Aflatoxin in Food: What Role for Biotechnology?

OFAB 38 JUNE 2010 Aflatoxin in Food: What Role for Biotechnology? Dr Maina began his talk by thanking the OFAB-PC for an opportunity to invite him at the forum as a guest speaker. He added that it was a privilege for him to share with the OFAB fraternity on such a timely topic. He pointed out that Aflatoxin should be recognized as a threat to food security in Kenya which is why it is important to understand it and eventually eliminate it from our food. Causes of Aflatoxin With the help of micrographs and pictures, Dr Maina revealed to the audience that Aflatoxin is caused by species of Aspergillus such as Aspergillus flavus and Aspergillus parasiticus. He added that Aflatoxins can be identified in colonized corn kernels by viewing in UV light. Definition Dr Maina defined Aflatoxin as a type of Mycotoxins. Mycotoxins are naturally occurring secondary toxic fungal metabolites that contaminate various agricultural products, either pre- or post- harvest. A world wide problem Mycotoxin contamination of foods and animal feedstuff is a worldwide problem, in such an extent that the Food and Agriculture Organization (FAO) of the United Nations has estimated 25% of the world s food crops and a higher percentage of the world s animal feedstuffs to be significantly contaminated by mycotoxins and thus represent risk to human safety. In sub-saharan Africa, the aflatoxin, fumonisin and ochratoxin mycotoxins are the most common, economically important mycotoxins. Health hazards for humans and domesticated animals due to both chronic and acute toxicological manifestations of Aflatoxin have been reported in this part of the world our countries. What are AFlatoxins? Aflatoxins are polyketide-derived, toxic, and carcinogenic secondary metabolites produced primarily by two fungal species, Aspergillus flavus and A. parasiticus, on crops such as maize, peanuts, groundnuts, cottonseed, and treenuts. At least 13 different types of aflatoxin are produced in nature; Aflatoxin B1 is considered the most toxic and is produced by both Aspergillus flavus and Aspergillus parasiticus whereas Aflatoxin G1 and G2 are produced exclusively by A. parasiticus. Types of Alfatoxins Aflatoxin B1 & B2: produced by Aspergillus flavus and A. parasiticus. Aflatoxin G1 & G2: produced by Aspergillus parasiticus. Aflatoxin M1: metabolite of aflatoxin B1 in humans and animals Aflatoxin M2: metabolite of aflatoxin B2 in milk of cattle fed on contaminated foods Aflatoxin Toxicity 1

Toxicological manifestation: include cancer, mutagenicity, birth defects (teratogenic) and estrogenic, gastrointestinal, urogenital, vascular, kidney and nervous system disorders. The compromised immune response that accompanies chronic exposure to some mycotoxins reduces resistance to infectious disease and may be the most important and the most underappreciated of the health problems associated with mycotoxins Toxicological effects on children include reduction of growth. Development of children exposed to aflatoxins may reduce quality of life and limit an individual s ability to reach their potential. Aflatoxin Pathogenicity Dr Maina discussed some of the diseases inflicted by Aspergillus. Toxicological manifestations inflicted by the fungus include cancer, birth defects, gastrointestinal, urogenital, vascular, kidney and nervous system disorders. He affirmed that the compromised immune response that accompanies chronic exposure to some mycotoxins reduces resistance to other infectious diseases as well. He was of the opinion that the later is one pathogenicity aspect which may be the most important and the most underappreciated of the health problems associated with mycotoxins. Exposing some of the strains responsible to cause illnesses, Dr Maina listed A. flavus as second only to Aspergillus fumigatus among Aspergillosis strains causing Aspergillosis. He added A. flavus as the most frequently reported Aspergillus species causing keratitis, an inflammation of the eye cornea. Detection of Aflatoxigenic fungi Dr Maina shared the methods used so far in detecting Aflatoxigenic fungi, including the use of UV light, Polymerase Chain Reaction, Transcription Polymerase chain reaction and Multiple Transcription Polymerase chain reaction Detection of Mycotoxigenic Fungi by Fluorescence under UV light Aflatoxins can be identified in colonized corn kernels by viewing under UV light. However, De Maina pointed out that the detection of Aflatoxins as judged by fluorescence of fungal colonies is not conclusive, in that non-aflatoxigenic strains of Aspergilli, such as A. parasiticus and A. niger fluoresce also under UV. This could lead to wrong interpretation and conclusion that there is presence of Aflatoxins. Further diagnostic test for the presence of Aflatoxins in fungal colonies can be determined by observation of continued phosphorescence of Aflatoxins at room temperature, after switching off the UV light, the fluorescence will last for 0.5 seconds. Without the UV light, Non-Aflatoxigenic Aspergilli do not phosphoresce. With the help of pictures, Dr Maina took time to illustrate, by photos, the difference between the appearance of non-aflatoxigenic and Aflatoxigenic colonies under UV light and after switching off the UV lamp. Detection of Mycotoxigenic Fungi by Polymerase Chain Reaction Another way to detect mycotoxins was found to be by Polymerase Chain Reaction (PCR). 2

