Molecular approaches for the early detection of mycotoxigenic fungi in feeds and feed matrix

Feeds are substances or products, including additives, that are either processed, unprocessed or semi-processed and used for feeding animals (Comisión Europea, 2011).

Livestock feed is mainly designed with combinations tailored to the nutritional needs of animals with minimum costs (Pereira et al., 2019).

Cereal and cereal-based products are a major component of animal feed, supplying enormous quantities of nutrients to livestock (Deepthi y Sreenivasa, 2020).

In many developed countries, nearly 70% of harvested cereals are destined to be included in animals’ daily feed, whereas in developing countries they are primarily consumed by humans (Dass et al., 2007).

Globally, feed industries include cereals such as wheat, maize, sorghum, barley, and oats. Among these, wheat and maize are global key commodities in agriculture for animal and poultry feed (FAO, 2009).

A global survey reported that the demand for agricultural commodities is expected to experiment an 84% growth from 2000 to 2050 (FAO y OMS, 2007).

According to Directive 2002/32/EC, poultry and animal feed safety should be prominent, both for governments and producers, since the consumption of feed can pose certain risks to animal health, with hazardous consequences for the human food chain and livestock production industry (FAO 2009; Comisión Europea, 2015).

Due to poor agricultural practices during the pre-harvest, harvest, post-harvest, and storing stages, a high level of mycotoxin-producing fungal contaminants are mainly associated with the cereals that serve as a major ingredient of feed manufacturing (Deepa y Sreenivasa, 2017a, Deepa y Sreenivasa, 2017b) (Figure 1).

Figure 1. Processing of feed commodities.

The risk of mycotoxins

Mycotoxins are naturally produced by filamentous fungi from the genus Fusarium, Aspergillus, Penicillium, Alternaria, Stachybotrys and Claviceps. Approximately 450 mycotoxins have been reported so far (CAST, 2003).

Based on their occurrence in feed and their consequences on livestock health, certain groups of mycotoxins, such as aflatoxins (AF), fumonisins (FB), ochratoxins (OTA), trichothecenes (TRC), zearalenone (ZEN), and deoxynivalenol (DON) have been targeted in practice through management strategies (Smith et al., 2016).

⇰ Aspergillus species mainly produce AF and OTA, while Penicillium species produce patulin (PAT) and fumonisins, ZEN, DON and trichothecenes are produced by Fusarium species (Iqbal et al., 2015).

The presence of mycotoxigenic fungi in feed has become a significant problem (Kovalsky et al., 2016) as the consumption of the mycotoxins present in the feed has negative health effects ranging from chronic to acute and sometimes even death, as well as affecting animal performance which indirectly affects profitability (CAST, 2003) (Table 1).

Worldwide organizations and institutions have restricted the level of mycotoxin acceptance in poultry and animal feeds as mycotoxin-free feed is considered impossible to guarantee (Smith et al., 2016).

Certain standard limits on the level of mycotoxin contamination have been set by legislation, varying in each country since many economic, scientific, and political issues are involved in the decision framing sessions.

For example, the maximum content of AFB₁ in feed has been set to 0.02-0.05 ppm and, based on studies, limits for deoxynivalenol, fumonisins, zearalenone and ochratoxin A in feed have been recommended (Comisión Europea 2015; Comisión Europea, 2006).

The importance of mycotoxin detection

The presence of moulds in feed does not directly correlate with the occurrence of its mycotoxin.

Classical culture-based methods used in the detection and identification of mycotoxin producing fungi have various limitations as:

• ⇰ They are labor-intensive, environmentally dependent on the target, and time-consuming.
• ⇰ They have poor reliability of results and difficulties in standardization, among other aspects.

Currently, there is a great demand for molecular technologies to detect mycotoxigenic fungi in feed entering food chain at early stages (Figure 2).

Polymerase chain reaction (PCR) is a rapid, specific, sensitive, and safe method that ensures rapid detection of fungal contamination in feed matrices in comparison with conventional, time consuming, laborious microbiological analysis (Rahman et al., 2020).

Researchers have been increasing the use of various molecular techniques in feed industries for the early detection of mycotoxigenic fungi, such as polymerase chain reaction (PCR), nested PCR, quantitative PCR (qPCR), real-time PCR, magnetic capture hybridization PCR (MCH-PCR), immunocapture PCR, in situ PCR, PCR restriction fragment length polymorphism (PCR-RFLP), droplet digilet PCR, cooperational PCR, multiplex PCR, reverse transcriptase PCR (RT-PCR), loop-mediated isothermal amplification (LAMP), DNA microarrays, and fluorescence in situ hybridization (FISH) (Deepa et al., 2021; Deepa et al, 2018) (Figure 3).

