1 Bacteriological study of Avian Salmonellosis (S. pullorum, S ... - Abef

Bacteriological study of Avian Salmonellosis (S. pullorum, S. gallinarum. S. Enteritidis y S.
Typhimurium) in Latin America
Dr. Horacio Raúl Terzolo, Med. Vet., Ph.D
Instituto Nacional de Tecnología Agropecuaria (INTA), Área de Producción Animal,
Casilla de Correo 276, (7620) Balcarce, Argentina.
E Mail: hterzolo@balcarce.inta.gov.ar
This review is intended to be practical and comprehensive, taking into consideration not
only general aspects of bacteriology of the genus Salmonella but also the relationship of
Salmonella pullorum, S. gallinarum, S. Enteritidis and S. Typhimurium with the different
diseases they cause. Its is expected that this presentation will be useful as an introduction to
understand the complexity of these diseases in order to confer practical information that will
enable us to provide tools to select and effectively enforce the best available control measures.
Zoonotic Salmonella in Latin America
Salmonellosis is a mayor cause of bacterial enteric illness in both human and animals.
Salmonella paratyphoid infections are important zoonoses and therefore this subject has to be
approached focusing in both the infection in animals and the public heath issues. In Latin
America human bacterial infections account for circa 57% of the diseases transmitted by food
and circa 10% of these infections are caused by ingestion of food prepared using poultry
ingredients. The prevalence of Salmonella serovars in poultry varies in different countries and
during different periods of time. Certain serovars emerge within a country or region for a period
and then disappear with no obvious cause or intervention measure. Historically, in Latin America
and also all around the world, S. Typhimurium has been the most prevalent serovar from both
humans and poultry. However, during the 1980’s, S. Enteritidis emerged as the predominant
serovar, overwhelming the isolation rates of S. Typhimurium. Nowadays, in Latin America S.
Enteritidis is the predominant isolate from humans, animals, food and environment, representing
between 25 to 60% of the overall Salmonella isolates.
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Salmonella is a ubiquitous environmental bacterium
It has to be pointed out the increasing importance of Salmonella contamination of foods
prepared with crops, vegetables and seeds, among other foods prepared from plants; the
contamination of soybeans and especially soybean meal in bulk that is transported by ship is a
serious worldwide problem that becomes very relevant because this product is the basis of many
balanced foods destined to feed various domestic animal species including poultry. Salmonella is
hugely widespread because is able to grow between 7 and 45°C, is only destroyed at 65°C during
10 to 15 minutes, resists very acid pH (even below 4), resists salt added in food up to 20%,
survives in frozen food, remains viable during years in the environment and in certain foods is
able to survive during years (for instance in honey), etc. Bacteria from the Salmonella Genus
make up a versatile and fascinating group of microorganisms that are successfully adapted to live
in very different environmental conditions and some of them are also adapted to colonize
different animal hosts. In each ecological niche Salmonella will develop different phenotypical
adaptations; for instance if the bacteria live in a river many flagella will be developed to allow
movement of the microorganisms in a liquid medium but if they need to grow over a kitchen
chopping board they will have no flagella but instead will be organized into biofilms, which are
very resistant to mechanic cleaning and disinfection.
Biofim formation
The ability of Salmonella to form complex surface-associated communities, called
biofilms, contributes to its resistance and persistence in both host and non-host environments and
is especially important in food processing environments. Salmonella can be attached to different
types of abiotic (plastic, glass, cement, rubber, and stainless steel) or biotic surfaces (plant
surfaces, epithelial cells, and gallstones) as well as plastics used in the laboratory (polystyrene).
Different structural components of Salmonella are involved in biofilm formation: curli and other
fimbriae, protein BapA, flagella, cellulose, colanic acid, anionic O-antigen, capsule and fatty
acids. Biofilm formation is strongly influenced by different environmental signals via a complex
regulatory network. Biofilm formation allows Salmonella to resist against different stress factors
such as desiccation or disinfectants (e.g. hypochlorite, glutaraldehyde, cationic tensides and
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triclosan) and also some antibiotics (e.g. ciprofloxacin). Dome biofilm inhibitors may be used
such as surfactin, glucose, halogenated furanones, 4(5)-aryl 2-aminoimidazoles, furocoumarins
and salicylates. Also it is possible to combine therapy and disinfection (e.g. combinations of
triclosan and quaternary ammonium salts or halogenated furanones and treatments
antibiotics/disinfectants) and micro-emulsions such as soybean oil-in water. The halogenated
furanones are secreted by various algae and plants and specifically act by interfering with quorum
sensing.
