Volume 10 No 1 March 1989

Publlstl ed as a service to mic robiology by Oxold Limited Oxold IS a registered trade mark.

Listeria monocytogenes: attributes and prevention of transmission by food

EH Kampelmacher, DVM PhD, Professor-Emeritus, Food Microbiology and Hygiene, Agricultural University, Wageningen and OM Mossel, 8M, MA, PhD, Chair of Medical Food Microbiology, Faculty of Veterinary Medicine, Department of the Science of Food of Animal Origin, Neth. Gov!. University, Utrecht, The Netherl ands.

Pathogenic properties, clinical attributes of L, monocytogenes' always rely on the strictly quantitative prevalence and ecology Table 1: Lag times (days} of Listeria monocytogenes in comparison (i) the aUeged elevated heat resis· approach known as Holistic Risk 4oAI Listeria monocytogenes was discover­ to other psychrotrophic pathogens and spoilage agents, at tance,37 a point which we will Analysis. The essentials of the ed as a pathogen of animals and man temperatures ranging from 0 - 10 °C. discuss later: procedure will be summarlsed In the in the 1930'5,1 .2 As far as humans (Ii) the ability for relatively rapid next section. The result s allow an are concerned the organism was Temperature, °C growth at refrigeration tempera­ unbiased approach to the often initially Identified as a cause of tures, as Illustrated by the data in emotive subject of food·borne abortion In early pregnancy, stillbirth listeriOSIS, Orgamsms 0-1 2-3 5 7-8 9-10 Tables 1 and 2; Of a sepbCaeml(l (granulomatOSIS ~IO a marked tolerance of reduced Infanbsepuca) aher an unevenrful pH-values;38 Food transmitted listeriosis: blnh J ~ A hydrophila >22 6 -10 3-4 2 meningitis and flncephalitis In L. monocylogenes 3-33 2-8 1 -3 <1-2 < 1.5 5% sodium chloride.38 The consumption of every food. [ike Y enterocolitlca 3 2.4 <0,1 the newborn and pregnant women - - A rational approach to a clarification virtually aU other activities of man, appeared also 10 be caused by of the real hazard must take account entails a fisk, however sma!1. Hence, L. monocytogenes. M ore recently It Psychrotrophic spotlers 2.5 - 15 - 05 of two fundamental conSiderations:&! the often pursued target of 'zero fisk' was demonstrated that listerlal Infec­ 1. Many species of Listefla, closely IS, scientifically speaking. an unreal· 118 119 tions may occur In adults of both Data from Walker and Stnnger 1987 and Elliott and Michener 1965. related to L. monocytogenes, occur istic situation. ObViously In any sexes. The maJonty of patients were as Innocuous saprophytes ln the particular Instance the fisk Incurred IS debilitated when they contracted the environment. This makes It essential to be kept as low as is reasonably disease. This was caused by either character. This is illustrated by the cause. the risk of infection by to base all epidemiological studies on attainable and maintainable, ThiS immuno-incompetence or pre-existing data for lag time and growth rate, the L. monocytogenes should be L. monocytogenes sensu strictu , as leads to a 'low-risk' situation, which of conditions. e.g. diabetes, alcohol latter expressed In the reciprocal reduced to the lowest possible defined in Table 3. An occasional course, is not identical with a 'no-risk ' 64 abuse, drug addiction. Patients with parameter generation lime, shown in extenl. report in the literature describes position; details of this unbiased cardiac lesions or valve replace­ Tables 1 and 2. Epidemiological rather than circum­ L. seeligerii as a cause of human approach are summari sed In Table 4. 39 ments- an increasing part of the stantial evidence for a possible asso­ purulent meningitis and th is Assessment and subsequent manage­ population- are particularly at risk Transmission ciation between the ingestion of exception should be kept in mind. In ment of each accurately defined S from listerial endocarditis However, In the 1950's, circumstantial evid­ particular foods and listeriosis started contrast however, segregation of health hazard relies on the following 26 listeriosis is also observed in pre­ ence made it likely that ruminants to be published in the middle particular serotypes of L. monocyto­ steps: (i) identification of the risk of 6 viously healthy individuals. - 10 may acquire listeriosis from 80'S.33 - 36 These reports have led to genes, especially 1/2a and 4b, as transmission of L. monocyrogenes The reported incidence in the US contaminated feed, including silage speculations about the aetiological being of major pathological signifi­ caused by /he ingestion of a particular amounts to about 800 patients per mentioned above. Hence, it seems role of food generally. This has cance seems unjustified at present. 35 food; (ii) assessment of the size of the annum or under 4 cases per likely that the route of absorption in become a point of concern parti­ 2. Assessment of the health hazard of hazard by appropriate computations minion. I I This is not unlike the situa­ man is also through the intestinal cularly in view of the following four a given food commodity should of a holistic nature, i.e, taking into 27 tion in Britain where in 1987 about tract. Th is assumption is sub­ account al/ instances of contamination 260 patients were reported ,'2 or stantiated by the observation that up and proliferation that can occur roughly 4 per million.'3 The true to 20% of healthy individuals harbour Table 2: Growth rate of Listeria monocytogenes in comparison to throughout the fragile food chain, disease rate is, however. estimated at L monocytogenes or other species of thermotrophic, mesophilic, other psychrotrophic and psychrophific targeting at the food as ingested: (iii) 22 28 :s: 50 per million and the case fa tality the genus in their enteric tract . bacteria. evaluation whether the risk thus rate at about 30%. " .12 and. because listeriosis is also calculated is acceptable, when Ecological surveys have demon­ frequently observed in urban Generation times" in hours at measured against hazards generally strated that listeriae in general, and dwellers, 29 foods must in principle be considered acceptably low, as in L. monocytogenes in particular. are considered a source of infection. This Figure 1: (iv) intervention with naturally occurring in a wide variety mode of transmission of listeriosis had Organisms 10 - 13°C 4 -5°C 0 - 1° e necessary changes in design and of domestic and wild animals, parti­ been much debated up 10 1980, with other measures which will reduce the cularly sheep, goat, cow, alpaca, and European epidemiologists being con­ y, enterocolitica 20 25 risk to an acceptable level; (v) valida­ various birds. II. - 16 In these animals vinced much earlier of this possibility L. monocytogenes 5-9 13 - 25 62- 131 tion whether the risk reduction target 26 28 the bacterium leads to a particular than others. . ,30.31 A. hydrophila 4 -6 9 - 14 >49 has been attained. type of encephalitis, e.g. 'circling The alimentary route of infection does True psychrophiles 2-3 6 12 Acceptable risks in medical food disease' in sheep2 as well as mastitis not mean that ingestion of L. mono­ microbiology are- in a first order n ,a Psychrotrophic in cattle. . Upon recovery. affected cytogenes will always result in clinical approach- expressed in 'safe' cfu­ Enterobacteriaceae 2-4 8-12 16-20 animals often continue to void disease but as indicated above, and spoilers levels in the commodity as consumed. L. monocytogenes in faeces and milk. pregnant women and other groups of For instance. a risk level of 10- 7 is It is therelore not surprising that the the public whose antimicrobial generally accepted for Salmonella Salmonella spp. <8 >30 ~ bacterium is often isolated from soil, defence is decreased, are particularly species in milk products to be con­ mud, surface water and vegeta­ at risk. When previously completely sumed by heallhy adulls. On Ihe olher Data from Rosenow and Marth 1987,120 Walker and Stringer 1987,1 18 Mossel tion. 1619 - 22 It also occurs in healthy individuals contract listeriosis, hand only a level below 10 - 12 is, as 23 25 ~988 . 44 silage. - in which lactic acid it has been theorised that they have •. Generation times of 1, 6, 10, 30 and 90h correspond to times to attain a rule, considered sufficiently safe for 6 7 fermentation has been impeded. The been exposed either to excessive populations of 10 - 10 cfu of approximately 1, 5, 8, 24 and 72 days, a much more dangerous organism predominance of L. monocytogenes numbers 01 viable cells of L. mono­ when lag times are virtually nil. like Clostridium botulinum. Such first in these environmental habitats is cytogenes or to strains of unusually =: unknown. 9 32 favoured by its psychrotrophic high virulence. . Whatever thee CONTI NUED OVERLEAF 0.01 0.1 10 100 1000

