Eläinlääketieteellinen tiedekunta


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  • Kant, Ravi (Helsingin yliopisto, 2018)
    This thesis details an in silico exploration of the genetic potential of the Gram-positive genus Lactobacillus through the establishment and analysis of a pan-genome dataset. Lactobacilli are an intensively researched and studied group of bacteria, which is in part owed to their exploitation for various man-made food and industrial purposes and their advocated beneficial use as gut probiotics. Bacterial pan-genomics is an outgrowth of the comparative genomics field and by comparing the genomes from many strains of the same species the data obtained serves to catalogue the entire genetic repertoire available to a particular species. In effect, the pan-genome represents the complete assortment of putative genes in a species (or genus) of bacteria. The research presented in this thesis is comprised of three distinct studies (I, II, and III), each of which used the pan-genome approach to give a theoretical account of Lactobacillus genetics. In study I, a pan-genome was assembled at the genus level using 20 fully sequenced genomes from 14 different Lactobacillus species. Here, complete genome sequences were selected for creating a broader framework that would allow more comprehensive genomic comparisons. Varying aspects were apparent among the genome sequences used, including sizes that ranged from ~1.8 to ~3.3 Mbps and a G+C content of between ~33% and ~51%. The assembled pan-genome was sized at 14,000 protein-encoding genes, and out of which a small 383-gene Lactobacillus core genome (LCG) was derived. The genetic content of the LCG was used for reconstructing a molecular phylogeny, which then permitted a taxonomic grouping of the 20 genomes into three main clades. Additional classifications of the LCG involved identifying core group and signature group genes, as well as so-called ORFan genes that were further sorted as either LCG-specific or group-specific. In study II, a pan-genome of the Lactobacillus rhamnosus species was constructed from 13 different genomes. L. rhamnosus is a highly adaptable bacterium that thrives in a variety of hosts and environments. Presumably, there is little doubt that numerous genetic peculiarities are the source of the niche-related phenotypes that enable the inherent ecological adaptability of L. rhamnosus strains. For this, the genetic content of the assembled pan-genome was examined for those geno-phenotypic variations occurring at the cell-surface level and whether these correlate to a particular habitat preference of various L. rhamnosus strains. The L. rhamnosus pan-genome itself had an estimated size of 4,893 protein-encoding genes, which was further partitioned into the 2,095-gene core and 2,798-gene accessory genomes. Pan-genomic comparisons were benchmarked against the gut-adapted L. rhamnosus GG strain and focused primarily on seven functionally characterized surface-exposed proteins. Most notably, the operonic genes for the mucoadhesive SpaCBA pilus were part of the accessory genome and can be regarded as a genomic novelty in L. rhamnosus. Nonetheless, for those L. rhamnosus strains with a functional SpaCBA piliation trait, this would improve niche-specific fitness and presumably prolong transient (allochthonous) colonization of mucosal epithelial surfaces in the gut or elsewhere in the body. In study III, a pan-genomic appraisal was performed on the Lactobacillus ruminis species by compiling the genomes of nine different strains obtained from human, bovine, porcine, and equine digestive tracts. L. ruminis is a piliated and flagellated strict anaerobe and one of the few indigenous (autochthonous) lactobacilli in the gut. The pan-genome was utilized to pinpoint the molecular basis for the intractable colonization behavior of L. ruminis, where the focus was on those geno-phenotypes associated with cellular surface morphology and anaerobic fermentation and respiration. The size of the L. ruminis pan-genome was predicted to contain 4,301 protein-related genes, while the number of genes in the core and accessory genomes was 1,234 and 3,067, respectively. As inferred from the pan-genomic data, the presence of certain surface proteins and a substitute anaerobic energy-yielding metabolism might represent the adaptive phenotypes that help make L. ruminis a gut-autochthonic species.
  • Culebro Escandon y Perez, Alejandra (Helsingin yliopisto, 2018)
    Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastroenteritis, affecting approximately 96 million people around the world every year. Campylobacteriosis is characterized by diarrhoea, abrupt abdominal pain, and fever. Generally, the disease is self-limiting and resolves within a few days. Occasionally, neurological symptoms manifest clinically three weeks post-infection, indicating the onset of Guillain-Barré syndrome (GBS). GBS is a polyradiculoneuropathy characterized by an acute progressive and symmetrical motor weakness of the extremities with varying degrees of areflexia. GBS has been established as a post-infectious sequelae, whose principal aetiological agent is C. jejuni. The C. jejuni-GBS link was the first confirmed case of human autoimmune disease caused by ganglioside mimicry. C. jejuni lipooligosaccharides (LOS) have been established as the main structure responsible for ganglioside mimicry. Studies on the genetic basis of ganglioside-like LOS expression in C. jejuni have identified the genes associated with the addition of sialic acid (Neu5Ac) to the LOS chain; neuA, neuB, neuC, and a gene encoding a sialyltransferase from the glycosyltransferase CAZy family 42 (GT-42). These genes are found within the LOS biosynthesis locus of C. jejuni. The existence of neuA, neuB, neuC, and GT-42 genes in C. coli is unknown, despite the fact that this species has also been isolated from GBS patients. The primary aim of this study was to investigate the diversity of the C. coli LOS biosynthesis locus with particular interest in identifying genetic features associated with the synthesis and transfer of Neu5Ac. C. coli was found to possess a more diverse LOS biosynthesis locus than previously thought. A total of 27 different LOS locus classes containing a GT-42 encoding gene were identified. Among these, 16 are potentially able to synthesize sialylated LOS structures. Interestingly, several LOS locus classes resemble those of C. jejuni involved in ganglioside mimicry. Thus, bacterial factors implicated in GBS aetiology can cross species barriers. Also, C. coli has a larger GT-42 enzyme repertoire than C. jejuni. Two of the most common GT-42 encoding genes in C. coli were found to be associated to the presence of nonulosonic acid in LOS structures. Marked differences in diversity in the LOS locus were observed between C. coli clades, suggesting a potential role of this structures in niche adaptation. The importance of LOS to C. coli ecology and host-pathogen interaction remains to be explored.