Fowl Typhoid and Paratyphoid infections in Latin America
Most countries in Latin America have combined problems of Salmonellosis due to S.
Gallinarum (Fowl Typhoid) and S. Enteritidis (Paratyphoid) in laying hens, though in breeders
Paratyphoid usually has sporadic presentations and outbreaks are generally quickly controlled. In
the broiler industry, S. Enteritidis and S. Typhimurium are only sporadically encountered,
although other serovars may be found in either slaughter houses or chicken carcasses in retail
shops. There is more available information on human alimentary outbreaks, some of them related
with consumption of food from poultry origin.
The presence of Fowl Typhoid does not assure that the farm should be free from Paratyphoid
salmonellas as both diseases in the same farm have been simultaneously found.
The Kauffmann – White scheme
Species and subspecies of Salmonella are phenotypically classified using different
bacterial growth and biochemical tests while serovars or serotypes are classified according to the
Kauffmann – White scheme. First the "O" antigen type is determined based on polysaccharides
associated with lipopolysaccharides (LPS); the different “O” antigens are expressed by numbers
at the beginning of the antigenic formula. Then the "H" antigens are determined based on
flagellar proteins. Since Salmonella exhibit phase variation, exist either monofasic (single phase)
or bifasic (double phase) strains; besides there are non-motile and motile phenotypes and the
latter can express different "H" antigens. S. Gallinarum is always non-motile but retains the
genetic material coding for flagella and even a flagellar hook-basal body complex is seen by
electron microscopy, the protein is called “FliE”. Non-motile isolates can be induced to switch to
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the motile phase by means of cultures in Craigie tubes and also these tubes may be used to detect
a strain that is biphasic although unable to express one of the flagellar phases. The flagellar
antigens are expressed by combining letters and numbers. Pathogenic strains of S. Typhi as well
as some strains of S. Dublin carry the "Vi" antigen (Vi for virulence), which is related to a
protein capsule. Different variations of the strains may affect the serotyping results: S – R
(Smooth – Rough), M – N (Mucoid – Non-mucoid), form variations such as “L phase” when the
LPS are lost and the salmonella after loosing its cell-wall acquires a spheroplast shape, fimbriate
– Non-fimbriate, OH – O (Motile – Non-motile), and Monophasic – Biphasic flagellar antigen.
Salmonella species, subspecies and serovars
New serovars are constantly added to the list. Nevertheless only two bacterial species are
actually recognized within the Genus: S. enterica ( 2,443 serovars) and S. bongori (20 serovars).
S. enterica is composed by six subspecies: enterica, salamae, arizonae, diarizonae, houtenae and
indica. Nearly all pathogenic species from warm-blooded animals are included into Salmonella
enterica subsp. enterica (1,454 serovars). The rest of the other Salmonella enterica subspp. (989
serovars) either colonize cold-blooded animals or are environmental free-living salmonellas,
excepting some serovars of S. enterica subsp. arizonae that may cause disease (Arizonosis) in
turkeys.
Groups of Salmonella according to their host prevalence
The majority of outbreaks of Salmonellosis in humans and domestic animals are caused
by relatively few serovars and these serovars can be divided into three groups on the basis of host
prevalence. The first group consists of host-specific serovars that cause systemic diseases in a
limited number of phylogenetically related species. Examples of these serovars are the serovar S.
Gallinarum (which includes biovars S. gallinarum and S. pullorum) in poultry, S. Typhi and S.
Paratyphi A in humans, S. Typhi-suis in pigs, S. abortus-ovis in sheep, and S. abortus-equi in
horses. All these animal-adapted or human-adapted specific serovars cause disease in only certain
animal species or humans but causes no gastroenteric disease in humans by cross-infection of
Salmonella species adapted to an animal and therefore are not considered to be zoonotic serovars.
