The air-borne distribution of zoonotic agents from livestock waste spreading and microbiological risk to fresh produce from contaminated irrigation sources.

AIMS: To assess the risks of zoonotic agents in dissemination of livestock wastes into the environment by airborne distribution. To subsequently assess the survival time of zoonotic agents, introduced in irrigation water, on the phylloplane of produce. METHODS AND RESULTS: An Escherichia coli marker was introduced into pig slurry which was spread using a rain gun sprayer. Air sampling was undertaken to determine the distance reached by the marker. No recoveries were observed at a distance of 250 m. Borehole water, contaminated with zoonotic agents, was used to irrigate field plots sown with lettuce and spinach. Decline in bacterial numbers on the phylloplane was observed with time. After initial rapid decreases, we were unable to detect any pathogen from the phylloplane, 1 month after contamination. CONCLUSIONS: These preliminary results suggest that the risks to public health from the aerosolized spread of bacteria during slurry spreading by rain gun are low. Although, zoonotic agents on crop phylloplanes perish quickly, the risks of overhead irrigation of fresh produce 3 weeks before harvest should still be considered. SIGNIFICANCE AND IMPACT OF THE STUDY: These preliminary results improve our understanding on the fate of zoonotic agents in the environment. Spreading liquid livestock wastes by an airborne mechanism may not pose a significant public health risk. Detection of zoonotic agents 3 weeks after contamination of lettuce and spinach means that consideration should be given by the farmers until the time of harvest, when irrigating fresh produce with water that may have been directly or indirectly contaminated by livestock wastes.

Thermal death of Escherichia coli O157:H7 in cattle feeds.

AIMS: To determine if the temperatures used in feed manufacture are likely to destroy Escherichia coli O157. METHODS AND RESULTS: Two commercial feeds were ground and inoculated with E. coli O157 cells. The feeds were heated to 50, 55, 60, 65 or 70 degrees C. Heating produced quadratic survivor curves, with rapid initial decreases. The survival characteristics of E. coli O157 differed in the two feeds. The reductions observed in one feed may not have been due to heat alone. There was evidence that indigenous anti-E. coli O157 factor(s) in one feed acted with the heat and contributed to the observed rates of bacterial death. Heating at 70 degrees C for 20 or 120 s resulted in approx. 1.3 and 2.2 log reductions in E. coli O157 numbers respectively. Lesser reductions were observed at lower temperatures. CONCLUSIONS: The time/temperature combinations used in commercial pelleting processes would not effectively kill high numbers of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to look at the survival of E. coli O157 strains after heat treatment within concentrated animal feed. The study provides information on the likely risk of E. coli O157 surviving the animal feed manufacturing process.

Shiga toxin-producing Escherichia coli, faecal coliforms and coliphage in animal feeds.

AIMS: Animal feeds (n = 226), collected from pastures or feeding troughs on UK farms and from feed manufacturers' bulk stores, were analysed for Escherichia coli harbouring shiga-toxin genes (stx), faecal coliforms, coliphages and stx-harbouring bacteriophages. METHODS AND RESULTS: Samples comprised of 79 fresh grasses, 26 silages and 121 dried or heat-processed feeds (DPF). Five of the 79 (6.3%) fresh grass samples contained stx(2)-E. coli. stx-E. coli were not detected in the silages or DPF that were examined. Faecal coliforms were detected in 75/79 (94.9%) of fresh grasses, 19/26 (73.1%) of silages and 36/121 (29.8%) of processed feeds. Coliphages were detected in 63/79 (79.7%) and 18/26 (69.2%) of fresh grasses and silages, respectively. Coliphages were isolated at a significantly lower prevalence of 5% (6/121) from processed feeds. Although stx(2)-phage was isolated from the enrichment of a single grass sample, stx-phages were not detected in any of the silage or processed feeds. We did not detect stx(1)-phage in any of the samples collected. CONCLUSIONS: Pastures have the potential to act as transmission vectors for stx-harbouring E. coli for grazed livestock. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to report on the prevalence of E. coli harbouring stx genes, faecal coliforms, coliphages and stx-harbouring bacteriophages in a range of feedstuffs destined for consumption by UK livestock. This study provides information on the risk of feeds to the spread of stx-phages between livestock and/or the environment.

