Twenty years of active bat rabies surveillance in Germany: a detailed analysis and future perspectives.

SUMMARY: In Germany, active bat rabies surveillance was conducted between 1993 and 2012. A total of 4546 oropharyngeal swab samples from 18 bat species were screened for the presence of EBLV-1- , EBLV-2- and BBLV-specific RNA. Overall, 0·15% of oropharyngeal swab samples tested EBLV-1 positive, with the majority originating from Eptesicus serotinus. Interestingly, out of seven RT-PCR-positive oropharyngeal swabs subjected to virus isolation, viable virus was isolated from a single serotine bat (E. serotinus). Additionally, about 1226 blood samples were tested serologically, and varying virus neutralizing antibody titres were found in at least eight different bat species. The detection of viral RNA and seroconversion in repeatedly sampled serotine bats indicates long-term circulation of the virus in a particular bat colony. The limitations of random-based active bat rabies surveillance over passive bat rabies surveillance and its possible application of targeted approaches for future research activities on bat lyssavirus dynamics and maintenance are discussed.

Accuracy of peripheral quantitative computed tomography (pQCT) for assessing area and density of mouse cortical bone.

Peripheral quantitative computed tomography (pQCT) is increasingly used for measurement of cortical bone geometry and density in mice. We evaluated the accuracy of pQCT for area and density measurements of thin-walled aluminum phantoms and mouse femora. Aluminum tubes with varying wall thicknesses and femora from 1- to 6-month-old C3H/HeJ (C3H) and C57B1/6J (B6) mice (average cortical thickness 0.14-0.29 mm) were scanned at 70- or 90-microm resolution. pQCT values of area were compared to optical values determined after sectioning, while pQCT density (vBMD) was compared to solid aluminum density or correlated to bone ash content. For the aluminum phantoms, the error in pQCT area and density depended strongly on wall thickness, and density was consistently underestimated. For mouse femora, threshold values were found that produced zero error in bone area for each strain and age group, although the optimal threshold differed between groups. pQCT vBMD correlated strongly with ash content (r2=0.7), although the regression equations differed between strains and the magnitude of the inter-strain difference in vBMD was fourfold greater than the difference in ash content. This finding suggests that pQCT can overestimate the differences in volumetric mineral density between inbred mouse strains whose bones are of different thickness (e.g., C3H vs. B6). In conclusion, both area and density values obtained by pQCT depend strongly on specimen thickness, consistent with a partial volume averaging artifact. Investigators using pQCT to assess cortical bones in mice should be aware of the potential for cortical thickness-dependent errors.

Phenotypic characterization of mice bred for high and low peak bone mass.

In humans, peak bone mineral density (BMD) is a highly heritable trait and a strong determinant of subsequent osteoporotic fracture risk. To identify the genetic factors responsible for variation in peak BMD, investigators have turned to animal models. In this study we examined the heritability of BMD acquisition and characterized differences in skeletal geometry, histomorphometry, and biomechanical competence between two lines of mice artificially selected for extremes of peak whole body BMD. F2 progeny from a cross between C57BL/6 and DBA/2 inbred strains was used as the foundation population to develop lines selected for either high or low BMD. Whole body BMD was measured by dual-energy X-ray absorptiometry (DXA). By the third generation of selection, highest-scoring BMD (HiBMD) mice exhibited 14% greater peak BMD than lowest-scoring BMD (LoBMD) mice. The mean realized heritability of peak BMD was 36%. Femoral shaft cortical area and thickness and vertebral cancellous bone volume (BV) were significantly greater (16-30%) in the HiBMD line compared with the LoBMD line. Mean cancellous bone formation rates (BFRs) were 35% lower in HiBMD mice compared with LoBMD mice. Failure load and stiffness in the femoral shaft, femoral neck, and L6 vertebrae were all substantially greater (by 25-190%) in HiBMD mice. Thus, these divergently selected murine lines serve to illustrate some of the means by which genetic mechanisms can affect skeletal structure and remodeling. Identification of the individual genes influencing peak BMD in this experimental system will likely reveal some of the genetic determinants of overall bone strength.

Evidence for the tissue inhibitor of metalloproteinases-1 (TIMP-1) in human polymorphonuclear leukocytes.

A tissue inhibitor of metalloproteinases (TIMP)-1 was isolated from human polymorphonuclear leukocytes (PMNL) in a complex with latent 95-kDa gelatinase (matrixmetalloproteinase, MMP-9). It was separated from the enzyme by gel filtration in the presence of SDS. Using a competitive ELISA procedure, we determined that 10% of the isolated gelatinase was complexed with TIMP-1. The presence of the inhibitor in isolated PMNL could also be demonstrated by indirect immunofluorescence using a specific antibody against TIMP-1. Cellular mRNA was isolated from PMNL, which were highly purified by separation via formylMet-Leu-Pro-stimulated chemotactic migration in a Boyden chamber. Using reverse-transcription PCR and Northern blotting, TIMP-1 mRNA was shown to be present in PMNL, suggesting that these cells are also capable of synthesizing TIMP-1.

Granulocyte chemotaxis measured in a Boyden chamber assay by quantification of neutrophil elastase.

The human polymorphonuclear leukocyte reaction in response to chemotactic or chemokinetic stimulation is often assayed using the Boyden chamber technique. We present a quick and reliable method for evaluating Boyden chamber experiments, which avoids time-consuming cell counting and does not require expensive equipment. This method is based on assaying human neutrophil elastase, a serine protease derived from polymorphonuclear leukocytes. We tested the method in different types of Boyden chambers equipped with two superimposed filters or a filter amnion membrane combination. The chambers were incubated with the cells for 2 h then dismantled and the elastase activity in supernatant, filters or membrane was assayed. The results were compared with the results obtained by cell counting, or measured by determination of myeloperoxidase. There was a good correlation between the cell count and elastase technique (r = 0.90), but the elastase method achieved higher intra- and interassay precision. Myeloperoxidase and elastase results also correlated well (r = 0.94) and showed comparable intra- and inter-assay precision. With the elastase method it was also possible to quantify polymorphonuclear leukocyte reactions on an amnion membrane surface. In amnion membrane assays the percentage of cells which reacted in response to formyl-peptide stimulation was not altered by varied cell concentrations, and polymorphonuclear leukocytes showed little unstimulated adherence or migration.