Journal
PARATUBERCULOSIS
IN SAIGA
EXPERIMENTAL Thomas
TRANSMISSION
W. Dukes,’
and Mark
ANTELOPE
Gordon
of Wildlife
(SAIGA
TO DOMESTIC
J. Glover,2
Brian
W. Brooks,
28(2),
Diseases,
TATARICA)
1992,
pp.
161-170
AND
SHEEP
J. Robert
Duncan,’
Swendrowski3
‘Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario, Canada K2H 8P9 2Veterinarian, Assiniboine Park Zoo, 2355 Corydon Ave., Winnipeg, Manitoba, Canada R3P 0R5 Services Laboratory, Manitoba Dept. Agriculture, Agricultural Services Complex, 545 University Crescent, Winnipeg, Manitoba, Canada R3T 2N2 ABSTRACT: Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted. Key words: Paratuberculosis, saiga antelope, transmission, sheep, Saiga tatarica, natural infection.
INTRODUCTION While is
paratuberculosis
primarily
nants it
has
1969)
also
been
captive
1988;
ilies
et al.,
(Katic,
in
Bovidae)
This
to the in
and
clinical
expressed
in
investigate the in
virulence
material into
the
organism
and
newborn
one
the
was young
poor
cultural
possible different
frequently To
recovery
of
animal was
there
causative
animals. strain
antelope
domestic
tatari-
in Canada.
in cattle, of
disease
for from
disease
relatively
paratu-
(Saiga
isolation
fam-
Bovidae
reports
to a zoo
In comparison agent
and
paper
was
van 1982;
b) of the
antelope
imported
et al., and
1980,
1983a,
saiga
difficulty
free-ranging
Chiodini
Camelidae,
1961).
Bovidae,
(Baradel
Weber,
1979,
Cervidae,
berculosis
in
AND
in imported
METHODS
saiga antelope
Sixteen saiga antelope were imported into Canada in 1985 from a zoo in Germany. These animals had been negative on previous export testing for paratuberculosis in the country of origin. Serum was collected during quarantine for paratuberculosis testing in addition to other federal import test requirements. Two animals died in quarantine, two male and six female antelope were sent to the Assiniboine Park Zoo (Winnipeg, Manitoba, Canada R3P 0R5) where they were housed in a newly constructed exhibit area and six animals were sent to another facility in Canada for which no further information is available. Necropsies were performed at the Manitoba Veterinary Services Laboratory, Winnipeg, on all saiga antelope which were euthanized or died at the Assiniboine Park Zoo beginning 6 mo after the animals arrived. The lower small intestine and associated lymph nodes were examined. Histologic sections were stained by hemotoxylin and eosin and by Kinyoun’s acidfast method. Paratuberculosis lesions were classified microscopically on the basis of morphology and the number of acid-fast bacteria present
rumi-
family
1982; 1983;
disease)
domestic
the
ruminants
Commichau,
Williams
of of
observed
wild
Kruiningen,
Ca:
(Johne’s
a disease
(Katic,
and
MATERIALS
Paratuberculosis
variation species, inoculated
sheep. 161
162
JOURNAL OF WILDLIFE DISEASES, VOL. 28, NO. 2, APRIL
as described (Brooks et al., 1988). A diagnosis of paratuberculosis required demonstration of typical acid-fast bacilli in association with a granulomatous lesion in the lower small or large intestine or associated mesenteric lymph nodes. Demonstration of typical acid-fast bacilli in a direct smear was not by itself considered to be sufficient criteria for a diagnosis (Merkal, 1973). Fresh, frozen or fixed tissues and blood from antelope with mycobacterial enteritis and lymphadenitis consistent with paratuberculosis were sent to the Animal Diseases Research Institute (ADRI) (Nepean, Ontario, Canada K2H 8P9) for immunological testing, culture and histology. Information was requested from five North American zoos to determine if paratuberculosis had been observed in their saiga antelope.
Experimental
animals
Ewes were selected as potential dams to deliver lambs for oral challenge experiments or to serve as negative controls from a caesarian derived “minimal disease” sheep flock. The ewes were moved to isolation cubicles prior to parturition. Lambs were held with their dams until weaning at approximately 75 days of age at which time the ewes were sent for necropsy. Negative controls were housed in separate cubicles in an adjacent building. The cubicles were washed daily to remove fecal material. Necropsies were conducted with emphasis on examination of intestine and associated lymph nodes.
Inoculation
and microbiological
procedures
Intestinal scrapings (3 g) from the mucosal surface from a histologically diagnosed case of paratuberculosis (antelope 12, Table 1) was homogenized with a tissue grinder and suspended in 22 ml of 0.85% saline to form the inoculum. An air dried smear of the inoculum was stained using the Kinyoun acid-fast method and examined microscopically. Large numbers of acidfast bacilli were seen per oil immersion field. The inoculum was stored at -70 C. It was thawed at room temperature (RT) prior to administration to the lambs and kept at 4 C during the period between inoculations. Two of three triplet lambs (981 and 982) in the experimental group received 2.7 ml of inoculum orally on day 1 (day of birth) and day 3 of the study. The total inoculation dose for each lamb was approximately 0.65 g of mucosa. The third lamb (983) in this group served as an in-contact control. Feces and blood were collected from the lambs weekly from weeks three to eight and monthly thereafter to the time of euthanasia or to the end of the study at 18 mo postinoculation. Ewes
1992
were sampled prior to selection for the experiment and weekly until weaning and euthanasia. Clotted blood samples were allowed to stand overnight at room temperature before processing. Heparinized samples of blood were used for the lymphocyte stimulation test (LST) within 6 hr of collection. Direct smears were made from tissues collected at necropsy and from some fecal samples. Smears were stained by Kinyoun’s acid-fast method and examined for the presence of typical acid-fast bacilli. Antelope fecal samples were shipped fresh, chilled but not frozen, and processed on the day of arrival at the laboratory. Fecal samples from the lambs were processed within 6 hr of collection. In general the culture procedure described by Merkal (1973) was followed, modified by using cetylpyridinium chloride (CPC) as the decontaminant and centrifugation prior to inoculation. Briefly, 1 g of fecal material was transferred to a 50 ml sterile, conical centrifuge tube and 40 ml of sterile distilled water added. The tube and contents were shaken for 30 mm, and then placed in an upright position and left to stand undisturbed for 30 mm. The top 5 ml of the fecal suspension was transferred to another 50 ml sterile centrifuge tube containing 35 ml of sterile CPC. The final concentration of CPC was 1%. Following standing at RT for 18 to 24 hr, samples were centrifuged at 2,250 x g (15 C) for 0.5 hr. The supernatant was discarded and the pellet resuspended in 1 ml sterile saline and distributed equally amongst five tubes of Herrold’s egg yolk medium (HEYM) containing 2 mg/i mycobactin J (Allied Laboratories, Glenwood Springs, Colorado 81601, USA). The inoculated media was incubated for at least 24 wk at 37 C. Isolates were subeultured onto 1 tube of HEYM with and 1 tube HEYM without mycobactin to confirm mycobactin dependency. Samples of intestine and mesenteric lymph nodes were collected from lambs 981, 982, and 983 at sites immediately anterior to and approximately 60 cm anterior to the ICV. Intestinal samples were also collected at two additional sites from 983. Tissues from the lambs were held at 4 C overnight and processed the day after collection. Culture of tissue samples was performed as described (Brooks et al., 1988) with decontamination in 0.5% CPC for 18 to 24 hr including centnfugation at 2,250 x g (15 C) for the final 30 mm. Resuspension of the pellet, inoculation of media and incubation were the same as for fecal samples. Identification of an isolate as M. paratuberculosis was on the basis of survival of the decontamination procedure described above, slow growth on HEYM with mycobactin, no growth on HEYM without mycobactin, typical colonial and cellular morphology and acid fastness.
