Feed additives to control Salmonella in

poultry
F. VAN IMMERSEEL*, K. CAUWERTS, L.A. DEVRIESE, F. HAESEBROUCK
and R. DUCATELLE

Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary

Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium

Poultry meat and eggs are important sources of human pathogens. Salmonella is a
major cause of human foodborne infections following consumption of poultry
products. The original ambition of the EU to eradicate zoonotic agents from the
animal production chain has been tempered to reducing the infection pressure of
specified zoonotic agents at all levels of the production chain. This can be done by a
combination of pre-harvest, harvest and post-harvest measures. Feed additives
constitute an important group of pre-harvest measures which can help in controlling
Salmonella on the farm. Feed additives used for the control of Salmonella can be of
different types, including antibiotics, prebiotics, probiotics and synbiotics. Public
concerns regarding possible antibiotic resistance transfer lead to the ban of
antibiotics as growth promoters in monogastric diets within the EU. Experimental
and practical use of pre-, pro- and synbiotics, as well as volatile fatty acids as feed
additives are discussed in this review. The effects of these additives on resistance to
infection, on the extent of excretion and on the persistence of infection are reviewed.
Attention is paid also to possible undesirable effects of some of these products.
Taking into consideration the underestimated high level of contamination of poultry,
the feed additives reviewed in this article can certainly play a valuable part in control
strategies during the pre-harvest phase aiming at reducing the infection pressure and
thus limiting the risk of contamination of poultry products.

Keywords: Salmonella;feed additives; prebiotics; probiotics; fatty acids; poultry

Introduction
Poultry products represent an important source of pathogens that can enter the food chain
(Corry and Hinton, 1997; Todd, 1997). Bacteria constitute the most important category of
pathogens (Corry and Hinton, 1997). The incidence of bacteria, which are potentially
pathogenic for humans, such as Campylobacter and Listeria spp. on raw poultry meat is
high (Waldroup, 1996). Helicobacter pullorum represents a new pathogen that was
isolated from raw poultry meat (Corry and Atabay, 2001). Arcobacter butzleri may cause

*Corresponding author: e-mail: filip.vanimmersee1@rug.ac.be

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human enteritis and is frequently recovered from poultry carcasses (Wesley and Baetz,
1999; Houf et al., 2001). The leading cause of human foodborne infections in the world,
associated with consumption of poultry products, however still is Salmonella (Gomez et
al., 1997). Domestic fowl can become infected through numerous sources. Litter, faeces,
feed, water, dust, insects, rodents and equipment are only a few examples of infection
sources. The so-called ‘invasiveserotypes’constitute the greatest risk. These are serovars
known to pass from the intestine into the tissues of poultry. Worldwide, Salmonella
enterica serovar Enteritidis is one of the most common invasive serovars isolated from
poultry, causing human disease. Poultry can become carriers and asymptomatically
excrete Salmonella Enteritidis intermittently (Ducatelle et al., 2000). Re-excretion may be
induced by stress conditions. Vertical transmission can occur when follicles in the ovary
are infected or the developing eggs become infected in the oviduct (Poppe, 2000).
In 1992, the Council of the European Community issued a directive (EU-92/117 and
subsequent amendments) requiring member countries to monitor for zoonotic agents, to
specify measures for reducing the risk of introduction of Salmonella on the farm to control
Salmonella in parent and layer flocks. These instructions basically require a top - down
approach, which means that measures are very strict in grandparent and parent flocks.
These measures are expected to be of benefit to the general sanitary status of the whole
production chain. This top-down concept implies that zoonotic agents are transmitted
vertically in the poultry population, what is, to some extent, the case for invasive
serotypes, such as Enteritidis, vphimurium, Bertha, Thompson, Infantis and Hadar. For
non-invasive serotypes, only eggshell contamination can lead to vertical transmission.
Since horizontal transmission is as important as vertical transmission in Salmonella
infections in poultry, a top-down approach not including measures in the commercial
broiler and layer flocks is doomed with limited success. The original ambition of the EU
of eradicating zoonotic agents from the animal production chain and thus producing
animal products free from zoonotic agents has been considerably tempered. Plans now are
designed to reduce the infection pressure of specified zoonotic agents at the different
levels of the production chain.
Salmonella control schemes have been put in place in most EU countries in recent years.
These include pre-harvest, harvest and post-harvest measures. All of these are equally
important and each type of measure has a more or less important effect on reducing the
Salmonella incidence, but no measure is successful on its own. Harvest measures are
essentially hygienic measures during catching and transport, while post-harvest measures
include both hygienic measures and the application of decontaminatingtreatments on the
meat or eggs. However, carcass disinfectants are prohibited in the European Community
and decontamination of fresh table eggs is difficult. Therefore, prevention and
monitoringleradication during the live phase (pre-harvest) is of great importance in
Europe. he-harvest prevention and control measures include preventive hygienic
measures as well as physical and chemical decontamination treatments of feed, drinking
water and the environment of the birds. This review contains a description of feed
additives for controlling Salmonella in poultry, including the advantages and
disadvantagesof these products. The feed additives can be grouped in different classes or
types, namely the antibiotics, prebiotics, probiotics, synbiotics and short chain fatty acids.

