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Contamination of sliced cooked meat products with a Lactobacillus sake starter strain was suspected to cause spoilage in the products before the end of the expected shelf life. Slicing and vacuum packaging of the cooked products was done in the room in which the fermented product was handled. Since L. sake strains are known to be a dominant part of spoilage microflora associated with vacuum-pack-aged meat products, a contamination study was performed. One hundred and eighteen strains were isolated from 6 spoiled vacuum packaged meat products and from the surfaces of the packaging room and adjac-ent refrigerators. DNA was isolated from these strains and cleaved using Eco RI and Hind III restriction endonucleases to obtain characteristic ribotypes. Corresponding ribotypes of the L. sake starter strain were compared to the 14 different patterns obtained from the strains growing in spoiled products and on surfaces by Eco RI digestions. The L. sake starter strain was shown to contaminate the packaging room and it was also isolated from one of the products. However, it was not a dominant strain in this product and it could not be linked to the other products. Our results indicated that handling the fermented product in the refrigerating and packaging rooms together with cooked products was not the major cause of spoilage in these products.

SUMMARYThe first reported bovine botulism outbreak in Finland is described. Nine out of 90 cattle on a dairy farm died after being fed non-acidified silage contaminated by animal carcasses. Type C botulinum neurotoxin gene was detected in one heifer by polymerase chain reaction (PCR) and the neurotoxin was detected by the mouse bioassay.Clostridium botulinumtype C was isolated from liver samples. The isolated strain was identified with amplified fragment length polymorphism (AFLP) analysis as group IIIC. botulinum. To our knowledge, this is the first time that a type C bovine botulism outbreak has been diagnosed by PCR and confirmed by subsequent isolation and AFLP identification of the disease strain. The importance of the acidification process in silage production to inhibitC. botulinumtoxin production in silage and thus to prevent further botulism outbreaks is emphasized. Nevertheless, preformed toxin in the carcass is not destroyed by acid.

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (

A total of 312 samples of sliced, vacuum packaged, cold-smoked pork from 15 meat processing plants in Latvia and Lithuania, obtained over a 15-month period from 2003 until 2004, were analyzed for the presence of Listeria monocytogenes at the end of their shelf-life. Overall, 120 samples (38%) tested positive for L. monocytogenes. Despite the long storing period, the levels of L. monocytogenes in cold-smoked pork products were low. Manufacturing processes were studied at seven meat processing plants. A new approach with a logistic multivariable regression model was applied to identify the main factors associated with L. monocytogenes contamination during the manufacturing of cold-smoked pork products. Brining by injection was a significant factor (odds ratio 10.66; P0.05) for contamination of product with L. monocytogenes. Moreover, long cold-smoking times (or = 12 h) had a significant predictive value (odds ratio 24.38; P0.014) for a sample to test positive for L. monocytogenes. Pulsed-field gel electrophoresis results indicated that various sources of L. monocytogenes contamination existed over periods of time in several meat processing plants. In two meat processing plants, persistent L. monocytogenes strains belonging to serotypes 1/2a and 1/2c were found.

http://www.elsevier.com/locate/issn/01681605 Microbiological and sensory changes in vacuum-packaged charcoal-broiled river lampreys from three lamprey processing plants were monitored as a function of time at 8°C. The lampreys were examined every 7 days up to 8 weeks for aerobic plate count (APC) and lactic acid bacteria (LAB). The highest mean APC and LAB were 6.01 log CFU/g and 4.86 log CFU/g, respectively. Only 6 out of 15 lots reached an APC value of 7.0 log CFU/g during storage. The sensory scores remained at the baseline levels after 8 weeks´ storage. Twenty-seven isolates were randomly picked from MRS agar and identified to species level using a 16S and 23S rDNA HindIII RFLP (ribotyping) database and sequencing of the 16S rRNA gene if no database match was obtained. Twelve of the 27 isolates were identified as Lactobacillus curvatus subsp. curvatus, and two Leuconostoc mesenteroides and one Weissella halotolerans strain were also detected. Twelve isolates were not identified by the LAB database. However, they possessed very high (99.9%) 16S gene sequence similarity with either Staphylococcus warneri or Staphylococcus pasteuri type strains. The LAB detected, with the exception of W. halotolerans, have commonly been associated with spoilage of fishery products, but in these vacuum-packaged lampreys they were not the dominant organisms within the developing spoilage population.

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.

http://www.elsevier.nl/locate/01681605 A total of 176 lactic acid bacteria (LAB) isolated from a typical Spanish blood sausage called “morcilla de Burgos” were identified by means of phenotypic characteristics and 16S rDNA RFLP (ribotyping). LAB were isolated from “morcilla” of different producers and in different storage periods, that includes unpackaged, vacuum and modified atmosphere packaged “morcilla” and vacuum packed and pasteurised “morcilla”. The knowledge of specific spoilage bacteria of ”morcilla de Burgos” will be useful to design new preservation methods to extent the shelf-life of this product. Identification made according to phenotypic and biochemical characteristics shows the majority of the isolates were heterofermentative LAB (93.2%) and eight different bacterial groups could be distinguished (A-G). W. viridescens was the main species detected (42%). In addition, Leuconostoc spp. (23.9%), W. confusa (11.4%) and Lactobacillus fructosus (5.7%) species were found. Few strains were phenotypically missidentified as Lb. sanfrancisco, Pediococcus spp., Lb. sakei/curvatus and Carnobacterium spp. and 11 strains remained unknown. Most of the leuconostocs were identified as Lc. mesenteroides and Lc. carnosum species. Ribotyping shows a quite good correlation with phenotypic methods, although it has been possible to identify 15 different clusters. W. viridescens and leuconostocs were also the predominant LAB. Strains identified as W. confusa by phenotypic characteristics were resolved in W. confusa and W. cibaria by ribotyping. Neither Carnobacterium piscicola nor Lb. sanfrancisco were identified by means of genotypic method. All Lb. fructosus strains and some more included in different phenotypic groups (17 strains in total) could not be associated with any reference strain (cluster VII). Although some discrepancies exists the combination of phenotypic and genotypic methods led to a better identification and characterization of the strains isolated from “morcilla de Burgos”.

A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying ( cpe -positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe -positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe -positive C. perfringens was successfully isolated from both of these samples. The detection of cpe -positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe- positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe- positive C. perfringens could be isolated if the ratio of cpe- positive C. perfringens spores to total C. perfringens spores was 6 × 10 −5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe -positive C. perfringens . The method also offers a new approach in the investigation of the epidemiology of cpe- positive C. perfringens strains.

Isolates 302, 334, 356, 377 and 379, detected in modified atmosphere packaged (MAP) broiler meat, together with LMG 12317T and LMG 13617, detected in dog tonsils, were analysed in a polyphasic taxonomy study including numerical analysis of ribopatterns and whole cell protein patterns, 16S rDNA sequence analysis, DNA-DNA hybridisation and determination of some phenotypic properties. The results indicated that these isolates represent a new species in the genus Enterococcus. The isolates showed classical phenotypic reactions for the genus Enterococcus with the exception of not possessing the Lancefield group D antigen. Isolates 334, LMG 12317T, and LMG 13617 showed the highest 16S rDNA sequence similarity (98.3-99.0%) with the Enterococcus pallens type strain. In the distance matrix tree based on 16S rDNA sequences, the three isolates were located in the Enterococcus avium –group with E. pallens as their closest phylogenic neighbour. Numerical analyses of whole cell protein patterns and HindIII/EcoRI ribotypes placed all seven isolates together in a single cluster separated from the E. avium group reference strains. The DNA-DNA hybridisation level between strains 334 and LMG 12317T was 93.5% confirming that they represent the same species. Low hybridisation levels (12-30%) were, on the other hand, obtained with the E. pallens and Enterococcus raffinosus type strains. The name Enterococcus hermanniensis sp. nov. is proposed, with strain LMG 12317T (=CCUG 48100 T) as the type strain. We would like to thank Ms. Henna Niinivirta for her excellent technical assistance. Financial support from the Academy of Finland (project 100479) is gratefully acknowledged. T.C. and P.V are indebted to the Fund for Scientific Research – Flanders (Belgium) for a position as postdoctoral fellow and research grants, respectively. T.C. also acknowledges the support from Belgian Federal Government (Federal Office for Scientific, Technical and Cultural Affairs).

Background. The vehicles and sources of Yersinia pseudotuberculosis infection are unknown. In Finland, clinical microbiology laboratories routinely report Y. pseudotuberculosis isolations and submit isolates for serotype analysis. In October 1998, the number of serotype O:3 infections increased markedly. Methods. Case patients with culture-confirmed Y. pseudotuberculosis O:3 infection were identified by use of laboratory-based surveillance. We conducted a population-based case-control study. Healthy community control subjects were matched by age, sex, and postal code. Isolates were subtyped by pulsed-field gel electrophoresis (PFGE). Results. Nationwide, 47 case patients were identified (age range, 277 years; median, 19 years). One patient with bacteremia died; 5 underwent appendectomies. We enrolled 38 case patients and 76 control subjects in the case-control study. Seventy-one percent of case patients and 42% of control subjects reported having eaten iceberg lettuce (matched odds ratio, 3.8; 95% confidence interval, 1.39.4); a dose-response relationship was found for increasing frequency of consumption. Of the 27 isolates obtained from case patients and tested in the analysis, all had indistinguishable PFGE patterns. Four lunch cafeterias that had served iceberg lettuce were associated with clusters of case patients. The lettuce was traced back to originating farms. Conclusions. Iceberg lettuce was implicated as the vehicle of a widespread foodborne Y. pseudotuberculosis outbreak. Ongoing laboratory-based surveillance and serotype analysis were essential in the rapid detection of infection. Cases of yersiniosis, which appear to be sporadic, may be part of unrecognized outbreaks caused by contaminated fresh produce.

