Research project C3/022 (Research action C3)
Context
Silages are key sources of forages for over-wintering livestock in Belgium. Different crops are used for ensilage such as whole maize plant, corncob-mix, grass, immature cereals, etc. Ensilaged whole maize plant, which is produced on 160.000 ha/year in Belgium, takes the major part of cattle forages. The grass silages also represent a big part of the beef cattle and dairy heifers forages, with a yearly production of 250.000 ha (source: http://statbel.fgov.be/downloads/crp2004_fr.xls). Under anaerobic conditions, these crops can be successfully preserved by bacterial lactic acid fermentation. However, spoilage of silage material by the presence and development of moulds is frequently observed and can result in numerous problems for cattle feed and health. The development of these fungal contaminants can be favored by numerous factors including soil contamination, insufficient sugar content, failure to maintain anaerobiosis, …
Project description
Objectives
The research entails to study the biological and metabolic diversity of fungal contaminants in silage. The morphological characterization of the isolated fungal strains will be related to the molecular characterization and completed by phylogenic studies. The diversity of fungal strain isolated form the various silages will be analyzed through RAPD and AFLP profiles and DNA sequencing. The results of the morphological, taxonomic, phylogenic and diversity will allow extending the BCCMTM/MUCL -collection through the integration of subsets of well described and characterized fungal material (silage contaminating fungi).
Within this project, new methods will be developed for extraction and HPLC analysis of mycotoxins isolated for the different silages. The analysis of mycotoxins will also be realized on samples issued form the in vitro culture of fungal contaminants and particular attention will be devoted to the less-known mycotoxins and to other (potentially unknown) metabolites isolated from fungi. The information related to the mycotoxins and production of metabolites of the fungal isolated from the silages will be used to refine the phylogenic characterization of fungal strains. Toxicological analysis will be conducted on silage samples et on pure fungal isolates extracts in order to establish dangers linked to the presence of mycotoxines and other fungal toxic compounds poorly or unknown. The capacity of pure mycotoxins et contaminated silage extracts to induce genes will be evaluated on transgenic cell cultures.
Five partners (BCCM/MUCL, CERVA-CODA-VAR, Hogeschool Gent, CIPF, UA) will collaborate in the realization of this project.
Methodology
In a first step, different types of silages from all regions of the country will be collected in order to have a broad view of the situation of maize and herb silage in Belgium. Information will be collected about the raw material of silage (plant, variety, phenologic state of the plant at time of harvesting) and the ensilage system (type of silo, design etc…). In addition, information will be collated on the symptoms developed by cattle (diseases, fertility problems, allergic reactions etc...) fed with these silages to have some background information on the possible effects of mycotoxins.
In a second step, the selected silages will be analyzed through sampling in healthy as well as contaminated parts in order to (1) isolate and identify molds and yeasts, (2) analyze mycotoxins from silage samples and (3) evaluate the mycotoxin toxicity in silages.
(1) Isolation and identification of molds and yeasts
Molds and yeasts will be isolated from fresh samples and will be incubated on appropriate culture media. The fungal samples will be maintained on mineral oil, by cryopreservation and lyophilisation.
For an accurate identification of species, regions of taxonomic interest will be sequenced, based on the expertise of MUCL and on the literature. The diversity within the different species isolated from the different type of silages will be analyzed using RAPD and AFLP. This method will allow us to obtain a picture of the possible populations within species.
(2) Analysis of mycotoxins from silage samples
Preliminary mycotoxin markers analyses will be performed for all collected samples of silages: deoxynivalenol (DON) and Patulin (PAT) on Maize silage and PAT solely on grass silage. Depending on the preliminary results, further analyzes will be made to find other mycotoxins.
(3) Mycotoxin toxicity in silages: a gene expression and flow cytometric approach
The toxicity of mycotoxins will be evaluated on living cell lines.This project introduces a new and versatile technology to the assessment of mycotoxin detection and toxicity in the follow up of silages. Starting from detection of mycotoxin toxicity, the technology allows for identification of the mycotoxin family through determination of the molecular mode of action, comparison and ranking of different contamination events in one or several silages. The current assay consists of a battery of genetically transformed bacterial strains and transgenic human liver cell lines. The working principle of both systems is similar and is based on the induction of specific stress gene promoters.
