Toxin Type Research Articles

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus.

To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.

Food-borne botulism: review of five cases

Food-borne botulism is a disease caused by the ingestion of food contaminated with botulinum toxin, often present in smoked meat, canned food and preserved food; it can occur as sporadic case or as an outbreak. In the last decades there has been an increasing incidence of food-borne botulism in Portugal. The authors do a review of five cases of food-borne botulism, three isolated cases and 2 familiar. Four were associated with the ingestion of smoked ham and one of canned tunafish. The incubation period was 48 hours in one patient and 4 days in another, in the remaining patients it was not possible to determine this period. The clinical picture was dominated in all patients by diplopy, dysphagia, dizziness, blurred vision, dry mouth and constipation, and in two patients there were gastrointestinal complains. In one patient the electromyography findings were compatible with pre-synaptic neuromuscular blockage. A toxin type B was found in the serum of one patient and in the food involved in the two familiar cases. All patients experienced complete recovery with only symptomatic treatment. With this article the authors intend to call attention to this diagnosis, which is not rare, but difficult for someone not familiar with its presentation, being of notice that the diagnosis is essentially clinic with a strong epidemiological history, confirmed by typical electromyography findings and by the identification of the toxin involved. In Portugal there is only descriptions of clinical cases associated with the type B and the type E toxins, not being necessary the resource to the antitoxin therapy.

Botulinum toxin types A and B: comparison of efficacy, duration, and dose-ranging studies for the treatment of facial rhytides and hyperhidrosis

One of the most common etiologic forces for the persistence of facial rhytides is the repetitive contraction of the intrinsic muscles that are necessary for facial expression. These include the forehead lines, crow's feet, glabellar rhytides, and wrinkles in the lower face. Although filling agents such as collagen and laser procedures can help reduce the appearance of these lines, they do not address the underlying forces that cause these wrinkles to persist. Botulinum toxin type-A and type-B are neurotoxins that address these issues and result in the relaxation of the intrinsic facial muscles and subsequent resolution of these dynamic facial rhytides. This article will compare the efficacy, duration, dose ranging studies, and safety in the treatment of facial rhytides with both types of toxins. In addition, the treatment of hyperhidrosis with type-A and type-B botulinum toxin will also be discussed.

Botulinum toxin types A and B: comparison of efficacy, duration, and dose-ranging studies for the treatment of facial rhytides and hyperhidrosis

One of the most common etiologic forces for the persistence of facial rhytides is the repetitive contraction of the intrinsic muscles that are necessary for facial expression. These include the forehead lines, crow's feet, glabellar rhytides, and wrinkles in the lower face. Although filling agents such as collagen and laser procedures can help reduce the appearance of these lines, they do not address the underlying forces that cause these wrinkles to persist. Botulinum toxin type-A and type-B are neurotoxins that address these issues and result in the relaxation of the intrinsic facial muscles and subsequent resolution of these dynamic facial rhytides. This article will compare the efficacy, duration, dose ranging studies, and safety in the treatment of facial rhytides with both types of toxins. In addition, the treatment of hyperhidrosis with type-A and type-B botulinum toxin will also be discussed.

Toxins of the Helicobacter Genus and Their Roles in Pathogenesis

In the 20 years since the discovery of Helicobacter pylori the number of formally described Helicobacter spp. has increased dramatically. The majority of species in the genus have been associated with some form of pathology. Similar to other Gram-negative bacteria, all helicobacters have lipopolysaccharides or endotoxins in the outer leaflet of their outer membrane which is an important modulator of the immune system. H. pylori endotoxin has a number of roles in the pathogenesis of the bacterium. Its relatively low biological and immunological activity and molecular mimicry may contribute to the chronic nature of infection through avoidance of host defence mechanisms and adhesion. In addition to endotoxins, various helicobacters also secrete distinct exotoxins capable of host cell damage. H. pylori has been shown to possess a cytotoxin capable of inducing vacuoles in epithelial cells termed vacuolating cytotoxin or VacA. Although VacA has a number of roles in the pathogenesis of H. pylori its use as a predictor of clinical outcome has yet to be established. In addition, similar to the closely related Campylobacter jejuni, a number of enterohepatic helicobacters express a cytolethal distending toxin capable of inducing severe cell damage in vivo and in vitro. Both endotoxins and exotoxins may play a combined role in the pathogenesis of Helicobacter spp. Both toxin types have been identified as being present on outer membrane vesicles shed by H. pylori which may act as antigen delivery vehicles and contribute to pathogenesis.