DUKES
Immunological
ET AL.-SAIGA
ANTELOPE
procedures
Agar gel immunodiffusion (AGID) tests using antigen “D” and complement fixation (CF) tests using a crude carbohydrate antigen were conducted as previously described (Brooks et al., 1988; de Lisle et al., 1980a, b). Enzyme linked immunosorbent assay (ELISA) tests utilized an exclusion peak from S200 gel filtration of the crude polysaccharide as antigen to coat wells (NUNC Maxisorb Immuno modules, GIBCO, Burlington, Ontario, Canada L7P 1A1). The antigen was dissolved at 10 g/ml in 0.01 M carbonate buffer, pH 9.6, sonicated and then 100 zl placed in each well. The plates were sealed and placed at -20 C shortly after coating and stored frozen for an indefinite period. Plates were thawed at room temperature before use followed by washing four times in a 96 well plate washer (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2) using a 0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20 buffer (TT), pH 8.0. Test sera were diluted 1:200 in TT buffer containing 0.02% NaN3 and 100 d placed in the microwell modules in duplicate. Controls consisted of (a) a prediluted and frozen aliquot of a positive bovine serum that served as a target, (b) a positive and (c) a negative serum from sheep diluted in the same manner as the test samples, and (d) TT buffer. These were placed on each plate as paired duplicates in columns 1 and 7 for a total of 16 wells for the four controls. Following an overnight serum incubation at room temperature, plates were washed and 100 l of an alkaline phosphatase conjugated rabbit anti-sheep F(ab’)2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania 19390, USA) was added for a 3 hr incubation at room temperature. Plates were washed again and 200 sl of p-nitrophenyl phosphate (1 mg/ ml) substrate in diethanolamine buffer, pH 9.8, 0.5 mm MgCl2 was added and the plates shaken continuously between three successive readings of optical density (405 nm) at approximately 2 mm intervals on a microplate reader (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2). The three optical density readings were used to determine the slope value for each paired sample using the regression function of Lotus 123 (Lotus Development Corp., Boston, Massachusetts 02142, USA). The mean slope value of the four control samples was used to calculate a corrected optical density for each sample thus standardizing test results for between plate variation. A microwell modification of the whole blood stimulation test (de Lisle and Duncan, 1981) was conducted in flat bottomed wells (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2) using three drops of media (approximately
PARATUBERCULOSIS
AND TRANSMISSION
TO SHEEP
163
150 l) and one drop of whole blood. Twelve wells were used for each sample consisting of four wells of the control media and two wells each of PHA (Phytohemagglutmn), J-PPD (johnin purified protein derivative), B-PPD (bovine) and A-PPD (avian). Plates were incubated for 72 hr at 39 C followed by the addition of 0.5 1zCi of tritiated thymidmne per well for 18 hr and then frozen at -20 C until thawing for harvesting on a Titertek Cell Harvester (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2). The level of incorporation of the radiolabel was determined by the addition of 3 ml of scintillation fluid to minivials and counted on a liquid scintillation counter (Beckman Instruments, Toronto, Ontario, Canada L5T 1W5) using a wide tritium window. Results are expressed as net counts per mm (NCPM = mean of stimulated cultures minus mean of control cultures).
RESULTS in saiga
Paratuberculosis Two
imported
antine their
sent
The
to the
ropsied
Park
for
limited
intestine 1).
of
born
the
lymph
at
six
of
in
1989.
severity and
the
11
node first
zoo
in
second An
1987
cobacterial
of
of
ileal
phatics
with
lymph
nodes
imals.
Histologically,
matous matous
15
the white
enteritis in
the
for
or
animal in
Table
with
a
consisted
of
varying
thickening and
lym-
Mesenteric in
there
several
was
granulo-
numerous and and
mywith
dilated
nodules.
macrophages
lymphangitis
extent
each
enlarged with
in
animals
serosa
were
and born
is given
mucosal
on
2)
inflammation
enteritis tags
in
offspring
load the
(Table offspring
(Table
mortem
in
fibrous
done
granulomatous
Lesions
as the
confirmed
generation
post
examof
generation
mycobacterial
degrees
was also
estimation
of
examined
bacteria
not study
was
seven
necin four
were
histopathologic and
were Paratuber-
paratuberculosis
Paratuberculosis
five
Zoo
period.
others
de-
animals
histologically
two
specifically
only
being
eight
year
quarwith-
status
diagnosed and
in
trauma)
remaining a 6
was
antelope ined
and
Assiniboine
over
culosis
died
stress
paratuberculosis
termined.
3.
animals
(transport
out
antelope
acid-fast a
granulo-
lymphadenitis.
an-
JOURNAL
164
1.
TABLE
OF WILDLIFE
DISEASES,
VOL. 28, NO. 2, APRIL
of paratuberculosis
Summary
serological
1992
results
and
necropsy
findings
from
imported
adult
saiga
antelope. ACID’
Date
Animal number
10/85
10&
ND
07/86
07/89
09/88
Date of death2
04/90
Reason
12’s
-
-
13
-
-
-
-
-
07/91
14
-
-
-
-
-
07/91
15
-
-
-
-
-
05/91
death trauma trauma trauma killed killed death
16
-
-
02/88
hemolytic
17
-
-
06/87
18
-
clinical trauma
155’s 11
Agar 2
sampled
10/85
R
gel
during
Fecal
07/86
05/86 results:
R, reactor;
but was negative for death or euthanasia.
each
S. suspicious;
animal
at each
collected was
paratuberculosis ‘sIntestinal
tissue
during
this
quarantine
animal
were
While
revealed
a few
acid-fast
was
inoculated
observed
in
the
orally
into
with
2. spring
one
of
confirmed
1),
TABLE
unknown
positive unknown
paratuberculosis
not
done.
on
smears
The
complement
fixation
test
was
liver
of
four
imported
offspring
ani-
paratuberculosis
to the
AGID
all
confirmed
Summary 1987
bacteria
but
culture
for
Mycobacterium
sheep.
2,
only
a reactor
born
positive crisis
sampled.
animals.
(Table
ND,
unknown unknown positive positive negative negative
negative.
from
Granulomas
mals
negative;
‘ -,“
date
for necropsy
quarantine.
samples
some
03/87
immunodiffusion
also conducted Date of natural
Died
10/85
S
Paratuberculosis status
test
cases
in
ical
the
1987
serological
results
prior
1990,
remaining
of paratuberculosis and
May
to necropsy
detected to
reactors
or
AGID
33 AGID
findings
within
from
34) had
test. the
1
tested
and
offspring
the
(Table
less) When
(animals 1989
on
necropsy
25 necropsy.
two six
and
by
numbered
to 5 mo
was
prior
were
animals
in
of
the
serologThese
saiga
an-
offspring
1989.
AGID/CP Date
Animal number
2
09/88
Paratuberculosis status
07/89
04/90
Date of death
Reason for necropsy
S/S
R/R
07/90
thin
positive
12/88
died died
positive positive
19
14 (16)’s
-/-
20
17
R/S
21
13
-/-
R/R
22
16(14)
-/-
R/S
11/89
thin
positive
23
16(14)
-/-
R/S
08/89
died
positive
24
15
-/-
-/-
11/89
25
15
thin trauma
negative negative
07/89
07/87
29
19
-/-
07/91
killed
positive
30
21
-/-
03/91
31
21
-/-
09/90
positive positive
33
14
R/-
07/90
died pneumonia died
34
22
S/ND
04/90
died
positive
35
22
-/
07/91
killed
positive
‘Animals which Agar
Dam
sampled
19 to 25
were
born
in
1987.
died within 7 wk of birth. gel immunodiffusion/complement
No
Animals
evidence
-
of paratuberculosis
was
26 or higher were born in 1989. fixation: R, reactor; S. suspicious;
‘sDam
was
probably
animal
14 but
may
have
been
animal
16.
Dam
was
probably
animal
16 but
may
have
been
animal
14.
seen “-,‘
at necropsy negative.
of
animals
positive
(26,
27,
28,
32,
36)
DUKES
ET AL.-SAIGA
imals died within 2 mo of the test date and paratuberculosis nosed histologically in both. ing four 1989 offspring were have were
paratuberculosis killed over
cilli
the
although
were
3).
isolated
from ing
five the
of
acid-fast
fecal
of the
death
smears
the
paratuberculosis
quent cultural sues collected
was
animals
Salmonella juni, vey
spp.
from
and
animal
Cam
Two of three zoos responding had diagnosed paratuberculosis
ga antelope in their in which paratuberculosis received
their
rope. The zoo was not observed collections
in
within
Recovery experimental
11
+++
+++
+++
+++
12
+++
+++
+++
+++
13
-
-
ND
ND
14
-
-
ND
ND
15
++
++
++
++
16
-
-
-
ND
+++
were
colonies
isolated
from
of
+++
++
++
++
to tisanimals
23
++
++
+++
ND
24
-
-
ND
+
25
-
-
ND
ND
to je-
+++
+++
+++
+++
+++
+++
+++
+++
31
+++
+++
+++
+++
33
++
+++
+++
ND
34
+++
++
+++
++
35
+++
+++
+++
+++
No evidence other
successful.
paratuberculosis their saiga
29 30
of paratuberculosis
animals
(26,
A positive
direct
smear
for classification
died
or
of five
within
7 wk
of
lesions
sufficient
as positive
classified
for
mild;
as +,
evidence
paratuberculosis. + +,
moderate;
severe.
