Faculty of Veterinary Medicine

 

Recent Submissions

  • Korzyukov, Yegor (Helsingin yliopisto, 2020)
    The family Arenaviridae is a well-represented clade of RNA viruses. The genus Mammarenavirus is dominated by rodent-borne arenaviruses, several of which have been identified as the causative agents behind hemorrhagic fevers and neurological infections in humans. Despite having been studied for more than 90 years, mammarenavirus diagnostics, vaccines and antiviral compounds are only available for some mammarenaviruses. Another genus, Reptarenavirus, includes viruses linked to boid inclusion body disease (BIBD) in constrictor snakes. BIBD has been reported in captive constrictor snake species since the 1970s, but the etiological agents were only identified in 2012. BIBD can lead to the eradication of the entire affected snake populations. The range of possible host spectrum and the immune response against reptarenaviruses are not well characterized. This thesis aims to define the potential reptarenavirus host cell spectrum as well as expand understanding of the boid immune response. One of the hallmark signs of reptarenavirus infection in snakes is the formation of inclusion bodies (IB) in host cells. Snakes are poikilotherm and the replication of viruses is often susceptible to temperature variation. Reptarenavirus infection in mammalian, boid, and arthropod cells, incubated at 37 °C did not induce IB formation, whereas prominent IB formation occurred in all three phyla when incubated at 30°C. Reptarenaviruses replicated efficiently at 30°C, whereas at 37°C the replication efficiency reduced significantly. Many animal viruses take advantage of glycoproteins (GPs) to mediate binding and entry via attachment to host cell surface receptors. To study the ability of reptarenavirus GPs to mediate cell entry, a pseudovirus system based on reporter gene-bearing recombinant vesicular stomatitis virus (rVSV) was introduced. The pseudoviruses with reptarenavirus GPs served to demonstrate that the majority of arenavirus GPs could mediate entry to both mammalian and reptilian cells but at varying efficiencies. In order to validate the link between BIBD and reptarenavirus infection, constrictor snakes (Boa constrictor and Python regius) were experimentally infected. Despite transient central nervous system signs, IB were not detected in the infected snakes. The snakes were sacrificed and sera was collected to determine the magnitude of the humoral immune response. In order to assess the antibody response against reptarenaviruses it was necessary to develop reagents capable of detecting immunoglobulins in snake sera. The generated reagents were initially tested using sera from BIBD-positive snakes. IgY and IgM class antibodies binding reptarenaviruses were detected in serum samples, validating the functionality of the reagents. In the next study, these antibodies were used to detect IgM and IgY antibodies in experimentally and naturally infected snake populations. Extracted sera was further assayed using the rVSV-based pseudoviruses decorated with reptarenavirus GPs to show a neutralizing antibody response following reptarenavirus infection. This thesis adds to understanding of reptarenavirus infectivity across species barriers. The generation of novel diagnostic reagents will allow generation of serodiagnostic tools for reptarenavirus infection. Future studies of reptarenaviruses should aim to establish a virus-specific link between reptarenaviruses and BIBD that could serve in the development of effective and preventive treatment strategies.
  • Tamminen, Tuire (Helsingin yliopisto, 2020)
    This dissertation focuses on canine dystocia and is based on three individual studies conducted with client-owned pet animals. The aim was to elucidate the role of oxytocin receptors in uterine inertia. The suitability of human cardiotocography on parturition monitoring in bitches was tested. Oxytocin is one of the most potent uterotonic hormones; its effect is mediated through specific oxytocin receptors, thus increasing the uterine contractions to expel the puppies through the birth canal. Oxytocin receptor distribution and expression in canine uterus were investigated with immunohistochemistry and qPCR. Progesterone prevents uterine contractions and prostaglandin F2α has a key role in the initiation of parturition. Therefore, the role of progesterone, prostaglandin F2α, as well as, oestradiol and ionized calcium in uterine inertia were investigated. The preparing contractions in the first stage of parturition are not externally visible, and the second stage of normal parturition may last up to 24 hours, which complicates the follow-up of the parturition in the bitch. Human cardiotocography was tested in uterine contraction and foetal heart rate monitoring to improve diagnosis of dystocia. Oxytocin receptor expression and distribution were demonstrated in full-thickness uterine samples in non-pregnant, pregnant, and dystocic bitches; receptors were localized in all bitches in the endometrial luminal, superficial and deep glandular epithelium, in the stroma, and in the longitudinal and circular myometrium. The relative expression of oxytocin receptors was higher in pregnant than in non-pregnant bitches. Progesterone concentrations were significantly higher in samples taken prior to the first stage of parturition in group of elective Caesarean section bitches than in obstructive dystocia or combined dystocia groups. Prostaglandin F2α metabolite concentrations were significantly higher in bitches with normal parturition than in bitches of elective Caesarean section or combined dystocia groups. Ionised calcium concentrations did not differ between the groups and no hypocalcaemia was detected. The absence of progesterone as such cannot be the only stimulation for oxytocin receptor expression in uterus, because the anoestrous basal level progesterone did not upregulate the oxytocin receptors. The gradual decrease of progesterone in late pregnancy is more likely involved in oxytocin receptor upregulation than sudden prepartum drop, because the upregulation of oxytocin receptors seems to occur before the sudden prepartum decline of progesterone. The decline of oestrogens two days prior to parturition may have regulatory role in oxytocin receptor expression. Our results suggest that in complete primary uterine inertia the aetiology is not the absence or downregulation of oxytocin receptors but more likely in the uterine functional level. The cardiotocography complements clinical and ultrasonographic examination of periparturient bitches. It detects uterine contractions and can be used to confirm diagnosis of uterine inertia and to monitor the response to uterotonic medicine treatment. The cardiotocography’s ultrasonographic doppler probe appeared to be sensitive to artefacts, which may compromise foetal distress monitoring.
