Differential interaction of dendritic cells with Rickettsia conorii: impact on host susceptibility to murine spotted fever rickettsiosis

Spotted fever group rickettsioses are emerging and reemerging infectious diseases, some of which are life-threatening. In order to understand how dendritic cells (DCs) contribute to the host resistance or susceptibility to rickettsial diseases, we first characterized the in vitro interaction of rickettsiae with bone marrow-derived DCs (BMDCs) from resistant C57BL/6 (B6) and susceptible C3H/HeN (C3H) mice. In contrast to the exclusively cytosolic localization within endothelial cells, rickettsiae efficiently entered and localized in both phagosomes and cytosol of BMDCs from both mouse strains. Rickettsia conorii-infected BMDCs from resistant mice harbored higher bacterial loads compared to C3H mice. R. conorii infection induced maturation of BMDCs from both mouse strains as judged by upregulated expression of classical major histocompatibility complex (MHC) and costimulatory molecules. Compared to C3H counterparts, B6 BMDCs exhibited higher expression levels of MHC class II and higher interleukin-12 (IL-12) p40 production upon rickettsial infection and were more potent in priming na?ve CD4(+) T cells to produce gamma interferon. In vitro DC infection and T-cell priming studies suggested a delayed CD4(+) T-cell activation and suppressed Th1/Th2 cell development in C3H mice. The suppressive CD4(+) T-cell responses seen in C3H mice were associated with a high frequency of Foxp3(+) T regulatory cells promoted by syngeneic R. conorii-infected BMDCs in the presence of IL-2. These data suggest that rickettsiae can target DCs to stimulate a protective type 1 response in resistant hosts but suppressive adaptive immunity in susceptible hosts.     

Experimental murine model of disseminated Pseudallescheria infection.

Pseudallescheria boydii is found in soil and has a worldwide distribution. This fungus was initially identified as a pathogen targeting a variety of tissues. There are fragmentary data in the literature on the in vitro susceptibility of P. boydii to different antifungal compounds. P. boydii is highly refractory to antifungal treatments. In this study, a murine model of disseminated Pseudallescheria infection was developed to evaluate efficacy of different treatment regimens. A clinical strain of P. boydii was studied in normal and neutropenic outbred ICR mice. Several inocula were tested over a range from 1 x 10(3) to 5 x 10(6) cfu. Groups of eight mice were injected with a intravenous dose of one inoculum. Mortality correlated with the dose of the inoculum, and with immunosuppression. Quantitative cultures of various tissues showed initial dissemination of disease in immune competent mice. This was followed by, reduction of tissue burden, except in the brain. In contrast, disseminated infection persisted in most organs in immunosuppressed animals (p < 0.0001). This model should be appropriate for in vivo evaluation of antifungal chemotherapy.     

Transfer factor: a murine model.

Transfer factor has been studied extensively in humans, but a satisfactory subprimate model has not been established. Using BALB/c mice immunized with complete Freund adjuvant, we show that a low-molecular-weight substance derived from disrupted spleen cells transferred sensitivity to purified protein derivative (PPD) to recipient nonimmunized BALB/c mice. Transfer was confirmed by footpad swelling to PPD in vivo and by splenic lymphocyte transformation to PPD in vitro. In recipients of transfer factor, an inverse correlation was noted between the splenic lymphocyte response to PPD and to concanavalin A. Material obtained from spleens of saline-treated BALB/c mice did not transfer sensitivity to PPD to recipient mice.     

Effect of immunization on experimental Bacteroides gingivalis infection in a murine model.

BALB/c mice were immunized with an invasive (A7A1-28) or noninvasive (381) Bacteroides gingivalis strain, Bacteroides intermedius, or Ringer solution. All immunized mice were subsequently challenged with the invasive B. gingivalis strain and examined for septicemia or secondary spread of the infection or both. Mice immunized with the invasive B. gingivalis strain localized the infection to the challenge site. Mice immunized with the noninvasive B. gingivalis strain, B. intermedius, or Ringer solution developed spreading infections. These data suggest that immunization with an invasive B. gingivalis strain can alter the course of subsequent infections.     

