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SEROLOGIC SURVEY OF SELECTED ZOONOSES AND CANINE VIRAL PATHOGENS
IN GRIZZLY BEARS (URSUS ARCTOS) AND BLACK BEARS
(URSUS AMERICANUS) FROM ALASKA
Chomel B.B. 1, Chappuis G.2, Kasten R.W. 1, Soulier M.2, Kikuchi Y.', Zarnke R.L.3
Six cent quarante quatre Ochantillons de serum recoltes sur 480 ours grizzly et 40 ours bruns dAlaska entre 1988 et
1991 furent testes pour la presence d'anticorps contre diverses zoonoses et infections virales canines. La
prevalence chez les ours grizzly etait de 0% pour la parvovirose, 8.3% (40/480) pour la maladie de Cant, 14%
pour l'hapatite infectieuse, 16.5% pour la brucellose, 19% pour la tularOmie et 47% pour la trichinose. La
prevalence en anticorps variait chez les ours bruns de 0% pour la parvovirose et la maladie de Caffe a 27.5% pour
la trichinose et 32% pour la tularemie. La prevalence en anticorps anti-brucelliques et anti-tularOmiques etait
identique (2.5%) pour les grizzly et les ours bruns de la meme region. Des differences notables de prevalence
furent observees selon /'origin et Page des ours. Les anticorps anti-Gaffe et anti-hepatite infectieuse Otaient surtout
presents chez les ours de Kodiak et de la peninsule d'Alaska. Les anticorps anti-brucelliques etaient
principalement observes chez les ours de l'ouest et du Hord de !Alaska, et les anticorps anti-tularOmiques chez les
ours des regions centrales et arctiques. Un gradient croissant Sud-Nord en anticoprs anti-trichinosiques fut
observe. La seroprevalence augmentait avec rage pour la plupart des infections testees, mais pour quelques-unes
aucun anticorps n'Otait detectable chez les grizzly <2,5 ans. Les grizzly apparaissent etre d'excellentes sentinelles
pour la surveillance epiderniologique des zoonoses en Alaska.
INTRODUCTION
There are an estimated 30,000 grizzly bears (Ursus arctos) and 140,000 black bears (Ursus americanus) in Alaska
(USA). As predators and scavengers, bears may come in contact with agents of zoonotic diseases. Serologic
evidence of infection by Brucella spp. in Alaskan grizzly bears has been reported previously and in black bears
from other parts of North America. Bears can be infected by Francisella tularensis, the agent of tularemia, which is
endemic in Alaska. However, no extensive serosurvey of this infection in Alaskan bears has been performed.
Trichinosis is also known to be endemic in Alaska, and antibody prevalence >50% have been reported in brown
bears (Ursus arctos). Antibodies to canine distemper virus (CDV) and canine parvovirus (CPV) have been reported
from giant pandas (Ailuropoda melanoleuca) in China, and more recently for distemper from polar bears (Ursus
maritimus) from Alaska and Russia. Evidence of canine infectious hepatitis (CIHV) antibodies in Alaskan bears has
also been reported, but no information is available on seroprevalence in Alaskan grizzly and black bears of canine
distemper virus and canine parvovirus, infections which have been reported in Alaskan and Canadian wolves. The
objective of this study was to determine the seroprevalence of various zoonotic agents and canine viruses in
Alaskan bears.
MATERIAL AND METHODS
Personnel of the Alaska Department of Fish and Game and the U.S. Fish and Wildlife Service captured 480 grizzly
bears (GB) and 40 black bears (BB) in the course of performing population ecology studies between 1988 and 1991.
Sampling was opportunistic and some bears were captured more than once; 644 serum samples were available for
testing. 76 blood samples were collected from 40 BB in Interior Alaska on the Tanana Flats, south of Fairbanks.
The 568 GB blood samples were collected from 8 different areas : In southem Alaska, 79 samples were collected on
Kodiak Island, and 86 samples from the Alaska Peninsula (Katmai Coast (38 samples) and Black Lake (48
samples)). In Interior Alaska, 53 samples were collected in the Tanana Flats, Denali Park, and Fairbanks areas. In
Western Alaska, 40 samples came from Seward Peninsula and 99 from Noatak river drainage. In Northern
Alaska,133 samples came from northwestern Alaska, 6 from north central Alaska, and 72 from northeastern Alaska.
