Molecular techniques in the study and control of porcine circovirus type 2
Porcine circovirus type 2 (PCV2) is an emerging virus that may result in devastating disease that affects swine herds worldwide. Only a decade has passed since researchers began to study the characteristics of the virus and the resulting diseases, and limited information was available regarding the long term presence or persistence of the virus in healthy swine herds. Our research contributes to the knowledge of the persistence of the virus in serum and semen, and the PCV2 antibody profile in healthy pigs. In addition, we developed a novel quantitative polymerase chain reaction (PCR) assay and quantified the PCV2-shedding in healthy and PCVD (porcine circovirus disease)-affected pigs. Lastly, we determined the efficacy of a novel vaccine in a subset of pigs from a PCVD-affected herd. Porcine serum was assayed by two PCR protocols (nested polymerase chain reaction (nPCR) and non-nested PCR) and a competitive enzyme-linked immunosorbent assay (cELISA) to determine when PCV2 viremia and a rise in the serum level of PCV2-specific antibody (Ab) occurred in pigs raised in a large Canadian farrow-to-finish barn. Eight serial blood samples were collected from each of 40 pigs from 5 to 156 (±1.5) days of age; six pigs were removed from the study for various reasons at various times. Viremia was not detected in the samples collected before 72 days of age but was detected in those collected on or after 72 days: of 33 pigs, seven (21%) had only one serum sample positive for PCV2 deoxyribonucleic acid (DNA) by nPCR after day 72; 11 (33%) were intermittently positive by nPCR, non-nested PCR, or both between 72 and 156 days; and the remaining 15 (45%) were repeatedly positive (in two to four samples). The level of serum Ab against PCV2 declined after weaning and increased between 72 and 107 days of age, only after PCV2 was detected in serum. Our results show that PCV2 viremia persists in the presence of elevated levels of PCV2-specific Ab. In a separate study, we determined the long term presence or persistence of PCV2-shedding in semen from healthy boars and the effects of PCV2 on sperm quality. A nPCR protocol was applied to porcine semen to demonstrate the PCV2-shedding patterns and duration in naturally-infected boars. Sperm morphology analysis was performed on a subset of samples to determine if the presence of PCV2 DNA in semen was associated with reduced semen quality. Semen was collected serially from 43 boars representing six breeds, aged 33.9 to 149.3 weeks. Of the 903 semen samples collected, 30 samples (3.3%) were positive for PCV2 DNA by nPCR from 13 boars. Boars shedding PCV2 DNA in semen ranged between 35.9 and 71.0 weeks of age, and shedding occurred over a period of up to 27.3 weeks. A semen nPCR test was 2.6 times more likely to be positive when collected from pigs that were ≤52 weeks of age and 3.0 times more likely to be positive when collected from pigs that were ≤26 weeks from the time of entry into the stud main unit (Generalized Estimating Equations: P=0.02; 95% confidence interval (CI) of the Odd’s ratio (OR) 1.2 to 5.5 and P=0.01; 95% CI of the OR 1.3 to 6.9, respectively). PCV2 DNA was detected in semen from Duroc and Landrace boars only; however, the semen of the Hamline, Large White maternal, Large White paternal, and Meishan-synthetic boars were negative for PCV2 DNA. These results demonstrate a sporadic and long-term shedding pattern of PCV2 DNA in semen from naturally-infected boars. PCV2 DNA in semen did not have detrimental effects on sperm morphology; however, boar age and possibly breed may contribute to the persistence of PCV2-shedding in semen. To further our studies of PCV2-shedding from pigs, a PCV2 quantitative molecular assay was developed using SYBR green technology. This assay allowed for the simultaneous quantification of all the genotypes of PCV2. The emergence of multiple genotypes of PCV2, as demonstrated by phylogenetic analysis of whole genome or capsid sequences, makes it necessary to have quantitative diagnostic assays that perform equally well on all strains. The objectives of this study were to develop and validate a novel real-time PCR assay targeting the highly conserved replication-associated gene (Rep) open reading frame 1 (ORF1) and investigate the effects of diagnostic specimen choice on its performance. The assay was tested in naturally-infected conventional pigs, experimentally-infected gnotobiotic pigs, and plasmid-spiked negative serum, lung tissue, and feces and found to have a linear detection range of 2.2 × 10³ to 2.2 × 10¹⁰ copies