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Simple Summary: Campylobacter fetus subsp. venerealis (Cfv) is the causative agent of bovine genital campylobacteriosis (BGC), also known as bovine venereal campylobacteriosis, which is a disease relevant to international trade listed by the World Organization for Animal Health (WOAH). In Germany, BCG was already found to be endemic in the 1920s, shortly after the agent and the disease had been described for the first time. It can be assumed that BCG had already circulated uncontrolled for a long time in the predecessor states of Germany, influenced only by the political situation and trading networks of the time. A single genetic Cfv lineage was identified which probably emerged in the 19th century and diversified over time. Interestingly, no recurrent cross-border introduction became evident. Recent ambitious efforts of German agricultural policies made for animal welfare, conservation of environment and genetic resources and necessary structural adjustment must go hand in hand with control measures of veterinary public health. This review gives insight into the history of bovine genital campylobacteriosis considering the structural change in cattle farming in Germany and reflecting on the political background of the time. Contagious bovine genital campylobacteriosis (BGC), also known as bovine venereal campylobacteriosis, is a disease relevant to international trade listed by the World Organization for Animal Health (WOAH). It is caused by Campylobacter fetus subsp. venerealis (Cfv), one of three subspecies of Campylobacter fetus. Bulls are the reservoir but BGC may also be spread by artificial insemination (AI). BGC is characterized by severe reproductive losses such as infertility, early embryonic death and abortion with considerable economic losses. This significant economic impact has prompted several countries to adopt stringent eradication and surveillance measures to contain the disease. While there are commercial and autologous vaccines available, scientific evidence for the effectiveness of vaccination is still lacking. In Germany, BCG was already found to be endemic in the 1920s, shortly after the agent and the disease had been described for the first time. It can be assumed that BCG had already circulated uncontrolled for a long time in the predecessor states of Germany, influenced only by the political situation and trading networks of the time. After WW II, BCG was eradicated in the German Democratic Republic due to industrialized cattle production based on AI but it was still endemic at low levels in the Federal Republic of Germany with its diverse cattle production. There has been a steady decline in BGC incidence in re-unified Germany over the past 28 years. A single genetic Cfv lineage was identified which probably emerged in the 19th century and diversified over time. Interestingly, no recurrent cross-border introduction became evident. This review gives insight into the history of bovine genital campylobacteriosis considering the structural change in cattle farming in Germany and reflecting on the political background of the time. [ABSTRACT FROM AUTHOR]

The European brown hare (Lepus europaeus) is a quite adaptable species, but populations have been decreasing for several decades in different countries, including Germany. To investigate infectious diseases as possible influences on observed population decline in the German federal state Schleswig-Holstein, 118 deceased free-ranging European brown hares were collected between 2017 and 2020 and underwent detailed postmortem examination with extended sampling. Infectious diseases were a major cause of death (34.7%). The number of juveniles found exceeded the adult ones. The main pathomorphological findings were hepatitis (32.8%), pneumonia (22.2%), nephritis (19.1%), liver necrosis (12.9%), and enteritis (40.7%). An unusual main finding was steatitis (20.9%) of unknown origin. Animals were mainly emaciated and showed high infection rates with Eimeria spp. (91.3%) and Trichostrongylus spp. (36.2%). European Brown Hare Syndrome Virus reached an epidemic status with few fatal infections (4.2%) and high seroprevalence (64.9%), whereas the prevalence of Rabbit Haemorrhagic Disease Virus 2 was very low (0.8%) in hares in Schleswig-Holstein. Pathogens such as Yersinia pseudotuberculosis (5.9%), Pasteurella multocida (0.8%), and Staphylococcus aureus (3.4%) only caused sporadic deaths. This study illustrates the wide distribution of various infectious pathogens with high mortality and even zoonotic potential. Infectious diseases need to be considered as an important influence on population dynamics in Schleswig-Holstein. [ABSTRACT FROM AUTHOR]

