Traditionally, veterinarians and professionals who work in animal production were trained to isolate or detect the causative agents of the clinical cases reported. To achieve accurate identification, measured and monitored replication of the etiological agent was performed to evaluate the clinical signs and symptoms in animals. In the case of toxins, some feed factories maintain experimental poultry sheds where they can test whether a nutritional ingredient or contaminant is causing the reported poisoning.
Thanks to these prior studies and facilities, some benchmarks have been achieved and utilized to understand the role of ingredients or toxins in commercial practice. Of these studies, many have explored the impact (and solutions for) mycotoxins. Mycotoxins are not living microorganisms, they are metabolites produced by fungi. Unfortunately, it is not always possible to identify the mycotoxins that caused different symptoms, signs are often subclinical, and lesions in the animals are only observed after the production cycle.
There are many reasons preventing us from confirming the relationship between what we see in the band and the presence of mycotoxins in the analysis.
In other words, there is no clear way to demonstrate that mycotoxins were indeed present in the feed consumed by the affected animals.
Unlike the protein or moisture content in corn or soybeans, mycotoxins are not evenly distributed. The main reason is that fungi do not grow everywhere, only in specific places.
An accurate analysis means determining the average contamination within a batch of grain or formulated feed. If proper sampling procedures are not followed, analytical results are likely to underestimate the true mycotoxin concentration.
Things to consider when sampling include recognizing that:
Unfortunately, even in clinical cases of mycotoxicosis—where affected animals show typical lesions and samples are taken in the right way—we often do not identify mycotoxins during testing.
Results may vary depending on the type of test. Some tests are more specific than others with the ability to detect lower levels of mycotoxins (higher sensitivity). High-precision liquid chromatography (HPLC) has a higher sensitivity than an enzyme-linked immunoassay (Elisa). Depending on the needs of the operation (cost, speed, accuracy) different tests are available. Many operations have started with a layered strategy of testing, an initial rapid test to evaluate suspicions of mycotoxins followed by a slower, but more accurate, test like HPLC.
This includes techniques such as Elisa, thin layer chromatography (TLC), and immunogenic tapes (immunostrips) that are characterized by reporting preliminary results in less time. Elisa’s test is used to detect aflatoxins, T2 toxin, DON, HT-2, zearalenone, and fumonisins, among others. Thin layer chromatography is a technique that takes longer to perform than Elisa because it is necessary to clean the samples to obtain more accurate results.
These are tests with greater specificity (less chance of presenting false positives) and sensitivity (capable of detecting very low levels). Among these are HPLC and liquid chromatography coupled to tandem mass spectrometry (LC/MS). Liquid chromatography–mass spectrometry is highly advanced and simultaneously analyzes hundreds of metabolites and mycotoxins, something that cannot be done with HPLC or Elisa.
A new challenge that prevents determining the real concentration of toxins in the feed is masked mycotoxins.
Fungi that cause damage to plants at the level of fields or greenhouses are identified as phytopathogens. It is important to clarify that not all fungi that grow on plants affect their well-being. Masked mycotoxins occur when the chemical structure of the mycotoxin is changed during the growing period of the plant. This change creates mycotoxin derivatives that are not detectable with conventional analytical tests. The agents that catalyze the development of these variations are enzymes produced by plants that are intended to act as a detoxification process.
In this process, mycotoxins adhere to other nutrients such as sugars (glucose), fatty acids, or amino acids. Generally, mycotoxins adhere to a more polar substance. Once created, there is the possibility these conjugates will release their toxic precursors after hydrolysis occurs within the host (e.g. the animal’s digestive tract). For example, a metabolite of the T2 toxin is HT-2, which originates in plants as a defense mechanism to try to neutralize the toxic effect of this mycotoxin.
After reviewing the factors mentioned in this article, there is no doubt the detection of mycotoxins in ingredients or feed has certain limitations. For this reason, clinicians have resorted to other laboratory techniques to determine if mycotoxins are present in these clinical field cases.
The most useful technique used so far is histopathology or checking for lesions. This method allows one the ability to identify characteristic (non-pathognomonic) lesions in target organs caused by specific mycotoxins.
Another technique that is likely to be used more frequently in the future is the identification of markers or metabolites of mycotoxins. With this testing method, the objective is to demonstrate that the animals were in contact with the mycotoxins and the host produced chemical substances indicative that the exposure occurred.
In recent years it has become fashionable to test for mycotoxins. Unfortunately, depending on the type, it does not always offer consistent results. Moving forward, feed tests are likely to diminish in popularity as metabolite markers will become more reliable. In some cases, the relationship between markers and intoxication is already clearly reported. An example is when evaluating the relationship between the levels of sphingosine and sphingosine. These two markers are associated with intoxication with Fumonisin, which inhibits the enzyme responsible for the metabolism of blood sphingolipids (fats).
There are many methods to test for toxins. Unfortunately, currently, the primary challenge is collecting representative feed samples. With this is the choice between speed and accuracy, leading my producers to perform two-part evaluations of their feed. Another method is histopathology, but this form of evaluation requires skill and occurs after intoxication. In the near future, metabolite markers show promise in providing accurate evaluation, however, their limitation is that they also only measure a response once the animal is exposed.