Automobile ace ide nl 22 ~ Liver biop sy 200 Escherichia coli 0157 Latex Test Child beari ng 100

Having a fall 71 I Surgical anaesthes ia 40 I Drowni ng 38

Poisonl ng 30 I Fir es 29

Aeroplane trav el 8

Striking by falling obJe cl ... ,,,. Railway aeeiden ls 3

Cold shoc k 2

ElectrocutIOn at hom e 1 Vaccinatio n 1 A latex agglutination test for the identification of Escherichia. coli Serogroup 0 157. (Ordering Code: Suffocation in cradle or bed 1 DR 620 - 100 Tests) .

Lightnin 9 0.6 Tornado, flood, earthqua ke 0.5 Venomous insects. snak es 0.2 Botulis m :J 002\ - Salmonellas is 0.01 0.1 10 100 1000 <' "

Figure 1: Risk of death associated with accidents and medical procedures_ Data for the USA and UK assessed in the decades 1960 - 1980 and expressed on log scale per 106 cases. order estimates have. obviously, to be corrected ' by a purely scienlific elaborated more accurately by appli­ approach. In such instances risk cation of a delalled fiSk analysIs analysIs alone can achieve very little. model This takes Int o account, Only when the public has been re­ besides the virulence 01L. monocyto­ assured can a fisk analysis of the genes. parameters li ke (I) vulnerability situation serve a useful purpose .~4 of tile consumer: (Ii) frequency and amount Ingested of the food under Achievement of safe levels of study. and (III) tile fate of a given L. monocytogenes pathogen III tile food under custom­ A most important final facet of risk ary conditions of distnbutlOll. storage. analysis and management is to assess sale and culinary preparation In the the attainment and maintenance of the Ilome or restaurant. .1 ;>~:l A matrix to low level of contamination with be used In Ole assessment of the risk L. monocytogenes required by the of tranSmiSSion of listeriosis by any results of hazard analysis. Once it has particular food IS presented In been demonstrated by technological Table 5. intervention that this low level can be It IS essential to empllaslse that fisk reached. food producers have to analysIs models are based on (I) ensure tllat it is consistentfy attained JlIstdled concern: and (I I) tile attempt and maintained. Figure 2: The detection of Listeria monocytogenes by enrichment in PAl CAM (polymyxin acriflavin lithium chloride to protect the consumer adeQ.uately Since 1985 extensive surveys have ceftazidime aesculin mannitol) broth.93 relying on expenmental data. Realistic been carried out on the occurrence acceptability levels obtained In thiS of Listeria monocytogenes and closely pathogens is widespread. about 3kGy ('radicidation') is a good vegetables. in addition to cleaning 69 71 74 way are always open to discuSSion related bacteria In various foods. Listeria spp. have been isolated, in procedure to attain this goal. - and disinfection. measures to and Will ultimately lead to agreement These surveys have mainly covered some instances at isolation rates over particularly because radicidation can prevent post-treatment contamination between qualified segments of the commodities that were raw or pro­ 500f0. from raw staple foods includina be applied to the already hermetically are essential. If such post-process public and governments or between cessed by methods not involving chlcken ,~5 - ~9 red meat. ~8 . ~9,50 - 52 packaged product. This process measures are neglected. reintroduc­ 53 public health autllontles and food heat-treatment. Not surprisingly most seafood and. of course the avoids recontamination of processed tion of pathogens will occur from the 54 manufactUring InduSlf1es, '" of the commodities have been found classical habitats raw milk. - S6 soft commodities, a possibility which poorly controlled. and hence severely 56 58 59 73 On tile other Iland, concerns are often to be more or less severely contam­ curd cheese - and vegetables. always mars conventional contaminated, food environment. 73 expressed W~l lC ll are of a pllOblc Inated with listeriae. ThiS substantiates Numbers of cfu per gramme of food technologies. It has been maintained for a long time nature. I e are dictated by emotional much earlier views that this class of have mosti&, been low. about' - 10~ Purely emotional resistance to its that Listeria monocytogenes is usually factors wh ich cannot be Justified or bacteria. Including the human cfu g ' .52. - 61 L. monocytogenes application has so far, however. been heat resistant. More recently it was occurred only infr equently and an insurmountable hurdle." Pending demonstrated beyond doubt that Table 3: Key characteristics of Listeria monocytogenes (Aocourt et al serotypes 4b and 1/2a were often in acceptance of irradiation as safe for Bearns and Girard's observations in 121 1987 ) _ the minority systematic processing, no other 1958 were marred by the use of a Hence. the incidence of listeriae in means for- low level- consumer deficient laboratory technique for the 75 foods of animal and vegetable origin protection remain except: (i) meti· assessment of heat resistance. Not Morphology is comparable to that of the classical culous hygiene during preparation surprisingly therefore. work done Gram-positive non·sporing rod-shaped baclerium. wilh tendency 10 coccoid Gram-negative enteric pathogens: (ol/owed by protection from environ­ lately using meticulously validated pleomorphism. salmonellae. campylobacters and mental contamination : (ii) effective methodology has failed to indicate Motile by peritnchous !lageUae at 25°C. the enterovirulent types of the genera chilled storage and distribution. In any heat resistance of Listeria mono­ Escherichia. Edwardsiel/a. Yersinia view of the psychrotrophic character cytogenes that is beyond the normal Biochemical properties and Aeromonas. Consequently of L. monocytogenes this must be in range for Gram-rositive non-sporing. Catatase positive. (j-haemolysls - somellmes weak. potential measures of control. while the so-calted 'super chill' range. i.e. pathogens.61.76.7 .78,m This is illustrat­ 73 Glucose. maltose. aesculin and saliCin fermented by a homofermentative similar for all ttlese orgamsms, are between - 1 and +3°C. ed by the data in Table 6. pathway:"mannllol and xylose nOI anacked. essenllally different for raw and for On the other hand. recontamination Arginine hydrolysed. nitrate not reduced , gelatin not attacked. urea not processed commodities. 62 - 6~ Processed foods of adequately heat-processed hydrolysed Fortunately. means of control are fully products has been demonstrated to

