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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.

Vaccination of dams in a PCV2 subclinically infected farm 2 and 4 weeks before insemination, with a booster at 12 weeks of gestation did not influence fertility parameters of the dams. However, growth parameters of offspring of vaccinated sows improved significantly (+ 51 g/d), resulting in a shorter growing period of 9 days and a massively improved economy. Mortality of weaners and fattening pigs was not significantly influenced by dam vaccination. Nevertheless, compared to a period of 6 months before vaccination, the mortality rate declined in the weaning period by 0,3 % and in the fattening period by 5,5 %. The Return on Investment (ROI) was calculated with 1:9.5. Even, the historically low pork prices in 2011 led to a ROI of 1:7.

Postweaning multisystemic wasting syndrome (PMWS) was epizoozic between 2003 and 2008 in Switzerland. Nevertheless, infectious risk factors including porcine reproductive and respiratory syndrome virus (PRRSV) were missing at all or were seen only sporadically (enzootic pneumonia and actinobazillosis). In a case-control study, 30 farms with PMWS affected pigs were compared to 30 inconspicious farms ("matched pairs"). The case-control allocation was verified by PCV2 DNA measurements of 5 healthy weaned pigs in each control farm, 5 healthy and 5 PMWS affected weaners in each PMWS affected farm. Diseased pigs showed in average 1.8x10(8) DNA templates per ml serum significantly higher than healthy pigs from control farms with 1x10(6) DNA templates per ml serum. Virus load in healthy pigs did not differ between control- and PMWS affected farms. PMWS mainly emerged among affected pigs in the 5th to 8th week of age. In a logistic regression model risk factors were identified such as high occupancy in weaning pens (p = 0.002), large groups in gestation facilities (p = 0.03) as well as reduced birth weight < 1.3 kg (p = 0.04). We suggest these factors might have lead to chronic stress e.g. through influencing negatively social interaction in pigs or disturbances of the maturing immune system. Heavy fly and rodent infestation might not only be viewed as a vector for disease transmission, but, also as a stress factor. In den Jahren 2003 – 2008 entwickelte sich das «Postweaning Multisystemic Wasting Syndrome» (PMWS) in der Schweiz zu einer Epizootie, obwohl beschriebene infektiose Risikofaktoren fur das Angehen von PMWS, wie das «Porcine Reproductive und Respiratory Syndrome Virus» (PRRSV) fehlen oder bei der enzootischen Pneumonie (EP) und der Aktinobazillose eine untergeordnete Rolle spielen. In einer Fall-Kontroll-Studie wurden 30 PMWS-Problembetriebe und 30 Kontrollbetriebe («matched pairs») auf Risikofaktoren analysiert. Kranke Schweine wiesen mit durchschnittlich 1.8 x 108 DNA-Kopien/ml Serum signifikant hohere Virustiter auf als gesunde Schweine aus den Kontrollbetrieben, welche 1 x 106 DNA-Kopien/ml Serum aufwiesen. Der Virustiter von gesunden Tieren unterschied sich nicht signifikant zwischen PMWS- und Kontrollbetrieben. PMWS trat meist zwischen der 5.–8. Lebenswoche auf. In einer ogistischen Regressionsanalyse wurden Risikofaktoren wie hohe Belegdichten in den Absetzbuchten (p = 0.002), grosse Galtsauengruppen (p = 0.03) sowie ein vermindertes Geburtsgewicht < 1.3 kg (p = 0.04) identifiziert. Diese Faktoren konnen zu chronischem Stress fuhren, welche die soziale Interaktion storen und die Ontogenese des fetalen Immunsystems negativ beeinflussen. Auch Fliegen- und Schadnagerbefall sind nicht zu unterschatzende Risikofaktoren beziehungsweise Stressfaktoren.

