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Veterinary Pathology Online
http://vet.sagepub.com/content/early/2012/06/25/0300985812452579
The online version of this article can be found at:
DOI: 10.1177/0300985812452579
published online 25 June 2012Vet Pathol
J. Valdivia, F. Real, F. Acosta, B. Acosta, S. Déniz, J. Ramos-vivas, F. Elaamri and D. Padilla
With Ovine Cells in VitroCorynebacterium PseudotuberculosisInteraction of
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Interaction of Corynebacterium
Pseudotuberculosis With Ovine Cells in Vitro
J. Valdivia
1
, F. Real
1
, F. Acosta
1
, B. Acosta
1
,S.De
´niz
1
,
J. Ramos-vivas
2
, F. Elaamri
1
, and D. Padilla
1
Abstract
Caseous lymphadenitis is an infectious and contagious disease caused by Corynebacterium pseudotuberculosis, with a worldwide
distribution and high prevalence in small ruminant populations. This disease causes significant economic losses in small ruminants
through reduced meat, wool, and milk production. C. pseudotuberculosis can also affect horses, domestic and wild large ruminants,
swine, and man. It is considered an occupational zoonosis for humans. As part of in vitro investigations of the pathogenesis of
C. pseudotuberculosis, this study analyzed its capacity to adhere to and invade the FLK-BLV-044 cell line, derived from ovine
embryonic kidney cells. C. pseudotuberculosis showed a measurable capacity to adhere to and invade this cell line with no
significant differences between the four strains assessed. The incubation of the cell line at 4C, pre-incubation with sugars, com-
plete and heat inactivated antiserum, and heat-killed and ultraviolet-killed bacteria produced a significant (P< 0.05) decrease in
the invasion efficiency or inability to invade the cell line. Plate counting and fluorescence studies showed intracellular bacteria
for up to 6 days. Non-phagocytic cells may therefore act as a suitable environment for C. pseudotuberculosis survival and play a
role in the spread of infection and/or maintain a carrier state.
Keywords
cell line, Corynebacterium pseudotuberculosis, diffusion, invasion, persistence
Caseous lymphadenitis, also called pseudotuberculosis, is a pre-
valent disease with a worldwide distribution, causing pathologi-
cal lesions in sheep and goats.
6
This disease does not produce
high mortalities but causes important economic losses by reduc-
ing production of meat, wool, and milk.
1,7
This pathogen has
also been isolated from abscessed tissues in other species and
is associated with conditions such as ulcerative lymphangitis and
pigeon fever in horses, cattle, camels, buffaloes, and humans.
7
This disease shows three clinical forms—cutaneous, visceral,
and a mixed form—but the cutaneous and visceral forms are the
most frequent. The cutaneous form is characterized by abscess
formation in superficial lymph nodes and subcutaneous tissues,
while the visceral form is characterized by abscesses that can
develop internally in spleen, kidneys, lungs, and liver.
7,15
Little data exist on the pathogenesis of C. pseudotuberculosis
in animal tissues, and the potential for this pathogen to adhere to
and invade ovine cell lines has not been previously studied. The
precise nature of the mechanism responsible for adherence and
the way interacts with animal cell receptors to facilitate bacterial
entry into host cells may be of importance in understanding the
pathogenicity of C. pseudotuberculosis in small ruminants, and
recently an invasion-associated protein has been described in
corynebacterias.
19,27
The invasion and persistence in non-
phagocytic cells could be used by C. pseudotuberculosis as a
strategy to avoid hostile environments in the tissues and to
escape phagocytosis. This would aid progression of infection,
with a level of protection against host defenses, providing a
suitable environment for its proliferation.
3,9
We hypothesised that C. pseudotuberculosis may exploit
non-phagocytic cells to avoid the host immune defenses and
establish a favorable ecological environment for subsequent
dispersion of infection to other tissues. Investigations of intra-
cellular behavior using an in vitro model may be useful for
investigation of interactions between caprine and ovine patho-
gens and their host and assist development of new therapeutics.
