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Consistency and risk-basis are core elements of effective enforcement of food safety legislation. In Finland, inspections of food retail premises have been conducted since 2013 based on new national guidelines for evaluation and grading. According to the guidelines, food control authorities should initiate an administrative enforcement process to ensure compliance if the food business operator (FBO) is given the poorest grade in the inspection. In this study, we examined the consistency within and between local food control units on the threshold of initiating an enforcement process. The study was conducted through an analysis of inspection reports of FBOs and by an electronic survey and interviews of local food control officials. The results reveal that most officials consider the national evaluation guidelines as helpful in improving the consistency of using enforcement measures. However, inconsistencies exist between and within the local food control units in the alignments of initiating an enforcement process. Enforcement measures are mainly used on a risk-basis and gradually, as in most enforcement cases the FBO had multiple non-compliances and the FBO had been given a prior request to correct the non-compliance before initiating an enforcement process. The results, however, revealed rather weak compliance and repeated violations among some FBOs. Based on the observed persistence of non -compliances and the efficacy of enforcement measures in inducing compliance, a lower threshold of initiating an enforcement process towards FBOs with repeated violations appears beneficial in enhancing the correction of violations. Increasing the consistency of the enforcement process begins with unifying the practices within the local food control units by establishing clear procedures for enforcement and ensuring adequate orientation of personnel. Further strengthening of cooperation, peer-review and discussion on interpretations of required control actions between the units is needed for nationally consistent implementation of the evaluation and disclosure system and use of enforcement measures. (C) 2017 Elsevier Ltd. All rights reserved.

We surveyed Finnish local food control officials' opinions about the use and challenges of administrative enforcement measures. Responses to the questionnaire were received from a total of 129 food control officials, covering 72.6% of the 62 local food control units in Finland. In the opinion of 42.7% of the respondents, enforcement measures are not used often enough in their unit to ensure forod safety. Based on our results, large units have better practical tools such as templates for enforcement decisions and guidelines that facilitate the use of enforcement measures than small units, but uncertain practices and a lack of routine appear to impede the use of the measures in many units. Particular challenges highlighted by the officials were related to laboriousness and slowness of the administrative process and reasoning for using enforcement measures. Moreover, impairment of cooperation with the food business operator as a consequence of using enforcement measures was of concern for many officials. The officials assessed the expertise of the head of the unit more positively in the units in which enforcement measures were used than in the units in which enforcement measures were not used, and participation in trainings was the strongest explanatory factor for the use of enforcement measures among the inspectors. Our results indicate that development of operating procedures and provision of specific training on administrative procedure with a practical approach play a key role in strengthening officials' expertise and confidence in using enforcement measures. Moreover, merging the small units with the large ones may promote uniform practices and better capabilities to use enforcement measures in all units. (C) 2017 Elsevier Ltd. All rights reserved.

The competent authorities of the Member States of the European Union are required to perform animal welfare inspections on livestock farms. The data obtained from these official inspections performed in Finnish cattle and pig farms in 2010&ndash, 2015 were used with the aim of estimating the prevalence of the most common non-compliances and identifying underlying risk factors. The prevalence of non-compliant cattle and pig farms was 24.2% and 27.9%, respectively. In cattle, the most common problem was an inadequate lying area followed by deficient housing conditions for calves, in pigs, it was a lack of enrichment material. The non-compliances concerning cattle were most frequently detected in autumn and in farms with small herd size, with tie-stall housing and outdoor rearing year-round. The pig farms with a farrow-to-finish unit had a higher prevalence of non-compliances than other production types. The prevalence of the non-compliant farms differed notably between the regions. It can be concluded that the cattle welfare inspections should be performed with a focus on the cold and rainy seasons and at small farms, whereas the pig welfare inspections should mainly focus on farrow-to-finish units. The data received from official inspections should be efficiently utilized in the development of animal welfare inspection system, with the aim of risk-based, consistent and uniform inspections. In addition, the data should be utilized in targeting information for farmers.

