Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Persistent Idiopathic Dentoalveolar pain. Literature review and clinical case treated with intraoral application of Botulinum toxin / Dolor Idiopático Dentoalveolar Persistente (DIDAP). Revisión de literatura y caso clínico tratado con aplicación intraoral de toxina Botulínica

ABSTRACT: Persistent Idiopathic Dentoalveolar Pain (PIDAP) is an orofacial neuropathic pain, which can be difficult to diagnose and is usually accompanied by increasing anxiety from both the patient and the treating dentist. A case of a 38-year-old female patient is presented, and it is shown the diagnostic process and therapeutic approach. The interdisciplinary management accompanied by several pharmacological lines is highlighted: Botulinum toxin was used as an adjunctive treatment allowing it to decrease systemically administered medications dosing and therefore its possible side effects. This condition usually affects psychosocial aspects of the patient and has a major impact on his quality of life. It is very important before initiating an invasive clinical treatment, obtaining a clear differential diagnosis and assessing in some cases the presence of non-odontogenic pain, such as PIDAP.

RESUMEN: El Dolor Idiopático Dentoalveolar Persistente (DIDAP), es un dolor neuropático orofacial, que puede resultar difícil de diagnosticar y generalmente se acompaña de creciente angustia tanto de parte del paciente como también del odontólogo tratante. Se presenta un caso de una paciente femenina de 38 años en donde se demuestra el proceso diagnóstico y abordaje terapéutico. Se resalta el manejo interdisciplinario acompañado de varias lineas farmacológica: la toxina Botulínica se utilizó como tratamiento coadyuvante para disminuir la dosis de medicamentos administrados por vía sistémica y por ende sus posibles efectos secundarios. Esta condición habitualmente abarca aspectos psicosociales del paciente y tiende a verse sumamente afectada su calidad de vida. Es de suma relevancia antes de iniciar un tratamiento clínico invasivo, obtener un diagnóstico diferencial claro y valorar en algunos casos la presencia de dolor no ontogénico, como el DIDAP.

De novo design of modular and tunable protein biosensors.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Modification and validation of the Endopep-mass spectrometry method for botulinum neurotoxin detection in liver samples with application to samples collected during animal botulism outbreaks.

Botulinum neurotoxins (BoNTs) are the most potent toxins known and they cause the paralytic disease botulism in humans and animals. In order to diagnose botulism, active BoNT must be detected in biological material. Endopep-MS is a sensitive and selective method for serum samples, based on antibody capture, enzymatic cleavage of target peptides, and detection of cleavage products using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In many cases of animal botulism, serum samples are not available or they do not contain detectable amounts of BoNT and liver sampling is an alternative for postmortem examinations. However, the Endopep-MS method is impaired by the inherent protease activity of liver samples. In the presented study, the Endopep-MS method has been successfully modified and validated for analysis of cattle, horse, and avian liver samples, introducing a combination of a salt washing step and a protease inhibitor cocktail. These modifications resulted in a substantial decrease in interfering signals and increase in BoNT-specific signals. This led to a substantial improvement in sensitivity for especially BoNT-C and C/D which are among the most prominent serotypes for animal botulism. Botulism was diagnosed with the new method in liver samples from dead cattle and birds from outbreaks in Sweden. Graphical Abstract.

Proposed BoNT/A and /B Peptide Substrates Cannot Detect Multiple Subtypes in the Endopep-MS Assay.

Botulinum neurotoxins (BoNTs) are a family of protein toxins consisting of seven known serotypes (BoNT/A-BoNT/G) and multiple subtypes within the serotypes, and all of which cause the disease botulism-a disease of great public health concern. Accurate detection of BoNTs in human clinical samples is therefore an important public health goal. To achieve this goal, our laboratory developed a mass spectrometry-based assay detecting the presence of BoNT via its enzymatic activity on a peptide substrate. Recently, publications reported the use of new peptide substrates to detect BoNT/A and /B with improved results over other peptide substrates. However, the authors did not provide results of their peptide substrate on multiple subtypes of BoNT. In this work, we describe the results of testing the new substrates with multiple BoNT/A and /B subtypes and find that the substrates cannot detect many subtypes of BoNT/A and /B.