Decline of zoonotic agents in livestock waste and bedding heaps.

AIMS: To measure the rates of decline of zoonotic agents introduced into heaps of spent bedding and faecal wastes generated by commercially farmed livestock and managed in a similar way to that of a working farm. METHODS AND RESULTS: Livestock isolates of Salmonella, pathogenic Listeria, Campylobacter and Escherichia coli O157 were laboratory cultured and used to inoculate 5 m3 heaps of cattle, sheep or pig wastes mixed with bedding materials. Decline of each of the infectious agents was monitored with time as was the temperature inside each heap. Temperatures of >50 degrees C were typically achieved at the core of the heaps. Pathogen decline was rapid, typically <3 days for a 1-log reduction in levels. The longest time that zoonotic agents were isolated from the heaps was 93 days. CONCLUSIONS: Movement of heaps of livestock bedding waste from animal pens to a secondary store, and storing them under conditions conducive for increased temperature is a simple and cost-effective treatment for rapidly lowering levels of zoonotic agents in solid farm wastes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a simple and cheap treatment that can be used to help prevent the spread of zoonotic agents through agricultural environments.

Declines of zoonotic agents in liquid livestock wastes stored in batches on-farm.

AIM: To measure the decline rates of zoonotic agents introduced into liquid livestock wastes in on-farm storage tanks. METHODS AND RESULTS: Salmonella spp., Escherichia coli O157, Campylobacter jejuni, Listeria monocytogenes and Cryptosporidium parvum, propagated in laboratory-controlled conditions, were inoculated into 35,000-l volumes of fresh livestock wastes (pig slurries, cattle slurries and dirty waters). D-values for bacteria were six to 44 days, and for C. parvum were 133 to 345 days. Campylobacter jejuni declined significantly more rapidly than the other bacterial pathogens, while E. coli O157 declined significantly more slowly. On average, bacterial declines were not affected by the season of waste deposition and storage or by the dry matter content of the wastes, but were more rapid in dirty waters than in pig slurries. The physiciochemical composition of wastes in each category varied significantly. CONCLUSIONS: Zoonotic agents can survive for several months during storage of liquid livestock wastes. Livestock wastes should be batch-stored and not subjected to continuous additions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that batches of liquid livestock waste, if contaminated with bacterial pathogens, should be stored for 6 months to reduce contamination levels. Alternative strategies for reducing C. parvum levels in liquid livestock wastes should be explored.

Analyses of livestock production, waste storage, and pathogen levels and prevalences in farm manures.

Survey results describing the levels and prevalences of zoonotic agents in 1,549 livestock waste samples were analyzed for significance with livestock husbandry and farm waste management practices. Statistical analyses of survey data showed that livestock groups containing calves of <3 months of age, piglets, or lambs had higher prevalences and levels of Campylobacter spp. and Escherichia coli O157 in their wastes. Younger calves that were still receiving milk, however, had significantly lower levels and prevalence of E. coli O157. Furthermore, when wastes contained any form of bedding, they had lowered prevalences and levels of both pathogenic Listeria spp. and Campylobacter spp. Livestock wastes generated by stock consuming a diet composed principally of grass were less likely to harbor E. coli O157 or Salmonella spp. Stocking density did not appear to influence either the levels or prevalences of bacterial pathogens. Significant seasonal differences in prevalences were detected in cattle wastes; Listeria spp. were more likely to be isolated in March to June, and E. coli O157 was more likely to be found in May and June. Factors such as livestock diet and age also had significant influence on the levels and prevalences of some zoonotic agents in livestock wastes. A number of the correlations identified could be used as the basis of a best-practice disposal document for farmers, thereby lowering the microbiological risks associated with applying manures of contaminated livestock to land.

