characterization of pneumocystis carinii infection in sorex araneus ...

3 downloads 0 Views 696KB Size Report
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

AND D. Y. E. PEREY. lymphoid tissue. I. MorphologLaboratory Investigation 28:

P.

1957. The life of the shrew. Max London, United Kingdom, 199 pp. GROCOTT, R. G. 1955. A stain for fungi in tissue sections and smears. American Journal of Clinical Pathology 25: 975-979. HANSKI, I. 1992. Insectivorous mammals. In Natural enemies. The population biology of predators, parasites, and diseases, M. J. Crawley (ed). Blackwell Scientific Publications, Oxford, United Reinhardt,

Kingdom,

pp.

J. M.

HOPKIN,

163-187.

Pneurnocystis United

1991.

University

Press,

car/nil. Oxford Kingdom, 140

Oxford,

pp. HUGHES,

A.

W. T., R. A.

PRICE,

KAFATOS,

M.

G.

1974.

SMYTHE.

host

KIM,

for

American 128:

F.

W. S.

SISK0,

SCHONLAND,

HAVRON,

P.

AND

Protein-calorie

determinant

fection.

in-

than in S. araneus J. Laakkonen, un-

high prevalence may be a sign

of the

seem

to be

686-692. CROWCROFT,

tro-

the

and

awaits

N. JOHNSTON,

Bronchial ical characteristics.

of P. car-

phases

J.,

1973.

in-

studies about the

of cysts

to Voitto

Foundation

BIENENSTOCK,

dren

caecutiens, (Laakkonen

aptation

into

observed

the proliferating

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

abolic rate, relatively low energy reserves, and the lack of capacity to enter torpor (Vogel, 1976, 1980). We propose that shrews of the genus Sorex become with P. carunii without having

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

IN SOREX

M.

malnutrition.

Pneurnocystls

Journal

of

A

in-

car/nil

Diseases

of

Chil-

44-52.

C. K., J. M. FOY, M. T. CUSHION, D. STANFORTH, M. J. LINKE, H. L. HENDRIx, AND P. D. WALZER. 1987. Comparison of histologic and quantitative techniques in evaluation of therapy for experimental Pneumocystls car/nil pneumonia. Antimicrobial Agents and Chemotherapy 31: 197201.

J.,

LAAKKONEN,

A. SUKURA, 1993.

HENTTONEN.

V.

HAUKISALMI,

Pneurnocystls

AND

car/nil

helminth parasitism in shrews Sorex Sorex caecutiens. Journal of Wildlife

araneus

Diseases

H. and and 29:

273-277. LINDSEY,

J. R., H. J.

CASSELL, chronic Pathology

AND

BAKER,

C.

respiratory

64:

E. disease.

675-716.

R. G.

G. H.

OVERCASH,

HUNT.

1971.

American

Murine Journal

of

232

JOURNAL

PETERS,

S. E.,

OF WiLDLIFE

K.

DISEASES,

J.

ENGLISH,

VOL. 31, NO. 2, APRIL

LAAKKONEN,

J.

AND

DNA analysis of PneumocysI/s car/nil infecting Finnish and English shrews. Journal of Eukaryotic Microbiology 41: 5108. SErrNrs, 0. P., AND P.-E. LARSEN. 1979. Inhibition of toluidine blue 0 stain for Pneumocystls car/nil by additives in the diethyl ether. American Journal of Clinical Pathology 72: 493-494. ,AND J. LODAL. 1980. Prevalence of Pneumocystis car/nil Delano#{235} & Delano#{235}, 1912 in rodents in Denmark. Nordisk Veterinaermedicin 1994.

GURNELL.

32:

SHIOTA,

T., H.

KURIMOTO,

Y.

AND

1986.

YOSHIDA.

in wild rof#{252}r Bakteriologie, Series A 261: 381car/nil

389. AND

ed. Freeman,

F. J. ROHLF. San

1981.

Biometry,

1980. Metabolic levels and biological strategies in shrews. In Comparative physiology: Primitive mammals, K. Schmidt-Nielsen, L. Bolis, and C. R. Taylor (eds.). Cambridge University Press,

New

California,

Francisco,

859

A.,

J.

T.

LAAKKONEN,

HENTTONEN,

AND

Pneurnocystls

car/nIl

L.-A. in

H.

SOVERI,

LINDBERG.

corticosteroid-treated

York,

New

P. D., R. D.

York,

POWELL,

pp. JR.,

E. RUTLEDGE, ANDJ. E. MILDER. characteristics and pathogenesis Pneurnocystls car/nil pneumonia. Immunity 27: 928-937.

C., M. R.

1989.

pp. SUKURA,

206.

WANGxuE,

R. R., 2nd

voles: A comparison of three different staining methods. Journal of Wildlife Diseases 28: 121124. VOGEL, P. 1976. Energy consumption of European and African shrews. Acta Theriologica 21: 195-

WALZER,

17-27.

Prevalence of Pneumocystis dents in Japan. Zentralblatt Mikrobiologie, und Hygiene, SOKAL,

1995

Experimental

ALLEY,

AND

170-180.

K.

mice with Bordetella parapertussls sheep. Journal of Comparative

isolated Pathology

1992. for

publIcation

16 May

MANKTELOW.

of pneumonia

77-89. ReceIved

M.

Growth of experimental Infection and

B. W.

induction

YONEDA,

1980.

1994.

in

from 100: