Seroprevalence of tick-borne encephalitis virus in wild and domestic animals in northern Germany.

Tick-borne encephalitis virus (TBEV) is a tick-transmitted flavivirus, which can infect humans and animals, sometimes even with a fatal outcome. Since many decades, TBEV is endemic in southern Germany, while only sporadic occurrence has been noted in northern parts of the country so far. Nevertheless, autochthonous human clinical cases are increasing in the federal state of Lower Saxony in north-western Germany, and several natural foci of TBEV transmission have recently been detected in this federal state. In order to shed more light on the current distribution of TBEV in Lower Saxony, the present study examined blood samples from wild and domestic animals for antibodies against TBEV. Overall, samples from 4,085 animals were tested by ELISA, including wild boar (N = 1,208), roe deer (N = 149), red deer (N = 61), fallow deer (N = 18), red foxes (N = 9), nutria (N = 9), raccoon dogs (N = 3), raccoons (N = 3), badgers (N = 1), European pine martens (N = 1), horses (N = 574), sheep (N = 266), goats (N = 67), dogs (N = 1,317) and cats (N = 399). Samples with an ELISA result of ≥60 Vienna units (VIEU)/ml were subjected to confirmatory serum neutralization tests (SNT). In total, 343 of 4,085 (8.4%) animals tested positive for anti-TBEV-IgG by ELISA, of which 60 samples were confirmed by SNT. Samples of 89 animals showed a cytotoxic effect in the SNT and were excluded from seroprevalence calculation, resulting in an overall seroprevalence of 1.5% (60/3,996). Seroprevalence was higher among wild animals (wild boar: 2.9% [34/1,190], roe deer: 2.7% [4/149], red deer: 1.7% [1/60], fallow deer: 5.6% [1/18]) than among domestic animals (dogs: 1.1% [15/1,317], horses: 0.8% [4/505], sheep: 0.4% [1/266]). No anti-TBEV-antibodies were detected in the other wild animal species as well as goats and cats. A notable clustering of positive samples was observed in districts where TBEV transmission foci have been described. Further clusters in other districts suggest the existence of so far undetected transmission foci, underlining the fact that both wild and domestic animals are useful sentinels for monitoring the spread of TBEV.

Detection and Characterization of Alongshan Virus in Ticks and Tick Saliva from Lower Saxony, Germany with Serological Evidence for Viral Transmission to Game and Domestic Animals.

The newly discovered group of Jingmenviruses has been shown to infect a wide range of hosts and has been associated with febrile illness in humans. During a survey for Jingmenviruses in ticks from Lower Saxony, Germany, Alongshan virus (ALSV) was identified in Ixodes spp. ticks. Additional virus screenings revealed the presence of ALSV in the bodies and saliva of ticks collected at several locations in Lower Saxony. Vector competence studies that included Ixodes ricinus and Dermacentor reticulatus validated the replication of ALSV within those tick species. In vitro feeding experiments with ALSV-injected Ixodes ricinus demonstrated effective viral transmission during blood feeding. To evaluate the potential viral transmission during a natural blood meal, sera from wild game and domestic animals were investigated. One serum sample from a red deer was found to be positive for ALSV RNA, while serological screenings in game and domestic animals revealed the presence of ALSV-specific antibodies at different locations in Lower Saxony. Overall, those results demonstrate the broad distribution of ALSV in ticks in Lower Saxony and hypothesize frequent exposure to animals based on serological investigations. Hence, its potential risk to human and animal health requires further investigation.

Role of F1C fimbriae, flagella, and secreted bacterial components in the inhibitory effect of probiotic Escherichia coli Nissle 1917 on atypical enteropathogenic E. coli infection.

