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1999, 37(11):3634. J. Clin. Microbiol.
A. L. Frisk, M. König, A. Moritz and W. Baumgärtner with DistemperBlood, and Cerebrospinal Fluid from Dogs Transcription-PCR Using Serum, WholeNucleoprotein RNA by Reverse Detection of Canine Distemper Virus
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JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/99/$04.0010
Nov. 1999, p. 3634–3643 Vol. 37, No. 11
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Detection of Canine Distemper Virus Nucleoprotein RNA byReverse Transcription-PCR Using Serum, Whole Blood, and
Cerebrospinal Fluid from Dogs with DistemperA. L. FRISK,1 M. KONIG,2 A. MORITZ,3 AND W. BAUMGARTNER1*
Institut fur Veterinar-Pathologie,1 Institut fur Virologie, Fachbereich Veterinarmedizin,2 andMedizinische und Gerichtliche Veterinarklinik,3 Justus-Liebig-Universitat Giessen,
35392 Giessen, Germany
Received 21 April 1999/Returned for modification 10 June 1999/Accepted 26 July 1999
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 suspecteddistemper. Results were correlated to clinical findings, anti-CDV neutralizing antibody titers, postmortemfindings, and demonstration of CDV NP antigen by immunohistochemistry. The specificity of the RT-PCR wasensured by amplification of RNA from various laboratory CDV strains, restriction enzyme digestion, andSouthern blot hybridization. In 29 of 38 dogs, CDV infection was confirmed by postmortem examination andimmunohistochemistry. 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 ofprimers targeted to different regions of the NP gene of the CDV Onderstepoort strain. Expected amplicons wereobserved in 82, 53, and 41% of the 17 samples, depending upon the primer pair used. With the most sensitiveprimer 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 immunohistochemicallynegative dogs. Nucleotide sequence analysis of five RT-PCR amplicons from isolates from the field revealed fewsilent point mutations. These isolates exhibited greater homology to the Rockborn (97 to 99%) than to theOnderstepoort (95 to 96%) CDV strain. In summary, although the sensitivity of the RT-PCR for detection ofCDV is strongly influenced by the location of the selected primers, this nucleic acid detection system representsa highly specific and sensitive method for the antemortem diagnosis of distemper in dogs, regardless of theform of distemper, humoral immune response, and viral antigen distribution.
Canine distemper virus (CDV), which is closely related tomeasles virus and rinderpest virus, two other members of thegenus Morbillivirus of the Paramyxoviridae family, is a devas-tating, highly contagious pathogen that occurs worldwide (10,32). The host spectrum of CDV comprises dogs and manyother carnivores and noncarnivores as well as marine mammals(1, 3, 7, 10, 27, 45). A possible link between Paget’s disease ofbone in humans and CDV infection was shown by epidemio-logical studies and was substantiated by detection of CDVRNA in affected tissues (17, 30). CDV is also discussed as acandidate that might play a role in the initiation of multiplesclerosis (35). Recently, a new member of the Paramyxoviridaefamily was isolated from an outbreak of fatal respiratory andnervous disease in horses and humans in Australia. This newisolate, first classified as a morbillivirus, most likely representsa new genus within the Paramyxovirinae subfamily (26, 46).
In dogs, CDV infection can result in subclinical infection,gastrointestinal signs, and/or respiratory signs, frequently withcentral nervous system (CNS) involvement (3, 4, 22). Nervoussigns may also occur as a late manifestation of CDV infectionwithout any other signs (7, 22, 33). Following aerosol infection(4), the virus replicates in macrophages and lymphoid cells ofthe upper respiratory tract (4, 22). Systemic dissemination ismediated 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). Pathologiclesions are most prominent in the respiratory and gastrointes-tinal tracts, lymphoid tissues, and CNS (1, 2, 7, 14, 29).
A variety of clinical parameters and different types of assayshave been suggested for use for the definitive antemortemdiagnosis of distemper. However, due to the unpredictable andvariable course of distemper, e.g., length of viremia, organmanifestation, and a lack of or delayed humoral and cellularimmune responses, the final diagnosis for most animals re-mains uncertain. Various specimens including conjunctival andvaginal imprints, urinary epithelium cells, skin and stomachbiopsy specimens, cells from tracheal washings, blood smears,and cerebrospinal fluid (CSF) taps have been used for anetiological diagnosis (1, 6, 42). In addition, inoculation of ca-nine primary (lung macrophages or fibroblasts) or permanentcell lines with organ suspensions or cell explants from diseasedanimals, the ferret inoculation test, immunofluorescence, an-tigen immunocapture enzyme-linked immunosorbent assay,immunocytochemistry, and in situ hybridization have beenused for detection of CDV antigen and CDV RNA (3, 4, 6, 16,40). However, the majority of these methods are laborious andtime-consuming, and, more importantly, they are of limitedusefulness when they are applied to clinical specimens. Al-though immunohistochemistry represents a highly sensitiveand specific method for detection of CDV antigen in tissueobtained postmortem, it is suitable only within limits for thediagnosis of distemper in living animals (6). The determinationof CDV neutralizing antibodies in serum or CSF may be help-ful in some animals with of chronic CNS infection, but again,the results are variable and depend on the stage of the disease.
