To analyze a suspected case of Clostridium botulinum food poisoning in Bayingolin Mongolian Autonomous Prefecture, Xinjiang and to help validating the diagnosis and providing technical support for clinical treatment. The basic information and clinical manifestations of food poisoning cases were investigated by using the epidemiological method of food safety accidents. The botulinum toxin genes in the samples were detected by real-time PCR and inoculation of KM mouse. The enriched bacteria were further purified and validated. PFGE and cluster analysis were performed on five isolates. Clostridium botulinum type A was detected in two homemade fermented bean samples and stool lavage fluid samples of three patients from enriched samples by toxin test and real-time PCR, and were further validated after isolation of Clostridium botulinum. PFGE showed 100% homology among five isolates. Five isolates of bacteria isolated from the stool lavage fluid of three patients and two homemade fermented bean curd were identified as the same source through PFGE. The cause of this food poisoning cases is food pollution of Clostridium botulinum type A.
Animals ; Clostridium botulinum/genetics* ; Feces ; Foodborne Diseases/epidemiology* ; Gerbillinae ; Humans ; Mice

The aim of this study was an examination of 240 multifloral honey samples collected from Polish apiaries to determine Clostridium botulinum occurrence. Honey was collected from apiaries directly after the extraction process. Samples were inoculated by using the dilution and centrifugation method. Suspected isolates were examined by using mouse bioassay, polymerase chain reaction (PCR), and real-time PCR methods. C. botulinum type A and B strains were detected in 5 of 240 examined honey samples (2.1%). Bacterial strains were also detected that were phenotypically similar to C. botulinum but that did not exhibit the ability to produce botulinum toxins and did not show the presence of the botulinum cluster (ntnh and bont genes) or expression of the ntnh gene. The methods used in the examination, especially the expression analysis of ntnh gene, enabled specific analysis of suspected strains and could be used routinely in environmental isolate analyses of C. botulinum occurrence.
Animals ; Biological Assay ; Botulinum Toxins ; Centrifugation ; Clostridium botulinum ; Clostridium ; Honey ; Methods ; Mice ; Neurotoxins ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Spores

Laboratory-based pathogen isolation, identification, and toxicity determination were performed on samples from a suspected case of infant botulism. Mice injected with cultures generated from the enema sample and ingested Powered infant formula (PIF) presented typical signs of botulism. Antitoxins to polyvalent botulinum neurotoxins (BoNTs) and monovalent BoNT type B antitoxin had protective effects. Clostridium botulinum isolated from the enema and residual PIF samples were positive for type B toxin. Pulsed-field gel electrophoresis (PFGE) revealed that the two strains of C. botulinum isolated from the two samples produced indistinguishable pulsotypes. These findings confirmed this case of type B infant botulism associated with the ingestion of PIF contaminated by type B C. botulinum spores.
Animals ; Beijing ; epidemiology ; Botulinum Toxins ; isolation & purification ; toxicity ; Botulism ; diagnosis ; epidemiology ; Clostridium botulinum ; isolation & purification ; Gastrointestinal Tract ; microbiology ; Humans ; Infant ; Mice ; Toxicity Tests

OBJECTIVETo analyze the clinical characteristics and diagnosis of three cases with infant botulism.

METHODClinical data of three clinically diagnosed cases with infant botulism in May 2015 in Peking University First Hospital were retrospectively analyzed. Literature search at databases of PubMed, Wanfang, China National Knowledge Infrastructure and VIP with the key words"infant AND botulism". The date of literature retrieval was from the database founding to November 2015. The characteristics of infant botulism were summarized through review of literature.

RESULTThree patients were infants of 4-8 months of age, and all had acute onsets of anorexia and poor response. All of them had normal psychomotor development previously, and without clear history of exposure to poisons. The main findings on physical examination were reduced muscle strength and hypotonia, dullness or disappeared pupillary light reflex, reduced facial expression, weak crying and dysphagia. Unexpectedly their states of consciousness were relatively normal. Finally, through identification and PCR genotyping of bacteria in stool, 2 cases were confirmed as Clostridium (C.) botulinum type B infection. Totally 446 reports were retrieved from foreign language literature and 52 reports from Chinese literature. More than 3,000 cases of infant botulism cases were reported in the world. Rare cases were reported in China and only 1 case was reported in 2000.

