Hemolytic interactions of Dermatophilus congolensis.

The strains of Dermatophilus congolensis grew on blood agar with washed sheep erythrocytes with marked total hemolysis. In testing for hemolytic interactions they gave a significant synergistic effect of a characteristic shape with Rhodococcus equi and Streptococcus agalactiae, whereas with Staphylococcus aureus producing beta hemolysin and with Staphylococcus aureus producing delta hemolysin a simultaneous synergistic as well as antagonistic effect were observed. First of all a conspicuous inhibition of in the beta hemolysin zone began and then the hemolytic effect of D. congolensis was enhanced. A similar double reaction was also observed with Listeria ivanovii. With delta hemolysin there was an inhibition of the hemolytic effect of D. congolensis and at the same time a synergistic effect could be observed. Also D. congolensis gave a weak synergistic effect with Micrococcus lylae and Listeria monocytogenes, and a further weak antagonistic effect with alpha hemolysin of Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes and Micrococcus luteus. No interaction of D. congolensis was established with Corynebacterium pseudotuberculosis.     

新疆不同来源单核细胞增生李斯特氏菌血清型的多重PCR鉴定

为鉴定分离自新疆北疆绵羊单核细胞增生李斯特氏菌,本研究采用多重PCR方法,鉴定来自病发地区部分羊场的发病绵羊、健康绵羊、羊舍环境和乌鸦粪分离的30株李斯特氏菌分离株的8株单核细胞增生李斯特氏菌分离株血清型。结果为4株发病绵羊株有3株鉴定为单核细胞增生李斯特氏菌,血清型为1/2a或4b,1株为非单核细胞增生李斯特氏菌;5株健康绵羊株血清型为1/2a;其余来自羊舍水源的3株、乌鸦粪的2株及健康绵羊16株为非单核细胞增生李斯特氏菌,表明来自发病绵羊、健康绵羊及参考菌株LM血清型之间具有相关性。     

应用环介导等温扩增技术快速检测单核细胞增生李斯特菌的研究

目的建立环介导等温扩增技术快速检测单核细胞增生李斯特菌。方法根据单核细胞增生李斯特菌(LM)hlyA基因序列中的保守区域,采用在线引物设计软件Primer Explorer4.0进行设计,获得一套特异性的环介导等温扩增(LAMP)引物,对单核细胞增生李斯特菌hlyA基因进行LAMP扩增,并与常规PCR方法进行比较。结果建立的LAMP方法能成功扩增出梯形条带,LAMP检测单核细胞增生李斯特菌纯培养物和人工染菌的灵敏度为5.44×102cfu/mL,而对照PCR检测的灵敏度为5.44×104cfu/mL。对10株细菌进行LAMP扩增,仅单核细胞增生李斯特菌得到阳性结果。从DNA提取到报告结果,耗时仅1h。结论 LAMP检测单核细胞增生李斯特菌灵敏度高,特异性强,耗时短,方法简便,有望发展成为快速检测食品中单核细胞增生李斯特菌的有效手段。     

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.     

Listeria monocytogenes contamination of finishing pigs: an exploratory epidemiological survey in France

Listeria monocytogenes is a foodborne pathogen of major concern for public health in industrialised countries. Since L. monocytogenes carriage by pigs at the herd level could be a primary source for carcass contamination, control measures should be designed to reduce the L. monocytogenes load at the pre-harvest stage. For this purpose, an exploratory analytical survey was carried out in 2000-2001 in 93 French farrow-to-finish pig farms concerning L. monocytogenes contamination in pigs before they left for the slaughterhouse. On each farm, the L. monocytogenes status of a batch of contemporary fattening pigs housed in the same room was assessed on faecal material samples taken by means of gauze swabs wiped on the perianal region of the pigs. Fourteen percent of the batches studied had at least one contaminated sample and were therefore classified as L. monocytogenes contaminated batches. Two logistic regression models were used to assess the association between managerial and hygiene practices and the risk of L. monocytogenes contamination of the batch at the end of the finishing period on the whole data set (n = 93) and in the wet feeding farms only (n = 57). Wet feeding during the fattening period was identified as a risk factor for L. monocytogenes contamination. Risk factors related to the introduction of L. monocytogenes in pig facilities were identified for both the general and wet feeding farm data sets. Poor care paid to hygiene on the farms was found to increase the risk of being infected (boots cleaning, change room presence). When the duration of the empty period prior to the introduction of growing pigs was less than one day in the fattening section, the risk of L. monocytogenes contamination was significantly increased. For wet feeding farms, a distribution pipeline cleaning procedure including disinfection was found to be associated with a higher risk of contamination than no cleaning or a procedure consisting of rinsing with water only.     

