University of Pretoria etd – Jansen Van Rensburg, C (2005) CHAPTER 1 Introduction Fungi, developing from spores and found ubiquitously in the environment, can grow on almost any organic matter. These fungi produce structurally diverse metabolites, mycotoxins, that occur as both food and animal feed contaminants worldwide (Ramos & Hernández, 1996). The Fusarium, Penicillium and Claviceps species of fungi generally contaminate feeds prior to harvest with mycotoxins such as those belonging to the trichothecene family (T-2 toxin, HT-2 toxin and vomitoxin) and the ergot alkaloids, respectively. Aspergillus and Penicillium species are commonly found in stored grain or food and produce aflatoxins and ochratoxins, and citrinin, respectively (Marquardt, 1996). Mycotoxins can cause serious health problems and production losses in livestock (Ramos & Hernández, 1996). Aflatoxin is the most prevalent and economically significant mycotoxin. It is found in maize, peanuts, cottonseed, millet, sorghum and other feed grains (Phillips, 1999). Like many microbial secondary metabolites, the aflatoxins are a family of closely related compounds (Moss, 1996) that include aflatoxin B1, B2, G1 and G2, but aflatoxin B1 (AFB1) is usually in the highest concentration and is the most toxic. Aflatoxin is stable once formed in grain, and is not degraded during normal milling and storage (Brown, 1996). AFB1 is known as a potent hepatotoxin and hepatocarcinogen and the liver is considered to be the primary target for aflatoxins (Towner et al., 2000), but it also affects other organ systems (Coulombe, 1994). The immune system is a highly sensitive indicator of aflatoxicosis in poultry (Giambrone et al., 1985b) affecting both cellular and humoral immune reactions (Giambrone et al., 1978). Practical methods for detoxifying mycotoxin-contaminated grain on a large scale and in a cost- effective manner are not currently available. A variety of physical, chemical, and biological techniques have been employed but with limited success (Edrington et al., 1997). The most recent approach is the use of non-nutritive adsorptive materials (enterosorbents), which bind the aflatoxin molecule and reduce aflatoxin absorption. Ideally, the adsorbent should have a high affinity for the specific mycotoxins, resulting in the formation of a strong complex with a low possibility of dissociation and should also have a high capacity for binding to prevent saturation (Ramos & Hernández, 1996; Edrington et al., 1997). Ledoux & Rottinghaus (1999) are of opinion that the addition of adsorbents to contaminated feed to selectively bind the mycotoxin during the digestive 1 University of Pretoria etd – Jansen Van Rensburg, C (2005) process, allowing the mycotoxin to pass harmlessly through the animal is, at present, the most promising and practical approach. The major advantages of adsorbents include low cost, safety and the ease with which they can be added to animal feeds, but according to Ramos & Hernández (1997) this strategy would only be effective if these materials had the ability to adsorb a large number of chemically distinct mycotoxins. Research indicates that a number of adsorbents are capable of binding aflatoxin and reducing or preventing its toxic effects. However, not all adsorbents are equally effective in protecting livestock against the toxic effects of aflatoxin and several adsorbents have been shown to impair nutrient utilisation (Chung et al., 1990; Kubena et al., 1993; Scheideler, 1993). Dale (1998) noted that many of the adsorbents on the market today have not been adequately tested for in vivo efficacy, but are used based on in vitro adsorption data only. In vitro tests may not always be a reliable indicator of ability to bind a mycotoxin (Scheideler, 1993; Dwyer et. al., 1997; Ledoux & Rottinghaus, 1999). Therefore, it is important that adsorbents be subjected to extensive in vivo evaluation to determine both efficacy and impaired nutrient utilisation from the diet (Ledoux & Rottinghaus, 1999). Humic acids are substances widely distributed in nature and are present in soils, natural waters, river, lake and sea sediments, peat, brown and brown-black coals and other natural materials as a product of chemical and biological transformations of animal and plant residues (Novák et al., 2001). The humic acids in peat have been known since ancient times for their therapeutic properties such as anti-inflammatory, antiviral, oestrogenic and profibrinolytic activity (Schepetkin et al., 2002). Humic acids in peat caused stimulation of lymphatic system cells (Obminska- Domoradzka et al., 1993a & b), thymus activity (Madej et al., 1993a & b), neutrophil function (Riede et al., 1991) and phagocytic activity of granulocytes (Jankowski et al., 1993). The oxidative