Eläinlääketieteellinen tiedekunta


Recent Submissions

  • 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.  
  • Mustonen, Eeva (Helsingin yliopisto, 2015)
    Forage legumes such as red clover (Trifolium pratense L.) are used due to their ability to fix atmospheric nitrogen. Red clover has a high crude protein content and sufficient digestible fibre. Red clover silage tends to increase dry matter intake and milk yield in dairy cows. The use of clover silage can lead to desirable changes in milk fatty acid composition, promote growth and increase live-weight gain in ewes and lambs. The literature review focuses on the principal isoflavones in red clover, their metabolism in the rumen and conjugation and excretion. The role of equol is reviewed regarding concentrations in plasma and milk. Red clover varieties studied contained isoflavones; formononetin (6.0–7.9 mg/g in dry matter (DM)), biochanin-A (3.7–6.1 mg/g in DM), genistein (0.5-0.6 mg/g in DM) and daidzein (0.2–0.30 mg/g in DM). There were significant differences in isoflavone contents. The isoflavone content was highest in cultivar Ilte, which had the highest concentration of formononetin. The effect of growing conditions (year), growth stage (harvest) and habitat (site) were significant for formononetin concentrations. The total isoflavone and formononetin concentrations were highest under poor weather conditions, at the northern location and with later harvest. In the feeding experiment, the ewes were fed red clover (RC) silage that contained on average 6.8 mg/g of formononetin in DM. Timothy/meadow fescue (Phleum pratense L./Festuca pratensis Huds.) grass silage did not contain any isoflavones. In the RC group, estimated daily intake of isoflavones was 10.5 g formononetin. The major part of formononetin ingested was metabolized, contributing to the increase of equol in the serum (average level of 7.7 mg/l). In HPLC analyses, the serum of ewes contained solely (S)-equol. The ewes of RC group gained weight faster (P<0.001) and were significantly heavier (P<0.01) at the end of the experiment. All ewes became pregnant. There were no significant differences in the time of conception, numbers or weight of foetuses or numbers of ovulations. The mass of the uterus was significantly greater (P<0.01) in the RC group compared with that in the control group. This difference was mainly explained by the increase of foetal fluids in the RC group (P<0.01). In the dairy cow feeding experiment, the RC silages contained 3.0–6.5 mg/g formononetin in DM. Grass silage did not contain any isoflavones. The formononetin contents of the red clover were highest for the shortest growthperiod. The daily intake per cow of daidzein was 1.7–2.6 g and of formononetin 27–76 g. The equol concentration in plasma was 4.6–8.4 mg/l. Intake of formononetin was strongly associated with the equol concentration in plasma (R2 0.71). Equol contents in plasma were significantly higher (P<0.01) for the RC than for the cows fed grass silage. Plasma equol contents were significantly higher (P<0.001) for fed silages, where the growth period were shortest. Equol concentrations in the milk of cows fed RC were 458–643 µg/l and daily secretion of equol in milk was estimated to be 12–19 mg/day. Intake of formononetin was only weakly associated with equol concentration in milk (R2 0.20). Equol concentrations were also analysed from organic and conventionally produced commercial milk samples. Organic skimmed milk contained 411 µg/l of equol and conventionally produced 62 µg/l. The HPLC method provided excellent accuracy and sensitivity for quantification of isoflavonoids from fodder, blood, and milk samples. (S)-equol was the main isoflavonoid detected. It is suggested that the estrogenic effects of metabolic equol in sheep reported earlier are solely or predominantly due to (S)-equol. In Finnish landrace sheep, the fecundity of ewes was not reduced by feeding RC. The volume of foetal fluids, however, increased, which could increase the risk of vaginal prolapse before term. A strong association between formononetin intake and equol concentration in plasma was demonstrated. The equol content in cow’s milk can be as high as 600 µg/l with RC silage feeding, even though only a small part of the formononetin is secreted into milk as the metabolite equol. Milk equol is derived from the formononetin of RC silage and RC-fed cows’ milk can be considered as a source of equol in human nutrition.
