure was repeated twice. To
separate the porins from the peptidoglycan, the pellet was solubilized in a buffer of 50 mM Tris-HCl (pH
7.7) containing 2% SDS, 5 mM EDTA, 0.4 M NaCl,
and 0.05% β-mercaptoethanol for 2 h at 37°C and was
purified by size exclusion chromatography on Sephacryl
S-200 (GE Healthcare, Pittsburgh, PA). The protein
concentration was determined by the method of Lowry
et al. (1951). The quantity of lipopolysaccharide was
determined by measuring the concentration of 2-keto3-octulosonic acid as described previously (Karkhanis
et al., 1978). Sodium dodecyl sulfate-PAGE was carried
out on 12% gels under reducing conditions (Laemmli,
1970).
Antiporine Serums
New Zealand rabbits (1.5 kg to 2 kg) were intradermally injected with 1 mg/mL of Salmonella Typhi
porins or with Salmonella Gallinarum porins emulsified in complete Freund’s adjuvant. Fifteen days later,
rabbits were immunized (intradermally) with 1 mg/mL
of Salmonella Typhi or Salmonella Gallinarum porins
emulsifiied in incomplete Freund’s adjuvant. A third
(intradermal) immunization was performed 15 d after
the second immunization, with 0.5 mg/mL of Salmonella Typhi or Salmonella Gallinarum porins. Three
additional immunizations were done with 8-d intervals
between each immunization with 0.5 mg/mL of Salmonella Typhi or Salmonella Gallinarum porins. Ten days
after the last immunization, rabbits were bled. Serum
for each rabbit was collected and used for Western blot
analysis.
Immunoblot Analysis
Electrophoretic transfer of porins from polyacrylamide gels to nitrocellulose membrane was accomplished
in an electroblotting unit by means of the transfer buffer [25 mM Tris, 192 mM glycine, 0.02% (wt/vol) SDS,
20% (vol/vol) methanol] and a constant current of 0.1
V/15 h. The membranes were blocked with 5% milkPBS, pH 7.4. After being washed with PBS-Tween
0.1%, membranes were incubated 3 h at 25°C in a 1:100
dilution of the test sample serum samples in blocking buffer. After being washed with PBS-Tween 0.1%,
membranes were incubated 1.5 h at 25°C in a 1:2,000
dilution of peroxidase-conjugated goat anti-rabbit IgG
(Sigma-Aldrich). The membranes were revealed by a
quimioluminescence detection kit (Amersham Life Science, Arlington Heights, IL).
Protective Capacity of Salmonella
Gallinarum Porins in Chicks
From Vaccinated Broiler Breeder Hens
Groups of 200 broiler breeders hens (Ross 380), 53
wk old, were subcutaneously immunized with 10 μg
of Salmonella Gallinarum porins (group 1), 30 μg of
Salmonella Gallinarum porins (group 2), or PBS without porins as a control (group 3) on 0 and 10 d. Seven days after the last immunization, fertile eggs from
these groups were collected and stored at 4°C. Eggs
were hatched at 21 d. A total of 270 chicks were distributed randomly in environmentally controlled Petersime
batteries (Petersime Incubator Co., Gettysburg, OH).
Experimental design was completely random: 9 treatments, 30 chicks for each group. The chicks were orally
challenged with 20, 100, and 500 LD50 of Salmonella
Gallinarum. Protection was defined as the percentage
of survival during the 10 d after challenge. Additionally, 7 d after the last immunization, IgY was purified us-
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 17, 2016
yeast extract and 12.5% glucose and were incubated in a
shaker at 37°C for 8 h until the mid-exponential growth
phase was reached (previously determined by a growth
curve). To challenge chicks, culture of Salmonella Gallinarum was grown in Luria-Bertani broth (Gibco-BRL,
Grand Island, NY) to logarithmic phase (previously
determined by a growth curve). Briefly, bacteria were
then washed 3 times in sterile saline by centrifugation
at 1,864 × g. Concentrations of Salmonella Gallinarum
were retrospectively determined by spread-plating on
xylose lactose differential agar (Becton, Dickinson, and
Company, Sparks, MD) plates containing NO (25 µg/
mL) and NA (20 µg/mL). To determine the half-maximal lethal dose (LD50) of Salmonella Gallinarum, 136
Salmonella-free chickens were distributed into 8 groups
of 17 chickens each and were challenged with different
Salmonella Gallinarum doses (from 1 × 104 to 1 × 1010
cfu/chicken; data not shown). The LD50 of the culture
of Salmonella Gallinarum was determined according to
Reed and Muench’s equation (Reed and Muench, 1938).
