Demographic Expansions and the Emergence of Host Specialization in Genetically Distinct Ecotypes of the Tick-Transmitted Bacterium Anaplasma phagocytophilum.

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 (Ne) has tracked changes in the population of its roe deer host. Specifically, both host and bacterium have undergone substantial increases in Ne 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.

Evaluation of the Amplex eazyplex Loop-Mediated Isothermal Amplification Assay for Rapid Diagnosis of Pneumocystis jirovecii Pneumonia.

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.

Prosthetic Valve Endocarditis with Bartonella washoensis in a Human European Patient and Its Detection in Red Squirrels (Sciurus vulgaris).

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 vulgarisorientis) from China. A total of 39% (24/62) of red squirrel (S. vulgaris) samples from the Netherlands were positive for the B. washoensisgltA gene variant detected in the patient. This suggests that the red squirrel is the reservoir host for human infection in Europe.

Formyl-peptide receptor 2 governs leukocyte influx in local Staphylococcus aureus infections.

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.

Cerebrospinal fluid penetration of very high-dose meropenem: a case report.

BACKGROUND: Standard dosing of meropenem (2 g t.i.d.) produces CSF concentrations of only 1-2 mg/L which is inferior to the clinical breakpoint for most Gram-negative bacteria. There is therefore concern that dosing must be increased in order to achieve therapeutic CSF concentrations for bacteria with susceptibility close to clinical breakpoints. Yet, the effects of high-dose meropenem on CSF concentrations are not well described in literature. We therefore determined meropenem CSF-levels in a patient who was treated with 15 g/day of meropenem. CASE PRESENTATION: Our patient suffered from a brain trauma and an external ventricular drainage was implanted. Later, a carbapenemase-producing Acinetobacter baumannii (OXA-23, NDM-1) was isolated from blood cultures and CSF. The MIC for meropenem was > 32 mg/L (R), and we opted for a combination therapy of meropenem, colistin and fosfomycin. Meropenem was given at an unusual high-dose (15 g/day) with the aim of achieving high CSF concentrations. CSF concentrations peaked at 64 mg/L. Yet, the patient succumbed to an intracranial bleed into a preexisting cerebral contusion. CONCLUSIONS: High-dose meropenem can achieve CSF levels largely superior to those achieved with commonly recommended dosing regimens. Though our patient succumbed to an intracranial bleed which could be regarded as a severe adverse event, we suggest that meropenem dosing can be increased when pathogens with increased MICs are found in the CSF. More in vivo data are however needed to determine the safety of high-dose meropenem.

Varying influences of selection and demography in host-adapted populations of the tick-transmitted bacterium, Anaplasma phagocytophilum.

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.

Endophytic Streptomyces in the traditional medicinal plant Arnica montana L.: secondary metabolites and biological activity.

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.

Anaplasma phagocytophilum strains from voles and shrews exhibit specific ankA gene sequences.

BACKGROUND: Anaplasma phagocytophilum is a Gram-negative bacterium that replicates obligate intracellularly in neutrophils. It is transmitted by Ixodes spp. ticks and causes acute febrile disease in humans, dogs, horses, cats, and livestock. Because A. phagocytophilum is not transmitted transovarially in Ixodes spp., it is thought to depend on reservoir hosts to complete its life cycle. In Europe, A. phagocytophilum was detected in roe deer, red deer, wild boars, and small mammals. In contrast to roe deer, red deer and wild boars have been considered as reservoir hosts for granulocytic anaplasmosis in humans, dogs, and horses according to groESL- and ankA-based genotyping. A. phagocytophilum variants infecting small mammals in Europe have not been characterized extensively to date. RESULTS: We amplified the total ankA open reading frames of 27 strains from voles and shrews. The analysis revealed that they harboured A. phagocytophilum strains that belonged to a distinct newly described ankA gene cluster. Further, we provide evidence that the heterogeneity of ankA gene sequences might have arisen via recombination. CONCLUSIONS: Based on ankA-based genotyping voles and shrews are unlikely reservoir hosts for granulocytic anaplasmosis in humans, dogs, horses, and livestock in Europe.

Granulocytic anaplasmosis in cats from central Europe and molecular characterization of feline Anaplasma phagocytophilum strains by ankA gene, groEL gene and multilocus sequence typing.

