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A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type and type D neurotoxin is developed. Streptavidin-poly-horseradish peroxidase is used as a conjugate and a precipitating substrate allowed the direct semiquantitative readout of the assay and for a more accurate quantitative detection, the substrate can be eluted and measured in a photometer.

Background: In contrast to human medicine, only a small number of serum tumor markers are established in veterinary medicine even though they are a non-invasive diagnostic tool. Objectives: This study examined whether survivin could be suitable as a potential canine serum tumor marker. Methods: This study measured the serum survivin concentrations of dogs with mammary tumors (n = 33), squamous cell carcinoma (n = 9), soft-tissue sarcoma (n = 18) and multicentric lymphoma (n = 22), using a commercially available, competitive immunoassay kit (BlueGene). The serum survivin concentrations were compared with those of a healthy control group (n = 20) and a control group of dogs with non-neoplastic diseases (n = 17). Results: Dogs with malignant tumors had serum survivin concentrations between 15 and 5,906 pg/mL (median, 72 pg/mL), those in the healthy group ranged from 7 to 99 pg/mL (median, 21 pg/mL) and those in the group of dogs suffering from non-neoplastic diseases from 15 to 93 pg/mL (median, 42 pg/mL). The differences in the survivin concentrations between the healthy dogs and dogs with malignant tumors and between the dogs with nonneoplastic diseases and those with malignant tumors were significant (p < 0.001 and p = 0.006, respectively). Conclusions: The serum survivin concentrations in dogs with malignant tumors, with some exceptions, are higher than in dogs with benign tumors and dogs that do not suffer from a malignancy. Therefore, survivin can provide information on the presence of malignant tumors and be used as a tumor marker in dogs. [ABSTRACT FROM AUTHOR]

A multiplexed quantitative real-time PCR assay was developed for the simultaneous detection of the human pathogenic Clostridium botulinum serotypes A, B, E and F. The pentaplexed assay was found to show high sensitivity and specificity and facilitated for the simultaneous screening and differentiation of specimens for C. botulinum A, B, E and F.

The thermal inactivation of BoNT/A, BoNT/A complex, BoNT/B, and BoNT/B complex in cow's milk in a pasteurization process similar to the industry standard is described. The high-temperature short-time (HTST) pasteurization process in the dairy industry is found to remarkably reduce the risk of consumer harm even if larger amounts of BoNT were deliberately released into the milk supply chain.

In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D) producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens) in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and polymerase chain reaction (PCR) assays; subsamples were fixed for fluorescence in situ hybridization analysis in combination with confocal laser scanning microscopy. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with clostridial spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight) and an enlarged root system induced by the systemic colonization of clover by C. botulinum strain 2301. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover. [ABSTRACT FROM AUTHOR]

Abstract: The genetically modified maize MON89034 × MON88017 expresses different crystal (Cry) proteins with pesticidal activity against the European corn borer (Cry1.105; Cry2Ab2) and the Western corn root worm (Cry3Bb1). Non-target organisms, such as soil nematodes, might be exposed to the Cry proteins that enter the soil in course of crop growing. Therefore, the risk of those proteins for nematodes was assessed by testing their toxic effects on Caenorhabditis elegans. All three insecticidal Cry proteins showed dose-dependent inhibitory effects on C. elegans reproduction (EC50: 0.12–0.38 μmol L−1), however, at concentrations that were far above the expected soil concentrations. Moreover, a reduced toxicity was observed when Cry proteins were added jointly. A C. elegans mutant strain deficient for receptors for the nematicidal Cry5B was also resistant against Cry1.105 and Cry2Ab2, suggesting that these Cry proteins bound to the same or similar receptors as nematicidal Cry proteins and thereby affect the reproduction of C. elegans. [Copyright &y& Elsevier]

