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

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

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

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

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

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

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

The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg1, respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters

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

Botulism is diagnosed by detecting botulinum neurotoxin and Clostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102 cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 102 spore/g for types A, B, and F to 101 spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum

Diversity of proteolytic Clostridium botulinum strains, determined by a pulsed-field gel electrophoresis approach

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

Thermal inactivation of non-proteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products.

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 103. An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 106 spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed

Infant botulism acquired from household dust presenting as sudden infant death syndrome

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

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

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

Assessing the association between supplemented puppyhood dietary fat sources and owner-reported epilepsy in adulthood, among Finnish companion dogs

IntroductionEpilepsy is a serious and common neurological condition in dogs, despite the wide number of antiepileptic drugs available, in approximately one third of the patients, epilepsy remains unsatisfactorily controlled. We aim to analyze whether feeding dietary fat sources during puppyhood was associated with canine epilepsy in adulthood.MethodsA nested case–control study was compiled from the validated DogRisk food frequency questionnaire (DogRisk FFQ). DogRisk FFQ collected feeding, disease, and background data about the dog. The study sample consisted of 108 owner-reported epileptic cases and 397 non-epileptic controls. Each case was matched with up to four controls for the key confounding factors of sex, breed, and age. We analyzed associations between feeding as a puppy and owner-reported epilepsy as an adult dog using Cox regression. We tested 55 different food variables.ResultsWe found that feeding fish fat from dietary sources at least once a week during puppyhood was inversely associated with epilepsy in later life in the unadjusted analysis [OR 0.46 (95% CI 0.25–0.83), p=0.01], while when adjusting for keeping conditions and dog characteristics the association was [OR 0.45 (95% CI 0.23–0.88), p=0.02]. When adjusted for keeping conditions, dog characteristics, and other feeding factors, the association was of similar magnitude but not significance [OR 0.56 (95% CI 0.27–1.15), p=0.12].DiscussionThe study indicates possible protective associations of feeding the dog with dietary sources of fish fat against epilepsy, although the result could be confounded by other feeding factors. Findings are compatible with current knowledge regarding the role of omega-3 fatty acids and ketogenic diet, a low carbohydrate, high fat diet as supportive treatments of epilepsy. As our findings are based on observations, we suggest the possibility of causality but do not prove it. Dietary intervention studies should now be conducted to confirm our findings