Evaluation of farm management practices as risk factors for clinical listeriosis and fecal shedding of Listeria monocytogenes in ruminants

OBJECTIVE: To assess seasonal variation in prevalence of Listeria monocytogenes on ruminant farms and identify management practices associated with ruminant listeriosis and fecal shedding of L. monocytogenes. STUDY DESIGN: Case-control study. SAMPLE POPULATION: 2056 samples of feces, feed, soil, and water from 24 case farms with listeriosis and 28 control farms without listeriosis. PROCEDURE: Samples were collected and evaluated via bacterial culture for L. monocytogenes. Univariate associations between farm management practices and listeriosis and fecal shedding of L. monocytogenes were assessed. Multivariate models were developed to identify farm management practices associated with listeriosis and fecal shedding of L. monocytogenes. RESULTS: The prevalence of L. monocytogenes on cattle, goat, and sheep farms was seasonal, especially in fecal samples, with peak prevalence in winter. Although the prevalence of L. monocytogenes in feedstuffs from small-ruminant farms also peaked during winter, the bacterium was detected at a constant rate in cattle farm feedstuffs throughout the year. Farm management practices, animal health and hygiene, and feedstuff quality and storage were associated with ruminant listeriosis and fecal shedding of L. monocytogenes. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the prevalence of L. monocytogenes on ruminant farms is seasonal, management practices are associated with ruminant listeriosis and fecal shedding of L. monocytogenes, and the epidemiologic features of listeriosis differ in cattle versus small ruminants. Awareness of risk factors may be used to develop control measures to reduce animal disease and introduction of L. monocytogenes into the human food chain.     

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.     

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.     

A case of bovine raw milk contamination with Listeria monocytogenes

ABSTRACT: During routine sampling of bulk raw milk on a dairy farm, the pathogenic bacteria Listeria monocytogenes was found to be a contaminant, at numbers < 100 cfu/ml. A strain with an indistinguishable pulsed-field gel electrophoresis pattern was isolated from the bulk milk two months later. Environmental swabs taken at the dairy environment were negative for the presence of L. monocytogenes, indicating a possible case of excretion of the L. monocytogenes directly into the milk. Milk samples were collected from the individual cows and analysed, resulting in the identification of L. monocytogenes excretion (at 280 cfu/ml) from one of the 4 mammary quarters of one dairy cow out of 180. When the infected cow was isolated from the herd, no L. monocytogenes was detected from the remaining herd. The pulsed-field gel electrophoresis pattern of the strain from the individual cow was indistinguishable from that originally isolated from the bulk milk. The infected cow did not show any clinical signs of disease, nor did the appearance of the milk have any physical abnormalities. Antibiotic treatment of the infected mammary quarter was found to be ineffective. This study shows that there can be risks associated with direct contamination of raw milk with L. monocytogenes.     

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.     

Encephalitic listeriosis in two adult llamas (Lama glama): clinical presentations, lesions and immunofluorescence of Listeria monocytogenes in brainstem lesions

Encephalitic listeriosis was diagnosed in 2 adult llamas. Both had a multifocal suppurative encephalitis with mixed lymphocytic and neutrophilic perivascular infiltrates. Listeria monocytogenes was cultured from the brain stem of 1 llama using cold enrichment techniques; the other llama was culture negative. Formalin-fixed and paraffin embedded sections of brainstem lesions from both affected animals were labeled with a fluorescein-conjugated, anti-L. monocytogenes antibody. Using this technique, intralesional L. monocytogenes were identified in both llamas.     

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.     