CONCLUSIONMost cases of infant botulism had no clear exposure history. The main clinical manifestations are hypotonia, cranial nerve paralysis, flaccid paralysis, but different patients may have different presentations. Detection of C. Botulinum and its toxin in stool can help to confirm the diagnosis. Infant botulism is relatively rare in China, which may be related to the insufficient understanding and inspection level of the disease. It might be underestimated in China.

Botulism ; China ; Clostridium botulinum ; Feces ; Genotype ; Humans ; Infant ; Paralysis

OBJECTIVETo establish the genotype-specific targets plasmids and engineered E.coli strains of botulinum neurotoxins (BoNT) types B and E based on reverse genetics.

METHODSThe gene sequences of BoNT were obtained from GenBank and analyzed using DNAMAN, Lasergene, Vector NTI and BLAST. Two target fragments of BoNT/B and BoNT/E were anchored and then synthesized as 5 and 10 short DNA single strands, respectively. The full-length target sequences were amplified by overlapping PCR and subcloned into pMD 18-T vector, and the recombinant plasmids were identified by restriction enzyme digestion and sequencing.

RESULTSSixty full-length sequences of 4 types of BoNT, namely types A, B, E, and F, were available in GenBank. Two target fragments, BoNT/B of 215 bp and BoNT/E of 360 bp, and their specific primer pairs were anchored after sequence analysis. pMD 18-T-BoNT/B and pMD 18-T-BoNT/E containing these two target sequences were confirmed.

CONCLUSIONThe engineered plasmids and E.coli stains containing the genotype-specific target fragments of BoNT/B and BoNT/E have been constructed successfully.

Base Sequence ; Botulinum Toxins ; genetics ; Botulinum Toxins, Type A ; Clostridium botulinum ; genetics ; isolation & purification ; Gene Targeting ; Genotype ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

A capture enzyme-linked immunosorbent assay (capture ELISA) was developed to detect Clostridium botulinum neurotoxin type A (BoNT/A) in assay buffer and human serum. The assay is based upon affinity-purified rabbit polyclonal and biotinylated monoclonal antibodies directed against the BoNT/A complex purified from C. botulinum ATCC19397. For the capture ELISA, the optimized amount (2 microgram/ml) of rabbit polyclonal antibody was immobilized on ELISA plates to detect BoNT/A (ranging from 0 to 500 ng/ml), which was recognized by 2 microgram/ml of the monoclonal antibody. From three independent repeated experiments, standard curves were linear over the range of 0~31.25 ng/ml BoNT/A and the coefficients (r(2)) ranged from 0.9951~0.9999 for all assays. The inter-variations were typically 0.50~6.93% and the specificity was confirmed by showing no cross-reactivity against BoNT/B and /E. The detection limit of capture ELISA was 0.488 ng/ml, which was close to mouse LD(50). In addition, application with BoNT/A-spiking human sera showed a possibility to detect BoNT/A with capture ELISA from the contaminated human sera. Taken together, the newly developed capture ELISA could serve as a rapid and sensitive screening tool for detecting BoNT/A simultaneously from massive specimens.
Animals ; Antibodies, Monoclonal ; Clostridium botulinum ; Enzyme-Linked Immunosorbent Assay ; Humans ; Limit of Detection ; Mass Screening ; Mice ; Sensitivity and Specificity

