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Handbook of the 10 Symposium on Diseases in Asian Aquaculture(DAA10)
Abstract ID: 234C(Poster &Elevator Pitch) EVALUATION OF PRIMERS TO DETECT Streptococcus agalactiae William Leigh, Kim D. Thompson, Anita Jaglarz, Janina Z. Costa and Ruth N. Zadoks Moredun Research Institute, Pentlands Science Park, Penicuik EH26 0PZ, UK. Email:
[email protected] Streptococcosis is highlighted as a major disease problem for the tilapia aquaculture industry. The two main bacterial species associated with disease outbreaks are Streptococcus agalactiae and Streptococcus iniae. As well as being responsible for disease in tilapia, S. agalactiae is also an important pathogen in humans and cattle. The bacterium has a global spread in multiple hosts and extensive genetic lineages. Due to the importance of S. agalactiae as a pathogen, there are many published PCR primers which have largely been designed for the detection of S. agalactiae from dairy and human samples. The design of primers for PCR to confirm species identity of S. agalactiae can be problematic, with many primers designed for this purpose also cross reacting with S. iniae. The ability to differentiate between S. agalactiae and S. iniae ininfected samples derived from the aquaculture industry is much more important than from samples collected from other host species; as both species of bacteria are likely to be isolated from infected fish, cause very similar disease symptoms and are morphologically similar, therefore they are easily confused. S. iniae is unlikely to be isolated from humans and has not been found in cattle, so differentiation between these species of bacteria is not so important in samples collected from these host species. The aim of the present study was to design a set of primers, based on the groEL gene of S. agalactiae thatis specific for S. agalactiae in PCR, can detect a diverse range of S.agalactiae isolates from different hosts and it is capable of discriminating between S. agalactiae and S. iniae. The chosen SagroEL2 primers were shown to be epidemiologically sensitive to S. agalactiae. Ninety seven isolates of S. agalactiae, representing 11 clonal complexes, produced a very clear product of the correct size with this primer set. This demonstrates the value of these primers, not just for use in aquaculture, but also in other fields were S. agalactiae needs to be identified. The primers were shown to have high analytical sensitivity, detecting low levels of S. agalactiae DNA, between 1x10-5 and 1x10-6 ng/µl of reaction mixture or 10-100 copies of genomic DNA per reaction. This shows that the assay is sensitive over a wide range of DNA concentrations and is likely to produce a positive result even in the presence of a very low number of bacteria. The assay was shown to produce a clear product with every isolate of S. agalactiae grown in broth culture, and also with DNA extracted from infected tilapia brain. The high analytical sensitivity of the assay is particularly important when diagnosing the early stages of disease outbreaks as sampled fish may only have low levels of bacteria growing in the organs sampled. Finally, as it is essential that primers designed for aquaculture purposes do not cross react with S. iniae the SagroEL2 primer pair was tested with 9 isolates of S. iniae chosen from different geographical locations and no cross-reaction was observed.
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Handbook of the 10 Symposium on Diseases in Asian Aquaculture(DAA10)
About this Handbook th
Handbook of the 10 Symposium on Diseases in Asian Aquaculture (DAA10) 28 August – 1 September 2017 The ANVAYA Beach Resort, Kuta, Bali, Indonesia.
Citation: th
Sunarto, A., Maskur and R. Subasinghe. 2017. Handbook of the 10 Symposium on Diseases in Asian Aquaculture (DAA10), Bali, Indonesia. The Fish Health Section of the Asian Fisheries Society.
Disclaimer: This volume is the pre-symposium compilation of abstracts. Although oral presentations were selected from submitted abstracts by three independent reviewers, the contents are not peer-reviewed and part from lay-out changes, the contents have been printed as received from submitting authors. Some abstracts are also need an extensive English editing. Please consult with the authors before using information contained in any of the abstracts. The Fish Health Section of the Asian Fisheries Society does not guarantee that this Handbook is without flaws and therefore disclaims any liability for any error, loss or other consequence which may arise from persons relying on the information in this publication. The symposium programme is correct of the time of the printing, however the organisers reserve the right to make changes where necessary.
Copies available from: The Fish Health Section of the Asian Fisheries Society http://www.fhs-afs.net/
Publications: 1. Review papers by keynote speakers will be published in Journal of the World Aquaculture Society. https://www.was.org/View/Journal-of-the-World-Aquaculture-Society.aspx 2. Subject to review, papers presented by participants will be published as a special issue of Fishes journal. http://www.mdpi.com/journal/fishes/special_issues/10th_Symposium_Disease_Asian_Aquaculture
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