STUDIES ON AFLATOXIGENIC ASPERGILLUS FLAVUS AND AFLATOXINS B1 AND M1 AND THEIR OCCURRENCE IN DAIRY CATTLE FEEDS AND MILK PRODUCTS IN KADUNA STATE, NIGERIA

ABSTRACT

Nigeria has one of the fastest growing population in Africa. Increasing population and nutritional knowledge may put pressure of demand on the dairy industry, which may in turn compromise the quality of dairy products. The likelihood of heavy contaminations of dairy cattle feeds and milk products by fungi and aflatoxins, in this part of the world, may be encouraged by the prevailing climatic conditions. Such level of contamination could be of greater concern, with over 80% of dairy production in Nigeria, in the hands of pastoralists, who have little or no knowledge of the subject matter. Majority of Nigerians, mostly in the low and rarely of the medium economic status, depend largely on locally produced and processed milk and milk products for protein supplementation. It therefore, implies that, many consumers are exposed to the risks of aflatoxin and other health related issues through consumption of contaminated dairy products. The aim of this study therefore was to investigate the occurrence of aflatoxigenic strains of A. flavus and their aflatoxins B1 and M1 (AFB1 and AFM1) in dairy cattle feeds and milk products from Fulani herd groups and conventional dairy herds in Kaduna State of Nigeria. The major objectives of the study were to isolate and identify the toxigenic strains of A. flavus from feeds using phenotypic means and identify the strains by PCR methods. Their metabolic products were also detected and quantified from feeds, milk and milk products using Enzyme linked Immunosorbent Assay (ELISA) and and High Performance Liquid Chromatography (HPLC) techniques for screening and confirmation. Out of the total of 144 dairy feed samples collected and analyzed in this study, 86 (59.7%) had fungal contamination of which 48 (55.8%) and 16 (18.6.7%) were A. flavus and A. parasiticus respectively. Of these proportions, 12 (18.8%) and 4 (6.3%), were identified as aflatoxigenic strains of A. flavus and A. parasiticus respectively. The average fungal colony forming units (CFU) determined in this study was 4.5 Log₁₀ CFU/gram of feed. This level is below the EU recommendation of 5.0 Log₁₀ CFU/g for poorly preserved feeds. Feeds fortified with concentrates, feeds of cereal grains only and hay were evaluated for levels of contamination by both aflatoxigenic and non-aflatoxigenic strains of A. flavus. In any of the feed type, there were relatively higher proportions of non-toxigenic strains (66.3 – 79.1%) than the aflatoxigenic strains (20.9 – 33.7%). Out of 144 dairy feed samples collected for testing using comparative analytical methods, 86.8% and 91.6% samples tested positive for aflatoxins B1. Of the positive samples, 92.0% showed AFB1 contamination levels at ≥ 5 ppb of which 56 (49.0%) had AFB1 contamination levels of up to and above 20ppb. Significant proportions of these AFB1 positive samples were feeds fortified with concentrates (14.6%), feeds of grain origin (8.3%), stored feeds (16.7%) and feeds from small holder‘s farms (>43.8%). A total of 201 milk and milk products were tested for AFM1 in this study, of which 174 (86.8%) and 197 (98.0%) of all the products tested positive for AFM1 with HPLC and ELISA analytical methods respectively. In this study, factors such as size and type of dairy farms seemed to have influence on the level of aflatoxin contamination. For instance, small scale dairy farms, comprising mostly of Fulani herds, showed significant proportion (91.7%) of samples positive for AFM1 contamination. Other factors of influence studied amongst others included, effects of heat-treatment temperatures on AFM1 concentration, such as milk sterilization at 121^oC which showed reasonably lower AFM1 concentration of 142.09 ppb than the lower temperature treatment of 80^oC which showed an AFM1 concentration of 183.58 ppb, when compared with the original fresh unpasteurized milk (219.98 ppb). Locally fermented yoghurt (Kindirmo) and milk (Nono) displayed relatively lower mean AFM1 concentrations of 0.158±0.025ppb and 0.231±0.019 ppb. Findings from this study have indicated potential health risks associated with the consumption of dairy products, particularly those products processed locally. Therefore, adequate and all-inclusive measures are recommended to be put in place for routine monitoring of dairy products including the locally processed products before they are marketed for human consumption in Nigeria.

