1. Conociendo las colonias del sur de Torreón
1.2. Impulso Comunal
A standard approach for the investigation into cell permeability, transcytosis
mechanisms, adhesion and invasion of bacteria into eukaryotic cells is using an in vitro
assay (Friis et al., 2005). Investigations into the nature of host-bacterial interactions are
also carried out using in vivo animal models to demonstrate host-restriction in particular
host cells (Graves et al., 2012) however in vitro model investigations have contributed
much more knowledge of this interaction (Friis et al., 2005).
Infections by zoonotic Salmonella in human and animal hosts are associated with
invasion into the Peyer’s patches and necrosis of M-cell epithelia in the lower intestinal tract. In vivo the epithelial layers are highly polarised with the basolateral membrane
interfacing with cells in the lamina propria; apical membranes are organised facing the
luminal contents. Highly polarised cells are difficult to establish in vitro however they
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invasion. In vitro cultures however are non-polarised; fortunately the function of the
membrane is not dependant on the polarity of the cell and transport and cellular
localisation of surface components such as Toll like receptors (Backhed & Hornet,
2003; Gewirtz et al., 2001; Monstov et al., 2000). In vitro models also reduce the use of
animal models.
The Gentamicin Protection Assay (GPA) (invasion assay) is an established in vitro
method used for the investigation of bacterial invasion into eukaryotic cells (Byrne et
al., 2007; Cossart & Sansonetti 2004; Flentie et al., 2008). Typically Salmonella are
sensitive to the antibiotic gentamicin and it also has a limited penetration of eukaryotic
cells. Therefore when investigating bacterial invasion, gentamicin is used following
incubation of bacteria with the eukaryotic cells to kill non-invading, attached and extra
cellular, bacteria in the suspension whilst avoiding inhibition of invaded bacteria which
are protected inside the eukaryotic cells. Enumeration of invaded bacteria is carried out
following lysis of eukaryotic cells using detergent or water and the subsequent use of
standard bacterial plating techniques (Friis et al., 2005). Invasion assays have been
exploited to investigate intracellular survival and replication (Friis et al., 2005).
Modifications of the method are employed depending on aspects of the hypothesis
under investigation. In some investigations the adhesion properties of the bacterium are
omitted by a centrifugation step which physical pulls and associates the bacterium to
the cells in the monolayer (Friis et al., 2005).
Typically, the eukaryotic cell of choice for invasion assays is the human colon
adenocarcinoma (CaCo-2) cell line as they mimic in-vivo conditions best (MacCallum
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the same cylindrical polarized morphology and properties as enterocytes, expressing
microvilli, transporters, enzymes and tight junctions. They were developed by the
Sloan-Kettering Institute for Cancer Research through research conducted by Dr.
Jorgen Fogh and were isolated from colon carcinoma enterocytes (Fog & Trempe,
1975). Different cell lines originating from different organs and animals can be
established and manipulated to mimic their functions in vivo. For example, Dog
Intestinal Epithelial Cells (DIEC) originating from the small intestine of adult beagles
created by Andrea Quaroni, Cornell University, New York are a good cell line for
investigation of canine gut cells (Personal communication with WALTHAM® Centre
for Pet Nutrition). Primary cultures of DIEC are established and subsequently
immortalised using a temperatures sensitive mutant of the simian virus 40 large tumour
antigen for the use in these assays (SV40 T-Ag; these cells were provided by the
WALTHAM® Centre for Pet Nutrition).
Confluent monolayers of cells are established onto an adherent surfaces and a known
concentration of bacteria is inoculated onto the monolayer. The ratio of bacteria to
eukaryotic cells is an important variable, referred to as the Multiplicity Of Infection
(MOI), and studies have shown differences in invasion with different numbers of
inoculated bacteria up to a maximum (Kusters et al., 1993). Studies in Campylobacter
models have shown that invasion efficiency is higher at lower MOI’s and this efficiency decreases with increasing MOI (Friis et al., 2005). However, two or more
MOI’s are established for invasion assays as there is variability in invasion capabilities depending on strain and cell monolayer type. Invasion of epithelial cells by
Salmonellae has been studied extensively (Altmeyer et al., 1993; Elsinghorst et al.,
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Although these studies have increased insights into the invasion mechanisms used by
Salmonellae, some contradictory observations have been reported and essential
questions remain unanswered (Kusters et al., 1993).
