EE‘EZ as: L mszu «EEG =32? ma “ EEE (In; .‘C/‘n E‘E UE‘ {LUL EE‘E EZU ULEE NEW a'i’F’ TE-EEZ ENE? MEE HE “250:2"..EE‘E Tu: far Ehe Uwr 0E FEE. D. EEEEEIEEEGE‘EE STATE? E‘EWL“! Y EMEE. EIEE EEEE EMU! EEZEE 1968 w HEEL/712 EEEEEEEEEEE" . R F” Wan-An. AE :2 2 9 19:35,} i 12 A 1‘0 ABSTRACT sou: ECOLOGICAL cousmmnous or m mm or W 30mm]; m: x 11! m was HICKIGAN noosrsmt by rum-d n. mum-o: rho poooiblo involvonoot o! typo I botnlioo in Loko Hichipo votorbird oortolitioo oud tootoro influonoin. thio botdin oto ooooidorod. Voriouo votorbirdo voro tod egotridiuo botolinuo typo I toxin and it woo dotoroiood thot tho birdo con bo otlootod by toxin produood by oooo otroino of tho orgooioo. Typo l botuliool toxin Ivoo found in «loot! olovivoo tokoo tro- tho Loko Hiohigoo ooooyotoo ot lovoio oottioioot to kill or oiokoo o bird. DDT in tho oultnro oodiI- io lovolo up to 500 porto por billion hot no offoot on tho production of g. hotulioun typo l tooio. DD! ond bottliool tolio. whoa proooot in vhito oioo, intoroot whoa ottootiu tho ooioolo olthoogh littlo ooologiool oinitiooooo ooo bo ooooludod Iron tho DDT-bottliool toxin iltorootion. Gino o ooitoblo oodiu, ouo otroino of g. b inu- typol-oybooblotoproduo mum“ oftoxininthohko Hiohixoo ooooyotoo. Tho olovivoo in Lotto Hiohiuo ooy provido oooh o oooiu oo vol]. oo providing o potootiol vohiolo for tho intonation of fioh-ootin; votorbirdo. SON! mowcxcu. COHSIDERATIOHS 01 1'83 mm 0? CLOSTRIDIUM mummy; m: I 13 ‘51! “KB MICHIGAI ECOSYSTBH 3’ EK‘ (\Oq liohord a? Honhoi-or A THBIB Sub-ittod to Michiton Stoto Uhivoroity in portioi fulfill-out of tho roquirooooto tor tho dogroo o! Mi OF PHILOSOPHY Doportooot o! riohorioo ond Hildlifo 1968 ACKNOWLEDGMENTS The author would like to express his sincere appreciation to all the members of his guidance committee for their help and direction during the course of this study. Particular thanks go to Dr. Peter I. Tack, Department of Fisheries and Wildlife, and Dr. Richard V. Lechowich. Department of Food Science, for the many hours they spent helping me. Special appreciation is also expressed to Dr. L. Dale Fay, Michigan Department of Conservation. for his continued encouragement and support. A.opecial thanks is expressed to my wife, Winifred, for her understanding and endurance during the course of my education. This study was supported by a research tollowohip from the Research and Development Section of the Michigan Department of Conservation. ii miscreants mmwmm................. “W“DHBIBODS oooooooooooo Iooioloborotoryproooduroo....... "toot o! olovivoo ootoxioprodootioo . "toot olwl'ootooiaprodootioo. . . . 0 "foot of oooooooivo botoliool toxio end m oddxiotrotioo tovhitolioo oooooooooooooo “foot of botoliool toxin oo ooptivo ootorbirdo' “um“mnmmmoooooo mmmmswum............... toxiniothohkoliohig-ooooyot. oooo o [fleet of botoliool tooio oo ooptioo gollo Iolotiooohip of toxin production to tioo ond tenorotIro. "“.“M-‘fl’fl~“oooooooooooo luootololovivooootoeioproduotioo ......... um: o! ooeooooivo boulinol toeio and m oooioiotrotioo tovhitooioo..................... W:~0......OOOOOOIOOOOOOOOOOOO “mam...OOOOOOOOOOOOOOOOOOOOO iii no- outs-ur- 10 12 u M 1‘ 28 fl L18! 0! was run rogo 1- WWWIIIMn-mtn- thommohignooooyotoooo...............IS 2. Iooolto tron loodiog California fills lovolo of toxin ’Wb’mCuMO‘EoMW‘ oooooool’ 3. looulto from (ooding ling-bill“ Gulls “.000 m o! tonioproduoodbyuootroinoolg.b_gg;l_i_n_gtypot A. hoolto (roe (coding ling-billed cello voriouo lovolo “$.Mtypolotroin026-000x oooo'ooooooo" 5. looolto tron tooding voriouo ntorbirdo toxin produced . by§.hg§u11ngt”IlIm1I026-wa ...........21 U 6. l-adehudddadfimtypolonlm‘ grovoiomoootoioiogm,otuooo100...o.....23 7. Iooultootthoooolyoiootvoriooooofthotonioitioooi m£.m;mwlulunogrmnmontm MotUoodSOC oooooooooooooooooooooz‘ 8. B-oryotthotooioitioootfi.m§glimtnolooltuno, grown in 178'! oootoining lovolo o! olovito oxtroot ot ”u”Coooooooooooooooooooooooooz’ 9. Ioooltoof tho onolyoio otvori-ooo! tho tuieitioool tho§.mgi_n_lntypolenltnroogroooinm1oootoiniog lovoloolelovitooxtmtotwc.............o30 to. looulto of tho nolyoio o! vorionooond Donoon'o loo lultiplo Iongo root for tho tonioitioo of tho 9,. 123514;; typolulturoogruninmoootoininglovolootoloviio oxtroototlSC.......................32 ll. Inoryolthotoxioitiooolfi.mtypoloeltsroo green in M! oootoioiog 08 and 1‘! hosted oloviio “u“UCU”Cooooooooooooooooooooooo" ll. looulto of tho .olyoio oi vorionoo of tho tuioitioo of thofi.m”polultumgmioflltm “ulflhooudoluihoxtuototwc....o.uoo.” iv L18! 0' runs (tanned) mu Iago l3. Iooslts lroo injootisg sioovith m folio.“ 22 hours byinjsotinooffi.mtypsltdlooo.oo...fl u. loosltoottho analysisoivorionooolthosioo sitsotsd byosoooooivoisjootiooootbbrndtypolbotslisol tosio 0.0.9.000...COOOOOOOOOOOOOO” u. Ioonlto iron isjootiog oioo sithslxloo dilutisoot botslinol can prohotod sith ostioorI- opsoilio {or typsltoais. ooooooooooooooooooooooo‘1 1.18! or name new: Page i. corveo shaving the relationship of production o! £.Wtypelt¢in(non~setivsted)totins-d towerstnrosotlendloc.........o.......13 2. Curves shoving the nlstionship of production a! $th tonic (trypois-eetivstsd) to tins andtsqoretnresoll!end”¢.uo............20 3. hints at shieh $0 persont oi the nice bees-e attested shonisjootedrithlovolootnmmg.mmtype 'm OOOOOOOOOOOOIOOOOOOIOOOI00w 1811013131103 slang-Jug pomlinnn is a rod-shaped, spore-forning hactsrinn whose options growth tonperstuo is bemoan 25 and 37 c (nergoy's banal fa Dotsrninative Bacteriology. 1957). Although this organise is generally oonsidsrod to be an anaerobs. it has been shown that the species can constinoo grow in an environ-sot that is initially aerobic (heft-ans and Harshsll. 1965). As a culture of this organics grows. it produces an entresaly potent onetonin which see he lethal to selected vertebrates (unease. 1959). Oral ingestion of this sustain rather than an infection by the «ultra is that lakes 9.- M Ilene-rout. There are six distinct antigenic types of g. botulint- which are classified as A through 1'. The toxin produced by each type is neutralissd only by its typo-specific antitoxin and it is by a nontralisstion test that the species is identified to type. All types of 9,. m can cause both hoses and aninol hotslisn. hat types A. I, I and I are those nonally associusd with hues botulios nhile c. D and l are the types that are soot often associated with anissl botnlisn. Type c botclisn affects a large variety of sainsls bet is particularly noted for causing large nortslities anong sildfowl in the western prairies of the United states (Sciplo. 