CHAPTER ONE

1.0 INTRODUCTION

1.1         Background

Mycotoxins are natural contaminants in raw materials, food and feeds (Bosco and Mollea, 2012). They are toxic metabolites produced by different species of toxigenic fungi, especially by saprophytic moulds growing on foodstuffs or animal feeds. Not until the past 30 years, the effects of these moulds and their toxins have been largely overlooked and have been sources of hazard to man and domestic animals. Although poisonous mushrooms have been carefully avoided, moulds growing on foods have generally been considered to cause unaesthetic spoilage, without being dangerous to health (Abdel-Gawad and Zohri, 1993; Ahmad, 1993). Between 1960 and 1970 it was established that some fungal metabolites, now called mycotoxins, were responsible for some animal diseases and death (Blount, 1961). In the decade following 1970, it became clear that mycotoxins have been the cause of human illness and death as well (Alpert et al, 1971; Richard, 2003).

It is now well established that mycotoxicoses (the diseases caused by mycotoxins) have been responsible for major epidemics in man and animals at least during recent historic times (Fink-Gremmels, 2008). One of these important diseases has been ergotism, which killed thousands of people in Europe in the last thousand years (Smoragliewicz et al., 1993). Other diseases include alimentary toxic aleukia (ATA) which was responsible for the death of many thousands of people in the USSR in the 1940s (Smoragliewicz et al., 1993), stachybotryotoxicosis, which killed tens of thousands of horses and cattle in the USSR in the 1930s (Fung et al., 1998); and aflatoxicosis, which killed 100,000 young turkeys in England in 1960 (Quist et al., 2000) and has caused death and disease in many other animals and man. Each of these diseases is now known to have been caused by growth of specific moulds which produced one or more potent toxins, usually in one specific kind of commodity or feed (Reddy, 2009). The discovery of aflatoxins (AFs) dates back to the year 1961 following the severe outbreak of turkey ―X disease, in England, resulting in the deaths of more than 100,000 turkeys and other farm animals (Quist, et al., 2000). The cause of the disease was attributed to a contaminated feed. Thin-layer chromatography (TLC) revealed that a series of fluorescent compounds, later termed aflatoxins, were responsible for the outbreak (De-Iongh et al., 1962; Balzer et al., 1978). The disease was linked to a peanut meal, incorporated in the diet, contaminated with a toxin produced by the filamentous fungus Aspergillus flavus. Hence, the name aflatoxin, an acronym that was formed from the following combinations: the first letter, ―A for the genus Aspergillus, the next set of three letters, ―FLA, for the species flavus, and the noun ―TOXIN meaning poison (Rustom,1997). Aflatoxins (AFs) are difuranocoumarins produced primarily by two species of Aspergillus fungus which are especially found in areas with hot, humid climates (Criseo et al., 2001; Udom et al., 2012). Aspergillus Section Flavi contains a number of species capable of producing a wide array of mycotoxins among which aflatoxins are the most important in food safety. Aflatoxins are potent carcinogenic, mutagenic, and teratogenic secondary metabolites and are produced predominantly by Aspergillus flavus and Aspergillus parasiticus (Bennett and Papa, 1988). There exists basically two groups of Aspergillus, the aflatoxin-producing species such as Aspergillus flavus, A. parasiticus, A. nomius, A. niger and the recently described species, A. pseudotamarii and A. bombycis (Cary and Ehrlich, 2006). The other group includes the aflatoxin non-producing species: A. oryzae, A. sojae, and A. tamarii, which have been used for production of traditional fermented foods in Asia (Kumeda and Asao, 2001). Aflatoxins belong to the class of mycotoxins (Huang et al., 2010). Chemically they are defined as difuranocyclopentano-cumarines or difuranopentanolidocumarines, that is, aflatoxins containing a dihydrofuran or a tetrahydrofuran ring, to which a substituted cumarin system is condensed. Out of about 20 known aflatoxins, the moulds Aspergillus flavus and A. parasiticus produce exclusively aflatoxins B1, B2, G1 and G2, and all the other aflatoxins are derivates of these four (Arseculeratne et al., 1969; Huang et al., 2010). The derivates are developed either by metabolism in humans, animals and microorganisms or by environmental reactions. Among the 18 different types of aflatoxins identified, the major members are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), M1 (AFM1) and M2 (AFM2). Aflatoxin B1 is normally predominant (in amount) in cultures as well as in food products (Arseculeratne et al., 1969). Pure AFB1 is a pale-white to yellow crystalline, odorless solid. Aflatoxins are soluble in methanol, chloroform, acetone and acetonitrile (Asao et al., 1963). Aspergillus flavus typically produces AFB1 and AFB2, whereas A. parasiticus produce AFG1 and AFG2 as well as AFB1 and AFB2 (Huang et al., 2010). Four other aflatoxins M1, M2, B2A, G2A which may be produced in minor amounts were subsequently isolated from cultures of A. flavus and A. parasiticus (Gulyas, 1985; Huang et al, 2010). A number of closely related compounds namely aflatoxin GM1, parasiticol and aflatoxicol are also produced by A. flavus (Nesbit et al., 1962; Gulyas, 1985). The order of acute and chronic toxicity produced by these aflatoxins is AFB1 > AFG1 > AFB2 > AFG2. The degree of the severity is therefore reflecting the role played by epoxidation of the 8,9-double bond and also the greater potency associated with the cyclopentenone ring of the B series, when compared with the six-membered lactone ring of the G series. Aflatoxins M1 and M2 are hydroxylated forms of AFB1 and AFB2 (Dors et al., 2011). AFM1 and AFM2 are major metabolites of AFB1 and AFB2 in humans and animals and may be present in milk from animals fed on AFB1 and AFB2 contaminated feed (Gundinc and Filazi, 2009; Filazi et al., 2010). Furthermore, it may also be present in poultry eggs (Zaghini et al., 2005), corn (Shotwell et al., 1976) and peanut (Ren et al., 2007; Huang et al., 2010). Aflatoxins interact with the basic metabolic pathways of the cell disrupting key enzyme processes including carbohydrate and lipid metabolism and protein synthesis (Quist et al., 2000). The health effects of aflatoxins have been reviewed by a number of workers (Tang, 2001; Kensler et al., 2003; Pang et al., 2005). Aflatoxins are among the most potent carcinogenic, teratogenic and mutagenic compounds in nature (Shephard, 2005; Kirk et al., 2006; Jackson and Al-Taher, 2008). The International Agency for Research on Cancer (IARC) has concluded that naturally occurring aflatoxins belong to group 1 carcinogens to humans, with a role in the aetiology of liver cancer, notably among subjects who are carriers of hepatitis B virus surface antigens. In experimental animals, there was sufficient evidence for carcinogenicity of naturally occurring mixtures of aflatoxins and of AFB1, AFG1 and AFM1, limited evidence for AFB2 and inadequate evidence for AFG2. The principal tumours were in the liver, although tumours were also found at other sites including the kidney and colon. Aflatoxin B1 is consistently genotoxic in vitro and in vivo (EFSA, 2007). The Joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) concluded that AFM1 should be presumed to induce liver cancer in rodents by a similar mechanism to AFB1, and that estimates of the potency of AFB1 can be used for determining the risk due to intake of AFM1, including those for populations with a high prevalence of carriers of hepatitis B virus. The carcinogenic potency of AFM1 was estimated to be one-tenth that of AFB1, based on a comparative study in the Fischer rat conducted by Cullen et al. (1987). Humans can be exposed to aflatoxins by the periodic consumption of contaminated food, contributing to an increase in nutritional deficiencies, immunosuppression and hepatocellular carcinoma. Aflatoxins have a wide occurrence in different kind of matrices, such as spices, cereals, oils, fruits, vegetables, milk and meat among others (Dors et al., 2011b). About 4.5 billion people, mostly in developing countries, are at risk of chronic exposure to aflatoxins from contaminated food crops (Shuaib et al., 2010). Therefore, in order to avoid the toxicity, the levels of aflatoxins and similar toxic compounds in foodstuffs have to be monitored closely, and to be kept under control continuously. Otherwise, related health effects like acute and chronic intoxications, and even deaths, will still be an issue (Becer and Filazi, 2010). Mycotoxins can be acutely or chronically toxic, or both, depending on the kind of toxin and the dose (Richard, 2003; Kensler et al., 2003). Acute mycotoxicoses include among others ergotism, a condition caused by a metabolic product known as ergot produced by Claviceps purpurea, alimentary toxic aleukia (ATA), a condition caused by a group of mycotoxins known as trichothecene (T-2). Other mycotoxicoses of acute nature include acute cardiac beriberi caused by citreoviridin, a mycotoxin produced by the comparatively rare species, Penicillium citreonigrum (Uragochi, 1971). Onyala is another mycotoxicosis of acute significance. It was discovered that toxigenic isolates of Phoma sorghina were found to be common in millet consumed by affected population (Rabie et al., 1975).

