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DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae.

ABSTRACT: A DNA microarray system was prepared and shown to facilitate identification and typing of Actinobacillus pleuropneumoniae. The DNA microarray, composed of 18 DNA polymerase chain reaction (PCR) amplicons printed on glass slides and arranged in 3 subarrays, was developed. These target DNA included 1 or multiple fragments of the outer membrane lipoprotein, apx toxin, capsular polysaccharide, and disulfide bound formation protein E (dsbE)-like genes of A. pleuropneumoniae. These arrayed target DNA retained their expected hybridization properties. The hybridization signal intensities ranged from the least-intense to the most-intense, 4626 to 9789 arbitrary fluorescence units, respectively. Cy3-probes of A. pleuropneumoniae strains labeled with multiplex PCR were hybridized to the DNA microarray. A total of 51 different A. pleuropneumoniae strains representing serotype 1 to 12 reference strains and clinical isolates were detected and typed by the DNA microarray. Twelve reference serotypes produced 11 distinct target DNA hybridization patterns, and hybridization patterns of serotypes 1 (n = 7), 3 (n = 5), and 7 (n = 6) field isolates were identical to hybridization patterns of reference serotypes 1, 3, and 7, respectively. Non-serotyped isolates 4, 6, and 11 (out of 21) from diseased pigs had identical hybridization patterns to reference serotypes 3, 7, and 1, respectively. The results show that the DNA microarray system described in the present study is a valuable tool for identifying and typing reference strains and isolates of A. pleuropneumoniae, and enables relatively rapid identification of non-serotyped isolates.

SUBMITTER: Xiao G

PROVIDER: S-EPMC2705073 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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DNA microarray-based identification and typing of Actinobacillus pleuropneumoniae.

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