XXIV

Worlds Poultry Congress 5 - 9

August - 2012 Salvador - Bahia - Brazil

Avian Salmonellosis, vaccines and immune

mechanisms of protection: present and
future perspectives
DVM, MSc., PhD.
Prof. Ad. Postgraduate Unit
Faculty of Veterinary Medicine,
University of San Marcos, Lima - Per

Summary

Introduction
There is continuing interest in finding ways of
preventing flock infection and, hence, contamination
of poultry products with Salmonella enterica.
Control measures are difficult to use effectively
because there are numerous potential sources of
Salmonella infection and product contamination in
an integrated poultry enterprise [Revolledo et al.,
2006]. Control of Salmonella infections in poultry
farms needs to begin with good farming practices
and appropriate management associated with strict
sanitary measures. Preventive and curative strategies

Pathogenesis in poultry

Liliana Revolledo

fficial control for animal diseases is defined

by thSalmonellosis is one of the most
prevalent foodborne diseases worldwide.
Food animals have been identified as
reservoirs for non-typhoid Salmonella infections.
Several measures have been used to prevent
and control Salmonella infections in poultry
and vaccination is the most practical measure
and effective method to control and prevent
Salmonellosis. Salmonella vaccines can decrease
public health risk by reducing colonization and
organ invasion, including reproductive tissues, and
by diminishing fecal shedding and environmental
contamination.
This
presentation
discusses
present and future approaches based on scientific
information regarding killed and live attenuated
Salmonella vaccines and their immune mechanisms
of protection.

have been widely applied for reducing the incidence

of Salmonella colonization in chickens at the farm
level [Vandeplas et al., 2010]. Various prophylactic
measures have been employed to prevent and control
Salmonella infection in poultry production, and
vaccination is one of them. Salmonella vaccination
aims to mimic the development of naturally acquired
immunity by inoculation of non-pathogenic but
still immunogenic components of the pathogen,
reducing or eliminating the risk for the consumer.
Killed and live attenuated products have been used
for controlling Salmonella in poultry production,
thus vaccination with live attenuated products has
proved to be effective [Cerquetti & Gherardi, 2000].
This is a brief overview of Salmonella vaccines in
chickens, immune mechanisms of protection, and
the future perspectives in this area.

Most of the known Salmonella serotypes

are pathogenic for humans or animals or both.
Although Salmonella pathogenesis has been well
characterized in the mammalian model [Barrow,
2007], there is limited information of specific
mechanisms in avian species [Dunkley et al., 2009].
Poultry species can be infected by host-specific and
non-host specific Salmonella serotypes.
Salmonella Gallinarum (SG) and Salmonella
Pullorum (SP) cause severe disease and death of
birds compared with other known Salmonella
serotypes. Chadfield et al., (2003) suggested that
SG invades the bursa but the process is not time
dependent, and they demonstrated no selectivity
for a potential port of entry for the host specific
serotype. Avian systemic salmonellosis has three
phases: invasion, systemic infection and the
resolution of the infection [Sadeyen et al., 2004].
The third phase can have three results: the clearance
of the bacteria, death of the birds due to infection
and, partial clearance of the bacteria, which leads
to a subclinical carrier state, as shown in Figure
1. The biology of Pullorosis is markedly different
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Immune response against

Salmonella infection

when compared to Fowl Typhoid, which causes

high mortality. Pullorosis induces an increase of
Salmonella in the spleen resulting in an infection of
the reproductive tract [Wigley et al., 2005].

The immune response to Salmonella infections

is very complicated and involves the interaction of
many components of the immune system including
the innate and the adaptive immune system
[Nagarajan et al., 2009]. Although progress has
been made in understanding immune responses
against Salmonella infections, further research
is needed to understand the complete roles of
humoral and cell-mediated immunity because until
now, no consistent pattern has been observed.
Pathogenic bacteria have evolved mechanisms
to invade the epithelial cell barrier and survive
within host tissues. Salmonella maintains genes
organized within pathogenicity islands that encode
virulence factors that allow adherence, invasion
and dissemination in the host [Aziz et al., 2007].
TLR (Toll-like receptors) are cell receptors which
recognize structural motifs on pathogens and initiate
signaling cascades controlling the development of
innate immune response [Chauss et al., 2011].
These receptors contribute to host resistance to
microbial pathogens and can drive the evolution of
virulence mechanisms [Arpaia et al., 2011] and can