Aspergillus flavus is ubiquitous, favouring the aerial parts of plants (leaves, flowers) and produces B aflatoxins. Aspergillus parasiticus which produces both B and G aflatoxins, is more adapted to a soil environment and has more limited distribution (EFSA, 2007). Aspergillus bombysis, A. ochraceoroseus, A. nomius, and A. pseudotamari are also AFs producing species, but are encountered less frequently. From the mycological perspective, there are qualitative and quantitative differences in the toxigenic abilities displayed by different strains within each aflatoxigenic species. Forexample, only about half of A. flavus AFs-producing strains produce AFs- more than 106 g kg−1 (Turner et al., 2009).

1.2 Statement of Research Problem

Feeds remain an integral part of dairy production, where high economic yield is required. Problem arises when global standards of feed preservation are not kept. Worst scenarios are noticed with pastoralists purchasing concentrates to supplement cattle feed without proper preservation (Uko, 2004)). This situation could expose their cattle to mouldy concentrates. Consumption of milk or milk products from previously aflatoxin-exposed dairy cattle is of public health concern. Such concern is more serious in areas where routine monitoring of milk and milk products intended for human consumption is absent or inadequate. It is a common practice among the pastoralists, particularly their children, taking milk directly from the udder of cattle. This practice exposes them to aflatoxin toxicity and is of public health concern. Many Nigerians in the North Western develop the habit of taking dairy products often or almost on daily basis. Such people are exposed to the risk of developing aflatoxin toxicity or mycotoxigenic hazards should such milk or milk products be contaminated with aflatoxin. In developing nations, such as Nigeria, there is the general belief by people that heat treatment reduces the chances of food getting contaminated with pathogens and toxins. Pasteurization, a known standard heat treatment of milk at regulated and specified temperatures and time, may not achieve this significantly with respect to aflatoxins (Al-Delamyi and Mamoud, 2015). However, the impact of high temperature treatment on aflatoxin M1 especially in aqueous state has not been exploited. This leaves a gap in information regarding heat treatment of milk using the standardized pasteurization technique only. Humans are constantly suffering invasion from wide arrays of microorganisms including fungi and their metabolic toxins (Mwanza, 2011). Amongst other environmentally stressed-related immunosuppressants, aflatoxins play significant roles (Surai and Dvorska, 2005). Human immuno-virus (HIV) and acquired immune deficiency syndrome (AIDS) are highly endemic especially in developing countries, and are characterized by high level of immunosuppression. Nutritional management has aided immunity amongst those vulnerable to immunosuppression. Combined effect of immunossuppressants present worst pathologic scenarios. Acute or chronic aflatoxicosis from consumption of contaminated milk intended for protein supplementation may present adverse health conditions to immunocompromised patients.

