Toxin Type Research Articles

Cross-Reactivity of Anthrax and C2 Toxin: Protective Antigen Promotes the Uptake of Botulinum C2I Toxin into Human Endothelial Cells

Binary toxins are among the most potent bacterial protein toxins performing a cooperative mode of translocation and exhibit fatal enzymatic activities in eukaryotic cells. Anthrax and C2 toxin are the most prominent examples for the AB7/8 type of toxins. The B subunits bind both host cell receptors and the enzymatic A polypeptides to trigger their internalization and translocation into the host cell cytosol. C2 toxin is composed of an actin ADP-ribosyltransferase (C2I) and C2II binding subunits. Anthrax toxin is composed of adenylate cyclase (EF) and MAPKK protease (LF) enzymatic components associated to protective antigen (PA) binding subunit. The binding and translocation components anthrax protective antigen (PA63) and C2II of C2 toxin share a sequence homology of about 35%, suggesting that they might substitute for each other. Here we show by conducting in vitro measurements that PA63 binds C2I and that C2II can bind both EF and LF. Anthrax edema factor (EF) and lethal factor (LF) have higher affinities to bind to channels formed by C2II than C2 toxin's C2I binds to anthrax protective antigen (PA63). Furthermore, we could demonstrate that PA in high concentration has the ability to transport the enzymatic moiety C2I into target cells, causing actin modification and cell rounding. In contrast, C2II does not show significant capacity to promote cell intoxication by EF and LF. Together, our data unveiled the remarkable flexibility of PA in promoting C2I heterologous polypeptide translocation into cells.

Potential protective immunogenicity of recombinant Clostridium perfringens α–β2–β1 fusion toxin in mice, sows and cows

Clostridial toxins are main pathogenic virulence of Clostridium perfringens that have been associated with a wide range of diseases in both humans and domestic animals. Genetically engineered toxoids have been shown to function as potential vaccine candidates in the prevention of Clostridium derived infectious diseases. In this study, we have developed recombinant α-toxin (CPA), β2/β1-fusion toxin (CPB2B1) and α/β2/β1 trivalent fusion-toxin (CPAB2B1) as vaccine candidates that may be used to vaccinate against C. perfringens α, β1 and β2-toxins. Mice immunized with these recombinant toxoids demonstrated a strong protective immunological response when administered a lethal dose of C. perfringens type C culture filtrate with high titers of neutralizing antibodies to the toxins in the sera, as well as the intestinal mucosal s-IgA level. Specific neutralizing antibodies to the toxins were also detected in the sera and colostrum of sows and cows vaccinated with the toxoids. Furthermore, the CPA and CPB2B1 recombinant toxoid cocktail was capable of stimulating relatively higher levels of immune responses compared to that of CPA, CPB2B1 and CPAB2B1 alone. The CPAB2B1 trivalent fusion toxoid also displayed increased immunogenicity relative to CPA and CPB2B1 alone. These results suggest that recombinant toxoids are potential vaccine candidates against Clostridial toxins; the use of mixed cocktails and/or multivalent recombinant toxoids against different types of toxins may be an effective approach in the prevention of diseases caused by toxins produced by C. perfringens.

Spotlight on Tdap5 Vaccine (Covaxis®) as a Single-Booster Immunization for the Prevention of Tetanus, Diphtheria, and Pertussis

Covaxis® (also licensed as Triaxis® or Adacel® in individual countries) is a Tdap5 (i.e. combined tetanus toxoid, reduced diphtheria toxoid, five component acellular pertussis [namely detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3]) vaccine for the prevention of diphtheria, tetanus, and pertussis. It is approved for use in Europe as a single intramuscular booster dose in children (aged ≥4 years), adolescents, and adults, and in the US it is approved for use in individuals aged 11–64 years.

