An inherent T cell deficit in healthy males to C. neoformans infection may begin to explain the sex susceptibility in incidence of cryptococcosis.

BACKGROUND: Cryptococcus neoformans, the causative agent of cryptococcosis, causes ~ 181,000 deaths annually, with males having a higher incidence of disease than females (7M:3F). The reason for this sex bias remains unclear. We hypothesized that this disparity was due to biological differences between the male and female immune response. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated and infected with C. neoformans ± exogenous testosterone or 17-ß-estradiol. C. neoformans, B, T, and NK cell proliferation was quantified by flow cytometry. Cytokine analysis was conducted via protein array or ELISA. Serological testing was conducted to determine previous exposure to C. neoformans. RESULTS: C. neoformans proliferated more in male PBMCs. T cell percentages in both sexes were lower in infected versus uninfected cells. Male PBMCs had lower CD3+, CD4+, and CD8+ T cells percentages during infection compared to females. Cytokine profiles showed differences in uninfected male and female PBMCs, which subsided during infection. Only one donor was sero-negative for prior C. neoformans exposure. There was an effect of estrogen in one dataset. CONCLUSIONS: These results suggest that males show an inherent deficit in T cell response during infection, which may contribute to the increased incidence of disease in males.

Co-infection with feline retrovirus is related to changes in immunological parameters of cats with sporotrichosis.

Feline sporotrichosis due to Sporothrix brasiliensis is frequently severe and often correlated to zoonotic transmission. Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) cause immunodeficiency in cats; no association has been identified with critical cases of sporotrichosis. Moreover, the cytokine profile in Sporothrix-infected cats and a potential impact of retrovirus co-infections on their immunity is unknown. This study assessed immunological parameters in cats with sporotrichosis with and without FIV or FeLV co-infection. FeLV infection was detected by antigen ELISA and by provirus PCR. FIV infection was investigated through ELISA and Western blot. Cytokine transcription (IFN-γ, IL-4, IL-5, IL-6, IL-10, IL-12, TNF-α) was quantified using RT-qPCR and lymphocyte subpopulations (CD4, CD8, CD5 and CD21) were assessed by flow cytometry. Thirty cats with sporotrichosis were recruited to the study, including three FIV-positive and five FeLV-positive (progressive infection) cats. One cat with regressive FeLV infection was excluded from statistics. In comparison to retrovirus-negative cats, FIV-positive cats and FeLV-positive cats had higher IL-10 levels, FeLV-positive cats had lower IL-4 levels and FIV-positive cats had lower IL-12 levels and a lower CD4+/CD8+ ratio. Remarkably, all cats with poor general condition were FeLV (progressive infection) or FIV-positive, but the retrovirus status was not associated with the sporotrichosis treatment length or outcome. The immunological changes and the more severe clinical presentation observed in cats with retrovirus co-infections encourage future prospective studies that address the impact of these changes on prognostic determinants of feline sporotrichosis and the development of new therapy strategies that control disease spread.

Long-term intake of Lactobacillus paracasei KW3110 prevents age-related chronic inflammation and retinal cell loss in physiologically aged mice.

Age-related chronic inflammation is a major risk factor for the incidence and prevalence of age-related diseases, including infectious and neurodegenerative diseases. We previously reported that a lactic acid bacteria, Lactobacillus paracasei KW3110, activated macrophages and suppressed inflammation in mice and humans. In this study, we investigated whether long-term intake of heat-killed L. paracasei KW3110 modulated age-related inflammation and altered the gut microbiota in physiologically aged mice. Compared with age-matched control mice, fecal analyses of gut microbiota revealed that intake of L. paracasei KW3110 mitigated age-related changes of beneficial bacterial composition, including the Bifidobacteriaceae family. L. paracasei KW3110 intake also mitigated age-related immune defects by reducing the prevalence of interferon-gamma (IFN-γ) -producing inflammatory CD4-positive T cells in the lamina propia of the small intestine, and reduced serum levels of proinflammatory cytokines. Furthermore, L. paracasei KW3110 intake suppressed retinal inflammation by reducing proinflammatory cytokine-producing macrophage, and age-related retinal cell loss. Taken together, these findings suggested that L. paracasei KW3110 mitigated age-related chronic inflammation through modulation of gut microbiota composition and immune system functions in aged mice, and also reduced age-related retinal ganglion cell (RGC) loss. Further studies are needed to evaluate the effect in age-related senescent changes of the retina.

