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Journal of Clinical Microbiology, March 1999, p. 620-627, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Mycoplasma hyopneumoniae Potentiation of
Porcine Reproductive and Respiratory Syndrome Virus-Induced
Pneumonia
Eileen L.
Thacker,1,*
Patrick G.
Halbur,2
Richard F.
Ross,1
Roongroje
Thanawongnuwech,2,3 and
Brad J.
Thacker2
Veterinary Medical Research
Institute1 and
Veterinary Diagnostic and
Production Animal Medicine,2 College of
Veterinary Medicine, Iowa State University, Ames, Iowa 50011, and
Department of Veterinary Pathology, Faculty of Veterinary
Science, Chulalongkorn University, Bangkok, 10330, Thailand3
Received 12 August 1998/Returned for modification 19 October
1998/Accepted 11 December 1998
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ABSTRACT |
An experimental model that demonstrates a mycoplasma species acting
to potentiate a viral pneumonia was developed. Mycoplasma hyopneumoniae, which produces a chronic, lymphohistiocytic
bronchopneumonia in pigs, was found to potentiate the severity and the
duration of a virus-induced pneumonia in pigs. Pigs were inoculated
with M. hyopneumoniae 21 days prior to, simultaneously
with, or 10 days after inoculation with porcine reproductive and
respiratory syndrome virus (PRRSV), which induces an acute interstitial
pneumonia in pigs. PRRSV-induced clinical respiratory disease and
macroscopic and microscopic pneumonic lesions were more severe and
persistent in M. hyopneumoniae-infected pigs. At 28 or 38 days after PRRSV inoculation, M. hyopneumoniae-infected
pigs still exhibited lesions typical of PRRSV-induced pneumonia,
whereas the lungs of pigs which had received only PRRSV were
essentially normal. On the basis of macroscopic lung lesions, it
appears that PRRSV infection did not influence the severity of M. hyopneumoniae infection, although microscopic lesions typical of
M. hyopneumoniae were more severe in PRRSV-infected pigs.
These results indicate that M. hyopneumoniae infection
potentiates PRRSV-induced disease and lesions. Most importantly,
M. hyopneumoniae-infected pigs with minimal to
nondetectable mycoplasmal pneumonia lesions manifested significantly
increased PRRSV-induced pneumonia lesions compared to pigs infected
with PRRSV only. This discovery is important with respect to the
control of respiratory disease in pigs and has implications in
elucidating the potential contribution of mycoplasmas in the
pathogenesis of viral infections of other species, including humans.
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INTRODUCTION |
Traditionally it has been presumed
that viral infections potentiate and increase the susceptibility of a
host to bacterial infections. However, recent evidence indicates that
members of the class of Mollicutes, which includes the
mycoplasmas and ureaplasmas, may play a more significant primary role
in viral diseases than was previously realized. In 1989, it was
reported that a high proportion of AIDS patients were infected with
Mycoplasma fermentans and Mycoplasma penetrans
(4, 16). Since then, several other mycoplasmas, including
Mycoplasma genitalium and Mycoplasma pirum, have
been implicated in playing a synergistic role with human immunodeficiency virus (HIV) (2, 4). Evidence that supports the role that mycoplasmas play in accelerating the progression of HIV
to AIDS has included the enhanced cytopathic effects (CPE) on tracheal
epithelial cells (25) and human CD4+ lymphocytes
(2) infected with both HIV and M. fermentans in vitro. The possible mechanisms by which mycoplasmas could influence the
pathogenesis of HIV have not yet been elucidated, although mycoplasmas
have been shown to activate B and T cells polyclonally both in vitro
and in vivo (20, 24).
Recently, a new respiratory syndrome in swine, designated porcine
respiratory disease complex (PRDC), has emerged as a serious health
problem in most pig-raising regions of the world. Pneumonia in pigs
with PRDC is due to a combination of both viral and bacterial agents.
Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus
(5), are two of the most common pathogens isolated from pigs
exhibiting PRDC.
M. hyopneumoniae is recognized as the causative agent of
porcine enzootic pneumonia, a mild, chronic pneumonia commonly
complicated by opportunistic infections with other bacteria
(22). The primary clinical sign associated with M. hyopneumoniae infection is a sporadic, dry, nonproductive cough.
Other clinical signs, such as fever or impaired growth, are linked to
secondary invaders, especially Pasteurella multocida.
