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In a proof of concept, PCV2-specific IgG-antibodies from testicular tissue fluid of seven-day-old castrated piglets were measured to verify the vaccination status of their mothers. Twelve randomly selected sows were vaccinated twice during the last third of gestation with a PCV2 vaccine, while ten controls received only adjuvant. PCV2- specific IgG-antibody titers of serum and colostrum from the sows were correlated with PCV2-specific IgG-antibody titers of serum and testicular tissue fluid of their castrated male offspring. Vaccinated sows showed significantly higher average PCV2-specific IgG-antibody titers in serum (29250 ELISA units, EU) and colostrum (65410 EU) compared to 980 EU and 2630 EU of the control group, respectively. Moreover, significantly higher average concentrations of antibodies were also measured in the serum (9362 EU vs. 247 EU) and the testicular tissue fluid (4022 EU vs. 354 EU) of piglets from vaccinated compared to piglets from adjuvant administered sows. Importantly, a strong linear correlation between PCV2-specific IgG-antibodies in the serum of the piglets and in their testicular tissue fluid was found (rs = 0.9148). PCV2-specific IgG-antibody titers of testicular tissue fluid from five randomly selected piglets allowed the determination of the vaccination status of the herd with a reliability of 98% for vaccinated and 73% for unvaccinated sows. Furthermore, using castration waste products is a very animal friendly method to replace painful and time-consuming blood samplings for herd monitoring or to verify vaccination status.In einem „proof of concept“ wurden PCV2-spezifische IgG-Antikörper in Hodengewebe-Flüssigkeit von sieben Tage alten, kastrierten Ferkeln zur Verifizierung des Impfstatus von Muttersauen gemessen. Zwölf zufällig ausgewählte Muttersauen wurde zweimal während des letzten Trächtigkeitsdrittels ein PCV2-Impfstoff und zehn Muttersauen zur Kontrolle nur Adjuvans appliziert. PCV2-spezifische Antikörpertiter von Serum und Kolostrum der Sauen wurde mit Serum und Hodengewebeflüssigkeit ihrer kastrierten Nachkommen verglichen. Geimpfte Sauen hatten mit 29250 ELISA- Units (EU) im Serum und 65410 EU im Kolostrum signifikant höhere PCV2 spezifische IgG-Antikörpertiter verglichen mit den Serum- (980 EU) respektive den Kolostrumtitern (2630 EU) der Kontrollsauen. Ferkel von geimpften Sauen wiesen mit 9362 EU im Serum und 4022 EU in der Hodengewebeflüssigkeit ebenfalls signifikant höhere Antikörper-Konzentrationen auf als Ferkel von Adjuvans behandelten Kontrollsauen, welche mit 247 EU im ­Serum und 354 EU in der Hodengewebeflüssigkeit aufwiesen. Es bestand eine hohe lineare Korrelation zwischen PCV2-spezifischen IgG- Antikörper Titern im Serum und den Titern in der Hodengewebeflüssigkeit (rs = 0,9148). Der Nachweis von PCV2-spezifischen IgG-Antikörper-Titern in der Hodengewebeflüssigkeit von fünf zufällig ausgewählten Ferkeln erlaubte eine korrekte Identifizierung des Impfstatus einer Herde mit einer Zuverlässigkeit von 98% bei den geimpften und 73% bei den nicht geimpften Sauen. Die Resultate zeigen, dass anstelle zeitaufwändiger und belastender Blutprobenentnahmen, Hodengewebe von kastrierten Ferkeln für Monitoringzwecke oder zur Feststellung des Impfstatus bei den Sauen verwendet werden könnte.Dans une preuve de concept, les anticorps IgG spécifiques au PCV2 provenant de liquide tissulaire testiculaire de porcelets castrés âgés de sept jours ont été mesurés pour vérifier le statut vaccinal de leur mère. Douze truies sélectionnées au hasard ont été vaccinées deux fois au cours du dernier tiers de la gestation avec un vaccin PCV2, tandis que dix témoins n’ont reçu que l’adjuvant. Les titres d’anticorps IgG spécifiques au PCV2 dans le sérum et le colostrum des truies étaient corrélés avec les titres d’anticorps IgG spécifiques au PCV2 dans le sérum et le liquide tissulaire testiculaire de leur progéniture mâle castrée. Les truies vaccinées ont montré des titres moyens d’anticorps IgG spécifiques au PCV2 significativement plus élevés dans le sérum (29250 unités ELISA, UE) et le colostrum (65410 UE) par rapport à respectivement 980 UE et 2630 UE dans le groupe témoin. De plus, des concentrations moyennes d’anticorps significativement plus élevées ont également été mesurées dans le sérum (9362 EU contre 247 EU) et le liquide tissulaire testiculaire (4022 EU contre 354 EU) de porcelets vaccinés par rapport aux porcelets de truies n’ayant reçu que l’adjuvant. Il est important de noter une forte corrélation linéaire entre les anticorps IgG spécifiques au PCV2 dans le sérum des porcelets et ceux présents dans leur liquide tissulaire testiculaire (rs = 0,9148). Les titres d’anticorps IgG spécifiques au PCV2 dans le liquide tissulaire testiculaire provenant de cinq porcelets sélectionnés au hasard ont permis de déterminer le statut vaccinal du troupeau avec une fiabilité de 98% pour les truies vaccinées et 73% pour les truies non vaccinées. De plus, l’utilisation de déchets de castration est une méthode très respectueuse des animaux pour remplacer les douloureux et longs prélèvements sanguins pour la surveillance du troupeau ou pour vérifier le statut vaccinal.Con una prova di fattibilità, sono stati calcolati gli anticorpi IgG specifici per PCV2 nel liquido testicolare del tessuto testicolare di suinetti di sette giorni castrati, per verificare lo stato di vaccinazione delle scrofe. Dodici scrofe selezionate a caso sono state vaccinate due volte durante l’ultimo trimestre di gestazione con PCV2, mentre dieci hanno ricevuto il solo coadiuvante. I titoli anticorpali specifici PCV2 del siero e del colostro delle scrofe sono stati comparati a quelli del siero e del liquido tissutale testicolare della loro discendenza maschile castrata. Con 29250 unità ELISA (EU) nel siero e 65410 EU nel colostro, le scrofe vaccinate avevano titoli anticorpali IgG specifici per PCV2 significativamente più elevati rispetto ai titoli sierici (980 EU) e del colostro (2630 EU) delle scrofe di controllo. I suinetti provenienti da scrofe vaccinate avevano concentrazioni di anticorpi significativamente più elevate con 9362 EU nel siero e 4022 EU nel liquido tissutale testicolare rispetto ai suinetti provenienti dalle scrofe di controllo trattate con coadiuvanti, che avevano 247 EU nel siero e 354 EU nel liquido tissutale testicolare. È importante notare che è stata trovata una forte correlazione lineare tra gli anticorpi IgG specifici per PCV2 nel siero dei suinetti e nel loro liquido tissutale testicolare (rs = 0,9148). I titoli di anticorpi IgG specifici per PCV2 del liquido tissutale testicolare di cinque suinetti selezionati a caso hanno permesso di determinare lo stato di vaccinazione della mandria con un’affidabilità del 98% per le scrofe vaccinate e del 73% per le scrofe non vaccinate. I risultati mostrano che invece di un campionamento del sangue lungo e doloroso, il tessuto tissutale testicolare dei suinetti castrati potrebbe essere utilizzato per il monitoraggio o per determinare lo stato di vaccinazione delle scrofe.