+++,
AF,
was not considered
of an animal
‘sparatuberculosis acid-fast
organisms hard
bacilli;
ND,
associated
to find;
not done.
with
+ +, small
Quantitation
lesions
was
numbers;
or
graded
of acid-fast as +, rare
+ + +,
and
large
numbers.
in
direct
from
America.
and
small
smears
in
numbers
of
the
were
lymph
Mycobacterium
paratuberculosis of lamb
intestine not from
from fecal
scrapings
seen
node.
paratuberculosis
in low
numbers lamb samples
from
was
a sample
982 (inoculated), of 982 or from
of but fecal
and tissue samples from lamb 983 (in-contact control). Acid-fast organisms were not seen on direct smears of tissues from either animal.
and no gross tuberculosis lambs.
lymph node, bacilli were seen
of intestinal
on necropsy
which
birth.
and while
smears
observed
27, 28, 32, 36)
Clinical and pathological experimental animals
direct
+++
++
56 and only small numbers of an organism consistent with M. paratuberculosis were isolated from the two samples of intestine
on the
+++
++
981 (inoculated), collected at 47, 51, 54, and 55 wk after inoculation. Small numbers of acid-fast organisms were seen on direct smears of feces collected at week 55. This animal was necropsied at week
from one mesenteric masses of acid-fast
ND
+++
22
to the surin sai-
samples
+++
-
Subse-
Eu-
fecal
+++
-
+++
from
M.
+++
-
isolated A few
AF’
++
antelope
of M. paratuberculosis animals
lesions’s
+++
zoos had
North
Direct smear2 AF’
thology
++
Both observed
which acquired
Histopa
+++
collections. was
Saiga
for necropsy.’
++
Attempts including not
and in
+++
pylobacter
11 were
of pathological paratuberculosis
for
21
12 and 17. Only small numbers of M. paratuberculosis were recovered after incubation for 24 wk although high acid-fast bacterial loads were demonstrated in dihistology. pathogens,
165
20
11. limited from
TO SHEEP
Gross lesions’s
19
follow-
results
presented
18
diagnosis
animal
studies were at post mortem
rect smear and by culture other enteric
antelope
17
collected
confirmed in
AND TRANSMISSION
Summary of examinations
Animal number
(Table
samples
imported
and
saiga
ba-
paratuberculosis from
3. microbiological TABLE
collected durof the imports for M. paratu-
in direct
Mycobacterium
not
PARATUBERCULOSIS
The remainconfirmed to
typical
observed
serological was diag-
when they died or next year (Table 3).
Culture of a fecal sample ing quarantine from one (animal 11) was negative berculosis,
ANTELOPE
There
was
no
findings
clinical
or microscopic in the dams
In
evidence
of
illness
lesions of paraof experimental
JOURNAL
166
thin
OF WILDLIFE
Clinically, lamb but appeared
when
was
it
DISEASES,
VOL. 28, NO. 2, APRIL
981 (inoculated) healthy until week
anorexic
and
had
soft
was 53 feces.
The feces became watery and proved again to a soft pelleted week 54. No Salmonella spp.,
then imform by Camplyo-
bacter spp. or parasites from the feces but fecal low numbers of acid-fast
recovered contained By week
56
the
severe
were smears bacteria.
diarrhea
had
the was
animal was euthanized. thin and had extensive
the
distal
small
submucosal
intestine
edema
thickness.
There
foci
along
the
The were
ileocecal enlarged
and
and
increased
mucosal
small
tortuous
2 mm
serosal
focal
numerous primarily
medulla
of
nodes. Numerous ing many acid-fast
lymphatics.
con-
were evident in granulomas were serosal of
lymphatics. macrophages
the
mesenteric
lymph
macrophages, containbacteria, formed sheets
necrosis were present area of the bowel. were full of necrotic in
normal ileum
mucosa. areas of
in the Peyer’s patch Some mucosal crypts debris and lymphatic
the
submucosa
982
(inoculated)
were
granulomatous
ident on acid-fast
lymphangitis
A mesenteric
lymph
of macrophages one giant cell were difficult
in the paracortex was seen. Acid-fast to find.
clinical
the
ICV
mass
gut
Histologically, lomatous
contained
were
associated
of
in
with
the
associated an
lesion
on pathological contact control
terminal
occasional associated
Immunological
test
(ELISA,
AGID,
slaughter. Sequential results from the two 982)
and
the
(983) are test results
one
age.
and
AGID test lambs (981,
in-contact
control
lamb
1. The the three
ELISA lambs
of passive in titer at
titers
lambs level
in
rose than
the
antibody fol4 to 6 months
two
orally
more rapidly the in contact
approximately
submucosa acid-fast other focus cosal patches
of the bacteria
mucosa
and
ileocecal valve were seen. In
but no several
The
four
negative tive by
non-contact
by AGID ELISA with
gle
lamb
of macrophages was seen. This mulesion was associated with the Peyer’s
The the
LST responses to PHA two orally challenged
of
sections
the
ileum.
of ileum
only
Lymphangiectasia
in-contact
with
control
shown
were
as negaof a sin-
a non-persistent
are
200
lambs
classified exception
one
histologic
developed “D” anticlinical
control and the
inoc-
and to a control.
responded
the
LST)
shown in Figure indicate that
The
ulated higher
ELISA challenged
disease (981) days later.
in
non-
of dams of experimental lambs were normal limits on samples collected to selection and from lambing to
of the lung teria could
macrophages
seen
the
test results in
lambs to the developed
of
were of
challenged antibodies lamb that
foci
granuPeyer’s
granuloma acid-fast bac-
Both orally precipitating gen but the
bacwere
ileal
animals
experimental
of
acid-fast There
in-
tissue.
focal with
examinations lambs.
clinically
no
the
lymphoid
lymph nodes were enlarged with extensive thickening of the cortex. Histologically tiny granulomas were seen in the interstitium and liver but be demonstrated.
foci and only bacteria
seen
patches and one submucosal were seen but rarely were teria observed. No lesions of paratuberculosis
results within prior
ev-
lamb (983) but at necropsy (2 mm) were seen on the about 60 cm anterior to
had a low level lowed by a rise
was
node
signs
contact control two white foci intestinal serosa
markedly
but at necropsy the wall of the was thickened and the mesenteric
were
the serosa of terminal ileum and bacteria were only rarely found.
Immunological
acid-fast bacteria in the paracortical area
of cells in the intestinal and cecal Submucosal, partly mineralized
dilated. Lamb
white
granulomas
bacteria Similar
in the dilated diffuse aggregates
spaces
of
and mesenteric lymph nodes and had a thick cortex.
Histologically,
containing were seen
and
evidence
and
No
This animal thickening of with
were
taining acid-fast liver and lung. evident More
returned
1992
response. and lambs
J-PPD and
of the
in
Figure
2.
DUKES
ET AL.-SAIGA
ANTELOPE
PARATUBERCULOSIS
AND TRANSMISSION
TO SHEEP
167
09
0.5 > 0
C’
0.1
0
1.3 4 0
0.9 0
z 2
0.5
0. 0
0 ,
300 to reach responses that approximated exceeded those induced by PHA.
nai, but
days)
sensi-
control
lambs.
1922; this
Dorofev is the
of
paratuberculosis
In was
(Jar1949)
natural
antelope.
in
1922
Kalechev,
report
saiga
reported
since
and
first in
study
been
of antelope
parathe
pres-
confirmed
in three of eight (38%) of the saiga antelope imported into the zoo and in seven of 18 (39%) of their offspring. The prevalence of paratuberculosis have been higher 18 offspring died
in the offspring may since another six of the at less than six months
of age onstrate
may not evidence
culosis
or There
ent
has
species
tuberculosis
The antigen (J-PPD) and mitogen (PHA) stimulated responses demonstrated by lamb 981 were more variable than those of 982 and
mycobacterial
DISCUSSION
ini-
inoculated
PHA
of
non-contact
>10,000
to increased
sensitization
antigens at this demonstrated
generally
response
J-PPD
>20,000) post
was
the
in the
when one histologic because
early stages the negative
the
disease
of development. serological
readily demof paratuberwould
be
in the
If we accept status of the two
JOURNAL
168
adult
OF WILDLIFE
female
as absence tion
saigas
DISEASES,
VOL. 28, NO. 2, APRIL
numbered
13
of paratuberculosis,
of their
offspring
and
then
(19,21,33)
14,
infec-
M.
paratuberculosis
control itized tential
to
indicates
the
in
a
zoo.
