The role of phosphatidylinositol mannosides in the serological diagnosis of mycobacterial infections

Accurate diagnosis of mycobacterial infections, such as bovine tuberculosis and paratuberculosis, remains challenging. Available direct diagnostic tests aimed at detecting the pathogen are highly specific but lack sensitivity, depending on the stage of infection and the prevalence of infection in a population. The sensitivity of indirect diagnostic assays that measure the host immune response to infection is similarly affected by disease characteristics. The choice of antigen used to detect a host response to infection has a critical impact on test sensitivity and specificity. Many indirect tests rely on crude antigen preparations and cell-free extracts, of which the production is poorly standardized. Moreover, these preparations contain ample uncharacterized cross-reactive compounds. To enhance serological test specificity, existing assays depend on the pre-treatment of samples and a relatively high cut-off value, that in turn influences test sensitivity. Research therefore focuses on the identification of more specific, defined antigens to improve diagnostics. In the current study, we extracted phosphatidylinositol mannosides (PIMs) and investigated their potential use in antibody-based tests. Our results demonstrate that specific IgG class antibodies are generated against PIMs in cows, but this is unrelated to tuberculosis or paratuberculosis infection status, making these antigens unsuitable for diagnostic applications. In addition, we demonstrate that PIMs are widely present in crude antigen preparations and in serum pre-absorption buffer. Our results indicate that PIMs are cross-reactive compounds with immunodominant B cell epitopes that could impair serological test specificity

The role of phosphatidylinositol mannosides in the serological diagnosis of mycobacterial infections

Accurate diagnosis of mycobacterial infections, such as bovine tuberculosis and paratuberculosis, remains challenging. Available direct diagnostic tests aimed at detecting the pathogen are highly specific but lack sensitivity, depending on the stage of infection and the prevalence of infection in a population. The sensitivity of indirect diagnostic assays that measure the host immune response to infection is similarly affected by disease characteristics. The choice of antigen used to detect a host response to infection has a critical impact on test sensitivity and specificity. Many indirect tests rely on crude antigen preparations and cell-free extracts, of which the production is poorly standardized. Moreover, these preparations contain ample uncharacterized cross-reactive compounds. To enhance serological test specificity, existing assays depend on the pre-treatment of samples and a relatively high cut-off value, that in turn influences test sensitivity. Research therefore focuses on the identification of more specific, defined antigens to improve diagnostics. In the current study, we extracted phosphatidylinositol mannosides (PIMs) and investigated their potential use in antibody-based tests. Our results demonstrate that specific IgG class antibodies are generated against PIMs in cows, but this is unrelated to tuberculosis or paratuberculosis infection status, making these antigens unsuitable for diagnostic applications. In addition, we demonstrate that PIMs are widely present in crude antigen preparations and in serum pre-absorption buffer. Our results indicate that PIMs are cross-reactive compounds with immunodominant B cell epitopes that could impair serological test specificity.</p

Analysis of biobanked serum from a mycobacterium avium subsp paratuberculosis bovine infection model confirms the remarkable stability of circulating mirna profiles and defines a bovine serum mirna repertoire

Johne's Disease (JD) is a chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). Current disease control strategies are hampered by the lack of sensitive and specific diagnostic modalities. Therefore, novel diagnostic and prognostic tools are needed, and circulating microRNAs (miRNAs) may hold potential in this area. The aims of this study were twofold: (i) to address the stability of miRNA in bovine sera from biobanked samples, and (ii) to assess the potential of miRNAs as biomarkers for JD disease progression. To address these aims we used bovine sera from an experimental MAP infection model that had been stored at -20°C for over a decade, allowing us to also assess the stability of miRNA profiles in biobanked serum samples through comparison with fresh sera. Approximately 100â€"200 intact miRNAs were identified in each sample with 83 of these being consistently detected across all 57 samples. The miRNA profile of the biobanked sera stored at -20°C for over 10 years was highly similar to the profile o

The role of phosphatidylinositol mannosides in the serological diagnosis of mycobacterial infections