Typical mycoplasmal pneumonia lesions consist of well-demarcated
dark-red-to-purple (acute) or tan-grey (chronic) areas of cranioventral
consolidation. Microscopic examination reveals bronchopneumonia with
suppurative and histiocytic alveolitis with peribronchiolar and
perivascular lymphohistiocytic cuffing and nodule formation, typical of
hyperplasia of bronchoalveolar lymphoid tissue.
In contrast, PRRSV induces a severe, acute pneumonia with clinical
disease characterized by labored and accentuated abdominal respiration
and tachypnea. Coughing is rarely observed. In addition, pigs infected
with PRRSV exhibit elevated rectal temperatures, with pronounced
lethargy and anorexia. Gross lesions associated with PRRSV infection
consist of severe, multifocal to diffuse, tan-mottled consolidation of
the lung. Microscopic lesions include septal infiltration with
mononuclear cells, type II pneumocyte hypertrophy and hyperplasia, and
an alveolar exudate consisting of mixed inflammatory cells and necrotic macrophages.
The mechanisms by which PRRSV and M. hyopneumoniae cause
disease are quite different. PRRSV infects cells of the
macrophage/monocyte/dendritic lineage. Pulmonary alveolar macrophages
(PAMs) and pulmonary intravascular macrophages (PIMs) are the primary
sites of replication in the lung (12, 28). Infection of PAMs
and PIMs by PRRSV induces cell lysis, presumably resulting in a
decreased ability of the respiratory tract to defend against both
respiratory and systemic pathogens (9). In contrast,
M. hyopneumoniae attaches to the cilia of tracheal
epithelial cells, resulting in damage to epithelial cells and the
mucociliary apparatus (6). The mechanisms explaining the
lymphocytic infiltration observed with M. hyopneumoniae are unknown. The resulting lesions lead to consolidation of lung parenchyma and local invasion of opportunistic bacteria or viruses that can ultimately lead to systemic disease.
Consistent with other virus-bacterium interaction models, the most
commonly proposed hypothesis is that PRRSV is the primary pathogen and
bacteria are secondarily involved in the pathogenesis of PRDC. In the
study reported here, we found that infection with M. hyopneumoniae potentiated and prolonged PRRSV-induced pneumonia clinically, macroscopically and microscopically. This report describes an in vivo model that conclusively demonstrates a potentiating effect
on a viral infection by a mycoplasma species.
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MATERIALS AND METHODS |
Pigs.
One hundred forty PRRSV- and mycoplasma-free crossbred
(Landrace, large white, and duroc) pigs were obtained from a commercial herd at the ages of 10 to 14 days and were randomly assigned to seven
treatment groups. The study was conducted in accordance with the
guidelines of the Iowa State University Institutional Committee on
Animal Care and Use.
Inocula and experimental design.
The experimental design is
summarized in Table 1. The pigs were 6 weeks old at day 0 (the day on which three of the four PRRSV groups
were inoculated with PRRSV). An inoculating dose of 105
50% tissue culture infective doses (TCID50) of the
high-virulence PRRSV strain ATCC VR-2385, passage 6, in a 5-ml volume
was administered intranasally to pigs in groups A, B, C, and F
(12). A tissue homogenate containing a derivative of
M. hyopneumoniae 11 (105 color-changing units
[CCU] per ml) was administered intratracheally to pigs in groups A,
C, and E at a dilution of 1:100 in 10 ml of mycoplasmal Friis medium
(23). M. hyopneumoniae was administered at a
dilution of 1:50 in 5 ml of medium to pigs in groups B and D due to the
younger age (3 weeks) and smaller size of the pigs at the time of
inoculation.
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TABLE 1.
Experimental design infection status, sequence of
inoculation, and number of pigs necropsied on each of three days
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Serology.
Blood was collected periodically throughout the
trial in order to evaluate antibody production. All sera were tested
for antibodies against PRRSV with a commercially available
enzyme-linked immunofluorescent assay (ELISA) (HerdChek: PRRS; IDEXX
Laboratories, Inc., Westbrook, Maine) according to the procedures
described by the manufacturer. Samples were considered positive if the
calculated sample-to-positive (S/P) ratio was 0.4 or greater. M. hyopneumoniae antibody titers were determined by ELISA as
previously described (3). Known positive and negative sera
were included as controls in each plate. Readings more than 2 standard
deviations above the mean value of the negative control were considered positive.
