J. KIM, C. CHOI, D. U. HAN, C. CHAE. POSTWEANING multisystemic wasting syndrome (PMws), a new porcine disease which affects mainly nursery and early.
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SHORT COMMUNICATIONS
Simultaneous detection
of porcine circovirus type 2 and porcine parvovirus in pigs with PMWS by multiplex PCR
Primers AF AR BF BR AF PCV-2
Sequence (5' to 3) CAG TTC GTC ACC CTT TCC C GGG GGA CCA ACA MA TCT CT CAT ACA CTG GAC AAT CAC AAC AAA GCC TM TTG CTG TTG CIT CTG
Position Size (bp) 939-957 1466-1485 3451-3474 3760-3780
547
330
forward prmer, AR PCV-2 reverse primer, BF PPV forward
primer, BR PPV reverse primer
J. KIM, C. CHOI, D. U. HAN, C. CHAE ethidium bromide. Their lengths were verified by a digested POSTWEANING multisystemic wasting syndrome (PMws), lamda DNA standard run simultaneously. The PCR reactions a new porcine disease which affects mainly nursery and early were repeated three times. Positive-control DNA from refergrowing pigs, is clinically characterised by poor body condi- ence strains was included in each reaction. Negative-control tion, dyspnoea, pallor of the skin and occasionally icterus DNA extracted from spleen of a day-old gnotobiotic pig was (Allan and Ellis 2000). PMWS is being diagnosed with increas- also included in each reaction. ing frequency in swine herds in western Europe, North To evaluate the sensitivity of the PCR method in detecting America and east Asia (Ellis and others 1998, Kennedy and viral genomic DNA, PCV-2 and PPV primers were tested with others 1998, Allan and Ellis 2000, Choi and others 2000). 10-fold dilutions of DNA extracted from 100 [d of the purified Porcine circovirus type 2 (PCV-2) antigen and nucleic acid PCV-2 and PPV suspension (preadjusted to 106 viral partihave been detected consistently in the tissues of pigs with cles/ml, as determined by negatively stained electron PMwS (Allan and others 1998, Ellis and others 1998, Choi and microscopy), respectively. In the specificity studies, porcine Chae 1999, Choi and others 2000). The demonstration of Pcv- circovirus type 1 (Pcv-1), porcine reproductive and respiratory 2 antigen and nucleic acid, closely associated with lesions in syndrome virus (PRRSV), porcine epidemic diarrhoea virus a wide range of tissues from diseased pigs, has led to specu- (PEDV), transmissible gastroenteritis virus (TGEv) and porcine lation that PCV-2 may be an aetiological agent of PMws. respiratory coronavirus (PRCV) were tested independently However, there has been evidence of co-infection with PCV-2 with both sets of primers. and porcine parvovirus (Ppv) in experimental and natural PCR products were purified using a 30 kD cutoff memcases of PMWS (Allan and others 1999, Ellis and others 1999, brane ultrafiltration filter. The nucleotide sequences of the 2000, Choi and Chae 2000, Krakowka and others 2000). The purified PCR products were determined by use of BigDye aim of this study was to develop a multiplex PCR for the simul- chemistry with an ABI Prism Sequencer (Applied Biosystems). taneous detection of PCV-2 and PPV in clinical specimens from Sequencing was also performed in the purified PCR products pigs with PMws. from a positive control and a case which was positive by PCR Lymph node and spleen specimens from 53 50- to 70-day- but culture negative. old pigs with PMws from 53 farms were processed to attempt The oligonucleotide primers selected for amplification of virus isolation as described by Ouardani and others (1999). the ORF2 and VP2 gene for PCV-2 and PPv, respectively, were The selection of PMWS cases was based on the presence of analysed to ensure that they met the essential criteria for optigranulomatous inflammation with or without basophilic mal PCR primers, and they could be used together in a multiintracytoplasmic inclusion bodies, and the presence of PCV-2 plex reaction under similar conditions for amplification and nucleic acid within these lesions. The presence of PPV nucleic produce products which could clearly be differentiated on the acid and PCV-2 nucleic acid within the lesions was determined basis of their sizes. The optimal annealing temperature was by in situ hybridisation, as described by Choi and Chae (1999, found to be approximately 60°C. Annealing temperatures 2000). Pcv-free porcine kidney (PK)-15 cells were used to above or below the optimum frequently reduced the yield of isolate PCV-2 and PPv. The SNUVROO0463 strain and SNUVROO0464 the specific 547 base pair (bp) and 330 bp for PCV-2 and PPv, strain were used as standard strains for PCV-2 and PPv, respec- respectively, and increased the generation of nonspecific tively. amplification products (data not shown). The PCV-2 and A multiplex PCR assay was designed with primers based on primer sets, when used together in the multiplex reaction, the published sequences of the open reading frame 2 (ORF2) amplified only specific products of the expected sizes, which and viral protein 2 (VP2) gene from PCV-2 and Ppv, respectively could be easily distinguished by agarose gel electrophoresis (Ranz and others 1989, Hamel and others 1998). The speci- (Fig 1). ficities ofthese primers were checked against the GenBank. The A positive amplification reaction for PCV-2 and PPV was sequences and locations of the primers are given in Table 1. visualised with 10-6 dilution of the purified viral preparation, DNA was extracted from PCV-2- or Ppv-infected PK-15 cells