Incidence of Listeria monocytogenes in wild animals in Japan

Between 1991 and 1993, the intestinal contents and feces of wild animals in Japan were examined for the presence of Listeria. The wild animals examined included 623 mammals (11 species) and 996 birds (18 species). Listeria species were isolated from 38 (6.1%) of the 623 mammalian samples and 133 (13.4%) of 996 bird samples. The highest incidence of Listeria in the mammals was found in Japanese monkeys (20.0%) and that in birds was found in crows (43.2%). The incidence of Listeria in Japanese monkeys varied from 0 to 40.0% depending on the capture area. L. monocytogenes was isolated from II of these positive samples. Serovars 1/2a and 4b predominated in eight serotyped L. monocytogenes isolates.     

Prevalence of Listeria monocytogenes in intestinal contents of healthy animals in Japan.

A total of 1,705 fecal specimens or ileo-cecal contents of cattle, pigs, dogs, cats, chicken and rats were submitted for the isolation of Listeria monocytogenes by the use of the combination of Oxford-LPM agar plates after the cold enrichment in PBS at 4 degrees C for 4-6 weeks. Prevalence of L. monocytogenes was found to be 1.9% in cattle, 0.6% in pigs, 0.9% in dogs and 6.5% in rats. However, none of L. monocytogenes was isolated from chicken or cats. Among 26 isolates of L. monocytogenes, 13 strains (50%) were classified into types 1/2a (3 strains), 1/2b (5 strains) and 4b (5 strains) and were often associated with human listeriosis. The majority of the Listeria spp. other than L. monocytogenes isolated from these animals was found to be L. innocua.     

Occurrence of Listeria spp. in captive antelope herds and their environment.

Two juvenile scimitar-horned oryx (Oryx dammah) at the Wild Animal Park Planckendael died from acute septicemia caused by Listeria monocytogenes serovar 4b. Subsequently, Listeria spp. were isolated from the feces, food, and environment of seven antelope species and examined using a two-stage enrichment procedure in Fraser Broth, followed by isolation on PALCAM agar. A total of 40/170 samples (23.5%) was positive for Listeria spp. No organisms were cultured in 83/170 samples (48.8%), and 47 samples (27.6%) were overgrown with Bacillus spp. Nonpathogenic Listeria spp. were isolated from 16/70 fecal samples, 22/40 soil samples, and 2/60 feed samples. Listeria monocytogenes serovar 1/2b was isolated from two soil samples collected in the enclosure of the scimitar-horned oryx.     

Detection of pathogenic Listeria spp. in zoo animal faeces: use of immunomagnetic separation and a chromogenic isolation medium

Faecal samples, collected from 200 healthy animals in Antwerp Zoo, were examined for the presence of pathogenic Listeria spp. A two-stage standard isolation (ISO) method was combined with immunomagnetic separation (IMS). ALOA agar, a chromogenic isolation medium, differentiating Listeria spp. on the basis of beta-glucosidase and phosphatidylinositol-specific phospholipase C (PIPLC) activity, was compared with PALCAM agar. Confirmation of the isolates was based on conventional biochemical tests and a disc test, which detects a specific aminopeptidase produced by all Listeria spp. except Listeria monocytogenes. Listeria spp. were isolated from 42 (21.0%), L. monocytogenes from 14 (7.0%), and Listeria ivanovii from two (1.0%) faecal samples. The application of IMS after primary enrichment detected pathogenic Listeria spp. in 12 (6.0%) samples. The ISO method, combining primary and secondary enrichment, detected pathogenic Listeria spp. in 15 (7.5%) samples. The sensitivity of IMS compared to the ISO method was 73.3% and the specificity was 99.5%. ALOA agar was superior to PALCAM agar for isolation of Listeria spp. The disc test identified all L. monocytogenes isolates. IMS after primary enrichment was a suitable screening method, but secondary enrichment increased the number of positive samples.     

