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Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms.

ABSTRACT: BACKGROUND: In pigs little is known about the role of innate immune defence in bacterial infections of the respiratory tract, despite their major role in pig production. In the present study we characterized and compared in vitro and in vivo activation of immune markers of different pig breeds 7 days before, and 4 and 21 days after an experimental aerosol infection with Actinobacillus (A.) pleuropneumoniae. RESULTS: In vitro stimulation of bronchoalveolar lavage fluid (BALF) and blood leukocytes with A. pleuropneumoniae, Streptococcus suis, PMA and LPS led to production of different amounts of H2O2, NO and TNF-alpha, depending on the stimulus, individual, breed and time of infection. Generally, significant responses to in vitro stimulation were observed only in blood leukocytes, whereas the alveolar macrophages showed a high basal activation. In addition, the production of haptoglobin and cytokines (TNF-alpha, IFN-gamma and IL-10) in vivo was measured in plasma and BALF. Plasma haptoglobin levels mirrored the clinical manifestations at 4 days post-infection. In plasma and BALF TNF-alpha could not be detected, whereas variable levels of IFN-gamma were found at pre- and post-infection times. IL-10 was found in some plasma but in none of the BALF samples. The different expression levels in individuals within the breeds correlated for some markers with the severity of clinical manifestations, e.g. H2O2, plasma haptoglobin and BALF IFN-gamma for German Landrace pigs. CONCLUSION: Our findings revealed differences in the activation of the immune markers with respect to infection time, individuals and breeds. Moreover, results showed different correlation grades between the immune markers produced in vitro or in vivo and the clinical manifestations. Further analyses will have to show whether these markers may serve as correlates of protection against porcine respiratory infections.

SUBMITTER: Benga L

PROVIDER: S-EPMC2678107 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms.

Benga Laurentiu L Hoeltig Doris D Rehm Thomas T Rothkoetter Hermann-Josef HJ Pabst Reinhard R Valentin-Weigand Peter P

BMC veterinary research 20090421

<h4>Background</h4>In pigs little is known about the role of innate immune defence in bacterial infections of the respiratory tract, despite their major role in pig production. In the present study we characterized and compared in vitro and in vivo activation of immune markers of different pig breeds 7 days before, and 4 and 21 days after an experimental aerosol infection with Actinobacillus (A.) pleuropneumoniae.<h4>Results</h4>In vitro stimulation of bronchoalveolar lavage fluid (BALF) and blo ...[more]

PMID: 19383119

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Project description:Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, an important disease in the pig industry. Accurate and sensitive diagnostics such as DNA-based diagnostics are essential for preventing or responding to an outbreak. The specificity of DNA-based diagnostics depends on species-specific markers. Previously, an insertion element was found within an A. pleuropneumoniae-specific gene commonly used for A. pleuropneumoniae detection, prompting the need for additional species-specific markers. Herein, 12 marker candidates highly conserved (99 - 100% identity) among 34 A. pleuropneumoniae genomes (covering 13 serovars) were identified to be A. pleuropneumoniae-specific in silico, as these sequences are distinct from 30 genomes of 13 other Actinobacillus and problematic [Actinobacillus] species and more than 1700 genomes of other bacteria in the Pasteurellaceae family. Five marker candidates are within the apxIVA gene, a known A. pleuropneumoniae-specific gene, validating our in silico marker discovery method. Seven other A. pleuropneumoniae-specific marker candidates within the eamA, nusG, sppA, xerD, ybbN, ycfL, and ychJ genes were validated by polymerase chain reaction (PCR) to be specific to 129 isolates of A. pleuropneumoniae (covering all 19 serovars), but not to four closely related Actinobacillus species, four [Actinobacillus] species, or seven other bacterial species. This is the first study to identify A. pleuropneumoniae-specific markers through genome mining. Seven novel A. pleuropneumoniae-specific DNA markers were identified by a combination of in silico and molecular methods and can serve as additional or alternative targets for A. pleuropneumoniae diagnostics, potentially leading to better control of the disease. IMPORTANCE Species-specific markers are crucial for infectious disease diagnostics. Mutations within a marker sequence can lead to false-negative results, inappropriate treatment, and economic loss. The availability of several species-specific markers is therefore desirable. In this study, 12 DNA markers specific to A. pleuropneumoniae, a pig pathogen, were simultaneously identified. Five marker candidates are within a known A. pleuropneumoniae-specific gene. Seven novel markers can be used as additional targets in DNA-based diagnostics, which in turn can expedite disease diagnosis, assist farm management, and lead to better animal health and food security. The marker discovery strategy outlined herein requires less time, effort, and cost, and results in more markers compared with conventional methods. Identification of species-specific markers of other pathogens and corresponding infectious disease diagnostics are possible, conceivably improving health care and the economy.

Project description:The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kan(r)) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kan(r) gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Deltacps1N and strain 4074Deltacps1B, respectively. Strain 4074Deltacps1N produced no detectable CP, but strain 4074Deltacps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Deltacps1N to produce 4074Deltacps1N(pABcps101), 4074Deltacps1N(pJMLcps53), and 4074Deltacps1N(pABcps55), respectively. Strain 4074Deltacps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Deltacps1N(pJMLcps53) and 4074Deltacps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Deltacps1N(pABcps101) > or = strain 4074Deltacps1N > strain 4074Deltacps1B. Strain 4074Deltacps1N(pJMLcps53) was less virulent than strain 4074Deltacps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Dataset Information

Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms.

Publications

Expression levels of immune markers in Actinobacillus pleuropneumoniae infected pigs and their relation to breed and clinical symptoms.

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