Fungal and chemical diversity in hay and wrapped haylage for equine feed

  • Birgitte Andersen
  • Christopher Phippen
  • Jens C. Frisvad
  • Sue Emery
  • Robert A. EustaceEmail author
Original Article


The presence of fungi and mycotoxins in silage (fermented maize) for cattle and other ruminants have been studied extensively compared to wrapped haylage (fermented grass) for horses and other monogastric animals. The purpose of this work was to examine the fungal diversity of wrapped haylage and conventional hay and to analyse the forage sample for fungal metabolites. Faeces samples were also analysed to study the fate of fungi and metabolites. Fungal diversity of the samples was determined by direct plating on DG18, V8 and MEA and chemical analyses were done using LC-MS/MS. The results show that Sordaria fimicola was common in both hay and haylage, while Penicillium spp. was prevalent in haylage and Aspergillus spp. in hay. Communiols were found in all types of samples together with gliocladic acid. Roquefortines and fumigaclavines were found in haylage with no visible fungal growth, but not in hay. In haylage hot spot samples, a series of Penicillium metabolites were detected: Andrastins, fumigaclavines, isofumigaclavines, marcfortines, mycophenolic acid, PR toxins, and roquefortines. Penicillium solitum was found repeatedly in haylage and haylage hot spot samples and viridicatols were detected in a hot spot sample, which has not been reported before. Even haylage with no visible fungal growth contained more metabolites than hay. Individually, the metabolites detected in haylage may, in high doses, be mutagenic, neurotoxic or immunosuppressive; but the synergistic effect of small doses may also have other or greater negative health effects on equines than on ruminants.


Horses Ponies Mycotoxins Roquefortine Laminitis Metabolite profiling Adverse health effects 



The authors wish to thank The Laminitis Trust for their financial support and Lisette Knoth-Nielsen for technical assistance.

Funding information

This study was supported by The Laminitis Trust.

Compliance with ethical standards

Conflicts of interest

The last author is a Trustee of the Laminitis Trust.


