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In Europe, genetically distinct ecotypes of the tick-vectored bacterium Anaplasma phagocytophilum circulate among mammals in three discrete enzootic cycles. To date, potential ecological factors that contributed to the emergence of these divergent ecotypes have been poorly studied. Here, we show that the ecotype that predominantly infects roe deer (Capreolus capreolus) is evolutionarily derived. Its divergence from a host generalist ancestor occurred after the last glacial maximum as mammal populations, including roe deer, recolonized the European mainland from southern refugia. We also provide evidence that this host specialist ecotype’s effective population size (N(e)) has tracked changes in the population of its roe deer host. Specifically, both host and bacterium have undergone substantial increases in N(e) over the past 1,500 years. In contrast, we show that while it appears to have undergone a major population expansion starting ~3,500 years ago, in the past 500 years, the contemporary host generalist ecotype has experienced a substantial reduction in genetic diversity levels, possibly as a result of reduced opportunities for transmission between competent hosts. IMPORTANCE The findings of this study reveal specific events important for the evolution of host specialization in a naturally occurring, obligately intracellular bacterial pathogen. Specifically, they show that host range shifts and the emergence of host specialization may occur during periods of population growth in a generalist ancestor. Our results also demonstrate the close correlation between demographic patterns in host and pathogen for a specialist system. These findings have important relevance for understanding the evolution of host range diversity. They may inform future work on host range dynamics, and they provide insights for understanding the emergence of pathogens that have human and veterinary health implications.

Bacteria species that must obligately replicate in vertebrate host cells make up a large proportion of the prokaryotic pathogens with human and veterinary health implications. In such bacterial taxa, extrinsic processes play an important role in influencing the phylogenetic diversity of viable hosts (‘host range’). These processes include both changes in host population densities and shifts in host geographic distributions. In Europe, distinct genetic strains of the tick-vectored bacteriumAnaplasma phagocytophilumcirculate among mammals in three discrete enzootic cycles. To date, the factors that contributed to the emergence of these strains have been poorly studied. Here we show that the strain which predominately infects roe deer (Capreolus capreolus) is evolutionarily derived. Its divergence from a likely host-generalist ancestor occurred after the last glacial maximum as mammal populations, including roe deer, recolonized the European mainland from southern refugia. We also provide evidence that this host-specialist strain’s effective population size (Ne) has tracked changes in the population of its roe deer host. Specifically, both host and bacterium appear to have undergone substantial increases in Neover the past 1,500 years. In contrast, we show that while it appears to have undergone a major population expansion starting ∼3,500 years ago, in the past 500 years the contemporary host-generalist strain has experienced a substantial reduction in genetic diversity levels, possible as the result of reduced transmission opportunities between competent hosts.IMPORTANCEThe findings of this study are some of the first to examine specific events in the evolution of host specialization in a naturally occurring, obligately intracellular bacterial species. They show that host range shifts and the emergence of host specialization may occur during periods of population growth in a host-generalist ancestor. The results discussed here also show the close correlation between demographic patterns in host and pathogen for a specialist system. These findings have important relevance for our understanding of the evolution of host-range diversity. They may inform future work on host-range dynamics and provide insights for understanding the emergence of pathogens which have human and veterinary health implications.

Many of the human infectious pathogens—especially the zoonotic or vector-borne bacteria—are fastidious organisms that are difficult to cultivate because of their strong adaption to the infected host culminating in their near-complete physiological dependence on this environment. These bacterial species exhibit reduced multiplication rates once they are removed from their optimal ecological niche. This fact complicates the laboratory diagnosis of the disease and hinders the detection and further characterization of the underlying organisms, e.g. at the level of their resistance to antibiotics due to their slow growth. Here, we describe the current state of microbiological diagnostics for five genera of human pathogens with a fastidious laboratory lifestyle. For Anaplasma spp., Bartonella spp., Coxiella burnetii, Orientia spp. and Rickettsia spp., we will summarize the existing diagnostic protocols, the specific limitations for implementation of novel diagnostic approaches and the need for further optimization or expansion of the diagnostic armamentarium. We will reflect upon the diagnostic opportunities provided by new technologies including mass spectrometry and next-generation nucleic acid sequencing. Finally, we will review the (im)possibilities of rapidly developing new in vitro diagnostic tools for diseases of which the causative agents are fastidiously growing and therefore hard to detect.