Blood samples were collected by femoral, saphenous or cephalic venipuncture. Serum was separated by
centrifugation and stored at -20°C until tested. Samples were collected from 25 (63%) of the 40 BB more than once :
4 BB had blood taken 4 times; 3 BB, 3 times, and 18 BB twice. Among the GB, samples were obtained 3 times
from 11 GB and twice from 67 GB. Serologic tests were performed at the Veterinary Public Health Lab., Davis,
California (brucellosis, tularemia and trichinosis) and at the Virology Lab., Rhone-Merieux, Lyon, France (CDV,
CPV, CIHV). Sera were tested for evidence of antibodies to : (1) Francisella tularensis (tularemia) using a
commercial slide agglutination test. Any titer 3 1:20 was considered positive, and positive serums were retested in
order to eliminate non-specific reaction. (2) Brucella spp. by buffered positive serum acidified card antigen test,
and, and positive serumsamples were also retested. (3) Trichinella spiralis, by enzyme linked immunosorbent assay
(ELISA). The ELISA was based on the official USDA method for pseudorabies, modified as follows. Briefly, 50111 per
well of a Trichinella spiralis ES antigen (511g/m1) produced by the Vet. Diagnostics Lab. , Ames, Iowa, USA at
1:1000 dilution in carbonate bicarbonate buffer, pH 9.6, was bound to 96-well flat bottom microtitration plates
(Linbro/Titertek plates (Flow Laboratories, Inc., McLean, Va, USA) by overnight
incubation at 4°C. Bear sera were diluted 1:40 in tris buffer (pH 7.4) containing 5% skim milk and 0.05% tween 20
and 0.01% bovine serum albumine fraction V (Sigma Chemical Co., St. Louis, Mo, USA). The peroxidase conjugate
was a raccoon anti-bear antibody at 1:500 (raccoon anti-bear IgG, Antibodies Inc., Davis, Ca, USA). The substrate
1 Department of Population Health & Reproduction, School of Veterinary Medicine, University of Califomia, Davis, Ca. 95616.
USA.
2 Laboratoire de Virologie, Rhone-Merieux, 254 rue Marcel Merieux, BP 7009. 69342 Lyon Cedex 07, France
3 Alaska Department of Fish and Game, 1300 College road, Fairbanks, Alaska 99701. USA.
was 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid;ABTS) (Sigma Chemical Co.). The reaction was stopped
after 30 min with 100 pl of a 0.1 M solution of hydrofluoric acid (pH 3.3). Each plate contained known positive and
negative control sera. The positive control serum was selected from a bear which was both positive by bentonite
flocculation and ELISA (optical density (0.D.)=1.0), and the negative controls were selected from 3 bears both
negative by ELISA (0.D.<0.1) and bentonite flocculation. Each serum was tested in duplicate and the mean of the
two absorbance values calculated. Microtitration plates were read at 410 and 450 nm respectively for test and
reference on a microelisa autoreader (MR 5000, Dynatech Laboratories, Inc. Chantilly, Va, USA). The cut-off point
for a positive test was determined at 0.D.= 0.3, which was the mean O.D. of the negative control population (all
bears from Kodiak Island) plus 3 standard deviations (SD) .(4) canine parvovirus by hemagglutination inhibition. A
viral suspension of 4 hemagglutining units in a 0.05 ml buffer was added to the various dilutions of the serum
samples to be tested for 1 hr at room temperature. Then, 0.05 ml of a suspension of 3x107 SRBC were added in
the plate wells and left overnight at 4°C. The reaction was read the next day. (5) canine distemper virus by
competitive ELISA. Briefly, 100p1 per well of capture monoclonal antibodies at a 1:1,500 dilution in carbonate
bicarbonate buffer, pH 9.6, was bound to 96-well flat bottom microtitration plates (ELISA Nunc Maxisorp, Nunc Inc.,
Rochester, N.Y., USA) by overnight incubation at room temperature. On cell culture plates, 50 pl of distemper virus
and 50 pl of each serum dilution (0.9, 1.8 and 2.7) and respective controls were incubated for 1 hr at 37°C with
permanent shaking. After 3 washes, 50 pl of the virus-serum mix was transfered on the monoclonal antibody
sensitized plates and incubated with shaking for 1 hr at 37°C. Then, 50 pl of the monoclonal antibody marked with
peroxydase were added in each well and plates incubated with shaking for 1 hr at 37°C. The plates were washed 3
times before 100 pl of substrate (orthphenylene diamine, Sigma Co.) was added. The reaction was stopped after
25 min with 50 pl of 2.5 M H2SO4 solution. Microtitration plates were read at 490 nm wavelength. Results were
expressed as O.D. percent compared to the control without serum (100%). Serum titers were given as log of the
reciprocal dilution with a 50% O.D. In order to validate the ELISA test and define the cut-off point (COP) for
seropositivity, 23 serum samples of bears with an ELISA titer30.8 were also tested by the classical serum-
neutralization (SN) test [4]. Titers by S.N. were usually lower than by ELISA, which led to defining the COP at 31.0.
Furthermore, ELISA positive samples and a random sample of negative serum samples were also tested using an
immunoperoxidase (IP) antibody test, which is similar to a fluorescence test, using IP instead of fluorescein
isothyocyanate. (6) canine hepatitis virus by serum-neutralization, using CAV type 2 and dog kidney cell line MDCK
(105 cells/ ml). Cytopathic effect on culture plates was read 7 days after infection, and titers were expressed in log10
protective dose 50% (PD50) on MDCK. Ages were estimated by examining cementum annuli of premolar teeth for
BB and GB. BB were classified in 4 inclusive age groups : 0-2 yr old, 2.5-4 yr old, 4.5 -8 yr old and 39 yr old. GB
were classified into 5 inclusive age groups : 0.5-2 yr (young bears), 2.5-4 yr (end of puberty,i.e subadult), 4.5-8 yr
(reproductively mature bears), 8.5-12 yr (prime reproductive capability), and 313 yr (old bears). GB from the Seward
Peninsula and McKinley Park were reported as being adults (i.e. 34.5 years). 62% (297/480) of the GB and 55%
(22/40) of the BB were females. Among the GB, 70 (12%) were <2 year old, whereas 24 (31.5%) of the BB were <2
year old. Demographic data were analyzed using Epi Info 6.02. Frequency distributions were obtained and chi-
square were calculated to obtain measures of association, and the statistical significance of such associations.
