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M Y C O T O X I N S A N D T H E I R P O S S I B L E R E L A T I O N S H I P TO M E A T

A. D. CAMPBELL

FOOD AND DRUG A D M I N I S T R A T I O N W A S H I N G T O N , D. C.

(From T r a n s c r i p t i o n -- N o t E d i t e d b y Author ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I w a s going t o apologize f o r speaking primarily on a f la toxin when the subject i s mycotoxin. Bill has already done t h i s and a s anyone who ge ts i n t o t h i s problem w i l l quickly rea l ize , very l i t t l e i s known r e a l l y about t h e subject of mycotoxins. Quite a l i t t l e i s coming t o l i g h t on a f la toxin and t h i s i s the one t h a t we know most about and most of us end up speaking about a f la toxin when r e a l l y t h e subject i s myco- tox ins i n general . broad category of tox ins which encompass t h e toxic metabolites produced by molds and fungi i .

A s you a l l know, the term mycotoxin i s given t o a

It seems t h a t it i s t h e popular thing these days t o organize a symposium on mycotoxins and many of the t i t l e s w i l l bear t h e word "mycotoxins". However, t h e audience soon learns t h a t a f la toxin i s one of t he f e w mycotoxins f o r which there i s any appreciable amount of information and by far t h e majority of t h e work t h a t w i l l be recorded w i l l relate t o a f la toxins . I am going t o be g u i l t y i n t h e same respect , because the ones w e know most about r e a l l y a re t h e a f la toxins . You w i l l note t h a t I used the p lu ra l . ago and a t t h a t t i m e it w a s a f la toxin . Now it has been shown t h a t t he re are a t least 4 a f la toxins readi ly discernible and chemically characterized.

The word w a s only coined about four years

One can r e f e r t o the suspected mycotoxins as f a r back a s b i b l i c a l times and a l s o there have been a few researchers, who have been ac t ive i n t h i s f i e l d of mycotoxins f o r a good many years. D r . i s one of these researchers i n t h e United S ta tes . He has reported on s tudies involving t h e so-called moldy corn and moldy peanut disease i n c a t t l e i n the ea r ly 1950's and also has been report ing on mycotoxins as r e l a t e d t o feeds tuf fs i n general. f i e l d of mycotoxicosis as t h e neglected disease. Aflatoxins, on the other hand, ce r t a in ly are not present ly being neglected, nor have they been s ince they were f i r s t recognized i n 1960. raphy prepared by the Southern Ut i l i za t ion Research and Development Division i n New Orleans c i t e s 424 references and includes an addendum containing about 50 references which were received too l a t e t o be included i n the alphabet ical sequence. cited a l i t t l e over 180 references, so you can see t h a t much work i s present ly going on with t h i s pa r t i cu la r mycotoxin. Drug Administration are ac t ive ly engaged i n work with a f la toxins and although many of t h e comments I w i l l make per ta in t o mycotoxins i n general, most of t h e work which I w i l l be report ing w i l l be d i r e c t l y concerned with a f la toxins as I have already indicated.

Forgosh

D r . Forgosh has of ten re fer red t o the

A recent bibliog-

The bibliography a l i t t l e over a year ago

We i n t h e Food and

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The term af la toxin was coined from the name of a mold Aspergillus flavus. This mold was found by a Br i t i sh s c i e n t i s t l a t e i n 1960. It i s associated with tox ic feeds tuf fs which produced disease i n Turkey. lack of a b e t t e r term, t h i s disease was re fer red t o as t h e Turkey X disease. of t h e disease was found t o res ide i n peanut meal which had been con- taminated with a s t r a i n of Aspergil lus flavus which was capable of producing t h i s potent toxin. Further work by t h e Br i t i sh , t he Cutch, the staff at M.I.T., t h e Focd and Drug Administration and mny others, has shown there are a t l e a s t four af la toxins . The s t ruc ture of these have been establ ished by the M.I.T. research workers and i s as follows:

For

Through t h e excel lent work of t h e B r i t i s h s c i e n t i s t s , t he cause

