SUSCEPTIBILITY OF THE SIBERIAN POLECAT TO SUBCUTANEOUS AND ORAL YERSINIA PESTIS EXPOSURE
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Journal of Wildlife Diseases, 37(4), 2001, pp. 746–754
䉷 Wildlife Disease Association 2001
SUSCEPTIBILITY OF THE SIBERIAN POLECAT TO SUBCUTANEOUS
AND ORAL YERSINIA PESTIS EXPOSURE
Kevin T. Castle,1,5 Dean Biggins,1 Leon G. Carter,2 May Chu,2 Kim Innes,3 and
Jeffery Wimsatt4
1
USGS, Midcontinental Ecological Science Center, 4512 McMurray Boulevard, Fort Collins, Colorado 80525, USA
2
Bacterial Zoonoses Branch, Centers for Disease Control and Prevention, Fort Collins, Colorado 80521, USA
3
Department of Biometrics and Preventive Medicine, University of Colorado Health Sciences Center,
East Ninth Avenue, Denver, Colorado 80262, USA
4
Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado 80523, USA
5
Corresponding author (email: castlekt@lamar.colostate.edu), present address: 2411 Dalton Drive, Fort Collins,
Colorado 80526, USA
ABSTRACT: To determine if the Siberian polecat (Mustela eversmannii) represents a suitable
model for the study of plague pathogenesis and prevention in the black-footed ferret (Mustela
nigripes), polecats were exposed to 103, 107, or 1010 Yersinia pestis organisms by subcutaneous
injection; an additional group was exposed to Y. pestis via ingestion of a plague-killed mouse.
Plague killed 88% of polecats exposed to Y. pestis (71% mortality in the 103 group, 100% mortality
in the 107 and 1010 groups, and 83% mortality in the mouse-fed group). Within the challenged
group, mean day of death post-challenge ranged from 3.6 to 7.6 days; all polecats died on or
before day 12 post-challenge. Animals receiving the lowest parenteral dose survived significantly
longer than those receiving higher parenteral doses. Within challenged animals, mean survival
time was lower in those presenting with significant weight loss by day 3, lethargy, and low fecal
output; time to onset of lethargy and other signs was also related to risk of dying and/or plague
dose. Six polecats developed serum antibody titers to the Y. pestis F1 protein. Three seropositive
polecats survived the initial challenge and a subsequent exposure to a plague-killed mouse, while
two seropositive animals later died. This study confirms that the Siberian polecat is susceptible
to plague and suggests that this species will offer an appropriate surrogate for black-footed ferrets
in future plague studies and related vaccine trials.
Key words: Black-footed ferret, experimental infection, ferret, Mustela eversmannii, Mustela
nigripes, plague, Siberian polecat, Yersinia pestis.
INTRODUCTION from 12 to 1.2 ⫻ 107 Y. pestis organisms
and challenged two Siberian polecats (M.
The black-footed ferret (Mustela nigri- eversmannii) with 12 or 120 Y. pestis or-
pes) recovery program has successfully ganisms. None of the animals developed
employed a captive breeding strategy to clinical signs of plague, but domestic fer-
produce surplus animals for reintroduction rets exposed to 1.2 ⫻ 103 or more Y. pestis
into native habitat. Unfortunately, plague organisms developed serum antibody ti-
(caused by Yersinia pestis) is common ters. Based on information available at that
throughout many of the proposed black- time, ferrets and polecats were thought to
footed ferret release sites (Ubico et al., be resistant to Y. pestis, so the authors sug-
1988). Plague causes periodic and some- gested that black-footed ferrets are unlike-
times dramatic die-offs of prairie dogs ly to succumb to plague.