Salmonella enterica subspecies enterica serovar Derby (S. Derby) is one of the most frequent causes for salmonellosis in humans and animals. Understanding the genetic diversity of S. Derby, as well as the nature and origin of its resistance to antimicrobial treatment are thus the key to epidemiological control and surveillance. Here, we report an analysis of 15 S. Derby strains isolated from pig and cattle in slaughterhouses across Germany (2000–2015), which belonged to multilocus sequence types (ST) ST39, ST40 and ST682. Strains were compared to publicly available S. Derby sequence data of these three STs from Germany, comprising 65 isolates collected between 2004 and 2018 from different sources (i.e., pigs, humans, cattle, wild boar, and poultry). A total of 80 sequences (ST39 = 34, ST40 = 21, and ST682 = 25) were analyzed to assess genetic diversity, to identify virulence-associated and antimicrobial resistance genes (ARGs), and to characterize plasmid content. Strains belonging to all three STs were identified in each source examined. Strains with the same ST were closely related regardless of origin. Altogether, 72.5% of the isolates carried at least one resistance gene, furthermore ST40 carried most of the ARGs and the plasmid replicons. The IncI1 replicon was detected in eleven isolates, four of them carried IncI1 plasmid ST26 with clonal complex 2. The comparison of these four isolates with an IncI1 ST26 plasmid reported in 2010 from a German pig (JX566770), showed only variations in a region carrying different ARGs and mobile genetic elements. The strains of our collection had similar genetic diversity as the strains taken from the public database. Moreover, we found that strains harboring multidrug resistant IncI plasmid were found in different animal species, indicating that S. Derby may be implicated in the spread of antimicrobial resistance among animal species. Results may contribute to the knowledge about the diversity in S. Derby in Germany, which may be useful for the future surveillance and antimicrobial resistance of this serovar. [ABSTRACT FROM AUTHOR]

Aliarcobacter cibarius and Aliarcobacter thereius are two rarely detected Aliarcobacter species. In the study, we analyzed the antimicrobial susceptibility and provide detailed insights into the genotype and phylogeny of both species using whole-genome sequencing. Thermophilic Campylobacter species are the most common bacterial foodborne pathogens causing gastroenteritis in humans worldwide. The genus Aliarcobacter is part of the Campylobacteraceae family and includes the species Aliarcobacter butzleri, Aliarcobacter cryaerophilus, Aliarcobacter skirrowii , and the rarely described Aliarcobacter cibarius, Aliarcobacter faecis, Aliarcobacter lanthieri, Aliarcobacter thereius , and Acrobarter trophiarum. Aliarcobacter are emergent enteropathogens and potential zoonotic agents. Here, we generated, analyzed, and characterized whole-genome sequences of Aliarcobacter cibarius and Aliarcobacter thereius. They were isolated from water poultry farms in Germany, cultured and identified by MALDI-TOF MS. With PCR the identity was verified. Antibiotic susceptibility testing was carried out with erythromycin, ciprofloxacin, doxycycline, tetracycline, gentamicin, streptomycin, ampicillin, and cefotaxime using the gradient strip method (E-test). Whole-genome sequences were generated including those of reference strains. Complete genomes for six selected strains are reported. These provide detailed insights into the genotype. With these, we predicted in silico known AMR genes, virulence-associated genes, and plasmid replicons. Phenotypic analysis of resistance showed differences between the presence of resistance genes and the prediction of phenotypic resistance profiles. In Aliarcobacter butzleri , the nucleotide sequence of the gyr A gene (DQ464331) can show a signature mutation resulting in an amino acid change T85>I. Acrobarter cibarius and Acrobarter thereius showed the same gene as assessed by similarity annotation of the mutations 254C>G. Most of the isolates were found to be sensitive to ciprofloxacin. The ciprofloxacin-resistant Aliarcobacter thereius isolate was associated with the amino acid change T85>I. But this was not predicted with antibiotic resistance databases, before. Ultimately, a phylogenetic analysis was done to facilitate in future outbreak analysis. [ABSTRACT FROM AUTHOR]

By combining a reference-independent SNP analysis and average nucleotide identity (ANI) with affinity propagation clustering (APC), we developed a significantly improved methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella (F.) tularensis. Molecular epidemiology of F. tularensis is currently assessed mostly based on laboratory methods and molecular analysis. The high evolutionary stability and the clonal nature makes Francisella ideal for subtyping with single nucleotide polymorphisms (SNPs). Sequencing and real-time PCR can be used to validate the SNP analysis. We investigate whole-genome sequences of 155 F. tularensis subsp. holarctica isolates. Phylogenetic testing was based on SNPs and average nucleotide identity (ANI) as reference independent, alignment-free methods taking small-scale and large-scale differences within the genomes into account. Especially the whole genome SNP analysis with kSNP3.0 allowed deciphering quite subtle signals of systematic differences in molecular variation. Affinity propagation clustering (APC) resulted in three clusters showing the known clades B.4, B.6, and B.12. These data correlated with the results of real‐time PCR assays targeting canSNPs loci. Additionally, we detected two subtle sub-clusters. SplitsTree was used with standard-setting using the aligned SNPs from Parsnps. Together APC, HierBAPS, and SplitsTree enabled us to generate hypotheses about epidemiologic relationships between bacterial clusters and describing the distribution of isolates. Our data indicate that the choice of the typing technique can increase our understanding of the pathogenesis and transmission of diseases with the eventual for prevention. This is opening perspectives to be applied to other bacterial species. The data provide evidence that Germany might be the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade. Described methods allow generating a new, more detailed perspective for F. tularensis subsp. holarctica phylogeny. These results may encourage to determine phylogenetic relationships and clustering of other bacteria the same way. Author summary: By combining a reference-independent SNP analysis and ANI (average nucleotide identity) with affinity propagation clustering (APC), we tested a methodology allowing resolving phylogenetic relationships, based on objective criteria. These bioinformatics tools can be used as a general ruler to determine phylogenetic relationships and clustering of bacteria, exemplary done with Francisella tularensis. Francisella tularensis causes the zoonosis tularemia. We analyzed the relationships between Francisella tularensis subsp. holarctica isolates from Germany using whole-genome sequences. We chose open source, reference independent methods to optimize the level of discrimination. Using a recently described clustering algorithm, we exploit a novel approach to the clustering of bacteria. APC can be used for assigning clades and can be used for rapidly typing strains when they arise. Additionally, we detected two sub-clusters. The data provide evidence that Germany is the collision zone where the clade B.12, also known as the East European clade, overlaps with the clade B.6, also known as the Iberian clade. [ABSTRACT FROM AUTHOR]

