Mycotoxins
Estimating the social costs of the impacts of fungi aflatoxins in south east Asia
The program on mycotoxins and experimental carcinogenesis (PROMEC) of the South African medical research council
Aflatoxin, kwashiorkor, and morbidity
Summary of data on aflatoxin exposure in west Africa
The occurrence of exposure to aflatoxins and to hepatitis B and C viruses in Guinea
Breeding for resistance in cereals to prevent Pre- and Post-Harvest toxin contamination
Fungal ecology and potential biological control of Aspergillus flavus
Fungal infection and mycotoxins in maize in the different agro-ecological zones of Benin and Nigeria, west Africa
Possible utilization of plant products in grain storage
Mycotoxins in maize and fermented maize products southern Ghana
J. D. Miller
Agriculture and Agri-Food Canada
Mycotoxin problems are not specific to the developed or developing world; these are problems that affect the agricultural economies of many countries, interfere or even prevent trade, reduce animal and animal product production and in some countries affect human health.
Between 25% and 50% of all commodities, especially staple crops, are contaminated with mycotoxins (Bhat & Miller, 1991; Mannon & Johnston, 1985). There are five agriculturally-significant mycotoxins worldwide: aflatoxin, deoxynivalenol, zearalenone, ochratoxin and fumonisin. Contamination of food and feed is well documented in most economically-developed countries where tens of thousands of analyses are conducted per year. In Asia, Africa and South America, all of the analyses in the open literature do not total 1,000 (Beardall & Miller, 1994a; IARC, 1993; Jelinek et al., 1989). It is important to note that mycotoxins in commodities in the USA, Canada, and the European Economic Community create significant losses in animal production in the tens of billions of dollars per year (CAST, 1989). This is manifested in terms of reduced meat (pork, chicken) production, reduced milk and egg output, increased disease and lowered reproduction rates of herds or flocks. In addition, the application of different aflatoxin tolerances between the USA, the EEC, and other countries has a potential impact on trade especially of maize, copra and groundnuts. In Thailand, Indonesia, and the Philippines economic losses due to aflatoxin have been estimated at about US$ 400 million per year (Lubulwa & Davis, 1994).
In Africa, there is ample evidence of the direct effects of mycotoxins on human health, in that aflatoxin exposure results in increased incidence of liver cancer and is a potent synergist of hepatitis B (IARC, 1993). Aflatoxin has been reported as causing acute toxicosis in Africa (Maracas, 1988). Increasingly, the immunosuppressive properties of mycotoxins including aflatoxin and deoxynivalenol are being recognized. These properties are part of the basis for guidelines in the US and Canada on deoxynivalenol in grains. It is argued that the regulations on aflatoxin in these two countries are needed to minimize immunological consequences (Pestka & Bondy, 1994).
In Africa, there is ample evidence of the negative impact on trade of mycotoxins, particularly with respect to trade with the EEC. Imports of peanut meal by the EEC has declined by 50% from 1980 to 1990 due primarily to the stringent regulation of aflatoxin (Bhat, 1991). Although heavy emphasis has been placed on the trade aspects of mycotoxin contamination of commodities, this may need to change. The rise of regional trading blocks and the gradual elimination of subsidies will likely result in the large grain-growing countries dominating the market place after the turn of the century. The losses in animal production in domestic markets as well as the effects on human health justify stricter management of mycotoxin contamination. High quality domestic produce will assure entry into regional market opportunities.
The application of existing technologies to manage post-harvest contamination, plus attention to the location, agronomy and germ plasm used in staple crop production is required to reduce the risk of mycotoxin contamination and thus enable agriculture to meet regulatory targets.
Mycotoxins And Human Health
In the recent World Development Report, Investing in Health (Oxford University Press, 1993), the World Bank has discussed the costs of disease to economies. This refers to the direct costs and the much larger indirect costs of excess disease, disability and mortality expressed in terms of Disability Adjusted Life Years (DALY). Diseases were classified into more than 100 categories covering almost all causes of death and disability. Using mortality data where available, all causes of death were assigned by sex and age. For each death, the number of years of life lost was defined as the difference between age of death and life expectancy. These data were factored so as to illustrate the impact of each death in relation to age at death. Sub-Saharan Africa has the highest burden of disease of any of the regions examined. The contribution of mycotoxins in excess morbidity and mortality is not known but it is probably large primarily based on the immuno-toxicity of the principal mycotoxins and increased cancers. The economic analysis of Lubulwa & Davis (1994) suggested that most of the loss was in terms of effects on human health.
Discovered in the early 1960's, most is known about the human health effects of aflatoxin. It is a potent liver carcinogen in humans that synergizes other liver carcinogens including hepatitis B (IARC, 1993; Kuiper-Goodman, 1994). An important feature of aflatoxin-modulated liver carcinogenicity is that young men get this cancer. This would make a proportionately higher contribution to the DALY measure than, for example, lung cancer which generally occurs later in life. Aflatoxin exposure is high in most of the developing world. The determination of aflatoxin-adducts in human tissues has made possible precise exposure measurements. Such data from several countries are presented in Table 1 (adapted from IARC, 1993).
Aflatoxin is immuno-modulatory in domestic and laboratory animals with oral exposures in the ppm range. Cell-mediated immunity is more affected than humoral immunity (Pestka & Bondy, 1994). Children suffering protein energy malnutrition (PEM) in developing countries are also exposed to aflatoxin. In a study conducted in southern Africa it was found that aflatoxin-adducts were higher in PEM than in control children. Aflatoxin metabolism was affected with relatively higher serum concentrations in PEM children. A second study compared PEM children with high and low aflatoxin adduct concentrations. The aflatoxin-adduct positive group of PEM children showed a significantly lower hemoglobin level (p= 0.02), longer duration of edema (p=0.05), an increased number of infections (p=0.03) and a longer duration of hospital stay (p=0.008) (Adhikari et al., 1994).