Antibiotics
Next to their prophylactic and curative use, antibiotics have also been given in feed to
poultry for their growth promoting effects. Most antibiotic growth promoters act by
modifying the intestinal flora, especially targeting Gram-positive bacteria, which are

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associated with poorer health and performance of the animal (Bedford, 2000). Indeed,
many so-called non-pathogenic bacterial species reduce feed conversion efficiency and
growth in chickens due to competition with the host for the nutrients in the intestinal tract,
degradation of host enzymes and reduction of the absorptive surface area. In some
instances these bacteria can elicit an immune response, which, as a side effect, causes
reduction of appetite and catabolism of muscle protein (Bedford, 2000). One of the
mechanisms underlying these phenomena is the massive production of tumour necrosis
factor alpha (TNF-a).Some of these intestinal bacteria are considered as opportunistic or
facultative pathogenic bacteria. Clostridium perjiringens, associated with necrotic
enteritis, is the best known example of this (Hofacre et al., 1998). Antibiotics have been
used for many years as growth promoting agents. In recent years, concerns arose about the
use of antibiotics in livestock. Antibiotic feed additives were linked to the emergence of
multiple drug resistant bacteria (Wray and Davies, 2000). The use of avoparcin in poultry
for example has lead to vancomycin-resistant Enterococcus spp. in poultry and swine
(Kruse et al., 1999; Bager et al., 1997). The presence of undesired antibiotic residues in
meat and environmental contamination have largely added to the public concerns
regarding the use of antibiotics in the feed. As a consequence at the end of June 1999 the
majority of antibiotics as growth promoters in monogastric diets have been banned within
the E.U. Exceptions are the anticoccidial products with antibiotic activity salinomycin,
monensin and narasin. These three antibiotics belong to the ionophore group of antibiotics
(polyether antibiotics) and are active against protozoon, parasites, such as Eimeria and
Plasmodium (Gumila et al., 1997), and against bacteria, mainly Gram-positives (Dutta
and Devriese, 1984). Monensin is a monovalent polyether antibiotic produced by
Streptomyces cinnamonensis (Watanabe et al., 1981). Monensin had no effect on tissue
invasion and intestinal colonization of S. Enteritidis when 7-day-old chicks were
inoculated with 106 cfu (Manning et al., 1994). Salinomycin is a monovalent carboxylic
ionophorous polyether antibiotic, produced by the fermentation of Streptomyces albus
(Lindsay and Blagburn, 1995). Salinomycin had no effect on length of Salmonella
shedding, numbers of Salmonella bacteria shed on postdosing day 3 and on tissue invasion
after S. Typhimurium infection of 3-day-old chicks (Ford et al., 1981). However, Bolder
et al. (1999) detected a reduced incidence of Salmonella shedding at 6 weeks of age, when
broilers were infected with lo8cfu S. Enteritidis at day 11 and 12. Narasin is a monovalent
ionophore produced by a strain of Streptomyces aureofaciens (Droumev, 1983). No
differences of in vitro growth and survival of a live Salmonella vaccine were detected in
the presence of narasin (Martin and Meyer, 1994).
Next to the above-described anticoccidials, also flavomycin and avilamycin, two
antibiotics unrelated to therapeutically used products, can still be used in the EU.
Flavomycin (bambermycin)belongs to the group of glycolipid antibiotics and is produced
by a group of Streptomyces species (Huber and Nesemann, 1968). Flavomycin has a
mainly Gram-positive spectrum and inhibits the peptidoglycan synthesis in the cell wall
(Huber and Nesemann, 1968). When 2-day-old chicks were orally challenged with 104 to
105 cfu S. 'Qphimurium, there was no difference in the proportion of Salmonella positive
birds and caecal colonization, between birds receiving flavomycin in their feed and
control birds (Humpert et al., 1991). However, in another study, feeding flavomycin
reduced the degree and incidence of Salmonella shedding when broilers were orally
challenged with 108 cfu S. Enteritidis at days 10 and 11 of their age (Bolder et al., 1999).
Avilamycin is an oligosaccharide antibiotic of the orthosomycin group and is produced by
Streptomyces viridochromogenes (Buzetti et al., 1968). Avilamycin is only active against
Gram-positive bacteria (Fuchs et al., 1999) and acts through binding with the 30s
ribosomal subunit, thereby interfering with the protein-synthesizing function (Wolf,
1973). When feed was contaminated artificially with S. Kedougou, no significant