A an exception, a scientific paper described a human empyema caused by S. gallinarum in an
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immuno-compromised person; nevertheless healthy and immuno-competent human beings are
completely refractory to any disease caused by S. gallinarum.
The second group consists of two host-restricted serovars (S. Dublin in cattle species and
S. cholerae-suis in porcine species), however, these serotypes are potentially capable of infecting
other animal species and humans.
The third group consists of widespread serovars of Salmonella enterica subsp. enterica,
such as S. Enteritidis and S. Typhimurium that usually causes gastroenteritis in a wide range of
unrelated host species. Originally these two serovars are specifically adapted to infect rodents but
also have developed the ability to infect other animal species, including humans. Originally these
two serovars were in fact rodent-adapted specific serovars that evolved capacity to infect other
species, among them human beings.
Host susceptibility to S. Enteritidis and S. Typhimurium
Regarding the susceptibility of S. Enteritidis and S. Typhimurium for different animal
species we can state that rodents and humans are the most susceptible, followed by ruminants and
horses as having an intermediate susceptibility and, interestingly, aves, carnivorous mammal
species and pigs are the less susceptible animals. That birds and poultry are one of the less
susceptible animal species to acquire any detectable illness makes these animals more relevant as
unapparent carriers of paratyphoid salmonellas; as often both serovars remain unnoticed, they
may have more chances to infect humans throughout consumption of contaminated food products
or processed food products. Furthermore, the world increase of the consumption of poultry
products means more opportunities for these ubiquitous serovars to infect humans.
Maternal immunity and development of disease
Very young chicks, less than 2 or 3 days old, are vulnerable to get a paratyphoid evident
disease due to Salmonella septicemia; according to the maternal immunity passively transferred
from their breeder parent flock, the chick progeny may or not be prone to suffer different
mortality rates. Nowadays, as most breeder flocks are vaccinated with inactivated S. Enteritidis,
vaccines, either alone or combined with S. Typhimurium, nearly all flocks have some protective
degree provided by maternal antibodies that usually hides any symptoms becoming the disease
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clinically unapparent. When during the 1980’s the pathogenic S. Enteritis PT4 or its related PT34
appeared for the first time in naïve broiler breeder flocks of Argentina, mortality and disease
signs became apparent. Such symptoms consisted of an uneven chicken growth together with a
dwarfness appearance among some of the chickens of the flock, incomplete resorption and
induration of the yolk sac and variable mortality rates, which may reached up to 10% in the first
batch of chicks that were hatched from naïve unvaccinated broiler breeder flocks. Interestingly,
as maternal immunity increased in the broiler-breeder parent flock, the successive batches of
hatched chicks of the corresponding progeny gradually diminished the symptoms and lesions as
well as the mortally rates, until complete disappearance after the two weeks.
S. Enteritidis and S. Gallinarum are clonally related
Comparative genome analysis between S. Enteritidis and S. Gallinarum indicated that
both serovars are highly related and they probably share genes that might be acquired from a
common primitive bacterium ancestor. The close relationship between the poultry-adapted S.
Gallinarum and S. Enteritidis signify that the latter possesses some factors that made the serovar
Gallinarum successful as poultry pathogen. Both isolates belong to serogroup D1, indicating that
the structure of their LPS is very similar. Both serovars are part of the same clonal lineage, have
the same mechanisms of intracellular multiplication and both possess SEF14 fimbriae, which is
strongly involved in the colonization of the reproductive tissues. Therefore, acquisition of SEF14
was critically important to allow S. Enteritidis a successful colonization of the eggs.
