Amelioration of Aflatoxicosis in Broilers: A New Look

Fungi live everywhere, in the air, in water, in soil, on land and on plants and animals.  There are a million or more species of fungi and this includes both advantageous and toxigenic fungi. Toxigenic fungi are ubiquitous and, in some cases, apparently have a strong ecological link with animal feed and human food supplies. The natural fungal flora existing in conjunction with feed and food production is dominated by three genera: Aspergillus, Fusarium and Penicillium species. They grow on cereal crops and other commodities, and produce toxic secondary metabolites, the mycotoxins.  Mycotoxins are produced before, or immediately after, harvest and when ingested produce deleterious effects that range from mild toxicity to mortality (Murphy et al., 2006).

One of the most toxic group of mycotoxins are the aflatoxins (AFs), produced by various species of Aspergillus fungi. However, in agricultural commodities, aflatoxins are produced predominantly by the toxic species of Aspergillus fungi namely Aspergillus parasiticus and Aspergillus flavus. Aspergillus fungi grow on living plant tissues and on mouldy hay, organic compost piles, leaf litter, dung, human tissues as saprophytes. They are produced on a wide variety of substrates by aflatoxigenic moulds. Foods of plant origin are normally contaminated more frequently and with higher concentrations than foods of animal origin Besides producing aflatoxins, growth of these fungi alters the nutritional quality of the grain on which it grows, thus reducing the availability of nutrients (Bandyopadhyay et al., 2007).  

Aflatoxin contamination has been reported in a wide variety of cereals, oilseeds and  tree nuts (Bankole and Mobekoje, 2004; Koirala et al., 2005). 

The fungi require temperatures of 24°C-35°C and moisture content above seven percent to grow and produce aflatoxin (Williams et al., 2004). The extent of aflatoxin production varies depending on the crop, the soil (Brown, et al., 2001; Bankole and Mabekoje, 2004) and drought conditions that damage the crop, favoring fungal infection. A heavy rain during harvest increases the moisture content of the crop and the risk of infection. Damage of pods by insects, both prior to harvest and during storage helps in the colonization of fungi and toxin production more easily.  Above all, storing feeds without proper drying or in moist places readily attracts Aspergillus infection and toxin production (Hell et al., 2000; Ono et al., 2002 ; Hawkins et al., 2005 ; Turner et al., 2005).

When poultry are fed with feed contaminated with aflatoxins, they affect liver, kidney, immune system and thereby affects the performance of birds. Aflatoxin toxicity in poultry is related with biochemical, hematological, reproductive and pathological changes (Ortatatli and Oguz, 2001). Further, aflatoxin residues in meat and eggs pose a threat to the health of consumers. Many a times, acute exposure to aflatoxins causes mortality in birds. On the other hand, chronic exposure to aflatoxin under field conditions may go unnoticed. But chronic exposure may affect the biochemical status of the birds and reduce their overall performance. Further, the manifestation of chronic or acute toxicosis in broilers depends on strain, duration of exposure and rate of metabolism of aflatoxin to less toxic metabolites (Kermanshahi et al., 2009; Shabani et al., 2010).  

A number of studies have reported the effects of aflatoxin on feed conversion ratio but the results are not uniform. For example, in a study by Diaz and Sugahara, (1995) it was reported that  feeding 0.6ppm aflatoxin decreases FCR at the end of first week of age. In contrary the  reports by Celik et al. (2005) did not indicate any change in FCR by feeding 0.5ppm aflatoxins up to 1 week of age. Additionally, a report by Oguz and Kurtoglu (2000) did not show any reduction in FCR after feeding 2.5ppm aflatoxin B1 for 2 weeks.

Simialrly, the reports on effect of aflatoxin on FCR at three weeks of age of broilers are contrary. A significant increase in FCR by 17% to 24% has been observed in chicks fed a diet containing 2.5 ppm aflatoxin (Huff et al., 1986; Oguz 1997) and 5.0 ppm aflatoxin (Diaz and Sugahara, 1995) from 1 to 21days of age. On the other hand, Oguz and Kurtoglu (2000) did not observe any significant difference in FCR in birds fed 2.5 ppm up to three weeks of age. 

A reduction in FCR has been noted in broilers fed 0.1 ppm aflatoxin for 5 weeks or 8 weeks (Verma et al.,  2004), 0.5 ppm and 1.0 ppm aflatoxin for 4 weeks (Celik et al.,2005). But there are contrary reports that do not indicate any negative effect of feeding 0.5 and 1.0 ppm aflatoxin for six weeks (Edrington et al., 1997; Kermanshahi et al., 2009). In the report by Umaya et al. (2011), the authors have used meta-analysis approach to derive at the effects of aflatoxin on FCR. They have reported that the effect of aflatoxin on FCR was negligible, moderate and large at the end of first, second and third weeks respectively. Further, it was also shown that the effect of aflatoxin was adverse at the end of fourth, fifth and sixth weeks of age of broilers respectively. These ill effects will ultimately leads to economic loses to the farmers.