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differences were found in the number of S. Kedougou bacteria in caecal contents between
birds that received avilamycin-supplemented feed and control birds (Hinton, 1988).
However, in the same study, more birds given the control diet had Salmonella positive
faecal samples compared with the birds whose feed contained avilamycin, when a low
number of S. Kedougou bacteria was included in the feed (less than 5 cfdg).

Prebiotics
Prebiotics have been defined by Gibson and Roberfroid (1995) as “non-digestible food
ingredients that beneficially affect the host by selectively stimulating the growth and/or
activity of one or a limited number of bacterial species already resident in the colon, and
thus attempt to improve host health”. Based on this definition, Russell (1998) formulated
the criteria according to which a substance can be a prebiotic. First, prebiotics are always
feed ingredients that are not digested by the host, not or little used and / or metabolised as
they pass through the upper portion of the intestinal tract, so they can reach the flora of the
large intestine. Secondly, they have to be able to serve as a substrate for one or more
bacterial species with a potentially beneficial effect on the host. Finally they have to be
able to cause a shift in the microflora that improves the health of the host.
In principle only non-digestible, fermentable feed components are prebiotics. Most of
these prebiotics are carbohydrates. Besides carbohydrates, there are other substances
thought to have a positive effect on the intestinal flora and/or the intestinal environment,
for example, organic acids. Whether these belong to the prebiotics or not is not clear.
Therefore they will be treated separately (see below). The term prebiotics thus is restricted
here to indigestible carbohydrates. These carbohydrates are divided in groups based on
their molecular length: mono-, di-, oligo- and polysaccharides. Another possible
classification is based on their source of origin: natural or synthetic saccharides (Iji and
Tivey, 1998).

CLASSES OF PREBIOTICS
The most important monosaccharide prebiotics are hexoses (glucose, fructose,
galactose, mannose) and pentoses (ribose, xylose, arabinose). Some of these, like glucose
and fructose, are available as monomers. Monosaccharides such as glucose and fructose
are digestible as monomers and therefore not prebiotics according to the definition of
Russell (1998). Galactose is available mostly under the disaccharide form of lactose.
Mannose is by far the most commonly used monosaccharide feed additive (Allen et al.,
1997). These monosaccharidescan form the basis for enzymatically constructed oligo- or
polysaccharides.
The most important natural disaccharides are sucrose, lactose and maltose.
Isomerizationproducts of these compoundscan be used as prebiotics, e.g. lactulose (based
on lactose). Lactose (Corrier et al., 1993), lactulose (Iji and Tivey, 1998) and lactosucrose
(Terada et al., 1994) reportedly have prebiotic effects in chickens. A reduction in the total
number of positive Salmonella Enteritidis organ invasions was observed following 14
days and 19 days after infection in chicks fed with lactose (Corrier et al., 1993). Lactose
is not hydrolysed or absorbed intact from the intestinal tract of chicks, and as much as 50%
of ingested lactose in poultry diets may be excreted unchanged. Because of its lack of
digestion and absorption, lactose passes into the lower segmentsof the intestine and caeca.
The hydrolization of lactose that does occur is primarily the result of the microflora
utilisation of the disaccharide (Tellez et al., 1993). Lactose feeding of broilers changes the
consistency of caecal contents and can lead to mild scouring (Corrier et al., 1993).
Oligosaccharides are usually defined as glycosides that contain a limited number of

