Universal identification of lethal amanitas by using Hyperbranched rolling circle amplification based on α-amanitin gene sequences.

Hyperbranched rolling circle amplification (HRCA) with a padlock probe (PLP) targeting the α-amanitin (α-AMA) gene, as a screening tool for the universal identification of lethal amanitas, was established in this study. With the isothermal HRCA assay, all of the lethal Amanita species tested from Phalloideae (10) were positive, while the non-Phalloideae Amanita species (15) and three amanitin-containing Lepiota and Galerina species were negative. Furthermore, the PLP based on α-AMA sequences from lethal Amanita species was effective for Amanita α-AMA, but not Amanita ß-AMA or non-Amanita α-AMA. HRCA sensitivity was 100-fold higher than conventional PCR with a detection limit of 100 copies (recombinant plasmid containing α-AMA), and 0.2% lethal amanitas could be detected in dry mushroom blends. The HRCA method presented provided a rapid, specific, sensitive and low-cost identification tool for lethal amanitas.

Long-Term Resistance of Drosophila melanogaster to the Mushroom Toxin Alpha-Amanitin.

Insect resistance to toxins exerts not only a great impact on our economy, but also on the ecology of many species. Resistance to one toxin is often associated with cross-resistance to other, sometimes unrelated, chemicals. In this study, we investigated mushroom toxin resistance in the fruit fly Drosophila melanogaster (Meigen). This fruit fly species does not feed on mushrooms in nature and may thus have evolved cross-resistance to α-amanitin, the principal toxin of deadly poisonous mushrooms, due to previous pesticide exposure. The three Asian D. melanogaster stocks used in this study, Ama-KTT, Ama-MI, and Ama-KLM, acquired α-amanitin resistance at least five decades ago in their natural habitats in Taiwan, India, and Malaysia, respectively. Here we show that all three stocks have not lost the resistance phenotype despite the absence of selective pressure over the past half century. In response to α-amanitin in the larval food, several signs of developmental retardation become apparent in a concentration-dependent manner: higher pre-adult mortality, prolonged larva-to-adult developmental time, decreased adult body size, and reduced adult longevity. In contrast, female fecundity nearly doubles in response to higher α-amanitin concentrations. Our results suggest that α-amanitin resistance has no fitness cost, which could explain why the resistance has persisted in all three stocks over the past five decades. If pesticides caused α-amanitin resistance in D. melanogaster, their use may go far beyond their intended effects and have long-lasting effects on ecosystems.

Modulation of the spleen transcriptome in domestic turkey (Meleagris gallopavo) in response to aflatoxin B1 and probiotics.

Poultry are highly susceptible to the immunotoxic effects of the food-borne mycotoxin aflatoxin B1 (AFB1). Exposure impairs cell-mediated and humoral immunity, limits vaccine efficacy, and increases the incidence of costly secondary infections. We investigated the molecular mechanisms of AFB1 immunotoxicity and the ability of a Lactobacillus-based probiotic to protect against aflatoxicosis in the domestic turkey (Meleagris gallopavo). The spleen transcriptome was examined by RNA sequencing (RNA-seq) of 12 individuals representing four treatment groups. Sequences (6.9 Gb) were de novo assembled to produce over 270,000 predicted transcripts and transcript fragments. Differential expression analysis identified 982 transcripts with statistical significance in at least one comparison between treatment groups. Transcripts with known immune functions comprised 27.6 % of significant expression changes in the AFB1-exposed group. Short exposure to AFB1 suppressed innate immune transcripts, especially from antimicrobial genes, but increased the expression of transcripts from E3 ubiquitin-protein ligase CBL-B and multiple interleukin-2 response genes. Up-regulation of transcripts from lymphotactin, granzyme A, and perforin 1 could indicate either increased cytotoxic potential or activation-induced cell death in the spleen during aflatoxicosis. Supplementation with probiotics was found to ameliorate AFB1-induced expression changes for multiple transcripts from antimicrobial and IL-2-response genes. However, probiotics had an overall suppressive effect on immune-related transcripts.

Genotoxicity of agaritine in the lacI transgenic mouse mutation assay: evaluation of the health risk of mushroom consumption.

The mutagenic potency of the common mushroom Agaricus bisporus and crude agaritine extracted from mushrooms was determined in vivo using a new mutagenesis assay with lacI transgenic mice (Big Blue mice). Pairs of female lacI mice were fed one of three diets for 15 wk: (1) fresh mushrooms 3 days/wk followed by normal lab chow for 4 days/wk; (2) freeze-dried mushrooms mixed at 25% (w/w) into powdered chow; or (3) a mushroom extract containing 30% agaritine (w/w) mixed into powdered chow. The corresponding daily doses of agaritine were 30 (averaged over the whole week), 80 and 120 mg/kg body weight, respectively. Positive control animals received N-nitrosodimethylamine, N-nitrosomethylurea or urethane, mixed into powdered chow at concentrations corresponding to daily doses of 0.3, 3 and 130 mg/kg body weight, respectively. DNA of the forestomach, kidney, liver, lung and glandular stomach of the lacI mice was examined for increases in mutant frequency (MF). Control MFs ranged from 5 x 10(-5) to 10 x 10(-5). Positive control substances induced a two- to seven-fold increase in MF in their respective target organs. Of the mushroom diets, significant effects were seen only with the crude agaritine extract: it induced an increase in MF of 100% in the kidney and 50% in the forestomach. The other two A. bisporus diets, with lower agaritine doses, showed slightly but not significantly, raised MF values in the kidney alone. Thus, agaritine was weakly genotoxic in vivo; no genotoxic activity other than that attributable to agaritine was detected in A. bisporus. Substances or processes that might influence carcinogenicity by means of non-genotoxic mechanisms (e.g. increase in fibre, or decrease in calorie intake) are not detected in the lacI assay. Using a previously derived quantitative correlation between mutagenicity in the lacI test and carcinogenic potency, the carcinogenicity of agaritine in mushrooms was estimated: the average Swiss mushroom consumption of 4 g/day would be expected to contribute a lifetime cumulative cancer risk of about two cases per 100,000 lives.

A continuing common-source outbreak of botulism in a family.

In December, 1974, three cases of botulism occurred in a family; two were fatal. The first patient died after a 10-day illness without botulism being suspected. 4 days later, after a 2-day illness, the second patient was diagnosed as having botulism after a cardiorespiratory arrest; she died 3 days later. In the third patient, the only symptom was dysphagia. Clostridium botulinum type B was found in stool specimens from all three patients. Home-canned (bottled) mushrooms, which were found to contain C. botulinum type B and its toxin, were believed to be responsible for the outbreak; mushrooms were found at necropsy in the gastrointestinal tracts of both patients who died. Heat treatment of the mushrooms during canning had been inadequate.