Innate pro-inflammatory and adaptive immune cytokines in PBMC of vaccinated and unvaccinated pigs naturally exposed to porcine circovirus type 2 (PCV2) infection vary with the occurrence of the disease and the viral burden

Abstract

Pro-inflammatory (IL-8, TNF-α, IL-1β) and immune (IFN-γ, IL-10) cytokines were evaluated in PCV2-vaccinated and unvaccinated pigs exposed to natural PCV2 infection retrospectively selected according to the time of the onset of viremia and the viral burden, and the presence of PMWS clinical signs. In a farrow-to-finish herd with a history of PMWS in animals aged older than 15 weeks, at weaning (21 ± 3 days of age), vaccinated pigs were intramuscularly inoculated with one dose of Porcilis^® PCV vaccine + adjuvant whereas the adjuvant alone was administered to the control animals. Thirty animals bled at 16 weeks of age (before the occurrence of the natural infection and the onset of the disease) and then at 19, 20, 22 and 26 weeks of age, were categorized as: (a) vaccinated non-infected and non-PMWS-affected (PCV2-vac), (b) unvaccinated spontaneously infected/non-PMWS-affected (Ctrl) and (c) unvaccinated spontaneously infected/PMWS-affected (Ctrl-PMWS+) pigs.

A major evidence of this study is that PMWS-affected animals were not able to mount an efficient innate pro-inflammatory response to cope with PCV2 infection as demonstrated by the low levels of pro-inflammatory cytokines, namely IL-8, TNF-α and IL-1β, and IFN-γ. Conversely, significantly increased gene expression levels of IL-8, TNF-α and IL-1β were detected especially in the PCV2-vac group at the early phase of the infection.

Moreover, in PMWS diseased animals, a significant increase of IL-10 occurred at the early phase of infection, while, vaccinated pigs, in addition to the low viremia burden and its frequency and the absence of PMWS disease, showed a more stable IFN-γ response.

Introduction

Porcine circovirus type 2 (PCV2) is a single-stranded DNA virus, member of the family Circoviridae, that has been identified as the etiologic agent of the Postweaning Multisystemic Wasting Syndrome (PMWS) (Allan et al., 1998, Ellis et al., 1998), that is widely spread in swine farms and represents one of several Porcine Circovirus Associated Diseases (PCVD). The clinical signs of this syndrome include weight loss, severe growth retardation and death in weaned piglets. PMWS is also characterized by lymphocyte depletion, monocytic infiltration in lymphoid tissues and a large amount of viruses in these affected tissues (Segalés and Domingo, 2002). The most important strategy for preventing and controlling PCV2-associated diseases, apart from farm management, is vaccination of piglets or sows and gilts, which has been shown to provide a beneficial effect on the reproductive performance (Thacker et al., 2008).

There are currently three vaccines for piglets from 3 to 4 weeks of age available: one is based on a chimeric inactivated PCV1-2 virus with the immunogenic capsid gene of PCV2 cloned into the backbone of the non-pathogenic PCV type 1 (Fenaux et al., 2004), while the other two are based on the PCV2 Cap protein expressed in a baculovirus system (Fachinger et al., 2008). The effectiveness of these commercial vaccines has been demonstrated, both experimentally and under field conditions, to include reduced mortality rates, significantly increased average daily weight gain (ADWG) and reduction of disease and lesions associated with PCV2. A decreased frequency of co-infections has also been reported in herds affected with PMWS (Blanchard et al., 2003, Fenaux et al., 2004, Cline et al., 2008, Fachinger et al., 2008, Opriessnig et al., 2008a, Opriessnig et al., 2008b, Thacker et al., 2008, Fort et al., 2009a, Fort et al., 2009b, Pérez-Martin et al., 2010).

Vaccine efficacy is associated with the seroconversion against PCV2 (virus-specific neutralizing antibody arrival) and the reduction of viral replication. An increasing number of trials, under both experimental and field conditions, have shown that a cell-mediated immune response is involved in the control of PCV2 infection and the evolution of associated pathology (Fenaux et al., 2004, Kixmoller et al., 2008, Fort et al., 2009a, Fort et al., 2009b, Steiner et al., 2009, Pérez-Martin et al., 2010, Martelli et al., 2011).

The study of cytokines can be important to classify the host immune responses that occur during viral infections and can be useful for the evaluation of immune efficiency. In fact, for successful resolution of infection, efficient activation of innate/inflammatory and acquired immunity is required to block pathogen replication and invasion, as well as to promote tissue clearance of the pathogens and/or infected cells.

The production of pro-inflammatory cytokines (IL-1β, TNF-α, IL-8) and the balance between pro-immune (IFN-γ) and regulatory (IL-10) cytokines play a pivotal role in eliciting the innate response as well as in priming and coordinating the adaptive immune response. However, if production is impaired, the innate and adaptive responses are delayed and inefficient in clearing the viral pathogen (Darwich et al., 2003a, Stevenson et al., 2006, Petry et al., 2007, Borghetti et al., 2010, Lunney et al., 2010, Zhang et al., 2010, Chae and Choi, 2011).

The production of pro-inflammatory and immune cytokines has been recently investigated in different PMWS infection models but frequently results have appeared to be ambiguous perhaps due to the variability of the study conditions; particularly, few data are available on the modulation of these critical inflammatory and immune mediators under field conditions (Kekarainen et al., 2008a, Kekarainen et al., 2010, Shi et al., 2010, Chae and Choi, 2011).

In a previous study (Martelli et al., 2011), we reported the immunological response and the clinical evolution of naturally occurring infection by a PCV2 field strain in vaccinated and unvaccinated pigs. The results showed that vaccination conferred clinical protection and allowed the assessment of vaccination efficacy in terms of clinical protection. On this basis, the present study aims at investigating innate pro-inflammatory (IL-8, TNF-α and IL-1β) and adaptive immune (IFN-γ and IL-10) cytokines in PBMC from pigs vaccinated with one dose of a PCV2 subunit vaccine based on the PCV2 Cap protein expressed in a baculovirus system compared to unvaccinated pigs, to evaluate their role as markers of immune efficiency during PCV2 natural infection.