at 6-hr intervals; shrews were taken ... for 1 hr with constant stirring ..... 41: 5108. SErrNrs,. 0. P., AND. P.-E. LARSEN. 1979. Inhibition of toluidine blue 0 stain for ...
of Wildlife
Journal
CHARACTERIZATION SOREX Juha
OF PNEUMOCYSTIS
ARANEUS
Laakkonen1
FROM
and
Timo
SOUTHERN
Histologic
31(2), Disease
1995, pp. 228-232 Association 1995
INFECTION
IN
FINLAND
Soveri2
‘Department of Anatomy and Embryology, P.O. Box 6, 00581 Helsinki, Finland 2 Helsinki Zoo, 00570 Helsinki, Finland
ABSTRACT:
CARINII
Diseases, © Wildlife
and
College
quantitative
of Veterinary
techniques
Medicine,
were
compared
in an evaluation
of the
intensity
carinli infection in common shrews (Sorex araneus) at Espoo, southern Finland, from September 1992 to May 1993. The histological scores were comparable to the results of the cyst count technique. The number of P. carinll cysts found in common shrews was low compared to those reported by others in clinically ill laboratory rats. The inflammatory changes detected in the lung sections had no significant relation to the presence of P. carlnli infection. Key words: Pneumocystis carinli, Sorex araneus, histology, cyst count, histopathology. of Pneumocysti.s
INTRODUCTION
Pneumocystis pathogen
possible
carinii
causing
is
a fatal
munocompromised Subclinical P.
a
pulmonary
pneumonia
apparently healthy laboratory rats. The infection develops into pneumonia 6 to 8 wk after administration (Walzer eta!., 1980). infection
also
ratory alone
has
rats by (Hughes
festation
of corticosteroids Pneumocystiscarinii been
dietary et al.,
provoked
in-
sp. has
ob-
served (Settnes 1986).
in
various wild mammal and Lodal, 1980; Shiota More recently, Laakkonen
(1993) carinii
reported in wild
other carinii
pneumonia
Since
the
infection
as
we
to assess
od
For
shrews, to
fection which
assess
1992 were
the
used
the
with the
intensity
to
of
P.
detected. P. carinii
by and
comparison we
signs
cyst of
organism comparing cyst count the
com-
mine
shrews
were
collected
trapping
periods
1993. Snap-traps at 6-hr intervals;
at
Finland,
from
September
baited shrews
with cheese were taken
slide
in the
order
modification
Silver
(GMS)
cut,
of stain
and
stained
Gomori’s
(Grocott,
with
Methena1955).
Using
a blind study, slides were examined by light microscopy at 200 x and 400 x. The following scoring system was used to evaluate the intensity of P. carlnli infection: 0 = no cysts on entire slide, 1 = 80 cysts per slide. The distribution of the cysts in each section was recorded.
agrestis), prevalence
histopathologically
several
Grocott’s
of P. carinii in late fall in Finland (J. Laakkonen, unpubl.). Finally, we examined the lungs of S. araneus
the even
AND METhODS
common
to May checked
on one
count methP. carinii in-
in field voles (Mwrotus have a relatively high
Since high,
immediately after trapping to the laboratory for necropsy. The shrews were classified as adults and juveniles according to tooth wear and condition of the pelage (Crowcroft, 1957). Lungs of 44 shrews were assessed histologically. Lung tissue first was fixed in 10% buffered formalin; the right lobes (cranial, medial, caudal and accessory) of each sample were treated separately from the only lung lobe on the left side. Fixed tissues were dehydrated, embedded in paraffin and sectioned at 5 zm. Ten horizontal sections of each sample were cut at 20 m intervals so that all lobes were represented, placed
by histological
evaluated
burden of common shrews the scores of histological mon
were
of low-grade
is difficult
methods.
no
are
(60#{176}14’N, 24#{176}46’E), southern
during
species et a!., et al.
compared
but
(PCP)
intensity
examination,
Espoo
a high prevalence of P. populations of common
(Sorex araneus) wild mammals,
shrews
Ninety-two
been
changes. of shrews
MATERIALS
in labo-
protein deprivation 1974). Pulmonary
by Pneurnocystis
rates
minor inflammatory process may impair the gas exchange of infected shrews. The objective of these analyses was to determine whether the frequent pulmonary infestation by pneumocystis in S. araneus is harmful to the apparently healthy host.
in im-
hosts (Hopkin, 1991). infection occurs in
carinii
inflammatory
respiratory
for
The 228
other
48
shrews
were
evaluated
by
the
LAAKKONEN
cyst
method.
concentration
of the described sample
The
wk for counting
the cysts.