Novel plant-derived antimicrobials are of interest in dentistry, especially in the treatment of periodontitis, since the use of established substances is associated with side effects and concerns of antimicrobial resistance have been raised. Thus, the present study was performed to quantify the antimicrobial efficacy of crude plant extracts against

Quantitative PCR (qPCR) assays are the gold standard for diagnosis of Pneumocystis jirovecii pneumonia (PCP). However, they are laborious and require skilled personnel. Therefore, execution outside regular working hours of the molecular biology laboratory is limited. The eazyplex P. jirovecii assay (PJA) uses loop-mediated isothermal amplification for detection of P. jirovecii. It is performed directly with respiratory specimens, without the need for special skills, and delivers a result within 3 to 25 min. The goal of our study was to compare the performance of the eazyplex PJA with that of established P. jirovecii qPCR assays. All archived bronchoalveolar lavage fluid (BALF) samples that had previously tested positive for P. jirovecii by qPCR assay and 50 control samples (retrospective part), as well as all BALF samples received for P. jirovecii analysis over a period of 4 months (prospective part), were tested. Forty-nine patients with proven PCP and 126 patients without PCP were included. The sensitivity and specificity of the eazyplex PJA (95.7% and 96.5%, respectively) were comparable to those for three different P. jirovecii qPCR assays. The detection limit of the eazyplex PJA was analogous to 103 copies of the major surface glycoprotein gene per 25 μl of BALF, corresponding to 10 to 20 P. jirovecii cells. The eazyplex PJA reliably discriminated patients with PCP from patients with P. jirovecii colonization. It delivered a positive result within a mean of 9 min 38 s and required a hands-on time of 2 min 45 s. In summary, the eazyplex PJA showed identical performance for the diagnosis of PCP, compared to qPCR assays. However, in terms of time to result, practicability, and robustness, the eazyplex PJA is clearly superior and allows for around-the-clock molecular testing.

An amendment to this paper has been published and can be accessed via the original article.

Anaplasma phagocytophilum (AP) has vast geographical and host ranges and causes disease in humans and domesticated animals. We investigated the role of northward migratory birds in the dispersal of tick-borne AP in the African-Western Palearctic. Ticks were collected from northward migratory birds trapped during spring migration of 2010 at two localities in the central Mediterranean Sea. AP DNA was detected by PCR (gltA and 16S rRNA) and variant determination was performed using ankA sequences. In total, 358 ticks were collected. One of 19 ticks determined as Ixodes was confirmed positive for AP DNA. The tick was collected from a woodchat shrike (Lanius senator senator) trapped in Greece, and molecularly determined to belong to the I. ricinus complex and sharing highest (95%) 16S RNA sequence identity to I. gibbosus. The ankA AP sequence exhibited highest similarity to sequences from rodents and shrews (82%) and ruminants (80%). Phylogenetic analyses placed it convincingly outside other clades, suggesting that it represents a novel AP variant. The divergent Ixodes species harboring a novel AP variant could either indicate an enzootic cycle involving co-evolution with birds, or dissemination from other regions by avian migration. None of the 331 Hyalomma marginatum sensu lato ticks, all immature stages, were positive for AP DNA, lending no evidence for the involvement of Hyalomma ticks transported by birds in the ecology of AP.

