PhD in Veterinary Sciences for Animal Health and Food Safety

Contacts

Supervisor

Flavia Girolami

Curriculum vitae

Curriculum Vitae

Phd thesis

TITLE: CONTROL OF AVIAN MYCOTOXICOSIS

 

• Scientific background/state of the art 

 

Mycotoxins (MYC) are low molecular weight secondary metabolites of toxigenic fungi that can contaminate a wide variety of food and feed commodities of vegetable origin, resulting in an array of negative effects in both humans and animals ¹ . The most common MYC includes aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON), T-2 toxin (T-2), and fumonisins (FBs), which have a diverse range of toxic biological activities ² .

The poultry industry is a major consumer of cereals. MYC contamination of crops causes mainly low egg production and immunosuppression, resulting in an overall diminished performance in both broilers and laying hens ³ . Such adverse effects are mostly triggered by the generation of reactive metabolites by xenobiotic-metabolizing enzymes (XME) and the impairment of the antioxidant defense system, which induces oxidative stress and cytotoxicity ⁴ 

Recent innovative strategies for controlling the contamination by MYC include the use of cold atmospheric plasma, magnetic materials, and nanoparticles, and natural essential oils ^5,6,7 . However, despite a promising potential, such methods are not completely effective. Another approach is the use of dietary supplements, which could prevent MYC absorption (eg, binders) or reduce the oxidative stress induced by MYC (eg, natural antioxidants) ^8-9 . The possible modulation of tissue drug transporters is also envisaged.

 

• Aims 

 

The project will take advantage of an already performed in vivo experiment in broilers ⁹ . In brief, animals were fed diets containing FB1, OTA and ZEN at concentrations approaching the maximum limits allowed in the EU, in the presence or absence of two different binders for 10 days. The selected binders were a feed additive containing a tri-octahedral smectite mixed with lignocellulose (SeOX) and an organic adsorbent which is under registration (ADS), respectively. At slaughtering liver, kidney and intestine (jejunum) were collected and stored at -80°C for subsequent analyses.

The primary purpose of the project is to investigate the effects of each MYC on the gene and protein expression/activity of XME (phase I and phase II), antioxidant enzymes (eg, CAT, SOD and GPX) and drug transporters (eg, ABC and OAT families) in the collected organs. Moreover, the possible counteraction of such effects by the dietary binders will be explored.

 

A further aim is to elucidate through an in vitro approach the role of XME (especially the CYP family) in the metabolism and bioactivation of OTA in the chicken, as well as the OTA-mediated effects on the antioxidant system. OTA has been selected among other MYC since it is currently under evaluation by EFSA, and recent studies have revealed its key role in inducing oxidative stress ¹⁰ . Finally, we will investigate in the same cell model the possible mitigating effect of the antioxidant Curcumin (CURC) on OTA cytotoxicity.

 

• Materials and methods 

 

Analyzes on Liver, Kidney and Intestine

 

The expression of target genes will be measured through the Real-Time PCR (qRT-PCR) method. Briefly, specific primers for the genes of interest will be designed and mRNA will be extracted from each collected tissue and reverse-transcribed into cDNA. Protein expression will be evaluated through Western Blotting assays, using specific primary antibodies. Enzymatic activities, of both XME and antioxidant enzymes, will be investigated through in-house methods with specific substrates or commercially available kits, respectively.

 

In vitro study

 

A chicken hepatocellular carcinoma cell line (LMH) will be employed. Cells will be exposed to increasing concentrations of OTA in the presence or absence of CURC. The cell viability at different time-points will be evaluated by means of different cytotoxicity assays (ie, WST-1 and Neutral Red assays). The mechanisms of interaction with OTA metabolism and toxicity will be investigated through the analysis of XME and antioxidant systems (eg, SOD, CAT, and GPx, and levels of GSH) at both gene (Real-time PCR) and protein level (Western blot and spectrophotometric/spectrofluorimetric assays).  

