Reoccurring Bovine Anthrax in Germany on the Same Pasture after 12 Years.

The zoonotic disease anthrax, caused by the endospore-forming bacterium Bacillus anthracis, is very rare in Germany. In the state of Bavaria, the last case occurred in July of 2009, resulting in four dead cows. In August of 2021, the disease reemerged after heavy rains, killing one gestating cow. Notably, both outbreaks affected the same pasture, suggesting a close epidemiological connection. B. anthracis could be grown from blood culture, and the presence of both virulence plasmids (pXO1 and pXO2) was confirmed by PCR. Also, recently developed diagnostic tools enabled rapid detection of B. anthracis cells and nucleic acids directly in clinical samples. The complete genome of the strain isolated from blood, designated BF-5, was DNA sequenced and phylogenetically grouped within the B.Br.CNEVA clade, which is typical for European B. anthracis strains. The genome was almost identical to BF-1, the isolate from 2009, separated only by three single nucleotide polymorphisms (SNPs) on the chromosome, one on plasmid pXO2 and three indel regions. Further, B. anthracis DNA was detected by PCR from soil samples taken from spots in the pasture where the cow had fallen. New tools based on phage receptor-binding proteins enabled the microscopic detection and isolation of B. anthracis directly from soil samples. These environmental isolates were genotyped and found to be identical to BF-5 in terms of SNPs. Therefore, it seems that the BF-5 genotype is currently the prevalent one at the affected premises. The area contaminated by the cadaver was subsequently disinfected with formaldehyde.

Mycobacterium microti Infections in Free-Ranging Red Deer (Cervus elaphus).

Infections with Mycobacterium microti, a member of the M. tuberculosis complex, have been increasingly reported in humans and in domestic and free-ranging wild animals. At postmortem examination, infected animals may display histopathologic lesions indistinguishable from those caused by M. bovis or M. caprae, potentially leading to misidentification of bovine tuberculosis. We report 3 cases of M. microti infections in free-ranging red deer (Cervus elaphus) from western Austria and southern Germany. One diseased animal displayed severe pyogranulomatous pleuropneumonia and multifocal granulomas on the surface of the pericardium. Two other animals showed alterations of the lungs and associated lymph nodes compatible with parasitic infestation. Results of the phylogenetic analysis including multiple animal strains from the study area showed independent infection events, but no host-adapted genotype. Personnel involved in bovine tuberculosis-monitoring programs should be aware of the fastidious nature of M. microti, its pathogenicity in wildlife, and zoonotic potential.

Streptococcus castoreus, an uncommon group A Streptococcus in beavers.

Streptococcus castoreus is a rarely encountered beta-haemolytic group A Streptococcus with high tropism for the beaver as host. Based on 27 field isolates under study, evidence strongly suggests that S. castoreus behaves as an opportunistic pathogen in beavers. Although it belongs to the resident mucosal microbiota, this Streptococcus species is associated with purulent lesions in diseased animals. With few exceptions, isolates proved to be highly similar in a panel of phenotypic (including biochemistry, resistance pattern, MALDI-TOF mass spectrometry and Fourier transform-infrared spectroscopy) and classic molecular (16S rRNA and sodA gene) analyses, and thus did not show any specific pattern according to host species or spatio-temporal origin. Conversely, S. castoreus isolates were differentiated into a multitude of pulsed-field gel electrophoresis 'pulsotypes' that did not seem to reflect true epidemiologic lineages. In contrast, single reactions of genomic fingerprinting using BOX-, (GTG)5- and RAPD-PCRs revealed at least subclusters with respect to host species, geographic origin or year, and confirmed the co-colonization of individuals with more than one isolate. In addition to isolates from free-ranging Eurasian beavers (Castor fiber), this study includes S. castoreus from captive North American beavers (Castor canadensis) for the first time.