Porzine Circoviren Typ 2 (PCV2) sind in der Lage Fruchtbarkeitsstorungen zu induzieren. Sechs Jahre nach Beginn der Epizootie des «Postweaning Multisystemic Wasting Syndrome» (PMWS) in der Schweiz wurden 286 Feten von 113 Muttersauen aus 59 Betrieben auf infektiose Ursachen fur Fruchtbarkeitsstorungen mit Aborten, vermehrt mumifizierten oder totgeborenen und lebensschwachen Ferkeln untersucht. 14 % der Falle wurden anhand von Erregerisolation und histologischen Entzundungsanzeichen einer bakteriellen Infektion zugeordnet. In weiteren 12 % konnten histologisch Entzundungsreaktionen ohne plausiblen Erregernachweis gefunden werden. PCV2 wurde mittels Immunhistochemie (IHC) in nur 4 % der Falle nachgewiesen und scheint somit in der Schweiz bei Fruchtbarkeitsproblemen eine untergeordnete Rolle zu spielen. Infektionen mit dem porzinen Parvovirus (PPV) sind mit 3 % der Falle deutlich seltener als in fruheren Untersuchungen. Auf Enteroviren/Teschovirus wurde nur stichprobenweise bei atiologisch unklaren Fallen untersucht und eine Pravalenz von 11 % ermittelt. Unseres Wissens ist das der erste Nachweis von Enteroviren/Teschovirus in Feten in der Schweiz. Die atiologische Ursache blieb in uber 50 % aller Falle, auch unter Anwendung moderner diagnostischer Methoden, unklar. = Porcine Circovirus type 2 (PCV2) is able to induce reproductive failures. 286 fetuses from 113 sows of 59 farms with increased reproductive disorders which included abortions, mummies, stillborn and weak born piglets were studied six years after the beginning of the epizooty of postweaning multisystemic wasting syndrome (PMWS) in Switzerland. 14 % of the cases were bacterial infections based on histological signs of inflammation and pathogen isolation. 12 % further cases showed inflammatory reactions by histology without pathogen identification. PCV2 was identified in only 4 % of cases by immunohistochemistry (IHC). Thus, PCV2 infections are of minor importance in respect to pig reproductive failures in Switzerland. Porcine parvovirus (PPV) infections were found in 3 % of the cases and seem to occur more infrequently compared to former findings. Hitherto, Enteroviruses/Teschovirus were marginally studied in etiologically undefined cases with a prevalence of 11 %. To our knowledge this is the first identification of Enteroviruses/Teschovirus in fetal tissue from reproductive failures in Switzerland. The etiology remained unclear in more than 50 % of all cases in spite of modern diagnostic methods.

In the present study, ocular chlamydial infections in pigs that originate from two different farming systems were investigated. In particular, the aim was to test pigs with and without clinical ocular symptoms for the presence of Chlamydiaceae and for linked infections with Acanthamoebae spp. possibly acting as vectors for Chlamydia or Chlamydia-like organisms. In a total of 181 pigs, 102 from Germany (GER), representing the intensively kept animals and 79 from Switzerland (CH), which were kept extensively, were screened for the presence of different pathogens by PCR, including a new Chlamydiaceae-specific intergenic spacer rRNA gene PCR. Additionally, results of clinical examination and cytology were compared between the symptomatic and asymptomatic pigs of the two groups. Ocular symptomatic pigs showed a high prevalence of Chlamydia suis in both groups: CH 79%, GER 90%. Only 23% asymptomatic pigs from CH, but 88% asymptomatic pigs from GER were positive for C. suis by PCR. DNA of Chlamydia-like organisms were detected in 19% CH, but only in 2% GER pigs, whereas only 4% CH and 1% GER pigs were also positive for Acanthamoebae spp. A co-infection of Acanthamoebae spp. and C. suis was present in only 3% of the CH but 28% of the GER pigs. In general, the intensively kept pigs in our study seemed to be pre-disposed to ocular chlamydial infection and associated conjunctivitis. Infections with Chlamydia-like organisms alone and in combination with Acanthamoebae played no role for clinical findings within the tested pig groups, whereas a co-infection of Acanthamoebae and C. suis was able to cause serious ocular manifestations in half of the cases of intensively kept pigs being positive for these microorganisms.

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.

