Monitoring Changing Toxigenicity of a Cyanobacterial Bloom by Molecular Methods
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2002, p. 6070–6076 Vol. 68, No. 12
0099-2240/02/$04.00⫹0 DOI: 10.1128/AEM.68.12.6070–6076.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Monitoring Changing Toxigenicity of a Cyanobacterial Bloom
by Molecular Methods
Judith A. Baker,1 Barrie Entsch,1 Brett A. Neilan,2 and David B. McKay3*
University of New England, Armidale 2351,1 and University of New South Wales, Sydney 2052,2 New South
Wales, and University of the Sunshine Coast, Maroochydore DC 4558, Queensland,3 Australia
Received 10 June 2002/Accepted 18 September 2002
Cyanobacterial blooms are potential health hazards in water supply reservoirs. This paper reports analyses
of a cyanobacterial bloom by use of PCR-based methods for direct detection and identification of strains
present and determination of their toxigenicity. Serial samples from Malpas Dam, in the New England region
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of Australia, were analyzed during a prolonged, mixed cyanobacterial bloom in the summer of 2000 to 2001.
Malpas Dam has been shown in the past to have toxic blooms of Microcystis aeruginosa that have caused liver
damage in the human population drinking from this water supply reservoir. Cyanobacterial genera were
detected at low cell numbers by PCR amplification of the phycocyanin intergenic spacer region between the
genes for the  and ␣ subunits. The potential for microcystin production was determined by PCR amplification
of a gene in the microcystin biosynthesis pathway. The potential for saxitoxin production was determined by
PCR amplification of a region of the 16S rRNA gene of Anabaena circinalis strains. Toxicity of samples was
established by mouse bioassay and high-pressure liquid chromatography. We show that bloom components can
be identified and monitored for toxigenicity by PCR more effectively than by other methods such as microscopy
and mouse bioassay. We also show that toxigenic strains of Anabaena and Microcystis spp. occur at this site and
that, over the course of the bloom, the cell types and toxicity changed. This work demonstrates that PCR
detection of potential toxicity can enhance the management of a significant public health hazard.
In Australia, most drinking water comes from natural, un- dent RNA polymerase (rpoC1) gene (16). The use of PCR for
developed catchment areas and is stored in reservoirs. The amplification of the phycocyanin intergenic spacer has recently
water is monitored and managed to ensure good-quality drink- been adapted for direct analysis of environmental samples (2).
ing water, free from taste and odor problems and health risks, This technique has the advantage of being specific for cya-
such as toxins, pathogenic bacteria, and parasites. Bloom- nobacteria in the presence of other organisms.
forming cyanobacteria can be a serious problem for water Although these molecular techniques have improved the
managers, and bloom toxicity needs to be determined early in accuracy of strain identification, they have not been able to
the bloom development in a drinking water reservoir. Cya- distinguish toxigenic from nontoxigenic strains. The biosyn-
nobacteria have been shown to produce a wide range of toxic thetic pathway for microcystin production has now been elu-
compounds, including neurotoxins, such as the saxitoxins, and cidated (35), and this has enabled the development of specific
hepatotoxins, such as the microcystins (34). Several incidences oligonucleotide primers for genes common to production of all
of acute effects of hepatotoxins, causing toxic liver injury (13) microcystins (36). By using these primers for PCR amplifica-
or death (20), have been reported. Perhaps more seriously, tion, toxigenic strains in bloom samples can be distinguished
microcystins have been shown to be tumor promoters (14, 19, (36).
29) and pose a serious risk to populations exposed to chronic
Besides microcystins, saxitoxins are another important group
low-level doses.
of toxins produced by Australian cyanobacterial blooms (18).