(Cynomys spp.) (Menkens and Anderson, The death of a black-footed ferret due
1991), and may adversely affect reintro- to a Y. pestis infection (Williams et al.,
duction efforts by reducing the ferret prey 1994) demonstrated the susceptibility of
base and through direct mortality of black- the species to plague. Recent multiple
footed ferrets. deaths of black-footed ferrets at the Pueb-
The susceptibility of some Mustela spp. lo Chemical Depot (Colorado, USA; D.
and Mustela spp. hybrids to plague has Biggins, unpubl. data) due to ingestion of
been recently investigated. Williams et al. prairie dog meat contaminated with Y. pes-
(1991) challenged groups of domestic fer- tis have underscored the potential hazard
rets (M. putorius furo) with doses ranging of plague to the species. Two Siberian
746CASTLE ET AL.—EXPERIMENTAL PLAGUE IN SIBERIAN POLECATS 747
polecats also died from plague at Pueblo, sure as a simulated flea bite, (3) whether
illustrating that both species can suffer animals that survive exposure to Y. pestis
mortality from plague, and that species develop sufficient immunity to reduce
differences in plague susceptibility, if pre- their risk of dying from a second exposure,
sent, are relative. Two black-footed ferrets (4) the dynamics of clinical Y. pestis infec-
and two polecats developed serum anti- tion, and (5) the optimal challenge dose in
body titers to Y. pestis at Pueblo and sur- polecats to use during plague-vaccine eval-
vived, demonstrating that both species can uations.
survive some level of Y. pestis exposure (D.
MATERIALS AND METHODS
Biggins, unpubl. data). However, the min-
imum lethal dose, lethal routes of expo- General
sure, and clinical signs of illness have not All procedures were approved by the Animal
been characterized for pure-bred Siberian Care and Use and Biosafety Committees at
polecats or for black-footed ferrets ex- Colorado State University (CSU; Fort Collins,
posed to a Y. pestis strain with well-de- Colorado, USA). The experiment was conduct-
ed between 21 October and 21 December
fined virulence factors. 1997.
The Siberian polecat has been used as
an investigational model for a number of Animals
studies applicable to the black-footed fer-
Siberian polecats used in this study were
ret (Hill and Carpenter, 1982; Powell et captive-bred descendants (six generations in
al., 1985; Williams et al., 1991). The two captivity) of animals wild-caught in Eastern In-
animals are similar behaviorally and mor- ner Mongolia, China. Thirty-five animals (20
phologically, and conspecificity has been females and 15 males) from 1 to 3 yr-old were
used. Polecats were stratified based upon
debated (Anderson et al., 1986; O’Brien et
founder female and breeding pedigree, and
al., 1989). If, however, the Siberian polecat randomly assigned to five treatment groups of
has developed some resistance to Y. pestis seven animals.
due to its association with the bacillus over Polecats were moved from the Pueblo
evolutionary time, then it may be an un- Chemical Depot breeding facility to a biosafety
level-3 containment facility at Colorado State
suitable surrogate for black-footed ferrets
University (Fort Collins) 1 wk prior to the start
in plague-challenge studies. Yersinia pestis of the experiment. Polecats were housed indi-
is thought to have originated in the Old vidually in stainless steel rabbit cages (0.5 m ⫻
World (Poland and Barnes, 1979), and it 0.5 m ⫻ 0.5 m); pans of wood shavings were
was isolated from dead polecats in central placed under each cage to collect feces and
urine. Each cage contained a hiding tube con-
Asia as early as 1911 and 1929 (Peshkov,
structed of a 35 cm long ⫻ 10 cm diameter
1954). The polecat may be a secondary piece of plastic pipe fitted with an endcap. An-
carrier of plague over its natural range, imals were provided with 60 g of commercial
and may therefore have a role in the nat- mink chow each day, and had continual access
ural epidemiology of plague (Kydyrbaev, to water. Constant ambient temperature (21–
23 C) and photoperiod (12L:12D) were main-
1988).
tained.
To study the suitability of Siberian pole-
cats as surrogates for black-footed ferrets Body temperature
in studies of plague, we exposed Siberian
Body temperature (TB) of each animal (un-
polecats (M. eversmannii dauricus) to a anesthetized) was determined by placing the
well-characterized, virulent strain of Y. animal in an 8 cm diameter cylindrical wire-
pestis via subcutaneous injection or inges- mesh tube and inserting a K-J thermocouple
tion of a plague-killed mouse. Our objec- (Fluke Company, Inc., Everritt, WA, USA) 2.5
tives were to determine (1) the effect of cm into the rectum and allowing it to equili-
brate for approximately 15 to 20 sec. Temper-
dose and route of exposure on mortality, ature was read on a Fluke telethermometer
(2) whether eating Y. pestis-infected dead (Fluke Company, Inc., Everritt, WA, USA).