In response to the EFSA call New approaches in identifying and characterizing microbial and chemical hazards, the project INNUENDO (https://sites.google.com/site/theinnuendoproject/) aimed to design an analytical platform and standard procedures for the use of whole-genome sequencing in surveillance and outbreak investigation of food-borne pathogens. The project firstly attempted to identify existing flaws and needs, and then to provide applicable cross-sectorial solutions. The project focused in developing a platform for small countries with limited economical and personnel resources. To achieve these goals, we applied a user-centered design strategy involving the end-users, such as microbiologists in public health and veterinary authorities, in every step of the design, development and implementation phases. As a result, we delivered the INNUENDO Platform V1.0 (https://innuendo.readthedocs.io/en/latest/), a stand-alone, portable, open-source, end-to-end system for the management, analysis, and sharing of bacterial genomic data. The platform uses Nextflow workflow manager to assemble analytical software modules in species-specific protocols that can be run using a user-friendly interface. The reproducibility of the process is ensured by using Docker containers and throught the annotation of the whole process using an ontology. Several modules, available at https://github.com/TheInnuendoProject, have been developed including: genome assembly and species confirmation; fast genome clustering; in silico typing; standardized species-specific phylogenetic frameworks for Campylobacter jejuni, Yersinia enterocolitica, Salmonella enterica and Escherichia coli based on an innovative gene-by-gene methodology; quality control measures from raw reads to allele calling; reporting system; a built-in communication protocols and a strain classification system enabling smooth communication during outbreak investigation. As proof-of-concepts, the proposed solutions have been thoroughly tested in simulated outbreak conditions by several public health and veterinary agencies across Europe. The results have been widely disseminated through several channels (web-sites, scientific publications, organization of workshops). The INNUENDO Platform V1.0 is effectively one of the models for the usage of open-source software in genomic epidemiology.

Simple Summary According to EU legislation, violations involving farm animal welfare need to be sanctioned. With this study, our aim was to assess criminal sanctions by Finnish district courts concerning cattle and pig welfare crimes. We found that small cattle farms were over-represented in court statistics. The most common sanctioned violations of cattle and pig welfare were the lack of cleanliness of premises and animals and inadequate feeding and watering. The violations were shown to have been continuing for a long time before a defendant stood trial for their actions. The penalties were lenient, i.e., the violations were not perceived as particularly blameworthy by the court. Veterinarians have a major role in initiating criminal procedures, collecting evidence, and evaluating the relevance of violations. Therefore, to improve the efficacy of criminal procedures, the police and prosecutors should work in close collaboration with veterinarians. Abstract EU legislation requires the violations of animal welfare standards to be sanctioned. Our aim was to evaluate criminal sanctions concerning violations of cattle and pig welfare on Finnish farms. We analyzed 196 court cases heard in Finnish district courts from 2011 to 2016. Almost all the cases (95%) concerned the violations of cattle welfare, of which 61% occurred on small farms. The lack of cleanliness and inadequate feeding and watering were the most common reported violations. Median time span from the start date of an offending to a judgement was slightly less than two years. Of the cases, 96% resulted in conviction. The court did not perceive the violations as being highly blameworthy as a small fine and a short conditional imprisonment were the most often imposed sanctions. A ban on the keeping of animals was used as a precautionary measure in half of the cases. Veterinarians were shown to have an important role in the initiation of criminal procedures, providing evidence for the police, and acting as witnesses. Therefore, it is crucial to achieve a well-functioning collaboration between veterinarians and the police and prosecutors. The expertise of these authorities on animal welfare legislation should also be emphasized to improve the efficacy of criminal procedures.

The aim of this study was to broaden the understanding of Finnish cattle and pig farmers’ perceptions of the on-site animal welfare inspections carried out by official authorities in livestock farms. The study was conducted using an electronic questionnaire, aimed at 500 Finnish cattle and 500 pig farmers. Responses were received from 96 cattle farmers and 105 pig farmers, of which 20 and 55, respectively, had undergone an animal welfare inspection. It was found that most of the farmers recognised the need for animal welfare inspections, but also that a more negative attitude was prevalent among farmers who had undergone these inspections. The inspection itself was a far more negative experience if the farmer had not understood the reason for the inspection, no opportunity existed to be heard, or the findings of the report were found to be unclear. The results suggest that although the farmers generally approve of inspections, their own negative experiences affect their perceptions. Moving forward, efforts should be made by inspectors to enhance the level of communication, thereby ensuring the findings of the report are clear to the farmer.