PRRSV and M. hyopneumoniae isolation and
titration.
PRRSV isolation was performed by using bronchoalveolar
lavage (BAL) fluid obtained at necropsy by lavaging the bronchi with 50 ml of minimal essential medium (MEM) containing antibiotics (9 µg of
gentamicin/ml, 100 U of penicillin G/ml, and 100 µg of streptomycin/ml) (18). Virus isolation was then performed
according to an established protocol (17). Virus titration
was performed on 10% (wt/vol) homogenized lung tissues in MEM as
previously described with several modifications (8).
Briefly, 200 µl of 10-fold serial dilutions of lung homogenate was
inoculated onto a confluent monolayer of CRL11171 cells for 1 h at
37°C in a humidified atmosphere with 5% CO2. The
cultures were monitored daily for CPE. If CPE was not observed within 7 days, the cultures were frozen and thawed and blindly passaged three
times to be considered negative. Monolayers were stained with an
anti-PRRSV monoclonal antibody, SDOW-17 (South Dakota State University,
Brookings), followed by fluorescein isothiocyanate-conjugated
anti-mouse immunoglobulin and were viewed with a fluorescence
microscope for evidence of specific viral antigens (14).
M. hyopneumoniae was isolated from lung sections and
titrated as previously described (22). Mycoplasma-appearing colonies that developed were
specifically identified by using epiimmunofluorescence with conjugates
prepared from rabbit antisera to M. hyopneumoniae 11 (7).
Clinical evaluation.
Pigs were evaluated daily for at least
15 min for clinical signs, including appetite, cough, increased
respiration rate, or behavioral changes. A daily clinical respiratory
score was assessed on days 0 to 14 according to a previously described
system (14).
Pathologic examination.
Pigs were necropsied at either day
3, day 10, or day 28, as outlined in Table 1. The right rib cage was
reflected, and the lungs were removed and evaluated for macroscopic
lesions. A portion of lung was aseptically collected for M. hyopneumoniae and PRRSV isolation, fluorescent antibody assay
(FA), immunohistochemistry (IHC), and histopathologic examination. The
lungs were then lavaged, and BAL fluid was obtained. Lesions consistent
with mycoplasmal pneumonia (dark-red-to-purple consolidated areas) were
sketched on a standard lung diagram. The proportion of lung surface
with lesions was determined from the diagram by using a Zeiss SEM-IPS image analyzing system (23). In contrast to
mycoplasma-induced lesions, PRRSV-infected lungs were characterized by
parenchyma that was mottled tan and rubbery and failed to collapse. The
lung lesions were scored by using a previously developed system based on the approximate volume that each lobe contributes to the entire lung: the right cranial lobe, right middle lobe, cranial part of the
left cranial lobe, and caudal part of the left cranial lobe each
contribute 10% each of the total lung volume, the accessory lobe
contributes 5%, and the right and left caudal lobes each contribute
27.5% (13). These scores were then used to calculate the
total lung lesion score based on the relative contribution of each lobe.
Sections were taken from all lung lobes, fixed in 10% neutral buffered
formalin, and routinely processed and embedded in paraffin
in an
automated tissue processor. Lung sections were blindly examined
and
given a score (0 to 4) for peribronchiolar and perivascular
lymphoid
cuffing and nodule formation consistent with
M. hyopneumoniae-induced
pneumonia lesions. The severity of
PRRSV-induced and interstitial
pneumonia lesions was also scored (0 to
6). The scoring systems
are summarized in Table
4, footnotes a and
b.
PRRSV and M. hyopneumoniae antigen detection.
PRRSV-specific antigen was detected in lung tissues by a previously
described IHC method (10). IHC was performed on sections cut
from one paraffin-embedded lung tissue block which included three
pieces (1 by 2 cm) of lung, one each from the left cranial, accessory,
and caudal lobes. The number of PRRSV antigen-positive cells was
counted as described elsewhere. A direct immunofluorescence procedure
was used for detection of M. hyopneumoniae as described previously (1).
Statistics.
Data were subjected to analysis of variance
(ANOVA). If the P value from the ANOVA was less than or
equal to 0.05, pairwise comparisons of the different treatment groups
were performed by least significant difference at the P < 0.05 rejection level.
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RESULTS |
Clinical disease.