Unique amnesic shellfish toxin composition found in the South East Asian diatom Nitzschia navis-varingica

Nitzschia navis-varingica is a diatom that is known to produce significant levels of amnesic shellfish poisoning (ASP) toxins. A total of 33 N. navis-varingica strains were isolated from four brackish water localities in the Philippines and Indonesia, and cultured to characterize the toxins produced. The isolates were analyzed for domoic acid (DA) and isodomoic acids A (IA) and B (IB) by HPLC with fluorescence detection. Two toxin composition types were detected that have not been previously described: strains producing only IB and strains producing DA–IA–IB. These two types were isolated from two different localities. Eighteen strains were isolated from the Philippines (northern Luzon Island). Among them, 10 isolates from Alaminos produced only IB with an average toxin content of 3.05 pg cell −1, seven isolates from Bulacan produced DA and IB with average toxin contents of 0.68 pg cell −1 and 1.18 pg cell −1, respectively. One isolate from Cavite produced DA, IA, and IB with a toxin content of 0.58, 0.20, and 0.92 pg cell −1, respectively. Fifteen isolates from Indonesia (Bone, South Sulawesi) produced only DA (four isolates) or DA with trace amounts of IB (eleven isolates), with an average toxin content of 2.38 pg cell −1 and 0.06 pg cell −1, respectively. Sub-strains were established from strains producing either of the three toxin types: IB, DA–IA–IB, and DA-trace IB. Results showed that the toxin composition type was the same for parent and sub-strains, indicating that the toxin composition is a stable character for a strain.

Bacillus thuringiensis: a century of research, development and commercial applications

Bacillus thuringiensis (Bt) is a soil bacterium that forms spores during the stationary phase of its growth cycle. The spores contain crystals, predominantly comprising one or more Cry and/or Cyt proteins (also known as δ-endotoxins) that have potent and specific insecticidal activity. Different strains of Bt produce different types of toxin, each of which affects a narrow taxonomic group of insects. Therefore, Bt toxins have been used as topical pesticides to protect crops, and more recently the proteins have been expressed in transgenic plants to confer inherent pest resistance. Bt transgenic crops have been overwhelmingly successful and beneficial, leading to higher yields and reducing the use of chemical pesticides and fossil fuels. However, their deployment has attracted some criticism particularly with regard to the potential evolution of pest-resistant insect strains. Here, we review recent progress in the development of Bt technology and the countermeasures that have been introduced to prevent the evolution of resistant insect populations.

Histological and ultrastructural changes in the adult male albino rat testes following chronic crude garlic consumption

Several studies have presented Garlic (Allium sativum) as a restoring agent for testicular cells after exposure to different types of toxins, however, others have shown that it modified spermatogenesis and might have spermicidal effects. This work has been to assess the positive or negative effect of chronic crude garlic consumption on rat testes using light and transmission electron microscopy with reference to plasma testosterone and LH levels. Forty albino male rats were divided into group A (control) and group B (treated rats). Group B was further subdivided into B1, B2, and B3 subgroups which were administer crude garlic as 20% of their daily food for two, three and four months, respectively. The current study has been the first, to the best of our knowledge, to describe the apoptotic effect of chronic crude garlic consumption targeting Sertoli cells, germ cells and peritubular tissue including interstitial Leydig cells and myoid cells. This might be better explained by inflammatory than degenerative changes in the peritubular tissue and Leydig cells leading to a decrease in testosterone level. Consequently, Sertoli cells degenerate due to a decrease in testosterone and detachment from the basal lamina. Germ cells, which are completely dependent upon Sertoli cells and testosterone to complete there spermatogenesis will be affected. Testicular apoptosis with disruption in spermatogenesis following chronic crude garlic consumption could be correlated with two possible theories; being an anti-hypercholesterolemic agent, it might inhibit steroidogenesis resulting in a decrease in testosterone level and being one of the famous phytoestrogens it possibly has direct estrogen-like actions on adult male rat testes.

Clinical, pathological and microbiological profiles of spontaneous enteropathies in growing rabbits