To ensure the safety of meat, official control, including meat and food safety inspections in slaughterhouses, should be of high quality. The prerequisites for high-quality official control were examined by sending a questionnaire to meat inspection personnel, slaughterhouse representatives, and officials who guide and organize the official control of slaughterhouses at the central level in Finland. The questionnaire inquired about the sufficiency of meat inspection personnel resources and its effects on official control. The post-mortem inspection skills of the official auxiliaries (OAs) and steps taken by the official veterinarians (OV) to evaluate OAs’ performance in post-mortem inspection were explored. Furthermore, OAs’ independence from the slaughterhouses in meat inspection, and further training and guidance provided to meat inspection personnel were surveyed. Our results showed that in most slaughterhouses at least occasional shortage of OVs was experienced, and it decreased especially the time dedicated to food safety inspections, meat inspection personnel’s participation in further training, and guidance given to OAs by OVs. All but one chief OV considered the skills of the regular OAs in post-mortem inspection to be totally sufficient, whereas over a third of the chief OVs did not find the post-mortem inspection skills of the OA substitutes totally sufficient. In red meat slaughterhouses, the frequency of the observation of the post-mortem inspection performed by the OAs was variable, and one-third of the red meat OAs considered that their performance in post-mortem inspection was not sufficiently evaluated by the OVs. Although most of the meat inspection personnel agreed that OAs understand working independently from the slaughterhouse in meat inspection, a substantial number of poultry OAs in particular stated that the independency was not entirely clear to them. A majority of the chief OVs considered that the officials guiding meat and food safety inspections at the central level are unaware of the practical problems involved in OVs’ work. Our results highlight the importance of practical experience for the officials guiding the official control in slaughterhouses and increased feedback between OVs and OAs in slaughterhouses. An adequate number of OVs should be guaranteed in all slaughterhouses. The practices to evaluate the red meat OAs’ performance in post-mortem inspection should be standardized and the maintenance of the post-mortem inspection skills of OA substitutes improved.

We surveyed the opinions of Finnish food business operators (FBOs) about the uniformity of local official food control and its importance for dairy, fishery and meat plants. A total of 136 FBOs responded to the questionnaire. Most FBOs considered official food control to be important for food safety and were generally satisfied with its quality. However, they often did not perceive official food control as being uniform, and 23% even considered it arbitrary. Small-sized FBOs were particularly critical of the relevance of control actions. The better the FBOs assessed their cooperation with the inspector, the higher they assessed the quality, uniformity and benefits of official food control. The cooperative approach in control practices should be emphasized to support the positive views of FBOs about official food control, thus promoting food safety. Cross-audits among local food control units are recommended to improve the FBOs' experience of uniformity of food control.

The level of food safety is determined by the actions of the consumers, the food business operators (FBOs) and the authorities that undertake official food control. Food control requires substantial investments by the FBOs as well as by the society, and these investments should be proportionate to the benefits obtained. The aim of this study was to assess the magnitude of economic resources currently invested in food control by the local food control authorities and by the restaurant business operators (RBOs). We also analysed the determinants of the variation in costs and evaluated the implications of the variation for the efficiency and equity of food control. Our results revealed a large variation in the costs, which in relation to the RBOs may in some cases imply risk-based targeting of food control, whereas in relation to the local control units, it may simply lead to unequal treatment of the control objects. The personnel resources (work years devoted to food control) of the local control units varied widely, and there were large differences (at most fourfold difference) in the number of control objects per personnel work year. The units that were found to have high costs had high costs regardless of the measurement unit (cost per work year, cost per control object, cost per inhabitant). Furthermore, larger units were found to have higher costs per work year and per control object. Less food control revenue was collected from control objects in units with a higher number of control objects per inspector. The challenge of management increases with the unit size, and focusing on the management skills and the organizational functionalities of the control units seems necessary.

Norovirus (NoV) gastroenteritis outbreaks appear frequently in food service operations (FSOs), such as in restaurants and canteens. In this study the presence of NoV and adenovirus (AdV) genomes was investigated on the surfaces of premises, especially in kitchens, of 30 FSOs where foodborne gastroenteritis outbreaks were suspected. The objective was to establish a possible association between the presence of virus genomes on surfaces and a visual hygienic status of the FSOs. NoV genome was found in 11 and AdV genome in 8 out of 30 FSOs. In total, 291 swabs were taken, of which 8.9% contained NoV and 5.8% AdV genome. The presence of NoV genomes on the surfaces was not found to associate with lower hygiene level of the premises when based on visual inspection; most (7/9) of the FSOs with NoV contamination on surfaces and a completed evaluation form had a good hygiene level (the best category). Restaurants had a significantly lower proportion of NoV-positive swabs compared to other FSOs (canteens, cafeteria, schools etc.) taken together (p = 0.00014). The presence of a designated break room for the workers was found to be significantly more common in AdV-negative kitchens (p = 0.046). Our findings suggest that swabbing is necessary for revealing viral contamination of surfaces and emphasis of hygiene inspections should be on the food handling procedures, and the education of food workers on virus transmission. Electronic supplementary material The online version of this article (doi:10.1007/s12560-017-9291-7) contains supplementary material, which is available to authorized users.