* Corresponding author. Mailing address: Institut fur Veterinar-Pathologie, Justus-Liebig-Universitat Giessen, Frankfurter Strasse 96,35392 Giessen, Germany. Phone: 49 (0)641 99 38202. Fax: 49 (0)641 9938209. E-mail: [email protected].
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TA
BL
E1.
Age,sex,vaccination
record,clinicalformof
distemper
andhistologicaland
imm
unohistologicalfindingsfor
dogsw
ithnaturally
occurringdistem
pera
Dog
no.A
ge(m
o)Sex
Vaccinationrecord
Clinical
formof
distemper
Histologicalfindings
Imm
unohistochemistry
forC
DV
antigenb
CN
SN
on-CN
SC
NS
RT
GIT
SpleenU
TB
loodsm
earV
iremia
c
135
FV
acc.N
IF
reezingartifacts
BP
11
12
22
21
12
1412
MV
acc.C
NSM
LB
P,E,L
D1
21
to1
12
22
215
3F
Vacc.
CN
SML
BP
12
1to
11
22
21
163
MN
IN
NSM
L1
22
22
NA
217
3F
Vacc.
SN
SML
IP2
11
NA
(1)
NA
118
5M
Vacc.
SA
cuteencephalopathy
E1
11
1to
11
11
to1
11
11
11
NA
119
2M
No
vacc.S
Acute
encephalopathyIP,B
P,E1
11
11
1N
A1
11
21
204
FN
IC
Acute
encephalopathyL
D1
22
22
NA
221
18F
Vacc.
SA
cuteencephalopathy
BP,L
D1
11
11
11
1to
11
1(1
)1
21
224
FN
ovacc.
SA
cuteencephalitis
IP,E,L
D1
(1)
22
2N
A2
232
FN
IC
Acute
encephalitisIP,E
,LD
11
11
11
NA
124
12F
Vacc.
CSD
WO
IB
P,E,L
D1
to1
11
11
1to
11
11
to1
11
NA
125
NI
MN
IS
SDW
OI
IP,BP,L
D1
11
11
11
11
11
11
11
NA
126
8M
Vacc.
SSD
WO
IB
P,LD
(1)
11
2(1
)2
227
3F
Vacc.
CSD
WO
IB
P,E,L
D1
11
11
to1
11
to1
11
1N
A1
287
FV
acc.C
SDW
IB
P1
22
22
22
294
FV
acc.C
SDW
IB
P,LD
11
11
11
21
to1
12
130
7F
Vacc.
CSD
WI
BP,E
,LD
11
11
11
NA
231
5M
NI
SSD
WI
BP,E
1to
11
11
11
1N
A1
1to
11
1N
A2
322
FV
acc.S
SDW
IIP,B
P,E1
1to
11
11
11
11
11
11
12
133
3M
NI
CSD
WI
BP
11
11
22
NA
234
6F
Vacc.
SSD
WI
IP,BP
11
11
to1
11
1(1
)1
22
358
MV
acc.S
SDW
I1
11
11
11
11
21
363
MN
IS
SDW
IE
11
11
1N
AN
A2
137
5F
Vacc.
SSD
WI
11
1to
11
11
22
3836
MV
acc.N
Chronic
demyelination
BP
(1)
22
22
NA
239
9F
NI
SC
hronicdem
yelinationB
P(1
)2
12
22
240
14M
No
vacc.N
Chronic
demyelination
LD
11
22
22
NA
241
8F
NI
NC
hronicdem
yelination1
12
22
22
2
aA
bbreviations:F,F
emale;M
,male;N
SML
,nosignificant
microscopic
lesion;SDW
OI,subacute
demyelination
without
inflamm
ation;SDW
I,subacutedem
yelinationw
ithinflam
mation;R
T,respiratory
tract;GIT
,gastrointestinaltract;U
T,urinary
tract;IP,interstitialpneumonia;B
P,bronchopneumonia;E
,enteritis;LD
,spleniclym
phoiddepletion;C
,catarrhalic;S,systemic;N
,nervous;NA
,notavailable;V
acc.,vaccinated;NI,
noinform
ationavailable;N
ovacc.,no
vaccination.b
2,no
antigendetected;(1
),singleim
munopositive
cells;1
,singlefocus
ofim
munopositive
cells;1
1,m
oderatenum
berof
imm
unopositivecells;
11
1,num
erousim
munopositive
cells.cA
sdeterm
inedby
imm
unohistochemistry.
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In addition, a vaccine-induced immune response or the pres-ence of maternally derived antibodies cannot always be ex-cluded (3, 24).