Improved Discrimination of Bacillus anthracis from Closely Related Species in the Bacillus cereus Sensu Lato Group Based on Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Discrimination of highly pathogenic bacteria, such as Bacillus anthracis, from closely related species based on molecular biological methods is challenging. We applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) to a collection of B. anthracis strains and close relatives in order to significantly improve the statistical confidence of identification results for this group of bacteria. Protein mass spectra of 189 verified and diverse Bacillus strains of the Bacillus cereus sensu lato group were generated using MALDI-TOF MS and subsequently analyzed with supervised and unsupervised statistical methods, such as shrinkage discriminant analysis (SDA) and principal-component analysis (PCA). We aimed at identifying specific biomarkers in the protein spectra of B. anthracis not present in closely related Bacillus species. We could identify 7, 10, 18, and 14 B. anthracis-specific biomarker candidates that were absent in B. cereus, B. mycoides, B. thuringiensis, and B. weihenstephanensis strains, respectively. Main spectra (MSP) of a defined collection of Bacillus strains were compiled using the Bruker Biotyper software and added to an in-house reference library. Reevaluation of this library with 15 hitherto untested strains of B. anthracis and B. cereus yielded improved score values. The B. anthracis strains were identified with score values between 2.33 and 2.55 using the in-house database, while the same strains were identified with scores between 1.94 and 2.37 using the commercial database, and no false-positive identifications occurred using the in-house database.

Molecular analysis of Coxiella burnetii in Germany reveals evolution of unique clonal clusters.

The causative agent of Q fever, Coxiella burnetii, is a query agent occurring naturally all over the world. We studied 104 German Coxiella burnetii strains/DNA samples obtained between 1969 and 2011 using a 14 microsatellite marker Multiple-locus variable-number of tandem repeat (VNTR) analysis (MLVA) technique. We were able to divide our collection into 32 different genotypes clustered into four major groups (A-D). Two of these (A and C) formed predominant clonal complexes that covered 97% of all studied samples. Group C consisted exclusively of cattle-associated isolates/DNA specimens, while group A comprised all other affected species including all sheep-derived strains/DNA samples. Within this second cluster, two major genotypes (A1, A2) were identified. Genotype A2 occurred in strains isolated from ewes in northern and central Germany, whereas genotype A1 was found in most areas of Germany. MLVA analysis of C. burnetii strains from neighbouring countries revealed a close relationship to German strains. We thus hypothesize that there is a western and central European cluster of C. burnetii. We identified predominant genotypes related to relevant host species and geographic regions which is in line with findings of the Dutch Q fever outbreak (2007-2010). Furthermore three of our analyzed German strains are closely related to the Dutch outbreak clone. These findings support the theory of predominant genotypes in the context of regional outbreaks. Our results show that a combination of 8 MLVA markers provides the highest discriminatory power for attributing C. burnetii isolates to genotypes. For future epidemiological studies we propose the use of three MLVA markers for easy and rapid classification of C. burnetii into 4 main clusters.

Inactivation of bacterial and viral biothreat agents on metallic copper surfaces.

In recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. Microbes are rapidly inactivated by a quick, sharp shock known as contact killing. The underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. Pathogenic bacterial and viral high-consequence species able to evade the host immune system are among the most serious lethal microbial challenges to human health. Here, we investigated contact-killing mediated by copper surfaces of Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis tularensis and Yersinia pestis) and of Gram-positive endospore-forming Bacillus anthracis. Additionally, we also tested inactivation of monkeypox virus and vaccinia virus on copper. This group of pathogens comprises biothreat species (or their close relatives) classified by the Center for Disease and Control and Prevention (CDC) as microbial select agents posing severe threats to public health and having the potential to be deliberately released. All agents were rapidly inactivated on copper between 30 s and 5 min with the exception of B. anthracis endospores. For vegetative bacterial cells prolonged contact to metallic copper resulted in the destruction of cell structure.

Draft genome sequence of Bacillus anthracis BF-1, isolated from Bavarian cattle.

Bacillus anthracis BF-1 was isolated from a cow in Bavaria (Germany) that had succumbed to anthrax. Here, we report the draft genome sequence of this strain, which belongs to the European B2 subclade of B. anthracis. The closest phylogenetic neighbor of strain BF-1 is a strain isolated from cattle in France.

Draft genome sequence of Bacillus anthracis UR-1, isolated from a German heroin user.