Reanimação estática do lábio inferior na paralisia facial como complicação de preenchimento / Static lower lip reanimation as a filler complication of facial paralysis

Procedimentos de rejuvenescimento facial substitutos da cirurgia tradicional tornaram-se cada vez mais populares para promover uma aparência jovial com procedimentos minimamente invasivos, como toxina botulínica injetável, preenchimento de tecidos moles e peelings químicos. No entanto, complicações podem ocorrer mesmo na presença de um injetor habilidoso e experiente. Apresentamos o caso de uma paciente submetida a reanimação labial estática usando retalho dermoadiposo para lesão do nervo facial direito após remoção de nódulos como complicação de preenchimento. A "abordagem modificada de bull horn" foi realizada para elevação do lábio superior em torno das asas nasais e columela e ao longo do sulco nasolabial direito. O retalho foi desepitelizado e obtido. Usando a ponta aberta de uma pequena cânula de lipoaspiração, a porção distal do retalho foi encapsulada e fixada diretamente em C-loop e foram utilizados pontos U, transfixando o retalho para o periósteo do arco zigomático. Nos três anos de seguimento não foram observadas complicações significativas e a paciente não relatou nenhuma limitação funcional ou insatisfação com o aspecto das cicatrizes no sulco nasolabial e ao redor das asas nasais e da columela.

Facial rejuvenation procedures to circumvent traditional surgery have become increasingly popular to promote a youthful appearance with minimally invasive procedures such as injectable botulinum toxin, soft-tissue fillers, and chemical peels. Nevertheless, complications can occur even with an astute and experienced injector. Here we present the case of a patient who underwent static lip reanimation using a dermoadiposal flap for right facial nerve damage following nodule removal as a filler complication. A "modified bulls horn approach" to the upper lip lift was performed around the nasal wings and columella and along the right nasolabial fold. The flap was de-epithelized and harvested. Using the open tip of a small liposuction cannula, the distal portion of the flap was tunneled and fixed directly in a C-loop fashion using U stitches, transfixing the flap to the periosteum of the zygomatic arch. At 3 years follow-up, no significant complications were observed, and the patient reported no functional limitations or dissatisfaction with the scars in the nasolabial fold or around the nasal wings and columella.

Sensitive detection of type G botulinum neurotoxin through Endopep-MS peptide substrate optimization.

Clostridium botulinum produces botulinum neurotoxins (BoNTs) that are one of the most poisonous substances. In order to respond to public health emergencies, there is a need to develop sensitive and specific methods for detecting botulinum toxin in various clinical matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate BoNT serotypes A-G by immunoaffinity capture of toxins and detection of unique cleavage products of peptide substrates. To improve the sensitivity of the Endopep-MS assay for the detection of BoNT serotype G, we report here the optimization of synthetic peptide substrates through systematic substitution, deletion, and incorporation of unnatural amino acids. Our data show that the resulting optimized peptides produced a significant improvement (two orders of magnitude) in assay sensitivity and allowed the detection of 0.01 mouseLD50 toxin present in buffer solution.

Further optimization of peptide substrate enhanced assay performance for BoNT/A detection by MALDI-TOF mass spectrometry.

Rapid and sensitive detection of botulinum neurotoxins (BoNTs), which cause botulism, is essential in a public health emergency or bioterrorism event. We have previously developed a mass spectrometry (MS)-based functional method, Endopep-MS assay, for the fast detection and differentiation of all BoNT serotypes by affinity enriching the toxin and detecting the serotype-specific cleavage products of peptide substrates derived from the in vivo targets. To improve the performance of the Endopep-MS assay, we report here the further optimization of the peptide substrate for the detection of serotype A botulinum neurotoxins. An increased substrate cleavage was achieved by extending the original peptide N-terminus with optimized amino acid sequence, increasing the detection sensitivity of the method. In addition, the resistance of the substrate to nonspecific hydrolysis was dramatically improved by selectively substituting amino acids at the scissile bond and various other positions of the extended peptide. Moreover, incorporating the N-terminal hydrophobic residues dramatically improved the relative intensity of the cleavage products in the mass spectra. This allowed easy detection of the cleavage products, further enhancing the performance of the assay. The limit of detection for spiked serum sample was enhanced from 0.5 to 0.1 mouseLD50 and from 0.5 to 0.2 mouseLD50 for spiked stool. Graphical abstract Mass spectra of optimized and old peptide substrates with BoNT/A.

Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep-MS assay.

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep-MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A-G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep-MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD50/ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples.

Improved detection of botulinum type E by rational design of a new peptide substrate for endopeptidase-mass spectrometry assay.

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humans. Endopeptidase-mass spectrometry (Endopep-MS) is used as a specific and rapid in vitro assay to detect BoNTs. In this assay, immunocaptured toxin cleaves a serotype-specific peptide substrate, and the cleavage products are then detected by MS. To further improve the sensitivity of the assay, we report here the rational design of a new substrate peptide for the detection of botulinum neurotoxin type E (BoNT/E). Our strategy was based on previously reported structural interactions integrated with analysis method efficiency considerations. Integration of the newly designed substrate has led to a more than one order of magnitude increased sensitivity of the assay.

