Ram Singh Bibyan*, Nikita Mahna, Yamini Khatri, Jannat Saini, Harneet Kour, Sarita Kaushal, Balaga Sravani, Pramod Kumar, Priyanka Patir, Lovely Anant, Prajakta Kailas Sangale, Sruthy Ravi+, Kanmoni Goyari+, Divya Begari, Ankita Patel, Shweta, Somesh Rameshrao Gaikwad and Ashwani Kumar Saini#
Animal Nutrition Division
ICAR- National Dairy Research Institute
Karnal- 132001 (Haryana) India
+PhD Scholar, IVRI, Izatnagar
# STO, CIRB sub campus, Nabha
*Email: carirsingh@yahoo.co.in
+91-9457602079
Mycotoxins are toxic secondary metabolites produced by fungi in various agricultural commodities, resulting in a variety of ill health effects and posing a serious threat, both to livestock and humans. Scientifically, mycotoxins are polyketones compounds resulting from condensation reactions produced under specific physical, chemical, and biological conditions that occur when the reduction of the ketone groups in the biosynthesis of the fatty acids, carried out by the mould, is interrupted. These fatty acids are primary metabolites used by mould as an energy source. About 500 mycotoxins have been identified so far, the most frequently naturally contaminating animal feed and human food are: aflatoxins, ochratoxins, zearalenone, fumonisins, trichothecenes (T-2 toxin, deoxynivalenol or vomitoxin, nivalenol, monoacetoxyscirpenol, diacetoxyscirpenol, triacetoxyscirpenol, scirpentriol), patulin, penicillic acid, sterigmatocystin, alternaria toxins (alternariol, alternariol monomethyl ether, altenuene, altenuisol), rye ergot alkaloids (ergotamine, ergotoxin, ergometrine), tremorgenic toxins (penitrem A and B), rubratoxins A and B, rugulosin, luteoskyrin, islanditoxin, and citreoviridin (Singh et al., 2013). Ruminants are considered to be less susceptible to the negative impacts of mycotoxins due to their detoxification in the rumen. Despite rumen degradation of mycotoxins, ruminants are still at potential risk of mycotoxin toxicity. Generally, the symptoms of mycotoxicosis in dairy animals vaguely include: reduced feed intake; feed refusal, rough hair coat; poor body condition and reproductive problems. Owing to the high feed intake, high producing dairy animals are more sensitive to aflatoxicosis, which leads to faster passage of feed through the digestive tract resulting in lower time for aflatoxin detoxification by rumen microbes. Also, high producing dairy animals are fed on diet containing high amount of fermentable carbohydrates to meet their energy requirements, thereby increasing the risk of ruminal acidosis. Low ruminal pH is associated with lower aflatoxin detoxification by rumen microbes because of altered rumen population at the expense of those with higher aflatoxin detoxifying activity.
Types of aflatoxins: Aflatoxins are a group of closely related toxic substances produced by some fungi, especially Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. Although 18 aflatoxins have been isolated, only 4 of them (B1, B2, G1, G2) are well known and studied extensively from toxicological point of view. The toxic nature of aflatoxin is due to its chemical structure. Aflatoxin B1 (AFB1) is the most toxic compound. The toxigenicity among four (AFB1, AFB2, AFG1, AFG2) aflatoxin compounds has been rated in the following order: B1˃ G1˃ B2˃ G2 when tested across various animal species. Two other familiar aflatoxins are AFM1 and AFM2, because of their presence in milk of animals exposed to AFB1 and AFB2. AFB1 is the most acutely toxic to various species. Other metabolites AFB2a, aflatoxicol, aflatoxicol H1 and aflatoxins P1 and Q1 have been identified (Singh et al., 2013). AFM1 is a metabolite of AFB1 in humans and animals. AFM2 is a metabolite of AFB2 in milk of cattle fed on contaminated feed. Although AFB1, AFB2 and AFG1 are common in the same food sample, AFB1 predominates (50-80% of the total aflatoxin content). Generally, AFB2, AFG1 and AFG2 do not occur in the absence of AFB1.