A
survey
paratuberculosis was zoological collections
that
a problem in of saiga antelope
other em-
phasizes the need testing in the trade susceptible species.
for strict control of this species and
and other
M ycobacterium generally inactive ical tests (Chiodini,
para tuberculosis on traditional biochem1986) and considered
to be abolic
a homogeneous or biochemical
ular
species from perspective.
characterization
nuclease
by
analysis
demonstrated
paratuberculosis isolates igin) were very similar to the species reference de Lisle, 1986; Whipple
and cultural recovery with
techniques numbers onstrated Taylor, 1987;
(Dunkin 1951; Brooks
and
54 M.
where were
Balfour-Jones,
Gummarsson, et al., 1988;
1979; de Juste et al.,
present
culture bovine
sheep isolates
isolates tested.
DNA DNA
difficult
are distinct In addition,
from
lambs similar
However,
agnostic ELISA) ing
the
tests with
for the
bacterial
1935;
comparison bohydrate veloped
to all other
slow growing, mycobactin dependent Mycobacterium spp., such as the wood-pigeon bacillus (Matthews and McDiarmid, 1979) and isolates from humans with Crohn’s disease (Chiodini et al., 1984) have also been
were dif-
These obserthis antelope having char-
include
the agents
prepared
pobefrom
and no reliable meaof viable organisms the
experiment
con-
firms the potential for interspecies transmission of infection and disease. Infection of sheep by oral inoculation of this strain led to seroconversion to the standard di-
al., 1988). cipitating
Lisle, 1991;
have of M.
submitted the and
inoculum
mucosa, number
inoculated.
large dem-
hybridization that
in the
the intestinal sure of the
This strain for sheep
and
samples
These limitations for other unrecognized
tion
of genomic
in
freezing may poor recovery
were encountered. are consistent with of M. paratuberculosis
significance development
endonuclease
While in the
zoo, samples from without freezing
Carrigan and Seaman, 1990). Collins et al., 1990 demonstrated on the basis of restricpatterns
In the
paratuberculosis level of antibody load
or the
(AGID, reflect-
severity
lesions as previously observed ural disease in domestic sheep
including culture
from specimens of acid-fast organisms
identification
acteristics different from those of classical bovine isolates. There are several limitations to extrapolating the results of experimental paratuberculosis to transmission of the natural
ing
differences observed in Watt, 1954; (Saxegaard,
properties conventional
from the cultivated
disease. tential
(50 of bovine orto each other and strain (Collins and et al., 1989). How-
ever, in other studies, distinct between isolates have been pigmentation (Taylor, 1951; Stuart, 1965), pathogenicity 1989), poor
is
endothat
the
paratuberculosis.
paratuberculosis
ficulties vations isolate
a metMolec-
restriction
complicate
as M.
ed in smears. been a factor
in-contact
indicating
and
isolate
present study M. paratuberculosis was isolated only in low numbers from the antelopes and lambs although large numbers of organisms were frequently demonstrat-
lamb was observed in a daily sanenvironment and emphasizes the pofor interspecies spread of the dis-
ease
reported
of an
that horizontal transmission from animals other than infected dams was important. This is supported by the experimental study where transmission of the antelope isolate of
1992
bacteremia ical disease
The later antibodies
of the
in the (Brooks
development to antigen
of pre“D” by
to the ELISA response antigen in the lamb clinical disease may be from the perspective of resistance and may be particularly as exemplified by and the following
development a short,
natet
to carthat deof some of the diagnosis.
the
pathogenic apparent 11
of clinmo, in-
cubation period. Caseation of lesions similar to that seen in other tubercular mycobacterial diseases (M. avium, M. bovis) and paratuberculosis of domestic small ruminants
was
observed
in the
experimen-
DUKES
tal sheep of classical in
although bovine
evidence.
perimental leled our
ET AL.-SAIGA
ANTELOPE
the non-caseous paratuberculosis
Thus
the
disease observations
in
lesion was also
results
of
the
ex-
sheep have paralin the saiga antelope
ing
are
observed
to share
places (Alton, 1990). The control or eradication
terial require been
applied
in domestic
for example, index (including
has
water-
species than
animals.
been seen quantitation)
may have
Finally as ported around production spread of that
are
zoological
in
wild ruminants the world for
there strains
wild
for
collections
want
in en-
Airport,
this
study
the of
Dr.
Canada, Mirabel Mirabel, Quebec. His
valu-
antelope
examined
by them
at necropsy.
Boca
NAUDIE,
C.
PERREAU,
A.
BROOKS,
In
K.
Animal
Nielsen
(eds.).
Florida,
pp.
LE
383-
DUNCAN.
logical
response
bacterium
A.
of sheep
J.
AND
J.,
J.,
CORNER,
in
of
one
flock
by
crossed
Journal
AND
the to
seroMycoimmu-
of Veterinary
199-204.
of Johne’s Journal R.
Interna-
H.
Evaluation
Canadian
M.
pathology Veterinary
of
France.
C. TURCOTTE,
paratuberculosis
CARRIGAN,
Results in
Office
ROBERTSON, 1988.
52:
C.
873-883.
H.
noelectrophoresis.
TRAP,
1988.
M. M. GARCIA,
R.
P.
D.
animals
technique
Y.
B. LARE-
PASTORET,
THIRY,
wild
et 7:
M. BOUTIN,
DELORME,
KIHM,
P-P. E.
of
scientifique Epizootie R.
U.
P. VANNIER.
survey
B. W.,
D.
GOFF,
AND
J.
BLANCOU,
SCHWERS,
AND
T.
SEAMAN.
H.
J.
VAN
The
1990.
Disease in sheep. 67: 47-50.
Australian
KRUININGEN.
1983.
Eastern white-tailed deer as a reservoir of ruminant paratuberculosis. Journal of the American Veterinary Medical Association 182: 168169.
Interinitial
nant
paratuberculosis
rent
status
Stan
J.
BARRAT,
Pierre
interest and co-operation were the initial stimulus for this work. We thank B. Rapheal of the Dallas Zoo, M. T. Berrie of the Oklahoma City Zoological Park and S. Abildgaard of the San Diego Zoo for providing information regarding saiga antelope in their collections. J. L. Neufeld, R. J. Austin, J. M. Bystrom and G. Spearman are thanked for the use of the data from the
and Raton,
C. DANNACHER, M. GOURREAU,
J.
Research
ruminants agricultural
to acknowledge
able contributions to Vivier of Agriculture
J.
M,
CHASTEL,
GERARD,
J.
ACKNOWLEDGMENTS
national
J.
C.
CHI0DINI,
authors
Inc.,
B. S. SAMAGH,
of
melitensis.
Duncan
R.
409.
tional
terprises.
The
Press,
Revue
are transzoos and meat
other and
CRC
CITED
Brucella
J.
a serological
ru-
may be increased risk of M. paratuberculosis
pathogenic
1990.
UILENBERT,
vaccisanitation of para-
captive 1989).
loans. The help in
expertise.
C. C.
BARADEL,
serum
sampling in captive wild species as part of a test and cull program presents a problem of increasing capture stresses and may be of questionable value. Therefore nation in conjunction with good should be considered as a method
some interlibrary Health Laboratory’s
graphics
brucellosis,
Frequent
control (MacDiarmid,
us with Public
ALTON,
idence
tuberculosis minants
assisted Manitoba
LITERATURE
a reliable of infection
antelope culture of histologic ev-
infection.
much appreciated for their efforts on our We thank the serology section for providing the results for standard serological tests for paratuberculosis. G. M. Ruckerbauer is thanked for her assistance with some of the German references. Claude Turcotte assisted with the cultural work. P. Atherton and G. Eldridge
puter
in cattle but in these saiga was very difficult in spite of heavy
169
van Zyll de Jong made helpful comments on ungulate taxonomy. The technical staff of ADRI, especially Henry Barleben, Barbara Dupree, Jim Hodgson, Hilary Kelly, Nancy Lopes, Vita Radzius, Jeanette Jeffreys and Barbara Stewart are
Culture,
as
TO SHEEP
mycobacterial culture of two cases is acknowledged. We thank Joan Graham for word processing assistance and David Gall for his com-
of mycobac-
disease in non-domestic different approaches
TRANSMISSION
very
experiments suggests that interspecies transfer of this disease could occur in the native habitat of the saiga antelope where species
AND
behalf.
to date. The relative ease of transfer of M. paratube rculosis from antelope to sheep in these
these
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R.