 

• Expected results 

 

The project is expected to assess the modulation of XME, antioxidant enzymes and drug transporters in broiler target organs by 3 major MYC (ie, FB1, OTA and ZEN). As a result of the expected reduced MYC intestinal absorption by binders (SeOX, ADS), it is conceivable that a reduction in the biotransformation/bioactivation of MYC will occur, inducing an overall protective effect in broilers. Likewise, the in vitro model will clarify the XME involved in OTA metabolism and their potential role in its toxicity. Finally, CURC is expected to positively modulate the antioxidant enzymes, reducing OTA-cytotoxicity, possibly envisaging a future in vivo application.

Overall, the findings of this study will highlight the role of the modulation of XME, antioxidant enzymes and drug transporters in MYC toxicity both in vivo and in vitro, and hopefully provide new insights into the use of additives in poultry feeding to counteract MYC effects in a cost-effective and easy manner.

 

References:

 

1. Omotayo, Oluwadara Pelumi, Abiodun Olusola Omotayo, Mulunda Mwanza, and Olubukola Oluranti Babalola. "Prevalence of mycotoxins and their consequences on human health." Toxicological research 35 (2019): 1-7.
2. Richard, John L. "Some major mycotoxins and their mycotoxicoses—An overview." International journal of food microbiology 119, no. 1-2 (2007): 3-10.
3. Ochieng, Phillis E., Marie-Louise Scippo, David C. Kemboi, Siska Croubels, Sheila Okoth, Erastus K. Kang'ethe, Barbara Doupovec, James K. Gathumbi, Johanna F. Lindahl, and Gunther Antonissen. "Mycotoxins in poultry feed and feed ingredients from Sub-Saharan Africa and their impact on the production of broiler and layer chickens: a review." Toxins 13, no. 9 (2021): 633.
4. Mavrommatis, Alexandros, Elisavet Giamouri, Savvina Tavrizelou, Maria Zacharioudaki, George Danezis, Panagiotis E. Simitzis, Evangelos Zoidis et al. “Impact of mycotoxins on animals' oxidative status.” Antioxidants 10, no. 2 (2021): 214.
5. Cai, Jing, Rong Yan, Jichao Shi, Jia Chen, Miao Long, Wenda Wu, and Kamil Kuca. "Antifungal and mycotoxin detoxification ability of essential oils: A review." Phytotherapy Research 36, no. 1 (2022): 62-72.
6. Sen, Yasin, Baran Onal-Ulusoy, and Mehmet Mutlu. "Detoxification of hazelnuts by different cold plasmas and gamma irradiation treatments." Innovative Food Science & Emerging Technologies 54 (2019): 252-259.
7. Kihal, A., Rodríguez-Prado, M., & Calsamiglia, S. (2022). The efficacy of mycotoxin binders to control mycotoxins in feeds and the potential risk of interactions with nutrient: a review. Journal of animal science ,  100 (11), skac328. https://doi.org/10.1093/jas/skac328
8. Sorrenti, Valeria, Claudia Di Giacomo, Rosaria Acquaviva, Ignazio Barbagallo, Matteo Bognanno, and Fabio Galvano. "Toxicity of ochratoxin a and its modulation by antioxidants: a review." Toxins 5, no. 10 (2013): 1742-1766
9. Damiano, Sara, Watanya Jarriyawattanachaikul, Flavia Girolami, Consiglia Longobardi, Carlo Nebbia, Emanuela Andretta, Chiara Lauritano et al. "Curcumin supplementation protects broiler chickens against the renal oxidative stress induced by the dietary exposure to low levels of aflatoxin B1." Frontiers in Veterinary Science 8 (2022): 1659.
10. Abdelrahman, R. E., Khalaf, A. A. A., Elhady, M. A., Ibrahim, M. A., Hassanen, E. I., & Noshy, P. A. (2022). Antioxidant and antiapoptotic effects of quercetin against ochratoxin A-induced nephrotoxicity in broiler chickens. Environmental Toxicology and Pharmacology ,  96 , 103982.

Research activities

Co supervisor

Carlo Nebbia

Seminars and Conferences:

Oral Presentation:

International Congress for the European Association of Veterinary Pharmacology and Toxiology (EAVPT).

Presentation Title: Turmeric powder mitigates oxidative stress in broilers dietary exposed to Aflatoxin B1 at EU maximum tolerated levels