We evaluated the consistency and efficacy of inspection processes of 17 Finnish municipal food control units by analyzing inspection reports of 83 restaurants over the 2007–2011 period and by interviewing food business operators. Fifty-six officials of the food control units responded to an electronic questionnaire about their inspection activities. We found several factors related to the food establishment, the food control official and the working unit of the official that can affect the inspection processes and the efficacy of the controls. The use of checklists and templates for inspection reports enhanced the consistency and efficacy of the controls. The templates also reduced the time used for, and increased the quality of, reporting. Time limits for correcting non-compliances had a significant effect on the efficacy of controls.

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.

Food control systems are sometimes challenged when requirements set by law are intentionally violated by Food Business Operators (FBOs) deliberately putting food on the market with the intention of deceiving the consumer (food fraud/adulteration). There is also a growing concern that in some cases food frauds could be considered more risky than traditional food safety threats since their public health risks are often unconventional and difficult to detect. In this study, food frauds/adulterations published in the EU Rapid Alert System for Food and Feed (RASFF) (n = 376) in 2008–2012, notifications of recalls published by the Finnish Food Safety Authority (Evira) (n = 50) in 2008–2012 and local Finnish cases (n = 16) in 2003–2012 were analysed to determine the overall pattern of reported frauds/adulterations in 2008–2012. Products originating from outside the borders were most often reported by both RASFF (92%) and Evira (90%), whereas local Finnish cases mainly dealt with domestic production (69%). In RASFF, 33% of notification reasons reported illegal or unauthorized trade, practices or adulteration/tampering, whereas the rest (67%) concerned detected frauds or intention of fraud in documentation. Missing, fraudulent or improper documents were also most commonly reported in local Finnish cases (63%), but the pattern was very different in Evira's notifications, where unauthorized food, ingredients or processes comprised 98% of all fraud notifications reasons. Both in RASFF fraud notifications (50%) and in local Finnish cases (88%), the majority of cases concerned food of animal origin, whereas in Evira's fraud notifications food supplements dominated (44%) and food of animal origin was reported only once (2%). The FBO was accused and found guilty of a health crime in 83% of local Finnish cases processed in court (n = 12). Although the differences detected in patterns of fraud/adulteration cases may be a reflection of true differences in frauds on the market, it may also indicate a risk of an overly narrow scope in surveillance of frauds/adulterations at the international, national or local level. This study also highlights the need to incorporate different types of data sources when planning national and international control systems for food frauds and adulterations.

We surveyed the knowledge and attitudes of restaurant business operators (RBOs) with respect to their restaurants' hygiene. A total of 82 RBOs were interviewed and a hygiene evaluation was performed in each of the restaurants by one researcher. Fifty-six food control officials responded to an electronic questionnaire. Based on our results, positive correlations exist between hygiene knowledge of the RBOs, their attitudes toward food hygiene and official food control, and their restaurants' hygiene level. Proper justification of control measures used by food control officials and a negotiative approach appear to be highly important for improving restaurant hygiene.

We surveyed the perceptions of regional food control officials (auditors) and municipal food control officials (auditees) on food control audits during 2007–2010 in Finland. According to the responses, the perceptions of auditors and auditees differed significantly. The regional officials had experienced the audits as clearly more useful and positive than the municipal officials and also found the current auditing system to be more suitable for evaluating municipal food control. The regional officials did, however, state that the audit results had not been adequately utilized in planning the guidance and education of professionals working in official food control. The municipal officials, in turn, were not satisfied with the expertise of the auditors concerning official food control in practice and did not find the audits very useful. The results suggest that more emphasis should be placed on peer-auditing to enhance the impact of food control audits.

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.