of PCV2 per mL. The assay successfully detected and quantified PCV2 DNA in serum, buffy coat, feces, and multiple lymphoid (bronchial, mesenteric, and superficial inguinal lymph nodes, thymus, tonsil, ileal Peyer’s patches, and spleen) and non-lymphoid (myocardium, lung, kidney, liver, and gluteal muscle) tissues from naturally-infected pigs. Across all tissues and sera of naturally-infected pigs, the mean PCV2 concentration was 3.0 logs higher in pigs that were demonstrating weight loss, as a primary clinical sign of PMWS (postweaning multisystemic wasting syndrome) when compared with PMWS-nonaffected pigs. PCV2 concentration measured by tissue culture and immunohistochemical staining in homogenized liver samples of experimentally-infected gnotobiotic pigs were compared to the concentrations estimated by quantitative PCR. Similar trends were noted with increasing PCV2 concentration detected in subclinically-infected to severely PMWS-affected pigs across all assays. Our diagnostic assay was developed with a conserved target sequence, and performed efficiently in the quantification of PCV2 in a variety of tissues from naturally- and experimentally-infected pigs. With a quantitative assay, we then determined the amount of PCV2-shedding in feces between healthy and PCVD-affected herds. This study examined if pigs (n=100) in a PCVD-affected herd shed more PCV2 in their feces than pigs in a PCVD-nonaffected herd (n=101), and if differences in shedding among production stages within and between the herds existed. The PCV2-shedding was quantified by real-time PCR. The highest median PCV2-shedding was found in the nursery stage of the PCVD-affected herd and in the grower stage of the PCVD-nonaffected herd. The PCV2-shedding was significantly higher in earlier stages (newly weaned, nursery, and pregrower) in the PCVD-affected herd (Wilcoxon Rank Sum; P<0.001) compared with the PCVD-nonaffected herd. PCV2 DNA was not detected in a significant proportion of lactating sows (parity ≥3) in the PCVD-nonaffected herd (Fisher’s Exact Test; P=0.001) compared with the PCVD-affected herd. The results of this study suggest there may be an association between the presence of PCV2 in the feces of lactating sows and increased PCV2-shedding in younger pigs. The quantitative PCR assay was used in a field study to determine the efficacy of a novel transdermal immune stimulating complex (ISCOM) technology-based PCV2 virus-like particle (VLP) vaccine candidate administered at one and three weeks of age. Fifty-four pigs (vaccinates (VX) (n=27); non-vaccinated controls (CTRL) (n=27)) with variable levels of maternally-derived antibodies (MDAb) (high (hiMDAb) versus low (loMDAb)) in a PCVD-affected farm were assessed for PCV2-specific Ab levels by cELISA, PCV2 DNA concentration in serum and feces by quantitative PCR, mortality, and average daily gain (ADG) from 1 to 18 weeks of age. A significant reduction in mortality (P=0.05) was observed in the VX. PCV2 DNA concentration in serum was lower in the VX at 9 and 11 weeks of age (P=0.003 and 0.01, respectively). The VX (both hiMDAb and loMDAb groups) at nine weeks of age had a higher Ab response to natural PCV2 exposure, while the CTRL (both hiMDAb and loMDAb groups) had a significantly reduced Ab response in comparison. This suggests a priming of the immune response against PCV2 infection by use of the vaccine. During this period from 9 to 11 weeks of age, the loMDAb CTRL were susceptible to PCVD. PCV2-specific Ab in serum was significantly higher in the VX at the onset of PCVD-related mortality (P=0.001); however, no significant difference was observed in the PCV2 DNA concentration shed in feces, or in ADG between VX and CTRL. The benefit of the novel ISCOM vaccine was most evident in the loMDAb VX, as these pigs were protected from a PCVD-related death that was otherwise experienced in the loMDAb CTRL. However, producers and veterinarians using a vaccine against PCV2 would expect a significant improvement in ADG and reduced viral shedding in feces. Although the vaccine prevented a PCVD-related death in the highest risk population, the loMDAb group, modifications regarding the protein or ISCOM concentration, or PCV2 protein construction should be considered to potentially improve the vaccine’s efficacy. This study is the first report of the use of an ISCOM matrix (Matrix Q) mixed with PCV2 VLP protein administered transdermally for the prevention of PCVD in swine.
polymerase chain reaction (PCR), porcine circovirus type 2 (PCV2), porcine circovirus disease (PCVD), immune stimulating complex (ISCOM), swine.
Doctor of Philosophy (Ph.D.)