Different molecular approaches used for the detection and differentiation of mycotoxigenic fungi

Polymerase Chain Reaction (PCR) and its variants

PCR

METHODOLOGY

The PCR technique is used for detecting contamination with mycotoxigenic fungi in feed matrices and to define the potential risk for human and animal health.

⇰ It involves a single round of PCR with a certain set of primers, PCR reagents, and the DNA sample followed by a gel documentation system.

APPLICATION

This technique is mainly applied for the identification of fungi and specific gene detection.

• Fusarium poae producing nivalenol (NIV) was detected through a PCR assay representing the presence of the tri7 gene (Dinolfo et al., 2012).
• The involvement of F. verticillioides contamination in 51 feed matrices was confirmed through conventional PCR and 69 samples were associated with Fusarium species (Deepa et al., 2016a).
• Feed material cereal samples were infected with 103 Fusarium isolates and 64 fumonisin-producing F. verticillioides were associated with sorghum (Sreenivasa et al., 2007; 2008).
• Among 108 feed samples, 45.37% of feed mixtures were contaminated with Fusarium species, which was confirmed through a conventional PCR method (Deepthi et al., 2017).

• Dass et al. (2009) analysed 80 poultry feed mixtures and 114 animal feeds for Fusarium contamination, reporting 374 Fusarium species with primers and 244 isolates for fumonisinspecific strains with FUM1 set of primers.

MULTIPLEX PCR

METHODOLOGY

In a single PCR tube, it allows the combination of several species-specific primers used simultaneously to detect a variety of mycotoxigenic fungi. Further amplified products are analysed with gel electrophoresis.

⇰ With this technique a single PCR run is sufficient for the identification of more than one gene at a time.

APPLICATION

• Maize samples associated with F. verticillioides and F. subglutinans contamination were analysed in Brazil by developing gaoB gene coding for galactose oxidase (Faria et al., 2012).
• One forward and two reverse primers were used to detect fumonisin producing F. verticillioides through mPCR (Deepa et al., 2016c).
• Aflatoxin-producing and non-aflatoxin-producing Aspergillus species were detected in meju, two sets of primers were used to amplify the genes (omtB, omtA, aflR, ver-1), set I (omtB-1,2, aflR-1,2 and omtA-1,2) and set II (aflR-1,2, ver-1-F-R and omtA-1,2) (Kim et al., 2011). Simultaneously, five major genes, pks (ochratoxin A), pks13 (zearalenone), aflr (aflatoxin), tri5 (trichothecene) and fum13 (fumonisin) produced by Aspergillus, Fusarium and Penicillium were detected using a multiplex PCR assay for feed matrices (Priyanka et al., 2015).
• Four major mycotoxin-related genes, fum13 (fumonisin), nor1 (aflatoxin), Tri6 (trichothecene) and otanps (ochratoxin A) were simultaneously detected in a single test run with specific and sensitive multiplex PCR method (Rashmi et al., 2013).

• Fumonisins (FUM1, FUM13), and trichothecenes (tri5, tri6) were detected in feed matrices using mPCR (Ramana et al., 2011).

NESTED PCR

METHODOLOGY

This method involves two consecutive reactions in a single closed tube in which the external primer pair amplifies a large amplicon used as template for the internal primer that needs a higher temperature than the external primer and where the internal primer will not bind (Porter-Jordan et al., 1990).

The method is cost-effective and 100 times more sensitive than other methods. Although the risk of contamination may be higher due to the two cycles of amplification, this technique increases the yield and specificity of the target DNA amplification (Rahman et al., 2013).

⇰ The nested PCR method is a double confirmatory test, because if the first primer binds to and amplifies an unwanted DNA sequence, the second set of primers will also bind within the unwanted region.

APPLICATION

• Klemsdal y Elen (2006) detected Fusarium culmorum in cereal crop feed matrices where the external primer pair (FculAF, FculAR) was diluted 10,000 times compared to the internal primer pair (FculBF, FculBR) with a detection limit of 5-50fg of target DNA.
• Fumonisin producing F. verticillioides associated with feed matrix cereals was detected using one forward (VERF-1) and two reverse primers (VERTR and VERTF-2) (VERTR y VERTF-2) (Deepa et al., 2016b).

REAL-TIME PCR (q-PCR)

METHODOLOGY

This method detects the amplicons of targeted DNA molecules based on the emission of fluorescent signals, which eliminates the post-amplification steps needed in the conventional PCR method, which reduces the time invested. It detects small concentrations and is even useful in identifying in symptomless infections.

APPLICATION

• Even though it can help to detect symptomless infections, qPCR technology is not effective when applied to feed (Ginzinger, 2002).

RT-PCR

METHODOLOGY

This technique was developed to differentiate living and dead fungi as mRNA is quickly degraded in dead cells. During the process, mRNA is reverse-transcribed to produce cDNA as a template by a reverse transcriptase enzyme (Brown et al., 2011).

