## Chapter 28: Cyanobacterial poisoning in
**livestock, wild mammals and birds – an overview**

Ian Stewart 1,2 , Alan A. Seawright 1 , Glen R. Shaw 2,3

1 : National Research Centre for Envir onmental Toxicology, University of Queensland, Brisbane, Australia; 2 : Cooperative Research Centre for Water Quality and Treatment, Adelaide, Australia; 3 : School of Public Health, Griffith University, Brisbane, Australia



### Abstract

Poisoning of livestock by toxic cyanobacteria was first reported in the 19 th
century, and throughout the 20 thcentury cyanobacteria–related poisonings

of livestock and wildlife in all contin ents have been described. Some mass mortality events involving unrelated fauna in prehistoric times have also been attributed to cyanotoxin poisoni ng; if correct, this serves as a reminder that toxic cyanobacteria blooms predate anthropogenic manipulation of the environment, though there is probably general agreement that

human intervention has led to increase s in the frequency and extent ofcyanobacteria blooms. Many of the early reports of cyanobacteria poisoning were anecdotal and circumstantial, albeit with good descriptions of the appearance and behaviour of cyanobact eria blooms that preceded or coincided with illness and death in exposed animals. Early necropsy findings of hepatotoxicity were subsequently confirmed by experimental investigations. More recent reports supplement clinical and post–mortem findings with investigative chemistry techniques to identify cyanotoxins in stomach

contents and tissue fluids.614 I. Stewart et al.



### Introduction

Planktonic blooms and surface scums appear to have been recognised for over two thousand years, and indigen ous peoples of North America, Africa and Australia may have been aware ofthe poisonous nature of cyanobacteria (Codd et al. 2005). Detailed studies – including microscopy – of the behaviour of planktonic cyanobact eria blooms began in the late 19 thcentury. Surface scums that were concen trated by wind activity were observed, having a sudden onset and di ssipation (Phillips, 1884). The world’s first scientific report of the toxic nature of cyanobacteria was written by George Francis, who described in 1878 the rapid death of stock animals at Lake Alexandrina, a freshwater lake at the mouth of the Murray River in South Australia (Francis 1878). Francis described a wind–blown surface scum of the brackish water cyanobacterium Nodularia spumigena , incidental consumption of which resulted in the death of sheep, horses, dogs and pigs within periods of 1 to 24 hours. Francis also experimentally dosed asheep with fresh bloom material. Necropsy examination showed a significant pericardial effusion, “two pints of yellow serum” (i.e. most likely ascitic fluid) in the abdominal cavity, and normal–looking liver, lungs and kidneys (Francis 1878). Codd et al (1994) note that the observations of Francis are entirely in agreement with the modern understanding of Nodularia –associated intoxication through its associated cyanotoxin, nodularin. Beasley et al (1989a, 1989b) suggestthat another cyanotoxin may have been involved, given that Francis did not report any hepatic pathology characteristic of nodularin intoxication, however the liver lesion caused by nodularin may not have been apparentor obvious to the inexperienced observer. The description of ascitic fluid in this context is strongly suggestive of acute liver damage. Beyond Francis’s (likely) observation of ascitic fluid, and the necropsy findings of (po ssibly) a single animal, it is probably unwise to attempt a reinterpretation ofFrancis’s findings (Francis did report opening “many sheep that died”, but it is not clear from the reportwhether his examinations of those field deaths extended beyond his obvious interest in the apparent absence of bloom material from the stomachs) (Francis 1878). Since Francis’s report in the last quarter of the 19 thcentury there have been numerous descriptions of mammal and bird mortalities associated with exposure to cyanobacteria. The relevant literature throughout the 20 th
century represents all inhabited c ontinents. Anecdotal and case reports have been collated and/or reviewed by many authors, e.g. (Schwimmer and Schwimmer 1955, 1968; Schwimme r and Schwimmer 1964; Hammer 1968; Codd and Beattie 1991; Carmicha el and Falconer 1993; Ressom etChapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds 615

al. 1994; Duy et al. 2000; Briand et al. 2003; Codd et al. 2005; Falconer2005). Many of the earlier reports are circumstantial, noting a temporal association between the presence of cyanobacterial blooms in a waterbodyand otherwise unexplained mortalities. Early necropsy findings of hepatotoxicity were subsequently confirmed by experimental toxicology studies into isolated and purified cyanotoxi ns. More recent reports supplement clinical and post–mortem findings withinvestigative chemistry techniques to identify cyanotoxins in stomach contents and organs. Discussion of wild and domestic animal poisonings can be found in most reviews of toxic cyanobacteria, and mention of field deaths often served to introduce and contextualise reports of experimental toxicological investigations into cyanobacterial susp ensions, extracts or isolates. As thenumber of primary anecdotal and case reports and reviews into cyanobacteria–related mortality of birds and te rrestrial mammals is extensive, a systematic, comprehensive review has notbeen conducted in recent times.Such a task is also beyond the scope of this review; therefore we will present some selected discussion of early reports, including palaeontologic findings, then we will discuss more recent investigations that incorporate modern investigative chemistry tec hniques to supplement clinical and post–mortem findings in order to diagnose cyanotoxin poisoning.



### Early reports (pre–1960s)

Lake Alexandrina in South Australia, as mentioned in the Introduction,was a trouble–spot for cyanobacteria–related poisonings, with several hundred deaths associated with a number of bloom events in the latter part of

the 19 thcentury. The local Aboriginal people were apparently aware of poisonings in the lake at around 1850 (Codd et al. 1994). Another early locus was South Africa, where Steyn (1945) suggested that “many thousands” of cattle and sheep mortalities over the preceding 25–30 years were attributable to cyanobacterial poisoning; horses, mules,donkeys, dogs, hares, poultry and waterbirds found dead near affected waterbodies were also thought to be similarly affected. A toxic bloom on the Vaal Dam in 1942 covered an estimated 98 per cent of the reservoir, this being at the time 120km x 24km (at its broadest point) with a 1 teralitre capacity (Stephens 1949). Microcystis sp. were identified in the water; fulminant hepatic disease was observed insome animals, as were subacute cases with death ensuing after some two weeks and chronic cases with weeks or months of morbidity preced ing either death or recovery. Necropsy findings in fulminant cases in cluded pulmonary haemorrhage, hae-61

moperitoneum, hepatomegaly (with liver s dark red or black in colour, and friable), splenomegaly, and occasiona l “bloody patches...on the mucous membranes of [the stomach and intestines].” Findings in subacute caseswere haemo–serous exudate in the pericardium, thorax and peritoneal cavity, the liver being soft or friable and yellow–coloured. Experimental dosing of animals with dam water produced identical clinical signs and post– mortem lesions. Secondary photosensi tivity was described in detail: painful inflammation, fissuring of aff ected epithelium, purulent discharge, sloughing of skin. Unpigmented areas are mainly affected – muzzle, udder and teats, ears (Steyn 1943). Cyanobacteria–related poisonings were reported in North America also

from the latter half of the 19 thcentury – see citations in Codd et al (2005). Howard and Berry (1933) discussed Anabaena –related cattle and other