The economic impact of mycotoxins are many fold, affecting all sections of production and consumption of grain production viz. grain producers, handlers, processors, farmers, consumers and society as a whole. Grain producers are affected by limited yields, restricted end markets due to contamination and price discounts. Grain handlers are affected by restricted storage facilities, costs of testing grain lots and loss of end markets. Grain processors incur higher cost due to higher product losses, monitoring costs and restricted end markets. Farmers incur losses due to low productivity of birds and low product quality of ruminants. Consumers end up paying higher end product prices due to increased monitoring at all levels of handling and in extreme cases death problems due to consumption of contaminated products. On the other hand, society as a whole end up paying higher costs due to increased regulations, needed research, lower export costs and higher import costs. At present these costs are found at every level of grain and animal production system and almost impossible to estimate the exact extent of losses (Umaya et al., 2013).

The strategies to reduce the impact of aflatoxins include its control both at preharvest and post harvest stages and are discussed below.

To prevent aflatoxin contamination, preharvest strategies like maintenance of proper planting / growing conditions, antifungal treatments and adequate insect and weed prevention would be helpful. During harvest, use of functional harvesting equipment, clean and dry collection / transportation equipment and appropriate harvesting conditions are also recommended. Further, post harvest strategies like drying as dictated by the moisture content of the harvested grain, appropriate storage conditions, and use of transport vehicle that are dry and free of visible fungal growth helps to reduce aflatoxin contamination (Park, 2002; Strasnider  et al., 2006).

 Many decontamination methods have been tried to destroy already formed toxin from food or feed and reduce the toxic effects of the contaminated products. The decontamination methods include physical, chemical and biological methods.

Screening and sorting of aflatoxin contaminated seeds are sugegsted as the most effective approaches in the case of peanuts when the seeds were blushed. But there are reports that indicate feeding of such sorted nuts to animals or sometimes they are consumed by the poorest producers and labourers. Roasting is effective in reducing the aflatoxin cotnant of feeds in time and temperature dependent manner. In case of rice, pressure cooking and are effective in removing AFB1. Sun drying of afaltoin contaminated feed for two days reduced aflatoxin content of feeds. Also, drying of feed as dry heat at temperature of 80-100 C for 6-8 hours is also an effective method to reduce aflatoxin content in feeds. Further feeding such sunlight treated or dry heat treated feed failed to induce any adverse effects in sheep (Gowda et al.,2006)

Addition of adsorbents bentonite, hydrated sodium calcium aluminosilicate (HSCAS) to contaminated feeds had proved to be effective in reducing the bioavailability of aflatoxins in animals. Calcium montmorillonite, the adsorbent was demonstrated to be safe for humans (Wang et al., 2005), which would allow the use of this technique for products intended for human consumption. Microorganisms like yeasts and bacteria have been tested on their ability to modify or inactivate aflatoxins (Zaghini  et al., 2005).

Chemically, aflatoxins can be destroyed with calcium hydroxide, monomethyl amine, ammonia and ozone. Among all chemicals, ammoniation was extensively used for cotton seed meal, peanut meal or sunflower meals. But the main drawback using chemicals was found to be their carryover to animals that resulted in the deterioration of animal health (Galvano et al., 2001).

 Flavobacterium aurantiacum was shown to remove AFB₁ from liquid media and was used in peanut processing as biodegrader (Diarra et al., 2005).In the recent years lactic acid bacteris have been demonstrated to aflatoxin, however it has to be established by in vivo trials.

In the recent years there is an increased interest among poultry scientists on the use of antioxidants against aflatoxin toxicity. This is because aflatoxins were demonstrated to induce the production of reactive oxygen species and oxidative stress has been suggested as one of the underlying mechanisms for AFB1 induced cell injury and DNA damage (Yang et al., 2000).

During the normal metabolism of cells, free radicals are produced. Free radicals are molecules or molecular fragments containing one or more unpaired electrons in atomic or molecular orbitals. Many types of radicals exist but those of most concern in biological systems are derived from oxygen and are referred as reactive oxygen species (ROS). The sequential reduction of molecular oxygen leads to the formation of a group of reactive oxygen species superoxide anion (O₂^.-) hydrogen peroxide (H₂O₂), hydroxyl radical (^.OH) and singlet oxygen (¹O₂), eventually terminate in the formation of water. The uncoupled electrons of free radicals are very reactive with adjacent molecules such as lipids, proteins and carbohydrates and were suggested to cause cellular damage (Halliwel, 2000). As a result of the relative instability of free radicals and their potential to damage cells and tissues, there are both enzymes and small molecular-weight molecules with antioxidant capabilities that can protect against the adverse effects of free radical reactions.  However, a disturbance in the prooxidant - antioxidant balance in favour of the former, leads to a condition termed “oxidative stress”. The excess reactive oxygen species target polyunsaturated fatty acids of biological membranes and form lipid hydroperoxides, which are finally decomposed to form reactive lipid aldehydes (lipid peroxidation process). Such damage to cell membranes alters the permeability of the cell and cell function.