The genus Anaplasma (Rickettsiales: Anaplasmataceae) comprises obligate intracellular Gram-negative bacteria that are mainly transmitted by ticks, and currently includes six species: Anaplasma bovis, Anaplasma centrale, Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma platys, and Anaplasma ovis. These have long been known as etiological agents of veterinary diseases that affect domestic and wild animals worldwide. A zoonotic role has been recognized for A. phagocytophilum, but other species can also be pathogenic for humans. Anaplasma infections are usually challenging to diagnose, clinically presenting with nonspecific symptoms that vary greatly depending on the agent involved, the affected host, and other factors such as immune status and coinfections. The substantial economic impact associated with livestock infection and the growing number of human cases along with the risk of transfusion-transmitted infections, determines the need for accurate laboratory tests. Because hosts are usually seronegative in the initial phase of infection and serological cross-reactions with several Anaplasma species are observed after seroconversion, direct tests are the best approach for both case definition and epidemiological studies. Blood samples are routinely used for Anaplasma spp. screening, but in persistently infected animals with intermittent or low-level bacteremia, other tissues might be useful. These guidelines have been developed as a direct outcome of the COST action TD1303 EURNEGVEC (>European Network of Neglected Vectors and Vector-Borne Diseases>). They review the direct laboratory tests (microscopy, nucleic acid-based detection and in vitro isolation) currently used for Anaplasma detection in ticks and vertebrates and their application., This work was done under the frame of COST action TD1303.

Members of the genus Bartonella are fastidious Gram-negative facultative intracellular bacteria that are typically transmitted by arthropod vectors. Several Bartonella spp. have been found to cause culture-negative endocarditis in humans. Here, we report the case of a 75-year old German woman with prosthetic valve endocarditis due to Bartonella washoensis. The infecting agent was characterized by sequencing of six housekeeping genes (16S rRNA, ftsZ, gltA, groEL, ribC, rpoB) applying a multilocus sequence typing (MLST) approach. The 5097 bp of the concatenated housekeeping gene sequence from the patient were 99.0% identical to a B. washoensis strain from a red squirrel (Sciurus vulgaris orientis) from China. 39% (24/62) of red squirrel (S. vulgaris) samples from the Netherlands were positive for the B. washoensis gltA gene variant detected in the patient. This suggests that the red squirrel is the reservoir host for human infection in Europe.

Members of the genus Bartonella are fastidious Gram-negative facultative intracellular bacteria that are typically transmitted by arthropod vectors. Several Bartonella spp. have been found to cause culture-negative endocarditis in humans. Here, we report the case of a 75-year-old German woman with prosthetic valve endocarditis due to Bartonella washoensis. The infecting agent was characterized by sequencing of six housekeeping genes (16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB), applying a multilocus sequence typing (MLST) approach. The 5,097 bp of the concatenated housekeeping gene sequence from the patient were 99.0% identical to a sequence from a B. washoensis strain isolated from a red squirrel (Sciurus vulgaris orientis) from China. A total of 39% (24/62) of red squirrel (S. vulgaris) samples from the Netherlands were positive for the B. washoensis gltA gene variant detected in the patient. This suggests that the red squirrel is the reservoir host for human infection in Europe.