The temporal dynamics of humoral immunity to Rickettsia typhi infection in murine typhus patients

ObjectivesThis study examined individuals with Rickettsia typhi infection in the Lao People's Democratic Republic (Lao PDR) to (a) investigate humoral immune dynamics; (b) determine the differences in reference diagnostic results and recommend appropriate cut-offs; (c) determine differences in immune response after different antibiotic treatments; and (d) determine appropriate diagnostic cut-off parameters for indirect immunofluorescence assay (IFA).MethodsSequential serum samples from 90 non-pregnant, adults were collected at seven time-points (days 0, 7, 14, 28, 90, 180 and 365) as part of a clinical antibiotic treatment trial. Samples were tested using IFA to determine IgM and IgG antibody reciprocal end-point titres against R. typhi and PCR.ResultsFor all 90 individuals, reciprocal R. typhi IgM and IgG antibody titres ranged from <400 to ≥3200. The median half-life of R. typhi IgM was 126 days (interquartile range 36–204 days) and IgG was 177 days (interquartile range 134–355 days). Overall median patient titres for R. typhi IgM and IgG were significantly different (p < 0.0001) and at each temporal sample collection point (range p < 0.0001 to p 0.0411). Using Bayesian latent class model analysis, the optimal diagnostic cut-off reciprocal IFA titer on patient admission for IgM was 800 (78.6%, 95% CI 71.6%–85.2% sensitivity; 89.9%, 95% CI 62.5%–100% specificity), and for IFA IgG 1600 (77.3%; 95% CI 68.2%–87.6% sensitivity; 99%, 95% CI 95%–100% specificity).ConclusionsThis study suggests suitable diagnostic cut-offs for local diagnostic laboratories and other endemic settings and highlights antibody persistence following acute infection. Further studies are required to validate and define cut-offs in other geographically diverse locations.     

Immunologically mediated fatigue: a murine model.

Chronic fatigue syndrome (CFS) is an idiopathic disorder in which the chief symptoms is profound fatigue. To explore the relationship between immune stimulation and fatigue, we developed a murine model for quantifying fatigue: reduction in voluntary running and delayed initiation of grooming after swimming. Inoculation of female BALB/c mice with Corynebacterium parvum antigen or the relatively avirulent Me49 strain of Toxoplasma gondii induced fatigue: baseline running reduced to less than 50 and 30% for 8 and 14 days, respectively, and delayed initiation of grooming after swimming in both immunologically stimulated groups. A threefold evaluation of serum transforming growth factor-beta levels, a cytokine increased in CFS patients, was found in fatigued C. parvum- and T. gondii-inoculated mice. This murine model appears promising for investigation of the pathogenesis of immunologically mediated fatigue.     

Efficacy of caspofungin and posaconazole in a murine model of disseminated Exophiala infection.

Disseminated phaeohyphomycosis is an uncommon infection affecting immunocompetent and immunocompromised individuals in which response to older antifungal agents has been variable. We compared the effect of six days of therapy with caspofungin, posaconazole, and amphotericin B in parallel studies of survival and fungal burden in an immunocompromised mouse model of Exophiala infection. Mice immunocompromised with cyclophosphamide were treated for 6 days starting one day after initiation of infection. Treatment regimens included amphotericin B, caspofungin, and posaconazole. In the survival studies, experimental animals were observed for 14 days. In the fungal burden tests the experimental animals were sacrificed 7 days after infection and brain and kidney burden determined. Treatment with any agent decreased mortality (P < 0.05), with 40%, 30%, and 80% observed survival of the animals treated with amphotericin B, caspofungin, and posaconazole, respectively. Amphotericin B and posaconazole treatment resulted in a decrease in fungal burden compared to untreated controls (P < 0.05). No reduction in fungal burden was noted in the caspofungin group. All three antifungals evaluated improved survival of immunocompromised mice in this otherwise fatal disseminated phaeohyphomycosis. Amphotericin B and posaconazole reduced fungal burden. Posaconazole and caspofungin appear to have potential for use in treatment of this rare infection.     

Protective role of mannan-binding lectin in a murine model of invasive pulmonary aspergillosis

