Immunodiagnosis of primary Toxoplasma gondii infection in sheep by the use of a P30 IgG avidity ELISA

Abstract.: Toxoplasma gondii is a protozoan parasite with a worldwide distribution. In both sheep and humans, if the parasite is encountered during pregnancy, fetal infection and abortion can occur. Therefore, Toxoplasma infection in sheep has a major economic impact upon sheep farming. Clinically, there is a need to distinguish recent (acute) infections from longstanding (chronic) infections. However, current serological techniques, such as detection of anti-T. gondii IgG, cannot discriminate between acute and chronic infections. Increasing immunoglobulin avidity is a good determining factor of how recent an infection is. In this study, we describe the application and validation of a T. gondii IgG avidity ELISA, based on the use of an affinity-purified, native T. gondii P30 antigen. The assay was used to examine sera from eight sheep experimentally infected with T. gondii and found that all seroconverted within 21days post-infection (p.i.), beginning with avidities that were initially low but that increased over time, with all sheep reaching high IgG avidity within 10weeks p.i. In addition, sera from clinically healthy but T. gondii-seropositive lambs and ewes and seropositive ewes with a history of abortion were also subjected to a preliminary serological investigation. High IgG avidities were found in 80% of the seropositive lambs, in 90% of the clinically healthy ewes and in 97% of the ewes with abortion problems. These findings indicate that the animals had most likely contacted the parasite a longer time ag

Assessment of antibody avidity in aborting cattle by a somatic Neospora caninum tachyzoite antigen IgG avidity ELISA.

A Neospora caninum IgG avidity ELISA was carried out on the basis of a somatic N. caninum tachyzoite antigen. The test was validated using experimentally infected calves, where a clear maturation of the IgG avidity over time could be demonstrated. At a maximum of 82 days after infection (d.p.i.), all animals showed antibody avidities ranging above 35%, and respective sera were thus defined as highly avid. Sera of 103 naturally infected seropositive cows with abortion (N. caninum association was provided by a N. caninum PCR-positivity of the fetus in 40 cases) and 139 seropositive animals without abortion history were concurrently examined. Significantly lower avidities were observed in aborting cows when compared to animals without abortion problems (P<0.01). While the avidity of sera collected before abortion remained practically constant until abortion, a significant increase of avidity could be observed in samples collected weeks to months after abortion (P<0.01). The avidities of non-aborting animals from farms with or without abortion problems did not differ significantly with time and were mainly located in the high avidity area. These data indicate that low avidities are not necessarily linked to recent N. caninum infection but can also be an indicator for increased abortion risk in cattle

PD-1/PD-L1-Associated Immunoarchitectural Patterns Stratify Pancreatic Cancer Patients into Prognostic/Predictive Subgroups.

Immunotherapy, including PD-1/PD-L1 agonists, has shown limited efficacy in pancreatic ductal adenocarcinoma (PDAC). We examined the PD-1/PD-L1 expression and immunoarchitectural features by automated morphometric analysis using multiplex immunofluorescence and 118 microsatellite-stable, treatment-naïve, surgically resected PDACs (study cohort). Five microsatellite-instable cases were stained in parallel (MSI cohort). Molecular analysis was additionally performed. An independent PDAC cohort (n = 226) was immunostained for PD-L1 and used as a validation cohort. PD-L1 expression on tumor cells (TC) and/or immune cells (IC) was present in 32% and 30% of the study and validation cohorts, respectively, and assigned into one of four patterns: "adaptive-1" (TC: 0, IC > 1%), "adaptive-2" (TC > 1% to 1%), "constitutive" (TC ≥ 25%, IC: 0), and "combined" (TC ≥ 25%, IC > 1%). "Constitutive" tumors were characterized by reduced numbers of all ICs and poor outcome. In contrast, "adaptive-1" tumors exhibited abundant T cells, including high counts of cytotoxic CD3+CD8+ and PD-1+CD3+CD8+ cells, but low counts of PD-L1+CD3+CD8+ cells and associated with the best outcome. "Adaptive-2" tumors displayed higher proportions of PD-L1+CD3+CD8+ T cells and tumor-associated macrophages (CD68+ and CD68+CD206+) compared with "adaptive-1" tumors. In the "combined" pattern, extensive PD-L1 expression on TCs was accompanied by increased numbers of T cells and improved overall survival. ICs were closer to PD-L1- than to PD-L1+ PDAC cells. TP53 and PIK3CA alterations tended to be more frequent in PD-L1+ tumors. The 5 MSI cases were PD-L1- The distinct PD-1/PD-L1-associated immunoarchitectural patterns underpin the heterogeneity of the immunologic responses and might be used to inform patient outcomes and therapeutic decisions in pancreatic cancer