BACKGROUND: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness called granulocytic anaplasmosis primarily in humans, horses, dogs, sheep, cattle and goats. In comparison, clinically apparent disease has been described rarely in cats especially compared to dogs and horses. It is currently unknown whether cats are less susceptible to A. phagocytophilum or whether granulocytic anaplasmosis might be underdiagnosed in cats. METHODS: To address this question, we examined clinical signs and laboratory findings in seven A. phagocytophilum infected cats from Germany and Switzerland. We then genetically characterized feline A. phagocytophilum strains and compared them to those from other hosts showing clinically apparent disease. For this purpose, ankA-based, groEL-based and multilocus sequence typing (MLST) were applied. Furthermore, the concordance between these typing methods was assessed. RESULTS: Fever, lethargy and anorexia were the most common clinical signs in cats suffering from granulocytic anaplasmosis. The most frequent laboratory finding was thrombocytopenia. All three typing methods consistently indicated that the A. phagocytophilum strains found infecting cats are the same as those that cause disease in humans, dogs and horses. In general, the three typing methods applied exhibited high concordance. CONCLUSIONS: The genetic characterization of the feline A. phagocytophilum strains indicates that strain divergence is not the explanation for the fact that granulocytic anaplasmosis is much less frequently diagnosed in cats than in dogs and horses. Otherwise, it may be possible that cats are less susceptible to the same strains than dogs and horse are. However, due to the unspecific clinical signs, it should be considered that granulocytic anaplasmosis may be under-diagnosed in cats.

Towards interoperability in infection control: a standard data model for microbiology.

The COVID-19 pandemic has made it clear: sharing and exchanging data among research institutions is crucial in order to efficiently respond to global health threats. This can be facilitated by defining health data models based on interoperability standards. In Germany, a national effort is in progress to create common data models using international healthcare IT standards. In this context, collaborative work on a data set module for microbiology is of particular importance as the WHO has declared antimicrobial resistance one of the top global public health threats that humanity is facing. In this article, we describe how we developed a common model for microbiology data in an interdisciplinary collaborative effort and how we make use of the standard HL7 FHIR and terminologies such as SNOMED CT or LOINC to ensure syntactic and semantic interoperability. The use of international healthcare standards qualifies our data model to be adopted beyond the environment where it was first developed and used at an international level.

Phenotypic and Molecular Characterization of Carbapenem-Heteroresistant Bacteroides fragilis Strains.

Carbapenem-resistant Bacteroides fragilis strains usually emerge by an insertion sequence (IS) jump into the upstream region of the cfiA carbapenemase gene. However, intermediate or fully resistant cfiA-positive strains also exist. These do not have such IS element activations, but usually have heterogeneous resistance (HR) phenotypes, as detected by a disc diffusion or gradient tests. Heteroresistance is a serious antibiotic resistance problem, whose molecular mechanisms are not fully understood. We aim to characterize HR and investigate diagnostic issues in the set of cfiA-positive B. fragilis strains using phenotypic and molecular methods. Of the phenotypic methods used, the population analysis profile (PAP) and area under curve (AUC) measurements were the best prognostic markers for HR. PAP AUC, imipenem agar dilution and imipenemase production corresponded well with each other. We also identified a saturation curve parameter (quasi-PAP curves), which correlated well with these phenotypic traits, implying that HR is a stochastic process. The genes, on a previously defined 'cfiA element', act in a complex manner to produce the HR phenotype, including a lysine-acetylating toxin and a lysine-rich peptide. Furthermore, imipenem HR is triggered by imipenem. The two parameters that most correlate with the others are imipenemase production and 'GNAT' expression, which prompted us to suspect that carbapenem heteroresistance of the B. fragilis strains is stochastically regulated and is mediated by the altered imipenemase production.

Antimicrobial Activity of Eucalyptus globulus, Azadirachta indica, Glycyrrhiza glabra, Rheum palmatum Extracts and Rhein against Porphyromonas gingivalis.

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 Porphyromonas gingivalis, a pathogen associated with periodontitis. The minimal inhibitory concentrations (MICs) of Eucalyptus globulus leaf, Azadirachta indica leaf, Glycyrrhiza glabra root and Rheum palmatum root extracts were determined by broth microdilution for P. gingivalis ATCC 33277 according to CLSI (Clinical and Laboratory Standards Institute). The MICs for the E. globulus, A. indica and G. glabra extracts ranged from 64 mg/L to 1024 mg/L. The lowest MIC was determined for an ethanolic R. palmatum extract with 4 mg/L. The MIC for the anthraquinone rhein was also measured, as the antimicrobial activity of P. palmatum root extracts can be partially traced back to rhein. Rhein showed a remarkably low MIC of 0.125 mg/L. However, the major compounds of the R. palmatum root extract were not further separated and purified. In conclusion, R. palmatum root extracts should be further studied for the treatment of periodontitis.