  • Mattila, Mirjami (Helsingin yliopisto, 2020)
    Listeria monocytogenes is the causative agent of serious food-borne illness, listeriosis. The ability of L monocytogenes to survive and proliferate over a wide range of environmental conditions allows it to overcome various food preservation and safety barriers. To be able to control the risk of L. monocytogenes in the entire food chain, it is important to understand the mechanisms behind the stress tolerance of this pathogen. The aim of this study was to investigate the stress response mechanisms in L. monocytogenes strain EGD-e. Various cellular processes needed for growth and survival under unfavourable conditions are regulated through alternative sigma factors σB and σL. To gather deeper understanding about the role of these sigma factors at low temperatures, this study investigated the regulons of σB and σL, and the expression profile of the ΔsigBL double-mutant strain with a whole-genome expression analysis at 3°C and 37°C. This analysis revealed 198 and 86 genes positively regulated by σB during exponential growth at 3°C and 37°C, respectively. Altogether 29 genes were found to be under positive σB transcriptional regulation during exponential growth at both temperatures. At 3°C, 237 genes were detected to be under positive σL dependent transcriptional control, and at 37°C the number of down-regulated genes was 203. Only 47 of these genes exhibited positive σL transcriptional regulation at both temperatures. Of the 254 genes down-regulated in ΔsigBL at 3°C, 38% were detected also in ΔsigB or ΔsigL, and 62% were present only in ΔsigBL. At 37°C, 87% of the 139 down-regulated genes were present in either ΔsigB or ΔsigL and 13% were detected only in ΔsigBL. It thus appears that the growth at low temperature expands the specialized expression profile of ΔsigBL, but at optimal growth temperature the ΔsigBL expression profile resembles more the expression profiles of ΔsigB and ΔsigL. The phenotypic evaluation of the ∆sigB, ∆sigL, and ∆sigBL mutants revealed impaired growth in the presence of ethanol and organic acids at low growth temperature. The growth of the ΔsigB mutant was compromised in the presence of lactic acid, acetic acid, and ethanol, while both the ∆sigL and ∆sigBL mutants showed impaired growth in the presence of lactic acid, acetic acid, citric acid, and ethanol relative to the wild-type strain. The growth of the ∆sigL and ∆sigBL mutants was also slower at low temperature (4°C) in defined medium relative to the parental strain. In addition, phenotypic microarray analysis exposed significant differences in growth of the wild-type strain and the ∆sigB, ∆sigL, and ∆sigBL mutant strains on different carbon sources and in the presence of various chemical compounds, such as antibiotics targeting cell wall, membrane, DNA, or protein synthesis. Apparently, the deletion in both sigB and sigL increases the sensitivity of the L. monocytogenes towards different stress conditions and chemical compounds and exposes phenotypic characteristics absent from ∆sigB or ∆sigL mutant strains. The stress response mechanisms positively regulated by both sigB and sigL might thus be more relevant than has been previously suspected. Overall, this study provided an expanded insight into σB and σL phenotypic roles and functional interactions in L. monocytogenes. DEAD-box proteins are conserved RNA helicases that can be found in most living organisms. They are needed in RNA metabolism and other metabolic processes and have been linked with cold stress tolerance in L. monocytogenes and many other bacteria. To investigate the role of DEAD-box RNA helicase encoding genes under low growth temperature, transcriptomic and phenotypic analyses of lmo0866, lmo1246, lmo1450, and lmo1722 deletion mutants were performed at 3°C, 25°C, and 37°C. The relative expression levels of lmo0866, lmo1246, lmo1450, and lmo1722 were significantly higher during growth at 3°C than at 37°C and also 30 min, 3 h, and 7 h after cold shock from 37°C to 5°C. At 3°C, the growth of ∆lmo0866 and ∆lmo1722 was totally impaired, and ∆lmo1450 showed only slight growth. The minimum growth temperatures of these mutants were significantly higher compared to the wild-type strain. The results suggest that lmo0866, lmo1450, and lmo1722 play an important role in the cold stress tolerance of L. monocytogenes. The temperature-dependent induction of flagella is a well-characterized phenomenon in L. monocytogenes. However, the essentiality of increased flagellum production during growth at low temperatures is still unclear. The role of flagella synthesis and motility genes flhA and motA in the cold stress tolerance of L. monocytogenes was studied at 3°C, 25°C, and 37°C. The relative expression levels of flhA and motA were found to be higher at 3°C compared to both 25°C and 37°C. The growth of the ΔflhA and ΔmotA deletion mutant strains was compromised at 3°C relative to the wild-type strain. These results illustrate that flhA and motA have a role in the cold stress tolerance of L. monocytogenes, yet the exact functions and regulation of these genes at low growth temperatures remains unknown. Deletion mutant ∆sigL was completely non-motile at 3°C and 10°C, and un-flagellated at 3°C. The ∆sigB and ∆sigBL mutants showed similar swarming pattern compared to the wild-type at 3°C, but the flagella formation of ∆sigBL was slightly compromised at 3°C compared to the wild-type. The cold-sensitive deletion mutant strains ∆lmo0866 and ∆lmo1450 were completely non-motile in BHI at 3°C, while the swarming pattern of the ∆lmo1246 was similar to the wild-type strain. The deletion mutant strains ΔflhA and ΔmotA were completely non-motile at 3°C and 25°C, and flagella formation was deficient. The growth of these strains was also significantly compromised when grown in BHI at 3°C. These findings suggest that the motility and flagella formation may play a role in the cold response of L. monocytogenes.