Diagnosis methods
There are in the market many very efficient culture media and techniques to isolate
Salmonella spp. from poultry, food or environmental samples. Different isolation techniques have
been described including non-selective and selective enrichment broths and different agars to
identify and pick Salmonella-like colonies. Non-selective enrichment broths include peptone
water and lactose broth. The most common selective enrichment broths are Salmocyst®,
Tetrationate, Rappaport Vassiliadis and Selenite-cystine. For most paratyphoid Salmonella
Xilose-Lisine-Deoxycholate, Salmonella-Shigella, Hektoen Enteric or Rambach agars are all
suitable for detection and isolation of Salmonella colonies. As the colonies of S. Gallinarum are
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smaller than those from other Salmonella enterica serovars, faintly produce SH2 and usually may
be isolated in abundant pure culture from the organs of diseased birds, usage of MacConkey agar
for primary isolation of Fowl Typhoid outbreaks is a very convenient choice. Batteries of cultures
and biochemical tests are used to phenotypically characterize the isolates. For a rapid screening
of Salmonella spp. in food or food ingredients, a rapid ELISA test is commercially available.
Now there are also available in the market molecular tests designed for identification of some
most common pathogenic serovars of Salmonella enterica. These molecular methods have the
advantage to overcome some of the aforementioned variations of the Salmonella strains that may
affect the classical serotyping results.
Sampling methods
The most appropriate samples for septicemic cases due to fowl typhoid or paratyphoid
Salmonella outbreaks are: liver, gallbladder with bile contents and spleen. All Salmonella spp.
are able to survive an even grow in pure bile, sometimes developing adherent biofilms into the
gallbladder walls. Caecum contents are always suitable to isolate paratyphoid Salmonella but it is
not always a good sample to isolate S. Gallinarum because in acute outbreaks of diseased birds
frequently die before the intestine is being colonized. Bone marrow from tarsus-metatarsus is the
best choice when the bird is found already dead from Fowl Typhoid. Cloacal swabs from adult
birds are not always the best sample because most of the times excretion is intermittent. On the
contrary, sampling meconium by pressing the belly of 1-day-old chickens is the best sample to be
taken. Sometimes it is very difficult to isolate Salmonella enterica from the organs or feces from
infected breeders; in such cases it is better to cultivate samples from the chick progeny rather
than from their parents. When autopsy from very young chicks is undertaken, in addition to
meconium, egg-yolk sac and livers are also excellent samples. Cultures of environmental samples
from farms, hatcheries, and slaughterhouses as well as different implements for poultry rearing or
work with birds are important to know the extent of contamination and to take preventive
measures, but it has to be taken in account that other environmental serovars which are not
involved in the disease of the birds may also be isolated.
Drawbacks of the whole blood agglutination test
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During many years the rapid whole blood stained S. Pullorum antigen was used in farms
as a practical agglutination test to eradicate Fowl Typhoid by elimination of the positive reacting
birds. Nowadays, due to the widespread of the pathogenic S. Enteritidis within the poultry
industry, most breeders are vaccinated with inactivated vaccines based on S. Enteritidis and,
because Enteritidis and S. Gallinarum share common antigens, these birds may develop
antibodies that react against both serovars. Sometimes when the birds of the farm are infected
with S. Enteritidis and the animals are free from S. Gallinarum, some chickens may be erratically
positive to the whole blood pullorum test. In addtion, some outbreaks of Fowl Typhoid may be
caused by a S. gallinarum strain that have antigenic differences with the standard or variable S.
pullorum strains used to formulate the antigen; in this case only a few infected birds may be
detected as positive reactors. Besides, it has also to take into account that some
Enterobacteriaceae share common antigens with Salmonella spp., such as some strains of
Escherichia coli (named “Salmonella coli”); these strains may cause a relatively high rate of false
positive reacting birds even if the farm is free from Fowl Typhoid. It should be then indicated
that this test has only a bird’s population value and do not has any individual value; hence these
results should be interpreted very carefully because the final diagnosis must be based on the
isolation the of causative Salmonella spp.
Relationship among Salmonella virulence, chicken rearing and environmental conditions
The nature and severity of Salmonella infections varies enormously and is influenced by many
factors: infecting Salmonella serovar, virulence of the strain, infecting dose, age and immune
status of the bird, management of the flock and biosecurity measures and also has to be taken into
account the geographical region were the farm is located and the climate conditions. The genetic
background of bird is also important, although to a lesser degree. It is paramount to consider the
virulence factors of the serovar and also the attributes of virulence that carries and expresses the
infecting strain. Among them we have to consider: cellular wall with its LPS, cellular membrane
with its proteins, thermolabile enterotoxin (LT), cytotoxin, flagella, pili or fimbriae and also the
DNA that carries virulence determinants either in the chromosome or plasmids.
