Epizootic hemorrhagic disease virus in Montana deer and cattle
by Timothy Jay Feldner
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Veterinary Science
Montana State University
© Copyright by Timothy Jay Feldner (1980)
Abstract:
A cytopathic agent was recovered from the spleen and blood of a white-tailed deer that exhibited gross
lesions consistent with what has been called hemorrhagic disease (HD) in white-tailed deer. An
experimental white-tailed deer developed similar symptoms and gross lesions five days,
post-inoculation with a 10% spleen suspension from the original white-tailed deer.
The viral isolate was propagated in VERO cells and produced a cytopathic effect at 72 hours
post-infection consistent with that produced by bluetongue virus (BTV) or epizootic hemorrhagic
disease virus (EHD) when grown in the same cell system.
Electron microscopy allowed for a determination of viral morphology and size. The isolate was
approximately 56.5 nm in diameter with large doughnut shaped capsomeres consistent with orbivirus
morphology.
Results of the indirect fluorescent, antibody test indicated that the viral isolate was EHDV. Further
verification of identity was obtained in the plaque reduction neutralization test which indicated that the
isolate was EHDV of the Alberta serotype.
Serum samples were collected from free-ranging deer and from bovine populations in eastern Montana
to determine the activity of both EHDV and BTV in the two populations. Results indicated that 79% of
the bovine samples collected from areas in which HD had been reported within the last three years
were seropositive to EHDV.
On the other hand, only 3% of the same samples were BTV seropositive. Of the deer serum samples
collected from HD areas, 73% of the mule deer were found to be EHDV seropositive while only 5% of
the whitetailed deer were EHDV seropositive. The low percentage of EHDV seropositive white-tailed
deer was considered to be a result of the high mortality rate (90%) reported for EHDV infection in
white-tailed deer. Approximately 15% of the deer serum samples, obtained from HD areas were also
BTV seropositive.
Results of this study indicate that EHDV was the etiologic agent responsible for the recent HD
outbreaks in eastern Montana. These findings also suggest that EHDV may have been responsible for
the previous HD outbreaks that have occurred in the same area. The high EHDV exposure rate detected
in the bovine population indicated their active role in the epizootiology of EHDV in eastern Montana. STATEMENT OF PERMISSION TO COPY
In presenting th is the sis in p a r tia l f u lf illm e n t o f the
requirements fo r an advanced degree a t Montana State U n iv e rs ity , I
agree th a t the L ib ra ry s h a ll make i t fre e ly a v a ila b le f o r in sp e ctio n .
I fu rth e r agree th a t permission fo r extensive copying o f th is thesis
fo r s c h o la rly purposes may be granted by my major p ro fe sso r, o r, in
h is absence, by the D ire c to r o f L ib ra rie s .
I t is understood th a t any
copying or p u b lic a tio n o f th is the sis fo r fin a n c ia l gain s h a ll not be
allowed w ith o u t my w ritte n permission.
EPIZOOTIC HEMORRHAGIC DISEASE VIRUS IN
MONTANA DEER AND CATTLE
by
TIMOTHY JAY FELDNER
A the sis submitted in p a rtia l f u lf illm e n t
o f the requirements fo r the degree
of
MASTER OF SCIENCE
in
V eterinary Science
Approved:
MONTANA STATE UNIVERSITY
■Bozeman, Montana
May, 1980
ACKNOWLEDGEMENTS
The author would lik e to thank Dr. M. H. Smith fo r his support,
guidance, and, above a l l , his d a ily wisdom throughout the course o f
th is study.:
Thanks also go to his graduate committee and e s p e c ia lly
to Dp. Richard Mackie fo r his help in arranging fo r the c o lle c tio n
o f f i e l d samples.
The in te re s t and support o f members o f the Montana
Department o f Fish, W ild life , and Parks, in c lu d in g Mr. Jon Swenson,
Mr. Gary Dusick, and Mr. Ken Walcheck, helped to get th is study o f f
the ground.
A ppreciation also goes to the National Animal Disease
Center and the Arthropod Borne Animal Disease Research Center fo r
th e ir generous donation o f te s t reagents and reference v ir a l s tra in s
and e s p e c ia lly to Ruth Breckon o f the ABADRC fo r her help and
suggestions.
A special thanks go to the a u th o r's w ife , Karen, and to his
c h ild re n , J a rid and L in d sie , fo r th e ir patience and understanding.
TABLE OF CONTENTS
LIST OF TABLES.........................................................................................v
LIST OF FIGURES
.
................................................................ .....
vi
.......................................................................
I
I.
INTRODUCTION
2.
LITERATURE REVIEW
. / . .
C h a ra c te ris tic s o f the Virus
EHD H is to ry ..............................
Host Range .
Transmission
.................................................................
Pathology and P ath o g e n e sis...........................
Im m u n o lo g y ...........................................................
E pizootiology .
Diagnosis and Control
...............................................
OO cn -P=*
CHAPTER
13
14
18
22
25
27
3.
MATERIALS and METHODS .......................................................... 33
Cell C ulture and C ulture M e d i a ...................................33
V i r u s ..................................
33
Antibody
.......................................................................
34
Virus I s o l a t i o n ................................................................ 34
V ira l T i t r a t i o n s ................................................................ 36
E lectron Microscopy . .
. . ■. . . . .
37
In d ire c t Fluorescent Antibody Technique
. . .
37
S erological Study Areas ........................
. . . .
39
Serum C o l l e c t i o n ................................................................41
Plaque Reduction N e u tra liz a tio n Test
. . . . ' 41
Agar Gel P r e c ip itin T e s t .............................................. 42
4.
RESULTS........................................................................................44
Virus I s o l a t i o n ................................................................44
In d ire c t Fluorescent Antibody Technique
. . .
46
E lectron Microscopy .....................................................
46
Plaque Reduction N e u tra liz a tio n .............................. 49
S erological Survey
..................................................... .50
5.
DISCUSSION ...................................
6.
SUMMARY........................................................................................64
55
V
LIST OF TABLES
Page
Table
1.
Plaque Reduction Cross N e u tra liz a tio n
........................
.
4,
2.
BTV and EHDV Serology o f Montana Deer by Study Area .
.
53
3.
BTV and EHDV Serology o f Montana C a ttle by County
.
54
vi
LIST OF FIGURES
Figure
Page
1,
EHDV S p e c ific F lu o re s c e n c e .......................................................... 47
2,
IFAT Control S lide o f BTV In fe cte d C e l l s .............................47
3,
E lectron Micrograph o f EHDV-M78
4,
Plaque Reduction N e u tra liz a tio n P late
5,
Agar Gel P r e c ip itin P l a t e .......................................................... 51
.
.
.
................................
48
51
vii
ABSTRACT
A cyto p a th ic agent was recovered from the spleen and blood o f a
w h ite -ta ile d deer th a t e xh ib ite d gross lesion s co n siste n t w ith what
has been c a lle d hemorrhagic disease (HD) in w h ite -ta ile d deer. An
experimental w h ite -ta ile d deer developed s im ila r symptoms and gross
lesions fiv e days, p o s t-in o c u la tio n w ith a 10% spleen suspension
from the o rig in a l w h ite -ta ile d deer.
The v ir a l is o la te was propagated in VERO c e lls and produced
a cyto p a th ic e ffe c t a t 72 hours p o s t-in fe c tio n co n siste n t w ith th a t
produced by bluetongue v iru s (BTV) or e p iz o o tic hemorrhagic disease
v iru s (EHD) when grown in the same c e ll system.
E lectron microscopy allowed fo r a determ ination o f v ir a l morphol­
ogy and size . The is o la te was approxim ately 56.5 nm in diameter w ith
large doughnut shaped capsomeres c o n s is te n t w ith o r b iv iru s morphology.
Results o f the in d ir e c t flu o re sce n t, antibody te s t in d ica te d
th a t the v ir a l is o la te was EHDV. F urther v e r ific a tio n o f id e n tity
was obtained in the plaque reduction n e u tra liz a tio n te s t which
in d ica te d th a t the is o la te was EHDV o f the A lberta serotype.
Serum samples were c o lle c te d from fre e -ra n g in g deer and from
bovine populations in eastern Montana to determine the a c t iv it y o f
both EHDV and BTV in the two populations. Results in d ic a te d th a t
79% o f the bovine samples c o lle c te d from areas in w h ich .HD had been
reported w ith in the la s t three years were se ro p o s itiv e to EHDV.
On the oth er hand, only 3% o f the same samples were BTV s e ro p o s itiv e .
Of the deer serum samples c o lle c te d fro m .HD areas, 73% o f the mule
deer were found to be EHDV s e ro p o s itiv e w h ile only 5% o f the w h ite ­
ta ile d deer were EHDV s e ro p o s itiv e . The low percentage o f EHDV sero­
p o s itiv e w h ite -ta ile d deer was considered to be a re s u lt o f the high
m o rta lity ra te (90%) reported fo r EHDV infe ction., in ,w h ite -ta ile d deer.
Approximately 15% o f the deer serum samples, obtained from HD areas
were also BTV s e ro p o s itiv e .
. Results o f th is study in d ic a te th a t EHDV was the e tio lo g ic agent
responsible fo r the re c e n t.HD o u tb re a ks.in eastern Montana. These
fin d in g s also suggest th a t EHDV may have been responsible fo r the
previous HD outbreaks th a t have occurred in the same area. The
high EHDV exposure ra te detected in the bovine population in d ica te d
th e ir a c tiv e ro le in the e p izo o tio lo g y o f EHDV in eastern Montana.
CHAPTER I
INTRODUCTION
E pizootics o f what has been re fe rre d to as hemorrhagic disease
(HD) have occurred in w h ite -ta ile d deer ( Odocoileus v irg in ia n u s )
populations in various regions o f the United States fo r q u ite some
tim e,
The e a r lie s t reports o f HD in w h ite -ta ile d deer date back to
1890 (89).
Is o la tio n o f e t io lo g ic agents from natural outbreaks
as w ell as work done w ith experimental in fe c tio n s in w h ite -ta ile d
deer have ind ica ted th a t two very c lo s e ly re la te d v iru s e s , bluetongue
v iru s (BTV) and e p iz o o tic hemorrhagic disease v iru s (EHDV), are the
most lik e ly pathogens involved in these fa ta l HD outbreaks.
Both
o f these viruses have been shown to be tra n sm itte d by the same
arthropod v e c to r, C ulicoides v a riip e n n is .
The c lin ic a l disease
and the pathologic lesions caused by both viruses in w h ite -ta ile d
deer, as w ell as the pathogenesis o f both diseases, is in d is t in ­
guishable (93, 101, 103, 105).
,
While EHDV is considered, a t th is tim e , to a ffe c t only a few
species o f w ild ruminants, BTV was f i r s t recognized as a serious
pathogen in the ovine species.
BTV is now known to cause m orbidity
and m o rta lity in the bovine as w ell as in various species o f w ild
ruminants.
EHDV has re c e n tly been shown to have widespread a c tiv ity
in bovine populations re s u ltin g in i l l
defined consequences ( I ) .
The host range o f the two viruses and the ever increasing amount
o f contact occurring between w ild and domestic ruminants in the
2
United States establishes the e sse n tia l nature o f the i d e n t i f i ­
catio n o f the e tio lo g ic agent involved in any HD outbreak in a
w h ite -ta ile d deer population.
Sporadic outbreaks o f HD in w h ite -ta ile d deer have been
recorded in the s ta te o f Montana since 1961 (113).
Although these
outbreaks have seemingly caused m o rb id ity and m o rta lity in fre e
ranging deer o n ly , the e tio lo g ic agent involved and i t s in t e r ­
re la tio n s h ip w ith domestic ruminants w ith in the s ta te has never
been e lu cid a te d .
BTV has the p o te n tia l fo r having a great economic impact in
a s ta te such as Montana where beef production is a m u lti- m illio n
d o lla r in d u s try .
P re se n tly, absolute embargos o f United States
liv e s to c k are in e ffe c t in many cou ntrie s due to the a c t iv it y
o f BJV w hile
other countries w ill not allow im po rta tion o f United
States liv e s to c k w ith o u t expensive te s tin g f i r s t being done.
T h is, along w ith the d ir e c t e ffe c t o f BTV in sheep and c a ttle and
the congenital e ffe c ts shown to be associated w ith BTV in fe c tio n ,
are cause fo r concern.
S im ila rly , EHDV and BTV could have an obvious d ire c t e ffe c t
on a second economic resource in the s ta te o f Montana;- i t s w ild ­
l i f e popula tion.
The very close a s s o c ia tio n , in clu d in g a n tig e n ic
s im ila r it ie s , between EHDV and BTV, ra ise s questions as to the
e ffe c ts EHDV, although not pre vio u sly recognized, may have on
3
domestic liv e s to c k .
Indeed, Ibaraki v iru s , a v iru s which causes
a b lLjetongue-Iike disease in Japanese c a t t le , is now thought to
be a s tra in o f EHDV (1 ,1 3 ).
In order to fu r th e r understand the HD problem as i t occurs
in Montana, two major o b je ctive s were developed as a p a rt o f th is
study.
F ir s t, to id e n tify the e tio lo g ic agent involved through
v ir a l is o la tio n from natural HD cases c o lle c te d in the f i e l d and
through the c o lle c tio n o f se ro lo g ic a l data from fre e-rang ing
deer in areas where HD was or had been a c tiv e ; and second, to estimate
the involvement o f domestic ruminants in the HD outbreaks through
a se ro lo g ica l survey o f the bovine populations in and around areas
o f HD outbreaks fo r antibody to both EHDV and BTV.
This th e sis is a re p o rt on the re s u lts from studies designed
to achieve these aforementioned o b je c tiv e s .
CHAPTER 2
LITERATURE REVIEW
For a t le a s t the la s t two decades, a h ig h ly fa ta l disease
has been occurring p e rio d ic a lly in the Montana w h ite -ta ile d deer
(Odocoileus v irg in ia n u s ) p o pula tion.
The disease is sporadic in
■
i t s appearance and occurrs only in the la te summer and e a rly f a l l
suggesting transm ission by an arthropod v e c to r.
Deer observed
during the term inal stages o f the disease have been noted to have
a loss o f a le rtn e s s , drooping ears, and extended necks in d ic a tin g
dyspnea (89).
Some o f the diseased deer have also been observed
w ith blood flecked foam near the muzzle area.
During these outbreaks, many o f the deer carcasses have been
found near water re s u ltin g in three possible im p lic a tio n s .
F ir s t,
the. deer may be fe b r ile and be in s tin c tiv e ly try in g to cool th e ir
bodies in w ater.
One observer watched a d is o rie n te d w h ite - ta il
submerge i t s e l f up to the neck before f a llin g dead in the water (113).
Second, the weakened c o n d itio n o f the a ffe c te d animals may cause
them to choose dow nhill t r a i l s w hich, in most cases, eve ntu alIy
lead to water.
F in a lly , the a ffe cte d deer may simply be seeking
p ro te c tiv e cover from the heat o f the sun and fin d sudi cover along
streambeds and riverbeds (35).
Almost a ll carcasses found during
the outbreaks have been in good fle s h in d ic a tin g th a t the animals
died from an acute disease.
Post-mortem examinations o f w h ite -ta ile d deer found during
5
the disease outbreaks have yie ld e d f a i r l y consistent re s u lts .
Among the fin d in g s are small subperic a rd ia l and myocardial hemor­
rhages, massively edematous lungs w ith hemorrhages over the surface,
excessive amounts o f serous f lu id in the th o ra c ic c a v ity and
p e ric a rd ia l sac, and petechial hemorrhages on the lymph nodes,
thymus, and the g a s tro in te s tin a l tr a c t (83).
A ll o f these lesions
are in d ic a tiv e o f experimental bluetongue v iru s (BTV) or e p iz o o tic
hemorrhagic disease v iru s (EHDV) in fe c tio n in w h ite -ta ile d deer (23,
54, 103, 105, 108).
W ithout is o la tio n and id e n tific a tio n of
the e tio lo g ic agent in vo lve d , these outbreaks are simply re fe rre d
to as hemorrhagic disease (HD).
The f i r s t documented cases o f HD in Montana occurred in 1961
in p rim a rily the northeastern section o f the s ta te .
There were
scattered reports o f deer m o rta litie s during the summer months
o f 1962, 1970, and 1971.
In 1976, a larg e scale d ie - o ff occurred
w ith m o rta litie s reported in 34 areas in eastern Montana, p rim a rily
McCone and Powder River counties (113).
Again, in 1977, there
were heavy w h ite -ta ile d deer losses during the summer months along
the Yellowstone River drainage in Richland and Dawson counties.
Ip one study area, c o n s is tin g o f approxim ately 16 m iles o f w h ite ­
ta ile d deer h a b ita t along the Yellowstone R iver, i t was estimated
th a t 190 w h ite - ta ils , about one th ir d o f the e x is tin g population in
th a t area, had died o f HD (93).
F in a lly , in 1978, w h ite -ta ile d deer
6
m o rta litie s occurred in the southeastern section o f Montana near
the town. o f Ekalaka.
