Different genotypes of anatoxin-producing cyanobacteria coexist in the Tarn River, France

Abstract

Repeated dog deaths occurred in 2002, 2003, and 2005 after the animals drank water from the shoreline of the Tarn River in southern France. Signs of intoxication indicated acute poisoning due to a neurotoxin. Floating scum and biofilms covering pebbles were collected in the summers of 2005 and 2006 from six different sites along 30 km from the border of this river. The cyanobacterial neurotoxic alkaloid anatoxin-a and/or its methyl homolog, homoanatoxin-a, was detected in the extracts of most samples examined by gas chromatography-mass spectrometry. Fifteen filamentous cyanobacteria of the order Oscillatoriales were isolated and displayed four distinct phenotypes based on morphological characteristics and pigmentation. Three of the phenotypes can be assigned to the genus Oscillatoria or Phormidium, depending on the taxonomic treatises (bacteriological/botanical) employed for identification. The fourth phenotype is typical of the genus Geitlerinema Anagnostidis 1989. Eight strains rendered axenic were analyzed for production of anatoxin-a and homoanatoxin-a, and all strains of Oscillatoria/Phormidium proved to be neurotoxic. The genetic relatedness of the new isolates was evaluated by comparison of the intergenic transcribed spacer sequences with those of six oscillatorian strains from the Pasteur Culture Collection of Cyanobacteria. These analyses showed that the neurotoxic representatives are composed of five different genotypes, three of which correspond to phenotypes isolated in this study. Our findings prove that neurotoxic oscillatorian cyanobacteria exist in the Tarn River and thus were most likely implicated in the reported dog poisonings. Furthermore, they reemphasize the importance of monitoring benthic cyanobacteria in aquatic environments to fully assess the health risks associated with these organisms.

FIG. 1.

Locations of the sampling sites along the Tarn River, France. The locations of the sampling sites are indicated by black circles. (Inset) Map of France showing the two regions, Tarn River (white circle) and the La Loue River (small black circle), where dog poisonings were reported.

FIG. 2.

GC-MS analysis of extracts from environmental samples. (A) GC chromatogram of a sample from Le Soulio, France, in July 2005. (Inset) EI mass spectrum at a retention time (T_R) of 21.7 min, characteristic of anatoxin-a (molecular ion m/z 165 and fragments m/z 150, 136, and 122). (B) GC chromatogram of a sample from Ste Enimie, France, in July 2005. (Inset) EI mass spectrum at retention time of 24.9 min characteristic of homoanatoxin-a (molecular ion m/z 179 and fragments m/z 164, 150, 136, and 122).

FIG. 3.

Photomicrographs of six cyanobacterial strains examined in this study. (A to D) Strains PCC 10601 (A), PCC 10702 (B), PCC 10608 (C), and PCC 10602 (D) correspond to phenotypes 1, 2, 3, and 4, respectively, described in Table 2. (E and F) Strains Oscillatoria sp. strain PCC 9240 (E) and Oscillatoria formosa PCC 10111 (F) studied for comparison. Bars, 20 μm.

FIG. 4.

Alignment of the nucleotide sequences of the ITSs. Percentage identity is represented by three colors: blue for 100%, green for values above 80%, and red for those above 60%. Gaps (dashes) are treated as differences. The conserved domains (D1, D1′, D2, D3, and D4), the tRNA genes, and the antiterminator (box B and box A) are indicated. The ITS sequences of strains PCC 10609, PCC 10610, PCC 10611, and PCC 10612 and the impure isolates Fil.10 PR and Fil.2D1 FY, corresponding to phenotype 1 are 99 to 100% identical to that of strain PCC 10601 and are not included in the alignment. The full-length ITS of strain Fil.1 SE is representative of phenotype 3. It was preferred over the identical but slightly incomplete sequence of strain PCC 10608, which was excluded from the alignment.

FIG. 5.

Dendrogram depicting the overall nucleotide differences in the ITS sequences (see Fig. 4 and legend) of 10 isolates from the Tarn River (France) in comparison to five other PCC strains, including the reference strains of Oscillatoria clusters 1, 2, and 4 (Table 2). Given that accurate phylogenetic inference cannot be drawn from this representation (see Materials and Methods), no scale bar is given for the nucleotide substitution per site.