Materials and Methods
Bacterial Strains and Culture Conditions
Four C. pseudotuberculosis strains were used: IUSA-1, IUSA-2
and TARA-1 (clinical isolates from goats and sheep in the
1
Institute of Animal Health IUSA, University of Las Palmas de Gran Canaria,
Arucas, Las Palmas, Spain
2
Department of Microbiology and Immunology. Hospital Universitario Mar-
que
´s de Valdecilla. Instituto de Formacio
´n e Investigacio
´n Marque
´s de Valdecilla
(IFIMAV), Santander, Spain
Corresponding Author:
Daniel Padilla, Instituto Universitario de Sanidad Animal. Facultad de Veter-
inaria. Trasmontan
˜a s/n. Arucas. 35416. Spain
Email: dpadilla@dpat.ulpgc.es
Veterinary Pathology
00(0) 1-6
ªThe Author(s) 2012
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DOI: 10.1177/0300985812452579
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Canary Islands), and CECT-808-T (a reference strain from the
Spanish Type Culture Collection). Strains were cultured on
brain heart infusion broth (BHIB) (Pronadisa) or on blood agar
base (Pronadisa) supplemented with 5%(v/v) sheep’s blood
and incubated at 37C for 48 to 72 hours. In both media,
0.5%(v/v) Tween 80 was added to reduce cell aggregation. All
strains were stored at –80C in BHIB with 15%glycerol.
The strains were confirmed as C. pseudotuberculosis by
multiplex polymerase chain reaction (PCR) based on primers
targeting the 16SrRNA and pld genes.
4,14,20
The PCR was per-
formed at a final volume of 25 mL, containing 1.5 U Taq DNA
polymerase (Invitrogen), 1X PCR buffer, 15 mM MgCl
2
,2mM
dNTPs, 1 mM of each of the primers (16S-F/16S-R and PLD-F/
PLDR2), and 10 ng DNA extracted from cultured C.
pseudotuberculosis.
Ovine Cell Line
The fibroblast-like cell line FLK-BLV-044 (DSMZ catalog
code: ACC 153) derived from ovine embryonic kidney cells
was cultured using Dulbecco’s Modified Eagle’s Medium
(DMEM) (Sigma) supplemented with 10%fetal bovine serum.
The cells were incubated at 37Cin5%CO
2
, and before reach-
ing total confluence were subcultured after detaching them
with trypsin-EDTA (Sigma) solution. The cells were plated
at a density of 10
5
cells per well in 24-well tissue culture plates
(Corning).
Invasion and Adherence Assays
The adherence and invasion assays were based on a gentamicin
protection assay described previously.
12,23
Strains were incu-
bated for 48 hours in BHIB at 37C, and viable bacteria were
determined by serial dilutions in sterile phosphate buffered sal-
ine (PBS) followed by plate counting on sheep blood agar
(SBA). To determine the effect of bacterial concentration on
invasion efficiency (percentage of initial inoculum interna-
lized), the bacteria were added to each tissue-culture well to
give a multiplicity of infection (MOI) of 5, 25, 50, 100, and
150. The mixture was centrifuged for 5 minutes at 33gto pro-
mote adherence of the bacteria to the host cells. Four hours post
infection, the ovine cells were incubated for 2 hours with
DMEM containing gentamicin (200 mg/mL), an antibiotic that
cannot penetrate intact eukaryotic cells, so it will kill suscepti-
ble extracellular but not intracellular bacteria. Each well with
the ovine cells was washed three times with PBS and lysed with
100 mLof0.2%Triton X-100 (Sigma). An additional 400 mLof
PBS was added per well, mixed and serially diluted in PBS, and
internal bacteria determined by plate counting on SBA. The
noninvasive E. coli DH5-awas used as a negative control.
In order to analyze C. pseudotuberculosis invasion in the
FLK-BLV-044 cell line, the number of bacteria recovered from
the cells over time was assessed at a MOI of 100. Extracellular
bacteria were removed after 30, 60, 90, 120, 150, 180, 210, and
240 minutes post infection by three washes with PBS, and the
ovine cells incubated for 2 hours with DMEM containing
gentamicin (200 mg/mL) and then processed as described for
the invasion assay.