Anaplasma phagocytophilum (AP) has vast geographical and host ranges and causes disease in humans and domesticated animals. We investigated the role of northward migratory birds in the dispersal of tick-borne AP in the African-Western Palearctic. Ticks were collected from northward migratory birds trapped during spring migration of 2010 at two localities in the central Mediterranean Sea. AP DNA was detected by PCR (gltA and 16S rRNA) and variant determination was performed using ankA sequences. In total, 358 ticks were collected. One of 19 ticks determined as Ixodes was confirmed positive for AP DNA. The tick was collected from a woodchat shrike (Lanius senator senator) trapped in Greece, and molecularly determined to belong to the I. ricinus complex and sharing highest (95%) 16S RNA sequence identity to I. gibbosus. The ankA AP sequence exhibited highest similarity to sequences from rodents and shrews (82%) and ruminants (80%). Phylogenetic analyses placed it convincingly outside other clades, suggesting that it represents a novel AP variant. The divergent Ixodes species harboring a novel AP variant could either indicate an enzootic cycle involving co-evolution with birds, or dissemination from other regions by avian migration. None of the 331 Hyalomma marginatum sensu lato ticks, all immature stages, were positive for AP DNA, lending no evidence for the involvement of Hyalomma ticks transported by birds in the ecology of AP.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks and causes tick-borne fever in domestic ruminants such as sheep, cattle and goats. However, in contrast to sheep and cattle little is known about the clinical course of infection in goats. We report here on three cases of symptomatic infection with A. phagocytophilum in two goats (Capra aegagrus hircus) and one water buffalo (Bubalus bubalis). The animals showed symptoms and laboratory findings similar to sheep and cattle. To our knowledge, this is the first report on the symptomatic infection of water buffalos with A. phagocytophilum. The infecting strains were genetically characterized by 16S rRNA gene, ankA gene and multilocus sequence typing (MLST). Four other strains from asymptomatically infected goats were also included. The ankA sequences from five goats were part of the formerly described ankA gene clusters I and IV that are known to contain A. phagocytophilum strains from sheep and cattle. However, the sequences from one goat and from the water buffalo belonged to ankA gene cluster II that was formerly described to be restricted to roe deer. A similar observation was made for MLST as three goats clustered with sequences from sheep and cattle, whereas three other goats and the water buffalo were found to be part of the roe deer cluster. However, since most of the strains from sheep and cattle were distinct from the roe deer strains, roe deer might not represent major reservoir hosts for tick-borne fever in domestic ruminants. When differing parts of the 16S rRNA gene were used for typing the results were conflicting. This shows that the use of a standardized typing method such as MLST is highly desirable to generate easily comparable results.

Anaplasma phagocytophilum is a tick-transmitted Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It causes tick-borne fever in cattle and sheep. We report here the case of a 5-year-old cow from Germany with clinically overt granulocytic anaplasmosis presenting with fever, lower limb oedema and drop in milk-yield. The herd encompassed 10 animals, 8 other animals showed subclinical infection. The strains from the 9 A. phagocytophilum positive cows were molecularly characterized using ankA gene-based and multilocus sequence typing (MLST). Seven of 9 (78%) animals were infected simultaneously with different ankA variants belonging to ankA clusters I and IV. MLST analysis also revealed the presence of multiple strain types. This could be due to co-transmission or superinfection. Hosts harboring diverse A. phagocytophilum strains might enable the emergence of new ankA variants and/or MLST sequence types via bacterial recombination.

Following publication of the original article [1], we have been notified of a typing error under the “Laboratory test results” section.

Anaplasma phagocytophilum is a tick-transmitted bacterium that replicates in neutrophil granulocytes and elicits febrile disease in humans and animals; it is widely distributed in the Americas, Europe, Africa, and Asia. A. phagocytophilum is commonly regarded as a single species, but several genetic variants with distinct host distribution and geographical origin have been described. In a previous study, we used multilocus sequence typing (MLST) to characterize 25 A. phagocytophilum strains from Ixodes spp. ticks collected in the Asian part of Russia. The obtained concatenated sequences formed two separate clades reflecting their Asiatic origin and/or the vector species. As one of the clades was related to A. phagocytophilum strains from European voles and shrews, we here extended our analysis to seven samples from the northern red-backed vole Myodes rutilus and included 38 additional strains of Asiatic origin from Ixodes persulcatus, I. pavlovskyi, and their hybrids. Further, the ankA gene was sequenced in 59 A. phagocytophilum strains from ticks and voles. The Russian strains belonged to the two new MLST clusters 5 (38/70) and 6 (32/70), previously referred to as clades within clusters 1 and 3, respectively. The total number of sequence types (STs) found was 27 including 12 new STs. The ankA sequences were unique and formed two new clusters: cluster 8 (34/59) and cluster 10 (25/59). The concordance between MLST and ankA-based typing was 100%. This means that at least two distinct genetic groups of A. phagocytophilum circulate in the Asian Part of Russia whose reservoir hosts and transmission cycles have to be further elucidated.