Identification of a cyanobacterial genus by microscopic mor-
A unique molecular technique for detecting toxigenic strains is
phology and/or molecular analysis does not indicate the poten-
not yet available for saxitoxin production because the toxin
tial for toxin production. Different strains of one species can be
synthesis pathway has not yet been fully elucidated. New stud-
morphologically identical but differ in toxigenicity. For exam-
ies show that PCR amplification of regions of the 16S rRNA
ple, Microcystis aeruginosa has both toxic and nontoxic strains
gene can be useful for distinguishing strains within a genus or
(23). There have been attempts to refine the identification of
strains by using specific gene analysis. Examples include the species. For example, PCR techniques have allowed separation
use of PCR for amplification of the phycocyanin intergenic of strains of common bloom-forming genera, such as Microcys-
spacer (PC-IGS) between the  and ␣ subunits of the phyco- tis (27), Anabaena (6), Nodularia (24), and Cylindrospermopsis
cyanin operon (2, 7, 8, 26), the 16S–23S rRNA internally tran- (33). One of these techniques has segregated Anabaena strains
scribed spacer region (28, 30), or segments of the ribulose into mostly toxic versus mostly nontoxic strains (6).
bisphosphate carboxylase genes (11, 32) and the DNA-depen- In this study we report an analysis of a mixed bloom over the
summer of 2000 to 2001 in Malpas Dam, in the New England
region of the Northern Tablelands of New South Wales, Aus-
* Corresponding author. Mailing address: University of the Sun-
tralia. This dam has a history of severe cyanobacterial blooms,
shine Coast, Faculty of Science, Maroochydore DC QLD 4558, Aus-
tralia. Phone: 61 7 5430 2887. Fax: 61 7 5430 2887. E-mail: dmckay and a toxic bloom of M. aeruginosa in March 1981 was the basis
@usc.edu.au. for an epidemiological study that showed evidence of liver
6070VOL. 68, 2002 MOLECULAR MONITORING OF A TOXIC CYANOBACTERIAL BLOOM 6071
TABLE 1. Reference cultures, bloom samples and database entries used in this study
Reference or
Code Straina (toxicity)b Origin
(mo.day.yr)
Reference cultures
AWQC118C A. circinalis (S-P) Australia 6
AWQC131C A. circinalis (S-P) Australia 6
AWQC306A A. circinalis (NT) Australia 6
AWQC271C A. circinalis (NT) Australia 6
NIES 80 Anabaena solitaria (ND) Japan 38
PCC 7806 M. aeruginosa (M-P) The Netherlands 31
Bloom samplesc
T-96 (clone) Microcystis sp. Malpas Dam 2.12.96
MD-34 M. flos-aquae Malpas Dam 1.24.00
BD-1 Microcystis sp. Burrendong Dam 7.7.98
CD-1 Microcystis sp. Chaffey Dam 4.2.96
CaD-1 Microcystis sp. Cania Dam 5.23.00
WW-1 Microcystis sp. Wagga Wagga 1.14.99
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Database entries
AF195159 M. aeruginosa (⫺) United States 36
AF195161 M. flos-aquae (⫺) United States 36
AF195162 M. flos-aquae (⫺) United States 36
AF195167 M. aeruginosa (⫹) South Africa 36
AF195174 M. aeruginosa (⫹) United States 36
AF195175 M. aeruginosa (⫹) Canada 36
AF195176 M. aeruginosa (⫹) Scotland 36
AF195177 (PCC 7806) M. aeruginosa (⫹) The Netherlands 36
a
Species designation based on morphology.
b
Toxicity: S-P, saxitoxin-producing strain; NT, non-toxin-producing strain; M-P, microcystin-producing strain; ND, not determined; ⫺, absence of the microcystin
synthetase NMT domain; ⫹, presence of the microcystin synthetase NMT domain.
c
Australian bloom samples other than the 2000-to-2001 Malpas Dam samples.
damage in the population of Armidale supplied by water from of soluble reactive phosphorus. The pH of Malpas Dam water averages 8.8. High
this dam (13). Records from the local water-monitoring au- pHs (pH 7 to 9) favor proliferation of cyanobacteria over other phytoplankton
(37).
thority over the last decade show that blooms of cyanobacteria
Reference cultures. The specific cyanobacterial strains used as references are
on Malpas Dam may be monospecific or mixed, often showing listed in Table 1.
changes in dominance over a period of days, weeks, or months. Environmental water samples. The local water authority, Armidale Dumaresq
We demonstrate the value of using specific PCR methods to Council (ADC), takes monitoring samples at four sites in the dam, on the surface
analyze bloom samples directly. During the progression of this and at 1-m depth intervals at each site, on a thrice-weekly basis during the
summer. For this study, only surface samples at the site adjacent to the drinking
complex bloom, strains of Anabaena and Microcystis developed
water off-take tower have been examined and analyzed. Once a week, for the
and dominated. It was found that PCR-based techniques could duration of this study, two samples were taken at this site. One sample was used
identify toxigenic strains of Microcystis and potentially toxic for analysis in our laboratory, and the other sample (the regular monitoring
strains of Anabaena circinalis. Early detection of problem or- sample) was used for cell counts and mouse bioassay by the water authority.
