Cyanobacteria (Blue-Green Algae) Toxins

Transcription

1 APPLIED AND ENVIRONMEAL MICROBIOLOGY, June 98, p /8/068-06$0.00/0 Vol. 4, No. 6 Hemagglutination Method for Detetion of Freshwater Cyanobateria (Blue-Green Algae) Toxins WAYNE W. CARMICHAEL* AND PETER E. BE Department of Biologial Sienes, Wright State University, Dayton, Ohio 4545 Reeived 8 September 980/Aepted 8 Marh 98 Strains of the freshwater yanobateria (blue-green algae) Anabaena flosaquae and Miroystis aeruginosa produed toxins that aused intermittent but repeated ases of livestok, waterfowl, and other animal deaths. They also aused illness, espeially gastrointestinal, in humans. The most ommon group of toxins produed by these two speies were peptide toxins termed miroystin, M. aeruginosa type, and anatoxin-. A method was found to detet the toxins whih utilizes their ability to ause agglutination of isolated blood ells from mie, rats, and humans. The method ould detet the toxin in samples from natural algal blooms, laboratory ultures, and toxin extrats. The method onsists of: (i) washing lyophilized yanobateria ells with physiologial saline (0.9% NaCl), (ii) entrifuging the suspension and then mixing portions of the ell-free supernatant with equal volumes of saline-washed erythroytes in V-shaped mirotiter plates, (iii) allowing the mixture to stand for to 4 h, and (iv) soring the presene of the toxin as indiated by blood ell agglutination. Nontoxi strains, as determined by intraperitoneal mouse bioassay of yanobateria or green algae, did not produe an agglutination response. 8 Over the last 00 years, about speies belonging to 9 genera of freshwater yanobateria (blue-green algae) have been impliated in ase histories of animal poisonings. In most reports, however, the speies responsible have been Miroystis aeruginosa Kutz emend Elenkin, Anabaena flos-aquae (Lyngb.) de Breb, and Aphanizomenon flos-aquae (L.) Ralfs. These bloom-forming speies have aused deaths of livestok and other animals in western Canada, midwestem United States, South Ameria, Europe, Asia, South Afria, and Australia. These poisonings are of onern to veterinarians, wildlife biologists, environmentalists, and publi health offiials. There are several reviews on toxi yanophytes; the most reent are by Moore (), Collins (8), and Gorham and Carmihael (4, 5). Struturally, the known toxins produed inlude alkaloids, peptides, and pteridines. Although the greatest and most dramati animal losses have been due to alkaloid neurotoxins, deaths due to the peptide toxins are widespread and therefore of greater onern for animal and human health. It is now known that the peptide toxins are produed by strains of both M. aeruginosa and Anabaena flos-aquae. Some of the Anabaena toxins have been given names based on their gross toxiologial signs when injeted intraperitoneally into mie, rats, and hiks. At present, there are six toxins produed by different strains of A. flos-aquae; these are termed anatoxin (antx)-a, -a(s), -b, -b(s), -, and -d (7). Of these, only antx-a has undergone extensive toxiologial and pharmaologial examination. It is a biyli tertiary amine (9) having potent pre- and postsynapti effets on the niotini reeptor (5, 5). Antx-b and -d also have gross neuromusular ativity, but their pharmaology and struture have not been investigated. Antx is a mixture of peptide or peptide-ontaining toxins whih are produed by many strains of A. flos-aquae. M. aeruginosa also appears to produe a toxin like antx-, urrently alled M. aeruginosa type toxin. Although their amino aid sequenes are unertain, it is known that all of these toxins are small peptides, perhaps yli, with a moleular weight ranging from about 500 to,700 (, 4). Based on urrent evidene, antx- and M. aeruginosa type are related and perhaps are the same as the peptide toxin, termed miroystin, whih was desribed for M. aeruginosa strain NRC- (4). Signs of poisoning in affeted animals drinking from waters infested with these toxins have onsistently indiated that the liver is engorged with blood and that puntate hemorrhages are present (, 6, 9, 4, 6). Suh effets are also observed with laboratoryanimals given intraperitoneal and oral dosages of freezedried lonal isolates or the toxin extrat. Histopathology of mouse livers indiates that severe

