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

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 polymerase rTth, 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 degrees C for 10 min, enrichment in tryptone-peptone-glucose-yeast extract broth at 30 degrees 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 x 10(3) 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.     

Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France

The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France. A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C. botulinum spores per 25 g and 100% of samples inoculated with more than 30 C. botulinum spores per 25 g. The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did. The prevalence of C. botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C. botulinum most-probable-number counts, respectively, and is in the low range of C. botulinum contamination reported elsewhere. The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E. Type F was not detected. The high prevalence of C. botulinum type B in fish samples is relatively unusual compared with the high prevalence of C. botulinum type E reported in many worldwide and northern European surveys. However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism.     

Incidence of Mesophilic Clostrodium Spores in Raw Pork, Beef, and Chicken in Processing Plants in the United States and Canada

The anaerobic film pouch technique was used to quantitate and isolate clostridial spores in 2,358 samples of raw meat (1,078 of chicken, 624 of beef, 656 of pork). Of 19,727 putrefactive anaerobic (PA) sporeformers isolated, 1 was confirmed by mouse protection testing to be Clostridium botulinum type C. This isolate was obtained from a Western Canada chicken sample which contained 5.33 clostridia per gram. These data indicate a very low incidence of botulinal contamination in raw meats at the packing-plant level (0.042% of 2,358 samples) and an almost 20,000:1 ratio of nonbotulinal PA sporeformers to mesophilic C. botulinum spores. The mean level of PA contamination was 2.8 PA sporeformers per gram of meat; 77% of the samples contained three or less PA sporeformers per gram. Small but statistically significant differences in the incidence of clostridial spores were noted for season, geographical region, and type of meat.     

Genes that encode botulism neurotoxins A, B, E and F in neotropical bee honey identified with the polymerase chain reaction

Honey can be used for the treatment of wounds, sores and skin bums, but it might be contaminated with Clostridium botulinum spores. In order to evaluate Costa Rican raw honey samples, the detection of neurotoxin gene sequences (corresponding to the bacterium) C. botulinum A, B, E and F was done with the polymerase chain reaction. A total of 64 raw honey samples, coming from different Costa Rican sites were analyzed. Reference C. botulinum strains type A (ATCC 19397), type B (ATCC 7949), type E (ATCC 17786) and type F (ATCC 25764) were used as templates for testing the effectivity of the method. The process consisted in culturing the honey samples in prereduced triptose-peptone-glucose-yeast extract media (TGPY) for 5 days. After this, the bacteria lysate obtained was used for PCR. The amplicons, product of the reaction, were visualized using agarose gel 2%. From the 64 honey samples analyzed, none produced positive results in the PCR, since no amplicons were obtained. Even though, all the reference C. botulinum strains used as controls were visualized and showed the effectivity of the extraction method and of the PCR used. The results obtained show promising therapeutic uses for honey from Costa Rica, but further evaluations shall be done in order to be sure of the safety of the product.     

Contamination routes of Clostridium botulinum in the honey production environment

Factors influencing Clostridium botulinum contamination in the honey production environment were evaluated in a 3-year survey. A number of 1,168 samples from 100 apiaries and related facilities were analysed for the presence of C. botulinum types A, B, E and F, using multiplex polymerase chain reaction targeted to botA, botB, botE and botF genes. Production methods and environmental factors were registered using a questionnaire and by personal observation. Clostridium botulinum was shown to be a common finding throughout the whole honey production chain, and the type most frequently detected was group I type B. In a pulsed-field gel electrophoresis (PFGE) analysis of 202 group I type B isolates, only six different PFGE profiles were observed, of which two clearly distinct profiles predominated. This may indicate the existence of at least two different genetic lineages. The high prevalence of C. botulinum in soil and in samples associated with beeswax suggests the accumulation of soil-derived botulinal spores in wax. Additionally, according to Spearman's rank order correlation and multivariate analysis, production hygiene-dependent factors have a significant influence on the contamination, and thus the number and frequency of C. botulinum spores in honey could possibly be diminished by increasing hygienic level in honey production.     

