Aflatoxin in Feed Grains

Aflatoxins are toxic by-products produced by the fungi Aspergillus flavus and Aspergillus parasiticus. The Aspergillus fungus can germinate and grow on feed grains at moisture levels of 15 % or greater in the presence of warm (70 to 100° F) temperatures. Aflatoxin production by the fungus is optimal at moisture levels greater than 17.5 % and temperatures of 77 to 92° F. The toxin can infect a variety of grains but most often occurs in corn. Infection can occur while grain is standing in the field, at and soon after harvest and during storage before or after the grain is processed into feed.

Outbreaks of acute aflatoxin problems vary among different grain producing regions and depend on growing conditions within a region. Some years the problem will be non-existent but in other years major outbreaks will occur. Corn grown in the southeastern U.S. has the most frequent occurrence, but significant problems can occur in the Midwestern states as well. Drought conditions, heavy insect damage and other crop stress are associated with greater occurrence of aflatoxin in corn. After harvest delayed drying, inadequate drying and poor storage conditions can contribute to increased aflatoxin production.

There are four major types of the aflatoxin molecule referred to as B1, B2, G1, and G2. Aflatoxin B1 is by far the most prevalent and the most toxic. Swine and other livestock and poultry are susceptible to aflatoxins at very low levels measured in parts per billion (ppb). Low levels of aflatoxin (20 to 200 ppb) in the diet of pigs can result in decreased feed intake, slower growth rate and decreased ability to resist disease. In general younger animals are more susceptible than older market animals or breeding animals. With increasing levels of aflatoxin in the diet, depressions in feed intake and growth rate become severe. If aflatoxin levels are high enough, liver damage can occur.

When aflatoxin contaminated rations are fed to lactating animals, the toxin may be secreted in milk resulting in a negative impact on suckling young. Metabolites of aflatoxin excreted in milk have been designated M1 and M2.

Table1. FDA action levels for aflatoxins in food and feedstuffs

Commodity	Concentration, ppb
All products, except milk, designated for humans	20
Milk	0.5 (M1)
Corn for immature animals and dairy cattle	20
Corn and peanut products for breeding cattle, breeding swine, and mature poultry	100
Corn and peanut products for finishing swine	200
Corn and peanut products for finishing beef cattle	300
Cottonseed meal (as a feed ingredient)	300
All other feedstuffs	20

Source: CAST, 2003

The U.S. Food and Drug Administration (FDA) has established "action levels" for total aflatoxins in food and feed (Table 1). These levels are intended as a guideline to prevent problems in human food and animal feed products. These levels can also be used by FDA to determine if enforcement actions should be taken when aflatoxin contaminated grains or feeds are sold and transported commercially.

Determination of the presence of aflatoxin in a feed grain can only be accomplished by chemical assay. An original screening test used at grain elevators and buying stations was to visually inspect samples of corn under an ultraviolet light for the presence of fluorescent particles. This test is capable of detecting the presence of the Aspergillus fungi, but it does not confirm if aflatoxin, the toxic by-product of the fungi, is present. In sophisticated laboratory settings, thin-layer chromatography and high-performance liquid chromatography have been used to accurately detect and quantify aflatoxin in grain and feed samples. More recently, commercial laboratories have developed rapid chemical field test kits that are relatively inexpensive, simple to conduct and require a minimum of special reagents and equipment to conduct. When the directions are followed carefully, these assay kits are very reliable in detecting the presence of aflatoxin at a specified concentration in samples. These kits can be used to determine the presence of aflatoxin at minimum allowable levels. Positive samples may then be forwarded to commercial laboratories for quantification of aflatoxin. With some modifications and serial dilution techniques, the test kits can also be used to quantify aflatoxins. Test kits are also available for detection of other important mycotoxins such as deoxynivalenol (DON or vomitoxin) and zearalenone.

When a feedstuff such as corn is contaminated with aflatoxin, several strategies have been identified to reduce potential negative affects of using the grain in animal feeds. If the level of contamination is low, the addition of other dietary ingredients such as protein, mineral and vitamin supplements may be all that is needed to dilute the toxin level and eliminate negative impacts on animals. Higher contamination levels may require that other grain sources free of aflatoxin may need to be blended with the contaminated grain to lower the aflatoxin concentration. Research has shown that several clay-based anti-caking and pellet aid feed additives added to the diet can reduce the negative impact of aflatoxins in some swine, cattle and poultry feeding situations. Segregating the aflatoxin contaminated feedstuff and using it strategically on lower risk animals (Table 1) has also been used effectively.