2 84 CARMICHAEL AND BE erythroyte aumulation ours in the hepati lobule with hroni or aute dosages of lyophilized ells ontaining these toxins or of the toxin extrats. Erythroyte aumulation ours in the area of the afferent portal veins (4, 6, 6; W. W. Carmihael, unpublished data). Although almost all reports to date onerning toxiities from freshwater yanobateria involve livestok, waterfowl, and other small animals, there is evidene for their role in ertain waterborne human illnesses. These gastrointestinal illnesses an result from aidental drinking of raw surfae water (0) or water from muniipal water supplies (, 0; H. T. P. Sargunar and A. A. A. Sargunar, Pro. 4th Int. Symp. Myotoxins and Phytotoxins, 979, GIO). The mouse bioassay is urrently the best available test for detetion of toxiity in freshwater yanobateria, but other assay methods that are more sensitive and more disriminating among the different toxin types are needed. The onsistently observed heavy aumulation of blood in the hepati units of livers affeted by these peptide or peptideontaining toxins resembles signs of poisoning previously reported for ertain letins (7, 8, ). Sine one of the main properties of letins is their ability to agglutinate ells, espeially erythroytes, it was felt that this may also be part of the ation for yanobateria toxins. Erythroyte agglutination was therefore tried as a detetion method for these toxins. MATERIALS AND MErHODS Table lists laboratory-ultured strains of green and blue-green algae used. Algal bloom samples were olleted diretly from a surfae onentration or by onentration of the algae in the water by using standard plankton nets. AM field samples, exept those from Hastings Lake, were olleted by persons from Alberta Environment, Division of Pollution Control. These field samples of onentrated and unonentrated algal blooms were oarse filtered to remove vasular plant debris and then lyophfilied. Laboratory strains of yanobateria (nonaxeni) were maintained in srew-top test tubes and 5-ml Delong flasks, ontaining ASM--TR ulture medium (6), at ± C with ontinuous illumination from Vita-Lite fluoresent tubes. Light intensity was approximately loo ue/m/s of photosynthetially ative radiation. Cultures, grown in -liter Delong flasks, were lyophilized near the end of their log phase of growth (0' to 0' ells or filaments per ml). Lyophilized ell material (00 mg) was suspended in 0.9% NaCl (physiologial saline) to a onentration of 0 mg/ml. This onentration was hosen beause it is in the top range for a natural water bloom (heavy yanobateria blooms are designated as greater than g [dry weight] per liter [W. W. Carmihael and P. R. Gorham, unpublished data]) and provided enough material for a mouse bioassay. The suspension was washed twie with saline, using gentle entrifugation. After the last wash, the supernatant was used for the agglutination test. APPL. ENVIRON. MICROBIOL. Blood was obtained by ardia punture (using a heparinized syringe) from Sprague-Dawley rats (00 to 00 g) and ICR Swiss mie (0 to 5 g). For some tests, human type 0 rr and type AB RR paked erythroytes, obtained from the University of Alberta Hospital blood bank, were also used. All samples were washed three times with physiologial saline and resuspended to a onentration of.0%. Erythroytes ould be stored at 5 C for up to 4 weeks, using a : mixture of washed blood plus Alsever preservative (). Hemagglutination tests were run with Cooke mirotiter V plates. Four onentrations, in dupliate, of sample supematant-.0% erythroytes were used: :, :, :4, and :0. Volumes were kept onstant with saline. Volumes used were: :, 00 pl of sample and 00 id of blood; :, 50 u of sample, 50 p of saline, and 00 p of blood; :4; 5 pl of sample, 75 p of saline, and 00 pi of blood; :0, 0 p of sample, 90 p of saline, and 00 p of blood. The mirotiter plates were overed and allowed to sit for to 4 h at room temperature. A positive response (++) was sored when lumping of erythroytes ells was observed in the test well. A partial positive (+-) was sored when about one-half of the ells had settled to the bottom of the test well and the rest remained diffused. A negative response (--) was noted when a well-defined spot of erythroytes was present at the bottom of the well, along with a lear solution on top (Fig. B). Aute toxiity of the ultures and samples of different blooms was tested by intraperitoneal injetion of TABLE. Laboratory ultures offreshwater yanobateria and green algae used for testing erythroyte hemagglutination Culture strain Toxin typea Anabaena flos-aquae NRC-44- a M-0 a NRC-55 a(s) A-5 b S-UTH- b(s) S- d F- A- A-0 A-8 M- M-5 Miroystis aeruginosa NRC--SS-7 Miroystin A-4 M. aeruginosa type A-07 M. aeruginosa type A-7 M. aeruginosa type 8- M. aeruginosa type S-6 M. aeruginosa type A. flos-aquae 444 Lyngbya boregerti M-9 L. versiolor 09 Lyngbya sp. 6 Tolypothrix sp. Comarium sp. Euglena sp. Oedogonium sp. Senedesmus sp. Selenastrum apriornutum a As determined by intraperitoneal mouse bioassay (milligrams of lyophilized ells per kilogram of body weight)., Nontoxi at,500 mg/kg.