RESULTS
Overall seroprevalence in GB was 0% for CPV, 9% (43/480) for CDV, 14% (68/480) for CIHV, 16.5% (79/480) for
brucellosis, 19% (93/480) for tularemia and 47% (225/478) for trichinosis. Seroprevalence in BB ranged from 0%
for canine distemper and parvovirus to 32% (13/40) for tularemia (Table I). Canine parvovirus : None of the grizzly
and black bears tested had canine parvovirus antibody at a significant titer.
Canine distemper : Antibodies were found only in GB, with an overall prevalence of 8.3% (40/480). Antibody
prevalence was slightly higher in females (10%;29/294) than in males (6%; 11/186). Prevalence varied with the
origin of the bears (Table I). GB from Kodiak Island and the Alaskan Peninsula were more likely than GB from other
areas to be seropositive for canine distemper (RR=6.57, 95% C1=3.29, 13.12). Overall, among all 41 distemper
positive samples, 73% had titers 32.0, and of these, 77% (23/30) were from southern Alaska. Animals with
distemper positive samples were also more likely to be seropositive for CIH (RR=2.18, 95% CI=1.28, 3.72). Mean
age of seropositive GB was 12.5 ± 0.85 yrs, whereas mean age of seronegative GB was 8.4 ± 0.27 yrs. Age
prevalences for distemper antibody ranged from 1.5% in 0-2 yr-old GB to >10% in GB38.5 yr old. High titers were
mainly observed in GB >8 yr old. In southwest and western Alaska, all seropositive GB were >7 yr old, whereas, in
Northern Arctic, 40% of positive GB were £4 yr-old. Annualized prevalences for Kodiak Island were stable at 32%
(1988) and 29% (1989). They ranged from 2.6% to 7.6% in Northern Arctic.
Canine infectious hepatitis : CIHV antibody prevalence was 14 % (68/480) in GB and 7.5% (3/40) in BB.
Seroprevalence by sex was 12% (22/186) in males and 15.5% (46/294) in females for GB, and 5.5% (1/18) and 9%
(2/22) for BB. Prevalence also varied with the geographical origin and the age of the bears. For GB, it was the
highest on Kodiak Island (31%, 26/77), and averaged 10.5% in the other areas (range :7.5% to 12.5%). None of the
young GB (0-2 yr old) had CIHV antibodies. Mean age of seropositive GB was 12.23 ± 0.57 yrs, whereas mean age
of negative GB was 8.2± 0.28 yrs. Adult GB were more likely to be seropositive (RR=7.6, 95% C1=2.82,20.46) than
young and subadult bears. Conversely, none of the adult BB were seropositive. Annual prevalences varied for BB
from 0% in 1988 (0/9) and 1989 (0/22) to 4.5% (1/22) in 1990 and 8.7% (2/23) in 1991. For GB, overall prevalence
decreased from 19% (38/202) in 1988 and 21% (24/114) in 1989 to 11% (13/121) in 1990 and 8% (11/134) in 1991.
All BB tested more than once were seronegative. In GB, none of the 2 bears tested twice on Kodiak Island had CIHV
antibodies. In Interior Alaska, of the 11 GB tested several times, one adult was persistently positive and 2 other
adults were negative at the first collection and positive at the second. In Noatak river drainage area, of the 11 GB
tested more than once, one female adult was consistently positive, another GB was negative at the first blood
collection and strongly positive three years later. In the Arctic region, 5 adults were seropositive at both collections,
01.06.2
Epidemiol. sante anim., 1997, 31-32
two adults were positive at the first blood collection and negative at the next ones, and one GB was initially negative
and positive 3 years later.
Brucellosis : The overall prevalence of brucella antibodies was 16.5% (79/480) in GB and 2.5% (1/40) in BB.
Antibody prevalence was similar in males (17%) and females (14.5%) for both species. Brucella prevalence varied
widely by geographical areas (Table I). GB from westem and northern Alaska (59/277) were more likely to be
seropositive for brucellosis than GB from southwestern and central Alaska (20/203) (RR=2.16, 95% CI=1.35, 3.47).
Mean age of seropositive GB (8.7± 0.59) was similar to the mean age of seronegative GB (8.8 ± 0.29).
Seroprevalence increased from 6% in young GB (0-2yr old) to 17% in adults (34.5 yr old). Overall, the annual
prevalence decreased from 19% (39/202) in 1988 to 6% in 1990 (7/121), but increased in 1991 to 14.5% (19/131).