Fate of Escherichia coli O157 and detection of stx phage during fermentation of maize, an animal feedstuff.

AIMS: The fate of inoculated Escherichia coli O157, stx phages and the physico-chemical properties of maize were studied during laboratory-scale fermentation by naturally occurring lactic acid bacteria. METHODS AND RESULTS: Before fermentation, chopped maize was inoculated with 6.2 log(10) CFU g(-1) of a five-isolate mixture of E. coli O157. After fermentation, the silage contained 70.6 g kg(-1) dry matter (DM) lactic acid and 12.8 g kg(-1) DM acetic acid and was pH 4.0. Levels of E. coli O157 fell rapidly, and none of the five isolates could be recovered from the fermenting maize after 8 days. Using a resuscitation step did not consistently enhance recovery of E. coli O157. Stx phages were not isolated from the fermenting maize at any time. Pulsed-field gel electrophoresis (PFGE) genotyping showed that E. coli O157 2975 and 64a/01 survived better than the other three isolates studied. Escherichia coli O157 isolate 1474/00 was particularly sensitive to the laboratory procedures used to harvest the inocula and contaminate the maize. Some colonies recovered during the fermentation had one to three band alterations compared with the initial PFGE genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the five different E. coli O157 genotypes survived maize fermentation. Fermentation of maize produces an animal feedstuff that is unlikely to contain E. coli O157 or stx phages.

Effect of length of time before incorporation on survival of pathogenic bacteria present in livestock wastes applied to agricultural soil.

In response to reports that the contamination of food can occur during the on-farm primary phase of food production, we report data that describes a possible cost-effective intervention measure. The effect of time before soil incorporation of livestock wastes spread to land on the rate of decline of zoonotic agents present in the waste was investigated. Fresh livestock wastes were inoculated with laboratory-cultured Salmonella, Listeria, and Campylobacter spp. and Escherichia coli O157 before they were spread onto soil. Incorporation of the spread wastes was either immediate, delayed for 1 week, or did not occur at all. Bacterial decline was monitored over time and found to be significantly more rapid for all waste types when they were left on the soil surface. There were no significant differences in initial bacterial decline rates when wastes were spread in summer or winter. Our results indicate that not incorporating contaminated livestock wastes into soil is a potential intervention measure that may help to limit the spread of zoonotic agents further up the food chain. The implications of these findings are discussed in relation to current advice for livestock waste disposal.

Levels of zoonotic agents in British livestock manures.

AIMS: To determine the prevalence and levels of zoonotic agents in livestock wastes. METHODS AND RESULTS: A proportionally weighted survey was undertaken and livestock waste samples analysed quantitatively for Escherichia coli O157, pathogenic Listeria, Salmonella, Campylobacter, Giardia and Cryptosporidium. A significant proportion of wastes contained at least one zoonotic agent. Relationships were found between dry matter content and the presence and levels of some zoonotic agents. CONCLUSIONS: British livestock wastes contain measurable levels of the zoonotic agents that cause most cases of gastroenteritis in the UK. SIGNIFICANCE AND IMPACT OF THE STUDY: Animal wastes are disposed of by spreading to agricultural land used for the production of crops and livestock grazing. As British wastes are contaminated with significant levels of zoonotic agents, the practice may represent a way for pathogens to travel further up the food chain.

Pulsed field gel electrophoresis of related Escherichia coli O157 isolates associated with beef cattle and comparison with unrelated isolates from animals, meats and humans.