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hexose or pentose units. Sometimes they are used in their natural form but mostly they are
obtained through enzymatic synthesis or hydrolysis. Synthetic oligosaccharides can be
based on different hexose monosaccharides, for example glucose, fructose, galactose and
mannose (Iji and Tivey, 1998). The synthetic route varies, depending on the direct
polymerization of disaccharides or the fractionation of microbial cells to obtain the
material from the cell wall. Polysaccharides, when fermented, also yield oligosaccharides
(Iji and Tivey, 1998). Fructo-oligosaccharides(FOS) are short-chain polymers of p 1-2-
linked fructose units, which are produced commercially by hydrolysis of inulin or by
enzymatic synthesis from sucrose or lactose (Le Blay et al., 1999). FOS are the
oligosaccharidesmost extensively studied in chickens with respect to their prebiotic effect
and their activity against Salmonella (Fukata et al., 1999) and Campylobacter (Schoeni
and Wong, 1994). FOS lead to a reduction of colonization of the intestine by Salmonella
in chickens, especially when the animals also received competitive exclusion flora in
addition to FOS (Bailey et al., 1991). Galacto-oligosaccharides can be produced
industrially by the transgalactosidase activity of beta-galactosidases. These products
hitherto have not been tested in poultry (Durst, 1996). Mannan-oligosaccharides(MOS) or
mannose-based carbohydrates occur naturally in many products such as yeast cell walls
and gums. A commercial product is available for poultry, which contains yeast cell wall
fragments derived from Saccharomyces cerevisiae after centrifugation of a lysed yeast
culture (Spring et al., 2000). When 3-day-old chicks were orally challenged with 104 cfu
S. Typhimurium, chickens receiving 4000 ppm of dietary MOS had reduced caecal S.
Typhimurium concentrations compared with birds with no MOS in their feed. When S.
Dublin was used as challenge organism, the number of birds that was positive in their
caeca was less when MOS was part of the diet (Spring et al., 2000). Caecal contents from
hens fed these MOS protected chicks from colonization with Salmonella Enteritidis
(Fernandez et al., 2000).
The most commonly used polysaccharide prebiotic for chickens is guar gum, produced
from the seeds of the guar bean, Cyamopsis tetragonolobus. By selectively cleaving the
mannan backbone-chain of guar gum using endo-f3-D-mannanase, a mixture of
galactomannans is obtained, called partially hydrolysed guar gum (PHGG). When feed
supplemented with 0.025% PHHG was given to young hens, no Salmonella Enteritidis
bacteria could be detected from 14 days post-infection on, when the birds were infected
with 3.106 cfu. Moreover, the incidence of S. Enteritidis on the surface of the eggshell and
in egg white and yolk was decreased when the feed of laying hens was supplemented with
0.025% PHHG and the animals were inoculated with 3,4.106 cfu S. Enteritidis (Isihara et
al., 2000). This is thought to be due to improvements in the balance of intestinal flora
(Ishihara et al., 2000).

MECHANISMS OF ACTION OF PREBIOTICS

Prebiotics can have a direct effect either by direct binding of pathogens or by increasing
the osmotic value in the intestinal lumen. More often, however, their effects are indirect,
mediated by metabolites, which are generated by the intestinal flora that uses the
prebiotics for their own metabolism. Such metabolites include short-chain fatty acids,
lactate, polyamines and bactericins.
Q p e 1 (Fl) fimbriae are described in several bacterial enteric pathogens (Bengmark,
1998), including Salmonella (Oyofo et al., 1989). These type 1 fimbriae bind to mannose
residues of glycoproteins, present on the surface of eukaryotic cells. They are needed for
the adhesion of the bacteria to the mucosal surface, which is a prerequisite for the
colonization of the host. Indigestible carbohydrates with mannose residues may bind the
F1 fimbriae and therefore block the adhesion of the bacteria to the epithelial cells
(Finucane et al., 1999).According to these authors yeast MOS induce mannose sensitive