To assess intracellular persistence of C. pseudotuberculosis
in the FLK-BLV-044 cell line at MOI 100, at 4 hours post
infection the bacteria were killed by gentamicin (2 hours, 200
mg/mL), and the culture medium was replaced by a medium
containing 20 mg/mL of gentamicin, and incubated for a further
24, 48, 72, 96, 120, and 144 hours to give sufficient time for
potential intracellular survival or replication.
In order to study the influence of antibodies on bacterial
invasion, strains were pre-incubated at 1:1 (v/v) for 30 minutes
at 37C with a complete and heat inactivated polyclonal anti-
serum against C. pseudotuberculosis (IUSA-1) produced by
using previously described protocol.
21
After pre incubation, the
mixture at MOI 100 was used to infect the ovine cells as
described previously for the invasion assay.
The effect of the cell metabolism on bacterial invasion was
examined by performing C. pseudotuberculosis infections at
4C.
13
In order to investigate bacterial ligand blocking, the
strains were grown for 48 hours in BHIB with mannose and
glucose (1%). After incubation, bacterial inoculum was used
to infect the cells as described previously at MOI 100, which
were then processed in the standard assay. In all experiments,
the invasion efficiency was calculated as the average of the
total number of colony forming units recovered compared to
the initial inoculum.
In the adherence assay, bacteria were used to infect the
ovine cells at a MOI of 100, and at 4 hours post infection, exter-
nal nonadhered bacteria were removed by washing the wells
three times with PBS and ovine cells lysed by adding Triton
X-100. Then serial dilutions of the disrupted mixture were plated
on SBA and incubated for 48 hours at 37C to obtain the total
bacteria (adherent and invasive bacteria). Bacterial adhesion
efficiency was calculated as the difference between the total and
invasive bacteria determined by plate counting.
Fluorescence Microscopy of C. pseudotuberculosis
Interactions With the FLK-BLV-044 Cell Line
For immunofluorescence studies, FLK-BLV-044 ovine cells
were seeded on 12 mm diameter coverslips in 24 well plates
at a density of 10
5
cells per well and then infected with C. pseu-
dotuberculosis (IUSA-1) at MOI 100 for 4 hours, after which
the ovine cells were washed three times with PBS and fixed
in cold paraformaldehyde (4%in PBS) for 20 minutes at room
temperature. Bacteria were detected using polyclonal rabbit
antibodies against C. pseudotuberculosis strain IUSA-1 and
developed with two commercial conjugates containing fluor-
escent dyes. The antibody reagents were all diluted 1:500 in
PBS containing 1%bovine serum albumin (Sigma) (PBS-
BSA) and applied for 20 minutes at room temperature. In the
first stage, after the coverslips were carefully washed twice in
PBS, extracellular bacteria were identified by incubating with
anti–C. pseudotuberculosis antibody. This was followed by
careful washing of the coverslips in PBS and development
of bound antibodies by incubation with anti-rabbit IgG
2Veterinary Pathology 00(0)
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antibody conjugated with Alexa Fluor 594 dye (Invitrogen).
Following this initial fluorescent dye staining, the ovine
cells were permeabilized with detergent, to enable staining
of both intracellular and extracellular bacteria with the
second fluorescent dye. The ovine cells were thus subjected
to PBS containing 0.1%Triton X-100 for 4 minutes at room
temperature and carefully washed four times with PBS.
The coverslips were then incubated with polyclonal anti–C.
pseudotuberculosis antiserum as described previously, and
bound antibodies developed with anti-rabbit IgG antibody
conjugated with Alexa Fluor 488 dye (Invitrogen). After
washing, the coverslips were mounted on glass slides with
Prolong Gold antifade reagent with DAPI (Invitrogen). All
preparations were examined by epifluorescence microscopy
with a 40 objective. Digital images were acquired using a
Zeiss AxioCam HRc camera and merged using Photoshop
CS3 (Adobe) software.
In order to investigate the effect of bacterial inactivation
by high temperature or ultraviolet light on the ability of
C. pseudotuberculosis to invade ovine tissue culture cells, a
400 mL volume of a BHIB culture of strain IUSA-1 was heated
at 80C for 20 minutes in a thermoblock or exposed to UV light
for 18 hours. After both treatments, subculture to SBA con-
firmed the nonviability of the bacteria in each assay. Then
heat-killed and UV light-killed C. pseudotuberculosis at MOI
100 were used to infect the ovine cells and processed for immu-
nofluorescence as described before.
