Characterization of porcine-specific surface (S-) layer protein carrying Lactobacillus species, S-layer proteins and the adhesin of Escherichia coli F18 fimbriae : Potential applications for veterinary medicine

Lactobacilli, common members of porcine intestinal microbiota, have been considered to be an important group of bacteria in maintaining the stability of gastrointestinal tract (GIT), preventing intestinal infections and supporting intestinal health. Because several species of lactobacilli have GRAS (generally regarded as safe) status and some of them have an ability to interact with intestinal epithelial cells, their possible applications as mucosal vaccine vector and/or probiotics have aroused interest. Selection criteria for lactobacilli to be used as vaccine vector or probiotic include the abilities to adhere to the intestinal epithelium cells and colonize the lumen of the GIT. Bacterial adhesins are often found in hair-like appendages called pili or fimbriae that extend outward from bacterial surface. Alternatively, they can be directly associated with the microbial cell surface. Surface layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. In this study, S-layer carrying lactobacilli from the intestine and faeces of pigs were isolated and their ability to adhere was studied. Besides the putative binding properties of Slps, a very large number of Slp subunits present in an S-layer make the use of the S-layer structure a very interesting alternative to surface display antigens. Therefore, the aim was to characterize the S-layer proteins. Two new surface layer proteins with potential to be tested as antigen carriers were characterized, and three slp genes were isolated, sequenced, and studied for their expression. To identify the S-layer carrying lactobacilli strains of porcine origin, a polyphasic taxonomic approach was applied. These results indicated that strains from Finland and the related L. sobrius strains, originating from elsewhere, constitute a single species, L. amylovorus, and that the name L. sobrius should be considered as a later synonym of L. amylovorus. The F18 fimbriae carrying Escherichia coli strains cause post-weaning diarrhoea and edema disease in pigs. The adhesin FedF of E. coli F18 fimbriae was characterized. The work aims at developing lactobacilli as a live mucosal vaccine vector for pigs against diseases caused by F18+ E. coli. Oral immunization of weaned piglets with adhesins is known to induce a protective mucosal immune response. Naked FedF appeared to be very unstable but we could produce it as a fusion protein with maltose binding protein (MBP). Specific adhesion to isolated porcine intestinal epithelial cells was demonstrated with MBP-FedF fusions as well as the ability of anti-MBP-FedF antibodies to prevent binding of E. coli F18 to porcine epithelial cells.Julkaistaan myöhemmi

Characterization of porcine-specific surface (S-) layer protein carrying Lactobacillus species, S-layer proteins and the adhesin of Escherichia coli F18 fimbriae : Potential applications for veterinary medicine

Lactobacilli, common members of porcine intestinal microbiota, have been considered to be an important group of bacteria in maintaining the stability of gastrointestinal tract (GIT), preventing intestinal infections and supporting intestinal health. Because several species of lactobacilli have GRAS (generally regarded as safe) status and some of them have an ability to interact with intestinal epithelial cells, their possible applications as mucosal vaccine vector and/or probiotics have aroused interest. Selection criteria for lactobacilli to be used as vaccine vector or probiotic include the abilities to adhere to the intestinal epithelium cells and colonize the lumen of the GIT. Bacterial adhesins are often found in hair-like appendages called pili or fimbriae that extend outward from bacterial surface. Alternatively, they can be directly associated with the microbial cell surface. Surface layer proteins (Slps) of lactobacilli have been shown to confer tissue adherence. In this study, S-layer carrying lactobacilli from the intestine and faeces of pigs were isolated and their ability to adhere was studied. Besides the putative binding properties of Slps, a very large number of Slp subunits present in an S-layer make the use of the S-layer structure a very interesting alternative to surface display antigens. Therefore, the aim was to characterize the S-layer proteins. Two new surface layer proteins with potential to be tested as antigen carriers were characterized, and three slp genes were isolated, sequenced, and studied for their expression. To identify the S-layer carrying lactobacilli strains of porcine origin, a polyphasic taxonomic approach was applied. These results indicated that strains from Finland and the related L. sobrius strains, originating from elsewhere, constitute a single species, L. amylovorus, and that the name L. sobrius should be considered as a later synonym of L. amylovorus. The F18 fimbriae carrying Escherichia coli strains cause post-weaning diarrhoea and edema disease in pigs. The adhesin FedF of E. coli F18 fimbriae was characterized. The work aims at developing lactobacilli as a live mucosal vaccine vector for pigs against diseases caused by F18+ E. coli. Oral immunization of weaned piglets with adhesins is known to induce a protective mucosal immune response. Naked FedF appeared to be very unstable but we could produce it as a fusion protein with maltose binding protein (MBP). Specific adhesion to isolated porcine intestinal epithelial cells was demonstrated with MBP-FedF fusions as well as the ability of anti-MBP-FedF antibodies to prevent binding of E. coli F18 to porcine epithelial cells.Julkaistaan myöhemmi