Abstract: Four lateral flow assays (LFAs) were evaluated for the detection of purified botulinum neurotoxin A, toxin complex, and unpurified culture supernatant. They included the BioThreat (Tetracore, Rockville, MD), SMART (New Horizons Diagnostics, Columbia, MD), BADD (ADVNT Biotechnologies, Phoenix, AZ), and RAMP (Response Biomedical, Burnaby, BC, Canada) assays. BioThreat and SMART did not detect the purified toxin. The best sensitivity was achieved with the RAMP test (50 ng mL−1). BioThreat and SMART measured as low as 10 ng mL−1 of the toxin complex. Specificity data differed among the tests. BADD gave false-positive signals with uninoculated bacterial culture medium. BioThreat and RAMP were further evaluated with clinical sample matrices (serum, gastric, and rectum contents from pigs). Because of matrix effects and a generally low positive response, the assays are unsuitable for the direct detection of the toxin. However, the LFAs can be a helpful tool in screening bacterial cultures for toxigenic Clostridium botulinum, if further validated according to the laboratory needs. [Copyright &y& Elsevier]

In a recent study it could be shown that compost samples can contain Clostridium botulinum. It was investigated if C. botulinum introduced with compost into botulinum-free soil can persist and be translocated within the soil. Compost was spiked with two C. botulinum type D spore concentrations (103 and 105 spores g−1) and the composts were spread on an experimental site. Over a period of 939 days, samples were taken from the upper (0–5 cm) and the lower (10–30 cm) soil horizons. Physical and chemical as well as microbiological variables were measured. Clostridium botulinum spores were quantified in a culture MPN-PCR assay. On day 757 the last positive sample was obtained in the plots with the lower spore concentration (103 g−1). The bacteria were never detected in the samples taken from the lower horizons of these plots. Clostridium botulinum persisted over the whole investigation period in the plots which were treated with compost spiked with 105 spores g−1. The concentrations found were between 20 and 20 000 spores g−1 soil. The bacteria were vertically translocated and could be found in the lower soil horizons (20–2000 spores g−1 soil) starting 70 days after the compost was spread. [ABSTRACT FROM AUTHOR]

Abstract: An immunomagnetic beads assay for the simultaneous quantification of botulinum neurotoxin types C and D was developed. Specific monoclonal antibodies against the heavy chain of the toxin and affinity-purified biotinylated polyclonal antibodies (pAbs) were used. The antibodies were preincubated with the sample. The complex being formed was then captured by magnetic beads coated with antimouse IgG. Streptavidin–poly-horseradish peroxidase, a signal amplifier, bound to the biotinylated pAb. A maximum sensitivity of approximately 0.3 minimal lethal doses for mice per milliliter was achieved with culture supernatants of both toxin types. [Copyright &y& Elsevier]

Abstract: A highly sensitive combination of a membrane dipstick assay and a flatbed scanner for determination of botulinum toxin type D is described. The immunochromatographic assay is based on a sandwich format using two primary antibodies of distinct specificities and one secondary antibody. One of the primary antibodies was conjugated with colloidal gold (detector reagent), the secondary antibody (capture reagent) was immobilized within a defined detection zone (test line) on a diagnostic cellulose nitrate membrane. In combination with an effective sample pre-treatment, the gold conjugated antibody and the second distinct antibody formed a mobile sandwich complex with the toxin. Within the test line the mobile sandwich complex was immobilized and therefore concentrated by the secondary antibody resulting in a distinct red test line. The intensity of colour of the red test line (signal intensity), which correlated directly with the concentration of BoNT/D in the standard or spiked horse faecal sample, was assessed visually and by computer image analysis using a three-determination analysis. Thus, toxin concentrations down to 50pg/mL were determined within 3.5h. The mean relative error of the arithmetic mean of the signal intensity amounted to approximately 7%. [Copyright &y& Elsevier]