Salmonella infects poultry and other animals

by the oral route. Non-host specific Salmonella in
poultry is frequently involved in food-poisoning in
humans. In chickens, it produces systemic disease
in some special cases, such as the laying period,
in chicks in the first two weeks of life or after viral
diseases. The pathogenesis of non-host Salmonella
serotypes in poultry is summarized in Figure 1.
Salmonellas are not native members of the gut
microbiota, but young chicks are readily colonized,
and the organisms may persist in the host for some
weeks or during all of the rearing period [Revolledo
et al., 2006]. They become localized in the cecal
tonsils and can occur in the upper part of the small
intestine and in the gizzard and proventriculus
[Fanelli et al., 1971]. Because most birds infected
with Salmonellas become symptomless carriers,
they constitute a reservoir of the organisms, which
is a potential human health hazard. Additionally,
by contaminating the environment, these birds are
responsible for increasing the number of infected
individuals [Revolledo et al., 2006].
Liliana Revolledo

Worlds Poultry Congress 5 - 9

Salmonellosis in
poultry

Systemic disease host-specific

(S. Pullorum, S. Gallinarum)

Food-poisoning non-host-specific
(S. Enteritidis, S. Typhimurium,
etc.)

Colonize
intestine poorly

Noninflammatory
entry

Systemic disease
eventually

Not involved in
human cases

Absence
of carcass
contamination

in lay, young chicks,

after some viral
infections

Bacterial persistance
in liver and spleen
(carrier state)
Reproductive tract
infected and infected
egg laid

Clearance / Death

Fecal
excretion
Colonize the
alimentary
tract and
ceca without
disease

Carcass
contamination
at slaughter

Symptomless
carriers

Salmonella
entering the
human food chain

Figure 1 - Pathogenesis of host-specific and non-host specific Salmonella in poultry.

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August - 2012 Salvador - Bahia - Brazil

promote adaptive immunity through control of

dendritic cell maturation [Iwasaki & Metzhitov,
2004]. The consequences of Salmonella infection on
the expression of the different TLR, and particularly
TLR4, have been widely studied [Crhanova et al.,
2011].
Salmonella Gallinarum does not induce an
inflammatory response and may not be limited by
the immune system, leading to severe systemic
disease [Kaiser et al., 2000]. Invasion of SG results
in little or no production of IL-6 suggesting that the
pathogenesis and host specificity of the SG infection
in the chicken may be related to some extent to the
lack of an inflammatory response in the early stages
of the infection in the gut [Kaiser et al., 2000].

It has been suggested that intestinal SIgA

(secretory IgA) responses partially contribute to
the later elimination of the Salmonella Enteritidis
from the gut, and the humoral systemic and
local immune responses seem to be related to
the cecal colonization [Berthelot-Hrault, 2003].
Cell-mediated immunity is responsible for tissue
clearance, but how this mechanism could be
responsible for intestinal clearance remains unclear
[Zhang-Barber et al., 1999]. The role of T cell
responses in the clearance of enteric Salmonellae
has not been proven. However, in the absence of
an essential role for B cells (bursa-derived cells) and
with faster clearance of infection as a secondary
challenge, the responses are likely to be important
evidence for immune memory [Smith & Beal, 2008].
Recently studies of cytokines and chemokines

Interestingly, Berndt et al., (2007) evaluated

the chicken cecum immune response and showed
that low quantities of enteric bacteria were inside
the macrophages. These results indicated the
capability of paratyphoid Salmonella serovars to
enter and invade the cecal mucosa, affecting the
level and character of the immune response. The
expression of IL-12, IL-18, TNF- (tumor necrosis
factor alfa), and iNOS in cecum was correlated with
the invasiveness of serovars in the lamina propria.
In contrast, IL-2 mRNA expression, and changes in
the numbers of TCR2 (T-cell receptor 2) and CD4+
cells seem to be more dependent on the infection
of intestinal epithelial cells [Berndt et al., 2007].
Crhanova et al., (2011) found out that chickens
respond to natural colonization of caecum by and
increased expression of IL-8 and IL-17 in the first
week of life. These authors showed that chickens
infected with Salmonella Enteritidis before, during
and after the IL-8 and Il-17 induction, responded
through Th1 (T helper cell subset 1) inducing IL-8
and IL-17, while birds infected after this point
responded more through Th17 (T helper cells subset
17) branch of the immune response. These results
indicate that the gut microbiota and expression
of some cytokines increase the resistance to S.
Enteritidis infection.

