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Abstract

The knowledge about virulence mechanisms, resistance to antimicrobial agents and the biofilm formation ability of Salmonella spp. in poultry industry has been expanded over the years. However, in spite of the research efforts and significant investments to improve management systems in poultry industry, it has become evident that none of the methods applied in all stages of food production chain are 100% effective in eliminating Salmonella spp. Different serovars are manifesting different mechanisms of invasiveness which depend on their ability to invade lower zones of the lamina propria, their ability to gain accesses to parenchymatous organs and survive in macrophages. The ubiquitous nature of Salmonella spp. due to their adaptation to animal and plant hosts, as well as their survival in hostile environments and their enhanced capacity to produce biofilms, contribute to a long lasting contamination of the environment, feed and animals. The emergency and spread of antimicrobial resistances in Salmonella spp. raise additional concerns.
Journal of the Hellenic Veterinary Medical Society
Vol. 69, 2018
Salmonella spp. in poultry: a constant challenge
and new insights
VELHNER M. Department of clinical
microbiology, Scientific
Veterinary Institute “Novi
Sad”, Novi Sad, Serbia
MILANOV D. Department of clinical
microbiology, Scientific
Veterinary Institute “Novi
Sad”, Novi Sad, Serbia
KOZODEROVIĆ G. Faculty of Education in
Sombor, University of Novi
Sad, Serbia
http://dx.doi.org/10.12681/jhvms.18012
Copyright © 2018 M. VELHNER, D. MILANOV, G.
KOZODEROVIĆ2
To cite this article:
VELHNER, M., MILANOV, D., & KOZODEROVIĆ, G. (2018). Salmonella spp. in poultry: a constant challenge and new
insights. Journal of the Hellenic Veterinary Medical Society, 69(2), 899-910. doi:http://dx.doi.org/10.12681/jhvms.18012
http://epublishing.ekt.gr | e-Publisher: EKT | Downloaded at 29/08/2018 11:09:25 |
Salmonella spp. in poultry: a constant challenge and new insights
M. Velhner1, D. Milanov1, G. Kozoderović2*
1 Department of clinical microbiology, Scientic Veterinary Institute “Novi Sad”, Novi Sad, Serbia
2 Faculty of Education in Sombor, University of Novi Sad, Serbia
Corresponding Author:
Gordana Kozoderović
Faculty of Education in Sombor, University of Novi Sad,
Podgorička 4, Sombor 25000, Serbia
E-mail : gocakozoderovic@gmail.com
D a t e o f i n i t i a l s u b m i s s i o n : 3-6-2017
Date of revised submission: 21-6-2017
Date of acceptance: 14-7-2017
Review article
Ανασκόπηση
J HELLENIC VET MED SOC 2018, 69(2): 899-910
ΠΕΚΕ 2018, 69(2): 899-910
ABSTRACT. The knowledge about virulence mechanisms, resistance to antimicrobial agents and the biolm forma-
tion ability of Salmonella spp. in poultry industry has been expanded over the years. However, in spite of the research
efforts and signicant investments to improve management systems in poultry industry, it has become evident that
none of the methods applied in all stages of food production chain are 100% effective in eliminating Salmonella spp.
Different serovars are manifesting different mechanisms of invasiveness which depend on their ability to invade lower
zones of the lamina propria, their ability to gain accesses to parenchymatous organs and survive in macrophages. The
ubiquitous nature of Salmonella spp. due to their adaptation to animal and plant hosts, as well as their survival in hostile
environments and their enhanced capacity to produce biolms, contribute to a long lasting contamination of the envi-
ronment, feed and animals. The emergency and spread of antimicrobial resistances in Salmonella spp. raise additional
concerns.
Keywords: poultry, Salmonella, pathogenesis, biolm, resistance
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900 M. VELHNER, D. MILANOV, G. KOZODEROVIĆ
J HELLENIC VET MED SOC 2018, 69(2)
ΠΕΚΕ 2018, 69(2)
INTRODUCTION
Poultry farming presents one of the most important
food manufacturing industries around the
globe. Therefore, food safety standards are highly
demanding and are generally better maintained in
large scale production facilities than in small ones.
In developing countries, rearing of backyard chicken
flocks contributes to the continuous occurrence of
some viral and bacterial diseases that are less likely
present in well maintained farms. Except for a very
few countries in the world, Salmonella spp. are
detected in environmental specimens in practically
all stages of the food production chain. Out of more
than the 2600 serovars known today, only 10% are
found in the commercial poultry and egg industry.
Two of them, S. Enteritidis and S. Typhimurium, are
of paramount importance to human health and can
colonize the intestines of chickens (Velge et al., 2005).
In most cases, the infected chickens either do not have
clinical symptoms or the symptoms remain unnoticed.
With all this taken into account, it is evident that
control programs for Salmonella spp. have to be
implemented in all stages of the food production
chain, starting from animal farms. According to
European Directive 1003/2005, the occurrence of
S. Enteritidis and S. Typhimurium in adult breeder
flocks has to be < 1%, in EU member states.
However, this directive also targets serovars Hadar,
Virchow and Infantis which are of public health
significance in the EU (Carrique-Mas and Davies,
2008). It is very difcult to accomplish such a goal
in developing countries, since implementing good
management practice is expensive and requires the
participation of educated staff. Even if biosecurity
measures are well established on a farm, salmonellae
can still be found in poultry and premises.
Other available measures to cope with Salmonella
spp. in farms include the use of prebiotics and
probiotics, antimicrobial therapy and vaccination
of the birds. For serovars S. Enteritidis and S.
Typhimurium commercial inactivated and attenuated
vaccines have been developed and used widely. These
vaccines target serogroups D and B respectively,
but do not protect livestock against serovars from
other serogroups. Therefore, vaccination against S.
Enteritidis and S. Typhimurium could lead to the
elimination of these two serovars on farms, opening
a vacant ecological niche, enhancing, thus, the
emergence of new serovars, such as S. Kentucky or S.
Heidelberg (Foley et al., 2011).
The framework of National control programs
in European Union member states includes the
vaccination of layer flocks during rearing which
has to be mandatory in cases of 10% prevalence
of S. Enteritidis (EC No 1168/2006 and EC No
1177/2006). Live vaccines could be used only in
cases when the discrimination of vaccine versus wild
type Salmonella is possible and the ban of antibiotic
use in layers has been initiated (Carrique-Mas and
Davies, 2008). Such high demands have motivated
a number of research works aiming to nd the best
sampling strategy and the best monitoring systems for
Salmonella spp. control all around the world.
The most convenient methods of taking samples for
bacteriological analysis from poultry houses are using
boot swabs or the “step on a drag swab” method
(Buhr et al., 2007). Ofcial sampling is carried out
while birds are in the unit while own checks are
carried out not only while livestock is in the unit but
also after depopulation. Own check programs must be
approved by the competent authorities. The sampling
strategy aiming to detect and control Salmonella spp
in adult breeding ocks of Gallus gallus is dened
in Commission Regulation EU No 200/2010 and
for laying hens in Commission Regulation (EU) No
517/2011. Reduction of the prevalence of the serovars
Enteritidis and Typhimurium in flocks of turkeys
is required and the sampling strategy is dened in
Commission regulation (EC) No 584/2008. After
cleaning and disinfection, swabs are collected from
walls, floor, vents, drinking and feeding systems,
changing rooms and other areas that may be exposed
to external contamination. It is important to collect as
many swabs as possible to determine the success of
cleaning and disinfection. The same strategy applies
for hatcheries which may become contaminated
with the pathogen. In fact, Salmonella spp. can be
effectively disseminated in the hatchery cabinet and
chickens may become infected before removing from
the hatchery (Bailey et al., 1998).
According to a longitudinal study of environmental
Salmonella contamination in caged and free-range
layer ocks carried out by Wales et al. (2007), the
timing of taking samples has been shown to have a
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significant influence on Salmonella spp. isolation.