  • Kettunen, Karoliina (Helsingin yliopisto, 2018)
    Efficacious and consistent enforcement of food safety legislation is a cornerstone in protecting public health and preventing distorted competition between food business operators (FBOs). In case of non-compliance, food control authorities must provide the FBO with advice and requests to ensure that the FBO corrects the violation. If these actions do not induce compliance or the operations cause a health hazard, food control authorities are to employ administrative enforcement measures such as orders or prohibitions. The objective of this thesis was to explore the risk-basis, efficacy and consistency of using administrative enforcement measures in local official food control in Finland. In addition, the aim was to investigate the importance and uniformity of local official food control from the perspective of FBOs. The methods include analysis of administrative enforcement decisions and inspection reports made by local authorities, survey and interview of local food control officials and survey of FBOs in approved dairy, fishery and meat establishments under local official food control. The results indicate that administrative enforcement measures are important control actions undertaken to force FBOs to correct multiple, recurrent or critical food safety violations. The enforcement process was initiated more rapidly and the duration of the process was shorter in critical than in non-critical food safety violations. However, the use of enforcement measures was often preceded with repeated requests to correct non-compliances, and the duration of enforcement processes was often rather long. Food control officials perceived the process as laborious and slow, and the use of enforcement measures varied among and within the local food control units. Some units had a strong routine for using the measures, while others used them rarely or not at all. Unclear alignments and uncertainty in using enforcement measures decreased the efficacy and consistency of enforcement. Many officials perceived the guidelines of the new disclosure system of inspection reports, the Oiva system, as helpful in enhancing the consistency of using enforcement measures. Respondent FBOs in approved dairy, fishery and meat establishments considered official food control to be important for food safety and appeared to be generally satisfied with its quality. However, many FBOs perceived the consistency of official food control as poor, and particularly small-sized FBOs were critical of the relevance of control actions. The Oiva system raised early-stage concerns among FBOs regarding the fairness and consistency of control. A perception of good co-operation with the official by FBOs was associated with their positive views about official food control and its importance. The results of this thesis indicate that local food safety enforcement is generally risk-based and control actions are progressively adjusted. However, the efficacy and consistency of using administrative enforcement measures could be improved and further efforts are needed to establish the Oiva system and unify the interpretations. Provision of more practical training for officials and further development of operating instructions and peer-review systems between and within control units would likely increase the efficacy and consistency of enforcement. Improving the consistency of control actions would also enhance the trust of FBOs in the relevance and justness of control.
  • Mahiout, Selma (Helsingin yliopisto, 2018)
    The aim of this thesis study was to gain more information on the physiological and toxicological functions of the aryl hydrocarbon receptor (AHR). The effects of two novel selective AHR modulators (SAHRMs) were investigated in vitro and in vivo. In addition, the involvement of the AHR in the avoidance of novel food was examined. The AHR is an evolutionarily ancient, apparently over 600-million-year-old protein. It is a ligand-activated transcription factor that modulates the expression of various genes within cells. One of the most studied groups of compounds that activate the AHR are dioxins. They are environmental contaminants primarily formed as by-products of various industrial processes, and many of them are toxic. Dioxins are chemically very persistent and lipid soluble, and thus accumulate in the food chain. Therefore, humans are also exposed to small amounts from food. In Finland, the most common source of dioxins is fatty wild fish from the Baltic Sea. The AHR has been recognised as the mediator of dioxin-induced toxicity for decades. More recently, it has also been shown to be involved in several physiological functions of the body, including the regulation of reproduction, foetal development, the immune system and autoimmunity. However, our understanding of the mechanisms of both the toxicological and physiological functions of the AHR remains incomplete. As a consequence, for instance, human health risk assessment of dioxins is challenging. Furthermore, better understanding of the physiological effects of the AHR could help elucidate the aetiology and pathogenesis of certain diseases, and therefore also benefit the discovery of novel pharmacological therapies. As lead compounds for drug discovery, SAHRMs are particularly interesting. They only elicit subsets of AHR-mediated effects, often without the major toxic outcomes of dioxins. Moreover, they could be valuable tools in elucidating the so far incompetently understood, multifaceted physiological roles of AHR, and the underlying molecular mechanisms. This thesis research had two main objectives. The first was related to studying the in vitro and in vivo toxicity of two novel SAHRMs, which are intended as drug compounds for the treatment of autoimmune diseases. The aim was to determine whether they appear suitable for pharmacological use from the pre-clinical safety perspective. Furthermore, finding out the extent to which their effects resemble or differ from those of the most toxic dioxin, TCDD, was of interest. The second objective was to accumulate more knowledge on a peculiar novel food avoidance behaviour, previously characterised in rats and mice after exposure to TCDD. This behaviour resembles a recognised behaviour model, conditioned taste aversion (CTA), which is also exhibited in humans, for instance in conjunction with nausea related to cancer treatment. The aim here was to verify whether the aforementioned rodent response is a physiological effect of the AHR or, more specifically, a consequence of TCDD exposure. Based on the results, the novel SAHRMs are very effective AHR activators, both in vitro and in vivo, and are in fact comparable to TCDD. However, their toxicity profiles are distinct from that of TCDD, and they appear considerably less toxic in rats. Therefore, the novel SAHRMs appear promising as possible drug compounds, and also highly interesting as tools for AHR research. Despite the differences in toxicity, one of the novel SAHRMs, as well as all of the three other AHR activators tested in this study, induced a strong avoidance response resembling that previously observed to TCDD, but shorter lasting. In addition, the reaction was not inducible in AHR knock-out rats. Thus, this study confirmed that the novel food avoidance behaviour is mediated by the AHR. The effect appears protective against potentially harmful ingested foods, and is therefore another physiological function of the AHR.