Occurrence of aflatoxin in feed: Aflatoxins are natural contaminants of corn, oats, barley, wheat, rice, peanuts, cottonseed meal, groundnut cake, maize gluten meal, coconut meal, sesame cake, sunflower meal and DDGS (Singh, 2019; Singh and Shrivastav, 2011a; Singh and Shrivastav, 2011b; Singh et al., 2010; Singh et al., 2009). Aflatoxins often occur in crops in the field prior to harvest. Post harvest contamination can occur if crop drying is delayed and during storage of the crop if water is allowed to exceed critical values for the mould growth. Insect or rodent infestations facilitate mould invasion of some stored commodities. Aflatoxins are difuranocoumarin derivatives produced by a polyketide pathway primarily by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius (Singh and Shamsudeen, 2008; Singh and Shrivastav, 2012; Singh et al., 2017; Singh and Mandal, 2014; Shamsudeen et al., 2013a; Shamsudeen et al., 2013b; Shamsudeen et al., 2014a). Aspergillus Parasiticus produces four types of aflatoxins e.g. aflatoxin B1; B2; G1 and G2; and Aspergillus flavus usually produces B1 and B2 (Singh et al., 2017; Singh and Mandal, 2014). Aflatoxins are extremely toxic, mutagenic and carcinogenic compounds which differ significantly in their relative toxicity. Aspergillus are moulds that belong to storage flora. Generally, the necessary minimum temperature and water activity (aw) for developing and produce mycotoxins is 10 to 12ºC and 0.75 to 0.83, respectively. The optimum conditions for the growth of Aspergillus and the production of aflatoxins are 25ºC with water activity of 0.95. Nonetheless, there are strains of Aspergillus flavus, which grow on substrates such as rice, at temperatures between 7 and 45ºC with an optimal temperature of 37ºC, and the production of mycotoxins between 11 and 36ºC with a maximum production at 30ºC. In substrates such as peanuts, rice, sorghum, wheat, and corn, strains of Aspergillus parasiticus NRRL 3000 and NRRL 2999 produced 107, 107, 72, 72, 53 mg/kg and 104, 185, 88, 19, 47 mg/kg of aflatoxins, respectively. However, the production of aflatoxins in soybean meal was only 19 and 2.8 mg/kg, respectively. The strain NRRL 3145 produced 8.50, 10.60, 57.60, 7.10, and 5.50 mg/kg of aflatoxins in peanuts, rice, sorghum, wheat, and corn, respectively; and the production of aflatoxins in soybean meal was significantly lower (0.06 mg/kg). Therefore, under optimum temperature and aw conditions, the mycotoxin production depends on the genetic strain and the substrate composition. The Aspergillus flavus NRRL 3251, 3357, 3517, and 3353 produced aflatoxins. However, NRRL 1957 did not produce aflatoxins. In another study, the highest yield of AFB1 (0.995g/kg maize) by Aspergillus parasiticus NRRL 2999 was recorded at 35% moisture and 30°C temperature in 7 days of incubation period, whereas, in case of Aspergillus parasiticus MTCC 411, maximum AFB1 production (0.665g/kg maize) was recorded at 30% moisture and 30°C temperature in 14 days of incubation period. On rice substrate, the maximum production of AFB1 (0.835g/kg rice) by Aspergillus parasiticus NRRL 2999 was recorded at 30% moisture and 30°C temperature on 7 days incubation period, however in case of Aspergillus parasiticus MTCC 411, maximum yield of AFB1 (0.605g/kg rice) was recorded at 35% moisture and 30°C temperature on 14 days of incubation period. Therefore, the toxigenecity of mould depends upon several factors like substrate, temperature, moisture and strain of mould (Singh and Shrivastav, 2012). Using liquid medium, consisting of 2% yeast extract and 15% sucrose, Aspergillus parasiticus MTCC 411 reduced 100 mg and Aspergillus parasiticus NRRL 2999 produced 110 mg AFB1 per litre of medium. Under dark condition Aspergillus parasiticus MTCC 411 produced higher amount of AFB1 as compared to light condition (Singh and Shamsudeen, 2008). In another study, Aspergillus parasiticus NRRL 2999 produced 30.07 mg AFB1 per litre of liquid medium (Shamsudeen et al., 2013a).