800x600 Normal 0 21 false false false ES X-NONE X-NONE MicrosoftInternetExplorer4 800x600 Normal 0 21 false false false ES X-NONE X-NONE MicrosoftInternetExplorer4 Normal 0 false false false ES X-NONE X-NONE In a rabbit production facility, health monitoring for enteropathies was performed in 15 production cycles for 20 mo. For each cycle, up to a hundred 35 d old rabbits weaned the same day were randomly selected, reared in the same fattening unit, but separately from the source batch and fed with the same feed except for antimicrobial supplementation. Clinical symptoms and enteric lesions of the selected group were recorded, using two checklists with binomial response (yes/no answer to a list of 54 clinical and enteric variables). The day after weaning, one week later, at the beginning of the enteric symptoms and 4-5 d after the start of the symptoms, inocula from the small intestine and caecum of selected animals were subjected to microbiological, C. spiroforme , Eimeria oocyst and rotavirus antigen detection tests. Representative samples of E. coli and C. perfringens isolates were tested, respectively, for serotype, biotype, eae , afr/2 genes and for a , b 1, b 2, e , i and enterotoxin toxin genes. The answers to the clinical-pathological variables were subjected to statistical analysis with a cluster analysis programme in order to obtain homogeneous, statistically significant groups of diseased animals (clusters). Then, the clusters were statistically associated with the laboratory outcomes. The cluster to which the enterotyphlitis lesions significantly contributed was associated with E. coli detection, E. coli O103 serotype detection and C. spiroforme (“several elements” variable). C. spiroforme (“rare elements” variable) was significantly associated with a cluster, characterised by a pathological profile consisting of bloating/rumbling noise and liquid content in stomach and caecum, without enteric inflammation. C. perfringens was significantly associated with a cluster, characterised by a pathological profile consisting of dilation/liquid content of small intestine, caecal impaction and mucoid content in the colon. Eighteen out of twenty-fi ve C. perfringens strains, examined for their toxin genotypes, proved to be toxin type A, while 7 out of 25 strains showed the a and b 2 toxin genes in combination. The rotavirus antigen and Eimeria oocysts were detected from healthy rabbits (specimens of the day after weaning and one week later) in about 15% of specimens examined, but their presence in the sick animals was not significantly associated with any cluster.

Tdap5 Vaccine (Covaxis®)

Covaxis (also licensed as Triaxis or Adacel in individual countries) is a combined tetanus toxoid, reduced diphtheria toxoid, five component acellular pertussis (namely detoxified pertussis toxin, filamentous hemagglutinin, pertactin, and fimbriae types 2 and 3) vaccine for the prevention of diphtheria, tetanus, and pertussis. It is approved for use in Europe as a single intramuscular booster dose in children (aged ≥ 4 years), adolescents, and adults, and in the US it is approved for use in individuals aged 11-64 years. In large, randomized, controlled clinical trials conducted in the UK and North America, a single intramuscular booster dose of Covaxis induced robust immune responses for all of its component antigens when given to children (aged ≥ 4 years), adolescents, and adults. In addition, Covaxis vaccine was safe and generally well tolerated in terms of solicited and unsolicited local injection-site and systemic adverse events, most of which were of mild intensity and resolved without sequelae. Furthermore, the immunogenicity of each individual component and the reactogenicity of Covaxis vaccine in children, adolescents, and adults was generally similar to that of comparator vaccines. Despite being a vaccine-preventable disease and having >90% primary vaccination coverage worldwide, pertussis remains uncontrolled, particularly amongst adolescents and adults. Given the changing epidemiology of pertussis and the requirement to reduce infection in adolescents and adults (including healthcare workers) and thereby prevent transmission of the disease from these individuals to very young infants, the new 'cocoon strategy' recommended in current vaccination guidelines has become a key strategy in the management of morbidity and mortality associated with pertussis. This strategy focuses on the immunization of healthcare workers, and the parents and family members of infants who are too young to have undergone primary immunization, so as to prevent the transmission of pertussis to these young at-risk infants. The implementation of the 'cocoon strategy' may finally give countries the ability to control pertussis infections in these at-risk infants and ultimately provide the desired herd immunity against pertussis. In line with this strategy, a booster dose of Covaxis vaccine provides a valuable option to reduce pertussis morbidity and mortality, and to maintain seroprotection against diphtheria and tetanus in children (aged ≥ 4 years), adolescents, and adults.