Yearly prevalence was consistent from one year to the other on Kodiak Island (12% (5/41) in 1988 and 8% (3/37) in
1989). A decrease was observed in Noatak river drainage from 37% (11/30) in 1988 to 15% (5/34) in 1991. In the
Arctic region, yearly prevalence also decreased from 20% (14/69) in 1988 to 8% (5/65) in 1990, but re-increased in
1991 to 17% (13/76), mainly in the central and northwestern Arctic areas (38%, 8/21). Among the 78 GB tested
more than once, 4 GB (5%) were consistently positive, 10 (13%) seroconverted and 7 (9%) became negative
between the first and the following collections
Tularemia : The overall prevalence of F. tularensis antibody was 19% (93/480) in GB and 32% (13/40) in BB.
Seropositive bears were also more likely to be positive for brucellosis (RR=2.33; 95% CI=1.57, 3.48).
Seroprevalence was similar in males (22.5%) and females (19%). Prevalence varied from 4% (3/77) on Kodiak
island to 10-15% in the Alaska Peninsula and Western Alaska and >30% in Interior Alaska and in the Arctic regions
(Table I). Prevalence (32%) was identical in BB and GB from Interior Alaska. GB from Interior Alaska and from the
Arctic regions (62/190) were more likely than GB from Western and Southwestern Alaska (31/290) to be seropositive
for F. tularensis (RR=3.05, 95% C1=2.07, 4.51). In BB, antibodies were found only in BB <9 yrs old. Mean age of
seropositive GB (7.33± 0.58) was slightly younger than mean age of seronegative GB (9.08± 0.29). Antibody
prevalence in GB was the highest in the age group 2.5-4 yr old and the lowest in the 0-2 yr old group. Young and
subadult GB(41/167) were 1.64 times more likely to be seropositive than adult GB (60/401) (95% CI=1.15, 2.34). On
Kodiak Island, only 3 GB were seropositive with a low titer (1:20). GB with high titers ( 31:160) were mainly from the
Arctic regions, Noatak river drainage and Interior Alaska. In Interior Alaska, the prevalence of bears with high titers
was similar in BB (7/21) and in GB (4/15). In BB, annual prevalences decreased from 39% (12/31) in 1988-89 to
20% (9/45) in 1990-91. The same decrease was observed in GB from Interior Alaska (40% (6/15) in 1988-89 and
24% (9/38) in 1990-91). For all regions, the yearly prevalences in GB increased from 11% (36/322) in 1988-89 to
26% (65/252) in 1990-91. Among the 78 GB tested more than once, 42 (54%) were negative at all blood collections,
8 (10%) were positive at all blood collections, 10 (13%) were positive at the first blood collection and negative at the
following ones, and 18 (23%) were negative at the first blood collection and positive at the following ones. Among
the BB, 12 (48%) were negative and 5 (20%) were positive at all blood collections, 3 young BB were positive at the
first collection and negative at the next one. One 1-yr old BB seroconverted the next year.
Trichinellosis : Trichinella spiralis antibody prevalence was 47% (225/478) in GB and 27.5% (11/40) in BB.
Seroprevalence was higher (RR=1.21, 95% CI= 1.01, 1.46) in males (51%, 104/204) than in females (42%,
132/314). Prevalence varied geographically with an increasing gradient from Southwest Alaska to the Arctic. All the
GB from Kodiak Island were seronegative and only one adult male from Katmai Coast (Alaska Peninsula) was
positive, with a low titer (0.D.<0.6). Conversely, GB from Black Lake (Alaska Peninsula), Interior Alaska and
Seward peninsula had a prevalence of 33% (42/128). Antibody prevalence was similar in GB (35%, 14/40) and BB
(27.5%) from Interior Alaska. GB from Noatak river drainage had a prevalence of 57% (50/87) and GB from the
Arctic regions had an overall prevalence of 89% (132/148). 70% of the GB with high titers (0.D. 30.9) were from the
Arctic regions and Noatak river drainage. In GB, antibody prevalence was similar in all age groups, with a mean age
of seropositive GB (8.96 ±0.38 yrs) close to the mean age of seronegative GB (8.57±0.37). In BB, 10 of the 11
positive BB were <5 yr old. Yearly prevalences in GB were constant at Noatak river drainage (57% in 1988 and
1989, 59% in 1991) and decreased moderately in the Arctic regions (91% in 1988 to 80% in 1990-91). Of the 78 GB
collected more than once, 50 (64%) tested positive and 18 (23%) tested negative at all blood collections; 10 (13%)
were negative at one blood collection and positive at the other. For the 25 BB collected more than once, 17 (68%)
tested negative at all collections, 5 (20%) BB became positive and 3 (12%) BB became negative at the second
blood sample collection.
