Search

Previously, our laboratory established the role of small, noncoding RNA species, i.e., microRNA (miRNA) including miR-135a in anti-chlamydial immunity in infected hosts. We report here chlamydial infection results in decreased miR-135a expression in mouse genital tissue and a fibroblast cell line. Several chemokine and chemokine receptor genes (including CXCL10, CCR5) associated with chlamydial pathogenesis were identified in silico to contain putative miR-135a binding sequence(s) in the 3’ untranslated region. The role of miR-135a in the host immune response was investigated using exogenous miR-135a mimic to restore the immune phenotype associated with decreased miR-135a following Chlamydia muridarum (Cm) infection. We observed miR-135a regulation of Cm-primed bone marrow derived dendritic cells (BMDC) via activation of Cm-immune CD4+ T cells for clonal expansion and CCR5 expression. Using a transwell cell migration assay, we explore the role of miR-135a in regulation of genital tract CXCL10 expression and recruitment of CXCR3+ CD4+ T cells via the CXCL10/CXCR3 axis. Collectively, data reported here support miR-135a affecting multiple cellular processes in response to chlamydial infection.

The leading cause of sexually transmitted bacterial infection is Chlamydia trachomatis. The aim of this study is to investigate the early events in colonization of this bacterium within the murine genital tract. An in vivo animal body imaging technology was used to track fluorophore labeled C. muridarum elementary bodies (EBs) inoculated intravaginally in C57BL/6 mice during the first 24 h of infection. Ascension of viable EBs was observed (1) to be localized to the lower regions of the murine genital tract within the first 24 h post challenge and (2) was dose independent during this early exposure period. Molecular detection revealed enhanced bacterial load in lower regions of the genital tract with increasing bacterial load in the upper region beginning 12 h post inoculation. This study provides additional insight into chlamydial colonization in the murine genital tract during the first 12–24 h following inoculation.

Mast cells are now recognized as effective modulators of innate immunity. We recently reported that mast cells and secreted interleukin-4 (IL-4) effectively control intramacrophage replication of Francisella tularensis Live Vaccine Strain (LVS), and that mice deficient in mast cells or IL-4 receptor (IL-4R(-/-)) exhibit greater susceptibility to pulmonary challenge. In this study, we further evaluated the mechanism(s) by which mast cells/IL-4 control intramacrophage bacterial replication and host cell death, and found that IL-4R(-/-) mice exhibited significantly greater induction of active caspase-3 within lung macrophages than wild-type animals following intranasal challenge with either LVS or the human virulent type A strain SCHU S4. Treatment of LVS-infected bone-marrow-derived macrophages with a pancaspase inhibitor (zVAD) did not alter bacterial replication, but minimized active caspase-3 and other markers (Annexin V and propidium iodide) of cell death, whereas treatment with both rIL-4 and zVAD resulted in concomitant reduction of both parameters, suggesting that inhibition of bacterial replication by IL-4 was independent of caspase activation. Interestingly, IL-4-treated infected macrophages exhibited significantly increased ATP production and phagolysosomal acidification, as well as enhanced mannose receptor upregulation and increased internalization with acidification, which correlated with observations in mast cell-macrophage co-cultures, with resultant decreases in F. tularensis replication.

The need for an efficacious vaccine against Francisella tularensis is a consequence of its low infectious dose and high mortality rate if left untreated. This study sought to characterize a live attenuated subspecies novicida-based vaccine strain (U112ΔiglB) in an established second rodent model of pulmonary tularemia, namely the Fischer 344 rat using two distinct routes of vaccination (intratracheal [i.t.] and oral). Attenuation was verified by comparing replication of U112ΔiglB with wild type parental strain U112 in F344 primary alveolar macrophages. U112ΔiglB exhibited an LD(50)>10(7) CFU compared to the wild type (LD(50) = 5 × 10(6) CFU i.t.). Immunization with 10(7) CFU U112ΔiglB by i.t. and oral routes induced antigen-specific IFN-γ and potent humoral responses both systemically (IgG2a>IgG1 in serum) and at the site of mucosal vaccination (respiratory/intestinal compartment). Importantly, vaccination with U112ΔiglB by either i.t. or oral routes provided equivalent levels of protection (50% survival) in F344 rats against a subsequent pulmonary challenge with ~25 LD(50) (1.25 × 10(4) CFU) of the highly human virulent strain SCHU S4. Collectively, these results provide further evidence on the utility of a mucosal vaccination platform with a defined subsp. novicida U112ΔiglB vaccine strain in conferring protective immunity against pulmonary tularemia.

