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Enforcement measures are an administrative tool used by food control authorities for making food business operators (FBOs) comply with food safety regulations. This study evaluates the effectiveness and uniformity of the use of enforcement measures in local food control units in Finland. The study investigates whether and how rapidly the use enforcement measures leads to compliance with food safety regulations and whether the enforcement processes and their durations vary according to the seriousness of non-compliance. Enforcement decisions were requested from 29 (34%) local food control units, ten of which had used no enforcement measures during the study period. A total of 188 enforcement cases from 19 control units were analysed. The food safety violations were categorized into critical and non-critical, based on their likeliness to directly contribute to food contamination or foodborne outbreaks. The use of enforcement measures led to verified compliance in 87.2% of the violations. However, enforcement measures were used recurrently in 15.7% of the violations, due to repeated violations. The duration of the enforcement processes ranged from one day up to several years. The duration of the enforcement process was significantly shorter in critical violations than in non-critical violations (Mann–Whitney U, p Enforcement measures appear to be an effective way of ensuring that FBOs correct their food safety violations, although the enforcement processes were rather long in some cases and required recurrent enforcement. However, several control units had not used enforcement measures, which may be due to dissimilarities in food control actions. Use of enforcement measures should be uniform among food control authorities and equal for FBOs.

Perceptions of local food control officers on introducing control fees and quality systems into local food control systems were studied in 2010 and compared with the situation in Finland in 2006. Government-regulated intra-regional cooperation, which was a significant organisational change during this same time period, was also evaluated. Most (94%) of the officers supported control fees in 2010 and the fears in 2006 about an increase in the number of complaints about officers' work and less time for counselling food business operators (FBOs) were significantly decreased in 2010. Introducing control fees were perceived by 75% of officers to have had a positive influence on in-house control systems of FBOs. The hourly fees collected in 2010 (range 32–56 euros) were significantly higher than those anticipated in 2006 (range 21–45 euros). Although significantly more local food control units had quality systems in place in 2010 (41%) than in 2006 (24%), they were less common in intra-regional cooperation areas than in single municipalities. Based on our results, the introduction of control fees is not only a financial tool but may also improve food control at the local level.

The aims of this study were to provide data on the microbiological contamination of sheep carcases slaughtered in Finland, and to compare excision and swabbing methods for microbiological sampling of sheep carcases. The results were also compared to the requirements laid down in Regulation (EC) No 2073/2005. A number of 50 sheep carcases were sampled both by excision and by swabbing with gauze at four slaughterhouses. The samples were analysed for total viable counts (TVC's), Enterobacteriaceae, and Escherichia coli. The same carcases were also sampled for Salmonella spp. by swabbing. The mean levels of TVC's and Enterobacteriaceae, and the results for Salmonella spp. were in line with the requirements laid down in Regulation (EC) No 2073/2005. The mean levels of TVC's for samples by excision and by swabbing were 3.77 log CFU/cm2 and 3.16 log CFU/cm2, respectively. Enterobacteriaceae were recovered from 72% and 76% of the carcases, and E. coli from 48% and 61% by excision and swabbing, respectively. No carcases were found positive for Salmonella spp. The mean levels of TVC's, Enterobacteriaceae and E. coli were significantly higher for the samples by excision than for the samples by swabbing. When the relationship between the sampling methods were analysed, the results for excision samples and swabbing samples were related for the TVC's, and there was a significant correlation between the sampling methods for Enterobacteriaceae. The results suggest that swabbing by gauze can be used as an alternative sampling method to excision. In addition, a significant correlation was seen between the results for Enterobacteriaceae and E. coli for the samples collected by swabbing, suggesting that E. coli can be used as an indicator bacterium instead for Enterobaceteriaceae for sampling of sheep carcases by swabbing.

To determine the prevalence of Clostridium botulinum types A, B, E, and/or F in pig intestinal samples, different PCR-based methods were compared using artificially contaminated faeces. The methods included a multiplex PCR targeted to botulinum neurotoxin genes A, B, E, and F or a nested PCR targeted to toxin genes B, E, and F, combined with different pre-enrichment protocols and forms of templates. A method including the two-step enrichment followed by DNA extraction and multiplex PCR yielded the highest number of positives. This assay protocol was employed to investigate 100 pig intestinal samples. The sample materials studied included colon wall, intestinal content, and mucus peeled from the colon wall. Three pigs (3%) were positive for C. botulinum type B, and no other toxinotypes were detected in any sample. The number of positive samples was higher when colon wall or peeled mucus was analyzed compared to the intestinal content. C. botulinum was isolated from two PCR-positive samples and confirmed to be type B by PCR. Both isolates were shown to be proteolytic and thus to represent C. botulinum group I.

