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.     

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.     

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 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.     

Epidemiologic investigation of a silage-associated epizootic of ovine listeric encephalitis, using a new Listeria-selective enumeration medium and phage typing.

The role of silage feeding in the origin of an epizootic of encephalitic listeriosis in a sheep flock was investigated by use of a new direct Listeria-selective isolation and enumeration medium, in combination with serotyping and phage typing. The silage contained high numbers (about 10(6) cells/g) of a L monocytogenes strain indistinguishable with respect to serovar and phagovar from that isolated from the brains of sick sheep. These results provided unambiguous bacteriologic evidence of the epidemiologic link between silage consumption and listeriosis in ruminants.     

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.     

Contamination of raw milk by Listeria monocytogenes on dairy farms

Possible sources of exogenous contamination of raw milk by Listeria monocytogenes were examined on four dairy farms of different size and type of animal housing during morning milking. Feeds, including hay and concentrates, were found to be major sources of both pathogenic and nonpathogenic species of Listeria on the barns. L. innocua was the only species isolated from the grass silage, which was of good quality on all the farms. The numbers of Listeria were below 10(2)/g in all feed samples. Fecal shedding of listeriae was detected in 11.9% of the cows and the prevalences of L. monocytogenes among farms ranged from no detections to 8.7%. The number of Listeria isolations from constructions inside the barns and from the milking environment varied between the farms. Listeriae were detected almost everywhere on one of the farms whereas on another farm the only isolations were from feed passages and floors. 13.6% of the swab samples taken from the teats before washing and drying were Listeria positive, whereas no isolations were made after cleaning the udder. Good milking and barn hygiene is considered important for diminishing the risks of exogenous contamination of raw milk by listeriae.     

Respiratory infection of turkeys with Listeria monocytogenes Scott A

The pathogenesis of L. monocytogenes strain Scott A was studied by challenging day-old male turkey poults by air sac inoculation with tryptose phosphate broth containing 10(0) cfu (control), 10(4), 10(5), and 10(6) cfu (low challenge), or 10(7) and 10(8) cfu (high challenge) of the Scott A (serotype 4b) strain of L. monocytogenes. Mortality at 2 wk postinfection (PI) ranged from 25% for low challenge to 100% for high challenge (P= 0.0001). Gross and histopathological lesions were observed in heart, liver, spleen, lung, and bursa of Fabricius of mortalities at 4 days PI. Listeria monocytogenes challenge resulted in significantly decreased relative weight of the bursa of Fabricius and increased relative weight of the spleen, and L. monocytogenes was isolated by direct plating of liver, pericardium, brain, and both left and right stifle joint synovium (knee) cultures, as well as gall bladder, yolk sac, and cecal tonsil from transfer swabs onto Listeria-selective agar. Isolates were confirmed as positive using Gram stain, biochemical tests, and the Biolog system. High challenge resulted in confirmed L. monocytogenes isolation from 48% of left knee and 59% of right knee cultures. Low challenge resulted in isolation of L. monocytogenes from 11% of both left and right knee cultures. These results suggest that L. monocytogenes Scott A colonization of turkey knee synovial tissue can initiate in day-of-age poults and that L. monocytogenes Scott A can be invasive through air sac infection.     

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.     

Determination of the minimum enrichment time for the qualitative detection of Listeria monocytogenes in minced pork meat using multiplex-PCR, microarray and ELISA

A qualitative detection of Listeria monocytogenes was performed in spiked minced pork meat using ELISA, Multiplex-PCR, Microarray and cultural reference method. Minced pork meat in batches of 10 or 25 g was spiked with 25 cfu Listeria monocytogenes per gram and incubated in selective enrichment solutions. During enrichment samples were collected continiously and a Listeria monocytogenes-ELISA, a Multiplex-PCR with electrophoretical detection (Listeria duplex) and a PCR with detection by Microarray (NUTRI Chip) were performed.The enrichment time after which all sub-samples were positive was defined as minimum enrichment time. With the cultural reference method Listeria monocytogenes was detected in 100 % of the samples after a total analysis time of 5 days. With the ELISA kit used in this study positive results were achieved after enrichment for 24 h. Multiplex-PCR with electrophoretical detection was positive after only 16 h of enrichment.The most sensitive detection method was the microarray. Using this technique, an enrichment time of 10 h was sufficient to get positive results in all samples.     

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.     