In summary, none of the methods mentioned above fulfillsthe requirements of a sensitive and specific CDV detectionassay. Recent developments in molecular techniques revealedthe suitability of these methods for diagnostic purposes as wellas pathogenic and epidemiological studies (15, 39). In a recentinvestigation, a reverse transcription (RT)-PCR was used fordetection of CDV RNA in peripheral blood mononuclear cellsfrom dogs with suspected distemper (41). However, only 53%of the animal were positive by RT-PCR, and the diagnosis ofdistemper was not confirmed by or correlated with the resultsof other methods, including immunohistochemistry, histopa-thology, and in vitro virus isolation methods. To further inves-tigate the suitability of RT-PCR for the detection of CDVRNA in clinical specimens, serum, whole blood, and CSF fromdogs with spontaneous CDV infection were used as a source ofviral RNA in the present study and results were correlated withthe clinical, pathologic, serological, and immunohistochemicalfindings.
MATERIALS AND METHODS
Animals, tissue samples, and viruses. Three healthy dogs (dogs 1 to 3) and 38dogs with suspected CDV infection (dogs 4 to 41) were used in this study (Table1). Tissue specimens from CNS, respiratory tract, spleen, and urinary and gas-trointestinal tracts were collected at necropsy from 38 animals (dogs 4 to 41),fixed in 10% nonbuffered formalin, embedded in paraffin, and investigated forCDV antigen by routine histology and immunohistochemistry techniques (7).Depending on the size of the animals, approximately 250 to 3,000 ml of serum(n 5 38), 250 to 10,000 ml of whole blood (n 5 22), and 250 to 3,000 ml of CSF(n 5 22) were collected by venipuncture from living animals and/or from the leftventricle or vena cava and by puncture of the atlanto-occipital joint duringnecropsy. In addition, serum and whole-blood samples were obtained from threehealthy dogs (dogs 1 to 3) one day prior to and 2 and 16 days after vaccination(SHLT1P Candur; Rockborn strain, Hoechst, Marburg, Germany). Bloodsmears were taken from 15-infected CDV dogs before the dogs were killed(Table 1). Serum, whole blood (clotted, without the use of anticoagulants), andCSF (without centrifugation) were stored at 280°C until they were used.
For in vitro studies, the following CDV strains were used: Onderstepoort(Ond-CDV; kindly provided by A. E. Metzler, Institut fur Virologie, UniversitatZurich, Zurich, Switzerland), R252-CDV (kindly provided by S. Krakowka, OhioState University, Columbus, Ohio), Convac (kindly provided by C. Orvell, Cen-tral Microbiological Laboratory of Stockholm County Council, Stockholm Swe-den), 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 byB. K. Rima, Medical Biology Centre, University of Belfast, Belfast, UnitedKingdom), canine parainfluenza virus type 2 (8), and the Edmonston strain ofmeasles virus (kindly provided by C. Orvell, Central Microbiological Laboratoryof Stockholm County Council) were used.
RNA extraction. RNA was extracted from serum (150 ml), whole blood (250mg), and CSF (150 ml) with the RNaid PLUS KIT (Dianova, Hamburg, Ger-many) according to the manufacturer’s instruction. Briefly, cells were lysed withguanidinium thiocyanate, followed by RNA extraction with acid phenol andchloroform-isoamyl alcohol (24:1). RNA, which was present in the top aqueousphase, was purified by adsorption to an RNA matrix. Negative controls forcarryover contamination included RNA extracted from noninfected Africangreen monkey kidney (Vero) cells between the extraction of RNA from eachsample from the dogs. Vero cells infected with Ond-CDV served as a positivecontrol.
RT-PCR and restriction enzymes. The oligonucleotides used for amplificationof the CDV nucleoprotein (NP) gene sequences are shown in Fig. 1 and Table2. Positions are indicated according to the positions of Sidhu et al. (42), which areavailable from the GenBank-EMBL data bank under accession nos. AF014953,L13194, and L13195. The sequences of all CDV primers except the antisenseprimer at positions 1610 to 1587 were localized in the highly conserved region ofthe NP gene of the Ond-CDV strain, which shows great homology amongmorbilliviruses (11, 34, 36, 42). The expected amplicon lengths are 287, 260, and900 bp for primer pair I (PP-I), PP-II, and PP-III, respectively. RNA integritywas ensured by amplification of a sequence from a housekeeping gene thatencodes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (primers werekindly provided by T. J. Rosol, Ohio State University, Columbus, Ohio) (Table2). The amplification product has a length of 229 bp (18).
The total amount of RNA isolated from whole blood varied between 400 and1,000 ng/ml, and the RNA concentration in the CSF samples was between 20 to400 ng/ml. The total RNA in the serum samples was not measured. The isolated
RNA was transcribed into cDNA followed by PCR amplification with the RNAPCR Core Kit (Perkin-Elmer, Weiterstadt, Germany) according to the manu-facturer’s instruction (12, 18, 19). Briefly, RT was performed at 42°C for 15 minwith 2.5 U of murine leukemia virus reverse transcriptase and 50 mM randomhexamers. After inactivation of the murine leukemia virus reverse transcriptase,the PCR master mixture (0.15 mM each CDV oligonucleotide primer) wasadded, followed by denaturation at 94°C for 1 min and 40 cycles consisting ofdenaturation at 94°C for 1 min, annealing at 59.5°C for 2 min, extension at 72°Cfor 1 min, and final extension at 72°C for 5 min in a thermocycler (Biomed TC60/2). The PCR products were analyzed on a 2% agarose gel after staining withethidium 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).