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum . The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.

Whitefish eggs were confirmed by pulsed-field gel electrophoresis to cause type E foodborne botulism in a 54-year-old patient in Finland. Botulinum neurotoxin and/or nonproteolytic Clostridium botulinum type E organisms were detected in fecal and gastric samples from the patient and in suspected whitefish eggs. Apart from C. botulinum type E, proteolytic type B organisms were detected in the patient's gastric content. This was considered to be insignificant with respect to the clinical disease, suggesting botulinal spores to be occasionally present in the human gastrointestinal tract without any apparent clinical significance. This is the first domestic case of foodborne botulism in Finland.

Comparative genomic hybridization analysis of 32 Nordic group I Clostridium botulinum type B strains isolated from various sources revealed two homogeneous clusters, clusters BI and BII. The type B strains differed from reference strain ATCC 3502 by 413 coding sequence (CDS) probes, sharing 88% of all the ATCC 3502 genes represented on the microarray. The two Nordic type B clusters differed from each other by their response to 145 CDS probes related mainly to transport and binding, adaptive mechanisms, fatty acid biosynthesis, the cell membranes, bacteriophages, and transposon-related elements. The most prominent differences between the two clusters were related to resistance to toxic compounds frequently found in the environment, such as arsenic and cadmium, reflecting different adaptive responses in the evolution of the two clusters. Other relatively variable CDS groups were related to surface structures and the gram-positive cell wall, suggesting that the two clusters possess different antigenic properties. All the type B strains carried CDSs putatively related to capsule formation, which may play a role in adaptation to different environmental and clinical niches. Sequencing showed that representative strains of the two type B clusters both carried subtype B2 neurotoxin genes. As many of the type B strains studied have been isolated from foods or associated with botulism, it is expected that the two group I C. botulinum type B clusters present a public health hazard in Nordic countries. Knowing the genetic and physiological markers of these clusters will assist in targeting control measures against these pathogens.

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.

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 μm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of 10 units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of >2.3 log 10 units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.

Clostridium botulinum type B was detected by multiplex PCR in the intestinal contents of a suddenly deceased 11-week-old infant and in vacuum cleaner dust from the patient's household. C. botulinum was also isolated from the deceased infant's intestinal contents and from the household dust. The genetic similarity of the two isolates was demonstrated by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis, thereby confirming that dust may act as a vehicle for infant botulism that results in sudden death.

A total of 294 honey samples produced in Denmark, Norway and Sweden were studied for the presence of Clostridium botulinum types A, B, E and F by using a multiplex-PCR method. The samples consisted of honeycombs taken directly from beehives, and extracted honey representing several hives or apiaries. The prevalence of C. botulinum showed a significant variation between Denmark, Norway and Sweden, the proportions of positive samples being 26%, 10% and 2%, respectively. The major serotype detected was type B. When analysed with pulsed-field gel electrophoresis (PFGE) using restriction enzyme SacII, the 24 strains isolated produced eight different PFGE patterns. At a similarity level of 95%, four clusters were produced, three of which contained 20 of the 24 analysed strains. One of the clusters included isolates from both Denmark and Norway.

The largest reported outbreak of type C botulism in fur production animals is described. Epidemiological investigation of 117 out of 157 (response rate, 74.5%) farms revealed that 44,130 animals died or were euthanized, while 8,033 animals with milder symptoms recovered. The overall death rate in all animals at risk was 21.7%. The death rates were significantly higher in blue and shadow foxes (24.2 and 27.8%, respectively) than in silver and blue silver foxes and minks (below 4%). All minks had been immunized against botulinum toxin type C. Deaths were associated with feed manufactured by a local processor, 83 of whose customer farms (70.9%) reported dead or sick animals. Five feedlots out of 19 delivered to the farms on the day preceding the onset of the outbreak (day 2) were associated with a death rate higher than 40%. These feedlots consisted of fresh feed processed on day 2 and feed processed 1 day earlier (day 1). In laboratory analysis, the day 2 feed contained botulinum toxin type C (>600 minimum lethal doses/g), while the day 1 feed did not contain toxin. Toxin was not detected in feed raw-material samples. Clostridium botulinum type C was detected by PCR in some feed components and in feed. However, as the feed temperature was continuously 8°C or below and the pH was continuously 5.6 or below according to the manufacturer, it seems unlikely that spore germination and toxin formation occurred during overnight storage. Hence, the events leading to toxin formation were not determined.

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10 3 . An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10 6 spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum ( C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.

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 .