Mycotoxin research

This review article illustrates the efforts which were done throughout thirty  two years in the field of mycotoxins; whether for developing the detection methods of some mycotoxins, detection of natural occurrence of some mycotoxins, preserving methods against fungal invasion and mycotoxins production, describing the toxic symptoms of some mycotoxicoses by different animal species, or treating attempts to overcome some mycotoxicoses often found under Egyptian conditions.


INTRODUCTION

The actual begin of the mycotoxins' story was in the early 1960s in the United Kingdom by discovering the aflatoxins. Thereafter, many other mycotoxins were discovered through improving the detection methods ^{(1, 2, and 47)}.  So, intensive researches all over the world were carried out to detect these mycotoxins in various commodities ^{(3, 4, 5, 6, 7, 8, 9, 12, 14, 15,17,19,20, 22, 36, 50, 59, 60, 73, 80, 84, and 86)}.  Survey of toxigenic fungi in the environment was done too, besides studying the environmental conditions, which are required for these fungi to produce their toxins ^{(65, 68, 69, and 70)}.   This step was followed by studying the harm effects of mycotoxicoses in different animal species and human being, whether those caused by the individual or combined mycotoxins ^{(1, 10, 11, 13, 23, 24, 26, 29, 30, 33, 34, 37, 48, 49, 52, 53, 54, 57, 61, 71, and 79)}.  Recently, attempts were investigated to eliminate, detoxify or alleviate the toxic effects of mycotoxins ^{(16, 35, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 51, 55, 56, 58, 62, 63, 64, 66, 67, 81, 82, and 83)}.  

In the eighties and nineties of the last century mycotoxins were not really considered a threat, merely because the detection methods were not sufficient enough to track them. Nowadays however these secondary metabolites of various fungi and moulds have become an inextricable part of animal nutrition. Whether it has to do with the larger occurrence or better detection methods it is a problem in the whole animal feed industry and on a global scale. It is clearly recognized that these toxins, when present in raw materials and feeds, have clear detrimental effects on animal health and performance. One distinguishes two different categories of economic losses that apply regarding mycotoxins in animal feed. The first is direct market losses for feeds that are rejected, because the mycotoxin levels are too high for animal consumption, based on existing regulations for maximum allowable levels in animal feed (interesting is that there is quite a difference in these levels between European and American maximum tolerable levels). The second is indirect market losses caused by animals consuming mycotoxins in their feed that may cause a variety of adverse health effects, which would translate to losses to producers. Analysis of the US market revealed that as a result of mycotoxin contamination some US$342 million was lost due to rejected feedstuffs. Animal health losses due to mycotoxin intoxication were estimated at another $8.4 million. For maize alone, one estimated the annual costs in the US to be $163 million for aflatoxin-related losses, $40 million for fumonisin-related losses, and $52 million for DON-related losses. In less developed countries the economic losses are at the same or at higher levels, especially in the warmer, more humid Asian countries. The losses in these countries are more heavily weighted toward direct animal health effects rather than rejected feedstuffs. In terms of future risks, one recognizes the increased use of ethanol co-products such as DDGS in animal feeds as an important factor to take into account. DDGS contain even higher levels of mycotoxins than the original grain, due to the 'condensing' effect of ethanol distilling. Mycotoxins are not affected by the process and thus after the starch is removed from the corn to make ethanol the same volume of mycotoxins remains in a smaller volume of co-products. Wu also stated that "in the near future, there is reason to believe that increased climate variability associated with climate change trends may result in higher pre-harvest levels of mycotoxins in grains worldwide." All-in-all it is clear that mycotoxins have evolved from an underestimated and almost undetectable risk to a clear and present danger to animal and human health. Fortunately many companies have also recognized this threat and are well underway to combat this menace of the feed industry ⁽⁸⁹⁾.

