Characterization of Clostridium perfringens isolates from healthy turkeys and from turkeys with necrotic enteritis.

Clostridium perfringens is an important bacterial pathogen, especially in poultry, where it can lead to both subclinical and clinical disease. The aim of this study was to present data on pathological findings at outbreaks of necrotic enteritis (NE) in turkey production in Finland during the period from 1998 to 2012. Furthermore, C. perfringens isolates from healthy and diseased turkeys were characterized and their genetic diversity was investigated using pulsed-field gel electrophoresis (PFGE). Isolates (n = 212) from birds with necrotic gut lesions and from healthy flocks of 30 commercial turkey farms were characterized for the presence of cpa, cpb, iA, etx, cpb2, and cpe and netB genes. A total of 93 C. perfringens isolates, including 55 from birds with necrotic gut lesions and 38 from healthy birds from 13 different farms, were analyzed with PFGE. All contract turkey farmers (n = 48) of a turkey company that produces 99% of domestic turkey meat in Finland were interviewed about background information, management at the farm, and stress factors related to NE outbreaks. Pulsed-field gel electrophoresis analysis with SmaI restriction enzyme resulted in 30 PFGE patterns among the 92 C. perfringens isolates of high diversity. Out of all isolates, 212 (100%) were α-toxin-positive and one isolate (0.5%) was both α- and ß2 toxin-positive. Fourteen isolates (6.6%) were necrotic enteritis toxin B (NetB) positive; all were recovered from turkeys with NE. In none of the isolates obtained from healthy turkeys was the netB toxin identified. In conclusion, a high diversity of C. perfringens isolates from turkeys with different health status was shown. All isolates produced α toxin, whereas only low percentages of isolates carried the netB toxin gene. The role of the netB toxin in NE in turkeys needs to be further investigated.

Distribution of Campylobacter jejuni isolates from turkey farms and different stages at slaughter using pulsed-field gel electrophoresis and flaA-short variable region sequencing.

The aim of this study was to assess the diversity of thermotolerant Campylobacter spp. isolated from turkey flocks at six rearing farms 1-2 weeks prior to slaughter (360 faecal swab samples) and from 11 different stages at the slaughterhouse (636 caecal, environmental, neck skin and meat samples). A total of 121 Campylobacter isolates were identified to species level using a multiplex PCR assay and were typed by pulsed-field gel electrophoresis (PFGE) and flaA-short variable region (SVR) sequencing. All Campylobacter isolates were identified as Campylobacter jejuni. PFGE analysis with KpnI restriction enzyme resulted in 11 PFGE types (I-XI) and flaA SVR typing yielded in nine flaA-SVR alleles. The Campylobacter-positive turkey flocks A, C and E were colonized by a limited number of Campylobacter clones at the farm and slaughter. The present study confirms the traceability of flock-specific strains (PFGE types I, V and IX; flaA types 21, 36 and 161) from the farm along the entire processing line to meat cuts. It seems that stress factors such as high temperature of the defeathering water (54-56 °C), drying of the carcass skin during air chilling (24 h at 2 °C), and oxygen in the air could not eliminate Campylobacter completely. Campylobacter-negative flocks became contaminated during processing by the same subtypes of Campylobacter introduced into the slaughter house by preceeding positive flocks even if they were slaughtered on subsequent days. Proper and efficient cleaning and disinfection of slaughter and processing premises are needed to avoid cross-contamination, especially in countries with a low prevalence of Campylobacter spp. The majority of flaA SVR alleles displayed a distinct association with a specific PFGE type. However, a linear relationship for all strains among both typing methods could not be established. To specify genetic relatedness of strains, a combination of different genotyping methods, is needed.

Wines as possible meat marinade ingredients possess antimicrobial potential against Campylobacter.

This research studied the survival of high (7 log cfu/mL) and low (3 log cfu/mL) inoculum levels of Campylobacter in white and red wines and in grape and tomato juices, which could function as potential antimicrobial marinade ingredients. For comparison, survival was also studied in a commercial poultry meat marinade. White and red wines were shown to have very high bactericidal effects against Campylobacter. High counts were rapidly inactivated to undetectable numbers within 15 min in white wine and within 1 h in red wine, and low counts within 15 min in white wine and within 30 min in red wine. By contrast, grape and tomato juices did not possess high bactericidal effects against Campylobacter because even low counts were occasionally detected after 48 h. The commercial marinade had rather high bactericidal effects against Campylobacter; the high counts were inactivated in most cases within 48 h, and all the low counts were inactivated within 3 h. When testing chicken meat inoculated with Campylobacter and subsequently submerged in white or red wine, the antibacterial activity of the wine was largely reduced. Wines lowered the Campylobacter load inoculated on chicken meat by approximately 1 log cfu/mL over 48 h. The results suggest that wines could be used as antimicrobial ingredients together with the addition of further antimicrobial agents in meat marinades to reduce the numbers of Campylobacter in naturally contaminated poultry products, thus lowering the risk of Campylobacter cross-contamination and transmission through food.