  • Yu, Xia (2018)
    The mammalian gut microbiota is composed of autochthonous species that permanently colonize the host intestine, and of allochthonous species that are only transiently able to occupy the intestinal environment. In this thesis research, Lactobacillus ruminis and Lactobacillus rhamnosus GG were investigated as paradigms for each type of microbe–host interaction, with special emphasis on the in vitro characterization of their adaptation factors in the host GIT. L. rhamnosus GG has two pilus operons: spaCBA encoding the well-studied SpaCBA pili and spaFED putatively encoding SpaFED pili. The expression of SpaFED pili in L. rhamnosus GG under laboratory conditions has not thus far been reported. In this study, a nisin-induced expression system was used for the generation of SpaFED or SpaF-deleted pili in Lactococcus lactis NZ9000. The results revealed that SpaFED pili were essential in mediating lactococcal adhesion to intestinal cell lines, to certain extracellular matrix proteins, and to porcine mucins, the tip pilin SpaF playing a central role as a focal adhesion factor. With regard to immunomodulation, SpaFED pili appeared to dampen the immune responses, which was largely attributed to the SpaF pilin, in HEK 293-blue cells expressing TLR2, and in Caco-2 cells. While encountering human peripheral blood monocyte-derived dendritic cells, neither immune response enhancement nor immune dampening by SpaFED pili was observed. Consistent with genomic analyses, transmission electron microscopy demonstrated that pili in gut autochthonous L. ruminis ATCC 25644 of human origin consisted of the tip (LrpC), basal (LrpB), and backbone (LrpA) pilins. Recombinant L. lactis strains were constructed, producing either LrpCBA or LrpC-lacking pili of L. ruminis. Recombinant LrpCBA pili mediated lactococcal adherence to ECM proteins and intestinal epithelial cells, and also dampened TLR2-dependent NF-κB signaling and IL-8 production in HEK cells, whereas L. ruminis itself induced evidently elevated immune responses. When incubated with Caco-2 cells, L. ruminis and recombinant lactococcal constructs expressing pili with and without LrpC pilin lowered IL-8 production. Subsequently, a novel L. ruminis strain was isolated from porcine feces and demonstrated to be flagellated and piliated. We analyzed the abilities of this new isolate and three other L. ruminis strains to adhere to host cells and extracellular matrix proteins, to inhibit pathogen binding and growth, to maintain epithelial barrier functions, and to modulate immune responses in vitro. The results indicated that the strains shared several characteristics, such as binding to ECM proteins and HT-29 cells, inhibition of pathogen adhesion and growth, maintenance of epithelial barrier functions in epithelial cells, and activation of innate immune responses to various extents. In conclusion, this thesis study demonstrated the adhesiveness of SpaFED and LrpCBA pili, which may respectively promote the gut retention of L. rhamnosus GG and of L. ruminis. Moreover, pilus-mediated dampening of innate immune responses might be a strategy for these two gut bacterial species to induce immune tolerance in the host. Additionally, L. ruminis inhibited enteropathogen adhesion and growth, as well as maintained intestinal barrier function, which could be regarded as beneficial to the host and which may in turn favor the persistent residence of L. ruminis.
  • Salla, Kati (Helsingin yliopisto, 2017)
    Medetomidine (MED), an 2-adrenergic agonist, is widely used in veterinary practice as a sedative and premedication agent, as it provides reliable sedation. However, the marked haemodynamic changes limit its use to only healthy patients. MK-467, a peripheral 2 -adrenoceptor antagonist, has been demonstrated to attenuate the early haemodynamic disturbances induced by MED with intravenous co-administration in dogs. The main aim of this series of investigations was to evaluate the haemodynamic effects of MK-467 when it is combined with MED and selected sedative or anaesthetic agents that are commonly used in veterinary practice in dogs. More specifically, the concomitant administration of MK-467, MED and butorphanol via intravenous or intramuscular routes was studied and compared with sedation induced with MED-butorphanol. The haemodynamic effects after the various doses of MK-467 in relation to the standard dose of MED were evaluated prior to and during isoflurane anaesthesia. In addition, the haemodynamic effect of the administration of MK-467 as a constant rate infusion was assessed during MED-alfaxalone total intravenous anaesthesia. As a comparison, the haemodynamic effects of the co-administration of MK-467 and MED as a premedication regimen in relation to acepromazine-butorphanol or glycopyrrolate-medetomidine prior to and during isoflurane anaesthesia were assessed. Finally, the influence of MK-467 on plasma dexmedetomidine and alfaxalone concentrations was determined. After intravenous administration of MK-467 and MED with or without butorphanol heart rate, cardiac output and oxygen delivery were well maintained, and the increase in systemic vascular resistance and blood pressures was prevented. The profitable effects of MK-467 appeared more slowly after intramuscular than after intravenous administration. The initial MED-induced haemodynamic effects were dose-dependently attenuated by MK-467 during the premedication period but not during isoflurane anaesthesia with these studied doses of MK-467 in relation to a standard dose of MED as a premedication regimen. The addition of MK-467 to MED and alfaxalone produced a stable haemodynamic outcome when they were administered as constant rate infusions. Cardiac output and oxygen delivery were well maintained during both inhalation and total intravenous anaesthetic regimens when MK-467 was involved. The haemodynamic effects after the co-administration of MED-MK-467 or acepromazine-butorphanol mimicked each other prior to and during isoflurane anaesthesia. Pre-treatment with subcutaneous glycopyrrolate failed to improve the MED-induced reduction in cardiac output by means of increasing heart rate prior to or during isoflurane anaesthesia. Peripheral 2- adrenergic receptor blockade by MK-467 resulted in a reduction in plasma dexmedetomidine and alfaxalone concentrations, presumably by altering the haemodynamic function. In conclusion, the addition of MK-467 in a MED-contained sedation or anaesthetic regimen attenuates the rather dramatic MED-induced haemodynamic changes by blocking peripheral 2-adrenergic receptors and by decreasing the plasma concentrations of dexmedetomidine. The overall haemodynamic stability is well maintained during either an isoflurane anaesthesia or an alfaxalone constant rate infusion. The combination of MK-467 with MED may offer an alternative premedication regimen to the combination of acepromazine and butorphanol in healthy dogs.
