Listeria Monocytogenes Antigens Research Articles

Immunohistochemical and pathological changes in BALB/c mice immunized with whole sonicated Listeria monocytogenes antigens and the effect of probiotics

The current study was undertaken to investigate the role of macrophages as a cellular immune function against immunization with whole sonicated Listeria monocytogenes antigens (WSLMAgs) and the effect of probiotics. A preparation of WSLMAgs containing whole L. monocytogenes cell, after two subcutaneous immunization of BALB/c mice with 0.5ml WSLMAgs (0.5mg/ml) at an interval of two weeks. The bacterial identification was conducted by a conventional culture method using Listeria selective media PALCAM and confirmed by Polymerase Chain Reaction (PCR), As well as, the immunohistochemical and pathological change of it was studied in vivo by inoculating mice with pre-challenge WSLMAgs and post-challenge with virulent L. monocytogenes (1×108 CFU/mL). The results revealed the cellular immune function against pre- and post-immunization in spleen organ via lymphocytic hyperplasia in white pulp and coalescence of lymphoid follicles and marker F4/80+ show the immune-positive cells in aggregation adjacent to lymphoid follicle or focal aggregation of macrophages between follicles. In conclusion, the effectiveness of sonicated L. monocytogenes pre and post-immunization then challenge with virulent L. monocytogenes in the induction of cellular immune response, might serve as an immunization platform for applicants.

Effect of the optimized selective enrichment medium on the expression of the p60 protein used as Listeria monocytogenes antigen in specific sandwich ELISA

This paper presents the effects of the composition of different media (i.e., Tryptic soy broth (TSB), Brain heart infusion (BHI), Listeria enrichment broth (LEB), Fraser broth (FB) and University of Vermont medium (UVM)) on the detection of a short peptide fragment PepD specific to the p60 protein (p60) of L. monocytogenes by a monoclonal antibody (anti-PepD mAb). Expression of the p60 obtained was demonstrated to be proportional to the cellular growth of Listeria monocytogenes regardless of the tested growth medium. However, the early growth of L. monocytogenes and the expression of the p60 were negatively affected by the presence of selective agents present in LEB, FB and UVM. Among those three selective enrichment media commonly used for L. monocytogenes, LEB allowed a better expression of L. monocytogenes p60 after an incubation period of 18 h. Optimization of the LEB revealed that the dextrose concentration was the critical factor for improving the expression of p60 and promotes the early expression of p60. Moreover, an optimal dextrose concentration of 0.5% (w/v) in LEB, coupled with anti-PepD mAb immobilized to solid support, reduced the detection of p60 from 18 h to 9 h for an initial concentration of L. monocytogenes of 108 CFU/ml.

Distinct behavior of myelomonocytic cells and CD8 T cells underlies the hepatic response to Listeria monocytogenes

Background: The immune response to Listeria monocytogenes (LM) is characterized by formation of leukocyte rich foci of infection in liver and spleen. Although much has been gained in our understanding of immune response through the study of LM, little is known about spatio-temporal regulation of immune response to Listeria in liver. Methods: We utilize a combination of molecular, genetic and intravital microscopic approaches to gain insight into the dynamics of foci and leukocyte behavior during hepatic Listeriosis. Results: LM foci efficiently exclude blood flow, indicating the presence of a barrier separating the foci and healthy tissue. Despite this barrier, sinusoidal myelomonocytic cells readily enter or transiently interact with cells at the edge of foci of infection. Next, utilizing L9.6 transgenic CD8 + T cells specific for an endogenously processed LM antigen, p60 217-225, along with LM deficient in this epitope, we define the role of TCR in T cell migratory behavior in infected liver. Surprisingly, T cell behavior varies with micro-anatomic locale. Near foci, non-specific adhesion mechanisms dominate lymphocyte behavior. Antigen specific effects on motility became detectable only distal to foci. Conclusions: These data suggest that LM antigens act in a paracrine manner to mediate protection from Listeriosis in the liver.