Flocks that remained longer on the premises yielded
more isolates comparing to the new flocks. The
temperature and the season also had an influence
on Salmonella spp. populations, proving increased
isolation rate during summer. The role of other
animal reservoirs harboring Salmonella in and
outside the farms is also signicant (Guard-Petter,
2001). Salmonella spp. in wildlife vectors correlated
well with the status of the ock and the same serovar
and phage type could be found in wild predators
caught around the farm and poultry. Cleaning and
disinfection in cases when organic matter had
been substantially removed and disinfectants were
adequately applied and in proper concentration, had
a positive inuence on Salmonella control. However,
the wildlife reservoirs, multiage farming and lack
of “all in all out” strategy highlight the need for
vaccination and the use of probiotics in ocks with
high and low incidence of the pathogen’s load or even
in cases that it is absent (Wales et al., 2007). Another
study by Dewaele et al. (2012) which aimed to
examine the Salmonella enterica serovar Enteritidis
environmental contamination on persistently positive
layer farms in Belgium during successive laying
cycles showed that in contaminated poultry houses,
neither vaccination nor cleaning and disinfection
are considered as the only prerequisite for
successful elimination of Salmonella spp. from the
environment and that the chances for Salmonella spp.
elimination were better in less contaminated poultry
farms, comparing to those in highly contaminated
environments. This is even more pronounced
if rodents, flies and mites come into contact with
poultry or equipment. In addition the authors
concluded that there is a possibility that even if
poultry houses are separately cleaned and disinfected,
egg collection areas may still become a reservoir of
Salmonella spp. In fact, the egg collection areas may
become contaminated with a few serovars which are
present on the entire farm.
THE PATHOGENESIS, TISSUE INVASION AND
IMMUNE RESPONSES
Salmonella spp. possesses an arsenal of genetic
determinants responsible for colonization, adhesion,
invasion and proliferation in host cells, including
mbriae, agella, toxins, surface lipopolysaccharides
(LPS), etc. Virulence genes are organized in clusters
and spread throughout the chromosome, such as
Salmonella Pathogenicity Islands 1 and 2 (SPI-1,
SPI-2), or located on virulence plasmids, such as spv
genes (associated with invasive strains). Salmonella
pathogenicity genomic islands carry genes that are
required for successful infection in poultry (Wisner et
al., 2012). Noninvasive strains cause gastroenteritis,
while invasive strains may cause systemic bacteremia
in humans and animals. The outcome of infection
depends on virulence factors, the pathogenesis of
Salmonella spp. and their interaction with the host
organism (Foley et al., 2013). Unlike noninvasive
strains, invasive Salmonella strains penetrate through
the epithelial lining to the lower parts of the lamina
propria. Also, invasive strains are commonly isolated
from parenhymatous organs (spleen, liver, ovaries)
and a small number of bacteria become internalized
by macrophages (Berndt et al., 2007). The survival in
the acidic environment of the stomach is enabled by
the activation of more than 50 acid tolerance response
proteins (Bearson et al., 2006). The first phase of
the infection has to provide a chance for the bacteria
to invade intestinal epithelial cells. This process is
accomplished by proteins encoded by Salmonella
Pathogenicity Island (SPI-1) type III secretion
system (T3SS). These organelles produce a special
structure in the bacterial envelope called “the needle
complex” which delivers toxins and other effector
proteins and injects them into the host cells (Kubori
et al., 2000). Bacterial effector proteins modulate
the host actin cytoskeleton and initiate the signal
transduction pathways required for the internalization
of the bacteria. In addition, invasive strains recruit
their own systems responsible for survival in
macrophages. Salmonella spp. become internalized
in a specific membrane bound compartment called
“Salmonella containing vacuole” (SCV). The
maturation of the SCVs and their migration to the
basal membrane disable the destruction of the bacteria
by phagolysosomes. Such intracellular trafficking
and intracellular pathogenesis is also accomplished
by the activation of the second T3SS encoded by
the SPI-2. Hence, the type III secretory system
encoded by SPI-1 and SPI-2 enables the attachment,
invasion and survival of the pathogen within the
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host cell, as well as the avoidance of antimicrobial
compounds (Hensel, 2000; Foley et al., 2013). Most
of Salmonella serovars contain SPI-1 to -5, while
other pathogenicity islands are not so common. The
colonization of the gastrointestinal tract and of the
internal organs of poultry is enabled by the type
VI secretion system encoded by the SPI-19 locus
present in serovar Gallinarum (Blondel et al., 2010).
In mice infected with serovar Typhimurium, the
SPI-6 was necessary for the intracellular replication
of the pathogen in macrophages and its systemic
dissemination. The experimental work indicates that
T6SS encoded by both SPI-6 and SPI-19 gene clusters
are genetically involved in bacterial pathogenesis
and that T6SS-SPI-6 play a role in gastrointestinal
colonization and systemic spread of serovar
Typhimurium in chickens (Pezoa et al., 2013).
Besides Salmonella pathogenicity islands-1 and 2,
Salmonella strains involved in extraintestinal non-
typhoid disease with bacteremia carry additional
virulence genes in a spv locus, contained on virulence
plasmids (Guiney and Fierer, 2011). Genes spv
were found in serovars Typhimurium, Enteritidis,
Choleraesuis, Abortusovis, Dublin, Gallinarum/
Pullorum and in subspecies arizonae. The plasmid
genes in the spv locus include spvABCD operon
which is positively regulated by the upstream spvR
gene. Only spvR, spvB and spvC are responsible for
spv related virulence phenotype. In spite of having
different biochemical pathways of action, SpvB
and SpvC proteins are eventually involved in late
apoptosis of macrophages, enabling the intracellular
proliferation of Salmonella spp. Subsequent uptake of
apoptotic macrophages by surrounding macrophages,
facilitates cell to cell spread of Salmonella spp.
(Guiney and Lesnick, 2005; Derakhshandeh et al.,
2013). Consequently, it potentiates the systemic
spread of the pathogen instead of causing a self
limited gastroenteritis.
Salmonellae have different invading capacities in
the poultry intestine and parenchymatous organs.
They trigger systemic and local immune response
which is in good correlation with their virulence.
Experimental work was conducted by Berndt
et al., (2007) to measure the immune response in
cecum after the infection of White Leghorn day old
chickens with serovars Enteritidis, Typhimurium,
Hadar and Infantis. At 2, 4 and 7 days post infection
(pi) serovars Hadar and Infantis showed diminished
invading capabilities for liver, compared to serovars
Enteritidis and Typhimurium. S. Enteritidis was
the best invader of the lower zones of the lamina
propria, while S. Infantis was found in epithelial
lining and subepithelial region. The increase
of granulocytes, TCR1 gd and CD8α+ in chicken
cecum was most prominent for serovar Enteritidis,
followed by serovars Typhimurium and Hadar,
while Infantis provoked less significant immune
cell influx. In the same study the reorganization
of the extracellular matrix proteins, notably the
increase of total fibronectin and tanascin-C, has
been more pronounced after the infection of day
old chickens with serovar Enteritidis comparing
to the infection with the non invasive Salmonella
Infantis. Furthermore, enhanced Salmonella spp.
entry and the ability to disseminate in the gut
epithelium support the concept that the most virulent
strains utilize distinctive genetic mechanisms to
invade the intestine and disseminate through the
body, showing an important ability to provoke
better immune responses in infected birds, as well
(Berndt et al., 2009). It was experimentally shown
that S. Infantis was found in higher numbers in avian
macrophages in vitro comparing to S. Typhimurium,
but the number of viable cells inside macrophages
was higher for S. Typhimurium than for S. Infantis
(Braukmann et al., 2015). Both serovars trigger active
immune responses by activating genes involved in
regulating immunological processes. The infection
of avian macrophages with both serovars induced
the increased expression of the immune mediators
up to four hours post infection. The longer survival
of serovar Typhimurium in macrophages was
probably related to a higher and rapid SPI-2 genes
activation, which explains the better invasiveness
and the ability of causing systemic infection,
something observed in serovar Typhimurium, but
not in Infantis. The unmbriated state of Salmonella
spp. and Escherichia coli in chicken intestine are
manifesting good colonizing ability in the intestine
and oviducts of laying hens at 19 weeks of age as
described by De Buck et al. (2004). However, the egg
content, particularly the yolk and the egg shell, was
contaminated by the wild type strain more efciently.