Enteropathogenic Escherichia coli (EPEC) is recognized as an important intestinal pathogen that frequently causes acute and persistent diarrhea in humans and animals. The use of probiotic bacteria to prevent diarrhea is gaining increasing interest. The probiotic E. coli strain Nissle 1917 (EcN) is known to be effective in the treatment of several gastrointestinal disorders. While both in vitro and in vivo studies have described strong inhibitory effects of EcN on enteropathogenic bacteria, including pathogenic E. coli, the underlying molecular mechanisms remain largely unknown. In this study, we examined the inhibitory effect of EcN on infections of porcine intestinal epithelial cells with atypical enteropathogenic E. coli (aEPEC) with respect to single infection steps, including adhesion, microcolony formation, and the attaching and effacing phenotype. We show that EcN drastically reduced the infection efficiencies of aEPEC by inhibiting bacterial adhesion and growth of microcolonies, but not the attaching and effacing of adherent bacteria. The inhibitory effect correlated with EcN adhesion capacities and was predominantly mediated by F1C fimbriae, but also by H1 flagella, which served as bridges between EcN cells. Furthermore, EcN seemed to interfere with the initial adhesion of aEPEC to host cells by secretion of inhibitory components. These components do not appear to be specific to EcN, but we propose that the strong adhesion capacities enable EcN to secrete sufficient local concentrations of the inhibitory factors. The results of this study are consistent with a mode of action whereby EcN inhibits secretion of virulence-associated proteins of EPEC, but not their expression.

Antimicrobial susceptibilities and occurrence of resistance genes in bovine Helcococcus ovis isolates.

The aim of the study was to investigate antimicrobial susceptibilities of bovine Helcococcus ovis isolates and to detect genes encoding for H. ovis erythromycin and tetracycline resistance. Twenty-nine isolates were included and the minimal inhibitory concentrations (MICs) of seven antimicrobials were determined using test conditions as given in an approved CLSI guideline for the pyridoxal-dependent Abiotrophia spp. and Granulicatella spp. Furthermore, the macrolide resistance phenotype was examined by the erythromycin-clindamycin double-disk test (D-zone test). Erythromycin MICs of ≥ 8 µg/ml were found in three (10%) isolates which also presented the macrolide, lincosamide, and streptogramin B (MLS(B)) resistance phenotype, either constitutive or inducible. The erm(B) gene was detected in one of these isolates. Increased tetracycline MICs (≥ 8 µg/ml) were obtained for 24 (83%) isolates, mostly associated with the tet(M) gene alone (n=21) or both the tet(L) and tet(M) genes (n=2). The MICs determined for penicillin, ampicillin, amoxicillin-clavulanic acid, and cephalothin did not indicate resistance to these antimicrobials. The study suggests that resistance to MLS(B) antimicrobials and tetracycline is frequent in H. ovis. Moreover, this is the first report about occurrence of the resistance genes erm(B), tet(L), and tet(M) in the Helcococcus genus.

Avian pathogenic Escherichia coli MT78 invades chicken fibroblasts.

Avian pathogenic Escherichia coli (APEC) are responsible for extraintestinal diseases, called colibacillosis, in avian species. The most severe manifestation of the disease is colisepticemia that usually starts at the respiratory tract and may result in bird death. However, it is not yet clear how APEC cross the respiratory epithelium and get into the bloodstream. In this work, we studied the interaction between 8 APEC strains (UEL31, UEL17, UEL13, UEL29, MT78, IMT5155, IMT2470, A2363) and a chicken non-phagocytic cell, the fibroblast CEC-32 cell line. We investigated the association profile, the invasion capability, the cytotoxicity effect and the induction of caspase-3/7 activation in an attempt to understand the way the pathogen gains access to the host bloodstream. Association to cells was determined after 1 h of infection, while cell invasion was determined after 4 and 24 h of infection. The cytotoxic effect of bacterial infection was measured by lactate dehydrogenase (LDH) release and the activation of the apoptotic program was verified by caspase-3/7 activation. Also, the presence of genes for adhesins, invasins and other related virulence-associated factors was verified by PCR. All bacterial strains showed similarity in relation to adhesion, LDH release and caspase-3/7 activation. However, one APEC strain, MT78, showed high invasion capability, comparable to the invasive Salmonella typhimurium strain SL1344. Since an APEC strain was capable of invading non-phagocytic cells in vitro, the same may be happening with the epithelial cells of the avian respiratory tract in vivo. CEC-32 monolayers can also provide a useful experimental model to study the molecular mechanisms used by APEC to invade non-phagocytic cells.