Influence of bacteriocin-like substance, generation times, and genetic profiles of Listeria innocua on the isolation of Listeria monocytogenes

Inhibition of isolation of Listeria monocytogenes by bacteriocin-like substance (BLS)-producing Listeria innocua after enrichment culture was investigated. When 26 L. monocytogenes strains were examined in combination with eight L. innocua strains using the spot on lawn method, 52/208 (25.0%) combinations showed the growth inhibition of L. monocytogenes. When two Listeria species were cultured simultaneously in selective enrichment broth, inhibition of isolation of L. monocytogenes was observed in 12/52 of the combinations at 24h (23.1%), in 24/52 at 48h (46.2%) and in 30/52 (57.7%) after 7 days of incubation. The randomly amplified polymorphic DNA profiles showed no interstrain similarities between either strains of the BLS-producing L. innocua or the BLS-sensitive L. monocytogenes strains. Therefore inhibition by BLS-producing L. innocua of isolation of L. monocytogenes after enrichment culture is unlikely to be dependent upon a particular genetic profile.     

Epidemiological studies on the occurrence of Listeria monocytogenes in the feces of dairy cattle

Seasonal variation in the fecal shedding of Listeria spp. in dairy cattle was examined by collecting a total of 3,878 fecal samples during a period of two years. The prevalences of Listeria spp. and L. monocytogenes were higher during the indoor season (12.7% and 9.2%, respectively) than in samples collected from the animals on pasture (5.3% and 3.1%, respectively). The highest frequencies of Listeria spp. (19.4%) and L. monocytogenes (16.1%) were detected in December. Listeriae were isolated from at least one of the dairy cows from 45.8% of the 249 herds examined. 2.9% of the 314 milk samples collected from the farm bulk tanks on 80 dairy farms on four different occasions yielded L. monocytogenes. The seasonal occurrence of these bacteria in milk reflected the frequencies of Listeria in the fecal material but not those in the main roughage used; grass silage and pasture grass. Fecal material is considered to be a potential source of contamination of raw milk by L. monocytogenes. Investigation of the numbers of viable Listeria organisms in different animal fodders is considered essential in further epidemiological studies of these bacteria.     

Listeria monocytogenes in horses in Iceland

Twenty isolates of Listeria monocytogenes associated with five confirmed and four suspected incidents of listeriosis in horses in Iceland were characterised by serotyping, pulsed-field gel electrophoresis and ribotyping. Semiquantitative estimates of the numbers of L monocytogenes were made on faeces from horses with clinical signs of listeriosis and on grass silage fed to them. Large numbers of L monocytogenes were often found in the faeces of horses with severe signs of disease. The 20 isolates could be divided into six genotypes, each incident involving only one genotype. One serovar 1/2a genotype was associated with three confirmed incidents of listeriosis in 1991, 1993 and 1997. In one incident, the same genotype was isolated from the organs of a horse with listeriosis and from the spoiled grass silage fed to it.     

Isolation of Listeria monocytogenes from the skin of slaughtered beef cattle

We attempted to isolate Listeria monocytogenes from skin, contents of large intestines and carcasses of cattle introduced to a slaughterhouse in order to identify source of contamination for this pathogen. Sixty skin samples, 60 samples of the contents of large intestines and 30 carcass samples were colleted in June, August and November 2003 for use in this study. Listeria spp. and L. monocytogenes were isolated from 30 (50%) and 3 (5%) of the cattle skin samples, respectively. However, no Listeria spp., including L. monocytogenes, were isolated from intestinal contents or carcasses. Seven isolates were obtained, of which five and two strains were serotypes 1/2a and 1/2b, respectively. Genetic analysis suggested that there was persistent inhabitation of the pathogen around the area investigated in this study.     

A direct plating method for monitoring the contamination of Listeria monocytogenes in silage.

Twenty-two silage samples were analyzed for the presence of L. monocytogenes using five Listeria selective plating media, with and without previous selective enrichment step. L. monocytogenes was recovered from 3 samples by both procedures, but direct plating allowed the quantification of Listeria population. Two of these positive samples were implicated in outbreaks of listeriosis in sheep; the L. monocytogenes population in these samples was about 10(6) cells/g. The L. monocytogenes population in the other positive sample was 10(3) cells/g. Direct isolation of L. monocytogenes was only possible from LPM, PALCAM and LSAMm media. MOX and LSM media were not selective enough to allow direct Listeria isolation. In our hands, LSAMm was the most suitable plating medium for the direct isolation and specific quantification of L. monocytogenes from silage employing a red blood cells overlay technique.     