3 VoL. 4, 98 DETECTION OF CYANOBACTERLAL TOXINS w Downloaded from FIG.. (A) Exised mouse liver after an intraperitoneal aute dose (approximately 0 mg/kg) of lyophilized M. aeruginosa produing antx-. The liver an also be swollen with blood and appear muh darker than in this photograph. Similar signs would be seen using extrated toxins. (B) Cooke mirotiter plate showing the range of responses seen with different yanophyte strains and onentrations of sample. Repliates are seen as rows and, and 4, and 5 and 6. Highest onentration, i.e., 00-Ml sample, is in rows and. For example, ++ is reorded under rows C, F, and G for au onentrations. A -- response (nontoxi strain or sample) an be seen for au of row H. An example of the endpoint of a response an be found between D,4, and D5,6. A, is a negative ontrol (erythroytes plus saline). on April, 09 by guest male mie weighing 0 to 5 g. Several dilutions of eah sample were tested, using three to five mie eah, until a point was reahed at whih all the mie lived. The 50% lethal dose (milliams per kilogram of body weight) was then estimated as the midpoint between the onentration at whih the mie lived and the onentration at whih they all died. Survival times in the mie for antx- miroystin- or miroystis type - produing strains were all about to h. Liver damage was indiated by a swollen, enlarged, reddened ondition plus red spots revealing erythroyte aumulation in the hepati units (Fig. A).

4 86 CARMICHAEL AND BE RESULTS AND DISCUSSION Preparation of erythroytes and toxin extrats for hemagglutination. Two fators were found whih interfered with proper erythroyte agglutination. The first involved the salt onentration that the yanobateria, algal ells, or extrats were suspended or dissolved in. Laboratory ultures of the yanobateria strains were grown in ASM--TR medium. This ulture medium ontains 05 mg of inorgani salts per liter, or 0.0%. Therefore, it was neessary to wash all ells to be tested with physiologial saline (0.9% NaCl) to prevent lysis of erythroytes. This is also true for water bloom samples, sine salt onentrations in a typial eutrophi lake an average about 500 to 700 mg/liter, or 0.5 to 0.7%. Seond, it was found that antx- toxins are not sereted as muh as antx-a; therefore, lyophilized ells were used for the test sine this allowed the maximum amount of toxin to be present in the supernatant. It was not neessary to ompletely lyse the yanobateria or algal ells, however. Lyophilization was used to prepare the ells, sine it also allowed a given weight of biomass to be tested. More rapid preparation of ells suh as soniation or freeze-thawing of a field sample also worked. In a rapid field analysis where quantifiation of the ells was not needed, these methods of ell treatment or others suh as osmoti shok ould be used. Results of all blood agglutination tests. Tables and present the results for agglutination tests using laboratory ultures and waterbloom samples, respetively, of seleted toxi yanobateria and other algae. Whenever mouse toxiity ould be deteted, i.e., liver damage plus death within to h, there was a positive agglutination produed in rat, mouse, and human erythroytes. The method was speifi for miroystis type or antx- toxins and did not give false positives with antx-a, -b, or-d. Toxi frations from extrats of M. aeruginosa miroystin or type toxin and A. flos-aquae antx- gave a positive erythroyte agglutination, whereas nontoxi frations gave a negative agglutination (data not shown). This provided evidene that it was the intraperitoneal mouse toxi fration of the ells whih aused the hemagglutination. The endpoint for any hemagglutination response was about.5 mg of ells per ml (Tables and ). Therefore, to hek the toxiity of an algal bloom, the onentration should be about to g/liter, a heavy bloom. Lower algal bloom onentrations ould be tested provided the material was onentrated with a dip net or by another method that provided enough ells for the agglutination assay. The agglutination