Statistical analysis. By assuming that the immunohistochemistry method rep-resents a well-characterized and highly specific method for detection of CDVantigen, the RT-PCR results were correlated with the results obtained with thisprotein 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 thegroup of animals that were negative for CDV by the immunohistochemistrymethod. The computation of the 95% confidence limits for specificity and sen-sitivity of the RT-PCR was performed by using the statistical program packageBiAS.
Sequence analysis of PCR products. The 287-bp DNA fragment (PP-I) wasextracted from the agarose gel and was directly sequenced from samples fromfive animals (animals 13 [isolate 833-Gi95], 23 [isolate 1127-Gi95], 29 [isolate2852-Gi95], 30 [isolate 2153-Gi95], and 40 [isolate 2495-Gi95]) and the Rock-born CDV strain. Briefly, after electrophoresis the DNA bands were visualizedunder UV light (360 nm) and were excised from the agarose gel. Purification ofthe DNA was performed according to the standard protocol with the QIAEX IIDNA extraction kit (QIAGEN GmbH, Hilden, Germany). Direct sequencingwas performed with the Vent cycle sequencing kit by using p1 or p2. The DNAfragments were separated on a 5% polyacrylamide gel (Sequa gel; Biozym,Oldendorf, Germany) in 13 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 byusing the GCG package (Genetics Computer Group, Inc., Madison, Wis.).
Southern blotting. To ensure the specificities of the RT-PCR products, South-ern blotting was performed with each amplicon obtained by RT-PCR for CDVby using a digoxigenin (DIG)-labeled double-stranded DNA (dsDNA) probe(13). Briefly, DIG-11-dUTP (DIG-dUTP) was incorporated during PCR by usingthe PCR DIG Labeling mix (Boehringer Mannheim, Mannheim, Germany) andPP-I, resulting in a 287-bp dsDNA probe. For Southern hybridization, a standardcapillary blot was applied (37). Prehybridization and hybridization were per-formed at 42°C under constant, gentle agitation. The hybridization buffer con-tained 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 53 SSC [SSC is 750 mM NaClplus 75 mM sodium citrate]). After washing under stringent conditions, themembrane was incubated with an anti-DIG-alkaline phosphatase antibody(Boehringer Mannheim). To visualize the hybridization reaction, a colorimetricdetection system (nitroblue tetrazolium chloride, X-phosphate) was used.
Histology and immunohistochemistry. For histological examination, tissuesections 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
FIG. 1. Schematic drawing of the CDV genome and mRNA with location ofthe primers used for PCR. P/V/C, phosphoprotein; M, matrix protein; F, fusionprotein; H, hemagglutinin; L, large protein; nt, nucleotide. Arrows indicatedirections of primers. Numbers are molecular sizes (in base pairs). Moderate,high, and little or no, sequence homology of the NP gene within the genusmorbillivirus.
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determine the loss of myelin. Immunohistochemically, viral protein was demon-strated by the avidin-biotin complex method with a CDV NP-specific monoclonalantibody (monoclonal antibody NP-2; clone 3991) kindly provided by C. Orvell,Central Microbiological Laboratory, Stockholm County Council (7). The degreeof immunoreactivity was scored semiquantitatively as follows: (1), single positivecells; 1, single focus of immunopositive cells; 11, moderate number of immu-nopositive cells; and 111, numerous immunopositive cells.
Serum and CSF microneutralization test. To investigate the presence ofanti-CDV neutralizing antibodies in serum and CSF, a standard serum micro-neutralization test was performed in 96-well microtitration plates (8). Prior touse, serum and CSF samples were heat inactivated. Twofold serum dilutions of50 ml were prepared (starting dilution, 1:10 in Eagle’s minimum essential me-dium with 10% fetal calf serum) and were tested in quadruplicate. A total of 50ml of the Eagle’s minimum essential medium with 100 median tissue cultureinfective doses of the Ond-CDV strain was added to each well. Serum-virusmixtures were incubated at 37°C for 1 h. A total of 100 ml of the Vero cellsuspension was added to each well, and the titration plates were incubated at37°C in 5% CO2 for 3 to 5 days. The neutralizing capacity of the sera wasdetermined by inhibition of the Ond-CDV-induced cytopathogic effect (giant cellformation) and the neutralization titer was calculated by the Reed and Muenchmethod (8).
RESULTS
Histological and immunohistochemical findings. Accordingto the immunohistological findings the necropsied animalswere 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 includinganemia, bronchopneumonia, septicemia, cardiac dilatation,subaortic stenosis, and subdural hemorrhage in the spinal cord.CNS lesions were absent from three animals (dogs 4 to 6), andthe remaining dogs suffered from granulomatous meningoen-cephalitis, lymphohistiocytic meningitis, purulent choroiditis,nonsuppurative encephalitis, or a meningioma.