No formation of IIldole from tryphophane nor of H2S from cysteine. Raw foods attainable in the case of processed occur much more frequently than is ReducliOn of 0.05% tetlurite , Consistent decontamination of raw foods, particularly of animal origin. generally assumed. This applies to 79 s1 meat and poultry products by pro- First and foremost. the maxim of Sir pasteurised milk - as well as to Identification cesslng for safety. similar to the Graham Wilson66 should be heeded. soft cheeses manufactured from 65 67 First stage by serology. pasteurisation of raw milk - IS long that processing for safety includes pasteurised milk. 57 In a recent survey 12 In epidemiological surveys phage typing is indispensabte though results overdue 66 Technologies for this much more than just a heat treatment. of cook-chill meals in the UK most are not at ways conctuslve.106 purpose are certainly available. Treat- as in the case of pasteurised milk. items found to be contaminated by L. ment with gamma rays at a dose of e ')imilarly, when dealing with fresh monocytogenes were poultry dishes. Table 5: Acceptable final levels of contamination with listeria monocytogenes, expressed in NI - per portion of food eaten.

Determined from : , . The amount of food consumed: 2. SIze 01 the population at risk: 3. The minimal infectious dose (MID) · of the' pathogen concerned.

Level of consumer protection Q12 = 0.99. corresponding to exposure of 72% of the population to a pathogen not more than once in a lifetime (about 100 years). when eating one portion daily.

MID' Populalion (x I O ~ 15 250 NI per portiont

13 1 3.4 x 10 - ' 2 2.1 X 10 - 1 10 3.3 X 10 - ' 2.5 X 10 - 100 4.6 x 10 4.4 x 10 1000 7.9 X 10' 7.8 X 10'