Emergence and genetic diversity of El Tor Vibrio cholerae O1 that possess classical biotype ctxB among travel-associated cases of cholera in Japan

Vibrio cholerae O1 are classified into two biotypes, classical and El Tor, each encoding a biotype-specific cholera toxin. However, El Tor strains have recently emerged with a classical cholera-toxin genotype (El Tor variant). We characterized El Tor strains of V. cholerae O1 from travel-associated cases of cholera in Japan isolated from 1991 to 2006 by cholera toxin B subunit gene (ctxB) typing and by molecular epidemiological methods. ctxB in the biotype El Tor shifted from the El Tor-specific type to the classical-specific type around 1993, and this type fully dominated the later half of the 1990s. Based on the results of PFGE and multilocus variable-number tandem repeat analysis, strains of the classical biotype remained diverse from those of El Tor biotype. The El Tor biotype strains formed multiple minor clusters and intermingled with each other irrespective of their origins and toxin types. El Tor variant strains are widespread in Asian countries and show significant genetic diversity, indicating that their spread is a result of multiclonal expansion rather than spread from a single clone.

Removal of albumin-bound toxins from albumin-containing solutions: Tryptophan fixed-bed adsorption on activated carbon

Fixed-bed adsorption on activated carbon is a common blood detoxification technique implemented in liver support devices to remove different types of toxins, among which is tryptophan. In this paper, fixed-bed adsorption kinetics of tryptophan on activated carbon were investigated. Experimental tryptophan breakthrough curves were obtained at different feed flow rates, both in albumin-free and albumin-containing solutions. A mathematical model based on linear driving force mass transfer kinetics was used to correlate data obtained with albumin-free solutions; subsequently, the results were successfully used to predict tryptophan breakthrough curves obtained with albumin-containing solutions, accounting only for the effect of albumin over tryptophan adsorption equilibrium.

Clostridium perfringens isolate typing by multiplex PCR

Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

Cholinesterase and glutathione‐S‐transferase activities in freshwater invertebrates as biomarkers to assess pesticide contamination

Studies investigating the use of biomarkers in pesticide risk assessment have greatly increased in recent years; however, issues concerning the ecological meaning of enzymatic responses have proved controversial. Ideally a good biomarker response should be modulated by the environmental contaminants alone and demonstrate a predictable behavior towards certain types of toxins. As these premises are rarely observed, the present study aims to outline research that has contributed to an understanding of the behavior of two widely used biomarkers, cholinesterase and glutathione-S-transferase, describing environmental and biotic factors that affect their response in freshwater invertebrates. Studies were performed in the main classes of aquatic invertebrates with these biomarkers and conclusions were reached concerning their behavior towards the main classes of pesticides. Links between biomarker responses and conventional endpoints were evaluated so that ecological relevance could be attributed to enzymatic responses. Toxicity of mixtures was investigated, and cases of synergism and antagonism were pointed out as factors changing the expected toxicity of aquatic systems and leading to misinterpretations of biomarker responses. Finally, the use of biomarkers as a tool for biomonitoring and in situ assays was investigated, with discussion of advantages and disadvantages of their use.

Lineage and Host Source Are Both Correlated with Levels of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains

Escherichia coli O157:H7 strains fall into three major genetic lineages that differ in their distribution among humans and cattle. Several recent studies have reported differences in the expression of virulence factors between E. coli O157:H7 strains from these two host species. In this study, we wished to determine if important virulence-associated "mobile genetic elements" such as Shiga toxin 2 (Stx2)-encoding prophage are lineage restricted or are host source related and acquired independently of the pathogen genotype. DNA sequencing of the stx(2) flanking region from a lineage II (LII) strain, EC970520, revealed that the transcriptional activator gene Q in LI strain EDL933 (upstream of stx(2)) is replaced by a pphA (serine/threonine phosphatase) homologue and an altered Q gene in this and all other LII strains tested. In addition, nearly all LI strains carried stx(2), whereas all LII strains carried variant stx(2c) and 4 of 14 LI/II strains had copies of both stx(2) and variant stx(2c). Real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that LI and LI/II strains produce significantly more stx(2) mRNA and Stx2 than LII strains. However, among LI strains significantly more Stx2 is also produced by strains from humans than from cattle. Therefore, lineage-associated differences among E. coli O157:H7 strains such as prophage content, toxin type, and toxin expression may contribute to host isolation bias. However, the level of Stx2 production alone may also play an important role in the within-lineage association of E. coli O157:H7 strains with human clinical disease.