902 M. VELHNER, D. MILANOV, G. KOZODEROVIĆ
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Although the type 1 mbriae decient mutant caused
prolonged bacteraemia in laying hens, the reduced
egg shell contamination in mutant comparing to wild
type strain, has shown that mbriae are important for
causing egg contamination in serovar Enteritidis (De
Buck et al., 2004).
THE PREVALENCE OF SALMONELLA
SEROVARS IN POULTRY FLOCKS
The rise of S. Enteritidis during the 1980s and 1990s
coincided with the extensive measures undertaken
to eradicate S. Gallinarum. It is suggested that S.
Enteritidis has taken the ecologic niche previously
occupied by S. Gallinarum in poultry ocks, via the
mechanism of competitive exclusion, due to their
antigenic similarity (Rabsch et al, 2000). Clearing
the commercial flocks from S. Gallinarum enabled
S. Enteritidis to colonize chickens without signs of
disease (Andino and Hanning, 2015). In addition,
serovar Enteritidis has a wider spectrum of natural
reservoirs which makes it easier to persist on the farms.
It has been isolated from insects, rodents, nematodes,
wild birds and other animal hosts living in and around
hen houses. Thus, after adequate disinfection of
houses and stocking with culture-negative chicken, S.
Enteritidis can be reintroduced from hen house pests,
especially mice (Guard-Petter, 2001).
In the United States of America, S. Enteritidis
which was dominant in the 1990s, was supplanted
by serovar Heidelberg in the period 1997-2006, but
since 2007 S. Kentucky has been the most prevalent
serovar isolated from poultry (Foley et al., 2011).
However, these serovars are less common in humans,
with serovars Enteritidis and Typhimurium being
the leading causes of alimentary toxoinfections in
the USA. There are several possible reasons for the
prevalence of serovars Kentucky and Heidelberg:
ock immunity against S. Enteritidis gained due to
vaccination or exposure might have opened the space
for these two antigenically different serovars to which
the ocks were susceptible (Foley et al., 2011). The
ability of S. Heidelberg to colonize the reproductive
tract in chickens and enter eggs, poses a threat to
public health as another important egg transmitted
pathogen, besides S. Enteritidis and S. Typhimurium
(Gast et al., 2004). Although S. Kentucky is not
so commonly involved in human infections, it is
very successful in colonizing chicken. One of the
reasons might be the acquisition of the virulence
plasmids ColBM and ColV from the avian pathogenic
Escherichia coli (APEC) (Johnson et al., 2010).
In the past few years, the emergence of S. Kentucky
strains resistant to multiple antimicrobial drugs has
become a new threat to human and animal health.
The international trade has facilitated the spread of
those strains to the domestic poultry in the region of
Mediterranean basin (Le Hello et al., 2013).
The experimental infection of two day old broiler
chickens has revealed that serovar Kentucky persisted
longer in the cecum comparing to Typhimurium and
the peak was noted at 25 days pi (Cheng et al., 2015).
Compared to S. Typhimurium, the expression of
genes regulated by RNA polymerase sigma S factor
(rpoS) was more pronounced in serovar Kentucky in
the ceca content. The expression of genes from the
metabolic pathway and the role of curli production
seem to be in correlation with the ability of serovar
Kentucky to colonize and persist in poultry.
Unlike other serovars, S. Gallinarum biovars
Gallinarum and Pullorum are restricted to avian
species and do not pose a risk to human health.
However, among poultry, they cause septicemic fowl
typhoid and pullorum disease (respectively) with high
mortality and morbidity. Strict control programs using
serological tests and elimination of positive birds has
lead to the eradication of diseases from commercial
poultry in the United States of America, Canada
and most of Western Europe, although outbreaks
occasionally occur (Barrow and Freitas Neto, 2011).
In the European Union, harmonized Salmonella
control programs have lead to the overall decrease
in the prevalence of ve serovars (S. Enteritidis, S.
Typhimurium, S. Infantis, S. Hadar, S. Virchow) of the
public health relevance. However, in 2015 there was
a slight increase in S. Enteritidis incidence comparing
to 2014, but S. Infantis was the most prevalent serovar
among domestic fowl (EFSA 2016a).
BIOFILM FORMING CAPACITY OF
SALMONELLA SPP. IN POULTRY AND FEED
INDUSTRY
Because of the profound ability to irreversibly
bind to different types of biotic and abiotic surfaces,
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processing and storage facilities of poultry products
(Gradel et al., 2003; Gradel et al., 2004; McKee et
al., 2008; Díez-García et al., 2012).
Biofilm is an important risk factor in feed
contamination, and one of the critical points of
controlling Salmonella spp. on poultry farms, having
an increasing importance in the last decades (Cox
and Pavic, 2010). The contamination of feed with
Salmonella spp. may occur as a consequence of the
use of contaminated raw materials or it may occur
during the production process, by getting in contact
with contaminated surfaces in production facilities.
Biofilm formation is involved in both processes.
The main components of the Salmonella BF-matrix,
the protein surface aggregative fimbriae (curli)
and the extracellular polysaccharide cellulose, are
required for the colonization of plant surfaces and
for the attachment to the surface of the feed factory
environment. These biolms allow the persistence of
Salmonella spp. in feed and food factory environments
for months, and even years (Vestby et al., 2009;
Schonewille et al., 2012; Prunić et al., 2016).
In slaughterhouses and facilities for processing
poultry carcasses, Salmonella spp. are found
continuously, despite the regular use of strict
measures for the control and reduction of pathogens
(Rose et al., 2000; Joseph et al., 2001; Gradel et
al., 2004; Marin et al., 2009). Research shows that
conventional methods of disinfection are ineffective
in eliminating Salmonella spp. from surfaces on
which fresh meat processing is carried out (McKee
et al., 2008). It is also experimentally evaluated
that only two out of 13 commercially available
disinfectants based on sodium hypochlorite, sodium
chlorite and alkaline peroxide were effective against
Salmonella biofilms formed on galvanized steel
in the presence of organic matter (Ramesh et al.,
2002). In eld conditions, methods of cleansing and
disinfection are often insufcient for Salmonella spp.
elimination from poultry housing facilities (Marin et
al., 2011; Davies and Breslin, 2003). The BF-matrix,
particularly the extracellular polysaccharide
cellulose, is considered to be an important factor for
the protection against chemical agents.
The purpose of maintaining a dry environment in
feed and food factories and low water activity in the
finished product is to reduce pathogens, but these
the Most Prevalent Poultry-associated Salmonella
serotypes (MPPSTs) usually have a capacity of
biofilm (BF) formation on plant surfaces, in the
host organism, as well as in a variety of materials
commonly used in the poultry production and feed
industry (Steenackers et al., 2012; White and Surette,
2006). Hence, BF formation is a common feature
of bacteria and it is characterized as a complex
surface associated community of microorganisms.
Biofilm is defined as matrix-enclosed bacterial
populations adherent to each other and/or on
surfaces or interfaces (Donlan 2002; Donlan and
Costerton 2002). Bacteria with the ability to form
biolms express different genes comparing to their
planktonic counterparts, becoming increasingly
resistant to antibiotics and disinfectants. Indeed,
the resistance of bacteria in the BF may be 10 to
1000 times higher comparing to the bacteria in
suspension, which is most often used for the
examination of the effectiveness of disinfectants or
other antimicrobial compounds, such as antibiotics
(Mah and O’Toole, 2001). Hence, biofilm is the
perfect microenvironment for the horizontal transfer
of genetic material and the emergence of pathogens
with new virulence factors and mechanisms of
antibiotic resistance.
In a number of experimental studies, the ability
of Salmonella to form BF on a variety of materials
such as concrete, glass (Prouty and Gunn, 2003),
cement (Joseph et al., 2001), stainless steel
(Oliveira et al., 2007), plastic (Stepanović et al.,
2004; Solomon et al., 2005), granite and rubber
(Arnold and Yates, 2009) was confirmed (Solano
et al., 2002; Steenackers et al., 2012). Salmonella
spp. can rapidly colonize hydrophobic substrate,
such as plastic, and they commonly produce a
BF on them. Plastic materials are widely used on
farms, in slaughterhouses and in food industry for
the preparation of tanks, pipe-work, accessories
and cutting surfaces (Díez-García et al., 2012). The
microorganism easily forms a BF on galvanized
steel, which is used for making transport containers
for poultry (Ramesh et al., 2002). Various serovars
of Salmonella spp. are characterized by a good
ability to produce BF, which enables their persistence
in poultry facilities, hatcheries, the water supply
systems on farms, slaughterhouses, as well as in
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practice of using antibiotics in animal husbandry.
