ISRAEL JOURNAL OF
VETERINARY MEDICINE
INCREASED PREVALENCE OF BORNA
DISEASE VIRUS ELISA AND
IMMUNOFLUORESCENT ANTIBODIES IN
HORSES FROM FARMS SITUATED ALONG
THE PATHS OF MIGRATORY BIRDS
Vol. 58 (2-3)
2003
*Teplitsky, V.1, *Pitlik, S.1, Richt, J.A.2, Herzog, S.2, Meir, R.3,
Marcus, S.3, Sulkes, J.4, Weisman, Y.3 and Malkinson, M.3
1. Department of Internal Medicine “C”, Rabin Medical Center, Beilinson
Campus, Sackler School of Medicine,Tel Aviv University, Petach Tikvah, Israel
2. Institut für Virologie, Justus-Liebig-Universität Giessen, Giessen, Germany
3. Division of Avian Diseases, Kimron Veterinary Institute, P.O.Box 12, 50250
Beit Dagan, Israel
4. Epidemiology Unit, Rabin Medical Center, Beilinson Campus, Sackler School
of Medicine,Tel Aviv University, Petach Tikvah, Israel
Abstract
Borna disease virus (BDV) causes a clinical syndrome of
meningoencephalomyelitis in horses, sheep, ostriches and several other
species. Asymptomatic infection is much more common than symptomatic one
and may contribute to the circulation of infection. Immunofluorescense (IF)
study is usually used for the determination of BDV antibodies in the serum,
but a commercial kit was not developed for this purpose. We have examined
the prevalence and distribution of antibodies to Borna disease virus by the
ELISA method. For standartization of ELISA, known positive and negative
BVD serum from Germany and Israel and 78 randomly selected sera were
tested for the presence of specific antibodies to the three recombinant BDV
antigens: p18, p24 and p40 using four serial dilutions 1:100, 1:200, 1:400 and
1:800. The correlation coefficients for each dilution were established by
regression analysis. As a consequence, 1:400 dilution with the p24 antigen
(correlation coefficient=0.65) was then selected for screening the 365 sera.
All the sera were coded and the study was conducted without prior knowledge
of their geographic origin or identity. For comparison, 39 selected serum
samples were examined by IF. After comparing the results of the two assays a
cut-off ELISA OD value was established to distinguish positive from negative
sera. Antibodies to p24 antigen were found in 94 horses (23%). The regions of
highest seropositivity corresponded to those areas where migratory birds were
most prevalent (p<0.001). When ELISA results were compared to
immunofluorescence, 79% sensitivity and 80% specificity were obtained.
Considering the limitations of IF, ELISA is recommended for diagnosing and
surveying Borna virus infection.
Introduction
Borna disease has been recognized for over 150 years and causes a clinical
syndrome of meningoencephalomyelitis in horses, sheep, ostriches and several
other species.
Borna disease virus (BDV) is a non-segmented, negative-strand RNA virus,
the prototype of a newly recognized virus family Bornaviridae (order
Mononegavirales) with several different strains (1,2), and recently, the
complete genomic sequence was published (3). The clinical disease in horses
varies from overt disease ranging from excitation, ataxia, somnolence,
abnormal posture, opisthotonus, nistagmus, blindness, paralysis and death to
asymptomatic, long-term infection. Transmission of infection probably occurs
by aerosol penetration of the virus through the nasal mucosa and then
intraxonally to the brain. In addition to the neurochemical disturbances,
BDV infection induced differential responses to serotonin compounds in
the brain of the neonatal rat (4).
Antibodies to BDV have been found in humans such as ostrich - farm
workers (5) and animals such as healthy horses (6,7,8) and hospitalized cats
(9) without clinical signs of Borna disease, suggesting that asymptomatic
infection is much more common than the symptomatic one. The role of the
BDV in the etiology of human chronic and mentopsychiatric disease has been
investigated (10,11,12 and others) but no decisive conclusions concerning its
causative role have been made so far (13).
Borna disease is distributed unevenly throughout the world, existing only in
certain areas of Germany, Austria, Iran, Japan and Israel suggesting that
environmental factors such as rodent or bird vectors could play a role in the
transmission or expression of the disease.
In the Near East (Egypt, Syria) outbreaks of viral encephalomyelitis of
equines and ruminants, resembling BD were observed between 1949 and 1957
(14).In Israel, BDV infection has been recognized since 1987 when antibodies
to BDV were found in 15% of 78 asymptomatic horses (Abraham and
Davidson unpublished study) and in 1994, the first case in horses was
diagnosed in the Galilee region. During the 1980s, the ostrich industry grew
rapidly in Israel and in 1988, an outbreak of Borna disease occurred on ostrich
farms in the central and northern regions of the country (15). Since horses and
ostriches are often raised in close proximity, even on the same farm, another
source of infection or vector, common to both species, was conjectured.
As many as 94 avian species from Europe and Asia migrate through Israel,
mostly roosting in the warm and cultivated areas on the Coastal Plain and Rift
Valley rather than in the central Mountain Range.