This is possible since at least 25 genes are involved in the biosynthesis of Aflatoxins and its regulation. Primers pertaining to sequences of afl-2, afld, aflm and aflp, (apa-2, nor-2, ver-2, omt-2, respectively) have been used to detect and identify aflatoxigenic strains of A. flavus and A. parasiticus among isolated colonies or in DNA extracts from food and feedstuff. Briefly, DNA of Aspergilli is used as template for the amplification of genes involved in Aflatoxin biosynthesis. Sequencing of the amplified fragments therefore will confirm the identity of aflatoxin biosynthetic genes. However, the mere presence of the genes reflects only the potential of the fungus to produce aflatoxins. Application of Reverse Transcription Polymerase chain reaction Application of Reverse Transcription Polymerase chain reaction (RT PCR) for the characterization of aflatoxigenic Aspergilli relies on the presence of mrnas pertaining to Aflatoxin biosynthesis genes. RT PCR is indicative of the presence of the aflatoxigenic fungus and of the Aflatoxin biosynthetic enzymes Application of Multiple Transcription Polymerase chain reaction Multiplex RT PCR containing 4 5 primer pairs of various combinations of afld, aflo, aflp, aflq, aflr and afls (aflj) are used to detect toxigenic fungi. Non-aflatoxigenic strains lack one or some Aflatoxin biosynthesis genes and their mrna products Regulation of Aflatoxin Biosynthesis Determination of the molecular basis for the phenomenon of non-production of aflatoxin in certain members of the A. flavus group of the fungi with a view leads to a better understanding of the global regulation of toxin synthesis and the convergent evolution of aflatoxin biosynthesis. Gene Characterization The characterization of genes of the aflatoxin biosynthetic pathway has provided a foundation for a genomic investigation aimed at understanding the biochemical function and genetic regulation of aflatoxin biosynthesis Aflatoxin Metabolic Pathway Aflatoxins are synthesized by a polyketide metabolic pathway. Mapping of overlapping cosmid clones of A. parasiticus and A. flavus genomic DNA has established that the genes in the aflatoxin biosynthetic pathway are clustered. In general, the aflatoxin gene cluster in A. parasiticus and A. flavus consists of 25 genes spanning approximately 70 kb Role of biotech in controlling Aflatoxin contamination Gene Cluster With the characterization of the gene cluster, new insights into the cellular processes that govern the genes involved in aflatoxin biosynthesis have been revealed, but the signaling processes that turn on aflatoxin biosynthesis during fungal contamination of crops are still not well understood. Exploiting the Aflatoxin biosynthetic pathway The fully characterized aflatoxin biosynthetic pathway and gene cluster comprising genes that govern this pathway, including the key regulatory gene (aflr), provides a sound basis for studies 3

of the efficacy and mode of action of putative pathway-blocking compounds (e.g. Tex6 100 kda protein) Plants natural products effect Regulation of the genes clusters during invasion of the plant host is also investigated using a genomics approach. This approach is based on the fact that certain plant-derived natural products apparently have regulatory effects on aflatoxin biosynthesis. Successful inhibition of aflatoxin elaboration therefore, may require not only the action of antifungal compounds, but of compounds that block biosynthesis of toxins as well. Enhancement of host resistance to aflatoxin contamination of maize Host resistance is the most widely explored strategy for eliminating aflatoxin contamination by Aspergillus flavus. Breeding strategies for developing resistant maize germplasm have been enhanced by developing new screening tools for field inoculation and for laboratory screening Resistance-associated proteins Restriction Fragment Length Polymorphism (RFLP) analysis of maize populations has highlighted the possibility that different resistance traits can be successfully pyramided into agronomically useful germplasm, while proteomics has impacted the identification of proteins associated with resistance-associated proteins (RAPs) Development of resistant germplasm Control of aflatoxin contamination of maize will likely be dependent upon the development and introduction into the commercial market, of germplasm, resistant to the growth of aflatoxigenic species, and/or biosynthesis of toxins by these species The identification of chromosomal regions as well as proteins and their corresponding genes associated with resistance, and the subsequent confirmation of their role in resistance using biotechnological tools available such as RFLP analysis or RNAi should provide an efficient means for the development of this germplasm. However, to derive commercial benefit from this resistance and develop lines that can aid growers, markers need to be identified to facilitate the transfer of resistance into commercially useful genetic backgrounds without transfer of unwanted traits. Limiting the growth of aflatoxigenic fungi might at times not be enough to maintain Aflatoxins at acceptable levels in maize crops. Therefore, the identification of compounds that block aflatoxin biosynthesis may represent the magic bullet needed to insure resistance. Thus, the identification of resistance traits in maize can, through marker-assisted breeding, facilitate a more rapid development of resistant, commercially-useful germplasm. Conclusion Dr Maina concluded by saying that Biotechnology tools have provided researchers with a rapid and effective method for identification of genes potentially involved in aflatoxin formation and infection of crops by A. flavus 4