animal deaths at an Ontario lake in 1924. Storm Lake in Iowa experienced

dramatic bloom events in 1952: associated with Anabaena flos–aquae blooms were estimated deaths of 5–7,000 gulls, 560 ducks, 400 coots, 200 pheasants, 50 squirrels, 18 muskrats, 15 dogs, 4 cats, 2 hogs, 2 hawks, 1 skunk, 1 mink, plus “numerous” songbirds. These figures were taken from the recorded number of animals buried by a field worker at the time, so were most probably underestimates of the total number of deaths. Signs of neurotoxicity were seen: prostra tion and convulsions preceded death; milder cases displayed restlessness, weakness, dyspnoea and tonic spasms. 57 weak and partially paralysed mallards recovered following gastric lavage. A dog drinking the water when si ck birds were being gathered then died near the lake shore when trying to escape from workers. Botulism was excluded from the diagnoses, and necropsy findings were unremarkable.Experimental oral or parenteral dosing of bloom filtrate to laboratory rodents and chickens resulted in rapi d death – within minutes, though delayed mortalities were reported: one ch icken lived for 48 hours, and guinea pigs exposed to bloom filtrate orally or by i.p. injection died “within 24 hours” (Firkins 1953, Rose 1953). While some of the aforementioned repo rts were dramatic in terms of the number of animals affected by speci fic bloom events, equally compelling were early descriptions of the rapi dity with which large animals succumbed after exposure to (presumably) neurotoxic cyanobacteria. McLeod and Bondar (1952) noted that a horse, “several” calves, two pigs and a cat

all died within an hour of consumi ng cyanobacteria–affected water at Lake Dauphin, Manitoba in 1945. At the sa me site in 1951, nine dogs and ahorse died within one hour; the horse showed signs of progressive muscular weakness and paralysis, with prof use sweating. Experimental dosing of laboratory rodents was conducted using bloom material (99% Aphanizomenon flos–aquae ) collected from the lake six days after the last re6 I. Stewart et al.617

ported poisoning event. Interestingly, oral or i.p. injections of lake water filtrate were not toxic, whereas unfiltere d lake water, various preparationsof cyanobacterial residue or filtrate from disrupted cellular material resulted in deaths ranging from under 12 hours to around 65 hours. Clinicalsigns were those of “loss of equilibrium followed by progressive paralysis.” Later signs were clonic muscular spasms and respiratory dysfunctionleading to pronounced cyanosis. Post–mortem findings from these experimental animals were not reported (McLeod and Bondar 1952).



### Prehistoric animal mortalities

The first detailed descriptions of the behaviour of cyanobacteria blooms and cyanobacterial toxicity were written in the latter part of the 19 thcentury (see Introduction), but there is no reason to suppose that both phenomena were not present at earlier stages of our history. Codd et al (1994) suggest that Pliny the Elder may have described a cyanobacterial or algal scum on the River Dnieper in AD77, and Höger (2003) implies that an Old Testament reference may be a description of a piscicidal Planktothrixbloom in the River Nile. These contemporaneous descriptions reach modern researchers through the device ofwritten language. However, two reports from the palaeontology literature suggest that cyanobacterial toxicity may be a prehistoric phenomenon. Br aun and Pfeiffer (2002) present their hypothesis that at Neumark–Nord in Ge rmany, a Pleistocene (1.8 million – 11,000 years ago) lake assemblage of >70 deer, as well as forest elephant, rhinoceros, auroch (ox) and cav e lion skeletons may represent a cyanotoxin–related mass mortality event. The preservation features and antler development suggest rapid, cat astrophic death in autumn. Calcified layers in the sediment are thought to represent decomposed cyanobacteria, and absorption spectra of cyanobacter ia–specific carotenoids were identified in sediment extracts. The authors used HPLC with UV detection to determine the presence of microcystins in methanol extracts of sediment; a similar (but not identical) UV spectrum to that obtained from an extract of

a Microcystis aeruginosa strain with an unspecified microcystin congener profile was seen. Other possible explan ations for the deaths at Neumark– Nord – butchery by early humans, or a gas eruption – are discussed by the authors but thought to be unlikely (Braun and Pfeiffer 2002). Considering further the cyanotoxin hypothesis, it would be interesting to see further confirmatory investigations for microc ystins in the sediment extracts using a second method such as mass spectro metry. Looking beyond the search for microcystins in the sediment, it would seem that sudden, catastrophic Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds618

death due to ingestion of a cyanobacterial neurotoxin could equally explain

these events. An older mammal and bird assemblage from the Middle Eocene epoch(49 – 37 million years ago) also has features suggestive of a mass poisoning event. The Messel oil shale pit in Germany was an ancient freshwater lake. While the sediment layers are more compacted than those of Neumark–Nord, and chemical and physical transformation of sediments by diagenesis is likely, Koenigswald et al (2004) suggest that there are marked similarities across both sites, and thin layers of siderite at Messel probably represent the decomposed remains of sunken cyanobacteria blooms. Well–preserved skeletons of horses, turtles, bats and birds are found; most were in good health, and stomach contents show that they were well nourished. Apparent features of seasonal death are seen: of some 50 dawn horse skeletons, five are of pregnant mares. The foetuses are all in late stages of development, which again suggests seasonal mortality, as predator pressure on newborn ungulat es drives seasonal parturition, usually in late spring. These skeletons were found in different layers, so the picture is that of seasonal deaths o ccurring in different years. The frequent presence of winged animal skeletons at Messel, which is unusual in lake deposits, also supports a toxic waterblo om hypothesis (Koenigswald et al. 2004). The authors also discuss a finding of aquatic turtle specimens in which two animals are preserved as pairs in close proximity to each other. “At least” five slabs of shale featuring such pairs of turtles have been found; photographs of two pairs are presented in the paper. The authors cite another worker’s interpretation of this finding as representing sudden death during mating, which would support a toxic cyanobacteria bloom hypothesis (Koenigswald et al. 2004) . However the photographs show two pairs of turtles apparently fused posteriorly but facing away from each other, which is not the normal copulatory position adopted by turtles. Themale mounts the female, though infrequent reports of “belly–to–belly” position can be found in the literature (Goode 1967, Kuchling 1999). If these reports do indeed describe anci ent cyanobacteria–related intoxications, they serve as a reminder that toxic cyanobacteria blooms are natural events; they predate recent anthropogenic manipulation of the environment, and may indeed predate human arrival. However, most cyanobacteriologists would acknowledge that the problem of harmful cyanobacteria is exacerbated in m odern times due to cultural eutrophication, and may worsen in future with the additional impact of human activity–related climate warming and ot her population–related pressures. I. Stewart et al.619



### Recent developments

While many early reports and reviews of cyanobacteria–related poisoningsare impressive because of their comprehensive nature and empirical observations supplemented by simple experimental techniques, more recent reports of mammal and bird poisonings reveal the advances in analytical techniques that allow definitive id entification of well–characterised cyanotoxins. The following section will present discussion of recent case studies that investigated animal poisoni ngs caused – or likely to have been caused – by cyanobacteria that pro duce toxins from each of the main cyanotoxin groups that have been char acterised to date. Clinical presentation, necropsy findings that reflect the current understanding of the mechanisms of toxicity, and modern inves tigative chemistry procedures that confirm the diagnosis are discussed. These examples are by no means exhaustive, as different analytical approaches may be applied, and factors such as dose, time period of exposure and ingestion of more than one type of cyanotoxin may complicate the diagnosis. The experimental toxicology of these isolated cyanotoxins has been well documented, and many excellent reviews are available, e.g. (Ressom et al. 1994, Codd et al. 1999, Carmichael 2001, Falconer 2005). Peracute microcystin and nodularin poisoning presents principally with fulminant haemorrhagic liver injury; pathologic cha nges can also be seen in the gut, kidney, heart and lungs. Cylindrospermopsinintoxication is characterised by fatty degeneration of the liver, cholestasis, injury to renal tubules and thymic atrophy. Experimental dosing with cylindrospermopsin–producing cyanobacterial extracts results in pathological effects on other tissues and organ systems, including the gastrointestinal tract and spleen. Cyanobacterial neurotoxins cause deat h by respiratory failure through various mechanisms: anatoxin–a by a depolarising block at the neuromuscular junction; anatoxin–a(s) is an anticholinesterase, thus preventing theenzymatic degradation of acetylcholine which results in uncontrolled hyperstimulation of muscles. Hypersaliv ation and lacrimation, which aresigns of parasympathetic stimulation, also characterise anatoxin–a(s) poisoning. The saxitoxin group are sodiumchannel blocking agents, thus interfering with axonal conduction. Death is very rapid – within minutes – when purified cyanobacterial neurotoxins are administered parenterally, particularly from anatoxin–a or saxitoxins. Post–mortem and histology findings are generally unremarkable. Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds62



