BackgroundMolecular techniques that detect canine lymphoma cells by their clonal antigen receptor gene rearrangement play an increasing role for diagnosis as well as for monitoring minimal residual disease during and after cytostatic therapy. However, the methods currently available are time-consuming and/or cost-intensive thus impeding the use in clinical routine. The aim of the present study was to develop and evaluate a real-time polymerase chain reaction (PCR) with subsequent melting curve analysis (MCA) for the detection of clonally rearranged antigen receptor genes in dogs with B and T cell lymphoma on non formalin-fixed and paraffin-embedded lymph node samples.ResultsIn lymph node aspirates from 30 dogs with multicentric B cell lymphoma, real-time PCR with MCA detected clonal rearrangement in 100% and conventional PCR with polyacrylamide gel electrophoresis (PAGE) in 93% of samples. Both methods correctly identified clonality in 80% of lymph node aspirates of 10 dogs with T cell lymphoma. None of the two PCR systems detected clonal rearrangement in samples from 9 dogs with lymph node hyperplasia. Using a dilutional series with regular lymphoid desoxyribonucleic acid (DNA), detection limits of lymphoma DNA were as low as 0.8% and 6.25% for B and T cell clonal rearrangement with real-time PCR and MCA and at 3.13% and 12.5% with the conventional system. Median absolute detection limits of lymphoma DNA were shown to be at 0.1 ng and 1 ng for the B and T cell immunophenotype with the real-time PCR system and at 10 ng each with conventional PCR and PAGE.ConclusionsReal-time PCR with MCA is a convenient and reliable method with a good analytical sensitivity. Thus, the method may assist the detection of clonal antigen receptor gene rearrangement in canine lymphoma patients in a clinical setting also in the presence of small amounts of neoplastic cells.

The outcome of an udder infection (mastitis) largely depends on the species of the invading pathogen. Gram-negative pathogens, such as Escherichia coli often elicit acute clinical mastitis while Gram-positive pathogens, such as Staphylococcus aureus tend to cause milder subclinical inflammations. It is unclear which type of the immune competent cells residing in the udder governs the pathogen species-specific physiology of mastitis and which established cell lines might provide suitable models. We therefore profiled the pathogen species-specific immune response of different cell types derived from udder and blood. Primary cultures of bovine mammary epithelial cells (pbMEC), mammary derived fibroblasts (pbMFC), and bovine monocyte-derived macrophages (boMdM) were challenged with heat-killed E. coli, S. aureus and S. uberis mastitis pathogens and their immune response was scaled against the response of established models for MEC (bovine MAC-T) and macrophages (murine RAW 264.7). Only E. coli provoked a full scale immune reaction in pbMEC, fibroblasts and MAC-T cells, as indicated by induced cytokine and chemokine expression and NF-κB activation. Weak reactions were induced by S. aureus and none by S. uberis challenges. In contrast, both models for macrophages (boMdM and RAW 264.7) reacted strongly against all the three pathogens accompanied by strong activation of NF-κB factors. Hence, the established cell models MAC-T and RAW 264.7 properly reflected key aspects of the pathogen species-specific immune response of the respective parental cell type. Our data imply that the pathogen species-specific physiology of mastitis likely relates to the respective response of MEC rather to that of professional immune cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-016-0307-3) contains supplementary material, which is available to authorized users.

Monocytes and neutrophils are important players in the innate immune response and cooperate during infection and inflammation. In our study we analyzed the effects of neutrophil degranulation products (polymorphonuclear granulocytes degranulation products, PMN-DGP) on the activation, the adhesion and the migration of three bovine monocyte subsets, as well as their effects on monocyte-macrophage differentiation. Cross-linking of surface CD18 molecules on bovine PMN resulted in the release of primary, secondary and tertiary granules as well as of secretory vesicles. PMN-DGP induced a significant Ca2+-influx in classical (classical monocytes, cM) and intermediate monocytes (intermediate monocytes, intM) but not in non-classical monocytes (non-classical monocytes, ncM). A selective and up-regulated expression induced by PMN-DGP was only seen for CD11a and CD31 on intM. PMN-DGP induced a selective migration of intM in vitro. The presence of PMN-DGP during the differentiation of cM or intM into macrophages resulted in increased expression of membrane CD163 and reduced expression of MHC-II molecules. PMN-DGP-derived macrophages produced more IL-12 and IL-10 and showed enhanced phagocytosis and ROS production capacities. In conclusion, PMN-DGP selectively attract bovine intM and skew the functional maturation of cM and intM.

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