Resumen de Development of new methodologies for the study of pathogenesis and control of porcine circovirus type 2 (pcv2) infection
Porcine circovirus type 2 (PCV2) is the etiologic agent of postweaning multisystemic wasting syndrome (PMWS), a disease that affects nursery and growing pigs causing significant economical losses in pig producer countries. PMWS is clinically characterized by progressive weight loss and wasting, fever, paleness of the skin, respiratory distress and digestive alterations. Typical histopathological findings of PMWS affected pigs are lymphocyte depletion with histiocytic infiltration in lymphoid tissues, correlating with immunosuppression of the animal and the occurrence of secondary opportunistic co-infections.
The difficulty to experimentally reproduce PMWS with PCV2 alone, together with the fact that only a small proportion of PCV2-infected pigs develop PMWS in the field, complicate the study of PMWS pathogenesis and many aspects, such as the cells where PCV2 replicates in vivo, remain not clearly elucidated. Moreover, the high prevalence of PCV2 infection and PCV2 antibodies in pig farms (in some cases close to 100%) difficults PMWS diagnosis, today relying in post mortem histopathological studies. The ubiquitous nature of PCV2 also caused a delay in the design and implementation of vaccines as a control measure. Only recently this strategy has been successfully implemented in the field with very promising results; thus, opening expectations even about the eradication of PMWS in a near future.
The main purpose of this Thesis has been to develop new methodological tools useful for studying in more detail, some relevant aspects of PMWS pathogenesis and control. The results of this Thesis have been reported in three studies, each one describing the development and use of a single methodology.
In a first study a new oligonucleotide probe was designed to specifically label the replicating form of PCV2 (dsDNA), thus allowing the characterization of those cells supporting PCV2 replication in tissues from PMWS affected animals. In situ hybridisation with this new probe allowed: i) to confirm that PCV2 mainly replicates within the nuclei of epithelial/endothelial cells and macrophages from lung, inguinal and mesenteric lymph nodes, tonsil and liver; and ii) to demonstrate that, contrary to previously assumed, only a small percentage of cells scoring positive for either PCV2-antigen or PCV2-nucleic acid (using other probes), really support PCV2 replication.
The second study describes the development of two new ELISAs, rCap ELISA and rRep ELISA, useful for the detection of specific antibodies against the PCV2 capsid protein (Cap) and the PCV2 replicase proteins (Rep), respectively. These tools allowed, for the first time, to perform a longitudinal study of specific anti-Cap and anti-Rep antibodies in pigs from PMWS affected farms. Two main conclusions could be extracted from this study: i) PMWS affected pigs had significantly lower anti-Cap and anti-Rep antibody levels than the rest of the animal groups (non-PMWS but wasted pigs and healthy animals), at the moment of the occurrence of the disease in the farm, and ii) PMWS affected pigs could be collectively distinguished from the rest of the pig groups by their lower anti-Rep antibody levels before the appearance of PMWS clinical signs, at 11 weeks of age. These results demonstrate the potential of both ELISAs for large-scale serological studies in PMWS affected farms and open the possibility to differentiate infected from vaccinated animals (DIVA), at least when inactivated virus or Cap PCV2 subunit vaccines are used.
Finally, the aim of the third study was to test the potential use of recombinant Cap (rCap) protein expressed in baculovirus and produced in Trichoplusia ni insect larvae as a method for PCV2 vaccination in pigs. Results from this work demonstrated: i) that animals vaccinated with rCap were able to induce specific immune responses and were protected from PCV2 infection in a dose-dependant manner; ii) that rCap was immunogenic even in the absence of immunoadjuvant, although its presence helped to overcome maternal immunity; and iii) that protection correlated with the induction of PCV2 specific antibodies. In certain cases, however, protection was achieved in the absence of antibodies, suggesting that the cellular immune response probably plays a role in protection against PCV2. The efficacy and the cost-efficiency of this recombinant vaccine open new expectations about its future use in farmed pigs.