Liliana Revolledo

Chickens infected with enteric Salmonella

serovars show high levels of specific antibodies,
a T cell response, cytokines and chemokines.
Within cell populations, their function can be
further discriminated by the presence of cellular
determinants, such CD4+ (T helper cells) and
CD8+ (T cytotoxic cells), which are associated
with helper and cytotoxic functions respectively
[Jeurissen et al., 2002]. The local immune response
in the gut has been shown to be more effectively
involved in the clearance of Salmonella Enteritidis
from the gastrointestinal tract than in the systemic
response [Desmidt et al., 1998]. An important role
of local cell-mediated immunity in the defense of
chickens against Salmonella exposure has been
suggested [Berndt & Methner, 2001], describing
that modifications of T-cell populations, especially
CD8+TcR1+() (T-cell receptor-bearing cells) cells in
ceca, occur few days after the inoculation of oneday-old chickens with the serovar Typhimurium.

expression in vitro have confirmed previous

work
showing
that
paratyphoid
species
stimulate significant mRNA expression levels of
proinflammatory IL-6, inducible nitric oxide synthase
(iNOS) and chemokines [Setta et al., 2009]. It was
suggested that host gene expression as well as
differences between chicken lines in host responses
toward the Salmonella infection, are host dependent
[Van Hemert et al., 2006].

Vaccines against Salmonella

and immune mechanisms of
protection
The regulation and effectiveness of the avian
acquired immune response is comparable to that in
mammals [Erf, 2004]. For a better understanding of
these mechanisms, an extensive review of vaccines,
protection and the immune dynamics of the avian
digestive system are available [Barrow, 2007; Korver,
2006]. In chickens, vaccines should prevent intestinal
and cecal colonization resulting in diminished fecal
shedding, and should be effective against systemic
infection preventing vertical transmission and egg
contamination. Vaccinations with either inactivated
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XXIV
or live products have been applied for reducing the
susceptibility of poultry to Salmonella infections
[Gast, 2007]. Both inactivated and live vaccines
are used to protect against Salmonella challenge,
however, killed products increase humoral
immunity and reduce Salmonella prevalence but did
not significantly decrease Salmonella in the farm
environment [Berghaus et al., 2011].

Vaccines
Vaccines based on dead Salmonella bacteria
have been used to protect poultry and its progeny
against field challenges. Inactivation is usually
achieved by either heating or the use of formalin
and an adjuvant. However, there are two remaining
problems with these products, they fail to elicit a
cell-mediated immune response, which is considered
the most important for clearing the intracellular
pathogen [Lax et al., 1995], and they do not
stimulate secretory IgA (SIgA) responses at mucosal
surfaces, which is the key for protection against
intestinal colonization [Cerquetti & Gherardi, 2000].

Liliana Revolledo

Subunits vaccines, like outer membrane proteins,

porins, ribosomal fractions have been tested to
prevent Salmonella infections. The efficacy of these

Worlds Poultry Congress 5 - 9

August - 2012 Salvador - Bahia - Brazil

products is not good when administered without

an adjuvant or a delivery system [Gamazo & Irache,
2007].
Live and attenuated vaccines have been used
worldwide, and their efficacy has been demonstrated
in challenge trials [Atterbury et al., 2010; Papezova
et al., 2008]. The aim of a live attenuated vaccine
should be to reduce the bacterial virulence while
maintaining its immunogenicity. Live vaccines
include semi-rough strains, such as 9R [Silva et
al., 1981; Kwon & Cho, 2011]. Other attenuated
vaccines include auxotrophic and metabolic drift
mutants (Table 1). Mutant vaccines attenuated by
biological gene-deletion techniques include aroA
mutants, crp mutants and others as describe in Table
1, this genetically modified organisms are permitted
in the USA, and other countries, except Europe.
It is important to evaluate the behavior of the
attenuated bacteria strains in the environment,
focusing on the attenuated strain obtained by gene
deletions, because they can acquire genes from
other microorganisms and recover their virulence.
The SG 9R vaccine (Table 1) strain still results in
systemic disease, with pathology in the liver and

Table 1 - Strain vaccines in poultry.

