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Journal of Clinical Microbiology, November 1999, p. 3634-3643, Vol. 37, No. 11
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Detection of Canine Distemper Virus Nucleoprotein
RNA by Reverse Transcription-PCR Using Serum, Whole Blood, and
Cerebrospinal Fluid from Dogs with Distemper
A. L.
Frisk,1
M.
König,2
A.
Moritz,3 and
W.
Baumgärtner1,*
Institut für
Veterinär-Pathologie,1 Institut
für Virologie, Fachbereich
Veterinärmedizin,2 and
Medizinische und Gerichtliche
Veterinärklinik,3
Justus-Liebig-Universität Giessen, 35392 Giessen, Germany
Received 21 April 1999/Returned for modification 10 June
1999/Accepted 26 July 1999
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ABSTRACT |
Reverse transcription-PCR (RT-PCR) was used to detect canine
distemper virus (CDV) nucleoprotein (NP) RNA in serum, whole blood, and
cerebrospinal fluid (CSF) samples from 38 dogs with clinically
suspected distemper. Results were correlated to clinical findings,
anti-CDV neutralizing antibody titers, postmortem findings, and
demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR was ensured by amplification of RNA from
various laboratory CDV strains, restriction enzyme digestion, and
Southern blot hybridization. In 29 of 38 dogs, CDV infection was
confirmed by postmortem examination and immunohistochemistry. The
animals displayed the catarrhal, systemic, and nervous forms of
distemper. Seventeen samples (serum, whole blood, or CSF) from dogs
with distemper were tested with three sets of primers targeted to
different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons were observed in 82, 53, and 41% of the 17 samples,
depending upon the primer pair used. With the most sensitive primer
pair (primer pair I), CDV NP RNA was detected in 25 of 29 (86%) serum
samples and 14 of 16 (88%) whole blood and CSF samples from dogs with
distemper but not in body fluids from immunohistochemically negative
dogs. Nucleotide sequence analysis of five RT-PCR amplicons from
isolates from the field revealed few silent point mutations. These
isolates exhibited greater homology to the Rockborn (97 to 99%) than
to the Onderstepoort (95 to 96%) CDV strain. In summary, although the
sensitivity of the RT-PCR for detection of CDV is strongly influenced
by the location of the selected primers, this nucleic acid detection
system represents a highly specific and sensitive method for the
antemortem diagnosis of distemper in dogs, regardless of the form of
distemper, humoral immune response, and viral antigen distribution.
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INTRODUCTION |
Canine distemper virus (CDV), which
is closely related to measles virus and rinderpest virus, two other
members of the genus Morbillivirus of the
Paramyxoviridae family, is a devastating, highly contagious
pathogen that occurs worldwide (10, 32). The host spectrum
of CDV comprises dogs and many other carnivores and noncarnivores as
well as marine mammals (1, 3, 7, 10, 27, 45). A possible
link between Paget's disease of bone in humans and CDV infection was
shown by epidemiological studies and was substantiated by detection of
CDV RNA in affected tissues (17, 30). CDV is also discussed
as a candidate that might play a role in the initiation of multiple
sclerosis (35). Recently, a new member of the
Paramyxoviridae family was isolated from an outbreak of
fatal respiratory and nervous disease in horses and humans in
Australia. This new isolate, first classified as a morbillivirus, most
likely represents a new genus within the Paramyxovirinae
subfamily (26, 46).
In dogs, CDV infection can result in subclinical infection,
gastrointestinal signs, and/or respiratory signs, frequently with central nervous system (CNS) involvement (3, 4, 22). Nervous signs may also occur as a late manifestation of CDV infection without
any other signs (7, 22, 33). Following aerosol infection
(4), the virus replicates in macrophages and lymphoid cells
of the upper respiratory tract (4, 22). Systemic
dissemination is mediated by infected cells, such as lymphocytes,
monocytes, and platelets, and/or occurs through non-cell-associated
virus, leading to infection of various organs (5, 23, 44).
Pathologic lesions are most prominent in the respiratory and
gastrointestinal tracts, lymphoid tissues, and CNS (1, 2, 7, 14,
29).