Zoonotic pathogens that can be transmitted via food to humans have a high potential for large-scale emergencies, comprising severe effects on public health, critical infrastructures, and the economy. In this context, the development of laboratory methods to rapidly detect zoonotic bacteria in the food supply chain, including high-resolution mass spectrometry proteotyping are needed. In this work, an optimized sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteome profiling was established for Francisella isolates, and a cluster analysis, as well as a phylogenetic tree, was generated to shed light on evolutionary relationships. Furthermore, this method was applied to tissues of infected hare carcasses from Germany. Even though the non-informative data outnumbered by a manifold the information of the zoonotic pathogen in the resulting proteome profiles, the standardized evaluation of MS data within an established automated analysis pipeline identified Francisella (F.) tularensis and, thus, could be, in principle, an applicable method to monitor food supply chains. [ABSTRACT FROM AUTHOR]

Background: Tularaemia is a zoonotic disease caused by the bacterium Francisella tularensis. In Germany, the disease is still rare (e.g. 34 human cases reported in 2015). There is a lack of data about the susceptibility of F. tularensis strains to antibiotics, because many cases are diagnosed using serological assays only.Objectives: The antibiotic susceptibility in vitro of F. tularensis subsp. holarctica strains isolated in Germany was assessed to determine whether the currently recommended empirical therapy is still adequate.Methods: A total of 128 F. tularensis strains were investigated that were collected between 2005 and 2014 in Germany from wild animals, ticks and humans. All isolates were genotyped using real-time PCR assays targeting canonical SNPs, and antibiotic susceptibility was tested using MIC test strips on agar plates. MIC values were interpreted using CLSI breakpoints.Results: The strains were susceptible to antibiotics commonly recommended for tularaemia therapy, i.e. aminoglycosides (MIC90 values: gentamicin 1 mg/L; streptomycin 4.0 mg/L), tetracyclines (MIC90 values: tetracycline 0.5 mg/L; doxycycline 1.5 mg/L) and quinolones (MIC90 value: ciprofloxacin 0.064 mg/L). Chloramphenicol (MIC90 value: 3.0 mg/L) may be of value in treatment of tularaemia meningitis. Ninety-four isolates were susceptible to erythromycin, which defines biovar I (genotypes B.4 and B.6); 34 were resistant (biovar II; genotype B.12).Conclusions: The F. tularensis isolates investigated in this study showed the typical antibiotic susceptibility pattern that was previously observed in other countries. Therefore, recommendations for empirical antibiotic therapy of tularaemia can remain unchanged. However, antibiotic susceptibility testing of clinical isolates should be performed whenever possible. [ABSTRACT FROM AUTHOR]

Tularemia outbreaks in humans have recently been reported in many European countries, but data on the occurrence in the animal population are scarce. In North America, seroconversion of omnivores and carnivores was used as indicator for the presence of tularemia, for the European fauna, however, data are barely available. Therefore, the suitability of wild boars ( Sus scrofa) and red foxes ( Vulpes vulpes) as indicators for the circulation of F. tularensis in Germany was evaluated. Serum samples from 566 wild boars and 457 red foxes were collected between 1995 and 2012 in three federal states in Central Germany (Hesse, Saxony-Anhalt, and Thuringia). The overall rate of seropositive animals was 1.1% in wild boars and 7.4% in red foxes. In conclusion, serological examination of red foxes is recommended, because they can be reliably used as indicator animals for the presence of F. tularensis in the environment. [ABSTRACT FROM AUTHOR]

The article presents a study on the transmission of tularemia in hare and human in North Rhine-Westphalia, Germany in November 2012. The zoonotic disease is caused by Francisella tularensis subsp. holarctica which was detected using various polymerase chain reaction (PCR) based assays, single nucleotide polymorphisms (SNPs) and insertion/deletion mutations (INDELs). Doxycycline was an effective treatment for the disease.