. An MID is a parameter affected by various determinants. Including: (1) genotypic and phenotypic virulence attributes of the pathogenic species to which it applies: (ii) health condition of persons at risk, particularly with respect to Ihe digestive tract and immune status: (iii) mode of ingestion. e.g. on an empty stomach or not. and in the presence or absence of substantial amounts of load lipids. t The limit 01 acceptability of L. monocytogenes in foods is at present < 1/25g. i.e. the level 01 detection with available techniques. Figure 3: Cu lture of Listeria monocytogenes on PAlCAM agar.93 85 pointing again to post-cooking con­ related bacteria. When studying the Isolatlon. This shortcoming is The medium lacked the required monocytogenes. Phage-typing is no tamination from the severely con­ fate of these bacteria in foods during compounded by the failure of the cold selectivity, however. when used for doubt another useful technique. taminated environment where raw processing, storage and distribu­ enrichmenl technique when applied plating enrichment cultures. The However. when used for epidemio­ poultry was handled. tion. 42,4 3.83 challenge tests are to foods because the listeriae may addition of lithium chloride and logical pu rposes this type of investiga­ 5 Guided by the D-values in Table 6 generally used. relying on 'spiking' of occur at a level of 10 - of the natural ceftazidlme was needed to suppress tion is still marred by many un­ and assuming an MID of the order food samples with realistic numbers microbial flora. which IS ollen Enterococcus and an occasional certainties. 106 102 t - 10 as in Table 5. the matnx pre­ of suitable test organisms. These are composed of competing psychro­ Micrococcus strain. The latter 4 6 73 sented in the latter table allows the of the order 10 - 10 cfu g - I: hence trophic species. organisms revealed their presence Need for resuscitation treatments estimation of the required Nj'level. for this purpose plate counts are to be This prompted the Introduction of a utiliSIng mannitol, but nonetheless they As could be expected. cells of From those and the homologlsed carried out. broad variety of selective agents to be obscured the reading of plates when listeriae damaged by exposure to Initial levels 01 contamination of raw In elaborating techniques sUitable for used to replace cold enrichment when too many were present. This pheno­ adverse conditions were found to be products with listeriae. the required the two different purposes we have examining fresh foods and the food menon is also often seen on plain markedly inhibited in and on the process lethality. /I, = log No -log NI followed the approach adopted since environment. The mosaic 01 selective brilliant green agar plates used for the highly selective media described can be calculated and rrocesslng about 1960 lor the detection and agents su~gested within the last five IsolallOn of salmonellae from enrich­ previously.18 102 101 . 109 Attempts to designed accordlngly.4o.s enumeration of pathogens. such as years as - 8 demonstrates that these ment cultures. 103 overcome such defiCiencies by modi­ Salmonella spp. and Staph. aureus. ,nhibllors have been used With mixed flcalton of the media are futile, The detection and enumeration of In foods. success in attempts to isolate the Identification o f isolates because what mitigates a given type Listeria spp. in foods target group from contaminated foods TI1e isolates of pathogenic listeriae of damage, e.g. heating. will not Enrichment techniques or food environments. We have found occurring on the finally chosen necessarily remedy a different stress, Principles Ever since Gray et al 84 discovered lithium chloride90 an indispensable selective diagnostic medium, termed e.g. exposu re to a low pH. no Hence Enrichmentlisolati on procedures are the psychrotrophlc properties of Ingredient to suppress Enterococcus PALCAM agar (Figure 3) are readily a treatment aiming at repair of any 91 required when examining foods for Listeria monocytogenes. 'cold enrich­ spp. and ceftazidime or cefalo· identified using the ty pical character­ lesion, termed resuscitation, is requi r­ 92 'absence' or better 'failure to detect' ment' techniques have been used for sporin to be reqUired to Inhibit the Istics of L monocytogenes shown in ed to attain the necessary proouctivity I09 lll lisleriae in 25g quantities according 10 the isolation of this organism. When other common commensals of fresh Table 3. Closely related listeriae are of the analytical procedure. . Table 5, and when monitoring the applied to clinical specimens the fcxx:js, such as chicken. pork, seafcxx:j identified by the same tests. Recently Resuscitation in tryptone soya 93 l12 fcxx:j environment for the 'occurrence' method requi res from one week to and soft cheeses. nucleic acid hybridisation assays' Qt. peptone broth or solid medium of L. monocytogenes and closely several months to lead to successful Far more important than these and ELISA (monoclonal anlibody repair on tryptone soya yeast extract inhibitors. however, appears to be tests) 105 have become available to the use of indicator substrates, a further facilitate the detection of L. CONTINUED OVERLEAF Table 4: The strategy of Holistic Quantitative Risk Assessment (HQA) principle successfully used before in and subsequent risk management. the detection of Staph. aureus in foods.9Rotavirus c.50 termed RaPAMY-agar was used 6. The expenditure of risk management may not be trifling in some instances. However, it must be weighed against associated benefits successfully for the monitoring of . Observed once for a stationary phase population of Salm. senftenberg 775 W. such as (i) disease and death averted: (ii) avoiding undue financial foods which had been processed for t Ent faecium markedly more heat resistant than Ent. faecalis burdens to society at large; (iii) preventing unjustified anxiety amongst safety.' OI It determined whether the the public which is very hard to allay post-factum, to be achieved food contained less than the required Data from Donnelly et al 1987,]!' Bradshaw et al 1987. 122 Bunnin9 e/ al by expert risk communication. target value of IIstenae within 48 1988,61 Magnus et /1988.123 Mosse11988.44 D'Aoust et a/1988.'2 '------_hours. agar. 110113114 was found to repair. poultry and sh. cannot be decon­ 11 Gelhn. B G and Broome. C V (1989) e Lancet. II : 1022 91 Bannerman. E S and Bille. J (1988) mostly within 4-6 hours at 25°C. taminated effectively and consistently. J Amer Med Assoc . 261 : 50 Le GUlllou, M. (1980) Bull Soc, Vet Applied envlronm Mlcroblol . 54: 165 - 167 or the required technology for decon­ 1313 - 1320 France. 64 : 45-53 stressed L monocytogenes cells. to 12 Kerr, K. el a/ (1988) Lancet II: 37 - 38, 51 NICOlas. J.A and Vldaud, N. (1987). 92. CurtiS G.D W et al (1989) Letters the extent that they would be virtually tamination is rejected by the 1133 Gllbert.RJ etal (1989) Lancet. Rec Med Vet .. 163: 283-285 applied MlCrobloi 8: 95 - 98 44 70 totally recovered on or in the selective public then the policy with i: 363 - 364, Kaczmarski. E Band 52 Breuer, J and Prandl. O. (1988). Arch. 93 Van Nellen. P et a/ (1989) Proc. Tenth Internat Symp Llsteflosls. Pecz. diagnostic media ultimately used, respect to L. monocytogenes is not Jones, D M (1989) Lancel. I: 549 Levensm. Hyg .. 39: 28 - 30. 13 Gill. P (1988) J In/ecl/on . 17: 1 - 5 53 Weagan1. S.D et aI. (1988). J Food Hungary. Internal J Food Microbial . an unusual one. It should be identical Gray. M.L. (1963) Amer J Publ Protection. 51 : 655-657: Buchanan. 9: In press to the attempted management of " HeaJrh. 53: 554 - 563 R L et al (1989). Applied enVlfonm 94 Van Doorne. H. et al (1981). Anlonle Retrospect and prospects 15 Fenlon. D A (1985) J Applied Microbial .. 55: 599 - 603 van Leeuwenkoek. 47: 267 - 278 other serious lood-transmltt ed The clinical data presented amply Bactenol. . 59: 537 - 543 54 Schutz. G (1967) Monatsh. Vet Med.. 95 Rodnguez. LD et al (1984) Applied infections, like toxoplasmosis and 16 Rocourt. J and Seelrger, H P R (1985) 22: 766 - 768 enVlfonm. MICrobial.. 47: 1188 - 1190 justify the maxim that human trichinosis: the public should be ZbI Saki Hyg A, 259: 317 - 330. 55 Hayes, P.S. et al. (1986) Applied 96 Mossel, 0 A.A. (1986) Foodborne exposure to L. monocylogenes 17 De Vries. J and Stnkwerda. R (1956) envrronm MlcrobioJ.. 51 : 438-440. Microorgalllsms and Ihe" Toxms. persuaded to follow responsible eat­ should be reduced to the smallest Zbl Saki. Hyg. . I. Ong , 167: 56 Beckers. H.J. ef al. (1987). Internal. J. Developng Methodology., (ed Pierson. ing habits to protect itself against 229 - 232 Food Microbial. . 4: 249-256. M D and Sterne. N.J.) Marcel Dekker, possible extent. It follows that these listeriosis. Unfortunately. so far the 18 Gitter . M er al (1980) Vet Record, 57 Terplan. G. et al. (1986). Arch. 1- 22. bacteria. like other pathogens. have 107: 390-393 Lebensm Hyg.. 37: 129 - 1'56. 97 Ralovlch. B. et al. (1971), Zbl Bakt prospects of this approach are er al. Scl/welz Hyg. I. Ong .. 216: 88 - 91 no leg it imate place in foods, II!> 19 Seehger. H.P.R (1965), 58. De Boer. E. and Kuik. D. (1987), poor.1I6 The reasons for this are fairly A Pathol Bakl.. 28: 590 - 596. Nefherl, Milk Dairy J. . 44: 227 - 237. 98. Kampelmacher. E.H. and Van Ncorle The examples dealt with in the second obvious. Self control in eating and 20 Welshslmer. H.J. and Danker·Voe!. J 59 SIZffiur. K. and Walker. C,W. (1988). Jansen. L M (1972). Zbl Bakl I, Of/g. section illustrate that. as far as (1971) Applied Mlcroblol , 21 : Lancet. i: 1167 A. 221: 139 - 140. other matters of hfe style conflicts with processed foods are concerned. 516 - 519. 60 Lovell, J. et al. (l987). J. Food 99. Taylor. W.1. (1965). Amer J Glln both the classical phenomenon of 21 Wels, J. and Seellger. H.P.R (1975) Profectlon. 50: 188 - 192. Pathol. . 44: 471 -475 control of transmission of pathogenic reluctance to change established Applied Microbio/ . 30: 29 - 32 61 Bunning. V.K. et aI. (1988). Applied 100. Bailey. J.S et al. (1988) J Food risteriae is indeed well within reach , An 22 Kampelmacher. E.H, and Van Noorle enVlfonm. MICrobial.• 54: 364 - 370. Protecllon, 51: 391 - 396 eating habits and with the hedonistic 101 . VanNellen, P. etal. {1988) Internal J essential provision IS that recontam­ Jansen. L M. (1980) ZbI Bakt I, Of/g.. 62 Masse!. DAA. and Kampelmacher. altitude induced by the al/luence of 246: 211 - 227 E.H (1981). Lancet, I: 208. Food MICrobial.. 6: 187 - 198 ination of safe. freshly processed 44 most Western Societies. Con­ 23. Gray, M L (1960) J Nner Vet MOO 53 M"""', D.AA (1984), J. Food Sa/ely. 102. Van Netten. P. et al (1988) Letlers loads must prevented. ThiS recon­ Assoc.. 136: 205 - 208. 6: 89 - 104. appiledMlCrobtol., 7: 17-21 be sequently, as in the prevention of lung lamination may be caused by the 24 Blenden. DC and Sza!alowlcz. FT 64 Mossel. DAA. et aI. (1987). J Food 103 Mossel. DAA. et ai, (1983) J Applied cancer. traffic accidents and sexually (1967) J Amer Vet Assoc . 151 : ProtectIOn. 50: 894 - 895. Bacterroi . 54: 313 - 327 incoming raw matenals or the poorly transmitted diseases there is a long 1761-1766 65 Wilson. G.S (1933). Lancel. Ii: 104 Klinger . J 0 et al (1988) J Assoc. sanitised food plant and environment. NIColas. J.A et aI (1988) Rec. Moo 829 - 832 OIlIC Anal Cllem. 71: 669 - 673 way to go. Policies aiming at control 25 Experience in the dairy industry has Vel . 164: 203 - 206 66 Wilson. G S (1935) The 8acJ.enologrcal 105 Mal1lngly. J A el al (1988) J Assoc of food-Iransmilled liSleriosis shouJd 26 UrbaCh. H and Schabmskl. G (1955) Gradl/lg 01 Milk. Med. Research OIlIC. Anal Chern. 71 : 679 - 682 demonstrated that control of recon­ rely on information. education. Z Hyg Infektwnskrankh. 141 : Council Spec . Rep. Sec No. 206.. 106 Audurler. A et al (1979) Annis tamination below the order 1 cfu 1l7 239 - 248 HMSO. London. M lcroblol . 1308: 179 - 1B9 example and persuasion by teams 67 Amer McLauchlin, J et al (1988). Lancet. i: kg - 1 is almost Impossible to ensure, 73 27 Schlech . W F (1988) Food Technal .. Poner. M.E. et al. (1984). J. Moo. of fully independent unimpeachable 42 : (4) 176 - 178 Assoc .• 252: 2048 - 2052. 177-178 What can be attained and maintained profeSSionals having reached con­ 28 Kampelmacher . E.H and Van Noorle 68 Smulders, F.J,M. et al. (1986). J Food 107 Dallmler . A Wand Martin. S E (1988) by the best possible industrial Jansen. L M (1961) Wlen Tleraertzl Technal. . 21 : 419 - 436. Applied enVifonm Microbial. 54: sensus before going on publiC record. practices has to be determined in Monalschr. 48 : 442 - 448 69 Kampelmacher. E.H (1983), Food 581 - 582 14 Risk communication based on the 29 Seehger. H.P R (1987) Deut mOO/zm Techn .. 37: (4) 117 - 119: 169. 108. Golden. D.A et al. (1988), Applied each specific circumstance: More­ principle of addressing the com­ Wschr .. 112: 359 - 361 70 Mossel. D.AA (1987). Elimrnatlon of envrfonm. Microbial.. 54: 1451 - 1456: over. as illustrated in the third section. POlel. J (1955). Arch Hyg Bakterrol.. PathogeniC Organtsms from Meal and Food Microbial .. 5: 17 - 23. munity. in an atmosphere of respect 30 accurate monitoring techniques rely­ 139: 245-264 Poultry, (ed Smulders. F.J.M,) EIseY!er, 109. loven. J. (1988). Food TechnoJ" 42(4) ; and agreement. by the Public Health 31 Bottone, E J and Sierra. M F. (1977) po305 - 316. 172 - 175 ing on classical mlcrobiologlcal­ Professions provides the following MI Smal J Med. 44: 42 - 59 71 Stegeman, H (l988). Abstr. Tenth 110. Mossel. D.AA and Van NetJen. P analytical principles are available, In Marcy, G and Bockemuhl. J (1987) In/ernal Symp. VsteflQSfs. Pecz p.l 04. (1984) The ReVival oIlnJUled MICrobes. elements of control of food· transmitted 32 summary, there IS no less SCientific Deut Lebensm Rundschau, 83: 72 Karnpelmacher. E.H. (1985). Mill. Geb, (eds Andrew, M.H.E and Russell. listeriosis. (1) It Will allow the public to 219 - 220 Lebensm Unlersuch Hygiene. 76: AD) AcademiC Press, pp329 - 369 and technological knowledge In the protect Itself against the real risks of 33 Schlech, W.F et al (1983) New Engl 10 - 27 111 Munoa. F J and Pares. R (1988) area of food-borne listeriOSIs than J Med. 308: 203 - 206 Ho, J L et al 73 Massel, DAA (1987). Microbial. Applied enVlfonm. Mlcroblol.. 54: listeriosis while quelling unjustified there IS with any other food-trans­ (1986) Archs mternal Med . 146: AI,ments. Nutfltion. 5: 267 - 282. 1716 - 1718 concerns about perceived risks 520 - 524 74 Koek. PC et a/. (1983) Food MICro­ 112 Mossel, DAA etal (1980) J Applied milled pafhogen. fostered by cerlam newspaper Bannister. B.A (1987) J In/ecrlon. 15: biology Advances and Prospecrs, Bacterid . 49: 405 - 419 By contrast. foods of annnar as well 125 165 - 168. Roberts T A and Skinner. FA (Editors). 113 Speck, ML et al (1975). Applied accounts. (2) It wilt prompt the as vegetable ongln no/ subjected to 35 Llnnan. M.J et al (1988) New Eng! J London. AcademIC Press. pp.231 - MlCroblol" 29: 549 - 550 food industry to apply the wealth 01 Med.. 319: 823-828 240 114 Masse!. DAA el al (1985) Rapd heal processing present a continual Information on the management of 36 Schwartz. B et al (1988) Larcel. II: 75 Donnelly. C W. ef al. (1987) J. Food Methods and Automation m Micro­ potential fisk of Infection With L,stena 779 - 782 Protecl/on. 50: 14 - 17; 20. biology and Immunology. Habermehl. food-borne listeriosis as reviewed in monocylogenes and pOSSibly an 37 Beams. RE. and Gerald. KF (1958) 76 Twedt. R.M. (1986). J. Food Protection. K O . Edllor. Berlin. Sprlllger , this paper. thus maintaining a high Canad J Microbial . 4: 55 - 61 49: 849 Pp.605 - 607. occasional allied pathogen. 1Q As level of microbiological safety of food. 38 Conner. D.E et al (1986) Applied 77 Donnelly. CW and Briggs. E H. 115 I-iunltll , A.C. (1939) Fo.xJReseBJch. 4: discussed In the first section. thi S IS envrronm. Microbial. 52 : 59 - 63 (1986) J Food ProteellOn. 49: 531 -538 the which consumer is entitled to 39 Rocourt. J. et 'al. (1986) Schwerz 994 - 998 116. Benneu. J V et ai, (1987), The Surden likely to be less frequent than Infec­ 65. 115 126 expect.62 medezrn Wschr .. 116: 248 - 25 1 78 Norttlo11. M.D. et al. (1988) Nelherl Milk of Unnecessary Illness. (eo' Amler. RW. ti ons caused by salmonellae. 40 Massel, 0 AA and Orion . E F. (1979). Dairy J., 42: 207 -219. and Duft , H.B.) Oxtord Unlv. Press. campylobacters. yerslniae etc, but its Antonle van Leeuwenhoek, 45: 79 Black. R.E. et af. (1978). New Engl. J. Pp.l02 -1 14 . 117. Wil son, G.S. (1973). The MlCfobK)k)glcal medical Impact is markedly more 321 - 323 Med.. 298: 76 - 79 References 41 Warner. F. Edlror (1983) Risk 80 Rampling. A el al. (1 987) Lancet. II: Safety of Food. (ed. Hobbs. B C and seriOUS, These facts should not be • Assessment London Royal Society 1209 ChIlSl!an. J H B.) AcademIC Press . concealed from the public. Sub· Murray. EG D. el al (1926) J Pallial 42 Brackell RE. (1988) Food Technol . 81 Ryan, CA et al. (1987) J Amer. Med. pp.XI - )ut Bac/eflol . 29: 407 - 439 sequently. the support of the medical 42: (4) 162 - 164. 178 Assoc .• 258: 3269-3274. lIB. Walker. SJ and Silinger. M F (1987) 2 Gill, OA (1933) Veter J , 89 : 43 Doyle. M F (1988) Food Technol . 42: 82 Pflug. I.J (1987). J. Food Proteclion, J Applied Bacreflal. 63(6) xx and nursing professions has to be 258 - 270 (4) 169 - 171 50: 347 - 351 119. ElliOt. RP and MIChener. H D (1965) sought to launch an honest Informa­ 3 Spencer. J A 0 (t987) Bnl MOO J . 44 Mosse/, D A A and Thomas. G (1988). 83 Ryser, E.T. and Marth. E.H (1987), J U.S. Depl. Agncuh Tech. BuD.. no. 132 295 - 349 120 Rosenow. E.M. and Mooh. E H. (1987) lion campaign. ThiS should aim at MlCrobJo/oge. Aliments. Nutrrtlon. 6: Food Protection. 50 : 372-378. 11 Smyth . R Land Bamtord. M F M 289 - 309 Massel. D A A (1989) 84 Gray. ML et al. (1948) J Bac/enol.. J Food Pro/ocllon. 50: 452 - 459. 463 motivating the consumer to refram (1988) J /nlec/lon, 17: 65 - 70 Interr18t J Food MlCrobol . 9: In press. 55: 471 - 476. 121 . Rocourt. J et al. (1987). Internal J generally from the Ingestion of all raw 5 Carvajal. A and Fredeflksen. W (1988) 45 Nilsson, A. and Karlsson. K A (1959) 85 Donnelly. CW (1988). J Assoc. O/flC Systern Bac/errol.• 37: 266 - 270 Revs Infect Dtseases, 10: 616 - 623 122 Bradshaw. J G el al (1987) J Food foods unless they can be shown to Nord Vet Med . 11: 305 - 3 15 Anal Chern .. 71: 644 -646. 6 lvarsson. Seta/ (1977) Infecflon. 5 : 46 Grner. M (1976) Vet Record . 99: 336 86 Buchanan, RL et al. (1988). J . Assoc Protecllon. 50: 543 - 544, 556 have been effectively decontaminat­ 204 - 206 47 Plnl. P N and Gilbert. A J (1988) OIfIC. Anal Chem.. 71: 651-654. 123 Magnus. CA et al. (1988) Canad Insr ed. Proof of failure or success of 7 Nieman. R E. and Lober. B (1980) Inlernat J Food Microbial , 6: 87 Dalla, A R et al. (1988). J. Asscc. Offic Food SCI Tecflnol J.. 21: 209 - 212 Revs Infect Diseases. 2: 207 - 227 attaining 'absence' of L monocyto­ 3 17 - 326, Anal Chern .. 71 : 673-675. 124. D'Aoust J y , et al (1988) J. Dairy SCI . 8 Mahrwernl. R et al (1986) Eur J Clm 48 Skovgaard. N and Morgan. CA 88 Farbel. J M et al. (1988). J. Assoc. 71 : 3230 - 3236 genes In some raw food. e.g. ready­ Mlcroblo/ . 5: 169 - 17 1 (1988) Internat J Food Microbial . 6: Olfic. Anal Chern .. 71 : 656-678. 125. Kerr. K.G, and Lacey. RW. (1988), J to-eat raw vegetables. IS easy to 9 Stelma, G N el al (1987) J Clm 229 - 242. Skovgaard, N and NOfrung. 89 Lovell. J, (1988). J. Assoc. Olfic, Anal. Hasplallnfectlon, 12: 247 - 250. Mlcroblol , 25: 2085 - 2089 provide as illustrated In the third B (1989) Internal. J Food Microbial . Chern .. 71: 658-660. 126 Grithths. MW. (1989) J. Sci Food 10 Azadlan. B S et al (1989) Lancel. I: 8: 59 - 63 90. Ludlam. G.B. (1949). Monthly Bull. Min, Agrlcull.. 47: 133-158 section. Where raw foods like meat. 322 - 323 49 Breer. C and Schoplen. K (1988) Health London. 8: 15 - 20. The immunological approach to the detection of mycotoxins Maurice O. Moss, PhD , Department of Microbiology, University of Surrey, Guildford, Surrey, UK.

