scholarly journals Development of a Combined Selection and Enrichment PCR Procedure for Clostridium botulinum Types B, E, and F and Its Use To Determine Prevalence in Fecal Samples from Slaughtered Pigs

2001 ◽  
Vol 67 (10) ◽  
pp. 4781-4788 ◽  
Author(s):  
Maria Dahlenborg ◽  
Elisabeth Borch ◽  
Peter Rådström
Keyword(s):  
Sample Preparation ◽  
Fecal Sample ◽  
Clostridium Botulinum ◽  
Geographical Location ◽  
Type B ◽  
Fecal Samples ◽  
Pcr Assays ◽  
Combined Selection ◽  
Spore Load ◽  
Slaughtered Pigs

ABSTRACT A specific and sensitive combined selection and enrichment PCR procedure was developed for the detection of Clostridium botulinum types B, E, and F in fecal samples from slaughtered pigs. Two enrichment PCR assays, using the DNA polymeraserTth, were constructed. One assay was specific for the type B neurotoxin gene, and the other assay was specific for the type E and F neurotoxin genes. Based on examination of 29 strains of C. botulinum, 16 strains of other Clostridium spp., and 48 non-Clostridium strains, it was concluded that the two PCR assays detect C. botulinum types B, E, and F specifically. Sample preparation prior to the PCR was based on heat treatment of feces homogenate at 70°C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30°C for 18 h, and DNA extraction. The detection limits after sample preparation were established as being 10 spores per g of fecal sample for nonproteolytic type B, and 3.0 × 103 spores per g of fecal sample for type E and nonproteolytic type F with a detection probability of 95%. Seventy-eight pig fecal samples collected from slaughter houses were analyzed according to the combined selection and enrichment PCR procedure, and 62% were found to be PCR positive with respect to the type B neurotoxin gene. No samples were positive regarding the type E and F neurotoxin genes, indicating a prevalence of less than 1.3%. Thirty-four (71%) of the positive fecal samples had a spore load of less than 4 spores per g. Statistical analysis showed that both rearing conditions (outdoors and indoors) and seasonal variation (summer and winter) had significant effects on the prevalence of C. botulinum type B, whereas the effects of geographical location (southern and central Sweden) were less significant.

scholarly journals Neurotoxin Gene Profiling of Clostridium botulinum Types C and D Native to Different Countries within Europe

2012 ◽  
Vol 78 (9) ◽  
pp. 3120-3127 ◽  
Author(s):  
Cedric Woudstra ◽  
Hanna Skarin ◽  
Fabrizio Anniballi ◽  
Lucia Fenicia ◽  
Luca Bano ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Simultaneous Detection ◽  
Geographical Location ◽  
Gene Profiling ◽  
Animal Origin ◽  
Bacterial Strains ◽  
Content Type ◽  
Pcr Assays

ABSTRACTClostridium botulinumtypes C and D, as well as their mosaic variants C-D and D-C, are associated with avian and mammalian botulism. This study reports on the development of low-density macroarrays based on the GeneDisc cycler platform (Pall-GeneDisc Technologies) applied to the simultaneous detection of theC. botulinumsubtypes C, C-D, D, and D-C. The limit of detection of the PCR assays was 38 fg of total DNA, corresponding to 15 genome copies. Artificially contaminated samples of cecum showed a limit of detection below 50 spores/g. The tests were performed with a large variety of bacterial strains, includingC. botulinumtypes C (n= 12), C-D (n= 29), D (n= 5), and D-C (n= 10), other botulinum neurotoxin (BoNT)-producingClostridiumstrains (n= 20), non-BoNT-producing clostridia (n= 20), and other bacterial species (n= 23), and showed a high specificity. These PCR assays were compared to previously published real-time PCRs for the detection ofC. botulinumin 292 samples collected from cases of botulism events in four European regions. The majority of the samples originated from wild birds (n= 108), poultry (n= 60), and bovines (n= 56). Among the 292 samples, 144 were positive for either thebont/C-D or thebont/D-C gene by using the GeneDisc arrays. The reliability of the results tallied to 97.94%. Interestingly, only BoNT mosaics, types C-D and D-C, were found in naturally contaminated samples whatever their animal origin and their geographical location. Further investigations should now be performed in order to check that mosaic types dominate in Europe and that acquisition of mosaic types helps in survival or adaptation to particular niche.