Clinical respiratory disease data are
presented in Table 2. All groups
inoculated with PRRSV displayed signs of respiratory disease consistent
with PRRSV-induced pneumonia by day 3 postinoculation. Signs included
labored breathing and increased respiratory rate. Coughing was observed
in all M. hyopneumoniae-inoculated groups beginning at days
10 to 14 but not in pigs infected with PRRSV only (group F).
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TABLE 2.
Summary of average clinical respiratory disease scores of
pigs by group after inoculation with PRRSV, M. hyopneumoniae, or both
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Analysis of the respiratory disease scores indicated that pigs infected
with both
M. hyopneumoniae and PRRSV had more severe
clinical respiratory disease than the single-organism-infected
groups.
Group B, which was inoculated with
M. hyopneumoniae 21
days
prior to PRRSV inoculation, developed more severe respiratory
disease
within the first 3 days after PRRSV inoculation than any
other
PRRSV-infected groups. Overall, the pigs in groups A and
B, which had
received both PRRSV and
M. hyopneumoniae, had significantly
more severe clinical respiratory disease than pigs inoculated
with
PRRSV alone (group F) or
M. hyopneumoniae alone (groups D
and
E).
Respiratory scores from pigs in group C do not match those of the other
PRRSV-infected groups due to the 10-day interval between
the
inoculation of group C pigs with PRRSV and the first determination
of
clinical scores shown in Table
2. Prior to inoculation with
M. hyopneumoniae on day 0, the clinical scores of group C matched
those of group F for the first 10 days following PRRSV infection,
which
for group C would be days
10 to 0 (data not shown). The
respiratory
scores for group C in Table
2 were determined 10
additional days
post-PRRSV challenge. Thus, the score of 2.2 ±
0.8 for group C on
days 1 to 3 in Table
2 was actually determined
11 to 13 days post-PRRSV
challenge and could be compared to the
scores in the final column,
determined on days 11 to 14, for the
other PRRSV-challenged groups. If
the group C data on days 1 to
3 are compared to the data for the other
PRRSV-challenged groups
on days 11 to 14, the clinical disease in group
C is equivalent
to that in group F. However, even though there is no
comparable
PRRSV-only control that matches group C for more than 14 days
postinoculation, the clinical respiratory disease in group C shows
no evidence of regressing over the course of the monitoring period,
extending to 24 days after inoculation with
PRRSV.
Macroscopic lesions.
The mean percentages of lung tissue with
visible pneumonia, either PRRSV-induced or M. hyopneumoniae-induced pneumonia, are summarized in Table
3. At necropsy on day 3, PRRSV-infected
pigs were already exhibiting PRRSV-induced pneumonia. PRRSV-induced lesions consisted of lungs which were mottled tan or diffusely tan,
were firmer and heavier than normal lungs, and failed to collapse upon
removal from the chest cavity (Fig. 1b),
compared to a normal lung (Fig. 1a). The pneumonia was well developed
in group C, which had been infected with PRRSV 13 days prior to
necropsy. No visible M. hyopneumoniae-induced pneumonia was
observed at day 3.
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TABLE 3.
Percentage of lung with visible pneumonia lesions in pigs
infected with either M. hyopneumoniae, PRRSV, or both
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FIG. 1.
(a) Normal lungs from an uninfected pig. (b) Lungs from
a pig infected 10 days previously with PRRSV. The lungs fail to
collapse and are diffusely mottled and tan in appearance. (c) Lungs
from a pig infected 28 days previously with M. hyopneumoniae. Lungs have multiple well-demarcated, dark-red areas
of pneumonia in the cranioventral region. (d) Lungs from a pig dually
infected 28 days previously with PRRSV and M. hyopneumoniae,
exhibiting both the characteristic failure to collapse and mottled tan
appearance of PRRSV and the well-demarcated, dark-red consolidated
areas typical of M. hyopneumoniae.
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At day 10, the level of PRRSV-induced pneumonia were similar in groups
A, B, and F, all of which had received PRRSV on day
0. The percentage
of pneumonic lung lesions was lower in group
C at day 10. However, pigs
in group C, inoculated with PRRSV 10
days prior to receiving
M. hyopneumoniae on day 0, were 20 days
post-PRRSV challenge.