  • Niinistö, Kati (Helsingin yliopisto, 2020)
    Problems related to equine large colon sand accumulations have been known for at least a century, but evidence-based medicine on the subject remains scarce. The current medical treatments are largely based on case reports and small studies on research horses with artificial sand accumulation or without confirmed accumulation. Therefore, clinical studies on naturally acquired sand accumulation are needed. The main objectives of the present studies were to define clinical signs related to the presence and size of large colon sand accumulations and to determine the optimal way to medically treat horses with these accumulations. A questionnaire study was performed to determine whether certain clinical signs are linked to the amount of sand in the colon and to clarify associations between equine large colon sand accumulations and individual traits and management-related factors. Owners or caretakers of 447 horses that had been radiographed for suspected large colon sand accumulation, responded to an internet based survey on clinical signs and management of their horses. The most common clinical signs of large colon sand accumulation noted by the owner were colic, poor performance and changes in faecal consistency (diarrhoea, increased faecal water). Colic (odds ratio (OR) 2.03, confidence interval (CI) 1.42-2.91) and poor performance (OR 1.89, CI 1.33-2.68) alone were associated with the size of sand accumulation. Horses with combinations of clinical signs, such as diarrhoea and poor performance or colic and diarrhoea also had a higher probability of a large sand accumulation; OR 2.12, CI 1.42-3.44 for diarrhoea and poor performance, and OR 2.64, CI 1.69-4.13 for colic and diarrhoea. Dominant position in the herd was protective for large colon sand accumulation (Spearman rho 0.170, P=0.002), while greediness predisposed the horse to the condition (Spearman rho 0.184, P<0.001). No significant association was detected between the studied management-related factors and large colon sand accumulation. A retrospective clinical study compared the effectiveness of medical treatment of large colon sand accumulation with nasogastric intubation of psyllium and/or magnesium sulphate (MgSO4) (both 1 g/kg bodyweight (BW) once a day for 4 to 7 days, N=170 horses) with feeding psyllium (1 g/kg BW for 2 to 5 weeks, N=57 horses). The daily administration of psyllium and MgSO4 in hospital via a nasogastric tube removed the sand accumulation more efficiently than multiple weeks of feeding with psyllium alone (P<0.05). This finding raised the need to examine the effect of psyllium and MgSO4 further, using a comparative approach in a randomised prospective study. Based on the results of the aforementioned retrospective study, it was hypothesized that the combination treatment with psyllium and MgSO4 would remove more sand than either treatment alone, or no treatment. The effect of psyllium (1 g/kg BW, N=12) or MgSO4 (1 g/kg BW, N=10) alone or combined (1 g/kg BW each, N=12) was evaluated prospectively in hospitalized horses with naturally acquired sand accumulation treated utilising nasogastric intubation once a day for four days. There were significant differences between treatments. The combination of psyllium and MgSO4 removed accumulations over a four-day period more effectively than either medication alone (number of resolved horses: P=0.012 for psyllium, and P=0.03 for MgSO4, respectively). Of 12 horses treated with a combination of psyllium and MgSO4, nine fulfilled the pre-determined criteria for clearance of sand from the ventral colon within four days. In contrast, only 3/12 horses treated with psyllium and 2/10 horses treated with MgSO4 cleared the sand accumulation. Furthermore, the effects of combination of 1 g/kg BW psyllium and 1 g/kg BW MgSO4 in 20 horses were compared with 20 untreated, hospitalized control horses that were solely removed from the sand source. After four days, the treated horses had cleared their accumulation (median area of sand decreased from 250 cm2 to 0 cm2) significantly more (P<0.001) than the control horses (median area of sand decreased from 285 cm2 to 176 cm2). In conclusion, large colon sand accumulations should be considered a differential diagnosis not only when colic, poor performance or changes in faecal consistency are seen, but also when the horse presents with combinations of various clinical signs. The most effective treatment to remove sand accumulations that had developed naturally in the population studied was repeated nasogastric intubation of psyllium and MgSO4.
  • Vennerström, Pia (Helsingin yliopisto, 2020)
    Viral haemorrhagic septicaemia (VHS) was isolated for the first time in Finland in 2000 from a Finnish brackish water fish farm farming rainbow trout in net pens in the Province of Åland, Baltic Sea. The efforts to eradicate the disease from the Åland islands were not successful. Epidemical factors, needed for VHS management in viral haemorrhagic septicaemia virus (VHSV) positive brackish water fish farms, were studied in a 3-year project, the results of which are presented in this thesis. The study compared the ability of four different surveillance procedures and three diagnostic tests to reveal whether a fish population was infected with VHSV. The programme that was conducted as syndromic surveillance, where the farmers sent in samples for diagnostics if any signs of possible fish disease were noticed, clearly outperformed the other three programmes, which were based on active surveillance. A real-time reverse transcriptase polymerase chain reaction method proved to be at least as sensitive in detecting acute VHSV infections as virus isolation in cell culture, which is considered the gold-standard method for diagnosing VHSV. An ELISA method was used to test fish serums for antibodies against VHSV and was found to be a promising tool in VHSV eradication, particularly for screening populations during the follow-up period, before declaring an area free of infection. During the epidemics it was a common suspicion wild fish being the most likely source of the reinfections of VHSV in infected fish farms in the restriction area. Wild fish of 17 different species from VHS-positive fish farms were screened for VHSV during 2005-2008. In addition, uninfected wild perch, roach and farmed whitefish were introduced to a fish farm with rainbow trout experiencing a clinical outbreak of VHS. The wild fish did not test positive on any occasion, but whitefish were infected and started to replicate VHSV for a short time. The replication of the virus in whitefish was verified using a new qRT-PCR method that tests separately for positive- and negative-sense viral sequences in infected organ samples. The presence of VHSV in the environment on fish farms or processing plants farming or handling VHSV-positive fish was studied by testing samples for VHSV from wild blue mussels (Mytilus edulis) living in infected fish farms. Sea water and sediment from infected fish farms were also tested for VHSV. Wild uninfected blue mussels were also challenged with VHSV in two different challenge tests. Wastewater from a processing plant was tested before and after disinfection treatment. Blue mussels were not found to be carriers of VHSV on any occasion. Sea water tested positive for VHSV RNA more often during the wintertime when water temperature was close to 0°C and sunlight (UV light) sparse. Most wastewater samples collected before the disinfection treatment were positive for VHSV, but samples collected after disinfection were all negative regarding VHSV RNA. Contacts between the processing plants and the fish farms in the restriction area of VHS were very common during this study. Processing plants are usually the place where fish food and farming equipment are stored, including boats that are used for the daily servicing of the farming localities. According to the results of this study, this contact was considered a major risk for disease spread, especially during the cold part of the year when daylight is also short. Altogether, this thesis compiles the results of a series of studies targeting factors that could affect the infection pressure of VHSV.