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Mutations and genetic interchange of Salmonella
Because the generation time of Salmonella only lasts circa 22 minutes, duplication of
bacteria is very fast. Such a rapid genetic duplication of the bacterial DNA gives chances to the
bacterium to relatively quickly acquire genetic changes, either due to mutations or genetic
interchange. Genetic interchange may take place by transformation or transference of free DNA,
transduction or transference by means of specific Salmonella bacteriophages and conjugation in
which either genetic material from the chromosome or the plasmids may be transferred by a
sexual pillus from the donor to the recipient bacillus.
Phagetyping
Phagetyping has for decades been useful as a phenotypical, definitive method for
epidemiological characterization of Salmonella Typhimurium. Phagetyping has also been very
useful to classify strains of Salmonella Enteritidis from different regions; for instance, it was
found that in Europe predominates phagetype (PT) 1, 4 and 6, in USA PT8, in Spain PT14b and
in Argentina PT4 and its variants PT34 (from very virulent strains) and PT7 (from low virulent
strains). The system recommended by the World Health Organization (WHO) Collaborative
Centre for phage typing of Salmonella has, however, become rather complex, and there are
challenges of sufficient standardization for the interpretation of lysis to make sure that the same
strain is assigned to the same phagetype by different laboratories. In the future molecular typing
methods will replace phenotypic characterization methods, but despite of that phage typing will
remain for some time as a useful tool for Salmonella surveillance.
Virulence plasmids of Salmonella
Pathogenic Salmonella serovars contain autonomously replicating plasmids, being only
some of them transferable by conjugation. Some are involved in virulence or harbor antibiotic-
resistance genes, while others are cryptic with no recognized function. Some plasmids have been
sequenced, as for instance the S. Enteritidis 60kb virulence plasmid. Virulence type plasmids
vary between 50 and 100kb and are present in many isolates of different Salmonella serovars.
The virulence plasmids have a common ~8kb DNA region encoding the spv genes (spv =
Salmonella plasmid virulence) and are present in pathogenic strains of S. Enteritidis, S.
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Gallinarum (biovars S. gallinarum and S. pullorum), S. Typhimurium, S. Cholareasuis, S. Derby,
S. Dublin and S. Paratyphi B. Other Salmonella subspecies such as S. arizonae have the spv genes
included in the chromosome. The spv plasmid is required for the expression in vivo of a full
virulence phenotype. Strains that were cured from the spv plasmid loose their capacity to survive
and persist in the host. By means of challenge experiments with S. gallinarum in 2 week-old
chickens it was demonstrated the relationship between mortality and presence, absence or
reintroduction of the virulence plasmid. Thirty genes of the spv plasmid of S. Typhimurium
regulates responses to the internal environ and stressful conditions that might be encountered
within the host, including anaerobic growth, nitrogen and phosphate starvation, acid and osmotic
shock and oxidative stress. A well characterized response to environmental stress is the induction
of acid tolerance response; previous exposure to low pH (4.4 to 5.8) for a short period of time
initiates a bacterial response that increases resistance to even more acidic conditions (pH 3.3 to
3).
Antibiotic multi-resistant Salmonella
The use of antibiotics in human and veterinary medicine and crop protection provides a
selection pressure resulting in the preferential spread of resistant bacteria. Due to the
aforementioned genetic interchange, some molecular determinants may be selected and
propagated in bacteria, either in the same bacterial species or in other species from genetically
related genus, for instance Salmonella with other Enterobacteriaceae.
Conflicting opinions have been expressed on the large-scale use of antibiotics in the
poultry industry. It was believed that banning the extensive use of antibiotics in chickens could
reduce the spread of antibiotic-resistant bacteria but this action should be accompanied by other
measures because if not it may result in an increase incidence of antibiotic resistant Salmonella in
chickens due to an increased requirement of treatments. Nevertheless, in some countries with
good biosecurity measures in the farms, as for instance in Denmark, banning of antibiotics as
growth promoters in chickens was found beneficial without producing any increase of Salmonella
shedding.