Leukocytes express formyl-peptide receptors (FPRs), which sense microbe-associated molecular pattern (MAMP) molecules, leading to leukocyte chemotaxis and activation. We recently demonstrated that phenol-soluble modulin (PSM) peptides from highly pathogenic Staphylococcus aureus are efficient ligands for the human FPR2. How PSM detection by FPR2 impacts on the course of S. aureus infections has remained unknown. We characterized the specificity of mouse FPR2 (mFpr2) using a receptor-transfected cell line, homeobox b8 (Hoxb8), and primary neutrophils isolated from wild-type (WT) or mFpr2−/− mice. The influx of leukocytes into the peritoneum of WT and mFpr2−/− mice was analyzed. We demonstrate that mFpr2 is specifically activated by PSMs in mice, and they represent the first secreted pathogen-derived ligands for the mFpr2. Intraperitoneal infection with S. aureus led to lower numbers of immigrated leukocytes in mFpr2−/− compared with WT mice at 3 h after infection, and this difference was not observed when mice were infected with an S. aureus PSM mutant. Our data support the hypothesis that the mFpr2 is the functional homolog of the human FPR2 and that a mouse infection model represents a suitable model for analyzing the role of PSMs during infection. PSM recognition by mFpr2 shapes leukocyte influx in local infections, the typical infections caused by S. aureus.—Weiss, E., Hanzelmann, D., Fehlhaber, B., Klos, A., von Loewenich, F. D., Liese, J., Peschel, A., Kretschmer, D. Formyl-peptide receptor 2 governs leukocyte influx in local Staphylococcus aureus infections.

Medical leeches (Hirudo medicinalis) in plastic and reconstructive surgery are often used for the treatment of vascular failure after microvascular surgery. Leeches are a reservoir for bacteria of the Aeromonas group that help digesting the blood meal. In some cases these bacteria are able to cause severe wound infections that can lead to loss of tissue transplants. We report about a patient with a common microvascular forearm flap after resection of an oral squamous cell carcinoma which got infected by Aeromonas spp. after treatment with medical leeches. Most of these species are resistant for common antibiotic treatment after surgery. This report shows the importance of an early concomitant antibiotic prophylaxis in the treatment of venous congestion with medical leeches.

Helicobacter felis belongs to the fastidious gastric non-Helicobacter pylori helicobacter species that are typically found in the stomach of cats and dogs. These bacteria have the potential to colonize the human stomach and are then associated with gastritis, gastroduodenal ulcers, and MALT lymphoma. Strains cultured from the human stomach are rare. Here, we present the first isolation of H. felis from a gastric biopsy specimen of a 14-year-old girl who presented with persistent epigastric pain. The strain was cultured using our routine protocol for H. pylori and identified by phylogenetic analyses of partial urease AB and gyrB gene sequences.

Summary Mucormycosis is associated with high morbidity and mortality and is perceived as an emerging fungal infection. However, contemporary paediatric data are limited. We present a series of paediatric cases of mucormycosis reported from Germany and Austria collected within a voluntary epidemiological survey through standardised, anonymized case report forms. Twelve cases were reported between January 2004 and December 2008 (six men; mean age: 12.6 years, range: 0.1–17 years). Mucormycosis was proven in nine, and probable in three cases. Isolates included Lichtheimia (syn. Absidia pro parte, Mycocladus) (five), Rhizopus (three) and Mucor (one) species. Infection was limited to soft tissue in three cases, the lung in two cases, and an infected thrombus in one case; rhinocerebral disease was found in three cases, and pulmonarymediastinal, pulmonary-cerebral and soft tissue-cerebral involvement in one case each. All three patients with isolated soft tissue infection were cured, whereas seven of the remaining patients died (one patient without follow-up). The overall mortality rate was 67%. While these data cannot provide conclusive data on incidence and disease burden of mucormycosis in paediatric patients, they reflect the continuing threat of these infections to immunocompromised patients and the need for improved diagnosis and management.