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica , which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF -positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with Not I and Spe I enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.

http://www.elsevier.com/locate/01681605 A total of 296 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged ‘gravad’ rainbow trout stored at 3ºC and 8ºC were characterised and identified using a molecular approach. The isolates were initially grouped according to their HindIII restriction endonuclease profiles and further identified to species level using a rRNA gene restriction pattern (ribotype) identification database. Lactobacillus sakei, Lactobacillus curvatus and Carnobacterium piscicola were the three main species detected. Only one isolate was identified as Carnobacterium divergens. Most of the carnobacteria were found in the samples stored at 3ºC. The relative proportion of L. sakei was higher in the samples stored at 8ºC.

Ten low-capacity slaughterhouses were examined for Listeria by collecting a total of 373 samples, of which 50, 250, and 73 were taken from carcasses, pluck sets, and the slaughterhouse environment, respectively. Six slaughterhouses and 9% of all samples were positive for Listeria monocytogenes. Of the samples taken from pluck sets, 9% were positive for L. monocytogenes, the highest prevalence occurring in tongue and tonsil samples, at 14% and 12%, respectively. Six of 50 (12%) carcasses were contaminated with L. monocytogenes. In the slaughterhouse environment, L. monocytogenes was detected in two, one, one, and one sample originating from the saws, drain, door, and table, respectively. Carcasses were contaminated with L. monocytogenes in those two slaughterhouses, where the mechanical saws, used for both brisket and back splitting, were also positive for L. monocytogenes. A total of 58 L. monocytogenes isolates were characterized by pulsed-field gel electrophoresis typing. The isolates were divided into 18 pulsotypes, 15 of which were detected in pluck sets. In two slaughterhouses, where the carcasses were contaminated with L. monocytogenes, the same pulsotypes were also recovered from splitting saws. In addition, identical pulsotypes were recovered from pluck sets. Our findings indicate that L. monocytogenes of tongue and tonsil origin may contaminate the slaughtering equipment that may in turn spread the pathogen to carcasses. Thus, it is of the utmost importance to follow good manufacturing practices and to have efficient cleaning and disinfection procedures to prevent equipment being contaminated with L. monocytogenes.

http://www.elsevier.nl/locate/01681605 Eighteen earlier well characterised Lactobacillus sake strains representing different slime production capabilities in vacuum-packaged meat products were analysed using repetitive element sequence-based PCR (rep-PCR). The single primers BOXA1R and RW3A and the primer pair REP1R-Dt & REP2R-Dt were evaluated for their applicability in L. sake genotyping. The five different patterns produced by RW3A were least revealing, with the discriminatory power equalling to ribotyping. BOXA1R and REP-primer pair both produced six different banding patterns and the combination of these results yielded seven different rep-types. Rep-PCR was concluded to have approximately the same discriminatory power as randomly amplified polymorphic DNA (RAPD) analysis, but it was inferior to pulsed-field gel electrophoresis (PFGE). How-ever, if the results of rep-PCR and RAPD were combined, the discrimination was comparable PFGE, with the exception that within Ribogroup I the non-slime-producing strains were not distinguished from weak slime producers. It was concluded that the combination of the two PCR-based typing techniques, rep-PCR and RAPD, would be a valuable tool in large scale contamination studies at the meat processing plants, since results can be obtained rapidly and fewer isolates need further analysis by PFGE.

Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout ( Oncorhynchus mykiss ) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing L. monocytogenes was low. During processing, the frequency of fish contaminated with L. monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating L. monocytogenes pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an L. monocytogenes eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating L. monocytogenes . None of the control samples taken in the 5 months after the eradication program was implemented contained L. monocytogenes .

http://www.elsevier.nl/locate/01681605 The spoilage flora of vacuum-packaged, salted, cold-smoked rainbow trout fillets, with or without the addition of nitrate or nitrite, stored at 4°C and 8°C, was studied. Of 620 isolates, lactic acid bacteria were the major fraction (76%), predominating in all samples of spoiled product. However, the phenotypical tests used were insufficient to identify the lactic acid bacteria to the species level. Gram-positive, catalase-positive cocci, Gram-negative, oxidase-negative rods and Gram-negative, oxidase-positive rods were found in 6%, 16% and 2% of the samples, respectively. Of 39 Gram-positive, catalase-positive cocci, 29 were identified as staphylococci and 10 as micrococci. Eighty-five isolates were found to belong to the family Enterobacteriaceae, with 45 of those being Serratia plymuthica. Eleven isolates from the nitrate treated samples stored at 8°C were identified as Pseudomonas aeruginosa. The occurrence of P. aeruginosa and staphylococci in the nitrate-containing samples, stored at 8°C, may cause problems with respect to the safety of the product. The types of lactic acid and other bacteria in the spoilage flora were generally reduced by the addition of nitrate or nitrite to fillets.

Lactic acid bacteria are considered a major component of the microbial population found on various types of vacuum-packaged cooked sausages. Lactobacillus sake and Lactobacillus curvatus have been shown to be common species in these products. L. sake seems to form the predominant part of the spoilage population. Lactic acid bacterial growth on the surface of the sausages produces undesirable sensory attributes, such as sour aroma and taste. A specific spoilage phenomenon of commercial significance, characterized by long, stretchy, polysaccharide ropes between sausages or sausage slices, has also been found. L. sake strains play a major role in this spoilage phenomenon as well. Cooking of sausages during manufacturing destroys lactic acid bacteria on the surface of the sausages. Sausages are recontaminated with spoilage lactic acid bacteria mainly during the processing stages after cooking. During the chilling process, product contamination apparently results from exposure to airborne microorganisms. Workers and equipment are among the most likely sources of contamination during packaging and slicing. Spoilage strains originating from raw material may spread to other areas in the production facility (i.e., chill, slicing, and packaging rooms) via the air, workers and equipment. Decontamination methods can be used to prevent the growth of spoilage microorganisms and to extend the shelf life of vacuum-packaged sausages after packaging.

Hafnia alvei strains which possess the attachment-effacement gene (eaeA) may have clinical importance as new diarrhea-causing pathogens and should therefore be differentiated from other H. alvei strains. We characterized diarrheal H. alvei strains, which were positive in the PCR test for the eaeA gene, using biochemical tests not routinely used for identification of members of the family Enterobacteriaceae, and compared them with eaeA-negative strains isolated from different clinical and nonclinical sources to find characteristics useful for identification. Random amplified polymorphic DNA (RAPD)-PCR and partial sequencing of the 16S rRNA gene were utilized to study the genetic diversity of the isolates. The eaeA-positive strains were found to have many characteristic biochemical properties. Negative reactions in the 2-ketogluconate and histidine assimilation tests and a positive reaction in the 3-hydroxybenzoate assimilation test may be useful in routine diagnostics. Nearly identical RAPD-PCR profiles and identical 353-bp fragments of the 16S rRNA genes indicated little genetic diversity among the eaeA-positive strains. The low level of homology (92%) in the partial 16S rRNA genes of eaeA-positive and -negative H. alvei strains raises questions about the taxonomic positioning of eaeA-positive H. alvei.

Microbiological counts for 10 different sampling sites of 28 reindeer carcasses were studied in 3 reindeer slaughterhouses in Finland. On each carcass the hindshank, round, abdomen, flank, brisket, foreleg, shoulder, neck, foreback and back were sampled immediately after slaughter, using a nondestructive swabbing method. The overall mean bacterial count for 10 sampling sites of reindeer carcasses was 1.51 ± 0.51 log(10) cfu/cm(2). Statistically significant differences were detected between sampling sites. The back part of the reindeer carcass, i.e. hindshank, round, back and foreback, seemed to be relatively clean. The most contaminated parts were the foreleg, brisket and abdomen (2.05–2.95 log(10) cfu/cm(2)); these could be used for monitoring the hygiene of the reindeer carcass after slaughter. Differences between the 3 slaughterhouses were detected for some sampling sites, which may be due to differences in slaughter techniques and hygiene.