Proteolytic cleavage of hemagglutinin is required for cell entry by receptor-mediated endocytosis and plays a key role in pathogenicity of the influenza virus. Despite several studies describing relationships between bacterial proteases and influenza A viral activation in mammals, very little is known about the role of the normal bacterial flora of birds on hemagglutinin activation. We examined the indigenous intestinal microflora of 100 mixed-sex, 27-d-old Ross chickens from a commercial poultry facility for protease-secreting bacteria. Protease-secreting bacteria were isolated from 82 of 100 chickens with 50 birds exhibiting 2 or more protease-secreting bacterial species. A total of 20 protease-secreting bacterial species were identified: 17 gram-positive cocci, 2 gram-positive rods, and 1 gram-negative rod. Enterococcus faecalis, Enterococcus gallinarum, and Proteus mirabilis were the most frequently observed protease-secreting bacterial species. The presence of proteolytic bacteria in the intestinal tract of poultry in this study suggests the possibility of yet-to-be-described role(s) in cleavage of hemagglutinin that may alter the pathogenicity of avian influenza viruses.

Francisella tularensis is a gram-negative, facultative intracellular bacterium and is the causative agent of the zoonotic disease tularemia [1]. F. tularensis has been considered a potential biological weapon due to its low infectious dose and high mortality rate [2]. F. tularensis can be classified into several subspecies, including those relevant to human disease: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B); F. novicida and F. mediasiatica [3]. However, most of our knowledge about the pathogenesis of Francisella and the immune responses to the infection have come from studies of F. tularensis LVS (derived from holartica) and F. novicida [4–6]. Both organisms are attenuated in humans, while retaining virulence in mice. It is well established that Francisella enters and replicates within host cells, and a strong research focus has been on deciphering the mechanisms for intramacrophage growth. However, little is known about the extracellular phase of Francisella. F. tularensis has been cultured from blood of infected animals and humans, although the reported cases have been rare [7, 8]. Specifically, Long et al. [8] demonstrated that LVS could be recovered from plasma prepared from mice 72 h after intravenous infection. These extracellular bacteria accounted for less than 1% of total LVS within the blood. However, a recent study by Forestal et al. [9] indicated a much higher percentage (>50%) of extracelluar Francisella (type A SCHU S4 and LVS) within plasma of mice infected intradermally or intranasally. Here, independently, we confirm the presence of extracellular Francisella in plasma after intranasal F. novicida challenge in a murine model of pulmonary tularemia [6]. Moreover, we demonstrate that the extracellular Francisella in plasma is highly infectious and that this phase may contribute to the rapid dissemination of the bacterium from lungs to the liver after pulmonary infection. F. novicida strain U112 shares a high degree of antigenic and genetic similarity with F. tularensis type A and B [10] and is highly virulent in mice with a LD50 of approximately 10 colony forming units (CFU) by the intranasal (i.n.) route [6]. In this study, we monitored the replication and dissemination of F. novicida in mice. Bacteria were grown at 37°C in Trypticase Soy Broth (TSB) supplemented with 0.1% cysteine. The concentrations of the bacterial inocula were determined by serial dilution plating on Trypticase Soy Agar (TSA). Female BALB/c mice (6–8 weeks old, obtained from the Jackson Laboratory, Bar Harbor, ME) were challenged i.n. with 400 CFU (40 LD50). All animal care and experimental procedures were performed in compliance with the Institutional Animal Care and Use Committee guidelines of the University of Texas at San Antonio. Bacterial loads in the lungs and livers were assessed by plating tissue homogenates on TSA. Within 24 h of pulmonary challenge, a large number of F. novicida, approximately 1×10 CFU/g tissue, was observed in the lungs, which increased to 1×10 CFU/g tissue by 48 h and then decreased to 1×10 CFU/g tissue by 72 h. In the livers of infected mice, there was a progressive increase in bacterial load with levels rising from 100 CFU/g tissue at 24 h to approximately 1×10 CFU/g tissue by 72 h (Fig. 1a). These results demonstrate dissemination of F. novicida from the lungs to the secondary organs after pulmonary exposure. To determine a probable mechanism for the rapid spread to the secondary organs, blood was collected from the orbital plexus at 24, 48 and 72 h Eur J Clin Microbiol Infect Dis (2008) 27:323–325 DOI 10.1007/s10096-007-0434-x