  • Syrjä, Pernilla (Helsingin yliopisto, 2020)
    This thesis aimed at describing the pathology of a novel progressive neurologic disease in dogs of the Lagotto Romagnolo (LR) breed, at testing the hypothesis of altered autophagy in affected dogs, derived from genetic investigations, and at testing the hypothesis of a lysosomal storage disease affecting the dogs, derived from the histopathologic changes. Autophagy is a degradative cellular process, responsible for the turnover of damaged organelles and clearance of protein aggregates, that is disturbed in several neurodegenerative diseases in man. Histologically, altered autophagy commonly leads to neuronal proteinaceous inclusions as well as spheroid formation. Occasionally, neuronal vacuoles have been linked to altered autophagy. Cytoplasmic vacuolation is histologically more typical for lysosomal storage diseases, a group of inherited diseases characterized by defective lysosomal degradation, which often affect the nervous system. Compensatory rerouting of cargo during defective autophagy and lysosomal degradation may affect the content and release of extracellular vesicles (EVs) from cells. Post mortem examination of 13 affected LRs confirmed a distinct histopathological phenotype, consisting of intracellular vacuole storage in neurons, axonal damage and neuronal loss. Secretory epithelial cells and selected mesenchymal cells were also affected by vacuolation. In order to link the histopathologic findings to altered autophagy, affected organs and age- as well as breed matched control tissue, were immunohistochemically stained with markers for autophagosomal, lysosomal and endosomal membranous antigens, autophagy receptors and autophagic cargo. Electron microscopy and immunoelectron microscopy with the same membranous markers were used in order to obtain more specific results regarding the subcellular morphological changes. Autophagy was functionally monitored by the LC3 I/II conversion assay and by quantifying autophagosomes with immunofluorescence in cultured fibroblasts from affected dogs in comparison to control. The screening for a lysosomal storage disease included urine analysis to detect excessive oligosaccharide or glucosaminoglycan excretion. Histochemical staining and electron microscopy were used to examine the affected tissues for lysosomally stored material. In addition, the activity of three lysosomal enzymes were measured in fibroblasts from affected dogs and controls, as well as in culture medium, in order to identify alterations in lysosomal function. The size, amount and cargo of EVs released from fibroblasts of affected dogs were compared to those released from control cells, in order to detect changes in EV release or content that could support or contradict either hypothesis. Nano-particle tracking analysis of EVs, as well as mass spectrometry and functional enrichment analysis of the EV proteomes were performed. Both autophagosomal markers and autophagic cargo accumulated within the lesions of the nervous system in affected dogs. The stored vacuoles in affected tissues represented hybrid organelles of the autophagic and endolysosomal pathways, displaying membranous markers of both pathways. Fibroblasts showed changes in basal, but not starvation-induced autophagy, as both basal LC3II levels and basal number of LC3 positive spots were significantly elevated. In addition, the basal EV release was significantly increased and the basal EV proteome enriched in substrates of basal autophagy in the cells of affected dogs in comparison to control cells. These findings suggest altered basal autophagy as a pathogenetic mechanism in this novel disease. Lysosomal storage of a specific material or a lysosomal enzyme deficiency were not detected. The genetic groups in the collaborative research where this thesis was part discovered a novel candidate gene for neurodegeneration, ATG4D. In aspects of comparative pathology, altered basal autophagy was uncovered as an additional etiology and morphological differential diagnosis for cytoplasmic vacuolation reminiscent of lysosomal storage diseases in dogs, and functionally linked to the ATG4D variant through this thesis. The importance of basal autophagy in cells was also hereby shown, as disease occurred despite starvation-induced autophagy being functional in the dogs. For veterinary medicine, the characterization of this novel inherited progressive neurodegenerative disease provides a broader scientific knowledge as basis for veterinarians regarding prognostication of affected dogs, and for LR owners regarding breeding.
  • Törnqvist, Heini (Helsingin yliopisto, 2020)
    Canine cognition has been widely studied especially with behavioral methods. Behavioral studies have shown that dog’s social cognitive abilities are similar to preverbal human infants, and that dogs are excellent readers of human communicative gestures. However, behavioral studies cannot determine the cognitive processes and neuronal functions underlying the behavior. In addition, direct comparisons between humans and dogs have been rarely used in previous studies. The aim of this thesis was to evaluate the feasibility of two novel non-invasive methods of examining dog social cognitive functions, and also to compare human and dog cognitive abilities with eye gaze tracking. The feasibility of non-invasive electroencephalography (EEG) and eye gaze tracking in dog cognitive studies were studied in experiments I–IV. In an EEG experiment, the visual event-related potentials (ERPs) were measured while dogs were watching human and canine facial images. In the eye tracking experiments fixations and saccades towards the stimulus images were measured. Experiment I confirmed, for the first time, the usability of completely non-invasive EEG measurement in intact fully alert dogs. The early visual ERPs were detected at 75–100 ms from the stimulus onset. Experiments II–IV verified the feasibility of the eye tracking method in dogs. Experiment II showed that dogs preferred facial images of dogs and humans over inanimate objects. In experiment III, comparisons between the eye movements of humans and dogs revealed that both dogs and humans gazed longer social interaction images than non-social images. However, dogs gazed longer human interaction images and humans gazed longer at dog interaction images, which indicates that processing social interaction of another species might take more time. Also, family dogs gazed at images longer than kennel dogs, suggesting that kennel dogs’ limited social environment might have affected their processing of social stimuli. Experiment IV explored dogs’ gazing behavior towards images containing dogs, humans and wild animals. This study showed that dogs focused their gaze at living creatures and especially gazed at the biologically informative areas in the images, such as the head area. In conclusion, EEG and eye tracking are promising methods for studying dog cognition, and eye tracking can be used to compare responses between humans and dogs. EEG and eye tracking studies showed that dogs were focusing on the objects in the images and their gazing behavior depended on the image category. These studies highlight the importance of facial information to dogs, and also reflect their excellent skills in comprehending social and emotional cues in both conspecifics and non-conspecifics.