The occurrence of pathogenic Listeria monocytogenes and antibodies against listeriolysin-O in buffaloes

The occurrence of Listeria monocytogenes in meat and milk samples, and antilisteriolysin O (ALLO) antibodies in sera of buffaloes were studied. Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. The pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test and mouse inoculation test. Of 167 meat samples 2.4 and 10.17% were positive for L. monocytogenes and Listeria sp., respectively. Of the 64 milk samples 6.25 and 26.13% were positive for L. monocytogenes and Listeria sp., respectively. A total of 284 serum samples were tested by listeriolysin O (LLO)-based indirect enzyme-linked immunosorbent assay of which 25.35% were found to be seropositive. The culture positivity for L. monocytogenes and detection of ALLO did not show any agreement (kappa = 0.035). The prevalence of pathogenic L. monocytogenes in milk and meat and the occurrence of anti-LLO antibodies is of concern from the public health point of view.     

The diagnosis of Listeria encephalitis in ruminants using cultural and immunohistological techniques. I. Comparison of different selective media and culture techniques for the detection of Listeria from ruminant brains]

The selective L-PALCAMY differential enrichment broth, the Listeria enrichment broth of the International Dairy Federation, Oxford Listeria selective agar, and PALCAM Listeria selective agar were comparatively examined in the cultural isolation of Listeria spp. from ten ruminant brains. The L-PALCAMY medium proved to be superior to the IDF broth in both selectivity and productivity for Listeria spp. in the brain samples, which were also contaminated with other bacteria. The Oxford and PALCAM agars corresponded in their productivity for Listeria spp. The latter, however, was more selective than the Oxford agar. Bacterial counts of up to 1.2 x 10(9) CFU/g of brain stem sample were made from Listeria monocytogenes (L.m.), and up to 6.2 x 10(4) CFU/g from Listeria innocua. A total of 164 brains from ruminants showing CNS disturbances and/or pathoanatomical CNS alterations were examined using L-PALCAMY medium, and Oxford and PALCAM agar. L.m. could be isolated from 29 of the brains, and Listeria innocua from five. Cultural isolation of both Listeria spp. occurred in one brain. Of 27 brains containing L.m., which were also examined using cold enrichment, L.m. was isolated in 59.3% of the cases with direct culture, in 81.5% of the cases using selective warm enrichment, and in 77.8% of the cases by means of selective cold enrichment. Five cases each were identified solely by cold or warm enrichment, respectively. In investigations of further 69 ruminant brains the number of brains shown to contain L.m. could be increased from seven to 13 by means of selective cold enrichment for three months.     

Isolation of pathogenic Listeria monocytogenes and detection of antibodies against phosphatidylinositol-specific phospholipase C in buffaloes

The isolation of pathogenic Listeria spp. in bacteriological samples, and anti-phosphatidylinositol-specific phospholipase C (anti-PIPLC) antibodies in sera of buffaloes were studied. Isolation of the pathogen was attempted from the samples by selective enrichment in University of Vermont Medium and plating onto Dominguez-Rodriguez isolation agar. Pathogenicity of the isolates was tested by Christie, Atkins, Munch Petersen test and mice incoulation test. Listeria spp. and L. monocytogenes were isolated from 8.8 and 2.4%, and 4.8 and 1.6% of 125 each meat and blood samples, respectively. Out of the 125 samples each of feacal, nasal and vaginal swabs from buffaloes 8 and 4%, 13.6 and 2.4%, and 6.4 and 2.4% were positive for Listeria spp. and L. monocytogenes, respectively. L. ivanovii was confirmed from 0.8% vaginal sample. A total of 125 serum samples were tested by phosphatidylinositol-specific phospholipase C (PIPLC) based indirect ELISA of which 4.0% turned out to be seropositive.     

