Comparative profiling of the transcriptional response to iron restriction in six serotypes of Actinobacillus pleuropneumoniae with different virulence potential

Background Comparative analysis of gene expression among serotypes within a species can provide valuable information on important differences between related genomes. For the pig lung pathogen Actinobacillus pleuropneumoniae, 15 serotypes with a considerable variation in virulence potential and immunogenicity have been identified. This serotypic diversity can only partly be explained by amount of capsule and differences in the RTX toxin genes in their genomes. Iron acquisition in vivo is an important bacterial function and in pathogenic bacteria, iron-limitation is often a signal for the induction of virulence genes. We used a pan-genomic microarray to study the transcriptional response to iron restriction in vitro in six serotypes of A. pleuropneumoniae (1, 2, 3, 5b, 6, and 7), representing at least two levels of virulence. Results In total, 45 genes were significantly (p <0.0001) up-regulated and 67 genes significantly down-regulated in response to iron limitation. Not previously observed in A. pleuropneumoniae was the up-regulation of a putative cirA-like siderophore in all six serotypes. Three genes, recently described in A. pleuropneumoniae as possibly coding for haemoglobin-haptoglobin binding proteins, displayed significant serotype related up-regulation to iron limitation. For all three genes, the expression appeared at its lowest in serotype 3, which is generally considered one of the least virulent serotypes of A. pleuropneumoniae. The three genes share homology with the hmbR haemoglobin receptor of Neisseria meningitidis, a possible virulence factor which contributes to bacterial survival in rats. Conclusions By comparative analysis of gene expression among 6 different serotypes of A. pleuropneumoniae we identified a common set of presumably essential core genes, involved in iron regulation. The results support and expand previous observations concerning the identification of new potential iron acquisition systems in A. pleuropneumoniae, showing that this bacterium has evolved several strategies for scavenging the limited iron resources of the host. The combined effect of iron-depletion and serotype proved to be modest, indicating that serotypes of both moderate and high virulence at least in vitro are reacting almost identical to iron restriction. One notable exception, however, is the haemoglobin-haptoglobin binding protein cluster which merits further investigation

An evaluation of the ability of Dichelobacter nodosus to survive in soil

<p>Abstract</p> <p>Background</p> <p><it>Dichelobacter nodosus</it> is the causative agent of footrot in sheep. The survival of the bacterium in soil is of importance for the epidemiology of the disease. The investigation evaluates the survival of <it>D. nodosus</it> in soil with and without added hoof powder stored under different temperatures.</p> <p>Results</p> <p>An experimental setup was used with bacteriological culture and real-time polymerase chain reaction (PCR), and the results indicate that the bacteria can survive in soil for longer time than previously expected. The survival time was found to be dependent on temperature and the addition of hoof powder to the soil, with the longest survival time estimated to be 24 days in soil samples with hoof powder stored at 5°C.</p> <p>Conclusion</p> <p>Our findings indicate that the survival time of <it>D. nodosus</it> and its ability to infect susceptible sheep on pasture under different climatic conditions should be studied further.</p

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces

Within-day repeatability for absolute quantification of Lawsonia intracellularis bacteria in feces from growing pigs

Absolute quantification of Lawsonia intracellularis by real-time polymerase chain reaction (PCR) is now possible on a routine basis. Poor repeatability of quantification can result in disease status misclassification of individual pigs when a single fecal sample is obtained. The objective of the current study was to investigate overall variation within a day for fecal numbers of L. intracellularis bacteria determined by real-time PCR in growing pigs. From each of 30 pigs with an infection of L. intracellularis, 5 fecal samples were collected within 1 day. A total of 150 fecal samples were obtained and subjected to quantitative PCR (qPCR) testing. Mean fecal dry matter content was 14.3% (standard deviation = 4.5%). Two pigs (6.7%) alternated between being L. intracellularis qPCR positive and negative. For 28 pigs, the excreting levels of L. intracellularis were within the dynamic range of the qPCR assay at all sampling points. For these 28 pigs, the mean excretion level of L. intracellularis was 6.1 log10 bacteria/g feces (standard deviation = 1.2 log10 bacteria/g feces). The maximum observed difference between 2 fecal samples from the same pig was 1.1 log10 bacteria/g feces. The average standard deviation for individual pigs was 0.27 log10 bacteria/g feces. The average coefficient of variation within pigs was 0.04, ranging from 0.006 to 0.08. The results imply that absolute quantification of L. intracellularis by qPCR has acceptable repeatability within 1 day. However, a population-specific proportion of pigs alternating between positive and negative test results must be expected. </jats:p