Contemporary diagnostics for medically relevant fastidious microorganisms belonging to the genera Anaplasma,Bartonella,Coxiella,OrientiaandRickettsia.

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.

Self-Reported Reactogenicity After Different COVID-19 Vaccination Regimens.

BACKGROUND: In Rhineland-Palatinate, most COVID-19 vaccinations are centrally registered by the Rhineland-Palatinate Division of Vaccine Documentation, which includes self-reported vaccination reactions (SRVR) and their level of perceived intensity. We analyzed the occurrence of SRVR reported between 12/2020 and 12/2021 in relation to the different vaccination regimens involving BioNTech/Pfizer (BNT) and Moderna (m1273) mRNA vaccines and AstraZeneca (ChAd) and Johnson & Johnson (Ad26) viral vector vaccines. METHODS: Using sex-specific logistic regression models, we analyzed the occurrence of all local and systemic SRVR, as well as the occurrence of local and systemic SRVR that were self-rated as "severe" by the vaccinated persons, in relation to the vaccine of the first vaccination and the vaccination regimen of the second vaccination (BNT/BNT, ChAd/ChAd, m1273/m1273, ChAd/ BNT, ChAd/m1273). Vaccination with BNT or the BNT/BNT regimen formed the reference category for the estimated odds ratios (OR) with respective 95% confidence intervals. RESULTS: Of all those vaccinated, 40.7% provided valid information on SRVR after the first vaccination and 33.8% after the second vaccination. As a result, 887 052 individuals were included in the analyses. Their median age was 60 years, and 58% were women. The most common vaccination regimen was BNT/BNT (67.3%). The most common SRVR were pain at the injection site and fatigue. Self-reported reactogenicity after the first vaccination was lowest for BNT. Self-reported systemic reactogenicity was notably higher after vaccination with a vector vaccine. After the second vaccination, self-reported reactogenicity was lowest after a ChAd/ChAd regimen and highest after an m1273 second vaccination. CONCLUSION: With overall acceptable tolerability, differences in self-reported reactogenicity were evident depending on the particular COVID-19 vaccines and vaccination regimens in question.

Distinct host species correlate with Anaplasma phagocytophilum ankA gene clusters.

Anaplasma phagocytophilum is a gram-negative, tick-transmitted, obligate intracellular bacterium that elicits acute febrile diseases in humans and domestic animals. In contrast to the United States, human granulocytic anaplasmosis seems to be a rare disease in Europe despite the initial recognition of A. phagocytophilum as the causative agent of tick-borne fever in European sheep and cattle. Considerable strain variation has been suggested to occur within this species, because isolates from humans and animals differed in their pathogenicity for heterologous hosts. In order to explain host preference and epidemiological diversity, molecular characterization of A. phagocytophilum strains has been undertaken. Most often the 16S rRNA gene was used, but it might be not informative enough to delineate distinct genotypes of A. phagocytophilum. Previously, we have shown that A. phagocytophilum strains infecting Ixodes ricinus ticks are highly diverse in their ankA genes. Therefore, we sequenced the 16S rRNA and ankA genes of 194 A. phagocytophilum strains from humans and several animal species. Whereas the phylogenetic analysis using 16S rRNA gene sequences was not meaningful, we showed that distinct host species correlate with A. phagocytophilum ankA gene clusters.

Mucormycosis in paediatric patients: demographics, risk factors and outcome of 12 contemporary cases.

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.

Anaplasma phagocytophilum Induces TLR- and MyD88-Dependent Signaling in In Vitro Generated Murine Neutrophils.