Table 1 Aflatoxin Adducts from Human Sera
|
Country |
Subjects |
Subjects with aflatoxin adducts (pg B1-lysine/mg albumin) |
||||||
|
<5 |
5-25 |
26-50 |
51-75 |
76-100 |
>100 |
|||
|
Gambia |
||||||||
|
|
May |
323 |
7 |
53 |
76 |
49 |
40 |
98 |
|
|
November |
67 |
0 |
39 |
13 |
7 |
3 |
5 |
|
Senegal |
29 |
0 |
20 |
6 |
2 |
1 |
0 |
|
|
Kenya |
91 |
48 |
26 |
5 |
1 |
5 |
6 |
|
|
China |
||||||||
|
|
Guangxi |
93 |
28 |
35 |
13 |
6 |
2 |
9 |
|
|
Shandong |
69 |
69 |
0 |
0 |
0 |
0 |
0 |
|
Thailand |
84 |
73 |
10 |
1 |
0 |
0 |
0 |
|
Revision to the current regulation for aflatoxin in the USA also takes into account its immuno-toxicity, particularly in high risk groups (Toxicology Forum, 1994). A partial summary of the burden of disease from the World Bank report is shown in Table 2 (adapted from an IDRC summary). This reveals that about 40% of the DALYs lost in developing countries are due to diseases modulated by mycotoxins. As noted above, there are little systematic data on mycotoxins in Africa. Aside from aflatoxin, fumonisin appears to be widely distributed in maize (Beardall & Miller, 1994b; Doko et al., 1995; Viljoen et al., 1993).
Table 2 DALYs Lost in Developing Countries
|
Infectious Diseases |
|
Cancer |
Other |
|
|
respiratory infection |
117 |
53 |
mental illness |
68 |
|
diarrheal diseases |
108 |
|
maternity |
29 |
|
tuberculosis |
46 |
|
car accidents |
26 |
|
measles |
34 |
|
IAQ+AQ |
33 |
|
HIV |
28 |
|
tobacco |
19 |
|
DPT |
28 |
|
parasites |
32 |
|
STD |
19 |
|
respiratory |
16 |
|
polio |
5 |
|
anemia |
13 |
|
hepatitis |
2 |
|
malnutrition |
39 |
|
leprosy |
1 |
|
alcohol |
9 |
Agriculturally-Important Mycotoxins
Although there are many compounds given the label "mycotoxin", there are only four agriculturally-important fungal toxins in developing countries: deoxynivalenol (replaced in some areas by nivalenol), zearalenone, fumonisin and aflatoxin. This is based on extensive analytical results (summarized in IARC, 1993) and very detailed information on the distribution of fungi in staple crops. The fifth agriculturally-important mycotoxin, ochratoxin, is a problem in Europe. It is now recognized that the co-occurrence of mycotoxins is a substantial problem in food and feed safety. Mixtures of toxins in foods and feeds is the rule rather than the exception. This must be considered in relation to the following information (Miller, 1991; 1993).
The mycotoxins mentioned above are produced by a number of fungi (table 3). Some of these are plant pathogens, some are storage fungi. Some toxins (e.g. fumonisin, deoxynivalenol) are only produced in the field. Some (e.g. aflatoxin) are produced in the field and in storage. Deoxynivalenol is probably the most widely distributed mycotoxin in food and feed. It occurs virtually where ever cereals are grown, with the exception of dryland wheat production in Australia, Canada, and other similar areas (IARC, 1993). The domestic animals most affected by deoxynivalenol are pigs. Acute toxicosis is manifested as intestinal disorders and emesis. However, deoxynivalenol seldom causes acute toxicity in pigs because its presence in feed limits consumption. This anorexic effect typically results in decreased feed consumption and growth in pigs at concentrations of more than 1 m g/g in diets containing naturally-contaminated grains. There are also reproductive effects in pigs including abortion, still births, and weak offspring (Prelusky et al., 1994).
Deoxynivalenol was responsible for a large-scale incident of human toxicosis in the Kashmir Valley, India in 1988. Acute toxicosis has been reported in China, Japan, and Korea among other countries (Beardall and Miller, 1994a; Kuiper-Goodman, 1994). Humans appear to be quite sensitive to deoxynivalenol (Kuiper-Goodman, 1994).
Zearalenone primarily occurs in F. graminearum and F. culmorum-contaminated maize. In tropical maize, zearalenols appear to be produced by Acremonium species. Zearalenone is an estrogen analog and causes hyper-estrocism in female pigs. The symptoms in pigs include swelling and reddening of the vulva, uterine enlargement, vaginal and rectal prolapse and enlargement of mammary glands. It also causes anestrous or constant estrus and may decrease litter size and may cause the production of weak and still-born piglets (Prelusky et al., 1994). Zearalenone has been implicated in several incidents of precocious pubertal changes in children (Kuiper-Goodman et al., 1987). IARC (1993) has recently evaluated the carcinogenicity of Zearalenone and found it to be a possible human carcinogen.
Fumonisins were discovered in 1988 by two groups working independently. One was investigating the cause of human esophageal cancer in parts of southern Africa. The other was attempting to find the cause of a well-known disease of horses, equine leucoencephalomalacia (ELEM). Fumonisins have been found as a very common contaminant of corn-based food and feed in the USA, China, Europe, southern Africa, and South America. The disease of equine species now known to be caused by fumonisin, ELEM has been recognized to be associated with F. moniliforme-contaminated maize since the turn of the century. Pure fumonisin was demonstrated to cause ELEM in mid 1988. ELEM involves a massive liquefactive necrosis of the cerebral hemispheres, hence the disease involves neurological manifestations including abnormal movements, aimless circling, lameness, etc. At high exposures, death can occur within hours after the onset of visible symptoms (Prelusky et al., 1994).
Exposure to maize contaminated with F. moniliforme has been linked to the elevated rates of esophageal cancer in the Transkei for 15 years and this has since been directly linked to fumonisin exposure. Fumonisins have been demonstrated to exhibit cancer-promoting activity in diethylnitrosamine-initiated rats and are toxic. Fumonisin B1 has been shown to be hepato-toxic and hepato-carcinogenic in rats fed 50 mg/kg. Fumonisins are considered to be poor initiators, are not mutagenic but are apparently good cancer promoters. There is not enough information to determine whether Fumonisins are human carcinogens. However, IARC (1993) recently examined the human carcinogenicity of grain contaminated with F. moniliforme containing fumonisins and fusarin C and found them to be possible human carcinogens.