Clostridium perfringens enterotoxin (CPE) regularly causes food poisoning and antibiotic-associated diarrhea; therefore, reliable toxin detection is crucial. To this aim, we explored stationary and mobile strategies to detect CPE either exclusively by monoclonal antibodies (mAbs) or, alternatively, by toxin-enrichment via the cellular receptor of CPE, claudin-4, and mAb detection. Among the newly generated mAbs, we identified nine CPE-specific mAbs targeting five distinct epitopes, among them mAbs recognizing CPE bound to claudin-4 or neutralizing CPE activity in vitro. In surface plasmon resonance experiments, all mAbs and claudin-4 revealed excellent affinities towards CPE, ranging from 0.05 to 2.3 nM. Integrated into sandwich enzyme-linked immunosorbent assays (ELISAs), the most sensitive mAb/mAb and claudin-4/mAb combinations achieved similar detection limits of 0.3 pg/mL and 1.0 pg/mL, respectively, specifically detecting recombinant CPE from spiked feces and native CPE from 30 different C. perfringens culture supernatants. The implementation of mAb- and receptor-based ELISAs into a mobile detection platform enabled the fast detection of CPE, which will be helpful in clinical laboratories to diagnose diarrhea of assumed bacterial origin. In conclusion, we successfully employed an endogenous receptor and novel high affinity mAbs for highly sensitive and specific CPE-detection. These tools will be useful for both basic and applied research. [ABSTRACT FROM AUTHOR]

This paper presents the case of a six year-old, male, thoroughbred horse with clinical signs of inappetence, weakness, and incoordination when walking. Clinical examination showed that the horse staggered and leaned to the left side. Feedstuff was present inside and around its mouth. Salivation was increased and there was no reflex at the palpebrae and tongue. The horse had difficulty swallowing and the tone of its tail was reduced. Botulism was diagnosed based on the clinical signs. Antibiotic (ceftiofur) and fluid-electrolyte treatment was commenced. Next day, neostigmin was added to the horse's treatment, and it became recumbent. The horse's palpebral, tongue and tail reflexes returned partially after neostigmine methylsulphate treatment on the same day and it stood up on day four. However, it could not swallow anything during the whole week, so after getting permission from the owner, the horse was euthanized on day 10. Samples of the colonic content and blood serum were sent by courier to the laboratory for toxin neutralization, however, botulinum neurotoxins could not be detected. After that, serum samples from days 6 and 10 were sent to another laboratory for testing for botulinum neurotoxin antibodies by ELISA. Specific antibodies against botulinum neurotoxin type A were measured, indicating a previous, immuno-relevant contact with the toxin. This seroconversion for type A supports the clinical botulism diagnosis. Type A botulism is rarely seen in Europe and has been detected in a horse in Turkey for the first time. [ABSTRACT FROM AUTHOR]

Abstract: The adsorption of the insecticidal Cry1Ab protein of Bacillus thuringiensis (Bt) on Na-montmorillonite (M-Na) and soil clay fractions was studied. The aim of this study was not to find the adsorption capacity of the soils from the experimental field site, where Bt corn (MON810) was cultivated, but rather to characterize the adsorption behavior of the Cry1Ab protein at concentrations typically found at experimental field sites. In kinetic experiments, the Cry1Ab protein adsorbed rapidly (<60min) on M-Na. As the concentration of M-Na was varied and the added Cry1Ab protein concentration was kept constant (20 and 45ngml−1), the adsorption per unit weight of Cry1Ab protein decreased with increasing concentrations of M-Na. Adsorption of Cry1Ab protein on M-Na decreased as the pH value of the suspension increased. All adsorption isotherms could be described mathematically by a linear regression with the parameter k, the distribution coefficient, being the slope of the regression line. Although their mineralogical composition was nearly identical, the soil clay fractions showed different k values. The different k values were correlated with the physical and chemical properties of the soil clay fractions, such as the organic carbon content, the specific external surface area, and the electrokinetic charge of the external surfaces of the clays, as well as with the external surface charge density. An increase in the amount of soil organic matter, as well as an increase in the electrokinetic external surface charge of the soil clays, decreased the distribution coefficient k. An increase of the specific external surface areas of the soil clays resulted in a higher distribution coefficient k. Less than 10% of adsorbed Cry1Ab protein was reversibly adsorbed on the soil clays and, thus, desorbed. The desorption efficiency of distilled water was higher than that of a solution of CaCl2 (2.25mmol) and of dissolved organic carbon (50mgCl−1). [Copyright &y& Elsevier]