polymerization of o- and p-diphenols produced synthetic humic acids, which showed similar therapeutic activities (Klöcking, 1994). It was also demonstrated that humic acids have the ability to adsorb heavy metals (Madronovà et al., 2001), herbicides (Leone et al., 2001), mutagens (Sato et al., 1987a & b), monoaromatic compounds (Nanny & Maza, 2001), polycyclic aromatic compounds (Kollist-Siigur et al, 2001), minerals (Fein et al., 1999) and bacterial DNA (Crecchio & Stotzky, 1998). In recent years, it has been observed that dietary intake of humates promote growth in poultry (Bailey et al., 1996; Shermer et al., 1998). Kocabağli et al. (2002) found that dietary humates significantly improved body weight and feed conversion of broilers. 2 University of Pretoria etd – Jansen Van Rensburg, C (2005) A South African company, Enerkom, developed an effective large-scale regeneration process for humic acids from coal. This technology can economically regenerate large quantities of pure, high quality humic acids by reversing the process whereby coal was formed. Humic acids produced in this way are called oxihumic acids. Chemically oxihumic acids differ only marginally from humic acids obtained from other sources (Cloete et al., 1990; Dekker et al., 1990; Cronjé et al., 1991; Bergh et al., 1997). The hypothesis of this study was that, because of the adsorbing abilities of humic acids to a wide range of compounds, oxihumate would bind mycotoxins in the digestive system to ameliorate the toxic effect thereof on the animal. The effectiveness of oxihumate to adsorb mycotoxins was evaluated to determine the possibility of developing it as a commercial mycotoxin binder to be used in the preventative management of contaminated poultry feedstuffs. This was done by (1) evaluating the in vitro affinity and adsorption capacity of oxihumate to aflatoxin (AF) B1 and G2 by studying their Langmuir and Freundlich adsorption isotherms; (2) determining the efficacy of oxihumate as an aflatoxin binder in broiler feeds in vivo and (3) investigating the ability of oxihumate to prevent the inhibiting effect of aflatoxin on lymphocyte proliferation in vitro. 3 University of Pretoria etd – Jansen Van Rensburg, C (2005) CHAPTER 2 Literature Review 1. Mycotoxins Fungi develop from spores and are ubiquitous in the environment, being present in the soil, on the walls of storage bins, on grain handling equipment, and within the home environment. Fungi can grow on almost any organic matter and often produce highly toxic compounds referred to as mycotoxins. Fungi are able to invade the plant during growth and during storage and as a result a wide range of pre- and post-harvest fungi may contaminate feeds. The major genera of mycotoxin- producing fungi are Aspergillus, Claviceps, Penicillium and Fusarium. The Fusarium, Penicillium and Claviceps species of fungi generally contaminate feeds prior to harvest and are often referred to as “field fungi” while Aspergillus and also the Penicillium species are commonly found in stored grain or food and are referred to as “storage fungi”. These fungi are able to produce a wide array of different and related mycotoxins. Important toxins produced by field fungi are for example those belonging to the trichothecene family (T-2 toxin, HT-2 toxin and vomitoxin) and the ergot alkaloids. The toxins produced by storage fungi of greatest concern are the ochratoxins (mainly ochratoxin A), citrinin and the aflatoxins, particularly aflatoxin B1 (AFB1) (Marquardt, 1996). 1.1 Environmental factors affecting mycotoxin formation Fungal growth and mycotoxin production are consequences of an interaction between the fungus, the host and the environment. The appropriate combination of these factors determines the amount of colonization of the substrate and the type and amount of mycotoxin produced (Pitt et al., 2000). The synthesis of any particular mycotoxin depends not only on the species but also on the strain (Sweeney & Dobson, 1998). Under field conditions, stress and subsequently reduced vigour often predispose crop plants to infestation and colonization by toxigenic fungi. Water stress, high temperature stress and insect damage of the host plant are major determining factors in mould infestation and toxin production under field conditions. In stored grain, factors which are likely to affect mycotoxin formation include moisture content of the substrate, environmental temperature, exposure time, damage to seed, oxygen availability, carbon dioxide concentrations, composition of the substrate, fungal abundance, prevalence of toxigenic strains, spore loads, microbial interaction and invertebrate vectors particularly insects. Spoilage, fungal growth and mycotoxin formation result from the complex interaction of these factors (Marquardt,