Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II.

Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C. botulinum-like strains. All proteolytic C. botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C. sporogenes strains were identified as C. botulinum or C. sporogenes by the API 20 A. Reversely, all non-proteolytic C. botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains. All C. sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain. All non-proteolytic non-toxigenic strains were identified as C. botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A. The results show that these test systems do not provide a reliable method for identification of C. botulinum.

Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR.

Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism. Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions. Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively. Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C. botulinum serotypes at the strain level. Group I strains were mainly discriminated at the serotype level. The discriminatory power of rep-PCR was found to be inferior to that of RAPD. The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment. The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results. As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.

Sodium nitrite and potassium nitrate in control of nonproteolytic Clostridium botulinum outgrowth and toxigenesis in vacuum-packed cold-smoked rainbow trout.

The effect of sodium-nitrite (NaNO2) and potassium nitrate (KNO3) on the outgrowth and toxigenesis of nonproteolytic Clostridium botulinum in vacuum-packed cold-smoked rainbow trout stored for-six weeks was studied in two inoculation studies at slightly abusive storage temperatures of 4 degrees C and 8 degrees C. The depletion rate of nitrite and the reduction rate of nitrate to nitrite as well as the effect of nitrite and nitrate on the shelf-life of the product during eight weeks' storage period were also determined. The nitrite concentrations were reduced from 166 mg/kg +/- 9 (mean +/- SE), to a final concentration of 34 mg/kg +/- 2 and 11 mg/kg +/- 2, and the nitrate concentrations from 686 mg/kg +/- 67 to 465 mg/kg +/- 140 and 427 mg/kg +/- 33 at 4 degrees C and 8 degrees C respectively. The nitrite depletion rate was more rapid at 8 degrees C; nitrate depletion was not significantly affected by temperature. A considerable amount of nitrite was detected in the nitrate-treated samples in the latter half of the storage period. At 4 degrees C the aerobic plate counts were significantly lower in the samples treated with NaNO2 + NaCl and with KNO3 + NaCl as compared to the NaCl-treated controls, while at 8 degrees C the differences were smaller. The sensorial shelf-life of the product was considerably extended by nitrite and nitrate curing. The nitrite and nitrate concentrations used in the present study did not completely inhibit the toxigenesis of nonproteolytic C. botulinum during the six-week storage period, although the number of toxic samples was considerably reduced by nitrite and nitrate curing.

Detection of Clostridium botulinum in fish and environmental samples using polymerase chain reaction.

A test protocol for the detection and enumeration of Clostridium botulinum in fish and sediment samples with specific identification of neurotoxin types A, B, E and F was developed. Specific amplification products generated by polymerase chain reaction (PCR) formed the basis of identification of the toxin-producing organism, whereas quantification of the results was achieved with the most probable number (MPN) method. Twenty-six C. botulinum strains studied with PCR assays after enrichment in trypticase-peptone-glucose-yeast extract (TPGY) broth gave identical results as with the mouse bioassay. The suitability of the detection method for food and environmental surveys was assessed by running it on 32 samples of rainbow trout inoculated with spore loads ranging from 10(2) to 10(6) C. botulinum type E spores per kg. The organism was detected in all samples, and MPN estimates corresponded well to inoculum levels. In order to assess possible natural contamination, 16 fish and 16 visceral samples of rainbow trout, as well as ten aquatic sediment samples were tested. Of these, eight (80%) of the sediment samples were positive, with estimated spore counts of C. botulinum type E ranging from 95-2710 per kg sample.

Ribotyping as an identification tool for Clostridium botulinum strains causing human botulism.