  • Restitutti, Flavia (Helsingin yliopisto, 2015)
    This series of investigations aimed to evaluate in dogs the interaction between MK-467, a peripheral α2-adrenoceptor antagonist with poor penetration into the central nervous system, and dexmedetomidine, a selective α2-adrenoceptor agonist commonly employed in small animal clinical practice due its potent sedative effects. The objective of this study was to find an optimal dose-ratio of dexmedetomidine and the antagonist that could attenuate or prevent major cardiovascular changes without any significant effect on the sedation induced by the agonist. The effects on blood flow in abdominal organs and on plasma concentrations of glucose, insulin, non-esterified free fatty acids, lactate and cortisol of this optimal dose were then evaluated. The sedative effects were assessed subjectively by means of a composite sedation score. Simultaneously, hypnosis was evaluated through the bispectral index. Haemodynamic parameters that were evaluated comprised cardiac output, arterial blood pressure, heart rate, central venous pressure and systemic vascular resistance. Time-intensity parameters derived from contrast-enhanced ultrasound imaging were used to assess blood flow in selected abdominal organs. Three doses of MK-467 (250, 500 and 750 µg/kg) were tested against dexmedetomidine alone (10 µg/kg). All treatments were administered IV. Sedation was significantly lower and BIS significantly higher with the medium and highest doses of MK-467 than with dexmedetomidine. However, bioequivalence between dexmedetomidine and the combination was reached with all treatments for the two parameters analysed. Early cardiovascular effects of dexmedetomidine were not completely prevented with the lowest dose of MK-467, and the highest dose reduced mean arterial pressure. The middle dose of MK-467 (500 µg/kg) provided the best cardiovascular stability. Addition of the peripheral antagonist attenuated dexmedetomidine-induced changes in organ blood flow evaluated by the CEUS. An increase in plasma glucose was observed in dexmedetomidine-treated dogs, but not when MK-467 was added. Inversely, plasma insulin concentration was reduced with dexmedetomidine, but not when dexmedetomidine was combined with MK-467. Plasma non-esterified free fatty acids concentration decreased transiently with the combination, while with dexmedetomidine alone the reduction persisted throughout the observation period. Plasma lactate concentration increased with dexmedetomidine, but not with the combination. In conclusion, the addition of MK-467 attenuated or prevented the early cardiovascular effects of dexmedetomidine, not having clinically relevant effects on the sedation induced by the latter. Some metabolic changes induced by dexmedetomidine were halted by MK-467.
  • Knuuttila, Anna (Helsingin yliopisto, 2015)
    Aleutian mink disease virus (AMDV) is a widespread parvovirus mainly affecting American mink (Neovison vison). It can cause a progressive and persistent immune complex-mediated disease (Aleutian disease, AD) in adult mink and an acute and fatal pneumonia in mink kits. The virus has a wide geographical distribution both in farmed mink and in the wild. Aleutian mink disease virus poses a major economic threat to mink farmers and it may affect the conservation and management of indigenous mustelids and other species. Infected farms are difficult to sanitize as the virus is resistant to physical and chemical treatments, it can be transmitted through several vectors and routes, and no effective medications or vaccines currently exist. Since the 1970s, diagnosis on AMDV in farmed mink has been based on the identification of specific antibodies with a counter-current immunoelectrophoresis (CIEP) test. In 2005, the Finnish Fur Breeders Association implemented an eradication program that required the development of a new AMDV-detection protocol to screen ca. 600 000 samples per year. Although AMDV can infect and may cause disease in other mustelids and carnivores, little is known about the epidemiology and evolutionary relationships of AMDV strains in the wild in Finland and elsewhere. Thus, this study aimed to develop a modern automated test for the large-scale serodiagnosis of AMDV in mink and to elucidate the epidemiology and phylogeny of this virus in farmed mink and free-ranging mustelids in Finland. A new antigen for the serological test was developed with a recombinant DNA technique. The major capsid protein (VP2) gene of a Finnish AMDV strain obtained from a farmed mink was amplified, cloned into a baculovirus transfer vector with subsequent recombination to baculovirus genome, and expressed in insect cells. The antigen formed virus-like particles