  1. Alonso VA, Pereyra CM, Keller LAM, Dalcero AM, Rosa CAR, Chiacchiera SM, Cavaglieri LR (2013) Fungi and mycotoxins in silage: an overview. J Appl Microbiol 115:637–643. CrossRefPubMedGoogle Scholar
  2. Alonso V, Cavaglieri L, Ramos AJ, Torres A, Marin S (2017) Modelling the effect of pH and water activity in the growth of Aspergillus fumigatus isolated from corn silage. J Appl Microbiol 122:1048–1056. CrossRefPubMedGoogle Scholar
  3. Auerbach H, Oldenburg E, Weissbach F (1998) Incidence of Penicillium roqueforti and roquefortine C in silages. J Sci Food Agric 76:565–572.<565::AID-JCFA990>3.0.COI2-6 CrossRefGoogle Scholar
  4. Bashyal BP, Wijeratne EK, Faeth SH, Gunatilaka AL (2005) Globosumones A− C, cytotoxic orsellinic acid esters from the Sonoran desert endophytic fungus Chaetomium globosum. J Nat Prod 68:724–728. CrossRefPubMedGoogle Scholar
  5. Bashyal BP, Burns AM, Liu MX, Paranagama PA, Seliga CJ, Turbyville TJ, Wijeratne EMK, Zhan J, Gunatilaka MK, Arnold AE, Faeth SH, Whitesell L, Gunitilaka AAL (2007) Discovery of small molecule bioactive agents from endophytic fungi of the Sonoran Desert. 6th International Symposium on fungal endophytes, Grassland Research Practices Series No. 13, New Zealand Grassland Association, Dunedin, pp 211-214.Google Scholar
  6. Bentley R (2000) Mycophenolic acid: a one hundred year odyssey from antibiotic to immunosuppressant. Chem Rev 100:3801–3825. CrossRefPubMedGoogle Scholar
  7. Bills GF, Gloer JB, An Z (2013) Coprophilous fungi: antibiotic discovery and functions in an underexplored arena of microbial defensive mutualism. Cur Op Microbiol 16:549–565. CrossRefGoogle Scholar
  8. Che Y, Gloer JB, Scott JA, Malloch D (2004) Communiols A–D: new mono-and bis-tetrahydrofuran derivatives from the coprophilous fungus Podospora communis. Tetrahedron Lett 45:6891–6894. CrossRefGoogle Scholar
  9. Che Y, Araujo AR, Gloer JB, Scott JA, Malloch D (2005) Communiols E− H: new polyketide metabolites from the coprophilous fungus Podospora communis. J Nat Prod 68:435–438. CrossRefPubMedGoogle Scholar
  10. Chen AJ, Hubka V, Frisvad JC, Visagie CM, Houbraken J, Meijer M, Varga J, Rasine D, Jurjević Ž, Kubátová A, Sklenář F, Samson RA (2017) Polyphasic taxonomy of Aspergillus section Aspergillus (formerly Eurotium) and its occurrence in indoor environment and food. Stud Mycol 88:37–135. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Coblenz WK, Akins MS (2018) Silage review: Recent advances and future technologies for baled silages. Journal of Dairy Science 101:4075–4092. CrossRefGoogle Scholar
  12. Cole RJ, Cox RH (1981) Handbook of toxic fungal metabolites. Academic Press, New YorkGoogle Scholar
  13. De Vries RP, Frisvad JC, van de Vondervoort PJ, Burgers K, Kuijpers AF, Samson RA, Visser J (2005) Aspergillus vadensis, a new species of the group of black Aspergilli. Antonie Van Leeuwenhoek 87:195–203. CrossRefPubMedGoogle Scholar
  14. Domsch KH, Gams W, Anderson T-H (2007) Compendium of soil fungi, 2nd edn. IHW-Verlag, EchingGoogle Scholar
  15. Dong X-X, Fu J, Yin X, Cao S, Li X-B, Lin L, Cao S-L, Li X-C, Lin L-F, Huyiligeqi, Ni J (2016) Emodin: a review of its pharmacology, toxicity and pharmacokinetics. Phytother Res 30:1207–1218. CrossRefPubMedGoogle Scholar
  16. Dorner JW, Cole RJ, Hill R, Wicklow D, Cox RH (1980) Penicillium rubrum and Penicillium biforme, new sources of rugulovasines A and B. Appl Environ Microbiol 40:685–687PubMedPubMedCentralGoogle Scholar
  17. Dubey MK, Aamir M, Kaushik MS, Khare S, Meena M, Singh S, Upadhyay RS (2018) PR Toxin–Biosynthesis, Genetic Regulation, Toxicological Potential, Prevention and Control Measures: Overview and Challenges. Front Pharmacol 9:288. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fabian SJ, Maust MD, Panaccione DG (2018) Ergot Alkaloid Synthesis Capacity of Penicillium camemberti. Appl Environ Microbiol 84:e01583–e01518. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Frisvad JC, Thrane U (1987) Standardized High Performance Liquid Chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone indices and UV VIS spectra (diode array detection). J Chromatogr 404(195):214. CrossRefGoogle Scholar