The Use of Botulinum Toxins for Chronic Pain and Headaches.

The use of botulinum toxin in the management of various neurologic and non-neurologic disorders has grown considerably over the past decade. At the same time, new information regarding the mechanism of action of these toxins has evolved allowing for a greater understanding of the versatility of these agents. Although two types of botulinum toxin (type A Botox and type B Myobloc ) are commercially available in the US, most studies of the use of these toxins for the management of chronic pain and headache have been completed with type A. Data from open-label and retrospective studies as well as clinical practice suggest as strongly as possible that there is a role for these agents, especially Botox, in the management of several chronic headache disorders, including chronic migraine, chronic tension-type, cervicogenic, and cluster headache. Emerging data regarding the use of these agents for so-called "analgesic-rebound" headache also appear impressive; however, as of yet, no multicenter, randomized, controlled studies for any headache type have been published that confirm the results seen in noncontrolled studies. Nevertheless, the benefit that some patients experience from this agent is impressive, and this drug appears for many to modify the disorder in a very positive manner. In a similar fashion, data for other pain states are often restricted to open-label and case study approaches; however, clinical experience and some of the available studies (even small controlled studies) suggest a role for the toxins in the management of various chronic pain states, such as myofascial pain, low back pain, and neuropathic pain. One of the greatest challenges ahead for all interested in this area is confirming the benefit seen clinically through appropriately designed multicenter, randomized, controlled studies.

Peau, rides et toxine botulique

Objective. – To present an up-to-date analysis about the use of botulinum toxin for treating facial lines and wrinkles. Method. – A systematic search of the literature was conducted to select the most recent or relevant publications on this topic, through Medline. Results. – Out of the 583 articles retrieved, 90 were finally selected for the study. Discussion. – Validity of using botulinum toxin for cosmetic use is demonstrated, together with contra-indications and different methods to objectivate the results. The different available types of toxin are presented and compared. Modalities of preparation, conservation, and waste disposal are detailed. Anatomical bases of muscular facial balance are reviewed, with techniques of injection presented for each site, and also with adjunctive procedures. Complications and side effects are described and analysed. Most complications can be prevented through: • perfect knowledge of local anatomy; • use of small volumes; • orientation of the needle bevel towards the muscle body, injection within the muscle body if thick, more superficial if thin; • application of ice on the skin pre- and post-injecting. Adding epinephrin or diluting with xylocaïne and epinephrin is not commonly used. Conclusion. – Botulinum toxin has found its way as a major component of the therapeutic armamentarium. Its efficacy for facial rejuvenation has made it extremely popular, but its use does follow strict rules, and should be restricted to soundly trained practitioners.

24-well plate spectrophotometric assay for preliminary screening of metabolic activity.

A 90-min assay using 24-well plates was developed for screening overall metabolic effects of toxic chemicals by measuring extracellular acidification rate of mammalian cells. During the 90-min test, the pH of each well of a 24-well plate is monitored by measuring the absorbance of phenol red using a spectrophotometric plate reader. The acidification rate is then calculated from the change in pH divided by the time interval and cell density, which is also estimated non-invasively by spectroscopy. The assay was verified by testing the effects of five well-characterized chemical toxins on fibroblast cell cultures. As expected, the responses of the fibroblasts were dependent on the dose and type of toxin. They also corresponded with the established mechanisms of the toxins and with measured lactate production rates. The set-up is simple, inexpensive, and amenable to being automated. The method is easy to perform and rapid. Uses include screening compounds for gross metabolic effects in mammalian cell lines, determining preliminary metabolic dose-response curves for guiding further research, and designing and optimizing media for in vitro systems utilizing cell cultures.

Outbreak of Botulism Type E Associated With Eating a Beached Whale—Western Alaska, July 2002

Botulism is a neuroparalytic illness caused by toxins produced by the bacterium Clostridium botulinum, an obligate anaerobe found commonly in the environment. Intoxication with toxin type E is associated exclusively with eating animal foods of marine (salt or fresh water) origin. Persons who eat raw or fermented marine fish and mammals are at high risk for botulism from type E toxin. On July 17, 2002, the Alaska Division of Public Health investigated a cluster of suspected botulism cases among residents of a fishing village in Alaska. This report summarizes the findings of the outbreak investigation, which linked disease to eating raw muktuk (skin and a pink blubber layer) from a beached whale (Figure). To avoid delays in treatment, health-care providers evaluating patients suspected of having botulism should base treatment decisions on clinical findings. Public health authorities should be notified immediately about any suspected botulism case.