We report the draft genome sequence of Bacillus anthracis UR-1, isolated from a fatal case of injectional anthrax in a German heroin user. Analysis of the genome sequence of strain UR-1 may aid in describing phylogenetic relationships between virulent heroin-associated isolates of B. anthracis isolated in the United Kingdom, Germany, and other European countries.

Whole-Genome Investigation of Salmonella Dublin Considering Mountain Pastures as Reservoirs in Southern Bavaria, Germany.

Worldwide, Salmonella Dublin (S. Dublin) is responsible for clinical disease in cattle and also in humans. In Southern Bavaria, Germany, the serovar was identified as a causative agent for 54 animal disease outbreaks in herds between 2017 and 2021. Most of these emerged from cattle herds (n = 50). Two occurred in pig farms and two in bovine herds other than cattle. Genomic analysis of 88 S. Dublin strains isolated during these animal disease outbreaks revealed 7 clusters with 3 different MLST-based sequence types and 16 subordinate cgMLST-based complex types. Antimicrobial susceptibility investigation revealed one resistant and three intermediate strains. Furthermore, only a few genes coding for bacterial virulence were found among the isolates. Genome analysis enables pathogen identification and antimicrobial susceptibility, serotyping, phylogeny, and follow-up traceback analysis. Mountain pastures turned out to be the most likely locations for transmission between cattle of different herd origins, as indicated by epidemiological data and genomic traceback analyses. In this context, S. Dublin shedding was also detected in asymptomatic herding dogs. Due to the high prevalence of S. Dublin in Upper Bavaria over the years, we suggest referring to this administrative region as "endemic". Consequently, cattle should be screened for salmonellosis before and after mountain pasturing.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC) genes but is located on a chromosomal region distinct from the LRC and FcR gene clusters.

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to identify additional receptors with the capability to bind chicken immunoglobulins. Based on database searches, we cloned a novel chicken FcR, designated gallus gallus FcR (ggFcR), which was shown to bind selectively chicken IgY. The receptor consists of four extracellular C2-set Ig domains, followed by a transmembrane region containing arginine as a positively charged amino acid and a short cytoplasmic tail. ggFcR associates with the common gamma-chain, indicative for an activating receptor, and real-time RT-PCR revealed high expression on PBMC, thrombocytes, and macrophages. The genomic organization is similar to most Ig-like receptor genes, where each Ig domain is encoded by a separate exon. Additionally, the ggFcR signal peptide is encoded by two exons, the second of which is 36 bp, a hallmark for genes encoded in the leukocyte receptor complex. Phylogenetic analysis also showed a relationship to genes encoded in the leukocyte receptor complex. Surprisingly, ggFcR is not encoded in the leukocyte receptor complex, but it is located as a single isolated gene at the extremity of chicken chromosome 20.

Antimicrobial Resistance in Isolates from Cattle with Bovine Respiratory Disease in Bavaria, Germany.

Patterns of antimicrobial resistance (AMR) regarding Pasteurella multocida (n = 345), Mannheimia haemolytica (n = 273), Truperella pyogenes (n = 119), and Bibersteinia trehalosi (n = 17) isolated from calves, cattle and dairy cows with putative bovine respiratory disease syndrome were determined. The aim of this study was to investigate temporal trends in AMR and the influence of epidemiological parameters for the geographic origin in Bavaria, Germany, between July 2015 and June 2020. Spectinomycin was the only antimicrobial agent with a significant decrease regarding not susceptible isolates within the study period (P. multocida 88.89% to 67.82%, M. haemolytica 90.24% to 68.00%). Regarding P. multocida, significant increasing rates of not susceptible isolates were found for the antimicrobials tulathromycin (5.56% to 26.44%) and tetracycline (18.52% to 57.47%). The proportions of multidrug-resistant (MDR) P. multocida isolates (n = 48) increased significantly from 3.70% to 22.90%. The proportions of MDR M. haemolytica and P. multocida isolates (n = 62) were significantly higher in fattening farms (14.92%) compared to dairy farms (3.29%) and also significantly higher on farms with more than 300 animals (19.49%) compared to farms with 100 animals or less (6.92%). The data underline the importance of the epidemiological farm characteristics, here farm type and herd size regarding the investigation of AMR.

