Summary We compared the mortality of honeybee (Apis mellifera) drone and worker larvae from a single queen under controlled in vitro conditions following infection with Paenibacillus larvae, a bacterium causing the brood disease American Foulbrood (AFB). We also determined absolute P. larvae cell numbers and lethal titres in deceased individuals of both sexes up to 8 days post infection using quantitative real-time PCR (qPCR). Our results show that in drones the onset of infection induced mortality is delayed by 1 day, the cumulative mortality is reduced by 10% and P. larvae cell numbers are higher than in worker larvae. Since differences in bacterial cell titres between sexes can be explained by differences in body size, larval size appears to be a key parameter for a lethal threshold in AFB tolerance. Both means and variances for lethal thresholds are similar for drone and worker larvae suggesting that drone resistance phenotypes resemble those of related workers.

ABSTRACT: BACKGROUND: There is a major paradox in our understanding of honey bee immunity: the high population density in a bee colony implies a high rate of disease transmission among individuals, yet bees are predicted to express only two-thirds as many immunity genes as solitary insects, e.g., mosquito or fruit fly. This suggests that the immune response in bees is subdued in favor of social immunity, yet some specific immune factors are up-regulated in response to infection. To explore the response to infection more broadly, we employ mass spectrometry-based proteomics in a quantitative analysis of honey bee larvae infected with the bacterium Paenibacillus larvae. Newly-eclosed bee larvae, in the second stage of their life cycle, are susceptible to this infection, but become progressively more resistant with age. We used this host-pathogen system to probe not only the role of the immune system in responding to a highly evolved infection, but also what other mechanisms might be employed in response to infection. RESULTS: Using quantitative proteomics, we compared the hemolymph (insect blood) of five-day old healthy and infected honey bee larvae and found a strong up-regulation of some metabolic enzymes and chaperones, while royal jelly (food) and energy storage proteins were down-regulated. We also observed increased levels of the immune factors prophenoloxidase (proPO), lysozyme and the antimicrobial peptide hymenoptaecin. Furthermore, mass spectrometry evidence suggests that healthy larvae have significant levels of catalytically inactive proPO in the hemolymph that is proteolytically activated upon infection. Phenoloxidase (PO) enzyme activity was undetectable in one or two-day-old larvae and increased dramatically thereafter, paralleling very closely the age-related ability of larvae to resist infection. CONCLUSIONS: We propose a model for the host response to infection where energy stores and metabolic enzymes are regulated in concert with direct defensive measures, such as the massive enhancement of PO activity.

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 unexpected 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"(NVS) actually designated 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 activity. Based on the characteristics satellitism/pyridoxal dependency, hemolysis on blood agar and the API rapid ID 32 Strep results for arginine dihydrolase, alpha-galactosidase, beta-galactosidase, beta-glucuronidase and pyroglutamic acid arylamidase activity, 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 exsudate of the valvular thrombi. Our results demonstrate for the first time that H. ovis represents an emerging pathogen in bovine valvular endocarditis, frequently isolated if appropriate culture conditions are used.

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.

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.

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)).

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.

ABSTRACT: BACKGROUND: Shiga toxin producing Escherichia coli (STEC) are an important cause of human gastro-enteritis and extraintestinal sequelae, with ruminants, especially cattle, as the major source of infection and reservoir. In this study, the fecal STEC shedding of 133 dairy cows was analyzed over a period of twelve months by monthly sampling with the aim to investigate shedding patterns and risk factors. RESULTS: Overall, 24.7%(in total 407) of 1,646 fecal samples were tested positive for stx by PCR with inner-herd prevalences on the different farms of 11.1% to 32.3%. At individual levels, cows were stx-positive on zero to eight consecutive samplings. According to a strictly longitudinal definition of Super-Shedding, in the present study 14 cows were identified as Super-Shedders of non-O157 serotypes. Significant risk factors for the shedding of STEC were the month of sampling, the number of lactations and days in lactation, the nutritional condition, the somatic cell count and the content of protein in milk. Most notably, the presence of STEC Super-Shedding cows in the herd was a significant risk factor, revealing that STEC Super-Shedding is not restricted to STEC O157:H7 alone. CONCLUSIONS: These data have implications for possible interventions, as removing single non-O157:H7 STEC Super-Shedding cattle from farms would significantly reduce STEC burden.