Background The host range of a pathogenic bacterial strain likely influences its effective population size, which in turn affects the efficacy of selection. Transmission between competent hosts may occur more frequently for host generalists than for specialists. This could allow higher bacterial population densities to persist within an ecological community and increase the efficacy of selection in these populations. Conversely, specialist strains may be better adapted to their hosts and consequently achieve greater within-host population densities, with corresponding increases in selection efficacy. To assess these different hypotheses, we examined the effective population sizes of three strains of the bacterium Anaplasma phagocytophilum and categorized the varying roles of selection and demography on patterns of genetic diversity and divergence in these populations. A. phagocytophilum is a tick-transmitted, obligately intracellular pathogen. Strains of A. phagocytophilum display varying degrees of host specialization, making this a good species for exploring questions regarding host range, effective population size and selection efficacy. Results We found that a roe deer specialist harbored the most genetic diversity of the three A. phagocytophilum strains and correspondingly had the largest effective population size. Another strain that is ecologically specialized on rodents and insectivores had the smallest effective population size. However, these mammalian hosts are distantly related evolutionarily. The third strain, a host generalist, was intermediate in its effective population size between the other two strains. Evolutionary constraint on non-synonymous sites was pervasive in all three strains, although some slightly deleterious mutations may also be segregating in these populations. We additionally found evidence of genome-wide selective sweeps in the generalist strain, whereas signals of repeated bottlenecks were detected in the strain with the smallest effective population size. Conclusions A. phagocytophilum is a diverse bacterial species that differs among distinct strains in its effective population size, as well as how genetic diversity and divergence have been influenced by selection and demographic changes. In this species, host specialization may facilitate increased population growth and allow more opportunities for selection to act. These results provide insights into how host range has influenced evolutionary patterns of strain divergence in an emerging zoonotic bacterium. Electronic supplementary material The online version of this article (doi:10.1186/s12862-015-0335-z) contains supplementary material, which is available to authorized users.

Arnica montana L. is a medical plant of the Asteraceae family and grows preferably on nutrient poor soils in mountainous environments. Such surroundings are known to make plants dependent on symbiosis with other organisms. Up to now only arbuscular mycorrhizal fungi were found to act as endophytic symbiosis partners for A. montana. Here we identified five Streptomyces strains, microorganisms also known to occur as endophytes in plants and to produce a huge variety of active secondary metabolites, as inhabitants of A. montana. The secondary metabolite spectrum of these strains does not contain sesquiterpene lactones, but consists of the glutarimide antibiotics cycloheximide and actiphenol as well as the diketopiperazines cyclo-prolyl-valyl, cyclo-prolyl-isoleucyl, cyclo-prolyl-leucyl and cyclo-prolyl-phenylalanyl. Notably, genome analysis of one strain was performed and indicated a huge genome size with a high number of natural products gene clusters among which genes for cycloheximide production were detected. Only weak activity against the Gram-positive bacterium Staphylococcus aureus was revealed, but the extracts showed a marked cytotoxic activity as well as an antifungal activity against Candida parapsilosis and Fusarium verticillioides. Altogether, our results provide evidence that A. montana and its endophytic Streptomyces benefit from each other by completing their protection against competitors and pathogens and by exchanging plant growth promoting signals with nutrients.

Die humane Anaplasmose und die humane Ehrlichiose werden durch Zeckenstich auf den Menschen ubertragen. Es handelt sich um unspezifische akut fieberhafte Erkrankungen. An Laborveranderungen werden am haufigsten eine Leukopenie, eine Thrombozytopenie und erhohte Transaminasen gefunden. Hautmanifestationen sind bei der humanen Anaplasmose selten. Bei der humanen Ehrlichiose konnen makulopapulose Exantheme und Petechien auftreten. Die Therapie besteht bei beiden Erkrankungen in der Gabe von Doxycyclin.

