Eläinlääketieteellinen tiedekunta


Recent Submissions

  • Viitanen, Sanna (Helsingin yliopisto, 2017)
    Bacterial pneumonia (BP) is an acquired inflammation of the lower airways and lung parenchyma secondary to bacterial infection. BP is difficult to induce experimentally in healthy dogs; the pathogenesis is therefore considered complex, involving several underlying mechanisms. BP was first described in dogs decades ago, but it is still one of the most common systemic bacterial infections in dogs, with a significant morbidity and mortality. Several aspects of BP, including the applicability of inflammatory biomarkers in its diagnosis and follow-up as well as the role of respiratory viruses in its clinical picture and development, warrant further studies. This thesis aimed to describe clinical findings during the disease and recovery periods in dogs with BP and to evaluate the applicability of acute-phase proteins as diagnostic and follow-up markers in BP. The prevalence and role of viral co-infections in dogs with BP were also investigated. We evaluated the diagnostic applicability of serum C-reactive protein (CRP) and noted that CRP is significantly elevated in BP relative to dogs with other lower respiratory tract diseases, such as chronic bronchitis, bacterial tracheobronchitis, canine idiopathic pulmonary fibrosis, and eosinophilic bronchopneumopathy, as well as in cardiogenic pulmonary edema. Our results indicate that serum CRP concentration may be used as an additional biomarker in the diagnosis of canine BP. Serum CRP, serum amyloid A (SAA), and haptoglobin (Hp) were followed during the disease and recovery periods. The follow-up study showed that serum CRP and SAA reflected well the recovery process and declined rapidly after initiation of successful therapy and could therefore be used as markers of treatment response in dogs with BP. Currently, markedly longer antibiotic courses are recommended in dogs with BP than in humans with pneumonia. Since serum CRP is a sensitive inflammatory biomarker, it was hypothesized that normalization of serum CRP could be used as an indicator for the cessation of antimicrobial therapy. In our study, we treated a group of dogs according to conventional recommendations. In another group, antimicrobial therapy was ended 5-7 days after CRP normalization. When the normalization of CRP was used to guide antimicrobial therapy, treatment length was significantly reduced without increasing the number of relapses. According to these results, normalization of serum CRP may be applied to guide the length of antimicrobial therapy in dogs with BP. Respiratory viruses, primarily canine parainfluenza virus, were found frequently in lower respiratory tract samples in dogs with BP. This indicates that viruses may play an important role in the etiology and pathogenesis of BP. Viral co-infections did not affect disease severity or clinical variables. Our findings add new knowledge about the natural course of BP as well as about the possible applications of acute phase protein measurements in the diagnosis and follow-up of BP. The utilization of acute phase protein measurements may allow a more precise diagnosis of BP, enable the early identification of patients with a poor response to treatment, and diminish the use of antimicrobial drugs.
  • Cheng, Jing (Helsingin yliopisto, 2016)
    The interactions between a host and his/her microbiota have co-evolved over time and they exert profound effects on each other. Intestinal microbiota has been linked with a number of diseases, such as irritable bowel syndrome; it is considered to be a major etiopathological factor since it can alter intestinal homeostasis. However, the role of intestinal microbiota, especially commensals, is unclear in celiac disease. To date, most efforts for detecting potential microbial changes affected by celiac disease have focused on adult individuals and have examined fecal materials, although it is known that early life is the critical period for the microbiota to colonize and establish their niche in the human intestine. At this time in healthy individuals, there is continuing cross-talk with the host e.g. via the immune system, leading to the establishment of homeostasis in both metabolic and immunological programming. Since the intestinal epithelium is the main interface for host- microbe interactions, the role of mucosa-associated microbiota may be distinct from that of fecal microbiota, but both the normal fluctuations in intestinal microbiota and the composition of duodenal mucosa-associated microbiota are still not fully clarified. The aims of thesis were to characterize the development and stability of intestinal microbiota in healthy young children and to compare the microbial features between children and adults. Furthermore, the aim was to investigate host-microbe interactions in celiac disease by studying duodenal mucosa-associated microbial signatures and mucosal gene expression in healthy children and their counterparts with celiac disease. The microbiota profiles were characterized by using the human intestinal tract chip (HITChip), which is a bacterial phylogenetic microarray. The amounts of Bifidobacterium spp. in children and adults were verified with real time qPCR. The levels of mucosal gene expressions were quantified with reverse transcriptase quantitative PCR. The results showed that intestinal microbiota is not fully matured at the age of five in children. A common core microbiota, including several butyrate-producing bacteria, was identified in children and it was developing towards core microbiota found in adults. The different progression pattern of major bacterial taxa may reflect the physiological development steps in children. Moreover, differences were observed between healthy- and celiac disease- associated microbial signatures. The differences may reflect changes in epithelial integrity associated with the disease. On the other hand, the studies on both microbiota and mucosal gene expression indicated that the persistently enhanced Th1 type immune responsiveness in subjects with celiac disease after treating with gluten-free diet might result from the increased expression of TLR9, which recognizes unmethylated CpG motifs in bacterial DNA via the direct stimulation of immune cells and/or intestinal epithelial cells. The results of this thesis project suggest that specific symbiotic and dysbiotic microbial signatures may provide potential functional diagnostic or therapeutic targets for promoting healthy/natural microbiota development. Long-term studies in a controlled environment with an adequate number of participants will be necessary to decode the disturbed microbial signatures. These trials should be combined with systematic pathological surveillance to reveal how the changes in the microbiota influence the onset of disease.