Chromatographic methods of detection are the main means of mycotoxins estimation; so, two different methods were developed for quantitative determination of ochratoxin-A ⁽⁸⁸⁾ and fusariotoxins (zearalenone and vomitoxin) ⁽⁸⁷⁾, respectively using high performance liquid chromatography.  These methods are rapid and specific for various foods and feed stuffs.  The first one has an average recovery of about 50% in a range of 50 - 500 ppb and detection limit of 5 ppb ochratoxin-A ⁽⁸⁴⁾.  The second method has detection limit of 2 ppb zearalenone and 70 - 80% recovery, whereas the detection limit in mixed feed with interfering substances was 25 ppb with 25 - 35% recovery ⁽⁸⁷⁾. However, thin layer as well as high performance liquid (or gas) chromatographic methods ⁽²⁾ were developed for simple, rapid and accurate quantitative determinations of aflatoxins, ochratoxin, citrinin, zearalenone, vomitoxin, T₂ -toxin and diacetoxyscirpenol (Table 1). The optimum conditions for mycotoxins separation by thin layer chromatography were rationalized too ⁽¹⁸⁾. 

OCCURRENCE

About 16% of the tested samples 2009 from the Middle East were contaminated with Aflatoxin, 14% zearalenone, 37% desoxynivalenol, 39% fumonisin, and 67% ochratoxin. 55% of the positive samples from this region were contaminated with more than one mycotoxin ⁽⁸⁵⁾.The following Table 2 presents some cases of the naturally occurred mycotoxins in Egyptian foods and feeds as well as human biological fluids. Peanuts have the highest level of aflatoxins, the contamination rate between their pods' shells/beans was 7/1.  Aflatoxin B₁ may often occur alone (in 76% of the contaminated samples); so, the contamination rate by aflatoxins B₁; G₁: B₂ was 22.4: 2.3: 1 ^{(5 and 14)}.  About ninety percent of the aflatoxin, ochratoxin or zearalenone contaminated samples have a level lower than 100 ppb, whereas the majority (90%) of samples contaminated with vomitoxin have a level higher than 100 ppb ⁽¹⁴⁾. Zearalenone detection was an indicator for other fusariotoxins contamination ⁽⁸⁶⁾. Also, vomitoxin presence was a good indicator for existence of other fusariotoxins such as T-2 toxin, T-2 tetraol, diacetoxyscirpenol and fusarenon-X is a case of multi-contamination. Moreover, there were positive correlations between grains humidity on one side and zearalenone as well as citrinin contamination levels ⁽¹⁸⁾ on the other side.  However, substrate moisture content is the main environmental factor responsible for mycotoxin production ^{(14 and 65)}, besides the substrate it self ^{(14, 59, and 85)}, mechanical damage ⁽⁶⁵⁾ and the incubation period ⁽⁷⁰⁾, e.g. aflatoxin concentration in feedstuffs had started to fall after 3 weeks storage and by 6 weeks had began to disappear ⁽⁷⁰⁾.


Table (2):Natural occurrence of mycotoxins in Egyptian animal feeds and   human foods.

*Liver and kidneys from beef and buffalo calves as well as from sheep, but not camels.
**Blood, urine and vomitus of food borne mycotoxic (poisoning) patients.