AND
inarian
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218-262.
Biochemical characteristics of variMycobacterium paratuberculosis.
of
Journal
of
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G. W.
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1992
for
publIcation
5 December
1990.
PARATUBERCULOSIS
IN SAIGA
EXPERIMENTAL Thomas
TRANSMISSION
W. Dukes,’
and Mark
ANTELOPE
Gordon
of Wildlife
(SAIGA
TO DOMESTIC
J. Glover,2
Brian
W. Brooks,
28(2),
Diseases,
TATARICA)
1992,
pp.
161-170
AND
SHEEP
J. Robert
Duncan,’
Swendrowski3
‘Agriculture Canada, Animal Diseases Research Institute, P.O. Box 11300, Station H, Nepean, Ontario, Canada K2H 8P9 2Veterinarian, Assiniboine Park Zoo, 2355 Corydon Ave., Winnipeg, Manitoba, Canada R3P 0R5 Services Laboratory, Manitoba Dept. Agriculture, Agricultural Services Complex, 545 University Crescent, Winnipeg, Manitoba, Canada R3T 2N2 ABSTRACT: Mycobacterium paratuberculosis was isolated in low numbers from the small intestine and associated mesenteric lymph nodes of a saiga antelope (Saiga tatarica) using routine culture techniques in spite of histologic evidence of high numbers of acid-fast bacteria in these tissues. Two newborn domestic sheep were fed the ground intestinal tissue containing acid-fast bacteria and the progression of the experimental disease was followed by fecal culture, immunodiffusion (AGID) and lymphocyte stimulation (LST) tests. One experimentally infected sheep developed progressive clinical illness 1 yr postinoculation. Few M. paratuberculosis were isolated from feces or tissues although an extensive granulomatous mycobacterial enteritis, lymphadenitis and lymphangitis were observed containing large numbers of typical acid-fast organisms. No clinical illness was observed in the second inoculated sheep after 18 mo of observation, although infection was demonstrated at necropsy. Both sheep developed AGID and LST reactions indicative of paratuberculosis. This study demonstrated that a difficult to culture isolate of M. paratuberculosis was responsible for paratuberculosis in captive wild ruminants and was transmissible to domestic sheep. Diagnosis of paratuberculosis in four of eight of the imported saiga antelope and in eleven of their 18 offspring indicates the importance of this disease in management of captive wild ruminants and the ease with which this organism can be transmitted. Key words: Paratuberculosis, saiga antelope, transmission, sheep, Saiga tatarica, natural infection.
INTRODUCTION While is
paratuberculosis
primarily
nants it
has
1969)
also
been
captive
1988;
ilies
et al.,
(Katic,
in
Bovidae)
This
to the in
and
clinical
expressed
in
investigate the in
virulence
material into
the
organism
and
newborn
one
the
was young
poor
cultural
possible different
frequently To
recovery
of
animal was
there
causative
animals. strain
antelope
domestic
tatari-
in Canada.
in cattle, of
disease
for from
disease
relatively
paratu-
(Saiga
isolation
fam-
Bovidae
reports
to a zoo
In comparison agent
and
paper
was
van 1982;
b) of the
antelope
imported
et al., and
1980,
1983a,
saiga
difficulty
free-ranging
Chiodini
Camelidae,
1961).
Bovidae,
(Baradel
Weber,
1979,
Cervidae,
berculosis
in
AND
in imported
METHODS
saiga antelope
Sixteen saiga antelope were imported into Canada in 1985 from a zoo in Germany. These animals had been negative on previous export testing for paratuberculosis in the country of origin. Serum was collected during quarantine for paratuberculosis testing in addition to other federal import test requirements. Two animals died in quarantine, two male and six female antelope were sent to the Assiniboine Park Zoo (Winnipeg, Manitoba, Canada R3P 0R5) where they were housed in a newly constructed exhibit area and six animals were sent to another facility in Canada for which no further information is available. Necropsies were performed at the Manitoba Veterinary Services Laboratory, Winnipeg, on all saiga antelope which were euthanized or died at the Assiniboine Park Zoo beginning 6 mo after the animals arrived. The lower small intestine and associated lymph nodes were examined. Histologic sections were stained by hemotoxylin and eosin and by Kinyoun’s acidfast method. Paratuberculosis lesions were classified microscopically on the basis of morphology and the number of acid-fast bacteria present
rumi-
family
1982; 1983;
disease)
domestic
the
ruminants
Commichau,
Williams
of of
observed
wild
Kruiningen,
Ca:
(Johne’s
a disease
(Katic,
and
MATERIALS
Paratuberculosis
variation species, inoculated
sheep. 161
162
JOURNAL OF WILDLIFE DISEASES, VOL. 28, NO. 2, APRIL
as described (Brooks et al., 1988). A diagnosis of paratuberculosis required demonstration of typical acid-fast bacilli in association with a granulomatous lesion in the lower small or large intestine or associated mesenteric lymph nodes. Demonstration of typical acid-fast bacilli in a direct smear was not by itself considered to be sufficient criteria for a diagnosis (Merkal, 1973). Fresh, frozen or fixed tissues and blood from antelope with mycobacterial enteritis and lymphadenitis consistent with paratuberculosis were sent to the Animal Diseases Research Institute (ADRI) (Nepean, Ontario, Canada K2H 8P9) for immunological testing, culture and histology. Information was requested from five North American zoos to determine if paratuberculosis had been observed in their saiga antelope.
Experimental
animals
Ewes were selected as potential dams to deliver lambs for oral challenge experiments or to serve as negative controls from a caesarian derived “minimal disease” sheep flock. The ewes were moved to isolation cubicles prior to parturition. Lambs were held with their dams until weaning at approximately 75 days of age at which time the ewes were sent for necropsy. Negative controls were housed in separate cubicles in an adjacent building. The cubicles were washed daily to remove fecal material. Necropsies were conducted with emphasis on examination of intestine and associated lymph nodes.
Inoculation
and microbiological
procedures
Intestinal scrapings (3 g) from the mucosal surface from a histologically diagnosed case of paratuberculosis (antelope 12, Table 1) was homogenized with a tissue grinder and suspended in 22 ml of 0.85% saline to form the inoculum. An air dried smear of the inoculum was stained using the Kinyoun acid-fast method and examined microscopically. Large numbers of acidfast bacilli were seen per oil immersion field. The inoculum was stored at -70 C. It was thawed at room temperature (RT) prior to administration to the lambs and kept at 4 C during the period between inoculations. Two of three triplet lambs (981 and 982) in the experimental group received 2.7 ml of inoculum orally on day 1 (day of birth) and day 3 of the study. The total inoculation dose for each lamb was approximately 0.65 g of mucosa. The third lamb (983) in this group served as an in-contact control. Feces and blood were collected from the lambs weekly from weeks three to eight and monthly thereafter to the time of euthanasia or to the end of the study at 18 mo postinoculation. Ewes
1992
were sampled prior to selection for the experiment and weekly until weaning and euthanasia. Clotted blood samples were allowed to stand overnight at room temperature before processing. Heparinized samples of blood were used for the lymphocyte stimulation test (LST) within 6 hr of collection. Direct smears were made from tissues collected at necropsy and from some fecal samples. Smears were stained by Kinyoun’s acid-fast method and examined for the presence of typical acid-fast bacilli. Antelope fecal samples were shipped fresh, chilled but not frozen, and processed on the day of arrival at the laboratory. Fecal samples from the lambs were processed within 6 hr of collection. In general the culture procedure described by Merkal (1973) was followed, modified by using cetylpyridinium chloride (CPC) as the decontaminant and centrifugation prior to inoculation. Briefly, 1 g of fecal material was transferred to a 50 ml sterile, conical centrifuge tube and 40 ml of sterile distilled water added. The tube and contents were shaken for 30 mm, and then placed in an upright position and left to stand undisturbed for 30 mm. The top 5 ml of the fecal suspension was transferred to another 50 ml sterile centrifuge tube containing 35 ml of sterile CPC. The final concentration of CPC was 1%. Following standing at RT for 18 to 24 hr, samples were centrifuged at 2,250 x g (15 C) for 0.5 hr. The supernatant was discarded and the pellet resuspended in 1 ml sterile saline and distributed equally amongst five tubes of Herrold’s egg yolk medium (HEYM) containing 2 mg/i mycobactin J (Allied Laboratories, Glenwood Springs, Colorado 81601, USA). The inoculated media was incubated for at least 24 wk at 37 C. Isolates were subeultured onto 1 tube of HEYM with and 1 tube HEYM without mycobactin to confirm mycobactin dependency. Samples of intestine and mesenteric lymph nodes were collected from lambs 981, 982, and 983 at sites immediately anterior to and approximately 60 cm anterior to the ICV. Intestinal samples were also collected at two additional sites from 983. Tissues from the lambs were held at 4 C overnight and processed the day after collection. Culture of tissue samples was performed as described (Brooks et al., 1988) with decontamination in 0.5% CPC for 18 to 24 hr including centnfugation at 2,250 x g (15 C) for the final 30 mm. Resuspension of the pellet, inoculation of media and incubation were the same as for fecal samples. Identification of an isolate as M. paratuberculosis was on the basis of survival of the decontamination procedure described above, slow growth on HEYM with mycobactin, no growth on HEYM without mycobactin, typical colonial and cellular morphology and acid fastness.