  • Viljamaa-Dirks, Satu (Helsingin yliopisto, 2016)
    Crayfish plague is a severe disease of European crayfish species and has rendered the indigenous crayfish populations vulnerable, endangered or even extinct in the most of Europe. Crayfish plague is caused by an oomycete Aphanomyces astaci, a fungal-like water mould that lives its vegetative life in the cuticle of crayfish and infects other crayfish by producing zoospores. Zoospores swim around for a few days in search of crayfish, and when they find one they attach onto its surface, encyst and germinate to start a new growth cycle as new growing hyphae penetrate the crayfish tissues. Unrestricted growth of A. astaci leads to the death of the infected animal in just a few weeks. Crayfish plague induced mortalities started in Italy around 1860. Although the disease was known about since 1860 its cause remained unknown for several decades. Little was done to prevent the spread of the disease. A lively crayfish trade probably facilitated the spread of the crayfish plague, which reached Finland in 1893. The crayfish plague has remained the most important disease problem of the Finnish noble crayfish Astacus astacus, since then. The consensus was that the disease killed all infected animals in a short time, and it appeared almost impossible to restore the flourishing crayfish populations to the levels that existed before. Following the example of neighbouring Sweden, a North American crayfish species, the signal crayfish Pacifastacus leniusculus that appeared resistant to crayfish plague was introduced to Finland in 1960s. As expected, the signal crayfish slowly started to replace the lost populations of the noble crayfish to become an important part of the crayfish fisheries. The introduction of the signal crayfish significantly added to the management problems of the noble crayfish stocks left. Signal crayfish appeared to be a chronic carrier of the crayfish plague agent, and spread the disease to the dwindling vulnerable noble crayfish populations. Later research showed that the crayfish plague agent is a parasite of North American crayfish that in normal circumstances does not harm the host animal. Intriguingly, the crayfish plague agent carried by the signal crayfish, genotype Ps1, is different from the pathogen originally introduced into Europe, genotype As. The diagnosis of crayfish plague especially when based on the isolation of the pathogen is challenging and accordingly the genotype difference was mostly unrecognized until recently. In this study we determined the genotype of the causative agent from most of the detected Finnish crayfish plague cases between 1996 -2006. It appeared that most of the epidemics in the immediate vicinity of signal crayfish populations were caused by genotype Ps1, whereas genotype As was more prevalent in the noble crayfish areas. Interestingly, a difference was seen in the outcome of the infection. The Ps1 infection was always associated with acute mortalities, while As infections were also frequently found in existing but weak populations. The persistent nature of an As infection could be verified in noble crayfish from a small lake in southern Finland. This finding explained why many of the efforts to introduce a new noble crayfish population into a water body after a crayfish plague induced mortality were futile. The main conclusion from the field study data of this research was the difference in virulence between the Ps1 and As genotype strains. This was also verified in a challenge trial with noble crayfish. While the Ps1 strains did not show much variation in their growth behaviour or virulence, there was much more variation in the As strains. The As genotype arrived in Finland more than 100 years ago, and since that date it seems to have adapted to the novel host, the noble crayfish, to some extent. In order to gain insight into a possible vector of this genotype, we studied another North American crayfish species present in Europe, the spiny-cheek crayfish Orconectes limosus from a Czech pond. This crayfish species appeared to carry a novel genotype of A. astaci, named Orconectes genotype, designated Or . It seems possible that many of the North American crayfish species carry their own type of crayfish plague agent, with variable features such as virulence. These differences should be further tested in the future. The results of this study alleviate the necessity to study the noble crayfish mortalities for the verification of crayfish plague, including the study for the genotype of the A. astaci strain. Crayfish fisheries and conservation management decisions should not be made without a prior control of the donating population and the receiving water body for the eventual presence of a low-virulent A. astaci.
  • Kaartinen, Johanna (Helsingin yliopisto, 2016)
    The alpha-2 agonist, medetomidine (MED), and its pure active enantiomer dexmedetomidine (DMED), are in clinical use for small animal practice as potent sedatives, analgesic agents, muscle relaxants, and as an adjunct agents for balanced anaesthesia. However, their cardiovascular effects limit their use. The use of constant rate infusion (CRI) for administration of MED was studied in order to provide the sedation and analgesia while decreasing the cardiovascular adverse effects. The second avenue of investigation performed was addition of the peripheral alpha-2 antagonist, MK-467, to limit the haemodynamic effects of MED. The cardiovascular effects of MED CRI were investigated in a dose finding study. Six dose levels were administered during general anaesthesia from very low dose until a high, positive control dose in order to quantify dose-dependency. In order to elucidate an appropriate agonist-antagonist dose ratio a step-down infusion protocol of MED and addition of step-up infusion protocol of MK-467 in anaesthetised dogs was performed. The effects and interaction of both drugs during absorption, and distribution phase were studied during an intramuscular study protocol using a co-administration of both drugs, where three doses of MK-467 were investigated. Plasma concentration measurements provided pharmacokinetic parameter estimates. MED-CRI administration showed promising results, in demonstrating the dose-dependency of the cardiovascular effects. With the low doses of MED CRI, the adverse effects may be minimised, although not completely avoided. A complex pharmacokinetic and pharmacodynamic interaction in between the two molecules was revealed; after intramuscular (IM) co-administration of both drugs the absorption of MED was accelerated by the addition of MK-467 to the treatment. The step infusions revealed that MK-467 also increased the elimination of MED. The optimal dose ratio finding is complicated as the context in which the drugs are given (IM, IV, CRI, under general anaesthesia etc.) affects the disposition of MED. The combined pharmacokinetic and dynamic results provide good initial pharmacokinetic estimates for the future PK-PD modelling to predict the interaction of these two drugs, MED and MK-467, in dogs.
