aflatoxins

Aflatoxin produces adverse effects in poultry resulting in decreased immunity even leading to death (Sravanan et al., Reference SRAVANAN, SUKUMAR, ARULMOZHI and MOHAN2006). After being exposed to aflatoxin contaminated feed, broilers develop anorexia, listlessness, poor FCR, decreased body weight, depressed growth rate can be observed and birds are immunosuppressive and have increased susceptibility to stress (microbial and environmental) (Leeson et al., Reference LEESON, DIAZ and SUMMERS1995).

Aflatoxicosis causes increased liver size, paleness of liver, and increase in the size of the bile duct (Espada et al., Reference ESPADA, DOMINGO, GOMEZ and CALVO1992; Fernandez et al., Reference FERNANDEZ, VERDE, GASCON, RAMOS, GOMEZ, LUCO and CHAVEZ1994; Ledoux et al., Reference LEDOUX, ROTTINGHAUS, BERMUDEZ and ALANSO-DEBOLT1999; Ortatali and Oguz, 2001). Aflatoxin is manifested as lesions in kidney and spleen tissue (Glahn et al., Reference GLAHN, BEERS, BOTTJE, WIDEMAN, HUFF and THOMAS1991; Bilgic and Yesildere, Reference BILGIC and YESILDERE1992). Immunity is severely disrupted by aflatoxicosis, and can be measured by different assays including macrophage function assay and antibody response to sheep RBCs (Hassan, Reference HASSAN2010; Khan, Reference KHAN2010). Birds fed with contaminated feed are more prone to develop diseases (Ibrahim et al., Reference IBRAHIM, SHAREEF and AL-JOUBORY2000; Oguz et al., Reference OGUZ, HADIMLI, KURTOGLU and ERGANIS2003).

Aflatoxins cause fatty changes, the development of small vacuoles within the parenchyma of liver (vacuolar degeneration) and bile duct proliferation in portal areas. It affects the bursa of Fabricius, whereby this organ shows depletion of lymphoid cells. In kidneys; tubular degeneration has been reported for affected animals (Ortatali et al., Reference ORTATALI, OGUZ, HATIPOGLU and KARAMAN2005). Kumar and Balachandran (2009) described the gross and histopathological lesions produced by aflatoxin B1. Grossly, there was pale or yellowish discoloration of liver along with enlargement of its size and distention of gall bladder. There was enlargement of the kidneys and the colour was pale and congestion was observed along with sporadic petechial haemorrhages. Microscopically, vacuolar degeneration, fatty changes and hepatocyte degeneration were observed. With the advancement of the disease, fatty deposits were transformed into fatty cysts. There was an infiltration of different lymphocytic cells, such as heterophils and mononuclear cells. Congestion and hyperplasia of bile duct was reported.

ochratoxins

Ochratoxin is more of a nephrotoxin than a hepatotoxin, and is a potent and severe nephrotoxic in broilers. Ochratoxin A has teratogenic, mutagenic and immunotoxic effects (O'Brien and Dietrich, Reference O'BRIEN and DIETRICH2005). It causes reduction in growth (Huff et al., Reference HUFF, WYATT, TUCKER and HAMILTON1974; Dwivedi and Burns, 1984 a, b; Kubena et al., Reference KUBENA, HARVEY, FLETCHER, PHILLIPS, MOLLENHAUER, WITZELL and HEIDELBAUGH1985; Mohiuddin et al., Reference MOHIUDDIN, REDDY and AHMED1992; Verma et al., Reference VERMA, JOHRI, SWAIN and AMEENA2004), reduced feed intake (Elaroussi et al., Reference ELAROUSSI, MOHAMED, EL BARKOUKY, ATTA, ABDOU and HATAB2006) and poor body weight gain (Prior et al., Reference PRIOR, O'NEIL and SISODIA1980; Garcia et al., Reference GARCIA, AVILA, ROSILES and PETRONE2003; Kumar et al., Reference KUMAR, JINDAL, SHUKLA, PAL, LEDOUX and ROTTINGHAUS2003). Birds’ behaviour is depressed and symptoms include loose to watery faeces (Abidin, Reference ABIDIN2010; Hassan, Reference HASSAN2010). OTA causes severe immunosuppression (Chang et al., Reference CHANG, HUFF and HAMILTON1979, Dwivedi and Burns, Reference DWIVEDI and BURNS1984a, b; Stoev et al., Reference STOEV, ANGUELOV, IVANOV and PAVLOV2000, Reference STOEV, DJUVINOV, MIRTCHEVA, PAVLOV and MANTLE2002; Politis et al., Reference POLITIS, FEGEROS, NITSCH, SCHATZMAYR and KANTAS2005) which can be measured by antibody response to sheep RBCs and the carbon clearance assay (Hassan, Reference HASSAN2010; Khatoon, Reference KHATOON2010). It causes increased mortality in birds (Gibson et al., Reference GIBSON, BAILEY, KUBENA, HUFF and HARVEY1989; Elaurossi et al., 2006). Long term body residues of OTA is due to its binding with albumin fraction present in blood and recycling of the toxin in bile and kidney (Marquardt and Frohlich, Reference MARQUARDT and FROHLICH1992).