Interestingly, groups A and C had higher
percentages of pneumonic lung
lesions due to
M. hyopneumoniae than the
M. hyopneumoniae-only group (group E). In addition, the
mean
percentages of lung tissue with mycoplasmal pneumonia in
groups A and C
were greater than those in either group B or group
D, which had been
inoculated with
M. hyopneumoniae 31 days prior
to necropsy.
M. hyopneumoniae-induced lesions were easily differentiated
from the PRRSV-induced lesions, as they consisted of well-demarcated
areas of dark-red to purple firm parenchyma, located primarily
at the
ventral and cranial tips of the anterior and middle lobes
(Fig.
1c).
At day 28, PRRSV-induced pneumonia was observed in only two of the
eight pigs in the group inoculated with PRRSV only (group
F). In
contrast, PRRSV-induced lung lesions were observed in all
pigs in the
dually infected groups (groups A, B, and C) (Fig.
1d). The percentage
of lung tissue with PRRSV-induced pneumonia
lesions in group A was
significantly greater than that in group
B or C. The levels of
mycoplasmal pneumonia were similar in groups
A, C, and E, while groups
B and D had minimal mycoplasmal
lesions.
Microscopic data.
Histopathological evaluations are presented
in Table 4. Interstitial pneumonia
consistent with PRRSV-induced pneumonia was present in all
PRRSV-infected pigs at day 3. This pneumonia was characterized by type
2 pneumocyte hypertrophy and hyperplasia, septal infiltration with
monocytes, and increased alveolar exudate consisting of macrophages,
necrotic macrophages, multinucleated cells, and proteinaceous fluid.
Groups A and B had microscopic lesions equivalent to those observed in
pigs from group F (PRRSV only), whereas group C had more severe
PRRSV-induced lesions, as expected due to its longer interval following
PRRSV inoculation (13 days). M. hyopneumoniae-induced lung
lesions were not yet observed at day 3.
Microscopic lesions consistent with PRRSV-induced interstitial
pneumonia were similar in all PRRSV-infected pigs on day 10
(Fig.
2b). Peribronchiolar and perivascular
lymphohistiocytic
cuffing consistent with
M. hyopneumoniae-induced pneumonia was
observed and was significantly
more severe in groups A and C than
in group E at day 10 (Fig.
2c).
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FIG. 2.
(a) Microscopic section of a normal lung from a pig. (b)
Microscopic section of a lung from a pig infected 10 days previously
with PRRSV. There is moderate diffuse interstitial pneumonia
characterized by accumulation of necrotic debris and inflammatory cells
in alveolar spaces, septal infiltration by mononuclear cells, and type
2 pneumocyte hypertrophy and hyperplasia. (c) Microscopic section of a
lung from a pig infected 28 days previously with M. hyopneumoniae. There is peribronchiolar and perivascular lymphoid
hyperplasia characteristic of M. hyopneumoniae infection.
(d) Microscopic section of a lung from a pig infected 28 days
previously with PRRSV and M. hyopneumoniae. Lesions
characteristic of both M. hyopneumoniae- and PRRSV-induced
pneumonia are present.
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Groups A and C had significantly more severe PRRSV-induced interstitial
pneumonia than group F (PRRSV only) at day 28. Microscopic
lesions
consistent with
M. hyopneumoniae infection were also more
severe in the dually infected groups A and C than in group E (
M. hyopneumoniae only) at day 28 (Fig.
2d).
The mild peribronchiolar and perivascular lymphohistiocytic cuffing
reported in the negative-control group and group F (PRRSV
only) are
nonspecific changes. There was no evidence of
M. hyopneumoniae infection in either the negative-control group or
group F based
on culture, which is a more sensitive test for the
presence of
M. hyopneumoniae. Peribronchiolar lymphoid
hyperplasia is considered
the most characteristic lesion associated
with
M. hyopneumoniae infection; however, in cases of
mycoplasmal pneumonia, it typically
progresses to discrete
peribronchiolar lymphoid nodule or follicle
formation. However, these
changes are not found only in pigs infected
with mycoplasmas.
Peribronchiolar lymphohistiocytic cuffing has
been reported in pigs
experimentally infected with PRRSV (
13)
or may simply be
associated with chronic antigen stimulation from
the environment.
PRRSV-induced peribronchiolar lymphohistiocytic
cuffing does not
progress to discrete nodule formation and typically
resolves by day 28
postinfection.
PRRSV and M. hyopneumoniae isolation and
titration.