Innate immune molecules such as lung collectins and serum pentraxins have evolved as important host defence proteins against Aspergillus fumigatus, a medically important opportunistic fungal pathogen. Mannan-binding lectin (MBL), an opsonin and lectin complement pathway activator, constitutes another vital player of innate immunity against several pathogenic organisms in the serum. Studies have reported significant binding of MBL to A. fumigatus; however, the protective role of MBL against A. fumigatus-mediated invasive disease remains elusive. Henceforth, we investigated the contribution of externally administered recombinant human (rh) MBL towards anti-fungal defence in invasive pulmonary aspergillosis (IPA) by in vivo and in vitro studies. In murine models of IPA with corticosteroid-induced immunosuppression, rhMBL-treated mice showed 80% survival compared to untreated IPA mice with no survivors. Treated IPA mice also showed a marked increase in tumour necrosis factor (TNF)-alpha and interleukin (IL)-1alpha and a significant decrease in pulmonary fungal hyphae and IL-10. In vitro, rhMBL-bound A. fumigatus conidia showed a dose-dependent increase in the deposition of C4b, the first product of the lectin pathway. There was an enhanced uptake of A. fumigatus conidia by the polymorphonuclear cells (PMNs) in the presence of rhMBL that increased further in the presence of MBL supplemented with MBL-deficient serum. However, an increase in the oxidative burst of PMNs and A. fumigatus killing were observed only when MBL was supplemented with MBL-deficient serum. The study suggests a therapeutic role of ex vivo-administered MBL in host defence against aspergillosis, possibly through MBL-mediated complement activation and other protective mechanisms aimed both directly at the pathogen, and indirectly through modulation of the host inflammatory responses.     

Respiratory syncytial virus infection in a murine model of cystic fibrosis

Viral respiratory infections play an important role in the development and progression of pulmonary disease in cystic fibrosis (CF). The CF mouse model provides a tool to examine the relationship between the cystic fibrosis transmembrane conductance regulator (CFTR) defect and lung disease. This work investigates the cellular response to a common viral pathogen, respiratory syncytial virus (RSV) in the lung of CF mice. RSV was administered by intranasal inoculation of CFTR(tm1Unc)-Tg(FABPCFTR)1Jaw/J (CFTR-/-) and control mice. At day 5 post infection, viral titers, bronchoalveolar fluid nitrate levels (BALF) cell and differential counts, histology and studies on airway mechanics were performed. CFTR-/- mice had an impaired ability to clear RSV. This was associated with an exaggerated inflammatory response (increased lymphocytes and neutrophils) in BALF of RSV-infected CFTR-/- mice and a decreased ability to generate nitric oxide (NO) (measured as BAL nitrate). Lung histopathology of RSV-infected CFTR-/- mice demonstrated increased inflammation compared to RSV (-) CFTR-/- and control mice (regardless of RSV treatment). The airway response to methacholine was increased by RSV infection in CF mice when compared to controls. The CFTR-/- mouse exhibits an aberrant response to RSV infection. This model should be useful in providing further mechanistic information on the biology of respiratory viruses in mammalian models, and provide new insights into the pathogenesis of airway inflammation in patients with CF.     

Evidence of Rickettsia spp. infection in Sweden: a clinical, ultrastructural and serological study

Sweden is an area potentially endemic for spotted fever rickettsioses. Rickettsia helvetica has been isolated from its tick vector Ixodes ricinus, and in a handful of cases linked to human disease. This study demonstrates for the first time in Sweden the transmission of rickettsial infection after a tick bite and the attack rate in an endemic area. We present three cases of documented rickettsial infection and a prospective serological study of Swedish recruits who were trained in the area where the patients lived and showed seroconversion to spotted fever rickettsiae. All patients showed a four-fold increase in antibody titer to the spotted fever rickettsia, R. helvetica, and immunohistochemical examination revealed rickettsia-like organisms in the walls of skin capillaries and veins. Electron microscopy showed organisms resembling R. helvetica and immunogold labeling with two anti-rickettsial antibodies demonstrated specific labeling of the rickettsial organisms in the skin biopsy specimens. Eight of the thirty-five recruits showed a four-fold increase in IgG titer reflecting a high rate of exposure. The results of this study demonstrate that spotted fever rickettsioses should be taken into consideration in the diagnosis of tick-transmitted infections in Sweden.     

Detection of invasive infection caused by Fusarium solani and non-Fusarium solani species using a duplex quantitative PCR-based assay in a murine model of fusariosis

A duplex Real Time PCR (RT-PCR) assay for detecting DNA of members of the genus Fusarium has been developed and validated by using two mouse models of invasive infection. The duplex RT-PCR technique employed two specific molecular beacon probes targeting a highly conserved region of the fungal rDNA gene. This technique showed a detection limit of 10 fg DNA per μl of sample and a specificity of 100%. The sensitivity in a total of 48 samples from a murine model of Fusarium solani infection was 93.9% for lung tissues and 86.7% for serum samples. In comparison, the sensitivity in a total of 45 samples of a F. oxysporum murine model infection was 87% for lung tissues and 42.8% for serum samples. This molecular technique could be a reliable method for the quantification and the evaluation of the disease in animal models and for the clinical diagnosis of fusariosis.     