  • Dyggve, Hanna (Helsingin yliopisto, 2017)
    Doberman hepatitis (DH) is a rare and chronic inflammatory condition of the liver. The early diagnosis of DH is difficult, and Dobermans may be affected for months or years before the diagnosis. There is no curative treatment and the prognosis is poor if the condition is associated with clinical signs of liver failure. Unraveling the disease etiologyand pathogenesis would enhance the diagnostic approaches, and novel therapies could improve the prognosis for Dobermans. Our studies aimed to identify supporting evidence for the suspected autoimmune background of DH by satisfying more descriptive characteristic risk factors that are described in human autoimmune diseases. We focused on major histocompatibility complex (MHC) class II genes, MHC class II regulatory regions, circulating serum anti-nuclear antibodies (ANA), and possible liver-related autoantigens associated with DH. We identified a homozygous dog leukocyte antigen (DLA) risk haplotype and allele for DH. DLA-DRB1*00601/DQA1*00401/DQB1*01303 haplotype elevated the risk of DH susceptibility close to 15-fold. A homozygous risk DLA-DRB1*00601 allele was found in all DH dogs, while this allele was present in 56.8% of the healthy controls. DLA-DQA1*00901/DQB1*00101 and allele DLA-DRB1*01501 appear to protect against DH development. Overall, the DLA region showed very low variation among DH patients, with only two different haplotype combinations compared with seven in the control group. We defined the level of variation of the regulatory promoters in DH patients and controls with the homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 haplotype. Also, the potential polymorphism in the DLA-DRAp promoter and DLA-DRA exon 2 was examined. The promoter DLA-DRAp and exon 2 were identical to the dog reference sequences in both groups. The promoters DRBp, DQAp, and DQBp were monomorphic, with no explaining variation when comparing DH patients with healthy control Dobermans. DLA-DRB1*00601 allele was associated with DRBp*1, DLADQB1*01303 allele with DQBp*6, and DLA-DQA1*00401 allele with DQAp*2. Our result suggests that promoter variants are not associated as risk modifiers for DH in Dobermans with the risk haplotype, but the whole DLA block is associated with DH. The study revealed ANA in DH patients. ANA were further characterized as antihistone autoantibodies (AHA) that were significantly elevated in DH. Thus, immune tolerance has failed in DH. Our results suggest that elevated AHA in conjunction with high alanine aminotransferase (ALT) in a Doberman with or without clinical signs of hepatitis support the DH diagnosis. A negative serum AHA result does not rule out DH, and AHA is not a suitable biomarker for disease progression. We described two novel liver-related target autoantigens in DH. These could be identified as dehydrogenase enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and alcohol dehydrogenase (ADH). DH dogs had significantly elevated serum IgG immunoreactivity towards GAPDH and ADH antigens compared with controls, and the disease was associated with both autoantibodies. This provides evidence for an antigen-driven autoimmune process and elucidates the previously unknown DH pathogenesis. This thesis provides new insights into the suspected autoimmune etiology and pathogenesis of DH. We conclude that the study contributes to the development of useful biomarkers that could be used to improve the currently difficult early diagnosis of DH in Dobermans with elevated ALT levels. Our findings support the theory that DH has an autoimmune origin.
  • Nokireki, Tiina (Helsingin yliopisto, 2017)
    European bat lyssavirus type 2 (EBLV-2) was first isolated in Finland from a Daubenton’s bat (Myotis daubentonii) in 2009. Rabies in bats was already suspected in 1985, when a Swiss biologist died in Finland of lyssavirus infection, later identified as EBLV-2 infection. However, the origin of the infection could not be confirmed at that time. In 2010–2011, during active surveillance study, samples from 774 bats were analyzed for EBLV viral RNA. In addition, sera from 423 bats were analyzed for the presence of lyssavirus antibodies. Antibodies were detected in 2010 and 2011 from two locations and from one location, respectively. All seropositive bats were Daubenton’s bats. No EBLV viral RNA was detected in any of these bats. In 2016, EBLV-2 was detected from a diseased Daubenton’s bat for the second time. These data provide proof that EBLV-2 is endemic in the Finnish Daubenton’s bat population. In phylogenetic analysis, the Finnish EBLV-2 strains formed a monophyletic group separate from other bat-type lyssaviruses with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujand virus (KHUV). EBLV-2 showed limited diversity compared to rabies virus (RABV) and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years. The Finnish EBLV-2 strains and a Swiss strain (1993) were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the Finnish strains (1985 and 2009) appear to have evolved from a common ancestor during the last 200 years. Rabies vaccine is used to protect against rabies virus before or after potential exposure. Since all the currently available vaccines are based on RABV, the vaccines are also used to protect against other lyssaviruses, and additionally against EBLV-2 infection based on cross-protection. We assessed the level of protection afforded by two commercial rabies vaccines, one for humans and one for animals, against intracerebral challenge in mice with EBLV-2 isolated from a bat in 2009. We compared this with protection using the same mouse model against challenge with RABV isolated from a Finnish raccoon dog in 1989. When challenged with RABV, all the vaccinated mice survived. When challenged with EBLV-2, 75% to 80% of the vaccinated mice survived. All vaccinated mice developed sufficient to high virus-neutralizing antibody (VNA) titers against RABV, ranging from 0.5 to 128 IU/ml. RABV-based vaccines also appear to offer good cross-protection against EBLV-2 circulating in the Finnish bat population. To investigate the factors influencing the response to rabies vaccination, we assessed the success of vaccination measured from the antibody response in dogs (n = 10 071) and cats (n = 722) sampled during 2009–2013. We examined the factors influencing the response to vaccination when animals failed to reach a rabies antibody titer of ≥0.5 IU/ml.