Chickens challenged with 108 cfu per chicken (LD50) of
the Salmonella Gallinarum strain reached 52.9% mortality at the end of the assay, d 10. In all birds that
died, bacteriology of liver, heart, cecum, and spleen was
performed searching for the presence of Salmonella Gallinarum colonies, and each sample was streaked for isolation on xylose lactose differential agar plates containing 25 µg/mL of NO and 20 µg/mL of NA. The plates
were incubated at 37°C for 24 h and examined for the
presence or absence of antibiotic-resistant Salmonella
Gallinarum.
RESEARCH NOTE
497
ing the EGGstract IgY purification systems (Promega,
Madison, WI).
Humoral Response Against Salmonella
Gallinarum Porins
The presence of specific antibodies against Salmonella Gallinarum porins was determined by an ELISA
assay (Engvall and Perlman, 1971). All reagents were
added in 100 μL/well to flat-bottom end microtiter
plates (Nunc, Roskilde, Denmark), which were coated
overnight at 4°C with 30 μg/mL of Salmonella Gallinarum porins in bicarbonate buffer, pH 9.6. The plates
were blocked with 5% milk in PBS for 2 h at 37°C. The
serum from broiler breeder hens and IgY from eggs
were diluted 1:100 in 5% milk-PBS and incubated 1 h
at 37°C. These were then washed 3 times with PBS/
Tween 0.5%. Plates were then incubated with a dilution
1:1,000 of peroxidase-conjugated goat anti-IgY (Novus
Biologicals, Littleton, CO). Unbound enzyme was removed by washing 3 times with PBS/Tween 0.5% and
the substrate o-phenylenediamine (0.4 mg/mL) was
added to each well and the reaction was stopped after
10 min with H2SO4. Optical density was measured at
492 nm in a semiautomated ELISA reader. The mean
and SD of each triplicate were calculated and expressed
in optical density.
Statistical Analysis
Differences among treatments for Salmonella Gallinarum-specific antibodies of the IgG isotype in serum
or eggs from hens that received an s.c. injection of PBS
without porins (control) or 10 µg/hen or 30 µg/hen
of Salmonella Gallinarum porins on 0 and 10 d were
analyzed by ANOVA using the GLM procedure. Sig-
nificant differences (P < 0.05) were further separated
using Duncan’s multiple range test and commercial statistical analysis software (SAS Institute, 2002). Differences (P < 0.001) among treatments for percentage of
survival chickens challenged with Salmonella Gallinarum were determined using the χ2 test of independence
(Zar, 1984).
Figure 2. Purified porins from Salmonella Gallinarum analyzed
by SDS-PAGE. Fractions that correspond to peak A and peak B were
pooled and analyzed by SDS-PAGE. Lane 1 = peptidoglycan-associated material (15-μg sample boiled); lane 2 = peak A (15-μg sample
not boiled); lane 3 = peak A (15-μg sample boiled); lane 4 = peak B
(15-μg sample not boiled); lane 5 = peak B (15-μg sample boiled); lane
6 = without sample; lane 7 = peak A (5-μg sample boiled); lane 8 =
without sample; lane 9 = Salmonella Typhi porins (5-μg sample not
boiled). MW = molecular weight markers. After electrophoresis, the
gel was stained with Coomassie Blue according to standard protocols.
The thick arrow represents porin homodimer; the thin arrow represents monomeric porin.
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 17, 2016
Figure 1. Profile of size exclusion chromatography on a Sephacryl S-200 column (GE Healthcare, Pittsburgh, PA). Salmonella Gallinarum
porins were purified from peptidoglycan-associated material. Vo = void volume; BSA = volume that was eluting BSA; OVA = volume that eluting ovalbumin. OD = optical density.
498
Gómez-Verduzco et al.
RESULTS AND DISCUSSION
Figure 4. Salmonella Typhi porins recognized by polyclonal antiSalmonella Gallinarum porins. Lane 1 = peak A (15-μg sample not
boiled); lane 2 = Salmonella Typhi porins (5-μg sample boiled). Antiserum was diluted 1:100 and peroxidase-conjugated goat anti-rabbit
IgG was diluted 1:2,000.
Figure 3. Salmonella Gallinarum porins recognized by polyclonal
anti-Salmonella Typhi porins. Lane 1 = peak A (15-μg sample not
boiled); lane 2 = Salmonella Typhi porins (5-μg sample boiled). Antiserum was diluted 1:100 and peroxidase-conjugated goat anti-rabbit
IgG was diluted 1:2,000.