Report of two unlinked cases of infant botulism in the UK in October 2007

Infant botulism is a rare disease in the UK, with the first case being recognized in 1978 and only five subsequent cases being reported before 2007. This study reports two unlinked cases of infant botulism, caused by two distinct strains of Clostridium botulinum (toxin types A and B, respectively), that occurred within a single month in the south-east of England in October 2007. The use of real-time PCR to detect C. botulinum neurotoxin genes in clinical specimens to improve the diagnostic procedure and to follow carriage of the causative organism in the infant gut is described. The laboratory investigation of these two cases demonstrated that a combination of the mouse bioassay, real-time PCR assays and conventional microbiological culture can provide rapid confirmation of a clinical diagnosis and affect patient management. Both infants (aged 4 and 8 months) were previously healthy prior to the onset of symptoms, and in both cases, a diagnosis of infant botulism was delayed for at least 10 days after initial admission to hospital. Once diagnosed, one of the infants was the first in the UK to be treated with human-derived botulism immunoglobulin. Real-time PCR was used to demonstrate that C. botulinum was excreted in the infants' faeces for up to 68 and 81 days, respectively. Despite the infrequency of infant botulism in the UK, clinicians should be aware of this rare but serious condition and should seek microbiological advice when presented with young infants with compatible symptomologies.

Fusion and Retrotransposition Events in the Evolution of the Sea Anemone Anemonia viridis Neurotoxin Genes

Sea anemones are sessile predators that use a variety of toxins to paralyze prey and foe. Among these toxins, Types I, II and III are short peptides that affect voltage-gated sodium channels. Anemonia viridis is the only sea anemone species that produces both Types I and III neurotoxin. Although the two toxin types are unrelated in sequence and three-dimensional structure, cloning and comparative analysis of their loci revealed a highly similar sequence at the 5' region, which encodes a signal peptide. This similarity was likely generated by gene fusion and could be advantageous in transcript stability and intracellular trafficking and secretion. In addition, these analyses identified the processed pseudogenes of the two gene families in the genome of A. viridis, probably resulting from retrotransposition events. As presence of processed pseudogenes in the genome requires transcription in germ-line cells, we analyzed oocyte-rich ovaries and found that indeed they contain Types I and III transcripts. This result raises questions regarding the role of toxin transcripts in these tissues. Overall, the retrotransposition and gene fusion events suggest that the genes of both Types I and III neurotoxins evolved in a similar fashion and share a partial common ancestry.

Aggressiveness ofFusarium graminearum sensu strictoIsolates in Wheat Kernels in Argentina

This study was designed to investigate the degree of aggressiveness of Fusarium graminearum sensu stricto isolates and its relationship with trichothecene production. In order to characterize Fusarium strains aggressiveness, disease severity was visually assessed as the percentage of spikelets bleached per ear. The severity ranged from a minimum of 27.19% seven days after inoculation to a maximum of 84.73% at the end of the experiment. At maturity the ears were harvested and threshed for grain weight determination. All treatments showed significant differences in kernel weight with respect to the control plants, with a yield reduction of 35-85% in comparison with the yield of the control. Grains infected by F. graminearum may contain significant levels of mycotoxins like trichothecenes. No correlation was found between aggressiveness and the toxins in the kernels at maturity, taking into consideration not only the quantity but also the type of toxin (nivalenol, deoxynivalenol or their acetylderivatives). Kernel weight reduction was a better estimator of the presence of deoxynivalenol in the kernels than the area under the disease progress curve (AUDPC) calculated with severity ratings. The amplified fragment length polymorphism (AFLP) technique was used to establish genetic relationships between 18 Argentinean isolates and eight reference strains of the Fusarium graminearum complex. All the isolates studied grouped with the two F. graminearum s. str. reference isolates, with a similarity coefficient greater than 75%. The other reference strains of the F. graminearum complex were clearly separated, with similarities ranging between 55 and 73%. The AFLP groups had no relationship with toxin accumulation on kernels or with the geographical origin of the isolates. Great heterogeneity was found in the AUDPC, yield reduction and toxin accumulation values across the regions.