The main responsibility of the local food control lies in the local food and environmental health units. The producer is responsible for the safety of the food produced, and applies an in-house control system to achieve this goal. The local food safety authority evaluates the in-house control system and assures that it is targeted to the most critical steps in the production to efficiently minimize the food safety risks. The main challenges in implementing efficient food control system are the variability in the knowledge of the producer about food hygiene, inadequate resources in control units and harmonization of the control measures in national level.

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.

Infant botulism is a rare illness caused by spores of Clostridium botulinum, which are ubiquitous in nature and harmless for healthy adults. Due to the immaturity of infantile intestinal microflora, the spores carried into the intestinal canal may germinate and produce toxin in vivo. The course of the infant botulism can vary from a prolonged constipation to sudden death of an infant, but several child-dependent predisposing factors are known to influence susceptibility to infection. Honey was the only food item associated with infant botulism until 2005, when a suspected case of infant milk formula-derived infant botulism was described. This thesis is based on the previously recognized association between honey and infant botulism. The autopsy specimens of a suddenly deceased 11-week-old boy were studied with multiplex polymerase chain reaction (PCR), and Clostridium botulinum group I type B was detected in the intestinal contents and in the vacuum cleaner dust of the patient's household. Genetic similarity of these isolates was demonstrated with pulsed-field gel electrophoresis (PFGE) typing and randomly amplified polymorphic DNA (RAPD) analysis, confirming that dust can act as a vehicle for infant botulism, resulting in sudden death. This was the first reported case of infant botulism in Finland. To evaluate the prevalence of C. botulinum in honey, a test protocol for reliable PCR-based detection of spores was developed. The inhibiting effects of honey were overcome by using a supernatant filtration method of in the preparation of samples before enrichment and PCR. PFGE analysis was optimized to study C. botulinum group I isolates, and applied to analyse the diversity of 55 strains of C. botulinum types A, AB, B and F. A total of nine restriction enzymes were tested, from which SacII, SmaI and XhoI were chosen for the cluster analysis of the 55 strains. Of these three enzymes, SacII was shown to be superior for analysis of C. botulinum group I isolates. C. botulinum group I was found to be heterogeneous, and in the majority of cases, PFGE enabled discrimination between individual strains of types A and B. However, certain clustering of type B strains was observed, and type F strains were located in a single cluster. A total of 190 samples from honey sold in Finland were studied by PCR to determine whether C. botulinum spores of types A and B were present. Spores of C. botulinum were detected in 8 of the 114 Finnish (7%) and in 12 of the 76 imported honey samples (16%). In addition, 294 honey samples produced in Denmark, Norway and Sweden were studied using multiplex-PCR for the presence of C. botulinum type A, B, E and F spores. The prevalence of C. botulinum showed a significant variation between Denmark, Norway and Sweden, the proportions of positive samples in each country being 26%, 10% and 2%, respectively. Type B was most frequently detected. The 24 strains isolated were confirmed to be proteolytic and were analysed with PFGE using restriction enzyme SacII. Twelve different PFGE patterns were produced. Three clearly distinguishable clusters containing more than three strains were noted, one of which included strains isolated in both from Denmark and Norway. Factors influencing C. botulinum contamination in the honey production environment were evaluated in a three-year survey. A total of 1168 samples from 100 apiaries and related facilities in Finland were analysed for the presence of C. botulinum types A, B, E and F using multiplex-PCR. Production methods and environmental factors were registered using a questionnaire and personal observation. C. botulinum was shown to be common throughout the whole honey production chain, with the type most frequently detected being group I type B. A PFGE analysis of 202 group I type B isolates suggested the existence of different genetic lineages. Only six PFGE profiles were observed, two of which predominated, covering 96% of the isolates. The high prevalence of C. botulinum in soil and in samples associated with beeswax suggests the accumulation of soil-derived botulinal spores in wax. In addition, based on the most significant bivariate Spearman's rank order correlations, production hygiene and extent of production were shown to have significant influences on the prevalence of botulinal spores in extracted honey. According to a logistic multivariable regression model, the presence of hand-washing facilities in the extraction room had a significant impact on decreasing the number of spores. Based on the results it is concluded that the number of spores in honey may be diminished. However, due to the observed wide existence of C. botulinum spores in the environment, it is not possible to produce honey