Wild birds and silage as reservoirs of Listeria in the agricultural environment

A method for the isolation of listeria which enabled a more rapid detection of the organism was used to examine samples of silage and bird faeces. Faecal samples indicated that seagulls feeding at sewage works had a higher rate of carriage than those elsewhere. Faecal samples from rooks generally suggested a low incidence of listeria except on one occasion when eight of twenty samples contained Listeria monocytogenes: this coincided with the nesting season and the peak period for listeriosis in sheep. The incidence of L. monocytogenes in clamp silages ranged from 2.5-5.9%, but in samples of big bale silages the incidence was 22.2% and, when mouldy samples were selected, 44%.     

The rapid demonstration and presumptive identification of Listeria monocytogenes in food using monoclonal antibodies in a direct immunofluorescence test (DIFT)

Two monoclonal antibodies conjugated to fluorescein isothiocyanate (FITC) were successfully used in a direct immunofluorescence test (DIFT) to demonstrate listeria in seven samples of soft cheese where Listeria monocytogenes had been cultured by conventional techniques. Using DIFT, listeria was not detected in 20 cheese samples from which L. monocytogenes had not been isolated, or was present in low numbers (< 10²/g). The DIFT was also used to presumptively identify > 90% of strains of L. monocytogenes isolated from food and cultured on Modified McBride agar or Blood agar. Less than 10% of strains of other species of listeria would be misidentified when grown on these media. All tests were carried out within 2 h.     

Incidence of Listeria monocytogenes in an acidified fish product, ceviche

Thirty-two samples of ceriche , a South American acidified fish product, were tested for the presence of listeria. Listeria innocua was isolated from 24 samples (75%) and L. monocytogenes was isolated from three samples (9%) of those tested.     

Incidence of Listeria in tropical fish and fishery products

Thirty-five tropical fish and fishery products were tested for the presence of listeria. Listeria innocua was detected in three of 10 fresh samples and five of 14 frozen samples. No listeria were detected in dried, salted fish. Listeria monocytogenes was not detected in any of the samples tested.     

A technique for the direct identification of haemolytic-pathogenic listeria on selective plating media

A technique based on the addition of a red cells top layer to a selective plating medium after listeria growth is proposed in order to detect directly the haemolytic activity of pathogenic listeria colonies. It was applied to different selective plating media (modified McBride agar, lithium chloride-phenylethanol-moxalactam, listeria selective medium–Oxford formulation, polymyxin-acriflavine-lithium chloride-ceftazidime-aesculin-mannitol and LSAMM). The haemolytic activity of listeria colonies was more easily detected with the top layer than when red cells were incorporated in the selective plating medium. The LSAMM was the best medium for the recovery and identification of Listeria monocytogenes colonies by this technique (three Listeria monocytogenes colonies were distinguished among 2520 Listeria innocua colonies in raw milk).     

Methods for improved recovery of Listeria monocytogenes from cheese.

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

Methods for improved recovery of Listeria monocytogenes from cheese

Method of homogenization (Waring blender versus stomacher), type of diluent (tryptose broth [TB] versus aqueous 2% trisodium citrate), and temperature of diluent (20 versus 40 degrees C) were compared for recovery of Listeria monocytogenes from freshly made and ripened Colby cheese. By using direct plating on McBride listeria agar, significantly higher numbers of L. monocytogenes were recovered when cheese samples were (i) homogenized for 2 min with the blender rather than the stomacher (P less than 0.01), (ii) diluted in trisodium citrate rather than TB (P less than 0.01), and (iii) diluted in diluents at 40 rather than 20 degrees C (P less than 0.05). Based on these results, a new diluent/enrichment medium was developed by adding 2% trisodium citrate to TB (TBC). Despite superior results with the blender, biosafety concerns led to use of the stomacher for homogenization of cheese samples; hence, the stomaching time was increased to 3 min. Results obtained by direct plating indicated that recovery of L. monocytogenes from Colby cheese and from curd samples taken during manufacture of brick cheese increased when samples were diluted 1:10 in TBC at 45 degrees C and stomached for 3 min, as compared with similarly treated samples diluted in TB at 25 degrees C. A similar comparison of both diluents for recovery of L. monocytogenes from cold-pack cheese food yielded bacterial counts which were not significantly different. Recovery of L. monocytogenes from cold-enriched (at 4 degrees C for up to 8 weeks) samples of Colby cheese and cold-pack cheese food was generally similar for samples homogenized in TBC or TB.     

The potential use of nisin to control Listeria monocytogenes in poultry

Nisin (Nisaplin^TM) was added to scald water and used to treat Listeria monocytogenes which were either directly suspended in the water or attached to turkey skin. There was at least a 1 log decrease of listeria for the nisin treatments. This was followed by further reductions when the samples were stored under refrigeration. Heat exhibited a synergistic relationship with the nisin when the cells were directly suspended in the scald water. A 2 log decrease was observed immediately after this treatment and there was complete elimination of the listeria after 48 h of refrigeration. However, this relationship was not as obvious when the cells were first attached to the turkey skin. In these latter experiments, the nisin treatments resulted in reductions in the listeria levels but they were smaller than those observed when the cells were directly suspended in the scald water.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Sensitive and specific detection of Listeria monocytogenes in milk and ground beef with the polymerase chain reaction.