Aflatoxins were discovered over 30 years ago and it is an understatement that there has been much research on these important compounds. IARC (1993) has recently re-evaluated aflatoxins in terms of their carcinogenicity. Naturally-occurring mixtures of aflatoxins were classified as class 1 human carcinogens. IARC provided a second conclusion about aflatoxin B1 i.e. that it is also a class 1 human carcinogen. There was inadequate evidence of the human carcinogenicity of aflatoxin M1, the metabolite of aflatoxin B1 found in human and animal milk.
Recommendations of 1993 GASGA and FAO Working Groups on Mycotoxins
A technical working group of the Group of Assistance for Grain Afterharvest (GASGA) developed a series of recommendations in response to increasing requests for research assistance on mycotoxins in several developing regions (GASGA, 1993). A policy oriented group comprising 11 Asian countries and convened by the FAO has also produced a recent analysis of mycotoxin problems (Miller, 1995).
The GASGA committee recommended that research and development money be focused on two issues:
(1) the effects of mycotoxins on human health in terms of immuno-toxicity and the effects of concurrent exposure to several mycotoxins on cancer and other endpoints;(2) more upstream research devoted to mycotoxin elimination in the affected countries.
The GASGA committee felt that too many resources were being devoted to repeating work done elsewhere.
The Asian policy makers made several recommendations. There was a clear desire to inform senior government managers and politicians about this issue so that governments would develop the political will and commitment to address the mycotoxin problem in each country. This process should involve the health, agriculture and trade constituencies. The expected outcome of this would be support for an integrated set of policies to educate farmers, manage toxin concentrations in commodities brought into urban areas, as well as the export markets and would probably be based on price incentives and, appropriate regional and domestic research and development.
General agreement was reached that appropriate information, education, and communication packages would be produced and distributed to all levels/sectors in the food/feed industry with a view to implementing existing mycotoxin management strategies. Furthermore, the need to strengthen information exchange both in the country, and between countries was stressed. With respect to international assistance the principal need identified was access to information. Specifically, the participants reiterated their request that FAO, GASGA, etc. continue to support information exchange though regional workshops, preparation of reference manuals, networking with other countries and establishing/maintaining data bases.
Table 3 Mycotoxigenic Fungi
|
Mycotoxin |
Commodity |
Fungal source |
|
deoxynivalenol/nivalenol |
wheat, maize, barley |
Fusarium graminearum |
|
zearalenone |
maize, wheat |
as above |
|
ochratoxin |
barley, wheat |
Aspergillus ochraceous, |
|
fumonisin |
maize |
Fusarium moniliforme a |
|
aflatoxin B1, B2 |
maize, peanuts b |
Aspergillus flavus |
|
B1, B2, G1, G2 |
maize, peanuts |
A. parasiticus |
a and several other less common species
b and many other commodities
Literature Cited
Adhikari, M., G. Ramjee, and P. Berjak. 1994. Aflatoxin, kwashiorkor and morbidity. Natural Toxins 2:13.
Beardall, J., and J. D. Miller. 1994a. Natural occurrence of Mycotoxins other than aflatoxin in Africa, Asia and South America. Mycotoxin Research 10:21-40.
Beardall, J., and J. D. Miller. 1994b. Diseases in humans with Mycotoxins as possible causes. In: Miller J. D. and H. L. Trenholm (eds.) Mycotoxins in grain: compounds other than aflatoxin. Eagan Press, St. Paul, MN. p. 48740.
Bhat, R. V. 1991. Aflatoxins: successes and failures of three decades of research. In: ACIAR Proceedings 36: 80-85.
Bhat, R. V., and J. D. Miller. 1991. Mycotoxins and food supply. Food, Nutrition and Agriculture (FAO). 1:27-31.
CAST. 1989. Mycotoxins: economic and health risks. Report 116: Council for Agriculture Science and Technology. Ames, LA.
Doko, M. B., S. Rapior, A. Visconti, and J. E. Schjoth. 1995 Incidence and levels of fumonisin contamination in maize genotypes grown in Europe and Africa. J Agric Food Chem. 43:429-434.
GASGA 1993. Mycotoxins in food and feedstuffs. ACIAR, Canberra, Australia.
IARC 1993. Monograph volume 56: Some naturally-occurring substances: food items and constituents, heterocyclic amines and mycotoxins. IARC Lyon, France. 599 p.
Jelinek, C.F., A. E. Pohland and G. E. Wood. 1989. Worldwide occurrence of Mycotoxins in foods and feeds-an update. J AOAC 72:223-230.
Kuiper-Goodman, T. 1994. Prevention of human mycotoxicosis through risk assessment and risk management. In: Miller, J. D. and H.L. Trenholm (eds.) Mycotoxins in grain: compounds other than aflatoxin. Eagan Press, St. Paul, MN. p. 439-470.
Kuiper-Goodman, T., P.M. Scott, and H. Watanabe. 1987 Risk assessment of the mycotoxins zearlenanone. Regul. Toxicol. Pharmacol. 7:253-306.
Lubulwa A.S.G., and J.S. Davis. 1994. Estimating the social costs of the impacts of fungi and aflatoxins in maize and peanuts. In: Highley, E., E.J. Wright, H.J. Banks, and B.R. Champ (eds.) Stored product protection, vol. 1. CAB International. p. 1017-1042.
Mannon, J., and E. Johnson. 1985. Fungi down on the farm. New Scientist Feb. 28,1985:12-16.
Marasas, W.F.O. 1988. Medical relevance of mycotoxins in southern Africa. Microbiol. Alim. Nutr. 6: 1-5.
Miller, J.D. 1991. The significance of mycotoxins for health and nutrition. ACIAR Proceedings 36: 126-135.