  20. Frisvad JC, Thrane U (1993) Liquid column chromatography of mycotoxins. In: Betina V (ed) Chromatography of mycotoxins: techniques and applications. Journal of Chromatography Library 54. Elsevier, Amsterdam, pp 253–372CrossRefGoogle Scholar
  21. Frisvad JC, Smedsgaard J, Larsen TO, Samson RA (2004) Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Stud Mycol 49:201–241Google Scholar
  22. Frisvad JC, Andersen B, Thrane U (2008) The use of secondary metabolite profiling in chemotaxonomy of filamentous fungi. Mycol Res 112:231–240. CrossRefPubMedGoogle Scholar
  23. Galey FD, Whiteley HE, Goetz TE, Kuenstler AR, Davis CA, Beasley VR (1991) Black-walnut (Juglans nigra) toxicosis –a model for equine laminitis. J Comp Pathol 104:313–326. CrossRefPubMedGoogle Scholar
  24. Gallo A, Giuberti G, Frisvad JC, Bertuzzi T, Nielsen KF (2015) Review on mycotoxin issues in ruminants: occurrence in forages, effects of mycotoxin ingestion on health status and animal performance and practical strategies to counteract their negative effect. Toxins 7:3057–3111. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Gräbsch C, Wichmann G, Loffhagen N, Herbarth O, Müller A (2006) Cytotoxicity assessment of gliotoxin and penicillic acid in Tetrahymena pyriformis. EnvironToxicol 21:111–117. CrossRefGoogle Scholar
  26. Hanche-Olsen S, Teige J, Skaar I, Ihler CF (2008) Polyneuropathy associated with forage sources in Norwegian horses. J Vet Intern Med 22:178–184. CrossRefPubMedGoogle Scholar
  27. Kalai S, Anzala L, Bensoussan M, Dantigny P (2017) Modelling the effect of temperature, pH, water activity, and organic acids on the germination time of Penicillium camemberti and Penicillium roqueforti conidia. Int J Food Microbiol 240:124–130. CrossRefPubMedGoogle Scholar
  28. Kildgaard S, Mansson M, Dosen I, Klitgaard A, Frisvad JC, Larsen TO, Nielsen KF (2014) Accurate dereplication of bioactive secondary metabolites from marine-derived fungi by UHPLC-DAD-QTOFMS and MS/HRMS library. Mar Drugs 12:3681–3705. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Klitgaard A, Iversen A, Andersen MR, Larsen TO, Frisvad JC, Nielsen KF (2014) Aggressive dereplication using UHPLC-DAD-QTOF – screening extracts for up to 3000 fungal secondary metabolites. Anal Bioanal Chem 406:1933–1943. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Larsen TO, Gareis M, Frisvad JC (2002) Cell cytotoxicity and mycotoxin and secondary metabolite production by common Penicillia on cheese. J Agric Food Chem 50:6148–6152. CrossRefPubMedGoogle Scholar
  31. Le Bars J, Le Bars P (1996) Recent acute and subacute mycotoxicoses recognized in France. Vet Res 27:383–394PubMedGoogle Scholar
  32. Liesener K, Curtui V, Dietrich R, Märtlbauer E, Usleber E (2010) Mycotoxins in horse feed. Mycotoxin Res 26:23–30. CrossRefPubMedGoogle Scholar
  33. Lin S, He J, Jiang Y, Wu F, Wang H, Wu D, Sun J, Zhang D-D, Qu H-X, Yang B (2014) Production of nigragillin and dihydrophaseic acid by biotransformation of litchi pericarp with Aspergillus awamori and their antioxidant activities. J Funct Foods 7:278–286. CrossRefGoogle Scholar
  34. Malekinejad H, Aghazadeh-Attari J, Rezabakhsh A, Sattari M, Ghasemsoltani-Momtaz B (2015) Neurotoxicity of mycotoxins produced in vitro by Penicillium roqueforti isolated from maize and grass silage. Human Exper Toxicol 34:997–1005. CrossRefGoogle Scholar
  35. Marwah RG, Fatope MO, Deadman ML, Al-Maqbali YM, Husband J (2007) Musanahol: a new aureonitol-related metabolite from a Chaetomium sp. Tetrahedron 63:8174–8180. CrossRefGoogle Scholar
  36. Medentsev AG, Akimenko VK (1998) Naphthoquinone metabolites of the fungi. Phytochem 47:935–959. CrossRefGoogle Scholar
  37. Moss MO (1998) Recent studies of mycotoxins. J Appl Microbiol 84:62S–76S. CrossRefGoogle Scholar
  38. Müller CE (2005) Fermentation patterns of small-bale silage and haylage produced as feed for horses. Grass Forage Sci 60:109–118. CrossRefGoogle Scholar
  39. Müller CE (2018) Silage and haylage for horses. Grass Forage Sci 73:815–827. CrossRefGoogle Scholar
  40. Müller CE, Pauly TM, Udén P (2007) Storage of small bale silage and haylage–influence of storage period on fermentation variables and microbial composition. Grass Forage Sci 62:274–283. CrossRefGoogle Scholar