DIHYDROAFLATOXIN 6 , AFLATOXIN B I

M O L . W T . 3 1 4 17H 14'6

DESOXYD I HYDROAFLATOX I N B I

U

17H 12'6 MOL. WT. 312

AFLATOXIN G,

M O L . W T . 298 '17"14'5

M O L . WT. 328 C17H1 2'7

The difference between B and G a f la toxins res ides i n t h i s oxygen which makes t h e G a s i x membered ring, i n t h e terminal r ing, ra ther than a f i v e memkered r ing as i n t h e B af la toxins . There are two forms c lose ly r e l a t e d t o B and G. B forms, res ides i n the terminal posi t ion. t i o n at t h a t posi t ion.

I mentioned four a f la toxins . The difference between

You note t h a t there i s unsatura- Likewise, t h e same difference e x i s t s betweenG forms.

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Cne of t he very useful physical cha rac t e r i s t i c s of a f la toxins This i s t h e i r a b i l i t y t o f luoresce when exposed t o u l t ra -v io le t l i g h t .

phenomenon, i n conjunction with t h e i r t h i n layer chromatographic cha rac t e r i s t i c i s the bas i s f o r the chemical analysis of these toxic me- t a b o l i t e s . A mixture of four a f la toxins a r e resolved when chromatographed on s i l i c a g e l t h i n layer p l a t e s using a mixture of chloroform and methanol as t h e resolving solvents. After development, t he pos i t ions of t h e afla- toxins on the t h i n layer p l a t e are observed by exposing t h e p l a t e t o u l t r a -v io l e t l i g h t . The B a f la toxins f luoresce with a b lu ish hue and the G a f la toxins with a greenish blue hue. spot i s ind ica t ive of the amcunt of a f la toxin present i n t h e sample. provides a means f o r t he semi-quantitative e s t ina t ion of t he amcunt of a f la toxin i n the sample. t o four hours with present day methods.

The in t ens i ty of t he fluorescent It

Cne can car ry cut an a f la toxin assay i n three

Sl ides (not avai lable) were then shown t o i l l u s t r a t e the U.V. fluorescence of chrcmatographically - separated spots of af la toxins .

A s I indicated, t he i n t e n s i t y of t he f luorescent spots i s ind ica t ive of the amcunt. This i s determined by conparison of matching spots of equal i n t e n s i t y of an unknown with t h a t of a knmn sample.

Biological assays a r e a l s o car r ied out t o determine t h e t o x i c i t y of a f la toxins . t o small doses of a f la toxin , which br ing about death; i n smaller doses, one produces a pathological change i n the l i v e r known as b i l e duct pro l i fe ra- t i on . The duckling b i l e t e s t has been used extensively i n t h e pas t .

The day old duckling i s a very sens i t ive animal

More recently, t he chick embryo assay has been develcped. It

It has been i s considerably more sens i t ive than the duckling t e s t and i n addition, can produce an answer more rap id ly than the duckling tes t . shown t o be r e l i a b l e and i s considered by many t o be eas i e r t o car ry out than the duck 'do-assay.

A t i s s u e cu l ture tes t using human lung c e l l s has a l so been developed by Legator. not aware of i t s being used f o r other than research p r p o s e s a t t h i s time.

Although t h i s i s an extremely sens i t ive method, I am

Although my subject per ta ins t o neat, I wish t o speak t o YGU f rom a s l i g h t l y broader aspect i n the r e s t r i c t e d viewpoint of t h e l e g a l o r t rade de f in i t i on and I w i l l be discussing the a f f e c t s of mycotoxins a s they r e l a t e t o meat, f i s h , poultry, eggs and milk. I am doing t h i s f o r several reasons. Most i q o r t a n t l y , we can draw analogies frcm t h e scien- t i f i c information known abcut these r e l a t ed products; r e l a t e t h i s t o t h e meat products. I n many instances, t h e products are d i r e c t l y in t e r r e l a t ed with neat prcduction and th i rd ly , there i s not a great deal of l i t e r a t u r e on t h i s subject t ha t per ta ins t o meat per se. t ionship of mycotoxins t o meat f r c m t h e following aspects. The a f f ec t of a f la toxins on Eeat producing animals, t h e re ten t ion of t he tox ins o r toxic metabolites i n t i s s u e and then w i l l b r i e f l y mention t h e develoTment of a f l a - toxin or mycotoxin i n processing and the development i n f in i shed prcducts.