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agglutination in 5 1% of E. coli strains tested and 53% of Salmonella strains tested. Among
the Salmonella strains, 80% of the strains of the Enteritidis serotype and 67% of the
Typhimurium serotype strains possess these agglutinins.
Prebiotics may constitute a substrate for the growth of intestinal flora. This
multiplication of normal flora may inhibit the colonization with pathogenic bacteria. This
phenomenon of inhibition is called “competitive exclusion”. FOS have been shown to
promote growth of Enterococcusfaecium, Lactobacillus lactis and Pediococcus spp. in
vitro. The above mentioned growth promoting effects on the endogenous microflora
however have not been demonstrated yet in vivo. Indeed Salmonella challenge in MOS
supplemented broilers resulted in decreased numbers of Salmonella in caeca, but no
differences in numbers of lactobacilli, enterococci or anaerobes were observed (Spring et
al., 2000).
Another possible mechanism of action of prebiotics is through a modification of the
metabolic activity of normal intestinal flora. Saccharides, indigestible by the host, are
fermented by the flora into volatile fatty acids (VFA including acetate, propionate and
butyrate), lactate and several gases including carbon dioxide, methane and hydrogen
(Cummings, 1981). Administration of MOS in the feed, however, induced no change in
lactate, VFA or caecal pH in broilers (Spring et al., 2000).

Probiotics
A probiotic, by the generally accepted definition, is defined as a live microbial feed
supplement, which beneficially affects the host animal by improving its intestinal
microbial balance (Fuller, 1989). A few events during history have lead to the
development of these products. The use of probiotics for farm animals was stimulated by
the findings of the Schwann Committee in 1969 which recommended that antibiotics in
animal feeds should be restricted, hereby creating a void which prebiotics began to fill
(Fuller, 1999). A very crucial event in the development of probiotics was the finding that
newly hatched chickens could be protected against colonization by Salmonella Enteritidis
by dosing a suspension of gut contents derived from healthy adult chickens (Nurmi and
Rantala, 1973).This concept is called competitive exclusion. Another important historical
finding that has been at the basis of the development of probiotics was the finding that
ingested milk improved the health status and longevity of men, and that lactobacilli play
an important role in this phenomenon (Metchnikoff, 1907).

DEFINED CULTURES
Many research efforts have focused on the probiotic use of Lactobacillus species,
isolated from poultry (Gusils et al., 1999). Jin et al. (1996a,b) found growth-inhibiting
effects of Lactobacilli against Salmonella Enteritidis, but none of the tested Lactobacillus
species were able to reduce the attachment of Salmonella Enteritidis to chicken intestinal
epithelial cells in vitro, in contrast with Salmonella Pullorum and Qphimurium. However,
when lo*cfu Lactobacillus salivarius CTC2197 was dosed by oral gavage together with
106 cfu S. Enteritidis directly into the proventriculus in 1-day-old chicks, the Salmonella
bacteria were completely removed from the birds after 21 days (Pascual et al., 1999). The
inclusion of this strain into a commercial feed mixture is a good way to supply it on the
farm, although the strain showed sensitivity to the temperatures, used during the feed
mixture storage and in the chicken farm (Pascual et al., 1999). Special attention should be
drawn to Lactobacillus reuteri. This bacterium produces and secretes an intermediary
metabolite, reuterin, which has antimicrobial activity against Salmonella, E. coli and
Campylobacter (Mulder et al., 1997). As opposed to other probiotic bacterial cultures,

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inoculation of Lactobacillus reuteri in ovo does not affect hatchability and it decreases the
colonization by Salmonella and E. coli after hatch in poultry. Additionally, mortality due
to in-hatcher exposure to E. coli or Salmonella is reduced. Use of antibiotics in ovo may
preclude the co-administration of probiotics, but gentamicin and Lactobacillus reuteri
apparently are a compatible mixture when administered in ovo in separate compartments
(Edens et al., 1997). Lactobacillus plantarum has been shown to possess mannose-
sensitive receptors, an uncommon phenomenon in gram-positive bacteria. With these
receptors it can compete for the same adhesion sites in the intestine as the gram-negative
pathogens (Bengmark, 1998).
Enterococcusfaecium 596, isolated from the intestinal tract of a free-ranging chicken,
inhibited the growth of Salmonella hllorum, Gallinarum, Qphimurium and Enteritidis in
vitro (Audisio et al., 1999). The antibacterial action was due to the combined effect of
lactic acid and bacteriocin. When lo9 cfu Enterococcus faecium J96 were given to 30
hours old broilers, and the animals were challenged 24 hours later with lo5cfu Salmonella
hllorum, the chicks treated with the probiotics survived the challenge, in contrast to
chicks that did not receive the lactic acid bacteria (Audisio et al., 2000).
Line et al. (1998) investigated the effects of yeast supplementation on intestinal
colonization of broilers by Salmonella Typhimurium. Saccharomyces boulardii, a non-
pathogenic yeast used in several countries as a living oral biotherapeutic agent to treat
human patients suffering from diarrhoea, was given to day-old chicks at a dose of 1 and
~ at day 4. Of the
100 g k g feed, where after the animals were challenged with 3 . 2 ~ 1 0cfu
positive control birds, 70% were colonized with Salmonella, whereas only 20 and 5% of
the l x and lOOx yeast treated birds were colonized (Line et al., 1998).