Raising moisture content of maize up to 16 - 20% led to reduction of dry matter, nitrogen free extract and the bulk density of the moisten samples and elevation of ash and total count of fungi, even with the presence of different preserving agents; except when chamomile, cinnamon, pepper, maleic acid, potassium benzoate, sodium carbonate and multifungin were added at 1.5%.  These additives prevent fungi to produce aflatoxins as well as ochratoxin-A, while some samples were positive for citrinin and/or zearalenone.  Since the addition of chamomile, cinnamon, potassium benzoate and multifungin individually to corn samples prevented the formation and accumulation of citrinin and zearalenone as well as reduced the fungal population of Fusarium, Penicillium, Aspergillus and Rhizopus ⁽⁶⁵⁾. Autoclaving, drying, sun light, room temperature, cooling and freezing, respectively could decompose aflatoxins, in a descending order according to the destruction rate.  Decontamination tended to decrease by increasing the initial level of the aflatoxins before the treatment.  Yet, the destruction increased in the time course of the treatment or its temperature.  Loss of aflatoxins G was higher than that of aflatoxins B. But, autoclaving affects the appearance (led to hard-sticky food with dark coloration) and digestibility of the treated food ⁽¹⁶⁾. Moreover, autoclaving has restricted apparent destruction rate (41.8 - 42.2%) for aflatoxin B₁ (and lower rate for ochratoxin A, being 11.3 - 27.7%), since the biological test by fish revealed its toxic effects after autoclaving the aflatoxic aqua feed ⁽⁶⁰⁾. Adsorbents reduced aflatoxin level by about 16 - 44%, but autoclaving and using microwave reduced its concentration by 33 and 53%, respectively. High temperature negatively affected appearance, consistency and composition of a treated food. Hydrogen peroxide and ammonization led to reduction of aflatoxin concentration by 38 and 88%, respectively, whereas methanol extraction reduced it by 54% and led to reduction of crude protein ⁽⁴¹⁾.

Moldy diet decreased the feed intake and digestibility and increased the water consumption, particularly water/feed ratio. It led to atrophy of liver and spleen and heaviness of the empty stomach and female genital tract.  It also was responsible for decreased cholesterol / phospholipids ratio and increased calcium/phosphorus ratio in rabbit's serum ⁽¹³⁾. Moldy diet was rich in ash and silica contents, so reduced feed intake and nitrogen and calcium retention by sheep. It led also to decline blood total nitrogen, calcium, calcium/phosphorus ratio and cholesterol/calcium ratio. Urine analysis revealed increases in calcium, magnesium and vitamin C, whereas pH values, total nitrogen and phosphorus were declined.  So, moldy feeds seemed to have toxic, nephritic and hepatic effects ⁽⁵⁴⁾. Mycotoxins are found frequently in different feedstuffs ^{(5, 6, 7, 8, 9, 14, 17, 19, 20, and 32)} and threat not only plant crops, but also animal and human health ^{(21, 23, 24, 25, 27, 28, 29, 30, 31, 36, 37, and 50)}. Aflatoxin residues were found in 36.5% of the tested blood samples (63 samples) from mothers (at the age of 19-36 years) and their children (from Mansoura University Children's Hospital), although the undetectable aflatoxin residues of the mothers' breast milk samples. The mean ± SE of AFB₁ of positive children's blood samples is 66.735 + 16.376 ppb.  While the mean ± SE of AFB₁ of mothers of positive and negative children is (68.013 ± 11.520 and 15.780 ± 0.0 ppb, respectively). AFB₁ level was not affected by child's age, sex, residence (whether rural or urban), maternal age, maternal parity, maternal education nor maternal occupation. AFB₁ in breastfed patients was significantly lower than in non-breastfed (artificially-fed, cow's milk fed or fully weaned) ones (P= 0.034). Weight -z - score (WAZ) showed no significant difference between AFB1 negative and positive case (P= 0.422) while height - z - score (HAZ) was significantly lower in AFB₁ positive compared to AFB₁ negative cases (P= 0.001). A significant negative correlation between AFB₁ concentration and their height-z-score (P= 0.001) while correlation between AFB₁ concentration and WAZ was non-significant (P= 0.185). In conclusion, this study suggests that breast feeding results in lower AFB₁ exposure and that there is a strong association between aflatoxin exposure and impaired growth ⁽⁷⁵⁾.

Sterigmatocystin led to decrease growth of fish and their muscle protein but increase their mortality, serum aminotransferases activity and muscle fat content.  There were pathological changes and sterigmatocystin residues in fish muscles ⁽¹¹⁾.