Statistical Analysis
Statistical analyses were carried out using SPSS version 17.0.
Data were analyzed by one-way ANOVA and Student’s t-test,
considering P< 0.05 as significant. The experiments were
performed in triplicate, and numerical data and bars are shown
as mean values with standard deviations.
Results
The number of intracellular bacteria detected increased with
increasing initial inoculum to MOI 100, with significantly
(P< 0.05) higher invasion efficiency above an MOI of 25
(Fig. 1). The degree of invasion and adherence in the FLK-
BLV-044 cell line are shown on Fig. 2. Maximal and minimal
adherence efficiencies were observed for strains IUSA-1
(2.01%) and TARA-1 (1.5%) (Fig. 2a), but no statistically sig-
nificant differences were found between strains. Maximum
invasion efficiency was observed for strain IUSA-1 (0.17%),
whereas strain IUSA-2 showed the minimal invasion efficiency
of 0.10%(Fig. 2b), but no statistically significant differences
were observed between strains. The noninvasive control E. coli
Figure 1. Effect of bacterial concentration using different multiplicity
of infection (MOI) on invasion of C. pseudotuberculosis (IUSA-1) for
cell line FLK-BLV-044. Results from three independent experiments
are given as mean +SD (standard deviation) intracellular bacteria
expressed as a percentage of the original inoculum. In bars, different
letters (a, b, c) indicate statistically significant differences (P< 0.05)
between treatments.
Figure 2. Adherence (a) and invasion (b) of C. pseudotuberculosis
IUSA-1, IUSA-2, TARA-1, and CECT-808 T for cell line FLK-BLV-
044 at a multiplicity of infection of 100 in a gentamycin protection
assay for 4 hours. E. coli DH5-awas used as negative invasion control.
All assays were performed on three separate occasions in triplicate
wells and results are given as mean +SD expressed as a percentage
of the original inoculum.
Valdivia et al 3
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DH5-awas tested over a similar range of MOI and the invasion
efficiency was 0.005%. In addition, we were unable to obtain
intracellular E. coli from experiments carried out at 4C (data
not shown). The invasion process was time dependent, increas-
ing over 240 minutes (Fig. 3) with intracellular bacteria
detected after only 30 minutes. In intracellular survival experi-
ments, all strains were able to grow up to 24 hours post infec-
tion and then survive up to 6 days (Fig. 4). After this period, the
tissue culture cells began to show signs of degradation, and the
cellular monolayer detached.
Incubation of the cell line at 4C, pre-incubation in the pres-
ence of glucose-mannose (1%), and pre-incubation with a
complete and heat inactivated antiserum produced a significant
(P< 0.05) decrease in the invasion efficiency by C. pseudotu-
berculosis compared to the control assay (Fig. 5).
Immunofluorescence microscopy confirmed the intra-
cellular location of C. pseudotuberculosis (Fig. 6). The immu-
nofluorescence also demonstrated that heat-killed and
ultraviolet-killed bacteria were unable to invade the cell line
(data not shown).
Discussion
Studies of adherence and cell invasion are currently assisting
the understanding of different aspects of the pathogenesis of
infectious diseases. This study is the first to investigate the
kinetics of adherence, invasion, and intracellular survival of
C. pseudotuberculosis in non-phagocytic cells. The capacity
of some bacteria to invade non-phagocytic cells is considered
an important virulence factor of several animal and human
bacterial pathogens, including Yersinia,
11
Salmonella,
5
and
Shigella.
22,25
Our results from fluorescence microscopy and gentamicin
protection assay demonstrated C. pseudotuberculosis adherence
to and invasion of FLK-BLV-044 cells, with similar efficiencies
among the strains tested and a possible correlation between the
efficiency of adherence and invasion. The maximum invasion
efficiency observed in the present study is lower than showed
by Brucella
24
and Salmonella
18
but similar or higher than those
reported in Campylobacter,
2,10
Burkholderia,
17,26
Hafnia
alvei,
21
and Prevotella.