In four
sample
was prepared after necropsy without freezing. cysts, the wet weight (i ± SD g) of 48 left lobes was recorded centration
These
lobes
blades,
and
digested
ma Chemical
Co.,
incubating stirring
fuged pellet
right
lung
lobes
shrews were prepared for histology as previously. The left lung lobe of each was frozen at -20 C and used within 1
were
=
cut into small with
the con-
immediately For counting 0.036 ± 0.012 after thawing.
pieces
0.2%
St. Louis,
cases
with
was
washed
three
with
USA)
by
0.9%
NaCl
of 0.9% NaC1. Two 5 d were placed on a slide;
two slides were made of each sample. The slides were air-dried, fixed in ethanol and stained with toluidine blue 0 (Settnes and Larsen, 1979). All cysts in each crop were counted, and the mean of the four counts was taken as the final cyst count per microliter. For comparisons with other studies, the number of cysts per microliter was
transformed
to
the
number
of
cysts
per
gram (Sokal
of lung tissue. The Spearman Rank Test and Rohlf, 1981) was used to determine the correlation between the histologic assessment of the intensity of P. carinii infection and
the quantitative test (Sokal and the
mean
cyst count. Rohlf, 1981)
The
Kruskal-Wallis used to analyze
was cysts count groups. carinii positive (n = 24) 23) samples were examined
ranks
for
the
Pneumocystis (n = histopathological changes. All samples of histological assessment were included in this amination, and also the three samples with negative
highest
cyst
earlier
for
count.
of the right lung the slides were
eosin,
and
From
histologic were stained
placed with
slide
was
each
microscopy.
If the
(lymphocytes
the
samples
assessment,
number
or
the exthe
sections
on the same hematoxylin
examined
by
of mononuclear
was
slightly in one or some of the lungs the increase was categorized
and for
prepared
three
macrophages)
slide, and
often
associated
pathological chance twice;
changes
(Sokal first the
with
than
the
can
cells
increased
visual fields of as small, but
observed
histo-
be expected by This was done
and Rohlf, 1981). samples of lungs with
no changes
were analyzed against the samples of small and noticeable changes (analysis 1), and secondly, only
the
samples
with
noticeable
samples 2).
For comparison
with
of
ARANEUS
lungs
the common
229
without
shrews,
the
left lung lobes of two field voles known to be infected with P. carinii, were evaluated by the same cyst concentration method as used for shrews. These voles were caught in the same area as the shrews in November 1992. RESULTS
changes
Twenty-one (48%) of the 44 shrews ashistologically were infected with P. carinii. Ten of these had 80 cysts per slide (category nificant (P> 0.05) differences
around
only Besides
were
scattered
in the
more
the
one clusclusters, around heavily
in-
fected shrews. Generally, the approximate parasite burden was the same in each of the ten sections examined from a given slide; but in the very light infections (category 1) the number of cysts appeared to be greater in the sections close to the surface of the lungs. Generally, the intensity of the infection was similar left and the right lung lobes dividual
light
if their amount was highly increased in some of the visual fields or evenly (although slightly) through the section, the increase was categorized as noticeable. The Pearson’s Chi-square test or Fisher’s exact test was used to analyze the null hypothesis that P. carinii cysts were not more
against (analysis
IN SOREX
(Sig-
Missouri,
times
in 1 ml suspension
analyzed changes
CAR/Nil
razor
collagenase
the samples for 1 hr with constant at 37 C. The digested fluid was centriat 3,000 rpm for 7 mm at 23 C, and the
and suspended drops of this
AND SOVERI-P.
the in-
host.
Twenty
(42%)
examined od
between of each
by
of the 48
the
cyst
P. carinii.
had
lung
samples
concentration
These
meth-
samples
were
di-
vided into three groups based on the histological assessment of the same samples. The arithmetic mean (±SD) of the cyst count was 3.10 (± 1.51) x 10 (n = 8) for the per the 80
first group (histological slide), 5.52 (± 2.80) second cysts
group per
x
score 80 cysts per slide). Using the Spearman
Rank
correlation
test a significant (r5
=
0.85)
was
(P
found
20,
sections when
the
P. cyst et al.,
carunmi count 1987).
with
shrews.
earlier among
Since vo!es
facilities.
bebein in-
How-
intensity done only
cyst
maximum voles was
This
of P. on a
count
of
number lower than
difference
is con-
(J.
findings a large
the prevalence than in shrews
both
et
Laakkonen, number of field
also is much lower in (Sukura et a!., 1992;
al.,
prevalence
1993), and
we
the
In
the
count
associated
the cyst
burden. mononuclear
The
than
assessment, in
with
that number
is higher
histopatho!ogica! increase
believe
average
of cysts found in S. araneus those found in field voles.