ganisms can provide a means to improve the management of Within 24 h of collection, a 1-ml aliquot of each sample was examined micro-
scopically in a Sedgwick-Rafter chamber at a magnification of ⫻400 under phase
water quality and treatment to avoid public health risks.
contrast. The number of cells of each bloom species was estimated, and a ratio
of the component species was derived. Features, such as the presence of akinetes,
MATERIALS AND METHODS were also noted. Species identification was based on morphological characteris-
tics as described in the Australian reference text of Baker and Fabbro (3). A
Study site. Malpas Dam (30°16⬘S, 151°75⬘E) lies on the Gara River in the New
subset of the weekly samples (Table 2) was subjected to detailed analysis.
England region of the Northern Tablelands of New South Wales, Australia. The
dam is at an elevation of 1,178 m, and it has a catchment area of about 20,000 ha Sample preparation for PCR. The cyanobacterial cells in bloom samples were
and a capacity of 13,000 Ml. This dam is the water supply reservoir for the city concentrated by centrifugation, washed, and subjected to a freeze-thaw treat-
of Armidale, a town with approximately 22,000 inhabitants. Annual average ment for PCR template preparation (2). All PCRs described in this study were
rainfall in the catchment area is 890 mm. The rainfall pattern is variable, but carried out after this treatment, by using approximately 1,000 cells per reaction.
rainfall peaks in the summer months. Average summer temperatures range from This method is simple and quick and has been proven effective with fresh bloom
10 to 23°C, and the winter range is between ⫺1 and 10°C. Malpas Dam is material, when most cells are intact.
classified as eutrophic on the basis of phosphorus, nitrogen, and chlorophyll DNA amplification, analysis, and sequencing for identification. PCR amplifi-
levels (5). The plentiful nutrients are a product of agricultural land use and cation of the PC-IGS was performed as previously described (2, 26).
management and the basic geology of the catchment area. The soil landscapes of Samples of DNA from PCR amplification were purified using a QIAquick
the Malpas catchment area include basalt with a phosphorus content 2 to 3 times PCR Purification kit (Qiagen) and sequenced using the ABI Prism BigDye
higher than the worldwide mean (39). An analysis of the dam water in 1995 Terminator v3.0 Ready Reaction Cycle Sequencing kit (Perkin-Elmer Applied
showed that levels of soluble reactive phosphorus ranged from 87 to 95 g liter⫺1 Biosystems) and an ABI Prism 3700 DNA Analyzer (Perkin-Elmer Applied
(9), exceeding the level of 50 g liter⫺1 considered conducive to cyanobacterial Biosystems).
blooms (37). In 2001, levels of soluble reactive phosphorus ranged from 6 to 11 Sequence analysis was performed with the programs available through the
g liter⫺1 (P. Wilson, unpublished data), but total phosphorus levels in sedi- Australian National Genomic Information Service (ANGIS). “Pileup” was used
ments remained high (measured at 1.6 mg g⫺1 [dry weight], compared to 1.2 mg for multiple sequence alignments by use of a simplification of the progressive
g⫺1 [dry weight] in 1995 [9]). Improved catchment management is probably alignment method (15). PC-IGS nucleotide sequences were compared to entries
responsible for reduced nutrient inflow into the dam and thus for the lower level deposited in GenBank by using “BlastN” (1).6072 BAKER ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 2. Composition of selected bloom samples by microscopy the work of Falconer (12). When mice died within 15 min with neurotoxic
symptoms, the sample was recorded as “neurotoxic.” When mice died within 24 h
A. circinalis/ 103 cell counts ml⫺1b and showed enlarged, blood-filled livers, the sample was recorded as “lethal
Sampling date
Code M. aeruginosa/ hepatotoxic.” If the mice did not die within 24 h, the sample was recorded as
(mo.day.yr)
M. flos-aquae ratioa Anabaena Microcystis
“nontoxic,” or as “nonlethal hepatotoxic” if the mice had mottled, enlarged livers
MD-78 11.28.00 0:0:0 0 0 (⬎20% heavier than the livers of control mice).
