Elsevier

Toxicon

Volume 60, Issue 3, 1 September 2012, Pages 378-384
Toxicon

First report of (homo)anatoxin-a and dog neurotoxicosis after ingestion of benthic cyanobacteria in The Netherlands

https://doi.org/10.1016/j.toxicon.2012.04.335Get rights and content

Abstract

In April and May 2011, three dogs died and one dog became ill after swimming in Lake IJmeer (The Netherlands). At the time, the lake was infested with the benthic cyanobacterial species Phormidium. A Eurasian Coot (Fulica atra) and a Black-headed Gull (Chroicocephalus ridibundus) also died near Lake IJmeer in the same period. One of the dogs and both birds were subjected to a pathological investigation. Furthermore, the Phormidium mat; algal samples from the dikes; contents of the animals’ digestive systems and organ tissues were analysed for the following cyanobacterial toxins: (homo)anatoxin-a; (7-deoxy-)cylindrospermopsin; saxitoxins and gonyautoxins by LC-MS/MS. Samples were also analysed for the nontoxic (homo)anatoxin-a metabolites dihydro(homo)anatoxin-a and epoxy(homo)anatoxin-a. The dog necropsy results indicated neurotoxicosis and its stomach contained Phormidium filaments. Anatoxin-a was detected in the Phormidium mat (272 μg g−1 dry weight, stdev 65, n = 3) and in the dog’s stomach contents (9.5 μg g−1 dry weight, stdev 2.4, n = 3). Both samples also contained the anatoxin-a metabolite dihydroanatoxin-a, and a trace of homoanatoxin-a was detected in the Phormidium mat. The birds were in bad nutritive condition at the time of necropsy and their stomachs and intestines did not contain any cyanobacterial material. Furthermore, no cyanobacterial toxins were detected in their stomachs, intestines and organs and they both had lesions that are not associated with cyanobacterial intoxication. This is the first report of anatoxin-a and homoanatoxin-a occurrence in The Netherlands, these toxins have likely caused the deaths of three dogs. The birds probably died of other causes. Dutch recreational waters are at this moment only screened for pelagic cyanobacterial species, the current bathing water protocol therefore does not protect humans and animals from negative effects of blooms of benthic cyanobacteria.

Highlights

► In The Netherlands, three dogs died after swimming. ► Clinical symptoms and necropsy pointed towards neurotoxicosis. ► Cyanobacteria and the neurotoxins (homo)anatoxin-a were found in stomach contents. ► Ingestion of the cyanobacterium Phormidium has caused the dogs’ death. ► First report of (homo)anatoxin-a occurrence and poisoning in The Netherlands.

Introduction

In the Netherlands, implementation of the EU Bathing Water Directive (2006/7/EC) has resulted in a protocol that emphasises on the monitoring of five potentially toxic and common (Kardinaal and de Haan, 2010) cyanobacterial genera (Anabaena, Aphanizomenon, Microcystis, Planktothrix and Woronichinia). There is broad consensus that representatives of these predominantly pelagic genera can produce a wide variety of potent toxins (Carmichael, 2001, Sivonen and Jones, 1999). The Dutch protocol does not include benthic cyanobacteria even though increasing numbers of toxigenic freshwater benthic species have been documented. Representatives of the benthic genera Aphanothece, Arthrospira, Fisherella, Lyngbya, Nostoc, Oscillatoria, Phormidium, and Scytonema are now known to produce one or more toxins (Ballot et al., 2004, Carmichael et al., 1997, Dasey et al., 2005, Edwards et al., 1992, Fiore et al., 2009, Gugger et al., 2005, James et al., 1997, Seifert et al., 2007, Smith et al., 2011, Wood et al., 2007).

Mats of these cyanobacteria can accumulate on the shoreline, where cattle, pets, wildlife and humans can get exposed to their toxins. Benthic cyanobacteria therefore constitute a hazard to animal and human health (Codd et al., 2005). Indeed, benthic cyanobacteria have been identified as potential sources of animal poisonings (Edwards et al., 1992, Gugger et al., 2005, Krienitz et al., 2003, Mez et al., 1997, Puschner et al., 2008, Sivonen et al., 1990, Wood et al., 2007). Dogs in particular seem to be attracted by these mats and might ingest substantial amounts of detached cyanobacterial material accumulated on shorelines (Codd et al., 1992). Several fatal cases of intoxication in dogs after ingestion of benthic cyanobacteria (mostly dominated by Oscillatoria and Phormidium) have been documented (Edwards et al., 1992, Gugger et al., 2005, Puschner et al., 2008, Wood et al., 2007). Anatoxin-a or homoanatoxin-a poisoning was identified as the most likely cause of death in these cases.

In April 2011, three dogs showed neurological symptoms after swimming in Lake IJmeer (The Netherlands), near Muiderstrand. Two dogs died shortly afterwards, one recovered after veterinary treatment. In May 2011, another dog died soon after swimming from the opposite shore (Catamaran beach) of the same lake. Benthic cyanobacterial mats had accumulated on both beaches. In addition, a Eurasian Coot (Fulica atra) and Black-headed Gull (Chroicocephalus ridibundus) were found dead near Lake IJmeer. The dog that died in May and both birds were subjected to a thorough post-mortem investigation. Because the dog likely died of neurotoxicosis, algal material from Lake IJmeer dikes; a cyanobacterial mat from the beach; and the dog’s stomach contents and liver tissue were analysed for cyanobacterial neurotoxins and their metabolites by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The stomach and intestinal contents of the birds and tissue of their livers and brains were also analysed for cyanobacterial neurotoxins.

Section snippets

Animal necropsy

The dog that died in May, the coot and the gull were subjected to pathological examination and macroscopic findings of abnormalities in the organs were recorded. The dog was dissected three days after it died and its body was kept at 4 °C prior to necropsy. The birds were dissected two days after their presumptive death and the remains were stored at 4 °C from the moment they were found until necropsy. Tissue samples from the dog’s heart, lungs, kidneys, adrenal glands, liver and brain were

Animal necropsy

The dog was a healthy, 4 month old female Golden Retriever. After swimming, it had vomited once and showed acute neurological symptoms (muscular spasms in the limbs, epileptic seizures and collapse). The dog died at the veterinary clinic a couple of hours after swimming. A first examination showed that the stomach was well filled with partly digested dog food, a small quantity of dark green or black leaf like material, and a small quantity of sand and black granules. Pathological examination

LC-MS/MS analysis

LC-MS/MS analysis of anatoxin-a can be hindered by the presence of the phenylalanine amino acid in samples (Furey et al., 2005, Gugger et al., 2005), because both compounds have a similar molecular weight and show partly similar product ions after collision-induced dissociation. Some authors state that anatoxin-a and phenylalanine also show the same chromatographic behaviour (Furey et al., 2005, James et al., 2005, Puschner et al., 2008), in which case presence of phenylalanine interferes with

Acknowledgments

E.J.F. was supported by grant 817.02.019 from the Netherlands Organization for Scientific Research (NWO). Dr Tineke Burger and Dr Arnold Veen (Rijkswaterstaat) are thanked cordially for sending a sample of the cyanobacterial benthic mat. Furthermore, we are grateful to Dr Hans Terwee (DAP Muiden) for providing information on the three dogs that got ill after swimming near Muiderstrand.

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