A high prevalence of PCV2 and PRRSV co-infection is frequently observed in PRDC-affected pigs in many pig producing countries, including Taiwan [3, 10, 21, 22]. The present study was conducted to study co-infection of PCV2 and PRRSV in swine AMs in the pathogenesis of PRDC by mimicking possible infection conditions/situations in the field [23, 24]. Similar to our previous study , PCV2 was easily internalized in the cytoplasm of AMs but caused no noticeable cell death; and PRRSV displayed a low infectious rate but severe cytopathic effect and strong TNF-α induction in AMs. PCV2-induced IFN-α likely caused reduction in PRRSV infectious rate and PRRSV-related AMs dysfunction when AMs were co-inoculated with PCV2 and PRRSV simultaneously [14, 18]. Similar PCV2-induced IFN-α effects were seen in the PCV2/PRRSV group but not in the PRRSV/PCV2 group, despite that a significant amount of IFN-α was also induced in PRRSV/PCV2 group, indicating that the pre-existing or co-existing PCV2 could interfere or hinder, at least partially, PRRSV infection. Our previous [14, 18] and present studies have demonstrated that a significant amount of PRRSV antigens and PRRSV-induced cell death and dysfunctions in AMs could be detected within the first 18 HPI when inoculation of AMs with PRRSV. Those findings indicate that PRRSV replication and PRRSV-induced dysfunction and cytokine production in AMs should have occurred during the first 18 HPI after inoculation of AMs with PRRSV. Thus, it is reasonable to suggest that as long as PRRSV has established its infection in AMs prior to PCV2 inoculation or infection, the subsequent IFN-α production induced by infection with PCV2 later is incapable of curtailing those adverse effects caused by previous or pre-existing PRRSV infection.
In the study reported by Buddaert et al. , pigs infected with porcine respiratory coronavirus virus (PRCV) 2 days prior to infection with PRRSV showed a reduction in PRRSV titer but had no change in PRCV-induced IFN-α production. Similarly, no interference with PCV2-induced IFN-α production was seen in all groups with dual infection of PCV2 and PRRSV in the present study. On the contrary, the study of Albina et al.  showed that swine AMs pre-infected with PRRSV 6 h prior to the inoculation of swine transmissible gastroenteritis virus (TGEV) in vitro resulted in a complete inhibition in TGEV-induced IFN-α production. The above mentioned results indicate that the interactions among different viruses are complicated and may lead to different disease courses in the field.
Simultaneous infection of PCV2 and PRRSV is frequently encountered in pig herds worldwide [10, 27]. Various field and in vivo and in vitro experimental studies have been conducted to evaluate the effects of co-infection of PCV2 and PRRSV [9, 10, 12–14, 27, 28]. Simultaneous inoculation with PRRSV and PCV2 viruses [9, 10], or PRRSV inoculated one week prior to PCV2 , was commonly used in those in vivo studies. Results from these field and experimental studies have demonstrated that PRRSV could cause enhanced PCV2 replication evidenced by higher serum and tissue PCV2 loads, increased severity of the pathological changes and clinical manifestation of PCVAD, and higher incidence of PCVAD [9, 10, 27, 28]. Our previous study has demonstrated that PCV2 cannot efficiently replicate in AMs unless being activated such as by lipopolysaccharide in vitro, and this may explain why, in contrast to the above mentioned in vivo studies, no significant changes in PCV2 antigen-positive rate were seen in the present study. Owing to that no in vivo study with PCV2 inoculation first followed by PRRSV has been reported, it would be of interest to see whether those alterations seen in PCV2/PRRSV group in the present study can be reproduced in vivo.