A sensitive and specific method for detection of Listeria monocytogenes in milk and ground-beef samples is described. It consists of culturing samples in listeria enrichment broth (LEB) and subculturing them from LEB to listeria plating media, followed by DNA extraction and species-specific detection of the organism by using the polymerase chain reaction (PCR). In developing the L. monocytogenes PCR assay, five oligonucleotide primers complementary to the nucleotide sequence of the listeriolysin O gene were synthesized and used in amplification experiments. PCR products of the predicted size, based on nucleotide sequence information, were generated with DNA from all of 72 L. monocytogenes strains with five different primer pairs. DNA from Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeia murrayi strains and a panel of 47 bacterial strains representing 17 genera did not generate PCR products with the primer pairs employed. As little as 1 pg of L. monocytogenes DNA could be detected with the assay. To determine the most sensitive culture protocol to use in conjunction with the PCR assay, milk (10 ml) and ground-beef (25 g) samples were inoculated with L. monocytogenes at concentrations ranging from 0 to 10(5) CFU ml-1 or g-1, as appropriate for the sample. PCR assays on DNA extracted from growth on listeria plating media, inoculated with 24-h LEB samples cultures, were most sensitive, allowing detection of as little as 0.1 CFU of L. monocytogenes ml-1 or g-1 of milk and ground beef, respectively.     

Listeria monocytogenes in Faeces from Clinically Healthy Dairy Cows in Sweden

Faecal samples from 102 clinically healthy dairy cows, representing 34 farms in the Swedish province of Uppsala, were analysed for the presence of Listeria spp. using an enrichment procedure. Listeria monocytogenes was isolated from six (6%) and L. innocua from 2 (2%) cows. From each of the 6 samples positive for L. monocytogenes, 5 isolates were further characterised by restriction enzyme analysis using the 3 enzymes Apa I, Sma I, and Asc I, followed by pulsed-field gel electrophoresis. Three of the L. monocytogenes positive cows lived at the same farm, and they all harboured the same clonal type. One of these 3 cows also harboured a further clonal type of L. monocytogenes. The fact that one of the cows harboured 2 different clonal types of L. monocytogenes is important from an epidemiological point of view when routes of infection are to be investigated.     

Quantifying translocation of Listeria monocytogenes in rats by using urinary nitric oxide-derived metabolites

The urinary nitric oxide metabolites NO(2)(-) and NO(3)(-) (summed as NO(x)) are a noninvasive, quantitative biomarker of translocation of salmonella from the intestinal lumen to systemic organs. Listeria monocytogenes is a food-borne gram-positive pathogen that can also cross the intestinal epithelium. In this study, we tested the efficacy of urinary NO(x) as a marker of listeria translocation. Rats (eight per group) were orally infected with increasing doses of L. monocytogenes; control rats received heat-killed listeria. The kinetics of urinary NO(x) and population levels of listeria in feces were determined for 7 days. Another group of rats was killed 1 day after infection to verify translocation by culturing viable listeria from systemic organs. Oral administration of increasing doses of L. monocytogenes resulted in a time- and dose-dependent increase in urinary NO(x) excretion. Translocation was a prerequisite for inducing a NO(x) response, since heat-killed L. monocytogenes did not elevate NO(x) excretion in urine. Fecal counts of listeria also showed dose and time dependency. Moreover, the number of viable L. monocytogenes cells in mesenteric lymph nodes also increased in a dose-dependent manner and correlated with urinary NO(x). In conclusion, urinary NO(x) is a quantitative, noninvasive biomarker of listeria translocation.     

Characterisation of aerobically grown non-spore-forming bacteria from paper mill pulps containing recycled fibres

A total of 179 non-spore-forming bacteria aerobically growing on Nutrient Agar, Plate Count Agar or in specific enrichment conditions for salmonella, campylobacteria, listeria, yersinia or staphylococci, were isolated from 16 untreated paper mill pulps. After phenotypical screening the isolates were characterised by automated ribotyping and partial sequencing of the 16S rRNA gene. They could be divided into seven taxonomical classes representing 63 taxa (species): actinobacteria (11 species), bacilli (7), flavobacteria (3) alphaproteobacteria (10), betaproteobacteria (5), gammaproteobacteria (25) and sphingobacteria (2). Most of the gammaproteobacteria were enterobacteria, mainly species of the genera Enterobacter (7 species, 7 samples/3 mills) and Klebsiella (5 species, 6 samples/3 mills). Other commonly occurring bacteria were most closely related to Microbacterium barkeri (7 samples/3 mills), Cloacibacterium normanense (6 samples/2 mills), Pseudoxanthomonas taiwanensis (5 samples/2 mills) and Sphingobacterium composti (5 samples/1 mill). Sporadic isolates of Listeria innocua, L. monocytogenes, Enterococcus casseliflavus and Staphylococcus warneri were detected, from which only L. monocytogenes is considered to be a food pathogen. No isolates of the genera Campylobacter, Salmonella or Yersinia were detected. The detected bacteria may be harmful in process control, but the load of food pathogens with recycled fibres to paper machines is insignificant. Faecal contamination of the pulp samples was not indicated.     