  • Tähkäpää, Satu (Helsingin yliopisto, 2016)
    After a series of food incidents in the 1990s, the food business sector has become one of the most heavily regulated sectors in the European Union (EU), with ever-evolving regulations regarding both official food control and food business operators (FBOs). The regulatory framework is meaningless if the regulation is not implemented promptly according to transitional provisions and in a unified way. Dissenting implementation of food safety legislation may also endanger the equal treatment of FBOs and the principle of free trade of foodstuff in the EU. This research provides a new perspective on the challenges of implementing food legislation, with the phenomenon surveyed from the viewpoints of both control officials and FBOs. Resources and organization of food control affect actual control work and were thus included. Varying ways of reporting and handling food frauds on the local, national and EU levels were also investigated. Fulfilling food control requirements set in food legislation necessitates an adequate quantity and quality of personnel, whereas organization of food control can differ between countries or areas depending on socio-economic and political factors. In Finland, municipalities alone or as a joint control unit are responsible for local food control in their respective areas. According to the results, this may lead to varying implementation and interpretation of food legislation, endangering equal treatment of FBOs. There is an alarming shortage of food control personnel in some regions in Finland. Even when food safety is the responsibility of FBOs, scarce resources in food control result in a lower percentage of approved in-house control systems among FBOs. This research revealed a connection between the number of approved in-house control systems and the number of reported food- or waterborne outbreaks in the area, especially in regions with inadequate food control resources. EU legislation concerning quality systems, food control plans and food control fees are implemented in Finland at regionally different time points and with different contents directly influencing FBOs in regionally variable ways. Control officials support larger control units, with the rationale that they will increase equal treatment of FBOs. Both control officials and FBOs have problems in implementation of food legislation, and FBOs are also challenged with varying interpretation of legislation and requirements of control officials. As food safety is the responsibility of the FBOs, they need to understand and carefully comply with legislation. The challenges of fish and meat FBOs in implementation of legislation were therefore evaluated. According to this study, the most common problems concerning food safety legislation are related to layout of production premises and transport routes, control fees, requirements concerning in-house control and structures and maintenance of premises. Risk evaluation is problematic for both control officers and FBOs. Traditional food control measures are challenged, when requirements set by law are intentionally violated for financial gain by FBOs, with food deliberately placed on the market with the intention of deceiving the consumer (food fraud). Uniform methods to detect and report food fraud are needed. Hence patterns of food frauds published in the EU Rapid Alert System for Food and Feed (RASFF) in 2008-2012, recalls of notifications published by the Finnish Food Safety Authority Evira in 2008-2012 and local Finnish food fraud cases in 2003-2012 were analysed. Patterns of food fraud and manners of reporting frauds at the local, national and EU levels differ significantly. If the detection and reporting of frauds and the legal consequences incurred by FBOs for frauds differ among member states, it may create distortion of competition.
  • Läikkö-Roto, Tiina (Helsingin yliopisto, 2016)
    The primary legal responsibility for ensuring food safety in the European Union lies with food business operators. However, official controls shall also be implemented to ensure that food handling complies with the relevant requirements. Level of food safety is thus affected by several factors: the appropriateness of legislation in order to achieve food safety, the compliance of food businesses with legislative demands, and the efficacy of official food controls in verifying and enforcing compliance. The main objective of this work was to examine the factors behind efficacious local official food controls and the possibilities for improving the efficacy of the controls at different levels of the food control chain in Finland. The second objective was to investigate the consistency and quality of the local official food controls and ways to enhance these. To achieve these aims, studies were conducted on four different levels of the food control chain, i.e. level of food business operators, level of official inspecting staff, level of management for the official inspecting staff, and level of auditing of official food controls. Businesses that both prepared and served foods (in this work restaurants or restaurant business operators ) were chosen as representatives of food businesses. Positive correlations were found between the hygiene knowledge of restaurant business operators, their attitudes towards official food control, and the hygiene level of their operations. Proper justification of control measures used by food control officials, provision of guidance, and a negotiative approach in tasks of official food controls appear to be highly important for improving hygiene in food establishments. Several factors related to the food control official and the working unit of the official may affect the inspection processes and the efficacy of controls. The use of checklists and templates for inspection reports were noted to enhance the consistency and efficacy of controls. The templates also reduced the time used in preparing inspection reports and increased the quality of these reports. Time limits for correcting non-compliances had a significant impact on the efficacy of controls. Food control units had created adequate working conditions by providing their staff with guidance papers, templates, and possibilities to collectively hold discussions. However, poor orientation of new staff, non-systematic utilization of tacit knowledge through converting it to explicit knowledge and sharing it, and incomplete commitment among staff to quality systems remain challenges in the units. Insufficient human resources and the inability of heads of food control units to recognize problems in the workplace setting may impair the functional capacity of units. Poor workplace atmosphere and weaknesses in organization of work may be reflected in lesser appreciation of food businesses operators towards official food controls. Perceptions of the auditors (regional officials) and of the auditees (municipal officials) differed greatly regarding the adequacy of the auditing system. The regional officials had experienced the auditing visits as clearly more useful and positive than the municipal officials and also found the current auditing system to be more suitable for the purpose. The regional officials did, however, state that the auditing results had not been adequately utilized in planning the guidance and education of professionals working in official food control. Based on the results of this work, certain weaknesses exist in the efficacy and consistency of local official food controls in Finland. However, several means to improve the efficacy and consistency of the controls were identified on all studied levels of the food control chain. Some of the observed impact possibilities, such as using checklists during inspections and using templates for inspection reports, are relatively simple to implement. Other measures, such as fully implementing risk-based procedures during inspections and more systematic utilization of the tacit knowledge that is present among the official food control staff, would require a substantial amount of time and effort of the food control authorities.