Regarding gross lesions, OTA causes congested and swollen kidneys, an increase in the size of liver, and reduction in the size of lymphatic organs such as spleen and bursa of Fabricius (Kumar et al., Reference KUMAR, JINDALI, SHUKLA, ASRANI, LEDOUX and ROTTINGHAUS2004). It does not cause any type of lesions on the heart (Mohiuddin et al., Reference MOHIUDDIN, REDDY and AHMED1992; Kumar et al., Reference KUMAR, JINDALI, SHUKLA, ASRANI, LEDOUX and ROTTINGHAUS2004). Ochratoxin A causes infiltration of mononuclear cells in the parenchyma of liver tissue (Elaroussi et al., Reference ELAROUSSI, MOHAMED, ELGENDY, EL BLARKOUKY, ABDOU and HATAB2008) whereby the outer layer of liver becomes thickened. Portal areas show congestion and sinusoidal spaces are congested and distended. In later stages of the intoxication (due to continuous exposure) hepatic cords become degenerated and individual cell necrosis is observable. Vacuolar degeneration has been reported (Kumar et al., Reference KUMAR, JINDALI, SHUKLA, ASRANI, LEDOUX and ROTTINGHAUS2004; Abidin, Reference ABIDIN2010). OTA causes proliferation of epithelium of the biliary channel along with its degeneration. Degeneration of individual cells and DNA adduct formation (up to 30 individual adducts) due to oxidative stress has been reported (Solcan et al., Reference SOLCAN, COMAN and SOLCAN2008). In kidneys, OTA causes necrosis of tubular epithelial cells, glomerular infiltration and distended glomerular spaces have been noted (Kumar et al., Reference KUMAR, JINDALI, SHUKLA, ASRANI, LEDOUX and ROTTINGHAUS2004; Abidin, Reference ABIDIN2010). Carmen et al. (Reference CARMEN, COMAN, SOLCAN, MIRON and OPREAN2008) reported microscopic alterations within kidneys, including degeneration in the epithelium of convoluted tubules and Malphighi corpuscles, increase in the size of epithelial cells, distended and degenerated epithelium of proximal convoluted tubules and pyknotic nuclei in the cytoplasm of epithelial cells. Similar findings have been reported by Stoev et al. (Reference STOEV, ANGUELOV, IVANOV and PAVLOV2000) and Elaroussi et al. (Reference ELAROUSSI, MOHAMED, ELGENDY, EL BLARKOUKY, ABDOU and HATAB2008). In the bursa, OTA causes atrophy of follicles and reduction in the lymphocytic mass present in the medulla. Follicles of cortex and medulla also show vacuolar degeneration (Kumar et al., Reference KUMAR, JINDALI, SHUKLA, ASRANI, LEDOUX and ROTTINGHAUS2004; Khatoon, Reference KHATOON2010). Within the thymus, OTA exposure reduced lymphocytic mass in the parenchyma and localised congested areas were observed (Kumar et al., Reference KUMAR, JINDALI, SHUKLA, ASRANI, LEDOUX and ROTTINGHAUS2004; Khatoon, Reference KHATOON2010).