Porcine circovirus type 2 (PCV2) infections and resulting diseases are a worldwide threat to pig production. PCV2 bears a uniqueness that allows for us to understand more about chronic infections and the immune system in general. The virus can be phylogenetically subdivided into PCV2a to PCV2h genotypes. Although vaccination against PCV2 has been seen to prevent the manifestation of PCV disease, PCV2 still lingers as subclinical infections in all developmental stages of pigs. The &ldquo, slow and low&rdquo, tactic gives PCV2 a particular advantage in a host&rsquo, s immune surveillance. Since the inception of the PCV2 associated panzootic, research scientists have been trying to understand the pathogenicity of PCV2. Different research groups found that one genotype group member was more pathogenic than others. We found, in our weaner infection model with in vivo transfection of different recombinant PCV2 genotype group members that these viruses alter T cell maturation in the thymus, including host&rsquo, s central tolerance. Here, we extend these original observations by showing that PCV2 infected cells were also found in proximity within the female and male reproductive organs of stillborn pig fetuses. These PCV2 pools were sufficient in infecting three and half-day-old embryos in sows. Furthermore, the dominant PCV2 group member was more pathogenic in our weaner infection model. PCV2 pre-immunocompetence infection makes PCV2 recognized by central immune tolerance as belonging to the host. This also explains why pathogenicity is not a genetically intrinsic characteristic of PCV2, however, the dominance of any one PCV2 genotype group member leads to a more efficient deletion of the T cells against that specific genotype group member in the thymus.