The mycotoxlns are a chemIcally 01 chemICal structure (Figure 1) whICh Problems of analysis acceptable methods have now been the routine monitoring of foods for diverse group of relatively small In turn presents problems for the Many countries have set maximum published for a wide range of foods. 4 their presence, might require the molecular weight metabolites analyst. Although tOXIC metabolites are tolerated levels In foods and animal A typical procedure will include analysis of many samples. In some produced by moulds capable of produced by a wide range of species feeds because of the known carClno· gflndlng and mixing of the sample. parts of the world. such as the Indian growing on Jood. an imal teeds or the of fungi. three genera are of particular geniC activity of aflatOXin 8 ,.3 These extraction with an appropriate solvent. and African continents, acute and raw materia ls used III the ir import ance in their association Wi th may be set as low as 15~g kg' In a cleanup stage to remove as many chronic poisoning by mycotoxins manu!acture. The presence of these tile mould spOilage of foods and some cou ntries and their enforcement COextractlves as possible. concentra- such as afl atoxin is a reality. In other compounds In tood can cause Illness, animal feeds:- Penicifllum. Aspergillus requires routine monitoring of foods tlon and. finally, analYSIS using a parts of the world. such as the North or even death I 2 In humans and and Fusanum (Table 1). It is not and feeds. The necessity to quantita- chromatographic process such as American continent. the presence 01 animals and tile symptoms may be as unusual for a sample of food to be tively and reprodUCibly detect such TLC. HPLC or GLC coupled with a aflatoxin in maize and ground nuts. or dIverse as liver damage. kidney contaminated with several diff erent low levels of metabolites In the quantitative detection method. A of deoxynivalenol in wheal and barley. damage. Irfllahon of membranes. the species of mould Simultaneously and complex matnces of foods requires skilled analyst would be hard pressed can have enormous economic can· disturbance of the nervous system or Figure 2 shows strains of Penicillium conSiderable skill from the analyst. In to complete the analysis of quite a sequences. Thus the total cost ariSing of the hormonal system controlhng aurantlogflseum and Aspergillus the case of aflatoxins the detailed small number of samples in a working from the contamination of maize by reproduction ThiS diverSity of flavus lsolat" rom a sample of methodology depends on the nature day. However. a satisfactory survey aflatoxin In the south eastern United biological aCllvlty reflects the diverSIty pistachiO nu of the food although Internatlonalfe for the occurrence of mycotoxlns, or States In 1980 has been estimated at a mass spectrometer, have made It Table 1: Mycotoxlns produced by Penicillium, Aspergillus and possible to detect 10-201l-g kg 1 with Fusarium a considerable confidence In specifi· city. However, such capllal intensive Species Mycotoxin LD" (mg kg" ) equipmenl cannot be widely available for the routine analysIs and screening of foods and animal feed s. Aspergillus flavus Aflatoxin B1 7.2 (male rat)

