SEROLOGIC SURVEY FOR ACTINOBACILLUS CAPSULATUS IN FREERANGING SNOWSHOE HARES (LEPUS AMERICANUS) FROM ALASKA AND ALBERTA Author(s): Randall L. Zarnke, Jamie K. Morton, and Patrick J. Manning Source: Journal of Wildlife Diseases, 26(4):518-521. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-26.4.518 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-26.4.518

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Journal

of Wildlife

Diseases,

© Wildlife

SEROLOGIC

SURVEY

FOR ACTINOBACILLUS

FREE-RANGING

SNOWSHOE

FROM

AND

ALASKA

HARES

AMERICA

A plate

(Lepus Actinobacillus

and Pathology,

method

agglutination

americanus) capsulatus.

hares

pp.

518-521 1990

Association

IN

NUS)

ALBERTA

of Laboratory Medicine Minnesota 55455, USA

ABSTRACT:

1990,

Disease

CAPSULATUS

(LEPUS

Randall L. Zarnke, Jamie K. Morton,2 and Patrick J. Manning3 Alaska Department of Fish and Game, 1300 College Road, Fairbanks, Alaska 99701, 2Agricultural and Forestry Experiment Station, University of Alaska, Fairbanks, Alaska Department Minneapolis,

26(4),

captured

was Alaska

in

Antiserum

University

of Minnesota

developed (USA)

to test or

Alberta

A. capsulatus A. capsulatus

against

was

USA

99775, USA

Medical

sera

School,

from

free-ranging

(Canada) prepared

for in

snowshoe

antibody

a domestic

against rabbit.

A

formalin-killed was prepared for use as an antigen. Serum antibody prevalence for hares was 98 of 239 (41%) in Alaska and 51 of 111 (46%) in Alberta. Prevalence in Alaska peaked in 1981 corresponding to a peak in hare population density. Seasonal prevalence peaked in May in Alaska. Prevalence at one capture site in Alaska was significantly higher than at four other sites. There was no difference in sex-specific prevalence for either Alaska or Alberta. Key words: Actinobacillus capsulatus, serology, snowshoe hare, Lepus americanus, survey. concentrated

suspension

of

INTRODUCTION

Various gens

Actinobacillus

of domestic

1984). Members of causing lesions

in

spp.

animal

of this genus

patho(Phillips,

species

suppurative cattle,

are

purposes of the present study were to (1) develop a serologic test to aid in the diagnosis of exposure to A. capsulatus and, (2) utilize the test to conduct a serologic survey in Alaska and Alberta (Canada).

are capable

or granulomatous

sheep,

horses

and

swine

MATERIALS

(Phillips, 1984). Actinobacillus spp. have been implicated in diseases of wildlife species including cases of pneumonia in both bighorn pronghorn

sheep

(Ovis (Antilocapra

canadensis) americana),

was prepared by injecting 2 ml of a suspension of A. capsulatus in phosphate buffered saline (PBS) containing 2 x 10 colony forming units/ml intramuscularly into the thigh of a domestic rabbit (Oryctolagus cuniculus) on two occasions 3 days apart. The bacterial culture was originally isolated from a snowshoe hare (Zarnke and Schlater, 1988) and had been passaged several times on sheep blood agar. Serum samples were collected on 4, 7, 14, and 20 days following the initial injection. Actinobacillus capsulatus was grown on slant tubes of sheep blood agar from 24 to 36 hr. Bacterial growth was flushed from the surface

and

splenic abscesses in pnonghorn, and jaw abscesses in elk (Cervus elaphus nelsoni) from Wyoming, USA (Thonne, 1982). Actinobaccillus capsula tus was originally isolated from caged Angora and mixed breed rabbits in Sri Lanka (Arseculeratne, latus was mestic

1961, injected

rabbits,

1962). When intravenously

infection

spread

capsu-

A.

into

do-

of the

throughout

formalin merous

of

the

fection

is known means

about natural host range, of exposure on other aspects

epizootiology in free-ranging

of

A.

wildlife

capsulatus species.

agar

slant with

approximately

2 ml

of 1%

in PBS. Bacterial suspensions from nutubes were pooled and centrifuged at 5,000 x g in a refrigerated centrifuge (Sorval model RC-2B, Dupont Medical Products, Wilmington, Delaware 19880, USA) for 10 mm. The supernatant was discarded and the pellet was resuspended in approximately #{188} of the volume of formalin PBS as was used for the original suspension. The resultant suspension was again centrifuged at 5,000 x g for 10 mm. The supernatant was discarded and the pellet was resuspended in a quantity of formalin PBS sufficient to create a suspension with an optical

the body and death occurred within 3 wk (Arseculeratne, 1962). A closely related variant of A. capsulatus was isolated from lung, liven and spleen of free-ranging snowshoe hares (Lepus americanus) in Alaska (USA) (Zarnke and Schlaten, 1988). Little natural