A variety of clinical parameters and different types of assays have
been suggested for use for the definitive antemortem diagnosis of
distemper. However, due to the unpredictable and variable course of
distemper, e.g., length of viremia, organ manifestation, and a lack of
or delayed humoral and cellular immune responses, the final diagnosis
for most animals remains uncertain. Various specimens including
conjunctival and vaginal imprints, urinary epithelium cells, skin and
stomach biopsy specimens, cells from tracheal washings, blood smears,
and cerebrospinal fluid (CSF) taps have been used for an etiological
diagnosis (1, 6, 42). In addition, inoculation of canine
primary (lung macrophages or fibroblasts) or permanent cell lines with
organ suspensions or cell explants from diseased animals, the ferret
inoculation test, immunofluorescence, antigen immunocapture
enzyme-linked immunosorbent assay, immunocytochemistry, and in situ
hybridization have been used for detection of CDV antigen and CDV RNA
(3, 4, 6, 16, 40). However, the majority of these methods
are laborious and time-consuming, and, more importantly, they are of
limited usefulness when they are applied to clinical specimens.
Although immunohistochemistry represents a highly sensitive and
specific method for detection of CDV antigen in tissue obtained
postmortem, it is suitable only within limits for the diagnosis of
distemper in living animals (6). The determination of CDV
neutralizing antibodies in serum or CSF may be helpful in some animals
with of chronic CNS infection, but again, the results are variable and
depend on the stage of the disease. In addition, a vaccine-induced
immune response or the presence of maternally derived antibodies cannot
always be excluded (3, 24).
In summary, none of the methods mentioned above fulfills the
requirements of a sensitive and specific CDV detection assay. Recent
developments in molecular techniques revealed the suitability of these
methods for diagnostic purposes as well as pathogenic and
epidemiological studies (15, 39). In a recent investigation, a reverse transcription (RT)-PCR was used for detection of CDV RNA in
peripheral blood mononuclear cells from dogs with suspected distemper
(41). However, only 53% of the animal were positive by
RT-PCR, and the diagnosis of distemper was not confirmed by or
correlated with the results of other methods, including
immunohistochemistry, histopathology, and in vitro virus isolation
methods. To further investigate the suitability of RT-PCR for the
detection of CDV RNA in clinical specimens, serum, whole blood, and CSF
from dogs with spontaneous CDV infection were used as a source of viral RNA in the present study and results were correlated with the clinical,
pathologic, serological, and immunohistochemical findings.
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MATERIALS AND METHODS |
Animals, tissue samples, and viruses.
Three healthy dogs
(dogs 1 to 3) and 38 dogs with suspected CDV infection (dogs 4 to 41)
were used in this study (Table 1). Tissue
specimens from CNS, respiratory tract, spleen, and urinary and
gastrointestinal tracts were collected at necropsy from 38 animals
(dogs 4 to 41), fixed in 10% nonbuffered formalin, embedded in
paraffin, and investigated for CDV antigen by routine histology and
immunohistochemistry techniques (7). Depending on the size of the animals, approximately 250 to 3,000 µl of serum (n = 38), 250 to 10,000 µl of whole blood (n = 22),
and 250 to 3,000 µl of CSF (n = 22) were collected by
venipuncture from living animals and/or from the left ventricle or vena
cava and by puncture of the atlanto-occipital joint during necropsy. In
addition, serum and whole-blood samples were obtained from three
healthy dogs (dogs 1 to 3) one day prior to and 2 and 16 days after
vaccination (SHLT+P Candur; Rockborn strain, Hoechst, Marburg,
Germany). Blood smears were taken from 15-infected CDV dogs before the
dogs were killed (Table 1). Serum, whole blood (clotted, without the
use of anticoagulants), and CSF (without centrifugation) were stored at
80°C until they were used.
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TABLE 1.
Age, sex, vaccination record, clinical form of distemper
and histological and immunohistological findings for dogs with
naturally occurring distempera
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For in vitro studies, the following CDV strains were used:
Onderstepoort (Ond-CDV; kindly provided by A. E. Metzler, Institut für Virologie, Universität Zürich, Zürich,
Switzerland), R252-CDV (kindly provided by S. Krakowka, Ohio State
University, Columbus, Ohio), Convac (kindly provided by C. Örvell, Central Microbiological Laboratory of Stockholm County
Council, Stockholm Sweden), Rockborn, and four different field isolates
(isolates 2582/90, 2015/91, 1052/93, and 98/91 [1]).
In addition, a porpoise morbillivirus virus (kindly provided by B. K. Rima, Medical Biology Centre, University of Belfast, Belfast, United
Kingdom), canine parainfluenza virus type 2 (8), and the
Edmonston strain of measles virus (kindly provided by C. Örvell,
Central Microbiological Laboratory of Stockholm County Council) were used.
RNA extraction.