Cell migration is essential throughout embryonic and adult life. In numerous cell systems, the small GTPase Rac is required for lamellipodia formation at the leading edge and movement ability. However, the molecular mechanisms leading to Rac activation during migration are still unclear. Recently, a mammalian superfamily of proteins related to the prototype member Dock180 has been identified with homologues in Drosophila and Caenorhabditis elegans. Here, we addressed the role of Dock180 and ELMO1 proteins, which function as a complex to mediate Rac activation, in mammalian cell migration. Using mutants of Dock180 and ELMO1 in a Transwell assay as well as transgenic rescue of a C. elegans mutant lacking CED-5 (Dock180 homologue), we identified specific regions of Dock180 and ELMO1 required for migration in vitro and in a whole animal model. In both systems, the Dock180.ELMO1 complex formation and the ability to activate Rac were required. We also found that ELMO1 regulated multiple Dock180 superfamily members to promote migration. Interestingly, deletion mutants of ELMO1 missing their first 531 or first 330 amino acids that can still bind and cooperate with Dock180 in Rac activation failed to promote migration, which correlated with the inability to localize to lamellipodia. This finding suggests that Rac activation by the ELMO.Dock180 complex at discrete intracellular locations mediated by the N-terminal 330 amino acids of ELMO1 rather than generalized Rac activation plays a role in cell migration.

A central paradigm of T cell development is that CD4+8+ (DP) thymocytes differentiate into CD4+ or CD8+ T cells in response to intrathymic signals that extinguish transcription of the inappropriate coreceptor molecule. Contrary to this prevailing paradigm, we now demonstrate that signaled DP thymocytes initially terminate CD8 transcription even when differentiating into CD8+ T cells. Remarkably, thymocytes that have selectively terminated CD8 transcription can be signaled by IL-7 to differentiate into CD8+ T cells by silencing CD4 transcription and reinitiating CD8 transcription, events we refer to as "coreceptor reversal." These observations significantly alter our understanding of CD8+ T cell differentiation and lead to a new perspective ("kinetic signaling") on CD4/CD8 lineage determination in the thymus. These observations also suggest a novel mechanism by which bipotential cells throughout development can determine their appropriate cell fate.

Heavy metal-induced transcription in mammalian cells is conferred by the metal-responsive 70 kDa transcription factor MTF-1 which contains six zinc fingers and at least three activation domains. In previous cell transfection experiments we have shown that the zinc finger region confers an about 3 fold metal inducibility of transcription, due to its differential zinc binding. However, we also noted that human MTF-1 was more metal-responsive than the mouse factor (about 10 fold versus 3 fold, respectively). Here we analyze this difference in more detail by using chimeric human-mouse factors and narrow the critical region to a 64 amino acid stretch immediately downstream of the zinc fingers, overlapping with the acidic activation domain. A short human segment of this region (aa 313-377) confers efficient metal induction to the mouse MTF-1 when replacing the corresponding mouse region. However, high metal inducibility requires an unaltered MTF-1 and is lost when human MTF-1 is fused to the general activation domain of herpesvirus VP16. Wild type and truncation mutants of MTF-1 fused to VP16 yield chimeras of high transcriptional activity, some exceeding the wildtype regulator, but only limited (about 3 fold) heavy metal inducibility.

Metallothioneins (MTs) are small cysteine-rich proteins that bind heavy metal ions such as zinc, cadmium and copper with high affinity, and have been functionally implicated in heavy metal detoxification and radical scavenging. Transcription of metallothioneins genes is induced by exposure of cells to heavy metals. This induction is mediated by metal-responsive promoter elements (MREs). We have previously cloned the cDNA of an MRE-binding transcription factor (MTF-1) from the mouse. Here we present the human cDNA equivalent of this metal-regulatory factor. Human MTF-1 is a protein of 753 amino acids with 93% amino acid sequence identity to mouse MTF-1 and has an extension of 78 amino acids at the C-terminus without counterpart in the mouse. The factors of both species have the same overall structure including six zinc fingers in the DNA binding domain. We have physically mapped the human MTF-1 gene to human chromosome 1 where it localizes to the short arm in the region 1p32-34, most likely 1p33. Both human and mouse MTF-1 when produced in transfected mammalian cells strongly bind to a consensus MRE of metallothionein promoters. However, human MTF-1 is more effective than the mouse MTF-1 clone in mediating zinc-induced transcription.

