Short communication

Application of N-PCR for diagnosis of distemper in dogs and fur animals

A. Rzez˙utka, B. Mizak

Department of Carnivores and Fur Animal Diseases, National Veterinary Research Institute, Al. Partyzanto´ w 57, 24-100 Pulawy, Poland

Received 30 November 2001; received in revised form 15 April 2002; accepted 5 May 2002

Abstract

The immunofluorescence test, routinely used for laboratory diagnosis of canine distemper virus(CDV) in Poland, is not sufficiently sensitive and specific. Therefore, the application of reversetranscriptase polymerase chain reaction (RT-PCR), nested PCR (N-PCR) and Southern blot hybri-dization for detection of phosphoprotein (P) gene of CDV in peripheral blood mononuclear cells(PBMCs) or internal organs of dogs and fur animals was the aim of these studies. The optimalparameters for two-step PCR were elaborated for reference strains of distemper virus and used fortesting biological samples collected from dogs, foxes, ferret and mink with spontaneous distemper.PCR product of 1069 bp was obtained in one out of 10 dog blood samples, three out of 14homogenates of internal organs of dogs and one out of five homogenates of internal organs of fox. Reamplification with the use of CDVa and CDVb primers demonstrated the 429 bp fragment insix samples, negative by PCR: two samples collected from dogs, two from foxes, one from mink andone from ferret. The specificity of N-PCR was confirmed by Southern blot hybridization. Weconclude that two-step PCR is sensitive and specific method for diagnosis of CDV infection.

Keywords:

Canine distemper virus; RT-PCR; P gene

1. Introduction

In Poland, ﬁrst clinical cases of distemper in dogs and fur animals were observed in themid of 1940s. Their number increased in 1950s, when large mink and fox farms wereestablished. Despite the wide use of different distemper vaccines in dogs, atypical clinicalsigns of the disease have been reported. The accurate diagnosis of canine distemper virus(CDV) infection can be made with the use of sensitive and speciﬁc methods based on

Veterinary Microbiology 88 (2002) 95–103

Corresponding author. Tel.:

4881-8863051; fax:

4881-8862595.

E-mail address:

bmizak@piwet.pulawy.pl (B. Mizak).0378-1135/02/$ – see front matter

molecular biology techniques. The immuno

ﬂ

uorescence test, routinely used for laboratorydiagnosis of CDV in Poland, is not suf

ﬁ

ciently sensitive and speci

ﬁ

c.Several probes exist for detection of viral nucleic acid in tissue sections by Slot blot andin situ hybridization: single stranded RNA and DNA probes, double-stranded (ds) DNAprobes, radiolabelled antisense RNA probes, as well as digoxigenin (DIG)-labelled RNAand DIG-labelled dsDNA probes have been described in the literature (Oglesbee et al.,1986; Hass et al., 1991; Zurbriggen et al., 1993; Gaedke et al., 1997; Nesseler et al., 1997).Application of reverse transcriptase polymerase chain reaction (RT-PCR), N-PCR andSouthern blot hybridization for detection of P gene of CDVin PBMCs or internal organs of dogs and fur animals was the aim of the studies reported here.

2. Materials and methods

2.1. Reference strains of CDV

Three reference strains were used for the studies: Onderstepoort (CDV-OND) andSnyder Hill (CDV-SH) kindly provided by Dr. Max Appel (James A. Baker Institute forAnimal Health, Cornell University, USA) and Lederle (CDV-LED) provided by Biowet,Pulawy, Poland. CDV-OND of titre 10

3.0

TCID

/ml was propagated in African greenmonkey kidney cells (Vero) and CDV-LED of titre 10

3.5

TCID

/ml, CDV-SH of titre10

3.5

TCID

/ml were propagated in dog lymphocytes.

2.2. Samples used for the studies

Following biological material was used for the studies: 10 blood samples collected fromdogs with clinical signs of distemper (patients of the Military Institute of Hygiene andEpidemiology, Pulawy), as well as fragments of lungs, mediastinal lymph nodes andstomach collected post-mortem from 14 dogs and

ﬁ

ve blue foxes with manifestation of upperrespiratory tractinfection aswell asfreeze-dried internal organsofonemink andoneferret which died of distemper.Blood samples were collected into the EDTA tubes. The 3

–

5 ml of the blood was diluted1:1 with PBS (Sigma) and gently mixed. PBMCs were separated by centrifugation over a10 ml mixture of 8.5 ml of 8% Ficoll-paque and 1.5 ml of 75% uropoline at 2000 rpm for30 min at 4

C. PBMCs were collected, washed twice with ice-cold PBS and centrifugedfor 5 min at 2000

–

2500 rpm. The supernatant was discarded and lymphocytes weresuspended in 250

l of PBS and used for the studies.Ten per cent homogenates of internal organs were prepared in PBS, centrifuged at13 000 rpm for 15 min

ﬁ

ltered twice (0.45 and 0.22

m) and immediately used forexaminations.