Francisella tularensis is the causative agent of the zoonotic disease tularemia. In Germany, most human infections are caused by contact with infected hares. The aim of this study was to characterize Francisella tularensis subsp. holarctica strains isolated from hares in Germany and to develop bioinformatics tools to analyze their genetic relatedness. In total, 257 German isolates—obtained mainly from hares (n = 233), other vertebrate animals, and ticks, but also from humans (n = 3)—were analyzed within this study. Publically available sequence data from 49 isolates were used to put our isolates into an epidemiological context and to compare isolates from natural foci and humans. Whole-genome sequences were analyzed using core-genome Multi-Locus-Sequence-Typing, canonical Single Nucleotide Polymorphism (SNP) typing and whole-genome SNP typing. An overall conformity of genotype clustering between the typing methods was found, albeit with a lower resolution for canonical single SNP typing. The subclade distribution, both on local and national levels, among strains from humans and hares was similar, suggesting circulation of the same genotypes both in animals and humans. Whilst close to identical isolates of the same subclade were found distributed over large areas, small geographical foci often harbored members of different subclades. In conclusion, although genomic high-resolution typing was shown to be robust, reproducible and allowed the identification of highly closely related strains, genetic profiling alone is not always conclusive for epidemiological linkage of F. tularensis strains. [ABSTRACT FROM AUTHOR]

Background: Rodents are important reservoirs for zoonotic vector-borne agents. Thus, the distribution of rodents and their vicinity to humans and companion animals may have an important impact on human and animal health. However, the reservoir potential of some rodent genera, e.g. Microtus, has not yet been precisely examined concerning tick-borne pathogens in Central Europe. Therefore, we examined small mammals from Germany and the Czech Republic for the following vector-borne pathogens: Babesia spp., Bartonella spp., Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis" (CNM) and Coxiella burnetii. Spleen DNA from 321 small mammals belonging to four genera, Myodes (n = 78), Apodemus (n = 56), Microtus (n = 149), Sorex (n = 38), collected during 2014 in Germany and the Czech Republic were available for this study. DNA samples were examined for the presence of Babesia and Bartonella DNA by conventional PCR targeting the 18S rRNA gene and the 16S–23S rRNA intergenic spacer region, respectively. For the detection of CNM, A. phagocytophilum and C. burnetii real-time PCR assays were performed. Results: Bartonella spp. DNA was detected in 216 specimens (67.3%) with 102/174 (58.6%) positive in Germany and 114/147 (77.6%) in the Czech Republic. The prevalence in each genus was 44.9% for Myodes, 63.2% for Sorex, 77.2% for Microtus and 75% for Apodemus. Four Bartonella species, i.e. Bartonella sp. N40, B. grahamii, B. taylorii and B. doshiae, as well as uncultured bartonellae, were detected. The Bartonella species diversity was higher in rodents than in shrews. In total, 27/321 (8.4%) small mammals were positive for CNM and 3/321 (0.9%) for A. phagocytophilum (S. coronatus and M. glareolus). All samples were negative for Babesia spp. and Coxiella spp. Conclusions: While the detected high prevalence for Bartonella in Apodemus and Myodes spp. is confirmatory with previous findings, the prevalence in Microtus spp. was unexpectedly high. This indicates that individuals belonging to this genus may be regarded as potential reservoirs. Interestingly, only Sorex spp. and M. glareolus were positive for A. phagocytophilum in the present study, suggesting a possible importance of the latter for the maintenance of certain A. phagocytophilum strains in nature. [ABSTRACT FROM AUTHOR]

The importance of established and emerging tick-borne pathogens in Central and Northern Europe is steadily increasing. In 2007, we collected Ixodes ricinus ticks feeding on birds (n = 211) and rodents (n = 273), as well as host-seeking stages (n = 196), in a habitat in central Germany. In order to find out more about their natural transmission cycles, the ticks were tested for the presence of Lyme disease borreliae, Anaplasma phagocytophilum, spotted fever group (SFG) rickettsiae, Francisella tularensis, and babesiae. Altogether, 20.1% of the 680 ticks examined carried at least one pathogen. Bird-feeding ticks were more frequently infected with Borrelia spp. (15.2%) and A. phagocytophilum (3.2%) than rodent-feeding ticks (2.6%; 1.1%) or questing ticks (5.1%; 0%). Babesia spp. showed higher prevalence rates in ticks parasitizing birds (13.2%) and host-seeking ticks (10.7%), whereas ticks from small mammals were less frequently infected (6.6%). SFG rickettsiae and F. tularensis were also found in ticks collected off birds (2.1%; 1.2%), rodents (1.8%; 1.5%), and vegetation (4.1%; 1.6%). Various combinations of coinfections occurred in 10.9% of all positive ticks, indicating interaction of transmission cycles. Our results suggest that birds not only are important reservoirs for several pathogens but also act as vehicles for infected ticks and might therefore play a key role in the dispersal of tick-borne diseases. [ABSTRACT FROM AUTHOR]