The immunohistochemical and most important histologicalobservations for animals in group II are summarized in Table1. Five dogs lacked microscopic brain lesions. The remaining24 animals displayed white matter lesions characteristic ofacute to chronic distemper (Table 1 and Fig. 2). Interstitialpneumonia and/or purulent bronchopneumonia and lympho-cytic depletion in the spleen were also observed. Cytoplasmicand intranuclear inclusion bodies were found in the CNS,epithelium cells of the gastric mucosa, urinary bladder, renalpelvis, bronchi, and bronchioles of various animals.
Widespread distribution of CDV antigen indicating earlyCNS infection was found in endothelial, meningeal, andependymal cells, choroid plexus epithelium, and occasionally,Purkinje’s cells and astrocytes of eight dogs (Fig. 3). In 20animals (dogs 20, 22, and 24 to 41), CDV antigen was foundpredominantly in lesions, although some chronic lesions werecompletely devoid of viral antigen. At extracerebral sites, viralantigen was detectable in bronchial epithelium cells, bronchialglands, and alveolar macrophages of the respiratory tract (Ta-ble 1). CDV antigen was also observed in gastrointestinal andurinary tract epithelium cells, splenic lymphocytes, and inter-digitating follicular cells. Detection of virus antigen in vascularendothelium cells and/or intravascular leukocytes from of 13dogs indicated ongoing viremia (Fig. 3 and Table 1). Althoughblood smears were available for seven of these dogs, no virusantigen-positive cells were detected in these preparations (Ta-ble 1). The only animal (dog 13) with CDV antigen-positivecells in the blood smear showed no evidence of viremia intissue sections, underlining the highly variable and unpredict-able course of virus dissemination in animals with distemper.
TABLE 2. Nucleotide sequence and position of primer pairs used for RT-PCR
Primer Sequence (59-39)a Nucleotide positionb Direction
PP-Ip1c ACA GGA TTG CTG AGG ACC TAT 769–789 Sensep2 CAA GAT AAC CAT GTA CGG TGC 1055–1035 Antisense
PP-IIp3 AAC TAT GTA TCC GGC TCT TGG 941–961 Sensep4 CGA GTC TGA AGT AAG CTG GGT 1200–1180 Antisense
PP-IIIp5 CAA AGA CGT GTG GTC GGA GAA 711–731 Sensep6 CTT AGT AAG CAT CCT CAT CTT GGC 1610–1587 Antisense
GAPDH p7 GCC AAA AGG GTC ATC ATC TC Sense
GAPDH p8 GGC CAT CCA CAG TCT TCT Antisense
a Sequences are displayed as mRNA.b Primer position according to Sidhu et al. (42).c P, primer no.
TABLE 3. DNA restriction enzyme endonucleolytic cleavage sites and fragment sizes for three different RT-PCRproducts derived from CDV NP RNA
Restrictionenzyme
Sequence aroundcleavage site
(59-39)a
PP-I PP-II PP-III
Site(s) Fragmentsize (bp) Site Fragment
size (bp) Site(s) Fragment size (bp)
AluI AG2CT 129, 138 9, 129, 149 244 16, 244 187, 196, 474, 526, 545,567, 572, 621
5, 9, 19, 22, 49, 52, 187,278, 279
BsiMI T2CCGGA 207 80, 207 34 34, 226 264 264, 636
a The arrows indicate the cleavage sites.
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Clinical findings. The mean age of the two female and sevenmale animals (animals 4 to 12) in group I was 7.3 months (agerange, 2 to 18 months). One dog was vaccinated with unknownvaccines, and the vaccination records for the remaining eightanimals were not available. Clinically, two dogs (dogs 7 and 12)presented with neurological dysfunction, including partial andgeneralized seizures, hind-leg ataxia, and rhythmic tonic-clonicmovements. Four dogs (dogs 6 and 8 to 10) showed gastroin-
testinal and/or respiratory tract disease, and three dogs (dogs4, 5, and 11) displayed nervous system and gastrointestinalsigns.
The mean age of the 17 female and 12 male dogs in group IIwith different vaccination histories was 7.2 months (age range,2 to 36 months) (Table 1). According to their clinical findings10, 14, and 4 animals (Table 1) suffered from the catarrhalic,systemic, or nervous form of distemper, respectively. The ner-vous form of distemper was characterized by seizures, hind-legataxia, and rhythmic tonic-clonic movements. Dogs sufferingfrom the catarrhalic form of distemper showed gastrointestinaland/or respiratory tract disease, whereas animals with the sys-temic form of distemper displayed a mixture of both the ner-vous and catarrhalic forms, including nervous signs, fever, mu-copurulent conjunctivitis and rhinitis, and multifocal erosivedermatitis.
Serological results. Four of six animals in group I had a virusneutralization antibody titer higher than 1:100. Two of threehealthy control animals seroconverted 16 days after vaccina-tion. Among the animals in group II (Table 4), the virus neu-tralizing antibody titers in seven dogs were .1:100, those inseven animals were between 1:40 and 1:100, and those in theremaining nine dogs were ,1:40. The virus neutralization an-tibody titer in CSF samples from 10 dogs with confirmed CDVinfection was .1:40 in only 1 dog.