Anaplasma phagocytophilum is a tick-transmitted obligate intracellular Gram-negative bacterium that replicates in neutrophils. It elicits febrile disease in humans and in animals. In a mouse model, elimination of A. phagocytophilum required CD4+ T cells, but was independent of IFN-γ and other classical antibacterial effector mechanisms. Further, mice deficient for immune recognition and signaling via Toll-like receptor (TLR) 2, TLR4 or MyD88 were unimpaired in pathogen control. In contrast, animals lacking adaptor molecules of Nod-like receptors (NLR) such as RIP2 or ASC showed delayed clearance of A. phagocytophilum. In the present study, we investigated the contribution of further pattern recognition receptor (PRR) pathways to the control of A. phagocytophilum in vivo. Mice deficient for the NLR NOD2 had elevated bacterial loads in the early phase of infection, but were unimpaired in pathogen elimination. In contrast, animals lacking adaptor proteins of different C-type lectin receptors (CLR) such as DAP12, Fc-receptor γ-chain (FcRγ) and SYK controlled A. phagocytophilum as efficiently as wild-type mice. Further, we investigated which PRR pathways are involved in the sensing of A. phagocytophilum by in vitro generated Hoxb8 murine neutrophils. In vitro, recognition of A. phagocytophilum by murine neutrophils was dependent on TLR- and MyD88 signaling. However, it remained intact in the absence of the NLR NOD1, NOD2 and NALP3 and of the CLR adaptor molecules DAP12 and FcRγ. From these results, we conclude that TLR rather than NLR or CLR are critical for the detection of A. phagocytophilum by neutrophils although in vivo defective TLR-signaling is compensated probably because of the redundancy of the immune system.

Impairment of gamma interferon signaling in human neutrophils infected with Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of tick-borne human granulocytic anaplasmosis (HGA), is an intracellular bacterium which survives and multiplies inside polymorphonuclear neutrophil granulocytes (PMN). Increased bacterial burden in gamma interferon (IFN-gamma)-deficient mice suggested a major role of IFN-gamma in the control of A. phagocytophilum. Here we investigated whether infection of human PMN with A. phagocytophilum impairs IFN-gamma signaling thus facilitating intracellular survival of the bacterium. The secretion of the IFN-gamma-inducible chemokines IP-10/CXCL10 and MIG/CXCL9 was markedly inhibited in infected neutrophils. Molecular analyses revealed that, compared to uninfected PMN, A. phagocytophilum decreased the expression of the IFN-gamma receptor alpha-chain CD119, diminished the IFN-gamma-induced phosphorylation of STAT1, and enhanced the expression of SOCS1 and SOCS3 in PMN. Since IFN-gamma activates various antibacterial effector mechanisms of PMN, the impaired IFN-gamma signaling in infected cells likely contributes to the survival of A. phagocytophilum inside PMN and to HGA disease development.

Detection of "Candidatus Neoehrlichia mikurensis" in two patients with severe febrile illnesses: evidence for a European sequence variant.

Recently, a new genus of Anaplasmataceae termed "Candidatus Neoehrlichia" was discovered in ticks and rodents. Here, we report on two patients who suffered from febrile bacteremia due to "Candidatus Neoehrlichia mikurensis" associated with thrombotic or hemorrhagic events. 16S rRNA and groEL gene sequencing provided evidence of three groups of sequence variants.

Co-infection, reinfection and superinfection with Anaplasma phagocytophilum strains in a cattle herd based on ankA gene and multilocus sequence typing.

BACKGROUND: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness in humans and animals. We used multilocus sequence typing (MLST) and ankA gene-based typing to study the molecular epidemiology of the A. phagocytophilum strains circulating in a German cattle herd over one pasture season. The aim was to investigate whether co-infection with two distinct variants, reinfection with the same and/or superinfection by a different strain occurred during one pasture season. Eight genetic loci were sequenced in 47 PCR-positive samples from 15 animals. RESULTS: Five different sequence types (ST) and four ankA alleles were detected in the cattle herd. Three different ST caused clinically overt tick-borne fever in primary infected animals. The concordance between ST and ankA allele was 100%. Therefore, the housekeeping genes used for MLST and the highly variable ankA gene were concatenated to increase resolution. Co-infection could be proven because samples of chronologically close collection dates were included. Co-infecting A. phagocytophilum strains differed by 14 to 18 single nucleotide polymorphisms (SNPs). Most superinfecting variants varied by 14 SNPs from the previous strain and appeared in median after a free interval of 31 days. Thus, it is unlikely that superinfecting strains arose by in-animal evolution. Immunity against re- or superinfection was assumed because the cattle developed clinical signs only during primary infection. CONCLUSIONS: The tick-pathogen-vertebrate host interaction is probably much more complex than previously thought taking into account the frequently occurring events of co-infection, reinfection and superinfection. This complex situation could not be easily simulated in an experimental infection and underlines the value of field studies.