### Microcystins

**Case study: duck deaths in Japan, 1995 (Matsunaga et al. 1999)**

Shin–ike pond in Hyogo Prefecture had become eutrophic from the entry of untreated sewage following an earthquake in January 1995, when a sewage treatment plant was damaged. A bloom of M. aeruginosa was evident. In September of that year, some 20 ducks died at the site; Oo–ike pond, 1km distant, also had a cyanobacteria bloom, but no unusual bird deaths were reported from that site. Water samples were collected from both ponds. Necropsy of one of the aff ected ducks showed a liver that wasnecrotic and “severely jaundiced...” Preliminary toxicity testing with sonicated cell suspensions using a mouse bi oassay resulted in unspecified signs

of Microcystis toxicity and death within two hours from the Shin–ike pond material, whereas that from Oo–ike pond did not produce signs of acute toxicity. Quantification of microcystins by HPLC revealed that Shin–ike pond lyophilised bloom material contained 318 μ g/g MC–RR, and 161 μ g/g MC–LR. Oo–ike pond cyanobacteria contained 29 μ g/g MC–RR and no detectable MC–LR (Matsunaga et al. 1999). While tissues from affected birds were not analysed for microcystins, the combination of necropsy, bioassay and microcystin quantification, along with differential findings between the suspect pond and a nearby “control” site lend strong support to the presumptive diagnosis of acute mi crocystin intoxication in this case.



### Nodularin

**Case study: dog death in South Africa, 1994 (Harding et al. 1995).** 

In March 1994 a bull terrier was admitte d to a Cape Town veterinary hospital with a history of lethargy, vomiting and loss of appetite after drinking from an urban lake the previous day. The lake is known to be eutrophic and the phytoplankton is normally dominated by M. aeruginosa . Antibiotic, corticosteroid, antiemetic and c holeretic pharmacotherapy was administered, as well as vitamin B complex and intravenous fluids. Serum alanine transaminase was markedly el evated, indicating acute hepatic dysfunction. The dog died on the fifth day after admission; necropsy was notconducted, but liver samples were collected and prepared for histopathological examination. Water and scum samples from the lake were col0 I. Stewart et al.621

lected two days after the dog drank from it. Microscopic examination revealed a bloom of 95% Nodularia spumigena , with the remainder being M. aeruginosa . Scum material was acutely toxic in a mouse bioassay, withliver damage suggestive of cyanobacterial hepatotoxin poisoning. Methanol extracts of bloom material analysed by HPLC with photodiode array and UV detection showed a major peak that increased when spiked withnodularin standard, and a UV spectrum that matched that of nodularin. Lyophilised cyanobacteria contained 3.5mg/g nodularin; no evidence for thepresence of microcystins in the bloom material was found. Histology findings suggested toxic liver injury, withhepatocyte degeneration and indications of cholestasis (Harding et al. 1995). Again, while tissues were not examined for the presence of cyanotox ins, the combination of clinical presentation, diagnostic tools – liver f unction tests, histopathology, mouse bioassay – and investigative chemistry strongly supported the diagnosis of nodularin poisoning.



### Cylindrospermopsin

**Case study: cattle deaths in Australia, 2001 (Shaw et al. 2004).**

Two separate poisoning incidents were investigated in Central and Northwest Queensland involving a total of 55 cows. Affected animals were lethargic and recumbent for three or four days ante–mortem. Post–mortem findings were typical of cylindrosperm opsin intoxication, with pallid livers and cholecystomegaly. Histopathologyshowed hepatocyte degeneration and necrosis, nephrosis and multifocal cardiomyopathy. Farm water samples, rumen contents, liver, kidney and muscle were analysed by HPLCtandem mass spectrometry for cylindrospermopsin, which was found in allsamples except muscle. Water and rumen samples contained cylindrospermopsin concentrations in excess of 1mg/L (Shaw et al. 2004). The principal cylindrospermopsin–producing cy anobacterium in tropical and subtropical Queensland is Cylindrospermopsis raciborskii . Associated stock animal deaths and human water suppl y–related acute and chronic poisonings have long been suspected there (Hayman 1992, Thomas et al. 1998,Griffiths and Saker 2003), and livestock industry publications seek to inform farmers, veterinarians and governme nt workers of the risks from this cyanotoxin (Berry 2001). Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds6



### Anatoxin–a

**Case study: dog deaths in France, 2003 (Gugger et al. 2005).**

Two dogs died in separate incidents in September 2003 shortly after drinking from the shore of a river in the Jura region. Clinical signs were vomiting, hind limb paresis and respiratory failure preceding death. The smaller dog (2.5kg) sickened and died shortly after emerging from the water, whereas the larger dog (25kg) had a delayed onset of signs and died within five hours. Stomach contents, intestinal contents and liver were sampled as well as water column and benthic biofilm from the river; stomach contents and field samples were examined for phytoplankton identification. An initial screen for three potential cyanotoxins was conducted on biofilm samples: protein phosphatase 2A inhibition assay for microcystins, mouse

neuroblastoma in vitro assay for saxitoxins, and HPLC with photodiode array for anatoxin–a. No microcystins or saxitoxins were detected, but a compound with similar chromatographic characteristics to anatoxin–a was seen, so subsequent investigations were directed to that end. A total of seven Oscillatorealean cyanobacterial species and genera were identifiedacross field and gastric samples; three of these were isolated and one isolate, Phormidium favosum , was found to produce anatoxin–a. This benthic species was found in the biofilm covering river sediments, stones and macrophytes, as well as from the stomach contents of both dogs – P. favosum being the dominant cyanobacterium found in the smaller dog’s stomach. Identification of anatoxin–a in biofilm, liver and gastro–intestinal contents was confirmed by tandem mass spectrometry; the authors draw attention to the necessity for confirmation of identity by fragmentation ion spectral assignments to allow definitive identification of anatoxin–a, as the amino acid phenylalanine has an identi cal mass spectrum peak to that ofanatoxin–a when single ion monitoring is used. This was also the first report of anatoxin–a in French waters, and the first identification of an anatoxin–a–producing Phormidium species. The authors suggest that neurotoxic signs in 37 dogs (with 26 deaths) in 2002 and 2003 in the south of France may also have been associatedwith anatoxin–a intoxication (Gugger et al. 2005). This comprehensive investigation utilised well–equipped facilities, use of anatoxin–a and phenylalanine standards, and combinedexpertise in the fields of investigative chemistry, phytoplankton identification and isolation, so it probably repr esents the current state of the art for the diagnosis of anatoxin–a poisoning.