assay desribed here is not APPL. ENVIRON. MICROBIOL. quantitative under the onditions tested. Even with extrated toxi frations, there is enough variability in the agglutination reation that a reliable quantitative measure of toxiity, i.e., 50% lethal dose, annot be estimated for a given ulture or sample. We are working to improve the methods of extration to see if there is a quantitative use for the assay. The test reported here is sensitive for a toxin produed by strains of these yanophytes whih auses death in mie within to 4 h. The toxin has the ability to ause tissue damage, partiularly in the liver, giving a harateristi aumulation of erythroytes in the hepati units. The toxin also has erythroyte-agglutinating properties. Quantities of the toxin are released into the environment both naturally and after lyophilization of the algae, allowing a positive agglutination reation. There is evidene from toxin extrations of different strains that more than one toxi omponent is produed (, 4, 6; Carmihael, unpublished data). This indiates that the hepatotoxin may not be the only ause of aute toxiity. Extrats whih ause hemagglutination also ause death in mie within to h, indiating that the hepatotoxin is a major part of the toxi priniple in these yanobateria. It an be reemphasized that, in all ases studied, a positive hemagglutination indiates the TABLE. Hemagglutination results from laboratory ultures ofseleted yanobateria Hemagglutination response of supernatant Culturea onn (mg [dry wt] of Mouse ells/ml) ityb toxi- (mg/kg) A. flos-aquae NRC Antx-a M-0 Antx-a NRC-55. Antx-a(s) S-UTH- Anti-b(s) A Antx-b S Antx-d A A A M M F M. aeruginosa NRC--SS A A A S S a All other ultures listed in Table showed no agglutination or mouse toxiity. b Eipressed as intraperitoneal 50% lethal dose. Data are for signs of poisoning with antx-, miroystin, or miroystis type toxin. Anti-a, -b, -a(s), -b(s), and -d all have neurotoxi properties; therefore, toxiity is not reported here.

5 VOL. 4, 98 DETECTION OF CYANOBACTERIAL TOXINS 87 TABLE. Hemagglutination results from field material oueted in July and August 979 Sample onn (mg [dry wt] of ells/ml) Mouse toxi. Colletion site Bloom omposition' M tosi jtyb (mg4g) North Dakota 00:0:0: ,000 Hastings LakeC (July ) Hastings Lake (July ) La La Bih Pigeon Lake La La Nonne Gull Lake Wizard Lake 70:5:0:5 85:Tr.: 45:46::7 88:::9 86:Tr:0:4 7:8:8: 90::4:4 79::Tr.0 9:Tr.8:0 98:::0 78:4:7: 70::4:5 7:::5 7:6::8 8:Tr.8:0 8::: 8::6: 90::5: O:Tr.00:0 7:Tr:O:9 8:5:0: : :0:76:8 0:0.8:9 8:0:67:5 7:0:0:9 Tr.40:96 5:5:0.50 :::6 :8:7: d _ _ , , , 'M. aeruginosa/anabaena flos-aquae/aphanizomenon flos-aquae/other. Tr, Trae (<%). b EXpressed as in Table. 'These samples also tested positive against human type AB and 0 blood. d Mouse bioassay not done. presene of toxi fators whih an ause the aute toxiity; therefore, even if it is not diretly responsible for aute toxiity, its presene is an indiation of a toxi water bloom ondition. The hemagglutinating peptides produed by these toxi yanophytes are not the only known phytohemagglutinins, for many are produed by higher plants as well. These higher plant agglutinins are part of a broad group of sugar-ontaining proteins alled letins. Plant letins are produed primarily by the legumes suh as astor beans, navy beans, peanuts, and lentils (). Plant letins are also toxi, and some reports list effets on the liver similar to those seen with these yanobaterial toxins (7, 8). In addition, many marine algae produe hemagglutinating ompounds (). Higher plant letins, however, have moleular weights in the range of 00,000, whereas moleular weights of antx- and related peptides are reported to be in the range of 500 to,700 (4). Survival times with aute dosages of plant letin are typially muh longer ( to 4 days) than with these toxins of yanobateria. Similarity among the toxins is suggested, however, and the assay desribed here should help to determine the nature of these small peptide toxins, their role in aquati ommunities, and their effet on animal and human health. ACKNOWVLEDGMES W. W. C. thanks Paul R Gorham, University of Alberta, Department of Botany, for support from his NSE grant during

