Bull Vet Inst Pulawy 57, 319-322, 2013

DOI: 10.2478/bvip-2013-0055

In vitro cultivation and immunostaining

of Lawsonia intracellularis strains

Anna Szczotka-Bochniarz, Katarzyna Podgórska,

Agnieszka Nowak, Zygmunt Pejsak
Department of Swine Diseases,
National Veterinary Research Institute, 24-100 Pulawy, Poland
anna.szczotka@piwet.pulawy.pl

Received: July 5, 2013 Accepted: September 11, 2013

Abstract

The aim of the study was to implement in vitro cultivation of L. intracellularis strains using ATCC 55783 and vaccine
strains, and McCoy cells (ATCC CRL-1696). The infection was monitored by daily observations under phase contrast
microscope. Indirect immunostaining using monoclonal antibody was also performed. Large number of S-shaped, moving
bacteria were found in the cell medium in cultures infected with ATCC 55783 and vaccine strain. Immunostaining revealed a
high number of multiple cell-associated or intracellular red stained bacteria in the infected cultures. This study describes for the
first time in vitro cultivation of L. intracellularis in Poland, which creates further perspective for more advanced research on this
bacterium.

Key words: pigs, Lawsonia intracellularis, in vitro cultivation, McCoy cells, immunostaining.

Introduction cellularis field strains and their further characterisation,

Lawsonia intracellularis (L. intracelularis) is
known as an aetiological agent of porcine proliferative
enteropathy (PPE), an important disease of swine,
affecting growers and finishers (12). PPE is
characterised by thickening of the intestinal mucosa at
the distal part of the small intestine, due to proliferation
of enterocytes related with intracellular bacterial
infection. In severe cases, the proliferative lesions may
extend to the proximal part of the small intestine or
large intestine (12).
Although the disease and bacterium responsible
for its development have been known for many years –
it was first described in pigs in 1931 (1), there are still
certain limitations related to the diagnosis of PPE. The
bacterium does not grow on standard media and its in
vitro cultivation is possible only in cell cultures (6).
For this reason, diagnostic laboratories commonly use
non-cultivation methods, with necropsy followed
by immunohistochemical detection of the bacterium as
the gold standard (9, 14).
Currently, only a few laboratories in the world are
able to propagate L. intracellularis since the method
is sophisticated and requires specific conditions.
However, for some reasons, like isolation of L. intra-
in vitro cultivation of this bacterium is the method of
choice and cannot be replaced by other technique for
the studies of the disease (7).
Up to date, only a dozen isolates have been
obtained worldwide (4, 17). Just a few of them were
described as being able to develop clinical signs and
typical lesions after experimental inoculation (4).
Moreover, the availability of these strains still remains
very limited (2). For this reason, implementation of in
vitro cultivation method would increase possibilities to
obtain strains from PPE field cases. Before in vivo
trials, such isolates should be first characterised in vitro
to estimate possible differences in their virulence.
Therefore, the aim of the study was to develop and
to implement L. intracellularis in vitro cultivation
protocol in the laboratory of the Department of Swine
Diseases, as well as to assess strains growth rate and
ability to infect cell culture.
Material and Methods
Cell culture. McCoy cells (ATCC CRL-1696)
were grown in Dulbecco’s modified Eagle medium
(DMEM, Gibco) supplemented with 7% foetal calf