17.9 (female rat) Immunological techniques Mycotoxins are relal ivel y small Aspergillus versicolor Sterigmatocystin 120 (rat) molecular weight compounds and are not Ihemselves immunologically active Penicillium viridicatum Ochratoxin 20 (rat) but, after coupling them to a suitable macromolecule such as bovine serum Fusarium sporotrichioides T-2 toxin 4.0 (rat) albumin, they may present suitable epltopes to which specific antibodies Fusarium graminearum Zearalenone 5490 (rat) can be raised . Although the first applications of immunological methods for the study of mycotoxins were reported more than a decade ago, it is only during the last few years that such methods have become more widely available in the form of analytical kits manufactured on a commerCial scale. Aflatrem The methodology has been Tremorgenic o Aflatoxin 8 1 thoroughly reviewed recently by Figure 3: TLC 01 an extract 1rom a wheat sample contaminated with Carcinogenic steri gmatocystin, ochratoxin A and citrinin. Photograph (c) British Crown Chu6 who has provided a biblio· Copyright 1989. Courtesy of K Scudamore, ADAS Central SCience graphy for the development and Laboratories. application of both polyclonal and IotYCOTOXINS monoclonal antibodies. The first task meal, but It may be a nuisance when mice. Once suitable antibodies are ('y'yNHCO~ is to prepare an Immunologically the need is for a method to detect all available they can be used for rad iO V COOH Y0 act ive conjugate of the mycotoxi n to the afl atoxlns In a sample. The Im munoassays (R IA), enzyme linked be analysed to a protein or poly­ Ochratoxin CI trichothecenes are a large family of immunosorbent assays (ELISA). Nephrotoxic peptide carner and the manner in compounds which at first sight seem immunohistochemical studies of which the mycotoxin IS linked to the to be very simila r one to another. In mycotoxin residues in animal tissues Zearalenone carrier will have a profound effect on fact. an antibody raised to one and highly specific affinity column Oestrogenic the activity and spec ifiCity of any member of this particular family shows chromatography of mycotoxins CH, ~!O H O .. /O H antibody subsequently raised to It. very little cross reactivity with other extracted from complex loods. \ .' Some mycotoxlns. such as members 01 the family. These lH·C~·CO .o i OAe ochratoxin, already have a carboxyl molecules have a complex shape Radio immunoassay CH) C~.OAc group which can be reacted directly (Figure 5) which is very sensitive to By incubating the specific antibody T-2 Toxin with a carrier molecule, but the the nature of groups attached to with mixtures of the solut ion to be Cytotoxic majority need (0 be 10lned to the different parts of the molecu le and this analysed (or a known standard) and carner wrth a reactive hnker molecule. IS reflected In the remarkable a constant amount of radioactively The hnker molecule may be attached specifiCIty of monoclonal antibodies labeled mycotoxin it IS possible to Figure 1: The structures of a selection of mycotoxins and the biological to a mycotoxin at one of the several raised agaInst them Thus, a determine the concentration of toxin activity associated with them. Slles around the molecule, thus monoclonal antibody raised against In the unknown. ThiS requires the 3-acetyl deoxynlvalenol showed separation of the free and bound toxin negligible cross reactivity with and the determination of the amount deoxynivalenol, nivalenol or T-2 toxin. of radioactive label in each of these The development of an appropriate Iractions. Because of the need lor conjugate has to be done with radioactively labelled materials this considerable care and it may be method is not widely used especially necessary, for example. to use several In the food Industry although it IS disttnct linker molecules before one is possible to detect nanogram quantI· discovered which is not itself the site ties of mycotoxin of antibody recognition. and which presents the mycotoxin molecule In an Enzyme-linked Immunosorbent optimum configu ra tion for antibody Assay formation. Polyclonal antibodies can The sensitivity of ELISA methods is be raised in rabbits by a programme potentially 10- 100 times greater than of Immunisati on, using the conjugated RIA and the methodology is accept­ mycotoxin complex. in the traditional able in a wide range of medical and manner but moncelonals must be food analysis laboratories. Although produced by the formation and there are two distinct types of ELISA selection of hybridomas between the both are essentially competiti ve antibody produci ng spleen cells assays depending on the develop· produced after the im munisation ment 01 a colour reaction, the Intensity programme and myeloma cells, both of which is inversely related to the cell types usually being derived from concentration of toxin in the un known