新疆绵羊及其环境中李氏杆菌的生态分布多样性调查

对新疆健康绵羊及其相关环境如青贮饲料、饮水、土壤、鸟粪中李氏杆菌(Listeria)种群采用改良国标法(4789.30-2010)进行生态分布多样性调查,并对分离的单核细胞增多性李氏杆菌(L.monocytogenes)采用PCR方法进行溶血素基因(hly)和血清型检测。结果:514份样本中,共检出阳性样本26份,平均阳性率为5.1%,其中羊体分离率为6.2%(22/353),饮水分离率为33%(2/6),鸟粪分离率为3.0%(2/67),其余样本均为阴性;26份阳性样品中,检出单核细胞增多性李氏杆菌5株,塞氏李氏杆菌(L.seeligeries)21株,且5株单核细胞增多性李氏杆菌均检出溶血素基因,血清型为1/2a。     

Survival of Listeria monocytogenes in wilted and additive-treated grass silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10(6)-10(7) cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in foodborne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8 x 10(5)/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25 degrees C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10(2) and 10(6) cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

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.     

Selective effect of propolis in the isolation of Listeria monocytogenes (author's transl)]

Propolis is a substance produced by honeybees. It is inhibitory to some bacteria species, mainly Gram-positive bacteria, but less inhibitory to Listeria monocytogenes (L.m) than to the other Gram-positive bacteria tested. In order to obtain selective growth of L.m. from contaminated samples, the effect of propolis in plating media and broths on various strains of bacteria was examined. Table I shows the effect of increasing concentrations of propolis in tryptose-agar (TA). L.m. tolerated higher concentrations of propolis than Streptococcus viridans and Staphylococcus aureus. L.m. grew well in tryptosebroth (TB) that contained 0.15 mg propolis pr. ml medium, while Streptococcus viridans and Streptococcus agalactiae were completely inhibited as seen in Table II. Table III shows that when serum was added to the agar, the inhibitory effect was reduced. It can also be seen that Gram-negative bacteria grew quite well on media that contained 0.19 mg propolis pr. ml. To reduce the growth of Gram-negative bacteria, nalidixic acid was added to the medium. Table IV illustrates growth of various species of bacteria in tryptosephosphatebroth (TFB) with or without propolis and nalidixic acid. Most of the strains tested were inhibited, but Pseudomonas aeruginosa and to some extent faecal streptococci were able to grow in the medium that contained the selective substances. As a conclusion it seems that propolis may be a valuable additive to a medium for the selective isolation of L.m.     

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.     

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%.     

Septicaemia, encephalitis and abortions in a housed flock of sheep caused by Listeria monocytogenes type 1/2

One hundred and ninety-six housed pregnant ewes were fed poor quality silage for two days. Ewes are reluctant to eat the silage and within 48 hours they became dull and developed diarrhoea and lameness. Despite treatment with antibiotics and calcium borogluconate 19 ewes died, more than 60 developed vaginal discharges and at lambing 94 ewes were barren. Six developed nervous signs and two of these died, one with lesions typical of listeric encephalitis. Post mortem lesions and the isolation of Listeria monocytogenes type 1/2 from lung, liver, spleen and kidney are described. L monocytogenes was also isolated from blood samples from live ewes. The estimated gross financial loss to the farmer was 5130 pounds or, for the flock, 26 pounds per ewe.     

The occurrence of Listeria monocytogenes in imported ready-to-eat foods in Japan

Quantitative analyses of Listeria monocytogenes in imported ready-to-eat (RTE) foods sold at retail stores in Japan were performed. Of the 77 non-cooked meat products, 6 samples (7.8%) tested positive. The levels of contamination of 4 of the samples were below 100 colony-forming units (CFU)/g, which is the microbiological criterion for L. monocytogenes in RTE foods as determined by Codex. However, Listeria cells at levels of 100 and 400 CFU/g were detected in a salami sample and a raw ham sample, respectively. All of the 70 cheese samples and the 3 samples made from raw ham and cheese showed negative test results. These results suggest that imported RTE foods are potential sources of the causative agent of listeriosis.     

The marked increase of Listeria monocytogenes isolation from contents of swine cecum

The actual prevalence of Listeria monocytogenes from contents of swine cecum was investigated. The efficiency of Listeria enrichment broth (LEB) for isolation was examined by the recovery of artificially inoculated L. monocytogenes in contents of swine cecum. The numbers of organisms did not increase after 48 h incubation, but increased when the rapid decrease in pH of the LEB was adjusted. Between 1991 and 1993, 250 contents of swine cecum were examined for the prevalence of L. monocytogenes using LEB enrichment, either with or without pH adjustment. L. monocytogenes was isolated from 74 samples in 1993 with pH adjustment, however, no organisms were isolated in 1991 and 1992. It was suggested that the marked rise of the L. monocytogenes isolation was due to the spread of the organism among swine. Furthermore, 67 out of the 74 isolates were identified as 1/2c by serotyping. The serovar 1/2c strains showed genetic diversity by random amplified polymorphic DNA.