of the sample infections
in we
voles that the intensity of P. carmnui infection seldom is as high as was observed in the two field voles examined in this study.
an
P. carinii
the
in field
found
of the
found
num-
found methods,
quantitative
M. agrestis,
Laakkonen
laboratory
laboratory
the evaluation of the infection should be
sistent unpub!.)
examination
the quantitative cyst count, mu infections found in shrews compared to those of clinically and
tory
correlation histological of
correlation the two
of
rough scale: positive or exceptionally positive. Also, because of the differences in the sizes of lungs, an interspecific comparison of the intensity of the infection is difficult without a quantitative method.
those on
sections
a highly significant found between the of the intensity and quantitative et al., 1980; Kim
of cysts
Based
demonstrated
infection in ecological studies which require large samples sizes, and in which samples often are collected in the field
had
cysts
1, and P = 0.29, analyses 2). Mononuclear cells either were increased homogeneously in the interstitial space or in clusters peribronchially. Based on the quantitative cyst count, the two field vole samples had 1.75 x 106 and x
be
fected shrews. However, because the arithmetic mean of this group differed clearly from the other groups, we believe that histological examination can be used for the evaluation of the intensity of P. caruniu
histopatho0.18, anal-
yses
9.60
could
study if several were examined.
did not find significant differences tween the cyst count groups of shrews cause of one very low cyst count score the small group of the most heavily
lowest The
to 1 wk at 20 C before concentration. No significant differences (P = 0.082) were found between the cyst counts sessment
rats, been
assessment infection (Walzer
same The were
up
grouped
araneus
histological each sample
the
1.91 x 106. of cysts in
ones
Sorex
Despite the similar this study between
study.
without as in the
by
P. the
case, no cysts were assessment but the
was
in this
in
of case
second (four cysts) sections of the sample by the histological assessment. rest of the sections of this sample negative. found by
histo-
zero, but (19 cysts)
was
first
1995
mononuclear a
peribronchial cells, mainly
small
we cell
organism
clusters lymphocytes,
of
LAAKKONEN
resembled tissue scribed
bronchus-associated
!ymphoid
which Bienenstock et a!. (1973) in several laboratory animals
AND SOVERI-P.
permanent deand
CAR/Nil
host-parasite
of other hosts (Peters this species-specific
tological
pathogenity
detected
no significant relation P. carinii cysts. Without a constant sp.
shrews
because
will of
in this to
supply
starve
their
the
study
had
presence
of
of food,
Sorex
a few
hours
high
met-
within
exceptionally
infected a sympto-
matic from
would long
disease because this disease and
fore
PCP
develops.
nary infestation araneus found study that
the hosts starvation The
of in
(Laakkonen
P. this et a!.,
P. cariniu
infection
that
the
cysts
the
1993)
as it in-
fections remain in their nests and burrows, and thus are never caught. Starvation experiments in captivity might help in determining fections would
whether develop give detailed
variation
of the
phozoites,
mu,
PCP. information
number
the
various
mild Such
and
form
of
P. carinii
common
in
the
does smaller
S. minutus) et a!., 1993;
pub!.), the S. araneus
not Sorex
araneus
is
host the
to be species
of P. carinui of a good
to its parasite. numerically
as (S.
in ad-
Because dominant
shrew species in Europe and western Siberia, and has a longer starvation time than the smaller Sorex spp. (Hanski, 1992), it may have had a higher number of encounters,
and
more
time
to evolve
a more
Haukisalmi,
Heik-
also
is gratefully
ac-
CITED
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of the
seem
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tro-
the
and
awaits
N. JOHNSTON,
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J.,
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Foundation
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caecutiens, (Laakkonen
aptation
into
observed
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during
fection. Since
S.
the
isolates
infectivity
isolates
LITERATURE
very
acute
in
between
kar Oflund knowledged.
is evidence
with
from
et a!., 1994). Whether variation of P. carinim
differences
We are indebted
die be-
species. However, with wild animals animals
distinct
of Sorex
ki Henttonen and Antti Sukura for providing critical comments on the manuscript. Tuire Pankasalo and Hanna Valtonen gave valuable technical assistance. The financial support of Os-
in S. earlier
usually
to
with
carinim
studied.
pulmo-
cariniu and in is not
harmful in this host in all disease studies is possible
frequent
led
231
ACKNOWLEDGMENTS
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has
ARANEUS
relationship
P. carinii. Pneumocystis araneus is genetically
which increased in respiratory infections caused by bacteria (Lindsey et a!., 1971; Wangxue et al., 1989). However, the hischanges
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M.
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