Yersinia pseudotuberculosis is an animal pathogen widespread in nature. The bacterium has caused infections in humans occasionally. Most of the reported Y. pseudotuberculosis -infections are sporadic, and the sources and vehicles of Y. pseudotuberculosis remain obscure. Y. pseudotuberculosis has emerged as an outbreak -associated pathogen in Finland since 1997. Indirect evidence has indicated that Y. pseudotuberculosis is a potential food borne pathogen, but no specific food has been previously confirmed as a source of the human infections. The aim of this thesis was to determine the prevalence of Y. pseudotuberculosis in different animal species and to evaluate them as a possible source of Y. pseudotuberculosis. Clinically healthy pigs were found to carry Y. pseudotuberculosis. The prevalence of Y. pseudotuberculosis was 4 % and 14 % in fattening pigs at slaughterhouse and at farm level, respectively. All Y. pseudotuberculosis isolates from pigs were pathogenic. Migratory birds representing 57 different bird species were studied for pathogenic Y. pseudotuberculosis during spring and autumn migration. Pathogenic Y. pseudotuberculosis was recovered from two species of birds during spring migration. Y. pseudotuberculosis was first time confirmed to be the cause of a food borne outbreak through domestically produced iceberg lettuce in epidemiological study. Contamination of iceberg lettuce was investigated on farm level in a two-year survey. Several factors predisposing to Y. pseudotuberculosis contamination of lettuce were found on the farms, including a large population of deers and free access of wild animals to unfenced lettuce fields and to irrigation water sources. Pulsed-field gel electrophoresis (PFGE) analysis was optimized for efficient molecular typing method for Y. pseudotuberculosis isolates. PFGE was used to compare Y. pseudotuberculosis isolates from different origin. Y. pseudotuberculosis isolates of different sources showed high genetic diversity, while Y. pseudotuberculosis isolates found in same animal species representing the same serotype showed limited genetic diversity. Identification of environmental Y. pseudotuberculosis isolates was found to be incorrect based solely on biochemical tests. Commercial test kits misidentified some environmental Yersinia spp. isolates as Y. pseudotuberculosis. However, no specific virulence genes of Y. pseudotuberculosis were found in these isolates by polymerase chain reaction (PCR) targeting these gene areas. Yersinia pseudotuberculosis on ympäristössä yleisesti tavattava bakteeri, jota on pidetty lähinnä eläimille tautia aiheuttavana patogeenina. Ihmisillä bakteerin aiheuttamia yksittäisiä infektioita on raportoitu satunnaisesti, mutta tartunnanlähdettä ja välittäjää ei ole tarkemmin pystytty selvittämään. Vuodesta 1997 lähtien Y. pseudotuberculosis on noussut merkittäväksi humaaniepidemioiden aiheuttajaksi Suomessa. Y. pseudotuberculosista on epäilty mahdolliseksi ruokamyrkytysbakteeriksi, mutta mitään tiettyä elintarviketta ei ole aikaisemmin voitu osoittaa tartunnanlähteeksi. Väitöskirjatyössä selvitettiin Y. pseudotuberculosiksen esiintymistä eri eläinlajeilla ja arvioitiin näiden merkitystä Y. pseudotuberculosiksen lähteinä. Kliinisesti terveiden sikojen todettiin olevan Y. pseudotuberculosiksen kantajia. Teurastamoilla 4 % ja tiloilla jopa 14 % tutkituista lihasioista oli Y. pseudotuberculosis -positiivisia Kaikki sioista eristetyt Y. pseudotuberculosis -kannat olivat patogeenisia, tautia aiheuttavia. Tutkittaessa Y. pseudotuberculosiksen yleisyyttä muuttolinnuissa kevät- ja syysmuuton aikana, voitiin 57 tutkitusta lintulajista todeta patogeenista Y. pseudotuberculosista kahdesta eri lintulajista kevätmuuton yhteydessä. Tutkimuksessa voitiin myös ensimmäistä kertaa varmistaa Y. pseudotuberculosis -epidemian aiheuttajaksi elintarvikelähde. Kotimainen jäävuorisalaatti osoitettiin tartunnan aiheuttajaksi epidemiologisen tutkimuksen avulla. Jäävuorisalaatin saastumista kasvatustilalla kartoitettiin kaksivuotisella tutkimuksella. Tiloilla havaittiin useita mahdollisia kontaminaatiolähteitä, joista tärkeimpänä pidettiin alueen suurta peurapopulaatiota ja luonnoneläinten vapaata pääsyä suoraan aitaamattomille pelloille sekä kasteluveden ottoon tarkoitetuille vesilähteille. Tutkimuksessa eristettyjen Y. pseudotuberculosis -kantojen tyypittämiseen kehitettiin soveltuvaa molekyylibiologista tyypitysmenetelmää, pulssikenttäelektroforeesia (PFGE). Eri lähteistä eristettyjä Y. pseudotuberculosis -kantoja verrattiin PFGE-menetelmällä. Samasta eläinlajista eristetyt ja samaa serotyyppiä edustavat Y. pseudotuberculosis kannat olivat perimältään hyvin yhdenmukaisia. Tutkimuksessa ympäristönäytteistä eristettyjen Y. pseudotuberculosis -kantojen biokemiallisiin testeihin perustuva tunnistaminen osoittautui riittämättömäksi. Kaupalliset testisarjat tunnistivat osan ympäristönäytteistä eristetyistä Yersinia-suvun bakteereista tutkimuksessa virheellisesti Y. pseudotuberculosikseksi. Y. pseudotuberculosiksen virulenssigeenien osoittamiseen perustuvassa PCR-tutkimuksessa kannoilla ei kuitenkaan voitu todeta Y. pseudotuberculosikselle ominaisia geenialueita.

The drive for risk-based food safety management, systems and control has spread world-wide in recent decades. Since the term is still internationally undefined, its use and implementation vary, producing different realizations. In this Ph.D. thesis, microbiological risk assessment (MRA) was investigated as a basis for risk-based food safety management, which was defined as ‘food safety management based on risk assessment in order to achieve an appropriate level of protection (ALOP)’. Governments are responsible for commissioning MRAs and also for setting food safety targets up to a certain point, but the practical management measures that need to be in place in order to achieve the targets are to be addressed by the operators. On the plant level, food safety is usually managed through regulation, quality assurance systems and a hazard analysis and critical control point (HACCP) programme with its prerequisites. In Finland, food safety management on the food plant level is implemented through an HACCP-like regulated system termed an own-checking (OC) programme. A quantitative microbiological risk assessment (QMRA) was conducted on salmonella in the beef production chain according to the official standards of the Codex Alimentarius Commission (Codex Alimentarius), and utilized in determining the food safety metrics for beef production. The Finnish Salmonella Control Programme (FSCP) and the main official interventions due to it were examined in the light of risk-based food safety management. The targets set for beef processing plants by the government were converted into quantitative limits, and the results of salmonella monitoring included in the FSCP were examined by the QMRA. The goal of the FSCP was declared in 1994 to ‘maintain the present salmonella situation’, which was considered to refer to the salmonella incidence in humans at that time, and also the de facto ALOP. The requirement for a maximum salmonella prevalence of 1% at defined stages of the beef production chain was embodied in the FSCP. This statement was considered to convey performance objectives (PO) for the aforementioned stages. According to the QMRA, the de facto ALOP was achieved in the referred year 1999, and even the true prevalence levels in the FSCP were estimated to be clearly under the set PO limits with 95% credibility. However, the PO limits were set too high for the de facto ALOP to be maintained in practice. If the salmonella prevalence reached the PO limit of 1% or values near it, the public health risk would increase and overrun the de facto ALOP. The QMRA produced in this work has for the first time provided the possibility to quantitatively asses the relationships between targets set in the FSCP and their impact on public health. At present, imports of beef and beef-derived foods may impose on Finnish consumers a significantly greater exposure than domestic products. If their salmonella prevalence or their share of the foods consumed in Finland increase, the number of human cases could rapidly rise. The models for the QMRA were mainly Bayesian hierarchical models using Markov chain Monte Carlo (MCMC) techniques, which was found to be a flexible and appropriate method for this type of complex modelling. The resulting distributions were also regarded as an advantage compared to the results from models developed with the deterministic approach, because the presentation of results included the extent of the uncertainty, and also in this manner better illustrated the actual operational environment. Based on an inquiry, the personnel in food processing plants had a positive attitude towards food safety management systems, but the knowledge, training and involvement of those employees directly operating on the site with these systems were discovered to be deficient. Therefore, a generic semi-quantitative hygiene risk assessment model, Hygram®, was developed for small and medium-sized food enterprises to offer assistance in understanding, training, and, first of all, detecting the critical steps of the processes, and thereby to contribute to the development of their own-checking systems towards risk-based food safety management. Hygram® was not considered a risk-based tool as such, but whenever the critical limits of the process have been defined as equal to a risk assessment, Hygram® can be used as a risk-based management tool. It can also serve as a tool for systematic hazard analysis and CCP detection when establishing a food safety management system. To conclude, the development of risk-based food safety management is a process in which risk assessment is an essential tool. Scientific, technical, psychological and resource-bound barriers need to be overcome in order to put risk-based management systems into practice. This study showed that QMRA can be valuable in national risk management decision making, although few QMRAs are currently available. Appropriate tools for practical risk management decision making on the industrial level, such as Hygram®, need to be further developed. Elintarviketurvallisuutta on viime vuosina pyritty muuttamaan riskiperusteiseksi maailmanlaajuisesti. Väitöskirjassa riskiperusteisuus määriteltiin tieteelliseen riskinarviointiin perustuvaksi elintarviketurvallisuuden riskinhallinnaksi, jonka tavoitteena on kansallisesti hyväksytyn suojatason, ALOP -arvon, saavuttaminen. Elintarvikkeen aiheuttaman terveyshaitan riskiperusteista hallintaa tutkittiin riskinarvioinnin keinoin vallitsevan lainsäädännön ja omavalvontatoiminnan kautta. Naudanlihantuotantoa esimerkkinä käyttäen tehtiin mikrobiologinen riskinarviointi salmonellan esiintyvyydestä koko elintarvikeketjussa sekä sen vaikutuksesta kuluttajien salmonellatapausten määrään. Samalla esitettiin naudanlihan tuotantoketjulle ns. elintarviketurvallisuusmittareita, jotka määriteltiin salmonellavalvontaohjelman ja muiden säädösten mukaisista vaatimuksista. Valvontaohjelman tavoitteena on ollut 1995 lähtien säilyttää Suomen hyvä salmonellatilanne, mikä tulkittiin tosiasialliseksi ALOP -arvoksi. Toimitetun riskinarvioinnin perusteella osoitettiin, että asetetut tavoitteet on saavutettu koko naudanlihaketjun osalta. Tulosten mukaan naudanlihan kuluttajille aiheuttama salmonellariski (vuonna jota tutkimus käsitteli 3 salmonellatapausta per 100,000 asukasta) on Suomessa hieman suurempi kuin sianlihan, ja selvästi suurempi kuin broilerinlihan tai kananmunien aiheuttama riski. Naudanlihan osuus kuluttajan salmonellariskiin vaikuttaisi Suomessa merkittävämmältä kuin muualla. Tämä lienee seurausta siitä, että Suomessa salmonellaa valvotaan siipikarjassa järjestelmällisesti jo parvitasolla ennen lintujen ja niistä saatavien tuotteiden päätymistä elintarvikeketjuun, kun taas naudan- ja sianlihantuotannossa salmonellavalvonnan painopiste on teurastamoissa ja lihanleikkaamoissa. Väitöskirjassa esitetyn riskinarvioinnin avulla ensimmäistä kertaa laskettiin elintarviketurvallisuustavoitteiden ja salmonellavalvontaohjelman välisiä suhteita sekä arvioitiin niiden vaikutusta kansanterveyteen. Vaikka salmonellaa esiintyy Suomessa kansainvälisesti katsoen erittäin vähän, valvontaohjelman edellyttämä korkeintaan 1 %:n esiintyvyystavoite havaittiin liian korkeaksi ylläpitämään hyväksyttyä suojatasoa. Lisäksi naudanlihan ja siitä saatavien elintarvikkeiden tuonnin arvioitiin, riippumatta vähäisestä kulutusosuudesta, voivan altistaa suomalaisia kuluttajia salmonellatartunnalle merkittävästi enemmän kuin kotimaisten tuotteiden. Salmonellatapausten määrä voi kohota nopeasti, jos salmonellan esiintyvyys kyseisissä tuontielintarvikkeissa lisääntyisi tai niiden osuus kulutuksesta kasvaisi selvästi. Tutkimus osoitti tieteellisen riskinarvioinnin arvon kansallisessa riskinhallintaan liittyvässä päätöksenteossa ja se nosti esiin tarpeen kehittää yritystason riskinhallintaan soveltuvia työkaluja.

