An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome

Abstract

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Reproduction of PMWS in immunostimulated SPF piglets transfected with infectious cloned genomic DNA of type 2 porcine circovirus

“…However, the seroconversion profile matched that observed in SPF pigs after infection with a crude viral isolate [6] thus indicating a successful production of viral antigens and the consequent response of the immune system. A high number of GECN per gram of tissue (3.7 × 10 11 GECN per gram) was found in the lymphoid organs (tracheobronchial, inguinal, mesenteric, axillary and iliac LN, tonsil, spleen and ileum) as well as many infectious particles (10 4.2 TCID 50 per gram) at 32 dpt.…”

“…The PCV2 antibodies in the weekly-collected sera were detected and titrated using an ELISA test based on the recognition of a recombinant PCV2 capsid protein/GST fused protein and a GST protein [6]. Those samples with an OD ratio higher than 1.5 were considered positive for PCV2 antibodies.…”

Quantification of porcine circovirus type 2 (PCV-2) within- and between-pen transmission in pigs

“…Pigs were declared as infected as soon as PCV-2 genome load was detected in the serum. In addition, PCV-2 antibodies were monitored weekly with an ELISA test [5].…”

A double-antibody sandwich ELISA based on the porcine circovirus type 2 (PCV2) propagated in cell culture for antibody detection

“…However, the quantification of antibodies is very useful for studies on PCV2 infection dynamics and its relationship with PMWS, in the association with protection against PCV2 infection, in serological surveys, in the assessing the effectiveness of vaccines in development, in the adoption of vaccination programs analysing the level and decline of passive immunity, in the evaluation of the animal response to vaccination and as an indicator of viral exposure and circulation on farms (Nawagitgul et al 2002, Blanchard et al 2003, Gerber et al 2009.…”

“…For the detection of antibodies against PCV2, the systems of antigen production in Escherichia coli (Shang et al 2008, Marcekova et al 2009, Sun et al 2010, Ge et al 2012, insect cells (Nawagitgul et al 2002, Blanchard et al 2003, Liu et al 2004) and insect larvae (Pérez-Martín et al 2008) exhibit more restricted access to techniques when compared with traditional antigen production in cell culture. However, these systems exhibit high purified protein yields (Nawagitgul et al 2002, Spencer et al 2007), which could be used in indirect ELISA methods.…”