Search

Acute influenza virus (AIV) infection can manifest as a severe life-threating illness in patients who are not vaccinated, and furthermore, have comorbidities that place them at risk for rapid respiratory decompensation. Each year influenza causes death in individuals with high risk for contracting this infection, although the illness is preventable by vaccination. Complications of AIV infection, such as bacterial pneumonia are treatable, but other severe complications such as acute respiratory distress syndrome (ARDS) leading to diffuse alveolar damage (DAD) are limited to supportive therapy and self-resolution. In most cases, ARDS leading to DAD is fatal, due to the insidious severity of symptoms which lead to rapid oxygen desaturation without correction, and despite supportive therapy. Regardless of a poor prognosis, the clinical signs and symptoms are congruent with imaging and attest to the importance of vaccination, which protect against high mortality rates.

To evaluate the effects of ZIKV infection on non-human primates (NHPs), as well as to investigate whether these NHPs develop sufficient viremia to infect the major urban vector mosquito, Aedes aegypti, four cynomolgus macaques (Macaca fascicularis) were subcutaneously infected with 5.0 log10 focus-forming units (FFU) of DNA clone-derived ZIKV strain FSS13025 (Asian lineage, Cambodia, 2010). Following infection, the animals were sampled (blood, urine, tears, and saliva), underwent daily health monitoring, and were exposed to Ae. aegypti at specified time points. All four animals developed viremia, which peaked 3&ndash, 4 days post-infection at a maximum value of 6.9 log10 genome copies/mL. No virus was detected in urine, tears, or saliva. Infection by ZIKV caused minimal overt disease: serum biochemistry and CBC values largely fell within the normal ranges, and cytokine elevations were minimal. Strikingly, the minimally colonized population of Ae. aegypti exposed to viremic animals demonstrated a maximum infection rate of 26% during peak viremia, with two of the four macaques failing to infect a single mosquito at any time point. These data indicate that cynomolgus macaques may be an effective model for ZIKV infection of humans and highlights the relative refractoriness of Ae. aegypti for ZIKV infection at the levels of viremia observed.

International audience; Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p < 0.01 for all), and closely correlated (p < 0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1 beta (IL-1 beta), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p < 0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood. K. Otterdal, (C) 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Scrub typhus is a neglected tropical disease, caused by Orientia tsutsugamushi, a Gram-negative bacterium that is transmitted to mammalian hosts during feeding by Leptotrombidium mites and replicates predominantly within endothelial cells. Most studies of scrub typhus in animal models have utilized either intraperitoneal or intravenous inoculation; however, there is limited information on infection by the natural route in murine model skin or its related early host responses. Here, we developed an intradermal (i.d.) inoculation model of scrub typhus and focused on the kinetics of the host responses in the blood and major infected organs. Following ear inoculation with 6 x 104 O. tsutsugamushi, mice developed fever at 11–12 days post-infection (dpi), followed by marked hypothermia and body weight loss at 14–19 dpi. Bacteria in blood and tissues and histopathological changes were detected around 9 dpi and peaked around 14 dpi. Serum cytokine analyses revealed a mixed Th1/Th2 response, with marked elevations of MCP-1/CCL2, MIP-1α/CCL3 and IL-10 at 9 dpi, followed by increased concentrations of pro-inflammatory markers (IL-6, IL-12, IFN-γ, G-CSF, RANTES/CCL5, KC/CCL11, IL-1α/β, IL-2, TNF-α, GM-CSF), as well as modulatory cytokines (IL-9, IL-13). Cytokine levels in lungs had similar elevation patterns, except for a marked reduction of IL-9. The Orientia 47-kDa gene and infectious bacteria were detected in several organs for up to 84 dpi, indicating persistent infection. This is the first comprehensive report of acute scrub typhus and persistent infection in i.d.-inoculated C57BL/6 mice. This is a significant improvement over current murine models for Orientia infection and will permit detailed studies of host immune responses and infection control interventions., Author Summary Scrub typhus is a life-threatening disease that presents as a severe acute febrile illness. It is caused by mite-transmitted Orientia tsutsugamushi, a Gram-negative, obligately intracellular bacterium. Every year, approximately one million people are infected globally; however, there is no vaccine for the control of this infection. Mechanistic studies of host immune responses have been few, partially due to the limited availability of suitable animal models and research facilities. Here, we report our development of an intradermal inoculation mouse model that mimics the natural infection. We also examined the kinetics of immune and inflammatory responses during acute and chronic stages of O. tsutsugamushi infection. As the first comprehensive report of an intradermal inoculation murine model of scrub typhus, this study improves our understanding of host immune responses following cutaneous exposure to the bacteria and opens new avenues for future vaccine-based investigations.