RNA was extracted from serum (150 µl),
whole blood (250 mg), and CSF (150 µl) with the RNaid PLUS KIT
(Dianova, Hamburg, Germany) according to the manufacturer's
instruction. Briefly, cells were lysed with guanidinium thiocyanate,
followed by RNA extraction with acid phenol and chloroform-isoamyl
alcohol (24:1). RNA, which was present in the top aqueous phase, was
purified by adsorption to an RNA matrix. Negative controls for
carryover contamination included RNA extracted from noninfected African
green monkey kidney (Vero) cells between the extraction of RNA from
each sample from the dogs. Vero cells infected with Ond-CDV served as a
positive control.
RT-PCR and restriction enzymes.
The oligonucleotides used
for amplification of the CDV nucleoprotein (NP) gene sequences are
shown in Fig. 1 and Table
2. Positions are indicated according to
the positions of Sidhu et al. (42), which are available from
the GenBank-EMBL data bank under accession nos. AF014953, L13194, and
L13195. The sequences of all CDV primers except the antisense primer at
positions 1610 to 1587 were localized in the highly conserved region of the NP gene of the Ond-CDV strain, which shows great homology among
morbilliviruses (11, 34, 36, 42). The expected amplicon lengths are 287, 260, and 900 bp for primer pair I (PP-I), PP-II, and
PP-III, respectively. RNA integrity was ensured by amplification of a
sequence from a housekeeping gene that encodes
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (primers were kindly
provided by T. J. Rosol, Ohio State University, Columbus, Ohio)
(Table 2). The amplification product has a length of 229 bp
(18).
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FIG. 1.
Schematic drawing of the CDV genome and mRNA with
location of the primers used for PCR. P/V/C, phosphoprotein; M, matrix
protein; F, fusion protein; H, hemagglutinin; L, large protein; nt,
nucleotide. Arrows indicate directions of primers. Numbers are
molecular sizes (in base pairs). Moderate, high, and little or no,
sequence homology of the NP gene within the genus morbillivirus.
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The total amount of RNA isolated from whole blood varied between 400 and 1,000 ng/µl, and the RNA concentration in the CSF
samples was
between 20 to 400 ng/µl. The total RNA in the serum
samples was not
measured. The isolated RNA was transcribed into
cDNA followed by PCR
amplification with the RNA PCR Core Kit (Perkin-Elmer,
Weiterstadt,
Germany) according to the manufacturer's instruction
(
12,
18,
19). Briefly, RT was performed at 42°C for 15 min
with 2.5 U of
murine leukemia virus reverse transcriptase and
50 µM random
hexamers. After inactivation of the murine leukemia
virus reverse
transcriptase, the PCR master mixture (0.15 µM each
CDV
oligonucleotide primer) was added, followed by denaturation
at 94°C
for 1 min and 40 cycles consisting of denaturation at
94°C for 1 min,
annealing at 59.5°C for 2 min, extension at 72°C
for 1 min, and
final extension at 72°C for 5 min in a thermocycler
(Biomed TC 60/2).
The PCR products were analyzed on a 2% agarose
gel after staining with
ethidium
bromide.
DNA restriction enzymes
AluI (Advanced Biotechnologies Ltd.,
Hamburg, United Kingdom) and
BsiMI (Angewandte
Gentechnologie
Systeme GmbH, Hamburg, Germany) were used for further
characterization
of the amplicons (Table
3) (
37).
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TABLE 3.
DNA restriction enzyme endonucleolytic cleavage sites and
fragment sizes for three different RT-PCR products derived from CDV
NP RNA
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Statistical analysis.
By assuming that the
immunohistochemistry method represents a well-characterized and highly
specific method for detection of CDV antigen, the RT-PCR results were
correlated with the results obtained with this protein detection
system. Therefore, sensitivity refers to the number of RT-PCR-positive
probes for the group of immunohistologically CDV-positive dogs, whereas
specificity expresses the number of RT-PCR-negative probes for the
group of animals that were negative for CDV by the immunohistochemistry
method. The computation of the 95% confidence limits for specificity
and sensitivity of the RT-PCR was performed by using the statistical program package BiAS.
Sequence analysis of PCR products.
The 287-bp DNA fragment
(PP-I) was extracted from the agarose gel and was directly sequenced
from samples from five animals (animals 13 [isolate 833-Gi95], 23 [isolate 1127-Gi95], 29 [isolate 2852-Gi95], 30 [isolate
2153-Gi95], and 40 [isolate 2495-Gi95]) and the Rockborn CDV strain.
Briefly, after electrophoresis the DNA bands were visualized under UV
light (360 nm) and were excised from the agarose gel. Purification of
the DNA was performed according to the standard protocol with the QIAEX
II DNA extraction kit (QIAGEN GmbH, Hilden, Germany). Direct sequencing
was performed with the Vent cycle sequencing kit by using p1 or p2. The
DNA fragments were separated on a 5% polyacrylamide gel (Sequa gel; Biozym, Oldendorf, Germany) in 1× TBE (Tris-borate-EDTA) buffer. After
fixation (10% acetic acid) and drying, the gels were exposed to a
Biomax X-ray film (Kodak, Berlin, Germany) for 1 day. Analysis of
sequence data was performed by using the GCG package (Genetics Computer
Group, Inc., Madison, Wis.).