DNA-pohjaiset genotyypitysmenetelmät ovat nykyään tärkeitä työkaluja bakteerien tunnistamisessa ja sukulaisuussuhteiden selvittämisessä. Eri genotyypitysmenetelmien soveltuvuudessa eri bakteerilajeille sekä erilaisiin tutkimustarkoituksiin on kuitenkin eroja. Tässä väitöstutkimuksessa selvitettiin amplified fragment length polymorphism (AFLP) –menetelmän soveltuvuutta elintarvikevälitteisten tautia-aiheuttavien bakteerien Listeria monocytogeneksen, Clostridium botulinumin ja Clostridium perfringensin geneettiseen tyypittämiseen. Väitöstutkimuksessa kehitettiin AFLP-menetelmästä hyvin erotteleva sovellutus L. monocytogenes, C. botulinum ja C. perfringens kantojen karakterisointiin. Menetelmän todettiin olevan hyvin toistettava, nopea sekä helppokäyttöinen. Tutkimuksessa todettiin myös, että AFLP-menetelmää voidaan käyttää apuna eri listeria- ja klostridi-lajien tunnistamisessa. Menetelmästä on hyötyä etenkin klostridi-lajien diagnostiikassa, sillä nykyisin käytössä olevat diagnostiset menetelmät ovat osin puutteellisia. Kehitetyn AFLP sovellutuksen avulla luodut AFLP-tyypitystulokset voidaan kerätä tietokantoihin, joita voidaan hyödyntää mm. epidemiologisissa tutkimuksissa, elintarviketeollisuuden kontaminaatioreittien selvittämisessä, ruokamyrkytysepidemia selvityksissä sekä listeria- ja klostridi-lajien tunnistamisessa. Väitöstyössä tutkittiin AFLP-menetelmän avulla L. monocytogeneksen kontaminaatioreittejä eineksiä valmistavassa elintarvikelaitoksessa kahdeksan vuoden aikana. Siivousrutiinien, valmistettavan tuotteen ja osastoinnin todettiin vaikuttavan tuotantoympäristön kontaminaatiotilanteeseen kuumennettuja eineksiä valmistavilla osastoilla. Osastolla, jolla valmistettiin eineksiä, joiden valmistusprosessiin ei kuulu kuumennuskäsittelyä, raaka-aineiden todettiin saastuttavan valmiita tuotteita. Siten raaka-aineiden laatuun tulisi kiinnittää erityistä huomiota kun valmistetaan eineksiä, joita ei kuumennuskäsitellä. Lisäksi tutkimuksessa osoitettiin, että tuotantolinjan rakenteelliset muutokset voivat edesauttaa elintarvikelaitosta pääsemään eroon listeriakontaminaatiosta. Tutkimuksessa selvitettiin myös elintarvikkeiden tuotantoympäristössä pitkäkestoisen (persistoivan) kontaminaation aiheuttavien sekä satunnaisesti (sporadisesti) esiintyvien L. monocytogenes –kantojen välisiä eroja AFLP- ja pulssikenttäelektroforeesi-menetelmillä. Vaikka persistoivien kantojen todettiin eroavan sporadisista kannoista, ei persistoivilla kannoilla todettu olevan yhteistä geneettistä alkuperää. In epidemiological studies, techniques that effectively discriminate between individual bacterial strains are essential. Recent developments in molecular techniques necessitate an ongoing need to tailor new genotyping methods for optimal characterization of different bacterial species and to evaluate their performance and suitability for research purposes. In this thesis amplified fragment length polymorphism (AFLP) analysis was tailored for optimal characterization of Listeria monocytogenes and Clostridium botulinum. The suitability of the developed AFLP protocol to type L. monocytogenes, C. botulinum and Clostridium perfringens at strain level was evaluated. AFLP proved to be a highly reproducible, easy-to-use, relatively fast and highly discriminative approach. When AFLP was applied to L. monocytogenes strains, its discriminatory power was shown to equal that of PFGE, which is considered the current gold standard for molecular fingerprinting of L. monocytogenes. These features make AFLP analysis a useful alternative to other genotyping methods in, for example, outbreak investigations and contamination route studies. Since phenotypic identification of Clostridium isolates is laborious, the suitability of AFLP for genomic species identification was assessed. The AFLP technique was applied to 129 strains representing 24 different Clostridium species. AFLP differentiated all species tested, except for Clostridium ramosum and Clostridium limosum. AFLP also differentiated between six different Listeria species. If AFLP profiles of well-defined strains are collected in identification libraries, the database can be a valuable additional tool for identification of Clostridium and Listeria species. Due to high throughput of samples, AFLP proved to be especially suitable for screening large numbers of isolates. AFLP was also used to trace contamination routes of L. monocytogenes in a chilled food processing plant producing ready-to-eat and ready-to-reheat meals during an 8-year period. Cleaning routines, product type and degree of compartmentalization seemed to have an influence on the contamination status in compartments that produced cooked meals. In addition, raw materials were shown to cause contamination of uncooked meals. Thus, special attention should be paid to quality control of raw ingredients when uncooked ready-to-eat meals are produced. This work also demonstrated that structural adjustments of a production line may facilitate the eradication of L. monocytogenes from the food processing environment. AFLP and PFGE analysis of sporadic L. monocytogenes strains and strains that cause persistent plant contamination revealed that persistent strains differ from sporadic strains. However, no specific evolutionary lineage of persistent strains was observed.