I w i l l discuss t h e rela-

There a re a number of l i t e r a t u r e references on the adverse ef- f e c t s of mycotoxins on farm animals. I have already mentioned the work of Forgosh, Zipple, Burnside and others on t h e so-called moldy corn and moldy

34.

peanut problem i n t h i s country. t o a f la toxins . t i r e i n 1961, so any references t o a f la toxins p r i o r t o t h a t t i m e a r e more or less speculative. moldy peanut disease t h a t Forgosh has reported on I th ink i n 1954 and 1957. Cn the other hand, t he re are scme ra the r c lose relat ionships . Dr. Forgosh presented me with some of t he cu l tures i so l a t ed frcm some of these out- breaks and i n several of the instances, they had never been sub-cultured over t h i s period of t i m e and w e found them t o produce af la toxins . So it gives a r a the r c lose relat ionship. C n t h e other hand, it i s not proof tha t t h e problem i n moldy corn or moldy peanuts was af la toxins per se.

Nost of the more recent references per ta in I might menti'on t h a t af la toxin, t he word was coined scme

This i s t r u e i n the case of t h e moldy corn and the

Most of t h e more recent references pe r t a in t o a f la toxins which were shown t o be the causative agent i n t h e Turkey X disease l a t e i n the 6 0 ' s by the B r i t i s h workers. 100,000 or more Turkish people. Bluesmore and Harding published i n 1961 cn t h e adverse e f f e c t s of Brazi l ian peanut meal on pigs. published later i n t h e same year on t h e same e f f ec t i n c a t t l e . In t h e i r in- ves t iga t ion of nine outbreaks of disease i n calves, they found t h a t peanut meal was involved. Some of the calves died, others shcwed spptoms of le thargy and weight gains were reduced. They observed t h a t t he symptoms disappeared a f t e r removal of t h e peanut meal frcm t h e d i e t of t h e surviving animals. I n most of these instances, they fcund t h a t young animals were involved but i n a t least one instance, 18 t o 22 Eonth old s t e e r s were affected. The l i v e r s f rom these animals were found t o be pale, f i r m and f ibrous. They report t h a t a s i n the case with poul t ry and pigs , t he suscep t ib i l i t y of t h e c a t t l e seemed t o diminish with age. Craig and Bryson reported i n d e t a i l on experiments involving he i f e r s and s t e e r s which had been f ed a d i e t of peanut meal f o r approximately s i x months. They were found t o be i n poor condition and t h e use of the meal was discontinued. The herd was put on good pasture and a thorough examina- t i o n f c u r months a f t e r t h e meal had been removed from t h e i r d i e t showed the anircals t o be i n a considerably improved condition, but had grown very l i t t l e i n t h i s period of t i m e . cows i n a da i ry herd which had been f ed peanut meal. report t he r e s u l t s of t h e i r work with f a r m animals. t h a t there were considerable va r i a t i cns i n the suscep t ib i l i t y of var icus species and a l s o t h a t young animals were more susceptible than adul t s .

These cutbreaks resu l ted i n the deaths of

These authors

A year later

These authors a l s o report abortions of two Al lc rof t and Carnigan

They found indicat ions

I n pcultry, ducklings are t h e most susceptible and then turkey

The Br i t i sh poul t s followed by chickens which have been fcund t o be f a i r l y r e s i s t en t . Bobwhite qua i l have a l so been shown t o be qui te r e s i s t e n t . have reported tha t pheasant, I ' m not ce r t a in i f t h i s i s our ring-neck pheasant but t h i s was t h e term used, have been shcwn t o be qui te r e s i s t e n t .