22 I. Stewart et al.623



### Anatoxin–a(s)

**Case study: waterbird deaths in Denmark, 1993 (Henriksen et al. 1997, Onodera et al. 1997).** 

Cyanobacteria–associated an imal deaths have been reported at Lake Knudsø since 1981. In 1993, two grebes and a coot that died when a cyanobacterial bloom was evident were colle cted and frozen. Stomach contents were examined microscopically to identify cyanobacteria; Anabaena lemmermanniiwere found in all three birds. This material was lyophilised for further toxin analysis. Four neurotoxic strains of A. lemmermannii were isolated and cultured from bloom material collected from the lake. Bloom material, cyanobacterial cultures and st omach contents were analysed for anticholinesterase activity with a colori metric assay and for microcystins by ELISA. Only low levels of micr ocystins (ng/kg range) were found in bird gastric contents. Neither anatoxin–a nor saxitoxins were detected by HPLC. Intraperitoneal injection of bloom material into mice produced signs of acute neurotoxicity, including the muscarinic signs of salivation, lacrimation and urinary incontinence that typify anatoxin–a(s) intoxication. Anticholinesterase activity was signifi cant in bloom samples (2.3mg anatoxin–a(s) equivalents/g); A. lemmermannii isolates and stomach contents also showed anticholinesterase activity (29–743 μ g/g, and 2–90 μ g/kg body weight respectively). Subsequent wo rk using mass spectrometry, nuclearmagnetic resonance and circular dichroism definitively identified anatoxin–a(s) in bloom material (Henriksen et al. 1997, Onodera et al. 1997).



### Saxitoxins

**Case study: sheep deaths in Australia, 1994 (Negri et al. 1995).**

Thirteen ewes and one ram died next to or within 150m of a farm dam. Observed signs were trembling, recumbency and crawling. The ram, which was ataxic, was caught by the owner an d “immediately died in his arms.” Necropsy and histopathology findings were unremarkable. The dam was

found to contain a bloom of Anabaena circinalis . Mouse bioassay of bloom material caused death within 4–11 minutes, with clinical signs of staggering, gasping, leaping and resp iratory failure. High levels of saxitoxins were found in bloom extracts and intestinal contents of one sheep by HPLC with fluorescence detection; principal components were C–toxins Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds6

and gonyautoxins, with low levels of saxitoxin and decarbamoyl saxitoxin. Neither anatoxin–a nor microcystins were found using GC/MS and HPLC respectively; trace amounts of microcystin–like activity were found with a protein phosphatase assay (Negri etal. 1995). Saxi toxin–producing Anabaena circinalis is a particularly problematic cyanobacterium in Australia (Humpage et al. 1994). A 1000 km-long riverine bloom in the spring and summer of 1991 was associated with significant stock losses; one farmer alone reportedly lost over 1,100 sheep during the six–week period of the bloom (NSW Blue–Green Algae Task Force 1992, Smith 2000).



### Lesional neurological **disease and cyanobacteria?**

The case studies summarised above repr esent investigations into animal

deaths associated with the well–charact erised cyanotoxins. However, other less well–understood cyanotoxins may be capable of killing animals, and are being actively researched. Wilde et al. (2005) and Williams et al (2007) report on avian vacuolar myelinopathy (AVM), a recently–

described wildlife disease. AVM was re portedly responsible for more than 100 bald eagle deaths, and “untold numbe rs” of waterbirds. Clinical signs

of the disease are loss of coordination on land and in water, and difficulty in flying. Necropsy reveals a diffuse vacuolation of myelinated neuronaltissue. Epidemiological investigations suggest that a neurotoxin is responsible, with exposure being seasonal, li nked to dietary intake by herbivorous waterfowl from AVM–positive waterbodies, and moving up the food chain (i.e., from waterfowl prey to eag le predator). Current evidence suggests that AVM is not contagious. A common factor at AVM–positivesites – and absent or rare at AVM–negative sites – appears to be an epiphytic cyanobacterium in the order Stigonematales. The authors demonstrate the potential of veterinary ep idemiology to advance the understanding of cyanobacteria–related toxicity by use of experimental techniques that are not available in the field of human epidemiology. A mallard duck sentinel trial was conducted, where 20 farm–raised birds were tagged for identification, had wing feathers cli pped and were released into an AVM– suspect pond. The site was regularly monitored, symptomatic birds were captured and sacrificed, and at the end of the six–week trial, remaining sentinels were captured and sacrificed. Five symptomatic ducks and tenbirds captured at the end of the trial – these 15 being all birds able to be recaptured from the pond – all showed AVM lesions in brain tissues (Wilde et al. 2005). Williams et al (2007) have isolated and cultured the suspectcyanobacterium from environmental samples, and have developed PCR as24 I. Stewart et al.625

says to aid detection and identification. Further epidemiological field work using suitable control sites would be of great interest.



### “Unusual” animal deaths and cyanobacteria

The previous discussion has concentrated on cyanotoxin–related deaths in common domestic, stock and wild animals. Some other animals reportedly poisoned by cyanobacteria are:

• Flamingos: Cyanobacteria–related mortalities have been reported in three flamingo species, both wild and captive (see summary by Codd et al (2003)). Lesser Flamingos in Ke nyan soda lakes have been the subject of research interest, with four microcystin congeners and anatoxin–a found in cyanobacterial mats and stomach contents of dead birds at Lake Bogoria. Microcystins and anatoxin–a were also detected in faecal pellets collected from lake shorelines (Krienitz et al. 2003). The same cyanotoxins have been f ound in feathers taken from poisoned flamingos, with a dietary origin most likely (Metcalf et al. 2006). Mass

die–offs of tens of thousands of bi rds have been reported in these crater lakes, with implications for management and regional and national economies, as flamingos are a significa nt tourist attraction (Krienitz et al. 2003, Ndetei and Muhandiki 2005). Isolated strains of Arthrospira fusiformis from two crater lakes have been shown to produce both microcystin–YR and anatoxin–a, while an A. fusiformis strain from a third lake produces anatoxin–a (Ballot et al. 2004, Ballot et al. 2005). Similar Lesser Flamingo poisonings have been reported from alkaline lakes in Tanzania, with toxic A. fusiformis implicated (Lugomela et al. 2006). The implications of these findings are significant for several reasons, as A rthrospira sp. (also known as Spirulina ) are the principal food source of Lesser Flamingos, and Spirulina spp. are used as a dietary supplement by humans and as a feed additiv e for livestock. There is a significant body of literature that suggests that consumption

of Spirulina is not harmful (Ciferri 1983, Belay et al. 1993, Hayashi et al. 1994, Qureshi et al. 1996, Salazar et al. 1998, Abdulquader et al. 2000, Al–Batshan et al. 2001), thoug h presumably these studies refer to

non-toxic strains of Arthrospira used for both commercial mass production and from wild harvesting. Investigations to determine the

relative contribution of A. fusiformis to the production of cyanotoxins in Kenyan soda lakes would be of great interest. Some of these lakes are periodically dominated by more well–known toxigenic cyanobacteria

such as Anabaena and Microcystis spp. (Ndetei and Muhandiki 2005), Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds62

so it will be important to estimate the production capacity of cyanotoxins by various cyanobacteria in field situations when harmful levels of cyanotoxins are present.

• Insectivorous bats: Staff at a campground in Alberta, Canada, in the summer of 1985, counted 500 dead bats and estimated over 1,000 deaths

on the leeward side of a lake. At least 24 dead mallards were also reported. The lake area was covered with a “thick white scum” that had a blue–green sheen. Examined animals were covered with a green slime; necropsy did not reveal any abnormal ities. An alkaloid was extracted from the material covering the carcases. This alkaloid was identified by GC/MS and found to be anatoxin–a ( known at the time as Very Fast Death Factor) (Pybus et al. 1986).

• Rhinoceros: Four white rhinoceroses were introduced to a South African game reserve in May, 1979. Two months later, two of them

were found dead but were unable to be examined. Approximately one week later, another rhino was found dead after being seen to be active the previous day. Macroscopic and microscopic findings were typical of acute hepatotoxicity: hepatomegaly, ascitic fluid, coagulopathy seen in various tissues, severe hepatic necrosis and loss of hepatic architecture. At the time of death, a severe bloom of M. aeruginosa covered the park dam, with a surface scum of 4–12cm (Soll and Williams 1985).

