Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log₁₀ 6.37 cfu ml^-1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log₁₀ 8.0 cfu ml^-1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml^-1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.     

Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log₁₀?6.37 cfu ml^?1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log₁₀?8.0 cfu ml^?1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml^?1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.     

Superinfection by Listeria monocytogenes of cultured human enterocyte-like cells infected with poliovirus or rotavirus

A mixed infection with either rotavirus or poliovirus and Listeria monocytogenes was analysed in Caco-2 cells, a tumour-derived cell line, highly susceptible to these pathogens. The multiplication of these pathogens, whose usual site of entry and/or replication is the intestine, was also followed by electron microscopy. Results obtained showed an increase of L. monocytogenes internalisation in cells infected with rotavirus, whereas the preinfection with poliovirus had only a slight interfering effect on bacterial entry. Analysis of L. monocytogenes multiplication in virus-infected cells revealed that rotavirus also promoted bacterial replication, which poliovirus hampered replication. Concerning the effect of Caco-2 cell invasion by L. monocytogenes on viral replication, we observed an increase in rotavirus antigen synthesis but no significant effect on poliovirus yield under our experimental conditions. Received: 2 May 1996     

Antibiotic susceptibility of Listeria monocytogenes in Denmark 1958-2001

In order to see whether the susceptibility of Danish Listeria monocytogenes strains has changed over the years we examined a collection of human isolates from the period 1958-2001. We, furthermore, wanted to compare L. monocytogenes susceptibility testing using a disc diffusion assay with MIC measurements performed by the E-test. 106 strains isolated predominantly from blood cultures and cerebrospinal fluids were examined together with three reference strains. Susceptibility to the following antibiotics was tested by the E-test and by Oxoid discs using Iso-sensitest agar: penicillin G, ampicillin, meropenem, gentamicin, sulphamethoxazole, trimethoprim, ciprofloxacin, erythromycin, vancomycin, linezolid, chloramphenicol and tetracycline. The strains were in the main sensitive to all antibiotics examined using both methods, except for ciprofloxacin, where the strains were intermediate sensitive. However, for penicillin, ampicillin and sulphamethoxazole, while the disc diffusion assay found the strains to be sensitive, MIC measurements generally placed the strains one dilution above the breakpoint for sensitivity in the intermediate sensitive group. Based on the MIC measurements, the antibiotic susceptibility of L. monocytogenes has not changed in Denmark from 1958 to 2001, and the multiresistant strains found in human infections elsewhere have not been found in Denmark.     

The cellular niche of Listeria monocytogenes infection changes rapidly in the spleen

The spleen is an important organ for the host response to systemic bacterial infections. Many cell types and cell surface receptors have been shown to play role in the capture and control of bacteria, yet these are often studied individually and a coherent picture has yet to emerge of how various phagocytes collaborate to control bacterial infection. We analyzed the cellular distribution of Listeria monocytogenes (LM) in situ during the early phase of infection. Using an immunohistochemistry approach, five distinct phagocyte populations contained LM after i.v. challenge and accounted for roughly all bacterial signal in tissue sections. Our analysis showed that LM was initially captured by a wide range of phagocytes in the marginal zone, where the growth of LM appeared to be controlled. The cellular distribution of LM within phagocyte populations changed rapidly during the first few hours, decreasing in marginal zone macrophages and transiently increasing in CD11c⁺ DC. After 4–6 h LM was transported to the periarteriolar lymphoid sheath where the infective foci developed and LM grew exponentially.     

Lack of microbiota reduces innate responses and enhances adaptive immunity against Listeria monocytogenes infection

The intestinal microbiota influences not only metabolic processes, but also the mucosal and systemic immune systems. Here, we compare innate and adaptive immune responses against the intracellular pathogen Listeria monocytogenes in germfree (GF) and conventional mice. We show that animals without endogenous microbiota are highly susceptible to primary infection with impaired activation and accumulation of phagocytes to the site of infection. Unexpectedly, secondary infection with otherwise lethal dose resulted in survival of all GF animals which cleared bacteria more rapidly and developed a stronger antilisterial CD8⁺ memory T‐cell response compared to conventional mice. In summary, lack of the intestinal microbiota impairs early innate immunity, but enhances activation and expansion of memory T cells.     