Characterization of pathogenesis of and immune response to Burkholderia pseudomallei K96243 using both inhalational and intraperitoneal infection models in BALB/c and C57BL/6 mice.

Burkholderia pseudomallei, the etiologic agent of melioidosis, is a Gram negative bacterium designated as a Tier 1 threat. This bacterium is known to be endemic in Southeast Asia and Northern Australia and can infect humans and animals by several routes. Inhalational melioidosis has been associated with monsoonal rains in endemic areas and is also a significant concern in the biodefense community. There are currently no effective vaccines for B. pseudomallei and antibiotic treatment can be hampered by non-specific symptomology and also the high rate of naturally occurring antibiotic resistant strains. Well-characterized animal models will be essential when selecting novel medical countermeasures for evaluation prior to human clinical trials. Here, we further characterize differences between the responses of BALB/c and C57BL/6 mice when challenged with low doses of a low-passage and well-defined stock of B. pseudomallei K96243 via either intraperitoneal or aerosol routes of exposure. Before challenge, mice were implanted with a transponder to collect body temperature readings, and daily body weights were also recorded. Mice were euthanized on select days for pathological analyses and determination of the bacterial burden in selected tissues (blood, lungs, liver, and spleen). Additionally, spleen homogenate and sera samples were analyzed to better characterize the host immune response after infection with aerosolized bacteria. These clinical, pathological, and immunological data highlighted and confirmed important similarities and differences between these murine models and exposure routes.

[Effect of perinatal factors on postnatal development of lymphocyte subsets in early preterm infants].

OBJECTIVE: To study the postnatal changes in lymphocyte subsets in early preterm infants and the effect of perinatal factors on lymphocyte subsets. METHODS: A total of 61 early preterm infants were enrolled. Flow cytometry was used to measure the absolute counts of lymphocytes and lymphocyte subsets at 1, 7, 14, and 28 days after birth, as well as at 6 months after birth for 17 of these early preterm infants. The effects of perinatal factors, such as antepartum use of hormone, intrauterine infection, gestational age at birth, and Ureaplasma urealyticum (UU) colonization, on lymphocyte subsets were analyzed. RESULTS: The absolute counts of lymphocyte subsets except natural killer (NK) cells were lowest at birth, increased rapidly at 1 week after birth, and reached the levels in healthy infants at 6 months; the count of NK cells remained at a low level and increased significantly at 6 months after birth. Compared with those with a gestational age of <28 weeks, the early preterm infants with a gestational age of ≥28 weeks had significantly higher absolute counts of T cells, T helper (Th) cells, and NK cells at 7 days after birth, a significantly higher absolute count of T cells at 14 days after birth, and significantly higher absolute counts of lymphocytes and Th cells at 28 days after birth (P<0.05). Compared with the group not using hormone, the group using hormone showed a significantly higher absolute count of T cells at 7 days after birth and significantly higher absolute counts of lymphocytes and all subsets at 14 days after birth (P<0.05). There was no significant difference in lymphocyte subsets at 1 day after birth between the intrauterine infection and non-infection groups (P>0.05); the intrauterine infection group had significantly higher absolute counts of B cells at 7 and 14 days after birth than the non-infection group. Compared those without UU colonization, the infants with UU colonization had significantly higher absolute counts of lymphocytes, T cells, Th cells, and Ts cells at 1 day after birth and a significantly higher absolute count of B cells at 14 days after birth. CONCLUSIONS: Early preterm infants have deficiencies in innate immune cells at birth and normal levels at about 6 months after birth. Various perinatal factors including antepartum use of hormone, gestational age at birth, intrauterine infection, and UU colonization have long-term effects on lymphocyte subsets in early preterm infants.

Migratory and adhesive cues controlling innate-like lymphocyte surveillance of the pathogen-exposed surface of the lymph node.

Lymph nodes (LNs) contain innate-like lymphocytes that survey the subcapsular sinus (SCS) and associated macrophages for pathogen entry. The factors promoting this surveillance behavior have not been defined. Here, we report that IL7R(hi)Ccr6(+) lymphocytes in mouse LNs rapidly produce IL17 upon bacterial and fungal challenge. We show that these innate-like lymphocytes are mostly LN resident. Ccr6 is required for their accumulation near the SCS and for efficient IL17 induction. Migration into the SCS intrinsically requires S1pr1, whereas movement from the sinus into the parenchyma involves the integrin LFA1 and its ligand ICAM1. CD169, a sialic acid-binding lectin, helps retain the cells within the sinus, preventing their loss in lymph flow. These findings establish a role for Ccr6 in augmenting innate-like lymphocyte responses to lymph-borne pathogens, and they define requirements for cell movement between parenchyma and SCS in what we speculate is a program of immune surveillance that helps achieve LN barrier immunity.