totally free from spores. Clostridium botulinum hunajantuotannossa liittyen imeväisbotulismiin Clostridium botulinum on ympäristössä tavattava bakteeri, joka hapen läsnäollessa esiintyy lepomuodossa, erittäin kestävänä itiönä. C. botulinumin itiöt ovat terveelle aikuiselle vaarattomia, mutta kasvulle suotuisissa, hapettomissa olosuhteissa ne voivat muuttua eläviksi bakteerisoluksi tuottaen voimakkainta tunnettua biologista myrkkyä, botulinumtoksiinia. Toksiini aiheuttaa botulismina tunnetun sairauden, jonka oireena on etenevä velttohalvaus. Toisin kuin terveillä aikuisilla, alle vuoden ikäisillä lapsilla suolistoon joutuneet C. botulinumin itiöt voivat aiheuttaa imeväisbotulismiksi kutsutun sairauden. Lapsen suoliston kehittymätön mikrobisto sekä tämän lisäksi useat yksilöstä johtuvat altistavat tekijät voivat edesauttaa suolistoon joutuneiden itiöiden muuntumista toksiinia tuottaviksi bakteerisoluiksi. Imeväisbotulismin tyypilliset oireet vaihtelevat itsestään rajoittuvasta ummetuksesta lapsen äkilliseen, kätkytkuolemaa muistuttavaan menehtymiseen. Tyypillisimpinä itiölähteinä imeväisbotulismitapauksissa pidetään hunajaa ja pölyä, mutta usein tartunnan lähde jää selvittämättä. Hunajan on ainoana elintarvikkeena osoitettu liittyvän imeväisbotulismiin. Hunajan bakteerikasvua estävästä koostumuksesta johtuen C. botulinumin itiöt eivät pysty muuntumaan hunajassa kasvukykyisiksi soluiksi, mutta voivat säilyä hunajassa vuosikausia. Itiöitä ei voida tuhota hunajasta kuumentamalla, sillä vaadittava lämpökäsittely muuttaisi hunajan rakennetta heikentäen merkittävästi hunajan laatua. Koska imeväisbotulismin ja kätkytkuoleman välinen yhteys on aikaisemmin tunnistettu, tutkittiin väitöskirjatyön osana kätkytkuolleiden lasten elinnäytteitä C. botulinumin varalta. Äkillisesti menehtyneen, 11 viikon ikäisen poikalapsen näytteistä, sekä lapsen kodin pölynimurin pölypussista voitiin eristää C. botulinum bakteeri ja molekyylibiologisten tyypitysmenetelmien avulla voitiin osoittaa, että lapsesta ja pölystä eristetyt kannat olivat geneettisesti samankaltaiset. Lapsen tartunnan lähteenä voidaan tässä tapauksessa pitää ympäristöä, eikä tapaukseen liittynyt hunajan syöttämistä lapselle. Löydös osoittaa, että myös Suomessa, jossa imeväisbotulismitapauksia ei ole aikaisemmin diagnosoitu, voi C. botulinum olla osallisena joihinkin kätkytkuolemantapauksiin. Väitöstyössä kehitetyn, polymeraasiketjureaktioon (PCR) perustuvan analyysimenetelmän avulla tutkittiin C. botulinumin esiintymistä Suomessa kaupan olevassa tuontihunajassa, sekä suoraan tuottajilta kerätyistä hunajanäytteistä Norjassa, Ruotsissa, Suomessa ja Tanskassa. C. botulinumin itiöitä todettiin maakohtaisesti 0-26 %:ssa näytteistä. Pohjoismaista Tanskassa tuotetussa hunajassa todettiin itiöitä selvästi useammin muihin Pohjoismaihin verrattuna. Tutkimuksessa kehitettiin lisäksi geneettistä tyypitysmenetelmää, pulssikenttäelektroforeesia, soveltuvaksi sellaisille C. botulinum kannoille, jotka todettiin yleisiksi hunajantuotantoympäristössä. Menetelmän avulla havaittiin, että hunajantuotannosta kerätyistä näytteistä eristetyissä C. botulinum kannoissa oli vallitsevana kaksi genotyyppiä, jotka kuitenkin erosivat toisistaan merkittävästi. Onkin mahdollista, että saman C. botulinum ryhmän sisällä esiintyisi useita erilaisia geneettisiä sukulinjoja. Kolmivuotisessa seurantatutkimuksessa selvitettiin reittejä, joiden kautta itiöt kulkeutuvat hunajaan, ja arvioitiin mehiläistarhaajan mahdollisuuksia vaikuttaa itiöiden määrään hunajassa. C. botulinumin todettiin olevan erittäin yleinen löydös Suomen maaperässä. Mehiläispesässä C. botulinumin itiöiden esiintyminen oli selvästi yleisintä mehiläisvahaan liittyvissä näytteissä, ja on todennäköistä, että maa-aineksen sisältämät itiöt kulkeutuvat pölyssä mehiläisten mukana mehiläispesään ja kertyvät kennoja peittävään mehiläisvahaan. C. botulinumin itiöiden kierto hunajantuotannossa voitaisiin näin ollen katkaista sulattamalla tyhjiksi lingotut vahakakut riittävän korkeassa lämpötilassa, jotta vahaan kertyneet itiöt tuhoutuvat. Sulatetusta vahasta valetut uudet, pesiin laitettavat vahapohjukkeet olisivat tällöin tuotantokauden alussa itiöttömiä. Mehiläispesään kertyvien itiöiden määrää voidaan lisäksi vähentää sijoittamalla mehiläistarhat kasvillisuuden peittämille alueille, jolloin kasvillisuus voi rajoittaa maan pölyämistä. Tuotantohygieeniset tekijät nousivat analyysissä tilastollisesti merkittäviksi lingotun hunajan saastumisessa. On todennäköistä, että tuottajan sekä välineistön mukana kulkeutuu linkoomotiloihin ja sitä kautta lingottuun hunajaan pölyä, joka sisältää itiöitä. Itiöiden esiintymistodennäköisyys lingotussa hunajassa vähenee merkittävästi, mikäli tiloissa on käsienpesumahdollisuus sekä käytössä on erilliset sisäjalkineet. Lisäksi tilojen riittävä valaistus tehostaa tilojen puhtaanapitoa vähentäen pölyn ja sitä kautta itiöiden määrä tuotantotiloissa. Myös tuotannon laajamittaisuus vaikuttaa itiöpitoisuuteen: mitä suurempi on tuotannon kapasiteetti, sitä pienempi on todennäköisyys itiöiden kertymiselle hunajaan. Tämä viittaa siihen, että ammattimaisessa hunajantuotannossa hygieniatekijät huomioitaneen paremmin kuin harrastetyyppisessä toiminnassa. Tutkimus osoittaa, että C. botulinumin itiöiden määrää hunajassa voidaan vähentää. Koska itiöiden esiintyminen ympäristössä on hyvin yleistä, näyttää siltä, että täysin itiöttömän hunajan tuotanto ei kuitenkaan ole mahdollista.

