Mycotoxins are secondary metabolites produced by certain filamentous microscopic fungi, which occur naturally in the environment and can commonly grow on a variety of crops, including wheat, maize and soybean (Goswami and Kistler, 2004; Murphy et al. 2006; Marin et al. 2013). They comprise a group of several hundreds of chemically different toxic compounds (Sweeney and Dobson, 1998) and their occurrence in cereal grains and animal feed have been reported worldwide (Placinta et al. 1999). Food and feedstuffs prepared using mycotoxin-containing crops deteriorate nutritional content and represent a potential risk for animal and human health (Hussein and Brasel, 2001; Bennett and Klick, 2003). The mycotoxins are cytotoxic, disrupting various cellular structures such as membranes, and interfering with vital cellular processes such as protein, RNA and DNA synthesis (Guerre et al. 2000). They destroy the tissues by oxidizing proteins, have immunosuppressive effects and increase diseases incidence (Kumar et al. 2008). Mycotoxins may cause fever (Cannon et al. 1982), gastrointestinal problems, internal bleeding, haemorrhages or bruising, stomach ulcers (Aziz et al. 1995), mouth sores, kidney or liver damage (Szilágyi et al. 1994), central nervous system problems (Gabal et al. 1986), immune-suppression (Richard et al. 1991; Kumar et al. 2008), tumour-genesis, eye and lung problems, hypertrophy of the adrenal cortex, reproductive organ problems (Szilágyi et al. 1994), damage to the heart muscle, tachycardia, skin problems (Fairhurst et al. 1987), bone marrow and spleen problems (Niyo et al. 1988), blood abnormalities (Mizutani et al. 1997), rectal prolapses and increased vascular fragility. In addition to the toxic effects, a mycotoxin contaminated diet may lead to other consequences like feed refusal, poor feed conversion and diminished body weight gain (Council on Agricultural Science and Technology, 1989; Council on Agricultural Science and Technology, 2003; Kolpin et al. 2014) which are responsible for great economical losses. These effects, in both humans and animals, depend on a number of factors including intake levels, duration of exposure, toxin species, mechanisms of action, metabolism, and defense mechanisms (Galvano et al. 2001; Hussein and Brasel, 2001). The most frequent sources of mycotoxins are the fungal genera Aspergillus, Fusarium, and Penicillium (Bennett and Klich, 2003). While Fusarium species are destructive plant pathogens producing mycotoxins before or immediately post harvesting, Penicillium and Aspergillus species are more commonly found as contaminants of commodities and foods during drying and subsequent storage (Sweeney and Dobson, 1998). Weather conditions, including moisture level and temperature have greatest influence on mold growth and mycotoxin production: while Aspergillus prefers warmer tropical areas, Fusarium and Penicillium also grow in European temperate areas (Bryden, 2012). Rabbit feed ingredients that constitute complete feed products are derived from different raw materials and the contamination of feed materials would represent an important potential hazard. Contamination of animal feed with mycotoxins still occurs very often, despite great efforts in preventing it. Multitoxin studies on animal feed reported that 75-100% of samples analysed contained more than one mycotoxin (Streit et al. 2012; Schatzmayr and Streit, 2013). Two environmentally important mycotoxins, which have gained an immense importance due to their biological effects and widespread toxicity, are ochratoxin A (OTA) and aflatoxin B1 (AFB1). WHO-IARC, (1993) designated AFB1 as group-1 and OTA as group-2B carcinogen (Hussein and Brasel, 2001) and are most frequently detected in the agricultural commodities (Bennett and Klich, 2003; Gabarty and Abou, 2016; Jan et al. 2017). Aflatoxins are a family of extremely toxic, mutagenic and carcinogenic compounds produced by fungi belonging to genus Aspergillus flavus and Aspergillus parasiticus (Jacobsen et al. 2007; Cortes et al. 2010); ochratoxins, potentially as important as the aflatoxins (Bennett and Klich, 2003), are commonly produced by two species of fungi, Penicillium verrucosum Dierckx and Aspergillus ochraceus Wilhelm (Frisvad and Samson, 1991). The alarming feature of mycotoxins is their occurrence in combination and in low dose levels frequently in cereals / feedstuffs which may exert a greater degree of damage (additive or synergistic) in comparison to the individual effects (Roberts et al. 1981; Raina and Singh, 1991; Adebajo, 1993; Sundaram et al. 1999; Prabu et al. 2013). The toxicity and clinical signs observed in animals when more than one mycotoxin is present in feed are complex and diverse (Prabu et al. 2013). The effects observed during multiple mycotoxin exposure can differ greatly from the effects observed in animals exposed to a single mycotoxin (Huff et al. 1988). Animals are usually exposed to mycotoxins through their diet and depending on different factors such as age, sex, route of administration: this can result in acute, sub-acute or chronic mycotoxicosis. Rabbits are highly susceptible to mycotoxins and they can have very pervasive yet sub-clinical effects on their health which very often remain unnoticed: when the clinical symptoms of mycotoxin poisoning can be observed, significant damage has already occurred. The aim of this review is to summarize the toxico-pathological effects caused by the most widespread mycotoxins (aflatoxins and ochratoxin and their interactions), potentially hazardous ingredients of rabbit feed.