Salmonella enterica subsp. enterica serovar Dublin is a cattle-adapted serovar that causes enteritis and systemic diseases in animals. In Germany, S . Dublin is not detected or is very rarely detected in some federal states but is endemic in certain regions. Information on detailed genetic characteristics of S. Dublin is not available. An understanding of the paths and spreading of S. Dublin within and between regions and over time is essential to establish effective control strategies. Whole-genome sequencing (WGS) and bioinformatic analysis were used to explore the genetic traits of S. Dublin and to determine their epidemiological context. Seventy-four S . Dublin strains collected in 2005 to 2018 from 10 federal states were studied. The phylogeny was analyzed using core-genome single-nucleotide polymorphisms (cgSNPs) and core-genome multilocus sequence typing. Genomic clusters at 100 cgSNPs, 40 cgSNPs, and 15 cgSNPs were selected for molecular epidemiology. WGS-based genoserotyping confirmed serotyping. Important specific virulence determinants were detected in all strains, but multidrug resistance in German S. Dublin organisms is uncommon. Use of different thresholds for cgSNP analysis enabled a broad view and also a detailed view of the occurrence of S. Dublin in Germany. Genomic clusters could be allocated nationwide, to a limited number of federal states, or to special regions only. Results indicate both persistence and spread of S. Dublin within and between federal states in short and longer time periods. However, to detect possible routes of infection or persistence of S. Dublin indicated by genomic analysis, information on the management of the cattle farms and contacts with corresponding farms are essential. IMPORTANCE Salmonella enterica subsp. enterica serovar Dublin is a bovine host-adapted serovar that causes up to 50% of all registered outbreaks of salmonellosis in cattle in Germany. S. Dublin is not detected or is only rarely detected in some federal states but has been endemic in certain regions of the country for a long time. Information on genetic traits of the causative strains is essential to determine routes of infection. WGS and bioinformatic analysis should be used to explore the genetic characteristics of S. Dublin. Combining the genomic features of S. Dublin strains with information on the management of the cattle farms concerned should enable the detection of possible routes of infection or persistence of S. Dublin. This approach is regarded as a prerequisite to developing effective intervention strategies.

Whole-genome sequencing (WGS) has been established for bacterial subtyping and is regularly used to study pathogen transmission, to investigate outbreaks, and to perform routine surveillance. Core-genome multilocus sequence typing (cgMLST) is a bacterial subtyping method that uses WGS data to provide a high-resolution strain characterization. This study aimed at developing a novel cgMLST scheme for Bacillus anthracis, a notorious pathogen that causes anthrax in livestock and humans worldwide. The scheme comprises 3,803 genes that were conserved in 57 B. anthracis genomes spanning the whole phylogeny. The scheme has been evaluated and applied to 584 genomes from 50 countries. On average, 99.5% of the cgMLST targets were detected. The cgMLST results confirmed the classical canonical single-nucleotide-polymorphism (SNP) grouping of B. anthracis into major clades and subclades. Genetic distances calculated based on cgMLST were comparable to distances from whole-genome-based SNP analysis with similar phylogenetic topology and comparable discriminatory power. Additionally, the application of the cgMLST scheme to anthrax outbreaks from Germany and Italy led to a definition of a cutoff threshold of five allele differences to trace epidemiologically linked strains for cluster typing and transmission analysis. Finally, the association of two clusters of B. anthracis with human cases of injectional anthrax in four European countries was confirmed using cgMLST. In summary, this study presents a novel cgMLST scheme that provides high-resolution strain genotyping for B. anthracis. This scheme can be used in parallel with SNP typing methods to facilitate rapid and harmonized interlaboratory comparisons, essential for global surveillance and outbreak analysis. The scheme is publicly available for application by users, including those with little bioinformatics knowledge.

Aliarcobacter butzleri is the most prevalent Aliarcobacter species and has been isolated from a wide variety of sources. This species is an emerging foodborne and zoonotic pathogen because the bacteria can be transmitted by contaminated food or water and can cause acute enteritis in humans. Currently, there is no database to identify antimicrobial/heavy metal resistance and virulence-associated genes specific for A. butzleri . The aim of this study was to investigate the antimicrobial susceptibility and resistance profile of two A. butzleri isolates from Muscovy ducks ( Cairina moschata ) reared on a water poultry farm in Thuringia, Germany, and to create a database to fill this capability gap. The taxonomic classification revealed that the isolates belong to the Aliarcobacter gen. nov. as A. butzleri comb. nov. The antibiotic susceptibility was determined using the gradient strip method. While one of the isolates was resistant to five antibiotics, the other isolate was resistant to only two antibiotics. The presence of antimicrobial/heavy metal resistance genes and virulence determinants was determined using two custom-made databases. The custom-made databases identified a large repertoire of potential resistance and virulence-associated genes. This study provides the first resistance and virulence determinants database for A. butzleri .