DUKES
Immunological
ET AL.-SAIGA
ANTELOPE
procedures
Agar gel immunodiffusion (AGID) tests using antigen “D” and complement fixation (CF) tests using a crude carbohydrate antigen were conducted as previously described (Brooks et al., 1988; de Lisle et al., 1980a, b). Enzyme linked immunosorbent assay (ELISA) tests utilized an exclusion peak from S200 gel filtration of the crude polysaccharide as antigen to coat wells (NUNC Maxisorb Immuno modules, GIBCO, Burlington, Ontario, Canada L7P 1A1). The antigen was dissolved at 10 g/ml in 0.01 M carbonate buffer, pH 9.6, sonicated and then 100 zl placed in each well. The plates were sealed and placed at -20 C shortly after coating and stored frozen for an indefinite period. Plates were thawed at room temperature before use followed by washing four times in a 96 well plate washer (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2) using a 0.01 M Tris, 0.15 M NaCl, 0.05% Tween 20 buffer (TT), pH 8.0. Test sera were diluted 1:200 in TT buffer containing 0.02% NaN3 and 100 d placed in the microwell modules in duplicate. Controls consisted of (a) a prediluted and frozen aliquot of a positive bovine serum that served as a target, (b) a positive and (c) a negative serum from sheep diluted in the same manner as the test samples, and (d) TT buffer. These were placed on each plate as paired duplicates in columns 1 and 7 for a total of 16 wells for the four controls. Following an overnight serum incubation at room temperature, plates were washed and 100 l of an alkaline phosphatase conjugated rabbit anti-sheep F(ab’)2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania 19390, USA) was added for a 3 hr incubation at room temperature. Plates were washed again and 200 sl of p-nitrophenyl phosphate (1 mg/ ml) substrate in diethanolamine buffer, pH 9.8, 0.5 mm MgCl2 was added and the plates shaken continuously between three successive readings of optical density (405 nm) at approximately 2 mm intervals on a microplate reader (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2). The three optical density readings were used to determine the slope value for each paired sample using the regression function of Lotus 123 (Lotus Development Corp., Boston, Massachusetts 02142, USA). The mean slope value of the four control samples was used to calculate a corrected optical density for each sample thus standardizing test results for between plate variation. A microwell modification of the whole blood stimulation test (de Lisle and Duncan, 1981) was conducted in flat bottomed wells (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2) using three drops of media (approximately
PARATUBERCULOSIS
AND TRANSMISSION
TO SHEEP
163
150 l) and one drop of whole blood. Twelve wells were used for each sample consisting of four wells of the control media and two wells each of PHA (Phytohemagglutmn), J-PPD (johnin purified protein derivative), B-PPD (bovine) and A-PPD (avian). Plates were incubated for 72 hr at 39 C followed by the addition of 0.5 1zCi of tritiated thymidmne per well for 18 hr and then frozen at -20 C until thawing for harvesting on a Titertek Cell Harvester (Flow Laboratories Inc., Mississauga, Ontario, Canada L5S 1R2). The level of incorporation of the radiolabel was determined by the addition of 3 ml of scintillation fluid to minivials and counted on a liquid scintillation counter (Beckman Instruments, Toronto, Ontario, Canada L5T 1W5) using a wide tritium window. Results are expressed as net counts per mm (NCPM = mean of stimulated cultures minus mean of control cultures).
RESULTS in saiga
Paratuberculosis Two
imported
antine their
sent
The
to the
ropsied
Park
for
limited
intestine 1).
of
born
the
lymph
at
six
of
in
1989.
severity and
the
11
node first
zoo
in
second An
1987
cobacterial
of
of
ileal
phatics
with
lymph
nodes
imals.
Histologically,
matous matous
15
the white
enteritis in
the
for
or
animal in
Table
with
a
consisted
of
varying
thickening and
lym-
Mesenteric in
there
several
was
granulo-
numerous and and
mywith
dilated
nodules.
macrophages
lymphangitis
extent
each
enlarged with
in
animals
serosa
were
and born
is given
mucosal
on
2)
inflammation
enteritis tags
in
offspring
load the
(Table offspring
(Table
mortem
in
fibrous
done
granulomatous
Lesions
as the
confirmed
generation
post
examof
generation
mycobacterial
degrees
was also
estimation
of
examined
bacteria
not study
was
seven
necin four
were
histopathologic and
were Paratuber-
paratuberculosis
Paratuberculosis
five
Zoo
period.
others
de-
animals
histologically
two
specifically
only
being
eight
year
quarwith-
status
diagnosed and
in
trauma)
remaining a 6
was
antelope ined
and
Assiniboine
over
culosis
died
stress
paratuberculosis
termined.
3.
animals
(transport
out
antelope
acid-fast a
granulo-
lymphadenitis.
an-
JOURNAL
164
1.
TABLE
OF WILDLIFE
DISEASES,
VOL. 28, NO. 2, APRIL
of paratuberculosis
Summary
serological
1992
results
and
necropsy
findings
from
imported
adult
saiga
antelope. ACID’
Date
Animal number
10/85
10&
ND
07/86
07/89
09/88
Date of death2
04/90
Reason
12’s
-
-
13
-
-
-
-
-
07/91
14
-
-
-
-
-
07/91
15
-
-
-
-
-
05/91
death trauma trauma trauma killed killed death
16
-
-
02/88
hemolytic
17
-
-
06/87
18
-
clinical trauma
155’s 11
Agar 2
sampled
10/85
R
gel
during
Fecal
07/86
05/86 results:
R, reactor;
but was negative for death or euthanasia.
each
S. suspicious;
animal
at each
collected was
paratuberculosis ‘sIntestinal
tissue
during
this
quarantine
animal
were
While
revealed
a few
acid-fast
was
inoculated
observed
in
the
orally
into
with
2. spring
one
of
confirmed
1),
TABLE
unknown
positive unknown
paratuberculosis
not
done.
on
smears
The
complement
fixation
test
was
liver
of
four
imported
offspring
ani-
paratuberculosis
to the
AGID
all
confirmed
Summary 1987
bacteria
but
culture
for
Mycobacterium
sheep.
2,
only
a reactor
born
positive crisis
sampled.
animals.
(Table
ND,
unknown unknown positive positive negative negative
negative.
from
Granulomas
mals
negative;
‘ -,“
date
for necropsy
quarantine.
samples
some
03/87
immunodiffusion
also conducted Date of natural
Died
10/85
S
Paratuberculosis status
test
cases
in
ical
the
1987
serological
results
prior
1990,
remaining
of paratuberculosis and
May
to necropsy
detected to
reactors
or
AGID
33 AGID
findings
within
from
34) had
test. the
1
tested
and
offspring
the
(Table
less) When
(animals 1989
on
necropsy
25 necropsy.
two six
and
by
numbered
to 5 mo
was
prior
were
animals
in
of
the
serologThese
saiga
an-
offspring
1989.