The major aim of the present study was to determine whether the principle of good governance impacts food safety or the effectiveness of control actions or decreases the health hazards related to environmental health or food safety. The legal proceedings in Finland for the last ten years and over 200 decisions made by veterinarians as municipal administrators were analysed. The main focus in the analysis was legal rights. The veterinarians working as authorities at the municipal level were questioned about their doctrines, attitudes and opinions about good governance and compulsory actions and the new legislation concerning foodstuffs. The answers were analysed by a modified, qualitative grounded theory–based method. The veterinarians were divided into three categories according to their doctrines and attitudes. This thesis is the first academic study in Finland which contemplates the environmental health sector through the principle of good governance. In the study there is a demonstration of four models which describe the impact of the different factors on the good governance and the compulsory actions. In conclusion, it was concluded that the good governance impacts the elimination of health hazards, the impressiveness of compulsory actions and the efficiency of control. The most significant aspects of good governance were the hearing process, the argumentation of the decisions, the documentation of health hazards and direct enforcement of the decisions. In the view of legal protection the major factors were the hearing process, the argumentation of the decisions and the directions of the appeals. The veterinarians' doctrines and attitudes were found to impact the notifying of the contraventions to the police and the use of compulsory actions. The lack of information and uncertainty among the veterinarians as authorities at the municipal level prevented the use of compulsory actions when needed. There were remarkable deficiencies in carrying out administrative procedures, protecting legal rights and upholding the principles of good governance. The written and documented hearing process was carried out only in 56 and 65 per cents of the material. The legal arguments were presented in 84 and 75 per cents of the material. The directions of the appeals were lacking in one-third of the material. There were also many defective and false proceedings and arguments concerning the hearing process and the argumentation of the decisions. The most significant deficiencies with the legal principles were with the principles of equality and objectivity. Also the practice of compulsory actions was noticed to vary in different municipalities. That means that the owners of the companies are placed in non-equal position depending on their companies' geographical location. In some cases there were also practices which were classified as unused or passive authority. A major role for the application of equal rights and demands in compulsory actions at the local level was noticed to be within the responsibility of the central food safety administration. The central administration can influence both the good governance practice and the use of compulsory actions at the municipal level by nationwide, standardised directives and regulations. In this thesis, there are recommendations presented based on the results acquired in this study. The results can be used to develop the central food safety administration's supervision of municipal authorities in the future. Ympäristöterveydenhuollon viranomaistehtävissä toimii Suomessa yli 300 eläinlääkäriä mm. terveysvalvonnan johtajina. Ympäristöterveydenhuollon tehtäviä suorittaessaan virkaeläinlääkärit ovat myös merkittäviä julkisen vallan käyttäjiä. Erityisesti velvoittavan julkisen vallan, kuten pakkokeinomenettelyn käyttö edellyttää hyvän hallinnon vaatimusten, oikeusturvan ja yhdenmukaisuuden toteutumista. Tutkimusta hyvän hallinnon toteutumisesta tai vaikutuksista ympäristöterveydenhuollossa ei ole kuitenkaan aikaisemmin tehty. Tässä väitöskirjassa selvitettiin, vaikuttaako hyvän hallinnon periaate elintarviketurvallisuuteen, terveyshaittojen poistamiseen, pakkokeinomenettelyn vaikuttavuuteen ja valvonnan tehokkuuteen. Tutkimuksen katsotaan kuuluvan ympäristöterveysoikeuden alaan. Tutkimuksessa kartoitettiin suomalainen oikeuskäytäntö kymmenen vuoden ajalta ympäristöterveydenhuollon pakkokeinomenettelyn osalta. Lisäksi tutkimuksessa analysoitiin yli 200 kunnan ja valtion virkaeläinlääkärin tekemää pakkokeinopäätöstä. Virkaeläinlääkäreiden tietoja, asenteita ja mielipiteitä pakkokeinomenettelystä kartoitettiin kyselytutkimuksella. Tutkimustulokset osoittavat, että hyvän hallinnon periaatteen noudattaminen ympäristöterveydenhuollon pakkokeinomenettelyssä tehostaa elintarviketurvallisuutta, terveyshaittojen poistamista, valvonnan tehokkuutta ja pakkokeinomenettelyn vaikuttavuutta. Valvontaa suorittavien eläinlääkäreiden tiedollisten valmiuksien ja asenteiden todettiin vaikuttavan lainrikkomusten ilmoittamiseen ja pakkokeinojen käyttöön. Tiedon puute ja epävarmuus nostavat kynnystä ryhtyä käyttämään pakkokeinoja tarvittaessa tai tehdä ilmoitus epäillystä lainrikkomuksesta poliisille. Nämä tekijät vaikuttavat erityisesti valtion virkaeläinlääkäreiden keskuudessa. Oikeusturvan toteutumisessa pakkokeinomenettelyssä todettiin huolestuttavia puutteita. Kirjallinen kuuleminen oli toteutettu tutkimuksen ensimmäisessä, aiempaa lainsäädäntöä koskevassa aineistossa 56 prosentissa ja voimassa olevaa lainsäädäntöä koskevassa tutkimusaineistossa 65 prosentissa pakkokeinopäätöksiä. Päätösten perustelut oli esitetty ensimmäisessä aineistossa 84 prosentissa ja jälkimmäisessä aineistossa 75 prosentissa tapauksia. Muutoksenhakuohje puuttui noin kolmasosasta päätöksiä molemmissa tutkimusaineistoissa. Kuulemismenettelyssä ja perusteluissa havaittiin paljon virheellisiä käytäntöjä ja perusteita. Myös oikeusperiaatteiden toteutumisessa todettiin selviä puutteita. Erityisesti yhdenvertaisuus- ja objektiviteettiperiaatteiden toteutuminen havaittiin puutteelliseksi. Viranomaisten pakkokeinokäytännöissä oli suuria vaihteluja eri kuntien välillä. Joissain tapauksissa ilmeni viranomaisen passiivisuudeksi ja toimivallan alittamiseksi luokiteltavaa toimintaa. Ympäristöterveydenhuollon alan toimijat voivat siis joutua eriarvoiseen asemaan maantieteellisestä sijainnista riippuen. Merkittävään rooliin nousi keskushallinto. Keskushallinnon kautta tulevalla ohjauksella ja yhdenmukaisella, valtakunnallisella linjauksella ja ohjeistuksella voidaan vaikuttaa sekä hyvän hallinnon toteutumiseen että pakkokeinomenettelyn vaikuttavuuteen. Väitöskirjassa esitetään suosituksia keskushallinnon taholta tulevan ohjauksen kehittämiseksi tutkimustulosten perusteella.

Groups I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct groups of organisms, with both groups being involved with human botulism. Due to differences in spore heat resistance and growth characteristics, the two groups possess different types of human health risks through foods, drink, and the environment. The epidemiology of human botulism due to Groups I and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates, and warrants thorough outbreak investigation with a particular attention to discrimination between the different physiological groups of C. botulinum. In this study, a PCR assay was developed to discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control targeted to conservative regions of 16S rrn in Groups I and II C. botulinum. The assay correctly identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and inclusivity. The assay provides a substantial improvement in discriminating between the Groups I and II C. botulinum, which traditionally is based on a time-consuming and error-prone culture method. Differentiation between the physiological groups of C. botulinum is an essential step in investigation of human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve our epidemiological understanding of human botulism.

The microbial numbers and the lactic acid bacteria population of the raw materials of cooked ring sausages were studied in order to determine the presence of the spoilage lactic acid bacteria of the sausages. The highest aerobic plate counts and lactic acid bacteria counts (up to the level of 108 and 105 CFU/g, respectively) were found in the pork skin emulsion and the meat trimmings. Lactic acid bacteria were also commonly found in the skim milk powder and the potato flour. Lactic acid bacteria isolates able to multiply at 8°C were recovered from all the raw materials studied except for the commercial spice mixture. These isolates could be divided into 11 different groups according to their morphological and biochemical characteristics. Isolates resembling the lactic acid bacteria strains responsible for the spoilage of vacuum-packed cooked ring sausages were found in the pork skin emulsion, the meat trimmings, and the potato flour. These raw materials can thus form a source for the spoilage bacteria of the sausages at a sausage processing plant.

The contaminants and microbiological quality of European river lamprey Lampetra fluviatilis L. were examined. The residues of mercury, cadmium, lead, hexachlorocyclohexanes (alpha-HCH, beta-HCH, lindane), hexachlorobenzene (HCB), heptachloroepoxide, dieldrin, chlordanes (oxychlordane, trans- and cis-chlordane, transnonachlor TNCL), DDT compounds, and 15 PCB congeners were determined in raw lampreys to evaluate the food safety of lamprey products. The levels og Hg and Cd residues were higher in lamprey caught in the Bothnian Bay area than in those from the Bothnian Sea. Pb was detected in only one sample. The mean ΣPCB, beta-HCH, lindane, HCB, dieldrin, oxychlordane and DDT levels were significantly higher in samples from rivers flowing downstream to the Bothnian Sea in comparison to levels in the Bothnian Bay. There were no statistical differences between the areas, considering the concentrations of the other pesticides. All residue concentrations were below the limits with regard to legislation. The microbiological quality of charcoal-grilled lamprey in three processing plants was examined in products stored at 3 and 22 °C. On the day of production the mean aerobic plate counts (APC) in grilled lampreys were 2.29 log CFU/g in plant A and under the detection limit (2.0 log CFU/g) in plants B and C. At 22 C the mean APCs of samples from all plants after 6 days were 8.56, 5.04 and 6.23 log CFU/g, respectively. The numbers of bacteria in lamprey from plant A were higher than those from plants B and C. No significant increases in APCs were observed during storage at 3 C for 24 days. Microbiological and sensory changes in vacuum-packed product were monitored as a function of time at 8 °C. The mean aerobic bacterial and LAB levels developed differently in samples from different plants. In samples from plants A and C aerobic bacterial growth was detected after 2 weeks and growth of LAB after 3 and 5 weeks. In samples from plant B, aerobic bacterial growth was detected only after 6 weeks but no LAB growth during 8 weeks. The mean APC was higher after 4-8 weeks storage than the mean levels of LAB. Twenty-seven LAB isolates were identified by ribotyping to species level. Twelve were identified as Lactobacillus curvatus subsp. curvatus, and two Leuconostoc mesenteroides and one Weissella halotolerans strain were also detected. Twelve isolates were not identified in the LAB database and were therefore subjected to 16S rRNA sequence analysis. These isolates possessed very high (99.9%) 16S rRNA gene sequence similarity with either Staphylococcus warneri- or Staphylococcus pasteuri-type strains. The sensory scores remained at the baseline levels after 8 weeks storage. Microbial development in charcoal-grilled river lampreys is slow and the shelf life of the product is longer than that of some other heat-treated fishery products. The food-borne pathogenic bacterial species Clostridium botulinum was studied in raw lampreys and Clostridium perfringens, Listeria monocytogenes and Staphylococcus aureus in the charcoal-grilled product. One out of 67 raw lampreys (1.5%) was positive for the botulinum neurotoxin type E gene. Neither C. perfringens nor L. monocytogenes was detected in the product, while S. aureus was found in two out of 300 samples (0.67%). Nahkiaista pyydetään Suomessa sen kutunousun aikana vuosittain n 1,5 miljoonaa kappaletta lähinnä Pohjanlahteen virtaavista joista. Nahkiaisen kehitys on kaksivaiheinen: kuoriuduttuaan se viettää toukkana 4-6 vuotta syntymäjoessaan ja muuttaa muotoaan meressä täysikasvuiseksi palaten jälleen jokiin kutemaan muutaman vuoden kuluttua. Nahkiainen on paistettuna ruoka-erikoisuus, jota on tuoreeltaan tarjolla syysaikana. Nahkiaiset paistetaan uunissa, jossa lämmön antavat palavat leppähalot ja niiden hiillos. Paistoaika on n 20 min ja lämpötila nousee tuotteessa 90-114 ºC. Tuote myydään pääosin tuoreeltaan toreilla, kauppahalleissa ja hyvinvarustetuissa myymälöissä. Nahkiaisen rasvaprosentti on n 15 % raakana ja 20-25 % paistettuna. Tutkimuksessa selvitettiin nahkiaisten raskasmetallipitoisuuksia (elohopea, kadmium ja lyijy) sekä useita torjunta-ainejäämiä sekä PCB-pitoisuuksia. Koska nahkiainen syö paljolti kalaa, em aineiden pitoisuuden olivat samansuuntaiset Itämeren kalojen pitoisuuksien kanssa. Elohopeaa ja kadmiumia löytyi enemmän Perämeren nahkiaisista kuin Selkämereltä tai Merenkurkusta. Lyijypitoisuudet olivat merkityksettömiä. Myös torjunta-aine- ja vierasainepitoisuudet olivat yleensä suuremmat eteläiseltä alueelta pyydetyissä nahkiaisissa verrattuna Perämeren alueeseen. Tämä saattaa johtua siitä että eteläisten jokien valuma-alueella on on runsaammin maataloutta ja teollisuutta verrattuna pohjoiseen alueeseen, joka on suurelta osin erämaata. Koska paistettua nahkiaista myydään runsaasti esijäähdyttämättä, selvitettiin myös tuotteen pilaantumista ja mikrobiologisia riskejä. Tuote on säilyvämpi kuin hiillostetut ja savustetut kalat yleensä. Paistettu nahkiainen alkaa pilaantua huoneenlämmössä kahden vuorokauden jälkeen mutta +3 asteessa vasta 24 vrk:n jälkeen. Vakuumisäilytyksessä +8 asteessa säilytettynä pilaantuminen alkoi viiden viikon jälkeen. Eri valmistajien välillä oli eroja säilyvyysaikojen suhteen. Maitohappobakteerit eivät olleet dominoiva mikrobiryhmä vakuumipakatussa tuotteessa. Ruokamyrkytyksiä aiheuttavia mikrobeja, kuten Listeria monocytogenes tai Clostridium perfringens bakteereita ei paistetussa tuotteessa todettu. Staphylococcus aureusta havaittiin mahdollisesti jälkikontaminaationa kahdessa näytteessä (0.67 %). Yhdessä raakanahkiaisnäytteessä (1.5 %) todettiin Clostridium botulinum itiöitä.