There is evidence that some resistant Salmonella
strains have increased virulence, which could be a
result of the integration of virulence and resistance
plasmids and their co-selection or up regulation of
the virulence or the improved tness of the bacteria
(Mølbak, 2005).
It is widely considered that antibiotics used in
human medicine should be avoided for the therapy
of animals. Such practice is well established in
developed countries except for rare cases, as for the
treatment of infections caused by susceptible bacteria
(Garcia-Migura et al., 2014). However, travelling
and trade have a high impact on establishing MDR
microorganisms in their communities. Besides the
restrictive use of antibiotics in developed countries,
growth promoter use was also banned in the year
2006 and the overall resistance rate in commensal
and pathogenic bacteria from food producing
animals has been decreasing. In developing countries
resistance to fluoroquinolones and extended
spectrum beta lactames is still worrisome. It has been
recorded that multiple drug resistant S. Kentucky,
S. Typhimurium and S. Infantis have a worldwide
distribution and that poultry present permanent (S.
Infantis, S. Typhimurium) or transient reservoirs
(S. Kentucky). Emerging strains of S. Kentucky
resistant to carbapenems and fluoroquinolones
may cause life threatening disease in humans and
they are among the most dangerous Salmonella
serovars that have been diagnosed recently (LeHello
et al., 2013). The rst report of the occurrence of
extended spectrum β-lactamase (ESBL) resistant S.
Kentucky from poultry specimens (whole chicken,
farm dust and chicken neck skin) in Ireland was
attributed to blaSHV-12 and blaCMY-2 genes. Even though
cephalosporins are not applicable for the therapy
of chickens in Ireland, there is a possibility that
the use of amoxicillin has favored the selection of
β-lactamase producers over the time (Boyle et al.,
2010). Salmonella Kentucky designated CVM29188
isolated from a chicken breast sample in the
year 2003 has shown resistance to streptomycin,
tetracycline, ampicillin and ceftiofur. All the genes
determining resistances (strAB and tetRA, blaCMY-2,
sugE) were found on two large transmissible
plasmids. In addition, the pCVM29188_146 plasmid
measures are not effective in controlling Salmonella
spp. In some Salmonella strains, including those of
serovar Enteritidis, isolated from food products with
low water activity, an increase in virulence and the
reduction of the infective dose was found (Aviles et
al., 2013; Andino et al., 2014). It is believed that the
increasing virulence of Salmonella spp. in products
with low water activity is the result of rpoS activation
(the main stress response regulator), which directly
affects the activation of virulence genes such as the
invA, hilA and sipC (Aviles et al., 2013). However,
experimental studies show that genes invA and hilA in
S. Enteritidis are down regulated in low water activity,
but the exact reason for the increased virulence of this
serovar remains unknown (Andino et al., 2014).
Differences in the ability to produce the BF are
established among different serovars, or strains of
the same Salmonella serovar (Schonewille et al.,
2012). However, in vitro conditions used in research
on BF formation capacities, may not always reect
the conditions required for BF formation in the
environment. Bacteria express important features
that enable them to adapt under various challenges
and the formation of the BF communities presents an
important defense mechanism.
Biolm is a risk that has been recognized recently
as it causes long term contamination and persistency
of some Salmonella serovars in all cycles of the
poultry industry. It also presents actual research
challenge in raising food producing animals and
in safe food production. There are no effective
measures to prevent or remove BF. Starting with
the fact that multiple sources of contamination with
Salmonella spp. are recognized, the only way to cope
with Salmonella spp. in poultry production facilities
is good management practice and high biological
safety. Innovations in the eld of BF control refer to
the compounds that actually inhibit biosynthesis of
signal molecules in BF, but they are not applicable in
poultry and food industry at present.
RESISTANCE TO ANTIMICROBIAL AGENTS
IN SALMONELLA SPP. FROM POULTRY
SPECIMENS
Multiple drug resistance (MDR) of Salmonella spp.
in poultry is developing because of the established
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colisitin was recorded in S. Infantis. High rate of
multi-drug resistance was detected in some EU
countries in Salmonella spp. isolates from turkey
meat. In ocks of boilers, the most prevalent was
S. Infantis with extremely high resistance rate to
ciprooxacin (except in Denmark and Spain). Second
most frequently detected serovar in broilers was S.
Enteritidis with overall resistance to ciprooxacin
and nalidixic acid of 23.3 and 24.6% respectively.
Levels of resistance to ciprofloxacin were high
in Salmonella spp. isolates from layer flocks in
Cyprus, Hungary, Italy and Romania. However,
trends in multi drug resistance were much lower in
Salmonella spp. from layers comparing to broilers
in the EU member states (EFSA 2016b). In the
report of the National Antimicrobial Resistance
Monitoring System (NARMS) USDA of 2011, it
was documented that the most prevalent serovars
from poultry in the USA were: Kentucky, Enteritidis,
Heidelberg, Typhimurum var-5 and Infantis
(NARMS-USDA, 2014). Resistance to beta lactam/
inhibitor combination and cephems was found in
17.9% of serovar Heidelberg isolates and in 0.7%
of serovar Enteritidis isolates, regarding poultry.
Resistance to (fluoro)quinolones was not found,
while resistance to gentamicin was evident in 1.3%
of the serovar Kentucky isolates, 14.3% of the
serovar Heidelberg isolates and in 10.5% of the
serovar Typhimurium var-5 isolates from poultry in
2011 (NARMS-USDA, 2014).
Poultry meat and products therefore present
a significant reservoir of resistant Salmonella all
around the world. However, the resistance patterns
differ markedly from continent to continent and
among countries. In this respect, the highest concern
is the resistance of serovars Kentucky and Infantis
which become well established in poultry ocks and
frequently develop a multidrug resistant phenotype.
CONCLUDING REMARKS
Much effort has been put through to provide safe
poultry meat and products worldwide. In spite of
the fact that many biological, chemical products
and vaccines have been invented and implemented
in poultry production systems, it is still difficult
to eliminate Salmonella spp. from the food chain.
Different Salmonella serovars tend to take place
is genetically similar to the virulence plasmids found
in avian pathogenic Escherichia coli (APEC). These
APEC-like plasmids were probably exchanged
among the two bacteria species in the intestinal
environment and they also possess virulence
elements that have contributed to their establishment
in predominant Salmonella Kentucky strains in
chicken intestines and meat (Fricke et al., 2009).
Serovar Infantis is a typical poultry Salmonella
serovar. It is well established on poultry farms with a
tendency of clonal spread of the multidrug resistance
phenotype. Clonal spread of Salmonella Infantis
in poultry and poultry meat was reported in Japan
(Shahada et al., 2006), Hungary (Nógrády et al.,
2007), Israel (Gal-Mor et al., 2010), Italy (Dionisi
et al., 2011), Germany (Hauser et al., 2012), Serbia
(Rašeta et al., 2014; Velhner et al., 2014) but also in
humans in Argentina (Merino et al., 2003) and Brazil
(Fonseca et al., 2006). All these clonal strains were
resistant to three or more antimicrobials except for
Serbia, where the predominant resistance phenotype
was nalidixic acid (NAL) / tetracycline (TET), while
an approximate 30% of the isolates was showing
resistance to ciprooxacin (CIP), with the minimal
inhibitory concentration (MIC) of > 1mg/L (Velhner
et al., 2014). The resistance to CIP was also found in
some isolates of Salmonella Infantis from Hungary
which belonged to the different pulsotype (Nógrády
et al., 2007). The occurrence of novel multidrug
resistant clones from human, food and poultry
sources in Israel was established in 2007. These
clones were resistant to NAL, TET, nitrofurantoin and
trimethoprim/sulfametoxazole (SXT). It was evident
that the resistance to TET and SXT was encoded by
a 280kb self-transmissible plasmid (pESI) and that
new clones represented 33% of all Salmonella strains
isolated in Israel (Aviv et al., 2014).