Miller, J.D. 1993. The toxicological significance of mixtures of fungal toxins. In: Wild, C.P. (ed.) Proceedings of the Pan African Environmental Mutagen Society, Cairo. African newsletter of occupational health and safety, 3:32-38. Institute of Occupational Health, Helsinki, Finland.
Miller, J.D. 1995. Mycotoxins in Asia: policies for the future. ACIAR Postharvest Newsletter 32: 5-15.
Pestka, J.J., and G.S. Bondy. 1994. Immunotoxic effects of mycotoxins. In: Miller, J.D. and H.L. Trenholm (eds.) Mycotoxins in grain: compounds other than aflatoxins. Eagan Press, St. Paul, MN. p. 339-358.
Prelusky, D.B., B.A. Rotter, and R.G. Rotter. 1994. Toxicology of mycotoxins. In: Miller, J.D. and H.L. Trenholm (eds.) Mycotoxins in grain: compounds other than aflatoxin. Eagan Press, St. Paul, MN. p. 359-404.
Toxicology Forum 1994. Special Meeting on mycotoxin health risk, control and regulation. Toxicology Forum, Washington, D.C.
Viljoen, J.H., W.F.O. Marasas, and P.G. Thiel. 1993. Fungal infection and mycotoxin contamination of commercial maize. In: Taylor, J.R.N., P.G. Randall, and J.H. Viljoen (eds.) Cereal science and technology: impact on a changing Africa. CSIR, Pretoria. p. 837-853.
G. Lubulwa and J. Davis
Economic Evaluation Unit, ACIAR
Canberra, Australia
This paper discusses five important impacts of fungi and aflatoxin in maize and peanuts. It suggests a way of estimating the annual social costs of these impacts and summarizes these costs. In the estimation of costs in the food and feed sectors, the two sectors were analyzed separately, in order to ensure that there was no double counting of benefits between the two sectors.
The total annual cost, in Indonesia, Philippines and Thailand, due to fungi and aflatoxins in maize in 1991 was about $(Aus)319 million. Indonesia incurred 62% of this cost, Philippines 27%, and Thailand 11%. The total annual cost of fungi and aflatoxins in peanuts in 1991 was about $(Aus) 158 million. Indonesia incurred 84% of this cost, Thailand 13%, and Philippines 3%.
In estimating the costs of human health (primary liver cancer), effects on increased morbidity and livestock productivity, use was made of the best available data on aflatoxin contamination in maize and peanuts which was also consistent for all three countries (Indonesia, Philippines and Thailand).
The costs of human health effects do not include the additional costs that countries incur in order to provide hospital and medical services to those suffering from primary liver cancer. Neither do they cover the cost of intangibles (pain and suffering, anxiety, and reduction in quality of life) associated with the incidence of primary liver cancer.
It is assumed that the distribution of aflatoxins in maize and peanuts is the same for food and feed. If the distribution of aflatoxins shows a higher percentage of food in the low quality grade compared to that for feed, then the estimated costs will understate the human life cost and overstate the livestock costs, and vice versa.
There is insufficient data on the joint occurrence, in food and feed, of aflatoxins with other mycotoxins in the three countries. We assume that maize and peanuts contain only aflatoxins, or that if other mycotoxins are present they do not lead to synergistic effects on the incidence of cancer or on the feed utilization efficiency of livestock. In those cases where maize and peanuts contain other mycotoxins the cost estimates are lower than the true cost of aflatoxins.
In conclusion, the estimated costs are likely to be on the lower boundary of total costs attributable to fungi and aflatoxins in maize and peanuts in Indonesia, Philippines, and Thailand.
W.F.O. Marasas
Research Institute for Nutrition Diseases,
Medical Research Council,
Tygerberg, South Africa
The Program on Mycotoxins and Experimental Carcinogenesis (PROMEC) of the South African Medical Research Council conducts research on mycotoxins (i.e. toxic metabolites of fungi) in food and feeds and examines their relation to human diseases. Particular attention is given to food-borne mycotoxins that are carcinogenic, e.g. aflatoxin produced by Aspergillus flavus and associated with human liver cancer. PROMEC has a total of 24 employees (including seven research scientists) and this multidisciplinary team publishes 20-30 papers per year in international indexed journals. We have extensive networks of collaborators in South Africa, elsewhere in Africa, and world-wide.
During 1988, a major scientific breakthrough was made at PROMEC with the isolation and chemical characterization of the fumonisins, new food-borne carcinogenic mycotoxins produced by Fusarium moniliforme in maize. Since that time research at PROMEC on the chemistry, natural occurrence, toxicity and carcinogenicity of fumonisins has caused world-wide interest. The World Health Organization is currently conducting an in-depth risk assessment of fumonisins for human health, and the program leader of PROMEC is acting as an expert consultant. We have shown that high levels of fumonisins in home-grown maize is a risk factor in the high incidence of human esophageal cancer in the Transkei region of South Africa. Similar findings have subsequently been reported from other high incidence areas of esophageal cancer in China, Italy, and the USA.
In our research on esophageal and liver cancer, the development of techniques for the early diagnosis of these cancers are currently in progress. These include the use of esophageal brush biopsies and serum growth factors to detect early pre-cancerous lesions in humans at risk for developing these cancers. In addition, the possible synergistic effects of different risk factors will also be investigated in these population groups exposed to a variety of environmental factors, e.g. food-borne mutagens, carcinogens, and viruses. The modulating role of different dietary factors, e.g. dietary fatty acids, polyphenolic agents and other chemo-preventative agents of carcinogenesis, are currently under investigation to evaluate their possible use in intervention trials in humans. The ultimate aim is reduction of the incidences of esophageal and liver cancer by mass screening, treatment, and intervention to reduce levels of food-borne carcinogens in the staple diet of home-grown maize, as well as improvement in nutritional status by means of dietary supplementation.