2.3. RNA isolation and reverse transcription reaction

PBMCs and homogenates of internal organs of animals were digested with proteinase K (100 mg per 1 ml) in PK buffer (20 mM Tris

–

HCl, 10 mM EDTA, 1% SDS, pH 7.6) at

A. Rzez˙utka, B. Mizak/Veterinary Microbiology 88 (2002) 95–103

Cfor 3 h. Then samples were heated at 100

Cfor 3 min and cooled on ice. Isolation of viral RNAwas carried out by method described by Chomczyn

ski and Sacchi and modi

ﬁ

edbyRidpath et al. (1994). Isolated RNA was dried at room temperature for 15

–

30 min,resuspended in 8

l of redistilled water treated with diethyl pyrocarbonate (Sigma) (H

O,DEPC) and used for the reverse transcription reaction.The 1

l (250 ng) of pd(N)

random hexamer was added into an Eppendorf tubecontaining 8

l of RNA. The sample was denatured at 65

C for 5 min and cooled onice. The

ﬁ

rst strand cDNAwas synthesized in 50

l of reaction mixture containing: 9

l of annealed RNA

–

random hexamer mixture, 5

l of 10 mM dNTP (dATP, dCTP, dGTP,dTTP); 10

l RT buffer; 5

l of 0.1 M DTT; 0.5

l (20 U) RNA inhibitor (RNasin) and20.5

l H

O (DEPC). The mixture was incubated at 25

C for 10 min and 42

C for 2 min.The 1

l (200 U) of SuperScript II (Gibco BRL) was added into the tube and the samplewas gently mixed. Reverse transcription reaction was carried out in a volume of 50

l at42

C for 50 min. Reverse transcriptase was inactivated by heating the sample at 70

C for15 min.

2.4. Primer selection for PCR and N-PCR

Two sets of primers within the P gene were used for the studies: CDV-1 (5

-GGATGTG-GAGAACGCAATAC-3

, position 283

–

303) and CDV-2 (5

-GGAGGTCTCTCAA-TAGTTGA-3

, position 1332

–

1352), enabling ampli

ﬁ

cation of 1069 bp fragment, wereelaboratedonthebaseofnucleotidesequenceofPgeneofOnderstepoortstrainavailableinGene Bank (X 51869), with the use of computer program Dnasis, version 7.0. The secondpair of primers CDVa (5

-ATGTTTATGATCACAGCGGT-3

, position 372

–

391) andCDVb (5

-ATTGGGTTGCACCACTTGTC-3

, position 780

–

800) elaborated byBarrettet al. (1993)enabled the ampli

ﬁ

cation of 429 bp fragment of all CDV strains. The optimalconditions for PCR (reaction temperature, concentration of the enzyme, dNTP, primers)were elaborated with the use of Vero cells inoculated with CDV-OND.PCR with the use of CDV-1 and CDV-2 primers, was carried out in reaction mixturecontaining: 1

l (2 U) Taq polymerase (Terpol, Sieradz), 5

l cDNA, 5

l 10

buffer A(100 mM Tris

–

HCl, pH 8.3; 11 mM MgCl

; 500 mM KCl; 0.1% gelatine), 1

l 10 mMdNTP (dATP, dCTP, dGTP, dTTP); 1

l (10 mM) each primer and redistilled water up to50

l. The mixture was prepared on ice and transferred into thermocycler T3 (Biometra).After its incubation at 94

C for 5 min, the ampli

ﬁ

cation was carried out in 35 cycles withdenaturation at 94

C for 1 min, annealing at 50

C for 2 min and elongation at 72

C for2 min. The

ﬁ

nal elongation was done at 72

C for 6 min. The reaction mixture without thematrix was used as the negative control and cDNA of Onderstepoort strain propagated inVero cells was the positive control of the reaction.N-PCR was carried out with the use of internal set of primers (CDVa, CDVb) and 5