The most frequently detected serovars in poultry
meat in the EU were S. Infantis, S. Indiana and S.
Enteritidis. According to the epidemiological cut
off breakpoints (ECOFFs), multi-drug resistance
in Salmonella spp from broiler meat in the year
2014 uctuated from high (Hungary) to low (France
and Lithuania) or complete absence of resistance
(Ireland). Resistance to colisitin was 31.6% in S.
Enteritidis while resistance to ciprofloxacin and
nalidixic acid was 22.4%. No resistance toward
906 M. VELHNER, D. MILANOV, G. KOZODEROVIĆ
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J HELLENIC VET MED SOC 2018, 69(2)
ΠΕΚΕ 2018, 69(2)
eliminate Salmonella spp. from the food production
chain, travelling and trade still pose and will continue
to pose a substantial risk for infection of humans and
efcient dissemination of Salmonella spp. globally.
ACKNOWLEDGMENT
This paper was supported by a grant from the
Ministry of Education, Science and Technological
Development of the Republic of Serbia (Project
number TR 31071).
CONFLICT OF INTEREST STATEMENT
The authors declare no conict of interests.
in commercially produced poultry, as soon as an
ecological niche becomes vacant. In many developed
countries, where measures, such as vaccination, were
undertaken to eradicate certain Salmonella serovars,
other less immunogenic serovars emerge and become
dominant. Salmonella control programs in poultry
industry has to cover all the segments of food
production by implementing various procedures and
strategies in integrated poultry production systems.
It has to follow up new trends in raising free range
chickens with respect to new challenges regarding
food safety in upcoming years. In countries where
comprehensive programs have been implemented to
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... Additionally, these bacteria possess a diverse array of genetic mechanisms that contribute to their ability to colonize, adhere to, invade, and proliferate within, host cells. These mechanisms include the fimbriae, capsule, flagella, and toxins, and the virulence genes organized in their chromosome, such as pathogenicity islands, virulence plasmids, acid tolerance proteins, etc. [3,4]. ...
... Different hypotheses could explain the surge in this serovar in broilers. One possibility is the implementation of vaccination and control programs targeting S. Enteritidis and S. Typhimurium, which have led to a decrease in, or even elimination of, these serovars on farms [3]. This reduction created an ecological niche, which allowed the proliferation and spread of S. Infantis, particularly in broiler production, where it accounts for half of all Salmonella isolates. ...
... Notably, S. Infantis has been found to possess various genetic strategies that enhance its epidemiological fitness. These strategies include the acquisition and transmission of AMR, a resistance to heavy metals, the presence of mobile virulence genes, and the ability to form biofilms [3,4,[22][23][24][25][26][27][28][29][30][31][32][33][34][35]. ...
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Salmonella spp. has been globally recognized as one of the leading causes of acute human bacterial gastroenteritis resulting from the consumption of animal-derived products. Salmonella Enteritidis, S. Typhimurium, and its monophasic variant are the main serovars responsible for human disease. However, a serovar known as S. Infantis has emerged as the fourth most prevalent serovar associated with human disease. A total of 95% of isolated S. Infantis serovars originate from broilers and their derived products. This serovar is strongly associated with an elevated antimicrobial (AMR) and multidrug resistance, a resistance to disinfectants, an increased tolerance to environmental mercury, a heightened virulence, and an enhanced ability to form biofilms and attach to host cells. Furthermore, this serovar harbors genes that confer resistance to colistin, a last-resort antibiotic in human medicine, and it has the potential to acquire additional transferable AMR against other critically important antimicrobials, posing a new and significant challenge to global public health. This review provides an overview of the current status of the S. Infantis serovar in the poultry sector, focusing on its key virulence factors, including its virulence genes, antimicrobial resistance, and biofilm formation. Additionally, novel holistic strategies for controlling S. Infantis along the entire food chain are presented in this review.
... Th e exact reasons for persistent infection with serovars Enteritidis and Typhimurium can be revealed using experimental animals. It was shown that invasive serovars have more virulence factors, invade internal organs of the host more effi ciently and possess mechanisms prompting their survival inside the macrophages of the host (Velhner et al., 2018). For instance, the global spread of Salmonella Enteritidis phage type 4 (PT4) was attributed to successful infection of the hens' reproductive tract most likely by inheriting additional virulence factors which facilitate internal contamination of eggs (Velge et al., 2005). ...
... Th e exceptions are the above-mentioned sequence types, which persist in various niches and disseminate thanks to their enhanced fi tness. During the investigation of the diverse, multidrug-resistant commensal E. coli from a poultry farm practicing frequent use of antibiotics in Serbia, three independent clones with the common virulence type and resistotype were determined by pulse-fi eld gel electrophoresis (Velhner et al., 2018). Th e multilocus sequence type of only a small number of E. coli isolates from gulls was done as well. ...
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It has been established that some clones of pathogenic bacteria such as Salmonella spp., Escherichia coli ST131, and methicillin-resistant Staphylococcus aureus (MRSA) tend to spread worldwide. Therefore, epidemiological surveys have been conducted to identify the source of infection and to break the chain of infection. In this article, it was pointed out that common international clones of Salmonella are represented with the serotypes Typhimurium, Kentucky, Infantis and Enteritidis. Serovars Typhimurium and Kentucky display multidrug-resistant phenotypes more frequently. Several sequence types of E. coli and the international clone ST131 are described, including clades C1 and C2 with the extended-spectrum cephalosporin-resistance genes (blaCTX-M-15 or blaCTX-M-27). These pathogens are often found in both humans and animals. It is noted that Staphylococcus aureus became resistant to methicillin almost instantly after its introduction into clinical practice. Soon afterwards, MRSA found its way to farm animals and wildlife. The cycles of infection are bidirectional: humans can disseminate MRSA in the environment but animals may also be sources of infection for humans. Comprehensive work has been done by epidemiologists to introduce all necessary measures to eliminate MRSA from hospitals. Also, much effort has been made in MRSA control to prevent infections on animal farms and contamination in the primary food production chain. As the struggle with pathogenic bacteria continues, we face the incessant threat of new resistance and virulence mechanisms, which bacteria use to resist the hostile environment and enhance their survival in their natural habitats including humans and animals. Therefore, the capacity of certain bacteria to spread due to their virulence mechanisms and resistance phenotypes is presented, and a brief description of the research conducted in Serbia is included.
... Furthermore, the emergence and proliferation of antimicrobial resistance in Salmonella spp. presents further challenges (Velhner et al., 2018). In the Mekong Delta, there were a few large-scale chicken farms. ...
Article
Salmonella can carry multiple antibiotic-resistant and metalresistant genes and transmit these genes among strains worldwide. This study examined seventy-five Salmonella isolates from small-scale chicken farms (chicken feces, bedding, feed, wild animals) in Vinh Long province for the presence and relation of antibiotic and metal-resistance genes in these strains. The single PCR method was applied to detect seven antibiotic-resistance genes (blaampC, blaTEM, dfrA1, tetA, strA, sul2, mcr1) and four metal-resistance genes (pcoR, czcD, cnrA, silE). The results indicated that those Salmonella isolates harbored several patterns of antibiotic-resistance genes. Genes blaampC and tetA were the most prevalent (48.00%), while genes mcr1 and dfrA were the most minor (1.33%). Of those Salmonella isolates, 92.00% harbored one to five antibiotic-resistance genes, and the blaampC + strA pattern was frequently obtained (12.00%). Moreover, 30.67% of Salmonella isolates showed multidrug resistance to three or four antibiotic categories. Among metal-resistance genes, gene pcoR encoding for copper resistance was the most predominant (53.33%), and gene cnrA encoding for cobalt-nickel resistance was the lowest (5.33%). There were diverse patterns of metalresistance genes, and one Salmonella isolate carried four examined genes (1.33%). Furthermore, these Salmonella isolates had several combined patterns of metal-resistance and antibiotic-resistance genes. Among them, pcoR, czcD, and silE genes had a significant coefficient relation to the examined antibiotic-resistance genes. It indicated the correlation between metal resistance and antibiotic resistance genes and revealed the potential risk of increasing antibiotic resistance in Salmonella isolates in chicken farms in Vinh Long province.