PRRSV was isolated from BAL of all groups of
PRRSV-infected pigs. PRRSV was isolated from the greatest number of
pigs in groups A (12 of 20) and B (10 of 20), which had received
M. hyopneumoniae either concurrently with or prior to
inoculation with PRRSV, respectively (Table
5). Isolation of PRRSV from BAL peaked at
day 10. Interestingly, at day 28, virus was isolated from only three of
eight pigs in group A (concurrently infected with PRRSV and M. hyopneumoniae) and one of eight pigs in group B (infected with
M. hyopneumoniae prior to PRRSV), while all other pigs at
that time were negative for PRRSV.
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TABLE 5.
Isolation of PRRSV from BAL and titration of virus from
lung tissue and number of cells with PRRSV antigen, as determined
by IHC
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Titration of lung tissue for PRRSV revealed that the highest average
titers of virus were found on day 10 in groups A, B,
and F, with
individual pig titers ranging from a TCID
50 of
10
2 to 10
5.6. The highest percentages of pigs
with virus isolated from the
lung homogenate were found in groups A and
B; in each of these
two groups, 83% of infected pigs had detectable
PRRSV in lung
tissue at necropsy. No differences were observed in the
levels
of PRRSV between the various
groups.
PRRSV antigen was detected in macrophages throughout the lungs of the
PRRSV-infected pigs, as previously reported (
12).
The number
of pigs which had PRRSV-positive cells was greatest
at days 3 and 10 in
groups A, B, and F (Table
5). By day 28,
only one of eight pigs in each
of these groups had PRRSV-positive
macrophages. In group C, which had
been inoculated with PRRSV
10 days prior to the other groups, six of
six pigs were positive
for PRRSV antigen by IHC at day 3, no positive
cells were detectable
at day 10, and three of eight pigs were positive
by day 28. The
six pigs in group C that were necropsied at day 10 were
all negative;
however, PRRSV-like lesions were moderate and multifocal
(microscopic
score, 3.3 of 6), and PRRSV was isolated from BAL of five
of six
pigs. It is not uncommon for animals with subacute PRRSV to have
lesions consistent with PRRSV yet to have negative IHC. IHC is
not as
sensitive as virus isolation from BAL fluids. No statistical
differences in the number of IHC-positive cells were observed
between
the dually infected groups, A, B, and C, and group F,
infected with
PRRSV only. However, there was a large variation
in the number of
positive cells among the individual pigs. In
addition, no evidence of
increased numbers of cells containing
PRRSV antigen was observed in the
areas of the lungs with microscopic
lesions consistent with
M. hyopneumoniae.
M. hyopneumoniae was isolated from all
M. hyopneumoniae-inoculated groups. However, only 25% of pigs in
group B (receiving
M. hyopneumoniae on day
21 and PRRSV on
day 0) and 10% of pigs
in group D (receiving
M. hyopneumoniae on day
21) were positive
for
M. hyopneumoniae by culture (Table
6).
Groups A (receiving
PRRSV and
M. hyopneumoniae concurrently)
and C (receiving PRRSV
on day
10) had the largest numbers of pigs
positive for
M. hyopneumoniae.
However, no significant
differences were observed in the mean
titer of
M. hyopneumoniae in lung tissue between the dually infected
groups
and the groups infected with
M. hyopneumoniae alone (Table
6). All pigs in groups A, C, and E were positive for
M. hyopneumoniae as determined by FA at day 28, while only three of
eight and one
of eight were positive in groups B and D, respectively.
Serology.
All pigs challenged with PRRSV developed antibodies
to PRRSV by day 10 as determined by ELISA (data not shown). None of the pigs developed antibodies to M. hyopneumoniae during the
study, as expected from previous studies (23).