Effect of aflatoxin on rumen fermentation: The rumen is a complex microbial ecosystem that harbours diverse populations of bacteria, methanogens archaea, protozoa, fungi and bacteriophages that are involved in the anaerobic digestion of plant biomass. Several in vitro studies investigated the effects of aflatoxin on rumen fermentation. Singh et al. (2020a) reported that AFB1 contamination of feed at 100 to 300 ppb levels significantly affected the in vitro rumen fermentation in terms of reduced truly degradable dry matter, truly degradable organic matter, gas production, volatile fatty acids (acetate, propionate and butyrate) concentration, microbial biomass production, partitioning factor and increased acetate: propionate ratio. Similar results were also reported by Singh and Saini (2022); Singh et al. (2021); Singh and Saini (2020a); and Singh and Saini (2020b) when feed contaminated with 300 ppb AFB1 was evaluated in vitro. The cellulolytic activity of rumen bacteria is essential to produce volatile fatty acids (VFAs), which are the main energy source for ruminants. The exposure of animals to aflatoxin can impact the rumen microbial balance and impair the cellulolytic functions of rumen bacteria resulting in negative impact on rumen fermentation. The mechanism of ill impacts of aflatoxin on rumen bacteria is not fully understood, however, it may involve oxidative stress, membrane damage, enzyme inhibition, DNA damage and interference with metabolic pathways. Mojtahedi (2013) reported that in vitro experiment with AFB1@ 0, 300, 600, 900 ng AFB1/ml buffered rumen fluid resulted in reduced gas production, DM digestibility and NH3-N. Westlake et al. (1989) also reported decreased DM digestibility at AFB1 concentration of 1, 10 µg AFB1/ml buffered rumen fluid in vitro. However, Auerbach et al. (1998) reported no effect on rumen fermentation at 9.5 ng AFB1/ml buffered rumen fluid. Jiang et al. (2012) also reported decrease in gas production, rate of degradation, NH3-N and increased isobutyrate, valerate and isovalerate molar proportion.
Effect on biochemical parameters: When dairy cows and other ruminants consume aflatoxin contaminated feed, they metabolically biotransform AFB1 into a hydroxylated form called AFM1 by liver enzymes cytochrome P450. This hydroxylated (AFM1) derivative is water soluble and is easily excreted in body fluids, such as urine and milk, which represents a serious risk to human health. The liver is the target organ for toxic effects of AFB1 (Singh et al., 2015a; Singh et al., 2015b; Silambarasan et al., 2016; Singh et al., 2016; Singh et al., 2019), and the serum total protein level is an indicator for the protein synthesis (Singh, 2019; Silambarasan et al., 2016). Meanwhile, the increased activities of ALT, AST, and ALP are sensitive indicators of both hepatic tissues and biliary system impairment (Silambarasan et al., 2016). Aflatoxicosis is also reported to degenerate the hepatocytes leading to leakage of these hepatic enzymes into the circulation (Silambarasan et al., 2016; Singh, 2019) which describe the reason behind the elevated levels of these enzymes (Khatke et al., 2012). Aflatoxicosis resulted in decreased serum total protein, which was due to the inhibition of protein synthesis (Singh, 2019; Silambarasan et al., 2016). An increase in the serum ALT, AST, and ALP was also reported (Singh, 2019; Silambarasan et al., 2016). Several other studies also reported significant increase in the serum AST and ALT levels (Rastogi et al. 2001; Bingol et al. 2007) in AF-intoxicated animals. Increase in the serum AST level after 24 to 48 h of aflatoxin intoxication in steers was reported, however, these changes were dose-dependent (Cook et al., 1986). The AFB1 has been reported to provoke the liver function through the inducing of apoptosis plus disturbing the cellular enzymatic activities. Some researchers reported no significant changes in the TP and ALP after AF intoxication due to the fact that serum glycocholic acid level could be considered a sensitive indicator for biliary impairment or cholestasis than alkaline phosphatase or bilirubin (Cook et al., 1986).
Impact on production performance and aflatoxin excretion in milk: Production performance and health of dairy cattle was adversely affected at dietary aflatoxin levels above 100 ppb, which is considerably higher than the amount that produces illegal milk residues (Singh, 2020). Feed contaminated with 120 ppb aflatoxin fed to lactating dairy cows under field conditions resulted in poor reproductive efficiency, increased somatic cell counts in milk, reduced milk yield; removal of feed (feeding aflatoxin free diet) resulted in significant increase in milk production (Singh, 2020) In another study, feed contaminated with 433 ppb AFB1 fed to lactating dairy cows in their mid lactation during a period of seven days caused significant reduction in feed intake and milk production, and the concentration of AFM1 in milk fluctuated between 1.05 and 10.58 ppb . The AFM1 disappeared in the milk after four days of aflatoxin free feeding diet (Singh, 2020). Aflatoxin contaminated feed (2.0 to 