C alcula tions using known population data in
the Long Pines area, 30 m iles south o f Ekalaka, ind ica ted th a t one
th ir d o f the area's w h ite - ta il deer p o p u la tio n , or approxim ately
500 deer, may have died o f HO (19).
Many o f the dead deer in th is
area were reported by hunters as being a n tlere d males' in v e lv e t
in d ic a tin g th a t death occurred sometime during Ju ly or August.
In an attempt to id e n tify the causative agent o f the 1976
MD outbreak, blood and tis s u e samples from deer m o rta litie s were
sent to v ir o lo g is ts in A lb e rta , South Dakota, and Ohio; to the
N ational Animal Disease Center in Ames, Iowa; and to the U n iv e rs ity
o f W isconsin's V ete rinary Science Laboratory.
None o f these
la b o ra to rie s were able to is o la te the causative agent o f the HD
from tissues received.
Workers from Wisconsin, however, using a
macro agar gel p r e c ip itin te s t and employing tissu e suspensions as
antigens and reference BTV and EHDV a n tis e ra , ind ica ted th a t e ith e r
BTV, EHDV, or both, were involved in the outbreak (118).
C h a ra c te ris tic s o f the Viruses
BTV and EHDV are very c lo s e ly re la te d viruses o f the o r b ivirus
genus.
Ten d is t in c t o r b iv iru s subgroups are recognized based
upon se ro lo g ica l re s u lts in d ic a tin g shared antigens among c e rta in
members. (6, 47).
The presence o f shared antigens, or cross reacting
antigens, between BTV and EHDV has warranted th e ir placement in the
7
same se ro lo g ica l subgroup (72).
The o r b iv iru s genus is included w ith the reovirus and probable
ro ta v iru s genera in the re o v irid a e fa m ily since a ll members possess
double stranded RNA as th e ir n u cleic acid type (69).
Like the
reov]'ruses, BTV and EHDV have been shown to have a segmented genome
co n sistin g o f ten segments o f three d is t in c t size ranges (44, 56,
107, 111).
BTV and EHDV are m orphologically in d is tin g u is h a b le , precluding
d iffe r e n tia tio n by e le c tro n microscopy.
The capsids o f both range
in diameter from 55 to 65 nanometers and are s in g le layered s tru c ­
tures composed o f 32 capsomeres (21, 73, 99, 106, 109).
The
capsomeres are r e la tiv e ly Targe (10.5 to 11.5 nanometers in diameter)
w ith an a x ia l hole 4.5 nanometers in diameter re s u ltin g in a
"doughnut lik e " appearance (106).
Although n e ith e r v iru s has a
tru e envelope, confusion re su lte d from e a rly reports o f enveloped
p a rtic le s in p u rifie d v iru s preparations.
These s tru c tu re s are now
known as "pseudo envelopes" and are a c tu a lly c e llu la r membranes
which o cca siona lly enclose one o r more v irio n s .
The pseudo enve­
lopes may be removed through fu r th e r p u r ific a tio n procedures w itho ut
a ffe c tin g in f e c t iv it y and are not considered to be p a rt o f the
v ir io n (21, 99).
BTV and EHDV are also very s im ila r in terms o f t h e ir physiochemical p ro p e rtie s .
Both have been reported to be r e la tiv e ly
8
re s is ta n t to li p i d solvents and sodium deoxycholate w ith only s lig h t
losses in in f e c t iv it y occurring a fte r incubation w ith s o lve n t fo r
16 to 20 hours (6)..
U nlike the re o viru se s, BTV and EHDV are heat
la b ile as w ell as very acid la b ile w ith complete loss o f in f e c t iv it y
occurring below a pH o f 6.4 w ith in 30 minutes (24, 43, 57).
C ells in fe c te d w ith e ith e r BTV o r EHDV respond w ith the
production o f dense cytoplasm ic in c lu s io n bodies.
Wi.th BTV in fe c tio n ,
some o f these cytoplasm ic in clu sio n s contain s p e c ific BTV antigen
as demonstrated by the d ir e c t flu o re s c e n t antibody technique (11).
With EHDV in fe c tio n , the in clu sio n s appear as v ir a l m atrices con­
ta in in g more and more v ir a l progeny as in fe c tio n progresses (106).
These v ir a l progeny become associated w ith the newly formed v e s ic u la r
membranes w ith in the cytoplasm re s u ltin g , perhaps, in the form ation
o f pseudo envelopes around groups o f v ir a l p a rtic le s (99).
BTV
and EHDV conclude th e ir re p lic a tio n cycle w ith c y to ly s is which
re s u lts in th e ir release from the in fe c te d c e ll.
EHD H istory
. EHD was f i r s t described by Shope e t a l . (88) in 1955 a fte r
an outbreak in New Jersey w h ite -ta ile d deer.
They pointed out the
fa c t th a t a s im ila r disease had occurred as e a rly as 1890 and a t
irr e g u la r in te rv a ls since th a t tim e.
The outbreaks, u s u a lly reported
by hunters and woodsmen o f the area, had been v a rio u s ly diagnosed
as b la ckle g , blacktongue, mycotic s to m a titis , or hemorrhagic septicem ia.
9
Shope was able to is o la te a " f ilt e r a b le v iru s " from a natural Iy
in fe c te d deer and tra n sm it the disease to a healthy deer.
He ca lle d
the "new" v iru s e p iz o o tic hemorrhagic disease v iru s because o f the
s a lie n t c lin ic a l and p a tho lo gical features o f the disease (88, 89).
An almost simultaneous outbreak o f HD in Michigan w h ite -ta ile d deer
was reported by Fay e t a l . (22) as being caused also by a " f ilt e r a b le
v iru s " .'
During the la te summer and e a rly f a l l o f 1956, an e p iz o o tic of
HD occurred in southeastern South Dakota (80).
A v iru s is o la te d
during th is outbreak was reported to induce lesions s im ila r to those
produced by the New Jersey is o la te .
Using a series o f n e u tra liz a tio n
te s ts in w h ite -ta ile d deer, M e ttle r (71) came to the conclusion th a t
the South Dakota s tra in and the New Jersey s tra in o f the EHDV were
immunologicalIy re la te d but not id e n tic a l.
E pizootics o f EHD have
been reported p e rio d ic a lly in South Dakota since 1956.
In 1969,
s e ro lo g ica l evidence o f EHDV in deer was detected in 14 South Dakota
counties w hile evidence o f EHDV in porcine or bovine species was
detected in 34 counties (78).
EHD occurred in both western North Dakota and southeastern
A lberta in the summer and f a l l o f 1962.
Tissues c o lle c te d from a
diseased w h ite -ta ile d deer in North Dakota yie ld e d EHDV s e ro lo g ic a lly
id e n tic a l, as determined by n e u tra liz a tio n te s ts in mice, w ith the
New Jersey s tra in o f the v iru s (116).
During the outbreak, 200 w h ite -
/
10 ,
ta ile d deer, 18 mule deer (Odocoileus hemionus), and 11 antelope
(Antelocapra americana) m o rta litie s were a ttrib u te d to EHD (84)..
In 1970, an e p iz o o tic in the same area o f western North Dakota
caused an estimated 2000 w h ite -ta ile d deer deaths.
During th is
e p iz o o tic , EHDV was again is o la te d and was considered to be the
e tio lo g ic agent responsible fo r the outbreak.
S erological data
c o lle c te d in 1970 and 1971 in North Dakota ind ica ted th a t w hile only
6% o f the w h ite -ta ile d deer tested were se ro p o s itiv e fo r exposure
to EHDV, 43% o f the mule deer tested were EHDV s e ro p o s itiv e (35).
These data suggest the le t h a lit y o f EHDV fo r w h ite -ta ile d deer and
the gre a te r resistance o f mule deer to the disease.
During the 1962
A lberta outbreak, 450 w h ite -ta ile d deer, 20 mule deer, and 13
antelope m o rta litie s were reported (14).
The EHDV is o la te d in
A lberta was said to be id e n tic a l to the New Jersey s tra in but the
te s tin g procedures used were not mentioned (18).
Comparison o f the various EHDV is o la te s was g re a tly f a c i l i ­
tated in 1962 when M e ttle r (71) was able to succe ssfully propagate
EHDV in suckling mice and in HeLa c e lls .
Taking advantage o f these
new methods, T ra in e r and Wilhelm (116), using a mouse n e u tra liz a tio n
te s t and hyperimmune ra b b it a n tis e ra , compared the EHDV is o la te s from
fiv e geographical areas.
They concluded th a t the New Jersey, South
Dakota, Michigan, and two North Dakota s tra in s were a ll o f the same
serotype.
Hence, the work o f M e ttle r and the work o f Wilhelm and
11'
T ra in e r was in disagreement as to the existence o f more than one
serotype o f EHDV.
Further advances in d ia gno stic techniques,
however, allowed Barber and Jochim (5) to e s ta b lis h the existence
o f two d is t in c t EHDV serotypes using the plaque reduction n e u tra l­
iz a tio n te s t in BHK-21 c e lls .
They then proposed the present
nomenclature fo r the EHDV serotypes as EHDV type I , w ith the New
Jersey s tra in as- the p ro to typ e , and EHDV type I I , w ith the A lberta
s tra in as the prototype.
The o rig in a l South Dakota s tr a in , which
M e ttle r f i r s t proposed as being a d iffe r e n t serotype than the NewJersey is o la te , was lo s t through storage in the la b o ra to ry and was
never adequately serotyped.
E pizootics o f HO' in deer have, occurred in Washington s ta te in
1946, 1953, and 1967 (24).
During the 1967 e p iz o o tic 227 deer
m o r ta litie s , the m a jo rity o f which were w h ite - ta ils , were reported.
EHDV has since been is o la te d from the spleen o f one o f the 1967
m o rta litie s but has not been serotyped.
S erological surveys conducted in Idaho and Oregon have ind ica ted
the previous exposure o f b la c k -ta ile d deer and mule deer to EHDV.
In Idaho, 4 o f 80 serum samples tested were p o s itiv e fo r EHDV
antibody w hile 12 o f 524 samples were se ro p o s itiv e in a study
conducted in Oregon (55, 94).
EHDV has been is o la te d from w h ite -ta ile d deer.during HD o u t­
breaks in Wyoming, Kentucky, and North C arolina (39).
During a
12
1971 HD outbreak in the southeastern United S tates, EHDV se ro p o s itiv e
deer were id e n tifie d in F lo rid a and Georgia.
At the same tim e,
deer s e ro p o s itiv e fo r BTV were id e n tifie d in F lo rid a , North C arolina,
and South C arolina (81).
Is o la tio n o f both BTV and EHDV from one
deer m o rta lity during th is 1971 outbreak, as w ell as strong sero­
lo g ic a l evidence in d ic a tin g the a c t iv it y o f both v iru s e s , has
im plicated both BTV and EHDV in the outbreak (102). .
Further se ro lo g ica l surveys have in d ica te d the a c t iv it y of
EHDV in Nebraska, Texas, and Wisconsin (39, 115).
I t should, be
noted th a t a ll EHD outbreaks, or suspected EHD outbreaks, seem to
have been confined to the North American co n tin e n t.
A lso, most o f
these outbreaks have occurred west o f the M ississip p i R iver.
The
only evidence o f EHDV outside o f the North American c o n tin e n t was
the is o la tio n o f EHDV from pooled c o lle c tio n s o f C ulicoides spp.
in Ibadan, N igeria (58).
BT H isto ry
BT has been recognized as a disease e n tity for. ne arly a century
since su sce ptible European sheep were introduced in to South A fric a (42)
The f i r s t d e s c rip tio n o f the disease was made by Hutcheon in 1881
who c a lle d i t "e p iz o o tic , c a ta rrh " (45).
In 1906, T h e ile r (98) showed
th a t the causative agent was capable o f being f ilt e r e d through a
B erkfeld f i l t e r and was probably a v ir u s .
BTV seemed to be confined to the A fric a n con tinen t u n til 1943
13
when an outbreak in sheep on the isla n d o f Cyprus caused up to 70%
m o rta lity in some areas (30).
The disease q u ic k ly spread to P alestine
and S yria and e ve n tu a lly reached the United States where i t was
diagnosed in Texas in 1948 (33).
BT then appeared in C a lifo rn ia
in 1952 where a v ir a l is o la te was id e n tifie d as BTV (68).
Since
th a t tim e, BTV has become a widespread problem in domestic and w ild
ruminants throughout the world and has now been diagnosed in at
le a s t 30 states in the United States (3 ).
Host Range
Although BT was f i r s t recognized as a disease o f sheep, c a tt le ,
and goats, BTV is now known as a widespread pathogen in North
American and A fric a n w ild ruminants (9; 103).
S erological evidence
o f BTV has been found in North American moose, barbary sheep, e lk ,
antelope, w h ite -ta ile d deer, b la c k -ta ile d deer, and mule deer (104).
BTV has also been is o la te d from natura l outbreaks in cap tive w h ite ­
ta ile d deer and desert bighorn sheep (93).
Is o la tio n o f BTV has
been reported as w ell from two species o f A fric a n w ild rodents,
a f ie ld mouse (Rhabdomys p u m ilio ) and a swamp ra t (Qdomys irro ra d u s )
(103).
EHDV, on the other hand, was long believed to cause disease in
w h ite -ta ile d deer only.
EHDV has since been is o la te d from diseased
mule deer and antelope (14, 84).
The s u s c e p tib ility o f a v a rie ty
o f domestic and la b o ra to ry animals to EHDV has been tested under
14
experimental con dition s re s u ltin g in negative or inconclusive
re s u lts (31, 57).
seems unclear.
The re la tio n s h ip o f the bovine to EHDV, however,
S erological data gathered by various in v e s tig a to rs
in d ica te s th a t as much as 75% o f the bovine population in some
areas o f the United States are EHDV s e ro p o s itiv e ( I , 55, 78).
Furthermore, EHDV has been is o la te d from natural outbreaks in c a ttle
in a t le a s t two instances (5, 28).
Experimental in fe c tio n s in the
bovine, however, w hile re s u ltin g in a virem ia which may la s t fo r
up to 50 days post in fe c tio n , have not been shown to cause any
apparent c lin ic a l disease (31). ' A s im ila r s itu a tio n is seen in
experim entalIy in fe cte d e lk ( Cervus candadensis) where a c e ll
associated virem ia may be detected fo r up to 30 days post in fe c tio n
although no c lin ic a l disease is evident (37).
P rese ntly, both the
bovine and the e lk are considered as po ssible re s e rv o ir hosts fo r
EHDV due to the long la s tin g virem ia associated w ith experimental
in fe c tio n .
Transmission
H is to r ic a lly , outbreaks o f both EHD and BT have occurred during
the la te summer and e a rly f a l l and have ceased a b ru p tly w ith the
onset o f cool temperatures.
These observations led e a rly workers
to speculate th a t BTV was tra nsm itte d by an in s e c t vecto r (92, 98).
This speculation was substan tiated in 1934 when Nieschelz (76)
found th a t a mosquito, Aedes lin e a to p e n n is , was capable o f harboring
15
BTV fo r up to 19 days p o s t- in fe c tio n .
Is o la tio n o f BTV from
Culicoides species trapped around areas o f BT outbreaks was accom­
plishe d in 1944 and 1954 leaving only the actual mechanical or
b io lo g ic a l transm ission o f the v iru s to be demonstrated (2 0 ,-8 2 ).
DuToit made progress in th a t respect by tra n s m ittin g BTV to suscep­
t ib le sheep w ith C ulicoides p a llid ip e h n is (20).
Some doubt remained
as to b io lo g ic a l transm ission, however, since the insects th a t
DuToit used were not allowed an adequate period to d ig e st the blood
meal and because susce p tib le animals were not held in in s e c t fre e
in clo su re s.
Final proof o f b io lo g ic a l transm ission was made by
Foster (27) in 1963 by using experim entalIy in fe c te d sheep and
b io lo g ic a lly tra n s m ittin g BTV to su sce ptible sheep w ith C u licoides
v a riip e n n is .
Luedke (6 5 ), using the same b io lo g ic a l v e c to r, was
able to show transm ission from in fe c te d sheep to su sce ptible c a ttle
and vice -ve rsa .
A fte r takin g a meal o f in fe c tiv e blood, a minimum incubation
period o f 10 days is required before Culicoides can b io lo g ic a lly
tra n sm it BTV to a susce p tib le animal (27).
During th is incubation
period , BTV has been shown to m u ltip ly to a high t i t e r w ith in the
s a liv a ry glands o f the in s e c t vector reaching a peak t i t e r s ix to
seven days a fte r in fe c tio n and remaining s u ff ic ie n tly high to in fe c t
susce ptible mammalian hosts fo r up to 26 days (9. 12).
C u licoides
are capable o f taking numerous blood meals during th e ir life tim e ;
■
16
th u s , each in fe cte d f l y could conceivably tra n sm it BTV to several
susce ptible anim als.
Mechanical transm ission o f BTV has been accomplished e x p e ri­
mental Iy using the sheep ked (Melophagus ovinus) (61).