International audience; Background: The pathophysiological hallmark of Rickettsia conorii (R. ă conorii) infection comprises infection of endothelial cells with ă perivascular infiltration of T-cells and macrophages. Although ă interferon (IFN)-gamma-induced protein 10 (IP-10)/CXCL10 is induced ă during vascular inflammation, data on CXCL10 in R. conorii infection is ă scarce. ă Methods: Serum CXCL10 was analyzed in two cohorts of southern European ă patients with R. conorii infection using multiplex cytokine assays. The ă mechanism of R. conorii-induced CXCL10 release was examined ex vivo ă using human whole blood interacting with endothelial cells. ă Results: (i) At admission, R. conorii infected patients had excessively ă increased CXCL10 levels, similar in the Italian (n = 32, similar to ă 56-fold increase vs controls) and the Spanish cohort (n = 38, 68-fold ă increase vs controls), followed by a marked decrease after recovery. The ă massive CXCL10 increase was selective since it was not accompanied with ă similar changes in other cytokines. (ii) Heat-inactivated R. conorii ă induced a marked CXCL10 increase when whole blood and endothelial cells ă were co-cultured. Even plasma obtained from R. conorii-exposed whole ă blood induced a marked CXCL10 release from endothelial cells, comparable ă to the levels found in serum of R. conorii-infected patients. Bacteria ă alone did not induce CXCL10 production in endothelial cells, macrophages ă or smooth muscle cells. ă Conclusions: We show a massive and selective serum CXCL10 response in R. ă conorii-infected patients, likely reflecting release from infected ă endothelial cells characterized by infiltrating T cells and monocytes. ă The CXCL10 response could contribute to T-cell infiltration within the ă infected organ, but the pathologic consequences of CXCL10 in clinical R. ă conorii infection remain to be defined. (C) 2016 Elsevier Ltd. All ă rights reserved.