Tularemia is a zoonotic disease caused by the bacterium Francisella tularensis. The disease can be transmitted to humans through contact with infected animals such as the European brown hare (Lepus europaeus) and ticks as vectors. The aim of this study was to isolate F. tularensis from ticks and hares in North Rhine-Westphalia using cysteine heart agar to determine their genetic relatedness and to identify other bacteria that grow on this medium. 848 European brown hares and 1556 questing ticks (all Ixodes ricinus) from forests were tested using cultivation and MALDI-TOF mass spectrometry or partial 16S rRNA gene sequencing. The majority of F. tularensis isolates from hares (n=24; 96%) and genomic F. tularensis DNA recovered from ticks belonged to the basal genetic clade IV and subclade B.18. These isolates were sensitive to erythromycin and were assigned to biovar I. Only a single strain isolated from a hare was assigned to basal clade I (B.12/B.35). All isolates were sensitive to tetracycline, doxycycline, streptomycin, gentamicin, chloramphenicol, and ciprofloxacin. Only 4 tick pools were positive for F. tularensis and cultivation was not successful in any of the pools. Most of the other isolated bacteria belonged to the order Bacillales with 36 Staphylococcus isolates, 9 Bacillus isolates and 8 Paenibacillus isolates. Prominent members of Enterobacterales were represented by different genera like Pantoea, Erwinia, Raoultella etc. Several of the bacterial species were soil or plant-associated, but some of the bacterial species were found in I. ricinus for the first time. Our results showed that F. tularensis was detected only in few ticks of an endemic area, but ticks were also infected by several other bacteria with zoonotic potential. Therefore, a wider spectrum of pathogens should be considered if a patient was bitten by a tick., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

The popularity of food produced from animals kept under an organic regimen has increased in recent years. In Germany, turkey meat consumption has increased. Despite several studies assessing the susceptibility of campylobacters to various antibiotics in poultry, no sufficient data exists regarding the antimicrobial resistance of campylobacters in organic-reared turkeys. This study provides information about antibiotic resistance in Campylobacter isolated from turkeys reared on organic farms in Germany. Ninety-six Campylobacter strains (41 C. jejuni and 55 C. coli) were isolated from different free-range turkey flocks. In vitro antimicrobial sensitivity testing was done using a broth microdilution test, and the presence of resistance genes to antibiotics (ciprofloxacin, tetracycline) was investigated. All Campylobacter isolates from organic turkeys (n = 96) were phenotypically sensitive to gentamicin, erythromycin, streptomycin, and chloramphenicol. In this study, the antibiotic susceptibilities of C. jejuni to ciprofloxacin, tetracycline, and naladixic acid were 56.0%, 51.3%, and 56.0%, respectively. In contrast, 44.0%, 73.0%, and 74.6% of C. coli isolates were resistant to tetracycline, ciprofloxacin, and nalidixic acid, respectively. Replacement of the Thr-86→Ile in the gyrA gene, and the presence of the tet(O) gene were the mainly identified resistance mechanisms against fluoroquinolones and tetracycline, respectively.These results also reinforce the need to develop strategies and implement specific control procedures to reduce the development of antimicrobial resistance., (© 2015 Poultry Science Association Inc.)

Background: Tularemia is a zoonotic disease caused by Francisella tularensis that has been found in many different vertebrates. In Germany most human infections are caused by contact with infected European brown hares (Lepus europaeus). The aim of this study was to elucidate the epidemiology of tularemia in hares using phenotypic and genotypic characteristics of F. tularensis., Results: Cultivation of F. tularensis subsp. holarctica bacteria from organ material was successful in 31 of 52 hares that had a positive PCR result targeting the Ft-M19 locus. 17 isolates were sensitive to erythromycin and 14 were resistant. Analysis of VNTR loci (Ft-M3, Ft-M6 and Ft-M24), INDELs (Ftind33, Ftind38, Ftind49, RD23) and SNPs (B.17, B.18, B.19, and B.20) was shown to be useful to investigate the genetic relatedness of Francisella strains in this set of strains. The 14 erythromycin resistant isolates were assigned to clade B.I, and 16 erythromycin sensitive isolates to clade B.IV and one isolate was found to belong to clade B.II. MALDI-TOF mass spectrometry (MS) was useful to discriminate strains to the subspecies level., Conclusions: F. tularensis seems to be a re-emerging pathogen in Germany. The pathogen can easily be identified using PCR assays. Isolates can also be identified within one hour using MALDI-TOF MS in laboratories where specific PCR assays are not established. Further analysis of strains requires genotyping tools. The results from this study indicate a geographical segregation of the phylogenetic clade B.I and B.IV, where B.I strains localize primarily within eastern Germany and B.IV strains within western Germany. This phylogeographical pattern coincides with the distribution of biovar I (erythromycin sensitive) and biovar II (erythromycin resistance) strains. When time and costs are limiting parameters small numbers of isolates can be analysed using PCR assays combined with DNA sequencing with a focus on genetic loci that are most likely discriminatory among strains found in a specific area. In perspective, whole genome data will have to be investigated especially when terrorist attack strains need to be tracked to their genetic and geographical sources.