While some
in v e s tig a to rs have reported contact transm ission o f BTV between
penned animals, the m a jo rity o f in v e s tig a to rs disagree (87, 92,
98, 101).
Repeated oral exposure, however, re la te d to the frequency
and not to the amount o f inoculum used, has been shown by Luedke (51)
to cause c lin ic a l BT in sheep.
In te re s tin g ly , fiv e o f the seven
sheep th a t developed c lin ic a l BT in Luedke's study did not develop
s ig n ific a n t antibody t i t e r s to BTV.
Urine from in fe c te d sheep does
contain pathogenic BTV allow in g fo r possible oral exposure under
natural c o n d itio n s.
Prompted by m orphological, p a th o lo g ic a l, and c lin ic a l s im ila r *
I tie s between BTV and EHDV5 Boorman and Gibbs (7) c a rrie d out
studies to determine whether EHDV was capable o f m u ltip ly in g in
C u ljcoides. • They found th a t EHDV m u ltip lie s in C ulicoides v a riip e n n is
w ith v iru s increasing to 1000 times the amount o r ig in a lly ingested.
I f v iru s is inoculated d ir e c tly in to the hemocoele, the amount
o f v iru s present ris e s to almost 100,000 times the amount inoculated
by seven days post in o c u la tio n .
These re s u lts suggested th a t
C ulicoides v a riip e n n is could be in fe c te d w ith EHDV by in g e stio n
and th a t the in fe cte d midges could be capable o f tra n s m ittin g the
17
v iru s by b it e . '
Is o la tio n o f EHDV from w ild Culicoides in N igeria
enhanced speculation th a t Culicoides was involved in the natural
transm ission cycle o f EHDV.
Final in c rim in a tio n o f C ulicoides v a riip e n n is as a b io lo g ic a l
vecto r o f EHDV was obtained by Foster e t a l . (25) in 1977.
In a
series o f t r i a l s , EHDV was tra n sm itte d between w h ite -ta ile d deer
by C ulicoides v a riip e n n is .
The insects were la b o ra to ry reared and
given a blood meal, through a membrane, o f in fe c tiv e blood.
A fte r
incubation periods o f 18 to 20 days, the insects were allowed to
feed on a re c ip ie n t host deer.
R ecipient host deer were then used
as in fe c tiv e donors fo r fu r th e r b io lo g ic a l transm ission.
Using
these methods, EHDV was su cce ssfu lly tra n sm itte d through two s e ria l
passages and caused acute disease in re c ip ie n t host deer.
Evidence also e x is ts fo r the transm ission o f EHDV through
contact or oral exposure.
In 1964, D itc h fie ld and associates (18)
hypothesized th a t the normal route o f EHDV transm ission was through
oral exposure and showed th a t pathogenic EHDV was shed in the
feces o f an in fe cte d animal.
This oral exposure, they po stu la te d ,
caused an asymptomatic illn e s s although an occasional anim al,
because o f marked s u s c e p tib ility or because o f a massive dose of
the v iru s , succumbed to the in fe c tio n .
Mechanical or b io lo g ic a l
transm ission by insects was thought o f as an abnormal route o f
in fe c tio n which generally culminated in death o f the in fe c te d animal.
18
Pathology and. Pathogenisis
Upon e n try in to a susce ptible host, both' EHDV and BTV require
an incubation period o f approximately fo u r to twelve days before
the appearance o f c lin ic a l signs (23, 59).
Studies done by P ini (79)
in d ic a te th a t BTV enters the regional lymph nodes o f a susceptible
animal and from there is disseminated via lymph or blood to lymphoid
tissue s in other areas o f the body.
A fte r primary re p lic a tio n in
these areas, a virem ia develops (u s u a lly s ix days p o s t-in fe c tio n )
and v iru s is c a rrie d to the m a jo rity o f body tissues re s u ltin g in
the appearance o f macroscopic lesions by e ig h t days p o s t-in fe c tio n .
Viremia has been shown to be c e ll associated and contained p rim a rily
w ith in the e ry th ro c y tic fra c tio n o f the blood in the case o f both
BTV and EHDV in fe c tio n (4, 38, 62).
The association o f the v iru s
w ith the c e llu la r fr a c tio n , e s p e c ia lly in the case o f BTV, may be
responsible fo r the a b i li t y o f the v iru s to c o -e x is t w ith n e u tra l­
iz in g antibody leading to la te n t in fe c tio n s , p a r tic u la r ly in the
bovine (31).
During the virem ic stage o f experimental EHDV in fe c tio n s
in w h ite -ta ile d deer, evidence o f v ir a l re p lic a tio n w ith in the
vascular e n d o th e lia l c e lls has been observed u ltr a s tr u c tu r a lIy (17,
106, 108).
A p r o c liv it y o f BTV fo r vascular e n dothelial c e lls
has also been reported (32, 93).
The hemorrhagic m anifestations
o f EHD and BT in w h ite -ta ile d deer are thought to be the re s u lt
19
o f a major disturbance both o f vascular in te g r ity and o f homeo­
s ta s is o f coa gulative mechanisms.
In te rru p tio n o f the en d o th e lia l
c e lls through d ire c t v ir a l a ctio n may allow contact between pro­
coagulants o f the blood and collag en , a c tiv a tin g the in t r in s ic
pathway o f blood coagulation.
At the same tim e, damage to e n dothelial
c e lls may cause the release o f tis s u e fa c to r and the a c tiv a tio n o f
coagulation through the e x tr in s ic pathway (93, 108).
M icrothrom bi,
a d ir e c t re s u lt o f blood coagula tion, have been demonstrated
h is to lo g ic a lly in both experimental BTV and EHDV in fe c tio n in
w h ite -ta ile d deer and correspond to the thrombocytopenia observed
during the course o f both diseases (23, 53).
C lin ic a l signs o f EHDV or BTV in fe c tio n in w h ite -ta ile d deer
begin fo u r to fiv e days post in fe c tio n w ith a pyrexia which c o rre ­
sponds to peak virem ia (39, 115).
S h o rtly th e re a fte r, the animal
shows signs o f anorexia, weakness, hyperemia o f the c o n ju n c tiv a ,
the tongue, and the oral and nasal mucosa, dyspnea, and, o fte n ,
excessive s a liv a tio n .
A fte r fo u r to 48 hours o f the acute disease,
the body temperature may become subnormal preceding coma and
death (29, 54, 89, 101, 112).
The m o rta lity ra te fo r both EHDV
and BTV in fe c tio n in w h ite -ta ile d deer has been reported to be
close to 90% although BTV is the le a s t fa ta l o f the two (39).
G rossly, the overwhelming le sio n o f experimental EHDV or
BTV in fe c tio n in w h ite -ta ile d deer is th a t o f hemorrhage, ranging
20
from petechial to ecchym otic, in various lo c a tio n s .
Several ta rg e t
areas may also be a ffe c te d by thrombosis o f small vessels and
accompanying tissu e necrosis.
These areas include the o ra l and nasal
mucosae, the tongue, the forestomach mucosa and submucosa, the myo­
cardium, and the acinar c e lls o f the mandibular s a liv a ry glands (23).
Widespread microthrombi form ation throughout the microvas­
c u la tu re co n trib u te s to the development o f edema associated w ith EHDV
and BTV in fe c tio n s .
In te rfe re n ce w ith normal blood c ir c u la tio n re s u lts
in subcutaneous, intram uscular, and fa c ia l edema, as w ell as
excess edema f lu id in the p e ric a rd ia l, p e rito n e a l, and p le u ra l
c a v itie s .
Massive pulmonary in te rlo b u la r edema is also a very
co n siste n t fin d in g w ith EHDV and BTV in fe c tio n s in w h ite -ta ile d
deer (23, 53).
Hematological studies concerning experimental EHDV in fe c tio n
have supported the diagnosis o f disseminated in tra v a s c u la r coagulation
(DIG), o f consumptive co a g u la tio n , as a s ig n ific a n t mechanism in the
pathogenesis o f the disease.
Shope (89) noted in e a rly studies th a t
"blood remained f lu id fo r an unusually long time a fte r i t was shed",
and postulated th a t the in fe cte d deer had bled in to th e ir own tissues
accounting fo r the shock th a t norm ally preceded death.
Karstad et a l .
(54) found g re a tly prolonged blood coagulation times and prothrombin
times th a t p a ra lle le d the increase in body temperature.
A co rre ­
sponding increase in the e ryth ro cyte count during the term inal
21
stages o f the disease has been a ttrib u te d to hemoconcentration
as evidenced by the severe edema.
Although to ta l leukocyte counts
remain unchanged during EHD, a n e u tro p h ilia occurs w ith a c o rre ­
sponding lymphopenia during the term inal stages (117).
N e u tro p h ilia
may occur during a number o f disease con dition s which include
hem olytic c r is is and s ig n ific a n t blood loss (86)..
The u t iliz a t io n
o f fib rin o g e n and fa c to r V III la te in EHDV in fe c tio n fu r th e r im p lic a te
DIC in the pathogenesis o f EHDV in fe c tio n in w h ite -ta ile d deer (16).
Hemorrhage, due to the u t iliz a t io n o f coagulation fa c to rs , damage
to e n d o th e lia l lin in g s o f vascular beds, and the form ation o f .
m icrothrom bi, becomes the overwhelming re s u lt o f EHDV or BTV
in fe c tio n in w h ite -ta ile d deer.
BTV and EHDV may also cause reproductive problems in deer
as w ell as in sheep and c a ttle .
Congenital abnorm alities due to
BTV in fe c tio n in pregnant sheep were f i r s t noticed by S hultz
e t a l . (91) in 1954 when a m odified liv e vaccine was used on
pregnant ewes.
I t is now recognized th a t in utero BTV in fe c tio n
a t an e a rly fe ta l age is d ir e c tly responsible fo r congenital
encephalopathies in lambs as w e ll as being abortogenic and te ra ­
togenic in pregnant c a ttle exposed during the f i r s t o r e a rly
second trim e s te r o f pregnancy (62, 96).
Abortogenic e ffe c ts of
BTV have been reported in w h ite -ta ile d deer as w ell as tra nspla cental
passage and fe ta l involvement during experimental in fe c tio n s w ith
22
EHDV (17, 23, 100).
Immunology
The immunological aspects o f both BTV and EHDV in fe c tio n
are as complex as they are va rie d .
Other than the se ro lo g ic a l
and epidem iological aspects, most o f the research done concerns
BTV in fe c tio n .
This work covers everything from i n .U te ro 1in fe c tio n
and re s u ltin g immunological tolerance to po lyva le n t vaccine pro­
duction.
BTV and EHDV,' lik e oth er viruses possessing segmented genomes,
have the p o te n tia l fo r a n tig e n ic v a ria tio n leading to the appearance
o f new s tra in s o f the v iru s .
The presence o f m u ltip le a n tig e n ic
va ria n ts o f BTV was f i r s t suspected when vaccinal fa ilu re s occurred
a fte r using monovalent vaccines produced through v ir a l attenu ation
in embryonating chicken eggs.
N eitz (74) established the p lu r a lit y
o f a n tigenic a lIy d iffe r e n t s tra in s by ca rry in g out a serie s o f
cross p ro te c tio n te s ts in sheep.
Howell (41) was able to c la s s ify
the a n tig e n ic va rie n ts in to 12 d is t in c t serotypes by using an
in v it r o n e u tra liz a tio n te s t.
Since th a t tim e, the number o f BTV
serotypes has rise n to 20 (3, 15).
A ll o f the serotypes appear to
have a common a n tig e n ic component detecta ble by the complement
fix a tio n te s t , and an unknown number o f d iffe r e n t s p e c ific a n tigen ic
components detectable by the serum v iru s n e u tra liz a tio n te s t (42).
Recent work has in d ica te d th a t polypeptide. 2, a component o f the
23
v ir a l capsid, is responsible fo r the s tra in s p e c ific it y o f the
various BTV serotypes.'
At the same tim e , polypeptides 3 and 7
are thought to be responsible fo r not only the a n tig e n ic s im ila r it ie s
between the various BTV serotypes, but also the a n tig e n ic s im ila r it ie s
detected between BTV and EHDV (77, H O ).
To date, only two d is t in c t serotypes o f EHDV have been recog­
nized (5 ).
The lack o f a n tig e n ic v a ria n ts o f EHDV may simply
r e fle c t the small number o f is o la tio n s th a t have been made, in most
cases from w ild rum inants, since the i n i t i a l is o la tio n o f EHDV
in 1955.
As mentioned e a r lie r ; BTV and EHDV are c la s s ifie d w ith in the
same o r b iv iru s subgroup according to th e ir a n tig e n ic re la tio n s h ip .
Evidence o f th is close a n tig e n ic re la tio n s h ip is e x h ib ite d by a
cross re a ctio n in the complement fix a tio n te s t using immune mouse
a s c itic f lu id and in fe c te d suckling mouse brain antigen (6 , 72).
Lack o f a cross re action in the plaque reduction n e u tra liz a tio n
te s t in d ica te s th a t w hile EHDV and BTV may share a common group
antigen, each also has i t s own serotype s p e c ific antigen.
A th ir d member o f the BTV subgroup, Iba raki v ir u s , causes a
BT lik e disease o f c a ttle in Japan.
Although o r ig in a lIy thought
to be a s tra in o f BTV, se ro lo g ic a l comparisons w ith BTV serotypes
revealed no a n tig e n ic re la tio n s h ip .
Recent studies have now shown
th a t Ibaraki v iru s shares not only a common group antigen w ith both
24
serotypes o f EHDV, but also a serotype s p e c ific antigen w ith the
A lberta s tra in o f EHDV as evidenced by a cross reaction in the plaque
reduction n e u tra liz a tio n te s t (13).
Since EHDV and BTV may also
share a common group antigen , an in d ir e c t a n tig e n ic lin k a g e , o r an
e vo lu tio n a ry lin ka g e , is apparent between BTV and Iba raki v iru s .
The a n tig e n ic re la tio n s h ip s mentioned above leave room fo r
much e x c itin g speculation concerning the BTV subgroup o f the
o r b iv iru s c la s s ific a tio n .
F ir s t , the p o s s ib ility fo r a n tig e n ic
v a ria tio n through s h i f t o r d r i f t (immunological s e le c tio n ) w ith in
the segmented genome becomes apparent.
Dual in fe c tio n s w ith
d iffe r e n t s tra in s o f BTV o r w ith BTV and EHDV have been reported
and present the p o s s ib ility fo r recombination o f genetic in fo rm a tio n ,
or phenotypic m ixing, re s u ltin g in s tra in s presenting new a n tig e n ic
p ro p e rtie s (28, 95).
Second, v a ria tio n o f the v iru s through
passage in an uncommon host, as evidenced by the a tte n u a tio n o f
EHDV in suckling mice, may re s u lt in su b tle changes in the host
range o f the v iru s and subsequent pa th o lo g ica l changes w ith in a
host once re fra c to ry to the disease syndrome (90).
The pa th o lo g ica l consequences o f an in utero BTV or EHDV
in fe c tio n have been mentioned p re v io u s ly .
Further work oh in utero
BTV in fe c tio n has revealed th a t bovine fetuses in fe c te d a t 60 to
120 days o f g e statio n may re s u lt in the b ir th o f calves th a t are
la te n tly in fe cte d w ith BTV.
These calves u su ally experience BT
25
disease w ith in the f i r s t s ix months o f l i f e fo llo w in g the loss of
maternal antibody.
The calves may be immunologicalIy incompetent
(to le ra n t) o r immunologicalIy competent in terms o f BTV and, in
e ith e r case, may harbor the v iru s fo r periods up to f iv e years
(62, 63, 64).
The fa c t th a t la te n t v iru s can coe xist w ith n e u tra l­
iz in g antibody in immunocompetent animals thus holds tru e fo r BTV
as i t does fo r lymphocytic ch o rio m e n in g itis and la c tic dehydrogenase
v iru s in fe c tio n s in mice and A leutia n disease v iru s in mink (67).
With BTV in fe c tio n , resistance o f the v iru s to n e u tra liz in g antibody
may be a d ir e c t re s u lt o f the close asso ciation o f BTV, lik e EHDV,
w ith the c e llu la r fra c tio n o f the blood (50).
E pizootiology
The in se ct vector responsible, a t le a s t in p a rt, fo r the
transm ission o f BTV and EHDV has been id e n tifie d as C ulicoides
v a riip e n n is .
The method o f s u rv iv a l o f e ith e r v iru s during the .
in te re p iz o o tic period s, however, has not been completely explained.
While s u rv iv a l o f BTV w ith in the arthropod vector by transovarian
transm ission has been elim inated as an overw intering mechanism
fo r BTV, there are no comparable re ports fo r EHDV (39).
An accumulation o f evidence has suggested th a t the bovine
may be a re s e rv o ir host fo r BTV (10, 65, 75).
Luedke e t a l . (66)
reported the b io lo g ic a l recovery o f BTV from an in utero in fe c te d
hereford b u ll fo r up to fiv e years a fte r b ir t h .
Recovery was
26
f a c ilit a t e d by the b ite s o f non-infected C ulicoides variipenm 's
which re su lte d in a "showering e ffe c t" o f la te n t v iru s in to the
bloodstream o f the animal.