Survey of Clostridium botulinum toxins in Iranian traditional food products

A total of 131 traditional food product samples (57 cheese, 11 kashk, and 63 salted fish) were examined using a bioassay method for detection of Clostridium botulinum toxin. Standard monovalent antitoxins were used to determine the toxin types. C. botulinum toxins were detected in 4.58% of examined samples (3.51% of cheese samples and 6.36% of salted fish samples). Contamination with this toxin was not observed in kashk samples. C. botulinum types A & E were dominant in cheese and salted fish samples, respectively. The results indicate that some Iranian traditional foods (cheese and salted fish) are contaminated by different types of C. botulinum. Consumption of these traditional foods either raw or cooked may contribute to food-borne toxicity in Iranian populations.

Bioinformatic characterizations and prediction of K+ and Na+ ion channels effector toxins.

BackgroundK+ and Na+ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K+ and/or Na+ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K+ and/or Na+ channels.ResultsFour multilevel motifs, overrepresented and specific to each group of K+ and/or Na+ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K+ and Na+ toxins.Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K+ and Na+ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K+ or Na+ toxins.Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors.Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences.ConclusionThe phylogenetic results were in perfect agreement with those obtained by the CART algorithm.Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.

STRAIN VARIATION IN KARLODINIUM VENEFICUM (DINOPHYCEAE): TOXIN PROFILES, PIGMENTS, AND GROWTH CHARACTERISTICS(1).

Karlodinium veneficum (D. Ballant.) J. Larsen strains, 16 from the U.S. Atlantic eastern seaboard and two from New Zealand (CAWD66 and CAWD83), were used to characterize toxin profiles during batch culture. All 18 strains were determined as the same species based on ITS sequence analyses, a positive signal in a chloroplast real-time PCR assay and pigment composition. Five karlotoxin 1 (KmTx 1) containing strains were analyzed from the Chesapeake Bay, and 10 karlotoxin 2 (KmTx 2) strains were analyzed from Florida to North Carolina. One strain (MD5) from the Chesapeake Bay produced no detectable toxin. The two cultures from New Zealand contained both novel karlotoxins with lower masses and earlier elution times. Toxin type did not change during batch culture, although the KmTx phenotype did change in some strains under extensive (months) phototrophic growth in replete media. KmTx cell quota did not change during batch culture for most strains. The mass spectrum for every KmTx examined showed a pattern of multiple coeluting congeners within each HPLC peak, with masses typically differing by 16 amu. KmTx congeners tested showed nearly a 500-fold range in specific hemolytic activity, with KmTx 1 (typically occurring at lower cellular levels) most hemolytic and CAWD66 toxin least hemolytic, while KmTx 2 and the CAWD83 toxin had similar intermediate specific activity. Despite morphological, genetic, and photopigment indicators consistent with species homogeneity among the 18 strains of K. veneficum, the high degree of toxin variability suggests different functional roles among strains that likely coexist in situ.

New highly specific botulinum type C1 endopeptidase immunoassays utilising SNAP25 or Syntaxin substrates

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, C1 and E intra-cellularly cleave SNAP25 and/or Syntaxin (type C1 only) resulting in a flaccid paralysis. Although highly sensitive, robust in vitro endopeptidase immunoassays have been developed for some serotypes, an endopeptidase immunoassay for type C1 has not previously been described. The current studies utilised solid phase synthesized SNAP25(137–206) peptide substrate, and a new specific antibody to the SNAP25(191–198) octapeptide epitope that becomes exposed following cleavage by type C1 toxin. The highly specific nature of the detecting antibody was illustrated by the failure of anti-SNAP25(191–198) to recognise the type A cleavage product which differs by just one amino acid residue. Conversely, anti-SNAP25(190–197), which recognises the type A cleavage product, fails to cross react with the type C1 toxin cleavage product. Utilising Syntaxin(232–266) peptide substrate, and a specific antibody to the cleavage product epitope, Syntaxin(254–261), it was also possible to develop an endopeptidase immunoassay. Assay sensitivities allowed the detection of less than 0.1 LD 50/ml (25 pg/ml) of type C1 haemagglutinin-complexed toxin. The assay failed to detect toxin serotypes A, B, D, E, F or G and therefore also provides an alternative highly specific in vitro identity test. In the absence of trypsin inhibitors, the assay is also capable of detecting 2 pg/ml of trypsin activity, or trypsin like contaminants. These new immunoassays will therefore provide highly specific tools for monitoring botulinum toxin light chain endopeptidase activity and serotype identity.