  • Palander, Pälvi Avilla (Helsingin yliopisto, 2016)
    Tail biting in pigs is a phenomenon which has been known since the 19th century. The prevalence of tail biting damage is estimated to be between 3 10 % of pigs slaughtered, being somewhat higher in non-docked than tail-docked populations. Being tail bitten causes pain and a state of stress to the victim pigs, as well as general disturbance in pig groups. The aim of this thesis was to improve animal welfare by generating new knowledge for resolving the multifactorial mystery of tail biting behaviour. The precise etiology of tail biting behaviour is unknown, although many risk factors have been identified. The most supported theory of tail biting involves lack of opportunities for pigs to fulfil their innate need for exploration and foraging behaviours in modern production environments. Both these behaviours are mainly performed orally. Deficiencies in the nutritional state of the animal can increase the motivation for foraging behaviour. The three studies presented within this thesis used a case-control design, with questionnaires to investigate feeding and diet related, and environmental risk factors for tail biting damage at a population level, and animal studies to search for differences in intestinal cell wall structure, nutritional state, blood metabolites and different brain area concentrations of serotonin and dopamine and their metabolites between tail-biter pigs, victim pigs, control pigs in tail biting pens and control pigs in non-biting pens at the individual level. Results from the population level study identified environment-, feeding- and management-related risk factors that were similar to those reported in earlier published epidemiological studies of both long-tailed and tail-docked pigs. Interactions between different types of risk factors were depicted. Both the etiologies of tail biting which originates from exploratory and foraging behaviour can be supported by the risk factors found here. The similarities between the risk factors for tail biting and gastric ulceration needs more attention in future studies. Tail biting pigs were found to have elevated serotonin metabolism in the prefrontal cortex (PFC). The serotonin precursor, tryptophan, and its ratio to other amino acids in blood, correlated with serotonin metabolism in the PFC. Experience of stress is suggested to explain the findings in the brain. The influence of enhanced serotonin turn-over on amino acid metabolism needs further study. Tail bitten pigs were found to have decreased concentration of several non-essential amino acids in blood, and a higher ratio of metabolites of serotonin and dopamine in the striatum and in the limbic cortex. These findings are possibly associated with the experience of stress from being bitten, and a change in protein metabolism arising from the acute phase reaction. In a pen where tail biting was present, it was not only the biters and bitten pigs that were affected by this, but also the control pigs. Control pigs were found to have reduced intestinal villus height and changes in blood nutrient concentrations that indicate short term malnutrition, possibly resulting from changes in feed intake.
  • Kilpinen, Susanne (Helsingin yliopisto, 2016)
    The term tylosin-responsive diarrhea (TRD) stands for canine chronic, idiopathic, recurrent diarrhea that responds repeatedly to tylosin treatment. A specific feature of TRD is that diarrhea ceases within a few days of initiating tylosin treatment and the stool remains normal for as long as treatment continues. After discontinuation of tylosin, diarrhea reappears in many dogs within a short time. When tylosin is reintroduced, its effect does not diminish, even with numerous treatments. Despite its wide acceptance as a treatment for canine chronic enteropathies, few published studies have assessed the efficacy of tylosin treatment in chronic diarrhea disorders in dogs. To evaluate the efficacy, a prospective, placebo-controlled, double-blinded, randomized clinical trial was performed. The proportion of dogs with normal fecal consistency at the end of treatment was 85% in the tylosin group and 29% in the placebo group. Tylosin at a dosage of 25 mg/kg once daily for seven days was effective, compared with placebo, in treating diarrhea in TRD dogs. No changes specific to TRD were detected in clinical, laboratory, or histopathologic examinations. In this study, TRD had affected mainly middle-aged, large-breed dogs, and the diarrhea was of a mixed small and large intestinal pattern. Diarrhea recurred in 88% of the diagnostically confirmed TRD dogs at a median of day 9 after discontinuation of tylosin. Diarrhea ceased at a median of 2-3 days after reintroduction of tylosin at three different dosages. Tylosin lacks official oral dosage recommendations and an optimal treatment strategy has not been established. The results showed that 93% of the dogs included in the trial, suffering from recurrent diarrhea and previously responding to oral tylosin therapy at a dose of 25 mg/kg once daily for seven days, responded also to doses of 5 and 15 mg/kg once daily for seven days after diarrhea relapse. The exact mode of action contributing to the cessation of recurrent diarrhea in dogs after tylosin administration remains obscure. Known pathogenic bacteria have previously been excluded as the underlying cause of TRD. Therefore, the antibacterial effect of tylosin may play a minor role in terminating recurrent diarrhea, and the immunomodulatory properties of tylosin could explain the favorable effect. These could be mediated by a shift of intestinal microbiota towards potentially beneficial commensal bacteria. Data revealed that tylosin administration resulted at the time of cessation of diarrhea in a significant increase in the fecal levels of Enterococcus spp. of TRD dogs compared with levels during the diarrhea period following discontinuation of tylosin. Cessation of diarrhea in TRD dogs with tylosin treatment could thus be mediated by selection of a specific intestinal lactic acid population, the Enterococcus spp.. To conclude, this thesis provides evidence-based data that tylosin is an effective treatment of chronic, idiopathic, recurrent diarrhea in dogs. The results suggest that an optimal treatment strategy for TRD is to start an empirical treatment trial with tylosin at a dosage of 25 mg/kg once daily for seven days. In case of a relapse after discontinuation of tylosin, the dose could be tapered down to 5 mg/kg once daily at the time of reintroducing tylosin. Further, the findings provide new insight into the possible mechanism behind the cessation of chronic diarrhea. The probiotic potential of some enterococci strains is known and these enterococci may possess properties that attenuate inflammation in the gut mucosa and normalize fecal consistency. They may thus provide an alternative to the long-term use of antimicrobials in the treatment of TRD in dogs.