The pulsed field gel electrophoresis (PFGE) diversity of 51 related Escherichia coli O157 isolates, associated with beef cattle from a single-farm-to-single abattoir (SF-SA) chain of events was determined. The 51 related E. coli O157 isolates from hides, faeces or carcasses of SF-SA cattle produced 11 different PFGE profiles. Also, the PFGE diversity of 6 isolates, associated with a second cattle abattoir, was determined; only two PFGE profiles were found. On the other hand, the PFGE diversity of 136 unrelated E. coli O157 isolates (from healthy meat animals, retail meats and cases of human disease) was also determined. The 136 unrelated E. coli O157 isolates produced 78 different PFGE profiles, most of which (approximately 70%) comprised only one isolate. Overall, the results showed: (a) related E. coli O157 isolates (from both SF-SA events, and the second abattoir) had a markedly narrower clonal profile than the 136 unrelated E. coli O157 isolates; (b) the isolation of identical PFGE types from hide, lairage environment, and carcasses confirms the significance of cross-contamination (both pre-slaughter and during skinning) taking place at abattoirs; and (c) PFGE typing of isolates offers a good tool for tracking sources/routes of such cross-contamination. Such cross-contamination may lead to originally E. coli O157-free animals (and resultant carcasses) becoming contaminated during farm-slaughter-dressing chain of events, so development of efficient control strategies is required.

Fate of Escherichia coli originating from livestock faeces deposited directly onto pasture.

AIMS: To determine the fate of Escherichia coli deposited onto grassland via faeces, from naturally colonized cattle, sheep or pigs. METHODS AND RESULTS: Groups of cattle, sheep and pigs were penned outdoors on grass during November, and removed after 14 days. Escherichia coli populations in the ground declined over 134 days from initial average levels of 5.34, 4.31 and 4.96 log(10) CFU g(-1) in cattle, sheep and pig pens, respectively. The maximum Escherichia coli survival time was up to 162 days (190 days taking sampling interval and deposition time into account), but numbers varied significantly amongst the 20 replicates taken each day. Escherichia coli originating from cattle and sheep had average decimal reduction times (D-values) of 38 and 36 days, respectively; E. coli originating from pigs declined significantly faster (average D-value of 26 days). SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli from livestock faeces can survive on grass for at least 5-6 months, affording opportunity for pathogenic biotypes to contaminate animals, plants or water.

Escherichia coil O157 diversity with respect to survival during drying on concrete.

Shiga toxin (Stx)-producing Escherichia coli O157 isolates (n = 123) were divided into groups according to origin, genotype (pulsed-field gel electrophoresis [PFGE] type, or ribotype), type of Stx produced, or phage type (PT). The survival rate ([number of CFU after 24 h of drying/number of CFU before drying] x 100) for each isolate was determined in triplicate after drying on concrete for 24.0 h. The overall mean survival rate among the 123 E. coli O157 isolates studied was 22.9%, but there was a wide range of responses to drying on concrete, with a minimum of 1.2% and a maximum of 61.9% of the initial inocula being recovered after drying. Among the groups, those isolates that originated from cases of human disease were, on average, significantly more sensitive (P < 0.001) to drying (with a mean survival rate of 15.3%) than isolates from the other three sources (with mean survival rates of 27.7, 26.0, and 22.9% for meats, bovine or ovine feces, and bovine hides, respectively). When the isolates were grouped by genotype, three of the PFGE types were, on average, significantly more resistant to drying than two other PFGE types were, and similarly, significant differences in average resistance to drying between groups of E. coil O157 with different ribotypes were seen. There were no differences between the abilities of isolates producing different Stxs (Stx 1 or Stx 1 and Stx 2) to survive drying. E. coli O157 isolates of PT4, PT21/28, and PT32 survived drying on concrete better than groups of other PTs did. Since the E. coli O157 isolates had various abilities to survive drying on concrete, drying could contribute to a kind of E. coil O157 natural selection along the meat chain. This possibility may have significant meat safety implications if a range of E. coil O157 isolates are simultaneously exposed to drying at any point along the meat production chain. Those E. coil O157 isolates that are more able to survive drying could be more likely to pass farther along the meat chain and ultimately reach consumers.

Combined use of two genetic fingerprinting methods, pulsed-field gel electrophoresis and ribotyping, for characterization of Escherichia coli O157 isolates from food animals, retail meats, and cases of human disease.