Natural Killer-like B Cells Prime Innate Lymphocytes against Microbial Infection.

Natural killer (NK) cells and non-cytotoxic interferon-γ (IFN-γ)-producing group I innate lymphoid cells (ILC1s) produce large amounts of IFN-γ and cause activation of innate and adaptive immunity. However, how NKs and ILC1s are primed during infection remains elusive. Here we have shown that a lymphocyte subpopulation natural killer-like B (NKB) cells existed in spleen and mesenteric lymph nodes (MLNs). NKBs had unique features that differed from T and B cells, and produced interleukin-18 (IL-18) and IL-12 at an early phase of infection. NKB cells played a critical role in eradication of microbial infection via secretion of IL-18 and IL-12. Moreover, IL-18 deficiency abrogated the antibacterial effect of NKBs. Upon bacterial challenge, NKB precursors (NKBPs) rapidly differentiated to NKBs that activated NKs and ILC1s against microbial infection. Our findings suggest that NKBs might be exploited to develop effective therapies for treatment of infectious diseases.

PD-1 Expression and Cytokine Secretion Profiles of Mycobacterium tuberculosis-Specific CD4+ T-Cell Subsets; Potential Correlates of Containment in HIV-TB Co-Infection.

HIV co-infection is an important risk factor for tuberculosis (TB) providing a powerful model in which to dissect out defective, protective and dysfunctional Mycobacterium tuberculosis (MTB)-specific immune responses. To identify the changes induced by HIV co-infection we compared MTB-specific CD4+ responses in subjects with active TB and latent TB infection (LTBI), with and without HIV co-infection. CD4+ T-cell subsets producing interferon-gamma (IFN-γ), interleukin-2 (IL-2) and tumour necrosis factor-alpha (TNF-α) and expressing CD279 (PD-1) were measured using polychromatic flow-cytometry. HIV-TB co-infection was consistently and independently associated with a reduced frequency of CD4+ IFN-γ and IL-2-dual secreting T-cells and the proportion correlated inversely with HIV viral load (VL). The impact of HIV co-infection on this key MTB-specific T-cell subset identifies them as a potential correlate of mycobacterial immune containment. The percentage of MTB-specific IFN-γ-secreting T-cell subsets that expressed PD-1 was increased in active TB with HIV co-infection and correlated with VL. This identifies a novel correlate of dysregulated immunity to MTB, which may in part explain the paucity of inflammatory response in the face of mycobacterial dissemination that characterizes active TB with HIV co-infection.

Lack of cross-protection against Mycoplasma haemofelis infection and signs of enhancement in "Candidatus Mycoplasma turicensis"-recovered cats.

"Mycoplasma haemofelis" and "Candidatus Mycoplasma turicensis" are feline hemoplasmas that induce hemolytic anemia. Protection from homologous re-challenge was recently demonstrated in cats recovered from primary infection. Here, we determined if cats recovered from "Cand. M. turicensis" infection were protected against infections with the more pathogenic M. haemofelis. Ten specified pathogen-free cats were exposed to M. haemofelis. Five of the ten cats had recovered from "Cand. M. turicensis" bacteremia (group A), and five cats were naïve controls (group B). No cross-protection was observed. By contrast, the "Cand. M. turicensis"-recovered cats displayed faster M. haemofelis infection onset (earlier PCR-positive and anemic) than the controls. No "Cand. M. turicensis" was detected in any cat. M. haemofelis shedding was observed in saliva, feces and urine. In both groups, evidence of a Th1 response was observed (high IFN-γ, low IL-4), but IL-10 levels were also high. In group A, total, CD4+ and CD8+ T cells increased within days after M. haemofelis exposure. At times of maximal bacteremia, macrocytic hypochromic anemia, neutropenia, monocytosis and a decrease in leukocyte, eosinophil, and lymphocyte counts and subsets thereof (B- and T-cells, CD4+, CD8+ and CD4+CD25+ cells) were particularly significant in group A. Moreover, an increase in protein concentrations, hypoalbuminemia and a polyclonal hypergammaglobulinemia were observed. Five of ten M. haemofelis-infected cats subsequently cleared bacteremia without antibiotic treatment. In conclusion, the study suggests that a previous hemoplasma infection, even when the cat has ostensibly recovered, may influence subsequent infections, lead to an enhancement phenomenon and other differences in infection kinetics.

