Mastitis diagnostics: quantitative PCR for Staphylococcus aureus genotype B in bulk tank milk

A novel real-time quantitative PCR assay for detecting the pathogenic and contagious Staphylococcus aureus genotype B (GTB) in bulk tank milk was developed and evaluated. The detection of this pathogen in bulk tank milk would greatly facilitate its control, as it is responsible for great economic loss in Swiss dairy herds. The assay is based on the simultaneous detection of 3 GTB-typical target sequences, including 2 enterotoxin genes and a polymorphism within the leucotoxin E gene. A variety of mastitis-associated bacteria was used to validate the assays, resulting in an analytical specificity of 100% and high repeatability. The analytical sensitivity in milk was 40 cfu/mL. An exponential association between simulated cow prevalence and quantitative PCR result was observed. An initial field study revealed 1 GTB-positive herd among the 33 studied herds. This novel assay for bulk tank milk analysis is suitable for routine purposes and is expected to be an effective tool for minimizing Staph. aureus GTB in Swiss dairy herds.

Mapping material distribution in a heterogeneous sand tank by image analysis

Water flow and solute transport through soils are strongly influenced by the spatial arrangement of soil materials with different hydraulic and chemical properties. Knowing the specific or statistical arrangement of these materials is considered as a key toward improved predictions of solute transport. Our aim was to obtain two-dimensional material maps from photographs of exposed profiles. We developed a segmentation and classification procedure and applied it to the images of a very heterogeneous sand tank, which was used for a series of flow and transport experiments. The segmentation was based on thresholds of soil color, estimated from local median gray values, and of soil texture, estimated from local coefficients of variation of gray values. Important steps were the correction of inhomogeneous illumination and reflection, and the incorporation of prior knowledge in filters used to extract the image features and to smooth the results morphologically. We could check and confirm the success of our mapping by comparing the estimated with the designed sand distribution in the tank. The resulting material map was used later as input to model flow and transport through the sand tank. Similar segmentation procedures may be applied to any high-density raster data, including photographs or spectral scans of field profiles.

Combined effects of heterogeneity, anisotropy, and saturation on steady state flow and transport: A laboratory sand tank experiment

Field soils show rather different spreading behavior at different water saturations, frequently caused by layering of the soil material. We performed tracer experiments in a laboratory sand tank. Such experiments complement and help comprehension of field investigations. We estimated, by image analysis, the first two moments of small plumes traveling through a two-dimensional, heterogeneous medium with strongly anisotropic correlation structure. Three steady state regimes were analyzed. Two main conclusions were drawn. First, low saturation led to very large heterogeneity and to strong preferential flow. Thus the description of the flow paths and the prediction of the solute arrival times require, in this case, more accurate knowledge about the topological structure. Second, saturation-dependent macroscopic anisotropy is an essential element of transport in unsaturated media. For this reason, small structural soil features should be properly upscaled to give appropriate effective soil parameters to be input in transport models.

Bovine mastitis: the diagnostic properties of a PCR-based assay to monitor the Staphylococcus aureus genotype B status of a herd, using bulk tank milk

Staphylococcus aureus genotype B (GTB) is a contagious mastitis pathogen in cattle, occurring in up to 87% of individuals. Because treatment is generally insufficient, culling is often required, leading to large economic loss in the Swiss dairy industry. As the detection of this pathogen in bulk tank milk (BTM) would greatly facilitate its control, a novel real-time quantitative PCR-based assay for BTM has previously been developed and is now being evaluated for its diagnostic properties at the herd level. Herds were initially classified as to their Staph. aureus GTB status by a reference method. Using BTM and herd pools of single-quarter and 4-quarter milk, the herds were then grouped by the novel assay, and the resulting classifications were compared. A total of 54 dairy herds were evaluated. Using the reference method, 21 herds were found to be GTB positive, whereas 33 were found to be negative. Considering the novel assay using both herd pools, all herds were grouped correctly, resulting in maximal diagnostic sensitivities (100%) and specificities (100%). For BTM samples, diagnostic sensitivities and specificities were 90 and 100%, respectively. Two herds were false negative in BTM, because cows with clinical signs of mastitis were not milked into the tank. Besides its excellent diagnostic properties, the assay is characterized by its low detection level, high efficiency, and its suitability for automation. Using the novel knowledge and assay, eradication of Staph. aureus GTB from a dairy herd may be considered as a realistic goal.

Serosurveillance of Schmallenberg virus in Switzerland using bulk tank milk samples.

Infections with Schmallenberg virus (SBV), a novel Orthobunyavirus transmitted by biting midges, can cause abortions and malformations of newborns and severe symptoms in adults of domestic and wild ruminants. Understanding the temporal and spatial distribution of the virus in a certain territory is important for the control and prevention of the disease. In this study, seroprevalence of antibodies against SBV and the spatial spread of the virus was investigated in Swiss dairy cattle applying a milk serology technique on bulk milk samples. The seroprevalence in cattle herds was significantly higher in December 2012 (99.5%) compared to July 2012 (19.7%). This high between-herd seroprevalence in cattle herds was observed shortly after the first detection of viral infections. Milk samples originating from farms with seropositive animals taken in December 2012 (n=209; mean 160%) revealed significantly higher S/P% ratios than samples collected in July 2012 (n=48; mean 103.6%). This finding suggests a high within-herd seroprevalence in infected herds which makes testing of bulk tank milk samples for the identification farms with past exposures to SBV a sensitive method. It suggests also that within-herd transmission followed by seroconversion still occurred between July and December. In July 2012, positive bulk tank milk samples were mainly restricted to the western part of Switzerland whereas in December 2012, all samples except one were positive. A spatial analysis revealed a separation of regions with and without positive farms in July 2012 and no spatial clustering within the regions with positive farms. In contrast to the spatial dispersion of bluetongue virus, a virus that is also transmitted by Culicoides midges, in 2008 in Switzerland, the spread of SBV occurred from the western to the eastern part of the country. The dispersed incursion of SBV took place in the western part of Switzerland and the virus spread rapidly to the remaining territory. This spatial pattern is consistent with the hypothesis that transmission by Culicoides midges was the main way of spreading.

Prevalence of antibodies against bluetongue virus serotype 8 in bulk-tank milk samples from dairy cattle herds located in risk areas for bluetongue virus transmission after a vaccination programme in Switzerland.

Switzerland had been affected by the bluetongue virus serotype 8 (BTV-8) epidemic in Europe in the years 2007 to 2009. After three years of mandatory vaccination and comprehensive surveillance, Switzerland showed to be free of BTV-8 in 2012. In the future Elisa testing of bulk-tank milk (BTM) samples as a very sensitive and cost-effective method should be used for the surveillance of all serotypes of BTV. To determine the prevalence of seropositive herds, BTM from 240 cattle herds was sampled in July 2012. The results showed an apparent seroprevalence of 98.7% in the investigated dairy herds. Most plausible, the high prevalence was caused by the vaccination campaigns rather than by infections with BTV-8. In the outbreak the cumulative number of BTV-8 cases in Switzerland had been 75.Thus it is very likely that the used inactivated vaccines induced long-term antibody titres. Due to the high seroprevalence, investigating for BT-antibodies cannot be used for early recognition of a new introduction of BTV at the moment. Nonetheless, testing of BTM samples is appropriate for an annual evaluation of the seroprevalence and especially as an instrument for early recognition for incursions as soon as the antibody prevalence declines.To determine this decline the BTM testing scheme should be conducted each year as described in this work.