Toxins from Adriatic blue mussels. A decade of studies

Abstract A research program was initiated in 1990 to carefully examine the toxin profiles in mussels from the northern Adriatic Sea. Since then, a number of polyether toxins have been isolated and characterized, some of which represent new additions to the diarrhetic shellfish poisoning (DSP) class of biotoxins and seem to be peculiar to the Adriatic Sea. During our investigation of toxic Adriatic mussels, we also isolated a new type of toxin, whose structure was elucidated by extensive use of 1D and 2D NMR techniques. Some of them could represent a further alarm for public health owing to their cytotoxic activity. The recent application of liquid chromatography/mass spectrometry (LC/MS) methods for detection of Adriatic marine biotoxins allowed us to hasten the analysis of toxic samples and to advance effective structural hypothesis even when full structure elucidation of new toxins by NMR spectroscopy is hampered by the limited amount of available material.

Isolation and serotypes of Vero toxin-producing Escherichia coli (VTEC) from pigeons and crows

To clarify the source and route of infection with Vero toxin-producing Escherichia coli (VTEC) in humans, we sampled gastrointestinal contents and isolated VTEC from wild birds captured to exterminate harmful birds between August 1997 and January 1998. Pigeons were caught in Sagamihara-shi and crows were caught in Sagamihara-shi, Kawasaki-shi, Yokohama-shi, and the Tokyo metropolitan area. The following results were obtained. 1) VTEC was isolated from 32 of 521 birds (6.1%) examined. Among pigeons, VTEC was isolated from 25 of 262 birds (9.5%) captured in Sagamihara-shi. Among crows, VTEC was isolated from 7 of 184 birds (3.8%) captured in Sagamihara-shi, but not isolated from any bird of 11.4, and 60 birds captured in Yokohama-shi, Kawasaki-shi, and the Tokyo metropolitan area, respectively. 2) Toxin was typed in 33 isolates. There were four VT1-producing isolates (6.5%), 27 VT2-producing isolates (88.7%), and two VT1, VT2-producing isolates (4.8%). 3) The serotypes of the isolates were: O78: H-, 10; O152: H-, 7; O153: H19.2; O164: H-, 1; O128: H-, 1; O164/143: H-, and O1: HUT, 1. The serotype was unknown in 10 isolates. Among 10 isolates for which the serotype could not be determined, auto-aggregation was observed in one isolate. 4) EaeA was investigated in the 33 isolates, and 31 isolates (93.9%) possessed eaeA. The above findings showed that strains with same toxin types and serotypes of human diarrhea-derived VTEC were isolated from pigeons and crows, and the isolates frequently possessed eaeA, which is considered to have an important association with its pathology, suggesting that birds are involved in VTEC infection in humans as a source of infection.

Botulinum toxin A: its expanding role in dermatology and esthetics.

The use of botulinum toxin A in cosmetic dermatology has increased in popularity due to the efficacy and relative safety of the treatment. Botulinum toxin A is one of eight exotoxins produced by Clostridium botulinum, a Gram-positive, spore-forming anaerobe. Flaccid paralysis results from the denervation of muscle fibers at the neuromuscular junction after botulinum toxin A administration. While treating blepharospasm, the Carruthers incidentally found that botulinum toxin A improved glabellar frown lines. Dynamic rhytides occur in areas of dynamic motion. These types of lines may be improved with botulinum toxin A. There are two types of botulinum toxin A commercially available (BOTOX and Dysport); only BOTOX is currently available in the US. The efficacy and tolerability of BOTOX was best demonstrated with a multicenter, double-blind, randomized, placebo-controlled study of the efficacy and safety of botulinum toxin type A in the treatment of glabellar lines in 264 patients. There was a significantly greater reduction in glabellar line severity with BOTOX. The effect was maintained for the duration of the study (120 days). There was low occurrence (5.4%) of mostly mild blepharoptosis in the BOTOX group. In another prospective study, it was found that about 1% of BOTOX patients reported severe headache. Botulinum toxin A can provide an alternative treatment of palmar and axillary hyperhidrosis when options such as topical agents (aluminum chloride) and iontophoresis have failed.