Use of ultraviolet irradiation to reduce Campylobacter jejuni on broiler meat.

The effects of UV irradiation at a wavelength of 254 nm on the survival of Campylobacter jejuni on the surfaces of broiler meat, skin, and carcasses were studied. On broiler carcasses, the effects of UV were also studied in combination with activated oxygen. The surfaces were inoculated with varying counts of C. jejuni and treated with UV irradiation using doses ranging between 9.4 and 32.9 mW/s per square centimeter. The log reductions in C. jejuni counts were determined by dilution plating. The effects of both treatments on the sensory quality of broiler meat, including visual appearance, odor, and fatty acid composition, were also evaluated. On broiler meat, the maximum reduction achieved was 0.7 log and on broiler skin 0.8 log. On broiler carcasses, the maximum reduction using UV irradiation was 0.4 log, and using UV in combination with activated oxygen 0.4 log. No significant differences were found in the sensory quality between the samples and the controls. The use of UV irradiation alone or in combination with activated oxygen cannot be recommended as a primary decontamination method for C. jejuni on broiler carcasses. The use of these methods in combination with other decontamination techniques, and processing with proper processing plant sanitation and hygiene, might be more effective in reducing the C. jejuni counts on broiler carcass surfaces than the use of these methods only.

Lactobacillus alimentarius: a specific spoilage organism in marinated herring.

Spoilage characterised by bulging of lids and gas formation affected various product lots of different marinated herring types. Microbiological analyses resulted in growth on MRS and Rogosa SL agar. Altogether, 206 randomly selected colonies from two unspoiled and ten spoiled samples were characterised using phenotypical key tests and a 16 + 23S rRNA gene-based RFLP identification database. L. alimentarius was found to be the specific spoilage organism in all samples. All isolates obtained from the different product types were of the same clonal type. The slight rise in pH value together with marked gas production suggested a rare lactic acid bacteria spoilage type called 'protein swell'. L. alimentarius has not been previously associated with herring spoilage.

Microbiological quality and shelf-life of vacuum-packaged 'gravad' rainbow trout stored at 3 and 8 degrees C.

Microbiological and sensory changes of vacuum-packaged 'gravad' rainbow trout slices were studied during storage at 3 and 8 degrees C. At the time of spoilage, after 27 and 20 days of storage at 3 and 8 degrees C, respectively, both mesophilic viable counts (MVC) and psychrotrophic viable counts (PVC) reached 10(6)-10(7) cfu/g at 3 degrees C and 10(7)-10(5) cfu/g at 8 degrees C. H2S-producing bacteria constituted a high proportion of the PVCs and lactic acid bacteria (LAB) counts were lower than the other determined bacterial counts. Sensory scores decreased with increasing MVC and PVC. The judges considered samples unfit for human consumption at MVC and PVC levels exceeding 10(6) and 10(7) cfu/g for samples stored at 3 and 8 degrees C, respectively. At respective levels of 10(7) and 10(8) cfu/g, most of the samples were deemed unfit. The main reasons for sensory rejection at both storage temperatures were the lack of the typical product odour or an ammonia off-odour and colour change to dark violet. The shelf-lives of the rainbow trout slices based on microbiological and sensory analyses were 20 days and 18 days at 3 and 8 degrees C, respectively.

Characterisation of lactic acid bacteria from spoiled, vacuum-packaged, cold-smoked rainbow trout using ribotyping.

A total of 405 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged, salted, sodium nitrite- or potassium nitrate-treated, cold-smoked rainbow trout stored at 4 degrees C or 8 degrees C were characterised and identified using a molecular method. The isolates were initially classified according to their restriction endonuclease profiles using HindIII and EcoRI restriction endonucleases and further characterised by rRNA gene restriction patterns (ribotypes). Numerical analysis of these ribopatterns was performed together with 19 reference LAB strain patterns in order to identify the isolates to species level. The strains were divided with HindIII and EcoRI ribopatterns into ten and nine clusters at the similarity level of 65% and 50%, respectively. The Leuconostoc-clusters and the Lb. sakei/Lb. curvatus-clusters formed the two main groups. Only one isolate was identified as Lactobacillus plantarum and no Carnobacterium strains were discovered. For both enzymes, the 35 isolates possessing six individual ribotypes and forming five clusters could not be identified further with the reference strains used. The relative proportion of Leuconostoc mesenteroides subsp. mesenteroides was higher in all samples stored at 4 degrees C. Most of the Leuconostoc citreum were found in the samples stored at 8 degrees C, and particularly in the nitrite-treated samples.

The spoilage flora of vacuum-packaged, sodium nitrite or potassium nitrate treated, cold-smoked rainbow trout stored at 4 degrees C or 8 degrees C.

The spoilage flora of vacuum-packaged, salted, cold-smoked rainbow trout fillets, with or without the addition of nitrate or nitrite, stored at 4 degrees C and 8 degrees 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 degrees C were identified as Pseudomonas aeruginosa. The occurrence of P. aeruginosa and staphylococci in the nitrate-containing samples, stored at 8 degrees 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.