Survival of Listeria monocytogenes in cheese made of unpasteurized goat milk

Food-born infections with Listeria monocytogenes have been reported during recent years, cheese being mentioned as one of the foods responsible. A classical opinion is that cheese represents a very inhospitable environment for pathogens due to antagonism by the starter culture of lactic-acid-producing organisms. In order to study the survival of L. monocytogenes in goat cheese, cheeses were made with the addition of L. monocytogenes cultures. The maximum survival time for L. monocytogenes was 18 weeks in two of the cheeses. It is concluded that L. monocytogenes has the ability to survive in semi-soft cheese made of unpasteurized goat milk during normal curing (2–3 months).     

Risk factors for Listeria monocytogenes contamination in French laying hens and broiler flocks

The objective of this study was to identify potential risk factors for Listeria monocytogenes contamination in French poultry production. Eighty-four flocks of layer hens kept in cages and 142 broiler flocks were included in this study. For each production type, a questionnaire was submitted to farmers and fecal samples were taken to assess the L. monocytogenes status of the flocks during a single visit to the farm. Two logistic regression models (specific to each production) were used to assess the association between management practices and the risk of L. monocytogenes contamination of the flock. The prevalence of L. monocytogenes-positive flocks was 30.9% (95% CI: 21.0; 40.9) and 31.7% (95% CI: 24.0; 39.4) for cage-layers and broiler flocks, respectively. For layer flocks, the risk of L. monocytogenes contamination was increased when pets were present on the production site. When droppings were evacuated by conveyor belt with deep pit storage, the risk of L. monocytogenes contamination decreased significantly. Feed meal was found to be associated with a higher risk of L. monocytogenes contamination than feed crumb. For broiler flocks, the risk of L. monocytogenes contamination was increased when farmers did not respect the principle of two areas (clean and dirty) at the poultry house entrance. A first disinfection by thermal fogging and the absence of pest control of the poultry house before the arrival of the next flock was found to increase the risk of contamination. When litter was not protected during storage and when farm staff also took care of other broiler chicken houses, the risk of L. monocytogenes contamination increased significantly. In the case of the watering system, nipples with cups were found to decrease the risk of contamination.     

A combination of diagnostic tools for rapid screening of ovine listeriosis

A combined serological and PCR method for the detection of Listeria monocytogenes infection in symptomatic and asymptomatic ovine flocks was evaluated. Seventy-eight milk samples and 157 serum samples were analysed using a L. monocytogenes PCR detection kit and an anti-listeriolysin O IgG immunoassay kit. The combined use of these commercial kits allowed a rapid and effective detection of L. monocytogenes infection in both the early stage, before seroconversion, and in a later phase, even after antibiotic therapy.     

In vitro study of Listeria monocytogenes infection to murine primary and human transformed B cells

Immunity to Listeria monocytogenes is largely mediated by T lymphocytes. Recently, B lymphocytes or their secreted products are implicated to provide immunity against L. monocytogenes infection. To understand whether L. monocytogenes can infect and kill B cells as a possible strategy to initiate an infection, we examined the effects of L. monocytogenes on a human B lymphoma (Ramos RA-1) and mouse primary B cells in vitro. L. monocytogenes infection resulted in significantly (p相似文献   

Eye infections due to Listeria monocytogenes in three cows and one horse.

A retrospective study was conducted to determine case histories, microbiological characteristics, and molecular subtypes associated with Listeria monocytogenes infections of the eye in large animals. For selected cases, environmental L. monocytogenes contamination patterns on case farms were also evaluated to probe for potential sources and spread of listerial eye infections. Records of 170 L. monocytogenes isolates from animal infections were reviewed to determine the fraction of isolates associated with eye infections (conjunctivitis, keratitis, and uveitis) of animals and to gather information on the clinical history of these cases. Overall, 4 of 170 Listeria monocytogenes isolates were associated with eye infections; 3 of these had occurred in cows and 1 in a horse. Molecular subtyping (by EcoRI ribotying) showed that 4 different L. monocytogenes subtypes were isolated from these 4 cases; the same ribotypes had previously been found among invasive animal listeriosis infections. Although a variety of L. monocytogenes subtypes were isolated from environmental sources, on 1 farm, the same ribotype associated with the eye infection was also isolated from a fecal sample of a healthy animal and from a soil sample. The data reported in this study further suggest that L. monocytogenes can be a cause of eye infections in several animal species. Listerial eye infections do not seem to require specific pathogen-related virulence characteristics but rather seem to be a function of environmental or host factors, such as direct exposure of the eyes of susceptible animals to high numbers of the pathogen. Although listerial eye infections are rarely diagnosed because of its ubiquitous nature, L. monocytogenes may have to be considered more commonly as a causative agent of eye infections in ruminants and horses.     