1953). Botnlisn type D has secured prinsrily in teeth African cattle but it has also been shove to be lethal to cattle in Australia (Si-eons and 'rsnasnagi. 19“). Until recently. the aninsls soot cos-only attested by type I because sore donesticated sink (Scholtsns and Coshon. 1965). A review of the literature (Delano. "60) shows that type I botuliou has usually been associated with a series envirouut. Cases of hues botulisn have usually been traced to isproporly prepared narins foodstuffs. such as pickled herring. sslnon eggs and whale scat. Pedorsen (1955) concluded that g. bgtulfl type I is very prevalent on the sea botton and several recent investigations have shown the organics to be present in both the Atlantic and Pacific Oceans (Hard at al.. 1961; Craig at al.. 1968). Although Prevot and Inst (1951) shoved the presucs of Q. bosgnun type I in fresh water porch in hence. until recently there has been little reason to believe that g. baggin- type I could be a factor relevant to the ecology of the Great Lakes. The iirot evidence that g. bogglinun type I was present in the Great Lakes cans in l960. whu an outbreak of botulisn occurred in tunnoapolis. Bianssota. tucked ”ciseos" frou the Great Lakes served as the vehicle for the food poisoning (better. 196‘). Another outbreak of hunan botulisn occurred in 1963 which was attributed to sucked “chubs' taken fron Lake Michigan (Beholtons and Coohon. op. cit.). In the fall of 1963. a large nortality of gulls (gr-2g W 1.- W. 19m minim) and an!“ other species of watcrbird occurred on Lake Richigan (lay at al... 1965). Iinilar nortalitiec. of a greater or lesser extent. have occurred every year since l963 (Pay. l968) . Ixaninotion of blood and tissues tron s randon sampling of these dead birds have shoun the presence of varying accents of 9. 29m type I toxin (Kaufman and Pay. 19“; Pay. 1966). Subsequent investigations have shown the spores st 9,. M type I to be widespread throughout the Great lakes (’“m .‘ n1" 1965; 30“ .‘ cl" l96‘)s The details involving the Lake Hichigan ustorbird nortslitiss and the discovery of 9. M type I nicroorganisno in the Great Lakes present good. but only oircustsntisl. evidence that type I botulisn is the cause or the bird nortalitiss. To be reasonably sure that botulisn is causing the deaths. a susceptibility level for the birds not be dstsrnined and than a source of the toxin in a fora suitable for intoxication nust be found in nature. Honhsinsr (1965) studied the susceptibility of Ring-billed Gulls (y. W to type I botulinol toxin and although botulisn was induced in then. no definite level of susceptibility was deternined. Also. no direct oviduce of ”naturally occurring" type I botulinal toxin in the Lake Michigan ecooysten has yet been recorded. this study. in part. thus provides the uoosccry evidence to conclude that the waterbird nortelitiss are indeed caused by type I botulisn. the second objective of this study is to session ecological factors that uy have influnced shat appears to be the ”sudden occurrence” of type I botulisn in the Great Lakes. It is recognised that the last of investigation. rather than the absence of the organics. nay mount for this apparent sudden occurrence. however. several changes in the Great Lakes ecosysten in the past decade could possibly accent f. this elevation into proninsnco of 9. pm type I. Two of these changes are the invasion of the originally marine slowife (Along pseudoharengus) and the accumulation of the chlorinated hydro- carbon l,l,l-trichloro—2,2-bis(p-chlorophenol)ethane (DDT). Besides providing a new vehicle for intoxication of waterbirds, the slewivss may provide a suitable medium in which g. botulinum type E may grow and produce toxin. DDT and/or the chemical make-up of aleuives may affect the growth characteristics of g. botulinum type B so that it produces toxin more readily than under the previous conditions of the ecosystem. Also, DDT (O'Brien, 1964) and botulinal toxin (Brooks, 1964) both affect an animal's nervous system and there thus may be some sort of interaction between the two materials when both are present in an animal. This study, therefore, also examines these aspects of DDT and the alewives in respect to their possible role in the occurrence of type B botulisn in the Great Lakes. n‘ 'au'l l‘» *vo-n's‘ mA- ".'“ I MATERIALS AND METHODS Wheaten bosom. All cultures of Q. botulinun type I paused in the laboratory during this study uero grown in the case basis bacterial growth nsdiu. This nediu contained 3.02 'i'rypticeso (Isltinoxo Biological Laboratories), 0.32 poptone (Difco), 0.21 curses (Merck) and 1.0! yeast extract (Difoo). Anaerobic conditions were produced is the culture by the addition of 0.1! oodim thiogly- collate. Prior to sterilisation by sutoclaving, the pH of the nsdiun was adjusted to 6.0 by the addition of hydrochloric acid. hereafter, this bacterial nediun will be referred to as 1781. The strain of Q. botulinun type I used throughout this study use isolated by Er. 2.1» Johnston (Iced and cm Adninistrstion, Detroit) fron a fresh "vhitefish chub” tat. free Lake Michigan in 1963. this strain is identified by the Food and Drug Adninistration so 020-080x. A culture of g. bgtulinun type I was produced using the Vancouver Barring (VB) strain, which is a "co-on” laboratory organics, but this one used in only one phase of the gull feeding experiments. At the beginning of this study, 13 xi sores-cap culture tubes containing '1'"! had been inoculated with a spore suspension of the QMWL incubatndst300for13hoursandthonfrosen at -13 0. these frosen cultures, thovnd just prior to use, were the stock used so the initial inocnlu for all subsequent cultures. Actively growing starter cultures vars used for inoculating the nsdi- for each culturing superman. These starters were groun for 13 or 2‘ hon-s at 30 or 13 c respectively, depending upon the to-crsture at which the experiment was to be conducted. The length of tine that the cultures of each expsrinent were held in the incubators at each tenporature were deter-iced by the results of a pilot study. Cultures for this pilot study were incubated in three- oIsIco prescription bottles at 13 or 30 c. One bottle was renewed each day for 13 days fron each tenpersture and the level of toxin dotsrnined to ascertain the tine period for scrim- toxin production. Titration of the toxin produced by each culture was coco-pliohed by intraperitoneslly injecting Swiss-Unborn:- white nice with various dilutions of the culture broth. The nice, which weighed between 13 and 21 gran. were raised specifically for this study by the Wildlife Pathology Laboratuy of the hiohigsn Dopartnsnt of Conservation at the Icon Lake Wildlife Ioonarch Center. Dilutions of the toxic cultures were node with a buffer solution containing 0.21 gelatin and 0.