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Vol. 57 (2) 2002 |
WEST NILE VIRUS ENCEPHALITIS IN HORSES IN ISRAEL
S. Perl 1,
L. Fiette2,
D. Lahav1,
N. Sheichat1,
C.Banet3,
U. Orgad1,
Y. Stram4
and M. Malkinson3
1 Departments of Pathology, 3Avian Diseases and
4Virology, Kimron Veterinary Institute, 50250 Beit Dagan, Israel
2 Unitי de Recherche et d'Expertise en
Histotechnologie et Pathologie, Institut Pasteur, 25, rue du Docteur Roux,
75724 Paris Cedex 15, France
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Abstract
West Nile (WN) virus is a mosquito-borne flavivirus
that can cause a variety of neurological symptoms in mammals and birds.
The virus is considered to be endemic in many parts of Asia, Africa and
Europe, particularly in countries bordering the Mediterranean Sea, where
outbreaks of disease occur from time to time. During August through
November 2000, an outbreak of West Nile fever affected over 400 people in
Israel with 29 fatalities. At the same time an outbreak of
encephalomyelitis was diagnosed in the local equine population. We
describe here the clinical, histopathological and virological findings of
five terminally-affected equines presenting neurological signs that were
submitted between August and October 2000. The animals (two males and
three female) were 2 to 10 years old. Four of them were recumbent and all
had paraplegia of various degrees. Histologically, four had encephalitis
and two of these had WN viral antigen in sections of the brain and spinal
cord with a sparse distribution of positive cells. WN virus was isolated
from three of the horses.
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Materials and methods Results
Discussion back
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Introduction
West Nile virus (WNV) is a flavivirus belonging to the
Japanese encephalitis-St.Louis encephalitis serocomplex of the Flaviviridae
family. The virus is transmitted by mosquitoes, usually Culex spp., from avian
reservoir hosts to susceptible mammals, principally horses and man as well as to
wild and domestic birds (1,2). WN disease is a zoonosis and when favorable
ecological and climatic conditions are in synchrony, epidemics of encephalitis
occur in humans with a case-mortality rate of up to 10%, while a fatality rate
of 40% in young domestic geese has been encountered (3).
Since its original isolation in Uganda in 1937 (4), WNV
infections and fever have been recognized in humans in numerous countries in
Europe, Africa, western Asia since the 1960s (5). Equine disease was first
described in Egypt (6), France during the 1960s (7) and in Portugal in 1971(8).
The disease was experimentally reproduced in equines as encephalomyelitis
(6,9,10). West Nile disease has reemerged recently in man, horses and birds
during the 1990s with mild to severe outbreaks of human encephalitis including
mortality being reported in Algeria (11), Romania (12), Czech Republic (5),
Volgograd, Russia (13) and the Congo (14). The virus was isolated for the first
time in the New World in New York City in 1999 and has since caused illness in
humans and equines throughout the eastern USA and southern Canada (15).
Outbreaks affecting equines were also described in this decade in Morocco in
1996 (16), Italy in 1998 (17), France in 2000 (18).
In Israel, outbreaks of WN fever in humans were first reported in the 1950s
(19) and most recently in 2000 (20). WNV was recently isolated from domestic
geese and various wild birds (2,3). Between August and October 2000, an
unspecified number of horses from various regions of Israel were reported by
veterinary practitioners as showing neurological signs of weakness and ataxia of
the hind limbs (A.Steinman, personal communication). We
describe here the clinical, virological and pathological
findings of the disease affecting four horses and one pony.
Introduction
Results Discussion
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Materials
and Methods
The first equine case was referred to us on August 23,2000
and the last case on October 15. During this period five equines were submitted
for post-mortem examination to the Pathology Department of the Kimron Veterinary
Institute. A complete necropsy was carried out, and the brain and spinal cord
from all five cases and samples of the viscera of two horses were fixed in 10%
neutral buffered formalin and processed routinely for histopathology. Samples of
brain and spinal cord were also frozen and stored at -20oC for virological
investigation.
Histological sections were stained with hematoxylin and eosin
(H&E). Immunohistochemistry was performed on 5 µm sections of selected paraffin
blocks using the standard peroxidase-anti-peroxidase method with amplification
by the EnVision+ system (Dako) using paraffin sections. The primary antibody was
an anti-WNV mouse monoclonal antibody.
Virology
Brain and spinal cord were collected from necropsies of all
five animals.10% suspensions were prepared in PBS (pH 7.0), clarified and
filtered through 0.22µ Millipore filters. Six well plates containing
semi-confluent Vero cell monolayers were inoculated with filtrates. The plates
were incubated at 37
0C
in 5%CO2:95%
air and inspected daily for seven days for cytopathic effect. Two blind passages
were performed.
RT-PCR
RNA was extracted using QIAamp viral RNA kit (Qiagen). RT-PCR
was conducted with primers WN132 and WN240 (21). The PCR product was 327 bp in
size.