Effects of polyunsaturated fatty acids on isolated canine peripheral blood mononuclear cells and cytokine expression (IL-4, IFN-gamma, TGF-beta) in healthy and atopic dogs.

Polyunsaturated fatty acids (PUFA) have been used to treat dogs with atopic dermatitis but the mechanism of action has not been well understood. The aim of this study was to evaluate the in vitro influence of PUFA on canine peripheral blood mononuclear cells (PBMC). PBMC isolated from eleven dogs with atopic dermatitis and eleven healthy control dogs were stimulated with concanavalin A and Dermatophagoides farinae extract in the presence of linoleic acid (LA), gamma-linolenic acid (GLA), alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) and GLA/EPA/DHA. Subsequently, quantitative polymerase chain reaction (qPCR) for interferon (IFN)-gamma, interleukin (IL)-4 and transforming growth factor (TGF)-beta m-RNA was performed. In the presence of concanavalin A, only PBMC of healthy dogs showed a gradual reduction in proliferation index from incubation without PUFA to incubation with ALA, EPA/DHA and GLA/EPA/DHA, respectively. A similar reduction was seen in normal and in atopic dogs in the presence of D. farinae allergen after incubation with ALA, EPA/DHA and GLA/EPA/DHA. In both groups IL-4 and IFN-gamma but not TGF-beta gene transcription was upregulated, when cells were incubated with D. farinae. Allergen-induced upregulation was not influenced by incubation with PUFA. These findings suggest that PUFA are able to influence proliferation of peripheral blood mononuclear cells in healthy and atopic dogs but do not seem to influence gene transcription of IL-4, IFN-gamma and TGF-beta.

Outbreak of Tularemia in a Group of Hunters in Germany in 2018-Kinetics of Antibody and Cytokine Responses.

In November 2018, an outbreak of tularemia occurred among hare hunters in Bavaria, Germany. At least one infected hare was confirmed as the source of infection. A number of hunting dogs showed elevated antibody titers to Francisella tularensis, but the absence of titer increases in subsequent samples did not point to acute infections in dogs. Altogether, 12 persons associated with this hare hunt could be diagnosed with acute tularemia by detection of specific antibodies. In nine patients, the antibody and cytokine responses could be monitored over time. Eight out of these nine patients had developed detectable antibodies three weeks after exposure; in one individual the antibody response was delayed. All patients showed an increase in various cytokines and chemokines with a peak for most mediators in the first week after exposure. Cytokine levels showed individual variations, with high and low responders. The kinetics of seroconversion has implications on serological diagnoses of tularemia.

Characterization of the chicken CD200 receptor family.

The modulation of myeloid cells via inhibitory and activating immunoglobulin superfamily members has been a subject of intense study in mammals. One such example is the inhibitory receptor for CD200, which is shown to regulate the activation threshold of myeloid cells by interaction with the broadly distributed CD200 molecule. By looking at sequence homology and synteny conservation in the chicken genome, we identified two members of the CD200 receptor family in chicken on chromosome one. Cloning and further characterization of the protein sequence yielded a potentially inhibitory ggCD200R-B1 with a splice variant lacking a transmembrane region and a potentially soluble ggCD200R-S1. Both showed a typical V/C2-set Ig domain arrangement and we present evidence that these two genes have evolved by gene duplication. The inhibitory receptor displayed an uncharged transmembrane region and a long cytoplasmic tail encoding four tyrosine residues, one of them embedded in a motif similar to the mammalian NPxY motif. Further characterization of ggCD200R-B1 showed that it is expressed as a highly glycosylated protein and that its cytoplasmic tyrosine residues can be phosphorylated. Real-time RT-PCR analysis of various tissues and primary cells showed that ggCD200R-B1 is predominantly expressed in macrophages, whereas ggCD200R-S1 is highly expressed in peripheral blood mononuclear cells, but not macrophages. In summary, we showed that there is a homologue of mammalian CD200R conserved in chicken suggesting a similar function in avian species. Furthermore, the presence of potentially soluble CD200R molecules implies an important role for these in the regulation of myeloid cells in chicken.

Chicken TREM-B1, an Inhibitory Ig-Like Receptor Expressed on Chicken Thrombocytes.