APPLICATION

• F. graminearum was detected using the RT-PCR method in a feed matrix. The method is specific and highly sensitive for detecting mycotoxigenic fungi in feed. However, it is also more expensive and laborious than any other method (Guo et al., 2005).

PCR based methods

PCR-RFLP

METHODOLOGY

This method is useful to discriminate at a species level with accuracy, sensitivity, and specificity.

⇰ RFLP records genetic variability among different and same species exhibiting genetic differentiation corresponding with pathogenic and mycotoxicological profiles.

APPLICATION

• Aflatoxin-producing Aspergillus species, such as A. flavus and A. parasiticus, were detected in complex food and feed matrices (Ahmad et al., 2014).
• ITS-RFLP discrimination was described between F. verticillioides and F. proliferatum (Visenti et al., 2012).
• The PCR-RFLP method was applied to detect variations between 33 fumonisin-producing F. verticillioides strains from feed matrices (Patino et al., 2006).
• Genetic variations were studied among ten strains of F. verticillioides using six different enzymes (EcoRI, Xho I, Hae III, Kpn I, Hinf I, Hind III) isolated from cereal samples (Deepa et al., 2018).

PCR-AFLP

METHODOLOGY

Amplified fragment Length Polymorphism- PCR is a technique designed to detect DNA polymorphisms.

⇰ Mycotoxicological and fungal pathogen characterization can be complemented by fingerprinting. The method is highly reproducible, sensitive and has the capability to amplify 100 fragments at a time.

APPLICATION

The method was applied to study genetic variability among 3,840 maize kernels in Brazil and FUM1 and FUM8 were the essential genes responsible for fumonisin biosynthesis among F. verticillioides species (Silva et al., 2017).

PCR-RAPD

METHODOLOGY

Random amplified polymorphic DNA identifies genotypic differences using short oligonucleotide primers to distinguish mycotoxigenic fungal species and variations within the species (Zhang et al., 2018).

⇰ Even though the method is used as a diagnostic tool in laboratories, it is not much in practice as it lacks reproducibility (Blackwell et al., 1999).

APPLICATION

• F. subglutinans and F. verticillioides were identified in maize kernels based on RAPD sequences of fragments (Moller et al., 1999).
• The RAPD-PCR method was applied to ten strains of F. verticillioides associated with cereal samples to study the genetic variation among them (Deepa et al., 2018).

PCR coupled methods

In situ PCR

METHODOLOGY

This technique is a combination of PCR and in situ hybridization to detect mycotoxigenic fungi in tissues or cells (Long, 1998) that has become common for analyzing gene expression and identifying DNA or RNA in cells (Chu et al., 2019). Usually, amplification of DNA from raw materials leads to misinterpretation if the material contains more than one species.

PCR-DGGE

METHODOLOGY

Technique that detects fungal communities by amplifying target DNA, initially using PCR, and later subjected to electrophoresis, but not suitable to describe species present in the sample (Laforgue et al., 2009).

APPLICATION

• Trichoderma harzianum spores from feed matrices were detected through this method. The method is mainly applied in diversity studies and for detecting pathogenic fungi, though it is a time-consuming and poorly reproducible technique (Rytkonen et al., 2011).

Post-amplification methods

LAMP

METHODOLOGY

Loop-mediated isothermal amplification is a highly sensitive and successful technique which is directly applied on-site to ensure feed quality and safety, amplifying target DNA in less than an hour at a constant temperature by using four primers to identify six regions on the target DNA with special attention in the annealing step (Tomita et al., 2008).

APPLICATION

• Ochratoxin producing Penicillium nordicum was rapidly detected directly on-site in food and feed production chains (Ferrara et al., 2015).

DNA ARRAYS

METHODOLOGY

DNA array technology was developed to detect a large number of organisms at a time. It includes microarray and macroarray techniques in which discrete positioned species-specific detector sequences are fixed on nylon filters or a glass slide. This method is mainly applied in gene expression studies.

⇰ Microarrays consist of a DNA chip for analyzing thousands of mRNAs simultaneously and are mainly used for gene expression studies and applied for the identification of Aspergillus species (Singh y Kumar, 2013).

⇰ Macroarrays involve a DNA hybridization technique and mainly used for identification of A. alternata, F. solani and A. fumigatus in feed matrices (Singh y Kumar, 2013).

APPLICATION

• Kristensen et al. (2007) applied microarray technology for Fusarium species detection which seems to be easy, safe, fast and cost-effective among feed matrices and food. Fusarium species producing 14 trichothecenes and moniliformin detection was determined by DNA microarray.