A member of the family Circoviridae , porcine circovirus type 2 (PCV2), is associated with postweaning multisystemic wasting syndrome (PMWS), a recent emerging disease worldwide. PCV2 is also found in clinically asymptomatic animals. This paradoxical finding makes the syndrome etiology challenging. We developed new assays to study PCV2 with links to syndrome etiology. For analysis, we used PCV2-infected tissues from subclinically infected and diseased piglets. We compared antigen- and PCV2 DNA-derived signals for tissue localization and intensity. Oligonucleotides were designed to the signature motif of the PCV2 capsid open reading frame to discriminate experimentally between PCV2 genotype groups by PCR, in situ hybridization (ISH), and fluorescence in situ hybridization (FISH). Unexpectedly, all PCV2-infected animals carried both PCV2a and PCV2b genotype group members. Using confocal microscopy, genotype single-cell infections and cell superinfections were visible. Additionally, we discriminated replicative DNA from total PCV2 DNA isoforms with FISH. This aided in our inquiry into cellular genotype-specific replication. Importantly, single-genotype-group replication was not observed. In infected cells with replicating virus, both genotype groups were equally present. These findings suggest PCV2 genotype group members relaxed replication regulation requirements and may even point to PCV2 replication cooperativity in vivo . These observations explain the readily seen PCV2 DNA recombinations and the high overall PCV2 genome plasticity. Hence, we suggest a novel mechanism of syndrome etiology that consists of a continuously changing PCV2 genome pool in hosts and pig herds, posing a constant challenge to the individual maturing immune system.

Porcine circovirus type 2 (PCV2) is the obligate infectious agent in postweaning multisystemic wasting syndrome (PMWS) of pigs. To control PMWS, we vaccinated dams at 4 and 2 weeks before pregnancy and again in the 12th week of gestation with an inactivated PCV2 vaccine (Circovac). Two producer farms run under the control of Swiss Swine Health Organization were selected for the experiment. Previously, in one farm PMWS was diagnosed on pigs after weaning, whereas in the other farm, pigs wasted during the fattening period. For the experiments 113 dams were randomly vaccinated, and 111 dams were sham injected. Vaccination increased serum antibodies in dams 3- to 9-fold, accompanied by serum antibody titer increases in their offspring. In the sixth week of life, progeny from vaccinated dams had about the same IgG antibody titers as progeny of unvaccinated dams at the third day of life. In sera of vaccinated dams only low concentrations of PCV2 DNA were detected, and no progeny developed PMWS. Interestingly, at day 56 four progeny of unvaccinated dams tested positive for anti-PCV2 IgM antibodies, indicating a primary infection with PCV2. Of economic importance is the observation that progeny of vaccinated dams had a significantly higher daily weight gain in the fattening period (farm X, +51 g/day; farm Y, +30 g/day) and thus a shortened fattening period of about 6 days compared to progeny of controls. To our knowledge this is the first demonstration of subclinical circovirus infection and its effects on growth performance of fattening pigs by vaccination of dams.

Postweaning multisystemic wasting syndrome (PMWS) is among the most important emerging pig diseases worldwide. Initially, the insidious nature of the disease made it difficult to pinpoint the pathogen. The presence of porcine circovirus type 2 (PCV2) in all PMWS diseased animals led to its acceptance, possibly together with an unknown factor, as the causative agent for PMWS. Also, presence of PCV2 in healthy individuals did not facilitate the understanding of the disease. Phylogenetic classification separates PCV2 viruses into at least two major groups. With the aid of a signature motif, a short amino acid motif encoded within the capsid protein, the viruses are determined as belonging to PCV-2a or PCV-2b. Recently, this classification received more attention, as it seemed to define PCV-2b to be more virulent. This simplification, however, could not be confirmed experimentally. Hence, we investigated whether virus genetic shift was an initiator for the PMWS epizooty in Switzerland. Piglet lymphoid tissues from 1973 to 2005 were investigated by histology, immunohistochemistry (IHC) and PCR. For genotype classification, a sequence amplificate of 137bp was used encompassing the signature motif. The onset of Swiss PMWS epizooty exhibited a marked shift in PMWS diseased and subclinically infected piglets to PCV-2b and specifically to one genotype subgroup. Complementary to these observations, healthy piglets also defined by IHC as negative are positive in the PCR reaction and are void of any PCV-2b virus during epizooty. Consequently, our data support PCV2 genome plasticity as a major contributing factor for PMWS disease manifestation.

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.