AND METHODS

Antiserum

inThe 518

ZARNKE

ET AL.-ACTINOBACILLUS

reading of approximately 580 units as determined by a spectrophotometer (model 8003, Klett Manufacturing Co., Inc., New York, New York 10001, USA). The volume of diluent required to attain this reading was approximately 20 times the volume of the pellet. The antigen suspension was therefore considered to be a 1 :20 dilution. Based upon comparison to standard McFarland nephelometer turbidity solutions, this antigen suspension contained approximately 9 x 10 bacteria per ml. Antigen suspensions were tested for sterility before use. Agglutination reactions were performed on glass slides with 30 individual 2 cm inside diameter raised ceramic rings (American Scientific Products, McGaw Park, Illinois 60085, USA). A standard volume (0.04 ml) of antigen suspension was placed on the slide and mixed with the

collection were of the chi-square

serum

Corresponding values berta were 51 of 111 were not significantly

density

to

be

tested.

A combination

of

equal

vol-

umes (0.04 ml) of antigen and serum was selected as our screening dilution. This dilution was assigned a value of 1:40. After 4 mm of gentle rocking, the mixture was evaluated for degree of agglutination and assigned a value from negative (0) to four (4+). Sera which exhibited agglutination at 1:40 dilution were considered to provide evidence of previous exposure to A. capsulatus, and were classified as “positive.” All others were classified as “negative.” Sera from apparently healthy, free-ranging hares from interior Alaska were collected from 1979 to 1986 by Alaska Department of Fish and Game personnel (Fairbanks, Alaska 99701, USA) or wildlife professionals from other agencies. Trapping effort was not uniform between years or collection sites. Hare sera from near Rochester, Alberta, Canada (1 13#{176}30’W;54#{176}15’N)were collected during 1976 by staff and graduate students from the University of Wisconsin-Madison (Madison, Wisconsin 53706, USA). Nineteen hare sera (representing a broad range of agglutination titers) were tested by means of enzyme-linked immunosorbent assay (ELISA) and western blot methods (Manning et al., 1986). The purpose of these tests were to (1) confirm agglutination results and (2) evaluate the degree of cross-reactivity relative to Pasteurella multocida. Actinobacillus capsulatus antigen was tested for agglutination against Francisella tularensis antiserum (Difco Laboratories, Detroit, Michigali 48232, USA) and A. capsulatus antiserum was tested for agglutination against F. tularensis antigen (Difco Laboratories). All sera from freeranging hares were also tested against F. tularensis antigen (Difco Laboratories). A titer of 1:20 was considered positive. Differences in antibody prevalence related to sex, capture location, and month and year of

SURVEY

CAPSULATUS

tested test

IN HARES

519

for significance by means (Johnson, 1980).

RESULTS

Antibody domestic

titer rabbit

for the reached

postinoculation

(P1)

level 20 days ranged from inverse number titer;

hares

and

high

was

still

at

P1. Titers in snowshoe 1:40 to 1:3,200. There

relationship of specimens

frequency tibody

known-infected 1 :8,000 4 days

frequency

of high prevalence from

between titer which exhibited

and that

of low

low

titers. for

Alaska

this

hares was an

titers

Overall

was

and serum

A.

capsulatus

98

of

239

anin (41%).

for hares from Al(46%). These values different (P> 0.25).

Prevalences based upon year of capture (hereafter referred to as “annulized” preyalences) exhibited significant variation between

(P

years




C 0

0

50

60

0.C

>‘0

0.

50 0

40

30 0 0

n-37

E

20

30

S

Cl)

n=33

20

n=1

5

10

n=1

7

1981

Year

0

antibody prevalence for Actinobacillus capsulatus in snowshoe hares (Lepus americanus) captured in interior Alaska with relative hare abundance. 1.

FIGURE

(64#{176}50’N;

Eielson

Comparison

147#{176}45’W)

Air

Force

50% (9 of 18); (64#{176}40’N; 147#{176}00’W)

trapping

All sites (Alaska,

sites

were USA).

40 km wright

from and

ception

each other Creamer’s

There

and

was

no


0.25).

