AFLA TOXINS IN MIXED FEEDS FOR RABBITS

Ten samples of mixed feeds and one of lucern (alfalfa) were analysed. They were supplied by processiJig plants from Santa Fe Province, Argentina, and designed for feeding rabbits. Several breeding farms had e/aimed that these lots were the possible cause of intoxication. Tlie degree and type of fungal contaminatipn and the presence of the following mycotoxins were ana~vsed: aflatoxins (.4FI), ochratoxin A, citrinin, penicillic acid, zearalenone (ZE4) , and trichothecenes T-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON). The total fungdl coun/ was carried out by the dilution plating method,on dichloran rose bengal chloramphenicol agar (DRBC). Isolation ofspecies lI'as pelformed by means of direct plating on moist chambers aswell as thefolloll'ing media: Aspergiltusflavus-parasiticus agar (.4 FPA), chlorO/l/phenicol-potato-dextrose agar (PDAC) and dichloran18%g~vcerol agar (DG18). Detection and quantiflcation of mycotoxins were pelformed by means of thin layer chromatography (TLL) . lt is no/ possible to assure tha! the intoxication cases observed by the breeders were caused by mycoloxins. However, the aflatoxins present in 7/ 11 sO/l/pIes and the high levels found in t1l'O of lhem (200 and 300 J1g/kg) could have been the reason.


SUMMARY.
Ten samples of mixed feeds and one of lucern (alfalfa) were analysed.They were supplied by proces-siJig plants from Santa Fe Province, Argentina, and designed for feeding rabbits.Several breeding farms had e/aimed that these lots were the possible cause of intoxication.Tlie degree and type of fungal contaminatipn and the presence of the following mycotoxins were ana~vsed:
Since their discovery and up to now, there have been few reports regarding field diseases associated to mycotoxins (20).The most characteristic case was the outbreak linked to aflatoxins, which killed 100,000 turkeys (" Turkey X Disease") in England, in 1960 (1).Sorne other cases were reported afterwards, related to field diseases induced by aflatoxins in ducklings, swine, calves, dogs and trout (38).In European countries there were nephropaties in swine, inducedbyochratoxinA (19,39).Outbreaks linked to T -2 toxin in poultry, horses, livestock and swine, and emetic syndrome in swine associated to DON were reported in USA (3 9).Estrogenic syndrome in swine and fertility reduction in livestock due to exposure to zearalenone were reported from Europe,USA and Australia (19).
Considering aH that has been stated above, rabbit feeds manufactured in Santa Fe, Argentina, were mycotoxicologically analysed.The animals which had been affected, presented hepatic symptoms and a decrease of weight gain.Aflatoxins, ochratoxin A, citrinin, penicillic acid, ZEA, T -2 .toxin, DAS and DON were determined in the samples.The mycota present was quantitatively tested and the isolated fungi were identified as regards genus and species.Finally, an association between the potentially toxigenic species and the presence ofmycotoxins in the analysed samples was attempted.

MATERIALS AND METHODS
Ten samples of mixed rabbit feeds and one of dried ground luce m (alfalfa), used as an ingredient, were analysed.Every feeds corresponded to prestarter feed, starter feed, fattening feed and final feed.The samples.\Vere supplied by processing plants from Santa Fe, 116 Argentina, immediately after manufacturing and marketing, from April to ' October 1990.They were processed for mycotoxicological analysis between the second and the tenth days following their reception at the laboratory.They were kept at O°C during that period, hermetically, to avoid humidity reabsorption.a. Sample preparation for mycological analysis: a.l.Sample preparation for dilution plating method: Portions of 25 g were aseptically taken from each ofthe 500/800 g total weight samples.The first dilution was prepared in 225 mI ofO.l % aqueous pepto-ne (vI v),shaked in a stomacher for 2 minutes at room temperature.Successive dilutions were obtained from this first one, in the same diluent.Aliquots of 0.2 mI wete poured, in duplicate, onto the surface of the culture mediurn respectively, so as to be counted and isolated (4,17,18).a.2.Sample preparation for direct plating: 25 g portions of the products were sterilized on surface for 2 minutes by means of 10% cornmercial chlorine bleaching agent and washed several times with sterilized -distiHed water.Sterilized absorbent paper was used to dry the material before pouring it on sterile moistchambers (twolayer strata of cotton + filter paper) .