The emergence of antimicrobial resistance among Campylobacter isolates recovered from turkeys has increased dramatically. Monitoring the progress of this resistance becomes a growing public health issue. The aim of the present study was to provide information of the current status of antibiotic resistance patterns in Campylobacter jejuni from turkeys. Seventy-six C. jejuni isolates were recovered from 67 epidemiologically unrelated meat turkey flocks in different regions of Germany in 2010 and 2011. The isolates were typed by flaA genotyping and were investigated for antimicrobial susceptibility against 12 antibiotics by using a broth microdilution test as well as testing the genetic determination of ciprofloxacin, tetracycline, and erythromycin resistance. All isolates (n = 76) were sensitive to gentamicin and chloramphenicol. The numbers of isolates that were sensitive to streptomycin, erythromycin, neomycin, and amoxicillin were 69 (90.8%), 61 (80.2%), 58 (76.4%), and 44 (57.9%), respectively. Only one isolate was sensitive to all tested antibiotics. The emergence of a high resistance rate and multidrug resistance to three or more classes of antimicrobial agents were observed. The resistance against sulphamethoxazole/trimethoprim, metronidazole, ciprofloxacin, naladixic acid, and tetracycline was 58 (76.3%), 58 (76.3%), 53 (69.7%), 51 (67.1%), and 42 (55.3%), respectively. None of the isolates was resistant to all antibiotics. Multidrug resistance to three or more classes of antimicrobial agents was found and ranged from 3.9% to 40.8%. Replacement of the Thr-86-->Ile in gyrA gene and detection of the tet(O) gene were the main resistance mechanisms for fluoroquinolones and tetracycline, respectively, while the lack of mutation in position 2074 and 2075 on the 23S rRNA gene was responsible for macrolide resistance. The phenotypic and genotypic resistance profiles were compatible in the case of ciprofloxacin and tetracycline but were not completely congruent with respect to erythromycin.

Background: Burkholderia (B.) pseudomallei and B. mallei are genetically closely related species. B. pseudomallei causes melioidosis in humans and animals, whereas B. mallei is the causative agent of glanders in equines and rarely also in humans. Both agents have been classified by the CDC as priority category B biological agents. Rapid identification is crucial, because both agents are intrinsically resistant to many antibiotics. Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-TOF MS) has the potential of rapid and reliable identification of pathogens, but is limited by the availability of a database containing validated reference spectra. The aim of this study was to evaluate the use of MALDI-TOF MS for the rapid and reliable identification and differentiation of B. pseudomallei and B. mallei and to build up a reliable reference database for both organisms., Results: A collection of ten B. pseudomallei and seventeen B. mallei strains was used to generate a library of reference spectra. Samples of both species could be identified by MALDI-TOF MS, if a dedicated subset of the reference spectra library was used. In comparison with samples representing B. mallei, higher genetic diversity among B. pseudomallei was reflected in the higher average Eucledian distances between the mass spectra and a broader range of identification score values obtained with commercial software for the identification of microorganisms. The type strain of B. pseudomallei (ATCC 23343) was isolated decades ago and is outstanding in the spectrum-based dendrograms probably due to massive methylations as indicated by two intensive series of mass increments of 14 Da specifically and reproducibly found in the spectra of this strain., Conclusions: Handling of pathogens under BSL 3 conditions is dangerous and cumbersome but can be minimized by inactivation of bacteria with ethanol, subsequent protein extraction under BSL 1 conditions and MALDI-TOF MS analysis being faster than nucleic amplification methods. Our spectra demonstrated a higher homogeneity in B. mallei than in B. pseudomallei isolates. As expected for closely related species, the identification process with MALDI Biotyper software (Bruker Daltonik GmbH, Bremen, Germany) requires the careful selection of spectra from reference strains. When a dedicated reference set is used and spectra of high quality are acquired, it is possible to distinguish both species unambiguously. The need for a careful curation of reference spectra databases is stressed.