Triggering receptors expressed on myeloid cells (TREM) form a multigene family of immunoregulatory Ig-like receptors and play important roles in the regulation of innate and adaptive immunity. In chickens, three members of the TREM family have been identified on chromosome 26. One of them is TREM-B1 which possesses two V-set Ig-domains, an uncharged transmembrane region and a long cytoplasmic tail with one ITSM and two ITIMs indicating an inhibitory function. We generated specific monoclonal antibodies by immunizing a Balb/c mouse with a TREM-B1-FLAG transfected BWZ.36 cell line and tested the hybridoma supernatants on TREM-B1-FLAG transfected 2D8 cells. We obtained two different antibodies specific for TREM-B1, mab 7E8 (mouse IgG1) and mab 1E9 (mouse IgG2a) which were used for cell surface staining. Single and double staining of different tissues, including whole blood preparations, revealed expression on thrombocytes. Next we investigated the biochemical properties of TREM-B1 by using the specific mab 1E9 for immunoprecipitation of either lysates of surface biotinylated peripheral blood cells or stably transfected 2D8 cells. Staining with streptavidin coupled horse radish peroxidase revealed a glycosylated monomeric protein of about 50 kDa. Furthermore we used the stably transfected 2D8 cell line for analyzing the cytoplasmic tyrosine based signaling motifs. After pervanadate treatment, we detected phosphorylation of the tyrosine residues and subsequent recruitment of the tyrosine specific protein phosphatase SHP-2, indicating an inhibitory potential for TREM-B1. We also showed the inhibitory effect of TREM-B1 in chicken thrombocytes using a CD107 degranulation assay. Crosslinking of TREM-B1 on activated primary thrombocytes resulted in decreased CD107 surface expression of about 50-70%.

Unexpected genomic relationships between Bacillus anthracis strains from Bangladesh and Central Europe.

The zoonosis anthrax caused by the bacterium Bacillus anthracis has a broad geographical distribution. Active enzootic areas are typically located away from central and northern Europe where cases of the disease occur only sporadically and in limited numbers. In contrast, a few out of the 64 districts of Bangladesh are hyper-endemic for anthrax and there the disease causes major losses in live-stock. In this study we genotyped eight strains of B. anthracis collected from the districts of Sirajganj and Tangail in 2013. All these strains belonged to canSNP group A.Br.001/002 Sterne differing only in a few of 31 tandem-repeat (MLVA)-markers. Whole genome sequences were obtained from five of these strains and compared with genomic information of B. anthracis strains originating from various geographical locations. Characteristic signatures were detected defining two "Bangladesh" clusters potentially useful for rapid molecular epidemiology. From this data high-resolution PCR assays were developed and subsequently tested on additional isolates from Bangladesh and Central Europe. Remarkably, this comparative genomic analysis focusing on SNP-discovery revealed a close genetic relationship between these strains from Bangladesh and historic strains collected between 1991 and 2008 in The Netherlands and Germany, respectively. Possible explanations for these phylogenetic relationships are discussed.

Genome Sequence of Bacillus anthracis Strain Tangail-1 from Bangladesh.

Soil was collected in July 2013 at a site where a cow infected with anthrax had been the month before. Selective culturing yielded Bacillus anthracis strain Tangail-1. Here, we report the draft genome sequence of this Bacillus anthracis isolate that belongs to the canonical A.Br.001/002 clade.

Genome Sequence of Bacillus anthracis Larissa, Associated with a Case of Cutaneous Anthrax in Greece.

We report the genome sequence of Bacillus anthracis strain Larissa, isolated from a diseased sheep associated with a human case of cutaneous anthrax in Central Greece from 2012. Genome sequence analysis of strain Larissa may aid in describing phylogenetic relationships of B. anthracis isolates in Southeastern European countries.

Genome Sequence of Bacillus anthracis Isolated from an Anthrax Burial Site in Pollino National Park, Basilicata Region (Southern Italy).

A Bacillus anthracis strain was isolated from a burial-site in Pollino National Park where a bovine died of anthrax and was buried in 2004. We report the first genome sequence of B. anthracis isolated in the Basilicata region (southern Italy), which is the highest risk area of anthrax infection in Italy.