AGID/CP Date
Animal number
2
09/88
Paratuberculosis status
07/89
04/90
Date of death
Reason for necropsy
S/S
R/R
07/90
thin
positive
12/88
died died
positive positive
19
14 (16)’s
-/-
20
17
R/S
21
13
-/-
R/R
22
16(14)
-/-
R/S
11/89
thin
positive
23
16(14)
-/-
R/S
08/89
died
positive
24
15
-/-
-/-
11/89
25
15
thin trauma
negative negative
07/89
07/87
29
19
-/-
07/91
killed
positive
30
21
-/-
03/91
31
21
-/-
09/90
positive positive
33
14
R/-
07/90
died pneumonia died
34
22
S/ND
04/90
died
positive
35
22
-/
07/91
killed
positive
‘Animals which Agar
Dam
sampled
19 to 25
were
born
in
1987.
died within 7 wk of birth. gel immunodiffusion/complement
No
Animals
evidence
-
of paratuberculosis
was
26 or higher were born in 1989. fixation: R, reactor; S. suspicious;
‘sDam
was
probably
animal
14 but
may
have
been
animal
16.
Dam
was
probably
animal
16 but
may
have
been
animal
14.
seen “-,‘
at necropsy negative.
of
animals
positive
(26,
27,
28,
32,
36)
DUKES
ET AL.-SAIGA
imals died within 2 mo of the test date and paratuberculosis nosed histologically in both. ing four 1989 offspring were have were
paratuberculosis killed over
cilli
the
although
were
3).
isolated
from ing
five the
of
acid-fast
fecal
of the
death
smears
the
paratuberculosis
quent cultural sues collected
was
animals
Salmonella juni, vey
spp.
from
and
animal
Cam
Two of three zoos responding had diagnosed paratuberculosis
ga antelope in their in which paratuberculosis received
their
rope. The zoo was not observed collections
in
within
Recovery experimental
11
+++
+++
+++
+++
12
+++
+++
+++
+++
13
-
-
ND
ND
14
-
-
ND
ND
15
++
++
++
++
16
-
-
-
ND
+++
were
colonies
isolated
from
of
+++
++
++
++
to tisanimals
23
++
++
+++
ND
24
-
-
ND
+
25
-
-
ND
ND
to je-
+++
+++
+++
+++
+++
+++
+++
+++
31
+++
+++
+++
+++
33
++
+++
+++
ND
34
+++
++
+++
++
35
+++
+++
+++
+++
No evidence other
successful.
paratuberculosis their saiga
29 30
of paratuberculosis
animals
(26,
A positive
direct
smear
for classification
died
or
of five
within
7 wk
of
lesions
sufficient
as positive
classified
for
mild;
as +,
evidence
paratuberculosis. + +,
moderate;
severe.
+++,
AF,
was not considered
of an animal
‘sparatuberculosis acid-fast
organisms hard
bacilli;
ND,
associated
to find;
not done.
with
+ +, small
Quantitation
lesions
was
numbers;
or
graded
of acid-fast as +, rare
+ + +,
and
large
numbers.
in
direct
from
America.
and
small
smears
in
numbers
of
the
were
lymph
Mycobacterium
paratuberculosis of lamb
intestine not from
from fecal
scrapings
seen
node.
paratuberculosis
in low
numbers lamb samples
from
was
a sample
982 (inoculated), of 982 or from
of but fecal
and tissue samples from lamb 983 (in-contact control). Acid-fast organisms were not seen on direct smears of tissues from either animal.
and no gross tuberculosis lambs.
lymph node, bacilli were seen
of intestinal
on necropsy
which
birth.
and while
smears
observed
27, 28, 32, 36)
Clinical and pathological experimental animals
direct
+++
++
56 and only small numbers of an organism consistent with M. paratuberculosis were isolated from the two samples of intestine
on the
+++
++
981 (inoculated), collected at 47, 51, 54, and 55 wk after inoculation. Small numbers of acid-fast organisms were seen on direct smears of feces collected at week 55. This animal was necropsied at week
from one mesenteric masses of acid-fast
ND
+++
22
to the surin sai-
samples
+++
-
Subse-
Eu-
fecal
+++
-
+++
from
M.
+++
-
isolated A few
AF’
++
antelope
of M. paratuberculosis animals
lesions’s
+++
zoos had
North
Direct smear2 AF’
thology
++
Both observed
which acquired
Histopa
+++
collections. was
Saiga
for necropsy.’
++
Attempts including not
and in
+++
pylobacter
11 were
of pathological paratuberculosis
for
21
12 and 17. Only small numbers of M. paratuberculosis were recovered after incubation for 24 wk although high acid-fast bacterial loads were demonstrated in dihistology. pathogens,
165
20
11. limited from
TO SHEEP
Gross lesions’s
19
follow-
results
presented
18
diagnosis
animal
studies were at post mortem
rect smear and by culture other enteric
antelope
17
collected
confirmed in
AND TRANSMISSION
Summary of examinations
Animal number
(Table
samples
imported
and
saiga
ba-
paratuberculosis from
3. microbiological TABLE
collected durof the imports for M. paratu-
in direct
Mycobacterium
not
PARATUBERCULOSIS
The remainconfirmed to
typical
observed
serological was diag-
when they died or next year (Table 3).
Culture of a fecal sample ing quarantine from one (animal 11) was negative berculosis,
ANTELOPE
There
was
no
findings
clinical
or microscopic in the dams
In
evidence
of
illness
lesions of paraof experimental
JOURNAL
166
thin
OF WILDLIFE
Clinically, lamb but appeared
when
was
it
DISEASES,
VOL. 28, NO. 2, APRIL
981 (inoculated) healthy until week
anorexic
and
had
soft
was 53 feces.
The feces became watery and proved again to a soft pelleted week 54. No Salmonella spp.,
then imform by Camplyo-
bacter spp. or parasites from the feces but fecal low numbers of acid-fast
recovered contained By week
56
the
severe
were smears bacteria.
diarrhea
had
the was
animal was euthanized. thin and had extensive
the
distal
small
submucosal
intestine
edema
thickness.
There
foci
along
the
The were
ileocecal enlarged
and
and
increased
mucosal
small
tortuous
2 mm
serosal
focal
numerous primarily
medulla
of
nodes. Numerous ing many acid-fast
lymphatics.
con-
were evident in granulomas were serosal of
lymphatics. macrophages
the
mesenteric
lymph
macrophages, containbacteria, formed sheets
necrosis were present area of the bowel. were full of necrotic in
normal ileum
mucosa. areas of
in the Peyer’s patch Some mucosal crypts debris and lymphatic
the
submucosa
982
(inoculated)
were
granulomatous
ident on acid-fast
lymphangitis
A mesenteric
lymph
of macrophages one giant cell were difficult
in the paracortex was seen. Acid-fast to find.
clinical
the
ICV
mass
gut
Histologically, lomatous
contained
were
associated
of
in
with
the
associated an
lesion
on pathological contact control
terminal
occasional associated
Immunological
test
(ELISA,
AGID,
slaughter. Sequential results from the two 982)
and
the
(983) are test results
one
age.
and
AGID test lambs (981,
in-contact
control
lamb
1. The the three
ELISA lambs
of passive in titer at
titers
lambs level
in
rose than
the
antibody fol4 to 6 months
two
orally
more rapidly the in contact
approximately
submucosa acid-fast other focus cosal patches
of the bacteria
mucosa
and
ileocecal valve were seen. In
but no several
The
four
negative tive by
non-contact
by AGID ELISA with
gle
lamb
of macrophages was seen. This mulesion was associated with the Peyer’s
The the
LST responses to PHA two orally challenged
of
sections
the
ileum.
of ileum
only
Lymphangiectasia
in-contact
with
control
shown
were
as negaof a sin-
a non-persistent
are
200
lambs
classified exception
one
histologic
developed “D” anticlinical
control and the
inoc-
and to a control.
responded
the
LST)
shown in Figure indicate that
The
ulated higher
ELISA challenged
disease (981) days later.
in
non-
of dams of experimental lambs were normal limits on samples collected to selection and from lambing to
of the lung teria could
macrophages
seen
the
test results in
lambs to the developed
of
were of
challenged antibodies lamb that
foci
granuPeyer’s
granuloma acid-fast bac-
Both orally precipitating gen but the
bacwere
ileal
animals
experimental
of
acid-fast There
in-
tissue.
focal with
examinations lambs.
clinically
no
the
lymphoid
lymph nodes were enlarged with extensive thickening of the cortex. Histologically tiny granulomas were seen in the interstitium and liver but be demonstrated.
foci and only bacteria
seen
patches and one submucosal were seen but rarely were teria observed. No lesions of paratuberculosis
results within prior
ev-
lamb (983) but at necropsy (2 mm) were seen on the about 60 cm anterior to
had a low level lowed by a rise
was
node
signs
contact control two white foci intestinal serosa
markedly
but at necropsy the wall of the was thickened and the mesenteric
were
the serosa of terminal ileum and bacteria were only rarely found.