Campylobacter jejuni and C. coli are the most frequent causes of bacterial gastroenteritis in Finland. Most Campylobacter infections are sporadic and the sources of infection remain unidentified. Risk factors for Campylobacter infections include eating undercooked meat, especially chicken, drinking unpasteurized milk or contaminated water, having contact with pets and foreign travel. The asaccharolytic nature and inertness in traditional biochemical tests makes the identification of Campylobacter spp. difficult. We studied the phylogeny of 12 Campylobacter spp. based on partial 593-bp groEL gene sequences. In general, lower interspecies sequence similarities were observed for groEL (range from 65% to 94%) than for the 16S rRNA gene (range from 90% to 99%). However, the intraspecies groEL sequence similarities were high (range from 95% to 100%) making groEL gene sequencing and the PCR-RFLP method developed in our study valuable for the identification of Campylobacter species. The minimum growth temperature of around 30°C makes multiplication of Campylobacter in foods highly unlikely. However, the survival in cool and humid conditions, such as chicken meat stored refrigerated, has been shown to be good. The survival of C. jejuni was investigated on iceberg lettuce, cantaloupe, cucumber, carrot and strawberries. Survival on strawberries was significantly lower than on the other produce, as was survival at 21°C compared to 7°C. Survival on the other produce was comparable with earlier reports in water and milk, but not as good as that observed on chicken meat. The association of Penner HS serotypes and pulsed-field gel electrophoresis (PFGE) SmaI/KpnI genotypes of 208 human and 30 chicken caecal C. jejuni isolates was studied during the seasonal peak in 1999 in Finland. Of the strains from humans, 46% had overlapping sero/genotypes with those from chicken. During the seasonal peak in 2003, C. jejuni and C. coli isolates from human fecal samples showed 5.7% and 61% PFGE (KpnI) profile overlap with cattle fecal and poultry retail meat isolates, respectively, demonstrating the importance of genotypes circulating in chicken as compared to those isolated from cattle in human infections. However, in 1999, human cases were also isolated prior to the slaughter of the chicken flock colonized by the same Campylobacter sero/genotype, suggesting that common environmental sources may exist for both human infections and chicken flock contamination. Multilocus sequence typing analysis of 361 Finnish C. jejuni and C. coli isolates from human patients, cattle, chicken and turkey samples, provided new information on the potential association of some clonal groups of this diverse organism with source of isolation as well as demographic characteristics. Kampylobakteerit (Campylobacter jejuni ja C. coli) ovat yleisimpiä bakteeriperäisen ripulin aiheuttajia Suomessa ja monin paikoin ympäri maailmaa. Valtaosa sairastumistapauksista on yksittäisiä ja niiden lähteet jäävät selvittämättä. Riskitekijöiksi on tunnistettu mm. raa an tai huonosti kypsennetyn lihan, erityisesti broilerinlihan syönti, pastöroimattoman maidon tai käsittelemättömän veden juonti, läheinen kontakti lemmikkieläinten kanssa ja ulkomaanmatkat. Viimeisten vuosikymmenten aikana on kuvattu runsaasti uusia kampylobakteerilajeja, joista osan kliininen merkitys on yhä epäselvä. Lajien tunnistus perinteisin menetelmin on erittäin haastavaa. Väitöskirjan ensimmäisessä osatyössä tutkittiin 12 Campylobacter -lajin diversiteettiä groEL geenin sekvenssin vaihtelun perusteella. Geenialueen havaittiin erottelevan lajeja toisistaan huomattavasti paremmin kuin perinteisesti käytetyn 16S rRNA geenin. Lisäksi työssä kehitettiin polymeraasiketjureaktioon ja monistettujen tuotteiden restriktioanalyysiin perustuva menetelmä lajien tunnistuksen helpottamiseksi. Tuoreiden vihannesten ja hedelmien merkitys elintarvikevälitteisten infektioiden aiheuttajana on viime vuosina ollut kasvussa. Kampylobakteerit eivät normaalioloissa lisäänny elintarvikkeissa (alle 30°C), mutta nämä bakteerit säilyvät hyvin hengissä viileissä ja kosteissa olosuhteissa. Väitöskirjan toisessa osatyössä tutkittiin kampylobakteerien käyttäytymistä jäävuorisalaatissa, cantaloupe-melonissa, tuorekurkun leikkauspinnalla, porkkanaraasteessa ja mansikoiden pinnalla. Mansikoiden pinnalla ja korkeammassa lämpötilassa bakteerit kuolivat merkittävästi nopeammin. Muiden tuoretuotteiden pinnalla bakteerit säilyivät hengissä huonommin kuin broilerinlihassa, mutta yhtä hyvin kuin vedessä ja maidossa. Näiden tulosten perusteella tuoretuotteiden kampylobakteerisaastumisen välttäminen on ensiarvoisen tärkeää ja hyvää keittiöhygieniaa tulisi noudattaa erityisesti käsiteltäessä samanaikaisesti raakaa lihaa ja tuoretuotteita. Kolmessa viimeisessä osatyössä selvitettiin kampylobakteerien molekyyliepidemiologiaa. Siipikarjanlihassa todettiin esiintyvän selvästi nautakarjaa enemmän myös ihmisistä eristettyjä kampylobakteerien alatyyppejä. Mielenkiintoinen uusi havainto oli, että potilaista eristettiin samoja alatyyppejä kuin broilereista jo ennen saastuneen erän teurastusta. Tämä tulos viittaisi mahdollisiin yhteisiin tartunnanlähteisiin ympäristössä.

Reprinted with permission from Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, U.S.A. Contamination routes of Listeria monocytogenes were examined in a chilled food processing plant that produced ready-to-eat and ready-to-reheat meals during an 8-year period by amplified fragment length polymorphism (AFLP) analysis. A total of 319 L. monocytogenes isolates were recovered from raw materials (n=18), the environment (n=77), equipment (n=193), and products (n=31), and 18 different AFLP types were identified, five of which were repeatedly found to be persistent types. The three compartments (I to III) of the plant showed markedly different contamination statuses. Compartment I, which produced cooked meals, was heavily contaminated with three persistent AFLP types. AFLP type A1 dominated, and it comprised 93% of the isolates of the compartment. Compartment II, which produced uncooked chilled food, was contaminated with four persistent and five nonpersistent AFLP types. The equipment of compartment III, which produced cooked ready-to-reheat meals, was free of contamination. In compartments that produced cooked meals, the cleaning routines, product types, and lack of compartmentalization seemed to predispose production lines to persistent contamination. The most contaminated lines harbored L. monocytogenes in coolers, conveyors, and packing machines. Good compartmentalization limited the flow of L. monocytogenes into the postheat -treatment area and prevented the undesired movement of equipment and personnel, thus protecting the production lines from contamination. In compartment II, grated cheese was shown to cause product contamination. Therefore, special attention should be paid to continuous quality control of raw ingredients when uncooked ready-to-eat foods are produced. In compartment II, reconstruction of the production line resulted in reduced prevalence rates of L. monocytogenes and elimination of two persistent AFLP types.

The prevalences of various genotypes of enterotoxin gene-carrying (cpe-positive) Clostridium perfringens type A in 24 different food poisoning outbreaks were 75% (chromosomal IS1470-cpe), 21% (plasmid-borne IS1470-like-cpe), and 4% (plasmid-borne IS1151-cpe). The results show that C. perfringens type A carrying the plasmid-borne cpe is a common cause of food poisoning.

