Toxigenicity Assessment of the Murray River Bloom Using a Rapid and Automated Multiplex Tandem- Polymerase Chain Reaction.
Nijoy John Jex Lab; WEHI Murray River Bloom- 2016
• History of bloom events: 1991, 2007, 2009, 2010 • 194 samples from 18 sites along the Murray River (Victoria)- upstream of Wahgunyah to Wemen • Samples collected from March 21 to May 4th 2016 • Multiplex Tandem-PCR conducted on all samples (retested by conventional PCR) • Bio volume, microscopic ID / cell count, Toxin concentration (HPLC/ELISA)
(source: http://www.theage.com.au/victoria/bluegreen-algal-bloom-chokes-murray-cuts-water-to-farmers-20160308-gndihv.html)
AIMS
• To understand the community composition of the bloom • Assess the toxigenicity • Understand toxin gene expression
(source: http://www.theage.com.au/victoria/bluegreen-algal-bloom-chokes-murray-cuts-water-to-farmers-20160308-gndihv.html)
Toxic cyanobacteria in Australia
• Major water quality issue • Threat to public health, animals, water ecosystem
Cyanotoxins Toxicity Genera/species
Microcystins Hepatotoxic Microcystis sp., Dolichospermum sp.
Nodularins Hepatotoxic Nodularia spumigena
Saxitoxins Neurotoxic Dolichospermum circinalis
Cylindrospermopsin Hepatotoxic/Neurotoxic/Cytotoxic Cylindrospermopsis raciborskii; Chrysosporum ovalisporum
M. aeruginosa N. spumigena C. raciborskii D. circinalis Methods of detection
• Cannot differentiate toxic from non-toxic blooms • Microbiology/taxonomic expertise
• Less sensitive, effective when toxin is present • Cross reactivity of antibodies against other toxin variants
• Quantitative and sensitive • Expensive, lengthy analytical time
HPLC Molecular techniques: Multiplex-Tandem PCR
• Simultaneous amplification • Automated, rapid and sensitive • 72 individual tests
MT-PCR data
• Diagnostic specificity and sensitivity: 100% and 97.7% respectively (Baker et.al., 2013)- WaterRA project 1043
• Simultaneous amplification of 6 gene targets WaterRA project 1043 Microscopy data
• Predominant species: Chrysosporum ovalisporum • Subdominant species: Planktolyngbya, Chroococcales, Cyanogranis sp., Aphanocapsa sp. • Small quantities : Cylindrospermopsis raciborskii, Microcystis sp., Dolichospermum sp. • Highest Bio volume: 73 mm3/L at Picola Raw inlet • Total BGA count: more than drinking and recreational water guideline
• Cylindrospermopsin producer • Aphanizomenonaceae family • Nitrogen fixing cyanobacteria
C. ovalisporum Murray bloom: MT-PCR Data
Prevalence Overall Abundance
7%
19% 31% 39%
74% 30%
• > 60,000 copies/ml for Microcystis like McyE at Piangil river Abundance (>1000 gene copies/mL)
• > 6,000 copies/ml for CyrC at Numurkah 7%
Saxitoxin
31% Cylindrospermopsin Which species is producing microcystin? McyE Microcystis 62% Progression of the bloom
Wahgunyah
Pental
Conventional PCR , ELISA and HPLC data
• ELISA and HPLC: Low level production MCY and CYNs • Overall toxin levels were low (never rising about 1µg/L), but trace levels were detected by ELISA and by HPLC
Murray bloom -summary
• Two different bloom patterns
• Bloom was a mixture of toxic and non-toxic C. ovalisporum ??
• Microcystin producers other than Microcystis sp. and Dolichospermum sp. ??
• Propose that the gene was not expressed or transcribed
• Environmental factors regulating toxin production- possibly Nitrogen
Murray Bloom-Next steps
• Assess toxin gene expression vs toxin production • Study the activity of NtcA on toxin biosynthesis
• Effect of environmental factor- Nitrogen Acknowledgements
Supervisors Water Research Australia • Assoc. Prof Aaron Jex (Primary supervisor) • Carolyn Bellamy • Dr. Nick Crosbie • Dr. Judy Blackbeard
Mentor Jex Lab, WEHI • Dr. Daniel Hoefel (SA Water)
• Dr. Louise Baker • Dr. Najoua Lalaoui (WEHI) • Dr. Sammantha Emery • Brendan Ansell • Katharina Stracke Industry Partners • Cammille Awburn • Goulburn Murray Water • Goulburn Valley Water ALS • Grampians Wimmera • Lisa Synnott • North East Water • Sussan Cassar • Coliban Water