Removal of apoptotic cells is a dynamic process coordinated by ligands on apoptotic cells, and receptors and other signaling proteins on the phagocyte. One of the fundamental challenges is to understand how different phagocyte proteins form specific and functional complexes to orchestrate the recognition/removal of apoptotic cells. One evolutionarily conserved pathway involves the proteins cell death abnormal (CED)-2/chicken tumor virus no. 10 (CT10) regulator of kinase (Crk)II, CED-5/180 kDa protein downstream of chicken tumor virus no. 10 (Crk) (Dock180), CED-12/engulfment and migration (ELMO) and MIG-2/RhoG, leading to activation of the small GTPase CED-10/Rac and cytoskeletal remodeling to promote corpse uptake. Although the role of ELMO : Dock180 in regulating Rac activation has been well defined, the function of CED-2/CrkII in this complex is less well understood. Here, using functional studies in cell lines, we observe that a direct interaction between CrkII and Dock180 is not required for efficient removal of apoptotic cells. Similarly, mutants of CED-5 lacking the CED-2 interaction motifs could rescue engulfment and migration defects in CED-5 deficient worms. Mutants of CrkII and Dock180 that could not biochemically interact could colocalize in membrane ruffles. Finally, we identify MIG-2/RhoG (which functions upstream of Dock180 : ELMO) as a possible point of crosstalk between these two signaling modules. Taken together, these data suggest that Dock180/ELMO and CrkII act as two evolutionarily conserved signaling submodules that coordinately regulate engulfment.

Chlamydiae infect male genital organs of ruminants. However, little is known about their prevalence. Hence, we investigated fresh and cryopreserved semen (bulls: n=304; rams: n=78; bucks: n=44) by polymerase chain reaction (PCR), as well as genital organs (bulls: n=13; rams: n=10; bucks: n=6) by immunohistochemistry (IHC) and PCR. Sera from bulls (n=104) and small ruminants (n=61) were tested by LPS and rMOMP (recombinant major outer membrane protein) ELISA and competitive ELISA (cELISA), respectively. Three PCR assays were compared in this study for detection of chlamydial DNA in semen: 16S rRNA, IGS-S (intergenic spacer 16S/23S-short), and IGS-L (intergenic spacer 16S/23S-long) PCRs. PCR sensitivity and inhibitory effects were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. In bull semen, detection limits of the 16S, IGS-S and IGS-L PCRs were 10, 10, 100 templates, respectively. However, PCR sensitivity was reduced in ram and buck semen suggesting the presence of potential PCR inhibitors. Of 304 bull semen samples, the 16S PCR revealed DNA of chlamydiae in 20 samples (6.6%), including Cp. abortus (n=2), Cp. psittaci (n=1), Chlamydia suis (n=2), and Chlamydia-like organisms (n=15). In rams, one semen sample was positive for Chlamydia-like organism. All investigated male genital organs were negative for Chlamydia. Serology revealed 47.1% (49/104) positive bulls by LPS ELISA. Of these, 30 samples were positive by rMOMP ELISA, predominantly for Cp. pecorum. In small ruminants, cELISA displayed 34.8% (16/46) and 60% (9/15) positivity for Cp. abortus in rams and bucks, respectively. There was no correlation between serology and PCR of semen. The presence of chlamydiae in semen suggests the possibility of venereal transmission, although risk may be low in Switzerland.

One of the conserved functional pathways linked to engulfment of apoptotic corpses involves two membrane proteins low density lipoprotein receptor-related protein-1 (LRP) and ABCA1 and the LRP adapter protein GULP. Because LRP and ABCA1 play roles in cellular lipid trafficking and efflux, here we addressed whether the third member, the LRP adapter protein GULP, also affects cellular lipid transport. Several lines of evidence show that overexpression of GULP causes glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment that is accompanied by down-regulation of ABCA1 and decreased efflux. Conversely, knockdown of endogenous GULP expression promoted cholesterol flux through the late endosomes and up-regulation of ABCA1, even in the context of a disease state such as Niemann-Pick Type C disease. Mechanistically, we were able to show that trafficking of the LRP ligands alpha2-macroglobulin and prosaposin, a protein cofactor necessary for glycosphingolipid degradation, are impaired in cells expressing full-length GULP protein, resulting in glycosphingolipid and free cholesterol accumulation in the late endosome/lysosome compartment. On the other hand, knockdown of endogenous GULP results in enhanced targeting of prosaposin and enhanced clearance of glycosphingolipids and cholesterol from the late endosomes. Taken together, these data reveal that GULP/LRP/ABCA1 represents a triad of molecules involved in engulfment and cellular lipid homeostasis.