• Honeybees: In the summer of 1971, “almost total” mortality of bees

from 84 hives was associated with the insects watering on the leeward edge of a lake in New South Wales, Australia. That area of the lake was affected by a windborne scum of A. circinalis ; an apiary on the windward shore was unaffected (May and McBarron 1973).



### Exposure routes, **avoidance behaviour**

Exposure to cyanotoxins in the field is presumably either through contaminated drinking water or by directconsumption of benthic cyanobacteria. Codd et al (2005) also note suggestions that cyanotoxins may movealong the food chain to poison wild animals. This would seem to be a plausible interpretation: there is a signifi cant section of the literature devoted to experimental and observational studi es of cyanotoxin bioaccumulation and trophic transfer. Much of the literature is concerned with potential human exposure to cyanotoxins through cons umption of molluscs and fish; shortterm (days, weeks) accumulation of hazardous levels of cyanotoxins in aquatic prey is certainly feasible, and predator animals may be more at risk 6 I. Stewart et al.627

than humans in some circumstances, si nce they are more likely to consumethe viscera in which cyanotoxins are found in highest concentrations. Several bioaccumulation studies are inc onclusive; for some positive examples see: (Negri and Jones 1995, Saker and Eaglesham 1999, Magalhães et al 2001, Sipiä et al 2002, Saker et al 2004, Sipiä et al 2004, Smith and Haney 2006). Experimental work has shown that the inhalation route may be an efficient method for microcystin–LR to access the circulation (Creasia 1990, Fitzgeorge 1994, Ito 2001), but there is no evidence that cyanotoxins can be effectively aerosolised to toxic concentrations under field conditions. The topic of inhalational exposure to cyanotoxins is clearly under– researched at present.

Codd et al (1992) outlined the premise that exposure to toxic cyanobacteria may not always be coincidenta l; dogs may actively seek out and consume benthic cyanobacteria. Some descriptions in the report of Hamill (2001) also suggest that dogs may actively consume toxic cyanobacteria. The work of Gugger et al (2005) also appears to support this concept: while descriptions from the field were that two dogs died “soon afterdrinking water from the shoreline of the...river...” analysis of phytoplankton in the dogs’ stomachs revealed the presence of biofilm–associated cyanobacteria that were not seen in water column samples. This thickbiofilm reportedly covered sediment, stones and macrophytes at the water’s edge (Gugger et al. 2005); it is at least conceivable that those animals were also actively consuming toxic cyanobacteria for their final meal. As for planktonic cyanobacteria, there are suggestions that wild and domestic animals will avoid drinking from cyanobacterial scums if less–affected water is accessible. Falconer (2005 p.80) notes that detailed investigations of cyanobacteria–related stock animal deaths often reveal that

cleaner water was unavailable because offence lines, thus forcing the animals to consume toxic water. Other workers have reported avoidance behaviour in cattle (Codd 1983) and fl amingos (Ndetei and Muhandiki2005), but Carbis et al (1994) report that sheep did not discriminate between hepatotoxic cyanobacteria–contaminated water and a readily available alternate supply. Lopez Rodas and Costas (1999) report observing ungulates, birds, wasps and unspecified wildlife failing to demonstrate avoidance behaviour, consuming concentrated M. aeruginosa scum in Spanish reservoirs. Their subsequent experiments showed that laboratory mice demonstrated an ultimately fatal preference for dense cultures of microcystin–producing M. aeruginosa over tap water, and they did not discriminate between toxic and non–toxic strains of M. aeruginosa in their drinking water. Steyn (1943) noted th at pregnant animals and dairy cows displayed a preference for consuming Microcystis scums, suggesting that a Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds628

relative nutritional deficiency may explain higher mortalities than seen in dry cows.



### Treatment of affected **animals; research needs**

As no complete pharmacological antagonists against cyanotoxins are available, treatment of affected anim als is largely supportive. Purgatives and enemas may be helpful; unguents and restriction to shaded areas

should be considered for animals suffering secondary photosensitization(Steyn 1943). Other supportive therapies include emesis, activated charcoal and bathing to remove bloom material from the fur (Corkill et al.1989, Roder 2004). Blood transfusion and correction of electrolyte imbalance has been recommended (Beasley et al. 1989a). Aggressive intervention with fluid replacement and steroids has been suggested for cases ofcyanobacterial hepatotoxin poisoning (Roder 2004). While such an approach was unsuccessful in the case (d iscussed above) of the bull terrier presumed to have died from nodularin intoxication (Harding et al. 1995), there is clearly a need for controlled experiments to evaluate interventionaltherapies. Pre–treatment with pharmacologic doses of hydrocortisone prevented acute and delayed MC–LR–relate d mortality in mice (Adams et al. 1985), and dexamethasone and indometh acin pre–treatment blocked renaltoxicity caused by MC–LR (Nobre et al. 2001). Pre–treatment with steroids is obviously not a practical therapeutic intervention, but these findings suggest that there is much more to be learned about the immunotoxicology of the cyanobacterial hepatotoxins. Artificial ventilation has been sugg ested in cases of cyanobacterial neurotoxin poisoning (Beasley et al. 1989a, Beasley et al. 1989b, Carmichael and Falconer 1993, Roder 2004). While th e practical application of this technique in the field would present significant challenges, experimental

work to evaluate the benefits of intermittent positive pressure ventilation and other intensive therapy may have implications for the field of human medical care as well as for economically valuable equine and livestock

animals. Work conducted in the 1970s on calves and rodents dosed with anatoxin–a–producing cyanobacteria sugg ested that artificial ventilation is unlikely to be a practical intervention, with one calf failing to achieve effective spontaneous respiration after being ventilated for 30 hours, and rats similarly afflicted after maintenance ve ntilation of up to eight hours (Carmichael et al. 1975, Carmichael et al. 1977). However, anatoxin–a doses producing up to 95% neuromuscular bloc kade in rats were reportedly reversible with respiratory support (Val entine et al. 1991). Artificial ventila- I. Stewart et al.629

tion was conducted on three rats duri ng experimental administration of anatoxin–a(s); they survived a greater than 4–fold lethal dose in the short term (less than one hour), but the study was not designed to follow through to recovery (Cook et al. 1990). Interventional strategies to treat saxitoxin poisoning using antitoxin or the potassium–channel blocker 4– aminopyridine and artificial ventilation have shown promise, though the toxicodynamics of saxitoxin are complex and the combined neurological, cardiovascular and respiratory effects are incompletely understood (Changet al. 1993, Benton et al. 1994, Chang et al. 1996, Chang et al. 1997, Benton et al. 1998). Management of seizures, presumably anticonvulsant pharmacotherapy, has been suggested in cases of cyanobacterial neurotoxin poisoning (Roder2004), though this may be a moot point if, as suggested at least for anatoxin–a, seizures result from hypoxia–related cerebral ischaemia (Carmichael 1994). Cyanobacterial neurotoxin–related seizures are not well described in the literature, mostly be ing referred to as “convulsions”, though there are references to clonic spasms (McLeod and Bondar 1952), clonic seizures (Cook et al. 1989, Cook et al. 1998, Carmichael 2001), tonic spasm (Firkins 1953) and tonic seizures (Beasley et al. 1989a, Beasley etal. 1989b). The electrophysiology of these events has not been investigated. Opisthotonus is often described (Carmichael et al. 1975, Gorham and Carmichael 1988, Cook et al. 1989, Carmichael 2001, Codd et al. 2003); this increase in tone of the musculature of the neck and shoulders has some similarity to the clinical manifestation of a tonic seizure, though again the priority from a management perspective would appear to be restoration of oxygen to the tissues. Anticonvulsants have also been administered for management of seizures in cases of cyanobacterial hepatotoxin poisoning, but with little apparent benefit (Corkill et al. 1989).Further empirical studies are warranted to assess various emergency interventions, including pharmacotherapies, for cases of cyanotoxin poisoning. However, these are potent, rapidly acting toxins, so prevention will likely remain the most effective interv ention for the foreseeable future. Inthe short term this involves restricting access to cyanotoxin–affected drinking water, with longer–term strategi es of nutrient reduction to mitigate

blooms. Dissemination of informati on to farmers and the livestock industry is always important; beyond the aca demic research literature there are some industry and government partners hips that provide helpful resources, e.g. Australia’s Animal Health Surv eillance Report (see for example Berry 2001, Elliott 2001), and the Animal He alth Expositor from Canada (now Animal Health Perspectives) (Yong 2000a, Yong 2000b). Comprehensive economic analyses of the costs of cyanotoxin–related livestock and wild Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds63

animal poisonings to the agricultural and tourist industries would be valuable.