Southern blotting.
To ensure the specificities of the RT-PCR
products, Southern blotting was performed with each amplicon obtained
by RT-PCR for CDV by using a digoxigenin (DIG)-labeled double-stranded
DNA (dsDNA) probe (13). Briefly, DIG-11-dUTP (DIG-dUTP) was
incorporated during PCR by using the PCR DIG Labeling mix (Boehringer
Mannheim, Mannheim, Germany) and PP-I, resulting in a 287-bp dsDNA
probe. For Southern hybridization, a standard capillary blot was
applied (37). Prehybridization and hybridization were
performed at 42°C under constant, gentle agitation. The hybridization
buffer contained 6 ng of dsDNA probe in 10 ml of prehybridization
buffer (2% [wt/vol] blocking stock solution, 50% [vol/vol]
formamide, 0.1% N-lauroyl sarkosine-NaCl, 0.02% [wt/vol]
sodium dodecyl sulfate, and 5× SSC [SSC is 750 mM NaCl plus 75 mM
sodium citrate]). After washing under stringent conditions, the
membrane was incubated with an anti-DIG-alkaline phosphatase antibody
(Boehringer Mannheim). To visualize the hybridization reaction, a
colorimetric detection system (nitroblue tetrazolium chloride,
X-phosphate) was used.
Histology and immunohistochemistry.
For histological
examination, tissue sections were cut to a thickness of 2 to 4 mm and
were stained with hematoxylin-eosin. In addition, CNS sections were
stained with luxol fast blue-cresyl-violet to determine the loss of
myelin. Immunohistochemically, viral protein was demonstrated by the
avidin-biotin complex method with a CDV NP-specific monoclonal antibody
(monoclonal antibody NP-2; clone 3991) kindly provided by C. Örvell, Central Microbiological Laboratory, Stockholm County
Council (7). The degree of immunoreactivity was scored
semiquantitatively as follows: (+), single positive cells; +, single
focus of immunopositive cells; ++, moderate number of immunopositive
cells; and +++, numerous immunopositive cells.
Serum and CSF microneutralization test.
To investigate the
presence of anti-CDV neutralizing antibodies in serum and CSF, a
standard serum microneutralization test was performed in 96-well
microtitration plates (8). Prior to use, serum and CSF
samples were heat inactivated. Twofold serum dilutions of 50 µl were
prepared (starting dilution, 1:10 in Eagle's minimum essential medium
with 10% fetal calf serum) and were tested in quadruplicate. A total
of 50 µl of the Eagle's minimum essential medium with 100 median
tissue culture infective doses of the Ond-CDV strain was added to each
well. Serum-virus mixtures were incubated at 37°C for 1 h. A
total of 100 µl of the Vero cell suspension was added to each well,
and the titration plates were incubated at 37°C in 5%
CO2 for 3 to 5 days. The neutralizing capacity of the sera
was determined by inhibition of the Ond-CDV-induced cytopathogic effect
(giant cell formation) and the neutralization titer was calculated by
the Reed and Muench method (8).
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RESULTS |
Histological and immunohistochemical findings.
According to
the immunohistological findings the necropsied animals were divided
into CDV antigen-negative animals (group I; dogs 4 to 12) and CDV
antigen-positive animals (group II; dogs 13 to 41).
Animals in group I displayed a variety of changes including anemia,
bronchopneumonia, septicemia, cardiac dilatation, subaortic
stenosis,
and subdural hemorrhage in the spinal cord. CNS lesions
were absent
from three animals (dogs 4 to 6), and the remaining
dogs suffered from
granulomatous meningoencephalitis, lymphohistiocytic
meningitis,
purulent choroiditis, nonsuppurative encephalitis,
or a
meningioma.
The immunohistochemical and most important histological observations
for animals in group II are summarized in Table
1. Five
dogs lacked
microscopic brain lesions. The remaining 24 animals
displayed white
matter lesions characteristic of acute to chronic
distemper (Table
1
and Fig.
2). Interstitial pneumonia
and/or
purulent bronchopneumonia and lymphocytic depletion in the
spleen
were also observed. Cytoplasmic and intranuclear inclusion
bodies
were found in the CNS, epithelium cells of the gastric mucosa,
urinary bladder, renal pelvis, bronchi, and bronchioles of various
animals.