A completely synthetic gene encoding the He domain of Clostridium botulinum neurotoxin serotype A (AHc, 1287 bp, 429 aa, -50 kD) was constructed with oligonucleotides. After expressed in Escherichia coli, soluble product AHc was gained and verified by SDS-PAGE and Western blot analysis. The expressive level of recombinant AHc in E. coli was very high (36%-53% of soluble total proteins) and the purified yield was more than 30 mg/L by one-step purification. Then, the purified AHc was used to vaccinate Balb/c mice, which developed a strong and specific immune response as expected following administration of AHe protein via the subcutaneous route. Results from BoNT/A neutralization assay showed that the serum from mice vaccinated with AHc contained high titer protective antibody. These results showed that the soluble, stable and high-levelly expressive AHc not only could be produced by the prokaryotic expression system built in our lab, but also owned strong immunogenicity to prepare antitoxin for treatment and as sub-unit candidate vaccine for prophylaxis against botulinum toxin serotype A.
Animals ; Antibodies, Bacterial ; blood ; Bacterial Vaccines ; genetics ; immunology ; Botulinum Toxins, Type A ; biosynthesis ; genetics ; immunology ; Botulism ; immunology ; prevention & control ; Clostridium botulinum type A ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; T-Lymphocytes ; immunology ; Vaccines, DNA ; genetics ; immunology

Botulism is a life threatening disorder caused by a neurotoxin produced from the anaerobic, spore-forming bacterium Clostridium botulinum. There are seven antigenically distinct types of botulinum neurotoxins (types A through G), and the human botulism is primarily caused by toxin types A, B, and E. Four clinical forms of botulism occur in humans: foodborne botulism, wound botulism, infant botulism, and adult infectious botulism. Botulism is characterized by symmetric, descending, flaccid paralysis of motor and autonomic nerves, usually beginning with the cranial nerves. Dry mouth, blurred vision, and diplopia are usually the earliest neurologic symptoms. Botulism should be suspected in a patient with an acute onset of gastrointestinal, autonomic, and cranial nerve dysfunction. Confirmation of the diagnosis of botulism depends on the detection of the toxin or the organism in the patient. The most reliable method for the detection of the toxin is the mouse inoculation test. The mainstay of treatment for severe botulism is supportive therapy with mechanical ventilation. The administration of antitoxin is the only specific pharmacologic treatment available for botulism. Botulism is a rare but potentially fatal illness, so timely recognition of the clinical symptoms plays an important role in decreasing the mortality rate.
Adult ; Animals ; Autonomic Pathways ; Botulism* ; Clostridium botulinum ; Cranial Nerves ; Diagnosis ; Diplopia ; Humans ; Mice ; Mortality ; Mouth ; Neurologic Manifestations ; Neurotoxins ; Paralysis ; Respiration, Artificial

Designed a pair of primers through modifying N-terminal bases (5bps) of gene after ATG but not changing amino acid, and amplified a smaller mutated gene sequnce (468bp) containing two protective antigenic determinants from pBlue-BoNTaHc, N-terminal codon of mutated gene fragment is changed from low to high frenqence in E. coli. Mutated gene was ligated into pGEM-T vector and sequenced, then, cloned into a expression plasmid pBV220. As a result, cloned gene was expressed in insoluble form by temperature inducing (from 30 degrees C to 42 degrees C) in E. coli. Expression product is 40% of total proteins and is of specific binding activity to antibody in ELISA. The successful modification and high level expression of protective fragment of botulinum neurotoxin serotype A(BoNTaHc468) gene is conducive to further study on antitoxin and vaccine.
Bacterial Vaccines ; immunology ; Botulinum Toxins, Type A ; genetics ; immunology ; Clostridium botulinum type A ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis

Botulinum toxin type A is a polypeptide neurotoxin derived from the anaerobic bacterium Clostridium botulinum. Safe and effective Botulinum type A toxin has found clinical application as a treatment for strabismus, blepharospasm, palmar hyperhidrosis. Now this toxin is widely used for improving the facial rhytides cosmetically. Author applicated this toxin not only for facial rejuvenation but also for improving for masseter muscle hypertrophy, facial asymmetries after orthognathic surgery, dysmorphis caused by facial nerve paralysis, TMD and habitual TMJ luxation. Author report clinical results with literature review.]]>
Blepharospasm ; Botulinum Toxins ; Botulinum Toxins, Type A ; Clostridium botulinum ; Facial Asymmetry ; Facial Nerve ; Hyperhidrosis ; Hypertrophy ; Masseter Muscle ; Orthognathic Surgery ; Paralysis ; Rejuvenation ; Strabismus ; Temporomandibular Joint