Emerging Novel technologies in detection of mycotoxins

Other than Chromatographic methods (Thin Layer Chromatography, Gas Chromatography, and Liquid Chromatography) and Immunoassay-based methods (flow-through membranes, ELISA, dipsticks, Fluorescence Polarization Immunoassay (FPI), and lateral flow immunoassay (LFA)) (Agriopoulou et al., 2020), many more emerging techniques can be applied for detection of mycotoxins in feed.

BIOSENSORS

METHODOLOGY

This fast, easy, reproducible, stable, accurate, and inexpensive on-site-testing method requires transducers (optical, piezoelectric, or electrochemical) for the detection of mycotoxins (Schulz et al., 2019).

APPLICATION

• Oliveria et al. (2019) reported using biosensors for detecting mycotoxins produced by mycotoxigenic fungi in feed and food.
• The method detected AFB₁, OTA, AFM₁, PAT, ZEN, FB₁, T-2, and DON in corn powder, corn, barley, wheat, maize, and corn products (Hossain et al., 2019).
• Impedimetric sensors detected mycotoxigenic fungi producing AFB₁, AFM₁, OTA, and PAT in food and feedstuffs (Khan et al., 2019).
• Photentiometric sensors with a two or three-electrode system were used for the successful testing of AFB₁ and ZEN in corn powder and maize respectively (He et al., 2019).
• Amperometric sensors based on a two or three-electrode system have also been used to detect ZEN in corn and corn-based products (He et al., 2019).
• Surface Plasmon Resonance sensors are sensitive, and cost-effective with high specificity to detect DON, ZEN, and T-2 toxin in wheat (Hossain et al., 2019).
• Quartz crystal microbalance sensors detected AFB₁ in rice and wheat (Gu et al., 2019).

ELECTRONIC NOSE

METHODOLOGY

E-nose involves non-specific detectors that capture volatile organic compounds (VOCs) and detects qualitative volatile fingerprints associated with toxigenic fungi.

⇰ Odor is the preliminary information to detect metabolite produced on identifying specific VOCs related to fungi on feed matrices.

APPLICATION

• The method detects mycotoxigenic fungi-produced mycotoxins, such as aflatoxins, fumonisins in maize, and DON in wheat bran, which is the major ingredient in feed (Ottoboni et al., 2018).

FLUORESCENT POLARIZATION (FP)

METHODOLOGY

FP works with the analyte and the tracer for the antibody binding site. The tracer binding to the antibody influences the tracer molecule’s rotation increasing the FP value.

⇰ The amount of tracer is inversely proportional to the free analyte in the sample, which results in a polarization value inversely proportional to the analyte concentration.

APPLICATION

• The method detects Fusarium species producing ZEN in maize and DON in wheat-based products, as well as Aspergillus species producing AFB₁ in maize, and OTA in rice (Huang et al., 2020).
• The method is accurate in comparison to HPLC with limited sensitivity (Zhang et al., 2018).

QUANTITATIVE NMR AND HYPERSPECTRAL IMAGING

METHODOLOGY

Nuclear magnetic resonance (NMR) spectroscopy is used for the identification of organic compounds and metabolomics. Recently, 2D and multidimensional NMR has been applicable.

Hyperspectral imaging is a technique for the assessment of fungal mycotoxins that works with less cost without destroying the sample, being quick and operating on locating spectral data.

APPLICATION

• NMR-based metabolomics has been used to elucidate the rearrangement mechanism of the Fusarium mycotoxin, Fusarin C, in feed due to its high sample throughput and metabolomics with rich structural information (López-Ruiz et al., 2019).
• Hyperspectral imaging has been mainly used to detect DON and Fusarium pathogen contamination in bulk wheat kernels (Femenias et al., 2020).

CONCLUSIONS

Certain agricultural commodities, such as wheat, maize, soybeans, bajra, sorghum, and rice, are mainly used for manufacturing animal feed, which they are in higher demand for livestock industries.

Hence, animal feed safety is fundamental as feed consumption is a potential route for hazardous chemicals to enter the human food chain, including mycotoxins.

Due to the threat that mycotoxins pose to animal and human health, certain legislations have been applied in the EU to feed-based products intended for livestock with the establishment of verified limitation criteria, as well as the development of sensitive and consistent methods to detect mycotoxins.

Enormous PCR-based technologies and novel analytical techniques are applied to detect mycotoxins and mycotoxigenic fungi.

They are rapid, cost-effective, accurate, sensitive, portable, and safe for detection in food and feed matrices.

Currently, many more molecular methods, such as NASBA, FISH, SAGE, Northern blotting, DNA/RNA probe-based method, PCR-ELISA, MCH-PCR, Next generation PCR, Co-operational PCR, RNA-interference are in practice. However, they have not yet been applied for the detection of mycotoxigenic fungi associated with food and feed matrices.

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