PROMEC, with its internationally acknowledged track record for excellence in scientific research, has the infrastructure and multidisciplinary expertise, to help solve human health problems in Africa. In the face of steadily declining funding by the South African government, our main impediment is the shortage of funds to execute our research and implement our results to the benefit of human health in South Africa and elsewhere in Africa.
G. Ramjee
Department of Pediatrics and Child Health,
University of Natal, South Africa
Kwashiorkor was first recognized in tropical Africa in the 1930's. The etiology and pathogenesis of kwashiorkor remains obscure. One of the factors in the pathogenesis includes inadequate protein and energy intake. Aflatoxins have been reported by various authors to be present in blood, urine and livers of children with kwashiorkor. The aim of this study was to establish whether an association exists between aflatoxin poisoning and kwashiorkor. Aflatoxin analysis was performed on children in four groups: protein energy deficiencies, i.e. kwashiorkor (n=45); marasmus (n=20); underweight (n=11); and similar age-matched children (n=35) were the control. All children were aged between 0 and 2 years. All four study groups had presence of aflatoxins in blood and urine. However, children with kwashiorkor had a high concentration of serum aflatoxin, but low urinary aflatoxin. This difference was significant when compared with the controls (p<0.01). The same association was not observed between controls and other forms of malnutrition. All children had been exposed to dietary aflatoxins as detected in the serum and urine samples. Children with kwashiorkor had much higher serum aflatoxin but low urinary levels suggesting that the liver in the child with kwashiorkor was unable to metabolize the toxin. A second study was conducted to elucidate some of the effects that aflatoxins may have on the morbidity of children with kwashiorkor. Retrospective analysis of hospital data was conducted on aflatoxin positive and aflatoxin negative children who had kwashiorkor. Aflatoxin-positive children showed: significantly greater severity of edema (p = 0.002); more complications (p = 0.037); lower hemoglobin levels (p = 0.002) and longer duration of hospital stay (p = 0.007). In conclusion, aflatoxins may be a contributory factor in increasing the morbidity in children suffering from kwashiorkor.
C.P. Wild
International Agency for Research on Cancer,
Lyon, France
Primary hepato-cellular carcinoma (HCC) is the most common cancer in males in The Gambia (age standardized rate 1986-1992,33 6 per 100,000 per year). Major risk factors for HCC in West Africa are chronic infection with hepatitis B virus (HBV) and exposure to aflatoxins. There is some evidence that HBV can alter aflatoxin metabolism and, in this way, alter the risk associated with exposure to a given level of aflatoxin. This could be one mechanism of interaction between HBV and aflatoxin in the evolution of HCC.
A sensitive immunoassay has been developed which allows detection of aflatoxin bound covalently to albumin in peripheral blood (aflatoxin-serum albumin adducts). This assay can be performed on as little as 0.1 ml serum or plasma. This is a useful assay for screening populations to establish prevalence and levels of exposure. The aflatoxin-serum albumin marker can also facilitate the study of the health effects through epidemiological studies of aflatoxin exposure.
Exposure to aflatoxin has been shown to be widespread in West Africa (The Gambia, Guinea Conakry, Nigeria, Senegal) using the assay of aflatoxin-serum albumin adducts. Over 98% of all subjects tested are positive for this marker of exposure in this region. Exposure occurs throughout the lifetime of the individual, starting in utero.
Genetic polymorphisms in some carcinogen metabolizing enzymes may contribute to individual susceptibility to aflatoxins.
Mamadou Samba Diallo, INRANG, Kindia, Guinea & C.P. Wild, IARC,
Lyons, France
The prevalence of exposure to aflatoxin B1 and the hepatitis B (HBV) and hepatitis C (HCV) viruses, three major risk factors implicated in cellular hepatocarcinoma, was examined in Guinea, West Africa.
In total, 75 blood serum samples were collected from men living in organized collectives in the Prefecture of Kindia (Lower Guinea).
The samples were analysed using the ELISA technique for aflatoxin bound covalently to serumal albumin as a marker for exposure to aflatoxin. More than 90% of the samples contained detectably high levels for adults. The highest level was equivalent to 358 pg (picogram) aflatoxin-lysine per mg of albumin.
Eleven patients (14.7%) were positive for hepatitis B (surface antigen) and the samples in this group had a tendency to higher levels of aflatoxins-albumin adduct than the others (mean total of 70.4 pg/mg compared with 44.1 pg/mg). This difference was not significant (P=0.23).
Eight patients were positive to antibodies of HCV antigen and oddly, seven of these came from the same ethnic group (Malinke 25% occurrence). These data show that exposure to all three infections is prevalent in Guinea and that the occurrence of these risk factors is comparable to that observed in other West African countries.
It is important to evaluate the impact of this on public health in these countries.
Table 1 Distribution of viral infections patients by age, ethnic group (Hepatitis B and C)
|
|
No. |
% |
|
|
Age |
|||
|
|
<35 |
23 |
30.6 |
|
|
35-50 |
35 |
46.7 |
|
|
>50 |
17 |
22.7 |
|
Ethnic group |
|||
|
|
Malinke |
28 |
37.3 |
|
|
Peulh |
23 |
30.7 |
|
|
Soussou |
17 |
22.7 |
|
|
Others |
7 |
9.3 |
|
Infection with hepatitis B virus (HVB) |
|||
|
|
Recent infection |
28 |
37.3 |
|
|
Previous infection |
- |
- |
|
|
Anti-HBC |
36 |
48.0 |
|
Infection with hepatitis C virus (HVC) |
|||
|
|
Anti-HVC positive |
8 |
10.7 |
|
|
Anti-HVC negative |
67 |
89.3 |
Table 2 Infection by ethnic by three major factors: aflatoxin, HBV, and HCV
|
|
Malinke |
Peulh |
Soussou |
|
Aflatoxin-Albumin-Adduct |
65.3 (84.7) |
41.4(49.5) |
40.1 (48.6) |
|
HBC positive |
18.0(64.3) |
n of % 13.0(56.5) |
11.0(64.7) |
|
HB surface antigen positive |
4.0(14.3) |
3.0(13.0) |
2.0(11.8) |
|
anti-HVC positive |
7 (25.0) |
1.0(4.3) |
0.0 (0.0) |
There are two other projects of great medical importance in Guinea (collection of biomaterial). The first project deals with the expression of the cytochrome P450 and infection by hepatitis B virus in Guinea (36 patient volunteers). The second project deals with the mutations which affect minisatellite sequences in men exposed to aflatoxin B1.