The aforementioned virulence plasmid spv encoded genes associated with antibiotic
resistance in S. Typhimurium, S. Dublin and S. Choleraesuis. This plasmid acquired a toxin –
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antitoxin system that ensures its survival. Both toxin and antitoxin are encoded by the plasmid
and the two proteins bind to form a harmless dimmer in the bacterial cell. The antitoxin is labile
but is continually replaced by the gene encoded by the plasmid. If a bacterium loses the plasmid
during bacterial replication, the bacterium will still be intact complex Toxin – Antitoxin but the
antitoxin is continuously deteriorating and can not be replenished, so the toxin is released and
accumulates and eventually the cell lacking the plasmid will die. The problem of plasmid
mediated resistance in poultry is mainly limited to some multi-resistant strains of S.
Typhimurium that emerged in 1960’s; these strains are resistant to ampicillin, streptomycin,
sulfonamides, tetracyclines, and furazolidone. On the other hand, S. Enteritidis, despite having
the spv plasmid, rarely exhibits genetic acquired antibiotic resistance; its plasmids only confer a
limited resistance phenotype. During 1995-2001 it was undertaken a study that included 258 S.
gallinarum strains; it was demonstrated an augmented resistance for some antibiotics which had
been increasingly used and a diminished resistance for others that had been either completely
withdrawn from the farms or very sporadically administered.
Pathogenicity islands and virulence
Salmonella have evolved strategies to colonize various anatomical niches of the chicken.
The pathogenicity islands are large and distinct entities located on the bacterial chromosome.
They harbor one or more virulence genes. They may be genetically stable or more frequently
unstable due to genetic DNA-mobility by direct repeats, integrases, transposases and
bacteriophages genes. Many of these islands contain a %C+G (percentage of Cytosine plus
Guanine) that is higher or lower than the core genome. These genes have anchored points of
tRNA acquired from lisogenic phages. The Salmonella Pathogenic Island 1 (SP-1) is a stable
DNA that is required for invasion of non-phagocitic cells allowing the salmonellas to invade the
gut tissues after oral infection. In addtion SP-1 induces apoptosis of the macrophages and cleaves
two pro-inflammatory cytokines: interleukin (IL) IL1β and IL18. Both events allow the escape of
the internalized salmonellas from infected macrophages and at the same time attracts novel
phagocytes; hence the inflammatory focus increases by replication of the salmonellas into the
intercellular space and engulfment of the free bacillus by the recently arrived phagocytes; by
means of apoptosis Salmonella is able to produce new inflammatory focus. Another important
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function of SP-1 is the acquisition by Salmonella of the combined iron from the internal environ
of the chicken, allowing extra-cellular multiplication of bacteria within the intercellular spaces of
the chicken tissues.
SP-2 is necessary for the protection of the Salmonella cells contained within an
intracelullar vacuole of the bird because it prevents the action of the oxidase and nitric oxide,
powerful oxidizing agents that are released by a synthase produced by the phagocyte. Also
encodes for the intracellular translocation of effector proteins of Salmonella through the
formation of tubular aggregates in the endosomes of the cells of the bird. Another function of the
SP-2 is to codify for anaerobic respiration by means of the enzyme tetrathionate reductase.
SP-3 encodes for intracellular survival and replication of Salmonella within the nutrient
poor environment of the vacuoles using limited concentrations of purines, pyrimidines, particular
amino acids and Mg 2+ . SP-4 genes encode for the type I secretion system involved in production
of bacterial toxins and survival inside macrophages. SP-5, also called “Salmonella Centrisome 7
Genomic Island”, codifies for effector proteins of SP-1 and SP-2. SP-6 encodes for fimbriae that
contribute to virulence in S. enterica. SP-7 is also called “Mayor Pathogenicity Island” contains
genetic elements encoding virulence for pili that contribute to invasion of epithelial cells and the
Vi polysaccharide capsule of S. Typhi, S. Dublin and S. Parayphi C. More research is needed to
know the functions of the other islands, some are disclosed as follows: SP-8 encodes for
bacteriocins of S. Typhi; SP-9 for a citotoxin of S. Typhimurium; and SP-10 for the Sef
adherence fimbriae of S. Enteritidis and S. Typhi. Finally “Salmonella Genomic Island 1”
encodes for antibiotic resistance.