8
In the time-course analysis, at
Figure 3. Invasion of C. pseudotuberculosis strains in the cell line FLK-
BLV-044 at a multiplicity of infection of 100. Results of gentamycin
protection assays in three independent experiments per strain using
different periods of infection are given as mean +SD intracellular bac-
teria expressed as a percentage of the original inoculum.
Figure 4. Intracellular survival of C. pseudotuberculosis strains in the
cell line FLK-BLV-044 over time at a multiplicity of infection of 100.
Cells were infected for 4 hours, treated with gentamicin and incubated
for up to 6 days. Each invasion assay was performed on three separate
occasions, and results expressed as mean +SD intracellular bacteria
expressed as a percentage of the original inoculum.
Figure 5. Effect of pre-incubation with sugars, reduced cell metabo-
lism, and anti–C. pseudotuberculosis antisera on the invasion of FLK-
BLV-044 cells by C. pseudotuberculosis strain IUSA-1 at a multiplicity
of infection of 100. Bacteria in brain heart infusion broth were incu-
bated in the presence of 1% mannose and glucose, heat inactivated
or whole antiserum for 30 minutes at 37C before infection, or added
to the monolayers previously incubated at 4C. Results of the mean +
SD intracellular bacteria expressed as a percentage of the original
inoculum from three independent experiments per treatment are
shown. Asterisks denote significant differences (P< 0.05) between
treated and nontreated control organisms.
4Veterinary Pathology 00(0)
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30 minutes post infection a few internal bacteria were
detected in FLK-BLV-044 cells, but the internalization was
significantly (P< 0.05) higher after 240 minutes. The inva-
sion process was also dependent on bacterial quantity and
also suggested saturation of cellular receptors above MOI
100.
16
In addition, after invasion, C. pseudotuberculosis
remained viable for up to 144 hours, raising the possibility
that C. pseudotuberculosis may persist in non-phagocytic
cells in different tissues of sheep and goats, facilitating pro-
gression of the infection in the animals.
The invasion process was dependent on temperature, sug-
gesting that cell metabolism plays an active role in the invasion
process. Finally, the incubation of bacteria prior to invasion
with sugars, inactivated and whole antiserum, appeared to
block specific bacterial receptors and/or cellular ligands.
Immunofluorescence demonstrated that heat- or UV-
killed bacteria were unable to invade the FLK-BLV-044
cell line, suggesting that invasion requires either viable
C. pseudotuberculosis or intact bacterial surface components
to interact with the ovine cell surface. Further studies of the
invasion and persistence of C. pseudotuberculosis should aid
the development of therapeutics and vaccines and may assist
in new aspects relevant to responses against C. pseudotuber-
culosis or other facultative intracellular pathogens.
In conclusion, these in vitro findings raise the possibility
that C. pseudotuberculosis can persist and disseminate in vivo
through non-phagocytic cells, facilitating diffusion of the
infection and providing a certain level of protection against ani-
mal defenses and a suitable environment for its proliferation,
facilitating the establishment of a carrier state.
Acknowledgements
We would like to thank Spanish Agency for International Development
(AECID) for their program of PhD scholarships for foreign students for
J. Valdivia.
Figure 6. Adherence and invasion of C. pseudotuberculosis IUSA-1 at a multiplicity of infection of 100 for 4 hours in the cell line FLK-BLV-044
under fluorescence microscopy. (a) Extracellular bacteria were detected with anti–C. pseudotuberculosis sera and Alexa Fluor 594 conjugate on
non-permeabilised cells (arrows). (b) Total bacteria were detected on permeabilized cells after bound anti–C. pseudotuberculosis sera was devel-
oped with Alexa Fluor 488 conjugate (arrows). (c) The DNA of tissue culture cell nuclei was stained in blue with DAPI. (d) In the merged images,
extracellular bacteria are shown in orange or yellow (mixture of red and green), intracellular bacteria in green, and DNA of tissue culture in blue.
Arrows indicate some intracellular bacteria.
Valdivia et al 5
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Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship,
and/or publication of this article.
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