A Functionally Different Immune Phenotype in Cattle Is Associated With Higher Mastitis Incidence.

A novel vaccine against bovine viral diarrhea (BVD) induced pathogenic antibody production in 5-10% of BVD-vaccinated cows. Transfer of these antibodies via colostrum caused Bovine neonatal pancytopenia (BNP) in calves, with a lethality rate of 90%. The exact immunological mechanisms behind the onset of BNP are not fully understood to date. To gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (BNP cows) and non-responders. After in vitro stimulation of peripheral blood derived lymphocytes (PBL), we detected distinctly deviant expression levels of several master regulators of immune responses in BNP cells, pointing to a changed immune phenotype with severe dysregulation of immune response in BNP cows. Interestingly, we also found this response pattern in 22% of non-BVD-vaccinated cows, indicating a genetic predisposition of this immune deviant (ID) phenotype in cattle. We additionally analyzed the functional correlation of the ID phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. The significantly increased prevalence of mastitis among ID cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections.

A New Nanobody-Based Biosensor to Study Endogenous PARP1 In Vitro and in Live Human Cells.

Poly(ADP-ribose) polymerase 1 (PARP1) is a key player in DNA repair, genomic stability and cell survival and it emerges as a highly relevant target for cancer therapies. To deepen our understanding of PARP biology and mechanisms of action of PARP1-targeting anti-cancer compounds, we generated a novel PARP1-affinity reagent, active both in vitro and in live cells. This PARP1-biosensor is based on a PARP1-specific single-domain antibody fragment (~ 15 kDa), termed nanobody, which recognizes the N-terminus of human PARP1 with nanomolar affinity. In proteomic approaches, immobilized PARP1 nanobody facilitates quantitative immunoprecipitation of functional, endogenous PARP1 from cellular lysates. For cellular studies, we engineered an intracellularly functional PARP1 chromobody by combining the nanobody coding sequence with a fluorescent protein sequence. By following the chromobody signal, we were for the first time able to monitor the recruitment of endogenous PARP1 to DNA damage sites in live cells. Moreover, tracing of the sub-nuclear translocation of the chromobody signal upon treatment of human cells with chemical substances enables real-time profiling of active compounds in high content imaging. Due to its ability to perform as a biosensor at the endogenous level of the PARP1 enzyme, the novel PARP1 nanobody is a unique and versatile tool for basic and applied studies of PARP1 biology and DNA repair.

Direct and dynamic detection of HIV-1 in living cells.

In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA) at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

Association between floor type and behaviour, skin lesions, and claw dimensions in group-housed fattening bulls.

Rubber mats covering concrete slatted flooring are a developing market in dairy barns but remain rare in beef cattle facilities. The aim of this study was to investigate the effects of covering slatted concrete floor with perforated rubber mats on behaviour and occurrence of skin and claw lesions in fattening bulls. The groups of six bulls each with a total average age of 9.8 months were kept over 1 year on either slatted concrete (CONCRETE PEN) or on slatted concrete covered completely (RUBBER PEN) or partially (CHOICE PEN) with perforated rubber mats. Every quarter-year, behaviour (preference of flooring, lying, aggression, mounting) was recorded. In two-weekly intervals the incidence of skin lesions was examined. At 12 and 18 months of age the rising time of the bulls was measured. At the beginning of the study and after slaughter claw dimensions were recorded. Bulls in the CHOICE PEN preferred (P<0.01) the rubber coated area throughout the experiment. Animals in the RUBBER and the CHOICE PENS showed more lying periods (P<0.01) and had a lesser incidence of skin lesions (P<0.01) compared to bulls in the CONCRETE PEN. Bulls in the CHOICE PEN needed less time for rising (2.7+/-0.3s) than bulls in the CONCRETE PEN (4.4+/-0.5s, P<0.01). Net claw growth differed significantly between all pens (RUBBER>CHOICE>CONCRETE; P<0.05). In conclusion, the results of this study indicate that rubber coated slatted flooring has a positive influence on the housing conditions of beef cattle.

Mapping of the bovine spinal muscular atrophy locus to Chromosome 24.

A hereditary form of spinal muscular atrophy (SMA) caused by an autosomal recessive gene has been reported for American Brown-Swiss cattle and in advanced backcrosses between American Brown-Swiss and many European brown cattle breeds. Bovine SMA (bovSMA) bears remarkable resemblance to the human SMA (SMA1). Affected homozygous calves also show progressive symmetric weakness and neurogenic atrophy of proximal muscles. The condition is characterized by severe muscle atrophy, quadriparesis, and sternal recumbency as result of neurogenic atrophy. We report on the localization of the gene causing bovSMA within a genomic interval between the microsatellite marker URB031 and the telomeric end of bovine Chromosome (Chr) 24 (BTA24). Linkage analysis of a complex pedigree of German Braunvieh cattle revealed a recombination fraction of 0.06 and a three-point lod score of 11.82. The results of linkage and haplotyping analysis enable a marker-assisted selection against bovSMA based on four microsatellite markers most telomeric on BTA24 to a moderate accuracy of 89-94%. So far, this region is not orthologous to any human chromosome segments responsible for twelve distinct disease phenotypes of autosomal neuropathies. Our results indicate the apoptosis-inhibiting protein BCL2 as the most promising positional candidate gene causing bovSMA. Our findings offer an attractive animal model for a better understanding of human forms of SMA and for a probable anti-apoptotic synergy of SMN-BCL2 aggregates in mammals.