Dietary aflatoxin negatively affected rats' body gain, feed intake and efficiency, survival rate, blood picture, and relative weights of the internal organs ^{(51 and 66)}. It increased the blood albumin/globulin ratio, hepatic nucleic acids and blood urea concentrations, white blood cells count, and activity of blood alkaline phosphatase and aspartate amino transferase but reduced blood acid-phosphatase activity, iron concentration, and hemoglobin level ⁽⁶⁷⁾. Aflatoxins were causative of decreased feed intake, body weight and survival rate of rabbits.  Relative weights of liver, kidneys, heart and adrenal glands were increased but hemoglobin content, packed cell volume percentage and sedimentation rate were decreased. Aflatoxin led also to increase blood lipids and water content of rabbits' muscles and liver but reduced bone ash and volume.  There were aflatoxin residues in muscles, serum, liver, heart and kidneys with concentration relationships of 51: 24: 3: 2: 1, respectively.  Aflatoxin caused paralysis, disorder of fat deposition, hemorrhages, and pathological changes ⁽⁵³⁾. Aflatoxicosis by rabbits characterized by felt fur, high body temperature, loss of appetite, weakness, bloody diarrhea, nervous movements, paralysis and death. The post-mortem examination revealed presence of internal hemorrhages and congested-enlarged organs.  It lowered the body weight, feed utilization and blood values of hemoglobin, packed cell volume, red blood cells count, glucose and cholesterol. It increased the relative weight of liver and kidneys, the activity of blood transaminases and concentrations of blood urea, uric acid and creatinine. Its residues accumulated in liver > muscles > heart > kidneys at levels proportional to those consumed in the diet ⁽⁶⁴⁾. Low level of dietary aflatoxin negatively affected chicks' body weight, feed conversion, digestibility of nutrients, dressing percentage, relative weights of liver and kidneys, mortality rate, internal gross pathology ^{(46 and 60)} and biochemical characteristics of muscles, liver, bone and blood as well as semen quality ^{(43 and 47)}, besides symptoms of low viability, anorexia, peruses, ataxia, scattered feathering, hemorrhage, cramps, stinked excrements, edema, pale-yellowish liver, friable kidneys, dark colored gall bladder and congestion of glands, lungs and gonads ⁽⁴⁵⁾. Aflatoxic patients suffered from food poisoning ⁽²⁰⁾ with abdominal colic, vomiting, diarrhea and pallor; particularly with groups of younger age, female, students, and from rural areas of low economic status ⁽⁵⁰⁾, and nephropathy ⁽³⁶⁾. Dietary aflatoxin decreased body gain, growth rate, survival rate, feed conversion, nutrients utilization, and muscular protein, but increased carcass fat of intoxicated fish ^{(38, 58, 63, and 66)}.  It increased the internal organs' indices and white blood cells count besides activity of alkaline phosphatase and transaminases and the aflatoxin residues, but decreased hemoglobin concentration, packed cell volume, total protein and red blood cells' count.  Its residues were found in the whole body and tended to decrease after freezing period ^{(39, 58, 63, and 66)}. Aflatoxin reduced the area of fish muscles, caused chromosomal aberrations and lower mitotic index.  Severity of its harmful effects correlated positively with its dietary levels.  Its effects varied between fish sizes ⁽⁶¹⁾. However, all patients and their relatives, unexpectedly, showed negative AFB₁ in their blood. It can be concluded that AFB₁ is not necessarily present in all cases of HCC as a predisposing factor and that other factors as viral hepatitis and bilharziasis are more commonly correlated to HCC ⁽⁷²⁾. Although of the negative blood samples as regard to aflatoxin B₁, it cannot exclude the role of aflatoxin as a contributing factor may causing female infertility, since the toxin has been proved to produce deleterious effects on the reproductive system on animals, and more studies included aflatoxin analysis of ovarian biopsies are recommended to ascertain the generalizability and impact of this relationship ⁽⁷⁴⁾.