and was confirmed to be antigenic with several serological methods. Subsequently, an enzyme-linked immunosorbent assay (ELISA) was designed for the antigen and automated. Because the small glass capillaries used to collect blood samples in CIEP could not be utilized in the ELISA test, a wicking technique using a filter paper blood comb was developed. The performance of this test was compared to CIEP (an imperfect gold standard) by testing blood/serum samples from farmed mink. The results were analyzed with Bayesian modelling allowing for conditional dependence. The new automated ELISA test was found to be accurate with a diagnostic sensitivity of 96.2% (95% probability interval [PI], 91.5 99.0) and specificity of 98.4% (95% PI, 95.3 99.8), and was therefore determined suitable for the serodiagnosis of AMDV. The epidemiology and phylogenetics of AMDV were inferred from organ and/or blood samples from farmed mink and free-ranging mustelids. The samples were screened with the newly-developed ELISA (described above) or CIEP test for anti-AMDV antibodies and with previously described or newly-developed PCR assays for AMDV DNA. Test results were studied with statistical, phylogenetic, and sequence analysis methods. Aleutian mink disease virus was found to be prevalent in the wild in Finland. A new host species, the European badger (Meles meles), with a prevalence of 27% (7/26; 95% confidence interval [CI], 13 46), was identified. In addition to the badger, infection markers were found in 54% (31/57; 95% CI, 42 67) of feral American mink and in one European polecat (Mustela putorius) (1/14; 95% CI, 1 29). No infection was found in Eurasian otters (Lutra lutra) (24; 95% CI, 0 10), European pine martens (Martes martes) (183; 95% CI 0 1), least weasels (Mustela nivalis) (2; 95% CI, 0 67), stoat (Mustela erminea) (1; 95% CI, 0 85), or wolverine (Gulo gulo) (1; 95% CI, 0 85). Positive animals were distributed throughout western, southern, and eastern Finland (10/17 of sampled regions). American mink (odds ratio [OR], 335) and badger (OR, 74) had higher odds of infection compared to other species. Also, animals sampled during the first sampling period (2006 2009; OR, 5) had higher odds of infection compared to the second period (2010 2014). No significant association was detected between infection and age, sex, or region. Furthermore, mink farms were not associated with higher odds of AMDV infection nor appeared to serve as a major source of infection for free-ranging mustelids at the municipal or regional level. Based on these results, it appears that domestic and sylvatic transmission pathways are largely decoupled, but it seems probable that infections occasionally move between the farmed and wild populations (e.g., via infected escapees/intruders). A phylogenetic analysis, including Finnish, Estonian, and global strains, indicated that AMDV strains form at least five main clusters. It also inferred that the virus has been introduced to Finnish farms on at least three occasions. Unfortunately, it could not be discerned whether the occurrence of AMDV in Finland is natural or a consequence of the global mink trade. In addition to its main hosts (farmed and wild mink), similar strains of AMDV were found in pine martens, polecats, and badgers. Interestingly, Estonian badgers carried a divergent strain, possibly representing a new amdoparvovirus. Other than the strain found in Estonian badgers, and the tendency of strains from Finnish farmed and feral mink to diverge into separate clusters, AMDV strains did not cluster according to location, year, species, or pathogenicity. The nucleotide differences between Finnish AMDV sequences, based on partial non-structural protein 1 gene, ranged from 0% to 14% and similar levels of variability were observed in farmed and natural populations. As a result of these studies, an automated ELISA test for the serodiagnosis of AMDV was developed and validated with high diagnostic sensitivity and specificity. The test offers a low cost, easy sampling, rapid throughput of large sample numbers, reduced processing time, and automated data management. The new test can be utilized for the monitoring, control and eradication of the virus, calculating the seroprevalence, and confirming the infection status of farms or individual mink. In addition, new information on AMDV epidemiology and genetic variation in Finnish farmed mink and free-ranging mustelids were established with potential impact on the biosecurity of farms, outbreak investigations, and the conservation of threatened mustelid species. Moreover, the new diagnostic tools and additional sequence data generated in this study can be utilized in the future research on the epidemiology of AMDV.