  • Heikkilä, Helka (Helsingin yliopisto, 2017)
    Osteoarthritis (OA) is considered to be the leading cause of chronic pain in dogs. Oral medication is the mainstay of pain treatment in canine OA, but it can provide insufficient pain relief and intolerable adverse events. Therefore, new treatment modalities are needed for dogs suffering from osteoarthritic pain. Intra-articularly injected botulinum toxin A (IA BoNT A) has shown efficacy in treatment of joint pain in arthritic human patients. The mechanism of the antinociceptive action of IA BoNT A has not been studied in vivo and its adverse effects have not been thoroughly investigated. Our aim was to study the antinociceptive efficacy of IA BoNT A in osteoarthritic dogs and to investigate the effect it has on their pain mediators. Also, we wanted to compare the pain mediators between osteoarthritic and non-osteoarthritic joints and reveal any associations with osteoarthritic pain. Our aim was also to investigate the adverse effects of IA BoNT A and to study whether the toxin spreads from the joint. We investigated the antinociceptive efficacy of IA BoNT A in a randomized, double-blinded, placebo-controlled, 12-week clinical trial in 35 client-owned osteoarthritic dogs after an injection of either IA BoNT A or placebo into the stifle, hip, or elbow joint. We detected significant improvement in the ground reaction forces and Helsinki Chronic Pain Index in the IA BoNT A group. There was also a significant difference in the improvement of the ground reaction forces between the IA BoNT A and placebo groups. We measured substance P (SP), prostaglandin E2 (PGE2), and tumour necrosis factor-alpha (TNF-α) from the synovial fluid (SF) and serum of the 35 osteoarthritic dogs and also analysed SF SP and PGE2 from 13 non-osteoarthritic control joints. IA BoNT A did not affect SF or serum SP or PGE2. TNF-α was not detectable in our samples. SF PGE2 correlated with osteoarthritic joint pain and was significantly higher in osteoarthritic than in non-osteoarthritic joints. We investigated the adverse effects of IA BoNT A in a randomized, placebo-controlled, blinded trial in six healthy laboratory beagle dogs after IA BoNT A and placebo injections into the stifle joints. No significant clinical, cytological, or histopathological adverse effects were detected 12 weeks after the injections, but changes in the electrophysiological recordings in two dogs suggested possible spread of the toxin. Our results indicate that IA BoNT A has efficacy in the treatment of osteoarthritic joint pain in dogs. The antinociceptive effect of IA BoNT A inside the joint seems not to be related to the inhibition in the release of SP or PGE2. SF PGE2 could be a marker of chronic OA and pain in dogs. IA BoNT A does not cause clinical, cytological, or histopathological adverse effects in healthy dogs, although the toxin may spread from the joint.
  • Nordgren, Heli (Helsingin yliopisto, 2017)
    In 2007 Finnish fur farmers detected new kind of signs in farmed mink, foxes and raccoon dogs (Finnraccoon). Mink had severe pyoderma in the head around eyes, mouth or ears or in the feet around the nailbeds. Finnraccoons developed painful abscesses in the paws between the toes. Foxes had severe keratoconjunctivitis that occasionally spread to the eyelids and to facial dermatitis. The disease caused severe symptoms and even increased mortality on the farms. The spread of the disease was typical to an infectious disease. Similar signs have been detected in fur animals in other pelt producing countries as well. Similar signs in mink have been reported for the first time in U.S.A at 1970´s and in Canada 1996. North-American farmers and later researchers linked the onset of the symptoms to the start of feeding mink with feed containing seal byproducts. The University of Helsinki (UH), the Finnish Fur Breeders´ Association (FFBA), and the Finnish Food Safety Authority Evira initiated a collaborative project in 2009 to describe the pathology of the disease, identify the causative organism(s), describe the occurrence of the disease in Finland and to find out possible sources and risk factors for the disease. This thesis includes these studies. Ninety nine fur animals underwent necropsies with complete microbiological examinations, including both diseased and healthy control animals. Due to the common lesions, in diseased animals of all fur animal species, the disease was named Fur animal epidemic necrotic pyoderma (FENP). The bacterium Arcanobacterium phocae was isolated from all diseased animals, but not from healthy controls. The finding of A. phocae is particularly interesting, because this bacterium causes skin inflammations and abscesses in marine mammals such as seals, and there had been found a temporally connection between the onset of the symptoms in mink and the use of seals as feed source in North America. However, the disease occurs also in the countries where seal meat is not used as a feed raw material. Thus, it is probable that there has been a species shift of the bacteria A. phocae from seals to fur animals via feed, and the disease is currently spreading by diseased/carrier animals. The results indicate that the presence of the bacteria A. phocae alone is not enough to cause the disease, as predisposing factors, such as skin or mucosal trauma is needed to evoke the disease. In addition, new Streptococcal species, closely related to Streptococcal species of seals, was detected in these investigations. Furthermore, the infection trial proved that A. phocae caused FENP-like symptoms when mink were experimentally infected. The mail survey study showed that FENP had spread to all areas where fur farming is practiced in Finland and signs of FENP was detected on 40% of responding farms. The results indicated that the disease was introduced to Finland by imported carrier animals and it spread further in the country via domestic purchases. The disease seems to spread on the farms by bird and other wild life contacts. The study revealed also other possible risk factors, such as the farm type; FENP was seen more on larger size farms and on mixed farms (farms farming more than one fur animal species). Also some other predisposing factors were relieved. The results provide a basis for developing preventive methods and treatment of FENP diseased animals to improve animal welfare.