1.3 Justification for The Study

Large scale animal production is a pre-requisite requirement for sustainable economy and development of any nation. The dairy industry plays a significant role in this respect. In Nigeria, for example, the dairy industry contributes up to 38% (about 12.7% of the agricultural GDP) to agriculture in the form of milk production, which has annual growth rate of 4.3% (Uko, 2004; Udom et al., 2012). However, threats from mycotoxins are capable of distorting this pattern through high rates of disease occurrence and subsequent reduced production efficiency in cattle (Coulombe, 1993). CAST (2003) published a list of major research areas needed, which included amongst others, qualitative and quantitative mycotoxin studies. Several observations have been made with respect to aflatoxins as residues in tissues (Bintvihok et al., 2002), but mycotic and mycotoxic studies in dairy establishments have been under studied, thus creating paucity of data for public health interventions. More so, most of the few observations made were based on commercial dairies in Nigeria. Emphasis has not been placed on the local dairy industry, which is currently playing a major complementary role in meeting the daily milk demands on commercial dairies. Fear of risk of exposure to aflatoxins based on reports. Researches have indicated high risk of exposure to aflatoxins among the people in developing countries (Shephard 2005; Strosnider et (al., 2006). Higher incidences of liver cancer caused by aflatoxin consumption have been reported in Asia and sub- Saharan Africa (Kirk et al., 2006; Strosnider et al., 2006). A study carried out in the Eastern Nigeria showed that people in the tropics and subtropics have higher exposure risk to AFB1 (Okonkwo and Obionu, 1981). These have informed the urgent need for continued investigation of the potential public health risk associated with the consumption foods of animal origin particularly milk in Nigeria.There is increasing interest in western culture among the Nigerian elites on the use of dairy products like cheese, butter among others. Such habit could be extended to indigenous dairy products that might not have been well prepared, thereby exposing the consumers to the risk of aflatoxicosis.

Nigeria is one of the fastest growing countries in Africa. The increasing population and knowledge of nutrion have put pressure on the dairy industry with the consequences of increasing springing up of local yoghurt producing outfits and compromised quality of dairy products.

1.4 Aim of Research

To study the occurrence and the toxins produced by aflatoxigenic strains of Aspergillus flavus in dairy cattle feed and dairy products among dairy herds in Kaduna State.

1.5 Research Objectives

1. Isolate Aspergillus flavus and other Aspergillus spp and identify the aflatoxigenic strains of Aspergillus flavus from dairy cattle feeds using phenotypic method.
2. Determine the strains of Aspergillus flavus that encode specific genes, IGS(aflR-aflJ), 18S rRNA and fla by PCR.
3. Differentiate flavus from A. parasiticus, the major producers of aflatoxins using restriction fragment site analysis.
4. Detect the presence of genes in the Aspergillus spp isolates from dairy cattle feeds encoding aflatoxin production using PCR.
5. Determine the diversity of the flavus isolates by sequencing of the IGS region.
6. Determine the levels of aflatoxin (AFB1) by Enzyme immunoassay and HPLC in dairy feeds that are fed to cattle.
7. Determine the levels of aflatoxin metabolite (AFM1) in raw milk and locally fermented milk products (Kindirmo and Nono) sold for human consumption using ELISA and HPLC techniques.
8. Determine the effects of moisture content of feed on flavus and heat treatments on aflatoxin levels in milk.

1.6 Research Questions

• What proportion of dairy feeds is contaminated by fungi, particularly flavus?
• What proportion of flavus isolates in dairy feeds are toxigenic?
• What roles do type and size of dairy herd, type of feed, fresh and stored cattle feed play on the occurrence of toxigenic strains of flavus and aflatoxins?
• How genetically diverse are the flavus isolates identified in this study?
• What proportion of dairy feeds are contaminated with AFB1?
• What proportion of dairy products sold for human consumption are contaminated by AFM1?
• Are there differences in the concentrations of AFM1 between locally prepared and commercially produced dairy products?
• What is the effect of heat treatment and fermentation on AFM1 concentration in milk?

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