Table I
Prevalence of Brucella, Francisella tularensis, Trichinella spiralis, canine distemper virus and canine
infectious hepatitis virus antibodie
s in black bears and Grizzly bears, Alaska, by geographical origin. 1988-1991
www.oie.int/doc/ged/D9225.PDF
IN GRIZZLY BEARS (URSUS ARCTOS) AND BLACK BEARS
(URSUS AMERICANUS) FROM ALASKA
Chomel B.B. 1, Chappuis G.2, Kasten R.W. 1, Soulier M.2, Kikuchi Y.', Zarnke R.L.3
Six cent quarante quatre Ochantillons de serum recoltes sur 480 ours grizzly et 40 ours bruns dAlaska entre 1988 et
1991 furent testes pour la presence d'anticorps contre diverses zoonoses et infections virales canines. La
prevalence chez les ours grizzly etait de 0% pour la parvovirose, 8.3% (40/480) pour la maladie de Cant, 14%
pour l'hapatite infectieuse, 16.5% pour la brucellose, 19% pour la tularOmie et 47% pour la trichinose. La
prevalence en anticorps variait chez les ours bruns de 0% pour la parvovirose et la maladie de Caffe a 27.5% pour
la trichinose et 32% pour la tularemie. La prevalence en anticorps anti-brucelliques et anti-tularOmiques etait
identique (2.5%) pour les grizzly et les ours bruns de la meme region. Des differences notables de prevalence
furent observees selon /'origin et Page des ours. Les anticorps anti-Gaffe et anti-hepatite infectieuse Otaient surtout
presents chez les ours de Kodiak et de la peninsule d'Alaska. Les anticorps anti-brucelliques etaient
principalement observes chez les ours de l'ouest et du Hord de !Alaska, et les anticorps anti-tularOmiques chez les
ours des regions centrales et arctiques. Un gradient croissant Sud-Nord en anticoprs anti-trichinosiques fut
observe. La seroprevalence augmentait avec rage pour la plupart des infections testees, mais pour quelques-unes
aucun anticorps n'Otait detectable chez les grizzly <2,5 ans. Les grizzly apparaissent etre d'excellentes sentinelles
pour la surveillance epiderniologique des zoonoses en Alaska.
INTRODUCTION
There are an estimated 30,000 grizzly bears (Ursus arctos) and 140,000 black bears (Ursus americanus) in Alaska
(USA). As predators and scavengers, bears may come in contact with agents of zoonotic diseases. Serologic
evidence of infection by Brucella spp. in Alaskan grizzly bears has been reported previously and in black bears
from other parts of North America. Bears can be infected by Francisella tularensis, the agent of tularemia, which is
endemic in Alaska. However, no extensive serosurvey of this infection in Alaskan bears has been performed.
Trichinosis is also known to be endemic in Alaska, and antibody prevalence >50% have been reported in brown
bears (Ursus arctos). Antibodies to canine distemper virus (CDV) and canine parvovirus (CPV) have been reported
from giant pandas (Ailuropoda melanoleuca) in China, and more recently for distemper from polar bears (Ursus
maritimus) from Alaska and Russia. Evidence of canine infectious hepatitis (CIHV) antibodies in Alaskan bears has
also been reported, but no information is available on seroprevalence in Alaskan grizzly and black bears of canine
distemper virus and canine parvovirus, infections which have been reported in Alaskan and Canadian wolves. The
objective of this study was to determine the seroprevalence of various zoonotic agents and canine viruses in
Alaskan bears.
MATERIAL AND METHODS
Personnel of the Alaska Department of Fish and Game and the U.S. Fish and Wildlife Service captured 480 grizzly
bears (GB) and 40 black bears (BB) in the course of performing population ecology studies between 1988 and 1991.
Sampling was opportunistic and some bears were captured more than once; 644 serum samples were available for
testing. 76 blood samples were collected from 40 BB in Interior Alaska on the Tanana Flats, south of Fairbanks.
The 568 GB blood samples were collected from 8 different areas : In southem Alaska, 79 samples were collected on
Kodiak Island, and 86 samples from the Alaska Peninsula (Katmai Coast (38 samples) and Black Lake (48
samples)). In Interior Alaska, 53 samples were collected in the Tanana Flats, Denali Park, and Fairbanks areas. In
Western Alaska, 40 samples came from Seward Peninsula and 99 from Noatak river drainage. In Northern
Alaska,133 samples came from northwestern Alaska, 6 from north central Alaska, and 72 from northeastern Alaska.
