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.

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.

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.

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.

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.

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.

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.

Correction to: The absence of the drhm gene is not a marker for human-pathogenicity in European Anaplasma phagocytophilum strains.

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

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.

The absence of the drhm gene is not a marker for human-pathogenicity in European Anaplasma phagocytophilum strains.

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. The geographical distribution of A. phagocytophilum spans the Americas, Europe, Africa and Asia. However, human disease predominantly occurs in North America but is infrequently reported from Europe and Asia. In North American strains, the absence of the drhm gene has been proposed as marker for pathogenicity in humans whereas no information on the presence or absence of the drhm gene was available for A. phagocytophilum strains circulating in Europe. Therefore, we tested 511 European and 21 North American strains for the presence of drhm and compared the results to two other typing methods: multilocus sequence typing (MLST) and ankA-based typing. RESULTS: Altogether, 99% (478/484) of the analyzable European and 19% (4/21) of the North American samples from different hosts were drhm-positive. Regarding the strains from human granulocytic anaplasmosis cases, 100% (35/35) of European origin were drhm-positive and 100% (14/14) of North American origin were drhm-negative. Human strains from North America and Europe were both part of MLST cluster 1. North American strains from humans belonged to ankA gene clusters 11 and 12 whereas European strains from humans were found in ankA gene cluster 1. However, the North American ankA gene clusters 11 and 12 were highly identical at the nucleotide level to the European cluster 1 with 97.4% and 95.2% of identity, respectively. CONCLUSIONS: The absence of the drhm gene in A. phagocytophilum does not seem to be associated with pathogenicity for humans per se, because all 35 European strains of human origin were drhm-positive. The epidemiological differences between North America and Europe concerning the incidence of human A. phagocytophilum infection are not explained by strain divergence based on MLST and ankA gene-based typing.

A divergent Anaplasma phagocytophilum variant in an Ixodes tick from a migratory bird; Mediterranean basin.

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.

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.

Phylogeographical diversity of Anaplasma phagocytophilum in the Asian part of Russia based on multilocus sequence typing and analysis of the ankA gene.

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.

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.

Genetic characterization of Anaplasma phagocytophilum strains from goats (Capra aegagrus hircus) and water buffalo (Bubalus bubalis) by 16S rRNA gene, ankA gene and multilocus sequence typing.

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.

Simultaneous infection of cattle with different Anaplasma phagocytophilum variants.

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.

Correction to: Congenital infection with Anaplasma phagocytophilum in a calf in northern Germany.

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