Figure 2: Pistachio nuts contaminated with Aspergillus flavus and Penicillium aurantiogriseum. nearly 238 billion dOllars5and the toxicological test such as the one day exposing differe nt parts of the severe drought of 1987 in the US old chick test tor afl atoxin or the rabbit mycotoxin molecule against wh ich maize belt has given rise to consider­ skin test for trichothecenes. With the different antibodies may be formed. able problems of aflatoxin contamin­ huge improvement in extraction, Thus . an active derivative ot aflatoxin ation. In Iowa more than one third of cleaning and separation techniques 82 may be form ed through the the crop IS contaminated with levels for mycotoxins the need for biological pentenone function via oxime forma· greater than those permitted for assays Involving live animals for tlon leaVing the difuran ring system human consumption. AU of these specilic mycotoxlns has declined. exposed. or a succiniC aCid denvative pressures have produced the need High resolution two dimensional TLC may be formed through the hem I- for rapid, sensitive and highly specific IS very effective In the analysis of acetal leaving the pentenone end of methods for the analysis of mixtures of mycotoxins in complex the molecule exposed (Figure 4). mycotoxins. subst rates. Figure 3 shows the With the possibility of generating analysis of an extract from a sample of monoclonal ant ibodies the precise Traditional analytical methods wheat contaminated With sterigmato- derivative used Will determine the In the early days of mycotoxin work cyst in, och rat OXin A and citrinin and specificity of these antibodies to the there was little confidence in the demonstrates clearly how complex shape of a part of the mycotoxin specificity and sensitivity of physico· such extracts may be. The tri- molecule. Such specificity may be chemical methods and the chothecenes have always presented deSirable wh en developing an confirmation of the presence of a considerable difficulties tor the analytical method for a Single mycotoxin in a food or animal feed analysis but capillary gas chromato- mycotoxin, such as aflatOXin M 1 In Figure 4: Two m.~d S for linking aflatoxin Bl to a Protein for the depended on an appropriate graphy. especially when coupled ('_ milk or aflatOXin 8 1 in groundnut production of an' ically active conjugates. II