... S. Infantis has been described as an emerging serotype in the poultry industry [26], as it generally does not produce symptoms in poultry. This specific serotype is, therefore, difficult to identify [27]. In Colombia, this serotype has been isolated in commercial laying hen farms in the department of Antioquia and samples of litter, feed, drinking water, cloacal swabs, cecal content, and ovaries [28]. ...
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Background and aim: Salmonella has been identified as one of the most widely distributed zoonotic pathogens in broiler litter. Multidrug-resistant strains have been isolated from salmonellosis outbreaks, compromising the success of their treatment. This study aimed to isolate and identify Salmonella spp. serovars in healthy broiler litter in Tolima (Colombia), determine their resistance to different antimicrobials, and detect genes associated with b-lactam resistance that could be useful to control Salmonella spp. in poultry. Materials and methods: In total, 45 broiler litter samples were collected. Salmonella spp. was isolated and identified using selective and differential culture media and biochemical tests. Molecular confirmation of the pathogen was performed with the invA gene and serotyping by Kauffman-White scheme. Antimicrobial susceptibility to 15 antibiotics was determined by Kirby-Bauer method. In cefotaxime-resistant strains, blaCTX-M-F, blaCTX-M-1, blaCMY, and blaTEM genes were evaluated by polymerase chain reaction (PCR). Results: In total, 817 presumptive strains were obtained from xylose lysine deoxycholate and Salmonella Shigella agars and subcultured on xylose-lysine-tergitol 4 and MacConkey agars, from which 150 strains were isolated; 29 of these strains were presumptive for Salmonella spp. after performing biochemical tests and 16 were confirmed by PCR as Salmonella Infantis (15) and Gallinarum (1). All strains were found to be multiresistant to antibiotics, showing three different profiles and isolates resistant to cefotaxime, and the blaCTX-M gene was detected. Conclusion: This is the first study to isolate S. Infantis from broiler litter in Colombia. All isolates exhibited resistance to the evaluated antimicrobials, suggesting the misuse of antimicrobials in small- and medium-sized poultry farms. The presence of Salmonella enterica serovar Infantis is a public health problem. Thus, regular monitoring of poultry litter is recommended, as these bacteria can be transmitted to humans through animal products or contaminated environments.
... Uncontrolled and indiscriminate use of antibiotics for growth promotion or prophylaxis, especially in low-and middle-income countries, can lead to the emergence of antibiotic resistance to fluoroquinolones and extended-spectrum beta-lactam antibiotics in the poultry sector (Badr et al., 2015;Velhner et al., 2018). MDR salmonellae are significant for both human and animal health because they can lead to illness that is unresponsive to antibiotic treatment (Chen et al., 2013). ...
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Abstract | Monitoring of imported 1-day-old poultry is mandated in Egypt to prevent the possible introduction of new Salmonella serovars into the country’s poultry industry. Such serovars are considered to be a major public health threat. We examined 391 imported poultry flocks for the presence of salmonellae (231 duckling, 84 chick, and 76 turkey poult), serotyped all isolated salmonellae, and performed antimicrobial susceptibility testing. Molecular profiles were also constructed based on results of conventional polymerase chain reaction assays to detect virulence genes (stn, avrA, and sopB) and antibiotic resistance genes (blaTEM, tetA(A), and qnrS) in the Salmonella isolates. Thirty Salmo- nella strains were isolated from the 391 samples (7.7%). By poultry type, salmonellae were isolated from 21 of 231 (9.1%) duckling samples, 6 of 84 (7.1%) of chick samples, and 3 of 76 (3.9%) turkey poult samples. Serotyping of the isolates identified 16 different serovars: S. Enteritidis, S. Typhimurium, S. Sinstorf, S. Muenster, S. Vejle, S. Cuckmere, S. Indiana, S. Infantis, S. Koenigstuhl, S. Macallen, S. Nchanga, S. Neftenbach, S. Newlands, S. Nigeria, S. Nyborg, and S. Regent. The isolates showed variable degrees of antibiotic resistance across species. All tested Salmonella strains harbored the virulence and antibiotic resistance genes, with the exception of qnrS, which was found in only 50% of the isolates. In conclusion, examination of imported poultry is a critical point of control to prevent poultry from becoming a reservoir for human health hazards, including salmonellae with antimicrobial resistance phenotypes.
... Uncontrolled and indiscriminate use of antibiotics for growth promotion or prophylaxis, especially in low-and middle-income countries, can lead to the emergence of antibiotic resistance to fluoroquinolones and extended-spectrum beta-lactam antibiotics in the poultry sector (Badr et al., 2015;Velhner et al., 2018). MDR salmonellae are significant for both human and animal health because they can lead to illness that is unresponsive to antibiotic treatment (Chen et al., 2013). ...
Article
Full-text available
Monitoring of imported 1-day-old poultry is mandated in Egypt to prevent the possible introduction of new Salmonella serovars into the country’s poultry industry. Such serovars are considered to be a major public health threat. We examined 391 imported poultry flocks for the presence of salmonellae (231 duckling, 84 chick, and 76 turkey poult), serotyped all isolated salmonellae, and performed antimicrobial susceptibility testing. Molecular profiles were also constructed based on results of conventional polymerase chain reaction assays to detect virulence genes (stn, avrA, and sopB) and antibiotic resistance genes (blaTEM, tetA(A), and qnrS) in the Salmonella isolates. Thirty Salmonella strains were isolated from the 391 samples (7.7%). By poultry type, salmonellae were isolated from 21 of 231 (9.1%) duckling samples, 6 of 84 (7.1%) of chick samples, and 3 of 76 (3.9%) turkey poult samples. Serotyping of the isolates identified 16 different serovars: S. Enteritidis, S. Typhimurium, S. Sinstorf, S. Muenster, S. Vejle, S. Cuckmere, S. Indiana, S. Infantis, S. Koenigstuhl, S. Macallen, S. Nchanga, S. Neftenbach, S. Newlands, S. Nigeria, S. Nyborg, and S. Regent. The isolates showed variable degrees of antibiotic resistance across species. All tested Salmonella strains harbored the virulence and antibiotic resistance genes, with the exception of qnrS, which was found in only 50% of the isolates. In conclusion, examination of imported poultry is a critical point of control to prevent poultry from becoming a reservoir for human health hazards, including salmonellae with antimicrobial resistance phenotypes. Copyright © 2021 Badr et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
... Several factors (e.g. feed (Hald et al., 2012b) and other sources such as, replacement animals, humans, domestic, wild and feral animals and birds, insects, contaminated equipment or water) ) can introduce infection into a poultry unit and salmonellas can further spread within and between holdings through movements of people, vehicles and equipment Velhner et al., 2018). After introduction, infection may be perpetuated in poultry houses, often without detection . ...
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An increase in confirmed human salmonellosis cases in the EU after 2014 triggered investigation of contributory factors and control options in poultry production. Reconsideration of the five current target serovars for breeding hens showed that there is justification for retaining Salmonella Enteritidis, Salmonella Typhimurium (including monophasic variants) and Salmonella Infantis, while Salmonella Virchow and Salmonella Hadar could be replaced by Salmonella Kentucky and either Salmonella Heidelberg, Salmonella Thompson or a variable serovar in national prevalence targets. However, a target that incorporates all serovars is expected to be more effective as the most relevant serovars in breeding flocks vary between Member State (MS) and over time. Achievement of a 1% target for the current target serovars in laying hen flocks is estimated to be reduced by 254,400 CrI95[98,540; 602,700] compared to the situation in 2016. This translates to a reduction of 53.4% CrI95[39.1; 65.7] considering the layer-associated human salmonellosis true cases and 6.2% considering the overall human salmonellosis true cases in the 23 MSs included in attribution modelling. A review of risk factors for Salmonella in laying hens revealed that overall evidence points to a lower occurrence in non-cage compared to cage systems. A conclusion on the effect of outdoor access or impact of the shift from conventional to enriched cages could not be reached. A similar review for broiler chickens concluded that the evidence that outdoor access affects the occurrence of Salmonella is inconclusive. There is conclusive evidence that an increased stocking density, larger farms and stress result in increased occurrence, persistence and spread of Salmonella in laying hen flocks. Based on scientific evidence, an impact of Salmonella control programmes, apart from general hygiene procedures, on the prevalence of Campylobacter in broiler flocks at the holding and on broiler meat at the end of the slaughter process is not expected.