rodents provides a similar level of expo- The thermocouple was decontaminated with a748 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001
10% bleach solution and thoroughly rinsed be- fed a 5-wk-old male Swiss-Webster outbred
tween animals. strain mouse (Centers for Disease Control and
Prevention; CDC) that had died of Y. pestis
Blood collection and physical examination (strain CO963188, cat isolate) infection under
standard CDC protocols, and will be referred
On the day of plague exposure (day 0), and
to as the ‘‘mouse-fed’’ group. The Y. pestis iso-
on days 3, 9, 15, and 21 post-exposure, all an-
late was chosen because its virulence factors
imals were anesthetized with isoflurane (AEr-
have been well-characterized by CDC and it
rane, Fort Dodge, Iowa, USA) prior to blood
has the same virulence features of an isolate
collection and physical examination. Anesthesia
used in a previous study conducted by CDC
was induced with 5% isoflurane and 4 l O2/min
researchers (Gasper et al., 1993).
while the polecat was in its plastic hiding tube,
and was maintained by mask with 2 to 3% is-
oflurane in oxygen at 1.5 l/min using a bain Daily observations
nonrebreathing circuit. While the animals were
anesthetized, 1 to 3 ml of blood were collected Animals were observed and scored twice dai-
by jugular venipuncture. Blood was also col- ly for the first week after inoculation, and once
lected from animals just prior to euthanasia daily thereafter. Each day, the following criteria
when possible. One ml of blood was placed into were subjectively scored: behavior (alert, ag-
a tube containing sodium EDTA, and a com- gressive, lethargic, non-responsive); physical
plete blood cell count (CBC) and differential status (coat groomed or not, molting, abnormal
white blood cell count were performed. The vocalizations, ataxic or not); amount and ap-
remaining sample was placed into a glass serum pearance of feces (normal, diarrheic, bloody)
tube, allowed to clot for approximately 2 hr, and respiration (rate, presence of cough,
then centrifuged to recover the serum. wheezing, or dyspnea); and the presence of oral
Aliquots of serum were analyzed immediate- or nasal discharge or other abnormalities. Food
ly or quick-frozen and stored at ⫺70 C until intake was subjectively assessed, but was not
analyzed. Hematologic parameters were deter- quantitated because many polecats dispersed
mined by the Clinical Pathology Section of the their food throughout their cage and onto the
Veterinary Teaching Hospital at CSU. Param- floor, making individual animal measurements
eters measured included red blood cell count difficult.
(RBC), white blood cell count (WBC), platelet Polecats were anesthetized with isoflurane as
count, differential count, hemoglobin concen- described above and euthanized with an intra-
tration (g/dl), and packed cell volume (PCV, cardiac dose of 3 ml sodium pentobarbital
%). Standard red cell indices were calculated, (Beuthanasia D, Shering-Plough Animal
including mean corpuscular volume (MCV, fl), Health Corporation, Union, New Jersey, USA)
mean corpuscular hemoglobin (MCH, pg), and if the animal exhibited any of the following:
mean corpuscular hemoglobin concentration body temperature higher than 41.7 C; no re-
(MCHC, g/dl). sponse to stimuli; blood from any orifice; cya-
Physical examinations were performed on notic mucous membranes; ataxia or other se-
anesthetized animals, and included observation vere neurological signs.
of the oral cavity and eyes for lesions, swelling,
mucosal injection, and icterus; palpation of the Necropsy
spleen, liver, retropharyngeal, axillary, inguinal,
and popiliteal lymph nodes, and inspection of Polecats that died or were euthanized were
the subcutaneous inoculation site for swelling frozen at ⫺70 C until necropsy. All necropsies
and necrosis. were conducted at the conclusion of the exper-
iment at the Bacterial Zoonose Branch diag-
Dosing regime
nostic laboratory of the CDC (Fort Collins,
After blood collection and physical exami- Colorado, USA). At necropsy, we noted and
nation on day 0, anesthetized polecats were subjectively scored external and internal le-
dosed with Y. pestis. Group 1 animals received sions, hemorrhage, swelling, lymphadenopathy,
no inoculation, and served as controls. Groups and changes in spleen and liver appearance and
2, 3, and 4 were inoculated subcutaneously size, relative to a polecat that had died of other
(s.c.) in the right inguinal region with 103, 107, causes. Spleen and liver samples were smeared
and 1010 Y. pestis (strain CO963188, cat isolate) onto two-well microscope slides for direct fluo-
organisms, respectively, and will be referred to rescent antibody (DFA) analysis (see below).