Expression, purification and development of neutralizing antibodies from synthetic BoNT/B LC and its application in detection of botulinum toxin serotype B.

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) neurotoxins (BoNTs). The mouse bioassay is the gold standard for the detection of botulinum neurotoxins, however it requires at least 3-4 days for completion. Most of the studies were carried out in botulinum toxin A and less on type B. Attempts have been made to develop an ELISA based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. In the present study, the synthetic BoNT/B LC gene was constructed using PCR overlapping primers, cloned in a pET28a+ vector and expressed in E. coli BL21DE3. The maximum yield of recombinant proteins was optimized after 16 hrs of post induction at 21°C and purified the recombinant protein in soluble form. Antibodies were raised in Mice and Rabbit. The IgG antibody titer in the case of Mice was 1: 1,024,000 and Rabbit was 1: 512,000 with alum as adjuvant via intramascular route. The biological activity of the recombinant protein was confirmed by in-vitro studies using PC12 cells by the synaptobrevin cleavage, the rBoNT/B LC protein showed the maximum blockage of acetylcholine release at a concentration of 150nM rBoNT/B LC in comparison to the control cells. When the cells were incubated with rBoNT/B LC neutralized by the antisera raised against it, the acetylcholine release was equivalent to the control. IgG specific to rBoNT/B LC was purified from raised antibodies. The results showed that the developed antibody against rBoNT/B LC protein were able to detect botulinum toxin type B approximately up to 1 ng/ml. These developed high titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.

Surto familiar de botulismo no estado do Ceará: relato de caso / Familiar outbreak of botulism at Ceará state, Brazil: case report

Relato de surto familiar de botulismo por intoxicação alimentar, envolvendo um óbito, onde foram encontradas lacunas no preenchimento do prontuário. O objetivo foi descrever a patologia chamando a atenção dos profissionais de saúde para o fornecimento adequado de informações relevantes para a investigação epidemiológica de doenças de notificação compulsória.

Report of a family outbreak of botulism food poisoning involving a death, where gaps in the completion of medical records were identified. The study aimed to describe the pathology and emphasize to health professionals the need to provide adequate information relevant to epidemiological investigation of compulsory notification diseases.

Survival of radio-marked mallards in relation to management of avian botulism.

Avian botulism outbreaks are frequently perpetuated by type C toxin produced by Clostridium botulinum proliferating in decomposing bird carcasses and consumption of toxic maggots from these carcasses by healthy birds. Therefore, removing bird carcasses has been advocated for disease management because availability of toxic maggots should be reduced, increasing duck survival. However, this management is expensive, and its effect on waterfowl mortality under field conditions is unknown. We radio-marked 419 molting mallards on 11 lakes in western Canada during July-August 1999-2001 and monitored them for 30 days, testing whether survival was higher on lakes with carcass removal. Botulism occurred on 10 lakes. On five carcass removal lakes, greater-than-normal effort was made to conduct early, thorough surveillance and immediately remove carcasses; on six nonremoval lakes, no carcasses were removed. In 1999, estimated 30-day survival probabilities ranged from 0.149 (95% CI=0.065-0.304) on one large lake with carcass removal to 0.466 (95% CI=0.270-0.674) and 0.618 (95% CI=0.443-0.767) on two nonremoval lakes. As a result, we conducted work on smaller wetlands thereafter, reasoning that any management benefit would be easier to detect. In 2000, estimated 30-day survival probabilities were 0.313 (95% CI=0.143-0.556) and 0.794 (95% CI=0.609-0.905) on two carcass removal lakes versus 0.525 (95% CI=0.362-0.682) and 0.743 (95% CI=0.564-0.866) on two nonremoval lakes. In 2001, botulism was detected on two nonremoval lakes where survival probabilities were 0.845 (95% CI=0.630-0.946) and 0.942 (95% CI=0.778-0.987), and on one removal lake where survival probability was 1.0 (95% CI=0.99-1.0), but not detected on the other removal lake where no marked birds died from botulism (1.0, 95% CI=0.99-1.0). Survival tended to be higher on lakes with lower carcass density, but when data were organized by carcass removal versus nonremoval, mallard survival was not consistently greater on lakes where carcasses were removed.