Helcococcus ovis, an emerging pathogen in bovine valvular endocarditis.

The initial isolation of Helcococcus ovis from a valvular thrombus prompted us to investigate the prevalence of this bacterium in bovine valvular endocarditis. Specimens from 55 affected hearts were examined by culture using Columbia blood agar and cross streaking the inoculated plate with a Staphylococcus aureus strain. As confirmed by 16S rRNA gene sequencing, H. ovis was isolated with an unexpectedly high frequency of 33%, predominantly as heavy growth and pure culture. The majority of H. ovis isolates showed distinct satellitism around S. aureus and pyridoxal dependency, resembling "nutritionally variant streptococci" (now assigned to the genera Abiotrophia and Granulicatella). Using the API rapid ID 32 Strep, API ZYM, and Rosco Diatabs systems, incongruent results were obtained for alkaline phosphatase, beta-galactosidase, beta-glucuronidase, and leucine aminopeptidase activities. Based on the satellitism/pyridoxal dependency; hemolysis on blood agar; the API rapid ID 32 Strep results for arginine dihydrolase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase, and pyroglutamic acid arylamidase activities; hippurate hydrolysis; and acidification of sucrose, a scheme for the identification of H. ovis and its differentiation from other members of the Helcococcus genus and the pyridoxal-dependent species Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans is proposed. By establishing specific fluorescence in situ hybridization, large H. ovis aggregates were specifically detected within the fibrinous exudate of the valvular thrombi. Our results demonstrate for the first time that H. ovis represents an emerging pathogen in bovine valvular endocarditis that is frequently isolated if appropriate culture conditions are used.

High prevalence of treponemes in bovine digital dermatitis-a molecular epidemiology.

To validate the epidemiology of Treponema spp. associated with digital dermatitis (DD) a large number of DD samples (n=56) were examined by DNA-DNA dot blot analyses using oligonucleotide probes specific for phylogenetic group I-VII of oral treponemes and DD-associated phylotypes DDKL-4, DDKL-12 and DDKL-20 as well as for T. brennaborense and T. socranskii. Positive hybridisation results were obtained for phylogenetic groups I, II and IV and phylotypes DDKL-4 and DDKL-12. While phylotype DDKL-4 was detected in 100% of the samples treponemes belonging to phylogenetic group TRE I, TRE II and TRE IV were prevalent in nearly 80% of the samples and phylotype DDKL-12 was detected in 66.1% of the samples. Analysis of Treponema groups present concurrently in the same sample revealed that a combination of TRE I-TRE II-TRE IV-DDKL-4 was most prevalent and could be detected in up to 71% of the samples. These data indicate that this combination of different Treponema spp. seems to be the most important one in the pathogenesis of DD. In contrast, T. brennaborense originally isolated from DD material this treponeme was not detected in any of the samples clearly indicating that this species is not absolutely associated with DD and therefore may represent only an incidental treponeme. Fluorescence in situ hybridisation (FISH) obviously highlights the invasive character of DD-associated treponemes. Mainly treponemes belonging to phylogenetic group TRE I and phylotype DDKL-4 were detected in high numbers compared to the total number of bacteria and also in deeper layers of the epithelium at the transition of unaffected and affected tissue. Our results confirm a high prevalence and diversity of Treponema spp. in DD lesions. In addition, our data indicate that certain combinations of Treponema spp. are detected much more frequently than others. Furthermore, Treponema spp. appears at the interface between healthy and diseased tissue underlining their importance for the pathogenesis of DD.

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera).

American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae-specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death.

Association of Treponema spp. with canine periodontitis.