Reprinted with permission from Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, U.S.A. Persistent Listeria monocytogenes contamination of food industry equipment is a difficult problem to solve. Ultrasonic cleaning offers new possibilities for cleaning conveyors and other equipment that are not easy to clean. Ultrasonic cleaning was tested on three conveyor belt materials: polypropylene, acetal, and stainless steel (cold-rolled, AISI 304). Cleaning efficiency was tested at two temperatures (30 and 45 degrees C) and two cleaning times (30 and 60 s) with two cleaning detergents (KOH, and NaOH combined with KOH). Conveyor belt materials were soiled with milk-based soil and L. monocytogenes strains V1, V3, and B9, and then incubated for 72 h to attach bacteria to surfaces. Ultrasonic cleaning treatments reduced L. monocytogenes counts on stainless steel 4.61 to 5.90 log units; on acetal, 3.37 to 5.55 log units; and on polypropylene, 2.31 to 4.40 log units. The logarithmic reduction differences were statistically analyzed by analysis of variance using Statistical Package for the Social Sciences software. The logarithmic reduction was significantly greater in stainless steel than in plastic materials (P

Contamination of fish products with Listeria monocytogenes, a bacterium causing listeriosis, presents a risk to consumer health. To better control this, the present study investigated the prevalence and sources of L. monocytogenes in different stages of fish production chain as well as the effects of a pasteurisation method on rainbow trout roe products. Farmed rainbow trout from different fish farms were found to contain L. monocytogenes at an average rate of 9%. L. monocytogenes prevalence varied greatly among fish farms from 0 to 75%. The bacterium occurred almost exclusively in the gills (96%) and only sporadically in the skin and viscera. Special effort should be focused on the isolation and removal of rainbow trout gills before L. monocytogenes contamination spreads further. The main L. monocytogenes contamination sources in the studied fish farm were the brook and river waters, as well as other runoff waters from environment. Rainy weather conditions were found to increase the probability of finding L. monocytogenes in the fish farm environment. The fish farm did not spread L. monocytogenes contamination, but suffered contamination from environmental sources. L. monocytogenes appeared on cleaned surfaces of one-third of the 23 studied fish processing factories, at least sporadically. The presence of Listeria spp. on the factory surfaces was indicative of increased possibility of occurrence in the fish products. Contamination of different ready-to-eat fish products with L. monocytogenes varied from 0 to 20%. The pulsed-field gel electrophoresis typing of L. monocytogenes isolates, from 15 fish factories, showed that the same pulsotypes of L. monocytogenes, isolated form fish products, occurred also in raw fish and fish production environments. Thus, raw fish materials and production environment are sources of L. monocytogenes contamination that both need to be properly controlled. Presence of L. monocytogenes in different Finnish fish species roe products in retail markets varied between 2 to 8%. Recovery of L. monocytogenes was significantly higher in fresh-bought roe products (18%) than in frozen or frozen-thawed (1%) roe products. Pasteurisation of rainbow trout roe, at 62 °C or at 65 °C for 10 minutes, eliminated inoculated 8 log units of L. monocytogenes. The quality of pasteurised vacuum packaged rainbow trout roe was found to be consistently good, in terms of microbial as well as sensory quality, for up to six months stored at 3 °C. Kalatuotteiden saastuminen Listeria monocytogenes -bakteerilla, joka aiheuttaa listerioosia, aiheuttaa kuluttajien terveydelle riskin. Tutkimuksessa selvitettiin L. monocytogenes -bakteerin esiintymistä ja lähteitä kalatuotantoketjun eri vaiheissa, sekä kirjolohenmädin pastöroinnin vaikutuksia, bakteerin aiheuttaman vaaran hallitsemiseksi. Eri kalankasvattamoista peräisin olevissa kirjolohissa todettiin olevan keskimäärin 9 %:ssa L. monocytogenes -bakteeria. Bakteerin esiintyminen eri kalankasvattamojen välillä vaihteli huomattavasti (0-75 %). L. monocytogenes -bakteereja löydettiin lähes yksinomaan kalojen kiduksista (96 %) ja vain satunnaisesti nahasta tai sisälmyksistä. Kidusten poistoon tai eristämiseen pitäisikin jatkossa kiinnittää huomiota saastumisen leviämisen estämiseksi. Tarkemmin tutkitussa kalankasvattamossa todettiin pääasialliseksi L. monocytogenes -bakteerien lähteeksi kasvattamon läheiset joki- ja purovedet sekä muut ympäristön valumavedet. Sateinen sää lisäsi todennäköisyyttä löytää L. monocytogenes -bakteeria kasvattamolta. Tutkittu kalankasvattamo ei levittänyt L. monocytogenes -bakteeria vaan pikemmin kärsi ympäristöstä peräisin olevasta saastutuksesta. Kolmanneksesta tutkituista 23 kalanjalostamosta todettiin puhdistetuilta pinnoilta L. monocytogenes -bakteeria, ainakin satunnaisesti. Jonkin Listeria -lajin löytyminen tuotantopinnoista indikoi kohonnutta todennäköisyyttä löytää niitä myös yrityksen kalatuotteista. Eri kalavalmistuotteista todettiin L. monocytogenes -bakteeria 0-20 %:ssa. Eri kalalaitoksista (15) todettujen L. monocytogenes -isolaattien tyypittäminen pulssikenttäelektroforeesilla osoitti, että samoja L. monocytogenes tyyppejä esiintyi valmiissa kalatuotteissa, raaka-ainekalassa sekä tuotantoympäristössä. Tuotantoympäristö sekä raaka-ainekalat ovat molemmat siten L. monocytogenes -saastutuksen lähteitä, joita pitää asianmukaisesti valvoa ja hallita. Suomalaisten kalalajien mädeistä todettiin vähittäiskauppatasolla 2-8 %:ssa L. monocytogenes bakteeria. Huomattavasti enemmän L. monocytogenes -bakteeria löydettiin tuoreena ostetuista mädeistä (18 %) kuin pakasteena tai pakasteesta sulatettuna (1 %) ostetuista mädeistä. Kirjolohenmädin pastörointi 62 tai 65 °C:ssa 10 minuuttia tuhosi mätiin lisätyt 8 logaritmista yksikköä L. monocytogenes -bakteereja. Pastöroidun vakuumiin pakatun kirjolohenmädin mikrobiologinen ja aistinvarainen laatu säilyivät hyvinä kuuden kuukauden ajan säilytettäessä 3 °C:ssa.

SUMMARY Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

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.

Systematiikka on tieteenala, joka tutkii eliöiden, esimerkiksi bakteereiden, sukulaisuussuhteita, määrittelyä ja luokittelua. Taksonomian voidaan ajatella olevan tämän tieteenalan käytännön sovellus: taksonomian tavoitteena on jakaa eliöt niiden sukulaisuussuhteita kuvaaviin, järkeviin ryhmiin, antaa jokaiselle ryhmälle sitä kuvaava nimi sekä löytää ne ryhmän ominaispiirteet, joiden avulla kyseinen ryhmä voidaan jatkossakin vaikeuksitta tunnistaa uudelleen. Taksonomia siis tarjoaa tutkijoille yhteisen kielen, jotta kaikki puhuisivat asioista niiden "oikeilla nimillä" ja vältyttäisiin väärinkäsityksiltä. Taksonomisten joukkojen perusyksikkö on laji. Suvullisesti lisääntyvillä eliöillä voidaan ajatella, että samaan lajiin kuuluvat ne eliöt, jotka pystyisivät keskenään tuottamaan lisääntymiskykyisiä jälkeläisiä. Asia tosin ei läheskään aina ole näin yksinkertainen. Suvullisen lisääntymisen puuttuessa bakteereiden perhesuhteita on vieläkin vaikeampi määritellä, ja siksi tiedon lisääntyessä aiempaan luokitukseen tulee jatkuvasti muutoksia. Jotta luokittelusta saataisiin mahdollisimman pysyvä ja pitkäkestoinen, on bakteereiden luokittelussa ryhdytty käyttämään niin sanottua polyfaasista (monivaiheista) taksonomiaa, menetelmää, jossa bakteerijoukon ominaisuuksia tutkitaan mahdollisimman monella tavalla ja lopuksi yhdistetään kaikki saatu tieto. Tässä tutkimuksessa polyfaasista taksonomiaa käytettiin hyväksi viiden broilerinlihavalmistesiin liittyvän maitohappobakteeriryhmän tunnistuksessa. Samalla arvioitiin polyfaasisen taksonomian soveltuvuutta maitohappobakteereiden luokittelussa. Tutkimuksessa käytettiin sekä fenotyyppisä (ilmiasuun perustuvia), genotyyppisiä (perimään perustuvia) että fylogeneettisiä (kehityshistoriaan eli evoluutioon perustuvia) menetelmiä. Lopuksi kunkin bakteeriryhmän tulokset koottiin yhteen. Useissa aiemmissa tutkimuksissa oli saatu viitteitä siitä, että Lactobacillus curvatus ja Lactobacillus sakei -lajien taksonomiassa on epäselvyyttä. Tämän tutkimuksen tulosten perusteella luokittelua korjattiin siirtämällä Lactobacillus curvatus- bakteerin melibiosus- alalaji osaksi Lactobacillus sakei- lajia, sen alalajiin carnosus. Polyfaasista lähestymistapaa sovellettiin myös neljän tuntemattoman bakteeriryhmän tunnistamisessa. Tulosten perusteella havaittiin että kaksi näistä, kokkimainen koiran nielurisoista ja suojakaasupakatusta broilerista eristetty ryhmä ja sauvamainen, pääosin pilaantuneesta, marinoimattomasta, suojakaasupakatusta broilerinlihavalmisteesta eristetty ryhmä, oli aiemmin kuvaamattomia lajeja. Molemmat uudet lajit kuvattiin, eli niille annettiin nimet ja niiden ominaisuudet listattiin. Kokkibakteeri sai nimekseen Enterococcus hermanniensis, eläinlääketieteellisen tiedekunnan Helsingin Hermannin kaupunginosassa sijainneen vanhan kampuksen mukaan, ja sauvabakteeri Lactobacillus oligofermentans, sen energiankäytön mukaan (bakteeri pystyy käyttämään eli fermentoimaan vain harvoja = oligo sokereita). Kaksi muuta ryhmää sen sijaan tunnistettiin jo olemassa oleviksi lajeiksi. Kokkimainen bakteeri, joka oli eristetty suojakaasupakatusta, marinoidusta ja marinoimattomasta broilerin koipireisivalmisteesta ja broilerinlihanleikkaamon ilmasta tunnistettiin Streptococcus parauberis- bakteeriksi. Osalla näistä bakteereista oli kuitenkin kaksi ominaisuutta, joiden perusteella ne erosivat muista samaan lajiin kuuluvista kannoista: ne eivät kyenneet hyödyntämään laktoosia energianlähteenään, ja galaktoosiakin vain huonosti. Poikkeuksellisiksi nämä ominaisuudet tekee se, että tätä lajia on pidetty lähinnä lehmän utaretulehdusta aiheuttavana bakteerina, ja siten olisi ollut odotettavissa että kaikki sen jäsenet pystyisivät käyttämään laktoosia ja galaktoosia, maidon yleisimpiä energianlähteitä. Toinen, sauvamainen, espanjalaisesta, vakuumi- ja suojakaasupakatusta "Morcilla de Burgos" verimakkarasta eristetty ryhmä tunnistettiin Weissella viridescens- bakteeriksi. Polyfaasinen lähestymistapa sopi hyvin näille maitohappobakteereille. Yksittäisten testien perusteella ei kestävää lajimääritystä olisi saatu aikaan. Vaikka fenotyyppisten testien tulokset voivat ollakin kirjavia, ne kuuluvat kuitenkin uuden lajin kuvaukseen uusien genotyyppisten menetelmien rinnalle. Tutkimuksessa havaittiin myös, että erilaisten testien tuloksista luodut kirjastot ovat luotettavia apuvälineitä bakteereiden tunnistuksessa, kunhan niitä päivitetään jatkuvasti.