Synbiotics
A synbiotic is, in its simplest definition, a combination of probiotics and prebiotics
(Collins and Gibson, 1999; Schrezenmeir and de Vrese, 2001). This combination could
improve the survival of the probiotic organism, because its specific substrate is available
for fermentation. This could result in advantages to the host offered by the live
microorganism and the prebiotic. Bengmark (2001) defines synbiotics as products
produced by fermentation. Since in mixtures of pre- and probiotics the prebiotics will be
fermented when the appropriate choice of products is made, also this definition is
plausible. Fermentation can lead to the production of short-chain fatty acids, which are
discussed below. Examples of synbiotics are FOS and b@dobacteria, and lactitol and
lactobacilli (Collins and Gibson, 1999). Bailey et al. (1991) used a combination of FOS
and competitive exclusion flora to reduce Salmonella colonization in chickens. The
combination was more effective in reducing Salmonella colonization than FOS or
competitive flora alone.

Short chain organic acids

Volatile fatty acids (VFA) (formic, acetic, propionic and butyric acid) are produced by the
normal anaerobic intestinal flora as end products of their metabolism (Mead, 2000). The
production of these VFA by the intestinal flora can be stimulated by adding fermentable
prebiotics to the feed (Cummings, 1981). It is clear that the use of probiotics also
stimulates fermentation and therefore the production of the VFA. VFA or short chain fatty
acids (SCFA), mainly formic and propionic acid, can also be added directly to the feed.
Commercial products consisting of propionic and formic acid encapsulated in silica beads

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are applied in the field. Encapsulation allows slow release of the acids, so that a sufficient
amount of product reaches the lower intestinal tract. The acids not only exert an
antibacterialeffect in the intestine, but also in the crop (Hinton and Linton, 1988). In most
experiments where single VFA were added to the feed, no protection against Salmonella
was found (Izat et al., 1990; Hume et al., 1993). Commercial products however contain
mixtures of propionic acid and formic acid, and proved to decrease shedding of
Salmonella Kedougou (Thompson and Hinton, 1997).
SCFA are bacteriostatic or bactericidal in vitro for gram-negativebacteria, provided that
there are sufficient undissociated acid molecules present and that they are in contact with
the bacteria for sufficiently long time (Thompson and Hinton, 1997). Within practical
limits, lowering the pH increases the concentration of undissociated molecules. Therefore
the products will be more efficient at low pH. This was proved in in vitm experiments by
a decreased growth rate of Salmonella Typhimurium at decreasing pH levels in the
presence of VFA (Durant et al., 2000). Formic acidpropionic acid 1% in the feed reduces
the caecal pH (Waldroup et al., 1995), which increases the proportion of undissociated
acids. The SCFA diffuse into the bacterial cell in undissociated form. Inside the bacterial
cell, the acid dissociates, resulting in reduction of intracellular pH and anion accumulation
(van der Wielen et al., 2000). Concentrations of VFA sufficient to cause growth inhibition
of E. coli in vitro immediately slowed the rates of RNA, DNA, protein, lipid and cell wall
synthesis (Cherrington et al., 1990). Since synthesis of macromolecules other than DNA
was not completely inhibited, and since there was no loss in membrane integrity, bacterial
mass increased without cell division in the presence of VFA (Chemngton et al.,
1990;1991).
VFA have been shown to increase cell differentiation and decrease cell proliferation in
human colonic epithelial cell lines (Siavoshian et al., 1997). It is known for Salmonella
Enteritidis that these bacteria bind to more differentiated chicken caecal epithelial cells in
virro and in vivo (Desmidt et al., 1998; Van Immerseel et al., 2002). It was recently
speculated that butyrate could activate chemokine secretion (e.g. IL-8) of human epithelial
cells through histone acetylation (Fusunyan et al., 1999). Consequences of this for
bacterial infections have not yet been investigated.
VFA also have undesirable effects, which are thought to be beneficial for bacteria. The
acid resistance of Salmonella Typhimurium was enhanced upon exposure to VFA (Kwon
and Ricke, 1998). While Salmonella bacteria normally do not survive at pH 3.0 in culture,
a high number of bacteria survived after exposure to any VFA for some hours. Since
bacteria can already be in contact with the VFA before ingestion, these bacteria could be
protected against the gastric acidity (pH 3.0). Also the acidificationof the phagolysosome
which Salmonella would encounter when phagocytosed by macrophages would be no
problem for bacteria that are pre-exposed to VFA, either in the feed or in the intestinal tract
(Kwon and Ricke, 1998).
In vitro studies suggest that VFA might have an undesirable effect possibly favouring
the invasiveness of Salmonella and the development of the carrier state. Indeed at pH 6, all
VFA induce hilA and invF expression (Durant et al., 2000). These genes are
transcriptional regulators of the Salmonella pathogenicity island I, needed for invasion of
host tissues. Moreover SCFA also induce spv expression in Salmonella Dublin (El-
Gedailly et al., 1997). Spv genes are common in most Salmonella serovars and are
involved in virulence, inducing systemic disease and macrophage survival (Libby et al.,
2000). In vitro data demonstrate that exposure of Salmonella Typhimurium to VFA affects
cell-association and invasion of cultured Hep-2 cells, an epidermal carcinoma cell line
derived from the human larynx (Durant et al., 1999; 2000).
All above described studies indicate that care should be taken when VFA are used to
control Salmonella in poultry. If the purpose is only to reduce Salmonella bacteria in