C.H.A. Snijders
CPRO-DLO, Wageningen, Netherlands
Agriculturally important fungal toxins in maize and other cereals in Africa are deoxynivalenol (DON) and zearalenone produced by Fusarium graminearum, fumonisin produced by Fusarium moniliforme and aflatoxin produced by Aspergillus flavus and A. parasiticus. Factors involved in the field outbreak of mycotoxicosis caused by a plant fungus are:
- the infection of a susceptible host plant;
- toxigenic potential of the pathogen;
- environmental factors favorable to disease development;
- conditions favorable to the production and accumulation of the toxins; and
- consumption of sufficient quantities of toxin-containing plant material.
The first two factors are involved in the host-plant interaction in which resistance breeding can intervene. Factors affecting Fusarium and Aspergillus infection in maize such as: bird and insect damage, husk looseness, drought stress, and stress caused by other diseases, can also be affected by breeding for resistance. This paper will be restricted to true resistance to the fungus Fusarium in maize.
The interaction of host, pathogen and environment greatly influences Fusarium in maize. An early season water deficit accelerates root senescence of maize plants and permanently increases the likelihood of chronic water stress, resulting in an earlier senescence, increased Fusarium infection, and its systemic colonization. As long as cells remain vigorous, most host genotypes have genetic components for resistance to the potential pathogens. Apparently synthesis of cellular resistance substances decreases with senescence.
Resistance to Fusarium ear-rot is quantitative; complete resistance has not been discovered. Fusarium graminearum is non-host specific, i.e. it is pathogenic without showing specialization for crop or crop genotype. F. moniliforme is less pathogenic, but is also non-host specific, as it is sporadically isolated from blighted wheat and sorghum. Prerequisites for breeding for resistance are genetic variation, the availability of experimental inoculation techniques and accurate assessment techniques. Inoculation methods can be divided into two types, namely: inoculation with mechanical injury, and inoculation without mechanical injury.
Method 1 refers to inoculation with Fusarium-encrusted toothpicks or needles, knives or pin-bars, by inserting the device through the husk perpendicular to the ear axis and midway between the butt and ear tip. With method 2, a spore suspension is sprayed onto the maize silks with an atomizer until it runs off; injected into the silk tuft; or toothpicks colonized by Fusarium are placed in the silk channel near the cob tip. The best differential infections are obtained when inoculations are made 4-17 days after silks have emerged.
Assessment techniques vary according to the incidence of infected plants or cobs, percentage of ear infection to kernel infection, and kernel plating. To obtain an accurate estimate of the amount of fungal biomass, it is advisable to make ergosterol analyses.
Resistance to Fusarium in maize consists of two components: 1) resistance to initial penetration; and 2) resistance to the spread of pathogen in host tissue. Inoculation method type 1 screens generally for resistance component 2 only Wound-type inoculations simulate insect attack to some degree as they bypass morphological barriers. Inoculation method type 2 closely simulates natural infection as the host plant is not wounded. In addition to local infections, corn plants can become systemically infected with F. moniliforme, and F. graminearum, even without showing symptoms. Resistance component 2 prevents the colonization of plant tissue by the fungus. Several studies have shown that there was no distinct correlation between endosperm type in quantity, and the concentration of kernel carbohydrates on the one hand, and infection by Fusarium on the other hand. However, kernels with certain endosperm types may have small cracks in the pericarp in which hyphae can grow and sporulate. In this case, the effects of endosperm on resistance are due to inherent morphological characters. Factor(s) for resistance to kernel infection after natural inoculation by F. moniliforme are not conditional on the genotype of the endosperm, embryo or cytoplasm, but on the genotype of the pericarp, the silk or of other maternal tissues.
DON and fumonisin are phytotoxic. They effect membrane stability which causes electrolyte leakage from the plant cells. Comparison of data on phytotoxicity, with the high concentrations of DON found in maize tissue, presupposes a phytotoxic effect of DON during development of the seed. DON and fumonisin produced with Fusarium stalk and ear-rot in maize can also have physiological effects in other parts of the plant. Both toxins are water soluble and translocation in the phloem as a bulk flow of solution is assumed. DON was found in tissues of the infected corn plant which were not invaded by F. graminearum. Both DON production in F. graminearum and fumonisin production in F. moniliforme segregates aggressively, though proportions for F. moniliforme are not absolute. DON and to some extent fumonisin are non-specific toxins and may be regarded as aggressiveness factors, i.e. they increase the extent of disease symptoms and colonization, but are not involved in the primary interaction which determines basic compatibility between host and pathogen. DON is produced in the cells immediately adjacent to the hyphal tip. An active defense mechanism of the host in reaction to hyphal invasion after penetration requires protein synthesis. Low concentrations of trichothecenes such as DON prevent protein synthesis and as a result further colonization will not be inhibited. Plants resistant to these toxins will not show complete resistance, but an increased resistance to colonization. Germ plasm of various crops, including maize, has been shown to be highly tolerant to trichothecenes. This is based on trichothecene degradation, increased membrane stability, and a modified peptidyl transferase. In wheat it was demonstrated that DON is transported from the chaff to the young kernel, which the pathogen later colonizes. A Fusarium resistant wheat-line which possessed resistance to colonization appeared to inhibit DON translocation from chaff to kernel. Evidently this is a membrane-based trichothecene tolerance which resulted in inhibition of systemic colonization. As DON and to a lesser extent fumonisin are regarded as aggressiveness factors, it is suggested that DON and fumonisin tolerance in maize will result in an increased level of resistance to F. graminearum and F. moniliforme, respectively, and inherent prevention of toxin accumulation.