To evaluate the association of oral Treponema (T.) spp. with severity of canine periodontitis, subgingival plaque samples of dogs of various breeds undergoing surgery were investigated. A wide range of oral Treponema spp. was analysed by a molecular and culture-independent approach applying DNA-DNA dot blot hybridization analysis and fluorescence in situ hybridization using Treponema specific oligonucleotide probes specific for phylogenetic groups I-VII of oral treponemes as well as probes specific for T. socranskii and T. denticola. To assess the periodontal status of affected dogs clinical parameters were measured and the periodontal status was classified from grade 0 (physiological periodont) to 3 (severe periodontitis). The periodontal status correlated significantly with an increasing concentration of volatile sulfur compounds (VSC, r=0.854) determined with a Halimeter, indicating a positive correlation between the presence of VSC-producing bacteria and periodontitis. In this study Treponema spp. of phylogenetic groups III, V-VII were not detected in any sample, whereas T. denticola-like treponemes were found only in 2 of 51 animals. However, treponemes belonging to phylogenetic groups I, II and IV of oral treponemes or T. socranskii were found in up to 64.84% of the dogs. The detection rate of Treponema spp. was significantly associated with an increased periodontal status. Treponemes present in periodontal lesions were also visualized by fluorescence in situ hybridization of gingival biopsies showing Treponema spp. not only in the microbial biofilm but also within the gingival tissue. The data presented here indicate that oral Treponema spp. are associated with canine periodontitis. Similar to human periodontitis, treponemes of groups I, II and IV and T. socranskii were found more frequently the higher the degree of periodontitis was.

Involvement of Guggenheimella bovis in digital dermatitis lesions of dairy cows.

Digital dermatitis (DD) of cattle leads to lameness and a decrease of milk production and is responsible for major economic losses worldwide. Although a bacterial aetiology is generally accepted, it still is unclear which microorganisms cause and/or maintain the disease. Recently, a previously undiscovered bacterial species, Guggenheimella bovis, has been isolated from the front of two DD lesions in Swiss cattle and suggested as a potential pathogen. The aims of the present study were to determine the prevalence of G. bovis in 58 German cows suffering from DD via dot blot hybridization, and to analyse the spatial distribution of G. bovis within the affected tissue by fluorescence in situ hybridization (FISH). A species-specific probe, GUBO1, was designed and evaluated. In none of the 58 samples Guggenheimella could be detected, while cultured G. bovis was reliably identified by GUBO1. Further FISH experiments were carried out on two additional biopsies of Swiss cattle tested positive for G. bovis by quantitative PCR and permitted visualization of the newly discovered bacteria in situ. In these biopsies G. bovis proved to be tissue invasive forming characteristic spherical microcolonies not only within the bacterial biofilm but also in seemingly unaffected parts of the tissue not yet reached by the advancing bacterial front. Although the presence of G. bovis does not constitute an essential premise for DD, it seems likely that the bacterial species involved in DD vary, and that in some cases G. bovis is crucial for the development of DD lesions.

Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine.

In vitro studies on the pathogenesis in swine have been hampered by the lack of relevant porcine cell lines. Since many bacterial infections are swine-specific, studies on pathogenic mechanisms require appropriate cell lines of porcine origin. We have characterized the permanent porcine intestinal epithelial cell line, IPEC-J2, using a variety of methods in order to assess the usefulness of this cell line as an in vitro infection model. Electron microscopic analyses and histochemical staining revealed the cells to be enterocyte-like with microvilli, tight junctions and glycocalyx-bound mucin. The functional integrity of monolayers was determined by transepithelial electrical resistance (TEER) measurements. Both commensal bacteria and important bacterial pathogens were chosen for study based on their principally different infection mechanisms: obligate extracellular Escherichia coli, facultative intracellular Salmonella and obligate intracellular Chlamydia. We determined the colonization and proliferation of the bacteria on and within the host cells and monitored the host cell response. We verified the expression of mRNAs encoding the cytokines IL-1alpha, -6, -7, -8, -18, TNF-alpha and GM-CSF, but not TGF-beta or MCP-1. IL-8 protein expression was enhanced by Salmonella invasion. We conclude that the IPEC-J2 cell line provides a relevant in vitro model system for porcine intestinal pathogen-host cell interactions.

Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer.