4 and 5). Accordingly, both boiled fractions (A and
B) were located in the same molecular weight regions
as the Salmonella Typhi porins (Figure 2, line 9). Lipopolysaccharide present as contaminant in the purified
preparation was 0.04%, which is similar to the amount
found in purified porins from Salmonella Typhi (Isibasi
et al., 1992). In addition, these proteins showed crossreactivity with a specific rabbit IgG antibody against
the Salmonella Typhi (Figure 3) and also rabbit IgG
against these proteins reacted with Salmonella Typhi
porins (Figure 4). This cross-reactivity must be due to
the high degree of homology between Salmonella porins.
Thus, we consider these proteins as Salmonella Gallinarum porins. These results suggest that Salmonella
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 17, 2016
A crude preparation of porins was isolated from peptidoglycan-associated material and purified by size exclusion chromatography on Sephacryl S-200 columns;
this methodology has been used to isolate porins from
Escherichia coli, Salmonella Typhimurium, and Salmonella Typhi. Figure 1 shows the porins purified to
homogeneity. The first peak (A) was eluted with an
estimated molecular weight above 66 kDa and a second
peak (B) was obtained with an estimated molecular
weight below 45 kDa. When peak A was analyzed by
SDS-PAGE under reducing conditions and the sample
was boiled, only a protein of 34 kDa was observed,
(Figure 2, line 2 and 7, thin arrow line); nevertheless,
if the sample was not boiled, a band with molecular
weight above 66 kDa was observed (Figure 2, line 3,
thick arrow line). On the other hand, peak B shows a
protein with a molecular weight of 34 kDa that does
not change if the sample was not boiled (Figure 2, line
499
RESEARCH NOTE
Table 1. Presence of specific IgY anti-Salmonella Gallinarum
porin antibodies in serum and fertile eggs from immunized
breeders with Salmonella Gallinarum porins1
Treatment
Serum (optical
density 492 nm)
Egg (optical
density 492 nm)
Control
10 μg
30 μg
0.011 ± 0.32b
0.164 ± 0.0033a
0.173 ± 0.0031a
0.028 ± 0.057b
0.157 ± 0.117a
0.198 ± 0.122a
a,b
Values within columns with different lowercase superscripts differ
significantly (P < 0.05).
1
Groups of 200 breeders, 53 wk old, were immunized with 10 and 30
µg/hen of Salmonella Gallinarum porin on d 0 and on d 10 via s.c. injection. The control group was injected with PBS without porins. Presence
of IgY specific antibodies against Salmonella Gallinarum porin in serum
and in fertile eggs of immunized breeders (15 samples for each group)
were quantified by ELISA as described in Materials and Methods. Data
expressed as means ± SD.
Table 2. Protecting capacity of anti-Salmonella Gallinarum
porin antibodies in chicks challenged with Salmonella Gallinarum1
Salmonella
Gallinarum
20 LD50
100 LD50
500 LD50
Control
10 g
b,2
0/30 (0%)
0/30 (0%)b
0/30 (0%)b
30 g
a
16/30 (53.3%)
17/30 (56.6%)a
21/30 (70%)a
21/30 (70%)a
20/30 (66.6%)a
18/30 (60%)a
a,b
Values within rows with different lowercase superscripts differ significantly (P < 0.001).
1
Groups of 200 breeders, 53 wk old, were immunized with 10 and 30
µg/hen of Salmonella Gallinarum porin on d 0 and on d 10 via s.c. injection. Seven days after last immunization, eggs were collected and incubated for 21 d. At d 1, chicks were distributed in groups of 30 chicks and
were orally challenged with 20, 100, and 500 half-maximal lethal dose
(LD50) of Salmonella Gallinarum. The control group was integrated with
chicks from broiler breeder hens immunized with PBS without porins,
challenged with the same LD50.
2
Numbers of survivor progeny chicks/total (%).
REFERENCES
Achouak, W., T. Heulin, and J. M. Pages. 2001. Multiple facets of
bacterial porins. FEMS Microbiol. Lett. 199:1–7.
Blanco, F., A. Isibasi, C. González, N. V. Ortiz, J. Paniagua, C. Arreguin, and J. Kumate. 1993. Human cell mediated immunity to
porins from Salmonella Typhi. Scand. J. Infect. Dis. 25:73–80.