C ulicoides feeding on the b u ll a fte r
these s tim u la tin g b ite s were then in fe c te d and, a fte r a s u ita b le
incubation period, were able to b io lo g ic a lly tra n sm it the v iru s to
su sce ptible animals.
A mechanism such as th is fo r e ith e r BTV or
EHDV would allow fo r not only a s u ita b le re s e rv o ir ho st, but also
fo r the reappearance o f the v iru s a t a time when the in s e c t vector
has again become a c tiv e and b io lo g ic a l transm ission has once
again become possible.
-
The bovine has also been suggested as a re s e rv o ir host fo r
EHDV (31).
EHDV, as mentioned e a r lie r , has been is o la te d from
natural in fe c tio n s in the bovine in a t le a s t two instances (5, 28).
Although no c lin ic a l disease is apparent in experim entalIy in fe c te d
c a tt le , virem ia has been detected fo r as long as 50 days post
in fe c tio n (31).
Furthermore, se ro lo g ic a l evidence o f EHDV, exposure
in the bovine has been detected by more than one in v e s tig a to r
and in one instance was detected in 75% o f the c a ttle tested in a
p a rtic u la r area ( I , 55, 78).
S erological surveys in both domestic and w ild ruminants
have id e n tifie d geographical areas which are considered to be
enzootic fo r EHDV, BTV, or both.
Antibody to both v iru s e s , fo r
example, has been found in a large percentage o f the w h ite -ta ile d
27
deer on the Welder W ild life Refuge in Texas (34, 40, 115).
Recog­
niza b le outbreaks o f e ith e r disease, however, have not occurred in
th is area which may in d ic a te the enzootic nature o f the diseases.
Although sub-species d iffe re n ce s in the Texas deer may play a ro le
in th e ir resistance to BTV or EHDV outbreaks, the determ ining fa c to r
as to whether an area is enzootic or has p o te n tia l fo r an e p iz o o tic
area is probably the mode o f transm ission o f the v iru s .
E pizootic
areas may be defined as areas in which a large susce ptible population
is a v a ila b le and ap propriate conditions e x is t fo r the ra pid tra n s ­
mission o f the v iru s (115).
Oral exposure to EHDV has been reported
to cause s u b -c lin ic a l disease in w h ite -ta ile d deer w ith the re s u lta n t
b u ild up o f immunity (17, 18. 115).
In enzootic areas, th is mode
o f transm ission may produce immunity in a large enough propo rtion
o f the population to prevent an e p iz o o tic from occu rring .
Diagnosis and Control
The s im ila r it ie s th a t e x is t in the gross and h is to lo g ic a l
lesions produced in w h ite -ta ile d deer by not only BTV and EHDV,
but also mucosal disease v iru s , m alignant ca ta rrh a l fe ve r v iru s ,
and a number o f b a c te ria l pathogens, emphasize the need to is o la te
and id e n tify the e tio lo g ic agent involved in any HD outbreak.
Is o la tio n o,f EHDV and BTV may be accomplished most e a s ily by the
in o c u la tio n o f susceptible deer or sheep w ith tis s u e suspensions
obtained from diseased animals.
Virus may be recovered from a
28
v a rie ty o f tissue s w ith spleen and the c e llu la r fra c tio n o f the
blood being the two best sources (71, 85, 89).
While BTV may also be is o la te d through the in o c u la tio n .iof
embryonating chicken eggs, there are c o n flic tin g reports concerning
the success o f c u ltiv a tin g EHDV in avian systems (25, 36, 89).
Use o f embryonating chicken eggs or natural susceptible hosts
fo r v ir a l is o la tio n is advantageous in th a t p re lim in a ry adaptation
o f the v iru s to the system is g e nerally not required.
BTV and EHDV may both be propagated in suckling mice or
hamsters.
In th is system, however, s e ria l passage is necessary
fo r the adaptation o f the v iru s .
The same adaptation problems
are encountered when is o la tio n is attempted d ir e c tly onto c e ll
c u ltu re .
In most cases, a series o f b lin d passages must be c a rrie d
out before cyto p a th ic e ffe c t (CPE) becomes v is ib le .
C ell c u ltu re
systems s u ita b le fo r the propagation o f BTV include prim ary newborn
ovine kidney, Marmosa m itis embryo fib r o b la s ts , BHK-21 (clone 13),
and A frica n green monkey kidney (VERO) c e lls .
Systems which have
been s u ita b le fo r the propagation o f EHDV include primary fe ta l
w h ite -ta ile d deer spleen, HeLa, BHK-21 (clone 13), and A fric a n green
monkey kidney (VERO) c e lls (2 ).
The most s u b sta n tia l problem in is o la tin g the e tio lo g ic agent
from a HD outbreak in w h ite -ta ile d deer lie s in o b tainin g appropriate
samples from f ie ld cases.
Peak v ire m ia , in both BTV and EHDV
29
in fe c tio n s , is reported to correspond to the temperature spike
seen in the diseased animals (39, 116).
Hence, samples c o lle c te d
from animals which have not y e t succumbed to the in fe c tio n are
the most f r u i t f u l fo r is o la tio n attem pts.
The d i f f i c u l t y o f
obtainin g f i e l d samples from a w il d l i f e population during a s p e c ific
stage o f a disease is apparent.
A number o f te s ts may be applied in the id e n tific a tio n o f
BTV o r EHDV once they have been is o la te d .
Group s p e c ific te s ts ,
which w i l l not d iffe r e n tia te between the various BTV serotypes and
which, in some cases, may cross re act w ith a common a n tig e n ic
component o f EHDV include the m odified d ir e c t complement fix a tio n
te s t and the agar gel p r e c ip itin (AGP) te s t (8, 46).
The in d ir e c t
flu o re s c e n t antibody te s t (IFAT) is a group s p e c ific te s t but may be
used to d iffe r e n tia te between BTV and EHDV provided th a t cross
reactions are elim in ated by d ilu tio n o f the respective a n tise ra (47).
BTV and EHDV may be d iffe re n tia te d by the use o f the serum v iru s
n e u tra liz a tio n te s t (SN) or the plaque reduction n e u tra liz a tio n
(PRN) te s t (49).
The SN and PRN te s ts are both serotype s p e c ific
and w i l l d is tin g u is h not only between EHDV and BTV, but also
between the various serotypes o f each w ith the use o f serotype
s p e c ific a n tis e ra .
A number o f s e ro lo g ica l te s ts have been employed fo r the
detectio n o f group s p e c ific or type s p e c ific antibody to EHDV and
30
BTV in the serum o f w ild and domestic ruminants.
The PRN and SN
te s ts , being serotype s p e c ific , have lim ite d value in BTV s e ro lo g ica l
surveys or in the c e r t if ic a t io n o f BT disease fre e animals due ,
to the s tra in v a ria tio n s o f BTV (9 ).
The highest t it e r s o f n e u tra l­
iz in g antibody to BTV, as determined by the SN te s t, are reached
a t about fo u r weeks p o s t-in fe c tio n and are reported to p e rs is t
fo r as long as two years (65).
Group s p e c ific te s ts , on the o th e r hand, may de tect antibody
to any o f the BTV serotypes.
Because o f the group s p e c ific it y
o f these te s ts , however, cross reactions may occur between BTV
and EHDV antibody.
The m odified d ire c t complement fix a tio n te s t
is now the o f f ic i a l and most commonly used te s t to detect antibody
to BTV.
This te s t, however, is r e la tiv e ly in s e n s itiv e fo r the
d e tectio n o f antibody and, in some instances, may not be able to
d is tin g u is h between EHDV and BTV antibody (70, 72).
Complement
fix in g antibody peak t it e r s are reached a t fiv e weeks post in fe c tio n
but disappear from the c irc u la tio n a ft e r s ix to e ig h t weeks fu rth e r
in h ib itin g the meaningful ness o f the complement fix a tio n te s t (42).
The AGP te s t, adapted fo r the d e te ctio n o f BTV antibody by
Jochim and Chow (4 8 ), is also a group s p e c ific te s t.
This te s t
is a m o d ific a tio n o f the Ouchterlony te s t and employs a nonin fe c tio u s soluble antigen placed in a c e n tra l w ell w hile serum
samples to be tested fo r p r e c ip ita tin g antibody are placed in
31
peripheral w e lls .
Improved s e n s itiv it y may be obtained by placing
reference BTV antiserum in peripheral w e lls adjacent to the serum
samples to be tested (46).
BTV soluble antigen was reported by
Wang (114) as being derived from the v ir io n surface.
The actual
composition o f the solu ble antigen has been disputed and may, in
fa c t, be a m etabolic by-product o f v ir a l re p lic a tio n w ith in the
in fe c te d c e ll (48).
Although cross reactions may occur in the AGP
te s t, only high t it e r in g EHDV antiserum w ill cross re act w ith BTV
solu ble antigen and even then form only a f a in t p r e c ip itin l i n e . (46).
BTV antiserum , on the o th e r hand, does not cross react w ith the
major component o f EHDV soluble antigen but may cross re a c t w ith
what is termed a minor component o f the soluble antigen (52).
P re c ip ita tin g antibody can be detected in experim entalIy in fe c te d
sheep and c a ttle three to fo u r weeks post in fe c tio n and p e rs is ts '
fo r a t le a s t two years (48).
Control o f e ith e r BT or EHD in domestic or w ild populations
is a very d i f f i c u l t problem.
Vaccines using attenuated s tra in s
o f the v iru s have been produced fo r both BTV and EHDV.
The existence
o f m u ltip le s tra in s o f BTV, however, makes id e n tific a tio n o f the
s p e c ific s tra in a c tiv e in a p a rtic u la r geographic area e sse ntial
in order fo r a vaccine to be e ffe c tiv e .
BTV vaccine a v a ila b le in
the United states is produced using the BTV 8 (type 10) s tra in o f
the v iru s (3 ).
There are a t le a s t fo u r s tra in s or a n tig e n ic types
32
o f BTV a c tiv e in the United States (3, 60).
These a n tig e n ic types
e x h ib it varying degrees o f cross p ro te c tio n in sheep cross immunity
challenge te sts (9 ).
The s tra in v a r ia tio n , along w ith the fa c t
th a t vaccin ation o f pregnant ewes may cause congenital a b n o rm a litie s,
exem plify the problems encountered in a vaccination program fo r
BTV.
To com plicate matters fu r th e r , Foster e t a l. (26) has reported
th a t BT vaccine v iru s may hot only cause c lin ic a l disease in sheep,
but also may re v e rt back to it s more v ir u le n t form through fu r th e r
passage in hemophagous in se cts.
Immunization studies done by Shope and associates (90) i.ndicate
th a t EHDV may be attenuated.by s e ria l passage in suckling mouse
brain and w i l l produce a s o lid and durable immunity in w h ite ­
ta ile d deer as shown by challenge w ith homologous v iru s .
Again,
however, the problems encountered w ith BTV vaccines are encountered
w ith EHDV vaccines (87).
A d d itio n a lly , the problems o f vaccinating
a w ild population make vaccination fo r EHD an improbable method
o f co n tro l in the fu tu re .
Some hope may reside in the development
o f an oral vaccine th a t could be d is trib u te d in a p a la ta b le form
in areas where se ro lo g ic a l data and ve cto r presence may in d ic a te
the p o s s ib ility o f fu tu re EHD e p iz o o tic s .
Id e n tific a tio n o f the
re s e rv o ir host fo r EHDV may lead to fu tu re p o s s ib ilitie s fo r the
co n tro l o f EHD in fre e -ra n g in g deer populations.
CHARTER 3
MATERIALS AND METHODS
C ell C ulture and C ulture Media
A frica n green monkey kidney (VERO) c e lls 12 were used e x c lu s iv e ly
fo r v iru s is o la tio n , propagation, and t i t r a t i o n procedures.
VERO
c e ll c u ltu re s were grown in 90% medium 199 (M-199), pH 7.2 , and
10% fe t a l c a lf serum and maintained in serum fre e Eagle's basal
medium in E a rle 's balanced s a lt s o lu tio n (EBME).
One ml o f a c e ll
suspension containing approxim ately 150,000 c e lls per ml was used
to seed each 16 mm diameter w ell in tis s u e c u ltu re c lu s te r plates
2
3
o f 24 . Four chambered s lid e s
were seeded w ith 0.5 ml o f the
same suspension fo r the production o f monolayers.
Monolayers were
u su ally complete a fte r one day o f incubation a t 37°C in an hum idified
atmosphere o f 5% COg in a ir .
Vi rus
Reference v ir a l s tra in s used were EHDV-New Jersey, EHDVA lb e rta , and BTV type 13.
Al I three s tra in s were obtained from the
Arthropod Borne Animal Disease Research Center in Denver, Colorado
where they had been adapted pre vio u sly to VERO c e ll c u ltu re .
Stock v iru s pools were produced from in fe c te d VERO monolayers
grown in 250 ml p la s tic tis s u e c u ltu re fla s k s w ith a surface area
1
^Arthropod Borne Animal Disease Research Center, Denver, Colo.
2
Costar, D iv is io n Data Packaging Corp., Cambridge, Mass.
3
Lab-Tek Products, D iv is io n Miles Lab. I n c . , Kankakee, I l .
34
o f 75 cm^.
When cyto p a th ic e ffe c t (CPE) reached 70% to 100% in
the in fe c te d monolayers, media and c e ll debris were harvested by
low speed c e n trifu g a tio n (150 X g).
The c e ll debris was sonicated
in a small amount o f supernate, added back to the to ta l supernate,
and ce n trifu g e d again a t 1000 X g fo r one hour.
Cell debris was
then discarded and the supernate mixed w ith an equal volume o f
'o
buffered lactose peptone and stored in one ml a liq u o ts a t -70 C.
Antibody
Reference EHDV and BTV antibody o f bovine o rig in were obtained
from the National Animal Disease Center in Ames, Iowa.
Homologous
antibody against the v ir a l is o la te was prepared in A fric a n pygmy
I
goats.
Two goats were i n i t i a l l y in fe c te d through subcutaneous
4
in o c u la tio n o f fiv e ml o f in fe c tiv e deer blood in OPG . At .50
days post in o c u la tio n (D PI), the goats were re -in o c u la te d w ith
fiv e ml of a tis s u e c u ltu re prepared v iru s suspension containing
approxim ately 10^ plaque forming u n its (PFU).
Hyperimmune sera was
c o lle c te d 14 days la te r a t 64 DPI.
Virus Is o la tio n
Blood and tis s u e samples fo r v iru s is o la tio n attempts were
obtained w ith the cooperation o f the Montana Department o f Fish,
W ild life , and Parks.
Department personnel were contacted so th a t
^Anticoagulant p reservative s o lu tio n c o n s is tin g o f potassium oxa late,
5 g ; phenol, 5 g ; g ly c e rin 500 ml; and d is t i l l e d w ater, 500 ml.
35
f ie ld samples from any suspected HD m o rta lity would be shipped, on
ic e , to the V ete rinary Research Laboratory in Bozeman, Montana.
Is o la tio n was attempted both d ir e c tly in c e ll c u ltu re and
through in o c u la tio n o f an experimental w h ite -ta ile d deer.
Inocula
fo r experimental in fe c tio n consisted o f a 10% spleen suspension .
prepared in EBME containing 5000 u n its o f p e n ic illin and 5 mg o f
streptom ycin per ml o f suspension.
A fte r subcutaneous in o c u la tio n
w ith e ig h t ml o f the spleen suspension, the temperature and c lin ic a l
signs o f the experimental animal were monitored d a ily .
Heparinized
blood samples fo r is o la tio n attempts in c e ll c u ltu re were c o lle c te d
from the experimental animal when c lin ic a l signs in d ica te d an
ongoing virem ia.
Virus is o la tio n in c e ll c u ltu re was done according to the
method o f Bando (2) w ith s lig h t m o d ific a tio n s .
Whole blood from
natural HD cases, or heparinized blood from an experimental anim al,
was f i r s t mixed w ith an equal volume o f s t e r ile water.
The m ixture
was sonicated a t a s e ttin g o f 4 kc/second fo r one minute and
d ilu te d 1:5 w ith M-199 containing p e n ic illin (200 u n its /m l) and
streptom ycin (200 mcg/ml).
The d ilu te d blood preparation was
adsorbed in two ml q u a n titie s onto drained VERO monolayers th a t
had been established in 250 ml tis s u e c u ltu re fla s k s .
A fte r a one
hour adsorption period a t 37°C, the inocula were removed, the c e ll
sheets were rinsed w ith a calcium and magnesium fre e s a lin e s o lu tio n ,
36
and the maintenance media was replaced.
Infected fla s k s were observed d a ily fo r the appearance o f CPE.
CPE negative samples were taken through three consecutive b lin d
passages a t fiv e day in te rv a ls before being considered negative.
CPE p o s itiv e samples were harvested when 70% to 100% o f the monolayer
was a ffe c te d and processed in the same manner as the stock v iru s
pools.
V ira l T itr a tio n s
A m o d ific a tio n o f the plaque assay method described by Jochim
e t a l . (49) was used fo r v ir a l t it r a t io n s .