### Concluding remarks

Cyanotoxins in the environment continue to be hazardous to livestock and wild animals throughout the world. While much is known about the most potent, acutely toxic neurotoxins and hepatotoxins, there is still more to learn about the toxicokinetics and toxicodynamics of even the well– characterised cyanotoxins. Cyanobacteria are rich sources of biologicallyactive compounds, and it is likely that some yet–to–be–described cyanobacterial products will be found to be harmful. Current investigations into the subacute disease avian vacuolar my elinopathy hint at this possibility. While controlled dosing with purifie d cyanotoxins under laboratory conditions has been an essential endeavour that allows these materials to be characterised and understood, exposures in the field may be much more complex. Subacute or chronic exposures, particularly to hepatotoxiccyanobacteria, may result in clinical presentations characterised by concomitant degenerative and regenerative changes. Expert veterinary clinicians and pathologists, as well as specialist investigative chemistry laboratory services, are invaluable resources for the diagnosis and management of these complex poisonings.



### Acknowledgements

Thanks to Wasa Wickramasinghe for helpful discussion. This work was supported by the Cooperative Research Centre for Water Quality and

Treatment. The National Research Centre for Environmental Toxicology is co– funded by Queensland Health, The University of Queensland, Griffith University and Queensland University of Technology.



### References

Abdulqader G, Barsanti L, Tredici MR ( 2000) Harvest of Arthrospira platensis from Lake Kossorom (Chad) and its household use among the Kanembu. J Appl Phycol 12(3–5):493–498 0 I. Stewart et al.631

Adams WH, Stoner RD, Adams DG, Slatkin DN, Siegelman HW (1985) Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa. Toxicon 23(3):441–447Al–Batshan HA, Al–Mufarrej SI, Al–Homaidan AA, Qureshi MA (2001) Enhancement of chicken macrophage phagocytic function and nitrite production by dietary Spirulina platensis. Immunopharmacol Immunotoxicol 23(2):281–

289 Ballot A, Krienitz L, Kotut K, Wiegand C, Metcalf JS, Codd GA, Pflugmacher S (2004) Cyanobacteria and cyanobacterial toxins in three alkaline Rift Valley lakes of Kenya––Lakes Bogoria, Na kuru and Elmenteita. J Plankton Res 26(8):925–935 Ballot A, Krienitz L, Kotut K, Wiegand C, Pflugmacher S (2005) Cyanobacteria and cyanobacterial toxins in the alka line crater lakes Sonachi and Simbi, Kenya. Harmful Algae 4(1):139–150 Beasley VR, Cook WO, Dahlem AM, Hooser SB, Lovell RA, Valentine WM (1989a) Algae intoxication in livestock and waterfowl. Vet Clin North Am Food Anim Pract 5(2):345–361Beasley VR, Dahlem AM, Cook WO, Valentine WM, Lovell RA, Hooser SB, Harada K, Suzuki M, Carmichael WW (1989b) Diagnostic and clinically important aspects of cyanobacterial (blue–gree n algae) toxicoses. J Vet Diagn Invest 1(4):359–365Belay A, Ota Y, Miyakawa K, Shimamatsu H (1993) Current knowledge on potential health benefits of Spirulina. J Appl Phycol 5(2):235–241 Benton BJ, Keller SA, Spriggs DL, Capacio BR, Chang FCT (1998) Recovery from the lethal effects of saxitoxin: a therapeutic window for 4–aminopyridine (4–AP). Toxicon 36(4):571–588Benton BJ, Rivera VR, Hewetson JF, Chang FCT (1994) Reversal of saxitoxin induced cardiorespiratory failure by a burro–raised alpha–STX antibody and oxygen therapy. Toxicol Appl Pharmacol 124(1):39–51 Berry J (2001) Blue green algae. Animal Health Surveillance Quarterly Report 6(3):11 http://www.animalhealthaustralia.com.au/shadomx/apps/fms/fmsdownload.cf m?file_uuid=2FC528AC-013A-817F-983F-2932E47F83C5&siteName=aahc Braun A, Pfeiffer T (2002) Cyanobacterial blooms as th e cause of a Pleistocene large mammal assemblage. Paleobiology 28(1):139–154 Briand JF, Jacquet S, Bernard C, Humbert JF (2003) Health hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems. Vet Res 34(4):361–377Carbis CR, Simons JA, Mitchell GF, Anderson JW, McCauley I (1994) A biochemical profile for predicting the chro nic exposure of sheep to Microcystis aeruginosa, an hepatotoxic species of blue–green alga. Res Vet Sci 57(3):310–

316 Carmichael WW (1994) The toxins of cyanobacteria. Sci Am 270(1):64–72 Carmichael WW (2001) Health effects of toxin–producing cyanobacteria: "The CyanoHABs". Hum Ecol Risk Assess 7(5):1393–1407 Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds6

Carmichael WW, Biggs DF, Gorham PR (1975) Toxicology and pharmacological action of Anabaena flos–aquae toxin. Science 187(4176):542–544 Carmichael WW, Falconer IR (1993) Di seases related to freshwater blue–green algal toxins, and control measures. In: Algal toxins in seafood and drinking water. Edited by Falconer IR. London: Academic Press 187–209 Carmichael WW, Gorham PR, Biggs DF (1977) Two laboratory case studies on the oral toxicity to calves of the fre shwater cyanophyte (blue–green algae) Anabaena flos–aquae NRC–44–1. Can Vet J 18(3):71–75 Chang FCT, Bauer RM, Benton BJ, Keller SA, Capacio BR (1996) 4–aminopyridine antagonizes saxitoxin– and tetrodotoxin–induced cardiorespiratory depression. Toxicon 34(6):671–690 Chang FCT, Benton BJ, Lenz RA, Capacio BR (1993) Central and peripheral cardio–respiratory effects of saxitoxin (STX) in urethane–anesthetized guinea–pigs. Toxicon 31(5):645–664 Chang FCT, Spriggs DL, Benton BJ, Keller SA, Capacio BR (1997) 4– aminopyridine reverses saxitoxin (STX)– and tetrodotoxin (TTX)–induced cardiorespiratory depression in chronically instrumented guinea pigs. Fundam Appl Toxicol 38(1):75–88 Ciferri O (1983) Spirulina, the edible microorganism. Microbiol Rev 47(4):551–

578 Codd GA (1983) Cyanobacterial poisoning hazard in British freshwaters. Vet Rec 113(10):223–224 Codd GA, Beattie KA (1991) Cyanobacteria (blue–green algae) and their toxins: awareness and action in the United Kingdom. PHLS Microbiol Dig 8(3):82–