RESULTS
Table 1, shows the outcome of lhe mycological analysis.Fungal counts range from 9.0 x102 to 3 x104 CFU/g.Four samples contained a wider diversity of species, some of them considered "field species" (Acremo,'¡um strictum, Epicoccum purpllrascens, Cladosporium c1adosporioides, Phoma eupyrena, Mucor sp.).Aspergilllls flavus was isolated from a11 samples, including the one of dry ground lucern (alfalfa).c.Mycotoxin analysis: .Goliñsky et al. multi-method (lO) was employed to analyse ochratoxin A, citrinin and penicillic acid.For aflatoxins and ZEA, the BF modified toluene technique was used, according to the Argentinian IRAM standards (24).To analyse trichothecenes techniques employed were that ofKamimura et al. (15) and that ofTrucksess et al. for DON in particular (35).Toxin detection and confirmation was carried out by thin layer chromatography: quantification being made by means of visual corilparison agai nst the corresponding reference standards.
From ad the analised toxins, onl)' AFI B 1 and AFI B2 were detected.Table 2. shows the results of this analysis.Four out of eleven samples were free from aflatoxins in detectable levels.Two samples contained both AFI B 1 and AFI B2 in levels ranging from traces to 8 ¡.tglkg.Two samples contained AFI B 1 in a high level: 200 and 300 ¡.tglk~.Polland (1982 and1985) led.scientists to set Up warnings regarding fungal counts in mixed animal feeds of 100.000CFU/g (4).This figure is taken into account when it referskeeping quality in the commercial sense.However. it can be concluded that when figures represent fungal species with healih risk for animals.theyare also linked to the biological and nutritional quality of the feeds .The samples examined gave maximum CQunts of 10 4 CFU/g.being the level below the standard ofreference.but in all of them species of recognized potential toxigenicity .such as Aspergillus and Penicillium species were detected and 118 identified (5 . 9. 11).The only association observed between toxigenic species and their toxins was the simultaneous presence ofA.flavus andAFI B 1 and AFI B2 in 7 and 2 out of 11 samples, respectively.There is a variety of toxic secondary metabolites that can be synthetized in lab cultures by many ofthe species from the mentioned genera aboye (2. 5, 14, 21, 31 , 39).However, a precise prediction of their behaviour in food and feeds is not possible.This is due to thefact that these are complex ecosystems influenced by a number of factors namely substratum matricial effect, fungal metabolite interaction (including their synergistic and antagonistic effects) among them and with other organisms, environmental factors (temperature.water activity, CO/0 2 , pH) and the external agents such as pesticides (3, 6. 22 . 32) .
The presence of AFI B 1 alone, or AFI B 1 and B2 in feeds could have caused the symptoms observed in• the animals, even when these toxins had been found in trace levels.since rabbits are one ofthe most sensitive animal.It has been observed that rabbits and ducklings are the most sensitive 10 the action of aflatoxins.",ith an oral LD50 of only 0.3 mg/kg body weight for AFI B 1 (20,38).
Taking into account that mycotoxins -especialIy aflatoxins-can be heterogeneously distributed in feed matrixes (13). it is possible to state the hypothesis that suspicious feeds (those in which aflatoxins ",ere not detected) could ha\'e been not conveniently sampled and that the samples taken foi the analysis were not representative of the portio n eaten by the animals.When food orfeed iskept in a wrong \Vay .m~inly under .humidityand temperature conditions thatfa~(>ur fungal .development and the production of mycotoxins.the latter can develop in certain points and not in the total volume of the 101.Sorne years ago it was proved that these toxins can be found in the fields.",hen A. flavus in\'ades peanuL cottonseeds and corn under certain conditions (6, 18.22.34).The bibliography indicates tha!. even in those products which undergo intensive mixing.the distribution of aflatoxins is heterogeneous enough to allow variations in the contamination levels reach two orders between t\Vo different portions ofthe same lot (25).The presence of A. flavus in all ofthe samples place s them in the category of "suspicious feeds ••.Nowadays.over 40 countries -including the EEC-have .currentor proposed legislatioon for the regulation of aflatoxins in animal feeds.S.everál countries (mainl)' those which need to import raw material or manufactured products) have quite high tolerance levels.These values are almost exclusively applied to certain ingredients of mixed feeds tha!. in turno musl be mixed so as to obtain the final product.Thus.an important reduction ofthe initial contamination 15 achieved (from peanul.soya and cotton seeds byproducts.mainly) .The type and age ofthe animals to be fed are also taken into account.In China, the maximum tolerance is of 1 ,000 f.lg/kgfor total aflatoxins in feedstuff ingredient~, with a maximum of 4% in final products.In Japan, the maximum toIeranceis of 1 ,000 f.lg/kg of Ail B 1 for import peanut meal (maximum : 2 to 4% of this material in final products).TheEEC acceptsa maximum of200 f.lg/kg of AFI B 1 in certain ingredients.France and Senegal aBo\\' 300 f.lg/kg of AFI B 1 in general ingredients and peanut based feedstuffs respectively.Nevertheless.most couritrie5 have set a maximu111 tolerance level ranging from 10 to 50 f.lg/kgmfor bolh.AFI B l and the lOlal aflatoxins in fina l producls.exceptions being not higher than !OO f.lg/kg (33 . 37).From alllhat has been stated above.it is considered lhalAFl B 1 values of200 f.lg/ kg in prestarter feed and 300 f.lg/kg in fattening feed detected in this work are alarming levels.taking into accOllllt the fact that they are final products for animal comsumption, especialIy when these animals are highly susceptible as rabbits are.