scholarly journals Genomic Markers for Differentiation of Francisella tularensis subsp. tularensis A.I and A.II Strains

2007 ◽  
Vol 74 (1) ◽  
pp. 336-341 ◽  
Author(s):  
Claudia R. Molins-Schneekloth ◽  
John T. Belisle ◽  
Jeannine M. Petersen
Keyword(s):  
Suppression Subtractive Hybridization ◽  
Francisella Tularensis ◽  
Geographical Location ◽  
Subtractive Hybridization ◽  
Clinical Isolates ◽  
Type B ◽  
Tularensis Subsp ◽  
Genomic Markers ◽  
Pcr Assays ◽  
Genomic Regions

ABSTRACT Tularemia is caused by two subspecies of Francisella tularensis, F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). F. tularensis subsp. tularensis is further subdivided into two genetically distinct populations (A.I and A.II) that differ with respect to geographical location, anatomical source of recovered isolates, and disease outcome. Using two human clinical isolates, suppression subtractive hybridization was performed to identify 13 genomic regions of difference between A.I and A.II strains. Two PCR assays, one to identify A.I and A.II as well as to discriminate between F. tularensis subsp. holarctica and F. novicida and another specific for A.I, were developed. This is the first report to identify and characterize conserved genomic differences between A.I and A.II.

Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

2019 ◽  
pp. 60-66
Author(s):  
Viet Quynh Tram Ngo ◽  
Thi Ti Na Nguyen ◽  
Hoang Bach Nguyen ◽  
Thi Tuyet Ngoc Tran ◽  
Thi Nam Lien Nguyen ◽  
...  
Keyword(s):  
Bacterial Meningitis ◽  
Real Time ◽  
Real Time Pcr ◽  
Diagnostic Methods ◽  
Type B ◽  
E Coli ◽  
Pcr Assays ◽  
Set Up ◽  
Etiological Agents ◽  
Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

scholarly journals Identification of protein receptor for Clostridium botulinum type B neurotoxin in rat brain synaptosomes.

1994 ◽  
Vol 269 (14) ◽  
pp. 10498-10503 ◽  
Author(s):  
T. Nishiki ◽  
Y. Kamata ◽  
Y. Nemoto ◽  
A. Omori ◽  
T. Ito ◽  
...  
Keyword(s):  
Rat Brain ◽  
Clostridium Botulinum ◽  
Type B ◽  
Rat Brain Synaptosomes ◽  
Protein Receptor ◽  
Botulinum Type ◽  
Brain Synaptosomes

Potential for Growth of Nonproteolytic Types of Clostridium botulinum in Pasteurized Restructured Meat Products: A Review1

1985 ◽  
Vol 48 (3) ◽  
pp. 265-276 ◽  
Author(s):  
J. SIMUNOVIC ◽  
J.L. OBLINGER ◽  
J.P. ADAMS
Keyword(s):  
Heat Resistance ◽  
Causative Agent ◽  
Clostridium Botulinum ◽  
Meat Products ◽  
Type B ◽  
The U.S ◽  
Restructured Meat

Type E and nonproteolytic type B strains of Clostridium botulinum can grow and produce toxin at temperatures below 5°C. Recent publications describing the greater heat resistance of nonproteolytic type B C. botulinum spores than type E spores are discussed in relation to suitable proess lethalities required for a safe pasteurized product. The incidences of botulism in Europe caused by nonproteolytic type B spores were compared to the lack of such incidences in the U.S. and to published procedures for isolating the causative agent for botulism. The incidence of C. botulinum spores in meat products in the U.S. also is reviewed.

scholarly journals Study on potential Clostridium botulinum growth and toxin production in Parma ham

2016 ◽  
Vol 5 (2) ◽  
Author(s):  
Giuseppe Merialdi ◽  
Mattia Ramini ◽  
Giovanni Parolari ◽  
Silvana Barbuti ◽  
Maria Angela Frustoli ◽  
...  
Keyword(s):  
Clostridium Botulinum ◽  
Toxin Production ◽  
Limiting Factor ◽  
In Vitro Test ◽  
Type B ◽  
Potential Growth ◽  
Parma Ham ◽  
Nacl Concentration ◽  
Vitro Test

