Silja Åvall-Jääskeläinen

Purpose: Two internet-based learning tools have been developed at the Faculty of Veterinary Medicine at the University of Helsinki. One of the tools offers interactive cases related to veterinary epidemiology, the other offers interactive cases related to veterinary bacteriology. This study aimed to investigate the effect of these learning tools on learning outcomes of veterinary students for the cognitive and generic academic skills. Methods: Over several academic years, when second-year students solved bacteriology cases or third-year veterinary students solved epidemiology cases, they filled in a learning survey. The survey consisted of a cognitive skills assessment and a self-evaluation questionnaire related to generic skills. The cognitive assessment was divided into pre- and post-exercise sections, each comprising a logical organization test and a narrative question. The data derived from the narrative responses of the learning surveys were evaluated using the Structure of the Observed Learning Outcome (SOLO) method. A Wilcoxon signed rank test for paired groups was used for statistical analysis. Results: Between 2015 and 2018, 104 students that used the epidemiology learning tool, and between 2016 and 2020, 191 students that used the bacteriology learning tool were included in the study. Both learning tools enhanced the students’ cognitive and generic skills. Both logical reasoning and the SOLO-based evaluation of the narrative responses showed a slight but statistically significant (p from 0.001 to 0.01) improvement in cognitive skills. Of the generic skills, reasoning, problem-solving and information-processing skills improved in particular. Most students also noted an improvement in their critical thinking, creativity and continuous learning skills. Most of the students also felt that the epidemiology learning tool had taught them ethical practice skills. Conclusion: This study provides evidence that internet-based interactive cases have beneficial effects on learning outcomes in veterinary education and its results can help further develop these learning tools.

The COVID-19 (coronavirus disease 2019) pandemic has forced universities to find new ways to conduct learning and teaching, as traditional face-to-face teaching has been prevented or restricted to an absolute minimum in many instances. Therefore, we redesigned and taught second-year veterinary student microbiology laboratory exercises (labs) with a hybrid learning approach. For this, a novel ‘remote partner’ model was implemented in which students present on-site in the laboratory worked synchronously pairwise with their remote partner present online. A student feedback survey revealed that in this remote partner model, both on-site and online participation in the labs were experienced as being useful in improving their laboratory skills. The students' overall performance in hands-on microbiological laboratory skills and safe working practices was similar in the hybrid learning approach (the 2021 class) and in the traditional on-site participation approach (the 2018–20 classes). This study suggests that the remote partner model is an effective way to acquire microbiological laboratory skills. This learning approach can be used in the non-pandemic future and/or also be applied to other fields.

Non-aureus staphylococci (NAS) are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS speciesStaphylococcus agnetis, S. chromogenes,andS. simulans, and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of fourS. aureusisolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searchedin silico. We compared the presence of putative virulence factors in the NAS species andS. aureusand evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical). The NAS isolates had much less virulence gene orthologues than theS. aureusisolates. One third of the virulence genes were detected only inS. aureus. About 100 virulence genes were present in allS. aureusisolates, compared to about 40 to 50 in each NAS isolate.S. simulansdiffered the most. Several of the virulence genes detected among NAS were harbored only byS. simulans, but it also lacked a number of genes present both inS. agnetisandS. chromogenes. The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or subclinical), indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis.

Lactobacillus ruminis, an autochthonous member of the gastrointestinal microbiota of humans and many animals, is a less characterized but interesting species for many reasons, including its intestinal prevalence and possible positive roles in host–microbe crosstalk. In this study, we isolated a novel L. ruminis strain (GRL1172) from porcine feces and analyzed its functional characteristics and niche adaptation factors in parallel with those of three other L. ruminis strains (a human isolate, ATCC 25644, and two bovine isolates, ATCC 27780 and ATCC 27781). All the strains adhered to fibronectin, type I collagen, and human colorectal adenocarcinoma cells (HT-29), but poorly to type IV collagen, porcine intestinal epithelial cells (IPEC-1), and human colon adenocarcinoma cells (Caco-2). In competition assays, all the strains were able to inhibit the adhesion of Yersinia entercolitica and enterotoxigenic Escherichia coli (ETEC, F4+) to fibronectin, type І collagen, IPEC-1, and Caco-2 cells, and the inhibition rates tended to be higher than in exclusion assays. The culture supernatants of the tested strains inhibited the growth of six selected pathogens to varying extents. The inhibition was solely based on the low pH resulting from acid production during growth. All four L. ruminis strains supported the barrier function maintenance of Caco-2 cells, as shown by the modest increase in transepithelial electrical resistance and the prevention of dextran diffusion during co-incubation. However, the strains could not prevent the barrier damage caused by ETEC in the Caco-2 cell model. All the tested strains and their culture supernatants were able to provoke Toll-like receptor (TLR) 2-mediated NF-ĸB activation and IL-8 production in vitro to varying degrees. The induction of TLR5 signaling revealed that flagella were expressed by all the tested strains, but to different extents. Flagella and pili were observed by electron microscopy on the newly isolated strain GRL1172.