Ochratoxicosis-A by rabbits reflected clinical symptoms including feed refusal, increased pulse rate, hard breathing, land lay, and slack. The post-mortem examination revealed presence of blue spots on the lungs; hemorrhagic patches on the esophagus, stomach, intestine, heart, liver, and kidneys; and dilated kidneys. The blood analysis indicated increases in concentrations of creatinine, urea, uric acid, cholesterol and transaminases activity; whereas decrease of blood total proteins concentration was recorded.  Residues of ochratoxin-A were found at highest level in the kidneys followed by liver, blood, muscles, spleen, heart and feces, respectively with higher concentrations in females than in males' tissue.  Yet, lungs, bran, and gonads were ochratoxin-A free. The histological test cleared that the liver suffered from congestion of the central vein, cellular infiltration, and nodular hyperplasia and vacuolization of the hepatocytes. The kidney showed also hyalinization and degeneration of the glomeruli with tubular cell necrosis and degeneration of the proximal and distal tubules ⁽⁵²⁾.

Oxalic acid mycotoxin decreased the rabbits' body weight gain and feed intake and efficiency but increased its level in animals' blood proportionally to its dietary level.  It led to stress symptoms, which tended to be severer by elongation of the feeding period (at the height levels) followed by death.  Post-mortem test revealed presence of inflammation and congestion along the digestive system; blue spots on the esophagus, stomach and intestine, hemorrhagic patches on stomach, intestine, liver, lungs and kidneys; pale lungs; enlarged-dark kidneys and liver; and white crystalline precipitations (gout) in the stomach.  It increased the weights of kidneys and liver but decreased the carcass weight.  It increased also the activity of blood transaminases and the concentrations of blood cholesterol, creatinine and uric acid, but decreased the blood total protein level.  Oxalic acid accumulated in muscles, liver, and kidneys, respectively in descending order with higher levels in male than female organs.  Histopathologically, it led to mild congestion of the portal vein in the portal tract.  Kidneys showed a degeneration of the glomeruli, polymorphonuclear leucocytes infiltration, interstitial nephritis, and oxalate deposition ⁽³⁷⁾.

Zearalenone elevated body weight gain, feed intake, water consumption, digestibility, hemoglobin level, packed cell volume and concentrations of serum calcium, phosphorus and vitamin C in young rabbits.  Yet, the opposite trend was reported in old rabbits fed on diets contaminated with zearalenone.  Zearalenone caused noticeable histopathological changes in liver, kidneys, lungs, heart, adrenal glands, spleen and uterus ⁽⁵⁶⁾. Zearalenone naturally contaminated diet of sheep reduced nitrogen and calcium retention; yet, it increased digestibility of dry matter, crude fiber and energy.  It had toxic, nephritic and hepatic effects ⁽⁵³⁾.

The fumonisin contaminated diet led to neuropathic behavior of the rabbits, reduced muscular content of protein and increased the muscular fat and ash contents. It increased also blood Hb, PCV, RBCs, albumin, AST, ALT, cholesterol, and creatinine. It led to histological alterations in liver, brain, lung and kidney ⁽⁷⁶⁾. A collective scheme of most common negative effects by mycotoxicoses is given in the following Table 3.

ANBi: aflatoxin Bi, ANs: aflatoxins B & G, ONA: ochratoxin A, CN: citrinin, ZN: zearalenone, PA: penicilic acid, RNB: rubratoxin B, SN: sterigmatocyctine, MNs: mixture of mycotoxins, OA: oxalic acid, FN: fumonisin, Clin.Symp.: clinical symptoms, FC: feed conversion, ALP: alkaline phosphatase, RBCs: red blood cells, WBCs: white blood cells, Hb: hemoglobin, Ht: hematocrite, S.: serum, vit.: vitamin, M.: muscular, B.: bone, Phys. Char.: physical characteristics (lean meat %, texture index, feder value).