Two genetic fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and ribotyping, were used to characterize 207 Escherichia coli O157 isolates from food animals, foods of animal origin, and cases of human disease (206 of the isolates were from the United Kingdom). In addition, 164 of these isolates were also phage typed. The isolates were divided into two general groups: (i) unrelated isolates not known to be epidemiologically linked (n = 154) and originating from food animals, foods and the environment, or humans and (ii) epidemiologically related isolates (n = 53) comprised of four related groups (RGs) originating either from one farm plus the abattoir where cattle from that farm were slaughtered or from one of three different English abattoirs. PFGE was conducted with the restriction endonuclease XbaI, while for ribotyping, two restriction endonucleases (PstI and SphI) were combined to digest genomic DNAs simultaneously. The 207 E. coli O157 isolates produced 97 PFGE profiles and 51 ribotypes. The two genetic fingerprinting methods had similar powers to discriminate the 154 epidemiologically unrelated E. coli O157 isolates in the study (Simpson's index of diversity [D] = 0.98 and 0.94 for PFGE typing and ribotyping, respectively). There was no correlation between the source of an isolate (healthy meat or milk animals, retail meats, or cases of human infection) and either particular PFGE or ribotype profiles or clusters. Combination of the results of both genetic fingerprinting methods produced 146 types, significantly more than when either of the two methods was used individually. Consequently, the superior discriminatory performance of the PFGE-ribotyping combination was proven in two ways: (i) by demonstrating that the majority of the E. coli O157 isolates with unrelated histories were indeed distinguishable types and (ii) by identifying some clonal groups among two of the four RGs of E. coli O157 isolates (comprising PFGE types different by just one or two bands), the relatedness of which would have remained unconfirmed otherwise.

Pulsed-field gel electrophoresis characterization of Shiga toxin-producing Escherichia coli O157 from hides of cattle at slaughter.

Contamination of the brisket areas of the hides of healthy adult cattle with Shiga toxin-producing Escherichia coli O157 at slaughter in England was studied. In total, 73 cattle consignments comprising 584 animals delivered to one abattoir over 3 days during 1 week in July 2001 were studied: 26 cattle consignments arriving on Monday, 32 consignments arriving on Wednesday, and 15 consignments arriving on Friday. Consignment sizes ranged from 1 to 23 animals, with a mean consignment size of 8. The hide of the first animal to be slaughtered in each consignment was sampled by using a sponge swab moistened with 0.85% saline to rub an unmeasured brisket (ventral) area (ca. 30 by 30 cm). The process of isolating E. coli O157 from the swabs consisted of enrichment, screening with immunoprecipitation assay kits, and immunomagnetic separation. E. coli O157 was found on 24 of 73 (32.9%) cattle hides examined, and 21 of these 24 isolates produced Shiga toxins. The 24 E. coli O157 isolates produced six different pulsed-field gel electrophoresis profiles, and 18 (75%) of the isolates were of one prevalent clone. The high prevalence of one E. coli O157 clone on the hides of cattle at slaughter could be due to a high prevalence of that clone on the 18 farms involved (not investigated in the current study), in the postfarm transport or lairage environments, or both. Since the lairage environment, but not the farm of origin or the postfarm transport vehicle, was a factor common to all 18 cattle consignments, it could have played an important role in spreading the prevalent E. coli O157 clone to the cattle hides. Lairage pen floors and the stunning box floor were identified as the most probable sites along the unloading-to-slaughter route at which the brisket areas of cattle hides could become contaminated.

Potential for the spread of Escherichia coli O157, Salmonella, and Campylobacter in the lairage environment at abattoirs.