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FIG. 2.
Cervical spinal cord of dog 38 showing chronic myelitis
with malacia, demyelination, and moderate perivascular
lymphohistiocytic cuffs. Hematoxylin and eosin stain was used.
Magnification, ×35.
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Widespread distribution of CDV antigen indicating early CNS infection
was found in endothelial, meningeal, and ependymal cells,
choroid
plexus epithelium, and occasionally, Purkinje's cells
and astrocytes
of eight dogs (Fig.
3). In 20 animals
(dogs 20,
22, and 24 to 41), CDV antigen was found predominantly in
lesions,
although some chronic lesions were completely devoid of viral
antigen. At extracerebral sites, viral antigen was detectable
in
bronchial epithelium cells, bronchial glands, and alveolar
macrophages
of the respiratory tract (Table
1). CDV antigen was
also observed in
gastrointestinal and urinary tract epithelium
cells, splenic
lymphocytes, and interdigitating follicular cells.
Detection of virus
antigen in vascular endothelium cells and/or
intravascular leukocytes
from of 13 dogs indicated ongoing viremia
(Fig.
3 and Table
1).
Although blood smears were available for
seven of these dogs, no virus
antigen-positive cells were detected
in these preparations (Table
1).
The only animal (dog 13) with
CDV antigen-positive cells in the blood
smear showed no evidence
of viremia in tissue sections, underlining the
highly variable
and unpredictable course of virus dissemination in
animals with
distemper.
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FIG. 3.
Cerebellum of dog 32 showing a strong positive signal
for NP antigen in endothelial cells and intravascular lymphocytes
interpreted as ongoing viremia. The avidin-biotin complex method was
used. Magnification, ×350.
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Clinical findings.
The mean age of the two female and seven
male animals (animals 4 to 12) in group I was 7.3 months (age range, 2 to 18 months). One dog was vaccinated with unknown vaccines, and the
vaccination records for the remaining eight animals were not available.
Clinically, two dogs (dogs 7 and 12) presented with neurological
dysfunction, including partial and generalized seizures, hind-leg
ataxia, and rhythmic tonic-clonic movements. Four dogs (dogs 6 and 8 to
10) showed gastrointestinal and/or respiratory tract disease, and three
dogs (dogs 4, 5, and 11) displayed nervous system and gastrointestinal signs.
The mean age of the 17 female and 12 male dogs in group II with
different vaccination histories was 7.2 months (age range,
2 to 36 months) (Table
1). According to their clinical findings
10, 14, and 4 animals (Table
1) suffered from the catarrhalic,
systemic, or nervous
form of distemper, respectively. The nervous
form of distemper was
characterized by seizures, hind-leg ataxia,
and rhythmic tonic-clonic
movements. Dogs suffering from the catarrhalic
form of distemper showed
gastrointestinal and/or respiratory tract
disease, whereas animals with
the systemic form of distemper displayed
a mixture of both the nervous
and catarrhalic forms, including
nervous signs, fever, mucopurulent
conjunctivitis and rhinitis,
and multifocal erosive
dermatitis.
Serological results.
Four of six animals in group I had a
virus neutralization antibody titer higher than 1:100. Two of three
healthy control animals seroconverted 16 days after vaccination. Among
the animals in group II (Table 4), the
virus neutralizing antibody titers in seven dogs were >1:100, those in
seven animals were between 1:40 and 1:100, and those in the remaining
nine dogs were <1:40. The virus neutralization antibody titer in CSF
samples from 10 dogs with confirmed CDV infection was >1:40 in only 1 dog.
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TABLE 4.
Detection of CDV NP nucleic acid by RT-PCR and
virus-specific neutralizing antibody titers in dogs with distemper
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RT-PCR results and restriction enzymes.
The GAPDH-specific
amplification product was demonstrated in all whole-blood samples,
whereas amplification of GAPDH was possible for only 63% of the CSF
samples. Amplification from the GAPDH housekeeping gene was not
possible for two and four CSF samples from animals in group I (dogs 4 and 8) and group II (dogs 15, 19, 33, and 34), respectively.
RT-PCR with CDV RNAs from various CDV strains resulted in amplicons of
the expected length for each primer pair. The specificities
of PCR
amplification products were ensured by restriction enzyme
digestion and
positive hybridization by Southern blotting. Porpoise
morbillivirus RNA
was amplified only by PP-I and not by PP-II
and PP-III. No
amplification products or hybridization signals
were detected with
canine parainfluenza type 2 and the Edmonston
strain of the measles
virus (Fig.