Interaction between the different partners
CIPF and HGent partners will collect about the ensilages and all informations relative to these materials. HGent will isolate fungal contaminants from silage samples, and will send the isolates to MUCL for identification. MUCL will identify (morphologically and molecularly) and cultivate the fungi in liquid medium. The culture supernatants will be sent to CERVA for mycotoxin analyses. CERVA will extract and analyze the mycotoxin from the silage samples and liquid medium cultures. UA will use the extracts for the toxicological analyzes. MUCL, -as coordinator- will collect and integrate the results.
Expected results and/or products
Fungal and mycotoxin contamination in maize and grass silage feeds will be studied to gain an overview on their effect on the forage quality in Belgium. The study will let us to estimate the toxicological effects of mycotoxins. In details, the project will yield the following results:
- Development of new methods for mycotoxin extraction and analysis from maize silage matrix and grass silage matrix
- An overview on the most important silage contaminating fungal species
- Effect of inoculants and/or additives application on the quality of the silage and on the fungal and mycotoxin contamination
- The implementation of an on-line database on molds and mycotoxins of the ensilages, being of use as support to the farmers for the management of the problems of contamination of the ensilages by detailing the risks of production and the toxicity of mycotoxins
Isolation of fungal species from moldy silage and characterization of those strains is predisposed to valorize the biological material available in the BCCM collections by improving and expanding the existing knowledge on the characteristics of the material. Characterization will include morphological data, molecular data (RAPD, AFLP, sequences), metabolite production (mycotoxins) and toxicological data. The private partners will gain a better insight in the overall problematic of fungal contamination and consequent mycotoxin production in silage. This will eventually lead to improved quality of silage feed, reduction of contamination events and hence reduction of costs.
Partners
Activities
Mycothèque de l'UCL
Project coordinator. Isolation, identification (morphological and molecular) of the isolates. Storage of isolates.
CERVA-CODA-VAR
Mycotoxin analyzes with HPLC method, from silages and liquid culture medium.
Hogeschool Gent, Department of Biotechnological Sciences (BIOT)
Silage sampling in collaboration with CIPF. Isolation of microbes from silage samples.
Centre Indépendant de Promotion Fourragères (CIPF)
Silage sampling in collaboration with Hogeschool Gent.
University of Antwerp, Department of Biology, Ecophysiology, Biochemistry and Toxicology Group
Toxicological analyzes of silage contaminants, using transgenic bacterial strains and human liver cells lines.
Contact information
Mycothèque de l'Université catholique de Louvain (MUCL)
Unité de Microbiologie (MBLA)
Croix du Sud, 3 bte 6
B-1348 Louvain-la-Neuve, BELGIUM
Tel : +32-10-473084
Fax : +32-10-451501
Stéphane Declerck (declerck@mbla.ucl.ac.be), François Van Hove (vanhove@mbla.ucl.ac.be), Péter Harcz (harcz@mbla.ucl.ac.be)
URL: http://www.belspo.be/bccm/
CERVA-CODA-VAR
Leuvensestenweeg 17
3080 Tervuren
Tel:+32(2) 769 22 00
Fax:+32(2) 769 23 05
Luc Pussemier (lupus@var.fgov.be)
URL: www.var.fgov.be
Hogeschool Gent
Department of Biotechnological Sciences (BIOT)
Volkenslaan 270
B-9000 Gent
Tel : +32(0)9 242 42 75
Fax: +32(0)9 242 42 79
Geert Haesaert (geert.haesaert@hogent.be)
URL: www.hogent.be
Centre Indépendant de Promotion Fourragères (CIPF)
Place Croix du Sud, 2 Bte 11
1348 Louvain-la-Neuve
Tel : 010/ 47.34.62 - 010/ 47.38.40
Fax : 010/ 47.20.21
Jurgen Depoorter (j.depoorter@cipf.be)
University of Antwerp
Campus Middelheim
Department of Biology,
Ecophysiology, Biochemistry andToxicology Group
Groenenborgenlaan 171/U7
B-2020 Antwerp
Tel: +32(0)3 265 33 47
Fax: +32(0)3 265 34 97
Ronny Blust (Ronny.Blust@ua.ac.be)
Users committee
- M. Damien Vanoystaeyen, Agence Fédérale pour la Sécurité de la Chaîne Alimentaire (AFSCA)
- M. L. Fabry, Association Wallone de l’élevage (AWE)
- M. Ignace Dewaele, Tiense Suikerraffinaderij
- M. Guy Vandepoel, Belgische Boerenbond
- Mme Geertrui Vlaemynck, Agricultural Research Centre, Dept. of Animal Product Quality and Transformation Technology
- M. Pierre Frankinet, ALFRA sa