Neuropathogenesis of Naturally Occurring Encephalitis Caused by Listeria monocytogenes in Ruminants

Listeriosis is a serious food‐borne disease with increasing frequency in humans and ruminants. Despite the facts that in both hosts, listeriosis can occur as rhombencephalitis and ruminants are a reservoir of Listeria monocytogenes (LM) strains pathogenic for humans, little work has been done on the pathogenesis in ruminants. This study investigates the neuropathogenesis of listeric encephalitis in over 200 natural cases in cattle, sheep and goats by analyzing anatomical distribution, severity, bacterial load and temporal evolution of the lesions. Our results suggest that LM gains access to the brainstem of all three species via axonal migration not only along the trigeminal nerve, but also along other nerves. The ensuing encephalitis does not remain restricted to the brainstem. Rather, LM spreads further from the brainstem into rostral brain regions likely by intracerebral axonal migration. Significant differences in severity of the lesions and bacterial load were found between cattle and small ruminants, which may be caused by species‐specific properties of antibacterial immune responses. As histopathological lesions of human rhombencephalitis caused by LM strongly resemble those of ruminants, the disease likely has a similar pathogenesis in both hosts.     

Diagnostic Ultrastructure of Listeria monocytogenes in Human Central Nervous Tissue

The pathogenesis of Listeria monocytogenes infection of the human central nervous system is poorly understood and ultrastructural aspects are not documented. A case of fatal human L. monocytogenes meningoencephalitis is described, in which diagnosis was confirmed by blood culture and in which special staining (Brown's) showed slender gram-positive bacilli in inflamed areas of the brainstem at autopsy. Electron microscopy of blocks rescued from formalin revealed rods, up to 2.5 μm long × 0.4 μm in diameter, with grampositive type cell walls and distinctive conic ends, the latter being apparent in axial section only. The organism was either free within the cytoplasm or within endosomes or phagosomes of macrophages, smooth muscle cells, and endothelial cells. In one instance, one was seen adhering to the luminal aspect of the vascular endothelium. Central nervous system parenchymal cell infection was suspected but not confirmed. Intracytoplasmic bacteria were surrounded by an electron-transparent halo and, beyond that, a cloud of host-derived microfilaments, as previously described by others in vitro and in animals. The morphology of L. monocytogenes is compared with that of other intracellular pathogens and is found to be distinctive, suggesting the value of ultrastructural study in diagnosis. Such work could also help to solve outstanding pathogenetic questions of central nervous system invasion and spread.     

The defined attenuated Listeria monocytogenes Δmpl2 mutant is an effective oral vaccine carrier to trigger a long-lasting immune response against a mouse fibrosarcoma

Listeria monocytogenes has been proposed as a carrier to elicit major histocompatibility complex class-I restricted immune responses able to protect against tumor challenge. In this study the properties of the attenuated L. monocytogenes Δmpl2 mutant has been evaluated in vivo against a highly aggressive mouse fibrosarcoma which expresses β-galactosidase (β-gal) as a tumor-associated antigen (TAA). Immunization with the vaccine prototypes resulted in both elicitation of specific antibodies and generation of cytotoxic lymphocytes (CTL). Oral vaccination protected 55–64% of the immunized animals from tumor take (p < 0.01) and strongly reduced the average size of the tumor in the other 34–45% (p < 0.01). Vaccinated mice developed a long-lasting response, which resulted in 100% protection from a subsequent tumor challenge. Substitution of the whole TAA by its CTL-defined immunodominant epitope resulted in 43% protection, suggesting a contribution of the humoral response to the observed antitumor effect. No statistically significant differences were observed in the antitumor response when mice were immunized with strains expressing the immunodominant TAA epitope in the context of carrier proteins which were either exported or restricted to the bacterial cytoplasm. This suggests that the topology of the recombinant antigen does not play a major role in the outcome of the protective response.     

转录调控蛋白PrfA对两组新近发现的单核细胞增生李斯特菌基因的体外转录作用的研究

目的研究转录调控蛋白PrfA对两组新近发现的单核细胞增生李斯特菌基因的体外转录作用。方法利用本室近年来建立的体外转录系统,对两组基于转录基因组体内研究发现的5个可能的受PrfA不同调节的单核细胞增生李斯特菌基因进行了体外转录活性的研究。结果第一组中的hpt基因的体外转录活性受PrfA正调节,而其它4个基因既不被PrfA正调节也不被负调节。结论除hpt基因外,其它4个基因体外转录结果与体内实验不相一致,说明PrfA在体内可能通过复杂多样的非直接方式、或者还需要一些目前未知的因子来调控这些新近发现的基因的表达。     

Rat dorsal root ganglia neurons as a model for Listeria monocytogenes infections in culture