To ascertain the health risks that may be posed by the land application of sewage sludges, a scheme was devised to determine the types and numbers of pathogenic and potentially pathogenic bacteria present in sludges. A processing treatment was adapted to sludge to give a homogenate which yielded the greatest numbers of viable bacteria. Conventional methods were successful in enumerating Klebsiella, Staphylococcus, gram-negative enteric bacteria, and commonly used indicator organisms. Modifications of conventional methods improved the enumeration of Salmonella, Mycobacterium sp., fluorescent Pseudomonas sp., and Clostridium perfringens. However, Shigella methodology yielded only one isolate. Utilizing the proposed scheme, the population densities of these organisms were estimated in three domestic wastewater sludges. In light of these results, the potential impact of land application of sewage sludges is discussed.

The susceptibility of suckling mice to oral infection with several different Vibrio cholerae was determined. Mice up to 10 days of age were uniformly susceptible to oral infection with 10 8 colony-forming units of virulent organisms. Age-dependent resistance occurred thereafter to a maximum at about 15 days of age. The efficacy of selected vaccines was compared by oral challenge of 8-day-old, passively immunized CFW mice. An Ogawa-derived ribosomal antigen was found to be superior to a commercial whole-cell vaccine or to purified cholera enterotoxin. The ribosomal antigen was 50- to 100-fold more protective than the other vaccines on a weight basis against otherwise lethal challenge with Ogawa, Inaba, or El Tor Ogawa serotypes.

Oral-intragastric inoculation of infant CFW mice with Candida albicans, leading either to lethality or to persistent infection of long duration, provides a useful model for study of the host-pathogen interrelationships in candidosis. Mice were most susceptible to the lethal effects of challenge when 4 to 6 days of age, increasingly resistant up to 10 to 11 days, and then resistant to doses of C. albicans lethal for the younger animals. Older mice harboring persistent infections of the gastrointestinal tract, originally initiated when the animals were 6 days old, were used to study the effects of agents which commonly are administered to cancer patients or which are known to predispose to candidosis. The broad-spectrum antibiotic chloramphenicol, cortisone acetate, X-irradiation, or single high doses of cyclophosphamide (Cytoxan) resulted in markedly enhanced levels of C. albicans in the gastrointestinal tract without systemic spread. Repeated smaller doses of Cytoxan, or treatment with methotrexate or a combination of cortisone acetate and Cytoxan, produced gastrointestinal candidosis associated with invasion and systemic spread. The data indicate that the persistently infected animals provide a realistic model for studying treatments that precipitate candidosis in humans.

The ability of motile strains of the Ogawa and Inaba serotypes of classical Vibrio cholerae and of the El Tor biotypes to kill suckling mice after oral challenge with 10-8 colony-forming units (representing at least 100 to 1,000 minimal lethal doses) was compared to that of nonmotile derivatives of the same strains. Loss of motility, in each case, resulted in a marked reduction in virulence. The mortality (at 36 h) caused by 10 of the 13 nonmotile strains was 32% or less. whereas the motile wild-type strains resulted in nearly 100% deaths. The reduced virulence of the nonmotile strains was associated with reduced capacity to adsorb to the surface of segments of mouse intestine. The mutants were tested for alterations in enterotoxin production and surface properties. The results suggest that motility may contribute to virulence by increasing the chance for association of the vibrios with the intestinal mucosa.

