Sequence Analysis of the msp4 Gene of Anaplasma phagocytophilum Strains

The causative agent of human granulocytic ehrlichiosis was recently reclassified as Anaplasma phagocytophilum, unifying previously described bacteria that cause disease in humans, horses, dogs, and ruminants. For the characterization of genetic heterogeneity in this species, the homologue of Anaplasma marginale major surface protein 4 gene (msp4) was identified, and the coding region was PCR amplified and sequenced from a variety of sources, including 50 samples from the United States, Germany, Poland, Norway, Italy, and Switzerland and 4 samples of A. phagocytophilum-like organisms obtained from white-tailed deer in the United States. Sequence variation between strains of A. phagocytophilum (90 to 100% identity at the nucleotide level and 92 to 100% similarity at the protein level) was higher than in A. marginale. Phylogenetic analyses of msp4 sequences did not provide phylogeographic information but did differentiate strains of A. phagocytophilum obtained from ruminants from those obtained from humans, dogs, and horses. The sequence analysis of the recently discovered A. phagocytophilum msp2 gene corroborated these results. The results reported here suggest that although A. phagocytophilum-like organisms from white-tailed deer may be closely related to A. phagocytophilum, they could be more diverse. These results suggest that A. phagocytophilum strains from ruminants could share some common characteristics, including reservoirs and pathogenicity, which may be different from strains that infect humans.Fil: de la Fuente, J.. Consejo Superior de Investigaciones Científicas; España. Oklahoma State University; Estados UnidosFil: Massung, R. F.. Centers for Disease Control and Prevention; Estados UnidosFil: Wong, S. J.. Wadsworth Center; Estados UnidosFil: Chu, F. K.. Wadsworth Center for Laboratories and Research; Estados UnidosFil: Lutz, H.. University of Zurich; SuizaFil: Meli, M.. University of Zurich; SuizaFil: von Loewenich, F. D.. University Clinic of Freiburg; AlemaniaFil: Grzeszczuk, A.. Medical University of Bialystok; PoloniaFil: Torina, A.. Istituto Zooprofilattico Sperimentale della Sicilia; ItaliaFil: Caracappa, S.. Istituto Zooprofilattico Sperimentale della Sicilia; ItaliaFil: Mangold, Atilio Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Naranjo, Ana Verónica. Instituto de Investigación en Recursos Cinegéticos; EspañaFil: Stuen, S.. Norwegian School of Veterinary Science; NoruegaFil: Kocan, K. M.. Oklahoma State University; Estados Unido

The IFN-γ-Inducible GTPase, Irga6, Protects Mice against Toxoplasma gondii but Not against Plasmodium berghei and Some Other Intracellular Pathogens

Clearance of infection with intracellular pathogens in mice involves interferon-regulated GTPases of the IRG protein family. Experiments with mice genetically deficient in members of this family such as Irgm1(LRG-47), Irgm3(IGTP), and Irgd(IRG-47) has revealed a critical role in microbial clearance, especially for Toxoplasma gondii. The in vivo role of another member of this family, Irga6 (IIGP, IIGP1) has been studied in less detail. We investigated the susceptibility of two independently generated mouse strains deficient in Irga6 to in vivo infection with T. gondii, Mycobacterium tuberculosis, Leishmania mexicana, L. major, Listeria monocytogenes, Anaplasma phagocytophilum and Plasmodium berghei. Compared with wild-type mice, mice deficient in Irga6 showed increased susceptibility to oral and intraperitoneal infection with T. gondii but not to infection with the other organisms. Surprisingly, infection of Irga6-deficient mice with the related apicomplexan parasite, P. berghei, did not result in increased replication in the liver stage and no Irga6 (or any other IRG protein) was detected at the parasitophorous vacuole membrane in IFN-γ-induced wild-type cells infected with P. berghei in vitro. Susceptibility to infection with T. gondii was associated with increased mortality and reduced time to death, increased numbers of inflammatory foci in the brains and elevated parasite loads in brains of infected Irga6-deficient mice. In vitro, Irga6-deficient macrophages and fibroblasts stimulated with IFN-γ were defective in controlling parasite replication. Taken together, our results implicate Irga6 in the control of infection with T. gondii and further highlight the importance of the IRG system for resistance to this pathogen

Metagenomic Profile of the Bacterial Communities Associated with Ixodes ricinus Ticks