Neurotropism of Listeria monocytogenes was studied in rat dorsal root ganglia (DRG) and hippocampal neurons in culture. Using a system in which the DRG neurons can grow relatively free from other cells, it was observed that such DRG neurons, in contrast to hippocampal neurons, can be effectively infected by L. monocytogenes. The bacteria aligned along DRG axons, but not along hippocampal neurites. A mutant deficient in internalin, a protein required for entry into E-cadherin-expressing cells, did not interact with DRG neurons. Axonal migration of bacteria was studied in the DRG neurons grown in a double-chamber system, where either the neurites or the nerve cell bodies were exposed to the bacteria. The data suggest that L. monocytogenes can infect both axons and DRG nerve cell bodies, and that the bacteria can migrate in a retrograde as well as anterograde direction. These results support the notion that L. monocytogenes can spread via primary sensory neurons to the central nervous system. Infection of DRG primary sensory neurons, as employed in the present study, provides a model for analysis of bacterial and neuronal factors of importance for neurovirulence of L. monocytogenes. Received: 3 April 1999     

Diversity of Listeria monocytogenes isolates of human and food origin studied by serotyping, automated ribotyping and pulsed-field gel electrophoresis

Automated ribotyping, pulsed-field gel electrophoresis (PFGE) and serotyping were evaluated for the epidemiological study of isolates of Listeria monocytogenes collected in Finland in 1997-1999 from human blood (n = 116) and the food industry (n = 72). The isolates divided into six serotypes, 23 EcoRI ribotypes, 54 AscI PFGE types, and 57 final subtypes if all results were combined. The discrimination index of ribotyping was lower (0.873) than that of PFGE (0.946). Two final subtypes dominated among human isolates, and identical subtypes were also found among food industry isolates. All PFGE types were serotype-specific, whereas two ribotypes included isolates of two serotypes. Isolates of serotype 3a, involved in an outbreak in Finland in 1999, matched one of these ribotypes, which also included some food industry isolates of serotype 1/2a. Ribotyping with EcoRI would not have been sufficient to define the outbreak in Finland caused by serotype 3a isolates. Although ribotyping is applicable as the first method in outbreak situations, human and food isolates with identical ribotypes should be investigated further by PFGE.     

Interleukin-4-producing CD4+ NK1.1+ TCRα/βintermediate liver lymphocytes are down-regulated by Listeria monocytogenes

Experimental infection of mice with the intracellular bacterium, Listeria monocytogenes, provides a paragon model for immune defence dominated by T helper type 1 (Th1) responses. Potent production of interleukin (IL)-12 by infected macrophages is considered the determining factor in Th1 cell development. In contrast, it is assumed that IL-4 producers remain virtually unstimulated in listeriosis. In the liver, the major target organ of listeriosis, an unusual T lymphocyte population exists with the intriguing phenotype CD4⁺NK1.1⁺ TCRα/β^intermediate (TCRα/β^int). Here we show that IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes are down-regulated early in listeriosis. We assume that curtailment of IL-4-producing CD4⁺NK1.1⁺TCRα/β^int liver lymphocytes promotes unconstrained development of Th1 cells which are central to protection against intracellular bacteria.     

IFN‐γ protects from apoptotic neutrophil‐mediated tissue injury during acute Listeria monocytogenes infection

Listeria monocytogenes (LM) is a foodborne Gram‐positive intracellular pathogen that can cause listeriosis in humans and animals. Although phagocytes are known to be involved in the response to this infection, the role of neutrophils is not entirely clear. Here, we have demonstrated that soon after LM infection, a large number of IFN‐γ‐producing neutrophils quickly accumulated in the spleen, blood, and peritoneal cavity. Both in vivo and in vitro experiments demonstrated that neutrophils were an important source of IFN‐γ. IFN‐γ played a critical protective role against acute LM infection, as demonstrated by the poor survival of Ifng^?/? mice. Moreover, IFN‐γ promoted bacterial clearance by the neutrophils, thereby inhibiting LM‐induced neutrophil apoptosis and spleen damage. In addition to this, IFN‐γ could effectively drive macrophage‐mediated phagocytosis of apoptotic neutrophils, which was accompanied with TGF‐β secretion and was involved in protection against tissue injury. Importantly, by phagocytizing apoptotic neutrophils, macrophages obtained myeloperoxidase, an important bactericidal molecule only produced by neutrophils, which further promoted the antibacterial activity of macrophages. These findings demonstrate that neutrophils are an important source of IFN‐γ at the early stage of LM infection, which is characterized by both LM elimination and tissue‐protective effects.     