ochratoxin analysis

Biro et al. (Reference BIRO, SLTI, VETRO, BAGO and GLAVIS2002) explained the estimation of levels of ochratoxin within the tissues after OTA administration by using High Performance Liquid Chromatography (HPLC) and Enzyme Linked Immunosorbent assay (ELISA). When contaminated feed was given to layers, OTA residues were found in eggs, as determined by Frye and Chu (Reference FRYE and CHU1977) using radioactivity technique. Residues of OTA were examined in male and female bird tissues by Prior et al. (Reference PRIOR, O'NEIL and SISODIA1980). An advanced technique using HPLC along with the method of extraction of OTA from tissues and eggs has been put forward by Sizoo and Egmond (Reference SIZOO and EGMOND2005).

aflatoxins

Thin layer liquid chromatography (TLC) technique has been used for estimation of aflatoxin B1 (AFB1) by Fermandez et al. (1994) in broiler tissues. Hussain et al. (Reference HUSSAIN, KHAN, KHAN, JAVED, SALEEMI, MEHMOOD and ASI2010) determined AFB1 in tissues after oral administration of AFB1 using HPLC. The same technique was used by Micco et al. (Reference MICCO, MIRAGLIA, ONORI, IOPPOLO and MANTOVANI1988) and Khan (Reference KHAN2010). Omurtag et al. (Reference OMURTAG, ATAK, KESKIN and ERSOY2002) used HPLC for aflatoxin estimation and levels more than 1 ppb were estimated by TLC.

In general, Fluorescence Polarization Immunoassay (FPIA) can be used for determination of mycotoxin levels.

mycotoxin decontamination

If the levels of mycotoxins are high in feed, then their removal or decontamination is preferred. However, it is difficult to remove the contaminated portion from feed. It has been proposed that cereals can be decontaminated by ammoniation, but there is no method to detoxify the toxins present in forages. Chemical, biological and physical methods of decontamination of diet can be explained by different reviews (Lopez-Garcia and Park, Reference LOPEZ-GARCIA and PARK1998; Sinha, Reference SINHA1998). A de-epoxidising enzyme isolated from pure strain of bacteria has also been identified (Binder et al., Reference BINDER, HEIDLER, SCHATZMAYR, THIMM, FUCHS, SCHUH, KRSKA and BINDER2000).

use of mycotoxin binders

Various reviews (Ramos et al., Reference RAMOS, FINK-GREMMELS and HERNANDEZ1996; Ramos and Hernandez, Reference RAMOS and HERNANDEZ1997; Huwig et al., Reference HUWIG, FREIMUND, KAPPELI and DUTLER2001) have explained the importance of toxin binders. Mycotoxin binders prevent adverse effects of mycotoxins by binding to the toxins, thus preventing their adsorption within the digestive system. The use of binders is considered to be a preventative than a therapeutic approach.

Different binders are available, and include activated carbon, complex indigestible carbohydrates (e.g. cellulose, glucomannans, peptidoglycans), synthetic polymers such as polyvinylpyrrolidone, cholestryamine and alluminosilicates such as bentonite, clay, zeolite, montmorillonite and phyllosillicates (Avantaggiato et al., Reference AVANTAGGIATO, SOLFRIZZO and VISCONTI2005).

Activated carbon has excellent quality of adsorption, due to its large surface area, and is considered to be a general binder for a variety of toxicities of digestive system (The Merck Veterinary Manual, Ninth Ed.). The first study on activated carbon showed a reduction in aflatoxicosis in goats (Hatch et al., Reference HATCH, CLARK, JAIN and WEISS1982). Activated charcoal is associated with reducing the levels of aflatoxin residues within cow's milk (Galvano et al., Reference GALVANO, PIETRI, BERTUZZI, FUSCONI, GALVANO, PIVA and PIVA1996), however it is needed in higher quantities to facilitate this (Diaz et al., Reference DIAZ, HAGLER, BLACKWELDER, EVE, HOPKINS, ANDERSON, JONES and WHITLOW2004). Silicate is another class of mycotoxin binders (Phillips et al., Reference PHILLIPS, SARR, CLEMENT, KUBENA and HARVEY1991; Bingham et al., Reference BINGHAM, PHILLIPS and BAUER2003). The mechanism of action of silicate binders is to chelate the β-dicarboxyl moiety of aflatoxin with free metal ions present in clay material (Phillips et al., Reference PHILLIPS, SARR, CLEMENT, KUBENA and HARVEY1991). Santin et al. (Reference SANTIN, MAIORKA, KRABBE, PAULILLO and ALESSI2002) reported a toxin binding effect of hydrated sodium calcium aluminosilicate against the effects of ochratoxicosis in broilers. Use of cell wall yeast as an adsorbent of mycotoxins in broiler diets can negate the effects of OTA (Santin et al., Reference SANTIN, PAULILLO, NAKAGUI, ALESSI, POLVEIRO and MAIORKA2003). Sodium bentonite can be used to bind AFB1 in broiler feed and has been shown to reduce the symptoms produced due to aflatoxicosis (Kermanshahi et al., Reference KERMANSHAHI, HAZEGH and AFZALI2009; Eraslan et al., Reference ERASLAN, ESSIZ, AKDOGAN, SAHINDOKUYUCU, ALTINTAS and HISMIOGULLARI2005).