Aflatoxin as a small molecule is rapidly absorbed in the gastrointestinal tract by passive diffusion and transported to liver. In the liver, AFB1 is metabolized by cytochrome P450 enzymes to form AFB1-8, 9- exo-epoxide (AFBO). This epoxide  readily binds with the N7 position of guanine and forms AFB-DNA adduct, a compound mainly responsible for the mutagenic and carcinogenic effects of aflatoxin. However, in poultry carcinogenic effects of aflatoxin have not been recorded but such binding of epoxide to macromolecules alter cell function.  Secondly, aflatoxins, especially AFB1, produce reactive oxygen species (ROS) such as superoxide radical anion, hydrogen peroxide and lipid hydroperoxides; though these do not appear to interact with DNA, they are precursors to the hydroxyl radical. These hydroxyl radicals interact with DNA and produce mutations. The overproduction of reactive oxygen species by AFB1 has been shown to be due to generation of free radicals in liver disproportionately via cytochrome P 450 enzymes and an iron-mediated redox mechanisms. Aflatoxin also induces expression of inducible nitric oxide synthase and in turn nitric oxide (NO), peroxynitrite, nitrogen dioxide or nitrate which cause protein and DNA damage (Ozen et al., 2009). Besides increasing lipid peroxidation, aflatoxin reduces the antioxidant enzyme activities on tissues of birds affected with aflatoxin and in vivo trial in broilers has confirmed the same. (Umaya and Parvatham, 2009). Earlier studies on aflatoxin focused on performance and biochemical and pathological changes. But with the advent of molecular biology, few studies have unveiled the molecular changes induced by aflatoxin in broilers and have generated information that are useful for researchers and poultry industry. Aflatoxin has been shown induce alterations in gene expression patterns. Feeding 2.0ppm aflatoxin for 21 days to day old chicks has resulted in an up regulation of genes related to biotransformation and cellular proliferation and down regulation of genes involved in immunity, antioxidant function and oxidative phosphorylation (Yarru et al., 2009).

Use of plants as a source of medicine has been inherited and is an important healthcare system in India. In the recent years, a more universal approach followed to brazen out aflatoxins is the use of antioxidants. This approach is based on the counteractive effect of antioxidants against the oxidative stress induced by aflatoxin.

Reports on the protective effect of antioxidants during aflatoxicosis in poultry were published in the last few years. The first report was on silymarin, bioactive compound from Silybum marianum (milk thistle). Silymarin contains a mixture of flavonolignans that acts as an antioxidant, cell membrane stabilizer and permeability regulator, as well as a promoter of DNA, RNA, and protein synthesis. Feeding studies conducted using silymarin phytosome showed  protection against the negative effects of 0.8ppm AFB1 on performance of broiler chicks. The protective effect of silymarin was attributed to its inhibitory effect on oxidative damage (Tedesco et al., 2004).

Curcumin and the curcuminoids are the antioxidant principles of C.longa. These compounds significantly overcame the negative effects of 1.0ppm AFB1 on feed intake, body weight gain, lipid peroxidation and antioxidant status and hepatocellular degeneration in broiler chicks (Gowda et al., 2009). Alpha-lipoic acid, a prosthetic group present in dihydrolipol transacetylase parts of dehydrogenase compounds in mitochondria, possesses antioxidant capacity. Alpha-lipoic acid and its reduced form, dihydrolipoic acid, can scavenge free radicals, bind metals such as copper and iron, and regenerate antioxidants such as glutathione (GSH), ascorbate and vitamin E. This compound has been demonstrated to inhibit the nitric-oxide-dependent lipid peroxidation and the subsequent cellular damage in aflatoxicosis. LA supplementation in water at 60 mg/kg/body weight reduced the degenerative changes in several organs, including liver and kidney. It was partially effective in preventing the molecules involved in the AF-mediated cellular damages and lipid peroxidation both in liver and kidney (Karaman et al., 2010).

In the study by Sirajudeen et al. (2011), broiler chicks fed aflatoxin-contaminated diets (0.5 or 1 ppm diet) exhibited in a significant increase in lipid peroxidation  in the liver and erythrocytes accompanied with suppression of superoxide dismutase and catalase enzyme activities of erythrocytes. Simultaneous administration of melatonin, with AFB1 resulted in an obvious improvement in the tested parameters. The protective action of melatonin was attributed its antioxidant properties. Further, it was observed that long-term rather than short-term administration of MEL was more effective in rendering protection against AFB1 induced toxicity. The leaves of M.oleifera, are rich sources of flavonoids, quercetin and kaempferol- the powerful antioxidants. Supplementation of 1% M.oleifera leaves significantly reduced the lipid peroxidation products and increased the antioxidant enzymes in liver; thus counteracted the oxidative stress and offered protection against aflatoxin toxicity in broilers (Umaya et al., 2012).

Right from identification of aflatoxins, various efforts have been taken to control its exposure to livestock and humans by researchers and administrators worldwide. However, reports on aflatoxin keep coming till date. Newer approaches are needed to identified and evaluated. With respect to antioxidants, further studies needs to be conducted to establish the molecular mechanism of action of effective plant products and the stability of such products needs to be validated under filed conditions.

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