Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)

Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome. These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism. Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes. Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness. These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution. Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer. PAIs are the fascinating proof of the plasticity of bacterial genomes. PAIs were originally described in human pathogenic Escherichia (E.) coli strains. In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants. The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli. In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei. The LEE is an important virulence feature of several animal pathogens. It is an obligate PAI of all animal and human enteropathogenic E. coli (EPEC), and most enterohaemorrhegic E. coli (EHEC) also harbor the LEE. The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system. The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium. Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens. Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations. However, the evolution of the LEE, its origin and further spread in E. coli, are far from being resolved.     

Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status

To learn more about the molecular biology of Pasteurella multocida 289 strains isolated from various clinically healthy and diseased hosts were examined for capsule biosynthesis genes (capA, B, D, E, and F) and 14 virulence associated genes by PCR and DNA-DNA-hybridization. As expected, capsule type A strains were highly adapted to bovines (92.3%) and poultry (85.7%) while we mainly found capA (34.9%)- and capD (58.1%)-positive strains in swine. A noticeable amount of capD-positive strains also originated from small ruminants (34.9%) and capF was detected in wild type strains from diseased cattle (2.2%) and cats (7.4%). None of the isolates harboured capE, while capB was exclusively found in all strains from buffaloes. Nearly all isolates showed a combination of genes encoding outer membrane proteins, colonization factors, iron aquisition factors and superoxid-dismutases without any clue for host specificity. In contrast, the transferrin binding protein encoding gene tbpA (31.5%) was limited to ruminant strains and only 37.0% of all P. multocida strains harboured pfhA, coding for a filamentous hemagglutinin, supposed to be a putative adhesion- und serum resistance factor. PfhA revealed a strong positive association to the outcome of disease in bovine hosts and in combination with toxA to that in swine. The dermonecrotoxin encoding toxA, present in 12.5% of all strains, was detected in isolates from swine, small ruminants, cattle, and poultry. A significant association to the disease status, however, was only existent in swine, although with 66.7% we found a notably high prevalence of the toxin gene among strains from small ruminants. The genes toxA, tbpA and pfhA as well as capsule biosynthesis genes are supposed to be important epidemiological marker genes for characterizing P. multocida field strains.     

Solar wind neon from Genesis: implications for the lunar noble gas record

Lunar soils have been thought to contain two solar noble gas components with distinct isotopic composition. One has been identified as implanted solar wind, the other as higher-energy solar particles. The latter was puzzling because its relative amounts were much too large compared with present-day fluxes, suggesting periodic, very high solar activity in the past. Here we show that the depth-dependent isotopic composition of neon in a metallic glass exposed on NASA's Genesis mission agrees with the expected depth profile for solar wind neon with uniform isotopic composition. Our results strongly indicate that no extra high-energy component is required and that the solar neon isotope composition of lunar samples can be explained as implantation-fractionated solar wind.     

On the Current Situation of Glanders in Various Districts of the Pakistani Punjab

Glanders is a highly infectious and zoonotic disease of solipeds caused by Burkholderia mallei. Progressive loss of efficiency and fatal outcome resulted in massive economic losses, which forced veterinary authorities throughout the world to implement disease control measures; these measures included mass testing using the complement fixation test and/or malleinization, and the culling of positives. This led to the eradication of glanders from Western Europe and North America in the 1950s. However, in the last decade, the number of outbreaks in Asia and South America increased steadily, and glanders regained the status of a re-emerging transboundary disease. Pakistan has been an endemic country for the past 120 years, but concise data on the presence of disease are not available. A total of 533 serum samples were collected from draught equines, a suspected risk group for glanders, from various districts of Punjab in Pakistan. The complement fixation test and the highly sensitive Western blot technique were used for serodiagnosis. No animal (horse, mule, and donkey) was found to be positive for infection. Glanders seems to be restricted to remote, sporadic pockets of endemicity and may cause outbreaks after being introduced into naive populations by (asymptomatic) shedders.     

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.     