4 and
5). To investigate the
influence of the
selected primer pairs on the RT-PCR results,
samples from 17 CDV
antigen-positive animals were tested with
the three primer pairs. For
samples from 14 (82%), 9 (53%), and
7 (41%) dogs, specific RT-PCR
bands were observed with PP-I, PP-II,
and PP-III, respectively, by use
of serum, whole blood, and CSF
(Table
5).
Although the number of RT-PCR-positive animals was
not increased by
using all three primer pairs for amplification
of CDV RNA in the same
body fluid, the number of positive animals
was increased when all three
body fluids from one animals were
used. By using PP-I in RT-PCR tests
with the remaining tissues,
CDV NP RNA was detected in 25 serum samples
(sensitivity, 86%;
95% confidence interval, 68 to 96%) and 14 whole-blood and CSF
samples (sensitivity, 88%; 95% confidence
interval, 62 to 98%)
(Fig.
6 and Table
4). CDV RNA was not detected in serum
samples
(specificity, 100%; 95% confidence interval, 72 to 100%) or
whole-blood
and CSF samples (specificity, 100%; 95% confidence
interval, 61
to 100%) from immunohistologically CDV negative dogs. All
samples
from 11 (85%) of 13 dogs with virus antigen in the vascular
endothelium
and/or in the intravascular space showed specific RT-PCR
products.
CSF samples from 2 of 22 animals with a systemic
antigen distribution
lacked amplification products, but a specific
amplicon was detected
in serum and whole-blood samples. Samples from
animals in which
the virus antigen distribution restricted to the
CNS showed variable
RT-PCR results (Table
4). In one animal (dog 37) a
strong hybridization
signal was obtained by Southern blotting, even
though no band
was visible in the ethidium bromide-stained agarose gel.
Although
CDV RNA was detected in most samples, it appeared that
negative
results or only weak bands were more frequently found in gels
for serum samples from dogs with nervous distemper and that virus
antigen expression was restricted to the CNS (Table
4). All RT-PCR
products were cleaved with the
AluI restriction enzyme.
Surprisingly,
digestion with the restriction enzyme
BsiMI was observed in only
six dogs (dogs 15, 17, 23, 28, 33, and 40), indicating nucleotide
substitutions in most isolates
between positions 975 and 980.
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FIG. 4.
RT-PCR results (top panels) obtained with PP-I from CDV
laboratory strains, CDV field isolates, Edmonston strain of measles
virus, porpoise morbillivirus, and canine parainfluenza virus RNA and
results obtained by Southern blot analysis (bottom panels). (A) Lanes
1, CDV-Convac; lanes 3, canine parainfluenza virus type 2; lanes 5, porpoise morbillivirus; and lanes 7, R252-CDV. (B) Lanes 1, Ond-CDV;
lanes 3, Edmonston strain of measles virus; lanes 5, field isolate
98/91; lanes 7, field isolate 2582/90. Lanes with even numbers,
negative controls (noninfected Vero cells); lanes M1,
DIG-labeled molecular size marker; lane M2, molecular size
marker (100-bp ladder). Numbers on the left and right are molecular
sizes (in base pairs).
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FIG. 5.
RT-PCR results (top panels) with PP-III from CDV
laboratory strains, CDV field isolates, the Edmonston strain of the
measles virus, porpoise morbillivirus, and canine parainfluenza virus
RNAs and results obtained by Southern blotting analysis (bottom
panels). (A) Lanes 1, CDV Convac; lanes 3, canine parainfluenza virus
type 2; lanes 5, porpoise morbillivirus; and lanes 7, CDV R252. (B)
Lanes 1, Ond-CDV; lanes 3, Edmonston strain of measles virus; lanes 5, field isolate 98/91; and lanes 7, field isolate 2582/90. Lanes with
even numbers, negative controls (noninfected Vero cells); lanes
M1, DIG-labeled molecular size marker; lanes
M2, molecular size marker (100-bp ladder). Numbers on the
left and right are molecular sizes (in base pairs).
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TABLE 5.
Detection of CDV NP RNA in serum, CSF, and whole blood by
RT-PCR with three different primer pairs in 17 dogs with
immunohistologically confirmed CDV infection
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FIG. 6.
RT-PCR amplification of CDV NP nucleic acid (A) and
Southern blot analysis (B) of CSF, serum, and whole blood from dog 32 with PP-I. Lanes 1, 3, and 5, amplicons in CSF, serum, and whole blood,
respectively; lanes 2, 4, and 6, controls (noninfected Vero cells);
lanes M1, DIG-labeled molecular size marker; lanes
M2, molecular size marker (100-bp ladder). Numbers on the
left and right are molecular size markers.
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Sequence analysis.