PJ. Cotty
Food and Feed Safety Unit, USDA,
New Orleans, USA
Products are not currently available for preventing aflatoxin contamination in the field. Certain cultural practices may limit contamination but these are unreliable and often not economically feasible. To protect crops fully from contamination, procedures must be active in the field under hot, dry conditions that are not very conducive to crop development but which are often near optimal for Aspergillus flavus group fungi. Controls must be effective during both crop development and in storage. Furthermore, damaged grains sustain large amounts of aflatoxin contamination, and therefore controls must also protect damaged portions of crops.
Seeding of agricultural fields with atoxigenic A. flavus may meet the above criteria. Greenhouse and field experiments in which developing crops were wound-inoculated with various A. flavus strain combinations, showed that atoxigenic strains can reduce contamination (80 to 90%) during crop development. In laboratory tests, atoxigenic strains have also been shown to reduce aflatoxin contamination of maize in storage. Atoxigenic strains reduce contamination both by spatially excluding aflatoxin producing strains and by competing for resources. In theory, seeding fields with atoxigenic strains may permit seeded strains to compete with other resident strains for crop-associated resources. During environmental conditions which favor aflatoxin contamination, the seeded strains may thus increase in population size while competitively excluding aflatoxin production. This theory has been tested in cotton in field plot experiments in the irrigated desert agricultural regions of the United States. Strain seeding resulted in large and significant reductions in the aflatoxin content of the crop at maturity, and aflatoxin content was inversely correlated with the incidence of the seeded vegetative compatibility group of the fungus. Atoxigenic strain application was associated with neither increased crop infection, nor increased A. flavus populations on the crop at maturity. Applied strains spread from points of application within the field and beyond. Applied strains also survive between crops and influence the composition of A. flavus populations in subsequent years. Atoxigenic strains may be particularly useful in community-wide aflatoxin management programs directed at reducing contamination on multiple crops throughout an area.
The use of atoxigenic strains seeks to limit neither the amount of crop infection by the A. flavus group, nor the quantity of these fungi associated with the crop. The procedure merely selects which fungi become associated with the crop. Aspergillus flavus group fungi typically become associated with crops in the field during crop development and remain associated with the crop during harvest, storage and processing. Thus, seeding of atoxigenic strains into agricultural fields prior to crop development many provide post-harvest protection from contamination by associating the harvested crop with high frequencies of atoxigenic strains.
K. Hell, J. Udoh, M. Setamou, K.F. Cardwell & A. Visconti
Plant Health Management Division, IITA, Benin
Instituto Tossine e Micotossine da Parassiti Vegetali, Bari, Italy
Pre-harvest and stored maize produced by small scale farmers in various agro-ecological zones of Benin and Nigeria was analyzed from 1993 to 1995, to assess the fungal contamination and mycotoxin levels. Analysis of farming systems from which the maize samples were taken indicated which practices increased, and which decreased, aflatoxin levels.
In Benin during the 1993-1994 sampling period, the mean percentage of 100 kernels from each of 300 stores infected with Aspergillus spp. was 25% one to three months after harvest. This level increased significantly to 75% infected kernels after six to eight months in storage. The Southern Guinea Savanna (SOS) (the middle zone of the country) had the highest percentage of stores containing aflatoxin positive samples during the first three months of storage; however, these levels did not increase during the subsequent storage period. All other zones showed a significant increase in the number of stores contaminated after six to eight months in storage. In the two southern zones, Southern Forest Mosaic (SFM) and SGS after six to eight months, 25% of the stores were contaminated with a mean of 100 ppb aflatoxin. The northernmost zone, the Sudan Savanna showed an increase from 5% to 56% of stores contaminated with aflatoxins, with mean aflatoxin levels increasing from 10 ppb after three months storage to a mean of 220 ppb six months later.
In 1994, sampling of maize standing in the field revealed the highest percentage infection of pre-harvest maize cobs in the northern savannas. In samples of pre-harvest maize, 98% of the Aspergillus spp. were A. flavus. The high level of field infection was reflected in the infection levels of the maize in the stores 2 to 3 months after harvest.
In Benin the 1994-1995 sampling period gave a different profile from the previous year. Across agro-ecological zones, 35-70% of the stores were contaminated at one to three months after storage, with much higher initial levels than the previous years, but there was no increase in contamination during the storage period. The mean aflatoxin levels per zone during this period ranged from 10 to 150 ppb.
One to three months after storage in the 1993-1994 sampling period, fumonisin analysis was conducted on twenty of the samples collected from stores from each of the four agro-ecological zones. Fumonisin levels decreased significantly from south to north, but were significantly correlated with aflatoxin levels, indicating that, as grain degrades it is likely to be simultaneously contaminated by various toxins (Table 1).
In Nigeria, crop husbandry practices which significantly reduced aflatoxins in maize samples taken from 125 small farm stores were: use of fertilizer, pesticides, and seed protectants. Aflatoxin levels increased significantly when farmers said they used "improved varieties," and when they harvested late after crop maturity. Each processing step before and during storage decreased aflatoxin levels. Steps included measures such as: drying, sorting, cleaning, dehusking, degraining, sunning, and smoking. Insect control in the field and store decreased aflatoxin levels significantly. Storage of maize on the floor in a room resulted in high aflatoxin levels. When farmers complained of having storage problems, they had significantly higher levels of aflatoxin in their maize, though few of them were aware of moldiness.
Table 1 Correlation among maize grain inhabiting fungi, mycotoxins, and grain humidity across four ecological zones in Benin Republic
|
|
Zone |
FUS |
PEN |
ASP |
HUM |
FUMON |
AFLATOX |
|
Zone |
1.00 |
0.05 |
0.28** |
0.01 |
0.50*** |
-0.25* |
0.17 |
|
FUS |
|
1.00 |
0.07 |
0.09 |
0.14 |
0.15 |
0.06 |
|
PEN |
|
|
1.00 |
0.28** |
0.34** |
0.14 |
0.38*** |
|
ASP |
|
|
|
1.00 |
0.05 |
0.22* |
0.39*** |
|
HUM |
|
|
|
|
1.00 |
0.13 |
0.23* |
|
FUMON |
|
|
|
|
|
1.00 |
0.31** |
|
AFLATOX |
|
|
|
|
|
|
1.00 |
*, **, and *** coefficients significant at P = .05, P = .01, and P = .001. Zone = 1) SFM (south). Southern Forest Mosaic, 2) Southern Guinea Savanna, 3) Northern Guinea Savanna, 4} Sudan Savanna (North). FUS = % Fusarium spp., PEN = % Penicillium spp., and ASP = % Aspergillus spp., HUM = % grain humidity, FUMON = Fumonisin, AFLATOX = Aflatoxin B.