Members of the genus Candida, particularly C. albicans, are frequent human pathogens that have increased in importance with advances in modern medicine and chemotherapy. Candida albicans in the yeast form may occur in small numbers on the mucous membranes of the alimentary tract or vagina of healthy human hosts; but, they may be the etiologic agent of disease in virtually any tissue of the body of the compromised host (186). The most common manifestations of candidiasis (or candidosis) involve superficial infections of the skin or the mucous membranes of the mouth and vagina. Odds (186) for example, in his extensive review of the literature, has estimated that oral thrush may occur in approximately 5% of neonates, candidal-associated palatal stomatitis in as many as 60% of elderly denture wearers, and vaginal thrush in approximately 5% of obstetric and gynecologic outpatients. Advances in the medical sciences have led to increases in both the incidence and spectrum of severity of candidiasis. For example, Candida is a frequent cause of either superficial lesions of epithelial surfaces or a generalized systemic mycosis in patients who are undergoing chemotherapy for cancer (55, 218, 315), immunosuppressive treatment for organ transplants (43), surgery (67), and prolonged oral antibiotic medication (74, 257); or, in patients suffering from immune deficiency diseases (34, 43, 210, 212, 302), including the newly recognized epidemic illness of acquired immunodeficiency syndrome (AIDS) (73).

Persistent infection of the gastrointestinal tract of CFW mice with Candida albicans was produced by the oral-intragastric inoculation of 6-day-old infants. Other intraabdominal organs (liver, kidneys, and spleen) were usually free of the organism in survivors at 20 days of age. However, all survivors retained high levels of the organism in the stomach and intestinal tract at 30 days of age. The possible utility of these persisting C. albicans infections of the gastrointestinal tract for the study of the efficacy of short-term antifungal therapy was studied. Drug treatment was initiated for a 2-week period when the survivors were 15 to 19 days old. Some representative antifungal agents in current use (i.e., amphotericin B, 5-fluorocytosine, and miconazole) effected significant reductions in the numbers of C. albicans in homogenates of gastrointestinal organs.

Infant mice have been shown previously to be a useful model for the study of gastrointestinal (GI) and systemic candidosis. In this study, the virulence of four strains of Candida albicans was compared in intragastrically inoculated infants and in adult mice inoculated intravenously. The four strains differed in their ability to kill both infant and adult mice. A smaller inoculum was required to kill adult mice inoculated intravenously. Neonates could not be inoculated intravenously. The ability of the strains to spread systemically from and to persist for long periods of time in the digestive tract was also examined in intragastrically inoculated infants. The yeast cells spread to liver, lungs, kidneys, and spleen within 30 min postinoculation. Yeast were not detectable in the lungs or in blood from the pleural cavity up to 15 min post-inoculation, thus making it unlikely that systemic spread resulted from faulty inoculation or from aspiration. The region where C. albicans crossed the GI tract of infant mice was visualized histologically in the upper third of the small intestine. The four strains varied in their ability to persist for long periods in the GI tract, in the rate at which they appeared systemically, and in ability to kill infant mice. Three of the four strains colonized the gut for up to 10 weeks postinoculation without use of any compromising agents.

Nonmotile mutants of Vibrio cholerae isolated from Ogawa, Inaba, and El Tor strains were less virulent than parent wild types when administered to adult mice intraperitoneally. The cells were suspended in 5% hog gastric mucin. Antitoxic immunity did not protect mice against this type of challenge, but a ribosomally derived vaccine did. Intraperitoneal injection of 10 50% lethal doses of enterotoxin (based on intravenous doses) was without toxic manifestations as were 10(10) heat-killed vibrios similarly administered, regardless of strain. Virulent organisms killed with formalin or ultraviolet irradiation were significantly lethal at a dose of 10(10) cells. Mice made tolerant to endotoxin were protected from death caused by an injection of 3 X 10(10) boiled cells, but they did not survive an injection of formalin-killed cells. It is believed that the cause of death in this animal model of cholera is dependent, at least in part, on a toxic heat-labile moiety closely associated with the vibrios.

Infant mice 8 days of age were infected orally with virulent, motile, classical or El Tor strains of Vibrio cholerae and with nonmotile mutants of low virulence derived from the same strains. At intervals of 8 and 12 h postinfection, frozen thin sections of the ileum were prepared, stained with fluorescein isothiocyanate-labeled rabbit anti-vibrio antibody, and examined with the fluorescence microscope. The motile organisms were present in larger numbers, especially at 12h, and had penetrated the intervillous spaces and crypts of Lieberkuhn more completely than nonmotile vibrios. Dilution counts were made on various regions of the intestines of infant mice challenged orally 12 h previously with either motile or nonmotile strains of V. cholerae. Greater numbers of organisms were found, especially in the upper intestinal regions, when motile organisms were used. Low numbers of vibrios, limited mostly to the lumen, were seen in the ileum of infant mice infected with motile organisms when the infants were the offspring of mothers that had been immunized with crude flagellar vaccine or a vesicular preparation derived from the vibrio cell surface. The distribution of vibrios in this case was similar to that found in infected infants of unvaccinated mothers challenged with nonmotile organisms. Motility appears to enable the bacteria to better populate the upper regions of the intestinal tract and to avoid the washing effects of secretions and peristalsis. Antibacterial immunity may function, at least in part, by making it impossible for motile vibrios to accomplish this widespread distribution within the ileum.