RT-PCR products from samples from five
animals (dogs 13, 23, 29, 30, and 40) were sequenced. For three of
these animals (dogs 13, 29, and 30), endonucleolytic cleavage sites for
BsiMI were lacking. The alignment of the nucleotide
sequences revealed a small number of nucleotide substitutions (2 to 12)
compared to the numbers for the Rockborn and Ond-CDV (42)
strains; the substitutions were most frequently observed at the third
positions of the codons (Fig. 7). In two
sequences the first base of the codon was substituted. A transversional
substitution was observed in only one sequence; the remaining
substitutions were transitional replacements. None of these resulted in
a change in the deduced amino acid sequence. Nucleotide sequence
analyses revealed 97 to 99% homology with the Rockborn CDV strain and
94 to 95% homology with the Ond-CDV strain (36, 42). A
substitution of cytosine for thymine at position 977 compared to the
sequence of Ond-CDV was found in the sequence of the RT-PCR products
from samples from animals which were not cleaved with BsiMI.
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|
FIG. 7.
Alignment of the nucleotide sequence of the NP gene of
different distemper virus strains (five CDV isolates and the Rockborn
strain) compared to that of the NP gene of Ond-CDV GenBank-EMBL data
bank accession numbers are as follows: strain 13, AF166268 (isolate
833-Gi95); strain 23, AF166269 (isolate 1127-Gi95); strain 29, AF166270
(isolate 2852-Gi95); strain 30, AF166271 (isolate 2153-Gi95); strain
40, AF166272 (isolate 2495-Gi95); CDV Rockborn (CDV-ROCK), AF166273.
|
|
| |
DISCUSSION |
The present study confirms and extends previous observations on
the usefulness of RT-PCR as a fast, sensitive, and specific method for
the diagnosis of CDV infection in dogs. CDV RNA was detected by RT-PCR
in 86% of serum samples and 88% of whole-blood and CSF samples from
dogs with immunohistochemically confirmed distemper. The nucleic acid
detection system applied in the present study proved to be highly
sensitive and specific, regardless of clinical signs, pathological
findings, neutralizing antibody titers, and virus antigen distribution.
However, the sensitivity of the RT-PCR varied between selected primers,
depending on their position in the gene.
The three different primer pairs investigated recognized various CDV
strains but not closely related morbilliviruses and paramyxoviruses, such as the Edmonston strain of measles virus and canine parainfluenza virus type 2. Surprisingly, despite congruent results with laboratory strains, the three primer pairs differed in their sensitivities when
they were applied in tests with clinical specimens, indicating that
isolates from clinical specimens might display higher degrees of
nucleotide substitutions. RT-PCR with all three primer pairs did not
increase the sensitivity of the assay. However, by use of RT-PCR with
all three different body fluids (serum, CSF, and whole blood),
sensitivity was increased, showing a heterogeneous distribution of CDV
RNA in different body compartments. Similar results with respect to the
role of the selected primer pair for the sensitivity of RT-PCR for
detection of CDV has been described by others (41).
Furthermore, the activities of endogenous RNases and lack of
accessibility of partially degraded RNA may influence the sensitivity
of RT-PCR. Serum, whole blood, and CSF appeared to be equally suitable
as substrates for the RT-PCR. We found no evidence of inhibition of the
Taq DNA polymerase by hemoglobin, as described elsewhere
(31). The false-negative results with whole blood from two
animals from group II were not due to inadequate RNA isolation, as
demonstrated by GAPDH amplification. Amplification of GAPDH was not
possible for two and four CSF samples from animals in groups I and II,
respectively, indicating a lack of cells in these preparations.
However, CDV RNA could still be amplified from the CSF of two of these
dogs, suggesting that CDV RNA might not be always associated with CSF
cells; alternatively, single cells carry very large loads of CDV RNA.
CDV RNA was not detected in immunohistochemically CDV antigen-negative
animals or in dogs following vaccination, supporting previous
observations that a previous vaccination does not cause false-positive
results (43). In contrast, Shin et al. (41)
obtained positive RT-PCR results until 10 days after vaccination,
indicating that under certain circumstances vaccination may cause
false-positive results for dogs. To rule out false-positive results due
to cDNA contamination, DNase treatment prior to RT (data not shown) or
PCR without preceding RT was performed in some cases; however, there
was no evidence of CDV cDNA, as has been described for murine
lymphocytic choriomeningitis virus infection (21). The
negative RT-PCR results for four serum samples and for whole blood and
CSF samples from four and two animals, respectively, might be due to a
complete lack of CDV RNA or to the presence of only low levels of CDV
RNA in the samples. Autolytic degradation of CDV RNA due to released
endogenous RNases should be considered a possible source of
false-negative results; however, in the present and previous studies,
CDV transcripts were also found in animals with advanced autolytic
changes (12, 13, 19), indicating that postmortem changes
play an inferior role as a cause of false-negative results.