Botulism in Brazil, 2000-2008: epidemiology, clinical findings and laboratorial diagnosis / Botulismo no Brasil, 2000-2008: epidemiologia, achados clínicos e diagnóstico laboratorial

Botulism is a rare and potentially lethal illness caused by Clostridium botulinum neurotoxin. We describe the findings of a laboratorial investigation of 117 suspected cases of botulism reported to the surveillance system in Brazil from January 2000 to October 2008. Data on the number and type of samples analyzed, type of toxins identified, reporting of the number of botulism cases and transmission sources are discussed. A total of 193 clinical samples and 81 food samples were analyzed for detection and identification of the botulism neurotoxin. Among the clinical samples, 22 (11.4 percent) presented the toxin (nine type A, five type AB and eight with an unidentified type); in food samples, eight (9.9 percent) were positive for the toxin (five type A, one type AB and two with an unidentified type). Of the 38 cases of suspected botulism in Brazil, 27 were confirmed by a mouse bioassay. Laboratorial botulism diagnosis is an important procedure to elucidate cases, especially food-borne botulism, to confirm clinical diagnosis and to identify toxins in food, helping sanitary control measures.

Botulismo é uma doença rara e potencialmente letal, resultante da ação de uma neurotoxina produzida pelo Clostridium botulinum. No presente estudo, estão descritos os resultados da investigação laboratorial de 117 casos suspeitos de botulismo notificados ao sistema de vigilância, ocorridos no Brasil no período de janeiro de 2000 a outubro de 2008. Os dados obtidos sobre as fontes de transmissão, os tipos de toxina identificados e de amostras analisadas serão discutidos. Foram analisadas 193 amostras clínicas e 81 amostras de alimentos para detecção e identificação de neurotoxina botulínica. Entre as amostras clínicas, 22 (11,4 por cento) amostras apresentaram resultado positivo para toxina (nove do tipo A, cinco do tipo AB e em oito o tipo não foi identificado) e entre as amostras de alimentos, oito (9,9 por cento) foram positivas (cinco do tipo A, uma do tipo AB e em duas o tipo não foi identificado). Dos 38 casos considerados positivos para botulismo, 27 foram confirmados pelo bioensaio em camundongo. O diagnóstico laboratorial de botulismo é importante para elucidação dos casos, principalmente de botulismo alimentar, para confirmação dos diagnósticos clínicos e identificação das toxinas nos alimentos, provendo subsídios para as medidas de controle sanitário.

Linden flower (Tilia spp.) as potential vehicle of Clostridium botulinum spores in the transmission of infant botulism / El té de tilo como vehículo potencial de esporas de Clostridium botulinum en la transmisión del botulismo infantil

Infant botulism is an intestinal toxemia caused principally by Clostridium botulinum. Since the infection occurs in the intestinal tract, numerous food products have been investigated for the presence of C. botulinum and its neurotoxins. In many countries, people use linden flower (Tilia spp) tea as a household remedy and give it to infants as a sedative. Therefore, to help provide a clear picture of this disease transmission, we investigated the presence of botulinum spores in linden flowers. In this study, we analyzed 100 samples of unwrapped linden flowers and 100 samples of linden flowers in tea bags to determine the prevalence and spore-load of C. botulinum. Results were analyzed by the Fisher test. We detected a prevalence of 3% of botulinum spores in the unwrapped linden flowers analyzed and a spore load of 30 spores per 100 grams. None of the industrialized linden flowers analyzed were contaminated with botulinum spores. C. botulinum type A was identified in two samples and type B in one sample. Linden flowers must be considered a potential vehicle of C. botulinum, and the ingestion of linden flower tea can represent a risk factor for infant botulism.

El botulismo del lactante es una toxiinfección causada, principalmente, por Clostridium botulinum. Debido a que esta infección ocurre en el tracto intestinal, la presencia de esta bacteria y sus neurotoxinas ha sido investigada en numerosos alimentos. En muchos países se utiliza el té de tilo (Tilia spp.) como sedante natural, el que se administra incluso a los lactantes. A fin de contribuir al esclarecimiento de la transmisión de esta enfermedad, se investigó la prevalencia y la carga de esporas botulínicas en esta hierba. Se analizaron 100 muestras de tilo comercializado a granel y 100 muestras de tilo industralizado en “saquitos”. Los resultados de prevalencia fueron analizados por el test de Fisher y la carga de esporas por la técnica del número más probable. Se halló una prevalencia de esporas de C. botulinum del 3% en el tilo comercializado a granel, con una carga de 30 esporas/100 g de hierba. En tanto, ninguna de las muestras en saquitos acusó la presencia del patógeno. Se identificaron tres cepas de C. botulinum, dos tipo A y una tipo B. En virtud de estos resultados, el tilo podría considerarse un potencial vehículo de esporas de C. botulinum y la administración de sus infusiones a menores y lactantes, un riesgo para la transmisión de la enfermedad.