RT-PCR results and restriction enzymes. The GAPDH-spe-cific amplification product was demonstrated in all whole-
FIG. 2. Cervical spinal cord of dog 38 showing chronic myelitis with malacia,demyelination, and moderate perivascular lymphohistiocytic cuffs. Hematoxylinand eosin stain was used. Magnification, 335.
FIG. 3. Cerebellum of dog 32 showing a strong positive signal for NP antigenin endothelial cells and intravascular lymphocytes interpreted as ongoing vire-mia. The avidin-biotin complex method was used. Magnification, 3350.
TABLE 4. Detection of CDV NP nucleic acid by RT-PCR andvirus-specific neutralizing antibody titers in dogs with distemper
Dogno.
Clinicalform of
distempera
RT-PCR result with PP-I Neutralizingantibody titer
Serum CSF Whole blood Serum CSF
13 NI 1 NAb NA 1:80 NA14 C 1 NA NA 1:280 NA15 C 1 2 1 1:67 NA16 N 1 NA NA NA NA17 S 1 NA NA NA NA18 S 1 NA NA 1:540 NA19 S 1 1 1 1:113 NA20 C 1 NA NA ,1:80 NA21 C 1 1 1 1:56 ,1:1022 S 1 NA NA ,1:40 NA23 C 1 1 1 1:80 1:2024 C 1 NA NA 1:320 NA25 S 1 1 1 NA ,1:1026 S 1 1 1 1:190 ,1:1027 C 1 1 1 ,1:10 ,1:1028 C 2 1 2 1:10 NA29 C 1 1 1 NA ,1:1030 C 1 1 1 ,1:10 NA31 S 1 1 1 1:20 ,1:1032 S 1 1 1 ,1:10 ,1:1033 C 1 2 1 1:23 ,1:1034 S 6 1 1 NA NA35 S 1 NA NA NA NA36 S 2 NA NA 1:134 NA37 S 2c NA NA 1:40 NA38 N 6 6 2 1:80 NA39 S 6 NA NA ,1:10 NA40 N 1 1 1 1:28 1:4041 N 2 NA NA 1:190 NA
a NI, no information available; C, catarrhalic; N, nervous; S, systemic.b NA, not available.c Negative on the ethidium bromide-stained agarose gel but positive by South-
ern blotting.
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blood samples, whereas amplification of GAPDH was possiblefor only 63% of the CSF samples. Amplification from theGAPDH housekeeping gene was not possible for two and fourCSF samples from animals in group I (dogs 4 and 8) and groupII (dogs 15, 19, 33, and 34), respectively.
RT-PCR with CDV RNAs from various CDV strains re-sulted in amplicons of the expected length for each primer
pair. The specificities of PCR amplification products were en-sured by restriction enzyme digestion and positive hybridiza-tion by Southern blotting. Porpoise morbillivirus RNA wasamplified only by PP-I and not by PP-II and PP-III. No am-plification products or hybridization signals were detected withcanine parainfluenza type 2 and the Edmonston strain of themeasles virus (Fig. 4 and 5). To investigate the influence of the
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 virustype 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; lanes7, field isolate 2582/90. Lanes with even numbers, negative controls (noninfected Vero cells); lanes M1, DIG-labeled molecular size marker; lane M2, molecular sizemarker (100-bp ladder). Numbers on the left and right are molecular sizes (in base pairs).
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 virustype 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; andlanes 7, field isolate 2582/90. Lanes with even numbers, negative controls (noninfected Vero cells); lanes M1, DIG-labeled molecular size marker; lanes M2, molecularsize marker (100-bp ladder). Numbers on the left and right are molecular sizes (in base pairs).
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selected primer pairs on the RT-PCR results, samples from 17CDV antigen-positive animals were tested with the threeprimer 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-positiveanimals was not increased by using all three primer pairs foramplification of CDV RNA in the same body fluid, the numberof positive animals was increased when all three body fluidsfrom one animals were used. By using PP-I in RT-PCR testswith the remaining tissues, CDV NP RNA was detected in 25serum samples (sensitivity, 86%; 95% confidence interval, 68to 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-bloodand 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 thevascular endothelium and/or in the intravascular space showedspecific RT-PCR products. CSF samples from 2 of 22 animalswith a systemic antigen distribution lacked amplificationproducts, but a specific amplicon was detected in serum andwhole-blood samples. Samples from animals in which thevirus antigen distribution restricted to the CNS showed vari-able RT-PCR results (Table 4). In one animal (dog 37) astrong 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 mostsamples, it appeared that negative results or only weak bandswere more frequently found in gels for serum samples fromdogs with nervous distemper and that virus antigen expressionwas restricted to the CNS (Table 4). All RT-PCR productswere cleaved with the AluI restriction enzyme. Surprisingly,digestion with the restriction enzyme BsiMI was observed in onlysix dogs (dogs 15, 17, 23, 28, 33, and 40), indicating nucleotidesubstitutions in most isolates between positions 975 and 980.