a)

• Mycotoxin y Rabbit Antibody Alkaline CJ phosphatase Goat anti- rabbit IgG

Hemocyanm b) ... " protein

Figure 6: Diagrammatic outline of (a) Direct and (b) Indirect ELISA ana lysis of mycotoxins.

sample. In the direct ELISA the be eluted subsequently with a solvent antibody IS bound to a solid surface such as aqueous methanol and such as a microliter plate. It is analysed by ELISA or phYSICO­ Figure 5: A computer generated model of deoxynivaleno1. Courtesy of D. Lewis, Department of Biochemistry, necessary to prepare a conjugate of chemical methods. The immuno­ University of Surrey. the mycotoxin to be analysed with the affinity column makes it possible to enzyme to be used for colour effect a considerable concentrat ion development, usually phosphatase or and clean up 01 a mycotoxin from a peroxidase The sample or standard complex matrix containing large IS mixed with the enzyme-li nked amounts of im purities wh ich would mycotoxin and the two compete for otherwise interfere in the analysis. It OSITIVE ••• antibody bound to fhe plate. After is ideall y suited to the rapid analysis washing soluble material away the of aflatoxin 8 1 , In extracts from quantity of enzyme bound to the commodities such as peanut butter. plate. which Will be Inversely and of afl atoxin M2 in milk. although proportional to the mycotoxin con· the methods are still being validated centration In the unknown sample, is by the use of collaborative trials.12 determined by adding its chromo­ In conclusion. there can be li ttle doubt geniC substrate (Figure 6a). that immunological methods are Although the In direct competitive going to be increasingly used for the ELISA (Figure 6b) seems more analysis and detection of mycoloxins. complex there are several advantages They will particularly have a role to to its use. In this method a mycotoxin play in situations where sensitivity and conjugate IS prepared using a protein specificity are required and where such as keyhole limpet hemocyanin precision is not critical. Such situations or a synthetic polypeptide such as include the analysis of large numbers polylysine. and it is this which is used of samples in screening programmes to coat the surfaces of the microtitre to determine the occurrence of plates. The plates are Incubated with mycotoxins in foods and animal leeds mixtures of a specific rabbit antibody on the one hand and in animal and and the unknown or standard human tissues, serum and milk solutions of mycotoxin. Again soluble samples on the other hand. matenal IS washed away and anyh antibody bound to the plate is allowed to react with goat anti rabbit IgG­ References enzyme complex and subsequent Smith. J.E. and Moss. M 0 (1985). colour development. The advantages Mycotoxlns. Formation. AnalySis and of the Indirect method are that SIgnificance .. Chichester. John Wi ley mycotoxin protein complexes can be 2. Marasas. W.F.O. and Nelson. P.E. (1987). Mycotoxlcology. In/roductlon to the prepared which are more stable than Mycology. Plalll Pa thology. Chemistry. m ycotoxin enzyme complexes. Toxicology and Pathology of Naturally Indeed. plates coated with mycotoxi n Occurring MycotOXIcoses In Animals and Man .. UniverSily Park. Pennsylvania State hemocyanin conjugate remain stable Unlverslly Press. for many months after preparation if 3. Schulter. P.L. . van Egmond. H P. and carefully stored. The goat anti· rabbit Stoloff. L. (1983). PrOCeedings 01 the International SympoSium on Mycotoxms .. IgG -enzyme complex IS usually Naguib. K.. Naguib. M.M .. Park. D.l. and commerCially ava ilable and this Pohland. A.E. (Eds.) Cano. Egypt. method IS very much less demanding (NIDOC) DOkkl. pp.ll 1 - 129 4 Wi ll iams. S. (Ed.). (1985) OffICIal Methoos on precIous specific mycotoxin of AnalySiS of the ASSOCIation of OffiCial antibody A full deSCription of the Analytical Chemists. Arl ington . VA . development and application of such A.O A.C. Chap. 26:. 5 Nichols. T. E. (1983), Aflatoxin and an assay for ochratOXin A IS given by Aspergillus flavus In Corn. Diener. U.L .. 7 Morgan et al. ELISA methods have AsqUith. R.L. and Dockens. JW (Eds.) also been developed for T-2 toxins. Alabama Agile. Exp1. Stalion. Alabama pp.67,71 ROrldln A9 . aflatoxin 8 10 . and 6 Chu. F.S (1985).MycoloxlnsandPIJyco, aflatOXin M i l . and those for the IOXInS Steyn. P.S. and Vleggaar. R aflatoxlns have been Incorporated Into (Eds.) AmSlerdam. ElseVier pp.277-292. 7 Morg.1n. M.R.A.. MCNerney. R. and commerCially available analytical kits. Chan. HW-S (1983). J Assoc Off Anal Chern .. 66' 1481 -1484. Immuno-affinity columns 8 Hunter. K.W. Jr el ai, (1985). Appl EnVifOn. Microblol.. 49: 168 - 172 Once an antibody specific to a 9 HaCk. R. . Martbauer. E and Terplan. G. particular mycotoxin is available it can (1988). Appl. EnViron Mlcroblol . 54 be immobilised onto an inert solid 2328 - 2330 10. Sun. T .. Wu . Y and Wu. S (1983) elm substrate. packed into a column. and J. Oneol.. 5 401 - 405 the column used to absorb mycotoxin 11 . Woychlk. N.A. Hlnsdili. A 0 and Chu. from a known volume of an extract, F.S. (1984) Appl EnVifon Microbial. 48 1096-1099. or a liquid such as milk. The 12. Mortimer. 0 N el al (1988) Fd Add mycotoxin bound 10 the column can Con/am . 5' 601 - 608