Bouzoubaa, K., K. V. Kabbaj, J. A. Newman, and B. S. Pomeroy.
1989. Feasibility of using proteins from Salmonella Gallinarum
vs. 9R live vaccine for the prevention of fowl typhoid in chickens.
Avian Dis. 33:385–391.
Bouzoubaa, K., K. V. Nagaraja, J. A. Newman, and B. S. Pomeroy. 1987. Use of membrane proteins from Salmonella Gallinarum
for prevention of fowl typhoid infection in chickens. Avian Dis.
31:699–704.
Cervantes-Barragan, L., C. Gil-Ruiz, R. Pastelin-Palacios, K. S.
Lang, A. Isibasi, B. Ludewig, and C. Lopez-Macias. 2009. TLR2
and TLR4 signaling shape specific antibody responses to Salmonella Typhi antigens. Eur. J. Immunol. 39:126–135.
Diaz-Quiñonez, A., N. Martin-Orozco, A. Isibasi, and V. Ortiz-Navarrete. 2004. Two Salmonella OmpC K(b)-restricted epitopes
for CD8+-T-cell recognition. Infect. Immun. 72:3059–3062.
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 17, 2016
Gallinarum porin might form homodimers (peak A)
at the outer membrane instead of homotrimers as has
been reported for other porins (Nikaido, 2003). Peak
B might be a monomer, but further studies need to be
done to establish whether the Salmonella Gallinarum
porins described in this paper are indeed porins and to
identify to which porin family they belong.
Hen immunization was performed employing very low
concentrations of the porin (peak A) in relation to the
weight of the birds (10 and 30 μg; an average weight
of 3.5 kg per breeder) and without any adjuvant. We
avoided the use of adjuvant because previous studies, in
mouse and humans, have showed that Salmonella Typhi
porins are potent inductors of the immune response (Isibasi et al., 1992; Salazar-Gonzalez et al., 2004). Table
1 shows the presence of specific IgY anti-Salmonella
Gallinarum porin antibodies in serum and fertile eggs
from immunized breeders with Salmonella Gallinarum
porins. These results corroborate that porins are potent
immunostimulatory molecules and agree with the fact
that porins can be used as carrier molecules to induce
high antibody titer against T-cell-independent antigens
such as the Vi capsular polysaccharide (Singh et al.,
1999). Moreover, porins from other pathogens have
been described as activators of innate immunity. These
proteins are agonists for toll-like receptor 2 and induce
the expression of co-stimulatory molecules, upregulate
MHC class II molecules, promote cytokine release by
macrophages, and increase antibody production by B
cells (Massari et al., 2002; Galdiero et al., 2003; Ray
and Biswas, 2005).
Protection conferred by antibodies against Salmonella Gallinarum porin is observed in Table 2. All of
the chickens used in this work were negative for Salmonella spp. at day of hatch. Chickens hatched from
immunized breeders survived the challenge with different half-maximal lethal doses. Survival ranged from
53 to 70%, which agrees with the level of protection
observed in the mouse model (Isibasi et al., 1992). On
the contrary, chicks hatched from the control group and
injected with buffer died after the challenge with 20
LD50. All inoculated birds became ill from the fifth day
postchallenge. Mortality started at d 5, and at the end
of the experiment, all surviving chickens were seriously
ill. Chickens from control breeder hens that were injected with PBS markedly reduced their food intake and
showed weakness, somnolence, and fever 4 d after challenge. At this stage, many of them died, and the peak
of mortality occurred at the sixth day postchallenge.
The main lesions found in sick chickens were hepatitis
and splenitis, with hepatomegaly and splenomegaly in
most birds; some chickens had enteritis, and their ceca
presented a firm caseous material. Bacteriologic analysis showed that mortality in all groups was from fowl
typhoid. These results indicate the usefulness of Salmonella Gallinarum porin for induction of antibodies in
the hens that provided protective maternal antibody in
the progeny and due to the similarities between porins,
it is likely that Salmonella Gallinarum porin might induce protection against other Salmonella strains of importance to the poultry industry.
500
Gómez-Verduzco et al.
Nikaido, H. 1983. Proteins forming large channels from bacterial and
mitochondrial outer membranes: Porins and phage lambda receptor protein. Methods Enzymol. 97:85–100.
Nikaido, H. 2003. Molecular basis of bacterial outer membrane permeability. Microbiol. Mol. Biol. Rev. 67:593–656.