To the Editor: We report a case of Rickettsia sibirica mongolitimoniae strain infection associated with lymphangitis (1). A 44-year-old man was admitted to Sao Bernardo Hospital in Setubal, Portugal, on August 21, 2006. Twelve days previously while on vacation at Troia Peninsula, he noted malaise, insomnia, and dry buccal mucosa. The next day he observed a small erythematous pruritic lesion on the lower right forearm that 2 days later developed into an eschar. He also had fever and sought medical care. After treatment with topical bacitracin, floxacillin, and acetaminophen for 2 days, fever (38.7°C) continued with lymphangitis extending from the right wrist to the elbow. The medication was changed to nimesulide. Three days later a rash developed on the trunk and arms, and lymphangitis extended to the axilla. Fever and chills continued, leading to hospital admission. No history of tick exposure was reported. Physical examination showed blood pressure 128/73 mm Hg, pulse 96/min, and a rubbery, nontender right supraclavicular lymph node ≈1 cm in diameter. Several 5- to 10-mm maculopapular erythematous lesions were observed on the patient’s palms. He had inflammation on the right forearm suggestive of lymphangitis and an eschar with surrounding edema and erythema on the dorsal lower right forearm (Figure). Admission evaluation showed platelets 117,000/μL, total bilirubin 0.42 mg/dL, albumin 3.42 g/dL, creatinine 1.1 mg/dL, alanine aminotransferase 244 U/L, aspartate aminotransferase 54 U/L, alkaline phosphatase 1061 U/L, creatine phosphokinase 87 U/dL, lactate dehydrogenase 784 U/L, C-reactive protein 7.1 mg/dL, radiographic pulmonary diffuse reticular pattern, arterial pO2 68 mm Hg, O2 saturation 94%, pCO2 22 mm Hg, and arterial blood pH 7.35. The differential diagnoses included rickettsiosis, pneumonia, and cellulitis. Treatment with vancomycin, cefatriaxone, and 100 mg of doxycycline twice a day was begun. On the day after hospitalization, a heparinized blood sample and 2 skin biopsy samples were collected. Vancomycin and ceftriaxone were discontinued at 48 hours when rickettsial infection was confirmed by PCR on skin biopsy; 48 hours later, the patient was afebrile. Figure Lymphangitis extending from the right forearm to the axilla and (inset) eschar on right forearm, caused by Rickettsia sibirica mongolitimonae strain. Arrows indicate lymphangitis. Immunofluorescence assay for antibodies that used R. sibirica mongolitimonae strain as antigen demonstrated seroconversion with no antibodies in the serum sample collected on August 21 and immunoglobulin G (IgG) and IgM antibodies at a titer of 256 in serum collected on August 30. DNA was extracted from 1 skin biopsy sample by using a DNeasy Tissue Kit (QIAGEN, Hilden, Germany). The products of nested PCR showed 100% similarity with gltA (353/353) and ompA (350/350) nucleotide sequences of R. sibirica mongolitimonae strain (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"DQ423368.1","term_id":"90657239"}}DQ423368.1 and {"type":"entrez-nucleotide","attrs":{"text":"DQ423367.1","term_id":"90657237"}}DQ423367.1) (1). Cutaneous biopsy indicated epidermal and dermal necrosis with extensive lymphocyte- and macrophage-rich inflammatory infiltrates involving the papillary and reticular dermal blood vessels characteristic of rickettsial infection. Relatively scant intracellular organisms were observed in the reticular dermis by spotted fever group rickettsia–specific immunohistochemistry (2). Because of the presence of shared protein and lipopolysaccharide antigens in spotted fever group rickettsiae, distinguishing infections with closely related rickettsiae such as R. conorii, R. africae, and R. sibirica by serologic or immunohistochemical methods is very difficult. However, isolation and/or PCR detection followed by genetic characterization can determine the genotype of the organism to the level of genus, species, and strain. The incidence of R. sibirica mongolitimonae strain infection in Portugal is not known because the usual laboratory confirmation by serologic methods does not distinguish these cases from Mediterranean spotted fever. Both cases of R. sibirica infection that have been recognized in Portugal occurred in August during the seasonal peak of Mediterranean spotted fever (1). This epidemiology differs from that in other countries (3–6). Perhaps differences in seasonal activity, population dynamics, or species of the vectors are the basis for the varying epidemiology. In Portugal, R. sibirica has been detected in Rhipicephalus pusillus (1). Rickettsia sibirica mongolitimonae strain was first isolated from a Hyalomma asiaticum tick from Inner Mongolia in 1991 (7) and subsequently from H. truncatum in Niger and from H. excavatum removed from a Greek patient (4,8). Lymphangitis in some patients with R. sibirica mongolitimonae strain infection is a potentially useful diagnostic sign. Nevertheless, half of the patients with reported cases have not had lymphangitis, and infections caused by other Rickettsia spp. can also cause lymphangitis (e.g., patients with African tick bite fever and R. heilongjiangensis infections) (1,3–6,9,10). Thus, the diagnosis of any rickettsiosis should not be based solely on clinical manifestations. The pathogenic role of rickettsiae in lymphangitis remains to be determined. R. sibirica may possibly infect the endothelium of the lymphatic vessel along the pathway from the rickettsial portal of entry at the eschar inoculation site to regional lymph nodes. However, rickettsiae have not been observed in lymphatic vessels, and the lymphatic vessel lesion has not been characterized. The possibility of another agent or pathogenic effectors cannot be excluded. Currently, this clinical manifestation is the strongest evidence that rickettsiae may initially spread by a lymphogenous route before hematogenous dissemination.

We investigated 2 fatal cases of Rocky Mountain spotted fever that occurred in 2003 and 2004 near the same locality in Colombia where the disease was first reported in the 1930s. A retrospective serosurvey of febrile patients showed that >21% of the serum samples had antibodies against spotted fever group rickettsiae.

Background: Based on their essential role in concerting immunological and inflammatory responses we hypothesized that the homeostatic chemokines CCL19 and CCL21 may play a pathogenic role in rickettsiae infection. Methods: Serum levels of CCL19 and CCL21 in patients with R. africae and R. conorii infection were analyzed by enzyme immunoassays. Lungs from R. conorii infected mice were examined for CCL19, CCL21 and CCR7 expression by immunohistochemistry. Results: We found that patients with R. africae infection (n = 15) and in particular those with R. conorii infection (n = 16) had elevated serum levels of CCL19 on admission, with a decline during follow-up. While a similar pattern was seen for CCL21 in R. africae infection, patients with R. conorii infection showed persistently increased CCL21 levels during follow-up. In experimental R. conorii infection, we found strong immunostaining of CCL19 and CCL21 in the lungs, particularly in individuals that had received lethal doses. Immunofluorescence showed co-localization of CCR7 to endothelial cells, macrophages and fibroblasts within the lung tissue of R. conorii infected mice. Conclusions: Our findings suggest that the CCL19/CCL21/CCR7 axis is up-regulated during R. africae and in particular during R. conorii infection, which may potentially contribute to the pathogenesis of these disorders. © 2014 Astrup et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