  • Murros, Anna (Helsingin yliopisto, 2017)
    Yersinia genus includes currently 18 species: Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia bercovieri, Yersinia mollaretii, Yersinia rohdei, Yersinia ruckeri, Yersinia aldovae, Yersinia aleksiciae, Yersinia massiliensis, Yersinia similis, Yersinia entomophaga, Yersinia nurmii, Yersinia pekkanenii and Yersinia wautersii. The history of the genus Yersinia can be dated back to 1883. In 1965, the genus consisted of only three species: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis. Since then the taxonomy of the genus has been under tremendous change over the years, and especially the taxonomy of Y. enterocolitica, of which many new species have been separated from. Still Y. enterocolitica is a group of very heterogeneous bacteria, which can be divided into 6 biotypes and about 30 serotypes and into pathogenic and non-pathogenic strains. This variability makes the identification of Y. enterocolitica very challenging. In the thesis, two Yersinia species; Y. nurmii and Y. pekkanenii are described. These species were characterized by polyphasic taxonomic methods, including of 16S rRNA gene analysis, multilocus sequence analysis (MLSA) of housekeeping genes glnA, gyrB, recA and HSP60, DNA-DNA hybridization studies, 16S and 23S rRNA gene restriction fragment length polymorphism (RFLP), and phenotyping. Y. nurmii was isolated from broiler meat packaged under modified atmosphere, and Y. pekkanenii from water, soil and lettuce samples. In the 16S rRNA gene analysis and the 16S and 23S rRNA gene RFLP analysis, these two species grouped with other Yersinia, but in separate clusters. In the phylogenetic analysis of separate or concatenated housekeeping genes, the species formed unique monophyletic groups in all phylogenetic trees constructed. Y. nurmii had a phenotypic profile most similar to that of Y. ruckeri. Y. pekkanenii could not be differentiated from Y. pseudotuberculosis using phenotypic tests. Methods of polyphasic taxonomy were also used to estimate the taxonomic position of European Y. enterocolitica strains of non-pathogenic biotype 1A. Y. enterocolitica has been divided into two subspecies; Y. enterocolitica subsp. enterocolitica and Y. enterocolitica subsp. palearctica. Both subspecies consist of pathogenic and non-pathogenic biotypes. In this thesis, 212 Y. enterocolitica strains were characterized by numerical analysis of HindIII ribopatterns (16S and 23S rRNA gene RFLP). These strains consisted of 162 strains of biotype 1A and 50 strains of biotypes 2 to 4 isolated from different sources in Europe during years 1997-2013. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). The biotype 1A strains studied were found to form a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and Y. enterocolitica subsp. palearctica, with suggestion of the existence of a subspecies formed by non-pathogenic Y. enterocolitica biotype 1A strains. Finally, while studying Enterobacteriaceae in cold-stored (6 °C), modified atmosphere-packaged (MAP) pig cheek (musculus masseter) and hind leg meat (musculus semimembranosus), it was noticed that the pathogenic Y. enterocolitica subsp. palearctica bioserotype 4/O:3 multiplied into high numbers from a non-detectable level in (MAP) pig cheek meat. The Enterobacteriaceae isolated in this study were identified by 16S and 23S rRNA gene RFLP using the HindIII enzyme. Y. enterocolitica bioserotype 4/O:3 is the most common pathogenic bioserotype word-wide, and it can be transmitted to humans through raw or undercooked pork. Usually the growth of Enterobacteriaceae is inhibited by a modified atmosphere with 20% or more CO2 at refrigerated temperatures. However, in this study, high numbers of Y. enterocolitica bioserotype 4/O:3 was observed in MAP cold-stored pig cheek meat, with a concentration of 30% CO2 and 70% O2 in the packages.