Mechanisms of immunity in typhus infection: some characteristics of Rickettsia mooseri infection of guinea pigs.

Rickettsia mooseri infection has been studied in syngeneic guinea pigs inoculated intradermally with the objective of developing a model for the study of immune mechanisms. Characterization of infection included the following: a study of replication, dissemination, and clearance of rickettsiae; measurement of the antibody response with different rickettsial antigens and tests; and attempts to measure the cell-mediated immune response using the correlate of delayed-type hypersensitivity skin reactions. Following intradermal inoculation, rickettsiae replicate locally and then spread to the draining lymph nodes and subsequently cause systemic infection. Spread to draining lymph nodes occurred before the appearance of circulating antibody, whereas systemic infection occurred afterwards. Two distinct patterns of acquired resistance developed. The first was marked by a cessation of rickettsial growth within a given organ and the second by a clearance of rickettsiae. The duration of each of these phases differed markedly from one organ to another. Delayed-type hypersensitivity was not demonstrated by skin testing.     

Chlamydial infection increases gonococcal colonization in a novel murine coinfection model

Genital tract infections caused by Neisseria gonorrhoeae and Chlamydia trachomatis serovars D to K occur at high incidence in many areas of the world. Despite high rates of coinfection with these pathogens, investigations of host-parasite interactions have focused on each pathogen individually. We describe here a coinfection model in which female BALB/c mice were first infected with the mouse Chlamydia species C. muridarum and then inoculated with N. gonorrhoeae following treatment with water-soluble 17β-estradiol to promote long-term gonococcal infection. Viable gonococci and chlamydiae were recovered for an average of 8 to 10 days, and diplococci and chlamydial inclusions were observed in lower genital tract tissue by immunohistochemical staining. Estradiol treatment reduced proinflammatory cytokine and chemokine levels in chlamydia-infected mice; however, coinfected mice had a higher percentage of vaginal neutrophils compared to mice infected with either pathogen alone. We detected no difference in pathogen-specific antibody levels due to coinfection. Interestingly, significantly more gonococci were recovered from coinfected mice compared to mice infected with N. gonorrhoeae alone. We found no evidence that C. muridarum increases gonococcal adherence to, or invasion of, immortalized murine epithelial cells. However, increased vaginal concentrations of inflammatory mediators macrophage inflammatory protein 2 and tumor necrosis factor alpha were detected in C. muridarum-infected mice prior to inoculation with N. gonorrhoeae concurrently with the downregulation of cathelicidin-related antimicrobial peptide and secretory leukocyte peptidase inhibitor genes. We conclude that female mice can be successfully infected with both C. muridarum and N. gonorrhoeae and that chlamydia-induced alterations in host innate responses may enhance gonococcal infection.     

External layers of Rickettsia prowazekii and Rickettsia rickettsii: occurrence of a slime layer.

Using a simple specific-antibody stabilization procedure on organisms gently liberated from their host cells, we have demonstrated by electron microscopy that Rickettsia prowazekii and Rickettsia rickettsii possess a coat of variable thickness, external to the outer leaflet of the cell wall and the structure designated by others as a "microcapsule," which corresponds most closely to the slime layer of certain other bacteria. Reactions in the methenamine silver and ruthenium red staining procedures and the failure to be visualized by standard procedures suggest that the slime layer is largely polysaccharide in nature. It is postulated that this slime layer accounts in large part for the large, electron-lucent, halo-like zone which is found by electron microscopy to surround organisms of the typhus and spotted fever groups in the cytoplasm of their host cells, that it may be the locus of some major group-specific antigens, and that it may function as an antiphagocytic mechanism, as an aid for attachment of rickettsiae to potential host cells, or both. Moreover, because the attenuated E strain of R. prowazekii has been shown to possess a substantial slime layer, the basis for attenuation is not likely to be a simple smooth-to-rough variation.     