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Listeria monocytogenes (in the meat, fish and seafood, dairy and fruit and vegetable sectors), Salmonella enterica (in the feed, meat, egg and low moisture food sectors) and Cronobacter sakazakii (in the low moisture food sector) were identified as the bacterial food safety hazards most relevant to public health that are associated with persistence in the food and feed processing environment (FFPE). There is a wide range of subtypes of these hazards involved in persistence in the FFPE. While some specific subtypes are more commonly reported as persistent, it is currently not possible to identify universal markers (i.e. genetic determinants) for this trait. Common risk factors for persistence in the FFPE are inadequate zoning and hygiene barriers; lack of hygienic design of equipment and machines; and inadequate cleaning and disinfection. A well‐designed environmental sampling and testing programme is the most effective strategy to identify contamination sources and detect potentially persistent hazards. The establishment of hygienic barriers and measures within the food safety management system, during implementation of hazard analysis and critical control points, is key to prevent and/or control bacterial persistence in the FFPE. Once persistence is suspected in a plant, a ‘seek‐and‐destroy’ approach is frequently recommended, including intensified monitoring, the introduction of control measures and the continuation of the intensified monitoring. Successful actions triggered by persistence of L. monocytogenes are described, as well as interventions with direct bactericidal activity. These interventions could be efficient if properly validated, correctly applied and verified under industrial conditions. Perspectives are provided for performing a risk assessment for relevant combinations of hazard and food sector to assess the relative public health risk that can be associated with persistence, based on bottom‐up and top‐down approaches. Knowledge gaps related to bacterial food safety hazards associated with persistence in the FFPE and priorities for future research are provided.
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Enhanced Salmonella surveillance programmes in poultry were implemented in all European Member States, with minimum prevalence targets for a list of targeted serotypes to safeguard food and public health. Based on the Belgian Salmonella surveillance programme and focusing on poultry, the overarching aim of this study was to highlight possible Salmonella transmissions across the food chain (FC). For this purpose, firstly, the prevalence patterns of Salmonella (targeted and the most prevalent non‐targeted) serotypes along the FC were described over time. Secondly, the effectiveness of the control measures against vertical transmission (breeders to 1‐day‐old broiler and layer chicks) was indirectly assessed by looking into the odds of targeted serotypes detection. Thirdly, it was appraised if Salmonella prevalence can significantly increase during broilers and layers production. In addition, it was tested if being tested negative at the end of production in broilers when tested positive at the entrance is serotype dependent (targeted vs. non‐targeted serotypes). Results showed that, firstly, the prevalence patterns of the listed serotypes were inconstant over time and across the FC. Secondly, the odds of Salmonella targeted serotype detection in 1‐day‐old broiler and in 1‐day‐old layer flocks were lower than in breeder flocks while, thirdly, infection during broiler and layer production can lead to significant increase in positivity in subsequent samples. Finally, being infected by a targeted or by non‐targeted serotype at the entrance of the flock poorly reflects the Salmonella status at the end of production. Note that this study did not make a distinction between the different sources of contamination and the effects of sampling methods and isolation methods should be subject to further investigation.
Article
Salmonella is a major cause of foodborne illness across Europe but there has been little recent research on its control in broiler production in Great Britain. Investigations of Salmonella presence on 20 broiler farms and a separate exploratory risk factor analysis involving 36 Salmonella-positive farms and 22 Salmonella-negative farms were carried out to investigate Salmonella contamination and control on broiler farms in Great Britain. Sources of Salmonella persistence on farm and potential risk factors for on-farm contamination were identified, enabling provision of up-to-date advice on Salmonella control to farmers. Twenty broiler farms across England and Wales were intensively sampled over time. Most farms were included in the study after routine testing as part of the Salmonella National Control Programmes (NCPs) identified regulated Salmonella serovars or potential associations with outbreak cases of significance for human health. Across all farms and visits, the highest proportion of Salmonella-positive samples were from areas exterior to broiler houses compared to anterooms or house interiors. Exterior Salmonella-positive samples were primarily collected from the immediate areas around the houses, with the highest proportions being from drainage, farm tracks/driveways, and pooled water. Elimination of Salmonella was variable but was most successful inside affected houses (compared to exterior areas) and for regulated Salmonella serovars under the Salmonella NCPs and high priority Salmonella strains with multi-drug resistances. It is likely that the financial and reputational concerns associated with regulated Salmonella serovars and those of greater public health significance underlie the reason that these serovars were more effectively controlled at farm level, as effective elimination of Salmonella can involve a considerable investment in infrastructure, time and resources. Without perceived direct benefits in eliminating non-regulated Salmonella serovars at farm level it can be challenging to maintain the required motivation and investment. A separate farm-level risk factor analysis was carried out using data collected from 58 broiler farms representing six GB broiler companies. Risk of testing positive for Salmonella via NCP sampling in the previous year was greater in the absence of house-specific anterooms and if at least some poultry houses were surrounded by soil/grass compared to if all were surrounded by concrete or a mixture of concrete and stones/gravel. Odds of testing positive for Salmonella in the previous year was also greater for farms whose maximum holding capacity was >100,000 birds, and farms where the usual number of visitors per day was 0−1 compared to 2−3. The analysis was exploratory and caution is required with interpretation, but results provide preliminary insight into aspects of farm management that may be important, practicable targets for Salmonella control on broiler farms in GB.
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This report of EFSA and the European Centre for Disease Prevention and Control presents the results of the zoonoses monitoring activities carried out in 2015 in 32 European countries (28 Member States (MS) and four non-MS). Campylobacteriosis was the most commonly reported zoonosis and the increasing European Union (EU) trend for confirmed human cases since 2008 continued. In food, the occurrence of Campylobacter remained high in broiler meat. The decreasing EU trend for confirmed human salmonellosis cases since 2008 continued, but the proportion of human Salmonella Enteritidis cases increased. Most MS met their Salmonella reduction targets for poultry. More S. Enteritidis isolates were reported and S. Infantis was confirmed as the most frequent serovar isolated from domestic fowl. In foodstuffs, the EU level Salmonella non-compliance for minced meat and meat preparations from poultry was low. Despite the significant increasing trend since 2008, the number of human listeriosis cases stabilised in 2015. In ready-to-eat foods, Listeria monocytogenes seldom exceeded the EU food safety limit. The decreasing EU trend for confirmed yersiniosis cases since 2008 continued. Positive findings for Yersinia were mainly reported in pig meat and products thereof. The number of confirmed shiga toxin-producing Escherichia coli (STEC) infections in humans was similar to 2014. In food, STEC was most frequently reported in meat from ruminants. A total of 4,362 food-borne outbreaks, including waterborne outbreaks, were reported. Bacteria were the most commonly detected causative agents, followed by bacterial toxins, viruses, other causative agents and parasites. The causative agent remained unknown in 33.5% of all outbreaks. As in previous years, Salmonella in eggs continued to represent the highest risk agent/food combination. The report further summarises trends and sources for tuberculosis due to Mycobacterium bovis, Brucella, Trichinella, Echinococcus, Toxoplasma, rabies, Coxiella burnetii (Q fever), West Nile virus and tularaemia.