corresponding to approximately 10 viral particles/ml. The and 25 mg of sample tissue using a commercial kit (Qiagen; nucleotide sequences for the PCR products of PCV-2 and PPV Santa Clarita) according to the manufacturer's instructions. were determined. Comparison of determined sequences with Samples (10 pl) of each of the supernatants containing the those of previous reports (Ranz and others 1989, Hamel and extracted DNA were used as PCR templates. Amplification was others 1998) revealed sequence homologies were identical in performed in 50 pl of reaction mixture containing 1 25mM both PCR products. Strain SNUVROO0463 of PCV-2 and strain magnesium chloride, 1 X PCR buffer, 02mM of each deoxyri- SNUVROO0464 of PPV reacted with the primers of PCV-2 and the bonucleoside-5'-triphosphate, I OOpM of each primer and 2-5 primers of PPv, respectively. The primers of PCV-2 did not U of Taq DNA polymerase. The amplifications were performed react with PPV and vice versa. There was no difference in specifor 35 cycles at 94°C for 30 seconds, 60°C for 30 seconds and ficity and sensitivity when single PCR methods for each virus 720C for one minute, followed by a 10 minute extension at was used. In addition, the primers of both PCV-2 and PPV did 720C. The amplified product was visualised by standard gel not react with other swine viruses (Pcv-i, PRRSV, PEDV, TGEV electrophoresis of 10 [d of the final reaction mixture on a two and PRCv). per cent agarose gel. Amplified DNA fragments of specific sizes Of the 53 samples, 12 were positive for both PCV-2 and were located by ultraviolet fluorescence after staining with PPV by in situ hybridisation and multiplex PCR. Among these 304
The Veterinary Record, September 8, 2001
Veterinary Record (2001) 149, 304-305 J. Kim, DVM, MS, C. Choi, DVM, MS, D. U. Han, DVM, MS, PhD, C. Chae, DVM, MS, PhD, Department of Veterinary Pathology, College of Veterinary Medicine and School of Agricultural Biotechnology, Seoul
National University, Suwon, 441-744, Republic of Korea
Correspondence to Dr Chae
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FIG 1: Detection of porcine circovirus type 2 (PCV-2) and porcine parvovirus (Ppv). Lane M 100 bp DNA ladder, Lane 1 Negative control, Lane 2 PCV-2 standard strain, Lane 3 PPV standard strain, Lane 4 PCV-2 and PPV standard strain, Lane 5 Pcv-2-positive sample, Lane 6 Ppvpositive sample, Lane 7 PCV-2- and PPv-positive sample
Ml1 23456 7 bp
500-
Virus isolation In situ hybridisation Multiplex PCR PCV-2 and PPV PCV-2 only PPV only
6 36
12 41
12 41
0
0
0
12 positive samples, six were culture negative for both PCVand PPv. Six multiplex PCR products which were culture negative for both viruses were sequenced, and their identity was confirmed as PCV-2 and PPV. Forty-one samples were positive for PCV-2 only by in situ hybridisation and multiplex PCR. Among these 41 positive samples, five were culture negative and sequenced, and their identity was confirmed as 2
(Table 2). The multiplex PCR assay, targeting the ORF2 and VP2 gene of PCV-2 and PPv, respectively, was very sensitive, detecting 100 per cent of all culture positive tissue samples. Multiplex PCR results were identical to those of in situ hybridisation; thus, sequencing could be a good system to confirm positive PCR results which are culture negative. By sequencing confirmation, 100 per cent specificity was accomplished compared with the results of virus isolation. Therefore, the multiplex PCR assay described here can also be used to directly diagnose PCV-2 and PPv infection from pig tissues without virus isolation. The bulk of the available evidence for both naturally occurring disease and the experimental data (Allan and others 1999, Choi and Chae 1999, Ellis and others 1999, 2000, Krakowka and others 2000) indicate that PCV-2 is the primary infectious viral agent causing PMWS. Twelve of 53 pigs with PMWS in the present study were co-infected with PPv. PPV has an in vivo tropism for monocytes and lymphocytes (Oraveerakul and others 1993, Choi and Chae 2000). PPV replicates in porcine immune cells and inhibits some of their functions (Harding and Molitor 1988). It is possible that PPvinduced immune dysfunction facilitates the replication of PCV-2. It cannot be ruled out that PPV may only be a concurrent infection with no impact on PMWS or may have some impact in only a few cases of PMWS. Further studies are required to clarify the interaction between PCV-2 and PPv in the pathogenesis of PMWS. PCV-2
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Notes for contributors
ACKNOWLEDGEMENTS
A FULL version of notes for contributors can be found on The Veterinary Record/In Practice website at:
The authors would like to thank Dr Keith West at Prarie Diagnostic Services, Saskatchewan, Canada, for the PK- 15 cells. This research was supported by the Ministry of Agriculture, Forestry and Fisheries Special Grants Research Program and Brain Korea Project, Republic of Korea.
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Downloaded from veterinaryrecord.bmj.com on April 18, 2012 - Published by group.bmj.com
Simultaneous detection of porcine circovirus type 2 and porcine parvovirus in pigs with PMWS by multiplex PCR J. Kim, C. Choi, D. U. Han, et al. Veterinary Record 2001 149: 304-305
doi: 10.1136/vr.149.10.304
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References
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