  • Mäkitaipale, Johanna (Helsingin yliopisto, 2020)
    The main function of vitamin D is the maintenance of serum calcium concentration and skeletal calcium balance. Diet, vitamin D supplements, and endogenous synthesis are possible sources of vitamin D in pet rabbits, but studies regarding the vitamin D concentrations, main sources of vitamin D, and vitamin D deficiency levels in rabbits are lacking. Vitamin D deficiency and the existence of metabolic bone diseases in pet rabbits are topics of intermittent debate. The question regarding the existence of metabolic bone diseases in rabbits arose 20 years ago, after decreased bone density was observed in prepared skulls and radiographs of pet rabbits with advanced stages of dental disease. Dental disease is still one of the most common health disorders in pet rabbits. Observations that rabbits with dental disease had higher parathyroid hormone and lower total calcium concentrations compared to rabbits in free-range conditions, and that pet rabbits housed in hutches had low 1,25-dihydroxyvitamin D3 concentrations, were associated with the theory of vitamin D and calcium deficiency as a common problem in pet rabbits, and a possible predisposing factor for dental disease. This thesis aimed to study serum 25-hydroxyvitamin D (25(OH)D) concentrations and sources of vitamin D in 140 pet rabbits aged 0.1 to 9.3 years. Several tibial bone parameters, including cortical bone density, were measured in 87 rabbits using peripheral quantitative computed tomography. Possible breakpoints were searched in 139 rabbits for serum 25(OH)D concentration when compared to parathyroid hormone concentration and cortical bone density, which indicates a threshold for vitamin D deficiency. Dental disease was diagnosed after examination of the oral cavity and a lateral skull radiograph. Association between dental disease and serum 25(OH)D concentration and tibial bone parameters were compared. Serum 25(OH)D concentration varied from 4.5 to 67.5 ng/ml (mean 26.0 ng/ml). According to GLM analysis adjusted for body weight, age, and season of sample collection, diet was significantly associated with serum 25(OH)D concentration, but outdoor access was not. Serum 25(OH)D concentration was higher in rabbits that received diets with lots of hay and commercial rabbit food, >1dl daily, compared to rabbits with lower amounts of these vitamin D–containing diets. The suppression of parathyroid hormone concentration occurred at a serum 25(OH)D concentration of 17 ng/ml, whereas the breakpoint for cortical bone density occurred at a serum 25(OH)D concentration of 19 ng/ml. No breakpoints were found for ionised calcium, total calcium, or phosphorus. Results suggest a serum 25-hydroxyvitamin D concentration of 17 ng/ml as a threshold for vitamin D deficiency in pet rabbits. One-third of the rabbits participating in this study had serum 25(OH)D concentrations below this threshold, which raises concern regarding the high prevalence of vitamin D deficiency in Finnish pet rabbits. The mean diaphyseal tibial cortical density in rabbits was high (about 1400 mg/cm3). There was no tendency for an age-related decrease in trabecular or cortical bone density, at least up to six years of age. No statistically significant group differences were observed in bone parameters between intact females and males or between intact and castrated males after controlling for body weight and age. Of 140 rabbits, 47 (33.6%, 95% CI: 25.5-41.7) were diagnosed with dental disease. Bone parameters were measured from 87 rabbits, of which 26 rabbits (29.9%, 95% CI 20.3-39.5) had dental disease. After controlling for body weight, age, and season of sample collection, no differences existed in serum 25(OH)D concentrations between healthy rabbits and rabbits with dental disease. Additionally, no statistically significant group differences in tibial bone parameters occurred between healthy rabbits and rabbits with dental disease after controlling for body weight and age. Our results conclude that vitamin D deficiency, diagnosed as serum 25(OH)D concentration below 17 ng/ml, is common in Finnish pet rabbits. Diet is the main source of vitamin D, as outdoor access is too limited to provide them with adequate vitamin D synthesis. Vitamin D deficiency may increase their risk for metabolic bone disease and other health disorders. This study failed to support the theory of vitamin D deficiency as a predisposing factor for dental disease. The results of this study should be used to increase pet rabbits’ health and well-being by preventing vitamin D deficiency and should advocate for further studies regarding the supply of adequate concentrations of vitamin D and consequences of chronic vitamin D deficiency in pet rabbits. Further prospective case-control studies are needed to evaluate the aetiology of dental disease.