Introduction
Materials and methods
Discussion back
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Results
Table 1:
Summary of clinical, histopathological , immunohistochemical (IHC) and
virological findings in five horses
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Horse
No. |
Age
(yrs) |
M/F |
Clinical signs |
Duration
of illness |
Histology |
IHC
|
Virus
isolation |
|
1 |
2 |
F |
Ataxia,
fever,
paresis,
recumbency |
4 days |
No lesions |
Negative |
+ |
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2
(pony) |
8 |
F |
Ataxia,
paraplegia
hind legs,
fever,
recumbency
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3 days,
euthanized |
Encephalitis |
A few
positive
cells |
+ |
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3 |
10 |
F |
Ataxia,
quadriplegia,
recumbency
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2 days,
euthanized |
Very discrete
encephalitis |
Negative |
- |
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4 |
10 |
M |
Ataxia,
quadriplegia,
circling,
fever
|
7 days |
Encephalitis |
A few
positive
cells |
+ |
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5 |
7 |
M |
Ataxia,
paresis,
recumbency |
2 days |
Encephalitis |
Negative |
- |
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Clinical findings:
The clinical findings are summarized in Table 1. All five
horses were ataxic and became recumbent in the final stages of the illness. In
two horses (case nos. 1 and 2) only the hind limbs were affected while
quadriplegia was present in the other three horses. The duration of the illness
ranged from 2 to 7 days and two horses had to be euthanized 2 and 3 days after
they became recumbent.
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Macroscopical findings
In one horse (case no.1) there were multifocal subcutaneous
haemorrhages in the subcutis and endocardium. In horse no.3, a granulomatous
focal lesion in the mandible was noted and focal dark areas in the gray matter
of the lumbo-sacral region of the spinal cord were seen (Fig.1). In horse no.4
we observed a few haemorrhagic infarcts and edema was seen in the lungs,
multifocal haemorrhages on the epicardium and endocardium, and multifocal dark
areas in the gray matter of the lumbar-sacral region of the spinal cord. In
horse no.5, only the head was submitted for pathological examination and no
pathological changes were visible
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Figure 1.
Lumbar spinal cord; horse.
Multifocal haemorrhages in the gray matter.
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Microscopical findings
In 4 of the 5 horses a non-suppurative, moderate to severe
polio-encephalomyelitis was observed. The lesions were located mainly in the
lumbar-sacral region of the spinal cord and in the brain stem. Multifocal
perivascular to diffuse haemorrhages were present in some of the cases in the
gray matter of the spinal cord. (Fig.2). In other cases perivascular mononuclear
cell infiltrations were present in the gray matter. These were composed of
lymphocytes, plasmocytes and macrophages (Fig. 3). The lesions were
symmetrical and bilateral and involved the gray matter and particularly the
ventral horns of the spinal cord. Slight inflammation and edema of the meninges
and spinal cord were also present in some horses.
No significant histopathological
lesions were detected in the lung, heart muscle, liver, spleen and kidney of any
of the horses.
Viral antigen:
We found viral antigen in two cases (Nos.2 and 4). Positive cells were very
sparse and associated with foci of inflammation in the gray matter. They had the
morphology of pyramidal neurons in the ventral horns (Fig.4). In addition, we
saw labeled cellular debris in the neuropil or microglial cells (Fig.5).
Virological findings
Three WNV isolates were made from the brain and spinal cord (horse no. 1, 2 and
4). The isolates were identified by RT-PCR on Vero cell supernatants and mouse
brain (Banet, personal communication ). Partial sequence analysis of the E gene
showed that the isolates were 99.8% homologous with WNV- ISR98 isolated from a
goose (22).
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Figure 2.
Lumbar spinal cord. Multifocal and diffuse
haemorrhages in the gray matter. H&E. x5.
Figure 3. Lumbar
spinal cord; severe lymphocytic perivascular infiltration. H&E. x20.
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Figure 4.
Spinal cord: Immunoperoxidase-staining of WN viral antigen in the cytoplasm
of a neuron and multifocal glial cell proliferation (gliosis).
x20.
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Figure 5.
Spinal cord: Immunoperoxidase-staining of WN viral antigen in glial cells.
x40.
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Introduction
Materials and methods Results
back to top
Discussion
In the five equine cases described here, the
clinical and histopathological findings are similar to those that have been
described previously in spontaneous or experimental WNV infection of horses and
ponies. In the majority of affected horses, WNV infection usually causes a
sub-clinical illness, associated with the appearance of specific serum
antibodies. Only a small number of infected horses develop acute neurological
signs varying from 20% to 40% in different studies, with a mortality rate of
about 45% (1). Ataxia, weakness, paresis, paraplegia or tetraplegia are the most
consistent signs; while fever is not (9,11,17,18).
Lesions induced by WNV are limited to the central nervous
system. They consist of a moderate to severe meningo-encephalitis associated
with haemorrhages. They are preferentially observed in the brain stem and
ventral horns of the lumbo-sacral spinal cord, whereas the brain and cerebellum
show few changes. The preferential location and microscopic appearance has to be
considered in the differential diagnosis of equine viral encephalitides, and it
is essential to examine the spinal cord where WNV is suspected.
West Nile disease has then to be confirmed by appropriate tests, e.g.
MAC-ELISA and RT-PCR. As described previously in cases from Italy and New York
(23,24), viral antigen is detected in the gray matter in morphologically normal
or degenerate neurons or fibers, and in glial cells. In our cases,
immunohistochemistry was positive only in two cases so this method is not very
sensitive. In our series, we used IHC and virus isolation in parallel. Three
cases were positive by both methods, in one case (No.1), the virus was isolated
but lesions were not found in the samples, while in horse no.4 we observed
inflammatory lesions similar to the other cases but virus isolation was
negative. Considering the low level of antigen that was found,
immunohistochemistry does not seem to be the most reliable method to confirm the
diagnosis of WNV
Introduction
Materials and methods Results
Discussion back
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References
1.