The objective of this study was to investigate <em>Clostridium botulinum</em> growth and toxin production in the industrially manufactured Italian Parma ham. The study focuses on the Parma ham production phase identified as maximum risk to <em>C. botulinum</em> proliferation, <em>i.e.</em> the transition from cold phase (salting and resting) to a phase carried out at temperature between 15 and 23°C (drying). A preliminary in vitro test was carried out in order to verify the capability of 6 <em>C. botulinum</em> strains (1 type A, 4 type B, and 1 type E strains) to grow in conditions of temperature, pH and NaCl concentration comparable to those of the beginning stage of ham drying. Five <em>C. botulinum</em> strains grew at 20°C and pH 6, four strains produced toxin when inoculated at a concentration equal to 10<sup>3</sup> cfu/mL at NaCl concentration of 4%, while when the inoculum concentration was 10 cfu/mL, NaCl concentration of 3% resulted the toxin-genesis limiting factor. An experimental contamination with a mixture of the 5 <em>C. botulinum</em> strains selected by the preliminary in vitro test was performed on 9 thighs inoculated at the end of the resting phase. The study was designed to evaluate the potential growth and toxin production in extremely favourable conditions for the bacterium. Type B proteolytic <em>C. botulinum</em> toxin was produced after 14 days of incubation at 20°C in 2 thighs characterised by high weight, low number of days of resting and anomalous physiochemical characteristics [one for very low NaCl concentration (1.59%), the other for elevated pH (6.27) and both for high water activity values (&gt;0.970)]. The results of this research confirm that the cold resting step is a critical phase in the production process of Parma ham for the investigated hazard. Based on the present study, the long resting phase adopted in the manufacturing of Parma ham is proven effective to prevent the growth of <em>C. botulinum</em>, an event which could not otherwise be excluded if the hams were processed under less stringent technological conditions.

Differentiation between Types and Strains of Clostridium botulinum by Riboprinting

2000 ◽  
Vol 63 (10) ◽  
pp. 1347-1352 ◽  
Author(s):  
GUY E. SKINNER ◽  
STEVEN M. GENDEL ◽  
GEOFFREY A. FINGERHUT ◽  
HAIM A. SOLOMON ◽  
JODIE ULASZEK
Keyword(s):  
Clostridium Botulinum ◽  
Hazard Analysis ◽  
Control Point ◽  
Egg Yolk ◽  
Type B ◽  
Critical Control Point ◽  
Microbial Characterization ◽  
Automated Ribotyping ◽  
Degree Of Differentiation ◽  
Foodborne Botulism

The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System. Pure spores of C. botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35°C. Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remove vegetative cells that were heated for 10 min at 80°C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI. Riboprint patterns were obtained for 30 strains of the four major types of C. botulinum most commonly involved in human foodborne botulism. Proteolytic strains yielded the best and most consistent results. Fifteen ribogroups were identified among the 31 strains tested. Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridium lineages. This degree of differentiation between strains of C. botulinum may be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.

scholarly journals Quantitative Interaction Effects of Carbon Dioxide, Sodium Chloride, and Sodium Nitrite on Neurotoxin Gene Expression in Nonproteolytic Clostridium botulinum Type B

2004 ◽  
Vol 70 (5) ◽  
pp. 2928-2934 ◽  
Author(s):  
Maria Lövenklev ◽  
Ingrid Artin ◽  
Oskar Hagberg ◽  
Elisabeth Borch ◽  
Elisabet Holst ◽  
...  
Keyword(s):  
Gene Expression ◽  
Carbon Dioxide ◽  
Growth Rate ◽  
Sodium Chloride ◽  
Optical Density ◽  
Sodium Nitrite ◽  
Clostridium Botulinum ◽  
Lag Phase ◽  
Type B ◽  
Phase Duration

ABSTRACT The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO2 in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO2 was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO2 concentration; the cntB mRNA level was fivefold greater in a 70% CO2 atmosphere than in a 10% CO2 atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng · ml−1 · unit of optical density−1 in the 10% CO2 atmosphere and 126 ng · ml−1 · unit of optical density−1 in the 70% CO2 atmosphere.

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