Assessment of the microbial diversity residing in arthropod vectors of medical importance is crucial for monitoring endemic infections, for surveillance of newly emerging zoonotic pathogens, and for unraveling the associated bacteria within its host. The tick Ixodes ricinus is recognized as the primary European vector of disease-causing bacteria in humans. Despite I. ricinus being of great public health relevance, its microbial communities remain largely unexplored to date. Here we evaluate the pathogen-load and the microbiome in single adult I. ricinus by using 454- and Illumina-based metagenomic approaches. Genomic DNA-derived sequences were taxonomically profiled using a computational approach based on the BWA algorithm, allowing for the identification of known tick-borne pathogens at the strain level and the putative tick core microbiome. Additionally, we assessed and compared the bacterial taxonomic profile in nymphal and adult I. ricinus pools collected from two distinct geographic regions in Northern Italy by means of V6-16S rRNA amplicon pyrosequencing and community based ecological analysis. A total of 108 genera belonging to representatives of all bacterial phyla were detected and a rapid qualitative assessment for pathogenic bacteria, such as Borrelia, Rickettsia and Candidatus Neoehrlichia, and for other bacteria with mutualistic relationship or undetermined function, such as Wolbachia and Rickettsiella, was possible. Interestingly, the ecological analysis revealed that the bacterial community structure differed between the examined geographic regions and tick life stages. This finding suggests that the environmental context (abiotic and biotic factors) and host-selection behaviors affect their microbiome

A review on the eco-epidemiology and clinical management of human granulocytic anaplasmosis and its agent in Europe

Anaplasma phagocytophilum is the agent of tick-borne fever, equine, canine and human granulocytic anaplasmosis. The common route of A. phagocytophilum transmission is through a tick bite, the main vector in Europe being Ixodes ricinus. Despite the apparently ubiquitous presence of the pathogen A. phagocytophilum in ticks and various wild and domestic animals from Europe, up to date published clinical cases of human granulocytic anaplasmosis (HGA) remain rare compared to the worldwide status. It is unclear if this reflects the epidemiological dynamics of the human infection in Europe or if the disease is underdiagnosed or underreported. Epidemiologic studies in Europe have suggested an increased occupational risk of infection for forestry workers, hunters, veterinarians, and farmers with a tick-bite history and living in endemic areas. Although the overall genetic diversity of A. phagocytophilum in Europe is higher than in the USA, the strains responsible for the human infections are related on both continents. However, the study of the genetic variability and assessment of the difference of pathogenicity and infectivity between strains to various hosts has been insufficiently explored to date. Most of the European HGA cases presented as a mild infection, common clinical signs being pyrexia, headache, myalgia and arthralgia. The diagnosis of HGA in the USA was recommended to be based on clinical signs and the patient’s history and later confirmed using specialized laboratory tests. However, in Europe since the majority of cases are presenting as mild infection, laboratory tests may be performed before the treatment in order to avoid antibiotic overuse. The drug of choice for HGA is doxycycline and because of potential for serious complication the treatment should be instituted on clinical suspicion alone

Contact sensitizers induce skin inflammation via ROS production and hyaluronic acid degradation.

BACKGROUND: Allergic contact dermatitis (ACD) represents a severe health problem with increasing worldwide prevalence. It is a T cell-mediated skin disease induced by protein-reactive organic and inorganic chemicals. A key feature of contact allergens is their ability to trigger an innate immune response that leads to skin inflammation. Previous evidence from the mouse contact hypersensitivity (CHS) model suggests a role for endogenous activators of innate immune signaling. Here, we analyzed the role of contact sensitizer induced ROS production and concomitant changes in hyaluronic acid metabolism on CHS responses. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed in vitro and in vivo ROS production using fluorescent ROS detection reagents. HA fragmentation was determined by gel electrophoresis. The influence of blocking ROS production and HA degradation by antioxidants, hyaluronidase-inhibitor or p38 MAPK inhibitor was analyzed in the murine CHS model. Here, we demonstrate that organic contact sensitizers induce production of reactive oxygen species (ROS) and a concomitant breakdown of the extracellular matrix (ECM) component hyaluronic acid (HA) to pro-inflammatory low molecular weight fragments in the skin. Importantly, inhibition of either ROS-mediated or enzymatic HA breakdown prevents sensitization as well as elicitation of CHS. CONCLUSIONS/SIGNIFICANCE: These data identify an indirect mechanism of contact sensitizer induced innate inflammatory signaling involving the breakdown of the ECM and generation of endogenous danger signals. Our findings suggest a beneficial role for anti-oxidants and hyaluronidase inhibitors in prevention and treatment of ACD