Ribotyping was used for characterisation of 68 Clostridium botulinum strains and five related Clostridium species to determine the applicability of this method for identification of species causing human botulism. Thirteen restriction enzymes were initially tested for suitability for ribotyping of C. botulinum, of which EcoRI and HindIII were selected. Both enzymes clearly differentiated between proteolytic (group I) and a nonproteolytic (group II) strains of C. botulinum, and can be recommended for Group/species identification. Using a commercial software package (GelCompar), a numerical analysis of the discriminatory abilities of EcoRI and HindIII ribotyping within and between the two C. botulinum groups was performed. EcoRI had the higher discriminatory index (0.982), but the ribopatterns generated with group II strains were partly muddled and difficult to interpret. All HindIII ribopatterns were easy to analyse and the discriminatory index for all strains was almost equally high (0.954), whereas this enzyme did not discriminate well between group I isolates. The Clostridium strains diverged at 35+/-13% (mean+/-standard deviation) Dice similarity in dendrograms based on cluster analysis of the ribotyping results. These findings are in good agreement with taxonomical ribotyping studies with other bacterial genera, indicating that ribotyping is a highly suitable method for C. botulinum species identification.

Predicted and observed growth and toxigenesis by Clostridium botulinum type E in vacuum-packaged fishery product challenge tests.

The observed growth and toxigenesis by Clostridium botulinum type E in vacuum-packaged unprocessed, raw pickled and cold-smoked rainbow trout stored at slightly abusive temperatures were compared to predictions generated by two currently available predictive microbiological programs, Food MicroModel and Pathogen Modelling Program. In unprocessed fish there was only a 2 log increase in type E cell count at the time the toxicity first occurred after 2 weeks storage at 8 degrees C. Neither growth or toxin production was observed in raw pickled fish with a NaCl concentration of 6.7% (w/v) during 6 weeks storage at 6 degrees C. In cold-smoked fish with a NaCl level of 3.2% (w/v) toxic samples were detected after 3 and 4 weeks storage at 8 degrees C and 4 degrees C, respectively, without any increase in type E count. Both models were hampered by limitations to controlling environmental factors set by the programs which also had an adverse effect on the reliability of predictions. Most predictions generated by the models were inconsistent with the results observed in the challenge studies. In certain situations, the models seemed to be 'fail-safe', in that, the growth rate predicted from the model was faster or a predicted time to toxicity shorter than that which actually occurred in the food. In other situations, the predictions showed the product to be safe when it was not. The results demonstrate the need for further development and rigorous validation of the models before they are accepted for wider use by inspecting officials and the food industry.

Type E botulism associated with vacuum-packaged hot-smoked whitefish.

On January 16, 1997 two Germans got botulism after eating hot-smoked Canadian whitefish produced in Finland. The serum sample of one of the patients contained 6 MLD/ml of botulinum toxin. The type of toxin was identified as E by the toxin neutralization test and the botulinum neurotoxin type E (BoNT/E) gene was also amplified from the serum by polymerase chain reaction (PCR), but C. botulinum could not be isolated from the positive serum sample. The remains of the hot-smoked whitefish eaten by the patients contained botulinum toxin detected by the mouse bioassay and the BoNT/E gene as determined by PCR. C. botulinum was isolated from the fish sample and it was confirmed to be type E by the mouse bioassay and by PCR. Eleven other fish samples from the same lot did not contain botulinum toxin nor any BoNT gene. The incriminated food was processed on the 9th and 10th of January, 1997 from frozen whitefish imported to Finland from Canada. The pulsed-field gel electrophoretic pattern of the isolated C. botulinum strain resembled a reference strain of North American origin. It did not match any C. botulinum strains isolated from the Baltic sea-bottom or from the fish caught in the area indicating that the fish was contaminated by C. botulinum in Canada. The conditions resulting in toxin production could not be identified. The safety problems associated with vacuum-packaged hot-smoked fish seem to be of utmost concern and the product is one of the most important botulism food vehicles processed on an industrial scale. Temperature monitoring and the use of time-temperature indicators are to be recommended in order to ensure adequate storage temperature from processing through to consumption. Allowing the use of nitrate and nitrite together with sufficiently high NaC1 concentration in this particular product should also be considered.

High prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland.

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.