6 88 CARMICHAEL AND BE field olletions made in 979. We also thank him for his areful ritique of the manusript. The assistane of E. Dale Allen, Department of Botany, and the ooperation of David Beliveau, Environmental Protetion Servies, Alberta Environment, is also appreiated. Publiation support was provided by the Biologial Sienes Department, Wright State University, and a Publi Health Servie biomedial seed grant to W.W.C. from the National Institutes of Health. LITERATURE CITED. Alam, M., Y. Shimizu, M. Ikawa, and J. J. Sasner Reinvestigation of the toxins from the blue-green alga, Aphanizomenon flos-aquae, by a high performane hromatographi method. J. Environ. Si. Health A: Aziz, K Diarrhea toxin obtained from a waterbloom-produing speies, Miroystis aeruginosa. Kuetzing Si. 8: Bent, P. E., and W. W. Carmihael Isolation and haraterization of peptide toxins from freshwater yanophytes Anabaena flos-aquae and Miroystis aeruginosa. Ohio J. Si. 80: Bishop, C. T., E. F. L. J. Anet, and P. R. Gorham Isolation and identifiation of the fast-death fator in Miroystis aeruginosa, NRC-. Can. J. Biohem. Physiol. 7: Carmihael, W. W., D. F. Biggs, and M. A. Peterson Pharmaology of anatoxin-a, produed by the freshwater yanophyte Anabaena flos-aquae NRC-44-. Toxion 7: Carmihael, W. W., and P. R. Gorham An improved method for obtaining axeni lones of planktoni blue-green algae. J. Phyol. 0: Carmihael, W. W., and P. R. Gorham Anatoxins from lones ofanabaena flos-aquae isolated from lakes of western Canada. Mitt. Int. Ver. Limnol. : Collins, M Algal toxins. Mirobiol. Rev. 4: Devlin, J. P., 0. E. Edwards, P. R. Gorham, N. R. Hunter, R. K. Pike, and B. Stavri Anatoxina, a toxi alkaloid from Anabaena flos-aquae NRC-44 h. Can. J. Chem. 55: Dillenberg, H. O., and M. K. Dehnel Toxi waterbloom in Saskathewan, 959. Can. Med. Asso. J. 8: Elleman, T. C., L. R. Faloner, A. R. B. Jakson, and M. Runnegar Isolation, haraterization and APPL. ENVIRON. MICROBIOL. pathology of toxin from a Miroystis aeruginosa (=Anaystis yanea) bloom. Aust. J. Biol. Si. : Farnham, W. F Survey of British seaweeds for hemagglutinins. Lloydia 8: Garvey, J. S., N. E. Cremer, and D. H. Susdorf Methods in immunology, rd ed. W. A. Benjamin, In., Reading, Mass. 4. Gorham, P. R., and W. W. Carmihael Phyotoxins from blue-green algae. Pure Appl. Chem. 5: Gorham, P. R., and W. W. Carmihael Toxi substanes from freshwater algae. Prog. Water Tehnol. : Heaney, S. L. 97. The toxiity of Miroystis aeruginosa Kutz from some English reservoirs. Water Treat. Exam. 0: Ikegwuonu, F. I., and 0. Bassir Effets of phytohemagglutinins from immature legume seeds on the funtion and enzyme ativities of the liver, and on the histopathologial hanges of some organs of the rat. Toxiol. Appl. Pharmaol. 40: Kakade, M. L., K. K. Keahey, C. K. Whitehair, and R. J. Evans Morphologial hanges in rats fed Navy beans. Pro. So. Exp. Biol. Med. 9: Konst, H., P. D. MKerher, P. R. Gorham, A. Robertson, and J. Howell Symptoms and pathology produed by toxi Miroystis aeruginosa NRC- in laboratory and domesti animals. Can. J. Comp. Med. Vet. Si. 9: Lippy, E., and J. Erb Gastrointestinal illness at Sewikley, PA. J. Am. Water Works Aso. 68: Moore,.R. E Toxins from blue-green algae. Biosiene 7: Sharon, N., and H. Lis. 97. Letins: ell-agglutinating and sugar-speifi proteins. Siene 77: Shimizu, Y Dinoflagellate toxins, p. -4. In P. J. Sheuer (ed.), Marine natural produts: new perspetives, vol.. Aademi Press, In., New York. 4. Skulberg, 0. M Toxi effets of blue-green algae. Norwegian Institute for Water Researh, Oslo. 5. Spivak, C. E., B. Witkop, and E. X. Albuquerque Anatoxin-a: a novel, potent agonist at the niotini reeptor. Mol. Pharmaol. 8: Toerien, D. F., and W. E. Sott Miroystis toxins: isolation; identifiation, impliations. Water S. Afr. : 60-6.