An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.

Documentary sources to investigate multidecadal variability of droughts

Droughts are probably the natural hazard with the highest socioeconomic impact. Simultaneously, they are a very complex phenomenon; they are triggered by a diversity of physical factors and occur at a variety of time scales. Consequently, the instrumental record currently available is too short and the characterization of its multidecadal variability requires the use of natural proxies (tree rings, sedimentary records) or documentary sources. In this paper we analyse three documentary sources with potential to analyse the long-term variability of droughts: chapter acts, logbooks and chronicles. The chapter acts recorded discussions and decisions made during the assemblies of the local authorities and provide continuous and direct evidence on drought impacts. They are especially useful to study droughts between the 15th and the 19th centuries in Europe and the 17th to 18th in the former colonies. Logbooks recorded the meteorological conditions and the incidents occurred during navigation. They provide indirect information through the circulation indices that can be very helpful to understand the mechanisms and teleconnections associated to droughts. Finally, the chronicles are historiographical documents describing political and social events. They are secondary sources and the references to climatic events are discontinuous, thus their analysis must be extremely careful, but they are especially useful to study specific drought events especially prior to 15th century when no other sources are available.