2.4 ppm) given to cows during a period of seven months resulted in serious hepatotoxicity causing significant reduction in milk yield (Singh, 2020). Oral administration of 0.3 mg/kg AFB1 to dairy cows resulted in reduced appetite, decreased body weight, decreased milk production and significant enzymatic variation during 1-3 weeks after exposure to AFB1. The bacterial count in the milk increased due to the consumption of the aflatoxin. AFM1 was detected in the milk within 3-6 hours after the administration of AFB1 and remained detectable for a period of 72 hours after administering the last dose of AFB1. AFB1 and AFM1 were detected in the animal’s urine after administration of AFB1 and remained detectable upto 72 to 120 h after administering the last dose of AFB1 (Singh, 2020). Sheep appeared to be more resistant to the toxic effects of aflatoxin. Feeding diets contaminated with 2.5 ppm or 5.0 ppm of total aflatoxins consisting of 79% AFB1, 16% AFG1, 4% AFB2, and 1% AFG2 for a period of 35 days resulted in hepatotoxicity in ewe lambs. In another study, lambs fed 2.5 ppm aflatoxin contaminated feed daily for 21 days exhibited sign of clinical aflatoxicosis including hepatic and nephritic lesions, altered mineral metabolism, and increased size and weight of liver and kidney. AFM1 distribution in milk is not homogeneous, probably due to its semi-polar character. Cream separation can affect AFM1 distribution, since 80% is partitioned in the skim milk portion because of AFM1 binding to casein (Singh, 2020). An amount of 30% of AFM1 is estimated to be associated with non-fat milk solids particularly with casein (Singh, 2020). The deleterious effects of AFB1 were more serious in cows receiving impure aflatoxin compared to those receiving pure aflatoxin of same concentration (Singh, 2020). Dairy cows, having 550 kg BW, with induction of mammalian infection using Streptococcus agalactiae, Staphylococcus aureus, and Staphylococcus hyicus, during the lactating period, and subsequently receiving an oral dose of AFB1 @ 0.3 mg/kg BW/day during a period of 12 to 14 days, consuming 30 kg ration/day contaminated with 5500 ppb AFB1. The animals exhibited lack of appetite, weight loss, decreased milk production and significant enzymatic variation during 1 to 3 weeks after ingesting AFB1. No sign of acute mastitis was observed, however, the bacterial count in milk was increased due to consumption of AFB1. There was increase in the incidences of mastitis following the last administration of the mycotoxin (Singh, 2020).
Due to risk of human health owing to milk consumption, the AFB1 contamination in feed should not be more than 24 ppb to keep the level of AFM1 below permissible level of 0.5 ppb (Bibyan et al., 2023; FSSAI, 2021). It validates the permissible level of 20 ppb in the feed of dairy animals in India. AFB1 is a carcinogen and is excreted in milk in the form of AFM1. Therefore, the presence of AFM1 residues in milk and dairy products is a public health concern, taking into account their potential for additional aflatoxin exposure of humans through the diet. For this reason, several countries established maximum levels for AFM1 in milk and milk products, although, other mycotoxins may also be excreted into milk. Recent study showed that aflatoxicol is also excreted with milk, aflatoxicol is the major metabolite of AFB1 produced by microorganisms of the rumen flora, however, AFM1 is from hepatic origin. The toxicological properties of AFB1 are generally comparable to that of AFM1. On account of carcinogenicity of AFB1, the only mycotoxin legislated in milk is its metabolite, AFM1. The World Health Organisation proposes a maximum permissible level of 0.5 mg/kg in milk, which most countries have adopted. Cows consuming diets containing 30 ppb aflatoxin can produce milk containing aflatoxin residues above the WHO action level of 0.5 ppb (Bibyan et al., 2023). In US, the regulatory level of aflatoxins are 20 ppb for dairy feeds and in Europe 0.05 ppb in milk; therefore, an illegal milk residue can occur when feed contains more than 30 ppb of aflatoxin (Bibyan et al., 2023). After the ingestion of aflatoxin, the AFM1 metabolite is detected for the first time in milk after about 6h and peaks in the excretion can be observed after 24h of continuous exposure (Bibyan et al., 2023). Toxin clearance from the milk is usually achieved 3 days after the contaminated diet is withdrawn. Carry-over rates in dairy ruminants usually ranges from 0.6 to 6%. Bibyan et al. (2023) reported that the carry-over rate ranged from 0.48 to 0.76% with an overall carry-over rate of 0.59% when the AFB1 intake ranged from 500 to 2000 ppb per animal per day in Nili Ravi buffalo. However, the extent of transfer from feed to milk is influenced by various nutritional and physiological factors, including feeding regimens, rate of ingestion, rate of digestion, health of the animal, hepatic biotransformation capacity, and milk yield. Therefore, the rate of absorption of aflatoxins, and the excretion of AFM1 in milk, varies from animal to animal, from day to day, and from one milking to the next (Singh, 2020).