Study the influence of some Listeria monocytogenes antigens on the side effects of Mitomycin C

For studying in vivo influence of immune responses on side effects of Mitomycin C (MMC),Sixty-five male-white mice were divided to nine groups ;1st group inoculated MMC,I.P,0.4mg/ml,2 doses weekly/4 weeks;2nd to 7th groups immunized S/C with LM Ags(25mg/ml) protein concentration 0.4 ml, 2doses/2 weeks intervals;2nd and 3rd groups immunized CFLMAgs and CFLMAgs-MMC in 3rd group; also 4th and fifth groups but immunized with WSLMAgs; 6th and 7th groups inoculated with 0.4 ml Attenuated LM (LaLMAgs), 7th group immunized-MMC treated. Post-immunization, 5 animals sacrificed from 1-7groups and negative control to study the cellular and humoral immunity as well as bone marrow taken to cytogenic examination ,other animals from 1-7 groups and 8th group challenged with 0.4 ml of (ViLM) containing(1x109 CFU/ml) of), the animals of negative group inoculated 0.4 ml of sterile PBS,S/C.Results; showed high mean values of IFN-y and IgG1 in animals immunized with LM.Ags, while MMC decreased levels of IFN-y and IgG1and MI%, increased chromosomal aberrations and micronucleus while decreased in immunized also immunized–MMC treatment , immunized animals show increased MI%.The suppurative pathological lesions and suppurative granulomatous lesions were appeared clear in tissue sections of internal organs in animals of positive control,decreased severity in immunized,immunized-MMCtreated especially with LaLM immunization.

Matrix metalloproteinase expression in sheep with listerial meningoencephalitis

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of several central nervous system (CNS) diseases. In this study, we investigated the presence of Listeria monocytogenes antigens and detected the expression of MMP-9 and MMP-7 in the brains of 22 sheep with clinical signs and histopathological findings characteristic of listerial meningoencephalitis. Archived sections from the brainstem, cerebrum, and cerebellum were stained for immunohistochemistry. L. monocytogenes antigens were located mainly in the cytoplasm of neutrophils and some macrophages and/or extracellularly within microabscesses of the brainstem. MMP-9 was mainly immunolocalised in the endothelial cells, microglial cells, and neurons especially in inflammatory areas. MMP-7 immunoreactivity was detected in perivascular cuffs, microglial cells, and only a few neurons. Overall, immunohistochemistry in formalin-fixed, paraffin-embedded tissues is a useful tool for the diagnosis of encephalitic listeriosis caused by L. monocytogenes, and MMP-9 and MMP-7 may contribute to the pathogenesis of listerial meningoencephalitis.

Expression of two Listeria monocytogenes antigens (P60 and LLO) in Lactococcus lactis and examination for use as live vaccine vectors

Listeria monocytogenes is a food-borne intracellular pathogen that mainly infects pregnant and immunocompromised individuals. The pore-forming haemolysin listeriolysin O (LLO), the main virulence factor of Listeria monocytogenes, allows bacteria to escape from the harsh environment of the phagosome to the cytoplasm of the infected cell. This leads to processing of bacterial antigens predominantly through the cytosolic MHC class I presentation pathway. We previously engineered the food-grade bacterium Lactococcus lactis to express LLO and demonstrated an LLO-specific CD8(+) response upon immunization of mice with the engineered L. lactis vaccine strains. In the present work, we examined the immune response and protective efficacy of an L. lactis strain co-expressing LLO and a truncated form of the listerial P60 antigen (tP60). Oral immunization revealed no significant protection against listeriosis with L. lactis expressing LLO, tP60 or the combined LLO/tP60. In contrast, intraperitoneal vaccination induced an LLO-specific CD8(+) immune response with LLO-expressing L. lactis but no significant improvement in protection was observed following vaccination with the combined LLO/tP60 expressing L. lactis strain. This may be due to the low level of tP60 expression in the LLO/tP60 strain. These results demonstrate the necessity for improved oral vaccination strategies using LLO-expressing L. lactis vaccine vectors.