  • Ala-Kurikka, Eve (Helsingin yliopisto, 2020)
    Lameness of sows is a very common problem in the modern pig industry. It impairs both health and welfare of sows and production economics of farms. Lameness is a clinical sign caused by various problems associated with the locomotor system. However, the exact cause of lameness often remains undetermined and lame sows do not always get the necessary attention and treatment. The overall aim of this thesis was to gather information and add to the scientific knowledge on sow lameness. The specific aims of the studies were to investigate pathological-anatomical findings in the locomotor system of euthanized and spontaneously dead sows, to examine the effect of oral ketoprofen in treatment of lameness, and to study the effect of lameness on behavior of the sows. The pathological-anatomical findings in the locomotor system were examined in a descriptive study. The convenience sample consisted of 38 euthanized and 27 spontaneously dead sows from 15 Finnish farms. The majority (91%) of sows had one or more lesions in their locomotor system. The most common primary pathological-anatomical diagnosis (PAD) was inflammatory arthritis, with a prevalence of 9%. Degenerative joint disease (DJD) was diagnosed in 71% of the animals. DJD was the most frequent incidental finding, and its association with lameness remains in question. Lameness was however the most commonly reported clinical sign observed by the farmer within 30 days preceding death. The effect of oral ketoprofen in treatment of non-infectious lameness was evaluated in a randomized, double-blinded, controlled field trial. Altogether 141 lame sows were allocated to three groups: two dose rates of oral ketoprofen (2 mg/kg and 4 mg/kg) were compared with placebo treatment over five consecutive days. Lameness was assessed using a five-grade scoring system prior to and on the last day of the treatment. Treatment of lameness was considered successful for 54% of the sows in the ketoprofen 4 mg/kg group, for 53% in the ketoprofen 2 mg/kg group and for 21% of the sows in the placebo group. Parallel with the above-mentioned study, a randomized, double-blinded, controlled field trial was conducted to evaluate the effects of lameness and ketoprofen treatment on behavior of sows. Thirteen lame sows were allocated to treatment groups and paired with a healthy, non-lame control sows. Behavior was observed using a scan-sampling method prior to and after the treatment. Before the treatment on day 0, lame sows were observed to be more passive, lie down more and stand and explore pen fixtures less than the healthy control sows. On day 5 of the treatment, the behavior of sows with relieved lameness did not differ from that of healthy control sows, indicating that these sows had most likely been in pain before treatment. Some behavioral differences were apparent between sows with relieved and non-relieved lameness, showing that the latter sows were still in pain. The sows with non-relieved lameness were in contact with the wall and lay down significantly more often and moved and stood less than healthy control sows on day 5 of the treatment. In conclusion, pathological-anatomical findings in the locomotor system were very common in on-farm-dead sows. In future, more research is needed to evaluate the clinical relevance of these diagnoses and their association to lameness. Oral ketoprofen proved to be an effective treatment in alleviating the non-infectious lameness of sows. The differences in behavior between lame and healthy control sows and also between the ones with relieved and non-relieved lameness indicated that most of the lame sows were in pain and that the treatment of lameness is important from the animal welfare point of view.
  • Haen, Silke (Helsingin yliopisto, 2020)
    The hypothalamic-pituitary-gonadal axis is the endocrinological pathway of reproduction, where fertility is adjusted and fine-tuned with respect to intrinsic and extrinsic information. The pituitary gland receives the information that determines secretion of luteinizing hormone (LH) from the hypothalamus in the form of gonadotropin releasing hormone (GnRH), it is the frequency and amplitude of the GnRH pulses that regulate LH release. LH transports the information from the brain to the gonads, which feeds back to the hypothalamus and the pituitary gland in the form of steroids. This thesis work focused on the orchestration of GnRH, LH and progesterone during the early pregnancy of the pig. Therefore, gonads refer to the ovary with their functional bodies, the corpora lutea (CLs) and their principal steroid, progesterone. CLs form from the follicles after ovulation and they are either maintained, and pregnancy continues, or they regress, enabling follicle growth and new ovulations and a new opportunity for reproduction. The corpus luteum is independent of pituitary LH support for the first eleven days after ovulation. Subsequently the maternal organism is informed of the presence of embryos and the need to maintain the CLs. Thus, dependency of the CLs on LH begins at the same time as the sow receives the positive signals that indicate pregnancy. Progesterone is the main hormone of pregnancy and its concentration is highest around the time of maternal recognition of pregnancy. LH supports the CLs, but it remains unclear for exactly how long during pregnancy the LH stimulus directly triggers secretion of progesterone. The objective of this thesis work was to study the relationship between the two hormones, LH and progesterone, during early pregnancy in pigs. Firstly, we investigated the physiological release of LH and ovarian progesterone in inseminated gilts on Day 11 after oestrus, before the CLs are thought to be dependent on LH. Secondly, we looked at the pattern of progesterone release after the CLs had become dependent on LH. We studied the pattern of progesterone release from the ovary and were interested to ascertain if that pattern is related to the LH pattern. To explore a possible causal relationship between LH and progesterone further, a GnRH agonist model was used to nullify LH pulsatility around implantation of embryos. Thirdly, we were interested to know if parity, sow age and maturity affect the relationship between the two hormones. In our studies, a catheter was inserted through the vena saphena lateralis, enabling blood sampling in the vena cava caudalis next to the venous drainage of the ovary and uterus. LH and progesterone concentration were assessed after frequent blood sampling for 8–12 hours on Day 11 after oestrus in inseminated gilts, and on Days 16 and 21 in pregnant gilts. A slow-release GnRH agonist was inserted into pregnant gilts on Day 11 and LH and progesterone were studied on Day 16 and Day 21. Additionally, a vena jugularis catheter was inserted into pregnant primiparous sows and progesterone concentrations in local (vena cava caudalis) and peripheral (vena jugularis) blood samples were compared on Day 14. In gilts that were inseminated and classified post mortem whether they were pregnant or not, the progesterone release pattern measured in the vena cava caudalis had similar basal and mean progesterone concentrations, and frequency and amplitude of pulses on Day 11. On Day 11 and Day 21 of pregnancy, there was no relation between LH pulsatility and progesterone pulsatility. In gilts that were treated with a slow-release GnRH agonist on Day 11, LH pulsatility ceased but progesterone release remained pulsatile on Day 16 and Day 21 of pregnancy. On Day 21 we found that LH pulsatile release was active when progesterone release was basal. In primiparous sows, a progesterone pulse followed 60.8% of LH pulses within one hour. Mean progesterone concentration was approximately twice as high in the vena cava caudalis than in the vena jugularis in primiparous sows and differed significantly before and after feeding in the vena jugularis. GnRH-agonist-treated gilts had an elevated progesterone concentration on Day 21 compared with the control gilts. Progesterone concentration declined from Day 11 via Day 16 to Day 21 in gilts. LH pulse amplitude declined during those days, but no decline in mean or basal LH concentration or LH pulse frequency was recorded. LH pulsatility ceased on Day 16 and Day 21 in gilts treated with the GnRH agonist. LH release was synchronized in such a way that gilts under the same management conditions exhibited LH pulses around the same time during twelve hours, indicating a synchronized rhythm of secretion. We observed that progesterone release of CLs is similar in non-pregnant gilts and pregnant gilts on Day 11, before maternal recognition of pregnancy. During early pregnancy in gilts the pulsatile progesterone release of CLs on Days 11 and 16 is not responsive to – and on Days 16 and 21 it is not dependent on – LH pulsatile secretion. On Day 21 there is an alternate pattern of activity in pulsatile release of LH and progesterone in individual gilts. In primiparous sows, we found a temporal relationship between LH pulses and progesterone pulses already on Day 14 of pregnancy. We conclude that the progesterone release of the CLs functions without LH pulsatile stimulus from Day 11 to Day 21, but associations of these hormones at an individual level are evident.