Neurosecretion is catalyzed by assembly of a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-complex composed of SNAP-25, synaptobrevin and syntaxin. Munc 18-1 is known to bind to syntaxin in vitro. This interaction prevents assembly of the SNARE-complex, but might also affect intracellular targeting of the proteins. We have fused syntaxin and Munc 18 to the yellow- (YFP) or cyan-fluorescence-protein (CFP) and expressed the constructs in CHO- and MDCK-cells. We have studied their localization with confocal microscopy and a possible protein-protein interaction with fluorescence-resonance energy transfer (FRET). YFP-syntaxin localizes to intracellular membranes. CFP-Munc 18 is present in the cytoplasm as expected for a protein lacking membrane targeting domains. However, Munc 18 is redirected to internal membranes when syntaxin is coexpressed, but only limited transport of the proteins to the plasma membrane was observed. An interaction between Munc 18 and syntaxin could be demonstrated by FRET using two methods, sensitized acceptor fluorescence and acceptor photobleaching. A mutation in syntaxin (L165A, E166A), which is known to inhibit binding to Munc 18 in vitro, prevents colocalization of the proteins and also the FRET signal. Thus, a protein-protein interaction between Munc 18 and syntaxin occurs on intracellular membranes, which is required but not sufficient for quantitative transport of both proteins to the plasma membrane.

Treponema berlinense sp. nov. and Treponema porcinum sp. nov., novel spirochaetes isolated from porcine faeces.

Limit-dilution procedures were used to isolate seven, helically coiled bacterial strains from faeces of swine that constituted two unidentified taxa. Comparative 16S rRNA gene sequence analysis showed highest similarity values with species of the genus Treponema indicating that the isolates are members of this genus. Strain 7CPL208(T), as well as five further isolates, and 14V28(T) displayed the highest 16S rRNA gene sequence similarities with Treponema pectinovorum ATCC 33768(T) (92.3%) and Treponema parvum OMZ 833(T) (89.9%), respectively. Polar lipid profiles distinguished 7CPL208(T) and 14V28(T) from each other as well as from related species. Based on their phenotypic and genotypic distinctiveness, strains 7CPL208(T) and 14V28(T) are suggested to represent two novel species of the genus Treponema, for which the names Treponema berlinense sp. nov. and Treponema porcinum sp. nov. are proposed. The type strain for Treponema berlinense is 7CPL208(T) (=ATCC BAA-909(T)=CIP 108244(T)=JCM 12341(T)) and for Treponema porcinum 14V28(T) (=ATCC BAA-908(T)=CIP 108245(T)=JCM 12342(T)).

[Incidence and significance of treponemes in animals]. / Vorkommen und Bedeutung von Treponemen beim Tier.

The genus Treponema consists of various species. Currently most of them are not cultivable because respective cultivation conditions are unknown. Therefore the biodiversity of treponemes was only appreciated recently by applying comparative 16S rRNA sequence analysis. Treponemes are mainly representatives of the gastrointestinal autochthonal flora, especially in termites, but they have also been described in swine and cattle. On the other hand treponemes are involved in different infectious diseases, the most well known being syphilis in humans or venereal spirochetosis in rabbits. Furthermore, treponemes are associated with several infectious periodontal diseases, e.g. gingivitis or periodontitis, where they can be detected regularly. Culture has not been successful for most of the oral treponemes, so the major part can only be identified by their 16S rRNA sequence. Similar to these oral disorders treponemes are also associated with digital dermatitis (DD), a chronic inflammatory disease of the bovine skin, where different treponemal phylotypes were found in large numbers. Treponema brennaborense was first identified and isolated in DD biopsies. Unravelling the pathogenic potential and aetiological significance of treponemes in chronic infectious diseases like peridontitis or DD remains a costly task. Although treponemes can be frequently detected in such lesions, it is often unclear to what extent treponemes are involved in pathogenesis of these diseases. The possession of various virulence features like high motility, the ability to adhere and invade as well as to cause cytopathic effects in eukaryotic cells are highly indicative of the aetiological relevance of treponemes.