Immunological
acid-fast bacteria in the paracortical area
of cells in the intestinal and cecal Submucosal, partly mineralized
dilated. Lamb
white
granulomas
bacteria Similar
in the dilated diffuse aggregates
spaces
of
and mesenteric lymph nodes and had a thick cortex.
Histologically,
containing were seen
and
evidence
and
No
This animal thickening of with
were
taining acid-fast liver and lung. evident More
returned
1992
response. and lambs
J-PPD and
of the
in
Figure
2.
DUKES
ET AL.-SAIGA
ANTELOPE
PARATUBERCULOSIS
AND TRANSMISSION
TO SHEEP
167
09
0.5 > 0
C’
0.1
0
1.3 4 0
0.9 0
z 2
0.5
0. 0
0 ,
300 to reach responses that approximated exceeded those induced by PHA.
nai, but
days)
sensi-
control
lambs.
1922; this
Dorofev is the
of
paratuberculosis
In was
(Jar1949)
natural
antelope.
in
1922
Kalechev,
report
saiga
reported
since
and
first in
study
been
of antelope
parathe
pres-
confirmed
in three of eight (38%) of the saiga antelope imported into the zoo and in seven of 18 (39%) of their offspring. The prevalence of paratuberculosis have been higher 18 offspring died
in the offspring may since another six of the at less than six months
of age onstrate
may not evidence
culosis
or There
ent
has
species
tuberculosis
The antigen (J-PPD) and mitogen (PHA) stimulated responses demonstrated by lamb 981 were more variable than those of 982 and
mycobacterial
DISCUSSION
ini-
inoculated
PHA
of
non-contact
>10,000
to increased
sensitization
antigens at this demonstrated
generally
response
J-PPD
>20,000) post
was
the
in the
when one histologic because
early stages the negative
the
disease
of development. serological
readily demof paratuberwould
be
in the
If we accept status of the two
JOURNAL
168
adult
OF WILDLIFE
female
as absence tion
saigas
DISEASES,
VOL. 28, NO. 2, APRIL
numbered
13
of paratuberculosis,
of their
offspring
and
then
(19,21,33)
14,
infec-
M.
paratuberculosis
control itized tential
to
indicates
the
in
a
zoo.
A
survey
paratuberculosis was zoological collections
that
a problem in of saiga antelope
other em-
phasizes the need testing in the trade susceptible species.
for strict control of this species and
and other
M ycobacterium generally inactive ical tests (Chiodini,
para tuberculosis on traditional biochem1986) and considered
to be abolic
a homogeneous or biochemical
ular
species from perspective.
characterization
nuclease
by
analysis
demonstrated
paratuberculosis isolates igin) were very similar to the species reference de Lisle, 1986; Whipple
and cultural recovery with
techniques numbers onstrated Taylor, 1987;
(Dunkin 1951; Brooks
and
54 M.
where were
Balfour-Jones,
Gummarsson, et al., 1988;
1979; de Juste et al.,
present
culture bovine
sheep isolates
isolates tested.
DNA DNA
difficult
are distinct In addition,
from
lambs similar
However,
agnostic ELISA) ing
the
tests with
for the
bacterial
1935;
comparison bohydrate veloped
to all other
slow growing, mycobactin dependent Mycobacterium spp., such as the wood-pigeon bacillus (Matthews and McDiarmid, 1979) and isolates from humans with Crohn’s disease (Chiodini et al., 1984) have also been
were dif-
These obserthis antelope having char-
include
the agents
prepared
pobefrom
and no reliable meaof viable organisms the
experiment
con-
firms the potential for interspecies transmission of infection and disease. Infection of sheep by oral inoculation of this strain led to seroconversion to the standard di-
al., 1988). cipitating
Lisle, 1991;
have of M.
submitted the and
inoculum
mucosa, number
inoculated.
large dem-
hybridization that
in the
the intestinal sure of the
This strain for sheep
and
samples
These limitations for other unrecognized
tion
of genomic
in
freezing may poor recovery
were encountered. are consistent with of M. paratuberculosis
significance development
endonuclease
While in the
zoo, samples from without freezing
Carrigan and Seaman, 1990). Collins et al., 1990 demonstrated on the basis of restricpatterns
In the
paratuberculosis level of antibody load
or the
(AGID, reflect-
severity
lesions as previously observed ural disease in domestic sheep
including culture
from specimens of acid-fast organisms
identification
acteristics different from those of classical bovine isolates. There are several limitations to extrapolating the results of experimental paratuberculosis to transmission of the natural
ing
differences observed in Watt, 1954; (Saxegaard,
properties conventional
from the cultivated
disease. tential
(50 of bovine orto each other and strain (Collins and et al., 1989). How-
ever, in other studies, distinct between isolates have been pigmentation (Taylor, 1951; Stuart, 1965), pathogenicity 1989), poor
is
endothat
the
paratuberculosis.
paratuberculosis
ficulties vations isolate
a metMolec-
restriction
complicate
as M.
ed in smears. been a factor
in-contact
indicating
and
isolate
present study M. paratuberculosis was isolated only in low numbers from the antelopes and lambs although large numbers of organisms were frequently demonstrat-
lamb was observed in a daily sanenvironment and emphasizes the pofor interspecies spread of the dis-
ease
reported
of an
that horizontal transmission from animals other than infected dams was important. This is supported by the experimental study where transmission of the antelope isolate of
1992
bacteremia ical disease
The later antibodies
of the
in the (Brooks
development to antigen
of pre“D” by
to the ELISA response antigen in the lamb clinical disease may be from the perspective of resistance and may be particularly as exemplified by and the following
development a short,
natet
to carthat deof some of the diagnosis.
the
pathogenic apparent 11
of clinmo, in-
cubation period. Caseation of lesions similar to that seen in other tubercular mycobacterial diseases (M. avium, M. bovis) and paratuberculosis of domestic small ruminants
was
observed
in the
experimen-
DUKES
tal sheep of classical in
although bovine
evidence.
perimental leled our
ET AL.-SAIGA
ANTELOPE
the non-caseous paratuberculosis
Thus
the
disease observations
in
lesion was also
results
of
the
ex-
sheep have paralin the saiga antelope
ing
are
observed
to share
places (Alton, 1990). The control or eradication
terial require been
applied
in domestic
for example, index (including
has
water-
species than
animals.
been seen quantitation)
may have
Finally as ported around production spread of that
are
zoological
in
wild ruminants the world for
there strains
wild
for
collections
want
in en-
Airport,
this
study
the of
Dr.
Canada, Mirabel Mirabel, Quebec. His
valu-
antelope
examined
by them
at necropsy.
Boca
NAUDIE,
C.
PERREAU,
A.
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Nielsen
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and Raton,
C. DANNACHER, M. GOURREAU,
J.
Research
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to acknowledge
able contributions to Vivier of Agriculture
J.
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some interlibrary Health Laboratory’s
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ALTON,
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LITERATURE
a reliable of infection
antelope culture of histologic ev-
infection.
much appreciated for their efforts on our We thank the serology section for providing the results for standard serological tests for paratuberculosis. G. M. Ruckerbauer is thanked for her assistance with some of the German references. Claude Turcotte assisted with the cultural work. P. Atherton and G. Eldridge
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169
van Zyll de Jong made helpful comments on ungulate taxonomy. The technical staff of ADRI, especially Henry Barleben, Barbara Dupree, Jim Hodgson, Hilary Kelly, Nancy Lopes, Vita Radzius, Jeanette Jeffreys and Barbara Stewart are
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TRANSMISSION
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