use of Silymarin, L-carnitine and vitamins

Products like Silymarin, L-carnitine and vitamins can combat the harmful effects of mycotoxins and can improve the immune system of birds. L-carnitine (LC) is a quaternary ammonium (small water soluble) compound. It is naturally synthesised from the amino acids methionine and lysine. The basic role of LC is to generate energy during lipid breakdown, whereby it transports fatty acids from the cell cytosol to the mitochondria. It has antioxidant qualities and protects against lipid peroxidation (Kopple et al., Reference KOPPLE, DING, LETOHA, IVEANYI, QING, DUX, WANG and SONKODI2002). L-carnitine has the ability to bind AFB1, thereby limiting toxin interactions with tissue proteins and DNA (Yatim and Sachan, Reference YATIM and SACHAN2001). An adverse effect of toxin exposure is oxidative stress and this can be reduced by using LC as a supplement (Citil et al., Reference CITIL, GUNES, ATAKISI, AYLA, TUZCU and DOGAN2005). Similar effects have been reported by Sener et al. (Reference SENER, PASKALOGLU, SATIROGLU, ALICAN, KACMAZ and SAKARCAN2004) in rats. L-carnitine has beneficial uses in liver disorders (Yapar et al., Reference YAPAR, KART, KARAPEHLIVAN, ATAKISI, TUNCA, ERGINSOY and CITIL2007) and long lasting impact upon the immune responses and lymphoid organs of Leghorn birds (Deng et al., Reference DENG, WONG and NOLAN2006). It ameliorates the toxicological changes induced by ochratoxicosis in white Leghorn cockerels (Abidin, Reference ABIDIN2010), as well as improving immune responses (Bhatti, Reference BHATTI2010). The compound may improve other metabolic disorders of broilers (Buyse et al., Reference BUYSE, JANSENS and DECUYPERE2001).

Silymarin is an extract of Silybum marianum (milk thistle plant or lady's thistle plant). Silymarin has been used as remedy for the treatments of cancers and hepatic disorders in humans (Wen et al., Reference WEN, DUMAS, SCHRIEBER, HAWKE, FRIED and SMITH2008; Luper, Reference LUPER1998; Johnson et al., Reference JOHNSON, HE, OSUCHOWSKI and SHARMA2003). It has various beneficial effects including promoting DNA and RNA synthesis and regeneration of liver tissue (Fraschini et al., Reference FRASCHINI, DEMARTINI and ESPOSTI2002). It has been reported to stimulate immune status (Wilasrusmee et al., Reference WILASRUSMEE, KITTUR, SHAH, SIDDIQUI, BRUCH, WILASRUSMEE and KITTUR2002) and can inhibit the release of tumour necrosis factor (TNF)-α induced by OTA from isolated Kupffer cells and perfused liver of rats (Al-Anati et al., Reference AL-ANATI, ESSID, REINEHR and PETZINGER2009). Silymarin can help negate the toxic effects produced by AFB1 in broilers (Tedesco et al., Reference TEDESCO, STEIDLER, GALLETTI, TAMENI, SONZOGNI and RAVAROTTO2004). As for LC, it has antioxidant activity (Katiyar, Reference KATIYAR2005), which, as discussed above, can be useful in mycotoxicosis.