I n la rge farm anirrals, p igs f r o m t h r e e t o twelve weeks of age seem t o be the most susceptible. They a l s o fGund t h a t pregnant SGWS were comonly affected. I n a few older CGWS that were studied, they found no e f f ec t upon conception, gestat ion and pa r tu r i t i on but a s ign i f icant reduc- t i o n i n milk occurred i n a s m a l l group of cows. These cows were being fed a s t r m g l y tox ic peanut meal. L%eep have been found t o be qui te r e s i s t e n t and scme animals have been f ed highly tox ic meals f o r several years with aprarent ly l i t t l e or no e f f ec t .

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Allcrof t and Lewis , i n a comercia1 research group reported s tudies on the e f f e c t of tox ic Feanut meal i n c a t t l e and pig experiments with e s s e n t i a l l y the same f indings as Bluesmore acd others report the d r a s t i c e f f e c t of a f la toxin on milk production of a da i ry herd i n Br i ta in . They found tha t the milk y i e ld f e l l 5051, t o 7510 i n a herd of 40 da i ry cows. I bel ieve t h i s took place i n a matter of several weeks. This herd was not receiving peanut meal i n the d i e t b u t a cottonseed meal concentrate was being used. Aflatoxin was confirmed i n the samples of the cottonseed meal by both chemical and bio-assays.

Halver repor t s t h e i r f indings on the e f f e c t of a f la toxins i n the production of rainbow t r o u t hepatoma, as one-half pa r t Fer b i l l i o n of a f la toxin B i n synthet ic d i e t w i l l produce hepatoma i n t r o u t .

He has reported t h a t as l i t t l e

From t h i s brief review it i s c l ea r t h a t a f la toxins have a pronounced adverse e f f e c t on farm animals and therefore w i l l have a d i r e c t re la t ionship t o meat production and i f nothing more it w i l l have a tendency t o increase production cos ts .

I w i l l now t a l k about the re ten t ion of a f la toxins i n t i s s u e of animals which have been fed aflatoxin-containing ra t ions . Al lc rof t , Carnigan and L e w i s have re-ported on the presence of tox ic materials i n milk as the re- s u l t of feedicg a f la toxin containing materials t o l ac t a t ing COWS. DeYoung and others i n t h e i r work i n the Netherlands has shown t h a t the tox ic mater ia l found i n milk i s not one of the four recognized af la toxins b u t i s i n f a c t , a metabolite of a f la toxin B. metabolite i s a l so produced i n the milk of l ac t a t ing rats. leaves a typ ica l l i v e r l e s ion i n the b i l e duct. whether o r not t h i s metabolite i s a carcinogen as i s the case with i t s precussor. mined a t t h i s time. We f ind here an example where not only does the tox ic r ra ter ia l adversely e f f e c t t h e milk production b u t a tox ic material i s car r ied through i n t o the potable t i s s u e . car r ied out fu r the r s tud ies t o determine the f r a c t i o n of milk associated w i t h the tox ic material. Their exFeriments showed t h a t the tox ic was only present i n the renet prec ip i ta ted case i n f r ac t ion o f milk. These workers have a l s o reported experiments which show t h a t the toxin i s not car r ied through i n t o chicken eggs; they did t h i s by feeding ducklings eggs t h a t had been produced from layers which had received about 15% toxic peanut meal i n t h e i r feed, They a l s o report f indings on the feeding of l i v e r , blood serum and c lo t ted blood from a cow which had been fed tox ic Feanut meal i n the experiment where they were producing the tox ic metabolite i n milk. None of these three above mentionedt issuesproduced typ ica l l i v e r les ions i n ducklings.

He has shown t h a t t h i s tox ic This metabolite

It has not been determined

The chemical s t ruc ture of t h i s metabolite has not been de ter -

Pillcroft and Carnigan have

Fer re t s and mink have been shown t o be sens i t ive t o a f l a tox in poisoning and f e r r e t s have been e f f ec t ive ly used as t e s t animals f o r analysing raw meat because of t he i r s e n s i t i v i t y and carnivorous nature. Platenow and Beauregard of the Canadian Department of Agriculture have recent ly reported work where chickens were fed r a t ions containing up t o 3G$ tox ic peanut meal. f o r a period of s ix months.. .s ix weeks. The meat of these chickens was fed t o f e r r e t s and the l i v e r s were fed separately.