Salmonella
Serotype

Salmonella
Gallinarum

Salmonella
Enteritidis

Strain vaccine

Reference

Semi-rough lipopolysacharide structure

[Smith, 1956]

aroA mutant

[Griffin & Barrow 1993]

nuoG mutant

[Zhang-Barber et al., 1998]

crp mutant

[Rosu et al., 2007]

metC mutant

[Shah et al., 2007]

cobS and cbiA mutant

[Penha Filho et al., 2010]

Auxotrophic double marker mutant

[Meyer et al., 1993; Springer et al., 2000]

Metabolic drift mutation

[Linde et al, 1998]

aroA mutant

[Cooper et al., 1990; Cooper et al., 1992]

Outer membrane protein (ompR)

[Meenakashi et al., 1999; Khan et al., 2003]

Iron restricted

[Woodward et al., 2002]

Temperature sensitive spontaneous mutant

[Cerquetti & Gherardi, 2001]

Auxotrophic double marker mutant (chemical [Meyer et al., 1993; Springer et al., 2000]
mutagenesis)
Salmonella
Typhimurium

Metabolic drift mutants

[Linde et al., 1998]

aroAmutant

[Tan et al., 1997]

galE mutant

[Pritchard et al., 1978]

dam mutant

[Dueger et al., 2003]

phoP rpoS double deletion

[Methner et al., 2004]

cya crp mutants

[Hassan & Curtiss III 1990]

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spleen, and bacteria persist for several weeks at

these sites [Silva et al., 1981]. Lypopolysaccharide
(LPS) defect may be one of the major mechanisms
of SG 9R attenuation [Kwon & Cho, 2011] but this
defect could induce partial recognition by TLR4.
Possession of intact SPI-2 (Salmonella pathogenicity
island 2) and spvC (Salmonella plasmid virulence),
B, A and R virulence genes may be associated
with residual SG 9R virulence [Kwon & Cho, 2011]
Mutations should be introduced to increase safety
by reducing the risk of reversion [Linde et al., 1998].

Immune mechanisms of protection:

present and future perspectives
Despite the fact that many studies have
been carried out to demonstrate the efficacy of
inactivated [Woodward et al., 2002], sub-units
[, live attenuated [Atterbury et al., 2010; Kwon &
Cho, 2011], bacterial ghost [Peng et al., 2011],
genetically derivative strains [Nandre et al., 2011;
Methner et al., 2011] used as vaccines, the efficacy
of these product is demonstrated by challenge
trials and recovery of the challenge strain. Little
and isolated information explains how do vaccines
work and the immune response depending on the
antigen and route of vaccination.

The route of vaccination is important in

influencing immune responses at the initial site
of pathogen invasion where protection is more
effective [Belyakov & Ahlers, 2010]. Mucosal
dendritic cells play an important role in the
induction and maintenance of protective immunity
against pathogens like Salmonella. Dendritic cells
are responsible for antigen presentation following
mucosal vaccination and systemic immunization
have a limited effect on the delivery of antigen to
mucosal dendritic cells [Coombes & Powrie, 2008;
Kelsall, 2008].
An important difference must be established
in Salmonella attenuated vaccines regarding
the immune response: the administration route.
Parenteral vaccines stimulate a strong humoral
response, while oral live attenuated vaccines

Attenuated live bacteria vaccines applied by oral

route are excellent tool for mucosal immunization
[Husseiny & Hensel, 2005], because mucosal surfaces
are the first interface between Salmonella and the
host. The first step to initiating an immune response
in the gut surface by oral vaccines is based on the
signals sent by receptors for pathogen-associated
molecular patterns (PAMP) via pathogen recognition
receptors (PRR), such as TLR. TLR in chickens are
very similar to those in mammals; however, some
differences in recognition patterns related to TLR5,
which recognize flagellin are observed in hostspecific Salmonella and non-host-specific Salmonella
strains [Salazar-Gonzales & McSorley, 2005]. In
response to flagellin lamina propria dendritic cell,
the differentiation of nave B cells into IgA plasma
cells occurs via a mechanism that is independent of
the gut associated lymphoid tissue (GALT) [Uematsu
& Akira, 2009]. Recognition of S. Typhimurium is
largely mediated by TLR2, TLR4 and TLR5 [Uematsu
et al., 2006; Vasquez-Torres et al., 1995] and TLR9
is involved in the regulation of the replication of the
bacterium. Lypopolysaccharide (LPS) of Salmonella
is recognized by TLR4 expressed at the surface of
the immune cells and in the cytoplasm of intestinal
epithelial cells [Crhanova et al., 2011]. The second
step in the immune response is related to the ability
of the antigen to cross the epithelial barrier and to
be presented to antigen-presenting cells, especially
dendritic cells. Mucosal dendritic cells play a central
role in the induction of protective immunity against
invasive pathogens. Unique dendritic cells subsets
are responsible for antigen presentation following
mucosal vaccination [Belyakov & Ahlers, 2009]. In
the chicken, multiple lymphoid follicles exist, and
they are made up of B cells embedded in a network
of follicular dendritic cells [Brisbin et al., 2008]. The
chicken epithelial barrier can be crossed using three
mechanisms: endocytosis in the intestinal epithelial
cells, transcytosis crossing M cells (microfold cells),
and directly through the intraepithelial lymphocytes.
Area: Poultry Health and Biosecurity August 07