Control of adaptive immunity by the innate immune system.

Microbial infections are recognized by the innate immune system both to elicit immediate defense and to generate long-lasting adaptive immunity. To detect and respond to vastly different groups of pathogens, the innate immune system uses several recognition systems that rely on sensing common structural and functional features associated with different classes of microorganisms. These recognition systems determine microbial location, viability, replication and pathogenicity. Detection of these features by recognition pathways of the innate immune system is translated into different classes of effector responses though specialized populations of dendritic cells. Multiple mechanisms for the induction of immune responses are variations on a common design principle wherein the cells that sense infections produce one set of cytokines to induce lymphocytes to produce another set of cytokines, which in turn activate effector responses. Here we discuss these emerging principles of innate control of adaptive immunity.

Contribution of Thy1+ NK cells to protective IFN-γ production during Salmonella typhimurium infections.

IFN-γ is critical for immunity against infections with intracellular pathogens, such as Salmonella enterica. However, which of the many cell types capable of producing IFN-γ controls Salmonella infections remains unclear. Using a mouse model of systemic Salmonella infection, we observed that only a lack of all lymphocytes or CD90 (Thy1)(+) cells, but not the absence of T cells, Retinoic acid-related orphan receptor (ROR)-γt-dependent lymphocytes, (NK)1.1(+) cells, natural killer T (NKT), and/or B cells alone, replicated the highly susceptible phenotype of IFN-γ-deficient mice to Salmonella infection. A combination of antibody depletions and adoptive transfer experiments revealed that early protective IFN-γ was provided by Thy1-expressing natural killer (NK) cells and that these cells improved antibacterial immunity through the provision of IFN-γ. Further analysis of NK cells producing IFN-γ in response to Salmonella indicated that less mature NK cells were more efficient at mediating antibacterial effector function than terminally differentiated NK cells. Inspired by recent reports of Thy1(+) NK cells contributing to immune memory, we analyzed their role in secondary protection against otherwise lethal WT Salmonella infections. Notably, we observed that a newly generated Salmonella vaccine strain not only conferred superior protection compared with conventional regimens but that this enhanced efficiency of recall immunity was afforded by incorporating CD4(-)CD8(-)Thy1(+) cells into the secondary response. Taken together, these findings demonstrate that Thy1-expressing NK cells play an important role in antibacterial immunity.

First human model of in vitro Candida albicans persistence within granuloma for the reliable study of host-fungi interactions.

BACKGROUND: The balance between human innate immune system and Candida albicans virulence signaling mechanisms ultimately dictates the outcome of fungal invasiveness and its pathology. To better understand the pathophysiology and to identify fungal virulence-associated factors in the context of persistence in humans, complex models are indispensable. Although fungal virulence factors have been extensively studied in vitro and in vivo using different immune cell subsets and cell lines, it is unclear how C. albicans survives inside complex tissue granulomas. METHODOLOGY/PRINCIPAL FINDING: We developed an original model of in vitro human granuloma, reproducing the natural granulomatous response to C. albicans. Persistent granulomas were obtained when the ratio of phagocytes to fungi was high. This in vitro fungal granuloma mimics natural granulomas, with infected macrophages surrounded by helper and cytotoxic T lymphocytes. A small proportion of granulomas exhibited C. albicans hyphae. Histological and time-lapse analysis showed that C. albicans blastoconidia were located within the granulomas before hyphae formation. Using staining techniques, fungal load calculations, as well as confocal and scanning electron microscopy, we describe the kinetics of fungal granuloma formation. We provide the first direct evidence that C. albicans are not eliminated by immunocompetent cells inside in vitro human granulomas. In fact, after an initial candicidal period, the remaining yeast proliferate and persist under very complex immune responses. CONCLUSIONS/SIGNIFICANCE: Using an original in vitro model of human fungal granuloma, we herein present the evidence that C. albicans persist and grow into immunocompetent granulomatous structures. These results will guide us towards a better understanding of fungal invasiveness and, henceforth, will also help in the development of better strategies for its control in human physiological conditions.

Evaluation of peripheral blood lymphocyte subsets in family-owned dogs naturally infected by Ehrlichia canis.