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320 A. Szczotka-Bochniarz et al. / Bull Vet Inst Pulawy / 57 (2013) 319-322
serum (FCS, SIGMA), 1% L-glutamine (Gibco),
and 1% amphotericin B (SIGMA), in the atmosphere of
8% O2, 8.8% CO2, and 83.2% N2, at 37°C. The cells
were trypsinised at weekly intervals and seeded at a
concentration of 0.5 × 105 cells/mL in the cell culture
medium.
Infection of cell culture. McCoy cell cultures were
inoculated with avirulent L. intracellularis ATCC 55783
strain and modified-live avirulent L. intracellularis strain
from a commercially available live vaccine:
Enterisol®Ileitis (Boehringer Ingelheim), containing
1×104.9 – 1×106.1 TCID50 of the bacterium. The recons-
titution of the ATCC 55783 strain was performed
according to the ATCC instructions. The freeze dried
vaccine was dissolved in 10 mL of sterile water and
centrifuged (5000 × g, 10 min). The pellet was suspended
in 5 mL of sucrose potassium glutamate (SPG) (5) with
10% foetal calf serum (SIGMA). One millilitre of the
suspension was used to infect T-25 flask with McCoy
cells of 30% confluency, which corresponded to 24 h
culture.
Monitoring of infection. The infection was
monitored by daily observations of the inoculated cell
cultures, as well as the negative control cells under
phase contrast microscope (Optiphot-2, Nikon, Japan,
up to 600x). The observations included the assessment
of cell culture morphology, as well as search for
comma-shaped bacteria actively moving in the cell
medium. To estimate the level of infection, indirect
immunostaining with monoclonal L. intracellularis
antibody (Jeno) was performed before each passage
(14). Briefly, one loop of scraped cells was spread on a
microscope slide, dried, and fixed in methanol. The
cells were incubated for 30 min with monoclonal
antibody diluted in PBS (1:10), washed with PBS, and
incubated for 45 min with the secondary antibody
conjugated with HRP – EnVision System Labelled
Polymer – HRP Anti-Mouse (Dako). Afterwards, the
cells were stained with the enzyme substrate -
aminoethylcarbazole (AEC, Dako) for 20 min, washed
twice with PBS, mounted, and checked under the
microscope (Optiphot-2, Nikon, Japan, up to 600x) for
the presence of red-brownish stained bacteria (positive
result).
Passage of L. intracellularis. For the passage of
the bacterium, 100% confluent McCoy cells were
scraped and lysed by forcing the cell suspension
through a 3.5 inch 22 Gauge spinal syringe (Nunc). The
suspension was centrifuged at 1500 × g for 10 min to
remove cell debris. Then, the centrifugation was
repeated at 5000 × g, for 20 min to obtain the bacteria.
The pellet was suspended in 3 mL of medium and
examined under microscope (Optiphot-2, Nikon, Japan,
up to 600x) for the presence of live, moving bacteria
and possible McCoy cell leftovers. Afterwards, the
suspension was transferred into new flasks (either 25 or
75 cm2) with cell culture and incubated up to 7 d,
depending on the growth rate of the bacteria. During
each passage the number of microorganisms was
counted in the highest countable dilution under light
microscopy, in duplicate, as described earlier (4). Six
passages were done for each isolate.
Freezing L. intracellularis strains. The bacteria
were harvested as described above. The pellet was
suspended in SPG with 10% foetal calf serum and
mixed. A drop of the suspension was examined under
microscope (magnification up to 600x) to assess
morphology and viability of the bacteria. The
suspension was then aliquoted into small, screw-cap
tubes (Corning) and stored at -80°C until the next
usage.
Results
Daily observations revealed large number of
S-shaped, moving bacteria in the cell medium.
The infected cells had a tendency to clump together,
creating infected foci of several cells. However, the
cells’ morphology was not affected by the infection
compared to the negative control cells.
The cell suspension preparations, observed under
phase contrast microscopy, showed McCoy cells
floating freely in the medium, with the presence of
numerous, tiny, rod-shaped, S-shaped, or curved
bacteria of L. intracellularis morphology. In the
subsequent cultures, no visible inhibition of cell growth
was observed in flasks containing cells infected either
with the ATCC 55783 strain or with the vaccine strain.
The average number of bacteria in the flasks with cells
infected with ATCC 55783 strain was 1,5 × 108
bacteria/mL, while in flasks containing cells infected
with vaccine strain, the average concentration was
3,4 × 105 bacteria/mL. However, these differences may
be insignificant, since the initial number of L. intra-
cellularis ATCC 55783 remained unknown.
The immunostaining of the infected cell cultures
revealed intracellular multiple cell-associated, red
stained bacteria, (Figs 1A, 1B). It was especially
intense in the case of cells infected with the ATCC
55783 strain, which formed large foci (Fig. 1A). On the
contrary, the cells inoculated with the vaccine strain
presented less intense staining, suggesting a lower
number of bacteria (Fig. 1C). There was a clear
difference in the number of intracellular and cells
associated bacteria in McCoy cells, infected with
ATCC 55783 strain and the vaccine strains. Visibly
less microorganisms were seen in the case of the
vaccine strain. Non-infected, negative control cells
remained unstained and detached from each other.
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 A. Szczotka-Bochniarz et al. / Bull Vet Inst Pulawy / 57 (2013) 319-322
Fig. 1. L. intracellularis immunostaining. A -
Numerous McCoy cells (blue), heavily infected with
multiple L. intracellularis ATCC 55783 strain bacteria
(red-brownish), 200x. B - Single cell of stellate
morphology heavily infected with L. intra-cellularis
ATCC 55783 strain (red to brownish staining), 400x.
C - A few McCoy cells (blue) infected with L.
intracellularis vaccine strain (red-brownish staining),