Blood samples were collected by femoral, saphenous or cephalic venipuncture. Serum was separated by
centrifugation and stored at -20°C until tested. Samples were collected from 25 (63%) of the 40 BB more than once :
4 BB had blood taken 4 times; 3 BB, 3 times, and 18 BB twice. Among the GB, samples were obtained 3 times
from 11 GB and twice from 67 GB. Serologic tests were performed at the Veterinary Public Health Lab., Davis,
California (brucellosis, tularemia and trichinosis) and at the Virology Lab., Rhone-Merieux, Lyon, France (CDV,
CPV, CIHV). Sera were tested for evidence of antibodies to : (1) Francisella tularensis (tularemia) using a
commercial slide agglutination test. Any titer 3 1:20 was considered positive, and positive serums were retested in
order to eliminate non-specific reaction. (2) Brucella spp. by buffered positive serum acidified card antigen test,
and, and positive serumsamples were also retested. (3) Trichinella spiralis, by enzyme linked immunosorbent assay
(ELISA). The ELISA was based on the official USDA method for pseudorabies, modified as follows. Briefly, 50111 per
well of a Trichinella spiralis ES antigen (511g/m1) produced by the Vet. Diagnostics Lab. , Ames, Iowa, USA at
1:1000 dilution in carbonate bicarbonate buffer, pH 9.6, was bound to 96-well flat bottom microtitration plates
(Linbro/Titertek plates (Flow Laboratories, Inc., McLean, Va, USA) by overnight
incubation at 4°C. Bear sera were diluted 1:40 in tris buffer (pH 7.4) containing 5% skim milk and 0.05% tween 20
and 0.01% bovine serum albumine fraction V (Sigma Chemical Co., St. Louis, Mo, USA). The peroxidase conjugate
was a raccoon anti-bear antibody at 1:500 (raccoon anti-bear IgG, Antibodies Inc., Davis, Ca, USA). The substrate
1 Department of Population Health & Reproduction, School of Veterinary Medicine, University of Califomia, Davis, Ca. 95616.
USA.
2 Laboratoire de Virologie, Rhone-Merieux, 254 rue Marcel Merieux, BP 7009. 69342 Lyon Cedex 07, France
3 Alaska Department of Fish and Game, 1300 College road, Fairbanks, Alaska 99701. USA.
was 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid;ABTS) (Sigma Chemical Co.). The reaction was stopped
after 30 min with 100 pl of a 0.1 M solution of hydrofluoric acid (pH 3.3). Each plate contained known positive and
negative control sera. The positive control serum was selected from a bear which was both positive by bentonite
flocculation and ELISA (optical density (0.D.)=1.0), and the negative controls were selected from 3 bears both
negative by ELISA (0.D.<0.1) and bentonite flocculation. Each serum was tested in duplicate and the mean of the
two absorbance values calculated. Microtitration plates were read at 410 and 450 nm respectively for test and
reference on a microelisa autoreader (MR 5000, Dynatech Laboratories, Inc. Chantilly, Va, USA). The cut-off point
for a positive test was determined at 0.D.= 0.3, which was the mean O.D. of the negative control population (all
bears from Kodiak Island) plus 3 standard deviations (SD) .(4) canine parvovirus by hemagglutination inhibition. A
viral suspension of 4 hemagglutining units in a 0.05 ml buffer was added to the various dilutions of the serum
samples to be tested for 1 hr at room temperature. Then, 0.05 ml of a suspension of 3x107 SRBC were added in
the plate wells and left overnight at 4°C. The reaction was read the next day. (5) canine distemper virus by
competitive ELISA. Briefly, 100p1 per well of capture monoclonal antibodies at a 1:1,500 dilution in carbonate
bicarbonate buffer, pH 9.6, was bound to 96-well flat bottom microtitration plates (ELISA Nunc Maxisorp, Nunc Inc.,
Rochester, N.Y., USA) by overnight incubation at room temperature. On cell culture plates, 50 pl of distemper virus
and 50 pl of each serum dilution (0.9, 1.8 and 2.7) and respective controls were incubated for 1 hr at 37°C with
permanent shaking. After 3 washes, 50 pl of the virus-serum mix was transfered on the monoclonal antibody
sensitized plates and incubated with shaking for 1 hr at 37°C. Then, 50 pl of the monoclonal antibody marked with
peroxydase were added in each well and plates incubated with shaking for 1 hr at 37°C. The plates were washed 3
times before 100 pl of substrate (orthphenylene diamine, Sigma Co.) was added. The reaction was stopped after
25 min with 50 pl of 2.5 M H2SO4 solution. Microtitration plates were read at 490 nm wavelength. Results were
expressed as O.D. percent compared to the control without serum (100%). Serum titers were given as log of the
reciprocal dilution with a 50% O.D. In order to validate the ELISA test and define the cut-off point (COP) for
seropositivity, 23 serum samples of bears with an ELISA titer30.8 were also tested by the classical serum-
neutralization (SN) test [4]. Titers by S.N. were usually lower than by ELISA, which led to defining the COP at 31.0.
Furthermore, ELISA positive samples and a random sample of negative serum samples were also tested using an
immunoperoxidase (IP) antibody test, which is similar to a fluorescence test, using IP instead of fluorescein
isothyocyanate. (6) canine hepatitis virus by serum-neutralization, using CAV type 2 and dog kidney cell line MDCK
(105 cells/ ml). Cytopathic effect on culture plates was read 7 days after infection, and titers were expressed in log10
protective dose 50% (PD50) on MDCK. Ages were estimated by examining cementum annuli of premolar teeth for
BB and GB. BB were classified in 4 inclusive age groups : 0-2 yr old, 2.5-4 yr old, 4.5 -8 yr old and 39 yr old. GB
were classified into 5 inclusive age groups : 0.5-2 yr (young bears), 2.5-4 yr (end of puberty,i.e subadult), 4.5-8 yr
(reproductively mature bears), 8.5-12 yr (prime reproductive capability), and 313 yr (old bears). GB from the Seward
Peninsula and McKinley Park were reported as being adults (i.e. 34.5 years). 62% (297/480) of the GB and 55%
(22/40) of the BB were females. Among the GB, 70 (12%) were <2 year old, whereas 24 (31.5%) of the BB were <2
year old. Demographic data were analyzed using Epi Info 6.02. Frequency distributions were obtained and chi-
square were calculated to obtain measures of association, and the statistical significance of such associations.