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.

The antibacterial properties of 13 essential oils, derived from spices grown in Finland, were examined with an agar diffusion method against 12 bacterial strains. The organisms tested included both spoilage and pathogenic bacteria. The gram-positive bacteria appeared to be more sensitive than the gram-negative organisms, Clostridium botulinum and Clostridium perfringens being the most sensitive. Oregano, savory, and thyme showed the broadest antibacterial activity by distinctly inhibiting the growth of all the organisms tested. By gas chromatography-mass spectrometry analysis, differences were noted in the composition of oregano and thyme oils in comparison to previous reports.

A test protocol for reliable detection of Clostridium botulinum types A and B spores in honey by polymerase chain reaction (PCR) was developed and used for a prevalence survey of C. botulinum spores in 190 honey samples. The inhibiting effects of honey on microbial growth and PCR analysis were overcome by using a method of supernatant filtration (SF) in the preparation of the samples before enrichment and PCR. By using this method, an inoculum of 0.1 spore of C. botulinum/g honey could be detected. In the prevalence survey, spores of C. botulinum were detected in 8 (7%) of the 114 Finnish and in 12 (16%) of the 76 imported honey samples. The quantity of spores in PCR-positive samples varied from less than 18 to 140 spores/kg. Neurotoxin gene sequences corresponding to C. botulinum type A were detected in 17 samples and proteolytic type B in 12 samples by PCR analysis. Both types A and B were detected in nine samples. Strains of C. botulinum type A were isolated from 14 and type B from 2 of the 20 PCR-positive samples. This is the first report of type A spores of C. botulinum being detected and isolated in Fennoscandia.

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 .