Polymerase chain reaction for detection of Clostridium botulinum types A, B and E in food, soil and infant faeces

The application of the polymerase chain reaction (PCR) for detection of Clostridium botulinum types A, B and E in foods, environmental and clinical samples was evaluated and compared to the mouse bioassay. Samples inoculated with 10, 100 and 1000 spores of Cl. botulinum types A and B included pasteurized milk, UHT milk, infant formula, infant faeces, meat juice, canned tuna, mushrooms, blood sausage and soil. Clostridium botulinum type E spores were inoculated into fish eggs, canned tuna, picked herring, raw fish and soil at similar levels. Spores were added to 2.5 g of each sample with the exception of soil which was inoculated in 10 g samples. The presence of Cl. botulinum in sample enrichments was determined by both PCR and the bioassay. An overall correlation of 95.6% was observed between PCR results and the mouse bioassay. Of the total of 114 samples tested there was disparity between the mouse bioassay and the PCR in three samples of soil inoculated with 100 type A or E spores and 10 type B spores per 10 g, respectively, and two samples of infant faeces inoculated with 10 type A or B spores per 2.5 g. All of these samples gave negative animal results and positive PCR results.     

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

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 polymerase rTth, 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.     

Storage Stability of Clostridium botulinum Toxin and Spores in Processed Cheese

Growth initiated from detoxified spores of Clostridium botulinum 62A resulted in toxin production of 50 to 10,000 mouse lethal doses (MLD) per gram of processed soft surface-ripened cheese. Regular assays during subsequent storage of toxic samples at 2 to 4 C revealed a characteristic two- to fivefold increase in toxin titer during the initial 1 week to 12 months of storage. Thereafter, the toxin titer remained constant for 2 to 4 years, after which the toxicity declined rapidly. At the end of 6 years of storage at 2 to 4 C, the samples still contained 20 to 5,000 MLD of toxin per gram, with the usual toxin level at 200 to 500 MLD. Toxic culture filtrates of C. botulinum incorporated into cheese and stored at 30 C for 60 days showed no decline in toxin in processed type I cheese, but toxin decreased slightly in processed type II and type III cheese. The surface flora of these cheeses did not attack but, on the contrary, protected C. botulinum toxin during storage at 30 C. Initial difficulties in recovering C. botulinum organisms from type I cheese were traced to growth inhibitory activity which could be removed by washing with distilled water in a centrifuge. Viable spores or vegetative cells could be recovered from all samples after 4 to 5 years of storage at 2 to 4 C. After 6 years, organisms were recovered from all except three samples of type I cheese. Two other samples showed a large decrease in viable organisms. In type III cheese, spores remained remarkably stable for 6 years at the level of the initial inoculum, i.e., approximately 10(5) spores per gram.     

American foulbrood and African honey bees (Hymenoptera: Apidae)

We have taken samples of honey from individual beekeepers (N = 64), and of domestic (N = 35) and imported honey (N = 15) retailed in supermarkets in several sub-Saharan countries and cultivated these samples for Paenibacillus larvae subsp. larvae Heyndrickx et al. causing American foulbrood in honey bee colonies. The results are compared with samples of similar backgrounds and treated the same way but collected in Sweden (N = 35). No P. larvae subsp. larvae spores were found in any honey produced in Africa south of the Sahara although honey imported into this region frequently contains the pathogen. Swedish honey frequently contains P. larvae subsp. larvae spores although the general level of visibly infected bee colonies is low (roughly 0.5%). The results suggest that large parts of Africa may be free from American foulbrood. Behavioral studies (hygienic behavior) on Apis mellifera subsp. scutellata Lepeletier in Zimbabwe suggest that hygienic behavior of African bees could influence the apparent low level, or even absence of American foulbrood in large parts of Africa.     

An unusually heavy contamination of honey products by Clostridium botulinum type F and Bacillus alvei

Many spores (1-60/g) of Clostridium botulinum type F were detected in different containers of honey products of the same brand. Microbiological and physicochemical properties of the contaminated honey were compared with those of the negative one. No difference in pH, hydroxymethyl furfural contents or diastase activity was found between them. The total counts of anaerobes other than C. botulinum and of yeast were also similar, whereas the aerobe counts, which were proportionally related with the C. botulinum counts, were higher in the positive honey than in the negative one. Motile colony-forming Bacillus alvei was predominant among the aerobes. B. alvei stimulated the toxin production by C. botulinum type F in culture medium incubated under aerobic conditions. The high count of C. botulinum in the honey might have been due to the possible stimulation of growth by B. alvei or some other microorganisms at some stage of honey ripening.     