Aflatoxins are a group of naturally occurring carcinogens that are known to contaminate different food stuffs causing serious consequences in human and animal health. Aflatoxins have been reported to affect the various body organs like liver, kidneys, lungs, brain, testes and many endocrine and exocrine organs, the heart, skeletal muscles and the different body systems (Bbosa et al. 2013). They are produced by fungi belonging to genus Aspergillus flavus and Aspergillus parasiticus (Jacobsen et al. 2007; Cortes et al. 2010). The four major known aflatoxins include AFB1, AFB2, AFG1, and AFG2 where the “B” and the “G” refer to the blue and green fluorescent colors produced under UV light on thin layer chromatography platres, while the subscript numbers 1 and 2 indicate major or minor compounds (Thrasher, 2012; Bbosa et al. 2013). The aflatoxins display potency of toxicity, carcinogenicity, mutagenicity in the order of AFB1> AFG1> AFB2>AFG2 (Cortes et al 2010). AFG2 occurs in high quantities though less toxic while AFB1 is the most toxic of all the aflatoxin. The World Health Organization (WHO) classifies AFB1 as a class 1 carcinogen (WHO-IARC, 2000). Aflatoxicosis is a condition caused by aflatoxins which can result in acute, sub-acute or chronic mycotoxicosis. The acute primary aflatoxicosisis is produced when moderate to high levels of aflatoxins are consumed; the chronic primary aflatoxicosis results from ingestion of low to moderate levels of aflatoxins (USAID, 2012) Animals get exposed to aflatoxins by two major routes: (a) by direct ingestion of aflatoxin-contaminated foods (Agag, 2004) and (b) by inhalation of dust particles of aflatoxins, especially AFB1 in contaminated foods in industries and factories (Coulombe, 1994). The lethal toxicity of AFB1 varies in different animals. Rabbits are highly susceptible to aflatoxins having the least median lethal dose (LD50) of any animal species studied (Prabua et al. 2013; Cardona et al. 1991; Lakkawar et al. 2004). AFB1 as low as 15 mg/kg feed causes high levels of morbidity and mortality (Makkar and Singh, 2009; Mezes and Balogh, 2009). Experimental AFB1 toxicosis is known to cause alterations in enzyme levels along with patho-anatomical changes in vital organs (Clark et al. 1980; Abdelhamid et al. 1990; Sahoo et al. 1993). Short exposure to large doses of aflatoxins produces acute toxicity including fever oedema, vomiting, abdominal pain, inappetance, lethargy ataxia, rough hair coat and pale potentially fatal liver failure (Lakkawar et al. 2004). The prolonged rabbit feeding of AFB1 in a diet, beyond 40 days, results in cumulative toxicosis which is manifested by altered clinical signs. Symptoms are characterized by anorexia, diarrhoea, decrease in body weight gain, lethargy, fur chewing and dehydration (Clark et al. 1980; Abdelhamid et al. 1990; Prabu et al. 2013; Lakkawar et al. 2004). It has been reported that a decrease in body weight is one of the earliest indicators of clinical aflatoxicosis in animals (Clark et al. 1980). The gastrointestinal tract (GIT) is the main route of entry of aflatoxins as a result of consumption of aflatoxin-contaminated foods especially AFB1. Orally-ingested AFB1 is most efficiently absorbed from the small intestine at the duodenum (Kumagai, 1989) and the effects of AFB1 on the GIT are indirect following alterations in the liver’s detoxification mechanism and a possible reduction in nutrient uptake (Stetinova et al. 1998). The liver and kidneys are the most affected organs followed by stomach, intestine, lungs, heart, spleen, gonads, thyroid and brain (Lakkawar et al. 2004; Prabu et al. 2013). AFB1 is a potent hepatotoxic and hepato-carcinogenic mycotoxin which requires bio-activation to an active AFB1-8,9-epoxide (Essigmann et al. 1982) which binds to DNA resulting in abnormal cellular proliferation, leading to mutagenesis and carcinogenesis (Guengerich, 2001). AFB1 is metabolized in the liver by the cellular cytochrome-P450 enzyme system to form the reactive intermediate, AFB1-8, 9- epoxide, which in turn reacts with macromolecules