Wild rodents can be carriers of antimicrobial resistant Escherichia coli. As rodents are known to be involved in the transmission of bacteria of human and animal health concern, they could likewise contribute to the dissemination of antimicrobial resistant bacteria in the environment. The aim of this study was therefore to get first insights into the antimicrobial resistance status among E. coli isolated from wild small mammals in rural areas. We tested 188 faecal isolates from eight rodent and one shrew species originating from Germany. Preselected resistant isolates were screened by minimal inhibitory concentration (MIC) testing or agar diffusion test and subsequent PCR analysis of resistance genes. The prevalence of antimicrobial resistant isolates was low with only 5.5% of the isolates exhibiting resistant phenotypes against at least one antimicrobial compound including beta-lactams, tetracyclines, aminoglycosides and sulfonamides. These results suggest a minor role of wild rodents from rural areas in the cycle of transmission and spread of antimicrobial resistant E. coli into the environment. Nevertheless E. coli with multiple antimicrobial resistances were significantly more often detected in wildlife rodents originating from areas with high livestock density suggesting a possible transmission from livestock to wild rodents.

IbeA (invasion of brain endothelium), which is located on a genomic island termed GimA, is involved in the pathogenesis of several extraintestinal pathogenic E. coli (ExPEC) pathotypes, including newborn meningitic E. coli (NMEC) and avian pathogenic E. coli (APEC). To unravel the phylogeny of GimA and to investigate its island character, the putative insertion locus of GimA was determined via Long Range PCR and DNA-DNA hybridization in 410 E. coli isolates, including APEC, NMEC, uropathogenic (UPEC), septicemia-associated E. coli (SEPEC), and human and animal fecal isolates as well as in 72 strains of the E. coli reference (ECOR) collection. In addition to a complete GimA ( approximately 20.3 kb) and a locus lacking GimA we found a third pattern containing a 342 bp remnant of GimA in this strain collection. The presence of GimA was almost exclusively detected in strains belonging to phylogenetic group B2. In addition, the complete GimA was significantly more frequent in APEC and NMEC strains while the GimA remnant showed a higher association with UPEC strains. A detailed analysis of the ibeA sequences revealed the phylogeny of this gene to be consistent with that obtained by Multi Locus Sequence Typing of the strains. Although common criteria for genomic islands are partially fulfilled, GimA rather seems to be an ancestral part of phylogenetic group B2, and it would therefore be more appropriate to term this genomic region GimA locus instead of genomic island. The existence of two other patterns reflects a genomic rearrangement in a reductive evolution-like manner.

Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.

Until the late 1980s, specific viral infections of the honey bee were generally considered harmless in all countries. Then, with the worldwide introduction of the ectoparasite mite Varroa destructor, beekeepers encountered increasing difficulties in maintaining their colonies. Epidemiological surveys and laboratory experiments have demonstrated that the newly acquired virulence of several viruses belonging to the family Dicistroviridae (acute bee paralysis virus, Kashmir bee virus and Israeli acute paralysis virus) in Europe and the USA had been observed in relation with V. destructor acting as a disseminator of these viruses between and within bee colonies and as an activator of virus multiplication in the infected individuals: bee larvae and adults. Equal emphasis is given to deformed wing virus (DWV) belonging to the Iflaviridae. Overt outbreaks of DWV infections have been shown to be linked to the ability of V. destructor to act not only as a mechanical vector of DWV but also as a biological vector. Its replication in mites prior to its vectoring into pupae seemed to be necessary and sufficient for the induction of a covert infection in pupae developing in non-viable bees with deformed wings. DWV in V. destructor infested colonies is now considered as one of the key players of the final collapse. Various approaches for combating bee viral diseases are described: they include selection of tolerant bees, RNA interference and prevention of new pathogen introduction. None of these approaches are expected to lead to enhanced bee-health in the short term.