  • Rasinkangas, Pia (Helsingin yliopisto, 2016)
    The present thesis studied the biogenesis of the pili of Lactobacillus rhamnosus GG, and examined whether pili influence the in vivo persistence and the ability to modulate faecal microbiota in mice and humans. L. rhamnosus GG is one of the most extensively studied lactic acid bacteria, and it is commonly present in so-called probiotic products. Heterotrimeric proteinaceous pili, which are able to bind for example to mucus, collagen and β-lactoglobulin, have been found and characterised on the surface of L. rhamnosus GG during recent years. Pili are formed by the major pilin SpaA, which forms the shaft of the pilus structure, the mucus-binding tip pilin SpaC and basal pilin SpaB. The pili are synthesised on the cell membrane by the pilin-specific transpeptidase sortase C (SrtC), and attached to the cell wall by housekeeping transpeptidase sortase A (SrtA). As the pilus is the major mucus adhesin of L. rhamnosus GG and, according to various studies, a crucial factor in the L. rhamnosus GG adherence and signalling to the host, it was deemed important to characterise the pilus biogenesis pathway and the functions of the produced pili. Random chemical mutagenesis was used to obtain derivatives of L. rhamnosus GG with variable pilus production capacities to study factors affecting pilus biogenesis. Enrichment schemes for the isolation of the derivatives were devised, and consequently 10 pilus-deficient, 13 highly adherent and one pilus-secreting derivative were characterised. Genotypic and phenotypic characterisation of the derivatives revealed that a functional SrtC is essential for pilus biogenesis, as well as the sufficient expression of the major pilin SpaA. Additionally, the role of the insertion sequence-rich genomic region, containing also the pilus gene cluster, in the adaptation of L. rhamnosus GG to varying environments was revealed, as several derivatives were obtained in which the region was deleted. The adhesion capacity of L. rhamnosus GG was found to be increased either due to a mutation in the tip pilin SpaC or as a result of increased pilin production. One of the highly adherent derivatives with high pilin production harboured a mutation in the SpaA pilin, likely increasing the derivative s capacity to secrete this pilus backbone-forming subunit. In addition, a pilus-secreting derivative was characterised, and noted to harbour a mutated housekeeping sortase SrtA, demonstrating its essential role in the attachment of pili to the cell wall. Bioinformatic analysis revealed that the C-terminal sortase recognition motif of pilins, with the sequence LPxTG, was variable between the proteins recognised by pilin-specific transpeptidase SrtC and housekeeping transpeptidase SrtA. This was found not only for the pilins of L. rhamnosus GG, but also for pilins of other sortase-dependent pili-harbouring Gram-positive bacteria, indicating a role for this motif in the regulation of pilus biogenesis. In a subsequent functional characterisation of L. rhamnosus GG pilins, it was demonstrated that SrtC indeed recognised the shaft pilin SpaA through a conserved triple glycine (TG) motif, and was likely to recognise also the tip pilin SpaC, which harbours the same motif. The basal pilin SpaB, containing a single glycine (SG) motif, was recognised by SrtA, leading to the attachment of pilus to the cell wall. Finally, the in vivo effect of the L. rhamnosus GG pili was evaluated in intervention trials in mice and humans. By performing mouse and human intervention trials with one of the described pilus-deficient derivatives, L. rhamnosus GG-PB12 and the parental strain, it was possible to demonstrate that the presence of pili increased the persistence of L. rhamnosus GG in the gastrointestinal tract and led to significant changes in the faecal microbiota composition, especially in humans. Species richness increased significantly due to the consumption of piliated L. rhamnosus GG in the human trial, indicating potential beneficial effects for the host, as a high species richness of gut microbiota has been associated with stability and resilience towards exogenous species. In conclusion, the research described here provides new insights into pilus biogenesis in L. rhamnosus GG and highlights the importance of the pili in the in vivo adherence capacity and gut microbiota modulation ability of L. rhamnosus GG.