Sequence analysis. RT-PCR products from samples fromfive animals (dogs 13, 23, 29, 30, and 40) were sequenced. Forthree of these animals (dogs 13, 29, and 30), endonucleolytic
cleavage sites for BsiMI were lacking. The alignment of thenucleotide sequences revealed a small number of nucleotidesubstitutions (2 to 12) compared to the numbers for the Rock-born and Ond-CDV (42) strains; the substitutions were mostfrequently 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 se-
FIG. 6. RT-PCR amplification of CDV NP nucleic acid (A) and Southernblot analysis (B) of CSF, serum, and whole blood from dog 32 with PP-I. Lanes1, 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 sizemarker; lanes M2, molecular size marker (100-bp ladder). Numbers on the leftand right are molecular size markers.
TABLE 5. Detection of CDV NP RNA in serum, CSF, and whole blood by RT-PCR with three different primer pairs in 17 dogs withimmunohistologically confirmed CDV infection
Dog no.
RT-PCR resultsa
PP-I PP-II PP-III
Serum CSF Whole blood Serum CSF Whole blood Serum CSF Whole blood
13 1 ND ND 1 ND ND 1 ND ND15 1 2 1 1 2 1 2 2 217 1 ND ND 1 ND ND 1 ND ND20 1 ND ND 2 ND ND 2 ND ND22 1 ND ND 1 ND ND 2 ND ND23 1 1 1 1 ND 1 1 ND 125 1 1 1 ND ND 1 ND ND 128 2 1 2 2 2 2 2 2 229 1 1 1 1 1 ND 1 1 ND30 1 1 1 1 1 ND 1 1 ND33 1 2 1 2 2 2 2 2 236 2 ND ND 2 ND ND 2 ND ND37 2 ND ND 2 ND ND 2 ND ND38 1 1 2 2 2 2 2 2 239 1 ND ND 2 ND ND 2 ND ND40 1 1 1 ND 1 ND ND 1 ND41 2 ND ND 2 ND ND 2 ND ND
a ND, not determined; 2, negative; 1, positive.
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quence; the remaining substitutions were transitional replace-ments. None of these resulted in a change in the deducedamino acid sequence. Nucleotide sequence analyses revealed97 to 99% homology with the Rockborn CDV strain and 94 to95% homology with the Ond-CDV strain (36, 42). A substitu-tion of cytosine for thymine at position 977 compared to thesequence of Ond-CDV was found in the sequence of the RT-PCR products from samples from animals which were notcleaved with BsiMI.
DISCUSSION
The present study confirms and extends previous observa-tions on the usefulness of RT-PCR as a fast, sensitive, andspecific method for the diagnosis of CDV infection in dogs.CDV RNA was detected by RT-PCR in 86% of serum samplesand 88% of whole-blood and CSF samples from dogs withimmunohistochemically confirmed distemper. The nucleic aciddetection system applied in the present study proved to behighly sensitive and specific, regardless of clinical signs, patho-logical findings, neutralizing antibody titers, and virus antigendistribution. However, the sensitivity of the RT-PCR variedbetween selected primers, depending on their position in thegene.
The three different primer pairs investigated recognized var-ious CDV strains but not closely related morbilliviruses andparamyxoviruses, such as the Edmonston strain of measlesvirus and canine parainfluenza virus type 2. Surprisingly, de-spite congruent results with laboratory strains, the threeprimer pairs differed in their sensitivities when they were ap-plied in tests with clinical specimens, indicating that isolatesfrom clinical specimens might display higher degrees of nucle-otide 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, andwhole blood), sensitivity was increased, showing a heteroge-neous distribution of CDV RNA in different body compart-ments. Similar results with respect to the role of the selectedprimer pair for the sensitivity of RT-PCR for detection ofCDV has been described by others (41). Furthermore, theactivities of endogenous RNases and lack of accessibility ofpartially degraded RNA may influence the sensitivity of RT-PCR. Serum, whole blood, and CSF appeared to be equallysuitable as substrates for the RT-PCR. We found no evidenceof inhibition of the Taq DNA polymerase by hemoglobin, asdescribed elsewhere (31). The false-negative results with wholeblood from two animals from group II were not due to inad-equate RNA isolation, as demonstrated by GAPDH amplifi-cation. Amplification of GAPDH was not possible for two andfour CSF samples from animals in groups I and II, respectively,indicating a lack of cells in these preparations. However, CDVRNA could still be amplified from the CSF of two of thesedogs, suggesting that CDV RNA might not be always associ-ated with CSF cells; alternatively, single cells carry very largeloads of CDV RNA. CDV RNA was not detected in immu-nohistochemically CDV antigen-negative animals or in dogsfollowing vaccination, supporting previous observations that aprevious vaccination does not cause false-positive results (43).In contrast, Shin et al. (41) obtained positive RT-PCR resultsuntil 10 days after vaccination, indicating that under certaincircumstances vaccination may cause false-positive results fordogs. To rule out false-positive results due to cDNA contam-ination, DNase treatment prior to RT (data not shown) orPCR without preceding RT was performed in some cases;however, there was no evidence of CDV cDNA, as has beendescribed for murine lymphocytic choriomeningitis virus infec-
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 thatof the NP gene of Ond-CDV GenBank-EMBL data bank accession numbers are as follows: strain 13, AF166268 (isolate 833-Gi95); strain 23, AF166269 (isolate1127-Gi95); strain 29, AF166270 (isolate 2852-Gi95); strain 30, AF166271 (isolate 2153-Gi95); strain 40, AF166272 (isolate 2495-Gi95); CDV Rockborn (CDV-ROCK),AF166273.