Antioxidant treatment inhibits HA degradation by ROS <i>in vitro</i> and <i>in vivo.</i>

<p>(A) High molecular weight HA was incubated with ROS inducing compounds in combination with RF-40s (R) or solvent controls (S) at corresponding concentrations. MW of HA after incubation was analyzed by SDS gel electrophoresis and staining with Stains all. The untreated HA control is shown in lane 1, CuSO₄ or H₂O₂ only treated HA controls are shown in lanes 2 and 3. One representative gel out of three is shown. (B) High molecular weight HA was incubated with ROS inducing compounds as in (A) with or without addition of different concentrations of the antioxidant NAC. MW of HA after incubation was analyzed by SDS gel electrophoresis and staining with Stains all. One representative gel of three is shown. (C) Staining of HA in murine ears treated topically with acetone, TNCB or TNCB and NAC on the back side of the ear skin (upper side in panels). Samples from ears were fixed as described and paraffin sections (3 µm) were stained with bHABP with subsequent AEC (3-Amino-9-ethylcarbazole) staining and haematoxylin counterstaining. HA is stained brown/red and cell nuclei in blue. Pictures are representative of three independent experiments with three mice each. Magnification = 200×; scale bars  = 50 µm.</p

Source and kinetics of TNCB induced ROS production <i>in vitro.</i>

<p>(A) DCF fluorescence after incubation of BMDC with different concentrations of TNCB for 2 h either with or without NAC treatment was analyzed by flow cytometry. Data show mean fluorescence intensity of DCF +/− SD of triplicate stimulations. One of two independent experiments is shown. (B) Pam212 or L929 cells were left untreated (−) or stimulated with TNCB (300 µM) after pre-treatment with antioxidants NAC or RF-40s for 1 h. In addition, the influence of the mitochondria specific antioxidant APDC and the NADPH oxidase specific antioxidant DPI was analyzed under the same conditions. DCF fluorescence was analyzed 5, 10, 15, 20, 25, 30, 35, 40 and 45 min after TNCB addition. Data show mean fluorescence as calculated comprising all timepoints (B) or kinetics of fluorescence (C) +/− SD of quadruplicate wells from one representative experiment out of three. (D) gp91^phox −/− mice were sensitized with TNCB (3%) or mock treated with acetone. 5 days later, mice were challenged with TNCB (1%) and increase in ear thickness was measured 24 h later. Data shows mean increase in ear thickness +/− SD of n = 3 mice/group. One representative of three independent experiments is shown.</p

Contact sensitizers induce ROS production <i>in vivo</i> and in mitochondria <i>in vitro.</i>

<p>(A) Mice were pre-treated by topical application of antioxidants (NAC 5 mM or PBS as solvent, RF-40s 5.24 mM or solvent) on the ears. NAC/PBS was applied 1 h before and RF-40s/solvent 15 min before induction of ROS production by topical application of TNCB (7%). Acetone treatment served as solvent control for TNCB. 15 min later, ears were taken after euthanasia and incubated <i>ex vivo</i> with DHE (5 mM) in DMSO for 30 min before analysis of ROS production by fluorescence microscopy. Fluorescence was set for minimal background staining with the acetone control to optimize visualization of differences in ROS production in the other samples. Same acquisition times were used for all samples of one experiment. Results shown are representative of three independent experiments. Magnification  = 200×, scale bar  = 50 µm. (B) Pam212 or L929 cells were incubated with TNCB, LPS or left untreated for 1 h before addition of MitoSOX™. ROS production was observed by red/orange fluorescence of MitoSOX™ by fluorescence microscopy. Nuclei were visualized by DAPI staining (blue). Pictures shown are representative of three independent experiments. Magnification  = 400×, scale bar  = 20 µm. (C) Primary human fibroblasts or keratinocytes were incubated with DNCB or left untreated for 1 h before addition of MitoSOX™. ROS production was observed by red/orange fluorescence of MitoSOX™ by fluorescence microscopy. Pictures shown are representative of three independent experiments. Magnification  = 1000×, scale bar  = 50 µm.</p