http://www.elsevier.com/locate/issn/01681605 Lactic acid bacteria (LAB) isolated from marinated or non-marinated, modified atmosphere packaged (MAP) broiler leg products and air samples of a large-scale broiler meat processing plant were identified and analyzed for their phenotypic properties. Previously, these strains had been found to be coccal LAB. However, the use of a 16 and 23S rRNA gene RFLP database had not resulted in species identification because none of the typically meat-associated LAB type strains had clustered together with these strains in the numerical analysis of the RFLP patterns. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA–DNA hybridization experiments were done. The 16S rRNA gene sequences of three isolates possessed the highest similarities (over 99%) with the sequence of S. parauberis type strain. However, in the numerical analysis of HindIII ribopatterns, the type strain did not cluster together with these isolates. Reassociation values between S. parauberis type or reference strain and the strains studied varied from 82 to 97%, confirming that these strains belong to S. parauberis. Unexpectedly, most of the broiler meat-originating strains studied for their phenotypical properties did not utilize lactose at all and the same strains fermented also galactose very weakly, properties considered atypical for S. parauberis. This is, to our knowledge, the first report of lactose negative S. parauberis strains and also the first report associating S. parauberis with broiler slaughter and meat products.

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.

Unidentified lactic acid bacterium (LAB) isolates which had mainly been detected in spoiled, marinated, modified atmosphere packaged (MAP) broiler meat products during two previous studies were identified and analyzed for their phenotypic properties and the capability to produce biogenic amines. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA-DNA hybridization experiments were done. Unexpectedly for a meat spoilage associated LAB, the strains utilized glucose very weakly. According to the API 50 CHL test, arabinose and xylose were the only carbohydrates strongly fermented. None of the six strains tested for production of histamine, tyramine, tryptamine, phenylethylamine, putrescine and cadaverine were able to produce these main meat-associated biogenic amines in vitro. Polyphasic taxonomy approach showed that these strains represent a new Lactobacillus species. The six isolates sequenced for the 16S rRNA encoding genes shared the highest similarity (95.0 to 96.3%) with the sequence of the Lactobacillus durianis type strain. In the phylogenetic tree, these isolates formed a distinct cluster within the Lactobacillus reuteri -group which also includes L. durianis. Numerical analyses of HindIII/EcoRI ribotypes placed all isolates together in a cluster with seven subclusters well-separated from the L. reuteri group reference strains. The DNA-DNA hybridization levels between Lactobacillus sp. nov. isolates varied from 67 to 96% and low hybridization levels (3-15%) were obtained with the L. durianis type strain confirming that these isolates belong to same species different from L. durianis. The name Lactobacillus oligofermentans sp. nov. is proposed, with strain LMG 22743T (=DSM 15707T =AMKR18 T) as the type strain. Academy of Finland (project 100479)

http://www.elsevier.com/locate/issn/01681605 In order to show which of the initial lactic acid bacteria (LAB) contaminants are also causing spoilage of a modified-atmosphere-packaged (MAP), marinated broiler leg product at 6ºC, LAB were enumerated and identified on the 2nd and 17th day following manufacture. A total of 8 fresh and 13 spoiled packages were studied for LAB levels. In addition, aerobic mesophilic bacteria and Enterobacteriaceae were determined. The average CFU/g values in the 8 fresh packages were 1.3 × 103, 9.8 × 103 and 2.6 × 102 on de Man Rogosa Sharpe agar (MRS), Plate Count Agar (PCA) and Violet Red Bile Glucose agar (VRBG), respectively. The commercial shelf life for the product had been set as 12 days and all packages analyzed on the 17th day were deemed unfit for human consumption by the sensory analysis. The corresponding CFU/g averages in the spoiled product were 1.4 × 109, 1.1 × 109 and 3.9 × 107 on MRS, PCA and VRBG agar, respectively. For characterization of LAB population, 104 colonies originating from the fresh and 144 colonies from the spoiled packages were randomly picked, cultured pure and identified to species level using a 16 and 23S rDNA HindIII RFLP (ribotyping) database. The results showed that enterococci (35.7% of the LAB population) were dominating in the fresh product whereas carnobacteria (59.7%) dominated among the spoilage LAB. Enterococcus faecalis, Carnobacterium piscicola and Carnobacterium divergens were the main species detected. In general, when the initial LAB population is compared to the spoilage LAB, a shift from homofermentative cocci towards carnobacteria, Lactobacillus sakei/curvatus and heterofermentative rods is seen in this marinated product.

The taxonomic position of two bovine strains, LMG 13603 and LMG 14595, assigned to the species Enterococcus raffinosus on the basis of biochemical features, was reinvestigated. Both reference strains and two other isolates, 6/1 (=LMG 22829) originating from a charcoal-broiled river lamprey and IE38.4 (=LMG 22830) from the air of a poultry slaughter by-product processing plant, occupied a clearly separate position, on the basis of sequence analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase α-subunit), relative to the type strain of E. raffinosus and all other enterococcal species with validly published names. 16S rRNA gene sequencing of strains LMG 13603, LMG 14595, 6/1 and IE38.4 confirmed their phylogenetic position in the Enterococcus avium species group, there being more than 99 % 16S rRNA gene sequence similarity to most members of the group, including E. raffinosus, and revealed Enterococcus pseudoavium as the closest phylogenetic relative (99·8–99·9 %). Further phenotypic and genotypic analyses using whole-cell-protein electrophoresis, (GTG)5-PCR fingerprinting, ribotyping and DNA–DNA hybridization experiments demonstrated that all four strains represent a novel enterococcal species, for which the name Enterococcus devriesei sp. nov. is proposed. The type strain is LMG 14595T (=CCM 7299T).

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.

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.

www.elsevier.com/locate/meatsci Marinated meat products are consumed increasingly because they are convenient in meal preparing. In addition to sensory effects, marinating has been considered to increase product safety and shelf life quality. There are variations in meat marinating technologies around the world. In Finland, marinades are complex sauces which have a great effect on product appearance and taste. They are water-oil emulsions typically containing salt, sugar and acids (acetic, citric), rheology-improving additives (like xanthan gum and guar gum), antimicrobial agents (like sorbate and benzoate) spices and aroma strengtheners. The pH of these marinades is usually acidic, less than 5, so sugar is used to cut the edge of the acidic taste. Marinated meat products are usually packaged under modified atmospheres to prevent the growth of aerobic spoilage organisms. This results in the growth of psychrotrophic, anaerobic bacteria and lactic acid bacteria (LAB) are the dominating spoilage organisms in these products. Marinating did not increase the shelf life of Finnish poultry products and it strongly selects certain spoilage-associated novel LAB species. Surprisingly, it did not have a strong inhibitory effect on Campylobacter. This may be due to the buffering capability of meat quickly neutralizing the pH of the acidic marinade. The change in the acidic pH towards neutrality also results in dissociation of the lipophilic acids making their antimicrobial effect nonexistent.

Reprinted with permission from the Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, Iowa, U.S.A.