Similar to the findings of our previous studies [15, 18], transient decrease in phagocytosis but persistent reduction in microbicidal capability in the group inoculated with PCV2 alone and constant decrease in phagocytosis and microbicidal capability in all PRRSV-inoculated groups were noted in the present study. The significantly higher PRs of AMs in the PCV2/PRRSV and PCV2-PRRSV groups than those in the PRRSV group during 90 to 108 HPI indicate that PCV2 that was inoculated first or simultaneously not only hinder PRRSV replication but also reduce the PRRSV-induced adverse effects on the phagocytosis of swine AMs. Although with some variations, the constant and significant reduction in the capability of swine AMs to kill C. albicans in all PCV2- and/or PRRSV-inoculated groups suggests that PCV2 and/or PRRSV infection may lead to the survival and proliferation of the opportunistic or secondary pathogens and lesion development in pig lungs. This is in agreement with the findings in PRDC-affected pigs in the field [4, 30]. As suggested by the previous studies [15, 18], the impaired microbicidal capability in PCV2- and/or PRRSV-inoculated groups may be partially due to the reduction in reactive oxygen species production of swine AMs.
PCV2 can be further subdivided into two main subtypes, PCV2a and PCV2b . Although PCV2a has been considered as less virulent compared to PCV2b based on field observation , no experimental studies have been able to support this speculation by using PCV2 infection alone  or PCV2 and PRRSV co-infection . Our preliminary study with PCV2a also showed no difference from PCV2b on the reduction of the phagocytotic and microbial killing capabilities of AMs in vitro (unpublished data).
Fas (CD95)/Fas ligand (FasL) has been shown to play a major role in the induction of apoptosis in immune cells and bronchial epithelial cells [35–37] and mediation of neutrophil chemotaxis [38–40]. The ability of FasL to induce acute inflammatory response is even stronger than that of bacterial lipopolysaccharide (LPS) . The FasL expressed by AMs has also been suggested to participate in the pathogenesis of acute respiratory disease syndrome (ARDS) . To further elucidate the possible mechanism of pulmonary inflammatory response and tissue injury during PCV2 and/or PRRSV infection, Fas and FasL expressions were also analyzed in the present study. The results of FasL mRNA expression clearly showed that PCV2 but not PRRSV could stimulate swine AMs to produce FasL; however, PRRSV had the addictive effect on PCV2-related FasL mRNA expression in swine AMs when PRRSV co-existed with PCV2. Comparing with PCV2/PRRSV or PRRSV/PCV2 group, a significantly higher level of FasL mRNA expression was seen in the PCV2-PRRSV group. At 42 HPI, the duration for co-existence of PCV2 and PRRSV in the PCV2/PRRSV or PRRSV/PCV2 group was 18 h less than that in the PCV2-PRRSVgroup. The result suggests that the addictive effect of PRRSV on the enhancement of FasL mRNA expression by PCV2 corresponds with the duration for co-existence and interaction of PCV2 and PRRSV. A significant increase in FasL mRNA expression with no detectable Fas mRNA expression in all PCV2- and/or PRRSV- inoculated AMs suggests that Fas/FasL may not be directly involved in the apoptosis and other cytopathologies in the PCV2- and/or PRRSV-inoculated AMs.
Pigs naturally infected with PCV2 and/or PRRSV frequently show interstitial pneumonia [10, 42]. Inoculation of PCV2 or PRRSV alone has been shown to cause mild to moderate interstitial pneumonia in conventional pigs; however, severe interstitial pneumonia with occasional peribronchiolar mononuclear cell cuffing and scattered individual bronchiolar epithelial cellular necrosis could be induced in Cesarean-derived colostrum-deprived pigs with dual infection of PCV2 and PRRSV [9, 30]. Interferon-α activation in cellular immunity has been suggested to complicate interstitial pneumonia . Tumor necrosis factor-α can induce strong proinflammatory response, including the release of inflammatory mediators and chemokines . Interleukin 8 is an acute inflammatory chemokine for neutrophils and also considered as a key factor in the pathogenesis of interstitial pneumonia . In addition, FasL has been suggested to be associated with the process of pulmonary inflammation and vascular permeability and the induction of epithelial apoptosis when it ligates with the airway epithelial cells expressing Fas [39, 41]. Taken together, the increased expression of IFN-α, TNF-α, IL-8, and FasL mRNA in AMs from pigs in various infection orders by PCV2 and PRRSV observed in the present study, to some extent may contribute to pneumonia and bronchiolar epithelial damage in the lungs of PCV2- and/or PRRSV-infected pigs.