Determination of Mycotoxin Production of Fusarium Species in Genetically Modified Maize Varieties by Quantitative Flow Immunocytometry.

Levels of mycotoxins produced by Fusarium species in genetically modified (GM) and near-isogenic maize, were determined using multi-analyte, microbead-based flow immunocytometry with fluorescence detection, for the parallel quantitative determination of fumonisin B1, deoxynivalenol, zearalenone, T-2, ochratoxin A, and aflatoxin B1. Maize varieties included the genetic events MON 810 and DAS-59122-7, and their isogenic counterparts. Cobs were artificially infested by F. verticillioides and F. proliferatum conidia, and contained F. graminearum and F. sporotrichoides natural infestation. The production of fumonisin B1 and deoxynivalenol was substantially affected in GM maize lines: F. verticillioides, with the addition of F. graminearum and F. sporotrichoides, produced significantly lower levels of fumonisin B1 (~300 mg·kg-1) in DAS-59122-7 than in its isogenic line (~580 mg·kg-1), while F. proliferatum, in addition to F. graminearum and F. sporotrichoides, produced significantly higher levels of deoxynivalenol (~18 mg·kg-1) in MON 810 than in its isogenic line (~5 mg·kg-1). Fusarium verticillioides, with F. graminearum and F. sporotrichoides, produced lower amounts of deoxynivalenol and zearalenone than F. proliferatum, with F. graminearum and F. sporotrichoides. T-2 toxin production remained unchanged when considering the maize variety. The results demonstrate the utility of the Fungi-Plex™ quantitative flow immunocytometry method, applied for the high throughput parallel determination of the target mycotoxins.

Comparison and evaluation of seven different bench-top flow cytometers with a modified six-plexed mycotoxin kit.

Many bench-top flow cytometers (b-FCs) are compatible with microsphere-based multiplexed assays. Disciplines implementing b-FCs-based assays are expanding; they include monitoring and validating food quality. A multiplexed platform protocol was evaluated for poly-mycotoxin assays, which is compatible with a variety of b-FC models. The seven instruments included: BD FACSCalibur(™) , BD FACSArray(™) Bioanalyzer, Accuri C6, Partec CyFlow(®) Space, Beckman Coulter FC 500, Guava EasyCyte Mini, and Luminex 100 (™) . Current reports related to the food industry describe fungal co-infections leading to poly-mycotoxin contamination in grain (Sulyok M, Berthiller F, Krska R, Schuhmacher R, Rapid Commun Mass Spectrom 2006;20:2649-2659). It is imperative to determine whether b-FC-based assays can replace traditional single-mycotoxin enzyme-linked immunosorbent assay (ELISA). A six-plexed poly-mycotoxin kit was tested on seven different b-FCs. The modified kit was initially developed for the BD FACSArray(™) Bioanalyzer (BD Biosciences) (Czeh A, Mandy F, Feher-Toth S, Torok L, Mike Z, Koszegi B, Lustyik G, J Immunol Methods 2012;384:71-80). With the multiplexed platform, it is possible to identify up to six mycotoxin contaminants simultaneously at regional grain collection/transfer/inspection facilities. In the future, elimination of contaminated food threat may be better achieved with the inclusion of b-FCs in the food protection arsenal. A universal protocol, matched with postacquisition software, offers an effective alternative platform compared to using a series of ELISA kits. To support side-by-side evaluation of seven flow cytometers, an instrument-independent fluorescence emission calibration was added to the protocol. All instrument performances were evaluated for strength of agreement based on paired sets of evaluation to predicate method. The results suggest that all b-FCs were acceptable of performing with the multiplexed kit for five of six mycotoxins. For OTA, the detection sensitivity was consistent only for five of the seven instruments.

A new zearalenone biodegradation strategy using non-pathogenic Rhodococcus pyridinivorans K408 strain.

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17ß-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17ß-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.

A flow cytometry based competitive fluorescent microsphere immunoassay (CFIA) system for detecting up to six mycotoxins.

BACKGROUND: Exposure to multiple mycotoxins through the food chain represents a major potential health hazard to both humans and livestock. They can cause a variety of severe acute as well as chronic diseases. Eliminating mycotoxins from various grain crops is a global health priority. According to the Food and Agriculture Organization (FAO), world food production needs to double by 2050. Innovative solutions will be required to sustain toxin free grain supplies worldwide. METHODS: A competitive flow cytometry based multiplexed assay with fluorescent microspheres has been developed. The new multiplexed method can analyze simultaneously any one or all six major mycotoxins. They include: Ochratoxin A (OTA), Aflatoxin B1 (AFB1), Fumonisin B1 (FB1), T-2 toxin (T-2), Deoxynivalenol (DON) and Zearalenone (ZEA), which are all potential human health hazards. The CFIA described here includes a simplified single extraction step for mycotoxins from specimens and a comprehensive post acquisition software module. The new assay system was developed with a FACSArray™ BD Bioanalyzer flow cytometer (BD Biosciences, Belgium). RESULTS: The CFIA performs favourably when compared to commercial ELISA. Sensitivity range with CFIA increased between 13% and 100% with an average improvement of 50% for the six mycotoxins. CONCLUSIONS: The multiplexed assay presented here has the unique capacity to quantify up to six mycotoxins simultaneously from a single specimen extraction. CFIA's poly-mycotoxin detection sensitivity exceeds standard ELISA. CFIA may be part of a comprehensive assay system that will provide reliable and effective safeguard for agricultural commodities to be free of mycotoxins.