Evidence for involvement of peptidoglycan in the triggering of an oxidative burst by Listeria monocytogenes in phagocytes

We have shown previously that in listeric encephalitis of cattle and rats, nitrotyrosine was produced in microabscesses, implying that both superoxide anion (O(2) (-)) and nitric oxide (NO) are present and react with each other. Evidence of local synthesis of NO by macrophages was provided, but the source of O(2) (-) remained unknown. Here we have examined whether phagocytes exposed to viable and heat-killed Listeria monocytogenes (LMDelta) produce O(2) (-) and, if so, whether this results from direct interaction of phagocytes with the bacterial surface of L. monocytogenes or whether prior opsonization is required. Using lucigenin-enhanced chemiluminescence (LCL) for the measurement of O(2) (-), we show that LMDelta induces an oxidative burst in human neutrophils, monocytes and monocyte-derived macrophages (Mphi). Viability is not required, and opsonization by antibodies and/or complement does not enhance the LCL signal. As Toll-like receptors (TLR) were shown recently to mediate an oxidative burst, TLR agonists representative for pathogen-associated molecular patterns (PAMPs) were tested for their ability to elicit an oxidative burst. These included lipoteichoic acid (LTA), bacterial peptidoglycan (PGN), recombinant flagellin, CpG-containing DNA and double-stranded RNA. Only PGN and flagellin consistently elicited an LCL signal resembling that induced by LMDelta with regard to the kinetics and cell spectrum stimulated. However, flagellin was unlikely to be responsible for the LMDelta-mediated burst, as a flagellin-deficient mutant showed no decrease in LCL. We therefore assume that in LMDelta, core PGN acts as a PAMP and directly induces an oxidative burst in all phagocyte populations. We conclude that in cerebral lesions superoxide anion is generated locally by phagocytes recognizing bacterial PGN.     

IFN‐γ production by CD27+ NK cells exacerbates Listeria monocytogenes infection in mice by inhibiting granulocyte mobilization

Natural killer (NK) cells are key components of the immune system involved in several immune reactions, including the clearance of intracellular pathogens. When activated, NK cells rapidly secrete particular cytokines that activate innate immunity and facilitate development of adaptive responses. Conflicting reports on the role of NK cells during infection by Listeria monocytogenes can be found in the literature. Here, we demonstrate that during lethal infection by L. monocytogenes, activation of NK cells via the costimulatory molecule CD27 leads to excessive IFN‐γ production. This impairs innate anti‐bacterial host defenses by inducing downregulation of CXCR2 on granulocytes and consequently inhibiting their recruitment to the sites of infection. The use of antibodies to block CD27 signaling or to deplete IFN‐γ was sufficient to rescue mice from lethal challenge by L. monocytogenes. Our findings contribute to a better understanding of the importance of CD27 signaling in activation of NK cells and should provide new ways of interfering with infections.     

Effect of macrophage activation on killing of Listeria monocytogenes. Roles of reactive oxygen or nitrogen intermediates, rate of phagocytosis, and retention of bacteria in endosomes.

The role of macrophage activation in the killing of L. monocytogenes is unclear. Some studies suggest that activation for enhanced production of reactive oxygen and nitrogen intermediates may not be of central importance. Recent data have indicated an important role for interferon-gamma (IFN-gamma) induced retention of L. monocytogenes in endosomes. Data from the present study indicate that proteose peptone-elicited macrophages from DBA2/J, CD-1, and C3H/HeN mice are listericidal. Activation of these cells in vitro for 20 h by IFN-gamma (20 or 500 U/ml) increased H2O2 or nitrite production, but did not increase the number of L. monocytogenes killed during a subsequent 6-h or 7-h culture. Incubation of macrophages with IFN-gamma plus lipopolysaccharide (LPS) caused greater activation and increased the number of Listeria killed during a 6-h or 7-h culture. However, this seems primarily attributable to enhanced phagocytosis. Proteose peptone-elicited macrophages were significantly more effective than resident macrophages in preventing the escape of L. monocytogenes from endosomes into the cytoplasm. This capability was not significantly enhanced by IFN-gamma in vitro, but was enhanced by IFN-gamma plus LPS. This correlates well with the effects of these activation stimuli on killing of L. monocytogenes by proteose peptone-elicited macrophages. These results indicate that enhanced retention of L. monocytogenes in endosomes is induced by proteose peptone elicitation and that further macrophage activation in vitro by IFN-gamma does not improve listericidal activity.