Multiplex PCR assay for detection and identification of Clostridium botulinum types A, B, E, and F in food and fecal material.

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 10(2) cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10(-2) spore/g for types A, B, and F to 10(-1) spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Study of the presence of the spores of Clostridium botulinum in honey in Brazil

The isolation of Clostridium botulinum from honey samples is described. Botulism is characterized as an intoxication provoked by ingestion of contaminated foods with this toxin. Infant botulism happens by the ingestion of spores of C. botulinum together with food that in special conditions of the intestinal tract, such as those present in babies of less than 1 year old, will allow the germination and colonization of the intestine with production and absorption of botulinic toxin. The samples were subjected to dilution and to a thermal shock and cultivated in modified CMM (Difco). Cultures were subjected to Gram smears and toxicity tests in mice. The toxic cultures were purified in RFCA (Oxoid) plates and incubated in anaerobic jars. Positive samples were typed using the mouse assay neutralization test. From the 85 honey samples analyzed, six were positive for C. botulinum (7.06%), and identified as producers of type A, B, and D toxins.     

Interrelationship of Heat and Relative Humidity in the Destruction of Clostridium botulinum Type E Spores on Whitefish Chubs

Heat destruction of types B and E Clostridium botulinum spores on whitefish chubs was observed to be dependent upon the relative humidity (RH) in the chamber in which fish were heated. Experimental conditions were designed to simulate those attainable in commercial fish-smoking plants. Low numbers of type E spores were destroyed with regularity, within 30 min, on fish which were held at an internal temperature of 77 C (170.6 F) in an atmosphere of at least 70% RH. However, an internal temperature of 82 C (179.6 F) and a minimum RH of 70% were required to destroy several hundred thousand type E spores. Quantitative estimates of spore destruction were arrived at with a modified most probable number procedure. Type E spore populations were reduced by 2 to 4 logarithms at 77 C (170.6 F), by 5 to 6 logarithms at 82 C (179.6 F), and by more than 6 logarithms at 88 C (190.4 F) when fish were heated in an atmosphere of 70% RH. A 5 to 6 logarithm reduction of spores was also observed when fish inoculated with type B spores were processed at 82 C (179.6 F) in an atmosphere of 70% RH.     

PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp. and evaluation in food samples.

A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products. The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product. This assay was able to detect 1 C. botulinum type A or B and 10 C. botulinum type E strains per reaction. With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5%w/v) and polyphosphate (0.3%w/v) were either unheated or heated 80°C/5 min followed by 70°C/2 h before incubation at 15°, 20° or 27°C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl. botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD₅₀/ml) of the type B ELISA. No falsepositive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.     

Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting.     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80 degrees C/5 min + 70 degrees C/2 h, before storage at 15 degrees, 20 degrees or 27 degrees C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.     

Contribution to the study of fungal spores in honeys of Galicia (NW Spain)

We analysed 30 samples of Galician honey with the aim of quantifying and identifying the fungal spores contained in them. Using an optical microscope, we could identify 40 different types, among them Cladosporium, Penicillium/Aspergillus and the Basidiospores, which were identified in more than 80% of the samples. We calculated the relationship between fungal spores and amount of pollen grains in the samples. The maximum values were obtained in samples 21 (more than 208/100 grains of pollen) and 5 (153 spores/100 grains of pollen).     

Evaluation of a monoclonal antibody-based immunoassay for detecting type A Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system

A monoclonal antibody-based amplified enzyme-linked immunosorbent assay (ELISA) method for detecting Clostridium botulinum type A toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5–4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated, 80°C/5 min + 70°C/2 h, before storage at 15°, 20° or 27°C. The presence of specific toxin was confirmed by mouse bioassay and results compared with those of the amplified ELISA method. A total of 49 strains, 39 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), aiid 95 slurry samples were tested. Fourteen of 15 strains of type A Cl. botulinum and 34 of 36 slurry samples containing type A toxin were positive by ELISA. No false positive reactions occurred with Cl. botulinum types B, C, D, E and F, or with the 10 strains of Cl. sporogenes. However, toxin produced by one strain of Cl. botulinum type A (NCTC 2012) was not detected by the amplified ELISA.