Research Article: Molecular characterisation of the early response in pigs to experimental infection with Actinobacillus pleuropneumoniae using cDNA microarrays

Date Published: April 27, 2007

Publisher: BioMed Central

Author(s): Jakob Hedegaard, Kerstin Skovgaard, Shila Mortensen, Peter Sørensen, Tim K Jensen, Henrik Hornshøj, Christian Bendixen, Peter MH Heegaard.

http://doi.org/10.1186/1751-0147-49-11

Abstract

The bacterium Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a widespread, highly contagious and often fatal respiratory disease of pigs. The general porcine innate immune response after A. pleuropneumoniae infection is still not clarified. The objective of this study was hence to characterise the transcriptional response, measured by using cDNA microarrays, in pigs 24 hours after experimental inoculation with A. pleuropneumoniae.

Microarray analyses were conducted to reveal genes being differentially expressed in inflamed versus non-inflamed lung tissue sampled from inoculated animals as well as in liver and tracheobronchial lymph node tissue sampled from three inoculated animals versus two non-inoculated animals. The lung samples were studied using a porcine cDNA microarray with 5375 unique PCR products while liver tissue and tracheobronchial lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products.

A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus non-infected animals and 130 genes differed in expression in tracheobronchial lymph node tissue from infected versus non-infected animals. Among these genes, several have previously been described to be part of a general host response to infections encoding immune response related proteins. In inflamed lung tissue, genes encoding immune activating proteins and other pro-inflammatory mediators of the innate immune response were found to be up-regulated. Genes encoding different acute phase reactants were found to be differentially expressed in the liver.

The obtained results are largely in accordance with previous studies of the mammalian immune response. Furthermore, a number of differentially expressed genes have not previously been associated with infection or are presently unidentified. Determination of their specific roles during infection may lead to a better understanding of innate immunity in pigs. Although additional work including more animals is clearly needed to elucidate host response to porcine pleuropneumonia, the results presented in this study demonstrate three subsets of genes consistently expressed at different levels depending upon infection status.

Partial Text

Respiratory infectious diseases present a major problem in modern pig production with severe effects on both animal welfare and production economy [1]. The Gram negative bacterium Actinobacillus pleuropneumoniae is an inhabitant of the upper porcine respiratory tract and is the causative agent of porcine pleuropneumonia, a frequent respiratory infection which is highly infectious, often fatal and characterized by necrotizing, hemorrhagic bronchopneumonia and serofibrinous pleuritis [1]. Infection of the porcine lung with A. pleuropneumoniae has previously been reported to result in a local production of proinflammatory proteins or mRNA encoding the cytokines interleukin (IL) -1α, IL-1β, IL-6 and the chemokine IL-8 [2-5]. Likewise bioactive protein and/or mRNA coding for IL10, IL12p35, TNF-α˙
MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacuaHXoqygaqgaaaa@2E6B@ and INFα˙
MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacuaHXoqygaqgaaaa@2E6B@ have been shown to be up-regulated after infection with A. pleuropneumoniae in vivo or in vitro [2-8]. These studies have focused on a few selected genes using techniques such as quantitative real-time reverse transcriptase polymerase chain reactions (RT-PCR), northern blotting or in-situ hybridisation. The introduction of techniques for simultaneous measurements of gene expression for thousands of genes in a single analysis using microarrays allows a more comprehensive picture of the host response during infection with A. pleuropneumoniae. Using cDNA microarrays Moser and co-workers found 307 anonymous transcripts in blood leukocytes from pigs to be significantly affected after experimental infection with A. pleuropneumoniae [9].

Transcriptional profiling using DNA microarray technology has been extensively used for studying host response to pathogenic microorganisms [23,24]. Moser and co-workers [9] studied the gene expression in porcine peripheral blood leukocytes as a response to infection by A. pleuropneumoniae using cDNA microarrays. A total of 18 pigs were experimentally infected with A. pleuropneumoniae and based on principal components analyses of seven mainly phenotypic key performance measurements, two extreme-performing animals were selected and analyzed further using cDNA microarrays. Analysis of the gene expression change from 0 to 24 hours post-challenge revealed 307 anonymous genes to be significantly affected. The results presented here are in agreement with this as numerous genes were found to be significantly differentially expressed in liver, lung and tracheobronchial lymph nodes depending on infection status.

The gene expression response was characterised in pigs challenged with the respiratory tract pathogen A. pleuropneumoniae. Although additional work including more animals is clearly needed to study the host response to this infection, the obtained results demonstrate three subsets of genes consistently expressed at different levels depending upon infection status. Two-way cluster analysis of these subsets indicated that the expression profiles of the samples may be associated with the severity of pathological changes. In inflamed lung tissue, immune activating genes and other pro-inflammatory mediators of the innate immune response were found up-regulated. In the liver of infected animals, genes that are well known to be regulated as part of the acute phase response were found to be differentially expressed. A number of genes identified in this study to be affected by infection have not previously been associated with infection or are presently unidentified. Determination of their specific roles during infection may lead to a better understanding of innate immunity in pigs.

The author(s) declare that they have no competing interests.

KS and JH contributed equally to the work and should be considered as joint first authors. JH designed and carried out the microarray analyses, conducted the statistical analysis, participated in the biological interpretation and drafted the manuscript. KS designed and carried out the experimental infections, carried out the microarray analyses, participated in the biological interpretation and in drafting the manuscript. SM carried out the experimental infections and participated in the microarray analyses. PS and HH participated in the statistical analyses. TKJ carried out the experimental infections. CB participated in drafting the manuscript. PMHH participated in the biological interpretation and in drafting the manuscript. All authors read and approved the final manuscript.

 

Source:

http://doi.org/10.1186/1751-0147-49-11

 

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