Determination of botulinum toxins after peptic sample pre-treatment by multidimensional nanoscale liquid chromatography and nano-electrospray ion-trap mass spectrometry.

Botulinum neurotoxin types A to G are produced from different strains of Clostridium botulinum. The complex neurotoxins belong to the most toxic substances known and cause botulism both in humans and animals. Botulinum toxin complexes are produced with molecular weights of 300, 500 and 900 kDa. These large protein complexes contain beside the toxic zinc protease of 150 kDa, additional neurotoxin associated proteins, which are responsible for the extreme pH and protease stability. In this study we present for the first time a rugged detection method of botulinum toxins at femtomole levels in complex culture media after peptic sample pre-treatment and 2D-nano-LC-ESI-MS-MS-technique. In contrast to other studies, we used progenitor toxins directly from culture supernatant of C. botulinum strains A, B, E and F without further purification, to simulate complex, protein-containing sample conditions. We were able to demonstrate, that peptic pre-treatment is a great challenge in reducing ubiquitous proteins as well as proteins from suspicious samples. The study also found that multidimensional chromatography leads to significant better peptide differentiation and identification in protein loaded matrices than one dimensional nano-LC-ESI-MS.

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.

Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins.

Botulinum neurotoxins contain proteases that cleave specific intra-neural proteins essential for neurotransmitter release. Toxin types A, E and C1 intra-cellularly cleave SNAP25 resulting in a flaccid paralysis. As a consequence, various different endopeptidase assays have been developed to specifically detect the toxins enzymatic activity, however, many of these suffer from variability, low sensitivity or unwanted interference exerted by product specific excipients. The current studies utilised solid phase synthesized SNAP25(137-206) peptide substrate, and specific antibody to either the SNAP25(190-197) or (173-180) octapeptide epitopes that become exposed following cleavage by toxin types A or E respectively. Assay sensitivity was increased 50 fold by the use of an optimal 0.5% Tween 20 concentration in tandem to 0.1% albumin together with an improved, simplified assay design without a pre-activation / reduction step. Sensitivities capable of detecting 0.01 LD50/ml (40fg/ml or 0.3fM) of type A toxin was achieved with a linear dose response between 0.1 and 1 LD50/ml. This provides sufficient sensitivity and precision (inter assay GCV of < 2%) for monitoring activity within any current or newly marketed therapeutic products containing less units per vial and may also make it applicable for other applications. Both purified haemagglutinin free and complexed toxins could be detected equally. Unlike type A, type E activity could unexpectedly be detected in the complete absence of reducing conditions and the optimal assay had a limit of detection of 0.2LD50/ml (4.8pg/ml) with a linear dose response between 1 and 10LD50/ml. The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to other specific enzyme cleavage reactions in the future.

Conversion of a mouse Fab into a whole humanized IgG antibody for detecting botulinum toxin.

Antibodies serve as the gold standard in most immunodiagnostic assays. Recent advances in recombinant DNA technology have offered the production of antibody fragments or Fabs as promising alternatives. However, the lack of the Fc region of the antibody can be difficult in many standard diagnostic platforms. Therefore we sought to convert a murine Fab into a whole humanized IgG. The variable regions from an anti-botulinum Fab were cloned into human IgG heavy and light chain vectors and produced in myeloma cells. Purified humanized IgG demonstrated conversion to human IgG with no traces of mouse Fab as determined by Western blot analysis. In addition, the humanized IgG performed better as both a detection and capture reagent in an ELISA format and detected the botulinum toxoid at a lower concentration than the parental murine Fab. This technique offers the ability to convert various species of antibodies or antibody fragments into humanized antibodies with improved characteristics in immunodiagnostic assays, for use as human controls in serological assays, or for possible therapeutic benefit.

Methods for detection of Clostridium botulinum toxin in foods.

Botulism is a deadly disease caused by ingestion of the preformed neurotoxin produced from the anaerobic spore-forming bacteria Clostridium botulinum. Botulinum neurotoxins are the most poisonous toxins known and have been a concern in the food industry for a long time. Therefore, rapid identification of botulinum neurotoxin using molecular and biochemical techniques is an essential component in the establishment of coordinated laboratory response systems and is the focus of current research and development. Because of the extreme toxicity of botulinum neurotoxin, some confirmatory testing with the mouse bioassay is still necessary, but rapid methods capable of screening large numbers of samples are also needed. This review is focused on the development of several detection methods for botulinum neurotoxins in foods.