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DISCUSSION |
We have developed an experimental model demonstrating that a
mycoplasma can act as a cofactor in potentiating a viral pneumonia. Differentiating between the disease induced by the two pathogens was
simplified by the pronounced differences between their clinical manifestations and the resulting macroscopic and microscopic lesions. PRRSV infection is characterized by a severe, acute, diffuse, interstitial pneumonia, whereas M. hyopneumoniae induces a
chronic, mild, localized bronchopneumonia. These differences in
pathologic lesions enabled us to identify the increased severity and
duration of the PRRSV-induced pneumonia observed in all pigs infected
with both PRRSV and M. hyopneumoniae. M. hyopneumoniae
potentiated the viral pneumonia independent of the time of viral
infection. Surprisingly, even when M. hyopneumoniae did not
uniformly induce observable lesions typical of mycoplasmal pneumonia,
as observed in group B, the viral pneumonia was potentiated. This
finding indicates that it is not just an additive effect of the
different pathogens, but rather a potentiation of PRRSV-induced
pneumonia by M. hyopneumoniae. Specifically, pigs in group
B, which had received M. hyopneumoniae 21 days prior to
PRRSV inoculation, had a low level of mycoplasmal pneumonia yet had
PRRSV-induced lesions that persisted for 4 weeks after PRRSV
inoculation. PRRSV-induced lesions typically are resolved by 4 weeks
postinoculation, as confirmed by the group infected with PRRSV only
(group F) (12).
PRRSV infection did appear to increase the severity of the macroscopic
and microscopic M. hyopneumoniae-induced pneumonia at day 10 in groups A and C, which received PRRSV concurrently with or prior to
inoculation with M. hyopneumoniae. However, in both groups
of pigs, the percentages of lung tissue exhibiting macroscopic
pneumonia consistent with M. hyopneumoniae were not increased at day 28, which is our usual end point for studies of
mycoplasmal pneumonia. Microscopically, however, PRRSV and mycoplasmal
lesions were more severe in groups A and C at day 28. These results
were not observed in the pigs which were inoculated with M. hyopneumoniae prior to challenge with PRRSV (group B). These
findings suggest that the presence of PRRSV early in infection with
M. hyopneumoniae may increase the rate at which pigs develop mycoplasmal pneumonia and increase the damage induced at the cellular level but does not increase the overall percentage of lung tissue exhibiting macroscopic pneumonia.
Levels of either PRRSV or M. hyopneumoniae in tissue were
not increased in dually infected pigs. This suggests that the increased severity and duration of PRRSV-induced pneumonia was not due to increased PRRSV or M. hyopneumoniae replication. These
results support the hypothesis that the inflammatory response elicited in the course of mycoplasmal pneumonia may be a critical factor in the
potentiation of PRRSV-induced disease and lesions. M. hyopneumoniae-induced pneumonia is characterized by the
infiltration of the lung parenchyma by mononuclear cells consisting
primarily of lymphocytes and macrophages (26). In addition,
it has been demonstrated that M. hyopneumoniae has a
mitogenic effect on swine lymphocytes (19). These findings suggest that activation of the immune system may contribute to the
enhanced pneumonia observed in our model. M. hyopneumoniae's attraction of these inflammatory cells may
produce an ideal environment for PRRSV-induced inflammation to persist.
These findings are consistent with PRRSV infection of cells of the
monocyte/macrophage/dendritic cell lineage, which is one of the major
cell types responding to M. hyopneumoniae infection. The
steady influx of new monocytes/macrophages recruited by the relatively
chronic M. hyopneumoniae infection may allow PRRSV to
persist in the lungs at low levels for prolonged periods.
The specific cellular and subcellular mechanisms by which M. hyopneumoniae potentiates PRRSV are currently unknown. Recent studies conducted with humans have also identified mycoplasmas as
potentially important cofactors in a number of chronic disorders, including AIDS, malignant transformation, chronic fatigue syndrome, and
various arthritides (15, 21, 27, 29). These studies suggest
that our findings are not unique or without precedent. The mechanisms
by which mycoplasmas influence the pathogenesis of these diseases in
humans have not been elucidated. However, mycoplasmas are likely
candidates for initiating disease because they produce chronic, often
mild infections and have potent immunomodulatory properties.
Mycoplasmas could potentiate PRRSV by direct interaction with the
virus, by interaction with cells from the immune system, or by inducing
an inflammatory immune response by the secretion of cytokines.
| |
ACKNOWLEDGMENTS |
We thank F. C. Minion for editorial assistance. We also
thank N. Upchurch, B. Erickson, T. Young, R. Royer, T. Boettcher, and
T. Anderson for technical assistance.
This work was supported by a grant from the National Pork Producers Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: VMRI, Iowa State
University, 1802 Elwood Dr., Ames, IA 50011. Phone: (515) 294-5097. Fax: (515) 294-1401. E-mail: ethacker{at}iastate.edu.
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Abstract
Introduction