Meningoencefalite por Listeria monocytogenes em ovinos

São descritos sete casos de doença neurológica em ovinos por Listeria monocytogenes no Rio Grande do Sul e Paraná entre 2000 e 2007. Foram afetados ovinos com idades entre 12-24 meses. Os casos ocorreram no verão e início da primavera e os índices gerais de morbidade e letalidade foram de 3,15% e 100%, respectivamente. Quando essa informação estava disponível, nenhum dos ovinos afetados era alimentado com silagem. Em três propriedades havia contato próximo dos ovinos afetados com outras espécies. A evolução do quadro clínico foi de 12 horas a três dias e os sinais clínicos foram caracterizados por decúbito (7/7), desvio da cabeça (4/7), incoordenação (3/7), depressão (3/7), andar em círculos (2/7), cegueira unilateral, emagrecimento progressivo, febre, midríase, movimentos de pedalagem, nistagmo lateral, opistótono, paralisia flácida dos membros pélvicos ou dos quatro membros, salivação excessiva e tremores (1/7 cada). Histologicamente observou-se encefalite com microabscessos, predominantemente unilateral com variáveis graus de gliose e alterações degenerativas como esferóides axonais e infiltração de células Gitter. As lesões se estendiam desde a medula oblonga até o mesencéfalo. Antígenos de Listeria monocytogenes foram detectados por imuno-histoquímica em seções de tronco encefálico de todos os ovinos afetados. O diagnóstico foi realizado com base nos achados epidemiológicos e clinico-patológicos, e confirmado pela imuno-histoquímica (IHQ) utilizando anticorpo policlonal anti-L. monocytogenes.

Lactococcus lactis-expressing listeriolysin O (LLO) provides protection and specific CD8+ T cells against Listeria monocytogenes in the murine infection model

Lactococcus lactis has previously been proposed as a vaccine platform for the safe delivery of heterologous antigens. Here we utilized L. lactis as a live vector for expression of listeriolysin O (LLO), a major Listeria monocytogenes antigen and virulence factor. A variety of plasmid constructs were designed to permit either constitutive or nisin-inducible expression of secreted or non-secreted LLO in L. lactis. Recombinant strains were subsequently tested in a murine model for vaccination efficacy against L. monocytogenes infection. CD8+ T lymphocytes specific for the LLO91–99 epitope were detected when strains were administered via the intraperitoneal (IP) but not the oral route. Challenge with live L. monocytogenes revealed different levels of protection among the three vaccine strains tested with the nisin-inducible LLO-secreting L. lactis strain providing the greatest protection against secondary infection. This work highlights the usefulness of the GRAS (Generally Regarded As Safe) organism L. lactis as the basis of a live vaccine vector against L. monocytogenes. The work suggests that LLO-expressing L. lactis strains may also have the potential to act as a platform for directing other co-expressed antigens towards the cytosolic MHC class I pathway for enhanced stimulation of the CD8+ T-cell response.

Listeria monocytogenes Septicaemia and Concurrent Clostridial Infection in an Adult Alpaca ( Lama pacos)

A 10-year-old alpaca with a history of anorexia, weight loss and diarrhoea was humanely destroyed and shown to have a multifocal necrotizing hepatitis, splenitis and colitis, as well as an ulcerative to diphtheroid ileitis. Immunohistochemical examination revealed Listeria monocytogenes antigen in the liver and ileum. In addition, L. monocytogenes and Listeria sp.-specific gene fragments were detected by the polymerase chain reaction. L. monocytogenes was isolated from liver and small intestine and Clostridium perfringens type A with beta(2) toxin was found in the small intestine. It is suggested that the infection with C. perfringens type A facilitated the systemic spread of L. monocytogenes.

Vaccination with recombinant fusion proteins incorporating Toll-like receptor ligands induces rapid cellular and humoral immunity

