Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA

Schaller, A.; Djordjevic, S. P.; Eamens, G. J.; Forbes, W. A.; Kuhn, R.; Kuhnert, Peter; Gottschalk, M.; Nicolet, J.; Frey, Joachim (2001). Identification and detection of Actinobacillus pleuropneumoniae by PCR based on the gene apxIVA. Veterinary microbiology, 79(1), pp. 47-62. Amsterdam: Elsevier 10.1016/S0378-1135(00)00345-X

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The apxIVA gene, a recently discovered RTX determinant of Actinobacillus pleuropneumoniae, was shown to be species-specific. DNA hybridization experiments using probes for various regions of apxIVA revealed that the 3'-terminus of this gene was present in all 14 serotypes of A. pleuropneumoniae but absent from phylogenetically related species. A primer pair spanning this region specifically amplified a 422bp fragment in PCR experiments with DNA from the reference strains of the 14 serotypes and 194 field strains isolated from various geographic locations worldwide. DNA sequence analysis of PCR products derived from all serotypes were identical except in serotypes 3, 8, and 10, which showed minor differences. The PCR did not amplify any product when DNA from 17 different bacterial species closely related to A. pleuropneumoniae was used as template. In addition, the PCR was negative with DNA of several Actinobacillus sp. which were initially characterized as A. pleuropneumoniae using routine phenotypic and serological analyses but which were subsequently shown by 16S rRNA sequence analysis to belong to yet undefined Actinobacillus species. The sensitivity of the PCR was determined to be 10pg of A. pleuropneumoniae DNA. A set of nested primers amplified a 377bp fragment specifically with A. pleuropneumoniae DNA. DNA titration experiments using the flanking and nested primer pairs showed an improved level of sensitivity to approximately 10fg of genomic DNA. The nested PCR was used to monitor the spread of A. pleuropneumoniae in pigs experimentally infected with a virulent serotype 1 strain and housed in a controlled environment facility. A. pleuropneumoniae DNA could be detected by nested PCR in nasal swab samples of infected pigs receiving either a high dose (5x10(5)) or a low dose (1x10(4)) challenge and in unchallenged cohorts that were contact-infected by the inoculated animals. Furthermore, PCR confirmed the presence of A. pleuropneumoniae in 16/17 homogenates from necrotic lung lesions, while the bacterium was successfully recovered from 13 of these lesions by culture.

Item Type:	Journal Article (Original Article)
Division/Institute:	05 Veterinary Medicine > Department of Infectious Diseases and Pathobiology (DIP) > Institute of Veterinary Bacteriology
UniBE Contributor:	Kuhnert, Peter, Frey, Joachim
Subjects:	500 Science > 570 Life sciences; biology 600 Technology > 630 Agriculture
ISSN:	0378-1135
Publisher:	Elsevier
Language:	English
Submitter:	Peter Kuhnert-Ryser
Date Deposited:	05 Jun 2014 16:08
Last Modified:	05 Dec 2022 14:27
Publisher DOI:	10.1016/S0378-1135(00)00345-X
PubMed ID:	11230928
URI:	https://boris.unibe.ch/id/eprint/39154