Genetic characterization of porcine circovirus type 2 in piglets from PMWS-affected and -negative farms in Thailand
© Jantafong et al. 2011
Received: 3 December 2010
Accepted: 28 February 2011
Published: 28 February 2011
Porcine circovirus type 2 (PCV2) is the major swine pathogen associated with Porcine circovirus associated disease (PCVAD) including post-weaning multisystemic wasting syndrome (PMWS). Currently, there are 4 subtypes of PCV2 (PCV2a, b, c and d) and some epidemiological evidences demonstrated that virulence of PCV2 may relate to its subtypes. Recently, PMWS was observed more frequently in swine farms in Thailand; however, the information regarding to PCV2 subtype involved was limited. Therefore, this study was aimed to determine the association between occurrence of PMWS and PCV2 subtypes as well as genetically characterize PCV2 in Thailand. PCV2 DNA was isolated from faecal swabs and whole blood of piglets from PMWS-affected and -negative farms. The full length ORF2 sequences were compared using multiple alignment. The results showed that PCV2 DNA was detected more frequently in PMWS-affected farms. The nucleotide identities of the ORF2 from 9 PCV2 isolates representing each PMWS-affected farm and one from the negative farm ranged from 92.4 to 99.5% suggesting that there is some genetic variation of PCV2 in Thai swine. The 10 PCV2 isolates were classified into 2 clusters, in which the 7 isolates from PMWS-positive farms were in PCV2b cluster 1 A/B. The remaining isolates were separated in the new subtype called PCV2e. The results suggest the presence of new PCV2 subtypes in addition to PCV2a and PCV2b in Asian swine population. However, correlation between subtypes and virulence of PCV2 infection is not conclusive due to limited number of the PCV2 sequences from PMWS negative farms.
Postweaning multisystemic wasting syndrome (PMWS) is a major disease of swine, caused by porcine circovirus type 2 (PCV2). The major clinical signs of PMWS are wasting, paleness of the skin, enlargement of the lymph nodes, respiratory distress, and occasional diarrhea as well as icterus [1, 2]. PCV2 has a significant impact on the pig industry worldwide . PCV2 is a small non-enveloped DNA virus which contains a single-stranded circular genome of 1.7 kb [4, 5]. The PCV2 genome has three major open reading frames (ORFs), which encode replication-associated proteins (ORF1) , viral capsid protein (ORF2) , and apoptotic protein (ORF3) . The ORF2 is an essential determinant for the genetic typing of PCV2 isolates, since the capsid gene has more variability than the other ORFs . Phylogenetic analysis demonstrated two major subtypes of PCV2, which are PCV2a (Group2) and PCV2b (Group 1). Each subtype was classified into different clusters, 1A to 1C for the subtype PCV2b, and 2A to 2E for the subtype PCV2a . Furthermore, a third subtype (PCV2c), found only in the Danish pigs, was described . A novel subtype, PCV2d, which is dominant in Chinese pigs, was recently reported . A number of studies suggested that PCV2b is more virulent than PCV2a and predominant in PMWS-affected herds [12, 13]. However, an experimental inoculation of pigs with PCV2a and PCV2b demonstrated no divergence in the virulence of both subtypes . Thus, no association of the PCV2 subtype designations, the disease status, or the geographical distribution appears to be evident.
Since PMWS was first observed in Canada in 1991 , the prevalence of PCV2 and the occurrence of PMWS have been worldwide reported [15–25]. The first case of PMWS in 7 to 9 week-old pigs was described in Thailand in 1998 ; however, a retrospective study suggested that the PCV2 infection had been previously detected in 1993 . Although the infection has been widespread in Thailand since 1998, genetic information of PCV2 is limited with only one sequence (AY864814) deposited in the GenBank databases. Therefore, the aim of this study was to genetically characterize the PCV2 isolates from Thai swine and to determine the prevalence of the PCV2 infection in piglet from both positively and negatively PMWS affected farms.
Total DNA was extracted from the plasma and the suspension of faecal swabs with the application of a Nucleospin® Blood kit (Macherey-Nagel, Germany) in accordance with the manufacturer's instructions. The isolated DNA was amplified by means of PCR with the PCV2 specific primers . Descriptive statistics, with the employment of the NCSS 2007 program, were utilized to assess the overall data quality. Associations between the percentages of the PCV2 positive in PMWS-affected and PMWS-negative farms and the manifestation of PCV2 DNA in faecal swabs as well as in the whole blood samples were analyzed with the chi-square test. A P value of less than 0.05 was regarded as statistically significant. A positive PCR sample was selected from each farm for sequence analysis.
PCR amplification of PCV2 DNA from whole blood and faecal swab samples
Number of pigs in farms
In addition, the full length of the ORF2 of PCV2 was amplified with the PCV2-ORF2 specific primers [7, 30]. The PCR products were submitted for sequencing. The 10 ORF2 sequences (nine from PMWS-affected pigs and one from a negative pig) from the present study and 82 ORF2 sequences from GenBank were analyzed with the application of the ClustalW method provided in the DNASTAR software. The ORF2 gene sequence of PCV1 (AY699796) was used as the out-group. The phylogenetic tree was built by means of the neighbour-joining (NJ) method in MEGA 4 program with 1000 bootstrap replicates.
Classification of PCV2 strains based on nucleotides 262-267 of the ORF2 sequences
In conclusion, the present study constitutes the first genetic analysis of the Thai PCV2 isolates, which determines the correlation of the occurrence of PMWS and the PCV2 subtypes in the Thailand. This study also demonstrated that PCV2 infection associates with the occurrence of PMWS. In addition, the circulation of the PCV2 strains appears to be currently increasing in the Thai swine population. Two subtypes, PCV2e and PCV2b (1A/B), are at present existent in the Thai swine population, where PCV2b appears to be dominant. Phylogenetic analysis of the recent PCV2 strains from the present study has confirmed the existence of more PCV2 subtypes in addition to PCV2a and PCV2b. As new genetic clusters of PCV2 have continually emerged, the classification of the PCV2 subtypes and clusters needs to be reconsidered by the PCV2 taxonomists as to set standard criteria for the PCV2 genetic characterization.
This study was funded by the Charoen-Pokphand Foundation, Thailand. The authors greatly thank Dr. Sandro Kunz, English editor at Kasetsart University, Thailand, for final manuscript edition.
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