“ dibasic ssdiun phosphate. The pH of the buffer was adjusted to approxiastely 6.2 with hydrochloric acid. All injections used for titratiog toxin consisted of a 0.2 nl aliquot of the diluted culture. The results of the titrations are calculated in terns of sense LDSO per nilliliter of culture by the nothod of Reed and Huench (1938). Type I botulissl toxin is known to have the property of beesniag are toxic uh- afijectad to the onsynotic action of trypsin (Duff at al.. 19“). lessons sons fern of self activation nay occur in none cultures and not in others, a sore accurate essence of the total assent of toxin present in a culture my be the toxicity following trypsisisation. Therefore, all cultures also were titrated after the incubation of equal parts of culture and a 1.02 solution of trypsis (Difco, 1:250) at ‘0 c for one hour. The trypoin was is a solution of Sorsnson'o phosphate buffer (Clark, 1928) at a pH of 3.9. All cultures were exssinod nicroscopically to check for the occurrence of bacterial contasinaticn. Also, a specific toxin centralisation test was osploynd to ascertain that the lethal agent in each culture was Q. wing type I toxin. Mice were injected intraperitoneally with one unit (1,000 anti-Hm) of sonovalent g. 125% type I astitoxiu (obtained frou the Conunicable Disease Cater. Atlanta, Georgia) followed inediately by an injection of culture containing a lethal snout of toxin. This injection of culture, however, contained less than 1,000 souse sinisn lethal doses (m). Survival of the protected nice and deaths of unprotected nice identified the lethal agent as g. botulisn type I toxin. 51g.“ of Mus cg toxin prodggggn. Various asounts of a sterile slowifs honogenats were added to the basic TPSY sediu to detersine whether sons aspect of the chenistry of alswivns can affect the production of toxin by g. Etulinu type I. Trash-fros- alswivns that had be. tab. by trawl fros Lake Iichigan were thawed, out into pieces and added to an equal value of distilled water. This sisters was than placed into a Sorvnll Oni-nixnr and hosognnissd. The supernatant which resulted fros centrifugatiss of this hosogonste at 60,000n g for 30 sit-too was passed successively through silliporo filters with sosbrno porno sinus of 3.0;. 1.2}, 0.65; and 0.22}. The fluid was filtered through each siss nonbr-e several tines until it flowed freely. Pisal sterilisation of this alnwifn extract was unenliohod by passing it into a sterile disposable Ialguo filter with a sssbrsne pore sire of 0.20,. The sterile alnwifn extract was added aseptically to ass-ts of M! at a rate such that after the addition of one nilliliter of starter culture, the extract sado up either 2, 6, 0 or 16 percent of thststsl100slvol-s. Thsesntsisarsusedforthisoulturisg were three-ounce prescription bottles. Pivn replicate ultsros were run for cash percentage of fish extract as well as for controls containing an extract. this espsrisou was carried out at both 15 ad 30 c for incubation periods of tea and five days respectively. Titration of the toxin in those alnwifn-m cultures followed several schedules (the procedures were sodified several tisos during thisstudybeeauseofthe largonusbersofsicoandtissinvslvedis this typoofbiossoay). these cultures growsst300weroeossyedby isjoctisggroupsof tension. Dilutions of these culturesworessds suebthatousgruupofsioehadlsssthos502bntsorsthas02 sottaluy while another group had sue than 502 but less than 1002 ssrtality. The cultures incubated at 15 c were assayed by injecting groups of five sins. Dilutins used for this lot of cultures depended upon whether or not the culture was treated with trypoin. Ilsa-treated culture dilutions were undiluted, 1:10 and a two-fold serial dilution with the lowest being 1:50. Activated cultures were titrated using the case dilution schese for these cultures containing less than 50,000 sense USO/s1 toxin but for cultures with higher titers, a wo-fsld serial dilution beginning at 111,000 was used. “fact of gm: Q goxin pgdggfigg. the effect of ossll ascents of DDT onthsprsductionoffi.MtypeItoxiawesdetsruissdbyadding in levels of DU! (p.p' issuer. 1002; City Chen. Corp.. '3.) directly nun-mummummmnmr. mourn. first dissolved iaaostessand dilution prepared so that theedditiss ofosonilliliterofasotaswouldsdd5,50or500partopsrbillion (ppb)tho the TPIY. Aeostrol utnftubes reeoivodesssillilisor sfM-freoasstone. Theseuboowerethnutsclevodtostsriliss tossdi-ndtodrivooutthoacetose. Thsesslodtuboeth. roesivsd0.05slofiseeul-andworsinoubstsdatoithor15ec500 ferns-drivsdoyorespsctivoly. Pourreplicstss foreaohWTlsvol ondthoeestrolouerornsstesdtesperaturo. The botulisal toxin produced is these DDT-TN! cultures was assayed by injecting groups of three nice. A two-fold serial dilution was used, with the lowest dilution being 1:50 for non-trypsiaised cultures ad 131,000 for activated cultures. n. Interniauion of the possibility of an interaction between typo IbotuliseltosinudDDTiaasi-lownseceospliohedbyinjeotiag 10 DDT (p.p' is-sr. 1002) dissolved in corn oil iatrqorit-oally into four mo of 60 white nice at the rates of 5.0. 7.5, 10.0 or 15.0 q of DDT per sense. A consul group received - injection of corn oil only. Appronissuly 22 hours afar the DDT treats-st. the nine were injected istchritcusclly with dilutins of a pure altars of 9,. WWI. Pivo dilution sfculturnwero used, rangisgfr- 1:50 to 13300. A control group received . injection of the gelatin- pbespbato buffer diluut. All injections were of 0.2 al. This cupcrissnt thus contained 30sets ofnicowhich bedbecs injectedwith various lovolsefbotb nor-abounds: toxin. Inch soteoataissdtennies. Iontoftbosetseontaisedfivesalesosd five fancies, but in several isstsseu one sauce was clisisotsd fros agivos set dustoloshegeof isjsctsdsotoriel. Thesiesweighod bonIeonllendilgr-eosdthssstowerosohessgnso-eopossible withrnqccttsthsweigbtsofthsdes. Iadsotofsieewaohsld issnisdividnslbesandwaoobservedfersisdsysfsllswingthom injection. Deusiuwenressvedfrosthsbouoo. Thorecnltsare Mocmtsgsofsicsaffcctsd,sffcctsdniesbcingthcsc chotdieddcrisgthetsstsadthesethsthsdtypieslnnttrosoroat thsosdofthooixdqssstpsried. Thisencrisostwasrepliasted ascendtiss. W. 0m- mu m I- the prissry species of watcrbird a which a test the effect of 5,. mwltenisboeausssfthnrslotiveeasewithwhiehthsy 