The occurrence of Listeria in the production of processed meat, salami and mettwurst

In six Swiss meat-processing plants 206 samples of cured and air-dried beef (Bündnerfleisch), salami and Mettwurst were analyzed for the presence of Listeria spp. Samples were taken during the fabrication, fermentation and drying of the products. Out of 44.7% of all samples Listeria spp. could be detected. 6.8% turned out to be L. monocytogenes, 37.4% L. innocua and 0.5% L. seeligeri. Listeria spp. were found in all production stages of the tested foods. The concentration of L. monocytogenes was always less than or equal to 20 MPN/g. 86% of the isolated strains formed part of the serogroup 1/2 and 14% of the serogroup 4. Listeria spp. could only be found on the surface of Bündnerfleisch. Both, L. monocytogenes and L. innocua were able to survive the maturation process of salami, even when the initial concentration was very low. The ripening was more often survived by L. innocua than by L. monocytogenes. It appeared that Mettwurst had the highest contamination rate of Listeria spp. (94.4%), followed by salami (46.7%) and Bündnerfleisch (23.1%). The corresponding proportions for L. monocytogenes were 8.0% (salami), 5.8% (Bündnerfleisch) and 0% (Mettwurst). Listeria spp. positive samples were found in every examined plant, L. monocytogenes in five of therm. The Listeria spp. contamination rates moved from 10.0% to 86.2%, those of L. monocytogenes from 0% to 12.1%.     

Comparison of direct colony count methods and the MPN-method for quantitative detection of Listeria in model and field conditions]

In order to compare the plate count method for quantitating Listeria, as published in the "Official Collection of Testing Methods" in section 35 LMBG (L. 00.00-22), to an MPN-method for Listeria based on the same mediums, these two detection methods for Listeria were tested in three sets of experiments and a routine sample status evaluation. A pure broth culture of L. monocytogenes, artificially with L. monocytogenes contaminated ground meat, artificially contaminated and cold stored ground meat as well as 77 ground beef samples from Berlin retail food stores were used in the four trials. The detection limit of the MPN-method is about 66% lower than the plate count method allowing detection of a clearly greater number of Listeria-positive samples from naturally contaminated ground meat. The MPN-method yielded more Listeria spp.-positive samples (rel. 43%) and more L. monocytogenes-positive samples (rel. 21%) versus the colony count method based on the results from the field trial using ground beef samples from retail food stores in Berlin. Nevertheless the standardized colony count method is preferred over the MPN-method for routine use because of its slightly higher productivity and much smaller variation in the results. However, the MPN-method is preferable for epidemiological studies because of the significance of the lower detection level. The random sampling evaluation of ground beef from retail stores indicated that 39% of the samples were Listeria spp.-positive and 31% were L. monocytogenes-positive when using the colony count method. A total of 56% of the meat samples were found to be Listeria spp.-positive and 38% L. monocytogenes-positive when the MPN-method was used. Population levels ranged from 10 to 580 cfu/g (Listeria spp.-positive samples) and from 10 to 270 cfu/g (L. monocytogenes-positive samples) for the colony count method. The MPN-method yielded population levels of 3.6 to 930 MPN/g for Listeria spp.-positive samples and 3.6 to 150 MPN/g for L. monocytogenes-positive samples. L. monocytogenes strains isolated using the colony count method belonged to the following serovars: 1/2a (46%), 1/2b (13%), 1/2c (33%), 3b (4%) and 4c (4%). A similar serovar isolation pattern was found for L. monocytogenes-positive MPN-tubes. The most common serotype was 1/2a (43%), followed by 1/2c (32%) and 1/2b (14%). The serotypes 3c, 4b and 4c were all isolated 4% of the time.     

Prevalence of Listeria (spp.) in wild rats captured in the Kanto area of Japan.

In the Kanto area a total of 245 wild rats were captured. All rats captured in Ikebukuro (110 rats) and 9 out of 41 rats in Yokohama were Rattus rattus, and all other 126 rats were Rattus norvegicus. In Kashima and Ikebukuro, listeria was isolated from 28 rats (77.8%) and 27 rats (24.5%), respectively, but in the other 4 areas listeria was isolated from 0-7% rats. Listeria monocytogenes was isolated from 12 rats (10.9%) captured in Ikebukuro and 2 rats in Kashima and Numazu. The frequent isolation of L. monocytogenes in buildings suggests the possibility of R. rattus as a reservoir of L. monocytogenes and the continual environmental contamination in buildings by L. monocytogenes.     