Specific Detection of Clostridium botulinum Types A, B, E, and F Using the Polymerase Chain Reaction

Clostridium botulinum organisms generally produce 1 of 4 neurotoxin types (A, B, E, and F) associated with human illness. Neurotoxin type determination is important in identification of the bacterium. A polymerase chain reaction (PCR) method was developed to identify 24 h botulinal cultures as potential types A, B, E, and F neurotoxin producers as well as other clostridial species which also produce neurotoxins. Components of the PCR and amplification conditions were adjusted for optimal amplification of toxin gene target regions to enable simultaneous testing for types A, B, E, and F in separate tubes using a single thermal cycler. Each primer set was specific for its corresponding toxin type. A DNA extraction procedure was also included to remove inhibitory substances that may affect amplification. This procedure is rapid, sensitive, and specific for identification of toxigenic C. botulinum.

Type IV Longus Pilus of Enterotoxigenic Escherichia coli : Occurrence and Association with Toxin Types and Colonization Factors among Strains Isolated in Argentina

The longus type IV pilus structural gene (lngA) was sought among 217 clinical enterotoxigenic Escherichia coli (ETEC) strains isolated in Argentina. lngA was present in 20.7% of the isolates and was highly associated with ETEC producing heat-stable toxin and the most common colonization factors. The prevalence of longus among ETEC strains in Argentina was comparable to that of colonization factor antigen I (CFA/I), CFA/II, and CFA/IV in other regions of the world.

Botulinum toxins types A and B for brow furrows: preliminary experiences with type B toxin dosing

BACKGROUND : Facial lines resulting from hyperactivity can be misleading manifestations of negative emotions, fatigue and stress. They may also contribute to a perception of facial aging. A well established treatment is botulinum toxin type A (BTX-A). Recently, botulinum toxin type B (BTX-B) has become available for the treatment of cervical dystonia. There has been little comparison on the efficacy of the two different types of botulinum toxins, nor is there information on appropriate dosing of BTX-B for facial muscles. OBJECTIVES : The purpose of this pilot study was to observe the effects of BTX-B in comparison to BTX-A, on patients with brow furrows assessing initial efficacy and duration of effect. METHODS : Patients were injected with BTX-B in two different dose conversions against BTX-A to the corrugator-procerus complex. Some patients received a conversion of 50 units of BTX-B (total of 1000 units) to one unit of BTX-A while others received a conversion of 100 units of BTX-B (total of 2000 units) to one unit of BTX-A. The patients treated with BTX-A received a total of 20 units. These patients were clinically assessed prior to treatment and 3 days, 1 week, 4 weeks, 12 weeks and 16 weeks after treatment. RESULTS : Both types of botulinum toxin were effective at improving glabellar frown lines. The onset of actions occurred slightly sooner (2-3 days) with BTX-B than with BTX-A (3-7 days). Duration of effect with BTX-A was at least 16 weeks. With 1000 units of BTX-B, dose duration was 6-8 weeks and with 2000 units of BTX-B, duration was 10-12 weeks. SUMMARY : Both types of botulinum toxin are effective at correcting deep glabellar furrows. At least with the doses used, BTX-B has a quicker onset of action and BTX-A has longer benefit for glabellar wrinkles. These data strongly suggest that further dose ranging studies of BTX-B are necessary and indicated in controlled double blind studies in a larger patient population.

Does Baclofen Help Children with Tourette Syndrome

A recent open-label trial reported a decrease in tic severity in 95 percent of 264 patients treated with baclofen/botulinum toxin type A (see J Child

Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves

The enterotoxaemia syndrome in Belgian Blue calves is characterised by a high case fatality rate, sudden death, lesions of haemorrhagic enteritis of the small intestine and, quite often an absence of other clinical signs but its cause has not been yet identified. As a first step in this identification, the aerobic and anaerobic intestinal flora of a population of 78 calves, originating from farms located in southern Belgium and that died in circumstances defined as “calf enterotoxaemia” (study population) and of 64 calves that died in other circumstances (control population) were studied qualitatively and quantitatively. The colonies were identified after subcultures with appropriate API sugar sets. Anaerobically Clostridium perfringens was isolated in higher numbers (mean values of 10 7–10 7.5 colony forming units (CFU) versus 10 4–10 5 CFU per ml of intestinal content) and from more animals (79 versus 19%) in the study population than in the control population, although individual results from both populations could overlap. Other clostridial species, i.e. mainly urease-negative C. sordellii and C. bifermentans, were isolated in high numbers (>10 6 CFU per ml of intestinal content) from a few animals in the study population only. All but one of the 705 C. perfringens isolates from both populations belonged to the A toxin type and none of the urease-negative C. sordellii was toxigenic. Gram-negative anaerobes were not isolated in high numbers from any of the samples. Aerobically β-haemolytic E. coli were significantly more frequent among the study population, but were isolated from only 25% of the animals. Salmonella Typhimurium was isolated from only two animals in the study population. Less than 1% of the E. coli isolated were verotoxigenic and one-third were necrotoxigenic. At this stage only non-enterotoxigenic type A C. perfringens are thus statistically associated with the enterotoxaemia syndrome in Belgian Blue calves and fulfil the first of the Koch’s postulates.