Elevating dietary protein may alleviate sterigmatocystin toxic effects and residues in fish ⁽¹¹⁾.  Increasing dietary energy was beneficial to overcome the aflatoxic effects on the body weight, feed conversion, dressing percentage, relative weights of liver and kidneys and mortality of the chicks.  The withdrawal of aflatoxic diet improved the dressing percentage and, to some extent, the post-mortem examination ⁽⁴⁶⁾, as well as the biochemical characteristics of chicks' muscles and blood ⁽⁴⁷⁾. Dietary supplementation of selenium and vitamin E alleviated the toxic effects of the aflatoxic diets of mature cocks concerning body weight gain and feed intake due to increased aflatoxin excretion.  Also, vitamins-mixture supplementation and the withdrawal of the contaminated diets lowered the severity of aflatoxicosis by chickens ^{(43 and 45)}.  Dialysis of nephropathy patients led to low content of aflatoxin (and ochratoxin) in their urine, even still higher than in the control people ⁽³⁶⁾.  Dietary inclusion of charcoal in the aflatoxic diet may improve, to some extent, the severity of aflatoxicosis by rabbits ⁽⁵³⁾. Aluminosilicate and tafla addition to the aflatoxic diet lowered-to some extent- the severity of the aflatoxicosis by rabbits.  So, it become clear to what extent is the seriousness of food borne aflatoxicosis by animals and consequently by consumers.  Adsorbents still neither obstacle nor sufficient mean for removing aflatoxin and its toxic effects ⁽⁶⁴⁾. Dietary inclusion of adsorbent (like egg shells and shrimp shells) was useful in reducing the toxic effects of aflatoxin on fish, where it increased the body gain, growth, survival, feed conversion and nutrients utilization. It improved also carcass composition of the aflatoxicated fish ⁽³⁸⁾. Moreover, it increased hemoglobin concentration and red blood cells count and decreased white blood cells count, activity of alkaline phosphatase and transaminases and most of the internal organs' indices.  It alleviated the adverse effects of aflatoxin on the histopathological changes in different organs ⁽³⁹⁾.  Dietary inclusion of egg shell and clay proved to overcome some aflatoxic symptoms by fish ⁽⁶³⁾. Moreover, dietary addition of tafla, ammonia or hydrogen peroxide decreased the deleterious effects of aflatoxic diets on rats' performance and blood parameters.  The effective additive to partially protect against the negative effect of aflatoxin was tafla.  Aflatoxin excreted in feces was higher in rats fed the aflatoxic diet with different additives than in those fed the aflatoxic diet alone.  Yet, the different dietary additives did not remove the toxic effects of aflatoxin.  Therefore, it is to impose upon the concern of prophylaxis against fungal invasion of feed stuffs to prevent mycotoxin production ⁽⁵¹⁾. Dietary Biogen^® inclusion reduced severity of aflatoxin contaminated diet on fish but did not lead to its detoxification.  Thus, it is emphasized to hygienic control of feeds to avoid their fungal invasion by toxigenic fungi, since prophylaxis is more useful than medication ⁽⁵⁸⁾. Dietary ginger inclusion alleviated aflatoxicosis symptoms by fish, followed by aspirin and chamomile flowers, respectively as partial detoxifying agent of aflatoxin ⁽⁶⁶⁾.  However, the treatment with medical herbs, i.e. safflower, black cumin, ginger, thyme, garlic, rosemary and parsley improved the immunity of aflatoxic rats and prevented the existence of residual aflatoxin; yet, all these additives histologically altered each of liver and kidney of the animals.  No one of the tested medicinal herbs completely overcome the effects of food borne aflatoxicosis ^{(67 and 77)}. Glutathione and glutathione enhancer (as chemical antioxidants) have the potency of vanishing the aflatoxin residues ⁽⁷⁸⁾.

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