  • Kyöstilä, Kaisa (Helsingin yliopisto, 2015)
    Inherited diseases occur across different species. This thesis work has provided insights into the molecular genetic background of three autosomal recessive diseases that affect specific dog breeds. The studied phenotypes comprised two neurodegenerative diseases and a type of skeletal dysplasia. Genome-wide methods, such as SNP chip genotyping, were used to identify disease-associated gene variants. In all three disease phenotypes, the likely causative variant was found in a gene that had not been previously associated with a monogenic disorder. The Norwegian Elkhound and Karelian Bear Dog breeds are affected with inherited chondrodysplasia that causes short-stature dwarfism of varying severity. A genome-wide association study in Norwegian Elkhounds revealed a disease-associated locus on canine chromosome 17 and a nonsense mutation in the ITGA10 gene. The identified mutation was homozygous in all affected dogs from both breeds, and may have been introduced to Karelian Bear Dogs from Norwegian Elkhounds. The ITGA10 gene encodes an α10-integrin protein that assembles into a collagen-binding α10β1 integrin. The α10β1 integrin is a cell surface receptor, found in growth plate chondrocytes, where it mediates the cell s attachments with the surrounding matrix. Due to the mutation, a full-length α10-protein is not produced, disturbing the growth of long bones. Early-onset cerebellar degeneration occurs in the Finnish Hound dog breed. Neurological examination of affected dogs revealed quickly progressing cerebellar ataxia and failure to thrive. Genome-wide association analyses mapped the disease to a 1.5-Mb locus on canine chromosome 8. Sequencing of the SEL1L gene from the locus identified a homozygous missense mutation in all affected dogs. The mutation causes a serine to proline amino acid change within a highly conserved functional domain of the encoded SEL1L protein. The SEL1L protein is found in the endoplasmic reticulum, where it functions within a protein quality control and degradation pathway. Cerebellar tissue samples from affected dogs showed signs of endoplasmic reticulum stress, which may be the cause of premature cell death. A novel neurological disease, characterized by juvenile to adult onset cerebellar ataxia, was recognized in the Lagotto Romagnolo breed. Through linkage analysis, homozygosity mapping and whole-genome sequencing, the disease was associated with a homozygous missense change in the autophagy-related ATG4D gene. Pathological examination of affected dogs revealed progressive cerebellar degeneration, and intracellular vacuolar changes both in neuronal and extraneuronal tissues. The ATG4D gene encodes a cysteine protease, which is thought to function in the macroautophagy pathway. The autophagy process degrades and recycles damaged or obsolete cellular materials via membrane-enclosed autophagosomes. In line with this, the neuronal tissues of affected dogs showed signs of altered autophagic flow. Overall, this study has revealed three new disease-linked genes in dogs, which may be associated to similar disorders in other species. On the basis of the results, DNA-tests have been developed for veterinary diagnostic and breeding purposes. Importantly, by shedding light into disease-causing pathways, the results of this study could prove beneficial not just for canine health but for human medicine as well.