as the ‘‘103-,’’ ‘‘107-,’’ and ‘‘1010-dose’’ groups. Unfortunately, samples were not collected for
The 103 dose was given to simulate a flea bite histopathology prior to freezing of polecat car-
(Burroughs, 1947). Group 5 animals were each casses.CASTLE ET AL.—EXPERIMENTAL PLAGUE IN SIBERIAN POLECATS 749
Recovery of Yersinia pestis test in evaluating the effects of categorical var-
iables on dichotomous variables, and the AN-
To isolate Y. pestis from dead polecats, an
OVA test to examine the effects of categorical
asceptically collected section of liver was
variables on continuous outcomes. Multiple
streaked onto 6% sheep blood agar (SBA).
Cox regression was employed to determine the
Plates were incubated at 37 C for 24 to 48 hr,
independent effects of plague dose and route,
and examined for Y. pestis colonies, which were
and to assess the influence of potential con-
1 to 2 mm in diameter, gray-white to yellow in
founders, including gender and founder fe-
color, with raised irregular ‘‘hammered’’ shiny
male, on survival time. We also assessed the
surface and edge. Cultures were confirmed by
effect of Y. pestis dose and route on the devel-
bacteriophage lysis and supported by direct
opment of 11 clinical signs, including signifi-
fluorescent antibody (DFA) testing, following
cant weight loss (defined as ⬎10% from base-
standard CDC procedures (Chu, 2000).
line), fever (defined as maximum temperature
Yersinia pestis virulence
increase from baseline), lethargy, ataxia, un-
kempt fur, low fecal output, bloody diarrhea,
The virulence of the inoculating strain of Y. green diarrhea, wheezing, coughing, and dys-
pestis CO963188 was determined by eight ⫻ pnea. In the text, results are presented as the
eight titration in ICR outbred mice. A 24-hr mean ⫾ one standard error (in parentheses).
culture of CO963188 was diluted in normal sa-
line to McFarland’s turbidity standard #1 and RESULTS
then serially ten-fold diluted. Samples of each
dilution were spread on agar plates to deter- Because all 12 polecats initially chal-
mine colony forming units. Each of eight di-
luted inoculum, 100 to 107 cfu/ml, was injected lenged with a plague-killed mouse on day
s.c. at 0.1 ml volumes into eight mice/dilution. 0 (n ⫽ 7) or day 28 (n ⫽ 5) exhibited sim-
The LD50 was determined by the method of ilar responses, in terms of day of death
Reed and Meunch (1938) to be between 1 to (ANOVA; P ⫽ 1.0), maximum body tem-
100 cfu. CO963188 was also determined to perature, (ANOVA; P ⫽ 0.70) and subjec-
contain the full complement of Y. pestis plas-
mids by electrophoresis, and was positive by tively scored behavioral, physical, and fecal
Congo red agar (Chu, 2000). changes, results for those twelve animals
were pooled for all subsequent analyses.
Serology None of the control animals died during
Antibodies in serum of plague-exposed pole- the first 21 days of the experiment. One
cats were detected by passive hemagglutination control animal and one mouse-fed survivor
(PHA) test, following standard CDC methods died between days 21 and 28. The absence
(Chu, 2000). A titer of ⬎1:10 was considered
of clinical signs, gross lesions, and isolation
positive. All positive sera were verified by re-
peat titration by PHA and by inhibition for of Y. pestis indicated that plague was not
specificity. the cause of death in either case. All in-
fected animals that died during the exper-
Second plague challenge iment were Y. pestis culture positive and
Twenty-eight days after the initial Y. pestis phage lysis positive, providing strong evi-
challenge, five controls and three animals dence that plague was the cause of death.
which survived the initial challenge were each Mortality was significantly higher in
fed a plague-killed mouse (as above). Polecats
were sampled, monitored and scored on a daily
each experimental group than in the con-
basis for 21 d as described above. trols (Chi-square test, P ⬍ 0.016 for all in-
dividual comparisons), suggesting that
Statistical analyses even a relatively low Y. pestis does (103
All data were analyzed using the SAS statis- organisms) increased the risk of dying.