such as lipids and DNA, leading to lipid peroxidation and cellular injury (Stresser et al. 1994). The mitochondrial damage following aflatoxicosis can result in a decrease in the oxidation of fats by these organelles, with a concomitant accumulation of lipids, necrotic changes, fibroblastic proliferation, mononuclear cellular infiltration, bile duct hyperplasia (Sahoo et al. 1993; Mclean and Dutton, 1995; Lakkawar et al. 2004). Icterus observed in terminal stages may be due to an increase in the levels of bilirubin as a sequela to hepatic necrosis and cholestasis resulting from decreased cytochromed P450, increased heme oxygenase and biliverdin reductase activities along with an increase in heme catabolism in the liver (Guerre et al. 1997). AFB1 has been reported to cause pallor discoloration of liver and hepatomagaly, congestion of liver parenchyma, cytoplasmic vacuolation or fatty change of hepatocytes, necrosis of hepatocytes and newly formed bile ducts (Krishna et al. 1991; Sahoo et al. 1993; Churamani et al. 1995; Singh et al. 1999; Vinita et al. 2003; Lakkawar et al. 2004). Haemolytic anaemia and strong cytotoxic effects have been also observed (Verma and Metha, 1998). The renal lesions are secondary to those observed in the liver. The target site of action of AFB1 is the glomerular region causing reduction in the glomerular filtration rate and glucose reabsorption (Glahn et al. 1991). Kidneys appear congested, slightly enlarged and show a moderate degree of nephrosis (Ahmed et al. 2012). The urinary bladder appears distended with tick yellowish turbid urine (Lakkawar et al. 2004). Aflatoxin have reported to have serious acute effects on the respiratory systems. Besides the liver, the lung and trachea are also capable of activating AFB1 and rabbit lung and tracheal microsomes show high activity for this reaction (Daniels et al. 1990). Bio-activation related toxicity of AFB1 has also been observed in tracheal mucosa following intra-tracheal instillation of AFB1 in rabbits (Mezes, 2008; Coulombe et al. 1986). The pulmonary inflammation and oedematous changes observed might also be due to the production of eicosanoids stimulated by the AFB1. This might have also contributed to the manifestation of dyspnoea in the early stages of toxicosis (Massey et al. 1995; Lakkawar et al. 2004). Furthermore, aflatoxins have been reported to disrupt the reproductive system in both male and female animals after ingestion of aflatoxin-contaminated foods. Short exposure to large doses of aflatoxins produces acute toxicity; sub-symptomatic exposure to aflatoxins is known to produce male reproductive toxic effects with several manifestation. The principal target organ in causing male reproductive toxicity is the testis and various aspects of spermatogenesis and androgen biosynthesis are affected (Baker and Greene, 1987; Sahoo et al. 1993). However, the epididymis and vas deferens also are a target for the action of such reproductive toxicants (Akbarsha et al. 2000). The severity of pathological changes in epididymis are aflatoxins (AF) dose dependent and summarized as interstitial oedema and atrophy of epididymal tubules, associated with congestion of the blood vessels and capillaries. The seminiferous tubules show degeneration of the epithelium and a reduction in the number of mature spermatids in aflatoxin treated rabbits (Lakkawar et al. 2004). Salem et al. (2001) reported a relative decrease in testes weight and an increase in the number of abnormal/dead sperms following a 9-week administration of sublethal doses of AFB1 to mature male rabbits. Aflatoxins have reported to have serious acute effects on cardiovascular systems including vascular fragility, endothelial injury and haemorrhaging in tissues (Harriet, 2003). Heart can show epicardial congestion (Lakkawar et al. 2004). The coagulation defect and bleeding associated with aflatoxicosis has previously been attributed to either vitamin K antagonism, decreased hepatic protein and coagulation factor synthesis or disseminated intravascular coagulation secondary to hepatocellular