We have investigated the prevalence of BDV antibodies in horses from
farms located throughout the country. Our findings suggest that one possible
reason for the different prevalence among these geographically defined areas
can be linked to the flight paths taken by migratory birds.
Materials and Methods
Four hundred and forty three sera of healthy mares from all parts of Israel
were collected from 1988 through 1994 for pregnancy diagnosis. The sera was
stored at -200C. The recently described ELISA technique used for antibody
detection (16),was adapted in this study that included three phases:
a. Standardization of the ELISA; b. Screening of serum; c. Comparison of
ELISA results with indirect immunofluorescence (IF).
For standartization of ELISA, known positive and negative BVD serum from
Germany and Israel and 78 randomly selected sera were tested for the presence
of specific antibodies to the three recombinant BDV antigens: p18, p24 and
p40 using four serial dilutions 1:100, 1:200, 1:400 and 1:800. The correlation
coefficients for each dilution were established by regression analysis (Table 1).
As a consequence, 1:400 dilution with the p24 antigen (correlation
coefficient=0.65) was then selected for screening the 365 sera. All the sera
were coded and the study was conducted without prior knowledge of their
geographic origin or identity. The results of ELISA are expressed in terms of
optical density units (OD).
For comparison, 39 selected serum samples were examined with IF. After
comparing the results of the two assays a cut-off ELISA OD value was
established to distinguish positive from negative sera .
At this stage all the samples were identified and the results were analyzed
statistically. Forty-one sera were unidentified and excluded from further
statistical analysis. One serum was identified as being from a camel ; its OD
was consistently less than 0.07.
In order to evaluate the statistical significance in the distribution of positive
sera in the three geographical areas, chi-squared test was done. P-value less or
equal 0,05 was considered statistically significant.
ELISA
Ninety-six well plates (Nunc-Immuno Plate Maxisorp) were coated overnight
at 40C with 10ng of recombinant protein per well in 100nl of carbonatebicarbonate buffer (sodium carbonate 1.59g, sodium bicarbonate g, 93g,
sodium azide 0.2g, distilled water 1 liter, pH 9.5-9.7). Plates were washed
three times with washing buffer (0.05% Tween-20 in PBS) and incubated for 1
hour at room temperature with ELISA-diluent (0.5% bovine serum albumin
(BSA) fraction V (USB) Sigma in washing buffer).
Serial two-fold dilutions of serum were prepared in ELISA-diluent and 0.1ml
of each dilution(1:100 to 1:800) was then added to each well and incubated for
1.5 hours at 370C. Plates were washed three times with washing buffer. Then,
0.1ml of alkaline phosphatase conjugated goat anti-horse IgG (Sigma) diluted
1:1000 in ELISA- diluent was added to each well and incubated for one hour
at 370C. After washing the plates five times, 0.1ml of substrate solution was
added to each well. The substrate consisted of 4-nitrophenyl phosphate
disodium salt (hexahydrate) (Merck) 1 mg in 1ml of buffer substrate solution
(diethanolamine,pH 9.8). After incubation at room temperature for 30 minutes,
the absorbance at 405 nm was determined for each well using an ELISA reader
( Dynatech MR 5000).
Table 1: Correlation coefficients of optical density values of ELISA
performed on 78 sera with 1:100 to 1:800 dilution with p24, p18 and p40
recombinant Borna virus proteins. P value for all cells was 0.0001.
Antigen/Dilution
1:100
1:200
1:400
1:800
p24
0.58948
0.6113
0.64487
0.65845
p18
0.65933
0.67836
0.66706
0.66952
p40
0.59891
0.63932
0.67901
0.69217
Results
The cumulative frequency of the OD values
of all the sera examined is shown in Figure 1.
From the results of the comparison of ELISA
and IF using the latter as a “gold standard”
(Table 2) an OD value of 0.23 was chosen as
the cut-off point. This yielded 79%
sensitivity and 80% specificity for ELISA
with a positive predictive value of 69%, a
negative predictive value of 87%, overall
proportion of agreement 79% and the kappa
statistic 0.57. The total number of positive
sera was 94 (23%) (Figure 2). The annual
incidence of seropositivity was uneven and
varied from 27% in 1991 to 12% in 1993.
The 402 sera were identified as originating
from 125 stables that could be allocated to
three major geographical areas: Rift Valley,
Mountain Range and Coastal Plain. The
frequency of seropositivity was lowest in the
stables situated on the Mountain Range (6%)
(Figure 3,4) but was highest in the Rift
Valley (32%) and Coastal Plain (29%). These
differences
were
highly
statistically
significant (p<0.001).
In the rural districts, the percentage of
seropositive horses varied significantly from
year to year in two of the districts from
which substantial numbers of sera were
tested (Figure 5). For example in district A,
there was an almost twofold increase in 1992
and in district R, there was a two to five-fold
increase in the percentage of seropositives
during the years 1989. This could be
explained as due to repeated exposure to
BDV due to cyclic infection. Other rural
districts were represented by too small a
number of sera to permit annual analysis.