It has been suggested that broken and damaged kernels of corn are more susceptible to Aspergillus colonization than intact kernels. However, physical separation to reduce mycotoxin contamination in grains has produced mixed results. In the fall of 2002 we conducted a demonstration experiment at the Virginia Tech Tidewater AREC in Suffolk using corn that we had obtained from a local grain dealer. The corn (approximately 2800 bushels) was produced during the 2002 growing season, obtained by a local grain dealer and delivered to a storage bin at our swine research unit. Drought conditions during the growing season and reports from producers and grain dealers suggested that some risk for aflatoxin contamination existed in the 2002 crop. We used a commercial aflatoxin test kit (Agri-Screen®, Neogen Corporation, Lansing MI) on a single representative sample of corn from the bin. The result indicated that aflatoxin was present at a level of at least 20 ppb. The sample was forwarded to the toxicology laboratory at the Virginia-Maryland Regional College of Veterinary Medicine and analysis verified that aflatoxin was present and quantified at a concentration of 57 ppb.

Our demonstration experiment involved taking four replicate samples of 2 to 3 lbs. from each of three depth regions in the bin. We used a probe-type grain sampler and the sample depths were approximately 1, 3 and 5 meters deep from the top surface of the grain.

In the laboratory the samples were mechanically shaken over a metal screen with round openings 17/64 inches in diameter. Using this process finer particles (fines) passed through the screen and were separated from the intact corn kernels. After screening, fines made up 7.9 to 9.2% of the total sample weight. Dry matter and bulk density were determined for each sample fraction. The samples were ground in a laboratory grist mill and sent to the toxicology laboratory for determination of aflatoxin concentration.

Samples taken at 1 meter depth had slightly lower dry matter than those taken at 3 and 5 meter depths (87.7% vs. 88.7 and 88.6%). Whole kernels also had slightly less dry matter than fines (87.8% vs. 88.8%).

Differences were also observed in bulk density of the samples. Samples had bulk densities of 688.9, 702.3 and 717.5 grams/liter for bin depths of 1, 3 and 5 meters. The whole kernel fraction had greater bulk density than the fines fraction (755.8 vs. 649.9 grams/liter).

Furthermore the demonstration illustrated the fact that location within the bin can have a significant impact on aflatoxin concentration (Tables 2 and 3). The greatest concentration of aflatoxin in both whole kernels and fines was in samples taken at the shallow depth (1 meter). The bin was filled with farm truck loads of approximately 300 bushels each. It is possible that the final loads delivered from the elevator to top off the bin were higher in aflatoxin that the earlier loads. The moisture content in the shallow sampling depth was also slightly higher which may have been more conducive to aflatoxin production.

Table 2. Effects of gain fraction (whole kernel or fines) and sampling depth on aflatoxin concentration (ppb) in corn.

Grain
fraction*

Sample depth* in bin

	1 meter	3 meters	5 meters
Whole kernel	31	9	13
Fines	235	82	96

* Significant effects of depth, grain fraction and depth by grain fraction interaction.

Table 3. Main effects of grain fraction and sampling depth on aflatoxin concentration (ppb) in corn.

Main effect
of sample depth

Main effect
of grain fraction

Depth	Aflatoxin	Fraction	Aflatoxin
1 meter	133 ^a ppb	Whole kernel	18 ^x ppb
3 meters	46 ^b ppb	Fines	138 ^y ppb
5 meters	54 ^b ppb

^{a, b} means in the same column with different superscripts differ significantly.
^{x, y} means in the same column with different superscripts differ significantly.

Perhaps the most interesting finding was that the fines fraction contained substantially greater aflatoxin concentration than the whole kernel fraction (Tables 2 and 3). Based on this it appears that the fines fraction contributed most of the aflatoxin contamination problem in this particular bin of corn. We have no definitive explanation for the highly different aflatoxin concentrations in the two fractions. Visually, the fines fraction appeared to consist of a few weed seeds, small pieces of chaff, and bits and pieces of broken corn kernels. We speculate that greater surface area and starch exposure associated with broken pieces of grain provided a better medium for Aspergillus growth and aflatoxin production. Under the conditions of this demonstration, it appears that corn screening to remove fines would be effective in reducing aflatoxin to levels that pose little risk for livestock and poultry feeding.