A fte r e s ta b lis h in g
VERO monolayers in 24 w ell p la te s , growth medium was removed and
0.2 ml o f fiv e - f o ld v iru s d ilu tio n s made in serum fre e M-199 were,
added to appropriate w e lls .
Adsorption was allowed fo r one hour
a t 37°C, the d ilu tio n s were, removed, and the monolayers were
o v e rla id w ith 0.5% agarose in M-199.
A fte r incubation f o r s ix days
in humid a i r - t i g h t containers a t 37°C, the monolayers were fix e d
fo r 30 minutes a t room temperature by the a d d itio n o f 0.5 ml o f
10% buffered neutral fo rm a lin to each w e ll.
Agar plugs were then
removed and the monolayers were stained w ith c ry s ta l v io le t .
Plaques in each w ell were counted to determine the number o f plaque
forming u n its (PFU) and c a lc u la tio n s made to determine the number
o f PFU per ml of stock v iru s pool.
37
E lectron Microscopy
Al though d iffe r e n tia tio n between BTV and EHDV cannot be
achieved by morphological examination, e le ctro n micrographs were
produced o f v ir a l is o la te s to assure the presence o f an o r b iv iru s .
VERO c e lls were grown in 250 ml tis s u e c u ltu re fla s k s and in fe cte d
w ith stock pools o f the v ir a l is o la te .
Monolayers were fre eze-
thawed when CPE approached 50% monolayer d e stru ctio n and the
c u ltu re f l u i d and c e llu la r debris harvested.
C e llu la r debris was
p e lle te d by low speed c e n trifu g a tio n and the re s u ltin g supernate
discarded.
The p e lle t was resuspended in fiv e ml o f d i s t i l l e d
water and two drops o f the suspension added to a spot p la te w ell
containing three drops o f 4% phosphotungstic acid (PTA), 15 drops
o f d i s t i l l e d w ater, and two drops o f a fre s h ly prepared bovine
serurn albumin s o lu tio n .
Contrast and d ro p le t spreading were
optimized by varying the fin a l concentrations of PTA and albumin.
A fte r m ixing, the preparation was sprayed onto a carbon coated
5
fo r m y a r -fiImed g rid w ith a Pelco
a ll glass n e b u lize r. Grids
were examined immediately w ith a JEOL IOOCX transm ission e le ctro n
microscope operated a t 80 kv.
I n d ire c t Fluorescent Antibody Technique (IFAT)
The id e n tity o f the v ir a l is o la te was in ve stig a te d using the
IFAT as described by Jochim e t a l . (44).
^Ted P ella I n c . , T u s tin , C a lif.
VERO monolayers were grown
38
in fo u r chambered s lid e s and in fe c te d w ith the v ir a l is o la te or
w ith reference v iru s s tra in s .
S lides were fix e d at 24 or 48 hours
p o st-rin fe ctio n by removing one h a lf o f the media in each chamber
and replacing i t w ith cold acetone.
A fte r 15 minutes a t 4°C, the
acetpne media m ixture was replaced w ith pure acetone fo r a 30
minute fix a tio n period at -70°C.
The acetone was then removed and
the s lid e s stored a t -70°C u n til stained and examined.
The s ta in in g procedure was conducted a t room temperature.
Monolayers were rinsed w ith carbonate-bicarbonate b u ffe r (pH 9.0)
and 0.15 ml o f a f iv e - f o ld EHDV antibody d ilu t io n , prepared in
carbonate-bicarbonate b u ffe r, applied to appropriate chambers.
The s lid e s were held fo r 30 minutes, serum d ilu tio n s were discarded,
and the monolayers were rinsed w ith fo u r changes o f b u ffe r fo r
one, th re e , th re e , and three minutes.
A fte r rin s in g , 0.15 ml o f
a 1:16 d ilu t io n o f flu o re s c e in conjugated a n ti-b o vin e IgG ® was
added to the chambers.
A fte r 30 minutes, the monolayers were
rinsed once again using the same procedure.
P la s tic chambers and
gaskets were removed and covers! ips were mounted using Well come's
medium (pH 8 .9 ).
The s lid e s were examined using a L e itz Orthoplan
microscope equipped w ith a d a rk fie ld condenser and a 200-W mercury
vapor lamp.
^Miles Lab. I n c . , Research D iv is io n , E lk h a rt, In.
39
S erological Study Areas
Four separate study areas were included in the s e ro lo g ic a l
survey fo r BTV and EHDV antibody in the serum o f fre e ranging
deer,
The Long Pines, In ta ke , and M issouri Breaks areas had
experienced, or were p re se n tly experiencing, HD outbreaks in the
w h ite -ta ile d deer p o pula tion.
A fo u rth study area, the B ridger
Mountain area, had no h is to ry of HD problems in the fre e ranging
deer population.
Long Pines is located approximately 30 miles southeast of
Ekalaka in C arter county, Montana.
The area is p a rt o f Custer
National Forest and consists o f pine covered h i l l s w ith an e le ­
va tio n o f 4000 fe e t.
E levation drops adjacent to the Long Pines
area onto a g ric u ltu ra l lands involved in both beef and wool pro­
duction.
C a ttle are also grazed in the Long Pines area during the
spring and summer under lease agreement.
W h ite -ta ile d deer mortal
it ie s were reported in th is area in the summer and f a l l o f 1961,
1970, .1976, and 1978 (113).
The Intake area, co n sistin g o f about 80 square miles o f w hite
ta ile d deer h a b ita t, is located 10 miles northeast o f Glendive in
Dawson county, Montana.
The area is bordered on one side by the
Yellowstone River and includes both rive rb o tto m and a g ric u ltu ra l
lands w ith an e le va tio n o f approximately 3000 fe e t.
The estimated
population d e n sity o f 43.5 w h ite -ta ile d deer per square m ile f e l l
40
to 31 per square m ile a ft e r a HD outbreak th a t occurred the summer
and f a l l o f 1977 (97).
S im ila r outbreaks were reported in nearby
areas along the Yellowstone River in 1975 and 1976.
The Missouri Breaks area is in G a rfie ld and P h illip s counties
and consists o f high b lu ffs bordering the Missouri R iver as w ell
as dense th ic k e ts along the rive rb o tto m .
The area supports both
mule deer and w h ite -ta ile d deer and is grazed by lease agreement
during the spring and summer.
HD outbreaks have been reported in
the M issouri Breaks area in 1961 and in 1976 (113).
The fo u rth study area, the B ridger Mountain area, had no
h is to ry o f HD and was included because o f the a v a ila b ilit y o f samples
and use as a "negative c o n tro l" area.
The area is p a rt o f the
G a lla tin National Forest and is located approxim ately As m iles
north o f Bozeman in G a lla tin county, Montana.
E levation varie s from
■5000 to 9000 fe e t and liv e s to c k are grazed on both fo re s t land and
on p riv a te holdings adjacent to the G a lla tin National Forest.
Bovine serum samples were c o lle c te d from counties in eastern
Montana where HD had been reported w ith in the la s t three years.
These study areas included C arte r, Custer, Dawson, F a llo n , McCone,
Powder R iver, P r a ir ie , and Richland counties.
A small sample set
was also obtained from Lake county in western Montana fo r use as
a "negative c o n tro l" area.
41
Serym C o lle c tio n
Sera o f fre e ranging deer were c o lle c te d by ju g u la r venipuncture
from animals trapped during the 1978-79 liv e trapping program
conducted by the Montana Department o f F ish, W ild life , and Parks.
Serum samples were also obtained from hunter k ille d animals during
the 1978 Montana big game season.
Using these methods, 135 serum
samples were c o lle c te d from both mule deer and w h ite -ta ile d deer.
Bovine serum samples were obtained from the serology section
o f the Montana State D iagnostic Laboratory.
Portions o f samples
submitted to the la b o ra to ry fo r ro u tin e evaluation were c o lle c te d
and sto re d , lik e a ll serum samples in th is study, a t -20°C.
Approx­
im ately 340 bovine samples were c o lle c te d from the study areas, in
question.
Plaque Reduction N e u tra liz a tio n Test (PRN)
, Serum samples were screened a t a 1:50 d ilu tio n in the PRN te s t
fo r EHDV-Alberta n e u tra liz in g antibody.
Homologous antibody pre­
pared against the v ir a l is o la te was also reacted in a PRN te s t against
reference EHDV s tra in s fo r id e n tific a tio n purposes.
A m o d ific a tio n o f the PRN te s t described by Jochim e t a l . (49.)
was conducted on VERO monolayers grown in 24 w ell p la te s .
Heat
in a c tiv a te d serum d ilu tio n s were mixed w ith an equal volume o f virus
suspension ca lcu la te d to contain approxim ately 80 PFU per 0.1 ml
o f suspension and incubated a t 37°C fo r one hour.
A fte r in cu b a tio n .
42
0.2 ml o f each serum v iru s m ixture was applied to drained monolayers
in appropriate w ells and adsorbed fo r one hour a t 37°C.
The serum
v iru s m ixtures were then removed and the monolayers were o v e rla id
w ith 0.5% agarose in M-199 containing p e n ic illin (200 u n its /m l)
and streptom ycin (200 mcg/ml).
Plates were incubated on moistened
absorbent paper tow eling in a ir t ig h t containers a t 37°C fo r s ix
days.
Monolayers were fix e d arid stained as pre vio u sly described fo r
the plaque assay t i t r a t i o n and plaque counts were made.
Serum d ilu tio n s causing an 80% reduction in the number o f PFU
per w ell were considered p o s itiv e fo r EHDV s p e c ific n e u tra liz in g
antibody.
Al I samples were tested in d u p lic a te and the actual
ta rg e t number o f PFU used in the te s t was calcu la ted as described
by Jochim e t aI . (49).
Agar Gel P r e c ip itin Test (AGP)
Al I deer and bovine serum samples obtained during 1978 and 1979
were tested fo r BTV p r e c ip ita tin g antibody using the micro AGP te s t
as described by Jochim and Chow (46,48).
BTV soluble antigen was
provided by the National Animal Disease Center in Ames, Iowa and
the Arthropod Borne Animal Disease Research Center in Denver,
Colorado.
The AGP te s t is a m o d ific a tio n o f the Ouchterlony double
d iffu s io n te s t.
B r ie f ly , one inch square p le x ig la s s templates were
prepared by d r i l l i n g s ix w e lls set a t 60° to and 4 mm away, center
43
to cen ter, from a middle w e ll.
Pre-cleaned three inch by one inch
microscope s lid e s were coated w ith a 0.2% s o lu tio n o f Bacto-agar ^
and three s tr ip s o f double thickness e le c tr ic a l tape placed v e r tic a lly
down the w idth o f each s lid e .
The templates were then coated lig h t ly
w ith s ilic o n e stopcock grease and placed on the s lid e s using the
e le c tr ic a l tape fo r support.
The area between the surface o f the
s lid e and the template was f i l l e d w ith 0.9% agarose in is o to n ic
s a lin e using a three ml syringe and a 26 gauge needle.
Serum
samples to be tested were a lte rn a te d w ith appropriate reference
antiserum in periph era l w e lls and solu ble antigen placed in the center
w e ll.
A fte r incubation in humid, a i r - t i g h t containers f o r 72 hours
a t room temperature, the templates and e le c tr ic a l tape were removed
and the s lid e s were immersed in is o to n ic s a lin e fo r 24 hours.
The
s lid e s were then stained w ith th ia z in e red fo r seven minutes and
de-stained in two seven minute changes o f a 1.0% a c e tic acid s o lu tio n .
S lides were examined both before and a fte r the s ta in in g procedure
and samples recorded as e ith e r p o s itiv e or negative fo r BTV pre­
c ip ita tin g antibody.
D ifco Laboratories, D e tr o it, Michigan.
CHAPTER 4
RESULTS
Virus Is o la tio n
During the course o f th is study, blood and tis s u e samples
were c o lle c te d from three f ie ld cases th a t were HD suspects.
Two o f
the cases were mule deer which had been found in G a rfie ld county
in October o f 1978.
One o f these mule deer, a y e a rlin g female,
was a liv e when located.
She was found hunched over, convulsive,
foaming a t the mouth, fe v e ris h , and d ia rrh e ic , and was euthanized. .
A f ie ld necropsy revealed hemorrhages in the s k e le ta l muscle, abdom­
in a l w a ll, large and small in te s tin e , and cardiac muscle.
The second
mule deer was a male fawn th a t had apparently died in a convulsive
s ta te , evidenced by signs o f h y p e ra c tiv ity surrounding the carcass,
but showed no gross lesions on post-mortem examination.
The th ir d suspected HD case involved a three and one h a lf year
old female w h ite -ta ile d deer.
The animal had been recumbent and
was a c c id e n ta lly run over and k ille d in Rosebud county on Septemper
, 11, 1978.
Foam was observed around the muzzle area and a t necropsy
subcutaneous petechial hemorrhages as w ell as hemorrhages on the
surface o f the lung, l i v e r , and kidney were evident.
Straw colored
f lu id was present in the th o ra c ic and abdominal c a v itie s and in the
p e ric a rd ia l sac.
Blood vessels were engourged and, although the
f ie ld necropsy was conducted 12 hours a fte r death, v is c e ra l blood
s t i l l remained u n clo tte d .
45
An experimental w h ite -ta ile d deer, seronegative to EHDV, was
inoculated w ith spleen suspension prepared from the Rosebud county
w h ite -ta ile d deer m o rta lity .
At fiv e days post in o c u la tio n , the
experimental deer was found recumbent and in a shock-1 ike s ta te .
The animal was experiencing re s p ira to ry d i f f i c u l t y and convulsive
movements and had reddened conjunctiva and a cyanotic tongue.
Blood samples c o lle c te d before s a c r ific in g the animal took approx­
im ately 25 minutes to c lo t as opposed to the norm ally observed
c lo ttin g time o f two to fiv e minutes.
At necropsy, hemorrhages
were noted a t the apex o f the heart and on the serosal surfaces
o f the g a s tro in te s tin a l tr a c t.
There was abundant straw colored
f l u i d in the ple ura l c a v ity as w ell as in the p e ric a rd ia l sac.
The lungs were involved w ith a very d is t in c t in te rlo b u la r edema.
No b a c te ria l pathogens were cu ltu re d or id e n tifie d from blood
samples taken from the experimental animal a t necropsy.
A cyto p a th ic agent was is o la te d in c e ll c u ltu re from the
i n i t i a l passage o f tre a te d , heparinized blood from the experimental
w h ite -ta ile d deer.
A. cyto p a th ic agent was also is o la te d from the
whole blood o f the o rig in a l w h ite -ta ile d deer a fte r one b lin d
passage in VERO c e lls .
No is o la tio n s were made from tis s u e samples
c o lle c te d from e ith e r o f the G a rfie ld county mule deer m o rta litie s
a fte r three b lin d passages in VERO c e lls .
Cytopathic e ffe c t was observed in VERO monolayers in fe c te d
46
w ith the v ir a l is o la te s w ith in 48 to 72 hours p o s t-in fe c tio n .
C ells appeared more g ranu lar a t th a t time and began to detatch
from the surface o f the c u ltu re fla s k .
As in fe c tio n progressed,
u su a lly by 72 hours p o s t- in fe c tio n , the media became c lu tte re d
w ith c e llu la r debris and only about 50% o f the monolayer remained
attatched to the fla s k surface.
By 96 hours p o s t-in fe c tio n ,
CPE u su ally approached the maximum o f approximately 70% monolayer
d e s tru c tio n .
LFAT
EHDV s p e c ific fluorescence was observed in VERO monolayers
both 24 and 48 hours a fte r in fe c tio n w ith the v ir a l is o la te s .
Ir r e g u la r ly shaped aggregations o f v ir a l antigen , u su a lly p e r i­
nuclear in d is tr ib u tio n , were e a s ily v is u a liz e d a fte r flu o re s c e n t
s ta ip in g (fig u re I ) .
No s p e c ific fluorescence was seen in non-
in fe cte d monolayers or in monolayers in fe c te d w ith BTV type 13
(fig u re 2).
The two v ir a l is o la te s were considered id e n tic a l and
were designated EHDV-M78.
E lectron Microscopy
E lectron micrographs o f the v ir a l is o la te allowed fo r a
determ ination o f morphology and size .
N egatively stained p a rtic le s
were ca lcu la te d to be 56.5 nm in diam eter.
In d iv id u a l v irio n s
possessed cubic symmetry and r e la tiv e ly la rg e , doughnut shaped
capsomeres co n siste n t w ith o rb iv iru s morphology (fig u re 3).
Figure I .
EHDV s p e c ific fluorescence, p e rin u c le a r in d is tr ib u tio n ,
seen in VERO monolayer in fe c te d w ith the v ir a l is o la te
and fix e d a t 48 hours post in fe c tio n . (2000 X)
Figure 2.
VERO monolayer in fe cte d w ith BTV type 13 and fix e d
a t 48 hours post in fe c tio n . (2000 X)
48
Figure 3.
