Toxins2015, 7(9), 3497-3511; doi:10.3390/toxins7093497 (registering DOI) - published 31 August 2015 Show/Hide Abstract
Abstract: Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A–G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.
Toxins2015, 7(9), 3481-3496; doi:10.3390/toxins7093481 (registering DOI) - published 28 August 2015 Show/Hide Abstract
Abstract: Rice false smut is an emerging and economically-important rice disease caused by infection by the fungal pathogen Villosiclava virens. Ustiloxin B is an antimitotic cyclopeptide mycotoxin isolated from the rice false smut balls that formed in the pathogen-infected rice spikelets. A monoclonal antibody (mAb) designated as mAb 1B5A10 was generated with ustiloxin B—ovalbumin conjugate. A highly-sensitive and specific indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed. The median inhibitory concentration (IC50) of the icELISA was 18.0 ng/mL for the detection of ustiloxin B; the limit of detection was 0.6 ng/mL, and the calibration range was from 2.5 to 107.4 ng/mL. The LOD/LOQ values of the developed ELISA used for the determination of ustiloxin B in rice false smut balls and rice grains were 12/50 μg/g and 30/125 ng/g, respectively. The mAb 1B5A10 cross-reacted with ustiloxin A at 13.9% relative to ustiloxin B. Average recoveries of ustiloxin B ranged from 91.3% to 105.1% for rice false smut balls at spiking levels of 0.2 to 3.2 mg/g and from 92.6% to 103.5% for rice grains at spiking levels of 100 to 5000 ng/g. Comparison of ustiloxin B content in rice false smut balls and rice grains detected by both icELISA and high performance liquid chromatography (HPLC) demonstrated that the developed icELISA can be employed as an effective and accurate method for the detection of ustiloxin B in rice false smut balls, as well as rice food and feed samples.
Abstract: The mycotoxin zearalenone (ZEN) is frequently contaminating animal feeds including feed used in aquaculture. In the present study, the effects of dietary exposure to ZEN on carp (Cyprinus carpio L.) were investigated. ZEN at three different concentrations (low dose: 332 µg kg−1, medium dose: 621 µg kg−1 and high dose: 797 µg kg−1 final feed, respectively) was administered to juvenile carp for four weeks. Additional groups received the mycotoxin for the same time period but were fed with the uncontaminated diet for two more weeks to examine the reversibility of the ZEN effects. No effects on growth were observed during the feeding trial, but effects on haematological parameters occurred. In addition, an influence on white blood cell counts was noted whereby granulocytes and monocytes were affected in fish treated with the medium and high dose ZEN diet. In muscle samples, marginal ZEN and α-zearalenol (α-ZEL) concentrations were detected. Furthermore, the genotoxic potential of ZEN was confirmed by analysing formation of micronuclei in erythrocytes. In contrast to previous reports on other fish species, estrogenic effects measured as vitellogenin concentrations in serum samples were not increased by dietary exposure to ZEN. This is probably due to the fact that ZEN is rapidly metabolized in carp.
Abstract: Research has shown success using clay-based binders to adsorb aflatoxin in animal feeds; however, no adsorbent has been approved for the prevention or treatment of aflatoxicosis. In this study, growth and relative organ weights were evaluated along with a residue analysis for aflatoxin B1 in liver tissue collected from broiler chickens consuming dietary aflatoxin (0, 600, 1200, and 1800 µg/kg) both with and without 0.2% of a calcium bentonite clay additive (TX4). After one week, only the combined measure of a broiler productivity index was significantly affected by 1800 µg/kg aflatoxin. However, once birds had consumed treatment diets for two weeks, body weights and relative kidney weights were affected by the lowest concentration. Then, during the third week, body weights, feed conversion, and the productivity index were affected by the 600 µg/kg level. Results also showed that 0.2% TX4 was effective at reducing the accumulation of aflatoxin B1 residues in the liver and improving livability in birds fed aflatoxin. The time required to clear all residues from the liver was less than one week. With evidence that the liver’s ability to process aflatoxin becomes relatively efficient within three weeks, this would imply that an alternative strategy for handling aflatoxin contamination in feed could be to allow a short, punctuated exposure to a higher level, so long as that exposure is followed by at least a week of a withdrawal period on a clean diet free of aflatoxin.
Abstract: Pufferfish poisoning has not been well documented in the South Pacific, although fish and other seafood are sources of protein in these island nations. In this study, tetrodotoxin (TTX) and its analogues in each organ of the pufferfish Arothron hispidus and A. nigropunctatus collected in the Solomon Islands were investigated using high resolution LC-MS. The toxin profiles of the same two species of pufferfish from Okinawa, Japan were also examined for comparison. TTXs concentrations were higher in the skin of both species from both regions, and relatively lower in the liver, ovary, testis, stomach, intestine, and flesh. Due to higher TTX concentrations (51.0 and 28.7 µg/g at highest) detected in the skin of the two species from the Solomon Islands (saxitoxin was <0.02 µg/g), these species should be banned from consumption. Similar results were obtained from fish collected in Okinawa, Japan: TTX in the skin of A. hispidus and A. nigropunctatus were 12.7 and 255 µg/g, respectively, at highest, and saxitoxin was also detected in the skin (2.80 µg/g at highest) and ovary of A. hispidus. TTX, 5,6,11-trideoxyTTX (with its 4-epi form), and its anhydro forms were the most abundant, and 11-oxoTTX was commonly detected in the skin.
Abstract: Patients with multiple sclerosis (MS) often complain of urinary disturbances characterized by overactive bladder syndrome and difficulties in bladder emptying. The aim of the study was to investigate the pathophysiology of bladder dysfunction and the neurophysiological effects of intradetrusorial incobotulinum toxin A (BoNT/A) in patients with MS having both brain and spinal MS-related lesions. Twenty-five MS patients with neurogenic detrusor overactivity (NDO) underwent clinical evaluation and soleus Hoffmann reflex (H reflex) study during urodynamics. Of the 25 patients, 14 underwent a further session one month after intradetrusorial BoNT/A injection. Eighteen healthy subjects acted as the control. In healthy subjects, the H reflex size significantly decreased at maximum cystometric capacity (MCC), whereas in MS patients with NDO, the H reflex remained unchanged. In the patients who received intradetrusorial BoNT/A, clinical and urodynamic investigations showed that NDO improved significantly. Volumes at the first, normal and strong desire to void and MCC increased significantly. Despite its efficacy in improving bladder symptoms and in increasing volumes for first desire, normal and strong desire to void, BoNT/A left the H reflex modulation during bladder filling unchanged. In the MS patients we studied having both brain and spinal MS-related lesions, the H reflex size remained unchanged at maximum bladder filling. Since this neurophysiological pattern has been previously found in patients with spinal cord injury, we suggest that bladder dysfunction arises from the MS-related spinal lesions. BoNT/A improves bladder dysfunction by changing bladder afferent input, as shown by urodynamic findings on bladder filling sensations, but its effects on H reflex modulation remain undetectable.