11 csnbeobtaissdasdbocausocftheprovisuowsrkdosewiththss (medicines, op. cit.). ling-billedOnllo audIerring Cells 0,. Whichwsrethrestofivnwschooldverotakoafrcsagsll sootingeslosylooatedosnorthershflrnhsroansarlogsr'sCity, lichigcs. California Gulls Q.W wereanasyoung . the Deer liver Iigratery Iird Isfugc (sear tugbo- City, It‘) and shippcdtclaotl-nuingbycirexpress. Allgullswnrsholdispsco sttholesslakowildlife Research Center ntiltheywnrofullgroua. Their dict consisted prisarily of alswivns supploncstsd periodically withcaunoddegfsoderrussat. Adssscfthiasisshydrcchlorids was also given periodically to prevent vitasis deficiencies caused by thisniscsuintho alswivns. Tosinfsrfcsdingtcthegsllounooulturcdissss-litsrprco- criptisn bottles at 30 0 for five days. then held st -15 0 until used in the feeding trials. The toxinwco fed to the waterbirds using a syringe udablusted ill-inch lfgaugssccdlo. lhebeskwao'hald epss ad the needle placed into the throat as as to avoid getting the fluid into the trachea. The toxin was slowly injected into the esophagu- asd the bird was then held with its neck outstretched for qprouisstoly one sinute so that the fluid soured tho gastro-intsstissl tract .d could not be rcgurgitated. Ilisisotios of the possibility of oral irrigatiouduotslergovolanssefflaidwasscsqlishcdbyfesdisg thslargsrdoonoisnliquotoof10nlstistorvalocfesshour. no differences in the susceptibility of waterbirds to toxin produced by different strains of 9,. M type I, so Icons (1962) 12 found for chickens, was doeornined by force-feeding ting—billed Cells with 60,000 scene 10.!) toxin. Pour birds received the '11 strain of culture and four received the 026-wa strain. This culture was in a vol-ssflosl. TwogroqoofthrneCsliforniaonllowornalssfed thsdiffcrcntstrsissofcultnrn; onsbird incanhgroupwasfcd 60,000, 00,000 or 120,000 scuoo 141.0 in a volus of 10, 20 or 30 sl respectively. QMtypeIstraisou-waonltnrowao used udstonm the suosptibility level of ting-billed cello. Pour grows of 10 gulls were fed fros 1,500 ID to 30,000 In nu-trypsin activated toxin. Inurgroqscf fonrgnllowerofodfros67,500I.Dto250,000m trypois-octivatod toxin. All birds received a vol. of 10 n1. Cntrol groqoiselsdsdfivegellofod 10slof aeuniussulsted‘fnfasdfive gulls fed 10 n1 of tho gelatin-phosphate buffer which was used to bring he can doses to their final vol-s. Icvernl other species of waterbird were fed strain 026-000x alters in doses ranging fros 30,000 ILD to 150,000 10). These birds included 15Ioning0nllo,ono0reatllnoheroa(m_gw, onoCosnos mmm.umnmucuummunnuo. WW- 0- M- ". 1967. um dud aluivcs worn collsctu fron Lake lichigan just north of fadingtsn, Indigo. m of the fish were collected free the bottos of the late by scuba diving in 25 feet of water about 300 yards free the shore. 13 the ether sin iish were taken iron the beach, tee at the high-water line and teer trcn the water's edge. the water tenperatnre at the ties e1 eolleetien was apprainately 17 c (62 I) while the air uweretnre was sheet )0 c (mreninstely 80 I). All fish were yet iate platic be. which were placed an ice within 20 ninetea alter ecllectien. he tish were taken into the laboratory, held at l C everaight ad tented theiellewingdq fer tygatbctelinsl tcnin. leehiiehwes plasedsnebesrbantpeyerteraneveeneesaneistereend, tellewing weighing. ent inte neverel pieees. A yetate rieer wee need to erase theaeyieeaaendthejnisesthnssbtainedweredilntedwitbtbe gelatin-pheaybste bniier. tines the endileted jnisea at even iresb iiab bills injected nio, as lewest diletien need was 1310. lighsr dilntius need were a twe-ield aeriea beginning with moo. no jeises were alae treated with tryysia ad dilated in a siniler afier. teatingisr typelbstnlinalteniawaadenebypreteetiag the-ice with syeeitie antitsnia. he den were weed tor eeab diletien at each fish. RESULTS All) 0186388161 MW. sorghum-luau"- found to be present in the alswives collected frcn both theheach and water sf Lake Michigan (Table l). the tonic was present, therefore, in a vehicle seitahle for the intcnieaticn of fish-eating waterbirds. Increased taicity was not found after treat-ant with trypsin and it e- be eenslndad that the tccinwes present in the activated fore. the fish which were collected free the beach Qpeared to have been washed ashore the preceding day. tons fengi were growing on the fish bet they were set yet beesning daasieated. both of the fish eellsctsd enderwetar were considerably decayed. Dish ls. l was esqletely severed with fence and the head was nissing. fish lo. 2 was partially severed with fnagns hat was whole ad lees deceyed. ltienowevidentthflfimtypeltenindeesenistia the Lake lichig- esssysten and it is interesting to apeselats absnt theeselcgyofthis tuin. Althenghthstcninwcsfcnndinalysns location, there is no reason to believe that it is restricted to Inst pcgrnhisal area at even the tin of year at which it was fond. nemtmtmtfiinmfcndbownehuugbeedudhue water raises the prefsud pessibility of accidental been intuieatien. Miensly, it wen“ be heirable te have nose investigsti- into the eeslegy at this natnrally eecnrring tonic with respect to possible h-an invelv-snt. M 13 table 1. ‘glggggigigg,bc 53m 35 type I toxin in alswivns taken frcn the Lake hichigan ecesystnn. as: _‘:=‘ ~===£ w W Highest run In fish tits Height of dilation. lib tsnin tonic in I'm eellsctsd fish (gab to hill.niee - per gn fish fish 1 Underwater l! 1:000 d,000 10,000 2 ' ' 26 11100 500 13,000 3 leach at 30 0 -—- -- ‘watere edge 3 ' ' ll 0 -—- -—- s ' ' 31 o ... ... 7 bench at high 30 13100 500 15,000 water line 0 ' ' l1 1:100 500 13,500 1‘ WWW. nun-ah . mu n-ber of galls were fed tonic prodneed by different strains of 5. m type I, it is highly probable that gnlla do very in their susceptibility to tonia prodeaed by different strains. All of the California Gallo fed the 026-030: strain of tonic developed ante- of botulisn and died, while no sins of illness of an hind were observed in these that reeeivod the n tonic (table 1). statis- tical .alysis by the non—pcustrie Fisher tract Probability test indicates that, in doses fron “.000 to 120,000 10.0, the prsbdility is only 3.08 that California cells will be killed in eqnal n‘ors by thousstrainooftcsinnsod. Allof tholing-billedullsthat received 00,000 10.0 of the 020-0”: tcin booe- cbvionsly ill .d twosf thudiod,whilesnlycnegnllfedthontuinonhibited slight syntsns of betnlian. tens of the other ling-billed cells fed the w tsnin bosons visibly ill (table 3). The fisher Inset trebability test indicates only a 1.1: probability that the two strains of tonic tested have the a. effect on ling-billed Gulls. tonia feeding trials nsing varions doses of aon-estivatad tsnia strain 020-000! ednistered to the ling-billed Gallo indies“ that the 1.050 is apprui-tsly 12,000 nonso 10.0 (table 0). However, cslonletion by the nothod of bed ad bend: (op. cit.) gives o 1.050 of jest endsr 10,000nonsoll0. thalevelwhiaheaeseoSOporeantof thegnlls to been. ill is eslenlated to be approninately 12,000 nsnso 160. One of ths'llsfed galetin-pboaphatebefferae socntsel died, bet itdid 17 tile 1. tesnlts fron feeding California 0ells levels of tonin prcdncod by two strains of g. Engine type I. E; A rat: 42:: Does fed 3 n ed. (10.0) '8 026-000: 00,000 Lived Died 00,000 Lived Died 120,000 Lived Died 00esgnllreeaivodnolovelofasatraincf tonic. 