degeneration and necrosis (Baker and Greene, 1987). In the brain or central nervous system, the neurons have a high metabolic rate but little capacity for anaerobic metabolism and subsequently, inadequate oxygen flows to the brain kills the neuronal brain cells within minutes. Aflatoxins and its metabolites and other products such as the reactive oxygen species (ROS) like the AFB-8,9-epoxides may interfere with the normal functioning of the nerve cells by forming DNA adducts, protein adducts, oxidative stress factors, mitochondrial directed apoptosis of the nerve cells as well as inhibiting their synthesis of protein, RNA and DNA (Bbosa et al. 2013). AFB1 is known to alter the distribution of acetylcholine esterase (AChE) in the brain affecting cholinergic transmissions at the nerve endings and thus can result in manifestations of nervousness and behavioural deficiency (Egbunike and Ikegwuonu, 1984; Lakkawar et al. 2004). Sahoo et al. (1993) reported vascular congestion and focal mononuclear infiltration in the meninges along with perivascular cuffing, mild neuronal degeneration and gliosis following the oral administration of AFB1 to New Zealand White Rabbits at the rate of 0.0625 mg/day/animal for a period of 30 days. AFB1 is recorded to be teratogen in rabbits (El-Nahla et al. 2013). The dose of 0.1 mg/kg AFB1 body weight AFB1is the minimum teratogenic dose that interfere with intrauterine development during 6th-18th days of gestation (Wangikar et al. 2005).
Ochratoxin, commonly produced by two species of fungi, Penicillium verrucosum and Aspergillus ochraceus (Frisvad and Samson, 1991; Ostry et al. 2013; Saleemi et al. 2015; Valtchev et al. 2015), is potentially as important as the aflatoxins (Bennett and Klich, 2003). The most abundant and most toxic mycotoxin within the ochratoxins is ochratoxin-A (OT-A) (Marquardt and Frohlich, 1992), which occurs in maize, cereal grains such as wheat and barley and oil seeds such as soybean and peanuts (Manning et al. 2003). The OT-A is toxic to numerous animal species, the kidney being the main target organ in birds and mammals, but not adult ruminants (Pfohl-Leszkowicz and Manderville, 2007). The most consistent effect of ochratoxicosis in different species including rodents, guinea pigs, swine and poultry is growth depression (Thacker and Carlton, 1977; Dwivedi and Burns, 1985; Stein et al. 1985; Dwivedi and Burns, 1986; Rati et al. 1991; Harvey et al. 1994). OT-A is well known nephrotoxic, hepatotoxic, immunosuppressive, mutagenic, cardio-vascular toxic, teratogenic and possible human carcinogen (Ahmed et al. 2012; Hussain et al. 2016). In addition to these effects, OT-A is also considered as potent myelotoxic agent (Moura et al. 2004). OT-A is also extremely cytotoxic and may cause red blood cell haemolysis in rabbits (Zofair et al. 1996). OT-A at low doses influences energy metabolism such as carbohydrate, amino acid, cofactors and vitamins. However, in the high doses pathways influenced by OT-A are associated with the different body systems including circulatory, digestive, endocrine, excretory system (Jan et al. 2017). Rabbits are comparatively more susceptible to OT-A than mice, rats and guinea pigs (Ponnuchamy, 2000). OT-A has been observed to be acutely toxic to young rabbits with LD50 of 10 mg OT-A/kg body weight (Mir et al. 1999). Long term exposures have been observed to favor accumulation in the tissues and cause severe impairment of health, wide spread pathoanatomical alterations, and even death (Mir and Dwivedi, 2000; Mir and Dwiwedi, 2010). OT-A has been known to be actively reabsorbed through the proximal convoluted tubules (Stein et al. 1985). OT-A produces significant nephrotoxicity with the pale, soft and enlarged kidneys showing discoloured foci over the surface. The proximal tubule of the kidney is the primary site targeted in OT-A induced nephrotoxicity (Suzuki et al. 1975). The reason behind the high sensitivity of kidneys to the toxin might be correlated to the fact that the kidneys constitute the primary excretory pathway for OA and its