Different states of Salmonella poultry carriers
After Salmonella infection the birds may remain as carriers for a variable period of time or even
for life. S. enterica may remain viable inside macrophages and other cells of the Reticulo
Endothelial System. Due to the lisozyme content of some tissues from the host the cellular wall
of S. enterica may be completely or partially removed forming a protoplast or spheroplast or the
so called “L form”. In this state the bacteria generally remain undetected because do not elicit any
immune response; this is because the bacterial membrane has a composition that is very similar to
the one of the avian cell. Also can not be isolated by standard bacteriological methods because
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these procedures lack of osmotic protection prior to induce a reversal of “L form” (from L Phase
to Vegetative Phase with recovery of the cell wall).
Three types of carriers are described; Active: Salmonella have in vivo replication and high
numbers of bacteria are excreted and heavily contaminate the environment; Passive: Salmonella
is ingested but the bacterium is unable to multiply because of the host immune status or because
the bird is refractory to infection for this particular serovar or strain, in such case only the same
clone of bacteria that is ingested is excreted; and Latent: the bird does not excrete any salmonella
since intracellular bacteria remain hidden, at which stage the bacterial cultures that are commonly
used to detect the vegetative bacillus of Salmonella are all negative, a situation that continues
unabated until the animal is able to excrete bacteria, which usually occurs after a factor of stress
reactivates the disease. “Latent Carriers” may be turned into “Active Carriers” by the action of
different factors of stress, such as: transport, overcrowding, entering new birds into the farm,
sudden temperature changes, different climate alterations, food restriction, molting, intense
reproductive activity in breeders, production of eggs, and experimentally the stress can be
triggered by injecting corticosteroids.
Vertical transmission
S. Gallinarum, S. Pullorum, S. Enteritidis and S. Typhimurium are the most important
salmonellas that are transmitted through eggs. When this infection reaches a breeder farm, a
serious sanitary problem could be generated because this disease vertical spreads from the parent
flock to its chicken progeny. Moreover, once a pathogenic Salmonella strain is introduced into a
farm it is very difficult to eliminate infection because a single batch of infected breeding birds
can contaminate at the hatchery all the chicks from the other lots that so far have not been
infected. For that reason when contamination reaches the breeders the economic cost for the
elimination the disease is very high. Due to this reasons enormous efforts are made to raise
breeders that are free from pathogenic Salmonella. On the other hand, when laying hen farms are
infected with paratyphoid salmonellas, the infection usually becomes clinically unapparent and
the Salmonella can be inadvertently transmitted through eggs for human consumption. Also S.
gallinarum may be present in some farms and in this case the disease becomes clinically evident
and very frequently causes mortality and severe economic loses.
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Contamination of eggs by different serovars of Salmonella enterica
Some serovars of S. enterica subsp. enterica may cause human gastroenteric illness
through food prepared using contaminated undercooked or raw eggs. In USA S. Enteritidis was
described as the most frequent serovar (90%) isolated from table eggs whereas in the EU is the
most common environmental serovar (52%) in laying hen farms. In addition to S. Enteritidis,
other serovars have been described as a cause of human Salmonellosis due to consumption
contaminated eggs: S. Typhimurium, S. Infantis, S. Hadar, S. Newport, S. Stanley, S. Derby, S.
Agona, S. Kentucky and S. Virchow, among some others. The chicken adapted biovars S.
pullorum and S. gallinarum produce a high percentage of internally contaminated eggs (circa
6.5%) during the period of sexual maturity. On the contrary, S. Enteritidis is less successful,
being the percentage of internally contaminated eggs between 0.1% and 0.6 %. In addition to
systemic spread, other serovars of Salmonella enterica can also gain access to the oviduct
through ascending infection from the cloacae to the vagina. The rank order of the Salmonella
invasiveness in vaginal epithelium is dependent on the LPS type as follows: antigen O9 (S.