  • Björkman, Stefan (Helsingin yliopisto, 2017)
    We hypothesized that prolonged farrowing decreases subsequent fertility, that is, pregnancy rate. Furthermore, we hypothesized that prolonged farrowing causes retention of placentae and metritis, delays uterine involution and perturbs follicular growth after weaning. We also hypothesized that sows that undergo prolonged farrowing release less oxytocin at subsequent estrus in response to boar stimulation. We determined the farrowing duration (time between expulsion of first and last piglet) of sows and explored whether there is a negative effect on subsequent post-weaning pregnancy rate (n = 148). We explored the relationship between farrowing duration and placenta expulsion (n = 142), and postpartum uterine size and intrauterine fluid (n = 99). For that, placenta expulsion was observed until 24 h after birth of the last piglet and ultrasonography was used during the first week postpartum to examine the uteri of the sows. Uterine size and intrauterine fluid were used as indicators for initial uterine involution and puerperal metritis. Furthermore, we determined the farrowing duration of sows (n = 30) and monitored the subsequent follicular development using transrectal ultrasound twice a day between the third day after weaning and ovulation. At estrus, blood samples were collected in the presence of a boar in order to determine the endogenous oxytocin concentrations and release patterns. The results show that sows with a prolonged parturition (> 300 min) were 3.4 (P = 0.027) more likely not to be pregnant. Sows that experienced total (no expulsion of placental parts; 3%) and partially retained placentae (no expulsion of placental parts after birth of the last piglet; 3%) had longer farrowing durations (1009 ± 275 and 734 ± 136 min) than sows without retained placentae (369 ± 202 min; P = 0.021 and P = 0.004). Farrowing duration conformed to a quadratic relationship with the number of expelled placental parts (P = 0.001), placental expulsion duration (time between expulsion of first and last placental part; P = 0.002) and time between expulsion of last piglet and last placental part (P = 0.024). Use of oxytocin increased number of expelled placental parts (3.8 ± 0.2 vs. 2.9 ± 0.3, P = 0.035), decreased the placental expulsion duration (172 ± 44 vs. 328 ± 26 min, P = 0.011) and time between expulsion of last piglet and last placental part (148 ± 48 vs. 300 ± 24 min, P = 0.025). Furthermore, prolonged farrowing (β ± SE, Wald χ2, Odds; 2.0 ± 0.5, 13.1, 7.6; P = 0.001), obstetrical intervention (1.5 ± 0.7, 4.4, 4.3; P = 0.036) and two or more stillborn piglets (1.4 ± 0.7, 3.8, 3.9; P = 0.052) increased the risk of having enlarged uterine size whereas oxytocin administration (­ 1.5 ± 0.7, 4.7, 0.2, P = 0.040) decreased the risk. Two or more stillborn piglets (2.6 ± 0.9, 8.7, 13.7; P = 0.003), obstetrical intervention (1.8 ± 0.8, 5.0, 6.0; P = 0.025), prolonged farrowing (1.7 ± 0.8, 4.3, 5.7; P = 0.039) and impaired placenta expulsion (3.3 ± 1.7, 4.0, 26.9; P = 0.044) were associated with intrauterine fluid. After weaning, OT concentrations were higher in sows with prolonged farrowing than in sows with shortened farrowing (28.0 ± 7.7 vs. 20.6 ± 7.7, P < 0.01). OT concentrations correlated with diameters of the follicles measured 5 d after weaning and when the follicles reached their maximum size after weaning (r = 0.61, P < 0.01 and r = 0.57, P < 0.01, respectively).
  • Hallamaa, Raija (Helsingin yliopisto, 2017)
    Summer eczema is one of the most common diseases that causes discomfort and impairs the quality of life of horses worldwide. The lack of a feasible treatment has made this recurrent, insect hypersensitivity-linked allergic pruritus a challenge for veterinary medicine. The first aim of this study was to examine serum phospholipids and their use in the therapy of summer eczema by using an autologous serum preparation. The second aim was to delineate clinical features of summer eczema among Finnhorses. The efficacy of the therapy was investigated in 28 horses in a placebo-controlled study and was also evaluated according to long-term information collected from the owners of the 343 horses treated with this therapy over 12 years. Serum phospholipids and their changes after autoserum therapy were analysed in 10 horses with summer eczema and 10 matched healthy controls by LC-MS. Content of phospholipids in autoserum preparations of 10 affected and 6 healthy horses were analysed by ESI-MS. Horses in the placebo group showed significant aggravation in their clinical signs compared with horses treated with autoserum therapy at the same time (P=0.0329). According to long-term data, 70% of the horses benefited from this autoserum therapy (95% CI 0.64-0.75, P<0.0001) and 16% did not, and 14% of the owners did not provide a clear opinion. No harmful side effects were observed. Horses with summer eczema displayed significantly lower concentrations of phosphatidylcholine (P<0.0001) and sphingomyelin (P=0.0115) in their sera than healthy horses. After a 4-week autoserum therapy, no significant difference between these horses could be demonstrated. The change in clinical signs correlated significantly with the alterations in sphingomyelin concentrations (P=0.0047). Analysis of autoserum preparations revealed that these preparations contained major serum phospholipids, however, in significantly differing concentrations between eczema and healthy horses. Affected horses showed more abundant concentrations of phosphatidylcholine (P=0.042) and sphingomyelin (P=0.0017). In addition, concentrations of these phospholipids correlated significantly with the clinical status. Finnhorses formed the largest group. Most Finnhorses had become affected before the age of 5 years and showed moderate signs. Severity of the signs was not related to age at onset. No significant correlation existed between duration and severity of the disease. This study showed that an autoserum preparation containing serum phospholipids was a favourable method to treat summer eczema. Affected horses displayed significant differentiations in their serum phospholipid profiles and these alterations seemed to change according to the clinical status. Applications of this autoserum therapy for other allergic manifestations of horses should be explored.