MD-80 12.8.00 100:0:0 9.8 0 Toxins were identified and quantified by high-pressure liquid chromatography
MD-84 12.22.00 100:0:0 630 0 (HPLC) analysis. Samples (50 ml) of bloom material were analyzed for micro-
MD-87 1.5.01 99:0:1 11 0 cystins by using sonication, concentration on a C18 cartridge, and subsequent
MD-88 1.12.01 1:0:99 33 0 HPLC based on the method of Lawton et al. (22). Saxitoxins were identified and
MD-90 1.25.01 4:1:95 120 1,000 quantified by using a peroxide oxidation method of sample preparation, based on
MD-93 2.15.01 5:0:95 2,500 10 the work of Lawrence, Menard, and Cleroux (21). Samples (50 ml) of bloom
MD-96 2.26.01 5:85:10 380 65 material were sonicated, filtered, and then subjected to the peroxide oxidation,
MD-103 3.12.01 1:90:9 21 36 followed by HPLC.
MD-108 3.26.01 1:99:0 0.4 23 Nucleotide sequence accession numbers. The PC-IGS nucleotide sequences
MD-109 3.30.01 0:100:0 0 13 described in this study have been deposited in GenBank under accession num-
bers AY117039 to AY117047; A. circinalis AWQC118C has been assigned ac-
a
Obtained by microscopic examination of samples taken for molecular anal- cession number AF426004; and the accession numbers of Microcystis PC-IGS
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ysis in our laboratory (as described in Materials and Methods). sequences used in comparisons to Australian Microcystis bloom samples are
b
Samples taken separately, at the same time and place, by the Armidale listed in Table 1.
Dumaresq Council. These counts are from council records.
RESULTS
Assessing toxigenicity by PCR. PCR amplification of the N-methyltransferase
During the weekly monitoring of Malpas Dam, samples were
(NMT) region in the microcystin polyketide-peptide biosynthetic pathway in
cyanobacteria was performed as described previously (36). Thermal cycling was selected for molecular analysis, based on the occurrence of
carried out as published except that the annealing temperature was modified to particular cell types as they bloomed over the course of the
55°C to refine the method for conditions in our laboratory. This was confirmed summer. Samples can be identified by the dates in Fig. 1 and
with microcystin-producing cell lines. Table 2 and are indicated by arrows in Fig. 1.
Analysis of strains of A. circinalis was carried out to determine their toxic
grouping by PCR-based detection of a specific region of the 16S rRNA (6). The
Detection of cyanobacteria in selected samples. The samples
PCR conditions for this method were modified to use an annealing temperature listed in Table 2 were subjected to microscopic examination,
of 65°C. This was confirmed with Anabaena cultures known to produce saxitoxin. and the cyanobacterial species observed were recorded, to-
Electrophoresis of the PCR products was carried out in 1% agarose gels in gether with their relative frequencies. Table 2 also shows cell
TAE buffer (0.04 M Tris-acetate–0.001 M EDTA [pH 8.0]), and the DNA was
counts determined by the ADC on separate samples taken at
stained with ethidium bromide and visualized and photographed under UV
illumination. the same time and place. For samples MD-88, MD-93, and
Toxicity testing. A mouse bioassay was employed for rapid, broad-spectrum MD-96, there are obvious discrepancies between the ratios
toxin detection (12). Baker and Humpage (4) have reported the quantity of cells observed in our laboratory samples and the cell counts for the
of the common toxigenic bloom-forming cyanobacteria Anabaena, Microcystis, samples taken by the ADC. This has occurred where Micro-
Nodularia, and Cylindrospermopsis that gave a toxic response when extracted and
injected intraperitoneally into a 16- to 23-g mouse. Based on their results, our
cystis flos-aquae was present in the samples. In Malpas Dam
water samples were concentrated by centrifugation to achieve approximately 108 this species is very buoyant, floating right on the surface of the
cells ml⫺1 and then the extract from 1 ml was injected and assayed according to water. When replicate surface samples are taken, some sam-
FIG. 1. Cyanobacterial cell counts for site 1 surface samples, summer 2000 to 2001. Arrows indicate sampling dates shown in Table 2. Data are
summarized from ADC records.VOL. 68, 2002 MOLECULAR MONITORING OF A TOXIC CYANOBACTERIAL BLOOM 6073
FIG. 2. Identification of bloom samples by PC-IGS sequences. Key bloom samples MD-80, MD-88, and MD-108 were compared with a
reference culture and two database entries (see Tables 1 and 2 for more information). Sample MD-80 was confirmed as A. circinalis, and samples
MD-88 and MD-108 are Microcystis species.
