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An improved multiplex PCR for Actinobacillus pleuropneumoniae, Glaesserella australis and Pasteurella multocida
Xiaochen Sun1, P J Blackall1, Patrick Daniel2
1Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, Queensland 4067, Australia.
Abstract:
Glaesserella australis, a newly described bacterial species, has been isolated from pig lungs that displayed lesions very similar to those caused by Actinobacillus pleuropneumoniae, prompting the need for a validated diagnostic tool. In this work, we have altered a multiplex PCR used for the identification of cultures of G. australis, A. pleuropneumoniae and Pasteurella multocida to be more sensitive and then evaluated the use of the altered diagnostic tool on cultures and directly on tissues. The altered multiplex PCR was validated using 47 related species, both type/reference strains and field isolates. The sensitivity was assessed by serial dilutions and used a mixture of target bacteria in different concentrations. Further, 166 lung samples from 54 farms from four Australian States were used to validate the ability of the multiplex PCR to detect bacteria in lung swabs. The multiplex PCR was specific for the three target species. The assay could detect a minimum of 40 colony forming units (CFU) of G. australis, 786 CFU of A. pleuropneumoniae and 238 CFU of P. multocida. The multiplex PCR yielded more positives than coventional bacteriological examination. From a total of 166 lung samples, 51.9%, 51.9% and 5.6% of farms were PCR positive for P. multocida, A. pleuropneumoniae and G. australis, respectively. The results suggested that the new multiplex PCR was specific, sensitive and out performed traditional culture. The prevalence of G. australis was not very high, but it was the dominant pathogen in infected pigs.
Insights
A new multiplex PCR test accurately identifies Glaesserella australis, Actinobacillus pleuropneumoniae, and Pasteurella multocida in pig lungs. This sensitive diagnostic tool outperforms traditional culture methods for bacterial detection.
Area of Science:
- Veterinary Microbiology
- Animal Diagnostics
- Bacterial Pathogenesis
Background:
- Glaesserella australis, a novel bacterial species, causes pig lung lesions similar to Actinobacillus pleuropneumoniae.
- Accurate diagnostics are crucial for differentiating these pathogens and managing swine respiratory disease.
Purpose of the Study:
- To enhance and validate a multiplex PCR assay for the sensitive and specific detection of G. australis, A. pleuropneumoniae, and Pasteurella multocida.
- To evaluate the diagnostic performance of the multiplex PCR directly on pig lung tissues.
Main Methods:
- Modification of an existing multiplex PCR to improve sensitivity for G. australis, A. pleuropneumoniae, and P. multocida detection.
- Validation using 47 related bacterial species, serial dilutions, and mixed bacterial cultures.
- Application of the assay to 166 lung samples from 54 Australian pig farms.
Main Results:
- The multiplex PCR demonstrated high specificity for the three target species.
- The assay detected as few as 40 CFU of G. australis, 786 CFU of A. pleuropneumoniae, and 238 CFU of P. multocida.
- The PCR identified a higher prevalence of these bacteria in lung samples compared to conventional bacteriology, with P. multocida and A. pleuropneumoniae being most common.
Conclusions:
- The enhanced multiplex PCR is a specific and sensitive diagnostic tool for identifying G. australis, A. pleuropneumoniae, and P. multocida in pig lung tissues.
- This assay outperforms traditional culture methods, offering improved detection capabilities.
- G. australis, though less prevalent, was identified as the dominant pathogen in infected pigs in this study.