  • Kovanen, Sara (Helsingin yliopisto, 2017)
    Campylobacter jejuni is a leading cause of human bacterial gastroenteritis worldwide. This zoonotic pathogen transmits to humans mainly indirectly, via consumption of contaminated food or drinking water or from various environmental sources. In Finland, ~4500 confirmed campylobacteriosis cases are registered annually and most of the infections occur in the summer. Poultry has been generally identified as a major reservoir and source of human campylobacteriosis. Molecular epidemiology of C. jejuni has been widely studied. Especially multilocus sequence typing (MLST), which assigns isolates to sequence types (STs) and clonal complexes (CCs), has provided valuable information about existing C. jejuni genotypes in different sources worldwide. While MLST has advantages to compare larger data worldwide, it has limitations in further distinguishing isolates within STs for epidemiological purposes. Whole-genome sequencing (WGS) enables the investigation of whole bacterial genomes instead of few selected genes. Aim of this thesis was to study C. jejuni isolates in domestic human infections, poultry and water using MLST and further whole-genome (wg) MLST. The most commonly found STs belonged to ST-45 CC, ST-21 CC, ST-283 CC and ST-677 CC. In this thesis, we further explored C. jejuni isolates within same STs, using highly discriminatory wgMLST and also considering the temporal relationship between the isolates from humans and chickens in summer 2012. We were able to recognize human isolates that were both genetically and epidemiologically related, thus most likely sharing the same infection source. We also identified the association between C. jejuni isolates from chicken slaughter batches and human patients. Although 79% of MLST types of human C. jejuni isolates overlapped with the isolates from chickens, only 24% of domestic human infections were related to chickens using both wgMLST and temporal association. Hence, the origin of more than 70% of the infections remained unidentified suggesting other potential transmission routes such as other domestic animals or environment-associated sources. In addition, we used WGS data to characterize genomic features among C. jejuni ST-677 CC isolates. We recognized several genetic characteristics, which are typical for this lineage that has been frequently detected among Finnish patients and associated also with bacteremia.
  • Bennett, Rachel (Helsingin yliopisto, 2017)
    Background and rationale Alpha2-adrenoceptor agonists, such as medetomidine, are commonly used in veterinary medicine for the sedation and premedication of animals. Their use is associated with a range of undesirable pharmacodynamic effects most notably vasoconstriction, bradycardia (Pypendop and Verstegen 1998), hyperglycaemia (Hsu and Hummel 1981) and diuresis (Thurmon et al. 1978). MK-467 is a peripheral α2-adrenoceptor antagonist (Clineschmidt et al. 1988), which ameliorates the aforementioned side effects of (dex-)medetomidine whilst maintaining the sedative effects (Enouri et al. 2006; Honkavaara et al. 2008; Restitutti et al. 2012; Rolfe et al. 2012). Therefore MK-467 may offer some important clinical benefits, although some questions remain concerning its pharmacodynamic and pharmacokinetic interaction with other anaesthetic drugs. The main objectives of the following studies were to: determine the protein-binding fraction of MK-467, to assess the possible role of MK-467 as a P-glycoprotein substrate in vitro; and to evaluate the impact of MK-467 on the disposition of medetomidine in vivo. During in vivo studies, the impact of MK-467 on the centrally mediated effects: sedation and antinociception and peripherally mediated cardiovascular effects of medetomidine were assessed simultaneously. The protein-binding characteristics of MK-467 were investigated using the technique of equilibrium dialysis. Drug concentrations were measured using the technique of liquid chromatography and tandem mass spectrometry. Protein-binding fraction of MK-467 was approximately 70% and it was unaltered by the presence of medetomidine. Transcellular drug movement was determined using Madin-Darby Canine Kidney cells - wild type (WT-MDCKII) and cells transfected with the human gene encoding P-glycoprotein (MDR1-MDCKII). In addition to MK-467, acepromazine, a putative P-glycoprotein substrate, and dexmedetomidine were also investigated. Based on measured drug concentrations, apparent permeability of the cells was calculated and used to determine the role of active transport in the transcellular movement of the selected drugs. Passive movement of MK-467 was undetectable. Therefore, efflux ratios for MK-467 were not determined. However, movement in the basolateral to apical direction occurred in both cell lines. The identity of the possible transporter remains unclear. Transport ratios for acepromazine were 1.17:1:1.0 and 1.51:1.0 for WT-MDCKII and MDR1-MDCKII transfected cells, respectively. Currently acepromazine cannot be defined as a P-glycoprotein substrate based on these results. Whilst, dexmedetomidine transport ratios were 0.98:1.0 and 1.15:1.0 for WT-MDCKII and MDR1-MDCKII respectively. It does not appear to be a substrate for an active transport mechanism. In vivo drug concentration data underwent non-compartmental analysis. MK-467 increased the volume of distribution and clearance of dexmedetomidine and levomedetomidine, whilst area under the curve and elimination half-life were significantly decreased when compared with medetomidine alone. Medetomidine significantly decreased the clearance of alfaxalone during co-administration, whilst the additional administration of MK-467 counteracted the effect of medetomidine on alfaxalone clearance. The quality and duration of sedation were assessed using a composite sedation score, whilst hypnosis was evaluated by measurement of bispectral index or analysis of the electroencephalogram. Antinociception was determined by the measurement of limb withdrawal times and head lift times following the application of a nociceptive stimulus applied to the nailbed of a hind limb digit. Measured haemodynamic variables included arterial blood pressure, heart rate, cardiac output and systemic vascular resistance. Ventilatory effects of medetomidine and co-administered MK-467 were also assessed by the analysis of arterial and venous blood gas samples taken during these studies. The co-administration of MK-467 did not alter the initial quality of sedation but reduced the duration of sedation produced by medetomidine. MK-467 significantly diminished the antinociceptive action of medetomidine. MK-467 ameliorated the cardiovascular, haemodynamic and ventilatory effects of medetomidine prior to and during general anaesthesia. In conclusion, MK-467 is moderately protein bound and it is unlikely to be subject to drug-drug interactions in vivo. MK-467 shows little passive movement in MDCKII cells but may undergo active cellular efflux. It is unclear whether MK-467 is suitable for use in animals carrying the P-glycoprotein mutation. The addition of MK-467 alters the disposition of co-administered drugs resulting in lower plasma drug concentrations. The reduction in some pharmacodynamic effects may be attributed to the alteration in pharmacokinetics caused by the peripheral α2-adrenoceptor antagonist.