tical package (SAS Institute, Inc., Cary, NC, Overall, 29 of 33 (88%) polecats exposed
USA) or SPSS for the Macintosh (SPSS Inc., to Y. pestis died (Table 1). All polecats that
Chicago, Illinois, USA). Kaplan-Meier survival died, regardless of dose, died on or before
analysis (log rank test) was performed to deter-
mine the unadjusted effects of Y. pestis dose day 12 post-challenge. Considering only
and route of administration on survival time. In animals that died from Y. pestis infection,
our univariate analyses, we used the Chi-square the average day of death was significantly750 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001
TABLE 1. Numbers of Siberian polecats that died
after exposure to Yersinia pestis, and mean ⫾ SE days
to death.
Died/total
Route and dose exposed Day of deatha
Subcutaneous
103 5/7 7.61 ⫾ 1.7
107 7/7 3.62 ⫾ 0.3
1010 7/7 4.52 ⫾ 0.9
Mouse-fed 10/12 4.02 ⫾ 0.5
Total 29/33 Overall 4.6 ⫾ 0.5
a Means within a column followed by different numerical su-
perscripts are significantly different (Fisher’s protected least
significant difference, P ⬍ 0.05).
FIGURE 1. Comparison of the proportion survi-
higher in the 103 group than all other vorship of Siberian polecats exposed to Yersinia pestis
groups (Table 1). via subcutaneous injection of 103, 107, 1010 organ-
Within the subcutaneously challenged isms, or via ingestion of a plague-killed mouse. The
group, survival time fell progressively and proportion alive at the end of each day is presented.
See text for statistical comparisons among groups.
significantly with increasing Y. pestis does
(Fig. 1). Animals receiving the lowest dose
(103) had significantly longer survival times a lower maximum TB than those which
than those receiving the two higher doses died (ANOVA, P ⫽ 0.034). Similarly, the
(107 and 1010) and those ingesting plague- maximum increase in body temperature
killed mice (Kaplan Meier log rank test, P above baseline of animals that survived
⬍ 0.001). Gender and founder lines were was less than that of animals which died
not significantly related to survival times of (P ⫽ 0.008). Body temperature of three
Siberian polecats (log rank test, P ⬎ 0.05). survivors of the initial challenge increased
Clinical signs of illness that were signif- by about 1.4 C after the second challenge,
icantly associated with shorter survival whereas TB of the five polecats exposed to
times over all subcutaneous Y. pestis doses Y. pestis for the first time on day 28 in-
were greater than 10% weight loss by day creased by about 2.8 C; that difference in
3 post-challenge (log rank test, P ⫽ 0.012), TB increase approached significance (AN-
low fecal output (log rank test, P ⫽ 0.015) OVA, P ⫽ 0.06).
and lethargy (log rank test, P ⬍ 0.001). Among the challenged animals, Y. pestis
Maximum body temperature (TB) at- dose significantly affected the onset of
tained by experimental animals was signif- lethargy (log rank test, P ⫽ 0.026), occur-
icantly higher than that attained by the rence of low fecal output (log rank test, P
control group (ANOVA, P ⬍ 0.001), how- ⫽ 0.001), and green diarrhea (log rank
ever there were no significant differences test, P ⫽ 0.017), with onset of signs in an-
in maximum TB among the experimental imals receiving the 103 dose occurring lat-
groups (ANOVA, P ⫽ 0.42). Two animals er than in those receiving 107 or 1010 dos-
attained TB of over 41.7 C before they es of Y. pestis. Other signs, including
were euthanized. Of the 29 polecats that wheezing, coughing, dyspnea, bloody di-
died, 27 had TB over 40.6 C. arrhea, and ataxia, did not appear strongly
The average increase in TB above base- related to either survival time or Y. pestis
line (day 0) of the experimental groups was dose. At no time during the trial period
significantly higher than that of the control did any controls present with lethargy,
group (ANOVA, P ⬍ 0.001). Animals that ataxia, or significant weight loss, or show
survived the Y. pestis challenge exhibited any respiratory or gastrointestinal signs.CASTLE ET AL.—EXPERIMENTAL PLAGUE IN SIBERIAN POLECATS 751
Four of 33 Y. pestis-exposed polecats ex- rounded spleen (23 of 29 animals) and pe-
hibited nasal discharge prior to death; two techial hemorrhages on the skin (21 of 29
were in the 103-dose group and two were animals). Petechial hemorrhage occurred
in the 1010-dose group. Four of seven 103- in both subcutaneously injected animals
dose animals and three of seven 107-dose and those fed plague-killed mice. Other
animals developed red, swollen areas noteworthy abnormalities included hem-
around the inoculation site by day 3. All orrhage in the intestinal mesenteries (15
1010-dose animals developed similar le- of 29 animals) and yellow-tinged liver (9
sions by day 3, and one 1010-dose polecat of 29 animals). The inoculation site of
developed open sores around the injection most polecats was marked by hemorrhage
site. and necrosis (15 of 19 inoculated animals).