R.T. Awuah
Department of Crop Science,
University of Science and Technology,
Kumasi, Ghana
The problem of mold and aflatoxin contamination of stored grains in Ghana is not new, although it is only recently that efforts are being made to address the problem. Among the recommendations for mitigating the problem, rapid drying of the crop to achieve a low moisture content is emphasized. This recommendation however, is not easy to implement because farmers lack the requisite facilities. Thus, the crop is often stored at high moisture levels which encourage growth of mycotoxigenic fungi and mycotoxin synthesis. Among the mycotoxigenic fungi, Aspergillus parasiticus Speare and A. flavus Link ex Fries, are important in Ghana. These two fungi produce aflatoxins in a wide variety of grains.
Certain plants used in traditional medicinal practice in Ghana have recently been shown to possess fungitoxic properties. Some of these plants, notably Ocimum gratissimum, Cymbopogon citratus, Xylopia aethiopica, Monodera myristica, Syzigium aromaticum, Cinnamomum verum and Piper nigrum have also proven to be somewhat effective in inhibiting formation of norsolorinic acid (NOR), a precursor in the aflatoxin synthesis pathway (Table 1).
Table 1 Selected Plant Extracts and their Effects on NOR Production by A. parasiticus
|
Source of extract |
Intensity of NOR production* |
|
|
YES Medium |
PDB medium |
|
|
Ocimum gratissimum |
++ |
+(-) |
|
Cymbopogon citratus |
++ |
+(-) |
|
Xylopia aethiopica |
+++ |
- |
|
Monodera myristica |
+++ |
- |
|
Syzigium aromaticum |
+++ |
- |
|
Cinnamomum verum |
-? |
- |
|
Piper nigrum |
+++ |
- |
|
Control |
+++ |
++(+) |
* Intensity of NOR production:- = NOR absent,
+ = NOR slightly produced,
++ = NOR moderately produced,
+++ = NOR intensely produced.YES = Yeast Extract Sucrose.
PDB = Potato-Dextrose Broth.
These plants have some potential for use as preservatives against aflatoxigenic fungi and aflatoxin synthesis. Indeed, Ocimum leaf powder has been used with success in limiting mold development on soybean seeds during a 9 month storage period (Table 2).
Plans are underway to test a number of "botanicals" as storage protectants for a variety of grains. If this proves successful, it would be a cost-effective, easily implementable means of partly solving the aflatoxin (possibly the mycotoxin) problem.
Table 2 Occurrence of Fungi on Soybean (Cultivar Bengbie) Seeds Stored under Different Conditions
|
Storage condition |
Frequency of occurrence (%) |
||
|
3 months |
9 months |
||
|
Control |
|||
|
|
Polythene bag |
2.88 |
3.25 |
|
|
Paper bag |
6.50 |
9.38 |
|
Ocimum leaf powder |
|||
|
|
Polythene bag |
1.63 |
2.00 |
|
|
Paper bag |
2.75 |
4.38 |
|
Ocimum seed powder |
|||
|
|
Polythene bag |
3.13 |
5.50 |
|
|
Paper bag |
5.00 |
7.13 |
|
Dithane M-45 |
|||
|
|
Polythene bag |
1.38 |
2.13 |
|
|
Paper bag |
4.63 |
5.13 |
Source: Kwoseh, C. 1994 M. Phil thesis, University of Science and Technology, Kumasi, Ghana
K.A. Kpodo
Food Research Institute, Council for Scientific and Industrial Research (CSIR),
Accra, Ghana
Maize is a dietary staple for over 40% of the Ghanaian population. The bulk of the maize is grown by small-scale farmers mainly for the local markets. After harvesting and drying, storage is carried out in traditional cribs, silos, and warehouses to be resold to the public later. Maize is processed into fermented dough which is sold at most processing sites and in markets throughout the country.
Maize and maize products from silos, warehouses, processing sites, and markets were screened for aflatoxins B1, B2, G1, and G2 and in some cases, citrinin, ochratoxin A, zearalenone, and alpha-zearalenol using Thin Layer Chromatography and High Performance Liquid Chromatography. All the maize kernel samples from silos and warehouses contained aflatoxins at levels ranging from 20 ppb to 355 ppb (total aflatoxins). Quality of maize before storage was unknown.
Fermented maize doughs collected from major processing sites over a one and a half year period of time were all positive for aflatoxins with levels of 0.7 ppb to 313 ppb. No apparent relation between the time of year and aflatoxin level was observed. Twenty fermented dough samples from major markets all contained the mycotoxin citrinin (1.4 ppb - 584.5 ppb). Ochratoxin A was detected in low levels (<6.1 ppb) in five samples. Nineteen samples contained aflatoxins with aflatoxin B1 dominating. No zearalenone nor alpha zearalenol was detected.
Aflatoxin and citrinin levels of maize were not affected by the traditional steeping and fermentation processes as practiced in Ghana. Cooking of fermented maize dough for three hours, as done for "kenkey" (an end product of maize dough fermentation) production however resulted in 80% reduction of aflatoxins B1 and G1 and 35% reduction in aflatoxins B2 and G2 levels. Citrinin was no longer detectable after cooking. These preliminary studies showed that the mycotoxins, aflatoxins and citrinin are present in relatively high levels in maize and fermented maize products in southern Ghana and further research work needs to be carried out.
© 1996 International Institute for Tropical Agriculture (IITA)