E lectron
symmetry
Diameter
(640,000
micrograph o f EHDV-M78 demonstrating the cubic
and the larg e doughnut shaped capsomeres.
o f both v irio n s was approximately 56.5 nm.
X)
49
Plaque Reduction N e u tra liz a tio n (PRN)
A number o f PRN m anipulations were performed in an attempt
to serotype the v ir a l is o la te as EHDV-New Jersey or EHDV-Alberta.
F ir s t , hyperimmune goat antiserum prepared against the v ir a l is o la te
was tested fo r it s n e u tra liz in g a b i li t y against reference EHDV-New
Jersey and EHDV-Alberta v ir a l s tra in s and against the v ir a l is o la te .
The same b a tte ry o f tests, was performed using reference EHDV a n ti­
sera obtained from the National Animal Disease Center in Ames,
Iowa.
The re s u lts o f these te s ts are presented in ta b le I .
Table I .
Plaque Reduction Cross N e u tra liz a tio n ___________________
Antiserum
EHDV-M78
Virus
EHDV Reference
EHDV-M78
I : 640a
1:1280
EHDV-Alberta
1:1280
1:1280
EHDV-New Jersey
1:10
1:10
aValues expressed as highest d ilu tio n causing an 80% reduction in
thp number o f plaque forming u n its
Serum samples obtained from the se ro lo g ic a l survey th a t were
capable o f n e u tra liz in g EHDV-Alberta in a PRN te s t when d ilu te d
1:50 were selected from s ix deer and s ix c a ttle .
When these samples
were run in a PRN te s t , again a t a 1:50 d ilu t io n , against EHDVNew Jersey, no n e u tra liz a tio n was detected.
50
S erological Survey
The plaque reduction n e u tra liz a tio n te s t proved re a d ily
adaptable as a screening te s t fo r EHDV-Alberta antibody when serum
samples were run a t a 1:50 d ilu t io n .
A fte r fix in g and s ta in in g ,
the 24 w ell pla tes were e a s ily read and provided a permanent
record o f the immune status o f each animal (fig u re 4 ).
The AGP te s t proved to be a l i t t l e
less e ffe c ie n t in the
screening o f serum samples fo r BTV s p e c ific antibody.
\
In many
cases, the serum sample formed a "secondary reaction lin e " and did
not form a lin e o f id e n tity w ith the BTV reference antiserum
employed in the te s t.
Only samples forming lin e s o f id e n tity w ith
the reference antiserum were considered p o s itiv e fo r BTV s p e c ific
p r e c ip ita tin g antibody.
Again, however, the te s t was r e la tiv e ly
simple to perform, easy to read, and provided a permanent and
e a s ily stored te s t record (fig u re 5).
The re s u lts o f the s e ro lo g ic a l survey are presented in tables
2 and 3.
EHDV se ro p o sitive deer were, detected in a ll three o f the
study areas th a t had h is to rie s o f HD problems.
On the oth er hand,
n e ith e r mule deer nor w h ite -ta ile d deer from the negative control
Bridger Mountain area were found to possess EHDV-Alberta n e u tra l­
iz in g antibody.
The incidence o f BTV s e ro p o s itiv e deer was much
less than th a t o f -EHDV s e ro p o s itiv e deer.
Mule deer from the
Missouri Breaks area contained the highest number o f BTV se ro p o sitive
51
Figure 4.
An example o f a PRN p la te w ith each serum sample,
d ilu te d 1:50, run in d u p lic a te . Notice th a t three
samples on th is p la te did not contain EHDV-Alberta
n e u tra liz in g antibody.
Figure 5.
An example o f an AGP p la te on which s ix serum samples
have been tested fo r BTV p r e c ip ita tin g antibody. Lines
a t th re e , seven, and eleven o 'c lo c k are reference antiserum.
52
animals w ith 11 o f 31 samples g iv in g p o s itiv e reactions in the
AGP te s t.
Of the 314 bovine serum samples c o lle c te d from eastern Montana,
248, or 79%, were EHDV-Alberta s e ro p o s itiv e in the PRN te s t.
A small sample set c o n s is tin g o f 22 bovine samples from Poison,
Montana, in the extreme western section o f the s ta te , were run
in the PRN as a "negative c o n tro l" fo r EHDV exposure.
Only one
o f these samples showed any s p e c ific n e u tra liz in g a b i li t y against
EHDV-Albe rta.
.
,
i
In c o n tra s t to the high percentage o f EHDV-Alberta sero­
p o s itiv e bovine samples, only 3% o f the same samples reacted
p o s itiv e ly in the AGP te s t fo r BTV p re c ip ita tin g antibody.
Al I
o f the bovine sani.ples th a t were BTV s e ro p o s itiv e were also EHDV
s e ro p o s itiv e in d ic a tin g exposure to both v iru s e s .
The same was
tru e w ith the deer samples where a ll but three o f the samples
th a t were BTV s e ro p o s itiv e were also EHDV s e ro p o s itiv e .
53
Table 2.
BTV and EHDV Serology o f Montana Deer by Study Area
Study Area
Species3
BTV Pos.'
%
EHDV Pos.
%
'
Long Pines
MD
3/18b
17
14/18
78
Missouri Breaks
MD
11/31
35
2 2 /3 1
71
Bridger
MD
0/41
0
0/41
0
Long Pines
WTD
0/16
0
0/16
0
Intake
WTD
0 /2 6
0
2/2G A
7.5
Bridger
WTD
0 /3
0
0 /3
0
aMD = Mule.Deer
WTD - W h ite -ta ile d Deer
^Expressed as # p o s itiv e sera/# sera tested
54
)
Table 3.
BTV and EHDV Serology o f M ontana'Cattle by County
County
BTV Pos.
C arter
0/10a
Custer
%
EHDV Pos
%
0
4/10
40
7/60
12
59/60
98
Dawson
0/23
0
11/23
.48
Fallon
0/29 ■
0
25/29
86
McCone
1/45
2
33/45
73
Powder River
2/20
10
12/20
60
P ra irie
0/72
0
60/72
83
Richland
0/55
0
44/55
80
10/314
3
248/314
79
NTb
NT
Totals
Lake
1/22
aExpressed as # p o s itiv e sera/# sera tested
^Not tested
4
CHAPTER 5
DISCUSSION
A number o f v ir a l and b a c te ria l pathogens have the p o te n tia l
to produce a disease syndrome th a t may be confused c l i n i c a l l y w ith
BTV o r EHDV in fe c tio n in w h ite -ta ile d deer.
Some o f these pathogens
include^ mucosal disease v iru s , m alignant ca ta rrh a l fe v e r v iru s ,
and p a s tu re !la .
The e p iz o o tio lo g ic a l patterns e xh ib ite d by the HD
problems in the Montana w h ite -ta ile d deer, however, incrim inate d
two arthropod borne v iru s e s , BTV and EHDV, as the most lik e ly
e tio lo g ic agents o f the Montana HD problems'.
Hence, v iru s is o la tio n
and se ro lo g ic a l work were designed in th is study to c o n trib u te to a
fu r th e r understanding o f these two viruses in eastern Montana.
A cyto p a th ic agent was is o la te d from a 1978 HD m o rta lity and
id e n tifie d through e le ctro n microscopy, in d ir e c t flu o re s c e n t a n ti­
body te s ts , and plaque reduction n e u tra liz a tio n te s ts as EHDV.
The
is o la te was fu r th e r serotyped in our la b o ra to ry by re a c tin g homol­
ogous antiserum prepared against the is o la te in plaque reduction
n e u tra liz a tio n te s ts against reference EHDV serotypes.
Results o f
these "tests in d ica te d th a t the v ir a l is o la te was o f the A lberta sero
type.
The A lberta serotype was f i r s t is o la te d from a w h ite -ta ile d
deer in A lberta in 1962 and again during an outbreak in a captive
w h ite -ta ile d deer herd in Indiana in 1975 (14, 52).
While other
EHDV outbreaks in w h ite -ta ile d deer have been a ttrib u te d to the
New Jersey s tra in o f EHDV, the A lb e rta s tra in has been is o la te d
56
in recent years from the bovine (5 , 28).
The A lberta and New
Jersey s tra in s e x h ib it only a minimal cross reaction in the PRN
te s t (5 , 13).
Since a ll serum samples in th is study were screened
a t a 1:50 d ilu tio n against EHDV-Alberta, a ll reactors were considered
se ro p o s itiv e fo r EHDV-Alberta s p e c ific n e u tra liz in g antibody.
Furthermore, when PRN te s t reactors were tested fo r th e ir n e u tra l­
iz in g a b i li t y against EHDV-New Jersey, they c o n s is te n tly fa ile d
to show any n e u tra liz a tio n at a 1:50 d ilu t io n .
V ira l is o la tio n from one HD m o rta lity does not n e cessarily
imply th a t a ll HD e p izo o tics th a t have occurred in Montana were
caused by th a t p a rtic u la r v iru s .
The s e ro lo g ic a l survey c a rrie d
out in th is study d id , however in d ic a te recent EHDV-Alberta
a c t iv it y in not only the fre e -ra n g in g deer p o pula tion, but also
the bovine population in eastern Montana.
Taken c o lle c t iv e ly ,
the v iru s is o la tio n and s e ro lo g ic a l survey present a strong case
fo r the e tio lo g ic a l ro le o f EHDV-Alberta in the Montana HD e p i­
zo o tic s .
Of the fo u r separate deer study areas, three had recent
h is to rie s o f w h ite - ta il deer m o rta litie s a ttrib u ta b le to HD.
Samples were c o lle c te d in these areas from 91 deer in c lu d in g 42 "
w h ite -ta ile d deer and 49 mule deer;
While 36/49, or 73%, o f the
mule deer were s e ro p o s itiv e fo r EHDV, only 2/42, or 5%, o f the
w h ite -ta ile d deer were EHDV s e ro p o s itiv e .
The m o rta lity ra te in
57
natural and experimental EHDV in fe c tio n in w h ite -ta ile d deer is
approximately 90%.
Mule deer, on the other hand, are more re s is ta n t
to EHDV and b u ild up a s o lid and durable immunity.
This v a ria tio n in
s u s c e p tib ility is re fle c te d in the s e ro lo g ic a l re s u lts o f th is,
study and is in agreement w ith re s u lts obtained by other in v e s ti­
gators (34, 35).
Of p a rtic u la r in te re s t was the Long Pines study
area where serum samples were c o lle c te d in the f a l l a fte r a HD
e p iz o o tic th a t re su lte d in the loss o f approximately one th ir d o f
the lo ca l w h ite -ta ile d deer population.
Of the 18 mule deer, samples
c o lle c te d from th is area, 14, or 78% were EHDV s e ro p o s itiv e w hile
none o f the 16 w h ite -ta ile d deer tested possessed EHDV n e u tra l­
iz in g antibody.
W ithout p re -e p iz o o tic samples, the id e a l s itu a tio n
o f demonstrating seroconversion w ith in the mule deer population
was not possible.
The fo u rth deer study area, the B ridger Mountain area, had
no previous h is to ry o f HD w ith in the fre e ranging deer population.
This p a rtic u la r area could be described as a prime ta rg e t area fo r
fu tu re EHDV e p izo o tics due to the prevalence o f su sce ptible animals.
None o f the 44 serum samples c o lle c te d in th is area had any EHDV
n e u tra liz in g antibody.
In order fo r an e p iz o o tic to occur, however,
the vecto r fo r EHDV, C ulicoides v a riip e n n is , would have to e x is t'
in s u ff ic ie n t number to e ffe c ie n tly tra n s m it the v iru s .
The summer
range o f deer in the B ridger Mountain area lie s between 5000 and 9000
58
fe e t.
P erio dic cool n ig h t temperatures in th is area during the
summer may minimize o r prevent Culicoides breeding, re s u ltin g in
low vector in fe c tio n rates and l i t t l e
transmission, or perpetuation.
p o s s ib ility fo r disease
An analagous s itu a tio n was reported
in Oregon by K istn e r e t a l . (55) where the summer range o f mule
deer was between 7000 and 9000 fe e t in e le v a tio n w h ile major
C ulicoides breeding s ite s were 15 to 20 miles away a t the 4000
fo o t e le v a tio n .
Here ag ain, low BTV and EHDV in fe c tio n rates were
detected in the deer popula tion.
The lack o f BTV and EHDV sero­
p o s itiv e animals in the B ridger Mountain area was in te rp re te d in
th is study as adding credence to the e tio lo g ic a l ro le o f EHDV in
the eastern Montana deer population.
Of the bovine serum samples c o lle c te d from counties in
eastern Montana, 79% (248/314) were s e ro p o s itiv e fo r EHDV.
Anderson e t a l . ( I ) re c e n tly reported an average nationwide
exposure ra te in the bovine to EHDV-Alberta o f 49%.
The higher
exposure ra te obtained in th is study was p a r t ia lly expected since
Anderson's samples were randomly c o lle c te d from slau ghte r houses
whereas samples in th is study were c o lle c te d s p e c ific a lly from
a c tiv e HD areas.
The high EHDV incidence in the bovine may
in d ic a te th e ir a c tiv e ro le in the e p iz o o tio lo g y o f EHDV.
The d is tr ib u tio n o f EHDV in the United States was once thought
to p a ra lle l the range o f w h ite -ta ile d deer.
With recent evidence
59
o f almost ubiquitous EHDV a c t iv it y in United States c a tt le , the
range o f v iru s a c t iv it y must now be expanded.
The type 2 o r
A lberta
s tra in o f EHDV has been is o la te d from United States c a ttle and is ,
according to se ro lo g ic a l data, the s tr a in most prevalent in United
States c a ttle ( I ) .
This s tra in has the capacity to cause fa ta l
e p izo o tics in w h ite -ta ile d deer populations and has various im p li­
cations in bovine populations.
EHDV is not p resen tly recognized
as an im portant pathogen in the bovine species.
BTV, however,
was i t s e l f incrim inate d as a bovine pathogen many years a fte r it s
e ffe c ts on ovine species were recognized.
BTV is now known to cause
c lin ic a l disease ( d i f f i c u l t to reproduce exp erim e nta lly) in the
bovine as w ell as congenital defects in calves exposed during the
f i r s t trim e s te r o f g e sta tio n (62).
There is evidence th a t EHDV,
lik e BTV, may cross the placenta in in fe c te d deer re s u ltin g in
fe ta l a lte ra tio n s (17, 23, 100).
The question o f EHDV tra n s ­
placental passage and fe ta l changes in the bovine remains un­
answered.
A second question concerning the bovine, re la tio n s h ip to EHDV-is
whether o r not c a ttle may serve as re s e rv o ir hosts fo r EHDV.
Evidence o f la te n t in fe c tio n s in the bovine has incrim inate d them
as a re s e rv o ir host fo r the c lo s e ly re la te d BTV (66).
Although
s im ila r evidence does not e x is t fo r la te n t EHDV in fe c tio n in the
bovine, the question is appropriate because o f the s im ila r it ie s
60
between the two viruses and the amount o f EHDV a c t iv it y detected
in the bovine p o pula tion.
S im ila rly , mule deer may act as re s e rv o ir
hosts fo r EHDV as in d ica te d by th e ir gre a te r resistance and the large
number o f s e ro p o s itiv e mule deer as compared to w h ite -ta ile d deer.
Both EHDV and BTV e x is t p rim a rily in the c e llu la r fra c tio n o f the
blood during virem ia.
This c h a ra c te ris tic may enable e ith e r v iru s
to remain in the blood in s u f f ic ie n t ly high t i t e r to in fe c t hemophagous insects even a fte r the appearance o f s p e c ific n e u tra liz in g
antibody.
Experimental EHDV in fe c tio n in the bovine has resulted
in a virem ia demonstrable fo r up to 50 days p o s t-in fe c tio n (31).
These experimental in fe c tio n studies in the bovine add credence to
th e ir possible ro le as a re s e rv o ir host fo r EHDV and im p lic a te
them a t le a s t as an a m p lifyin g host f o r the v iru s .
Studies are
lacking concerning maintenance o f EHDV virem ia in mule deer.
E xperi­
mental in fe c tio n s o f w h ite -ta ile d deer w ith attenuated EHDV, however,
in d ic a te a disappearance o f c irc u la tin g v iru s concurrent w ith
the appearance o f s p e c ific n e u tra liz in g antibody (34).
Viruses possessing segmented genomes have the a b i l i t y to go
through various genetic acrobatics re s u ltin g in the emergence
o f new v ir a l s tra in s .
This process is exem plified by the various
in flu e n za v iru s s tra in s w ith which many o f us are a ll to fa m ilia r .
S im ila rly , i t has been suggested th a t dual in fe c tio n s w ith more
than one s tra in o f BTV, or w ith BTV and EHDV, may, by phenotypic
61
mixing and/or genetic recombination or reassortm ent, provide
the mechanism fo r the e vo lu tio n o f new v ir a l s tra in s (9 5).
Indeed,
dual in fe c tio n s w ith more than one s tra in o f BTV, or w ith BTV and
EHDV, have been reported in both c a ttle and deer (28, 95, 102).