heavy blood circulation (25% of cardiac output) (Marquardt and Frohlich, 1992). Testis, being an organ with rapid meiotic and mitotic cell divisions, could be a possible target of OT-A as it inhibits DNA, RNA and protein synthesis (Marquardt and Frohlich, 1992). OT-A (~99%) is bound to plasma proteins, mainly albumin, which can’t be excreted by glomerular filtrate. However, unbound portion (~1%) can be found in the urinary filtrate. The remaining OT-A is only execrated via organic anion transporter (OAT) route, which prone the proximal tubular epithelial cells to damage, by virtue of depletion of indigenous dicarboxylic acid (glutarate, ketoglutarate) on expense of OT-A internalization (Sekine et al. 2006; Khatoon et al. 2016). Several natural and experimental OT-A exposure studies have recorded similar changes in the kidney function (Khan et al. 2014). The increase in the serum biomarkers of kidneys damage, and the mechanistic nephrotoxicity associated with the OT-A, has also been augmented by histological alteration in the tubular cells of nephrons (Jan et al. 2017). Microscopically, kidneys reveal degeneration of the proximal convoluted tubules and the testes atrophic (Prabu et al. 2013). OT-A has been reported to cause direct effects on lymphocytes and plasma cells in primary and secondary lymphoid organs (Dwivedi and Burns, 1985). OT-A has also been reported to be teratogenic in rabbits (Wangikar et al. 2005), rats (Still et al. 1971; Brown et al. 1976; Mayura et al. 1982; Abdel-Wahhab et al. 1999), mice (Hayes et al. 1974; Arora and Fr¨ol, 1981), hamsters (Hood et al. 1976). Teratogenic effects were found among the 0.1 mg/kg dose group in the form of a significant increase in the incidence of gross anomalies (wrist drop, rudimentary tail, knuckling of fetlock and agenesis of tail), skeletal anomalies (agenesis of caudal vertebrae, incomplete ossification of skull bones and wavy ribs) and soft tissue anomalies (internal hydrocephalus, microphthalmia and kidney agenesis). Ochratoxicosis induces also biochemical alterations in the rabbits (Mir and Dwiwedi, 2010).
Aflatoxins and ochratoxins interaction
The alarming feature of mycotoxins is their occurrence in combination and in low dose levels frequently in cereals/feedstuffs which may exert a greater degree of damage to animal health (additive or synergistic) in comparison to the individual effects (Roberts et al. 1981; Raina and Singh, 1991; Adebajo, 1993; Sundaram et al. 1999; Prabu et al. 2013). The effects observed during multiple mycotoxin exposure can differ greatly from the effects observed in animals exposed to a single mycotoxin (Huff et al. 1988). Simultaneous occurrence of these two mycotoxins in feedstuffs under natural conditions was observed during screening of different food and feed stuffs (Mir, 1998). Although the scientific literature offers a broad variety of information on the effects of individual mycotoxins in various animal species, concurrent exposure to multiple mycotoxins is more likely in the livestock industry (Zaki et al. 2012). Occurrence of spontaneous aflatoxicosis and ochratoxicosis, individually and in interaction, the combined presence of low dose levels of AFB1 ranging from 0.03 to 2.06 ppm and OT-A ranging from 0.01 to 1.23 ppm in cereals/feedstuffs have been frequently reported in different species of animals (Raina and Singh, 1991; Dwivedi et al. 2004). There exists an interaction in terms of synergism in the toxicological effects when both the chemicals are fed together. Heavy mortalities of up to 75% in German Angora rabbits in Kangra district of Himachal Pradesh (India) were reported due to simultaneous spontaneous aflatoxicosis and ochratoxicosis with high levels of AFB1 and OT-A in the feed (Sharma, 1998). In experimental simultaneous exposure to OT-A e AFB1 in rabbits, during long term feeding of AFB1 and OT-A in combination, the intensity of clinical symptoms was comparatively more severe as than when either of these mycotoxins was given alone (Doerr and Huff, 1981; Wangikar et al. 2005; Prabu et al. 2013). The changes