Prevalences of Escherichia coli O157, Salmonella spp., and Campylobacter spp. were examined in 270 swabs taken from selected sites along the unloading-to-slaughter routes of animal movement in lairages of six commercial abattoirs, three for cattle and three for sheep. The overall prevalences of the pathogens in the respective lairage environments were compared with those for 270 swabs from the pelts of 90 lambs examined in the present study and 270 swabs from the hides of 90 cattle examined in a previous study that were slaughtered at the same abattoirs on the same days. Also, the results obtained were analyzed with the aim of identifying critical points at which animal-environment-animal transfer of the pathogens in lairages occurs. The results showed that (i) the overall prevalences of E. coli O157, Salmonella spp., and Campylobacter spp. were 27.2, 6.1, and 1.1%, respectively, in cattle lairages and 2.2, 1.1, and 5.6%, respectively, in sheep lairages; (ii) the overall prevalences of the three pathogens on cow hides (28.8, 17.7, and 0%, respectively) and sheep pelts (5.5, 7.8, and 0%, respectively) were higher than the overall prevalences in the respective lairage environments; (iii) the most frequently contaminated sites in cattle lairages were holding pen floors (50% of swabs positive for one or more pathogens), entrance gates of stun boxes (27.8% of swabs positive for one or more pathogens), and stun box floors (22.2% of swabs positive for one or more pathogens); (iv) the most frequently contaminated sites in sheep lairages were unloading ramp floors, holding pen floors, and water troughs (33.3, 22.2, and 22.2%, respectively); and (v) overall, cattle lairages and cow hides were more frequently contaminated with the pathogens than were lamb lairages and lamb pelts. Further research is needed to develop strategies for the incorporation of pathogen control in lairages into integrated microbial meat safety systems.

Can food-related environmental factors induce different behaviour in two key serovars, 4b and 1/2a, of Listeria monocytogenes?

Listeria monocytogenes isolates (81 in total; 42 isolated from cases of human listeriosis: 39 isolated from food), belonging to serovars 1/2a or 4b, were studied for any group differences between serovars to selected factors associated with foods (two bacteriocins and mild heat treatment), growth kinetics at 37 degrees C and pathogenicity for chick embryos. The isolates were tested for sensitivity to two bacteriocins at 4 degrees C and 37 degrees C, and were tested for the remaining parameters both before and after exposure to cold storage (4 degrees C) with starvation. In addition, the isolates were typed using pulsed field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE) and phage typing to find any correlation between the types and group differences in the chosen parameters. Considerable strain diversity within each L. monocytogenes serovar with respect to the chosen parameters was observed, especially after exposure to cold storage. Nevertheless, the serovar 1/2a isolates, as a group, tended to be more resistant to the two antilisterial bacteriocins at 4 degrees C than the group of serovar 4b isolates. In contrast, after cold storage at 4 degrees C, L. monocytogenes serovar 4b isolates, as a group, tended to be more resistant to heat treatment at 60 degrees C than the group of 1/2a isolates. In addition, the serovar 4b group tended to have shorter lag phases and higher pathogenicity, when transferred from cold storage to body temperature (37 degrees C), than the group of serovar 1/2a isolates. No correlation between PFGE-, MEE- and phage-types and the tested parameters was found. Although the above serovar-related differences were observed only when mean values of the groups were compared (not all isolates within each group followed the group pattern), the results indicate interesting directions for further research.

The case for routine HIV screening before IVF treatment: a survey of UK IVF centre policies.

The case for routine human immunodeficiency virus (HIV) screening of all couples seeking assisted reproductive treatment is so strong that it should be made obligatory for all couples entering IVF programmes to be given information about HIV transmission, and offered testing. In August 1999, questionnaires regarding routine HIV screening of couples seeking IVF treatment were sent to the medical directors of the 74 licensed assisted conception units in the UK. Of the 45 (60.8%) centres who responded, 19 (42.2%) routinely screen both partners for HIV antibodies, 25 (55.5%) do not screen and one centre selectively screens high-risk patients. There was no significant difference in the proportion of centres that routinely carried out screening with regards to the unit size: six out of 13 (46.2%) small units compared with 13/32 (40.6%) large units. In all, 17 centres (37.8%) rated HIV screening as essential, nine (20%) as desirable, 11 (24.4%) as not required, while eight (17. 8%) centres did not comment. Of the 19 centres that have a routine screening policy, 18 have management protocols in the event that the test is positive. Of these 18 centres, 12 adhere rigidly to the protocol, while five centres adhere to the protocol with few exceptions and the remaining one uses its protocol for guidance only. The main reasons for not employing routine HIV screening were: the lack of cost effectiveness, low prevalence of HIV infection in their population, necessity for and cost of counselling, uncertainty about the need for screening and potential delay to start of treatment.