  41. Müller CE, Hultén C, Gröndahl G (2011) Assessment of hygienic quality of haylage fed to healthy horses. Grass Forage Sci 66:453–463. CrossRefGoogle Scholar
  42. Nakamura T, Komagata D, Murakawa S, Sakai K, Endo A (1990) Isolation and biosynthesis of 3α-hydroxy-3, 5-dihydromonacolin L. J Antibiot 43:1597–1600CrossRefGoogle Scholar
  43. Nielsen KF, Frisvad JC, Sumarah M, Miller JD (2006) Production of metabolites from the Penicillium roqueforti complex. J Agric Food Chem 54:3756–3763. CrossRefPubMedGoogle Scholar
  44. Nielsen KF, Månsson M, Rank C, Frisvad JC, Larsen TO (2011) Dereplication of microbial natural products by LC-DAD-TOFMS. J Nat Prod 74:2338–2348. CrossRefPubMedGoogle Scholar
  45. Northolt MD, Bullerman LB (1982) Prevention of mold growth and toxin production through control of environmental conditions. J Food Prot 45:519–526. CrossRefPubMedGoogle Scholar
  46. O’Brien M, Nielsen KF, O’Kiely P, Forristal PD, Fuller HT, Frisvad JC (2006) Mycotoxins and other secondary metabolites produced in vitro by Penicillium paneum Frisvad and Penicillium roqueforti Thom isolated from baled grass silage in Ireland. J Agric Food Chem 54:9268–9276. CrossRefPubMedGoogle Scholar
  47. Ogunade IM, Martinez-Tuppia C, Qeiroz OCM, Jiang Y, Drouin P, Wu F, Vyas D, Adesogan AT (2018) Silage review: Mycotoxins in silage: Occurrence, effects, prevention and mitigation. J Dairy Sci 101:4034–4059. CrossRefPubMedGoogle Scholar
  48. Ostry V, Toman J, Grosse Y, Malir F (2018) Cyclopiazonic acid: 50th anniversary of its discovery. World Mycotoxin J 11:135–148. CrossRefGoogle Scholar
  49. Oswald IP, Marin DE, Bouhet S, Pinton P, Taranu I, Accensi F (2005) Immunotoxicological risk of mycotoxins for domestic animals. Food Addit Contam 22:354–360. CrossRefPubMedGoogle Scholar
  50. Panthama N, Kanokmedhakul S, Kanokmedhakul K, Soytong K (2011) Cytotoxic and antimalarial azaphilones from Chaetomium longirostre. J Nat Prod 74:2395–2399. CrossRefPubMedGoogle Scholar
  51. Pazhanivel N, Balachandran C, Muralimanohar B, Dhinakarraj G, Balakrishnan V, Kirubaharan JJ, Raja A (2015) Alleviative effect of gingerol on cell mediated and humoral immunity and immune organs against penicillic acid mycotoxicosis in broiler chickens. Int J Life Sci Pharma Res 5:L28–L34Google Scholar
  52. Pitt JI, Miller JD (2017) A concise history of mycotoxin research. J Agric Food Chem 65:7021–7033. CrossRefPubMedGoogle Scholar
  53. Polonsky J, Merrien MA, Scott PM (1977) Roquefortine and isofumigaclavine A, alkaloids from Penicillium roqueforti. Ann Nutr l’Aliment 31:963–968Google Scholar
  54. Qin JC, Gao JM, Zhang YM, Yang SX, Bai MS, Ma YT, Laatsch H (2009) Polyhydroxylated steroids from an endophytic fungus, Chaetomium globosum ZY-22 isolated from Ginkgo biloba. Steroids 74:786–790. CrossRefPubMedGoogle Scholar
  55. Rand TG, Giles S, Flemming J, Miller JD, Puniani E (2005) Inflammatory and cytotoxic responses in mouse lungs exposed to purified toxins from building isolated Penicillium brevicompactum Dierckx and P. chrysogenum Thom. Toxicological Sciences 87:213–222. CrossRefPubMedGoogle Scholar
  56. Rasmussen TB, Givskov M (2006) Quorum-sensing inhibitors as anti-pathogenic drugs. Int J Med Microbiol 296:149–161. CrossRefPubMedGoogle Scholar
  57. Rasmussen RR, Storm IMLD, Rasmussen PH, Smedsgaard J, Nielsen KF (2010) Multi-mycotoxin analysis of maize silage by LC-MS/MS. Anal Bioanal Chem 397:765–776. CrossRefPubMedGoogle Scholar
  58. Rasmussen RR, Rasmussen PH, Larsen TO, Bladt TT, Binderup ML (2011) In vitro cytotoxicity of fungi spoiling maize silage. Food Chem Toxicol 49:31–44. CrossRefPubMedGoogle Scholar
  59. Reisinger N, Dohnal I, Nagl V, Schaumberger S, Schatzmayr G, Mayer E (2016) Fumonisin B1 (FB1) induces lamellar separation and alters sphingolipid metabolism of in vitro cultured hoof explants. Toxins 8:89. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Samson RA, Houbraken J, Thrane U, Frisvad JC, Andersen B (2010) Food and indoor fungi. 1st edn CBS Laboratory Manual Series 2. CBS-Fungal Biodiversity Centre, Utrecht.Google Scholar
  61. Sarrocco S (2016) Dung-inhabiting fungi: a potential reservoir of novel secondary metabolites for the control of plant pathogens. Pest Manag Sci 72:643–652. CrossRefPubMedGoogle Scholar