18 table 3. lesnlts frcn feeding ting-billed 0ells d0,000 m of torinpredncadby twostrains «QMWI. W Strain of to. of gnlls lo. exhibiting lo. genie fed fed botnlisn amt” dead V11 0 1 0 020-0008 0 d 1 19 tale 0. lssnlts fro-feeding ting-billodcnllavarions levels of 3,. M type I strain 026-080n tonin. Due fed to. of gnlls lo. exhibiting 1-: llo. (10.0) fed A botulisn qutons desd__ 30,000 10 10 0 22,500 10 0 5 13,000 10 8 A 1,500 10 3 i hinoenlsted nadi. S 0 0 Gelatin-phosphate beffor 3 0 1 20 not staibit any sywtcns of botnlisn. tone of the gnlle receiving the trypoin-aetivstsd tonic or the uinocnlatod 1'08! boo-e ill or died. the resnlts frcn feeding toxin 026-080: to the other waterbirds indiccte that these species nay vary in their snecsptibility to the ruin (Table 5). inch larger nnfierofbirds wonldbo needed to deter-ins specific levels of tonin that are needed to prednen specific affects a the difference in s-eoptibility of the gnlls to the differnt strainesffi.mmtypeltoniabooeneeofgrestintnrestwhen consideringbeththetainlcnndinthedeedslewiveeendthemm seneeptibility levels of the ting-billed Gallo to the 026-000n strain oftsnia. Althoqhtho-snntsoftoninfoudinthealewivessbonld besefficienttosichenorkillnsnyofthognllsthatnightoattho fiah,tbegnestionnrisenastowhethertbisstrainefteniaistds tegnlls. thisspoeifistssiafronthefishweeaotfodtsgelle,bnt tny(1066)repertedthatssnedeedalswivespiehedqalengthebeaehsf Lekeliehigudidhillsoneoftheeaptivegnllewhichehthund thattypettssinwastbendeteetedintbnbloedfrontbeaegells. this difference in onsoeptibilityney also .wer the genetics arising frnthofeettbntthstcinfc-dinthofiehwasastiwoted-dthet gelleroertebeleaosuoeptiblstetrypsin-entivstsdtuin. the tdlfeudinthsfishisprdablyinsmweydiffetsntfrcethstuin neediathielebotsteryonperinent-dthestheuetuinennyaffest 21 table 5. losnlta frcn feeding varions waterbirds tonin prodnced by 5. M type 3 strain Old-000:. ==:_ “ m A- w A 4 “4—— :=== Dose fed No. of lo. exhibiting ls. lpecies (10.0) birds fed betnlisn synta- dead herring 0ell 05,000 5 5 1 herring Gull 90,000 5 d 0 Earring all 155,000 5 5 0 Great line Boron 150,000 1 1 1 0s.n 1e. 150,000 1 1 1 Ioraed Grebe 30,000 1 1 0 22 galls differently. The proteolytis processes of a gull's digestive systen nay denature the trypoin-actiwatsd tonic produced in the laboratory but not chngs the activated torin found in the fish. ch3 gfiggticn as a fgggcn of gigs god tumours. the results illustrating the relationship of tnin production to tine ad tenperatnre are shown in Figaro 1 for non-activated senles. figure 2 shows the curves for trypsia-astivated couples. Iron these curves, which are drawn by inspection, it was decided that oaltnres groweat150shonldboincubatedfsr10daysudthooogrowestloc should be incubated for five duo. the diletius for titrating the non-activated sanplea were a two-fold series while these for the activated senplen were a ten-fold series; this tn-fold dilation series ashes it difficult to ocqere the titers of the activated sales of each teqeratnre. We 'mdmmui-mfl typetgrowninmrconteininngerefo-dtobee-t-instedby other basteria .d all of these ultnros were typed positive for 5. Mag-type! rain. ttatisticel analysis of the toainitienef been altaresatiliasdonlythseelbwvelneeebtainedfronthotrypsia- astivetedhninfleblod). dun-aqunlysisefvarieneeefthees detnfleblenindiutethetthelevelsefw‘fanediathisefiem didnotaffeotthsnenntsftninpredneed. dlee,thorewasno interesti. in the cultures bone“ 00‘! ad teqeratase when occidering theeienreoftsniapreheed. Iowover,thie.elpsisdneeiediea. 3,000+ 2,0004 1,000. Botulinal toxin (MLD) x - toxin produced at 15 C. o - toxin produced at 30 C. 500‘ 250 1 50d Figure 1. Time (days) Curves showing the relationship of production of Q, botulinum type E toxin (non-activated) to time and temperatures of 15 and 30 C. 23 500,0004 0 - toxin produced at 15 C. x - toxin produced at 30 C. 50,0004 5,0004 x O A 500. c: S s 1-! x 0 U '3 a 50" .4 H n U o a: 5. f I I I I I I I I I I I I 1 I Ifi Time (days) Figure 2. Curves showing the relationship of production of g, botulinum type E toxin (trypsin-activated) to time and temperatures of 15 and 30 C. 24 25 table 0. s-ery of the tuicities of 3. type I snltares greeninmenntaininghb'fat15and300. m: r :5 ::=3 W _ 15 c 30 c 00! 30316307 (IDBO) ‘_“_ toxicity (0330) (m) lcn-trypsinised trypoiniaed lon-trypsiniaed trypoiniand 0 1,105 109,090 1,520 102,505 0 1,000 100,000 1,000 92,595 0 1,200 205,300 2,290 123,000 0 1,105 175,000 1,000 102,505 5 5,005 007,005 2,105 07,720 5 575 205,300 2,070 07,720 5 2,105 205,300 2,290 07,720 5 2,290 100,500 2,290 102,505 50 1,335 205,300 2,070 102,505 50 1,000 205,300 1,000 105,020 50 1,000 205,300 1,000 105,505 ‘50 1,200 205,300 2,070 102,505 500 1,105 205,300 1,335 07,720 500 2,070 100,500 2,290 50,035 500 2,290 200,305 2,290 123,000 500 1,335 175,000 2,290 07,720 20 table 7. Insults of the analysis of variance of theltenieities of theg. 003 at 15 and 30 0. type t cultures grown in trtt'esntaining tenperetura l 00! 3 Interaction 3 trrer 20 total 31 1,029,011,201 90,070,311 151,009,310 730,000,070 2,010,002,000 1,029,011,201 30.03 32,150,770 1.00 ”.‘1‘.“. 00“ ”p7” g“, '. 27 that, at the 992 signifienee level, there were differences in the “to of tuin produced at the different cornerstones. the neon velnnofthebnieitiesoftheoeltareeincnbatedstucwas 210,002 1.0” while that of the salt-es grown at 30 c was 97,029 1050. therefore, it can be concluded that 9,. ”mm mo 2 strain 020-0”: predates significantly nae tain when inctbeud in 200'! at 15 c then at 30 c. thefastthatthootraineffi.mgmtypstnsedinthis enperinsuproduesonntetsninatucthsnetJOCboldesignifieant eeelogisaliqlioatiens. lhsopti-ngrswtht-peretnreefthis mummmuuumuasnc.m epeneensutrntisassftbsverionsnntrisntsiathegrowth-di- (telnn, 1900). 