Adhesion of bacteria to the eukaryotic cell surface can also be investigated by similar
methods. Bacteria cells are washed with buffered reagents following an incubation
period and non- associated bacteria are removed. However the gentamicin or selected
antibiotic step is omitted and associated bacteria that have attached and invaded are
counted following eukaryotic cell lysis (Friis et al., 2005; Kusters et al., 1993). There
are many variables that can be targeted to investigate different aspects of bacteria host
interactions including incubation time (Finlay et al., 1989), bacteria load concentration
(Francis & Thomas, 1996), eukaryotic cell type and supplementing antibiotics use
(Kusters et al., 1993; Mroczenski-Wildey et al., 1989). These assays can be used to
investigate different capacities in invasion and adhesion of bacterial strains in different
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Aims and Objectives
Historical data has shown that 0-43% (McElrath, 1952), 1-35% (Finley et al., 2007),
1.2% (Gorham & Garner, 1951), 4.4% (Galton et al., 1952) and 15% (Shimi et al.,
1976) of non UK canines asymptomatically carry or shed non-typhoid Salmonella
species. More recently epidemiological investigation of Salmonella outbreaks has
revealed zoonotic transfer of Salmonella isolates from dogs to their owners (CDC,
2008). This highlighted the concern regarding the potential increase of zoonotic
infections from companion animals to their owners especially with approximately 50%
of western households owning a companion animal with cats and dogs being among the
most popular (PFMA, 2011). However, there are limited data on the asymptomatic
carriage of Salmonella in canines in the UK with the available data being from non-UK
sources that are some what dated. During this time the relationship between humans
and animals has changed to a much more intimate role and HAI and AAI’s are
becoming much more popular due to their positive benefits on individuals. Therefore,
the first objective of this study was to estimate the current incidence of asymptomatic
carriage of Salmonella in UK companion dogs.
Recent studies have identified Salmonella strains with a limited host specificity
suggesting host restricted characteristics (Pasmans et al., 2008). It is currently not
known if isolates from canines demonstrate any host specificity or are entirely zoonotic
to humans. For these reasons, Salmonella strains that cause clinical infections in
humans and canines were investigated for zoonotic potential and host restrictive
characteristics. In human hosts typhoid fever or enteric fever is caused by a host
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serotype for a canine host, if there are or is. This study therefore sought to determine if
any host adaptive strains of Salmonella exist by profiling isolates from canine and
human hosts using genotypic and phenotypic methods which were then analysed and
compared to reveal any significant associations.
The ultimate aim of this study was to investigate asymptomatic carriage of Salmonella
in dogs and to investigate their potential for zoonotic infection of humans.
More specifically the aims were to:
Devise a sampling protocol for the microbiological analysis of Salmonella in canine faeces including culture media, sensitivity of detection, storage
parameters and timescales for analysis.
Determine the prevalence of asymptomatic carriage of Salmonella species in a selection of UK canines by faecal culture using a validated isolation protocol.
Genotypically characterise Salmonella isolated from canines presenting with symptomatic disease using the HPA harmonised protocol (PulseNet), Pulsed-
Field Gel Electrophoresis (PFGE).
Compare canine PFGE patterns with human clinical PFGE profiles by the generation of dendrograms.
Characterise a panel of clinical canine and clinical human Salmonella isolates using phenotypic microarrays, API 20E, antibiograms and motility
confirmation.
Analyse complex phenotypic microarray data with Principal Component Analysis (PCA), to determine if differences exist between clinical canine and
human isolates in their biochemical profiles.
Determine any potential host adaptive, restrictive or competitive advantage of
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assays using CaCo-2and DIEC, human and dog intestinal epithelial cell lines,
methodology
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