Gallinarum, S. Enteritidis) > O4 (S. Typhimurium, S. Heilderberg), O4 > O7 (S. Infantis, S.
Montevideo), and O7 > O8 (S. Hadar).
Virulence factors associated with egg colonization
The process of colonization of the reproductive tract is complex and depends on many
factors including Sef fimbriae and manose dependent Type 1 fimbriae, flagellae, LPS, cell wall
structure and stress tolerance. S. Enteritidis is the best adapted paratyphoid serovar for the
colonization and persistance in the hen’s reproductive tract and it was demostrated that repeated
in vivo passages of a S. Enteritidis strain through the reproductive tissues of the hen increases its
ability to induce internal egg contamination, whereas serial in vivo passages through liver or
spleen did not affect the ability of the strain to cause internal egg contamination.
Contamination of eggs after oviposition
S. Enteritidis can contaminate eggshells and is able to migrate through the shell and
associated membranes thus contaminating the egg contents. This occurs more frequently if the
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eggshell is damaged or may also happen in undamaged recently laid eggs exposed to fecal
contamination of salmonellas. The fecal contamination could happen at the moment of
oviposition when the egg passes through the cloaca in an infected hen or if the egg is kept, for
instance, in a egg-pack, which have been previously used and was contaminated with feces. The
presence of chicken manure and other moist organic components in the shell facilitates the
survival and growth of Salmonella by providing nutrients and physical protection. Bacteria can
easily penetrate a cracked eggshell. However, the intact egg possesses three physical barriers that
are intended to avoid bacterial penetration: the cuticle over the pore openings, the crystalline
eggshell and the shell membranes. In addition to be a physical barrier the eggshell also acts as
chemical barrier having lisozyme, ovotransferin, avidin, and ovocalixin-36; the latter causes a
bactericidal-permeability increasing effect and belongs to a group of specific LPS binding
proteins. Because of such complete protection, the intact eggshell is only susceptible to be
penetrated immediately after the egg is laid. During the first few minutes after oviposition, the
cuticle is immature and some of the pores may be opened. At this very moment there is thermical
difference between the temperature of the recently laid egg and the environment; this difference
generates a negative pressure that sucks the Salmonella towards the inside of the egg.
Contamination of eggs before oviposition
The other possible routes are by direct contamination of the yolk, albumen, egg shell
membranes or the egg shell itself before oviposition. When infections are caused by invasive
Salmonella strains, infected macrophages migrate to the internal organs of poultry, particularly to
reproductive tissues: ovary, infundibulum, magnum, isthmus, and shell gland. Up to now all
available research suggests that contamination of the egg contents by S. Enteritidis is mainly a
consequence of the infection of the reproductive tissues. The bacterium is almost always present
in pure culture in the egg contents and different studies have demonstrated that there is no
association between the presence of S. Enteritidis on eggshells and egg contents. Furthermore, S.
Enteritidis can be isolated from the reproductive tissues of naturally infected hens even in the
absence of intestinal carriage. S. Enteritidis is mainly localized in the albumin-secreting region
where they persist for life without harming the hen. Less frequently S. Enteritidis may also infect
ovaries via bloodstream. S. Enteritidis generally does not produce the typical ovary lesions found
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in Fowl Typhoid but exceptionally few hens of a flock can have malformed ovules. The small
white immature follicles are more susceptible to S. Enteritidis invasion than the more mature
small and large yellow ones; the penetration of immature follicles might lead to contamination of
eggs after maturation meaning that a very few hens from a flock may have a continuous
transovarian infection of eggs lasting the complete reproductive cycle. It was found that S.
Enteritidis is able to colonize ovaries and preovulatory follicles at significant higher level than
other five non-related serovars after intravenous infections. S. Typhimurium may also be able to
colonize the ovaries but to a lesser extent than S. Enteritidis. In fact, S. Enteritidis colonizes more
ovaries and internal organs and these tissues habour more S. Enteritidis than S. Typhimurium per
gram.
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