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faeces and litter and to lower the infection pressure in the farm, VFA are effective
products. Based on the expression of virulence genes and changes in cell invasion patterns
after VFA exposure, it seems not unlikely that Salmonella bacteria are driven to
intracellular compartments of the intestine after exposure to VFA. In this case
bacteriological examination of cloacal swabs and litter samples could be negative while
the animals are carrying the bacteria inside their tissues. It is known that carrier animals
can excrete the bacteria intermittently under certain stress conditions and can contaminate
unexpectedly the other birds in the flock (Ducatelle et al., 2000). The effects of VFA on
bacterial invasion and on induction of the carrier state need to be further investigated. The
end point of an integrated control program, as described by the EU directives, can not only
be the reduction of Salmonella excretion in faeces.

Conclusion
This overview shows that a large spectrum of feed additives is available at present for the
control of zoonotic (intestinal) pathogens, such as Salmonella Enteritidis, in poultry. It is
not meant to be complete, and there is no doubt that there are more aspects to the
nutritional strategies. Not only the use of feed additives to prevent intestinal colonization,
but also the prevention of feed contamination itself has to be considered. Reduction of
Salmonella contamination of feed is possible by steam conditioning prior to pelleting, by
expanding and by high temperature - short time conditioning, which may result in a
reduction to about 0.001% of the level in the untreated material (Screenivas, 1998).
Elimination of Salmonella and other bacteria of zoonotic importance in feed can also be
achieved by gamma-irradiation (Campbell et al., 1986; Farkas, 1998) or chemical
treatment (Rouse et al., 1988). Nutritional strategies implementing the in this review
described feed additives become an increasingly important component of integrated
prevention and control plans at the level of individual companies as well as nation-wide.
Most of these tools undoubtedly can reduce the colonization and excretion of these
zoonotic agents, however probably none can eliminate these pathogens completely in a
field situation. Therefore such tools should never be used on their own. Moreover one
should realize, as mentioned above, that a number of the effects attributed to these tools
only have been shown in laboratory animal models or in vitro, and remain to be proven in
poultry under field conditions. Also any of these additives may slightly or considerably
add to the cost of feed production, thus requiring a thorough cost - benefit analysis. In
addition, further research is necessary on the possible undesirable side effects of feed
additives.
Taking into consideration, however, the underestimated high level of contamination of
the raw materials, it is clear that the feed industry has to take up its responsibility and its
share in the struggle against these pathogens that are a permanent threat to consumer
health. If the purpose is to decrease the infection pressure, the above-described feed
additives can positively contribute to solve the problem.

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