Liliana Revolledo

Vaccines should establish a long lasting immunity

by manipulating the cytokine milieu to induce the
appropriate effector mechanisms for each particular
pathogen and by creating a large pool of long lived
memory cells [Chabalgoity et al., 2007]. Vaccines
used in poultry against Salmonella infections have
been effective, but they were empirically designed,
and are not based on detailed information about the
immune responses of protection because efficacy is
frequently evaluated in challenge trials.

generate both mucosal and systemic immunity

[Bouvet et al., 2002; Mastroeni et al., 2000]. There is
a fundamental difference between inactivated and
attenuated live vaccines. Live attenuated Salmonella
vaccines stimulate the secretion of mediators
(cytokines, interleukin and interferon) inducing a
T helper 1 response and cell-mediated immunity;
in different animal species and in poultry this type
of immune response is not induced by inactivated
products. Additionally, CD8+T cells play a crucial
role in immune protection against intracellular
pathogens, such as Salmonella, and these cells
(CD8+T) response are exclusive after vaccination
using live attenuated vaccines.

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differ substantially depending on the regimen of

immunization, a secondary responses result in
preferential accumulation of memory T cells in
the lamina propria following mucosal vaccination
[Barnhart et al., 1991].
These cells might be exploited to develop new
live attenuated vaccines inducing abroad repertoire
of immune responses against intracellular pathogens
[Titball, 2008].
Vaccination is an excellent tool for handling
poultry Salmonella prevention programs in
developed and developing countries. Further
research is needed to evaluate immunological
interactions among the host and Salmonella,
avoiding empirical methods in developing new
vaccines and investigating ways to prevent the
infection. Vaccines against Salmonella infections in
chickens and other food-producing animals require
protection at both mucosal (gut) and systemic levels.
There is no ideal product, but regarding the immune
response, live attenuated Salmonella vaccines are
superior to inactivated, deleted or subunit vaccines.

Liliana Revolledo

The third step in the immune response produced by

oral vaccines is the processing by the dendritic cells
and the presentation to the T cells. Dendritic cells
in the gut can be activated by epithelial cells, which
produce cytokines based on the invasiveness of the
bacteria, and directly by noninvasive bacteria [Rimoldi
et al., 2004]. Salmonella vaccines administered orally
must induce Th1 and Th2 (T helper cell subset 2)
responses, stimulating cell-mediated immunity and
B cell activation for producing SIgA (Figure 2), which
block the attachment of the bacteria to mucosal
surfaces. To achieve protection, it is important that
memory cells are generated in enough numbers
and persist as a functional long-lived population
[Chabalgoity et al., 2007]. Some authors indicated
that it would seem crucial that the vaccine strains
retain capacity of invasiveness in order to stimulate
sufficient immunity to be protective [Barrow, 2007;
Van Immerseel et al., 2005], however, the immune
response in the intestinal mucosa has revealed new
possibilities because oral antigens induce effector
and memory cells that express certain receptors
only on lymphocytes of intestinal mucosa [Aziz et
al., 2007]. Mucosal memory T cells phenotypes

Worlds Poultry Congress 5 - 9

Figure 2 - Mucosal protection and CMI in the chicken gut conferred by oral live attenuated Salmonella
vaccines.

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Oral Salmonella vaccines provide an advantage over

parenteral products, because oral products are able
to mimic natural infection and to stimulate both the
mucosal and systemic immune responses, whereas
parenteral products are able to generate only
systemic immune responses. Advances and new
strategies in elucidating the mechanisms of action
of oral vaccines for poultry will facilitate the design
of multivalent and more effective products. New
design and delivery strategies for eliciting mucosal
and systemic immune response are needed in order
to develop more efficacious multivalent vaccines
against Salmonella for preventing infection in the
poultry and contamination in poultry products.

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