Although the l i v e r and kidney t i s s u e of t he tes t animals revealed abnor- Ealities, ne i ther the chicken meat nor the l i v e r s produced changes i n the tes t animals which could be a t t r i bu ted t o a f la toxin o r metabolites of af la toxin. Unfortunately, the cont ro l animals showed abnornralities, too. I am afraid t h a t t h i s work w i l l require repeating before any f i r m conclu- s ions can be drawn. tha t m c h fu r the r work w i l l be required before t h e question of re ten t ion of toxin i n edible t i s s u e s can ke answered.

It i s apparent frcm the research reported t o date,

NCW, l e t ' s b r i e f l y t u r n our a t t en t ion t o the aspect of s tor ing Th i s i s an a rea i n which very l i t t l e i s reported i n the and processing.

l i t e r a t u r e . The Russians have car r ied out scme s tudies on the t o x i c i t y of molds found on meat and meat products. This w a s s tored meat. This information was published i n a bcok by Bilay i n 1960. published infomat ion , 70% of the meat re jec ted i n Eussia was found t o be infec ted wi th mold during t ransportat ion. cases of mold in fe s t a t ion i n meat and meat products. The i so l a t ed molds were cul tured and the cul tured media was then t e s t ed i n dogs. of these mold cu l tures were found t o be toxic . ments do not shcw t h a t meat i tself was toxic but only t h a t t he meat prcducts harbor scme fungi capable of producing mycotoxins. It i s r ead i ly apparent t h a t e s sen t i a l ly no work has been reported on t h i s aspect of mycotoxins i n meat and experiments w i l l be required before any con- c lusions can be drawn on t h i s side of the subject.

According t o t h i s

They report a study of 26

A number These reported experi-

I th ink it i s in t e re s t ing t o note t h a t t he last two food addi t ive p e t i t i o n s per ta ining t o the use of enzymes contain a clause that states t h a t t he enzyme w i l l be produced. duced enzymes, produced frcm cul tures maintained i n such a lranner tha t they

these new enzyme preparations t h a t are being ccnsidered as food addi t ives a r e being invest igated as being as ac tua l ly s t a t ed -- regulated t o be free of tox ins before they a r e acceptable as food addi t ives . I have spoken about Aspergil lus f lavus producing af la toxin; it has been shown t h a t a number of other fungi a l s o produce the a f la toxins as we recognize them. Aspergil lus pa ras i t i cus has been reported by the Br i t i sh t o produce a f l a - toxin; Penicil l ium puberilium i s a l s o a producer. Several other p e n i c i l l i a have been reported t o produce af la toxin.

This per ta ins t o microbial pro-

w i l l be non-pathogenic and non-toxin-producing including af la toxin. So

We have i so l a t ed a foma and fuse r i a . I t ' s not been characterized pas t t h a t point , but indicat ions are t h a t these two organisms produce a f la toxins . So although when one reads t h e l i t e r a t u r e one wculd th ink t h a t i f you looked f o r Aspergil lus flavus, t h i s wculd be enough - t h i s i s not. You have t o look broader than t h a t because it would be unusual that only one organism would produce a Eetabol i te such as t h i s . So, as we f i n d i n nany other ne tabol i tes , it can be produced by several organisms.

I n sumarizing, it has been r ead i ly shown t h a t mycotoxins and af la toxins , i n pa r t i cu la r , have a lrarked adverse a f f e c t on meat producing animals. Regarding t h e re ten t ion of a f la toxins i n the t i s s u e of animals, which have ingested a f la toxins , m i l k i s the product which so far has been

37.

shown t o be affected. and eggs ind ica te t h a t t h e tox ic substances are not carried i n t o t h e products. t he re i s a need f o r fu r the r invest igat ion. it i s indicated t h a t s tored meats are po ten t i a l as mycotoxin producers. Thank you.