Inhibition of growth of nonproteolytic Clostridium botulinum type B in sous vide cooked meat products is achieved by using thermal processing but not nisin.

The safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum is under continuous evaluation. In the present study, mild (P7.0(85.0) values 0 to 2 min [P, pasteurization value; z-value 7.0 degrees C; reference temperature 85.0 degrees C]) and increased (P7.0(85.0) values 67 to 515 min) heat treatments were evaluated in relation to survival of nonproteolytic C. botulinum type B spores in sous vide processed ground beef and pork cubes. The use of two concentrations of nisin in inhibition of growth and toxin production by nonproteolytic C. botulinum in the same products was also evaluated. A total of 96 samples were heat processed and analyzed for C. botulinum by BoNT/B gene-specific polmerase chain reaction and for botulinum toxin by a mouse bioassay after storage of 14 to 28 days at 4 and 8 degrees C. Predictably, after mild processing all samples of both products showed botulinal growth, and one ground beef sample became toxic at 8 degrees C. The increased heat processing, equivalent to 67 min at 85 degrees C. resulted in growth but not toxin production of C. botulinum in one ground beef sample in 21 days at 8 degrees C: in the pork cube samples no growth was detected. The increased heating of both products resulted in higher sensory quality than the milder heat treatment. Nisin did not inhibit the growth of nonproteolytic C. botulinum in either product; growth was detected in both products at 4 and 8 degrees C, and ground beef became toxic with all nisin levels within 21 to 28 days at 8 degrees C. Aerobic and lactic acid bacterial counts were reduced by the addition of nisin at 4 degrees C. The study demonstrates that the mild processing temperatures commonly employed in sous vide technology do not eliminate nonproteolytic C. botulinum type B spores. The intensity of each heat treatment needs to be carefully evaluated individually for each product to ensure product safety in relation to nonproteolytic C. botulinum.

Comparison of the disease incidences of dairy cows kept in cold and warm loose-housing systems.

Finland's cold loose-housing systems for dairy cows were compared with the more traditional warm loose-housing systems regarding the incidences of ketosis, mastitis, metritis, parturient paresis and ovarian disorders. Approximately 5000 calvings on 210 farms during the years 1996 and 1997 were modelled, using multilevel Poisson regression and multilevel logistic-regression in a retrospective observational cohort study. Cows in a cold loose-housing system were at lower odds for developing late mastitis (15-305 days in milk), and metritis (Friesian breed); of the same odds for ketosis and early mastitis (0-14 days in milk); but at higher odds for developing parturient paresis and metritis (Ayrshire breed). The estimated odds ratio for ovarian disorders depended on the definition for exposure. Although one of the differences was statistically significant and many of them of veterinary interest, none of them appear to be substantial for the economy of a median-sized dairy farm in Finland.

Comparison of the breeding performance of cows in cold and warm loose-housing systems in Finland.

Finland's cold loose-housing systems for dairy cows were compared with the more traditional warm loose-housing systems regarding the number of days from calving-to-first-service, the first-service-pregnancy risk and the repeated-service-conception hazard. 3131 calvings registered during the indoor periods in 1996 and 1997 on 208 farms were modelled using multilevel survival analysis and logistic regression in a retrospective cohort study. Compared to cows in a warm loose-housing system, cows in a cold loose-housing system had the same period from calving-to-first-service, a significant 6% lower first-service-pregnancy risk and the same repeated-service-conception hazard.

Comparison of the disease incidences of Finnish Ayrshire and Finnish Black and White dairy cows.

Finnish Ayrshire and Finnish Black and White cows were compared regarding the incidences of early and late mastitis, parturient paresis, ketosis, ovarian disorders, metritis and the risk of having a test-day somatic-cell count >200,000 cells ml(-1) at any of the first three monthly test days in lactation. In a retrospective cohort study 101,793 cows from 5844 tie stalls and 11,811 cows from 437 loose-housing systems from all over Finland were followed from calving in 2000 until the end of lactation. The observed incidences of those cows were then analysed using generalised linear mixed models. Finnish Black and White cows had higher incidences of all diseases except ovarian disorders. Although the differences were statistically significant in all models except metritis and early mastitis in loose-housing systems, they were, in our view, only important on the national level (for the breeding organisations), and of little importance for the farmers.