86Codd GA, Bell SG, Kaya K, Ward CJ, Beattie KA, Metcalf JS (1999) Cyanobacterial toxins, exposure routes and human health. Eur J Phycol 34(4):405–415 Codd GA, Edwards C, Beattie KA, Barr WM, Gunn GJ (1992) Fatal attraction to cyanobacteria? Nature 359(6391):110–111 Codd GA, Lindsay J, Young FM, Morrison LF, Metcalf JS (2005) Harmful cyanobacteria: from mass mortalities to management measures. In: Harmful cyanobacteria. Edited by Huisman J, Matthijs HCP, Visser PM. Dordrecht: Springer 1–23 Codd GA, Metcalf JS, Morrison LF, Krieni tz L, Ballot A, Pflugmacher S, Wiegand C, Kotut K (2003) Susceptibility of flamingos to cyanobacterial toxins via feeding. Vet Rec 152(23):722–723Codd GA, Steffensen DA, Burch MD, Baker PD (1994) Toxic blooms of cyanobacteria in Lake Alexandrina, South Australia––learning from history. Aust J Mar Freshwater Res 45(5):731–736Cook WO, Beasley VR, Lovell RA (1989) Consistent inhibition of peripheral cholinesterases by neurotoxins from the freshwater cyanobacterium Anabaena flos–aquae: studies of ducks, swine, mi ce, and a steer. Environ Toxicol Chem 8(10):915–922 Cook WO, Iwamoto GA, Schaeffer DJ, Carmichael WW, Beasley VR (1990) Pathophysiologic effects of anatoxin–a(s) in anaesthe tized rats: the influence of atropine and artificial respiratio n. Pharmacol Toxicol 67(2):151–155 32 I. Stewart et al.633

Cook WO, Beasley VR, Dahlem AM, Dellinger JA, Harlin KS, Carmichael WW (1998) Comparison of effects of anatoxin–a(s) and paraoxon, physostigmine and pyridostigmine on mouse brain cholinesterase activity. Toxicon 26(8):750–753 Corkill N, Smith R, Seckington M, Pontefract R (1989) Poisoning at Rutland Water. Vet Rec 125(13):356 Creasia DA (1990) Acute inhalation toxic ity of microcystin–LR with mice. Toxicon 28(6):605Duy TN, Lam PKS, Shaw GR, Connell DW (2000) Toxicology and risk assessment of freshwater cyanobacterial (blue–gr een algal) toxins in water. Rev Environ Contam Toxicol 163:113–185 Elliott J (2001) Cyanobacterial toxicity. An imal Health Surveillance Quarterly Report 6(1):13 http://www.animalhealthaustralia.com.au/shadomx/apps/fms/fmsdownload.cfm ?file_uuid=2FC47F6C-9D1A-FA75-465E-DE8DCA75BBD4&siteName=aahcFalconer IR (2005) Cyanobacterial toxins of drinking water supplies: cylindrospermopsins and microcystins. Boca Raton: CRC Press Firkins GS (1953) Toxic algae poisoning. Iowa State Coll Vet 15(3):151–153 Fitzgeorge RB, Clark SA, Keevil CW (1994) Routes of intoxication. In: Detection methods for cyanobacterial toxins. Edited by Codd GA, Jefferies TM, Keevil CW, Potter E. Cambridge: The Royal Society of Chemistry 69-- 74 Francis G (1878) Poisonous Australian lake. Nature 18:11–12 Goode J (1967) Freshwater tortoises of Australia and New Guinea (in the Family Chelidae). Melbourne: Lansdowne Press Gorham PR, Carmichael WW (1988) Hazards of freshw ater blue–green algae (cyanobacteria). In: Algae and human affairs. Edited by Lembi CA, Waaland JR. Cambridge: Cambridge University Press 403–431Griffiths DJ, Saker ML (2003) The Palm Island mystery disease 20 years on: a review of research on the cyanotoxin cylindrospermopsin. Environ Toxicol 18(2):78–93 Gugger M, Lenoir S, Berger C, Ledreux A, Druart JC, Humbert JF, Guette C, Bernard C (2005) First report in a ri ver in France of the benthic cyanobacterium Phormidium favosum producing anatoxin–a associated with dog neurotoxicosis. Toxicon 45(7):919–928 Hamill KD (2001) Toxicity in benthic fr eshwater cyanobacteria (blue–green algae): first observations in New Zealand. N Z J Mar Freshwater Res 35(5):1057–1059 Hammer UT (1968) Toxic blue–green algae in Saskatchewan. Can Vet J 9(10):221–229 Harding WR, Rowe N, Wessels JC, Beattie KA, Codd GA (1995) Death of a dog attributed to the cyanobacterial (blue–green algal) hepatotoxin nodularin in South Africa. J S Afr Vet Assoc 66(4):256–259 Hayashi O, Katoh T, Okuwaki Y (1994) Enhancement of antibody production in mice by dietary Spirulina platensis. J Nutr Sci Vitaminol (Tokyo) 40(5):431–

441 Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds6

Hayman J (1992) Beyond the Barcoo – probable human tropical cyanobacterial poisoning in outback Australia. Med J Aust 157(11–12):794–796 Henriksen P, Carmichael WW, An J, Mo estrup O (1997) Detection of an anatoxin–a(s)–like anticholinesterase in natural blooms and cultures of cyanobacteria/blue–green algae from Danish lakes and in the stomach contents of poisoned birds. Toxicon 35(6):901–913 Höger SJ (2003) Problems during drinking water treatment of cyanobacterial loaded surface waters: consequences fo r human health. Doctoral Thesis Constance: Universität Konstanz http://www.ub.unikonstanz.de/v13/volltexte/2003/1071//pdf/SJHoeger_Thesis.pdf Howard NJ, Berry AE (1933) Algal nu isances in surface waters. Can Public

Health J 24:377–384 Humpage AR, Rositano J, Bretag AH, Brown R, Baker PD, Nicholson BC,Steffensen DA (1994) Paralytic shellfish poisons from Australian cyanobacterial blooms. Aust J Mar Freshwater Res 45(5):761–771 Ito E, Kondo F, Harada K (2001) Intratracheal administration of microcystin–LR, and its distribution. Toxicon 39(2–3):265–271 Koenigswald Wv, Braun A, Pfeiffer T (200 4) Cyanobacteria and seasonal death: a new taphonomic model for the Eocene Messel lake. Paläontol Z 78(2):417–

424 Krienitz L, Ballot A, Kotut K, Wiegand C, Pütz S, Metcalf JS, Codd GA, Pflugmacher S (2003) Contribution of hot spri ng cyanobacteria to the mysterious deaths of Lesser Flamingos at Lake Bogoria, Kenya. FEMS Microbiol Ecol 43(2):141–148 Kuchling G (1999) The reproductive biology of the Chelonia. Berlin: Springer Verlag Lopez Rodas V, Costas E (1999) Prefer ence of mice to consume Microcystis aeruginosa (toxin–produci ng cyanobacteria): a possi ble explanation for numerous fatalities of livestock and w ildlife. Res Vet Sci 67(1):107–110 Lugomela C, Pratap HB, Mgaya YD (2006) Cyanobacteria blooms–a possible cause of mass mortality of Lesser Flamingos in Lake Manyara and Lake Big Momela, Tanzania. Harmful Algae 5(5):534–541 Magalhães VF, Soares RM, Azevedo SMFO (2001) Microcystin contamination in fish from the Jacarepaguá Lagoon (Rio de Janeiro, Brazil): ecological implication and human health risk. Toxicon 39(7):1077-1085 Matsunaga H, Harada KI, Senma M, Ito Y, Yasuda N, Ushida S, Kimura Y (1999)Possible cause of unnatural mass death of wild birds in a pond in Nishinomiya, Japan: sudden appearance of to xic cyanobacteria. Nat Toxins 7(2):81–