Recognition of specific pathogen associated molecular patterns (PAMPs) is mediated primarily by members of the Toll-like receptor (TLR) family. Stimulation through these receptors results in quantitative and qualitative changes in antigen presentation and cellular activation, thereby linking innate and adaptive immunity. Consequently, the incorporation of TLR-ligands into vaccines could result in more potent and efficacious vaccines. To test this hypothesis, we employed a recombinant fusion protein strategy using the TLR5 ligand flagellin fused to specific antigens to promote protective immunity. These purified recombinant fusion proteins demonstrated potent TLR5-specific NF-κB dependent activity in vitro. Immunization of mice with the recombinant-flagellin-OVA fusion protein STF2.OVA resulted in potent antigen-specific T and B cell responses that were equal to or better than responses induced by OVA emulsified in Complete Freund's adjuvant. These included rapid and consistent antigen-specific IgG 1 and IgG 2a antibody responses that were detectable within 7 days of immunization, and the development of protective CD8 T cell responses. Moreover, the enhanced immunogenicity to OVA is dependant on the direct fusion to flagellin, as co-delivery of OVA with flagellin unlinked failed to augment antigen-specific responses in vivo. Similar results were obtained using a recombinant fusion protein comprised of flagellin and a novel polypetide sequence containing two immuno-protective epitopes derived from the Listeria monocytogenes antigens p60 and listeriolysin O. Animals immunized with this recombinant protein demonstrated significant antigen-specific CD8 T cell responses and protection upon challenge with virulent L. monocytogenes. We conclude that immunization with PAMP:antigen fusion proteins induce rapid and potent antigen-specific responses in the absence of supplemental adjuvants. Collectively our data demonstrate that PAMP:antigen fusion proteins offer significant promise for developing recombinant protein vaccines.

NOD/SCID/γcnull mouse: an excellent recipient mouse model for engraftment of human cells

AbstractTo establish a more appropriate animal recipient for xenotransplantation, NOD/SCID/γcnull mice double homozygous for the severe combined immunodeficiency (SCID) mutation and interleukin-2Rγ (IL-2Rγ) allelic mutation (γcnull) were generated by 8 backcross matings of C57BL/6J-γcnull mice and NOD/Shi-scidmice. When human CD34+ cells from umbilical cord blood were transplanted into this strain, the engraftment rate in the peripheral circulation, spleen, and bone marrow were significantly higher than that in NOD/Shi-scid mice treated with anti-asialo GM1 antibody or in the β2-microglobulin–deficient NOD/LtSz-scid (NOD/SCID/β2mnull) mice, which were as completely defective in NK cell activity as NOD/SCID/γcnull mice. The same high engraftment rate of human mature cells was observed in ascites when peripheral blood mononuclear cells were intraperitoneally transferred. In addition to the high engraftment rate, multilineage cell differentiation was also observed. Further, even 1 × 102 CD34+ cells could grow and differentiate in this strain. These results suggest that NOD/SCID/γcnull mice were superior animal recipients for xenotransplantation and were especially valuable for human stem cell assay. To elucidate the mechanisms involved in the superior engraftment rate in NOD/SCID/γcnull mice, cytokine production of spleen cells stimulated with Listeria monocytogenesantigens was compared among these 3 strains of mice. The interferon-γ production from dendritic cells from the NOD/SCID/γcnull mouse spleen was significantly suppressed in comparison with findings in 2 other strains of mice. It is suggested that multiple immunological dysfunctions, including cytokine production capability, in addition to functional incompetence of T, B, and NK cells, may lead to the high engraftment levels of xenograft in NOD/SCID/γcnull mice.

Demonstration of Listeria monocytogenes by immunohistochemistry in formalin-fixed brain tissues from natural cases of ovine and bovine encephalitis.

In the present work, evidence of Listeria monocytogenes antigens based on the avidin-biotin complex (ABC) immunoperoxidase technique was performed on formalin-fixed central nervous system tissues (CNS) from a total of 23 natural cases of encephalitis (four ovine and 19 bovine). Listeria monocytogenes serotype 4 was isolated from 10 of 17 cultured specimens. Meningoencephalitis characterized by focal necrosis, microabscesses, perivascular cuffing, and gliosis with presence of macrophages and/or neutrophils was observed at histological examination. Positive L. monocytogenes antigens were successfully identified by immunohistochemistry (IHC) in the CNS of all 23 cases. Paraffin-embedded tissues assayed were stored up for 17 years. Morbidity of the outbreaks was between 0.3-3% and 0.1-1% for ovine and bovine cases, respectively. In all the ovine cases, flocks involved were under extensive grazing conditions. In nine of the 19 bovine cases (47.3%), supplementation with corn silage was used. The ABC test can help as a practical tool for the diagnosis of natural cases of L. monocytogenes encephalitis on formalin-fixed specimens from ovine and bovine.