Mycotoxins have been a non-negligible problem in the livestock industry, and have an impact on food safety as well as human health due to their worldwide contamination, causing substantial economic losses. Impacts of aflatoxin on ruminants depend on several factors, such as (a) toxin-related (type and level of mycotoxin ingested as well as duration of intoxication period); (b) diet-related (as reported by Bibyan et al 2023: inclusion level of mycotoxin contaminated feeds, diet composition, forage to concentrate ratio, diet physical form, digestibility of dry matter or other nutrients, rate of passage, etc.); (c) animal-related (species, sex, age, breed, dry matter intake level, general health, immune status, nutritional strategies); and (d) environmental-related (farm management, hygiene, temperature, etc.) factors. Growth inhibition, decreased feed efficiency and increased liver and kidney weights at 700 and 1000 ppb level of aflatoxin in the diet of beef cattle was observed. Aflatoxin can also influence the reproductive efficiency of dairy animals. Several cases of third trimester abortions were already reported and correlated with the ingestion of mouldy peanuts contaminated with an average of 77 mg/kg AFB1 (Ray et al., 1986). However, Rossi et al. (2009), reported that aflatoxin will normally not have a direct impact on the oocyte or embryo; rather, they will alter material homeostasis by reducing dry matter intake, decreasing rumen motility, inducing liver damage and reducing immune response. This lack of nutrients and liver disorders will then interfere with ovarian and uterine activity. A case study conducted in Turkey related the contamination of Holstein cattle feeds with aflatoxin with incidence of AFM1 in milk, laminitis, lameness, cystic ovaries, clinical metritis and impaired fertility (Ozsoy et al., 2005). Feed contaminated with 2000 to 2400 ppb AFB1 given to cows during a period of 7 months resulted in serious hepatotoxicosis problems and significant reduction in milk production (Singh, 2020). Aflatoxicosis resulted in anorexia, depression, jaundice, photosensitization of unpigmented skin, decreased milk production, weight loss, lethargy, ascitis, icterus, tenesmus, abdominal pain, abortion submandibular edema, keratocojunctivitis, diarrhea with dysentery, hemorrhages in subcutaneous tissues, pericardium, beneath the epicardium, lymph nodes and skeletal muscles. Serora of the alimentary canal, enlarged hepatocytes, hepatocytes protoplasm was vacuolated and fat on vacuoles was also observed. Other signs of acute aflatoxicosis included blindness, walking in circles, ear twitching, frothy mouth and rectal prolapsed. Aflatoxicosis in one year old goats given a dose of 4 mg/kg body weight resulted in anorexia, depression, weakness, bleeding, head pressing, swaying, falling, blindness, convulsion, coma and death (Singh, 2020). Thus, Aflatoxin contaminated feed not only reduces animal performance and overall health, but it also creates risks of residues in milk (Bibyan et al., 2023). The carryover rate of aflatoxin can range widely among animals, day to day, and from one milking to the next. It is primarily influenced by factors such as diet composition; immune status of the animal, especially the status of the liver and its enzymatic activities; duration of exposure; physiological status of the animal; and level of aflatoxin in feed (Bibyan et al., 2023). For high-yielding dairy cows producing up to 40 kg of milk per day, Veldman et al. (1992) found a carryover percentage as high as 6.2%. In India, Bibyan et al. (2023) reported an average carryover rate of 0.59% in Nili-Ravi buffaloes fed on aflatoxin B1 (28 to 113 ppb) contaminated feed. Changes in the plasma-milk barrier and the consumption of large amounts of concentrated feeds in high yielding dairy cows may increase the carryover rate of AFM1 in milk.
The rumen is a complex ecosystem where microbial population degrade feed components and produce VFAs, ammonia, gases and microbial protein. Dairy animals exposure to aflatoxin results in altered rumen function and fermentation resulting in reduced fibre digestion; reduced VFAs production; reduced feed intake and efficiency; and impaired animal performance and health. Aflatoxicosis may contribute towards development of ruminal acidosis in dairy animals resulting in negative impact on their health, welfare, and productivity. To avoid mycotoxin occurrence in the food chain requires management strategies that would prevent contaminated commodities from entering food and feed processing facilities. Various methods to decrease or eliminate mycotoxins are being studied and several approaches such as physical methods of separation and detoxification, biological and chemical inactivation, and decreasing bioavailability to host animals are being investigated using various mycotoxin detoxifying agents. Further, it is also important to prevent or reduce the exposure of dairy animals to aflatoxins by using good agricultural practices, good feed storage and processing practices, and regular feed analysis and monitoring. It is also recommended to use effective aflatoxin deactivating feed additive(s) to reduce absorption or toxicity of aflatoxins in the gastrointestinal tract.
References are available on request from author: carirsingh@yahoo.co.in (+91-9457602079)