This review describes the application of SEM to the study of microorganisms in gastrointestinal (GI) pathobiology. SEM has proven to be a highly useful tool with a variety of applications in the biological and medical sciences. Examples in this paper are focused on the use of secondary electron imaging to study selected bacterial (cholera), fungal (candidosis), and protozoan (giardiasis) diseases in the GI tract of murine (mouse and rat) experimental models. The successful application of SEM to studies of microorganisms in GI pathobiology requires strict attention to optimal preparative techniques, to selection of the appropriate method and microenvironment(s) for study, and to awareness of the indigenous microorganisms characteristic of the system studied.

Scanning electron microscopy (SEM) was used in this study to investigate the mechanism of association of cholera vibrios with the intestinal mucosa. For this purpose, the interactions of three virulent, wild type strains (CA401, P, and 3083) of Vibrio cholerae with intestinal segments of infant mice were compared. The results of previous studies by different investigators using the three strains separately and in different assay systems had led to differing conclusions concerning the mechanisms of association with the mucosa. In this study, observations by SEM of the infant mouse intestinal segments, previously incubated in suspensions of the organisms, revealed that all three strains preferentially associated with intestinal mucin. The vibrios associated poorly with the mucosal epithelial surface even in areas of the segments free of mucin or in segments washed to remove mucin prior to the assay. In each case, the vibrios associated better with the serosal surface than with the epithelial surface of the mucosa, but not to the degree that they associated with mucin on the mucosal surface. The major difference observed by SEM of the strains was the markedly greater association of strain 3083 with the serosal surface as compared to strains CA401 and P.

Scanning electron microscopy (SEM) was used to observe the nature of association of mutant strains of Vibrio cholerae to infant mouse intestinal segments. Parental strain Inaba CA 401 was compared to the following mutant phenotypes: nonchemotactic, nonchemotactic reduced motility, flagellated nonmotile, nonflagellated nonmotile, and mucin nonutilizing. The virulence of the different strains was determined by oral-intra-gastric inoculation of seven-day-old mice. Localization of the parental strain during the course of infection in the patent gut of infant mice was followed by the fluorescent antibody (FA) technique. The highly virulent parental strain rapidly penetrated intervillus spaces in the upper and lower regions of the small intestine. By SEM the organism also associated markedly with the mucosal surface of intestinal segments. However, the vibrios were associated predominately with the intestinal mucin rather than the epithelial cell surfaces of villi. Nonchemotactic vibrios retained full virulence and ability to associate with the mucosa. The nonchemotactic strain with a reduced rate of motility was partially deficient in these properties. Nonmotile vibrios of either type were markedly reduced in both properties. The mucin nonutilizing strain associated normally but was weakly virulent. Low virulence of this strain may be due to a reduced ability of the organisms to multiply in the mucin.

Systemic and gastrointestinal infection can be established in infant mice after intragastric challenge with Candida albicans. Differences in virulence of the six strains tested were noted. As early as 3 h after infection, some but not all livers, spleens, and kidneys contained C. albicans, but the peak number of colony-forming units in these organs was seen at 6 h. The early colonization of the organs could not be attributed to aspiration of the inoculum since about 90% of lungs and livers tested yielded no colony-forming units at 10 to 15 min postinfection. In animals with systemic infections, lungs, livers, kidneys, and spleens showed similar numbers of colony-forming units within the organs during the first 6 h postinfection- and then the number declined progressively up to 72 h. The gastrointestinal tract was colonized throughout a 20-day period of study. Counts made at intervals beyond day 1 yielded between 10(5) and 10(6) colony-forming units in the stomach, ileum, and cecum. Preparatory techniques for scanning electron microscopy preserved the yeast, intestinal mucus layer, and epithelial surface and made it possible to visualize the association between the pathogen and host tissues within the digestive tract.