Accurate diagnosis of mycobacterial infections, such as bovine tuberculosis and paratuberculosis, remains challenging. Available direct diagnostic tests aimed at detecting the pathogen are highly specific but lack sensitivity, depending on the stage of infection and the prevalence of infection in a population. The sensitivity of indirect diagnostic assays that measure the host immune response to infection is similarly affected by disease characteristics. The choice of antigen used to detect a host response to infection has a critical impact on test sensitivity and specificity. Many indirect tests rely on crude antigen preparations and cell-free extracts, of which the production is poorly standardized. Moreover, these preparations contain ample uncharacterized cross-reactive compounds. To enhance serological test specificity, existing assays depend on the pre-treatment of samples and a relatively high cut-off value, that in turn influences test sensitivity. Research therefore focuses on the identification of more specific, defined antigens to improve diagnostics. In the current study, we extracted phosphatidylinositol mannosides (PIMs) and investigated their potential use in antibody-based tests. Our results demonstrate that specific IgG class antibodies are generated against PIMs in cows, but this is unrelated to tuberculosis or paratuberculosis infection status, making these antigens unsuitable for diagnostic applications. In addition, we demonstrate that PIMs are widely present in crude antigen preparations and in serum pre-absorption buffer. Our results indicate that PIMs are cross-reactive compounds with immunodominant B cell epitopes that could impair serological test specificity

Canonical and dominant isomiR differences.

<p>A) bta-miR-22-3p total abundance profile over time (left) and for 2 isomiRs (right), both are shorter 3’ variants. Analysing these isomiRs individually allows them to be detected as being differentially expressed over time. B) bta-miR-22-3p isomiR relative abundances. The canonical form, labelled <i>exact</i>, is only fourth most abundant. C) The canonical and dominant isomiRs differ in >50% of cases. Shown in the plot are the corresponding percentages between the 2 isoforms, when different. (By definition <i>dominant</i> means highest percentage of reads). For points in the top left of the plots, the actual canonical form is insignificant. Note the lower number of points for the 10–15 year (CVI) dataset.</p

Comparisons between miRNAs from fresh and biobanked serum.

<p>A) Fractions of small RNA identified in both fresh (-80°C/<1 year storage) and biobanked (-20^°C/>10 year storage) according to category using Bowtie mapping. UMD3.1 denotes mappings to the reference genome. miRNA fractions are shown inset in detail. Note these values slightly underestimate the total miRNAs present since they only include miRBase hairpins and not novel content. B) Overlap between fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) serum core miRNAs and a bovine macrophage dataset. C) Comparison of top 8 miRNAs in fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) samples shows large differences in the top 2 most abundant miRNAs. Counts are the means of normalised value per sample and error bars show the standard deviation over all n samples. For fresh samples n = 24, for biobanked samples n = 57. D) Correlation between log mean normalised counts for the same miRNA in fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) datasets. Error bars indicate the variance over all samples of mean read count.</p

Monitoring of the MAP experimental infection.

<p><b>(A)</b> Detection of anti-MAP antibodies in serum via ELISA across 49 months of the time-course. Six individual experimentally infected cattle (in red A-F) and six individual naturally infected cattle (in green G-L) are represented. Faecal culture data for <b>(B)</b> experimentally infected and <b>(C)</b> naturally infected animals at monthly intervals. 0 = negative, 1 = 1 cfu/ agar slant, 2 = <50 cfu/agar slant, 3 = <100 cfu/agar slant and 4 = >100 cfu agar slant. C = fungal contamination, resulting in an inability to accurately determine MAP cfus.</p

Normalised read variation for differentially abundant miRNA over 5 time points.

<p>Normalised reads are shown for distinct miRNA at the 0, 6, 43, 46 and 46-month time points. Note the x axis time interval is not to scale.</p

IsomiR abundances and variant representation in different datasets.

<p>A) IsomiR normalised counts compared between 1) fresh (-80°C/<1 year storage) and biobanked (-20°C/>10 year storage) bovine serum, 2) serum and macrophage (both bovine) and 3) bovine and human serum. B) Classification scheme for isomiRs using sRNAbench is hierarchical. <i>exactNucVar</i> means single nucleotide changes to the canonical sequence, most probably due to sequencing errors. <i>mv</i> indicates shifted sequences. non-templated addition is enzymatically addition of a nucleotide to the 3’ end and is given priority by sRNAbench since these changes may be of biological relevance. C) Plot shows the counts of dominant isomiRs categorised by class. The general trend of dominance is the same across all datasets, including non-serum.</p

isomiR abundance patterns compared between fresh and biobanked serum.

<p>A) Relative abundances of isomiRs with a single dominant form in the fresh (-80°C/<1 year storage) dataset but not in the biobanked (-20°C/>10 year storage) data. Note that many of the low abundance forms are not present in the biobanked data (green bars). B) Relative abundances (shown as fraction of total) of some miRNAs with multiple sub-dominant isomiRs compared between fresh and biobanked year samples.</p