1thaebesnfoued,bowever,uattbeopnsieswill predneesnell-o-tsoftsninattenperstaroeaslowasl.30 (Id-“total” 1901). lflergeaenntseftypetbotnlineltsnia seabeprodeeediaanterestuc,esieinplicdbytbiss.erinent, sesautbeniaforthatgivenasnitablenedin,aneneueftenins- bepredeeedinhehelicbig-(uciewithiathewaterteqoreturca achievedbyhsholichign). thieideeisinfoetsbstantiaudby thepmlydisueedfindhgdtypelbetalinsltuininthedeed alowivestahsnfrenthsbottcnsfhfisliehign. ltispesoiblothet thespti-growthseqeratareforfi.mmtypeldiffersfrse themataresfsptin-tonieprodastion,althonghthnprednotionof taisattholewts-srs-seesodiathisoqerinentnqbeoqneliw 28 of the particular strain of Q. botulinum type B. Strain 026-080x was isolated from the "cold" environment of Lake Michigan and it is not unreasonable to prOpose that this strain, as well as other strains present in Lake Michigan, produces "large" amounts of toxin at temperatures lower than is considered "optional" for the species. Effect of alewiveo on toxingproduction. None of the cultures of .9, botulinum type B grown in TPSY containing alnwifn extract were found to be contaminated by other bacteria and all of these cultures were positive for type E botulinsl toxin. Statistical analyses of the toxicities of these cultures utilised the L050 values from both trypoin-activated and non-activated samples, depending upon which treatment gave the higher value (Table 8). A onevway analysis of variance was used to analyse the toxin levels produced at each of the two temperatures because of the different methods used to titrate the two groups of toxin; if a two~way analysis of variance was used and a significant difference was found between the two temperatures, it could not be determined whether the difference was due to variations in toxin levels or to the variation of the titrating method. Due to lack of homogeneity of the variance terms of the L050 values of the cultures grown at 15 C, these values were transformed to logarithms before statistical analysis. The analyses of variance indicate that, at 30 C, there were no differences in the amounts of toxin produced at each level of alnwifn extract (Table 9). However, at 15 C, there was a significant difference, at the 97.52 level, between the amounts of toxin produced 29 table 0. lu-nry of the toxicities of g. beglinun type I cultures grown in 2902’ecnteining levels of alnwifn extract at 15 C“ 30 Co W ' A; “‘ 3:: a 2 u ___ 15 0 30 0 lercast . toxicity (103°) Toxicity (10:9) Icn-trypsinised trypoinised lon-trypsiniaed trypoinised 0 0 250 000 70,300 0 300 09,050 3,200 0,220 0 300 03,900 2,900 03,300 0 020 139,770 550 10,000 0 020 00,300 090 10,000 2 070 070 72,070 70,930 2 0,010 90,100 30,730 9,100 2 3,200 29,070 23,200 37,000 2 7,200 120,050 52,000 350 2 10 0 00,050 30,770 0 500 070 00,700 30,010 0 75 05 30,030 30,000 0 05 15 05,070 3,100 0 0,930 00,000 53,770 0,070 0 300 290 00,300 1,210 0 75 0 00,010 1,000 0 00 0 50,030 17,000 0 05 0 51,020 050 0 300 05 07,570 27,700 0 15 0 00,970 020 10 75 030 00,050 1,070 10 00 0 90,100 52,000 10 300 1,300 01,350 250 10 05 0 100,390 7,010 10 00 0 59,000 320 rcbls9. Insultsofthenalysisofvarienenofthetcaisitiessf the _c_. bgulinun type t cultures green in 270! cuteining levels of alewife extract at 30 c. m m touree of Degree of fun of variation f roedcn squares lean square 7 dlewife levels 0 5,590,995,700 1,119,799,157 2.11 31 at the different levels of alnwifn extract. Duncan's How ultiple tango rest (Duncan, 1955) indicates that the nounts of toxin decrease progressively as the levels of slewifs in the culture increase (table 10). the data presented on the toxicity of the THY-slewifo cultures tend to indicate that at warnsr teqeratures n alewife any be a suitsblenndinninwhichfi.b_p_tp_l_._i_ggtypt couldgrewsndprodues toxin. This is substantiated by the finding of toxin in the alswivns which had been lying in the sun on the bench. However, the data also indicate that at lower tenperaturos toxin production is inhibited by the fish extract and extrapolation of these data to an alnwifn as the entire gruth nediu, leads one to expect very little taxis to be produced at low tenperatures in an alnwifn. this, then, conflicts viththeidcathattcxinecnboproduendinanalnwifeinhahe Inchign, although toxin was fond in such a fish. toweralpoasiblo cnplnatisns cube given as towhy typo! botulinaltcxinwae foundinelewives taksnfronthobottouoflehe lichng while this experinent indicates that toxin levels should be very law. this eaporinent was carried out using pure cultures of §.Mtypetbut itwouldbohighly unlikely thatspurs culture wouldoeurinafishialehnuichigen. gagglggtypelheabeen shown to have different toxin producing detectnriatice wh- grown in associati- with other bacteria (Valeaasule et al., 1900). Also, toxin any be produced by strain 020-000n or author strain in an alnwifn atlswtsqeratursowhaniaonbated foraperiodlsngerthenwasused 32 table 10. Results of fin .nlysis of variance ad Duncan‘s how lbltipls tango test for the toxicities of the g. m type t cultures grown in.290!’conteiniag levels of alswife extract at 15 0. WW }#% toarce of Degree of I- of variation frendcn squares Mean square 7’ Alewife levels 4 22.20309 5.55002 ms trror 20 25.93703 1.29005 - tetal 20 00.10072 — .. Percent fish. . g _,_,..,, . _._1.0______L J..____..9... Othcselovelsoffishbeuoenwhichtherearenosignifieant differnoss are underlined . 33 inthisexperinent. Another possible explanationisthottoxinncy beproduesdindeadolewivcswhilothoyarocnthebocehandthat thasefiehcrethenwaehodbachiatofioweter duringastoru. the eslderlehewater would tendto"preserve"thetoxin. therearethas an-ber of factors that could influence toxiaproductiee in thelehe lichigan esosysten. in interesting phensnsncu occurred in the nit-alnwifn cultures incubated at 30 c; the toxicities of the trypsia-cstivatsd s-leo decreased progressively as fie level of fish extract in the cultures increased, while u.- toxicities of the na-cstivated sun-,1.- than.“ as the level of fish extract increased. ttstisticol analyses of these data by the non-paranetric Sign test (Siegel, 1950) indicates that, at the 992 significance level, trypeia increase the toxicity of a culture when no fish extract is present but when 102 fish extract is present in a culture, trypsin dnsrsueo the toxicity. lxanination of .tho unchanien of fiis phcnsnsnoswas acconplishod by repeating the eaperinsntct300nsiag firecreplicstseosfioftheoxandlfl levels of fish extract. However, prior to adding the fish extract to fie culture, the extract was heated at 121 0 for five uinutes to denature any protein present. the results (“table 11) show that heated fish entrust does not inversely affect the activation of toxin as does unheated fish. A ave-deny analysis of variance of fioae data (table 12) indicates that there are no significant differences in the anouets of toxinprodacedbctwoenthe02and102fishculturosorbotween fie trypoin-cstivated and the nee-estivated couples. the nsu-pcr-strie fabloll. 