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Introduction: Novel molecular techniques applied in biotechnology research have provided sound evidence on clonal persistence of distinct serovars of Salmonella in feed factory environments, over long periods of time (months, even years), which can be responsible for repeated in-house contamination of final products. In this study, we examined the possibility of clonal persistence of isolates of three Salmonella serovars that have been repeatedly identified in animal feed samples from three feed factories throughout a two-year period. Methodology: The isolates Salmonella enterica serovars Tennessee (n = 7), Montevideo (n = 8), and Infantis (n = 4) were tested for genetic diversity using pulsed-field gel electrophoresis (PFGE) and multicellular behavior patterns by applying the Congo red agar test. Results: SpeI and XbaI macro-restriction profiles indicated that isolates S. Montevideo and S. Infantis were identical, whereas isolates of S. Tennessee demonstrated greater genetic diversity, although the genetic differences did not exceed 10%. All Salmonella serovars demonstrated the ability to produce predominant matrix compounds essential for biofilm formation, curli fimbriae and cellulose. Conclusions: The identification of identical clones of S. Montevideo and S. Infantis, as well as the minor genetic diversity of S. Tennessee, which have been repeatedly isolated from animal feed in three production plants throughout a two-year period, indirectly suggests the possibility of their persistence in feed factory environments. Their ability to express the key biofilm matrix components further supports this hypothesis.
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Salmonella serovars are differentially able to infect chickens. The underlying causes are not yet fully understood. Aim of the present study was to elucidate the importance of Salmonella Pathogenicity Island 1 and 2 (SPI-1 and -2) for the virulence of two non-host-specific, but in-vivo differently invasive, Salmonella serovars in conjunction with the immune reaction of the host. Primary avian splenic macrophages were inoculated with Salmonella enterica sub-species enterica serovar (S.) Typhimurium and S. Infantis. The number and viability of intracellular bacteria and transcription of SPI-1 and -2 genes by the pathogens, as well as transcription of immune-related proteins, surface antigen expression and nitric oxide production by the macrophages, were compared at different times post inoculation. After infection, both of the Salmonella serovars were found inside the primary macrophages. Invasion-associated SPI-1 genes were significantly higher transcribed in S. Infantis- than S. Typhimurium-infected macrophages. The macrophages counteracted the S. Infantis and S. Typhimurium infection with elevated mRNA expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-12, IL-18 and lipopolysaccharide-induced tumor necrosis factor alpha factor (LITAF) as well as with an increased synthesis of nitric oxide. Despite these host cell attacks, S. Typhimurium was better able than S. Infantis to survive within the macrophages and transcribed higher rates of the SPI-2 genes spiC, ssaV, sifA, and sseA. The results showed similar immune reactions of primary macrophages after infection with both of the Salmonella strains. The more rapid and stronger transcription of SPI-2-related genes by intracellular S. Typhimurium compared to S. Infantis might be responsible for its better survival in avian primary macrophages.
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Data indicate that prevalence of specific serovars of Salmonella enterica in human foodborne illness is not correlated with their prevalence in feed. Given that feed is a suboptimal environment for S. enterica, it appears that survival in poultry feed may be an independent factor unrelated to virulence of specific serovars of Salmonella. Additionally, S. enterica serovars appear to have different host specificity and the ability to cause disease in those hosts is also serovar dependent. These differences among the serovars may be related to gene presence or absence and expression levels of those genes. With a better understanding of serovar specificity, mitigation methods can be implemented to control Salmonella at preharvest and postharvest levels.
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Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella's adaptation to its animal host and especially for S. Kentucky's emergence as the dominant serovar in poultry.
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During the year 2012 a study was conducted on the hygiene of the production of broiler carcasses at three abattoirs in the Republic of Serbia. A total of 150 samples of broiler neck skin were examined and 17 salmonella isolates were recorded. Isolates were, by using the corresponding monovalent and polyvalent sera, determined according to type as Salmonella enterica subspecies enterica serovar Infantis (S. Infantis 6, 7, r, 1, 5). In the case of the disease in humans, 5 Salmonella samples of identical serovars were isolated. After that, 22 samples were tested for antibiotic resistance by the disk diffusion test. Isolates showed resistance to ampicillin and nalidixic acid (95.5%), tetracycline (91%), cefotaxime/clavulanic acid (68.2%), but not to ciprofloxacin, gentamicin, and trimethoprim/sulfamethoxazole. The degree of genetic similarity of isolates from diseased humans and broiler carcasses was determined at a molecular level. Cluster analysis revealed the presence of 7 profiles, while all isolates have 92% genetic similarity. Although there are differences in the antimicrobial resistance of isolates originating from diseased humans and neck skin of tested broilers, can not be excluded an epidemiological link, because in the dominant genotype SINFXB0001, established in 8 isolates from diseased humans (3 isolates), and the neck skin of broilers (5 isolates), a genetic similarity of 100% was recorded. Based on these results, the presence of S. Infantis on broiler carcasses can be considered a hazard to human health. Key words: Salmonellosis, prevalence of Salmonella on broiler carcasses, Salmonella Infantis, antibiotic resistance, PFGE
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Adhesion of microorganisms to food processing surfaces and the problems it causes are a matter of strong concern to the food industry. Contaminated food processing surfaces may act as potential sources of transmission of pathogens in food industry, catering and in the domestic environments. Several studies have shown that adhesion of bacteria to surfaces partly depends upon the nature of the inert surfaces and partly upon the bacterial surface properties. The aim of this study was to compare the adhesion of four different strains of Salmonella Enteritidis to stainless steel 304 (SS 304). The effect of surface hydrophobicity and surface elemental composition on the adhesion process was also analysed. Hydrophobicity was evaluated through contact angle measurements using the sessile drop method. All the strains studied showed positive values of the degree of hydrophobicity (DGlwl) and so can be considered hydrophilic while stainless steel revealed a hydrophobic character. Bacterial cell surface composition was measured using X–ray photoelectron spectroscopy (XPS). The XPS results corroborated the similarity of the values of the degree of hydrophobicity obtained by contact angles. The different Salmonella strains showed similar elemental composition and cell surface physico–chemical properties. Nevertheless, S. Enteritidis MUSC presented higher adhesion ability to SS 304 (p<0.05). It can be concluded that the physico–chemical properties of the strain does not explain the ability of adhesion to stainless steel. Other factors like the production of polysaccharides must be considered.
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Feed components have low water activity, making bacterial survival difficult. The mechanisms of Salmonella survival in feed and subsequent colonization of poultry are unknown. The purpose of this research was to compare the ability of Salmonella serovars and strains to survive in broiler feed and to evaluate molecular mechanisms associated with survival and colonization by measuring the expression of genes associated with colonization (hilA, invA) and survival via fatty acid synthesis (cfa, fabA, fabB, fabD). Feed was inoculated with 1 of 15 strains of Salmonella enterica consisting of 11 serovars (Typhimurium, Enteriditis, Kentucky, Seftenburg, Heidelberg, Mbandanka, Newport, Bairely, Javiana, Montevideo, and Infantis). To inoculate feed, cultures were suspended in PBS and survival was evaluated by plating samples onto XLT4 agar plates at specific time points (0 h, 4 h, 8 h, 24 h, 4 d, and 7 d). To evaluate gene expression, RNA was extracted from the samples at the specific time points (0, 4, 8, and 24 h) and gene expression measured with real-time PCR. The largest reduction in Salmonella occurred at the first and third sampling time points (4 h and 4 d) with the average reductions being 1.9 and 1.6 log cfu per g, respectively. For the remaining time points (8 h, 24 h, and 7 d), the average reduction was less than 1 log cfu per g (0.6, 0.4, and 0.6, respectively). Most strains upregulated cfa (cyclopropane fatty acid synthesis) within 8 h, which would modify the fluidity of the cell wall to aid in survival. There was a weak negative correlation between survival and virulence gene expression indicating downregulation to focus energy on other gene expression efforts such as survival-related genes. These data indicate the ability of strains to survive over time in poultry feed was strain dependent and that upregulation of cyclopropane fatty acid synthesis and downregulation of virulence genes were associated with a response to desiccation stress.
Chapter
Many strains of Salmonella enterica represent zoonotic pathogens which are capable of causing disease in humans. One major source of food poisoning is Salmonella enterica serovar Enteritidis which is often associated with chicken meat, meat products and eggs. A number of virulence factors enable this bacterium to colonize and systemically infect poultry. In this review, we will concentrate on the S. Enteritidis Type III Secretion System (T3SS) located on Salmonella Pathogenicity Island-1 and highlight its role in invasion of chickens. The structural components of the complex secretion apparatus and the secreted effector proteins will be introduced. Aspects of T3SS assembly, its expression, regulation under different conditions, as well as its involvement in colonization and invasion of poultry will be discussed in the context of the recent findings.