  62. Schenk J, Djurle A, Jensen DF, Müller C, O’Brien M, Spörndly R (2018) Filamentous fungi in wrapped forages determined with different sampling and culturing methods. Grass Forage Sci 74:29–41. CrossRefGoogle Scholar
  63. Séguin V, Lemauviel-Lavenant S, Garon D, Bouchart V, Gallard Y, Blanchet B, Diquelou S, Personeni E, Gauduchon P, Ourry A (2010) An evaluation of the hygienic quality in single-species hays and commercial forages used in equine nutrition. Grass Forage Sci 65:304–317. CrossRefGoogle Scholar
  64. Séguin V, Garon D, Lemauviel-Lavenant S, Lanier C, Bouchart V, Gallard Y, Blanchet B, Diquelou S, Personeni E, Ourry A (2012) How to improve the hygienic quality of forages for horse feeding. J Sci Food Agric 92:975–986. CrossRefPubMedGoogle Scholar
  65. Seifert KA, Morgan-Jones G, Gams W, Kendrick B (2011) The genera of Hyphomycetes. CBS-Fungal Biodiversity Centre, UtrechtGoogle Scholar
  66. Shi Y, Zhang X, Lou K (2013) Isolation, characterization, and insecticidal activity of an endophyte of drunken horse grass, Achnatherum inebrians. J Insect Sci 13:151CrossRefGoogle Scholar
  67. Shotwell OL, Bennet GA, Goulden ML, Plattner RD, Hesseltine CW (1980) Survey for zearalenone, aflatoxin, and ochratoxin in U.S. grain sorghum from 1975 and 1976 crops. J Assoc Off Anal Chem 63:922–926Google Scholar
  68. Skóra J, Sulyok M, Nowak A, Otlewska A, Gutarowska B (2017) Toxinogenicity and cytotoxicity of Alternaria, Aspergillus and Penicillium moulds isolated from working environments. Int J Environ Sci Technol 14:595–608. CrossRefGoogle Scholar
  69. Stoev SD (2015) Foodborne mycotoxicoses, risk assessment and underestimated hazard of masked mycotoxins and joint mycotoxin effects or interaction. Environ Toxicol Pharmacol 39:794–809. CrossRefPubMedGoogle Scholar
  70. Stoev SD, Stefanov M, Radic B, Domijan AM, Peraica M (2004) Experimental mycotoxicosis in chickens induced by ochratoxin A and penicillic acid and intervention with natural plant extracts. Vet Res Commun 28:727–746. CrossRefPubMedGoogle Scholar
  71. Storm IM, Sørensen JL, Rasmussen RR, Nielsen KF (2008) Mycotoxins in silage. Stewart Postharvest Rev 6:1–12Google Scholar
  72. Storm IMLD, Kristensen NB, Raun BML, Smedsgaard J (2010) Dynamics in the microbiology of maize silage during whole-season storage. J Appl Microbiol 109:1017–1026. CrossRefPubMedGoogle Scholar
  73. Storm IMLD, Rasmussen RR, Rasmussen PH (2014) Occurrence of pre- and post-harvest mycotoxins and other secondary metabolites in Danish maize silage. Toxins 6:2256–2269. CrossRefPubMedPubMedCentralGoogle Scholar
  74. Sumarah MW, Miller JD, Blackwell BA (2005) Isolation and metabolite production by Penicillium roqueforti, P. paneum and P. crustosum isolated in Canada. Mycopathologia 159:571–577. CrossRefPubMedGoogle Scholar
  75. Thomson JR, McPherson EA (1983) Chronic obstructive pulmonary disease in the horse 2: therapy. Equine Vet J 15:207–210. CrossRefPubMedGoogle Scholar
  76. Vendruscolo CP, Frias NC, de Carvalho CB, de Sá LRM, Belli CB, Baccarin RYA (2016) Leukoencephalomalacia outbreak in horses due to consumption of contaminated hay. J Vet Int Med 30:1879–1881. CrossRefGoogle Scholar
  77. Wambacq E, Vanhoutte I, Audenaert K, De Gelder L, Haesaert G (2016) Occurrence, prevention and remediation of toxigenic fungi and mycotoxins in silage: a review. J Sci Food Agric 96:2284–2302. CrossRefPubMedGoogle Scholar
  78. Wang Y, Gloer JB, Scott JA, Malloch D (1993) Appenolides A, appendiculede B, appendiculide C: three new antifungal furanones from the coprophilous fungus Podospora appendiculata. J Nat Prod 56:341–344. CrossRefPubMedGoogle Scholar
  79. Wang HJ, Gloer KB, Gloer JB, Scott JA, Malloch D (1997) Anserinones A and B: new antifungal and antibacterial benzoquinones from the coprophilous fungus Podospora anserina. J Nat Prod 60:629–631. CrossRefPubMedGoogle Scholar
  80. Young JC, Miller JD (1985) Appearance of fungus, ergosterol and Fusarium mycotoxins in the husk, axial stem and stalk after ear inoculation of field corn. Can J Plant Sci 65:47-53. CrossRefGoogle Scholar

Copyright information

© Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biotechnology and BiomedicineTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.The Laminitis ClinicMead HouseWiltshireUK

Personalised recommendations