Background: Current epidemiological data on the situation of Coxiella (C.) burnetii infections in sheep are missing, making risk assessment and the implementation of counteractive measures difficult. Using the German state of Thuringia as a model example, the estimated sero-, and antigen prevalence of C. burnetii (10% and 25%, respectively) was assessed at flock level in 39/252 randomly selected clinically healthy sheep flocks with more than 100 ewes and unknown abortion rate., Results: The CHECKIT™ Q-fever Test Kit identified 11 (28%) antibody positive herds, whereas real-time PCR revealed the presence of C. burnetii DNA in 2 (5%) of the flocks. Multiple-locus variable number of tandem repeats analysis of 9 isolates obtained from one flock revealed identical profiles. All isolates contained the plasmid QpH1., Conclusions: The results demonstrate that C. burnetii is present in clinically inconspicuous sheep flocks and sporadic flare-ups do occur as the notifications to the German animal disease reporting system show. Although C. burnetii infections are not a primary veterinary concern due to the lack of significant clinical impact on animal health (with the exception of goats), the eminent zoonotic risk for humans should not be underestimated. Therefore, strategies combining the interests of public and veterinary public health should include monitoring of flocks, the identification and culling of shedders as well as the administration of protective vaccines.

To assess the general impact of endemic countries on the re-emergence of brucellosis in non-endemic regions of the European Union, the genetic fingerprints of Brucella melitensis strains imported to Germany were compared to ovine strains from Turkey in a molecular epidemiological study. Genotyping of 66 Brucella strains (based on Multiple Locus of Variable number of tandem repeats Analysis) isolated from German travellers and Turkish immigrants living in Germany revealed epidemiological concordance with 20 sheep isolates originating from Eastern Anatolia, Turkey. In summary, cross-border molecular tracing confirmed brucellosis being a zoonosis of concern for European public health., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Campylobacter fetus subsp. venerealis is the causative agent of bovine genital campylobacteriosis and is transmitted by asymptomatic carrier bulls via contaminated semen during artificial insemination. The aim of the present study was to determine the in vitro susceptibility of Campylobacter fetus subsp. venerealis isolated from bovine specimens in the years from 2000 to 2009 in Germany to antibiotics generally used in semen treatment. The susceptibilities of 50 strains to spectinomycin (10 microg), gentamicin (10 microg), streptomycin (25 microg), penicillin (10 microg), lincomycin (10 microg), ciprofloxacin (5 microg), erythromycin (30 microg) and tetracycline (30 microg) were determined using a disk diffusion susceptibility test. All strains were susceptible to gentamicin. A considerably reduced susceptibility to one or more antimicrobial agents was detected in seven out of 50 isolates (14%) with the most frequent reduction in susceptibility to lincomycin and spectinomycin. Furthermore, strains with reduced susceptibility to more than one antimicrobial agent were always associated with reduced susceptibility to lincomycin. It is recommended to determine the antimicrobial susceptibility of Campylobacter fetus subsp. venerealis isolates in order to evaluate the efficacy of the generally used antibiotic treatment of bull semen and to detect possible resistances.

A Chikungunya virus (CHIKV) epidemic emerged in the Indian Ocean islands of the Comores, Reunion, Mayotte, Mauritius, the Seychelles and Madagascar in 2005 resulting in the infection of about 250.000 inhabitants and travellers in only one year. Beginning in March 2006 increasing numbers of CHIKV-like febrile illnesses were reported from various parts of India. We investigated 70 consecutive German travellers returning from the affected areas and presenting with arthralgia and/or fever suggestive of CHIKV infection. Eleven patients had serological evidence of CHIKV infection. Real-time RT-PCR for CHIKV was positive in two cases, one who returned from Mauritius and the other who came back from Rajasthan, Northern India. In both cases CHIKV was isolated and sequencing of the entire viral genome was performed. The nucleotide sequence data obtained for both CHIKV strains revealed a high level of identity to CHIKV isolates from the ongoing epidemic. In detail, we found only 18 nucleotide exchanges between the isolates from Mauritius and Rajasthan, resulting in only six amino acid changes (nsP1 T128K, T376M, nsP3 S472N, capsid P23S, V27I and E1-protein A226V). Although the excessive dimension of the 2005/2006 outbreak in the Indian Ocean islands was at least in part accounted to the naïve population affected, our results of the Rajasthan isolate support that the emergence of this CHIKV subtype may rather be a result of a better viral fitness. This has been previously accounted to a A226V change in the E1 protein of the new CHIKV variant when compared to other CHIKV data available. This mutation, supposedly resulting in high-titred viremia in humans and/or an enhanced adaptation to the vector population resulting in increased transmission rates, was also found in our CHIKV isolate from Mauritius. The spread of an African CHIKV to Asia further demonstrates how fast viruses can emerge and establish in places where competent vectors are prevalent.