Development of a murine model to study the pathogenesis of rotavirus infection

A murine model to study enteritis induced by bovine (BRV) and murine rotavirus (MRV) has been developed. The course of infection was determined by clinical symptoms of diarrhea and virus isolation as well as histopathological, immunohistochemical, and electron microscopic methods. Both isolates were able to replicate and produce clinical symptoms in neonatal mice. Rotavirus-free neonates were orally inoculated with MRV or BRV and observed over a 192-hr postinoculation (HPI) period. Following infection with 10(4) PFU of virus, diarrhea and maximal intestinal dysfunction, as measured by xylose absorption, did not occur until beyond 20 hr postinfection even though maximal virus production occurred at 10-15 HPI. Immunohistochemically and by electron microscopy we were able to demonstrate viral antigen and virus particles in the enterocytes of villous tips at 5-8 HPI. The appearance of diarrheal symptoms was dependent on the virus dose and the type of virus isolate inoculated. The disease could be induced with doses as low as 1 x 10(2) PFU/mouse of BRV and 1 x 10(1) PFU/mouse of MRV. On the basis of these results, MRV was found to be more virulent than BRV in this model. The model should prove useful for studies designed to assess rotavirus virulence genes and for vaccine protection studies. This work emphasizes the need for early sample collection for critical evaluation of any vaccine or antiviral agent using this model.     

Therapeutic efficacy of an antibiotic-loaded nanosheet in a murine burn-wound infection model

Polymeric ultra-thin films (nanosheets) possess unique properties that make them suitable materials for various biomedical applications. In our previous study, we assessed the use of an antibiotic (tetracycline, TC)-loaded nanosheet (or "TC-nanosheet") for the treatment of gastrointestinal tissue defects. The nanosheet consisted of three functional layers: layer-by-layer nanosheet as a stable platform, TC as an antimicrobial agent with autofluorescence for tracing, and a poly(vinyl acetate) nanosheet to act as a protecting layer. The TC-nanosheet has high flexibility, adhesive strength and transparency. Here, we evaluated the effectiveness of the TC-nanosheet in preventing full thickness burn-wound infections. In an in vivo study, murine dorsal skin was injured by full-thickness burns and then infected with Pseudomonas aeruginosa (P. aeruginosa), a common bacterium causing burn-associated infections. The wound site was treated either with a TC-nanosheet, TC-unloaded nanosheet or left untreated. Wound management was facilitated by the high transparency of the TC-nanosheet. The TC-nanosheet significantly improved burn-wound infection by P. aeruginosa in mice. Indeed, all mice treated with the TC-nanosheet survived, whereas the other treatment groups displayed increased rates of mortality due to bacterial infection. According to histological analyses and viable bacterial counting in the liver (bacterial translocation), the TC-nanosheets were able to prevent not only the local inflammation but also systemic inflammation. We conclude that the TC-nanosheet can act as an effective treatment for full-thickness burn-wound infection. Hence, the TC-nanosheet is a promising therapeutic tool for burn-wound management in severely burn-injured patients.     

Mechanisms of immunity in typhus infection: analysis of immunity to Rickettsia mooseri infection of guinea pigs.

To study the mechanisms of immunity to Rickettsia mooseri (R. typhi) infection, sera and splenic cells collected from nonimmune and immune guinea pigs were inoculated separately into syngeneic nonimmune recipients which were subsequently challenged intradermally. Protection was measured by comparing the course of the challenge infections of recipients with infections initiated with the same rickettsial inocula in nonimmune animals. Recipients of splenic cells collected 21 days after donor infection were protected from lesion development at sites of intradermal challenge and showed fewer rickettsiae in their kidneys. Cells obtained from nonimmune donors did not protect against either skin lesion development at sites of challenge or kidney infection. Antibody-containing sera collected 21 days after donor infection, but not normal sera, reduced levels of kidney infection, but immune sera did not protect against the development of lesions at sites of intradermal challenge. It was concluded that both immune sera and immune splenic cells possess capacities to effect a partial control of the systemic phase of R. mooseri infection in guinea pigs, but that immune splenic cells possess a capacity not shared by immune sera, i.e., the capacity to protect from infection at local sites of intradermal inoculation.     

Vaccination against Chlamydia genital infection utilizing the murine C. muridarum model

Chlamydia trachomatis genital infection is a worldwide public health problem, and considerable effort has been expended on developing an efficacious vaccine. The murine model of C. muridarum genital infection has been extremely useful for identification of protective immune responses and in vaccine development. Although a number of immunogenic antigens have been assessed for their ability to induce protection, the majority of studies have utilized the whole organism, the major outer membrane protein (MOMP), or the chlamydial protease-like activity factor (CPAF). These antigens, alone and in combination with a variety of immunostimulatory adjuvants, have induced various levels of protection against infectious challenge, ranging from minimal to nearly sterilizing immunity. Understanding of the mechanisms of natural infection-based immunity and advances in adjuvant biology have resulted in studies that are increasingly successful, but a vaccine licensed for use in humans has not yet been brought to fruition. Here we review immunity to chlamydial genital infection and vaccine development using the C. muridarum model.