  • Tolvanen, Riina (Helsingin yliopisto, 2016)
    In this thesis contamination routes of L. monocytogenes were examined in a food establishment, the survival of L. monocytogenes strains was studied in dry-fermented sausages prepared using two different starter cultures, the acid and heat tolerance of L. monocytogenes strains were studied, and efficacy of ultrasonic cleaning was tested on conveyor belts contaminated with L. monocytogenes. Contamination routes of L. monocytogenes were examined during an 8-year period in a chilled food-processing establishment that produced ready-to-eat meals using amplified fragment length polymorphism (AFLP) analysis. Compartment I of the establishment, producing cooked meals, was heavily contaminated with three persistent AFLP types, and compartment II, producing uncooked chilled foods, was contaminated with persistent and non-persistent AFLP types. L. monocytogenes was isolated only once from compartment III. The persistent contamination appears to be influenced by the cleaning routines, product types and lack of compartmentalisation in facilities producing cooked meals. The reconstruction of the production line in compartment II resulted in the elimination of two persistent AFLP types. The survival of five L. monocytogenes strains was studied in dry-fermented sausages prepared using two different starter cultures with or without a bacteriocin-producing Lactobacillus plantarum DDEN 2205 strain. L. monocytogenes was detected throughout the ripening process in sausages containing no bacteriocin-producing strain. The use of both starters with bacteriocin-producing culture resulted in L. monocytogenes-negative sausages after ripening. Two of the L. monocytogenes strains survived in sausages with bacteriocin-producing cultures better than the other strains. Bacteriocin-producing strains provide an appealing hurdle in dry sausage processing, but differences in survival of L. monocytogenes strains require the use of other hurdles as well. The acid and heat tolerance of persistent and non-persistent L. monocytogenes strains were studied. L. monocytogenes strains exhibited large variation in both acid and heat tolerance. The persistent strains exhibited higher tolerance to acidic conditions than the non-persistent strains, but significant differences in heat tolerance between persistent and non-persistent strains were not detected. Due to the great differences in acid and heat tolerances between L. monocytogenes strains, preventive measures should be designed to be effective against the most tolerant strains. Ultrasonic cleaning was tested on three conveyor belt materials contaminated with L. monocytogenes strains. The ultrasonic cleaning was efficient for all materials, but the reduction of L. monocytogenes was significantly greater in stainless steel than in plastic materials. The ultrasonic cleaning was further studied by building a pilot-scale conveyor with an ultrasonic cleaning bath. The detachment of L. monocytogenes from the stainless steel conveyor belt caused by the ultrasonic treatment was significantly greater than without ultrasound. In both studies, lengthening of the treatment time did not significantly increase the detachment of L. monocytogenes. However, an increase in temperature improved the effect of the ultrasonic treatment, and 10 s at 50 °C reduced L. monocytogenes counts by more than 5 log units. These results indicate that the ultrasonic cleaning of conveyor belts is effective even with short treatment times.
  • Olkkola, Satu (Helsingin yliopisto, 2016)
    Campylobacteriosis is the most common cause of human bacterial gastroenteritis in the developed world. The most often isolated causative agent from diseased humans is C. jejuni, but also C. coli and C. upsaliensis, common colonizers of pigs and dogs, respectively, are known to cause disease. Campylobacteriosis is usually self-limiting but antimicrobial treatment is warranted in severe cases, with macrolides and fluoroquinolones being the first and second options, respectively. Intravenous aminoglycosides are indicated in Campylobacter bacteraemia. However, high rates of fluoroquinolone-resistant Campylobacter spp. have emerged in many parts of the world. Also, in several studies, high proportions of streptomycin-resistant C. coli or C. upsaliensis, have been found. Yet, the mechanisms of STR resistance have been only partially characterized in C. jejuni and C. coli and completely ignored in C. upsaliensis. The primary aim of this thesis was to investigate the molecular mechanisms of STR resistance in porcine C. coli and canine C. upsaliensis isolates. We were able to associate high level of STR resistance in porcine C. coli to mutations in the rpsL gene. In C. upsaliensis, a mutation in rpsL was also noted in all the low- and high-level STR-resistant isolates. All highly STR-resistant C. upsaliensis isolates had, in addition to the rpsL mutation, significant truncation of rsmG, encoding a conserved methyltransferase responsible for methylation of the ribosomal STR binding site. Even though STR resistance conferring mutations in rpsL and rsmG have been well documented in other bacterial species, they were first time described in Campylobacter spp. in the present study. Further, using genomics and insertional mutagenesis, a novel STR resistance-conferring gene was identified in the intermediately STR-resistant C. coli isolates. This gene is homologous, albeit at a low level, to other previously described aminoglycoside 6-adenylyltransferase encoding genes, and does not appear to originate from Gram-positive bacterial species. Based on our findings, we hypothesize that this gene could have evolved from a proto-resistance element in Campylobacter spp. Altogether these results provide a significant advance in understanding the mechanisms of STR resistance in Campylobacter spp. and will aid in predicting the phenotypic resistance from genome data. Fluoroquinolone resistance-associated mutations in the DNA gyrase-encoding gene gyrA were characterized in porcine C. coli treated with danofloxacin as well as among canine C. upsaliensis. The commonly described C257T mutation was found in both species. In C. coli this caused the amino acid change T86I in DNA gyrase and high levels of ciprofloxacin resistance, while in C. upsaliensis the predicted amino acid change was T86M causing only minor increase in CIP MIC but a high level of nalidixic acid resistance. Therefore, danofloxacin does not seem to induce novel mutations in C. coli in vivo but the same mutation appears not to be sufficient to cause a high level of fluoroquinolone resistance in C. upsaliensis.