  • Melamies, Marika (Helsingin yliopisto, 2015)
    Respiratory diseases, both chronic and acute, are relatively common in dogs. They often cause a dramatic reduction in quality of life, mainly due to repeated coughing, excessive secretion of mucus, respiratory distress and exercise intolerance, which also frequently results in substantial stress for the owner. If accurate diagnosis is not followed by appropriate treatment euthanasia of the dog is often considered. This means that there is a real need for reliable diagnostic methods and efficient, safe and easy to administer pharmacotherapy. The aims of this thesis were to study a diagnostic method, bronchoalveolar lavage (BAL), and a treatment modality, corticosteroid inhalation, in healthy dogs. BAL is used to collect epithelial lining fluid (ELF) and the lavage fluid volumes used affect the amount of ELF recovered. Different techniques were compared to determine the best method of recovering a consistent volume of ELF. The canine pharmacokinetics of a commonly used inhaled corticosteroid (ICS), budesonide (BUD), were described. Systemic adverse effects of inhaled BUD were assessed and compared to those of a conventional therapy, oral prednisolone, and another readily available ICS, fluticasone propionate (FP). We found that a BAL protocol adjusting for weight, i.e. adjusting the amount of fluid instilled according to the body weight of the dog, yielded more consistent ELF recovery than fixed-volume BAL. We therefore recommend use of a weight-dependent BAL protocol to ensure results are comparable and to quantify the constituents of bronchoalveolar lavage fluid (BALF). Following BUD (1.0 mg) inhalation the mean maximum plasma concentration (Cmax) was 0.89 ng/ml, time to peak plasma concentration (Tmax) was 16 minutes and area under the concentration curve (AUC0-6h) was 1.1 ng h/ml. Inhaled BUD had moderately low systemic and pulmonary bioavailability in healthy dogs. Pulmonary bioavailability was evaluated by administering oral charcoal prior to inhalation in order to block gastrointestinal (GI) absorption of BUD. A method to detect BUD in low-volume dog plasma samples was developed and validated. Instrumental analysis was carried out using high-pressure liquid chromatography tandem mass spectrometry (HPLC/MS/MS) in a positive ion electrospray mode, and overall the general method characteristics were excellent. Our results also indicated that in healthy dogs a four-week course of clinically effective doses of inhaled FP, or oral prednisolone, but not inhaled BUD, produced dose-related adrenal suppression according to the adrenocorticotropic hormone (ACTH) stimulation test. In summary, our findings contribute to knowledge of the BAL technique, and the pharmacokinetics and systemic adverse effects of inhaled BUD which provides valuable information for treating canine patients with pulmonary symptoms.
  • Omoruyi, Iyekhoetin Matthew (Helsingin yliopisto, 2015)
    Commercially processed food, drinking-water sources and effluent waters discharged into bodies of water from wastewater treatment plants are putative but yet poorly delineated sources of human exposure to chemical mutagens and oestrogen-like chemicals globally. To this end, this study was aimed at determining the current situation for a possible comparison between a European country (Finland) and an African country (Nigeria). A total of 116 commercially processed food items and ready-to-eat snacks (three lots each) were obtained from Finland (60) and Nigeria (36) for initial screening, as well as sachet-pure water (16 different brands) from Nigeria, bottled still and mineral waters (10 brands each), tap water (hot and cold collected over a 3-month period) and influent and effluent water samples from both a drinking-water treatment plant (collected over a 3-month period) and a wastewater treatment plant (collected over a 2-year period) in Finland. All samples were collected in their respective countries and extracted by established methods. The mutagenic potential of the food extracts was first determined by the standard plate incorporation assay (Ames test), using two strains of Salmonella enterica sv. Typhimurium (TA 100 and TA 98) in the presence and absence of metabolic activation (S9 mix), and subsequently by a methylcellulose overlay, as well as treat-and-wash assays, while the oestrogenicity of the water and food samples, as well as food packaging materials, was determined by a yeast bioluminescent assay, using two recombinant yeast strains (Saccharomyces cerevisiae BMAEREluc/ERα and S. cerevisiae BMA64/luc). The cytotoxicity of the food extracts was measured by the trypan blue and lactate dehydrogenase tests, using the HepG2 cell line, as well as by the boar sperm motility assay, while possible DNA damage was assessed by the comet assay. The mutagenicity of commercially processed food items in Finland was generally low: 60% or 73% were non-mutagenic in S. Typhimurium strains TA 100 and TA 