0.xryefthotoxicitiosdg.mlimtypetcaltares grown in.trt! containing 02 and 102 heated alnwifn entrant at so 0. ====~ _3: a======:' ‘ fife ~+A —:=:____=r Percent A f toxicity (“50’ alnwifn 'fiznetrypiinited ps #_J 0 070 22,005 0 335 2,000 0 335 13,335 10 335 2,000 10 000 13,335 10 000 13,335 table 12. Insults of fin analysis of variance of the toxicities of tugmtypotcultureagrowninMoontaining 02end102hcatodalcwifeextraetct300. W ...... of Degree of u- of veriatia frosdon squares Hoax square 9 Aluife levels 1 7,570,352 7,570,352 0.000 trypeia 1 302,005,002 302,005,002 3.103 Interaction 1 53,503,007 53,503,007 0.071 Irrer 2 227,009,035 113,700,017 - 30001 5 050,710,050 -- ... 30 test could not be used to analyse fiesc data because of the snall a-bor of replications used. the snall amber of replications is also the cost probable reason fist significant differences were not found baboon the activated and non-activated toxin. the conclusion that can he nude free file culturing experinsnt using heated alnwifn extract is fist fie protein present in . alnwifn extra“ obviously effects, in acne way, the toxin of 9,. 1m type 2. the protein probably affects the toxin at the nclefilcr level and it is possible that it activates the toxin in a cancer siniler to trypoin. This is substantiated by the fast that the toxin fed in the fish was activated. the unchnian of this “activation“ «not be exactly the scan as trypsin because the aolecule would not fies lose its tniciw when treated with trypoin. as it did in this experient. suc sive botuli l and adninis a to t mm. the results frcn injecting nice with both 003 and type 2 botulinal taxis are shown in table 13 as the actor of nice affected ever fie total saber of nice injected with each level of toxin and DD! for both replications of fie exporinent. Inspection of fines results indicated that sales and fennles reacted sinilarly and so the data in this respect were conbieod. ttctistical analysis of these results utilised a two-way analysis of variance, although fie data were first changed to percent affected and than transfer-ed wifi the Arcsin transfornstion. This nalysis (table 10) indicates that, at the 97.52 significance level, fisrs were differences between DD‘r levels aswellasbetwcnntsxinlevela, fidtherewascninterastienbefiesn 37 table 13. Insults free injecting nice with DD! followed 22 hours later by injections of 9,. M type I toxin. lignrea represent the anchor of nice affected over the total anchor of nice injected for each of the two replicates of the cxperincnt. arr —“‘ nor W “nan-n, (cg/noses) 1:50 1:100 1:150 1:200 1:300 buffer Oil only 9’10 0’10 0’10 0’10 0’10 0’10 5’10 0’10 0’10 0’10 0’10 0’10 5.0 10’10 0’10 1’10 0’10 0’10 0’9 sno sno 3’10 1110 0110 m 7.5 9’9 0’10 0’10 1’10 0’10 7’10 9’10 9’10 5’10 2’9 0’10 7’9 10.0 10’10 0’10 10’10 7’10 10’10 9’10 9’10 0’9 9’10 5’10 0’9 0’9 15.0 10’10 10’10 10’10 0110 9’10 9’10 10’10 10’10 10’10 9’10 0’10 9’10 table 10. Icealts ci the nelysic ct variuee cf the nice effected byeuceseivc iajacticaeelnh‘rcaitnclbetaliael rcaia levels 5 nor levels I. lateractica 20 lrrcr 50 tetel 50 0.005 00.750 5.907 0.821 090 115 10.19 50.27 2.30 39 Mend typclhotuliualteniawhcnaitcctin; nieces treatedin this superinsnt. this analysis also indicates that the 00‘! treat-ant levels affected all nice sinilerly iu the control group that received 00‘! enly (9 e 1.10). the slaps a! the DDT-haulinal tenia interaction is obvious iron the date; as the level a! train “uses“. it takes a greater cent at 00! to enact the nice. when an 0050 is coepated by the aethod at lead and liaannh (op. cit.) for these nine affected on set level olhctulincl toaiaendcadlcweloim. thcclepcaenhcgrqhieally illustrated (lip. 5). the slopes calculated by the least squares nthod (or both DDT and botulinal tonin are virtually identieal. It should be noted. however, that the 501 point computed tor each level oi 00‘: is dependent upon the level at torin and wine wares. to ascertain that it was the toain and net sons other netsricl inthceulturc thetea-cdthcintsraetioa. mmmdeu were injected with the different levels a! 00!. he of these groups was then injected with a 1:500 dilution of culture which had been treated with antiseru- speeiiio for 3. 19.92.11” type I tenia to inactivate the toxin iron the culture. he results (tile 15) alalyud by the parnetric paired t-hat show that. a apposed to the group at nine that received the 1:500 dilution oi tcaiu not treated with antiseru- (t I 1.700). a statistically siniler a-hcr a! nice at each level a! m was effected in hath grasps (t I 0.521). Botulinal toxin (ml/mouse) .0034J3 o - EDSO calculated for DDT levels. '00304 x - EDSO calculated for botulinal toxin levels. .0020« .00194 .0014‘ .0013: eOOIO“ .00086+ .00066« I I W T I I 5.0 5.6 7.4 7.5 9.5 10 10.5 DDT (mg/mouse) Figure 3. Points at which 50 percent of the mice become affected when injected with levels of DDT and Q, botulinum type B toxin. 40 table 15. Insults tron injecting nice with a 1:500 dilation of hetuliaal toxin protected with antiser- spseiiic tar type 8 toxin. figures represent the who: of nice attested ever the total nucr at nice injected with each DDT hula W 1:500 dilution ed Gelatin no! tuin treated with 9hcsphe. (ag’nase) specific antiserun latter 5.0 1’10 5’10 7.5 0’10 5’10 10.0 7’10 0’10 15.0 ‘ 0’10 0’10 01 A peseihls explanation of this interestiea lies with as nodes of anti. cf the two uteriala. the nervous systen of DDT-poisoned ninalsreapoads teasinglestiulcswithctrcinsfhigh—frequcasy inches. resulting in tracers in the animal (O'Brien. 1967). tycpteue dpsiscniaghavchccn cerrclstsdwith theconoentratisnsfm in the central nervous systsc (Dale at al., 1965). Ithe princry node of action dhetulinaltsnicaisprcveatingthsrelacsceiacctylaheliceat telinergically activated nerve syupccs (Brooks. 