Ray, A., and T. Biswas. 2005. Porin of Shigella dysenteriae enhances
toll-like receptors 2 and 6 of mouse peritoneal B-2 cells and induces the expression of immunoglobulin M, immunoglobulin G2a
and immunoglobulin A. Immunology 114:94–100.
Reed, L. J., and H. Muench. 1938. A simple method of estimating
fifty percent endpoints. Am. J. Hyg. 27:493–497.
Salazar-Gonzalez, R. M., C. Maldonado-Bernal, N. E. RamírezCruz, N. Rios-Sarabia, J. Beltrán-Nava, J. Castañón-González,
N. Castillo-Torres, J. A. Palma-Aguirre, M. Carrera-Camargo, C.
López-Macías, and A. Isibasi. 2004. Induction of cellular immune
response and anti-Salmonella enterica serovar Typhi bactericidal
antibodies in healthy volunteers by immunization with a vaccine
candidate against typhoid fever. Immunol. Lett. 93:115–122.
SAS Institute. 2002. SAS User’s Guide: Statistics. SAS Institute
Inc., Cary, NC.
Secundino, I., C. López-Macías, L. Cervantes-Barragán, C. Gil-Cruz,
N. Ríos-Sarabia, R. Pastelin-Palacios, M. Z. Villasis-Keever, I.
Becker, J. L. Puente, E. Calva, and A. Isibasi. 2006. Salmonella
porins induce a sustained, lifelong specific bactericidal antibody
memory response. Immunology 117:59–70.
Singh, M., H. Vohra, L. Kumar, and N. K. Ganguly. 1999. Induction
of systemic and mucosal immune response in mice immunized
with porins of Salmonella Typhi. J. Med. Microbiol. 48:79–88.
Vega, M. I., L. Santos-Argumedo, S. Huerta-Yepez, R. Luría-Perez,
V. Ortiz-Navarrete, A. Isibasi, and C. R. González-Bonilla. 2003.
A Salmonella Typhi OmpC fusion protein expressing the CD154
Trp140-Ser149 amino acid strand binds CD40 and activates a
lymphoma B-cell line. Immunology 110:206–216.
Zar, J. 1984. Biostatistical Analysis. 2nd ed. Prentice Hall, Englewood Cliffs, NJ.
Downloaded from http://ps.oxfordjournals.org/ at Pennsylvania State University on May 17, 2016
Engvall, E., and P. Perlman. 1971. Enzyme-linked immunosorbent
assay (ELISA): Quantitative assay of immunoglobulin G. Immunochemistry 8:871–879.
Galdiero, M., M. Vitiello, and S. Galdiero. 2003. Eukaryotic cell signaling and transcriptional activation induced by bacterial porins.
FEMS Microbiol. Lett. 226:57–64.
González, C. R., M. V. Mejía, J. Paniagua, V. Ortíz-Navarrete, G.
Ramírez, and A. Isibasi. 1995. Immune response to porins isolated from Salmonella Typhi in different mouse strains. Arch.
Med. Res. 26 Spec No:S99–S103.
Isibasi, A., V. Ortiz-Navarrete, J. Paniagua, R. Pelayo, C. R.
González, J. A. García, and J. Kumate. 1992. Active protection
of mice against Salmonella Typhi by immunization with strainspecific porins. Vaccine 10:811–813.
Isibasi, A., J. Paniagua, M. P. Rojo, N. Martín, G. Ramírez, C.
R. González, C. López-Macías, J. Sánchez, J. Kumate, and V.
Ortiz-Navarrete. 1994. Role of porins from Salmonella Typhi in
the induction of protective immunity. Ann. N. Y. Acad. Sci.
730:350–352.
Karkhanis, Y. D., J. Y. Zeltner, and J. J. Jackson. 1978. A new
and improved microassay to determine 2-keto-3-deoxyoctonate
in lipopolysaccharide of gram-negative bacteria. Anal. Biochem.
85:595–601.
Laemmli, U. K. 1970. Cleavage of structure proteins during the assembly of the head bacteriophage T4. Nature 227:680–688.
Lowry, O. H., N. J. Rosenbrough, A. L. Farr, and R. J. Randall.
1951. Protein measurement with folin phenol reagent. J. Biol.
Chem. 193:65–75.
Massari, P., P. Henneke, Y. Ho, E. Latz, D. T. Golenbock, and L. M.
Wetzler. 2002. Cutting edge: Immune stimulation by neisserial
porins is toll-like receptor 2 and MyD88 dependent. J. Immunol.
168:1533–1537.
Meenakshi, M., C. S. Bakshi, G. Butchaiah, 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.