98, respectively. While the majority of the initially positive samples proved negative in the complementary assays, cold cuts of cold-smoked beef, grilled turkey and smoked chicken (a single batch of each) were also mutagenic in all three assays with the TA 100 strain, with and without metabolic activation, indicating that the mutagenic effect was not secondary to histidine release from the food products. The low mutagenicity outcome of the Finnish food items was further confirmed by independent chemical analyses of similar food products for four polycyclic aromatic hydrocarbons. In contrast to the outcome in Finland, the majority of food items from Nigeria (75%) were mutagenic in the Ames test, either in the presence or absence of the S9 mix and in either of the strains. Chin-chin, hamburger, suya and bean cake were mutagenic in all three assays with the Salmonella TA 100 strain, either in the presence or absence of the S9 mix. However, none of the food samples caused DNA damage in the comet assay. They were also not cytotoxic in any of the three assays measuring this aspect. In all, 31% of the sachet-packed water samples in Nigeria were oestrogenic, with concentrations ranging from 0.79 to 44.0 ng/l oestradiol equivalent concentrations (EEQs), while the tap and bottled water samples from Finland showed no signs of oestrogenicity in the in vitro test. Similarly, the oestrogenic activity of the influent samples from the wastewater treatment plant in Helsinki were generally low (from below the limit of detection to 0.7 ng/l EEQ), except in March and August 2011, when relatively high levels (14.0 and 7.8 ng/l EEQ, respectively) were obtained. No oestrogenic activity was recorded in any of the treated effluent samples from the wastewater treatment plant, nor was any in the influent and effluent samples from the drinking-water plant. The outcome of this study implies that Nigerian food items and drinking-water sources are more likely to contain mutagenic and oestrogenic chemicals than their Finnish counterparts, and efforts should be made to reduce the level of human exposure to these chemicals in the diet.
  • Laurila (os. Heikkilä), Henna (Helsingin yliopisto, 2015)
    Canine idiopathic pulmonary fibrosis (CIPF) is a chronic interstitial lung disease of unknown origin mainly affecting West Highland white terriers (WHWT). No curative treatment exists. Differentiating CIPF from other chronic respiratory diseases is difficult. Therefore, a measurable biomarker would be helpful. CIPF shares clinical features with human idiopathic pulmonary fibrosis (IPF), but the histopathological resemblance of the two diseases has been unclear. We described the clinicopathological and diagnostic imaging findings in dogs with CIPF and compared them with those of healthy WHWTs. The most typical clinical signs were cough and exercise intolerance. Inspiratory Velcro crackles were characteristic and an abdominal breathing pattern was often present. Many dogs were hypoxemic. Bronchointerstitial opacity was the most common radiographic finding. In high resolution computed tomography, ground glass opacity was a consistent feature, whereas honeycombing and traction bronchiectasis were less common. Bronchoalveolar lavage fluid (BALF) total cell count was elevated in CIPF and bronchial changes were common. We investigated the serum and BALF concentrations of two potential fibrosis biomarkers, endothelin-1 (ET-1) and procollagen type III amino terminal propeptide (PIIINP) in dogs with CIPF, chronic bronchitis (CB), eosinophilic bronchopneumopathy (EBP) and healthy dogs. Serum ET-1 was higher in dogs with CIPF than in other groups. BALF ET-1 was measurable only in dogs with CIPF. BALF PIIINP was higher in dogs with CIPF than in dogs with CB or healthy dogs, but not different from dogs with EBP. Serum PIIINP was not useful. We defined the histopathological lesions and their distribution in WHWTs with CIPF and compared them with those of human usual interstitial pneumonia (UIP), which is the histopathological pattern of human IPF, and human nonspecific interstitial pneumonia (NSIP), which is an important differential diagnosis of human IPF. A diffuse mature interstitial fibrosis of varying severity, resembling human NSIP, was seen in the lungs of all CIPF dogs. The majority of CIPF dogs also had multifocal areas of accentuated subpleural and peribronchiolar fibrosis with occasional honeycombing and profound alveolar epithelial changes, reminiscent of human UIP. Interstitial fibroblastic foci, characteristic of UIP, were not seen in WHWTs. In this thesis we provide a detailed description of the clinicopathologic and diagnostic imaging features of CIPF and present quantitative values for arterial blood gases and BALF cytology. Serum ET-1 and BALF PIIINP are elevated in dogs with CIPF and could differentiate CIPF from CB. Histopathologically, CIPF is characterised by two types of interstitial fibrosis and shares features of both human UIP and NSIP.