Chlamydiae cause abortion and reproductive disorders in sows. Although organisms can infect the male genital tract, little is known about the disease situation in boars. Hence, we examined the prevalence of chlamydial infection in semen and genital tracts of boars. Samples collected from Swiss boars (group A: n=42), and boars from Germany (group B: n=39) were examined by bacteriology, LPS-ELISA, immunohistochemistry (IHC) and polymerase chain reaction (PCR). The latter methodology involved use of three PCR assays including 16Sig rDNA, IGS-S (intergenic spacer 16S/23S-Short) and IGS-L (intergenic spacer 16S/23S-Long) PCR for comparison methods. PCR sensitivity and the presence of potential PCR inhibitors were determined by spiking semen with Chlamydophila (Cp.) abortus DNA. Detection limits of the 16Sig and IGS-S PCR were 10 templates, while the IGS-L PCR was less sensitive (100 templates). Of 25 semen samples that were collected from group A, one semen sample was positive for Cp. psittaci and two were positive for Chlamydia-like organisms by 16Sig PCR. Screening of sera from Swiss boars revealed three animals with positive reactions in the LPS-ELISA, although we failed to detect chlamydiae within organs of these or sera-negative animals by IHC or IGS-S PCR. In group B, 10 ejaculates were positive for Chlamydia (C.) suis and two were positive for Chlamydia-like organisms by 16S PCR. The identification of DNA from Chlamydia-like organisms in semen from both groups of boars was surprising and a role for these bacteria in reproductive diseases requires further assessment. In conclusion, the prevalence of chlamydial infection was low in group A animals indicating that venereal transmission may not be significant for Chlamydia-associated reproductive diseases in pigs, although rare cases may occur.

The aim was to detect and characterize chlamydial infections in guinea-pigs (GP) with ocular disease, study their pathogenicity and zoonotic potential and to test for the presence of Acanthamoebae spp. in GP eyes and to investigate whether they could act as vectors for Chlamydia-like organisms. Overall 126 GP, of which 77 were symptomatic, were screened by clinical examination, cytology, gross pathology, histology, immunohistochemistry, polymerase chain reaction (PCR) and bacteriology. A new Chlamydiaceae-specific intergenic spacer rRNA gene PCR, designed to amplify this segment linking the 16S and 23S regions, was performed. DNA samples were also received from one owner including samples of his cat and rabbit. Guinea-pigs: 48 of 75 symptomatic, but only 11 of 48 asymptomatic GP were positive by PCR for Chlamydophila caviae guinea-pig inclusion conjunctivitis (GPIC) (P < 0.0001). Eighteen of 75 or 15/48, respectively, were positive for DNA from Chlamydia-like organisms. Acanthamoebae-DNA could be found in two GP, of which one was symptomatic. Owner, cat and rabbit: Samples of all three species were positive by PCR for C. caviae GPIC and the owner's one-day disposable contact lenses showed a positive PCR result for the Chlamydia-like organism Parachlamydia acanthamoebae. No Acanthamoebae-DNA could be detected. This study is the first to describe Chlamydia-like organisms in GP and to detect C. caviae GPIC in human, cat and rabbit. Therefore, C. caviae GPIC could pose a zoonotic potential. We believe that the finding of C. caviae GPIC in species other than GP is probably not unique.

The members of the Dock180 superfamily of proteins are novel guanine nucleotide exchange factors (GEF) for Rho family GTPases and are linked to multiple biological processes from worms to mammals. ELMO is a critical regulator of Dock180, and the Dock180-ELMO complex functions as a bipartite GEF for Rac. We identified a mechanism wherein the PH domain of ELMO, by binding the Dock180-Rac complex in trans, stabilizes Rac in the nucleotide-free transition state. Mutagenesis studies reveal that this ELMO PH domain-dependent regulation is essential for the Dock180-ELMO complex to function in phagocytosis and cell migration. Genetic rescue studies in Caenorhabditis elegans using ELMO and its homolog CED-12 support the above observations in vivo. These data reveal a new mode of action of PH domains and a novel, evolutionarily conserved mechanism by which a bipartite GEF can activate Rac.