ples may contain many M. flos-aquae colonies and other sam- Microcystin-producing potential in bloom samples. We em-
ples may contain few of this species. We have found no men- ployed the PCR technique for amplifying the NMT domain of
tion of this point in the literature. This was an unexpected the gene for microcystin synthetase A (mcyA) in bloom sam-
sampling problem, revealed by the discrepancies shown in Ta- ples to identify which samples contained potentially microcys-
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ble 2. tin producing strains (36). The results in Fig. 3 showed that
The samples in Table 2 were subjected to PCR amplification samples MD-90 and MD-93 (dominated by M. flos-aquae), and
of the PC-IGS, and products specific for cyanobacteria were samples MD-96 and MD-103 (dominated by M aeruginosa),
obtained for all samples except MD-78, which had no observ- each formed a PCR product identical in size to that formed
able cyanobacterial cells present. The results agreed qualita- from the toxin-producing reference culture. Thus, at least
tively with those of the microscopic examination. some of the Microcystis strains in each of these samples were
Identification of bloom species by using the phycocyanin capable of producing microcystin. Bloom samples MD-80,
region. Samples MD-80, MD-88, and MD-108 were taken at MD-84, and MD-87 (which were dominated by A. circinalis)
points during the bloom when only one cyanobacterial type was and sample MD-88 (dominated by M. flos-aquae) (data not
dominant. These samples were subjected to PC-IGS amplifi-
shown) did not produce PCR products. This suggested that the
cation, and the products were used directly in sequencing re-
A. circinalis strains in this bloom were not microcystin produc-
actions. The subsequent chromatograms showed single base
ing. The lack of product from sample MD-88 suggested that
peaks throughout, indicating that DNA from a single cya-
there was more than one strain of M. flos-aquae within this
nobacterial type dominated. Alignment of these sequences
bloom, some toxigenic (MD-90 and MD-93) and others not
with reference culture sequences allowed taxonomic identifi-
producing toxin (MD-88). There was no discernible PCR prod-
cation to the genus level, based upon both the length and the
sequence of the PC-IGS (2) (Fig. 2). Figure 2 shows that uct from sample MD-109, which contained M aeruginosa and
sample MD-80 was identical to the A. circinalis reference cul- had high numbers of the diatom Aulocoseira.
ture AWQC118C, in agreement with microscopy results. The Testing of A. circinalis in bloom samples by using 16S rRNA
sequences of samples MD-88 and MD-108, identified as Mi- analysis. In Australia, within the genus Anabaena, strains of
crocystis species by microscopy, were almost identical to the A. circinalis have been identified as producers of a neurotoxin
sequences of the reference culture M. aeruginosa PCC 7806 (4), which has subsequently been shown to be saxitoxin (18).
(AF195177) and an M. flos-aquae database entry (AF195162), To indicate which bloom samples may contain toxic A. circi-
respectively. nalis, we employed a set of PCR primers that were designed to
Alignment of the PC-IGS sequences of Malpas Dam Micro- anneal to specific sequences within the 16S rRNA gene. One of
cystis samples with those of overseas strains and Australian these primers was shown to be specific for a group of A. circi-
Microcystis bloom samples showed no grouping according to nalis strains which are mostly toxic (6). The results are shown
species or toxicity, as was found also by Tillett et al. (36). Thus, in Fig. 4. A PCR product was formed from all bloom samples
this method could not be used to differentiate species of Mi- except MD-78 and MD-109, which were not shown by micros-
crocystis in the bloom. copy to contain Anabaena (see Table 2). On the basis of this
FIG. 3. Detection of the NMT domain of the microcystin synthetase gene, mcyA, in Malpas Dam bloom samples (Table 2) and control samples
(Table 1). Of the control samples, only M. aeruginosa PCC 7806 (M.a. 7806) is a microcystin producer.6074 BAKER ET AL. APPL. ENVIRON. MICROBIOL.