Escherichia coli O157:H7 in healthy dairy cows.

Faecal samples were taken from 371 cows originating from 55 dairy farms and slaughtered at one slaughterhouse; tonsils were taken from 215 of these animals. Escherichia coli 0157:H7 was found in the faeces of only two animals and was not found in any tonsils. The farm supplying the first positive cow detected at the slaughterhouse was visited 3 months later and 160 animals (80 cows and 80 heifers) were tested by rectal swabs; E. coli 0157:H7 was not isolated.

Differences in pathogenicity for chick embryos and growth kinetics at 37 degrees C between clinical and meat isolates of Listeria monocytogenes previously stored at 4 degrees C.

Fifteen clinical strains of Listeria monocytogenes (eight strains of serogroup 4 and seven strains of serogroup 1) and 15 meat isolates (all serogroup 1) were stored with no growth in phosphate-buffered saline (pH 7.0) at 4 degrees C for 4 weeks. Pathogenicity for 14 day old chick embryos and growth kinetics in brain heart infusion (BHI) broth at 37 degrees C of the strains were determined before and after storage. Although no differences in pathogenicity between clinical and meat strains were found when tested as fresh cultures significant differences became apparent after cold storage. Firstly, the pathogenicity of clinical strains was not affected by storage, whereas the average mortality of embryos inoculated with meat strains decreased from 98.7 to 68.0%. Secondly, clinical strains subcultured at 37 degrees C had a significantly shorter average lag phase than meat strains after cold storage. The results of this study indicate that strains that caused human listeriosis have a higher resistance to the effects of unfavourable storage conditions than meat strains with respect to pathogenicity and lag phase duration at body temperature.

Insufficient antilisterial capacity of low inoculum Lactobacillus cultures on long-term stored meats at 4 degrees C.

Two of the 210 lactobacilli strains isolated from chilled meats produced antilisterial bacteriocins: Lactobacillus sake 265 (Lb 265) and Lactobacillus casei 52 (Lb 52). Factors affecting antilisterial effectiveness of these and two other bacteriocin-producing (Bac+) strains (Lactobacillus sake 706, Lb 706; and Lactobacillus sake 148, Lb 148) at refrigeration temperature (4 degrees C) were studied in laboratory media and meat systems. At both 4 degrees C and 25 degrees C, these Bac+ strains grown in buffered MRS broths (pH 5.4 or 6.5) showed longer lag phases and shorter generation times than Listeria monocytogenes (mixture of strains NCTC 7973 and two food derived strains, L70 and L72) when grown in buffered BHI broths at the same pH values. These differences were more significant at 4 degrees C than at 25 degrees C. The highest concentrations of bacteriocin in MRS broth were produced at 25 degrees C and 4 degrees C by strain Lb 265 and Lb 706, respectively. Generally, production of bacteriocins was more efficient at lower pH (in buffered MRS broths of pH 5.4 and unbuffered MRS broths), than at higher pH (in buffered broths of pH 6.5). On vacuum packaged, raw beef (pH 5.3-5.4) initial numbers of L. monocytogenes (10(3)/g) did not change significantly during 23-days storage at 4 degrees C, when inoculated either alone or in the presence of Bac+ strains inoculated at initial levels of 10(3)/g. On vacuum packaged emulsion-type of sausages (pH 6.4) inoculated with L. monocytogenes and stored at 4 degrees C for 23 days growth was not significantly affected by addition of Bac+ strains at initial levels of 10(3)/g. These results indicated that amounts of bacteriocins produced in situ by low initial numbers (10(3)/g) of the protective strains tested were not sufficient to inhibit and/or reduce L. monocytogenes on these chilled meats, where high initial numbers of lactic acid bacteria are not desirable for product quality resons. To achieve these effects, higher concentrations of active (free) bacteriocins in meats must be provided.