  • Argano, Martina (Helsingin yliopisto, 2020)
    The impact of perioperative factors on metastatic spread subsequent to surgery for primary cancer removal is of rising interest. Several studies show that anaesthesia for primary cancer surgery might impact cancer recurrence by influencing cell proliferation rate and regulating the expression of specific oncogenes like neuroepithelial transforming gene 1 (NET1), which has been associated with malignant behaviours and represents a novel prognostic marker in human epithelial cancers. This project investigates the effects of different concentrations of clinically available propofol and sevoflurane formulations on canine mammary tumour cell proliferation and on the expression of the NET1 gene in three prospective controlled in vitro trials. Primary (CIPp) and metastatic (CIPm) canine tubular adenocarcinoma cell lines were incubated with sevoflurane (1, 2.5 or 4 mM), propofol (1, 5 or 10 μg mL-1) or cell culture medium (control). Proliferation was assessed after 6 hours of sevoflurane and 6 - 12 hours of propofol exposures, while NET1 expression was evaluated after 6 hours of sevoflurane and 6, 12, 24 and 48 hours of propofol exposures. After 6 hours of propofol exposure, cell proliferation significantly increased in CIPp, while after 12 hours cell proliferation significantly increased in CIPp and decreased in CIPm. After 6 hours of exposure, propofol significantly augmented NET1 expression in CIPp, whereas in CIPm the highest propofol concentration significantly reduced gene expression. Propofol significantly decreased gene expression after 12 and 24 hours of exposure. No significant differences were found in CIPm after 48 hours, while the highest concentration of propofol significantly increased gene expression in CIPp after 48 hours. Sevoflurane significantly increased cell proliferation in CIPp, while it significantly decreased cell proliferation in CIPm. The NET1 gene expression was significantly increased in CIPm after exposure to the highest sevoflurane concentration. In conclusion, exposure to both propofol and sevoflurane mainly augmented proliferation in primary cells and diminished proliferation in metastatic ones. Conversely, propofol mostly decreased NET1 expression, while sevoflurane increased NET1 expression in metastatic cells exposed to high concentrations. A profound comprehension of the molecular mechanisms underlying cancer spread is necessary, however, the much more variable environment in the clinical setting makes direct translation from in vitro to in vivo conditions difficult. Even though the lack of similar trials in veterinary medicine limits the chances for comprehensive comparisons, some promising results were observed in our study. Future projects assessing the influence of anaesthetic techniques on cancer recurrence are warranted.
  • Savolainen, Saila (Helsingin yliopisto, 2020)
    Cats are increasingly popular companion animals, and their owners are willing to provide good-quality health care for their pets. However, while cats are considered to be difficult to medicate with orally administered pharmaceuticals at home, studies on medication compliance in cats have been scarce. Palatable pharmaceuticals would most likely increase compliance in feline medication, and the pharmaceutical industry would prefer synthetic flavours over organic ones. Cats’ taste receptors are sensitive to amino acids, and certain amino acids are considered to be palatable to cats. The methods of testing the palatability of pharmaceuticals in cats have varied. The objective of the present thesis was, firstly, to investigate the difficulties encountered by owners when medicating their cats with orally administered pharmaceuticals at home. Secondly, the aim was to find solutions for making feline oral medication easier by focusing on the development of methods for testing the palatability of pharmaceutical products in cats, in addition to screening synthetic flavours for future taste masking of feline pharmaceuticals. Furthermore, behavioural indicators were investigated to distinguish between a palatable food item, an unpalatable food item, and a palatable food item containing a pharmaceutically inactive placebo mini-tablet in pet cats. As a result of the included online survey, the compliance in feline medication was 76%, and most of the difficulties seemed to be related to the aversive taste of the pharmaceutical. Off-label use of pharmaceuticals was common, as approximately half of the medications were registered for use in cats. Pharmaceuticals registered for feline medication were significantly easier to administer to the cats than other pharmaceuticals. However, the voluntary acceptance rate was low. Most of the owners preferred a solid formulation for medicating their cat, and a mini-tablet was therefore selected to be used in the palatability trials. The acceptability of flavoured, pharmaceutically inactive mini-tablets was tested in pet cats with a rapid three-portal acceptance test. If the mini-tablets were not accepted voluntarily from a table or a hand, they were offered to the cat hidden inside a palatable food item. The acceptance of a placebo mini-tablet was also tested as administered inside a palatable food item. The synthetic flavours tested included amino acids, which were chosen based on the literature and because they are meat precursors, as well as synthetic meat aromas and d-(+)-maltose monohydrate, which is the predominant sugar in Cat Malt -products, in addition to thiamine hydrochloride, which is found in large amounts in yeast. The synthetically flavoured mini-tablets were not palatable to the cats, as the cats refused to consume the mini-tablets if the tablets were not hidden inside a palatable food item. Five behavioural patterns differentiated palatable food from unpalatable food: 'flick ears backwards', 'lick nose, not eaten', 'flick tail' and 'groom body' were more frequent with unpalatable food, whereas 'lick lips' was more frequent with palatable food. One indicator, 'drop item', was more frequent with palatable food containing the mini-tablet than with palatable food alone, indicating that the mini-tablet was not fully acceptable to the cats; or it is possible that the mini-tablet fell out more easily from some types of food. The feasibility of the palatability tests carried out with pet cats in their home environments was verified by the high inter-observer agreement. In conclusion, palatable and easily administered oral pharmaceuticals are needed for feline medication. Unfortunately, no promising synthetic flavours were identified for the taste masking of feline pharmaceuticals, and more investigations are required to discover synthetic flavours that are palatable to cats. The results of the palatability trials differed from the earlier literature as regards the taste preferences of cats concerning amino acids. However, the mini-tablets were acceptable by most of the cats when hidden inside a palatable food item. The investigated behavioural indicators and methods for testing palatability could be used in future palatability studies carried out in pet cats.