Our assessment of blood parameters
DISCUSSION
was hindered because most animals be-
came moribund rapidly and died. Data Plague killed a high percentage (88%)
suggest the development of anemia and of Siberian polecats in this study, regard-
thrombocytopenia, with lymphocytosis, less of dose or route of exposure. In pre-
neutrophilia and a left shift in Y. pestis- vious studies, all (n ⫽ 8) domestic ferrets
challenged animals. Those signs are con- survived subcutaneous challenge with up
sistent with a response to an acute bacte- to 107 Y. pestis organisms, as did two Si-
rial infection. berian polecats exposed to 12 or 120 or-
Yersinia pestis organisms were clearly ganisms (Williams et al., 1991). More re-
visible on blood smears of three polecats cently, E. S. Williams (pers. comm.) ex-
(one each from the 107, 1010, and mouse- posed black-footed ferret ⫻ Siberian pole-
fed groups); all three of animals died on cat hybrids to Y. pestis by subcutaneous
day 3 post-challenge, after blood collec- injection or by feeding plague-killed mice.
tion. All hybrids dosed with 103 Y. pestis sur-
All pre-infection serum samples were vived, as did three of four challenged with
negative. Six of 33 polecats exposed to Y. 104 organisms; nine of twelve orally-dosed
pestis developed antibody titers, including hybrids became infected and died, how-
four animals exposed to 103 organisms and ever. The mortality rate of orally-chal-
two animals that ingested infected mice. lenged hybrids was therefore very similar
Two of the seropositive 103-dose animals to that seen in our pure Siberian polecats.
subsequently died before the next sched- Subsequent work by E. S. Williams (pers.
uled bleeding; blood collected at the time comm.) resulted in 100% mortality of four
of death was seronegative. Titers were first black-footed ferrets exposed to about 800
detected 9 days after exposure (range: 1: organisms of Y. pestis by subcutaneous in-
64 to 1:2048, n ⫽ 6), and peaked 21 days jection.
after exposure (range: 1024 to 2048, n ⫽ Survival time of species susceptible to
4). Titers initially decreased after the sur- plague has been shown to vary with route
viving polecats were re-exposed to Y. pestis of exposure and dose (Poland and Barnes,
on day 28. On days 9 and 15 after the sec- 1979). Mean survival time (4.5 days) of Si-
ond exposure, all three polecats exhibited berian polecats in this study varied with
titers of 1:256. Titers again peaked on day dose, but was comparable to that observed
21 post-exposure (range 1:1024 to 1:2048, in a variety of susceptible species, includ-
n ⫽ 3). Titers for those three animals ing: black footed ferrets, black-footed fer-
ranged from 1:256 to 1:512 at the final ret ⫻ Siberian polecat hybrids (E. S. Wil-
blood draw, 53 days after the second ex- liams, pers. comm.), domestic cats (Gasper
posure. et al., 1993) rock squirrels (Spermophilus
The most common gross lesions ob- variegatus) (Quan et al., 1985), and grass-
served at necropsy were an enlarged, hopper mice (Onychomys leucogaster)752 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001 (Thomas et al., 1988, 1989). Mortality in 50 mo after the second challenge (Quan et humans typically occurs in 5 to 7 days in al., 1985). untreated cases of bubonic plague, and in The time frame of a second Y. pestis ex- 1 to 2 days following respiratory exposure posure may influence survival also. It is (Poland and Barnes, 1979). possible that if enough time elapses be- It has been suggested that a subcuta- tween exposures, antibody titers could neous injection of Y. pestis may not truly drop below the level at which they are pro- mimic a flea bite (Perry and Fetherstone, tective. Our animals were re-challenged 1997) independent of the possible differ- within 30 days of their initial exposure, so ence in the number of organisms injected. their antibody titers may have been un- At the typical body temperatures of a flea, naturally high. The minimum protective which roughly correspond to ambient tem- level is undetermined for most species, but perature less than 37 C, the bacterium titers of 1:128 appear to be protective in does not produce the F1 virulence factor, humans (Poland and Barnes, 