19“). tylsr (1905) noted that the tonic any effect the chelinergically activated inhibitory nuts. cf the spinal cord (leash- cells). therefore. if thesdlcthallsvelssfthshstalinaltuinuediathisenparicent ifiihit the idihitory cells cf the spinal card. the inpalscs caused hy the Mwouldhc trasnittednore freely through the central nervous sysa- .0 thus elicit a greater than nornal response. But. as the dessgs cf tonin decreases. the affect on these inhihitery neurons decreases although sons affect nay he present at the cyoaeural synapses. th. giving ”protection” free the Ill-caused trunsrs. Wthetceatisaustheenerciscdwhusntrcpslatiagdcta frudaeinjectediathslhoretcrytseuinalsastheysecurinthc wild. itdeesnetqpenrthet-yecelegical significancseanhesac- cladsdwh-rclatiagthcresultsefthisacperiacnttsthclchelichigan fibril“ ”Italians. At nut. lulevelsef hotelinaltcninuy pretestsueindividualhirdsfrcnm. hutitdcesnstscsnlihclythat lcrgsmhersefhirdswauldchtain depreciscquantitinsofthctss natarials that this superimt indicates is necessary for an antagoaiec. the results of this sudy iqu. quite conclusively. that the Lake lichisan waterhird nortalities are indeed «used hy type I hstulian. Q.Mtypelteninwes fonndintheeeesyetenine vehicle suitable for intuicatitn of waterhirds and the gull suscep- tibility ccpcrinents indicate that there was enough tonic in the dead alswivns to affect easy at the hirds that night cat th-. a teaching piece of infer-nation that would he dcsirblc is - observation of e free-living waterhird actually seasoning and than bacedeg effected hyancturallysccurriagtocicfich. thisweuldheeaentronely difficult tech. to say the least. It would also he desirable to study the effects of urisas environ-natal stresses upon hirds affected hy sch-lethal levels of 9,. 19535;. type I tonic. the second objective oi this study - to detercina the reason for thcepparent 'suddcasscurrenco'od typclhstulisninthooreat lahee - is. ufortcnatcly. still not ccqlctely covered. this study indicated that 00‘! prohOly has not directly influenced the occurrence of the discs“ hy either affecting the production of tonic or by acting synargiaticclly with the tonic in effecting urinals. However. Mcthnindirect influencchyhillicg fishwhieh thcaccy Necessuitahlc nsdiuinvhichfl.ng_l_imtypcl c-grovand produce tonic. lat. nestudieshsveyetprovedm tohe theceucc of any fish totality in the crest hahae. 03 M rhcrolethatthoalswivesplayintheoccurrcnoccftypcl hotelisa in the Great Lakes is still uncertain. This study has shown that at warner tenoratnres an alawifc a- he a suitahle growth nediuc for tonic production hut. at colder ucpcraturos. ea ale-rife would not he a suitahle cediun for the production of tonic. However. the fact still routine that type! toninwas (oundiadcad alewives tahen free hde Iiohigan. in any case. the noent advut of alswivns in the wpsr Great Lakes does provide a potentially care available vehicle of inteaieaticc of vatorhirds than was present haforc their invnsin. Another role that the alawivcs nay have played in relation to type I botulisn in the Great Lakes is the possihility that. since 9,. mm type I is considered to he of curiae origin. the elewives nay have introduced the srguisn into the Lakes who: they invaded free salt water. this is indeed an interesting hypothesis. unearuu m hergey's Kennel oi Deter-instive lacterislogy. 1957. 7th ed. no Hilli. and Uilhins “any. haltinore. “0‘. 0.1... Jo's MM. 'o m” .4 0.11. Poster. 196‘s W W ' t. in. ‘m m m We ’s 0.00. .1(”0 919-920. tracks. 0.). 19“. The pharaacslsgical action of hotnliun tonic. la Iotuliaa: freeesdinga d a synaiun. his. I... .d to M. J's M. M“. “a “M's ”a '.e ’9’.n"‘0 105-111. a“. 1.11.. .e a”. .‘ ‘s‘t “W's 0,6.c 1m. ‘ Mmelinsalnocandstherncrine fish in the ratific northwest. ippl. flicrohis. 16(01555-557. 3.0.0. 's'sp 2.3. m. go’s HI]. u “one We t,"e 0010.1]. by our: Ialation hcueea clinical signs ad concentrations in rat train. leienae 103100-1070. Del-n. 0.1. 1960. type I hotelisnt i hacard of the north. Arctic 15(0):”0—250. . 19“. 0rowth ad netaholic activities of 9,. Wm“. inlet-lint Proceedingsofasy'sein. hcwis. ‘a.e .0 ‘e ““1. he “Us “110 Nth 0“. ”a he ’99."-‘0Me “I. 0.1.. 6.0. m. d ‘s ”he 1’“s Activation .1 W W m. ‘ 0.1- h, .W‘Ie Jo last. 72:055-000. ”1 9.3. 1955. haltiple In. and aultiple 9-tests. lionetrics 0 Ih-us h. lab. 19“. Ilypc I hotelisn ic crest hehes watarhirde. true. 51st IA. Iildl. and let. 9asecroes 0d. latch 10-10: 159-109. a 0”.o “a ”‘s ‘ Oceans“ W lathelegist been“ c-cicatin). n 0.1!. Kanfnann ‘ ‘e‘o We 1905. ”I M” of weurhirdo in lute “an. Cost hehes he. Div.. the Hniv. d “fie Put. “a 0’0“.“s '00". 1.11.. Jo's ”Mg ’e‘e ‘9‘. .‘ to We 1903. W ”3“ food poisoning. d. lilh Iced reohnsl. 10:06-91. 00 I“. 's‘c 1902. 01010th “nu” .‘ 0'0 .u“ C! WMWH Action of toninon chickens. toe. Icpt. Iiol. ad Ind" tron. 50:112-110. mm. 0.9. m ‘sns Me 1"‘e mm W ‘ 0031- intissueeofdendleoesadgulls. uich. ItateUniv.dgri. llpeu. Itetin Quarterly Inlletln 07(2):230—201. ad I.I. hrshall. 1905. Develop-ant of W mw0ninatryptieaeccediaautoelavedinthc presence of lactose. 5. Dairy Science “(0):070-075. Iautter. D.A. 19“. WW ups I in cached fish. 5. Iced Boise. 29(0):“5-“9. lenana. 0. 1959. the nest poisonous poison. Ioiencs 150:705-772. Isnheiner. n.n. 1905. the effect of oral ingestion of W m type I on captive gulls. Inch. Itste Univ. hater a Mile t'hria. I.9. and I. unsure. 19“. M: A new hypothesis of the node of action. Iciaae 10000000057450. O'Brien. I.D. 1907. Insecticides: Action ad natehslisn. Accdcnic Dress. Ice fort ad London. Pcdereea. I.0. 1955. 0a type I hotelisn. J. Applied Iact. 10(5): 019-029. trovet. A.I. ad I. last. 1951. Iniotenee on trace du hstulisns 9.90. "9300‘“ 919091” 9' ‘9 W W '0 Inl. we '““s “a 155103-053. bed. Id. and I. teach. 1950. A siale nothod for astincting fifty percent and points. hear. .7. hygiene 27(5) 3095-097. .““. 0.0.. ‘c's “with C“ ’s’e 0e11neeeo. 09‘00 m d “-1- smut-i7 m0 Immm ‘0 '- .7. Iced lei. 20(0)r020-050. “CM. 0.0. .4 0.0. ms 0"’o W in ninale -4 nan. wiaspaialreferonatotypelmmg-W. am scientific Irocesdings. 101st Anal unsung: 220-250. Iciplc. 0.0. 1955. Avian hetelisns lefcrnetioa of earlier research. 0.9. Dept. of the interior special Ioiontific Ispert: wildlife lo. 25. 07 Iicgal. I. Iocparauetric statistics for the behavioral sciences. McCray-Hill Iooh Coaeny. Inc. 1950. license. 0.0. and 1.. tsunagi. 19“. WW type I as a cause of hovine botulisn in Queensland. act. Vet. door. 00 0 123'117 o 171st. IJ. 1905. Ihysiological observations in hunan botulisn. toicaee 159(5557):“7-“0. Valaneeula. 0.. 0.2.9.. lichcrson. 0. Caphell and I.A. 0oldblith. 1900. The effect of growth of other bacteria in radiation- sterilincd haddock tissues on outgrowth and tonic production by £1. m type I. Iotulisn 1900. Olav-II and Bell. ltd. 220-251. nICHIan STATE UNIV. LIBRQRIES l“WWWMWWWI1WWIMIWIHI‘WIV‘1| 31293106317070