84 May V, McBarron EJ (1973) Occurrence ofthe blue–green alga, Anabaena circinalis Rabenh., in New South Wales and toxicity to mice and honey bees. J Aust Inst Agric Sci 39(4):264–266 McLeod JA, Bondar GF (1952) A case of suspected algal poisoning in Manitoba. Can J Public Health 43(8):347–350 Metcalf JS, Morrison LF, Krienitz L, Ballo t A, Krause E, Kotut K, Pütz S, Wiegand C, Pflugmacher S, Codd GA (2006) Analysis of the cyanotoxins ana34 I. Stewart et al.635

toxin-a and microcystins in Lesser Flamingo feathers. Toxicol Environ Chem 88(1):159-167 Ndetei R, Muhandiki VS (2 005) Mortalities of lesser fl amingos in Kenyan Rift Valley saline lakes and the implications for sustainable management of the lakes. Lakes Reserv Res Manage 10(1):51–58 Negri AP, Jones GJ (1995) Bioaccumulation of paralytic shellfish poisoning (PSP) toxins from the cyanobacterium Anabaena circinalis by the freshwater mussel Alathryia condola. Toxicon 33(5):667-678Negri AP, Jones GJ, Hindmarsh M (1995) Sheep mortality associated with paralytic shellfish poisons from the cyanoba cterium Anabaena circinalis. Toxicon 33(10):1321–1329 Nobre AC, Coelho GR, Coutinho MC, Silva MM, Angelim EV, Menezes DB, Fonteles MC, Monteiro HS (2001) The role of phospholipase A(2) and cyclooxygenase in renal toxicity induced by microcystin–LR. Toxicon 39(5):721–724 NSW Blue–Green Algae Task Force (1992) Final report of the NSW Blue–Green Algae Task Force. Parramatta: NS W Department of Water ResourcesOnodera H, Oshima Y, Henriksen P, Yasumoto T (1997) Confirmation of anatoxin–a(s), in the cyanob acterium Anabaena lemmermannii, as the cause of bird kills in Danish lakes. Toxicon 35(11):1645–1648Phillips W (1884) The breaking of the Shropshire meres. Trans Shropshire Archaeol Nat Hist Soc 7:277–300Pybus MJ, Hobson DP, Onderka DK (1986) Mass mortality of bats due to probable blue–green algal toxicity. J Wildl Dis 22(3):449–450 Qureshi MA, Garlich JD, Kidd MT (1996) Dietary Spirulina platensis enhances humoral and cell–mediated immune functions in chickens. Immunopharmacol Immunotoxicol 18(3):465–476Ressom R, Soong FS, Fitzgerald J, Turczynowicz L, El Saadi O, Roder D, Maynard T, Falconer I (1994) Health eff ects of toxic cyanob acteria (blue–green algae). Canberra: National Health and Medical Research Council & Australian Government Publishing Service Roder JD (2004) Blue–green algae. In: Clinical veterinary toxicology. Edited by Plumlee KH. St Louis: Mosby 100–101 Rose EF (1953) Toxic algae in Iowa lakes. Proc Iowa Acad Sci 60:738–745 Saker ML, Eaglesham GK (1999) The accumulation of cylindrospermopsin from the cyanobacterium Cylindrospermopsis raciborskii in tissues of the Redclaw crayfish Cherax quadricarinatus. Toxicon 37(7):1065-1077Saker ML, Metcalf JS, Codd GA, Vasconcelos VM (2004) Accumulation and depuration of the cyanobacterial toxin cy lindrospermopsin in the freshwater mussel Anodonta cygnea. Toxicon 43(2):185-194 Salazar M, Martínez E, Madrigal E, Ru iz LE, Chamorro GA (1998) Subchronic toxicity study in mice fed Spirulina maxima. J Ethnopharmacol 62(3):235–

241 Schwimmer D, Schwimmer M (1964) Algae and medicine. In: Algae and man. Edited by Jackson DF. New York: Plenum Press 368–412 Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds63

Schwimmer M, Schwimmer D (1955) The role of algae and plankton in medicine.

New York: Grune & Stratton Schwimmer M, Schwimmer D (1968) Medical aspects of phycology. In: Algae,man, and the environment. Edited by Jackson DF. Syracuse: Syracuse University Press 279–358 Shaw GR, McKenzie RA, Wickramasinghe WA, Seawright AA, Eaglesham GK, Moore MR (2004) Comparative toxicity of the cyanobacterial toxin cylindrospermopsin between mice and cattle: human implications. In: Harmful Algae 2002. Edited by Steidinger KA, Landsberg JH, Tomas CR, Vargo GA. St Petersburg, Florida, USA: Florida Fish and Wildlife Conservation Commission, Florida Institute of Oceanogr aphy, and Intergovernmental Oceanographic Commission of UNESCO 465–467 Sipiä VO, Kankaapää HT, Pflugmacher S, F linkman J, Furey A, James KJ (2002)

Bioaccumulation and detoxication of nodu larin in tissues of flounder (Platichthys flesus), mussels (Mytilus edulis, Dreissena polymorpha), and clams (Macoma balthica) from the northern Baltic Sea. Ecotoxicol Environ Saf 53(2):305-311 Sipiä VO, Karlsson KM, Meriluoto JA, Kankaapää HT (2004) Eiders (Somateria mollissima) obtain nodularin, a cyanobacterial hepatotoxin, in Baltic Sea food web. Environ Toxicol Chem 23(5):1256-1260 Smith JL, Haney JF (2006) Foodweb transfer, accumulation, and depuration ofmicrocystins, a cyanobacterial toxin, in pumpkinseed sunfish (Lepomis gibbosus). Toxicon 48(5):580-589 Smith PT (2000) Freshwater neurotoxins: mechanisms of action, pharmacology, toxicology, and impacts on aquaculture. In: Seafood and freshwater toxins: pharmacology, physiology, and detection. Edited by Botana LM. New York:

Marcel Dekker 583–602 Soll MD, Williams MC (1985) Mortality of a White Rhinoceros (Ceratotherium simium) suspected to be associated w ith the blue–green alga Microcystis aeruginosa. J S Afr Vet Assoc 56(1):49–51Stephens EL (1949) Microcystis toxica sp. Nov.: a poisonous alga from the Transvaal and Orange Free State. Trans R Soc S Afr 32(1):105–112 Steyn DG (1943) Poisoning of animals by algae on dams and pans. Farming S Afr 18:489–492, 510 Steyn DG (1945) Poisoning of animals and human beings by algae. S Afr J Sci 1945, 41:243–244Thomas AD, Saker ML, Norton JH, Olsen RD (1998) Cyanobacterium Cylindrospermopsis raciborskii as a probable cause of death in cattle in northern Queensland. Aust Vet J 76(9):592–594 Valentine WM, Schaeffer DJ, Beasley VR (1991) Electromyographic assessment of the neuromuscular blockade produced in vivo by anatoxin–a in the rat. Toxicon 29(3):347–357 Wilde SB, Murphy TM, Hope CP, Habrun SK, Kempton J, Birrenkott A, Wiley F, Bowerman WW, Lewitus AJ (2005) Avian vacuolar myelinopathy linked to exotic aquatic plants and a novel cy anobacterial species. Environ Toxicol 20(3):348–353 6 I. Stewart et al.637

Williams SK, Kempton J, Wilde SB, Lewitus A (2007) A novel epiphytic cyanobacterium associated with reservoirs a ffected by avian vacuolar myelinopathy. Harmful Algae 6(3):343-353Yong C (2000a) Cyanobacteria (blue-green algae) poisoning. Animal Health Expositor 2(3):2 http://www.usask.ca/pds/I nformation/Sept%20%2700.pdfYong C (2000b) Blue–green algae poisoning in two dogs. Animal Health Expositor 2(3):3 http://www.usask.ca/pds/Information/Sept%20%2700.pdf Chapter 28: Cyanobacterial Poisoning in Livestock, Wild Mammals and Birds