The pathophysiological hallmark of spotted fever group rickettsioses comprises vascular inflammation. Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized that Dickkopf-1 (DKK-1), as a major modulator of Wnt signaling, could be involved in the pathogenesis in rickettsial infections. Our major findings were: (i) While baseline concentration of DKK-1 in patients with R. conorii infection (n = 32) were not different from levels in controls (n = 24), DKK-1 rose significantly from presentation to first follow-up sample (median 7 days after baseline). (ii) In vitro experiments in human umbilical vein endothelial cells (HUVECs) showed that while heatinactivated R. conorii enhanced the release of interleukin-6 (IL-6) and IL-8, it down-regulated the release of endothelialderived DKK-1 in a time- and dose-dependent manner. (iii) Silencing of DKK-1 attenuated the release of IL-6, IL-8 and growth-related oncogene (GRO)a in R. conorii-exposed HUVECs, suggesting inflammatory effects of DKK-1. (iv) Silencing of DKK-1 attenuated the expression of tissue factor and enhanced the expression of thrombomodulin in R. conorii-exposed HUVECs suggesting pro-thrombotic effects of DKK-1. The capacity of R. conorii to down-regulate endothelial-derived DKK-1 and the ability of silencing DKK-1 to attenuate R. conorii-induced inflammation in endothelial cells could potentially reflect a novel mechanism by which R. conorii escapes the immune response at the site of infection. © 2012 Antonov et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppAandlppB) located on the chromosome ofSalmonella entericaserovar Typhimurium contributed to bacterial virulence. In this study, we characterizedlppAandlppBsingle-knockout (SKO) mutants and compared them with anlppdouble-knockout (DKO) mutant using in vitro and in vivo models. Compared to thelppDKO mutant, which was nonmotile, the motility of thelppSKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT)S. entericaserovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with thelppSKO mutants compared to the cytotoxicity when cells were infected with thelppDKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with thelppBSKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WTS. entericaserovar Typhimurium, than the level in host cells infected with thelppASKO mutant. ThelppDKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with thelppBSKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-α and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in thelppASKO mutant-infected mice than in animals challenged with thelppDKO mutant. The increased cytokine levels observed with thelppBSKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with thelppASKO andlppDKO mutants. Moreover, thelppBSKO mutant-infected mice had increased susceptibility to death. Since thelppDKO mutant retained intact LPS, we constructed anS. entericaserovar Typhimurium triple-knockout (TKO) mutant in which thelppAandlppBgenes were deleted from an existingmsbBmutant (msbBencodes an enzyme required for the acylation of lipid A). Compared to thelppDKO andmsbBSKO mutants, thelpp-msbBTKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation toSalmonellapathogenesis.

By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene ( gidA ). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene ( act ) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act ::alkaline phosphatase gene ( phoA ) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila . The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila , Act.

We report cases of spotted fever rickettsiosis in Coronel Fabriciano Municipality of Minas Gerais State, Brazil. The cases occurred in May and June of 2000. During this period there were two deaths among children from an area named Pedreira in a periurban area of this municipality. In a boy who died with clinical manifestations of Brazilian spotted fever, a necropsy revealed the presence of a spotted fever group Rickettsia. The serological results confirm the difficulty in the differential diagnosis of patients with symptoms of rickettsial diseases. São reportados casos de rickettsioses do grupo das febres maculosas no município de Coronel Fabriciano, Estado de Minas Gerais, Brasil. Os casos ocorreram nos meses de maio e junho de 2000. Durante este período, ocorreram dois óbitos em crianças procedentes de uma área peri-urbana conhecida como Pedreira no município de Coronel Fabriciano. Em um menino, que faleceu com manifestações clínicas da febre maculosa brasileira, à necropsia foi revelada a presença de Rickettsia do grupo das febres maculosas. Os resultados sorológicos confirmaram a dificuldade no diagnóstico diferencial de pacientes com sintomas de doenças rickettsiais.

Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune toRickettsia conoriiexerted specific major histocompatibility complex (MHC) class I-matched CTL activity againstR. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells fromRickettsia australis-immune mice exerted specific CTL activity against anR. australis-infected macrophage-like cell line. Gamma interferon (IFN-γ) gene knockout mice were more than 100-fold more susceptible toR. australisinfection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome ofR. australisinfection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-γ. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-γ gene knockout mice intoR. australis-infected IFN-γ gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-γ. CTLs appear to be crucial to recovery from rickettsial infection.