Anaplasma phagocytophilum is an obligate intracellular tick-borne bacterium that propagates within neutrophils and causes human and animal granulocytic anaplasmosis (HGA). In the murine model of HGA, host immune response plays a more important role in histopathologic lesions than does pathogen load. We examined the role of CYBB, NOS2, and TNFalpha as effectors of innate immune-related injury. Our hypothesis is that the innate immune response to A. phagocytophilum results in inflammatory histopathology, but does not control the pathogen.

Anaplasma phagocytophilum is an obligate intracellular bacterium that is related to rickettsial organisms and replicates in the hostile environment of neutrophils. Previous studies with SCID mice suggested that T and/or B cells are required for its control in vivo. Here, we used mice deficient for Toll-like receptor (TLR)2 and TLR4, MyD88, tumor necrosis factor, inducible nitric oxide synthase, or phagocyte NADPH oxidase (gp91phox–/–) to define the pathways that are critical for the recognition and the killing of this pathogen. Whereas SCID mice developeda 60-fold higher bacterial load in the blood compared to wild-type mice and succumbed to infection, all other gene-deficient mouse strains were fully capable in overcoming a systemic infection withA. phagocytophilum. From these data we conclude that effector mechanisms that are crucial to the defense against numerous other intracellular pathogens are dispensable for the control of A. phagocytophilum. See accompanying Commentary http://dx.doi.org/10.1002/eji.200425250

Granulocytic anaplasmosis is a tick transmitted emerging disease in Europe and worldwide. The agent, Anaplasma phagocytophilum is transmitted by ticks of the genus Ixodes and causes infections in humans and domestic animals. The analysis of different target genes showed that in nature several genetic variants of A. phagocytophilum were present. The purpose of our study was to genetically characterize A. phagocytophilum strains from eight humans, 16 dogs, 12 wild boars, one bear and 18 tick pools from Slovenia. Therefore, the ankA and msp4 genes of A. phagocytophilum were chosen. The same genetic ankA and msp4 variant of A. phagocytophilum was detected in humans, wild boar and a part of the pooled ticks indicating that it circulates in a zoonotic cycle between wild boar and ticks. In dogs, three ankA variants of A. phagocytophilum were detected. One of them was identical to the one that was found in humans. In contrast, all dogs harboured the same msp4 variant as humans and wild boar. In ticks, numerous ankA and msp4 variants were present.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophils. It is transmitted via tick-bite and causes febrile disease in humans and animals. Human granulocytic anaplasmosis is regarded as an emerging infectious disease in North America, Europe and Asia. However, although increasingly detected, it is still rare in Europe. Clinically apparent A. phagocytophilum infections in animals are mainly found in horses, dogs, cats, sheep and cattle. Evidence from cross-infection experiments that A. phagocytophilum isolates of distinct host origin are not uniformly infectious for heterologous hosts has led to several approaches of molecular strain characterization. Unfortunately, the results of these studies are not always easily comparable, because different gene regions and fragment lengths were investigated. Multilocus sequence typing is a widely accepted method for molecular characterization of bacteria. We here provide for the first time a universal typing method that is easily transferable between different laboratories. We validated our approach on an unprecedented large data set of almost 400 A. phagocytophilum strains from humans and animals mostly from Europe. The typability was 74% (284/383). One major clonal complex containing 177 strains was detected. However, 54% (49/90) of the sequence types were not part of a clonal complex indicating that the population structure of A. phagocytophilum is probably semiclonal. All strains from humans, dogs and horses from Europe belonged to the same clonal complex. As canine and equine granulocytic anaplasmosis occurs frequently in Europe, human granulocytic anaplasmosis is likely to be underdiagnosed in Europe. Further, wild boars and hedgehogs may serve as reservoir hosts of the disease in humans and domestic animals in Europe, because their strains belonged to the same clonal complex. In contrast, as they were only distantly related, roe deer, voles and shrews are unlikely to harbor A. phagocytophilum strains infectious for humans, domestic or farm animals.

