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Additional file 1. Methods for sample size determination and sampling weights.

Additional file 5: Figure S5. StrainRecon reconstruction on laboratory-mixed samples given advance knowledge of n = 3 strains. Ground truth of barcodes (first row in each figure), the MAP estimate of the reconstruction matrix M and mixture vector v (second row), and the mean (third row) and standard deviation (fourth row) of the posterior density of candidate (M, v) solutions. Each block contains one row for each strain, ordered by decreasing frequency from top, with a SNP cell color ranging from purple (fraction of 0) to bright yellow (fraction of 1). The figure showcases the algorithm outputs with n = 3 strains on a variety of samples and mixtures, including cases of accurate and unique strain reconstruction (such as B33_PA_DBS).

Additional file 4: Figure S4. StrainRecon reconstruction on laboratory-mixed samples. Ground truth of barcodes (first row in each figure), the MAP estimate of the reconstruction matrix M and mixture vector v (second row), and the mean (third row) and standard deviation (fourth row) of the posterior density of candidate (M, v) solutions. Each block contains one row for each strain, ordered by decreasing frequency from top, with a SNP cell color ranging from purple (fraction of 0) to bright yellow (fraction of 1). Each reconstruction is shown as the input parameter n (of the number of strains) is varied from n = 1 and 4 (row-major order) to be reconstructed in StrainRecon on the B_24NOPA_DBS sample. The dominant strain is captured perfectly and with high confidence by considering the posterior statistics. The algorithm has difficulty reconstructing the other two less-prevalent strains, since their target range of

Additional file 1: Figure S1. RF classification using laboratory strains. Figure shows proportion of each unique amplicon relative to all amplicons from the targeted region on a plate from all laboratory strain samples based on RF classifier (when random selection determined the training set). X axis is the log10 count of SNP-specific reads in a Miseq run and y axis is log10 count of unique amplicon-specific reads in that individual run. Red points are amplicons classified as negative (False) for exclusion while green points are amplicons classified as positive (True) for further SNP frequency analysis.

Additional file 3: Figure S3. Plot SNP23 from Combination A mixes. Plot SNP23 from Combination A mixes was made separately from all other SNPs. Data is also separated by source material (DBS and frozen blood samples). Within each mixture, left to right, the colors are: blue—all strains identical, orange—dominant strain unique, green—intermediate strain unique, yellow—low strain unique, and gray—SNP23 in Combination A mixes. A pilot experiment was also conducted using a different culture source, and this difference at SNP23 was not observed. Based on the consistency among all other SNP sites and the difference between parasite batches, SNP23 from Combination A was excluded from the Fig. 3 analysis concerning two distinct SNP reads.

Mycobacterial diseases are persistent and characterized by lengthy latent periods. Thus, epidemiological models require careful delineation of transmission routes. Understanding transmission routes will improve the quality and success of control programs. We aimed to study the infection dynamics of Mycobacterium avium subsp. paratuberculosis (MAP), the causal agent of ruminant Johne&rsquo, s disease, and to distinguish within-host mutation from individual transmission events in a longitudinally MAP-defined dairy herd in upstate New York. To this end, semi-annual fecal samples were obtained from a single dairy herd over the course of seven years, in addition to tissue samples from a selection of culled animals. All samples were cultured for MAP, and multi-locus short-sequence repeat (MLSSR) typing was used to determine MAP SSR types. We concluded from these precise MAP infection data that, when the tissue burden remains low, the majority of MAP infections are not detectable by routine fecal culture but will be identified when tissue culture is performed after slaughter. Additionally, we determined that in this herd vertical infection played only a minor role in MAP transmission. By means of extensive and precise longitudinal data from a single dairy herd, we have come to new insights regarding MAP co-infections and within-host evolution.

International audience; AbstractThe objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large scale meta-analysis of both natural and experimental infections. Large difference in shedding patterns between experimentally and naturally infected cows were observed. Experimental infections are thus probably driven by different pathological mechanisms. For further evaluations of shedding patterns only natural infections were used. Within such infections, the transition to high shedding was studied as a proxy to the development of a clinical disease. The majority of studied cows never developed high shedding levels. Those that do, typically never reduced their shedding level to low or no shedding. Cows that eventually became high shedders showed a pattern of continuous shedding. In contrast, cows with an intermittent shedding pattern had a low probability to ever become high shedders. In addition, cows that start shedding at a younger age (less than three years of age) have a lower hazard of becoming high shedders compared to cows starting to shed at an older age. These data suggest the presence of three categories of immune control. Cows that are intermittent shedders have the infection process under control (no progressive infection). Cows that start shedding persistently at a young age partially control the infection, but eventually will be high shedders (slow progressive infection), while cows that start shedding persistently at an older age cannot effectively control the infection and become high shedders rapidly.