RESULTS
Overall seroprevalence in GB was 0% for CPV, 9% (43/480) for CDV, 14% (68/480) for CIHV, 16.5% (79/480) for
brucellosis, 19% (93/480) for tularemia and 47% (225/478) for trichinosis. Seroprevalence in BB ranged from 0%
for canine distemper and parvovirus to 32% (13/40) for tularemia (Table I). Canine parvovirus : None of the grizzly
and black bears tested had canine parvovirus antibody at a significant titer.
Canine distemper : Antibodies were found only in GB, with an overall prevalence of 8.3% (40/480). Antibody
prevalence was slightly higher in females (10%;29/294) than in males (6%; 11/186). Prevalence varied with the
origin of the bears (Table I). GB from Kodiak Island and the Alaskan Peninsula were more likely than GB from other
areas to be seropositive for canine distemper (RR=6.57, 95% C1=3.29, 13.12). Overall, among all 41 distemper
positive samples, 73% had titers 32.0, and of these, 77% (23/30) were from southern Alaska. Animals with
distemper positive samples were also more likely to be seropositive for CIH (RR=2.18, 95% CI=1.28, 3.72). Mean
age of seropositive GB was 12.5 ± 0.85 yrs, whereas mean age of seronegative GB was 8.4 ± 0.27 yrs. Age
prevalences for distemper antibody ranged from 1.5% in 0-2 yr-old GB to >10% in GB38.5 yr old. High titers were
mainly observed in GB >8 yr old. In southwest and western Alaska, all seropositive GB were >7 yr old, whereas, in
Northern Arctic, 40% of positive GB were £4 yr-old. Annualized prevalences for Kodiak Island were stable at 32%
(1988) and 29% (1989). They ranged from 2.6% to 7.6% in Northern Arctic.
Canine infectious hepatitis : CIHV antibody prevalence was 14 % (68/480) in GB and 7.5% (3/40) in BB.
Seroprevalence by sex was 12% (22/186) in males and 15.5% (46/294) in females for GB, and 5.5% (1/18) and 9%
(2/22) for BB. Prevalence also varied with the geographical origin and the age of the bears. For GB, it was the
highest on Kodiak Island (31%, 26/77), and averaged 10.5% in the other areas (range :7.5% to 12.5%). None of the
young GB (0-2 yr old) had CIHV antibodies. Mean age of seropositive GB was 12.23 ± 0.57 yrs, whereas mean age
of negative GB was 8.2± 0.28 yrs. Adult GB were more likely to be seropositive (RR=7.6, 95% C1=2.82,20.46) than
young and subadult bears. Conversely, none of the adult BB were seropositive. Annual prevalences varied for BB
from 0% in 1988 (0/9) and 1989 (0/22) to 4.5% (1/22) in 1990 and 8.7% (2/23) in 1991. For GB, overall prevalence
decreased from 19% (38/202) in 1988 and 21% (24/114) in 1989 to 11% (13/121) in 1990 and 8% (11/134) in 1991.
All BB tested more than once were seronegative. In GB, none of the 2 bears tested twice on Kodiak Island had CIHV
antibodies. In Interior Alaska, of the 11 GB tested several times, one adult was persistently positive and 2 other
adults were negative at the first collection and positive at the second. In Noatak river drainage area, of the 11 GB
tested more than once, one female adult was consistently positive, another GB was negative at the first blood
collection and strongly positive three years later. In the Arctic region, 5 adults were seropositive at both collections,
01.06.2
Epidemiol. sante anim., 1997, 31-32
two adults were positive at the first blood collection and negative at the next ones, and one GB was initially negative
and positive 3 years later.
Brucellosis : The overall prevalence of brucella antibodies was 16.5% (79/480) in GB and 2.5% (1/40) in BB.
Antibody prevalence was similar in males (17%) and females (14.5%) for both species. Brucella prevalence varied
widely by geographical areas (Table I). GB from westem and northern Alaska (59/277) were more likely to be
seropositive for brucellosis than GB from southwestern and central Alaska (20/203) (RR=2.16, 95% CI=1.35, 3.47).
Mean age of seropositive GB (8.7± 0.59) was similar to the mean age of seronegative GB (8.8 ± 0.29).
Seroprevalence increased from 6% in young GB (0-2yr old) to 17% in adults (34.5 yr old). Overall, the annual
prevalence decreased from 19% (39/202) in 1988 to 6% in 1990 (7/121), but increased in 1991 to 14.5% (19/131).