The major virulence factor which contributes to the survival of Neisseria meningitidis in the blood stream and the cerebrospinal fluid is the capsular polysaccharide. Expression of the capsule genes of N. meningitidis serogroups B, C, W-135 and Y is controlled by an intergenic region separating the capsule biosynthesis operon ( siaA-D ) and the capsule transport operon ( ctrA-D) . To further investigate capsule expression in N. meningitidis we amplified and sequenced the intergenic region of 42 meningococcal isolates of different serogroups. Sequence variations were found mainly in a repeat region preceding the siaA start codon. Correlation between sequence variation and serogroup could not be observed. To measure the transcriptional and translational activity of the respective intergenic regions we performed transcriptional and translational fusions with the lac Z gene integrated into the chromosome of N. meningitidis . Sequence variations preceding the siaA start codon had no effect on β-galactosidase activity. Different in vitro growth conditions such as temperature, glucose concentration, osmolarity, pH and iron concentration also did not influence β-galactosidase activity. Sequential deletions of the intergenic region showed that an Up-like element adjacent to the predicted -35 box is necessary for full transcriptional activity. The deletion of the untranslated region preceding the sia A start codon led to a threefold higher β-galactosidase activity compared with the full-length construct suggesting that the respective region may be involved in capsule regulation.

Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.

Anaplasma phagocytophilum is an obligate intracellular and tick-transmitted bacterium, which causes granulocytic anaplasmosis in animals and humans. Although infection with A. phagocytophilum in domestic animals and vector ticks is documented, there is sparse information on the occurrence of A. phagocytophilum in wild animals. Red foxes (Vulpes vulpes) as well as raccoon dogs (Nyctereutes procyonoides) are wildlife species highly abundant in certain areas of Germany and represent a potential wildlife reservoir for zoonotic diseases. To obtain data about the occurrence of A. phagocytophilum in these animals, red fox and raccoon dog carcasses (hunted or found dead) were collected from January to September 2009 in the Federal State of Brandenburg, Germany. Lung tissue samples were subjected to DNA extraction and were examined for the presence of A. phagocytophilum DNA by means of real-time PCR. Anaplasma phagocytophilum was detected in 10 out of 122 (8.2%) lungs of red foxes and in 3 out of 13 (23%) lungs of raccoon dogs. To the best of our knowledge, A. phagocytophilum was detected for the first time in red foxes and raccoon dogs in Germany.

BackgroundAllergic 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 findingsWe 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/significanceThese 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.

Mucormycosis is associated with high morbidity and mortality and is perceived as an emerging fungal infection. However, contemporary paediatric data are limited. We present a series of paediatric cases of mucormycosis reported from Germany and Austria collected within a voluntary epidemiological survey through standardised, anonymized case report forms. Twelve cases were reported between January 2004 and December 2008 (six men; mean age: 12.6 years, range: 0.1-17 years). Mucormycosis was proven in nine, and probable in three cases. Isolates included Lichtheimia (syn. Absidia pro parte, Mycocladus) (five), Rhizopus (three) and Mucor (one) species. Infection was limited to soft tissue in three cases, the lung in two cases, and an infected thrombus in one case; rhinocerebral disease was found in three cases, and pulmonary-mediastinal, pulmonary-cerebral and soft tissue-cerebral involvement in one case each. All three patients with isolated soft tissue infection were cured, whereas seven of the remaining patients died (one patient without follow-up). The overall mortality rate was 67%. While these data cannot provide conclusive data on incidence and disease burden of mucormycosis in paediatric patients, they reflect the continuing threat of these infections to immunocompromised patients and the need for improved diagnosis and management.