Previous research suggested that clinical manifestations, histopathological lesions, and even infection maintenance in the course of canine monocytic ehrlichiosis (CME) are directly related to the immune response developed by the host. In the present study, blood lymphocyte subsets were analyzed by multiparametric flow cytometry in 37 dogs with naturally occurring CME and 47 healthy dogs used as controls. T, T helper (Th), T cytotoxic (Tc), B, non-T, non-B lymphocytes and those that express MHC class II were characterized in every dog. Animals with CME showed higher relative values of T and Tc cells and a higher absolute number of Tc cells in peripheral blood. The percentage of Th cells and the absolute and relative values of B cells were higher in healthy animals than in CME-affected dogs. The significance of these changes on the pathogenesis of natural Ehrlichia canis infection in dogs needs further evaluation.

Probing CD8 T cell responses with Listeria monocytogenes infection.

CD8 T cells play a critical role in the control and eradication of intracellular pathogens. Increased understanding of CD8 T cell biology provides insight that can be translated into improved vaccination strategies. The intracellular bacterium, Listeria monocytogenes, has been used as a model organism to study every phase of the CD8 T cell response to intracellular bacterial infection. Infection of laboratory mice with L. monocytogenes has provided insight into the factors that are involved in primary T cell responses, memory CD8 T cell generation, maintenance, functionality, and diversification following repeated pathogenic challenges. In this review, we will focus on work from our laboratories utilizing the murine model of L. monocytogenes to investigate the characteristics of CD8 T cell responses to infection. This model has profoundly advanced our understanding of the CD8 T cell response to infection and is likely to continue to provide invaluable basic insights that can be translated into the development of effective vaccination strategies to protect against pathogens.

Repetitive antigen stimulation induces stepwise transcriptome diversification but preserves a core signature of memory CD8(+) T cell differentiation.

Repetitive antigen stimulation by prime-boost vaccination or pathogen reencounter increases memory CD8(+) T cell numbers, but the impact on memory CD8(+) T cell differentiation is unknown. Here we showed that repetitive antigen stimulations induced accumulation of memory CD8(+) T cells with uniform effector memory characteristics. However, genome-wide microarray analyses revealed that each additional antigen challenge resulted in the differential regulation of several hundred new genes in the ensuing memory CD8(+) T cell populations and, therefore, in stepwise diversification of CD8(+) T cell transcriptomes. Thus, primary and repeatedly stimulated (secondary, tertiary, and quaternary) memory CD8(+) T cells differed substantially in their molecular signature while sharing expression of a small group of genes and biological pathways, which may constitute a core signature of memory differentiation. These results reveal the complex regulation of memory CD8(+) T cell differentiation and identify potential new molecular targets to dissect the function of memory cells generated by repeated antigen stimulation.

Global suppression of mitogen-activated ovine peripheral blood mononuclear cells by surface protein activity from Mycoplasma ovipneumoniae.

Mycoplasma ovipneumoniae is associated with chronic non-progressive pneumonia of sheep and goats. As with many other mycoplasmas involved in animal diseases, protective immune responses have not been achieved with vaccines, even though antibody responses can be obtained. This study focuses on characterizing the interaction of M. ovipneumoniae with ovine PBMC using carboxy-fluorescein-succinimidyl-ester (CFSE) loading and flow cytometry to measure lymphoid cell division. M. ovipneumoniae induced a strong in vitro polyclonal suppression of CD4(+), CD8(+), and B blood lymphocyte subsets. The suppressive activity could be destroyed by heating to 60 degrees C, and partially impaired by formalin and binary ethyleneimine treatment that abolished its viability. The activity resided on the surface-exposed membrane protein fraction of the mycoplasma, since mild trypsin treatment not affecting viability was shown to reduce suppressive activity. Trypsin-treated mycoplasma regained suppressive activity once the mycoplasma was allowed to re-synthesize its surface proteins. Implications for the design of vaccines against M. ovipneumoniae are discussed.

The spectrum of systemic immune alterations after murine focal ischemia: immunodepression versus immunomodulation.