Porcine circovirus type 2 (PCV2) has a major economic impact on pig production. Vaccination against the virus appears to keep PCV2 disease (PCVD) manifestations in check. Nevertheless, PCV2 has not been eliminated. Several reports indicate that cattle also seem to be susceptible to PCV2 infection. In this study, we detected increased PCV2 cross-species transmission and suggest a strategy for identifying hidden PCV2 infection in cattle. We compared a postweaning multisystemic wasting syndrome (PMWS) distressed-farm with a subclinically-infected farm and investigated how differences in virus concentration would affect PCV2 transmission to a susceptible host such as cattle. The pig population of farm 1 had recurrent PMWS cases among weaners. The 34 cattle in proximity to the PMWS-affected pigs included 14 cattle (41%) that carried a PCV2 load of on average 2.5 x 105 virus genomes per ml blood. Among these was a 7 years old cow with chronic mastitis that was infected with the highest PCV2 load of 1.3 x 106 virus genomes per ml blood. These numbers contrasted with farm 2 that housed PCV2-subclinically infected pigs in proximity to 31 cattle of which six (19%) were infected with an average of 1.7 x 105 virus genome per ml blood. No PCV2-specific humoral response was measured in these cattle. Additionally, we encountered the problem of how to measure PCV2 latent/persistent infections. In pigs, after a one-shot vaccination, we have previously observed a direct anti-PCV2 IgG response instead of the expected IgM response. This indicated that the pig immune system was already primed with PCV2. We therefore hypothesized that cattle may also be PCV2- primed. Although the cattle’s humoral immune system hardly responded to vaccination, we detected PCV2-specific IgG antibody in one cow after the first vaccination shot. This, taken together with the PCV2 transmission rate in hybrid farms indicated sporadic infections in the cattle population. Thus, it can be concluded that PCV2 infections were not well established in cattle in comparison to the situation in swine

Until recently, knowledge of the pathogenicity of Circoviridae and Anelloviridae family members was limited. Our previous discoveries provided clues toward resolving this issue based on studies of the latent nature of porcine circovirus type 2 (PCV2) genotype group members. We developed a conventional pig infection model that indicated that weaners already harbored latent PCV2 infection in the thymus, which enabled the viruses to specifically modulate the maturation of T-helper cells. This finding raised the possibility that the thymi of normal fetuses were already infected with PCV2. The present findings further substantiate our hypothesis that PCV2 masquerades as the host by infecting fetuses before they acquire immune-competence. We provide the first demonstration that all domestic pig fetuses preferentially harbor latent PCV2-infected cells in their thymi. These PCV2-infected cells are different from thymocytes and are located in the medulla of the fetal thymus. These latent PCV2-infected cells in fetuses are found at the same location and share characteristics with the infected cells observed in adolescent pigs. Moreover, fetuses also harbor these infected cells in other lymph system organs. We provide the first demonstration that the fetal thymus virus pools are minimally affected by sow vaccination, highlighting the immune-privileged character of this organ. Furthermore, we found a striking reduction in virus-infected cells in the fetal spleen and an increase in PCV2-infected cells in the fetal intestine of anti-PCV2-vaccinated mothers. These data indicate that specific immune response interactions occur between mothers and their progeny that are not dependent on the humoral immunity of the mother and cannot be attributed to the rudimentary humoral responses of the fetuses because these pig fetuses do not have any PCV2-specific antibodies. These shifts in our understanding of the PCV2-infected cell pool will lead to different avenues in the search for effective vaccination strategies against latent and chronic pathogens.

Porcine circovirus type 2 (PCV2) diseases (PCVDs) have affected pig production worldwide over the last three decades. Since the advent of mass vaccination, manifestations of PCVDs have dramatically decreased. Nevertheless, persistent/latent PCV2 infections linger in the pig population. Therefore, we investigated whether conclusions can be drawn regarding the health status of a population based on humoral responses to a persistent pathogen such as PCV2. We examined the Swiss finisher population because in this population, time points associated with major events have been well documented. We measured PCV2- specific antibody titers of finishers before the Swiss epizootic in 1996-97 and compared these titers with antibody titers in 2006, during the peak of the epizootic, and in 2011, three years after the start of the mass vaccination of piglets. Two hundred serum samples from finishers were analyzed for each of these time periods, which correspond to before, during and after the Swiss epizootic. PCV2-specific IgG antibody titers were low to modestly positive during the pre-epizootic and post-epizootic periods. At the peak of the epizootic in 2006, almost all serum samples were positive, with higher average IgG concentrations than those detected in pre-epizootic and post-epizootic samples. Moreover, IgM antibodies were analyzed for 60 randomly chosen finishers in each time period. Of these 180 samples, only two serum samples from 2006 contained PCV2-specific IgM antibodies. Mass vaccination against PCV2 reduced PCV2-specific antibody titers in pigs with persistent/latent infection to pre-epizootic levels. Our data show prevalence for and concentrations of IgG-specific PCV2 antibodies, which appear to be correlated with PCVD. This result is counterintuitive because one would typically expect higher antibody concentrations to be associated with less disease; a possible interpretation is that elevated concentrations of immature anti-PCV2 antibodies provided pigs with a certain level of protection against PCVDs.

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.