Sortase-dependent surface pili (or fimbriae) in Gram-positive bacteria are well documented as a key virulence factor for certain harmful opportunistic pathogens. However, it is only recently known that these multi-subunit protein appendages are also belonging to the “friendly” commensals and now, with this new perspective, they have come to be categorized as a niche-adaptation factor as well. In this regard, it was shown earlier that sortase-assembled piliation is a native fixture of two human intestinal commensalics (i.e., Lactobacillus rhamnosus and Bifidobacterium bifidum), and correspondingly where the pili involved have a significant role in cellular adhesion and immunomodulation processes. We now reveal that intestinal indigenous (or autochthonous) Lactobacillus ruminis is another surface-piliated commensal lactobacillar species. Heeding to in silico expectations, the predicted loci for the LrpCBA-called pili are organized tandemly in the L. ruminis genome as a canonical fimbrial operon, which then encodes for three pilin-proteins and a single C-type sortase enzyme. Through electron microscopic means, we showed that these pilus formations are a surface assemblage of tip, basal, and backbone pilin subunits (respectively named LrpC, LrpB, and LrpA) in L. ruminis, and also when expressed recombinantly in Lactococcus lactis. As well, by using the recombinant-piliated lactococci, we could define certain ecologically relevant phenotypic traits, such as the ability to adhere to extracellular matrix proteins and gut epithelial cells, but also to effectuate an induced dampening on Toll-like receptor 2 signaling and interleukin-8 responsiveness in immune-related cells. Within the context of the intestinal microcosm, by wielding such niche-advantageous cell-surface properties the LrpCBA pilus would undoubtedly have a requisite functional role in the colonization dynamics of L. ruminis indigeneity. Our study provides only the second description of a native-piliated Lactobacillus species, but at the same time also involves the structural and functional characterization of a third type of lactobacillar pilus.

Staphylococcus aureus is a major causative agent of mastitis in dairy cows. The pathogenicity of S. aureus may vary; it is able to cause severe clinical mastitis, but most often it is associated with chronic subclinical mastitis. Here, we present the genome assemblies of four S. aureus strains from bovine mastitis.

Adhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) –layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins.

Background: Coagulase-negative staphylococci (CNS) cause usually subclinical or mild clinical bovine mastitis, which often remains persistent. Symptoms are usually mild, mostly only comprising slight changes in the appearance of milk and possibly slight swelling. However, clinical mastitis with severe signs has also been reported. The reasons for the differences in clinical expression are largely unknown. Macrophages play an important role in the innate immunity of the udder. This study examined phagocytosis and killing by mouse macrophage cells of three CNS species: Staphylococcus chromogenes (15 isolates), Staphylococcus agnetis (6 isolates) and Staphylococcus simulans (15 isolates). Staphylococcus aureus (7 isolates) was also included as a control. Results: All the studied CNS species were phagocytosed by macrophages, but S. simulans resisted phagocytosis more effectively than the other CNS species. Only S. chromogenes was substantially killed by macrophages. Significant variations between isolates were seen in both phagocytosis and killing by macrophages and were more common in the killing assays. Significant differences between single CNS species and S. aureus were observed in both assays. Conclusion: This study demonstrated that differences in the phagocytosis and killing of mastitis-causing staphylococci by macrophages exist at both the species and isolate level.

Lactobacillus brevis ATCC 8287 possesses a surface (S)-layer protein SlpA, the gene of which is very efficiently expressed. To study the expression signals of the slpA gene, several different reporter plasmids, based on the low-copy-number vector pKTH2121 derived from pGK12, were constructed. In the reporter plasmids, only one of the two consecutive slpA promoters (P1, P2) was placed upstream of the Lactobacillus helveticus proline iminopeptidase (pepI) gene, and defined parts of the sequences upstream of the promoter were deleted. As indicated by reporter enzyme activities, both promoters were efficiently recognized at different growth stages in L. brevis. An upstream region important for the full activity of P1 was identified. The quantification of pepI-specific mRNA in L. brevis and SDS-PAGE indicated that slpA expression is not regulated at the post-transcriptional level and revealed no regulation of slpA promoters under the conditions tested. The high expression levels of both slpA and the reporter gene in L. brevis were found to remain at a high level after the addition of bile or pancreatin in the growth medium or after a change of the carbon source, which is advantageous for the potential use of SlpA as a carrier in live oral vaccines.

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group. Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179–435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins. Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.