Effect of Selenium Deficiency on the Development of Central Nervous System Lesions in Murine Listeriosis

The effect of selenium (Se) deficiency, produced by feeding a Se-deficient diet, on the development of central nervous system (CNS) lesions was studied in mice infected with Listeria monocytogenes, administered in drinking water for 1 or 7 days in a daily dose of 109organisms, or for 7 days in a daily dose of 107. Se-deficient mice differed from Se-normal controls in developing CNS lesions significantly more frequently. Moreover, regardless of Se status, mice receiving repeated doses of 109organisms differed from those receiving a single 109dose in showing CNS lesions at least twice as often. The majority of animals with CNS lesions showed an inflammatory pattern of rhombencephalitis (17/24), while only two of 24 showed choroiditis-ventriculitis-meningitis; five of 24 animals showed both inflammatory patterns. Listeria monocytogenes antigen was identified within the areas of inflammation by an immunoperoxidase technique. Neuritis of the trigeminal nerve was present in eight animals. The relative lack of pathological changes in the liver and spleen validates this murine model for the study of CNS listeriosis.

CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon.

Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming. Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway. Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L. monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both. IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L. monocytogenes antigens. We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice. Despite their extreme susceptibility to primary L. monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L. monocytogenes antigens which were similar to responses of WT mice. Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection. These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L. monocytogenes antigen. In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L. monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen.

Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish

This study was conducted to evaluate the efficacy of the mini-VIDAS Listeria monocytogenes (LMO) system (BioMérieux Vitek, Inc., Missouri, USA) for detection of L. monocytogenes in environmental and fish samples from three Portuguese cold-smoking plants and from their fresh fish suppliers. Mini-VIDAS-LMO is a fully automated system that uses fluorescent ELFA (Enzyme Linked Fluorescent Assay) technology for detection of Listeria monocytogenes antigens in food. It can be a rapid screening method alternative to time consuming classical isolation and identification. Two hundred and ninety five samples were tested in mini-VIDAS-LMO and in parallel by the ISO 11290-1 traditional protocol. The mini-VIDAS-LMO detected 8 of the 11 confirmed positive samples and presented 11 false positive results. The specificity of the mini-VIDAS-LMO found in this experiment was 0.96 and the sensitivity 0.73.

Processing of Listeria monocytogenes antigens and the in vivo T‐cell response to bacterial infection

Presentation of antigens to T lymphocytes is a critical step in the clearance of pathogens from their hosts and in the establishment of protective immunity. Several animal models have been developed to study this process, but few have been as informative as the murine immune response to Listeria monocytogenes infection. Herein we review the presentation of L. monocytogenes proteins by the MHC class I antigen-processing pathway and the in vivo T-cell response to these bacterial antigens. These studies demonstrate the following: 1) The size of a peptide-specific T-cell response does not correlate with the amount of epitope presented by infected cells; 2) T cells specific for dominant epitopes do not, in the case of L. monocytogenes infection, inhibit responses to subdominant epitopes; 3) T cells responding to different epitopes presented by MHC class Ia molecules expand, contract and enter the memory pool synchronously; 4) Repeated in vivo expansion of antigen-specific T-cell populations results in a narrowing of their T-cell receptor repertoire and in an increase in their affinity for antigen; and 5) T cells restricted by H2-M3 MHC class Ib molecules constitute a major part of the primary response to bacterial infection, but appear to play a relatively smaller role in memory responses. These studies have provided a novel glimpse of the relationship between antigen processing and in vivo T-cell responses to infection, and provide a foundation for more detailed analyses of T-cell mediated adaptive immunity.

Effect of in vitro monocyte activation by Listeria Monocytogenes antigens on phagocytosis and production of reactive oxygen and nitrogen radicals in bovines