Almost a ll o f the animals th a t were BTV se ro p o s itiv e in th is study
were also EHDV s e ro p o s itiv e which may in d ic a te more dual in fe c tio n s .
Genetic reassortment has been shown to occur between temperature
s e n s itiv e mutants o f two re la te d o r b iv iru s e s , W allal v iru s and
M udjinbarry v iru s (8 1).
With the widespread nature o f EHDV in
United States c a ttle and th e ir p e rio d ic involvement w ith BTV, the
o p p o rtu n ity e x is ts fo r such genetic acrobatics to occur.
A ntigenic
s im ila r it ie s between EHDV and BTV may, in fa c t, be the re s u lt o f
ju s t such an occurrence in the past.
Ibaraki v iru s , also o f the
bluetongue v iru s subgroup o f o r b iv iru s e s , may be a fu r th e r v a ria n t
in th a t i t causes bluetongue lik e disease in Japanese c a ttle but
is now thought to be in d is tin g u is h a b le s e ro lo g ic a lly w ith EHDVA lberta ( I , 13).
According to the re s u lts o f the AGP te s t , BTV a c t iv it y in
Montana c a ttle and deer is much less than th a t o f EHDV.
Only 10
o f 314 bovine samples, or 3%, produced lin e s o f id e n tity to BTV
in the AGP te s t.
On the other hand, 14 o f 135 deer serum samples,
or 10% were determined to be BTV s e ro p o s itiv e .
When the mule deer
froni HD areas are considered alone, however, 29% were BTV se ro p o s itiv e
62
This fig u re may in d ic a te th a t BTV, as w ell as EHDV, was involved
w ith a t le a s t the more recent HD outbreaks in the eastern Montana
deer popula tion.
A s im ila r s itu a tio n occurred during a 1971 HD
outbreak in the southeastern United States where both EHDV and BTV
were is o la te d from a diseased w h ite -ta ile d deer (102).
In studying disease problems o f w ild lif e populations, i t is
d i f f i c u l t to a rriv e a t c le a r cut conclusions.
Problems are
encountered both in the observation o f and sample c o lle c tio n from
fre e -ra n g in g p o p u la tio n s.
Data c o lle c te d in th is study p o in t to
the involvement o f EHDV, and possibly BTV, in the HD outbreaks
th a t have occurred in Montana w h ite -ta ile d deer fo r the la s t two
decades.
Whether EHDV alone was responsible fo r the deer m o r ta litie s ,
or EHDV and BTV both, cannot be s a t is f a c t o r ily concluded.
The EHDV
is o la tio n , as w ell as the high ra te o f EHDV exposure in c a ttle and
mule deer in eastern Montana, however, do s o lid if y the ro le o f EHDV
in the e p iz o o tic s .
Disease problems have existe d in w i l d l i f e populations fo r
ce n tu rie s.
In many cases, these problems have been b e n e fic ia l in
terms o f the laws o f nature and have helped to m aintain a balance
between a s p e c ific w il d l i f e population and the lim ita tio n s o f it s
ecosystem.
R ecently, however, the increased amount o f contact
between w ild and domestic species and the decrease in h a b ita t a v a il­
able to w i l d l i f e , have made the study o f w ild lif e diseases an im portant
undertaking.
An understanding o f the pathogens common to w ild
and domestic species and the e ffe c ts o f these pathogens on in d iv id u a l
populations is e sse ntial i f the two populations are to e x is t side
by side in the fu tu re .
The dual involvement o f w ild and domestic
populations w ith the v iru s apparently responsible fo r the Montana
.HD problems serves to a m p lify th is p o in t.
CHAPTER 6
SUMMARY
A v iru s was is o la te d from a w h ite -ta ile d deer w hich, a t
necropsy, had gross lesions ty p ic a l o f what has been re fe rre d to
as hemorrhagic disease (HD).
The v ir a l is o la te was id e n tifie d
by e le ctro n microscopy, in d ir e c t flu o re s c e n t antibody te s ts , and
plaque reduction n e u tra liz a tio n te s ts as e p iz o o tic hemorrhagic
disease v iru s (EHDV) o f the A lberta serotype.
S erological studies in d ica te d th a t 73% (36/49) o f the mule deer
serum samples c o lle c te d from areas in eastern Montana where HD
problems had occurred were EHDV s e ro p o s itiv e .
Only 5% (2/42)
o f the w h ite -ta ile d deer serum samples c o lle c te d from the same areas
were EHDV se ro p o s itiv e in d ic a tin g the le t h a lit y o f EHDV in fe c tio n
fo r w h ite -ta ile d deer.
No EHDV s e ro p o s itiv e deer were found in a
sample set co n sistin g o f 44 samples Obtained in an area o f cen tral
\
Montana w ith no previous h is to ry o f HD.
The bovine was im plica ted in the e p izootiology o f EHDV due to
the large number (248/314) o f animals tested th a t were EHDV sero­
p o s itiv e .
Again, these animals were from eastern Montana where
HD problems in w h ite -ta ile d deer have been reported in the past.
Bovine serum samples c o lle c te d from an area in western Montana
w ith no HD h is to ry were found to be seronegative to EHDV.
' The v ir a l is o la tio n and id e n tific a tio n as w ell as the re s u lts
I
from the s e ro lo g ic a l survey im p lic a te EHDV as the e tio lo g ic agent
65
involved in HD d ie -o ffs th a t have been occurring in eastern Montana
w h ite -ta ile d deer fo r the la s t two decades.
LITERATURE CITED
1.
Anderson, A. A ., L. L. Smith, and C. H. Campbell.
1979.
Sero­
lo g ic a l survey fo r EHD and Iba raki viruses in the United
2.
S tates.
Abs. 60
Disease.
No. 4.
Bando, B. M.
th
1975.
Ann. Meeting Conf. Res. Workers in Anim.
Is o la tio n o f bluetongue and e p iz o o tic
hemorrhagic disease viruses in c e ll c u ltu re .
Vet. Lab. Diag.
3.
Barber, T. L.
1979.
18:
Proc. Am. Assn.
149-157.
Temporal appearance, geographic d i s t r i ­
b u tio n , and species o f o r ig in o f bluetongue v iru s serotypes
in the United S tates.
4.
Am. J. Vet. Res.
Barber, T. L ., and M. M. Jochim.
1973.
4Ck
1654-1656.
S erological cha racte r­
iz a tio n o f selected bluetongue v iru s s tra in s from the United
States.
Rroc. 7 7 ^ Ann. Meeting U.S. Anim. H lth . Assn.
:
352-359.
5.
Barber, T. L ., and M. M. Jochim.
1975.
Serotyping bluetongue
and e p iz o o tic hemorrhagic disease v iru s s tra in s .
Assn. Vet. Lab. Diag.
6.
J1S:
Proc. Am.
149-157.
Borden, E. C., R. E. Shope, and F. A. Murphy.
1971.
Physio-
chemical and morphological re la tio n s h ip s o f some arthropod
borne v iru s to bluetongue v iru s - A new taxonomic group.
Physiochemical and se ro lo g ic a l s tu d ie s .
261-271.
J . Gen. V ir o l.
13:
67
7.
Boorman, J . , and E: P. J. Gibbs.
1973.
M u ltip lic a tio n o f the
v iru s o f e p iz o o tic hemorrhagic disease o f deer in C u licoides
species.
8.
Arch, ges V iru sforsch.
Boulanger, P ., and J. F. Frank.
1975.
the diagnosis o f bluetongue.
Med.
10.
15:
Bowne, J . G.
c a ttle ?
11.
1971.
259-266.
S erological methods in
Aust. Vet. J.
Bluetongue disease.
SIl :
185-189.
Adv. V e t.S c i. Comp.
Ul
Bowne, J. G.
H-*
I
9.
41:
1973.
Is bluetongue an im portant disease in
J . A. V. M. A.
163:
911-914.
Bowne, J. G ., and M. M. Jochim.
1967.
Cytopathologi cal changes
and development o f in c lu s io n bodies in c u ltu re d c e lls in fe cted
w ith b luetongue v iru s .
12.
Am. J. Vet. Res.
Bowne, J. G., and R. H. Jones.
1966.
28:
1091-1105.
Observations on b luetongue .
v iru s in the s a liv a ry glands o f an in se ct v e c to r, Culicoides
v a ri ip e n n is .
13.
V ir o l.
_3Ck
127-133.
Campbell, C. H., I . L. Barber, and M. M. Jochim.
1978.
A ntigenic
re la tio n s h ip s o f Ib a ra k i, bluetongue, and e p iz o o tic hemorrhagic
disease viru se s.
14.
Vet. M icro.
Chalmers, G. A ., and H. N. Vance.
3;
15-22.
1964.
An outbreak o f e p iz o o tic
hemorrhagic disease in w ild ungulates in A lb e rta .
D is.
15.
42:
Davis, F. G.
Kenya.
W ild l.
1-6.
1978.
J. o f Hyg.
B luetongue studies w ith s e n tin e l c a ttle in
80:
197-204.
68
16.
Debbie, J. 6 ., and NI. K. Abeiseth.
e p iz o o tic hemorrhagic disease.
during v ir a l in fe c tio n .
17.
I.
Pathogenesis o f
Blood coagulation
J. In f. Dis.
124:
217-222.
Debbie, J. G., H. C. Roswell, L. H. Karstad, and J. D it c h f ield .
1965.
An approach to the pathogenesis o f v ir a l hemorrhagic
disease o f deer.
30:
18.
1971.
Trans. North Am. W ild l. Nat. Resour. Conf.
196-205.
D it c h f ie ld , J . , J.' G. Debbie, and L. H. Karstad.
1964.
v iru s o f e p iz o o tic hemorrhagic disease o f deer.
North
19.
Dusick, G.
Am. W ild l. Nat. Resour. Conf.
1979.
Personal communication.
Fish, W ild life , and Parks.
20.
D u to it, R. M.
1944.
21.
V ir o l.
7-16.
38:
in deer in Michigan.
23.
21:
Mont. Dept, o f
Onderstepoort J. Vet. S c i.
1969.
Morphology o f bluetongue
213-219.
Fay, I . D., A. P. Boyce, and W. G. Youatt.
Conf.
196-201.
The transm ission o f bluetongue and
E ls, H. J . , and D. W. Verwoerd.
v iru s .
22.
_19_:
Trans.
Broadus, Montana.
horse sickness by C u lic o id e s .
Anim. Ind.
29;
The
1956.
An e p izo o tic
Trans. North Am. W ild. Nat. Resour.
173-184.
F le tc h , A. L ., and I . H. Karstad.
1971.
Studies on the patho­
genesis o f experimental e p iz o o tic hemorrhagic disease of
w h ite -ta ile d deer.
Can. J . Comp. Med.
35;
224-229.
69
24.
Fosberg, S. A ., E. H. Stauber, and H. W. Renshaw.
1977.
Is o la tio n and c h a ra c te riz a tio n o f e p iz o o tic hemorrhagic disease
25.
v iru s from w h ite -ta ile d deer in eastern Washington.
Am.
J. Vet. Res.
,
38:
361-364.
Foster, N. M., R. D. Breckon, A. J . Luedke, R. H. Jones, and
H. E. M e tcalf.
1977.
Transmission o f 2 s tra in s o f e p iz o o tic
hemorrhagic disease viru s in deer by C ulicoldes v a riip e n n is .
J. W ild l, D is.
26.
13:
9-16.
Foster, N. M., R. H. Jones, and A. J . Luedke.
1968.
Trans­
mission o f attenuated and v ir u le n t bluetongue v iru s w ith
C ulicoides v a riip e n n is in fe c te d o r a lly via sheep.
'
27.
Vet. Res.
29:
Am. J .
275-279.
F oster, N. M., R. H. Jones, and B. R. McCrory.
1963.
P re lim in -
in a ry in v e s tig a tio n s on in se ct transm ission o f b luetongue
v iru s in sheep.
28.
Am. J . Vet. Res.
24:
1195-2000.
Foster, N. M., H. E. M e tca lf, T. L . Barber, R. H. Jones, and
A. J . Luedke.
1980.
B luetongue and e p iz o o tic hemorrhagic
disease v iru s is o la tio n s from ve rte b ra te and in v e rte b ra te
hosts a t a common geographic s it e .
J . A. V. M. A.
176:
126-129.
29.
Frank, J . F ., and N. G. W illis .
o f deer.
Aust. Vet. J .
51:
1975.
174-177.
B luetongue-Iike disease
70
30.
Gambles, R .•M.
1949.
Bluetongue o f sheep in Cyprus.
Comp. Path, and Therapeutics.
31.
59_:
Gibbs, E. P. J . , and M. J. P. Lawman.
J.
176-190.
1977.
In fe c tio n of
B r itis h deer and farm animals w ith e p iz o o tic hemorrhagic
disease o f deer v iru s .
32.
J . Comp. Path.
87:
335-342.
G le is e r, C. A ., E. L. S ta ir , and L. D. M cG ill.
1969.
Diagnosis
o f bluetongue in c a ttle by in tra v a s c u la r in n o cu la tio n o f
chicken embryos and immunofluorescence.
30:
33.
981-986.
Hardy, W. T ., and D. A. P rice.
J. A. V. M. A.
34.
Am. J . Vet. Res.
H o ff, G. L.
1972.
120:
1952.
Soremuzzle o f sheep.
23-25.
E p iz o o tio lo g ic a l studies on the viruses
o f bluetongue and e p iz o o tic hemorrhagic disease in w ild
ruminants.
35.
Ph.D. Thesis.
Univ . Wisconsin, Madison, Wisconsin.
H o ff, G. L ., S. H. Richards, and D. 0. T ra in e r.
o f hemorrhagic disease in North Dakota deer.
Mgmt.
36.
_37:
1973.'
E pizootic
J. W ild l.
331-335.
H off, G. L ., J . S patalin , and D. 0. T ra in e r.
1974.
Attempts
to propagate e p iz o o tic hemorrhagic disease v iru s o f deer
in avian host systems.
37.
Am. J. Vet. Res
H o ff, G. L ., and D. 0. T ra in e r.
1973.
35>:
817-819.
Experimental in fe c tio n
in North American elk w ith e p iz o o tic hemorrhagic disease
v iru s .
J. W ild !. D is .
9:
129-132.
71
38.
H o ff, G. L ., and D. 0. T ra in e r.
1974.
Observations on blue-
tongue and e p iz o o tic hemorrhagic disease viruses in w h ite ­
ta ile d d e e r:. ( I ) D is trib u tio n o f v iru s in the blood
C ross-challenge.
39.
J. W ild l. D is.
H off. G. L ., and D. 0. T ra in e r.
hemorrhagic disease viru se s:
species.
40.
Kk
1978.
(2)
25-31.
Bluetongue and e p iz o o tic
T h e ir re la tio n s h ip to w ild lif e
Adv. Vet. S c i. Comp. Med.
22:
111-132.
H off. G. L ., D. 0. T ra in e r, and M. M. Jochim.
1974.
Bluetongue
v iru s and w h ite -ta ile d deer in an enzootic area o f Texas.
J. W ild l. Dis.
41.
Howell, P. G.
10:
1960.
158-163.
A p re lim in a ry a n tig e n ic c la s s ific a tio n
o f s tra in s o f bluetongue v iru s .
Res.
42.
28:
357-363.
Howell, P. G., and D. W.- Verwoerd.
V ir o l. Monographs.
43.
Onderstepoort J. Vet.
9_:
1971.
37-74.
Howell, P. G., D. W. Verwoerd, and R. A. Oellermann.
Plaque form ation by bluetongue v iru s .
Vet. Res.
44.
Bluetongue v iru s .
34:
1967.
Onderstepoort J.
317-322.
Huismans, H., C. W. Brener, and T. L. Barber.
1979.
The
n u cle ic acid and p rotein s o f e p iz o o tic hemorrhagic disease
v iru s .
45.
Onderstepoort J. Vet. Res.
Hutcheon, D.
1881.
46_:
95-104.
Fever o f e p iz o o tic c a ta rrh .
from the College o f V e te rin a ry Surgery, 1880.
Report
p. 17.
72
46.
■
47.
Jochim, M. M.
1976.
Improvement o f the AGP te s t fo r bluetongue.
Proc. Am. Assn. Vet. Lab. Diag'.
19:
361-376.
Jochim, M. M., I . L. Barber, and B. M. Bando.
1974.
Id e n ti­
fic a tio n o f bluetongue and e p iz o o tic hemorrhagic disease
viruses by the in d ir e c t flu o re s c e n t antibody procedure.
Proc. Am. Assn. Vet. Lab. Diag.
48.
Jochim, NI. NI., and I . L. Chow.
bluetongue v iru s .
49.
17:
1969.
Am. J. Vet. Res.
Jochim, NI. NI., and S. C. Jones.
1976.
91-101.
Immunodiffusion o f
30:
33-41.
Plaque n e u tra liz a tio n
o f bluetongue v iru s and e p iz o o tic hemorrhagic disease viru s
in BHK-21 c e lls .
50.
Am.' J. Vet. Res.
Jochim, NI. NI., and S. C. Jones.. 1977.
37:
1345-1347.