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tion (21). The negative RT-PCR results for four serum samplesand for whole blood and CSF samples from four and twoanimals, respectively, might be due to a complete lack of CDVRNA or to the presence of only low levels of CDV RNA in thesamples. Autolytic degradation of CDV RNA due to releasedendogenous RNases should be considered a possible source offalse-negative results; however, in the present and previousstudies, CDV transcripts were also found in animals with ad-vanced autolytic changes (12, 13, 19), indicating that postmor-tem changes play an inferior role as a cause of false-negativeresults.
Interestingly, CDV RNA was also found in serum, wholeblood, or CSF from animals with subacute and chronic distem-per encephalitis. By immunohistochemistry, in some of theseanimals, viral antigen was restricted only to the CNS. Whetherthe detected CDV RNA represents intracellular degradationproducts or a mechanism of virus spread and persistence inthese animals remains to be determined. Similarly, the measlesvirus genome was detected in plasma, peripheral blood mono-nuclear cells, and CSF from patients with subacute sclerosingencephalitis (SSPE) and measles encephalitis (28, 38).
So far, confirmation of suspected canine distemper virusinfection in living dogs was unrewarding, mainly because of thelow level of sensitivity of the available methods (3, 6, 16). In thepresent study, detection of virus antigen in vascular endothelialcells and/or intravascular leukocytes was observed in 13 dogsindicating that these animals were still in the stage of ongoingviremia. Notably, in seven of these dogs no virus antigen wasdemonstrated in blood smears, supporting the observation ofthe low sensitivity of this assay (6). Interestingly amplificationof CDV RNA in five of seven serum samples and one of threewhole-blood samples from animals in which virus antigen ex-pression was restricted to the CNS was possible. These findingscannot readily be explained. Whether detection of CDV RNAin the absence of viral protein is the result of a restrictiveinfection as described for oligodendrocytes and neurons re-mains to be clarified in future studies (29, 48).
Detection of neutralizing antibodies did not correlate withthe form of distemper, antigen distribution, or RT-PCR re-sults, indicating the noncontributory role of neutralizing anti-body titers for the etiological diagnosis of distemper. Further-more, neutralizing activity against the Onderstepoort strainmay not correspond to neutralizing activity against field iso-lates and, therefore, may not be protective (25).
Comparison of the sequences of selected isolates to thesequences of vaccine strains demonstrated distinct silent pointmutations. Katayama et al. (20) found significant nucleotidesubstitutions with changes in the deduced amino acids amongmeasles and SSPE viruses in a highly conserved region of NPin brain tissues. Similar to SSPE virus, nucleotide transitionswere more frequent than transversions in CDV NP RNA (9).The CDV isolates showed greater nucleotide sequence homol-ogy to the Rockborn strain than to the Ond-CDV. Althoughthe database is too small and the significance of the observedsilent mutations remains unclear, most nucleotide substitutionswere found in CDV RNA from a dog with chronic brainlesions. It is tempting to speculate that these findings suggestthat there could be a correlation between an altered NP RNAsequence and viral persistence. However, the possibility thatthe observed mutations might have an impact on virus trans-lation and virus persistence remains speculative and needs tobe substantiated by further studies. Similarly, the biologicalsignificance of these mutations needs to be substantiated byfurther studies by using a broader database and by includingCDV genomic regions with known variability, such as the hem-agglutinin protein (10). Yoshida et al. (47) found that CDV
field isolates in Japan had one cluster of nucleotide substitu-tions that distinguished them from the laboratory Onder-stepoort strain. However, they also found no correlation be-tween sequence substitutions and differences in distemperpathology.
In summary, RT-PCR for detection of CDV represents asensitive and specific method for the early and safe antemor-tem 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 Forschungs-gemeinschaft (grants Ba 815/3-1 and Ba 815/3-2) and the Gemeinnutz-ige Hertie-Stiftung.
We thank Sandra Heinz and Annette Artelt for excellent technicalassistance; Ute Zeller for photographic support; Paul Becker, Institutfur Virologie des Fachbereichs Veterinarmedizin der Justus-Liebig-University, Giessen, Germany, for kind help and support with theanalysis of the sequence data; and K. Failing and H. Heiter, Arbeits-gruppe Biomathematik und Datenverarbeitung des Fachbereichs Vet-erinarmedizin der Justus-Liebig-Universitat Giessen, for performingthe statistical analysis. We also thank A. Lemmer for providing theserum and whole-blood samples from three of his dogs.
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