Some of t h e l imited experiments with l i v e r , blood

Cn the other hand the work with f e r r e t s i s inconclusive and I n t h e work of t h e Russians,

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Following A. D. Campbell's speech, due t o lack of time, a question and answering session was foregone. It was suggested t h a t i f there was scme specif ic question, anyone would be f r e e t o wri te t o the person or persons involved and perhaps they could help i n t h i s manner.

L W ! J. BFATZLER: Eefore the morning break, ... you heard D r . Cox mention t h a t Kansas State was the first Land Grant College ... I would l i k e t o take D r . Cox t o Michigan State and a t every entrance t o the campus he would f ind tha t a sign reads, The Pioneer Land Grant University -- Founded i n 1855.

Going t o the commercial problems i n meat processing, w e have Neil Webb, a s chairman of t h i s committee. Neil i s a product of West Virginia and, by the way, B i l l Sulzbacher i s very famil iar with Pittsburgh and I forgot t o mention t h i s when I introduced you, B i l l . After West Virginia, N e i l stopped i n I l l i n o i s , went t o Missouri, kept on rol l ing, went t o Michigan State and now N e i l is i n charge of the technology and food research with the Eckert Packing Company of Defiance, Ghio. Neil, i f you w i l l introduce your Committee and your speakers.

NEIL WEBB: I t ' s a pleasure t o be with you a t Kansas State . i n t e r e s t based on what w e have already heard f rom the previous sessions. F i r s t , I would l i k e t o introduce the c c m i t t e e who helped t o get the program together. t o stand at t h i s time, - Paul Goeser, Swift and Coqany, and Jack Gesler, California, Woody Aunan, American N e a t I n s t i t u t e , and Dale Huffman, Alabama. t h e i r e f f o r t s i n helping with the program.

I think the next two papers w i l l be of considerable

I ' d l i k e f o r the c c m i t t e e

I want t o thank the Comit tee f o r

Our first speaker i s a person t h a t I think, needs no He has been w i t h us a t nearly a l l introduction t o t h i s group.

of the R. M. conferences, and one whom we a l l enjoy hearing speak. morning on a subject tha t I think i s qui te important t o the Industry. we are studying these topics - I think the importance has been brought out very well by the previous sessions. D r . Strong mentioned, very well, some of the things I wanted t o have mentioned about why w e a re so concerned about packaging and bacteriology. In t he first place, today we are dealing with many, many new processes. The many new methods of packaging and processing technologies are generally, developed i n the laboratory or i n combination with p i l o t plant work. fortunately, we have d i f f i c u l t y i n acconiplishing the control i n our processing operations t h a t w e can i n the laboratory. Unfortunately, not a l l of us engaged i n the Industry can spend the t i m e it takes t o r e a l l y evaluate some of these developments under a l l s i tuat ions.

We asked John Ayres t o present a paper f o r us t h i s

I won't dwell too long on the importance as t o why

Un-

39.

The very best we can do, i n our Research Labs, whether it be Industry, University, U.S.D.A. o r what -- requires a l o t of t e s t ing and w e may cer ta in ly overlook some implication. area t h a t i s sometimes overlooked. t h i s e a r l i e r today and we a r e qui te ccncerned abcut introducing new processes without proper t e s t ing . was created on our conitnittee - the ccncern over some of the methods used today and what a re the microbiological implications. j u s t a word or two of background about D r . John A y r e s : University of I l l i n o i s graduate, f inished h i s Ph. D. there and then worked a t General Mills i n food micro-biology and f rom there went t o IGwa State where he i s Professor i n Charge of Food Technology i n t h e Dairy and Food Industry Group. I ' d l ike , a t t h i s time, t o turn the program over t o D r . Ayres.

I think t h a t bacteriology i s an John Ayres and I were ta lk ing about

This i s r e a l l y how t h i s whole subject

I 'll give you He i s a