Enhancement o f bluetongue
and e p iz o o tic hemorrhagic disease v iru s n e u tra liz a tio n w ith
a n ti gamma g lo b u lin .
Proc. Am. Assn. Vet. Lab. Diag.
20:
255-272.
51.
Jochim, M. NI., A. J. Luedke, and J. G. Bowne.
1965.
The
c lin ic a l and immunogenic response o f sheep to oral and in t r a ­
derma I a d m in istra tio n s o f bluetongue v iru s .
Res.
52.
26:
Kanit z , C. L.
1254-1260.
1977.
Immunodiffusion te s ts fo r bluetongue
and e p iz o o tic hemorrhagic disease:
fo r preparing so lu b le antigens.
Diag.
20:
Am. J. Vet.
291-302.
A rapid simple method
Proc. Am. Assn. Vet. Lab.
73
53.
Karstad, L. H., and D. 0. T ra in e r.
1967.
H istopathology o f
experimental bluetongue disease o f w h ite -ta ile d deer.
Can. Vet. J.
54.
8:
247-254.
Karstad, I . H ., A. W inter, and D. 0. T ra in e r.
o f e p iz o o tic hemorrhagic disease o f deer.
22:
55.
1961.
Pathology
Am. J . Vet. Res.
227-235.
K istn e r, T. P ., G. E. Reynolds, I . D. K o lle r, C. E. T ra in e r,
and D. L. Eastman.
1975.
C lin ic a l and s e ro lo g ic a l fin d in g s
on the d is tr ib u tio n o f bluetongue and e p iz o o tic hemorrhagic
disease viruses in Oregon.
18:
56.
Proc. Am. Assn. Vet. Lab. Qiag.
135-148.
Kontor, C. J . , and A. B. Welch.
1976.
C ha racte rizatio n o f
an e p iz o o tic hemorrhagic disease v iru s .
21:
57.
Res. in Vet. S c i.
190-196.
Lawman, M. J. P ., E. P. J. Gibbs, and J. A. Davis'.
1977.
E pizoo tic hemorrhagic disease o f deer v iru s in tis s u e c u ltu re
and la b o ra to ry animals.
58.
J. Comp", Path.
Lee, V. H ., 0. R. Causey, and D. L . Moore.
87:
345-352.
1974.
Bluetongue
and re la te d viruses in Ibadan, N ig e ria :
Is o la tio n and
p re lim in a ry id e n tific a tio n o f v iru s e s .
Am. J. Vet. Res.
35:
1105-1108.
74
59.
Luedke, A. J . , J . G. Bowne, M. M. Jochim, and C. Doyle.
1964.
C lin ic a l and pa th o lo g ica l features o f bluetongue in sheep.
Am. J. Vet. Res.
60.
25:
963-970.
Luedke, A. J . , and NI. NI. Jochim.
1968.
C lin ic a l and se ro lo g ic a l
responses in vaccinated sheep given challenge in o c u la tio n
w ith is o la te s o f bluetongue v iru s .
Am. J. Vet. Res.
29:
841-851.
61.
Luedke, A. J . , NI. NI. Jochim, and J. G. Bowne.
1965.
P re lim i­
nary bluetongue transm ission w ith the sheep ked Melophagus
ovinus (L ).
62.
Can. J. Comp. Med. Vet. S c i.
29/
Luedke, A. J . , M. M. Jochim, and R. H. Jones.
tongue in c a tt le :
229-231.
1977.
Blue­
E ffe cts o f C ulicoides v a riip e n n is tra n s ­
m itte d bluetongue viru s on pregnant h e ife rs and t h e ir calves.
Am,J. Vet. Res.
63.
38:
1687-1695.
Luedke, A. J . , M. M. Jochim, and R, H. Jones.. ,1977.
tongue in c a ttle :
E ffe cts o f vecto r tra nsm itte d bluetongue
v iru s on calves p re vio u sly in fe c te d in u te ro .
Res.
64.
38:
Am. J. Vet.
1697-1700.
Luedke, A. J . , M. M. Jochim, and R. H. Jones.
tongue in c a ttle :
1977.
38:
Blue­
Repeated exposure o f two ImmunologicaTly
to le ra n t calves to bluetongue v iru s by vector b ite .
Vet. Res.
Blue­
1701-1704.
Am. J.
75
65.
Luedke, A. J . , R. H. Jones, and M. M. Jochim.
1967.
Trans­
mission o f biuetongue v iru s between sheep and c a ttle by
C ulicoides v a riip e n n is .
66.
Am. J . Vet. Res.
28:
Luedke, A. J . , R. H. Jones, and I . E. Walton.
w in te rin g mechanism fo r b luetongue v iru s :
457-460.
1977.
Over­
B io lo g ic a l re ­
covery o f la te n t v iru s from bovine by b ite s o f CuTicoides
v a riip e n n is .
67.
M cG ill, L. D.
Am. J. Trop. Med. Hyg.
1972.
26:
313-325.
C ha ra cte riza tio n o f the immune response
and lesions o f the ce n tra l nervous system in b l uetongue
v iru s in fe c tio n o f mice.
Ph.D. Thesis.
Texas A & M Uni­
v e r s ity , College S ta tio n , Texas.
68.
McKercher, D. G., B. McGowan, J. A. Howarth, and J. K. Saito .
1953.
A p re lim in a ry re p o rt on the is o la tio n and id e n ti­
fic a tio n o f b l uetongue v iru s from sheep in C a lifo rn ia .
J. A. V. M. A.
69.
Meln ic k , J . L.
V ir o l.
70.
22:
122:
1976.
300-301.
Taxonomy o f v iru s e s , 1976.
211-221.
M e tca lf, H. E ., and M. M. Jochim.
E ffic a c y o f the AGP te s t.
71.
Prog. Med.
1970.
Bluetongue in c a ttle :
Am. J . Vet. Res.
31_:
M e ttle r, N. E ., L. G. MacNamara, and R. E. Shope.
1743-1749.
196.2.
The propagation o f the v iru s o f e p iz o o tic hemorrhagic disease
o f deer in newborn mice and HeLa c e lls .
665-678.
J. Exp. Med.
116:
76
72.
Moore, D. L ., and V. H. Lee.
1972.
A ntigenic re la tio n s h ip
between the viruses o f e p iz o o tic hemorrhagic disease o f
deer and bluetongue.
73.
Arch, ges V iru s fo rc h .
37:
282-284.
Murphy, F. A ., E. C. Borden, R. E. Shope, and A. H arrison.
1971.
Physiochemical and m orphological re la tio n s h ip s o f
some arthropod-borne viruses to bluetongue v iru s - A new
taxonomic group.
V ir o l.
74.
13:
Neitz , W. 0.
sheep.
75.
E lectron m icroscopic stud ies.
273-288.
1948.
Immunological studies on bluetongue in
Onderstepoort J . Vet. S c i. Anim. Ind.
N e v ill, E. M.
J. Gen.
1971.
2_3:
93-136.
C a ttle and C ulicoides b itin g midges
as possible ove rw inte ring hosts o f bluetongue v iru s .
step oo rt J . Vet. Res.
76.
38:
Onder-
65-72.
N ieschulz, O., G. A. J. Bedford, and R. M. DuToit.
1934.
. In v e s tig a tio n s in to the transm ission o f bluetongue in sheep
during the season 1931-1932.
Ind.
77.
2:
Onderstepoort J. Vet. S c i. Anim.
509-562.
Oellermann, R. A ., and P. C arter.
1977.
The immunological
response to in ta c t and disassociated bluetongue v iru s in
mice.
78.
Onderstepoort J. Vet. Res.
P arikh, G. C.
study.
1970.
44:
201-204.
E pizootic hemorrhagic deer disease
South Dakota Pittman-Robertson P ro je ct W-75-R-11.
77
79.
P in i. A.
1976.
A study on the pathogenesis o f bluetongue:
R e p lica tio n o f the v iru s in the organs o f in fe c te d sheep.
Onderstepoort J . Vet. Res.
80.
43/
P i r t l e 5 E. C., and J. M. Layton.
159-164.
1961.
E pizootic hemorrhagic
disease in w h ite -ta ile d deer - C h a ra c te ris tic s o f the South
Dakota s tra in o f the v iru s .
81.
Am. J. Vet. Res.
22:
104-108.
Prestwood5 A. K ., I . P. K is tn e r5 F. G. K e llo g 5 and F. A.
Hayes.
1974.
The 1971 outbreak o f hemorrhagic disease
among w h ite -ta ile d deer o f the southeastern United States.
J. W ild l. Dis .
82.
10:
217-224.
P ric e , D. A ., and W. T. Hardy.
1954.
Is o la tio n o f bluetongue
v iru s from Texas.sheep - C ulicoides shown to be a ve cto r.
J. A. V. M. A.
83.
Quinn5 W. J.
124:
1978.
255-258.
Personal communication.
Montana Dept.
o f Livestock - Animal Health D iv is io n 5 Bozeman5 Montana.
84.
Richards, S. H.
1964.
Epidemic hemorrhagic disease in deer
and a n te lo p e .. North Dakota Outdoors.
85.
Sawyer5 M. M., and B. I . Osburn.
1977.
26/
18-21.
Is o la tio n o f blue­
tongue vaccine v iru s by d ire c t in o c u la tio n onto tis s u e
c u ltu re .
86.
Cornell Vet.
67:
65-71.
Schalm5 0. W., N. C. J a in 5 and E. C. C a rro ll.
Hematology.
Lea and Febiger5 Phi Iid e lp h ia .
1975.
807 pp.
V eterinary
78
87.
Shope, R. E.
1967.
disease o f deer.
32:
88.
The e p iz o o tic !ogy o f e p iz o o tic hemorrhagic
Trans. North Am. W ild l . ;N at.. Resoun. Conf.
381-386.
Shope. R. E ., L. G. MacNamara, and R. Mangold.
1955.
Report
on the deer m o rta lity - E pizoo tic hemorrhagic disease o f
deer.
89.
New Jersey Outdoors.
6j
17-21.
Shope, R. E ., I . G. MacNamara, and R. Mangold.
1960.
A v iru s
induced e p iz o o tic hemorrhagic disease o f the V irg in ia w h ite ta ile d deer ( Odocoileus v irg in ia n u s ) .
J. Exp. Med.
Ill:
155-170.
90.
Shope, R. E ., I . G. MacNamara , and N. E. M e ttle r.
1963.
The
a tte n u a tio n o f the v iru s o f e p iz o o tic hemorrhagic disease
by it s s e ria l passage in the b ra in o f newborn mice.
Med.
91.
US:
J. Exp.
421-424.
S h u ltz, G., and P. D. Delay.
1955.
Losses in newborn lambs
associated w ith bluetongue vaccin ation o f pregnant ewes.
J. A. V. M. A.
92.
SpreulI , J.
1905.
127:
224-226.
M a la rial c a ta rrh a l fe ve r (bluetongue)
o f sheep in South A fric a .
18:
93.
J. Comp. Path. Therap.
321-337.
S ta ir , E. L ., R. M. Robinson, and I . P. Jones.
taneous bluetongue in Texas w h ite -ta ile d deer.
5:
164-173.
1968.
Spon­
Path. Vet.
79
94.
Stauber, I . H ., C. H. N e llis , R. A. Magonigle, and H. W. Vaughn.
1977.
Prevalence o f reactors to selected liv e s to c k pathogens
in Idaho mule deer.
95.
J. W ild !. Mgmt.
41:
515-519.
S to tt, J . L ., I . L . Barber, and B. I . Osburn.
1978.
Sero-
typ in g bluetongue v iru s : A comparison o f plaque in h ib itio n
(d is c ) and plaque n e u tra liz a tio n methods.
Vet. Lab. Diag.
96.
21:
Proc. Am. Assn.
399-410.
Svehag, S. E ., and 0. R. Gorham.
1963.
Passive tra n s fe r o f
immunity to bluetongue v iru s from vaccinated maternal
mice to t h e ir o ffs p rin g .
97.
Swenson, J. E.
1979.
Res. Vet. S c i.
4:
109-113.
E ffe cts o f a hemorrhagic disease e p i­
z o o tic on a w h ite -ta ile d deer population in eastern Montana.
Proc. Mont. Acad. S c i.
98.
T h e ile r, A.
1906.
38:
Bluetongue in sheep.
Transvaal fo r 1904-1905.
99.
:
Thomas, F. C ., and J . M ille r .
v iru s and EHD v iru s :
J . Comp. Med. . 35_:
100,
25-32.
Ann. Rep. D ir. A g ric.
110-121.
1971.
A comparison o f bluetongue
E lectron microscopy and serology.
22-27.
Thomas, F. C ., and 0. 0. T ra in e r.
1970.
Bluetongue v iru s :
( I ) In pregnant w h ite -ta ile d deer (2) A plaque reduction
n e u tra liz a tio n te s t.
101.
J . W ild l. D is.
Thomas, F. C ., and D. 0. T ra in e r.
in w h ite -ta ile d deer.
6_: 384-388.
1970.
Am. J. Vet. Res.
Bluetongue v iru s
31:
271-278.
Can.
80
102.
Thomas, F. C ., N. W illis , and G. Ruckerbauer.
1974.
Id e n ti­
fic a tio n o f the viruses involved in the 1971 outbreak o f
hemorrhagic disease in the southeastern United States
w h ite -ta ile d deer.
103.
T ra in e r, D. 0.
J. W ild l. D is.
1970.
Kk
"B luetongue".
187-189.
In In fe c tio u s Diseases.
o f Wild Mammals, ■Iowa State U n iv e rs ity Press, Ames, Iowa.
P. 55-59.
104.
T ra in e r, D. 0 ., and M. M. Jochim.
1969.
S erological evidence
o f bluetongue in w ild ruminants o f North America.
Vet. Res.
105.
30:
2007-2011.
T ra in e r, D. 0 ., and I . H. Karstad.
rhagic Disease".
1970.
T s a i, K ., and I . Karstad.
v iru s o f deer:
M icro.
107.
16:
"E p iz o o tic Hemor­
In In fe c tio u s Diseases o f Wild Mammals,
Iowa State U n iv e rs ity Press, Ames, Iowa.
106.
Am. J.
1970.
p. 50-54.
E pizoo tic hemorrhagic disease
An e le ctro n m icroscopic study.
Can. J.
427-432.
T s a i., K ., and I . Karstad.
1973.
U ltra s tru c tu ra l cha racte r­
iz a tio n o f the genome o f e p iz o o tic hemorrhagic disease
v iru s .
108.
In f . and I mm.
T s a i, K ., and L. Karstad.
8:
463-474.
1973.
The pathogenesis o f e p iz o o tic
hemorrhagic disease o f deer - An e le ctro n m icroscopic study.
Am. J. Path.
70:
379-389.
(
81
109.
Verwoerd5 D. W.
1970.
D ip lo rn a v iruses:
group o f double-stranded RNA viru s e s .
12:
HO,
Prog. Med. V ir o l.
192-210.
Verwoerd5 D. W.5 H. J . E ls 5 E. M. D e V illie r s 5 and H. Huismans.
1972.
10:
111.
A newly recognized
S tructure o f the bluetongue v iru s capsid.
J. V ir o l.
783-794.
Verwoerd5 D. W., H. Louw5 and R. A. Oellermann.
1970.
C haracte rizatio n o f the bluetongue v iru s rib o n u c le ic acid.
J. V ir o l.
112.
5/
1-7.
Vosdingh5 R. A ., D. 0. T ra in e r5 and B. C. Easterday.
1968.
Experimental bluetongue disease in w h ite -ta ile d deer.
Can. J. Comp. Med:
113.
Walcheck5 K.
1978.
Montana Outdoors.
114.
32_:
382-387.
E p izo o tics:
9h
30-35.
Wang5 C. S ., D. C. Lueker5 and I . L. Chow.
antigen o f bluetongue v iru s .
115.
Mystery deer diseases.
1972.
In f . and I mm.
Wilhelm, A. R ., and D. 0. T ra in e r.
1966.
5;
116.
30:
467-473.
A s e ro lo g ic a l
study o f e p iz o o tic hemorrhagic disease o f deer.
Mgmt.
Soluble
J. W ild l.
777-780.
Wilhelm, A. R ., and D. 0. T ra in e r.
1967.
A comparison of
several viruses o f e p iz o o tic hemorrhagic disease o f deer.
J. I n f . Dis .
117:
48-54.
82
117.
Wilhelm, A. R ., and D. 0. T ra in e r.
1969.
Hematological and
v iro lo g ic a l studies o f e p iz o o tic hemorrhagic disease o f
deer.
118.
B u ll. W ild l. Dis. Assn.
Y u i l l , I . M.
1978.
5^
77-94.
Personal communication.
Dept, o f Vet.
S c i., Univ. o f W isconsin, Madison, Wisconsin.
MONTANA STATE UNIVERSITY LIBRARIES
762 1001
7 6
F e l d n e r , T im o t h y J
F333
cop. 2
E p i z o o t i c h e m o r r h a g ic
d i s e a s e v i r u s i n M o n ta n a
d e e r and c a t t l e
DATE
IS S U E D
TO
-
//S M