Listeriosis in sheep. Isolation of Listeria monocytogenes from grass silage.

Two hundred and ninety-one grass silage samples from 113 farms with recent outbreaks of listeriosis were examined for the presence of Listeria monocytogenes (Lm). The frequency of Lm isolations increased with increasing pH. Lm was isolated from 22 % of the samples with pH < 4, from 37 % with pH 4–5 and from 56 % with pH > 5. Formic acid had been used as additive.A similar investigation was carried out on 32 samples from a farm with no outbreak of listeriosis during the investigation period. Lm was isolated from 9 samples.     

Isolation of Listeria monocytogenes from buffaloes with reproductive disorders and its confirmation by polymerase chain reaction

Listeria monocytogenes, a gram-positive, facultative intracellular pathogen was isolated from buffaloes with a history of reproductive disorders and polymerase chain reaction (PCR) analyses for the presence of virulence-associated genes were conducted. A total of 530 samples of faecal, nasal, vaginal swabs and blood samples from 135 buffaloes were screened. The prevalence of L. monocytogenes and other Listeria spp. was found to be 4.4 and 7.4%, respectively. All isolates were subjected to PCR for virulence-associated genes (prfA, plcA, hlyA, actA and iap) and to pathogenicity testing by the phosphatidylinositol phospholipase C (PI-PLC) assay and mice and chick-embryo inoculation. All L. monocytogenes isolates were hemolytic and positive for the hlyA gene. One L. monocytogenes isolate possessed all five virulence-associated genes and was also positive in the PI-PLC assay as well as in the in vivo pathogenicity tests. The remaining hemolytic L. monocytogenes isolates lacking the plcA gene and PI-PLC assay activity were, however, non-pathogenic via mice and chick-embryo inoculation tests, in spite of having the hlyA gene. The detection of multiple virulence-associated genes, in combination with in vitro pathogenicity tests, must be performed to identify pathogenic L. monocytogenes.     

Listeria spp. contamination in piggeries: comparison of three sites of environmental swabbing for detection and risk factor hypothesis

Listeria monocytogenes is a foodborne pathogen of major concern for public health in industrialized countries. Listeria carriage by pigs at the herd level could be a primary source for carcass contamination. Forty-seven finishing pig facilities were involved in the present study designed to compare three environmental swabbing sites in order to detect Listeria spp. in piggeries. Swabs were taken from the pen walls, the perianal regions of the pigs and the trough/feeder of the piggery premises. Listeria contamination of wet or dry feed given to the pigs was also investigated. The capacity of the various sampling sites for Listeria spp. detection were compared with a maximum likelihood estimation method. Listeria spp. were recovered in 74% of the pens studied and L. monocytogenes was detected in 15% of pens. With a specificity of 99%, sensitivity estimates (and 95% CI) of the Listeria spp. detection method were 93.4% (72.7-98.7) for pen walls, 73.1% (54.9-85.9) for pigs and 66.6% (48.6-80.7) for the trough/feeder. Listeria spp. were isolated from 84% of wet feed samples and 5% of dry feed samples. Listeria monocytogenes was found in 13% of wet feed samples. The type of feeding (wet versus dry) was associated (P < 0.001) with Listeria spp. contamination of both the pen and the feed. The results of this study confirm that Listeria spp., including L. monocytogenes, are present in pig facilities. Pen wall swabbing appears to be an effective way to assess Listeria spp. status of finishing pigs. The type of feeding (wet versus dry) could play a role in pig contamination.     

Rapid quantitative assessment of the distribution of Listeria in silage implicated in a suspected outbreak of listeriosis in calves

A silage clamp was sampled for the presence of Listeria species. Even though covered by a heavy duty black plastic sheet the top few centimetres of silage were heavily contaminated with Listeria species. In the areas nearest the edges of the sheet the pathogenic species Listeria monocytogenes was found in numbers in excess of 12,000 organisms/g silage. Using the methods of isolation described, pathogenic species of Listeria could be differentiated from non-pathogenic species in three to four days.     

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.