FIG. 4. Detection of potentially saxitoxin producing A. circinalis strains in Malpas Dam bloom samples by amplification of a region of the 16S
rRNA gene. For descriptions of strain and bloom samples, see Tables 1 and 2. Of the four reference strains of A. circinalis, AWQC118C and
AWQC131C are known saxitoxin producers while AWQC271C and AWQC306A are not known to produce saxitoxin, although AWQC271C gives
a positive result in this test (6).
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analysis, all of the samples that gave a PCR product contained from mid-December to mid-March. During this time cell
potentially toxic A. circinalis. counts continuously exceeded 10,000 cells ml⫺1. Changing lev-
Toxicity analysis of bloom samples. The bloom samples els of different species and toxins were observed over the
listed in Table 2 were analyzed for toxins by mouse bioassay course of the bloom. The untreated water of Malpas Dam
and HPLC (see Table 3 for results). remains a public health risk during such bloom periods.
Mouse bioassays showed that, from sample MD-90 onward, Determination of the toxigenicity of the cyanobacteria pres-
some degree of hepatotoxicity was apparent. This correspond- ent provides a warning of possible toxicity development and
ed with the presence of Microcystis strains in the samples (see allows early intervention to avoid health problems. We have
Table 2). Hepatotoxicity was not established for sample MD- demonstrated that PCR-based tests, combined with micros-
93 because the mice died rapidly with neurotoxic symptoms copy, quickly and simply monitored the succession of cyano-
before symptoms of hepatotoxicity were apparent.
bacterial types in this bloom and indicated their toxigenicities.
Measurement of saxitoxin by HPLC (Table 3) showed the
Previous work has shown that the NMT PCR assay reliably
presence of the toxins in the same samples that gave positive
indicates microcystin toxigenicity (36). We have adapted this
results in the PCR analysis (Fig. 4), and the sample with the
technique for use with bloom samples, and we have found that
highest toxin concentration was the sample that was neurotoxic
by mouse bioassay. Similarly, detection of microcystin by use of it provides a positive result with 1,000 toxigenic cells and that
HPLC was consistent with positive results in the PCR analysis environmental cell densities as low as 10 cells ml⫺1 can provide
(Fig. 3), and the highest microcystin concentration corre- sufficient cells for this assay. The NMT PCR assay signaled the
sponded with the strongly hepatotoxic mouse bioassay result. presence of toxigenic Microcystis strains as soon as Microcystis
was evident in samples by microscopy (Fig. 1; Table 3) and
demonstrated that toxigenic cells continued to be present
DISCUSSION
throughout the remainder of the bloom. By comparison, the
This study has shown that Malpas Dam, during the summer mouse bioassay (the method which has routinely been em-
of 2000 to 2001, contained toxigenic strains of cyanobacteria ployed to test Malpas Dam samples) showed lethal hepatotox-
TABLE 3. Summary of toxin assays and PCR results for selected Malpas Dam bloom samplesa
Concn (g/liter⫺1) of the
A. circinalis/M. aeruginosa/ Mouse bioassay PCR test resultd
Sample indicated toxin by HPLCc
M. flos-aquae ratio resultb
Microcystins Saxitoxin Anabaena Microcystins
MD-34 0:0:100 n-l H ND NT ND ⫹
MD-78 0:0:0 n-t ND ND ND ND
MD-80 100:0:0 n-t ND ND ⫹ ND
MD-84 100:0:0 n-t ND 33 ⫹ ND
MD-87 99:0:1 n-t ND 5 ⫹ ND
MD-88 1:0:99 n-t ND 7 NT ND
MD-90 4:1:95 n-l H 32 40 ⫹ ⫹
MD-93 5:0:95 N 1 135 ⫹ ⫹
MD-96 5:85:10 n-l H 8 6 ⫹ ⫹
MD-103 1:90:9 H 80 4 ⫹ ⫹
MD-108 1:99:0 NT ND ND NT ND
MD-109 0:100:0 n-l H ND ND ND ND
a
See also Table 2.
b
n-t, nontoxic; N, neurotoxic; H, hepatotoxic; n-l H, nonlethal hepatotoxic; NT, not tested.
c
The limit of detection was 10 g liter⫺1 because of interfering peaks. ND, peak(s) or band not detected.