Identification and molecular structural prediction analysis of a toxicity determinant in the Bacillus sphaericus crystal larvicidal toxin.

The operon containing the genes encoding the subunits of the binary crystal toxin of Bacillus sphaericus strain LP1-G, BinA and BinB (41.9 kDa and 51.4 kDa, respectively), was cloned and sequenced. Purified crystals were not toxic to Culex pipiens larvae. Comparison of the amino-acid sequences of this strain (Bin4) with those of the three other known toxin types (Bin1, Bin2 and Bin3) revealed mutations at six positions, including a serine at position 93 of BinA4, whereas all other types of BinA toxin from B. sphaericus had a leucine at this position. Reciprocal site-directed mutagenesis was performed to replace this serine in BinA4 from LP1-G with a leucine and the leucine in the BinA2 protein from strain 1593 with a serine. Native and mutated genes were cloned and overexpressed. Inclusion bodies were tested on C. pipiens larvae. Unlike the native Bin4 toxin, the mutated protein was toxic, and the reciprocal mutation in Bin2 led to a significant loss of toxicity. In vitro receptor-binding studies showed similar binding behaviour for native and mutated toxins. In the absence of any experimental data on the 3D structure of these proteins, sequence analysis and secondary-structure predictions were performed. Amino acid 93 of the BinA polypeptide probably belongs to an alpha helix that is sensitive to amino-acid modifications. Position 93 may be a key element in the formation of the BinA-BinB complex responsible for the toxicity and stability of B. sphaericus Bin toxins.

Germination, Growth, and Toxin Production of Nonproteolytic Clostridium botulinum as Affected by Multiple Barriers

ABSTRACT The effect of combinations of pH (6.5, 5.75), NaCl (0.25,1.75%), and incubation temperatures (7,13 °C) on spore germination, outgrowth, and time to toxicity of nonproteolytic Clostridium botulinum was examined in a broth system. Spores of four toxin type E and nonproteolytic type B were inoculated (104/ml) into Tryptone Peptone Glucose Yeast Extract (TPGY) broth. Cultures were monitored for three weeks, or until toxin was detected. A modified FSIS‐amplified ELISA (comparable in sensitivity to the mouse bioassay) was used to screen cultures for neurotoxin. Combinations of the most inhibitory level for each barrier reduced the degree and rate of germination, the lag and growth rate of vegetative cells, and the time to toxicity.

Regeneration of cultured midgut cells after exposure to sublethal doses of toxin from two strains of Bacillus thuringiensis

Toxin from two strains of Bacillus thuringiensis (Bt), AA 1–9 and HD-73, caused dose-dependent destruction of cultured midgut cells from Heliothis virescens larvae. HD-73 toxin was more effective although, at the doses used, not all cells were killed. After 2 days of exposure to 0.8 pg/μl AA 1–9 or 0.06 pg/μl HD-73, columnar and goblet cell numbers declined to ca 20% of controls. In contrast, stem and differentiating cells increased to 140–200% of controls. The dynamic of depletion and replacement depended on toxin type and concentration. Two days after toxin was washed out, ratios of cell types returned to approximate control levels, suggesting rapid population corrections in vitro. Regulation of the ratio of cell types in each population, and the rate of proliferation and differentiation of stem cells was induced by the cultured midgut cells themselves. Controls and cells treated with toxin from Bt strain AA 1–9 were stained using a polyclonal antibody to Lepidopteran midgut differentiation factor 1 (MDF1). With Bt toxin, 1.5 times more cells stained for MDF1, suggesting increased synthesis of this differentiation factor during increased stem cell differentiation. The response of cultured midgut cells to Bt toxin injury is similar to injured vertebrate tissues dependent on stem cells for replacement and healing.