Abstract
BackgroundActinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. However, research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample.ResultsHost and pathogen responses in pigs experimentally infected with A. pleuropneumoniae were analyzed by high-throughput RT-qPCR. This approach allowed concurrent analysis of selected genes encoding proteins known or hypothesized to be important in the acute phase of this infection. The expression of 17 bacterial and 31 porcine genes was quantified in lung samples obtained within the first 48 hours of infection. This provided novel insight into the early time course of bacterial genes involved in synthesis of pathogen-associated molecular patterns (lipopolysaccharide, peptidoglycan, lipoprotein) and genes involved in pattern recognition (TLR4, CD14, MD2, LBP, MYD88) in response to A. pleuropneumoniae. Significant up-regulation of proinflammatory cytokines such as IL1B, IL6, and IL8 was observed, correlating with protein levels, infection status and histopathological findings. Host genes encoding proteins involved in iron metabolism, as well as bacterial genes encoding exotoxins, proteins involved in adhesion, and iron acquisition were found to be differentially expressed according to disease progression. By applying laser capture microdissection, porcine expression of selected genes could be confirmed in the immediate surroundings of the invading pathogen.ConclusionsMicrobial pathogenesis is the product of interactions between host and pathogen. Our results demonstrate the applicability of high-throughput RT-qPCR for the elucidation of dual-organism gene expression analysis during infection. We showed differential expression of 12 bacterial and 24 porcine genes during infection and significant correlation of porcine and bacterial gene expression. This is the first study investigating the concurrent transcriptional response of both bacteria and host at the site of infection during porcine respiratory infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1557-6) contains supplementary material, which is available to authorized users.
Highlights
Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare
Histopathology At 6 h p.i. the lung tissue was affected by acute inflammation, characterized by hyperemia, bleeding, oedema, influx of neutrophils, numerous bacteria, fine threads of fibrin, and degeneration or necrosis of the alveolar septal cells (Figure 1B and C)
The observation of thickened alveolar septa in the control pigs is a common finding in conventional pigs and is considered to be a pulmonary response to environmental factors associated with swine production [48]
Summary
Actinobacillus pleuropneumoniae causes pleuropneumonia in pigs, a disease which is associated with high morbidity and mortality, as well as impaired animal welfare. To obtain in-depth understanding of this infection, the interplay between virulence factors of the pathogen and defense mechanisms of the porcine host needs to be elucidated. Research has traditionally focused on either bacteriology or immunology; an unbiased picture of the transcriptional responses can be obtained by investigating both organisms in the same biological sample. An unbiased picture of the interdependent transcriptional responses would be obtained by investigating both organisms in the same biological sample. Only a few studies employing a simultaneous characterization of concurrent host and pathogen gene expression during mammalian infection have been published [1,2,3,4]. The emergence of high-throughput qPCR systems, e.g. the BioMark from Fluidigm, offers a platform that is ideal for the focused, hypothesis-driven study of gene expression of even small quantities of RNA [6,7]. The high capacity of such platforms enables the researcher to cover diverse areas – as well as organisms – of interest in the same experimental setup
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