Figure 1. Cumulative frequency of optical density
the sera (433) examined.
Figure 2. Annual distribution of the sera.
Figure 3. Distribution of the sera according to geographic areas (p<0.001).
Figure 4. Graphical presentation of the sera distribution on the map of Israel according to geographic lo
Figure 5. Annual distribution of the sera in districts A and R.
Table 2: Indirect immunofluorescence (IF) results and optical density (OD)
values of indirect ELISA to p24 Borna virus recombinant protein of 39
selected sera.
Number
ELISA OD
value
IF titer
Number
ELISA OD
value
IF titer
1.
0.49
-
21
0.21
-
2.
0.44
1:10
22
0.20
-
3.
0.41
1:10
23
0.19
-
4.
0.38
-
24
0.19
-
5.
0.35
1:10
25
0.19
-
6.
0.35
1:20
26
0.18
-
7.
0.31
-
27
0.18
-
8.
0.30
1:40
28
0.18
-
9.
0.29
1:80
29
0.18
-
10.
0.29
1:40
30
0.17
-
11.
0.29
-
31
0.17
-
12.
0.28
-
32
0.11
1:40
13.
0.25
1:10
33
0.08
1:20
14.
0.23
1:40
34
0.08
-
15.
0.23
1:80
35
0.06
-
16.
0.23
1:40
36
2.06
-
17.
0.22
1:20
37
2.06
-
18.
0.22
-
38
0.05
-
19.
0.22
-
39
0.05
20.
0.21
-
- = IF negative (titer < 1:10)
Table 3: Borna disease virus seroepidemiological studies in horses
Country
Year
Number of
studied sera
Method
% of
positive sera
1.
Germany (6)
1985-6
1441
Indirect fluorescence
12%
2.
USA (17)
1993
295
Indirect
immunofluorescence,
2.7%
Western blot
3.
Japan (8)
1995
57
RT-PCR
29.8%
4.
Iran (7)
1996
72
Immunblotting RT-PCR
32.7%
5.
Turkey (18)
2002
323
ELISA
25%
6.
Israel*
1998
402
ELISA
23%
*Present study
RT-PCR = reverse transcriptase polymerase chain reaction.
Discussion
The ELISA has not previously been applied to the diagnosis of the BDV in
horses. Several serological surveys from different countries based on IF,
immunoblotting, and PCR have been published. The percentage positive
values ranged from 2.7% in USA (17), 25% in Turkey (18), 12% in Germany
(6) to 30% in Japan (8) and 33% in Iran (7) (Table 3). Thus, our value of 23%
was intermediate in this range. Compared with IF and immunoblotting, ELISA
is less expensive, quicker and an easier method to perform and is used widely
in clinical laboratories. These advantages make it even more convenient for
seroepidemiological studies of large numbers of sera. In our study, the ELISA
was found to be suitable for diagnosis and screening of Borna virus infection
of equines. The ELISA yielded good correlation coefficients and
reproducibility of results with all three BDV recombinant proteins. Of the 39
sera compared by ELISA and IF, five ELISA positive sera were not reactive in
IF. A similar discordance of reactivity of serum antibodies to recombinant
proteins in immunoblotting assay and to the intranuclear and intracellular
expressed viral antigens in IF was also noted in the study of Kao and others
(17).
It is likely that IF antibodies recognize the structural viral proteins whereas
immunoblotted antibodies bind to denatured protein (17). Another limitation
of IF noted by von Lange and others (6) is its poor correlation with clinical
infection. In that study, clinically normal horses displayed IF titres of 1:320 or
higher while animals with neurological signs of BD and infectious virus
demonstrated in the brain had very low titres (<1:10).
The mode of transmission of the disease in different countries is unknown,
although importation of the infection with latently infected horses is possible.
Our study suggests another possibility: transmission of infection by migrating
birds. There are 94 transient species that fly over Israel on route to Africa and
many of them stay in the warmer geographical areas such as the Coastal Plain
and the Rift Valley. For example ornithologists have observed flocks of
migrating starlings and doves feeding among ostriches during a recent winter
outbreak of BD at one farm in the Negev in 1998. The role of migrating birds
in the transmission of infectious diseases is suspected in Lyme disease (19),
Newcastle disease (20), Japanese encephalitis (21), Sindbis virus infection
(22) and West Nile fever (23, 24).
We have demonstrated an increased frequency of BDV antibodies in horse
farms situated in regions with large populations of migrating birds: the
Coastal Plain and Rift Valley, compared with the Mountain Range. Infected
birds could shed the virus during their migration while in close contact with
other birds and horses. To confirm our hypothesis of the role of migrating
birds in the transmission of BDV it would be necessary to trap birds for
extensive virological and serological examination.
Acknowledgments to Dr. A. Lublin, Dr. I. Davidson and Dr. M.Van Ham
for statistical assistance and to Dr. W.I. Lipkin for donating recombinant
proteins.
LINKS TO OTHER ARTICLES IN THIS ISSUE
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