In addition, with the availability of the A. oryzae whole genome sequence a close relative of A. flavus, which is used in industrial fermentation for enzyme production without producing Aflatoxins, and of A. fumigatus, a human pathogen and a non-aflatoxin producer will be very helpful to identify genes specifically used by A. flavus for aflatoxin formation, for fungal invasion of crops and for fungal survival in the field through comparative genomics. He was of the opinion therefore that using close relatives of a.flavus could help in mitigating Aflatoxin danger. He urged everybody to join hands and provide solutions to our families, societies and country. He suggested that the Kenya Bureau of Standards (KEBS), Kenya Plant Health Inspectorate Services (KEPHIS) and other regulatory agencies could use indigenous verities and explore if they can provide tools to induce resistance. Open discussions Comments: 1. Thank you for your good and timely presentation. I am from East African Grain Council (EAGC). Recently I attended a meeting where one of the recommendations was to form a national working group among those involved in fighting Aflatoxin. We took this resolution because we noticed that there are a number of disjointed efforts to tackle the problem. In order to achieve a concerted effort, I am encouraging you to get in touch with that committee because what is presented here is one option among many. A larger consortium of researchers can contribute mote to find solutions. 2. At the World Food Program we believe that scientists should join efforts. Reports have confirmed that there exist several approaches to fight Aflatoxin. It would be useful for scientists to link up and see what is being done and the various possibilities. Questions: 1. Question: Thank you for a good presentation. My question is not directly related to it but I would like to know why it is taking too long to get drought resistant maize? Answer: I would say that researchers have done much work not only for drought resistant maize but in other crops also. However, that is not enough. For example, to complete an invention every scientist needs a policy so that what he invents is protected. The NBA regulations which will cater for that need are not yet out. 2. Question: How are you positioned to link up with others who are fighting the same problem? Answer: I agree that we have to join efforts. JKUAT institution policy states that we have to link up with other research programs in order to get sustained support from the institution. From our side, we were tasked to come up with a policy document that brings together actors and restructures efforts towards fighting Aflatoxin. 3. Question: In Kenya it has been reported historically that he Aflatoxin is concentrated in Ukambani (Eastern province). Does it have anything to do with the soil? What is the reason? I would suggest that scientists link up with extension services to find a solution for the area. 5

Answer: In my view the problem is not only in Eastern province alone. However, the reason for such a situation might be the fact that donor agencies such as Red Cross supply large quantities of cereals in Eastern province while the province has no proper systems to handle storage of seeds. In Rift Valley where seed storage systems are working quite well, farmers don t know where to take their maize since they have harvested much surplus this season. This allows thinking that Aflatoxin is also extending in other areas of the country. 4. Question: It was found out that private companies find high levels of Aflatoxin in food and feed and yet national regulatory agencies find less. Also, other than maize, contamination by Aflatoxin has been reported in sorghum in Rift Valley province. Could you please comment on this? Answer: It is of concern that most of the products on the supermarket s shelves might be infected by Aflatoxin. It s not only in maize or in sorghum as you said. For detection, we should recognize that in our countries we have limited resources and we use outdated machinery for detection. KEBS and other regulatory agencies need more support to improve standards of control such as the ones used in Europe. Contribution from Dr Charity Mutegi, KARI-ICRISAT: On partnership: I think that this forum can help us to get linkages and work together. At KARI for example, we have a mandate to research but we are to link up with others. On Concentration of Aflatoxin in Eastern Province: Aspegillus is believed to be prevalent in Eastern.In addition, media reports point out that even in Kitale and Bungoma are affected but larger number of fatalities are reported more in eastern. However, we should bear in mind that if Aflatoxins leading to Acute Poisoning or death is reported more, while it is important to know that Chronic Poisoning which does not show directly is another reality. In addition to that, it is important to know that aflatoxin does not occur in isolation. It combines with others mycotoxins, whereby the need therefore, as researchers, to look at mycotoxins holistically. On Aflatoxin detection: Monitoring and regulation is a big problem in our country because most of the agriculturalists are small scale farmers, who do not necessarily come into contact with regulatory agencies so that their crops can be tested. This implies that KEBS, for example, tests a very small percentage of the national crop. The porosity of our borders with other countries also implies that the origin the Aflatoxin infection cannot be attributed solely to Kenyan crops. During crop testing, in research, expensive methods are used. The local regulatory agencies cannot use the same for routine tests. They mostly use rapid tests which allow high tolerance levels of aflatoxin. 6