单核细胞增生李斯特菌prfA基因重组菌的构建及其生物学特性研究

为制备单核细胞增生李斯特菌(LM)减毒的prfA基因重组菌,本研究通过构建重组质粒pKSV7-ΔprfA-gfp,电转化至LM TA感受态细胞中,将绿色荧光蛋白基因gfp插入LM的prfA基因第25 bp～93 bp之间,在42℃和10μg/mL氯霉素的双重选择压力下,筛选LM重组菌(rLM-ΔprfA-gfp),并对其生物学特性进行鉴定。试验结果表明,重组菌与亲本菌相比具有相似的体外生长特性,溶血素基因(hly)mRNA的转录水平降低;对小鼠的毒力显著减弱,其LD50由105.53升高到109.79,对肝、脾、肾的损伤不明显;免疫保护力试验显示重组菌与LM TA灭活疫苗的保护率分别为90%和35%,表明rLM-ΔprfA-gfp比传统疫苗具有更高的保护力。本研究构建的rLM-ΔprfA-gfp具有良好的遗传稳定性,并且具有较好的免疫原性,为LM活疫苗载体和分子标记疫苗的研制奠定了基础。     

Simultaneous occurrence of selected food-borne bacterial pathogens on bovine hides, carcasses and beef meat

The aim of this study was to determine the simultaneous occurence of Salmonella spp., L. monocytogenes, verotoxigenic E. coli (VTEC), and Campylobacter spp. in slaughtered cattle and in beef meat subjected for human consumption. A total of 406 bovine hides and 406 corresponding carcasses were used to collect the samples with a swab method after exsanguination and evisceration of animals, respectively. Furthermore, 362 beef meat samples were purchased in local retail shops over the same period of time as for the bovine samples. Food-borne bacterial pathogens were identified with standard ISO methods with some modification by the use of PCR for VTEC. The isolated bacteria were then molecularly speciated (Campylobacter), serotyped (L. monocytogenes) and characterized for the presence of several virulence marker genes (VTEC and Campylobacter). It was found that 49 hide (12.1%) and 3 (0.7%) carcass samples were contaminated with more than one bacterial pathogen tested. Most of the hides were positive for Campylobacter spp. and VTEC (27 samples) and Campylobacter spp. together with L. monocytogenes (12 samples). Eight bovine hides contained L. monocytogenes and VTEC while L. monocytogenes and Salmonella spp. were detected in one sample. Furthermore, 3 pathogens (Campylobacter spp., L. monocytogenes and VTEC) were simultaneously identified in one bovine hide tested. In case of bovine carcasses 2 samples contained Campylobacter spp. and VTEC whereas one carcass was positive for L. monocytogenes and VTEC. On the other hand, 10 out of 362 (2.8%) minced beef samples were contaminated with at least two pathogens tested. The majority of these samples were contaminated with L. monocytogenes and Salmonella spp. (6 samples). It was noticed that equal number of C. jejuni and C. coli were found, irrespective of the origin of the samples. Most of the strains possessed more than one pathogenic factor as identified by PCR. Molecular serotyping of L. monocytogenes revealed that the majority of the isolates (27 out of 31; 87.1%) belonged to 1/2a serogroup. It was found that most of the VTEC isolates possessed the Shiga toxin stx2 gene (12 strains) whereas only 2 strains were str1-positive. The eneterohemolysin and intimin markers were identified only in 7 and 2 isolates, respectively. PCR analysis revealed that 4 VTEC belonged to O91 serogroup, 2 strains were O145 and 1 isolate was identified as O113. None of the VTEC detected in the study was O157 serogroup.     

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%.