Effects of Bacillus cereus var. toyoi on immune parameters of pregnant sows

Changing immune parameters during pregnancy have previously been reported in humans and cattle, and have been suggested to contribute to increased susceptibility to infections. However, data regarding immune parameters during pregnancy in sows are rare. In this study, we investigated the peripartal immune status of sows using phenotypical (FACS analysis) as well as functional (proliferation assays, cytokine analysis) parameters of peripheral blood mononuclear cells (PBMCs) in pregnant sows. In previous studies, we reported a modulation of the immune system after feed supplementation of the probiotic Bacillus cereus var. toyoi in piglets [Schierack, P., Wieler, L.H., Taras, D., Herwig, V., Tachu, B., Hlinak, A., Schmidt, M.F., Scharek, L., 2007. Bacillus cereus var. toyoi enhanced systemic immune response in piglets. Vet. Immunol. Immunopathol. 118, 1–11]. Here, we extended these previous studies to include investigations of possible probiotic effects on the peripartal immune status of sows and their reproductivity. We show that immune parameters of sows change during pregnancy, the proliferative response of PBMCs to several bacterial antigens in control animals decreased from days 90 to 30 ante partum. Relative numbers (%) of CD3+CD8+, CD4+, cytotoxic T, CD14+ and CD21+ cells were reduced compared to non-pregnant sows. In contrast, the proliferative response of PBMCs of probiotic-treated sows increased during pregnancy. Bacterial antigens primarily stimulated the proliferation of naïve CD21+ cells and the relative CD21+ cell numbers were elevated in the probiotic group in the absence of effects on other immune cell populations. The clinical and microbial status of both control and probiotic sows was similar, excluding pre-existing health problems or infections as responsible for the immunological changes, and feed supplementation also had no significant effects on reproductivity. The results suggest that the probiotic B. cereus var. toyoi can alter the proliferative response of lymphocytes and affects the immune cell population ratios of pregnant sows. How and to what extent this may affect health and reproductivity should be the focus of further studies.     

Multidrug- and methicillin resistant Staphylococcus pseudintermedius as a cause of canine pyoderma: a case report

A case of a dog with a long-term inflammatory skin disorder due to infection with methicillin-resistant Staphylococcus pseudintermedius (MRSP) is described. After initial diagnostics of MRSP, follow-up swabs of the dog (nose, skin) were taken twice after four and seven weeks. MRSP was constantly isolated from the skin and once from the nose. Since infected humans might be a source of reinfection, the owners of the dog were screened (nasal) three times during their pet's therapy. Thereby, the male owner was found to be colonized with MRSP once in the first sampling round. Comparative typing of all MRSP-isolates by pulsed-field gel electrophoresis (PFGE), SCCmec typing, multilocus sequence typing (MLST), spa typing, PCR-detection of the leukotoxin encoding operon (LukI) and the Staphylococcus intermedius-exfoliative toxin (SIET) as well as antimicrobial resistance profiling by broth microdilution revealed that all five MRSP isolates from the dog and the single isolate from the owner were indistinguishable by any of the applied methods. All isolates were assigned to a certain strain, a multidrug-resistant MRSP belonging to sequence type (ST) 71, spa type (t)05, harbouring SCCmecIII as well as the genes encoding LukI and SIET. In this case, a number of reasons might have contributed to therapy failure and re-infection, respectively (e. g. contact to other MRSP-colonized dogs, contact to MRSP-colonized humans, refusal to clip the dog's fur). In addition, MRSP-contaminated objects or surfaces in the household, which were difficult to disinfect or simply not considered as a potential source of MRSP, might have served as a source of re-infection. These results envision the possibility of a dog-to-human transmission of MRSP and the relevance of this aspect as a potential source of re-infection in cases of bacterial-supported long-term skin disorders in canine patients. First cases of MRSP infections in humans have been described only recently. However, the general pathogenic potential of multidrug resistant MRSP in humans is unknown so far and needs further investigation.     

Comparative evaluation of three commercially available complement fixation test antigens for the diagnosis of glanders

The sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true-negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinically positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as the gold standard test. Highest sensitivity was shown for the CIDC antigen (100 per cent) followed by the c.c.pro antigen (99.39 per cent). However, the USDA antigen showed substantially less (p<0.05) sensitivity (62.19 per cent). Highest specificity was found for the USDA antigen (100 per cent) followed by the CIDC (97.5 per cent) and c.c.pro antigen (96.5 per cent). Positive and negative predictive values (assumed glanders prevalence of <0.1 per cent) for each antigen were calculated to be 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33 per cent (USDA), respectively. Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB.     

Comparative plasmid profile analysis of Salmonella typhimurium var. Copenhagen strains from a Salmonella outbreak in hospitalized horses]

From April 1990 through June 1991 clinical salmonellosis and asymptomatic faecal excretion of Salmonella spp. were seen in hospitalized horses at two veterinary hospitals. 76 Salmonella strains from hospitalized horses and 18 strains from horses without any clinical contact were characterized by serotyping and plasmid profile analysis. From April 1990 through January 1991 97.8% of the hospitalized horses were infected with strains of S. typhimurium var. Copenhagen, which were closely related according to their similar plasmid patterns. Other strains of S. typhimurium var. Copenhagen and serotype S. enteritidis were isolated not before February 1991. In the same period various plasmid profile types of S. typhimurium var. Copenhagen and strains of S. typhimurium, S. enteritidis and S. lexington were isolated from horses of the control group. Our results suggest that the high incidence of salmonellosis and latent salmonella infection in hospitalized horses was mainly due to the spread of one particular strain of S. typhimurium var. Copenhagen and that this strain was obviously acquired during hospitalisation.