Vitamin E is an excellent antioxidant with a major role in preventing lipid peroxidation. It has the ability to enhance the immune status of the birds which are immune-suppressed by mycotoxin exposure (Hoehler and Marquardt, Reference HOEHLER and MARQUARDT1996). This vitamin improves the overall performance of the birds (Hossain et al., Reference HOSSAIN, BARRETO, BERTECHINI, RIOS and SILVA1998), and negates cellular toxicity produced by OTA (Baldi et al., Reference BALDI, LOSIO, CHELI, REBUCCI, SANGALLI, FUSI, BERTASI, PAVONI, CARLI and POLITIS2004), as well as preventing the formation of toxin-DNA adducts (Grosse et al., Reference GROSSE, CHEKIR-GHEDIRAB, HUCA, OBRECHT-PFLUMIOC, DIRHEIMERC, BACHAB and PFOHL-LESZKOWICZ1997).

control of mycotoxicosis

The poultry feed industry suffers the greatest economic losses compared to other animal species due to mycotoxins (Fraga et al., Reference FRAGA, CURVELLO, GATTI, CAVAGLIERI, DALCERO and DA ROCHA ROSA2007), as it heavily reliant on cereals. Due to this, control strategies should be adapted to minimise the exposure of birds to mycotoxins. Control strategies are adapted at pre-harvest, harvest and post-harvest levels. The hazard analysis critical control point (HACCP) system can be used to manage the risk of mycotoxins throughout the production chain (Bricknell et al., Reference BRICKNELL, NG and BLANEY2006). A recent review by Chulze (Reference CHULZE2010) described different strategies to control mycotoxins from entering into food chain. At pre-harvest stage, use of antifungal peptides in crops is the most promising effort to combat aflatoxin contamination in feed (Rajasekaran et al., 2009).

There are three main methods to control mycotoxins: physical, chemical and biological (Huwig et al., Reference HUWIG, FREIMUND, KAPPELI and DUTLER2001). Physical methods include segregation of contaminated from sound grains and using UV light or gamma radiation (Basappa and Shanta, Reference BASAPPA and SHANTA1996). Use of mycotoxin binders are typically classified as physical methods for the control of mycotoxins (Huwig et al., Reference HUWIG, FREIMUND, KAPPELI and DUTLER2001).

Chemical methods include treatment of feed with ozone (Sousa et al., Reference SOUSA, FARONI, GUEDES, TOTOLA and URRUCHI2008), acidic and alkaline substances (Davegowda and Murthy Reference DAVEGOWDA and MURTHY2005), ammonia (Marth and Doyle Reference MARTH and DOYLE1979) and antioxidants (Nesci et al., Reference NESCI, FERRARI and ETCVHEVERRY2008). Ozone, ammonia and organic acids have been shown to be effective against aflatoxin and ochratoxin (McKenzie et al., Reference MCKENZIE, SARR, MAYURA, BAILEY, MILLER, ROGERS, NORRED, VOSS, PLATTNER, KUBENA and PHILLIPS1997: Marin et al., Reference MARIN, MAGAN, ABELLANA, CANELA, RAMOS and SANCHIS2000: Marth and Doyle, Reference MARTH and DOYLE1979). Antioxidants like butylated hydoxyanisole (BHA), butylated hydroxytoluene (BHT), praben (PP) and their mixtures have a negative impact on aflatoxins and ochratoxins (Torres et al., Reference TORRES, RAMIREZ, ARROYO, CHULZE S.N. and MAGAN2003).

Biological methods include use of microorganisms (probiotics) against mycotoxins present in feed. These probiotics form a chelated complex with mycotoxins and prevent absorption of mycotoxins by the body (Santillan et al., Reference SANTILLAN, BUJAIDAR, MARQUEZ and REYES2006). Probiotic bacteria such as Lactobacillus rhamnosus (Rawal et al., Reference RAWAL, KIM and COULOMBE2010) and Nocardia corynebacteroides (Tejada-Castaneda et al., Reference TEJADA-CASTANEDA, AVILA-GONZALEZ, CASAUBON-HUGUENIN, CERVANTES- OLIVARES, VASQUEZ-PELAEZ, HERNANDEZ-BAUMGARTEN and MORENO-MARTINEZ2008) can be used to reduce the lesions caused by aflatoxins and other mycotoxins in broilers.