Interestingly, CDV RNA was also found in serum, whole blood, or CSF
from animals with subacute and chronic distemper encephalitis. By
immunohistochemistry, in some of these animals, viral antigen was
restricted only to the CNS. Whether the detected CDV RNA represents intracellular degradation products or a mechanism of virus spread and
persistence in these animals remains to be determined. Similarly, the
measles virus genome was detected in plasma, peripheral blood mononuclear cells, and CSF from patients with subacute sclerosing encephalitis (SSPE) and measles encephalitis (28, 38).
So far, confirmation of suspected canine distemper virus infection in
living dogs was unrewarding, mainly because of the low level of
sensitivity of the available methods (3, 6, 16). In the
present study, detection of virus antigen in vascular endothelial cells
and/or intravascular leukocytes was observed in 13 dogs indicating that
these animals were still in the stage of ongoing viremia. Notably, in
seven of these dogs no virus antigen was demonstrated in blood smears,
supporting the observation of the low sensitivity of this assay
(6). Interestingly amplification of CDV RNA in five of seven
serum samples and one of three whole-blood samples from animals in
which virus antigen expression was restricted to the CNS was possible.
These findings cannot readily be explained. Whether detection of CDV
RNA in the absence of viral protein is the result of a restrictive
infection as described for oligodendrocytes and neurons remains to be
clarified in future studies (29, 48).
Detection of neutralizing antibodies did not correlate with the form of
distemper, antigen distribution, or RT-PCR results, indicating the
noncontributory role of neutralizing antibody titers for the
etiological diagnosis of distemper. Furthermore, neutralizing activity
against the Onderstepoort strain may not correspond to neutralizing
activity against field isolates and, therefore, may not be protective
(25).
Comparison of the sequences of selected isolates to the sequences of
vaccine strains demonstrated distinct silent point mutations. Katayama
et al. (20) found significant nucleotide substitutions with
changes in the deduced amino acids among measles and SSPE viruses in a
highly conserved region of NP in brain tissues. Similar to SSPE virus,
nucleotide transitions were more frequent than transversions in CDV NP
RNA (9). The CDV isolates showed greater nucleotide sequence
homology to the Rockborn strain than to the Ond-CDV. Although the
database is too small and the significance of the observed silent
mutations remains unclear, most nucleotide substitutions were found in
CDV RNA from a dog with chronic brain lesions. It is tempting to
speculate that these findings suggest that there could be a correlation
between an altered NP RNA sequence and viral persistence. However, the
possibility that the observed mutations might have an impact on virus
translation and virus persistence remains speculative and needs to be
substantiated by further studies. Similarly, the biological
significance of these mutations needs to be substantiated by further
studies by using a broader database and by including CDV genomic
regions with known variability, such as the hemagglutinin protein
(10). Yoshida et al. (47) found that CDV field
isolates in Japan had one cluster of nucleotide substitutions that
distinguished them from the laboratory Onderstepoort strain. However,
they also found no correlation between sequence substitutions and
differences in distemper pathology.
In summary, RT-PCR for detection of CDV represents a sensitive and
specific method for the early and safe antemortem diagnosis of
distemper by using serum, whole blood, and/or CSF regardless of
clinical signs, pathological findings, neutralizing antibody titers,
and virus antigen distribution.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the Deutsche
Forschungsgemeinschaft (grants Ba 815/3-1 and Ba 815/3-2) and the
Gemeinnützige Hertie-Stiftung.
We thank Sandra Heinz and Annette Artelt for excellent technical
assistance; Ute Zeller for photographic support; Paul Becker, Institut
für Virologie des Fachbereichs Veterinärmedizin der Justus-Liebig-University, Giessen, Germany, for kind help and support
with the analysis of the sequence data; and K. Failing and H. Heiter,
Arbeitsgruppe Biomathematik und Datenverarbeitung des Fachbereichs
Veterinärmedizin der Justus-Liebig-Universität Giessen, for
performing the statistical analysis. We also thank A. Lemmer for
providing the serum and whole-blood samples from three of his dogs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Veterinär-Pathologie, Justus-Liebig-Universität
Giessen, Frankfurter Strasse 96, 35392 Giessen, Germany. Phone: 49 (0)641 99 38202. Fax: 49 (0)641 99 38209. E-mail:
wolfgang.baumgaertner{at}vetmed.uni-giessen.de.
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Abstract
Introduction
