Testing for mycotoxin in poultry feed ingredients
Corn and other ingredients, including DDGS, must be assayed to verify the presence and the amount of mycotoxin present
Corn and dried distiller’s grain produced from corn are likely sources of potential contamination with mycotoxins. The principal species include the fungal pathogens of corn including Gibberella, Aspergillus, Fusarium and Penicillin. However, it is important to note that not all corn with ear-rot contains mycotoxins. Corn and other ingredients, including DDGS, must be assayed to verify the presence and the amount of mycotoxin present.
The mycotoxins that are most commonly associated with corn are aflatoxins, fumonisins, deoxynivalenol and zearalenone. Aflatoxins may occur with Aspergillus ear-rot disease, which is promoted by hot and humid growing conditions which occur seasonally in the Southern states. Aflatoxins are carcinogenic, affecting the liver and impair clotting of blood. Since aflatoxins can transfer into the milk of cows aflatoxicosis is also a food safety issue requiring screening of milk for the presence of the toxin.
Fumonisins are associated with Fusarium ear-rot disease of corn which is likely to occur when corn is damaged by ear-worm or by insects. Ideal conditions to promote production of fumonisin include a period of stressful hot and dry weather followed by high humidity. Fumonisin is a neurotoxin and causes leucoencephalomalacia in horses, which can be fatal. In swine it causes pulmonary edema.
Deoxynivalenol (DON) is elaborated by the pathogen that causes Fusarium-head blight in wheat and Gibberella-ear rot disease in corn. This infection is introduced during the stage of silking and is promoted by a cool, wet, environment. Although these conditions are unusual in many U.S. corn growing areas, the 2009 corn crop was subject to weather that promoted contamination. DON is commonly referred to as vomitoxin because ingestion results in feed regurgitation, feed refusal and weight loss in hogs, reduced milk production in dairy herds and immune suppression in most livestock species. Zearalenone contamination also may occur with Gibberella-ear rot disease.
Zearalenone production is promoted by a cool, wet climate and often occurs together with DON in corn. This is an estrogenic toxin, interfering with reproduction and it can affect the viability of neonates.
Mycotoxins are of particular concern for users of DDGS because levels in contaminated corn can be concentrated two to threefold during fermentation and processing. Mycotoxins remain with the grain residue, and are not extracted with ethanol. There is evidence that improper storage of wet DDGS can also result in elaboration of toxins.
Advisory levels of Mycotoxins that can be safely fed to poultry
The following Table summarizes current recommendations used by OISC (based upon FDA guidance) to determine safe-feeding levels for mycotoxins in poultry:
|Mycotoxin||Animal age||Feed||Advisory levels|
|Aflatoxins||Immature||corn / peanut||20 ppb|
|Mature||corn / peanut||100 ppb|
|Mature||Cottonseed meal||300 ppb|
|All||Other feeds||20 ppb|
|All||Total ration||50 ppb|
|Fumonisins||All||Grain <50% of diet||10 ppm|
|DON||All||Grain by-product <50% of diet||10 ppm|
The importance of sampling
Effective sampling is critical to an effective testing program and a sampling plan should be based on the intended outcome. To assay the contents of a bin of stored corn at an elevator, samples need to be obtained throughout the bin. The sampling procedure should not introduce bias through differences in particle size. Whatever is sampled and sent to the laboratory will be reduced to a smaller test portion, so that the sample must be representative of the batch.
Contaminated grain will not be uniformly distributed in a consignment. Multiple, composite samples will be necessary to evaluate a consignment. DDGs exhibits particle size segregation and research has shown that this will likely affect both mycotoxin assays and determination of nutrient content.
The test samples are ground to allow for the laboratory to obtain a representative test portion. For corn samples, the OISC lab pre-grinds the entire field sample using a 4mm screen on a Romer mill. The samples are riffled to fill a 16oz sample jar ¾ full. This sample is then ground using a Retsch mill and a 1.5mm screen. For DDG samples a 0.75mm screen is used. The samples are mixed by "tumble & roll" prior to removal of the test portion.
Qualitative test methods
The most common qualitative tests for mycotoxins are rapid test kits based on enzyme linked immunosorbent assay (ELISA). These test kits screen to a predetermined concentration, and reported results will be expressed as being more or less than the predetermined level. Many aflatoxin rapid test kits assay above or below 20ppb. Samples are extracted by shaking in a plastic cup, are filtered, diluted, mixed and then the strip test is dipped into the sample. The results are read visually as colored lines or may be scanned electronically.
Another common form of rapid qualitative test kits employs pairs of micro-titer wells. Standard toxin is put into the one well, and the test sample in the other. The samples are mixed and transferred to antibody coated wells. After a few simple wash steps a reaction with color reagents occurs. The reaction is stopped and the color of the sample well is compared to the standard well. Again, results are reported as more or less than the predetermined concentration of mycotoxin.
Quantitative test methods
Commercial rapid test kits based on ELISA are available to allow for testing over a range of toxin concentrations, including a lateral flow strip that meets the quantitative criteria for GIPSA verification. Charm Science use a heated incubator for the test strip with the sample extract added to a sample compartment at one end of the strip. After an incubation period the strips are read using an electronic reader to determine the concentration of mycotoxin.
Many quantitative rapid test kits based on ELISA use a micro titer well assay. Several manufacturers have test kits certified by GIPSA validated for a variety of mycotoxins in a range of ingredients. There are methods for specific mycotoxins using liquid chromatography in combination with UV, photo-diode array, fluorescence or mass spectrometric detection. Gas chromatographic methods apply flame ionization or mass spectrometric detection with specific protocols for single mycotoxins and for combinations of mycotoxins. In general, these procedures require trained analysts and complex instrumentation. Equipment is expensive and assays by both commercial and public sector laboratories may have extended turnaround times especially during seasonal demand for analytical services.
Adapted from a presentation made at the 2011 Midwest Poultry Federation Convention.