were more severe as evidenced by gross, histological, ultra structural, immunological and enzyme antioxidant changes observed both in liver and kidneys, suggesting an additive interaction of AFB1 and OT-A in rabbits (Prabu et al. 2013). Similar presence of changes both in the liver and kidney has been reported by several workers in guinea pigs (Richard et al. 1975). OT-A and AFB1, when administered in combination in pregnant rabbits, resulted in significant increase in the incidence of various fetal anomalies and post implantation losses and decreased fetal weights (Wangikar et al. 2005). Testis revealed severe disruption of the normal spermatogonial cell pattern and marked atrophic changes (Prabu et al. 2013). Similar results were observed in previous investigation with different combinations of OT-A and AFB1 in rats (Wangikar et al. 2004b; Wangikar et al. 2004c), suggesting that in combination, these toxins might have possibly antagonistic interaction. Mycotoxins comprise a group of several hundreds of chemically different toxic compounds (Moss, 1996; Rotter et al. 1996; Sweeney and Dobson, 1998) and their occurrence in cereal grains and animal feed have been reported worldwide (Placinta et al. 1999). Food and feedstuffs prepared using mycotoxin-containing crops deteriorate nutritional content and represent a potential risk for animal and human health (Hussein and Brasel, 2001; Bennett and Klick, 2003). Consumption of a mycotoxin contaminated diet may induce acute and long-term chronic effects resulting in teratogenic, carcinogenic, and oestrogenic or immune-suppressive effects. Direct consequences of consumption of mycotoxin contaminated animal feed include: reduced feed intake, feed refusal, poor feed conversion, diminished body weight gain, increased disease incidence (due to immune-suppression), and reduced reproductive capacities (Fink-Gremmels and Malekinejad, 2007; Morgavi and Riley, 2007; Pestka, 2007; Voss et al. 2007) which leads to economic losses (Huwig et al. 2001; Wu, 2004; Wu, 2006). It was widely reported that aflatoxins affect the various body organs like liver, kidneys, lungs, brain, testes and many endocrine and exocrine organs, the heart, skeletal muscles and the different body systems (Bbosa et al. 2013). In particular, rabbits are highly susceptible to aflatoxins having the least median lethal dose (LD50) of any animal species studied (Prabua et al. 2013; Cardona et al. 1991; Lakkawar et al. 2004). AFB1 as low as 15 mg/kg feed causes high levels of morbidity and mortality (Makkar and Singh, 2009; Mezes and Balogh, 2009). OT-A has been observed to be acutely toxic to young rabbits with LD50 of 10 mg OT-A/kg body weight (Mir et al. 1999). Rabbits are comparatively more susceptible to OT-A than mice, rats and guinea pigs (Ponnuchamy, 2000). Long term exposures have been observed to favor accumulation in the tissues and cause severe impairment of health, wide spread pathoanatomical alterations, and even death (Mir and Dwivedi, 2000; Mir and Dwiwedi, 2010). OT-A has been known to be actively reabsorbed through the proximal convoluted tubules (Stein et al. 1985). Multi-toxin occurrence may be one important explanation for divergences in effect levels described in the scientific literature, where defined, mostly purified mycotoxins are used in most studies. In field outbreaks, naturally contaminated feeds may contain multiple mycotoxins and thus apparently lower contamination levels of a single specific mycotoxin can be associated with more severe effects (Zaki et al. 2012). The presence of AFB1 and OT-A, both alone and in interaction at very low dose levels of 0.5-1 ppm in animal feeds and cereals can cause significant toxicity in rabbits with gross and histological changes and might be a potential threat to animal health (Prabu et al. 2013).
Based on literature review and data available, it has to be pointed out that far more work has to be done on this particular research field, especially in case of mycotoxins sub-acute contamination range as well as with combinations of more than two toxins.