  • Karikoski, Ninja (Helsingin yliopisto, 2016)
    Laminitis is a common, debilitating condition of equids that may have substantial effects on animal welfare. Laminitis may be related to endocrinopathic or inflammatory diseases, as well as uneven weight distribution. Until recently, research into the prevalence of endocrine disease amongst horses with laminitis has been overlooked, although it was anecdotally thought to be high. Most histological studies have been conducted on experimental carbohydrate overload or inflammatory models. However, more recently the histology of a hyperinsulinaemic model of laminitis was described with important differences apparent from the previous inflammatory models however, a more detailed study had not been performed. Furthermore, the histology of chronic, naturally occurring endocrinopathic laminitis has not been described and compared to that of healthy animals. The research presented in this thesis aimed to investigate endocrinopathic laminitis. In Study I, the prevalence of endocrine disease (either basal hyperinsulinemia or pituitary pars intermedia dysfunction, PPID) among horses that presented for laminitis at a first-opinion/referral equine hospital was shown to be 89%. In Study II, secondary epidermal lamellar elongation, narrowing and alteration in orientation were shown to be largely attributable to cell stretching in ponies with insulin-induced experimental laminitis (rather than proliferation as had been hypothesised), that occurred at the same time as an accelerated cell death-proliferation cycle. In Study III, the histomorphometry and pathological lesions of endocrinopathic laminitis were shown to be largely localized abaxially within the lamellar tissue including increased lamellar length and width, chronic abnormal keratinization, interlamellar epidermal bridging and apoptotic cell death with more acute lamellar tearing in some cases. Additionally, in all the laminitis cases, there were macroscopic pathological changes (divergent rings on the outer hoof wall and/or rotation of distal phalanx). However, both lamellar and macroscopic pathology varied in severity and were unrelated to the reported duration of laminitis. In Study IV, PPID animals with and without laminitis were compared. All the laminitic PPID animals also had hyperinsulinemia whereas all of the non-laminitic PPID animals were normoinsulinemic. In addition, all the PPID animals with laminitis had histological lesions in the lamellar region, but the lamellae of PPID animals without laminitis were normal when compared to the control group. In conclusion, endocrinopathic laminitis is by far the most common type of naturally occurring laminitis in horses and hyperinsulinemia is likely to be the primary cause in those horses, including those with PPID. In the acute phase, the pathophysiology behind the key lesion, SEL elongation, is proposed to be cellular (mechanical) compromise resulting in epidermal cellular stretching. In the chronic phase, the lamellar lesions are variable in severity in many cases allowing for a prolonged subclinical phase, and they are located predominantly abaxially close to the hoof wall.
  • Pääkkönen, Tarja (Helsingin yliopisto, 2016)
    Although the immature brain reportedly is more prone to seizure activity than the mature brain, there are no previous reports on well-defined juvenile epilepsy syndromes in dogs. This study describes a novel juvenile epilepsy syndrome in Lagotto Romagnolo (LR) dogs, namely benign familial juvenile epilepsy (BFJE). We studied the clinical characteristics of this novel syndrome in 25 affected dogs, while healthy littermates of the affected dogs served as controls. The mean age at onset of focal seizures is 6 weeks, and spontaneous remission of seizures usually occurs by the age of 4 months. Between the seizure episodes, most of the affected puppies are neurologically normal, but puppies with the most severe seizure episodes exhibit some neurological deficits interictally. These deficits also resolve with remission of seizures. Interictal electroencephalography (EEG) shows focal abnormalities, including sharp waves and spikes, in most (88%) of the affected dogs. Conventional imaging examinations, including magnetic resonance imaging (MRI), show no remarkable focal abnormalities in dogs with BFJE. Positron emission tomography (PET) is a nuclear neuroimaging modality that is able to detect abnormal metabolism in the epileptic focus of the brain. We investigated glucose metabolism of the brain in 6 affected and 5 control dogs using radiolabeled glucose, namely 2-[18F]fluoro-2-deoxy-D-glucose (FDG), as a tracer. In dogs with BFJE, FDG-PET shows areas of hypometabolism with good correspondence to focal EEG findings, thus supporting the area of abnormal metabolism being the epileptic zone. Furthermore, we performed a follow-up study by utilizing two previously validated questionnaires on impulsivity and activity levels in dogs, and additionally, we telephone-interviewed the owners of the affected dogs. We evaluated the results based on the data collected for 25 dogs with a history of BFJE and 91 control dogs. We utilized principal component analysis to explore the factorial structure of the questionnaire. Although the life span of affected dogs seems to be comparable with that of healthy control dogs and recurrence of seizures after remission is rare, the dogs with a history of BFJE exhibit abnormalities in behavior reminiscent of attention deficit hyperactivity disorder (ADHD) in humans. This study also reveals the mode of inheritance and the genetic defect behind BFJE. Based on pedigree analysis, we found that BFJE is inherited in a recessive Mendelian form. We further found that a mutation in the gene encoding for protein LGI2 is responsible for BFJE. LGI2, as well as LGI1, interacts with neuronal membrane proteins, namely ADAM22 and ADAM23, in synaptic transmission. LGI1, ADAM22, and ADAM23 have previously been shown to be important in the development of epilepsy, and this study reveals the importance of LGI2 in epileptogenesis of BFJE.