BACKGROUND AND PURPOSE: Therapeutic modification of the postischemic immune processes is a key target of current experimental stroke research. For successful translation into the clinical setting, experimental studies must account for the impact of different strokes on the immune system including susceptibility to infection. Herein, we characterize the impact of 3 ischemia models on systemic immunological and microbiological parameters. METHODS: In C57Bl/6 mice (n=235), the middle cerebral artery was occluded (MCAO) either permanently by distal coagulation or transiently by an intraluminal filament for 30 minutes or 90 minutes. Differential leukocyte counts were performed in blood and lymphatic organs. Lymphocyte subpopulations and apoptotic cells were characterized by flow cytometry. Blood cytokine concentrations were measured by ELISA. Microbiological cultures were grown from blood and lung samples. RESULTS: Only extensive infarcts induced leukopenia 24 hours, 3 days and 7 days after MCAO and decreased lymphocyte counts in spleen, lymph nodes and thymus. In contrast, small infarcts led to no significant changes in differential blood count or reduction of overall cell counts in lymphatic organs. Splenic lymphocyte apoptosis and blood cytokine production was significantly increased after extensive lesions compared to mild ischemia. Hypothermia and weight loss occurred only in mice with large infarcts which also suffered from pneumonia and sepsis. In contrast to infarct size, location and side of the infarct did not affect physiological parameters and immune cell alterations. CONCLUSIONS: Postischemic systemic immunomodulation and infectious complications differ substantially among stroke models. Translational studies of immunomodulatory therapies for stroke must account for this heterogeneity.

Reciprocal interactions between commensal bacteria and gamma delta intraepithelial lymphocytes during mucosal injury.

The intestinal mucosal surface is in direct contact with a vast beneficial microbiota. The symbiotic nature of this relationship is threatened when the surface epithelium is injured, yet little is known about how mucosal surfaces maintain homeostasis with commensal microbes following damage. Gammadelta intraepithelial lymphocytes (gammadelta IEL) reside at the gut epithelial surface, where they stimulate mucosal healing following acute injury. A genome-wide analysis of the gammadelta IEL response to dextran sulfate sodium-induced colonic damage revealed induction of a complex transcriptional program, including coordinate regulation of cytoprotective, immunomodulatory, and antibacterial factors. Studies in germfree mice demonstrated that commensal microbiota regulate key components of this transcriptional program, thus revealing a dialogue between commensal bacteria and gammadelta IEL in injured epithelia. Analysis of TCRdelta-deficient mice indicated that gammadelta T cells are essential for controlling mucosal penetration of commensal bacteria immediately following dextran sulfate sodium-induced damage, suggesting that a key function of gammadelta IEL is to maintain host-microbial homeostasis following acute mucosal injury. Taken together, these findings disclose a reciprocal relationship between gammadelta T cells and intestinal microbiota that promotes beneficial host-microbial relationships in the intestine.

Selective immunoglobulin M deficiency presenting with recurrent impetigo: a case report and review of the literature.

Selective immunoglobulin M (IgM) deficiency is a rare disorder defined by a decreased serum level of IgM and normal levels of other immunoglobulin classes. The disease has not been well described and the cause remains unknown. Patients with IgM deficiency may present with a wide spectrum of clinical manifestations, from asymptomatic to life-threatening infections, including recurrent respiratory and gastrointestinal infections, allergy and autoimmunity. Here, we report a 6.5-year-old otherwise healthy boy with selective IgM deficiency who presented with multiple recurrent impetigo. We reviewed the published data regarding selective deficiency of IgM.

Analysis of cells targeted by Salmonella type III secretion in vivo.

The type III secretion systems (TTSS) encoded in Salmonella pathogenicity island-1 and -2 (SPI-1 and -2) are virulence factors required for specific phases of Salmonella infection in animal hosts. However, the host cell types targeted by the TTSS have not been determined. To investigate this, we have constructed translational fusions between the beta-lactamase reporter and a broad array of TTSS effectors secreted via SPI-1, SPI-2, or both. Secretion of the fusion protein to a host cell was determined by cleavage of a specific fluorescent substrate. In cultured cells, secretion of all six effectors could be observed. However, two to four days following i.p. infection of mice, only effectors secreted by SPI-2 were detected in spleen cells. The cells targeted were identified via staining with nine different cell surface markers followed by FACS analysis as well as by conventional cytological methods. The targeted cells include B and T lymphocytes, neutrophils, monocytes, and dendritic cells, but not mature macrophages. To further investigate replication in these various cell types, Salmonella derivatives were constructed that express a red fluorescent protein. Bacteria could be seen in each of the cell types above; however, most viable bacteria were present in neutrophils. We find that Salmonella is capable of targeting most phagocytic and non-phagocytic cells in the spleen but has a surprisingly high preference for neutrophils. These findings suggest that Salmonella specifically target splenic neutrophils presumably to attenuate their microbicidal functions, thereby promoting intracellular survival and replication in the mouse.