Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP)

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay’s detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

Neurotoxic, Inflammatory, and Mucosecretory Responses in the Nasal Airways of Mice Repeatedly Exposed to the Macrocyclic Trichothecene Mycotoxin Roridin A

Macrocyclic trichothecene mycotoxins encountered in water-damaged buildings have been suggested to contribute to illnesses of the upper respiratory tract. Here, the authors characterized the adverse effects of repeated exposures to roridin A (RA), a representative macrocyclic trichothecene, on the nasal airways of mice and assessed the persistence of these effects. Young, adult, female C57BL/6 mice were exposed to single daily, intranasal, instillations of RA (0.4, 2, 10, or 50 μg/kg body weight [bw]) in saline (50 μl) or saline alone (controls) over 3 weeks or 250 μg/kg RA over 2 weeks. Histopathologic, immunohistochemical, and morphometric analyses of nasal airways conducted 24 hr after the last instillation revealed that the lowest-effect level was 10 μg/kg bw. RA exposure induced a dose-dependent, neutrophilic rhinitis with mucus hypersecretion, atrophy and exfoliation of nasal transitional and respiratory epithelium, olfactory epithelial atrophy and loss of olfactory sensory neurons (OSNs). In a second study, the persistence of lesions in mice instilled with 250 μg/kg bw RA was assessed. Nasal inflammation and excess luminal mucus were resolved after 3 weeks, but OSN loss was still evident in olfactory epithelium (OE). These results suggest that nasal inflammation, mucus hypersecretion, and olfactory neurotoxicity could be important adverse health effects associated with short-term, repeated, airborne exposures to macrocyclic trichothecenes.

Macrocyclic trichothecene production and sporulation by a biological control strain of Myrothecium verrucaria is regulated by cultural conditions

Myrothecium verrucaria is a pathogen of several invasive weed species, including kudzu, and is currently being evaluated for use as a bioherbicide. However, the fungus also produces macrocyclic trichothecene mycotoxins. The safety of this biological control agent during production and handling would be improved if an inoculum could be produced without concomitant accumulation of macrocyclic trichothecenes. Sporulation and trichothecene production by M. verrucaria was evaluated on standard potato dextrose agar (PDA) and a series of complex and defined media. Sporulation on PDA and on agar media with nitrogen as ammonium nitrate or potassium nitrate was more than ten-fold greater then sporulation on the medium with ammonium sulphate as the nitrogen source. Accumulation of macrocyclic trichothecenes was strongly affected by the media composition, with higher levels often associated with higher carbon content in the media. Overall, incubation in continuous darkness resulted in higher macrocyclic trichothecene concentrations. Results support the hypothesis that accumulation of macrocyclic trichothecenes by this fungus can be altered by manipulating carbon and nitrogen sources. Furthermore, the biosynthesis of these mycotoxins may be independent of sporulation, demonstrating that the bioherbicide can be readily produced on solid substrates while simultaneously yielding conidia that are less threatening to worker safety. A more detailed implementation of the concepts demonstrated in this study will facilitate the safe and economical production of this bioherbicide.