Among 97 diagnostic submissions from field cases collected in the USA, PCV2a/b co-infection was demonstrated by PCR in 25% of the clinical samples [18
]. We observed that PCV2 infection resulted in an anti-PCV2 IgM response followed by a strong anti-PCV2 IgG response in serum and a high prevalence of long-term PCV2 viremia. Individual PCV2 DNA-negative pigs were found throughout the study and some of those pigs (2/18) remained PCV2 DNA negative for up to 7 consecutive bleedings; however, at termination of the study, 13 of 18 PCV2 inoculated pigs were viremic. The observed intermittent PCV2 viremia pattern may be due to differences in replication kinetics, differences in replication sites, stressors, host susceptibility, or re-inoculation. Alternatively, it may be due to the detection limit of the PCR used. Samples were considered positive when the C
value was less than 40. Samples were considered negative when there was no observed C
value during the 40 amplification cycles. However, false positive results may arise due to overlap with background noise and accurate discrimination between positive and negative animals at high C
values may not always be possible.
Persistent chronic infection is defined as establishment of equilibrium between the virus and the host’s immune system, resulting in an infection of long duration. The present study demonstrated persistence of PCV2 through the 140 day observation period in conventional pigs. Previously, Bolin et al. [4
] described detection of viremia for 125 dpi in cesarean-derived, colostrum-deprived pigs inoculated at 3 weeks of age. PCV2 was also successfully isolated from several tissues obtained from the pigs on 125 dpi. The authors concluded that the extended duration, after experimental inoculation with PCV2, and isolation of infectious virus from multiple tissues indicated the establishment of a persistent infection [4
]. Likewise, in a study investigating persistence in sows with subsequent vertical transmission to piglets, PCV2 antigen was detected in lymphoid tissues, lungs, myocardium, and epithelial cells of the pinna of piglets born to sows inseminated with PCV2-spiked semen [26
The predominate genotype of PCV2 identified in the group with repeated heterologous PCV2 challenges (R-PCV2a/b) was the PCV2a used for the initial infection. PCV2b was only detected in two pigs on the day of necropsy. This is similar to what has been previously described [38
] and further confirms that PCV2 isolates are cross-protective and prior infection with PCV2a prevents or limits re-infection with a heterologous PCV2b strain. However, timing of concurrent or re-infection likely varies greatly in the field and may influence the development of viremia. In this study, re-infection occurred 5 weeks after the initial infection.
Although the anti-PCV2 antibodies present in the pigs were shown to have neutralizing capabilities, this was not correlated with a reduction of PCV2 viremia (correlation coefficient of 0.1368 on dpi 21 and 0.4012 on dpi 140). This is in contrast to what has been reported previously where neutralizing antibodies were found to be correlated with protection against replication and development of disease [28
]. Gnotobiotic or SPF pigs with high levels of neutralizing antibodies did not have detectable PCV2 replication in inguinal lymph nodes. However, the differences in the pig source (gnotobiotics or SPF pigs versus conventional pigs), age of the pigs at inoculation (3 weeks of age versus 11 weeks of age), sampling protocols (lymph nodes biopsies versus serum), differences in the assays used to measure neutralizing antibodies, and study outcomes (clinically affected pigs versus healthy pigs) between the studies were considerable and directly comparing the results is problematic. In addition, while in the previous studies the pigs were monitored until dpi 21 [28
] when neutralizing anti-PCV2 antibodies just start to appear in serum, in the current experiment the pigs were monitored for 140 days post initial infection. Also, virus isolation titration on lymph nodes was used to determine presence of replicating PCV2 [28
]. In the current study, we determined the amount of PCV2 DNA in serum by quantitative real-time PCR. The PCR assay used [32
] does not discriminate between viable and/or replicating PCV2 and non-viable PCV2 or DNA remnants. In addition, it needs to be considered that some of animals that were PCR positive had values close to the PCR cutoff and the biological significance of a low viremia level and the correlation with neutralizing antibody levels is unknown. However, PCV2 antigen was demonstrated by IHC in lymphoid tissues collected from pigs infected 140 days previously (Fig. 5). Others have estimated that a minimum viral load of 108
PCV2 genomes per 500 ng DNA is required in order to give visible IHC staining [5
]. A high tissue level of PCV2 140 dpi despite the presence of high levels of anti-PCV2 IgG suggests that replicating PCV2 was still present locally. In addition, more recently Fort et al. [15
] reported on co-existence of neutralizing antibodies and viral DNA in serum samples obtained from both clinically affected and non-affected pigs which reinforces that neutralizing antibodies alone are not sufficient for PCV2 clearance.
The results of the current study suggest that PCV2a and PCV2b co-infection administered 35 days apart is not sufficient to induce clinical disease in a conventional pig model. However, experimental infection of conventional SPF pigs with PCV2 results in persistent viral infection despite the presence of high levels of anti-PCV2-antibodies. A similar pattern was not only seen in re-infected animals but also in the PCV2a group which was inoculated only once 140 days prior to termination of the study.
All current commercially available PCV2 vaccines are killed products [36
] and have been shown to reduce the level and length of PCV2 viremia in experimental models [16
] and under field conditions [10
]. Reduction or elimination of PCV2 viremia after challenge was not observed in this study. Reasons for this may be that the initial viral infection results in persistence of the virus in cells of the immune system which may potentially interfere with antigen recognition or recall responses [22
]. Killed vaccine products, on the other hand, apparently are sufficient to induce a protective sterilizing immune response against infections with live PCV2 when the vaccine is given prior to virus challenge. The efficacy of PCV2 vaccines in PCV2 persistently infected pigs is currently unknown and needs to be further investigated.