Yearly prevalence was consistent from one year to the other on Kodiak Island (12% (5/41) in 1988 and 8% (3/37) in
1989). A decrease was observed in Noatak river drainage from 37% (11/30) in 1988 to 15% (5/34) in 1991. In the
Arctic region, yearly prevalence also decreased from 20% (14/69) in 1988 to 8% (5/65) in 1990, but re-increased in
1991 to 17% (13/76), mainly in the central and northwestern Arctic areas (38%, 8/21). Among the 78 GB tested
more than once, 4 GB (5%) were consistently positive, 10 (13%) seroconverted and 7 (9%) became negative
between the first and the following collections
Tularemia : The overall prevalence of F. tularensis antibody was 19% (93/480) in GB and 32% (13/40) in BB.
Seropositive bears were also more likely to be positive for brucellosis (RR=2.33; 95% CI=1.57, 3.48).
Seroprevalence was similar in males (22.5%) and females (19%). Prevalence varied from 4% (3/77) on Kodiak
island to 10-15% in the Alaska Peninsula and Western Alaska and >30% in Interior Alaska and in the Arctic regions
(Table I). Prevalence (32%) was identical in BB and GB from Interior Alaska. GB from Interior Alaska and from the
Arctic regions (62/190) were more likely than GB from Western and Southwestern Alaska (31/290) to be seropositive
for F. tularensis (RR=3.05, 95% C1=2.07, 4.51). In BB, antibodies were found only in BB <9 yrs old. Mean age of
seropositive GB (7.33± 0.58) was slightly younger than mean age of seronegative GB (9.08± 0.29). Antibody
prevalence in GB was the highest in the age group 2.5-4 yr old and the lowest in the 0-2 yr old group. Young and
subadult GB(41/167) were 1.64 times more likely to be seropositive than adult GB (60/401) (95% CI=1.15, 2.34). On
Kodiak Island, only 3 GB were seropositive with a low titer (1:20). GB with high titers ( 31:160) were mainly from the
Arctic regions, Noatak river drainage and Interior Alaska. In Interior Alaska, the prevalence of bears with high titers
was similar in BB (7/21) and in GB (4/15). In BB, annual prevalences decreased from 39% (12/31) in 1988-89 to
20% (9/45) in 1990-91. The same decrease was observed in GB from Interior Alaska (40% (6/15) in 1988-89 and
24% (9/38) in 1990-91). For all regions, the yearly prevalences in GB increased from 11% (36/322) in 1988-89 to
26% (65/252) in 1990-91. Among the 78 GB tested more than once, 42 (54%) were negative at all blood collections,
8 (10%) were positive at all blood collections, 10 (13%) were positive at the first blood collection and negative at the
following ones, and 18 (23%) were negative at the first blood collection and positive at the following ones. Among
the BB, 12 (48%) were negative and 5 (20%) were positive at all blood collections, 3 young BB were positive at the
first collection and negative at the next one. One 1-yr old BB seroconverted the next year.
Trichinellosis : Trichinella spiralis antibody prevalence was 47% (225/478) in GB and 27.5% (11/40) in BB.
Seroprevalence was higher (RR=1.21, 95% CI= 1.01, 1.46) in males (51%, 104/204) than in females (42%,
132/314). Prevalence varied geographically with an increasing gradient from Southwest Alaska to the Arctic. All the
GB from Kodiak Island were seronegative and only one adult male from Katmai Coast (Alaska Peninsula) was
positive, with a low titer (0.D.<0.6). Conversely, GB from Black Lake (Alaska Peninsula), Interior Alaska and
Seward peninsula had a prevalence of 33% (42/128). Antibody prevalence was similar in GB (35%, 14/40) and BB
(27.5%) from Interior Alaska. GB from Noatak river drainage had a prevalence of 57% (50/87) and GB from the
Arctic regions had an overall prevalence of 89% (132/148). 70% of the GB with high titers (0.D. 30.9) were from the
Arctic regions and Noatak river drainage. In GB, antibody prevalence was similar in all age groups, with a mean age
of seropositive GB (8.96 ±0.38 yrs) close to the mean age of seronegative GB (8.57±0.37). In BB, 10 of the 11
positive BB were <5 yr old. Yearly prevalences in GB were constant at Noatak river drainage (57% in 1988 and
1989, 59% in 1991) and decreased moderately in the Arctic regions (91% in 1988 to 80% in 1990-91). Of the 78 GB
collected more than once, 50 (64%) tested positive and 18 (23%) tested negative at all blood collections; 10 (13%)
were negative at one blood collection and positive at the other. For the 25 BB collected more than once, 17 (68%)
tested negative at all collections, 5 (20%) BB became positive and 3 (12%) BB became negative at the second
blood sample collection.
Table I
Prevalence of Brucella, Francisella tularensis, Trichinella spiralis, canine distemper virus and canine
infectious hepatitis virus antibodie
s in black bears and Grizzly bears, Alaska, by geographical origin. 1988-1991
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