Genetic variants of Anaplasma phagocytophilum from 14 equine granulocytic anaplasmosis cases

<p>Abstract</p> <p>Background</p> <p>Equine Granulocytic Anaplasmosis (EGA) is caused by <it>Anaplasma phagocytophilum</it>, a tick-transmitted, obligate intracellular bacterium. In Europe, it is transmitted by <it>Ixodes ricinus</it>. A large number of genetic variants of <it>A. phagocytophilum </it>circulate in nature and have been found in ticks and different animals. Attempts have been made to assign certain genetic variants to certain host species or pathologies, but have not been successful so far. The purpose of this study was to investigate the causing agent <it>A. phagocytophilum </it>of 14 cases of EGA in naturally infected horses with molecular methods on the basis of 4 partial genes (<it>16S rRNA</it>, <it>groEL</it>, <it>msp2</it>, and <it>msp4</it>).</p> <p>Results</p> <p>All DNA extracts of EDTA-blood samples of the horses gave bands of the correct nucleotide size in all four genotyping PCRs. Sequence analysis revealed 4 different variants in the partial <it>16S rRNA</it>, <it>groEL </it>gene and <it>msp2 </it>genes, and 3 in the <it>msp4 </it>gene. One <it>16S rRNA </it>gene variant involved in 11 of the 14 cases was identical to the "prototype" variant causing disease in humans in the amplified part [GenBank: <ext-link ext-link-id="U02521" ext-link-type="gen">U02521</ext-link>]. Phylogenetic analysis revealed as expected for the <it>groEL </it>gene that sequences from horses clustered separately from roe deer. Sequences of the partial <it>msp2 </it>gene from this study formed a separate cluster from ruminant variants in Europe and from all US variants.</p> <p>Conclusions</p> <p>The results show that more than one variant of <it>A. phagocytophilum </it>seems to be involved in EGA in Germany. The comparative genetic analysis of the variants involved points towards different natural cycles in the epidemiology of <it>A. phagocytophilum</it>, possibly involving different reservoir hosts or host adaptation, rather than a strict species separation.</p

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 Sensitzers Induce Skin Inflammation via ROS Production and Hyalurinic 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

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