1979). Do- and needs first to be phagocytized by the mestic ferrets maintained detectable anti- host’s WBCs. After reproducing in the bodies for at least 219 d post-exposure host’s cells at approximately 37 C, the bac- (Williams et al., 1991), and our surviving terium possesses the F1 protein, and be- polecats maintained detectable titers for at comes fully virulent. Our Y. pestis inocu- least 96 days post-exposure. lum was incubated at 37 C in the lab, so Generally, seroconversion after Y. pestis therefore may have caused a greater (or at exposure is associated with a greater least faster) mortality than may be seen af- chance of survival (Rust et al., 1971; Gasp- ter a true flea bite. Other Y. pestis viru- er et al., 1993); however, serological re- lence factors exist, and their production sponses are variable within and between may also differ between lab-reared and species. Siberian polecats that survived ex- flea-incubated Y. pestis. posure to Y. pestis in our study developed In our study, polecats with prior plague antibodies, and two animals that died also exposure appeared to be protected against seroconverted; seroconversion was not de- a second challenge; because of the small tected in the other 27 polecats that died. sample size these results must be viewed Three black-footed ferret ⫻ Siberian pole- cautiously. All three animals that survived cat hybrids that survived subcutaneous the first challenge with Y. pestis survived a challenge with 103 Y. pestis seroconverted, subsequent exposure via ingestion of a as did three of four hybrids dosed with 104 plague-killed mouse. A small and transient organisms; one orally-challenged hybrid increase in TB was the only measurable re- that died seroconverted, but the three sponse seen in animals after their second orally-challenged survivors failed to sero- exposure; there were no changes in behav- convert (E. S. Williams, pers. comm.). All ioral, physical, or fecal attributes. Antibody dogs exposed to Y. pestis seroconverted by titers in the pre-exposed animals were day 8 post-exposure, and all survived (Rust high before and after the second chal- et al., 1971). Two of five cats exposed by lenge, so it is likely that at least part of the Rust et al. (1971) seroconverted and sur- protection was conferred by adequate hu- vived, one cat seroconverted and died, and moral immunity. Few experiments (out- two cats died without seroconverting. Ten side of vaccine trials) have looked at the of ten cats that survived an oral challenge responses of previously plague-challenged seroconverted, while four of six animals animals to re-exposure. Two rock squirrels that died had seroconverted (Gasper et al., that were seropositive at capture survived 1993). Two cats died in that study without consecutive exposures to approximately seroconverting. Rodents (grasshopper 104 and 106 organisms; one of the squirrels mice and rock squirrels) that survived Y. maintained detectable antibodies at least pestis challenges were also more likely to
CASTLE ET AL.—EXPERIMENTAL PLAGUE IN SIBERIAN POLECATS 753
have antibodies than those that died, but had TB of 40.2 to 41.2 C, supporting this
some animals that survived did not sero- view. Results of the present and previous
convert (Quan et al., 1985; Thomas et al., studies suggest that body temperature
1988), a phenomenon also seen in black- alone should not be used as a criterion for
footed ferret ⫻ Siberian polecat hybrids euthanasia in laboratory plague-challenge
(E. S. Williams, pers. comm.) but not ob- trials. Animals that survived plague expo-
served in the present study. sure often exhibited body temperatures
Two polecats dosed with 103 Y. pestis similar to animals that died. Animals ex-
were seropositive early on day 9, but sub- hibiting high TB in combination with ab-
sequently died on days 9 and 12. Serum normal behavioral and/or physical signs
taken on the day of death, however, re- (e.g., lethargy and low fecal output) are
vealed no detectable PHA titer. This sug- not likely to survive, however, and inter-
gests that in the presence of circulating an- ventive treatment or euthanasia should be
tigen due to overwhelming disseminated considered in those cases.