(at 492 nm) of agglutinating

173).

where

>

multocida.

of

except Font Field Refuge

significant

prevalence (37%) and

female,

(78

Wainwnight,

was significantly locations (P

sex-specific 22 of 60

45%

5 km apart. at all locations,

of Fort

alence other

was

within 175 km of Fairbanks Collection areas were at least

are approximately were similar

MAY

were

Base

50% (20 of 39); Washington Creek (65#{176}10’N; 147#{176}55’W) 39% (33 of 84); and Delta Junction (63#{176}55’N; 145#{176}45’W) 26% (5 of 19). Total prevalence for these five major

APRIL

of serum

with

±

0.10 titers 1.14 0.09

test results pnoteinase hare

sera)

activity and few

to several A. P. multocida

occurred tested

when against

nor was there to F. tularensis hare

A. capF. tuany eviin any

sera.

DISCUSSION

(P (49%) were

Members serologically Rosendal, tibody

no signifisera with P.

ELISA mean optical density for five sera with no evidence antibody were 0.22 ± 0.06

of the

genus

Actinobacillus

are

cross-reactive (Maclnnes and 1987). It is possible that the anwhich

free-ranging in response

we

detected

hares was to exposure

in

sena

lus spp. other ever, isolation

than A. capsulatus. of A. capsulatus from

hares

and

(Zarnke

Schlater,

from

actually produced to an Actinobacil-

1988)

Howthree and

the

ET AL.-AC11NOBACILLUS

ZARNKE

results of ELISA and port our contention actions

reported

western blot tests that the serologic

here

indicate

supre-

exposure

to

A. capsulatus. A comparison Alaska (41%)

of and

that opportunity sulatus may America. body (Fig.

prevalences Alberta (46%)

for exposure be widespread

The

apparent

prevalence 2) may

as

LITERATURE

in

anti-

summer progresses the addition to the

reflect

population of large numbers of new-born hares which have not yet been exposed A. capsulatus.

Disease has long been considered one of many possible factors in the regular decline in hare 1978). The alized ulation exposure

antibody

density apparent

(Keith and correlation

prevalences

density suggests to A. capsulatus

and the

Windberg, of annuhare

pop-

possibility that may be density-

dependent. Effects of A. capsulatus infection on hare populations are difficult to evaluate. This bacterium is associated with mortality in hares (Zannke and Schlaten, 1988). However, the high antibody preyalences reported here (Fig. 2) indicate that large numbers of hares either (1) suffer no overt disease as a result of exposure, on (2) recover

from

infection. ACKNOWLEDGMENTS

The authors wish to thank Robert Mowrey for collecting and submitting sera for use in this study, Linda Schlater and Soren Rosendal for providing advice and reagents, and Jesse Venable for computer and statistical assistance. This

521

CITED

A preliminary report on actinobacillosis as a natural infection in laboratory rabbits. Ceylon Veterinary Journal 9: 5-8. 1962. Actinobacillosis in joints of rabbits. Journal of Comparative Pathology 72: 33-39. JOHNSON, R. R. 1980. Elementary statistics, 3rd ed. S. N.

ARSECULERATNE,

Duxbury

to

IN HARES

study was supported in part by Federal Aid in Wildlife Restoration Projects Number W-22-0 and W-23-1 and National Institutes of Health grant RRO 1234.

between suggests to A. capin North

decline

SURVEY

CAPSULATUS

Press,

1961.

North

Scituate,

Massachusetts,

pp. KEITH, L. B., AND L. A. WINDBERG. 1978. A demographic analysis of the snowshoe hare cycle. Wildlife Monograph 58. The Wildlife Society, Washington, D.C., 70 pp. MACINNES, J. I., AND S. ROSENDAL. 1987. Analysis of major antigens of Haemophilus (Actinobacillus) pleuropneumoniae and related organisms. 606

Infection

and

MANNING, L.

P.

J.,

Immunity M.

A.

1986.

LEARY.

55:

1626-1634.

NAASZ,

D.

DELONG,

Pasteurellosis

S.

AND

in

laboratory

rabbits: Characterization of lipopolysaccharides of Pasteurella multocida by polyacrylamide electrophoresis, immunoblot techniques and zyme immunoassay. Infection and Immunity

gel en53:

460-463.

J.

PHILLIPS,

manual

(eds.).

Williams

Maryland,

pp.

partment,

Cheyenne,

R. L.,

AND

L.

americanus)

Diseases for

from

24:

SCHLATER.

in

and

R.

Journal

1988.

Wyo-

Fish

De-

pp.

1988.

snowshoe

5 April

Wilkins

353

176-177.

publication

1, N.

and

wildlife

Game

Alaska.

Vol.

570-575.

of

Wyoming,

in free-ranging

bacillosis

Bergey’s

In

bacteriology,

E. T. 1982. Diseases 2nd ed. Wyoming

ming,

Received

Brucellaceae.

C. Holt

Baltimore,

THORNE,

ZARNKE,

1984.

J.

and

Krieg

Co.,

E.

of systematic

hares

of

Actino(Lepus Wildlife