100x
Discussion
Among various methods for L. intracellularis
identification, in vitro cultivation is still one of the
major challenges for diagnostic laboratories due to the
specific environmental requirements for the pathogen.
As far as we know, this is the first description of
in vitro cultivation of L. intracellularis in Poland. The
technique, described initially by Lawson et al. (13),
with former modifications by other authors (4, 18),
provides the possibility to isolate L. intracellularis
strains from field cases of proliferative enteropathy and
enables to propagate such strains for experimental
321
purposes (i.e. inoculation of pigs). The latter is crucial
for the studies on pathogenesis of L. intracellularis
infection, which to a large extent remains unexplored
(8). Besides, the method has a variety of applications.
Among them, isolation of L. intracellularis strains is
indispensable to monitor antibiotic susceptibility,
which remains one of the major concerns of the
successful therapy of animals (20). In vitro cultivation
technique also provides antigen for development of
quantitative laboratory diagnostic methods, like real-
time PCR. Moreover, this method is prerequisite for
discrimination of antigenic proteins of L. intracellularis
isolates (17).
For a long time, in vitro cultivation of L. intra-
cellularis has been performed in IEC-18, a rat small
intestine cell line (13). However, previous studies have
shown that many various intracellular bacteria could
be detected in the same samples using these cells,
therefore the passage of L. intracellularis was prone to
contaminations and therefore difficult to accomplish
(18). For this reason, different cells have been
implemented in this technique. Murine fibroblast-like
McCoy cells used in this study can be applied for
propagation of many microorganisms, which
development is dependent on eucaryotic host cell (3).
This modified technique improves and simplifies the
passage of L. intracellularis.
In the study, no cytopathic effect was observed
after infection. However, the development of
cytopathic effect in L. intracellularis infected cells
remians controversial. According to Lawson et al. (13),
heavily infected cells are rounded and detached from
the monolayers. Conversely, other authors did not
observe any morphological changes in the infected
monolayers, even using a high number of bacteria (2,
10, 11, 19).
This study provides information on in vitro growth
rate and ability to infect cells with L. intracellularis
ATCC 55783 strain and vaccine strain. For both of
them, a fast increase in the number of microorganisms
was observed at each passage. Immunostaining also
confirmed that both strains were able to infect McCoy
cells. However, more intense red-brownish staining,
corresponding to higher number of cell-attached or
intracellular bacteria, was found in the case of cells
infected with the ATCC 55783 strain, compared to
those inoculated with the vaccine strain. Similar
observations were described by Guedes et al. (5), who
compared the vaccine strain with field isolate.
Moreover, cells infected with the ATCC 55783 strain
were accumulated in bigger clumps, while those
inoculated with the vaccine strain were congregated in
groups of only a few cells. This observation could be
explained by the fact that in cell culture heavily
infected cells are present in such foci, indicating
transfer of infection between cells in its initial phase
(17).
As mentioned before, the number of isolated
L. intracellularis strains is very limited. Currently,
L. intracellularis ATCC 55783 strain used in this study
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322 A. Szczotka-Bochniarz et al. / Bull Vet Inst Pulawy / 57 (2013) 319-322
is the only commercially available isolate. However,
this strain is a patent deposit (Patent No. US 5885823),
therefore ATCC has no specific growth information,
and no product sheet is available. For this isolate, no
laboratory work was performed other than basic
viability testing, thus no strain specification is
accessible. The only information included in the patent
description (Patent No. US 5885823 A), states that the
strain is avirulent.
Enterisol Ileitis vaccine (Boehringer Ingelheim
Animal Health GmbH) comprises the B3903 strain
(ATCC PTA 4926) of L. intracellularis (15), which is
covered by the patent EP 1651260B1 until 2024
(personal communication - Dr. Bernd Grosse Liesner,
Boehringer Ingelheim Animal Health GmbH, 2013).
According to the previous studies, in vivo inoculation
using Enterisol Ileitis as a source of vaccine strain
results in slight clinical symptoms of the disease,
limited only to the sporadic cases of transient
diarrhoea, as well as later and shorter faecal shedding
(5). Since both strains used in this study are avirulent,
similar outcome could be expected after infection with
the ATCC 55783 strain. Both strains are also able to
infect cells, and to trigger the development of an
immune response (Patent No. US 5885823 A, 6),
without causing unnecessary suffering of animals.
These data indicates high usefulness of both strains
propagated in our laboratory for experimental trials,
and confirm the need for new isolates for research
purposes.
In conclusion, the described method and the
obtained pure cultures of both strains may be used
in the further projects, concerning an insight into the
pathogenesis and immunology of L. intracellularis
infections, based on experimental inoculations of
conventional pigs.
Acknowledgments: This study was supported by the
grant from the Polish Ministry of Science
and Higher Education N N308 075634. We would like
to thank Mrs. Lien Thi Minh Nguyen from the
Technical University of Denmark, National Veterinary
Institute, Section for Immunology and Vaccinology,
for her priceless support in this project.
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