bacterial infection, the antibody present Because high mortality rates were ob-
may not have been able to effectively pro- served in all the plague-challenged groups,
tect the animal. Furthermore, the anti- we were not able to calculate a minimum
body could have complexed with the mas- lethal dose or an LD50. However, the
sive amounts of free antigen, thus effec- LD50 is likely less than 103 with this de-
tively acting as an inhibitor to the PHA fined strain, and future plague-challenge
test. Unfortunately, the final serum sam- experiments should include much lower
ples from those animals were not of suf- doses if they are to determine an LD50.
ficient volume to test for the presence of Our results further indicate that a subcu-
free F1 antigen. High levels of circulating taneous exposure to 104 to 105 Y. pestis
antigen could also account for the initial organisms of the strain used in our study
decline in antibody titers observed after would be a reasonable challenge dose in
the three survivors were re-exposed to Y. plague vaccine trials, because exposure to
pestis. 107 Y. pestis was fatal to all polecats ex-
Few of the clinical signs of plague we posed to that dose, and a challenge dose
noted were significant predictors of im- should not kill all exposed animals.
pending death, and none were unique to Because Siberian polecats are highly
Y. pestis infection. Animals that lost ⬎10% susceptible to infection with the virulent
of their body mass in the first 3 d after strain of Y. pestis administered subcuta-
challenge, animals that had low fecal out- neously or via a plague-killed rodent, the
put, and those that became lethargic had Siberian polecat is a useful surrogate for
significantly shorter survival times. Those the black-footed ferret in plague-challenge
three signs are probably closely related, trials. The purebred polecats used in our
and may reflect anorexia after polecats study did not appear to exhibit resistance
were infected. Food intake was not quan- to Y. pestis postulated at the outset, but
tified during this study, but it was appar- succumbed to infection rapidly. Unfortu-
ent, based upon rate of refilling feed dish- nately, no published reports compare Si-
es, that Y. pestis-challenged animals usu- berian polecats and black-footed ferrets
ally decreased their food intake consider- exposed to the same doses of virulent Y.
ably. pestis at the same time. However, in light
Fever was an excellent, albeit nonspe- of the similar responses of black-footed
cific, indicator of plague infection, but did ferrets and Siberian polecats to accidental
not predict death. Gasper et al. (1993) re- ingestion of plague-infected prairie dog
ported that domestic cats dying from meat in Pueblo, the 100% mortality seen
plague attained a TB of 40.7 to 41.3 C, in parenterally challenged black-footed
while those that became ill but survived ferrets (E. S. Williams, pers. comm.), and754 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001
the high mortality seen in the present ferret. Yale University Press, New Haven, CT,
study, we expect black-footed ferrets to be pp. 21–33.
PERRY, R. D., AND J. D. FETHERSTON. 1997. Yersin-
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ACKNOWLEDGMENTS
CRC handbook series in zoonoses, Section A:
We would like to thank J. Steege, R. Heim- Bacterial, rickettsial, and mycotic diseases, J. H.
bichner, and K. Orlowski for their excellent and Steele (ed.). CRC Press, Boca Raton, Florida,
timely help with animal care and blood sample pp. 515–556.
collection, and L. Vap for help with clinical pa- POWELL, R. A., T. W. CLARK, L. RICHARDSON, AND
thology. We also thank E. S. Williams, M. Wild, S. W. FORREST. 1985. Black-footed ferret (Mus-
and two anonymous reviewers for helpful com- tela nigripes) energy expenditure and prey re-
ments on earlier versions of the manuscript. quirements. Biological Conservation 33: 1–15.
This research was supported by funds from QUAN, T. J., A. M. BARNES, L. G. CARTER, AND K.
U.S. Geological Survey/Midcontinent Ecologi- R. TSUCHIYA. 1985. Experimental plague in rock
cal Science Center. squirrels, Spermophilus variegatus (Erxleben).
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In Conservation biology and the black-footed Received for publication 17 November 1999.You can also read