  • Popova , Dina (Helsingin yliopisto, 2015)
    Dynamic modifications of synaptic connectivity enables the brain to adequately respond to environmental challenges. This ability, known as synaptic plasticity, peaks during the early postnatal period, yet it is maintained throughout life. Interestingly, antidepressants (ADs) and AD-like drugs can promote neuronal plasticity in the adult brain, a phenomenon recently suggested to contribute to the mood-improving effects of ADs. However, the mechanisms underlying AD-induced neuronal network refinement are still poorly understood. The main goal of this thesis was to advance our understanding of the mechanisms associated with pharmacologically-enhanced plasticity in the adult brain. Two pharmacologically distinct compounds with AD-like actions, namely the selective serotonin reuptake inhibitor fluoxetine (Flx) and the volatile anesthetic isoflurane were used to enhance synaptic plasticity in the rodent cortex and hippocampus. After drug exposure, behavioral, molecular, histological and in vitro electrophysiological approaches were utilized to investigate the effects of Flx and ISO on synaptic function and plasticity. Using electrophysiological recordings in brain slices, we show that chronic Flx treatment results in increased short- and long-term plasticity as well as enhanced basal transmission in excitatory CA3-CA1 synapses in the hippocampus. These changes were paralleled by an activity-dependent enhancement in the expression of proteins related to vesicular trafficking and release, such as synaptophysin, synaptotagmin 1, mammalian uncoordinated protein 18 (Munc 18) and syntaxin 1. Moreover, Flx treatment reduced the percentage of parvalbumin-expressing GABAergic neurons, increased the expression of polysialylated-neural cell adhesion molecule (PSA-NCAM) and decreased the expression of the potassium-chloride co-transporter 2 (KCC2) in the basolateral amygdala and in the medial prefrontal cortex (mPFC). All the above findings are likely to be attributed to increased dynamic range of synaptic plasticity induced by Flx. Our behavioral findings demonstrate that long term Flx administration in combination with extinction training results in long-term loss of fearful memories while the Flx treatment alone failed to influence fear behavior. These data suggest that behavioral training is indispensable for the guidance of Flx-induced network plasticity. Exposure to isoflurane promotes long-term synaptic plasticity and enhances basal synaptic transmission in excitatory CA3-CA1 synapses in the mouse hippocampus. These changes were correlated with increased tropomyosin receptor kinase B (TrkB) signaling through the mammalian target of rapamycin (mTOR) pathway in the prefrontal cortex and hippocampus and led to rapid antidepressant-like behavioral effects in the forced swim test. Taken together, our findings highlight that Flx and isoflurane enhance synaptic plasticity in hippocampal and cortical excitatory synapses, however, the underlying molecular mechanisms as well as behavior improvements were different. In conclusion, the results described in this work provide a mechanistic background for adult brain plasticity and network tuning, with high practical significance to the design of clinical therapy.
  • Llarena, Ann-Katrin (Helsingin yliopisto, 2015)
    During the last 40 years, Campylobacter has emerged as the number one cause of human gasteroenteritis in the developed world, of which C. jejuni accounts for the majority of cases. This Campylobacter species has an extremely broad host-range, and has been isolated from arctic penguins to cattle. Broiler chickens has traditionally been considered as the reservoir of importance for public health, but evidence suggests that other source, both known and unknown, are relevant. To decrease the number of human infections, targeted control efforts to reduce C. jejuni exposure to humans are needed. Such control efforts relies on knowledge on the nature of C. jejuni in both established and candidate sources such as broilers and wild birds, respectively, and reliable methods to trace and attribute human infections. Therefore, this thesis evaluated the usefulness of three metabolic markers in source attribution, resolved a Campylobacter outbreak using whole-genome sequence and characterized the dynamics and epidemiology of C. jejuni in Finnish chickens and barnacle geese. First, the possible host association of three metabolic markers, gamma-glutamyl transpeptidase, and the genes of secretory L-asparaginase and fucose permease, was investigated by examining their distribution among isolates collected from a variety of reservoirs and human patients, and by assessing their association with C. jejuni lineages as expressed by multilocus sequence typing. According to our results, the presence and absence of these three traits were linked to the lineages, and no evidence for host association independently of population structure was found. Therefore, these metabolic markers were deemed unsuitable as the sole subtyping scheme in source attribution. Secondly, in an attempt to identify possible new sources for human campylobacteriosis, the character, dynamics, and epidemiology of C. jejuni in barnacle geese and broiler chickens were investigated by multilocus sequence typing and whole-genome sequencing. In line with other studies, the C. jejuni population in barnacle geese was specific to that host, as certain genotypes (ST-702 and ST-1034 CC) were overrepresented in the collection. Furthermore, we proved that the C. jejuni in this wild bird species generally differed from the C. jejuni population found in agricultural animals and C. jejuni infecting humans. Therefore, barnacle geese are most probably not a major source for human campylobacteriosis. Tracing zoonotic pathogens from humans to the source of infection using whole-genome sequencing is a hot topic in the research community. To provide knowledge on how to utilize whole-genome data to profit future real-time investigations of Campylobacter outbreaks, next generation sequencing was used to reinvestigate a waterborne C. jejuni outbreak. Our results revealed that the pulsed-field gel electrophoresis performed in the original outbreak investigation overestimated the clonal relationship between some of the apparently related strains. Through the reanalysis, it became clear that the outbreak was caused by at least two different strains, or an unrelated, sporadic human case was mistakenly classified as part of the outbreak. Whole-genome sequence was therefore needed to infer the correct epidemiology of the studied human infections. To increase the knowledge on C. jejuni in a well-established reservoir, we characterized nearly 90% of all C. jejuni-positive chicken flocks by multilocus sequence typing and linked this information to the isolates metadata, such as farm and collection time-points. We found that the C. jejuni population on Finnish chicken farms was dominated by one genotype (ST-45 CC). Furthermore, our statistical analysis showed that slaughterhouse and year of collection affected the C. jejuni population in chickens mildly, while season influenced the presence of only one genotype (ST-45). Furthermore, the chicken farms were sporadically and infrequently colonized by C. jejuni, as is typical when no persistent colonization source is present on the farms. This thesis utilized an array of subtyping methods on both long- and short-time scales. The results highlight that more traditional methods, such as pulsed-field gel electrophoresis, still have their use in the current genomic era, and stress the usefulness of multilocus sequence typing as a preliminary screening tool to determine the population structure in the C. jejuni collection being investigated. However, we are convinced that whole-genome sequencing will be the subtyping scheme of choice in the near future, as it provides the full resistome, toxiome and virulome concurrently with the genotyping depth of need in a single operation.