Effects of gaseous environment and temperature on the storage behaviour of Listeria monocytogenes on chicken breast meat

Portions of skinless chicken breast meat (pH 5.8) were inoculated with a strain of Listeria monocytogenes and stored at 1, 6 or 15 degrees C in (1) aerobic conditions; (2) 30% CO2 + air; (3) 30% CO2 + N2; and (4) 100% CO2. When samples were held at 1 degree C the organism failed to grow under any of the test conditions, despite marked differences between treatments in spoilage rate and ultimate microflora. At 6 degrees C counts of L. monocytogenes increased ca 10-fold in aerobic conditions before spoilage of the meat, but only when the inoculum culture was incubated at 1 degree C rather than 37 degrees C. In CO2 atmospheres growth of L. monocytogenes was inhibited on meat held at 6 degrees C, especially under 100% CO2. By contrast, storage at 15 degrees C led to spoilage of the meat within 2 d, in all gaseous environments, and listeria levels increased up to 100-fold. Differences in the behaviour of L. monocytogenes on poultry and red meats are discussed.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland.

The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% (Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% (L. monocytogenes 33.3%); this was higher than that in samples from dairy farms (Listeria spp. 8.8%; L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples (L. monocytogenes) and 1 of 33 soft cheese samples (L. seeligeri). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Occurrence of Listeria species in raw milk and dairy products produced in Northern Ireland

J. HARVEY AND A. GILMOUR. 1992. The overall incidence of Listeria spp. in raw milk samples surveyed was found to be 25.0% ( Listeria monocytogenes 15.3%), with the incidence in samples from processing centres 54.0% ( L. monocytogenes 33.3%); this was higher than that in samples from dairy farms ( Listeria spp. 8.8% L. monocytogenes 5.3%). The FDA enrichment procedure was much more productive than cold enrichment and Oxford agar was superior to modified McBride agar for isolation of Listeria. Listeria monocytogenes was never isolated by direct plating of raw milk samples on Oxford agar at a detection level of 1.0 cfu/ml. Listeria spp. were isolated from 1 of 95 pasteurized milk samples ( L. monocytogenes ) and 1 of 33 soft cheese samples ( L. seeligeri ). Restriction fragment length polymorphism was more useful than sero- or phage-typing for typing of L. monocytogenes strains, and results suggest that specific L. monocytogenes strains may persist in both farm and processing environments.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

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.     

Listeria species escape from the phagosomes of interleukin-4-deactivated human macrophages independent of listeriolysin

Listeria monocytogenes is the causative agent of infections like sepsis and meningitis, especially in immunocompromised hosts. Human macrophages are able to phagocytose and digest L. monocytogenes but IL-4 prevents human macrophages from killing the bacteria, the mechanisms of which are unknown. In the present study, we examined various listeria species and strains including wild-type and deletion mutants in human macrophages pretreated with IL-4. To analyse the IL-4-mediated deactivation process, we combined quantitative infection assays with various morphologic methods. IL-4 facilitates survival and escape of the pathogenic L. monocytogenes wild-type strain 10403S from the macrophage phagosomes. In untreated macrophages, the isogenic listeriolysin deletion mutant strain DP-L2161 was killed and did not escape from the phagolysosomes. However, after macrophage deactivation with IL-4 DP-L2161 survived and escaped from the phagosomes. This was also the case, but to a lesser extent, even for the naturally avirulent L. innocua. As detected by confocal laser-scanning fluorescence microscopy and electron microscopy, IL-4 permitted the escape of all listeria species tested, including DP-L2161 and L. innocua from the phagosomal compartment of the macrophages. We conclude that escape from the phagosome and survival of the listeria species tested in IL-4-deactivated human macrophages is independent of the virulence factor listeriolysin.     

The incidence of Listeria monocytogenes in raw milk from farm bulk tanks in North-East Scotland

Over 180 farm bulk milk tanks were tested for the presence of Listeria monocytogenes on three separate occasions which included periods when cows were grazing and confined inside on a silage diet. The incidence of L. monocytogenes contamination was low, ranging from 3.8% in the summer samples to 1.0% in October. The level of contamination was estimated to be lower than one L. monocytogenes bacterium per ml in positive samples, as most required cold enrichment of 10–20 ml volumes before recovery. The distribution was sporadic; only one farm gave positive isolations on all three sampling occasions, one other on two, and all others were from different farms. No correlation between the presence of L. monocytogenes and hygiene standards or the feeding of silage was found.