Macrophages by virtue of their phagocytic and antibacterial activities play an important role in the host resistance to intracellular pathogens including Listeria monocytogenes. However, the precise killing mechanism adopted by macrophages in the case of L. monocytogenes and the role of macrophage activation in bacterial killing are still unclear. In the present investigation, different antigens of pathogenic L. monocytogenes and three non-specific activators, namely, Lipopolysaccharide (LPS), Phorbol Myristate Acetate (PMA) and Concanavalin A (ConA) supernatant were studied to adjudge their efficacy with regard to in vitro activation of bovine monocytes in terms of the production of reactive nitrogen intermediates (RNI), reactive oxygen intermediates (ROI) and their phagocytic index (PI). Of all the five antigens of L. monocytogenes, namely, viable bacteria used as live antigen (LA), heat-killed antigen (HKA), sonicated antigen (SA), culture filtrate antigen (CFA) and listeriolysin-O (LLO), LA turned out to be the best activator of monocytes for RNI as well as ROI production. In the PI assay, of the three antigens, that is, CFA, SA and LLO, CFA was found to be the best activator of phagocytosis followed by LLO and SA. Among non-specific activators, PMA induced the highest H 2O 2 production whereas LPS caused the maximum increase in RNI and PI production. On activation by different antigens of L. monocytogenes, the peripheral blood mononuclear cells (PBMCs) from infected animals produced significantly higher RNI than those from non-infected animals indicating the involvement of immune T-cells.

CTL epitope generation is tightly linked to cellular proteolysis of a Listeria monocytogenes antigen.

Abstract Listeria monocytogenes is a pathogenic intracellular bacterium that secretes proteins into the cytosol of host cells. A major secreted protein, p60, is processed by the host cell into the nonamer peptides p60 217-225 and p60 449-457, which are presented to CTL by H-2Kd MHC class I molecules. Herein, we use two membrane permeable peptide aldehyde protease inhibitors, LLnL and Z-LLF, to inhibit cytosolic proteolysis in L. monocytogenes-infected cells. These inhibitors, which have been shown to inhibit proteasomes, completely abrogate cytosolic p60 degradation. The effect of LLnL and Z-LLF on p60 epitope generation was determined by acid-eluting, HPLC-purifying, and quantifying p60 217-225 and p60 449-457 from infected cells. We show a direct linkage between p60 degradation and epitope generation. However, the two inhibitors have quantitatively different effects on the generation of the two epitopes. Our findings implicate proteasomes in the earliest stages of Ag degradation and suggest that different CTL epitopes can be generated by distinct proteolytic processes.

Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (lmaA) in Listeria spp. by the polymerase chain reaction

Two pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect targeted sequences in genes coding for listeriolysin O and Listeria monocytogenes antigen A (ImaA). Strains of Listeria spp. used in this study were isolated from clinical specimens, contaminated foods, and environmental sources. Primers were targeted to internal regions of the genes coding for listeriolysin ( hlyA) and Listeria antigen ( ImaA) and amplification fragments were detected after the PCR by agarose gel electrophoresis. PCR was performed using nucleic acids extracted from a collection of 74 strains of Listeria spp. including 18 reference strains, 41 L. monocytogenes, nine L. innocua, five L. seeligeri and one L. ivanovii, encompassing representative sources, serovars, and enzyme electrophoretic types. Although the listeriolysin gene was found exclusively in L. monocytogenes, some strains of serovar 4c were negative. Simultaneous presence of both genes was restricted to L. monocytogenes strains of serovars 1 2 , 3, and 4. The ImaA gene was identified in five of 10 L. innocua strains and one L. ivanovii isolated from pork. Strains of L. seeligeri, L. welshimeri, and L. grayi were negative for both genes. The detection limits in the PCR were found to be 10 pg of nucleic acids for the hlyA gene and 1 pg for the ImaA gene.

Particulate antigens may be reprocessed after initial phagocytosis for presentation to T cells in vivo.

Macrophages were pulsed with Listeria monocytogenes antigens by intraperitoneal injection prior to harvesting and thoroughly washing the cells. The pulsed macrophages were injected into the feet of Listeria-immune or naive mice to elicit a delayed hypersensitivity reaction. Where soluble Listeria antigen was used, presentation by donor macrophages to host T cells required identity within the I region of the H-2 complex. However, presentation of alcohol-killed Listeria organisms or of a living, temperature-sensitive mutant of Listeria was apparently not H-2 restricted. When T cells enriched in vitro for Listeria reactivity were injected into the feet of naive mice, they reacted in an H-2 restricted manner to antigen presented to them either by the pulsed macrophages or host cells apparently acquiring antigen from the original pulsed macrophages.