Evolutionary trends of European bat lyssavirus type 2 including genetic characterization of Finnish strains of human and bat origin 24 years apart

Erratum to: Evolutionary trends of European bat lyssavirus type 2 including genetic characterization of Finnish strains of human and bat origin 24 years apart Archives of Virology July 2015, Vol. 160, Issue 7, p 1875, 2015 10.1007/s00705-015-2475-2Among other Lyssaviruses, Daubenton's and pond-bat-related European bat lyssavirus type 2 (EBLV-2) can cause human rabies. To investigate the diversity and evolutionary trends of EBLV-2, complete genome sequences of two Finnish isolates were analysed. One originated from a human case in 1985, and the other originated from a bat in 2009. The overall nucleotide and deduced amino acid sequence identity of the two Finnish isolates were high, as well as the similarity to fully sequenced EBLV-2 strains originating from the UK and the Netherlands. In phylogenetic analysis, the EBLV-2 strains formed a monophyletic group that was separate from other bat-type lyssaviruses, with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV). EBLV-2 showed limited diversity compared to RABV and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years, and EBLV-2 diverged from KHUV about 8000 years ago. In a phylogenetic tree of partial N gene sequences, the Finnish EBLV-2 strains clustered with strains from Central Europe, supporting the hypothesis that EBLV-2 circulating in Finland might have a Central European origin. The Finnish EBLV-2 strains and a Swiss strain were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the two Finnish strains appear to have evolved from a common ancestor during the last 200 years.Peer reviewe

Molecular ecology of the yet uncultured bacterial Ct85-cluster in the mammalian gut

In our previous studies on irritable bowel syndrome (IBS) –associated microbiota by molecular methods, we demonstrated that a particular 16S rRNA gene amplicon was more abundant in the feces of healthy subjects or mixed type IBS (IBS-M) –sufferers than in the feces of individuals with diarrhea-type IBS (IBS-D). In the current study, we demonstrated that this, so called Ct85-amplicon, consists of a cluster of very heterogeneous 16S rRNA gene sequences, and defined six 16S rRNA gene types, a to f, within this cluster, each representing a novel species-, genus- or family level taxon. We then designed specific PCR primers for these sequence types, mapped the distribution of the Ct85-cluster sequences and that of the newly defined sequence types in several animal species and compared the sequence types present in the feces of healthy individuals and IBS sufferers using two IBS study cohorts, Finnish and Dutch. Various Ct85-cluster sequence types were detected in the fecal samples of several companion and production animal species with remarkably differing prevalences and abundances. The Ct85 sequence type composition of swine closely resembled that of humans. One of the five types (d) shared between humans and swine was not present in any other animals tested, while one sequence type (b) was found only in human samples. In both IBS study cohorts, one type (e) was more prevalent in healthy individuals than in the IBS-M group. By revealing various sequence types in the widespread Ct85-cluster and their distribution, the results improve our understanding of these uncultured bacteria, which is essential for future efforts to cultivate representatives of the Ct85-cluster and reveal their roles in IBS.Peer reviewe