Comparison of milk production of dairy cows kept in cold and warm loose-housing systems.

A retrospective cohort study was conducted to test whether the lactation curves of cows kept in cold loose-housing systems (CLHs) were the same as for cows in warm loose-housing systems (WLHs) in the Nordic countries. Approximately 40000 test-day records from 5366 Ayrshire or Black and White cows kept on 38 CLHs and 166 WLHs in Finland during 1996 and 1997 were used. Analysis used a random-coefficient model (correcting for parity, breed and calving-year-season and the correlation-structure between test-days of the same cow and cows of the same herd). Cows in a CLH produced up to 1l less milk per test-day, but this difference was not statistically significant. Surprisingly, the difference in milk yield was not affected by calving-year-season, parity or breed.

Prevalence of Clostridium botulinum type E in Finnish fish and fishery products.

The prevalence of Clostridium botulinum type E gene in fish and fishery products of commercial importance in Finland was determined using a quantitative PCR analysis. The contamination level in 438 raw fish samples from intestines, surface and whole fish and 208 fish roe samples varied from 10-40% and from 4-14% respectively, depending on the fish species studied. The presence of C. botulinum in European wild freshwater fish and roe was demonstrated for the first time by isolation of the organism from PCR-positive samples. Five percent of 214 vacuum-packed and 3% of 123 air-packed fishery product samples examined at retail level were positive for the botulinum neurotoxin type E gene. A contamination level of 10% in vacuum-packed hot-smoked whitefish was detected. The results demonstrate that C. botulinum type E poses a serious health risk for those consuming fishery products from the Baltic Sea area.

Genomic analysis of Clostridium botulinum group II by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (non-proteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, ApaI, MluI, NruI, SmaI, and XhoI gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of MluI, SmaI, and XhoI restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean +/- standard deviation = 3,890 +/- 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.

Prevalence of Clostridium botulinum in Finnish trout farms: pulsed-field gel electrophoresis typing reveals extensive genetic diversity among type E isolates.

The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg-1, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.

Evaluation of a molecular identification scheme based on 23S rRNA gene polymorphisms for differentiating canine and feline gastric Helicobacter spp.

A scheme for the rapid identification of Helicobacter spp. using restriction fragment length polymorphism digestion profiles of PCR amplified 23S rRNA genes is described. The efficacy of this scheme for speciation of the closely related gastric species H. felis, H. bizzozeronii and H. salomonis was evaluated. It was difficult to distinguish between some RFLP profiles obtained and often, more than one profile was seen with each species examined. Some evidence was found that the 23S rRNA gene copies of these species may not be identical. Moreover, the identification scheme was ineffective in discriminating these species from each other, although they could be differentiated, as a group, from other Helicobacter spp. The results indicate that this scheme should be carefully evaluated with a number of isolates if it is to be applied to additional, highly related Helicobacter spp.

A high prevalence of Clostridium botulinum type E in Finnish freshwater and Baltic Sea sediment samples.

The distribution of Clostridium botulinum serotypes A, B, E and F in aquatic environments of the Baltic Sea and Finnish mainland was examined. A total of 110 samples were tested with a neurotoxin-specific PCR assay. Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples. No other toxinotypes were found. Spore counts were quantified by the most probable number method, Cl. botulinum type E kg(-1) averaging 940 in sea and 370 in freshwater samples. The overall prevalence and spore counts of Cl. botulinum type E in aquatic sediments correlated significantly with offshore bottom oxygen content, depth, and bioturbation activity, whereas there was no correlation with bottom water temperature. These findings indicate the possibility of Cl. botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments.

Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products.

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.