Mammalian Dock180 and ELMO proteins, and their homologues in Caenorhabditis elegans and Drosophila melanogaster, function as critical upstream regulators of Rac during development and cell migration. The mechanism by which Dock180 or ELMO mediates Rac activation is not understood. Here, we identify a domain within Dock180 (denoted Docker) that specifically recognizes nucleotide-free Rac and can mediate GTP loading of Rac in vitro. The Docker domain is conserved among known Dock180 family members in metazoans and in a yeast protein. In cells, binding of Dock180 to Rac alone is insufficient for GTP loading, and a Dock180 ELMO1 interaction is required. We can also detect a trimeric ELMO1 Dock180 Rac1 complex and ELMO augments the interaction between Dock180 and Rac. We propose that the Dock180 ELMO complex functions as an unconventional two-part exchange factor for Rac.

Apoptosis or programmed cell death occurs in multicellular organisms throughout life. The removal of apoptotic cells by phagocytes prevents secondary necrosis and inflammation and also plays a key role in tissue remodeling and regulating immune responses. The molecular mechanisms that regulate the engulfment of apoptotic cells are just beginning to be elucidated. Recent genetic studies in the nematode Caenorhabditis elegans have implicated at least six genes in the removal of apoptotic cell corpses. The gene products of ced-2, ced-5, and ced-10 are thought to be part of a pathway that regulates the reorganization of the cytoskeleton during engulfment. The adapter proteins CrkII and Dock180 and the small GTPase Rac represent the mammalian orthologues of the ced-2, ced-5 and ced-10 gene products, respectively. It is not known whether CrkII, Dock180, or Rac proteins have any role during engulfment in mammalian cells. Here we show, using stable cell lines and transient transfections, that overexpression of wild-type CrkII or an activated form of Rac1 enhances engulfment. Mutants of CrkII failed to mediate this increased engulfment. The higher CrkII-mediated uptake was inhibited by coexpression of a dominant negative form of Rac1 but not by a dominant a negative Rho protein; this suggested that Rac functions downstream of CrkII in this process, which is consistent with genetic studies in the worm that place ced-10 (rac) downstream of ced-2 (crk) in cell corpse removal. Taken together, these data suggest that CED-2/CrkII and CED-10/Rac are part of an evolutionarily conserved pathway in engulfment of apoptotic cells.

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain, Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-B exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

Metallothioneins (MTs) constitute a class of low molecular weight, cysteine-rich, metal binding proteins which are regulated at the level of gene transcription in response to heavy metals and other adverse treatments. We have previously cloned a zinc finger factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in metallothionein promoters and shown that this factor is essential for basal and heavy metal-induced transcription. Here we report that the C-terminal part of MTF-1 downstream of the DNA binding zinc fingers harbours three different transactivation domains, namely an acidic domain, a proline-rich domain and a domain rich in serine and threonine. When fused to the heterologous DNA binding domain of the yeast factor GAL4 these activation domains function constitutively, i.e. transcription of a GAL4-driven reporter gene is not induced by heavy metals. In search of the region(s) responsible for metal induction, external and internal deletion mutations of mouse and human MTF-1 and chimeric variants thereof were tested with a reporter gene driven by a metal-responsive promoter. The N-terminal part of MTF-1 containing the zinc fingers, which are dependent on zinc for efficient DNA binding, can indeed confer a limited (3- to 4-fold) zinc-responsive transcription when fused to the heterologous activation domain of the viral VP16 protein. Another region containing the acidic and proline-rich activation domains also contributes to metal inducibility, but only in the context of intact MTF-1. This indicates that the activity of MTF-1 results from a complex interplay of different functional domains.

The bipartite DNA binding domain of the POU family of transcription factors contains a ‘POU-specific’ domain unique to this class of factors and a ‘POU homeodomain’ homologous to other homeodomains. We compared DNA binding of the Oct-2 factor POU domain and the Antennapedia (Antp) homeodomain with a chimeric Oct-2/Antp protein in which the distantly related Antp homeodomain was substituted for the Oct-2 POU homeodomain. The Oct-2/Antp chimeric protein bound both the octamer and the Antp sites efficiently, indicating that DNA binding specificity is contributed by both components of the POU domain.