d
⫹, band detected.VOL. 68, 2002 MOLECULAR MONITORING OF A TOXIC CYANOBACTERIAL BLOOM 6075
icity only in sample MD-103, which was shown by HPLC to This bloom showed a complex pattern of cyanobacterial
contain the highest level of microcystins (see Table 3). The species succession. Figure 1 shows that the bloom began with
mouse bioassay is not effective in indicating the presence of the development of large numbers of A. circinalis, which
microcystins when neurotoxins are also present because the peaked in mid-December and peaked again at even higher
neurotoxins rapidly kill the mice before microcystins cause numbers in late February. A Microcystis population suddenly
pathological effects (12). In mixed blooms such as the one in appeared in mid-January and was identified as M. flos-aquae by
this study, this bioassay could fail to detect the presence of microscopic examination. M. flos-aquae numbers declined and
microcystin producers. The negative result in the NMT PCR were replaced by M. aeruginosa (as identified by microscopy) in
assay of sample MD-109, which contained Microcystis (Table late February. The cells identified as M. flos-aquae and M.
2), was consistent with the negative result by HPLC and con- aeruginosa by microscopy were found to exhibit slightly differ-
trasted with the assessment of the possible presence of a low ent PC-IGS sequences (Fig. 2), which confirms that at least two
level of hepatotoxins in the mouse bioassay (Table 3). different Microcystis types were present in this bloom. Unfor-
HPLC analysis of these bloom samples clearly demonstrated tunately, the length and sequence of the PC-IGS are unable to
that at least some of the A. circinalis cells present throughout identify Microcystis species (2). Bloom sample MD-90 corre-
the bloom produced saxitoxins and were thus neurotoxic (see sponds to the peak identified as M. flos-aquae, was toxigenic as
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Tables 2 and 3). By comparison, the mouse bioassay showed detected by NMT PCR, and contained microcystins as mea-
neurotoxicity only in the sample (MD-93) which contained the sured by HPLC (see Table 3). A sample identified as M. flos-
very highest concentration of saxitoxin. The 16S rRNA-based aquae in a Malpas Dam bloom in 1999 to 2000, MD-34, gave a
PCR assay consistently agreed with the HPLC data, with the positive result in the NMT PCR assay, but microcystins were
exception of sample MD-80. This PCR test for toxic type A. not detected by HPLC. Sample MD-34 differed from sample
circinalis is not a definitive toxigenicity test, as it may give MD-88 (identified as M. flos-aquae by microscopic examina-
occasional false-positive or false-negative results (6). In the tion) in the sequence of the PC-IGS, indicating that these
absence of a PCR test to detect the presence of a gene in the samples may contain different strains of M. flos-aquae. This is
saxitoxin synthesis pathway, the 16S rRNA-based test is the the first study to indicate toxigenicity in blooms of M. flos-
best available method for rapid screening of environmental aquae, although this has been suggested in a Swedish study
samples. Based on the results in this report, the 16S rRNA- (10). Tillett et al. (36) tested the toxigenicities of many M.
based method was valuable in providing an early signal of flos-aquae strains by using the NMT PCR assay and the protein
potentially saxitoxin producing Anabaena. Like the NMT PCR phosphatase inhibition assay, and all strains examined were
assay for microcystins, the 16S rRNA-based assay for toxic nontoxic.
Anabaena types is effective at a level of 10 cells ml⫺1 and can
indicate the possible neurotoxicity of the bloom well before the ACKNOWLEDGMENTS
cell counts reach the local high-alert level of 20,000 cells ml⫺1.
J.A.B. acknowledges the support of an Australian Postgraduate
The local water authority responsible for Malpas Dam has an Scholarship. She also gratefully acknowledges the ADC both for fi-
action alert at a cell density of 2,000 ml⫺1, as recommended by nancial support and for help with collection of samples and access to
the Australian Drinking Water Guidelines (25), and a high- Council records.
alert level of 20,000 cells ml⫺1, where blooms may contain We thank Mandy Choice (ADC) for performing the mouse bioas-
sufficient toxin to be of concern for human health (17). says and Geoff Eaglesham and Brad Davis (Queensland Health Sci-
entific Services) for HPLC analysis.
In the work presented here, the PCR assays, applied directly
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