  • Kontturi, Miia (Helsingin yliopisto, 2020)
    Interdigital phlegmon (IP) is an infectious hoof disease of cattle. Typically, it causes severe clinical signs, such as lameness, and hence impacts cattle welfare. IP has been a well-known disease of both dairy and beef cattle all over the world for decades. In general, IP occurs as sporadic infections. Lately however, outbreaks of IP have been detected in dairy herds in Finland. Most of these outbreaks have occurred in recently built or renovated free stall barns. In these outbreaks morbidity in IP has been substantial, which has led to the extensive use of antimicrobials and heavy financial losses associated with affected herds. The aim of this thesis was to investigate the characteristics of the IP outbreaks in Finnish dairy herds and to explore the morbidity, clinical manifestation and degree of inflammation of the affected animals in these outbreaks. Moreover, we aimed to investigate the bacteriology in IP, i.e. to investigate several bacteria detected earlier from hoof diseases. An additional objective was to identify herd level risk factors behind these outbreaks. This thesis is based on three studies. The first two were observational, cross-sectional studies, which were performed on commercial free stall dairy herds. The majority of the herds suffered from an outbreak of IP and three herds were unaffected control herds. Altogether 100 cows with IP were clinically checked, diagnosed, and sampled for the bacteriological culture and PCR, and analysis of acute phase proteins; serum amyloid A, haptoglobin and albumin. Cows with other hoof diseases and control cows were sampled similarly for comparison. The third study was a survey of free stall dairy herds of ≥50 cows. We sought general herd data, barn characteristics, herd management details, and asked questions about leg and claw health of the herd. Based on the replies, the risk factors for an outbreak of IP to occur were investigated among farms that had experienced an outbreak and farms that had not. Fusobacterium necrophorum ssp. necrophorum is the main IP pathogen. The most common finding in IP samples in the early, acute stage was a combination of F. necrophorum and Dichelobacter nodosus. Trueperella pyogenes was frequently associated with IP at the later, healing stage. However, various bacterial combinations existed in IP samples. In outbreak herds, the morbidity was either high (≥50%), or moderate (9 – 33%), and no herd had intermediate morbidity. Strong acute phase response was detected among IP cows in the early stage of the disease; the values for serum amyloid A and haptoglobin were clearly elevated, and albumin decreased in comparison with the case for other hoof diseases or control cows in our study. The acute phase response was even greater in herds of high morbidity and with a bacterial combination of F. necrophorum and D. nodosus. The possible herd level risk factors for an outbreak of IP to occur were animal movement between herds, i.e. animal purchase or contract heifer rearing, enlargement of the barn within three years, and fields under organic farming. Mechanical ventilation in the barn seemed to lower the risk. Moreover, herds that had experienced an IP outbreak more often had other infectious hoof diseases. Based on our study results, the same cow may have several hoof diseases and a thorough clinical inspection is essential in diagnosing IP, also during an IP outbreak. Furthermore, IP causes severe clinical signs and a very strong APR. Thus, an anti-inflammatory should be included in the treatment of affected animals. Even though F. necrophorum is the key pathogen in IP, in the disease process several other bacteria play a role, such as D. nodosus, which may affect the severity of IP. To lower the risk of an IP outbreak, new cattle should be purchased very cautiously, if at all, and enlargement of the barn should be constructed without undue restrictions being placed on time and labour inputs.
  • Jaakkonen, Anniina (Helsingin yliopisto, 2020)
    Cattle are commonly asymptomatic carriers of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter jejuni, which cause gastroenteritis in humans. Especially STEC infections may lead to severe or fatal consequences. Both STEC and C. jejuni are intermittently shed in cattle feces and can contaminate bulk tank milk via fecal contamination during milking. These bacteria are effectively eliminated from milk by pasteurization, but the consumption of unpasteurized milk, or raw milk, poses a risk of infection. In recent years, the consumption of raw milk has become more popular, with public demand to relax legislation that restricts sales of raw milk. However, on-farm epidemiology of these pathogens have warranted further investigation to support the development of on-farm risk management practices and pathogen monitoring of dairy farms that sell raw milk to consumers. These studies investigated a milkborne outbreak caused by STEC (Study I) and obtained longitudinal data on the contamination of bulk tank milk by STEC and C. jejuni, and explored on-farm contamination routes of these pathogens (Study II). Furthermore, the studies revealed strain characteristics of C. jejuni that may affect survival and persistence of this pathogen in milk (Study III). The occurrence of STEC and C. jejuni, or C. jejuni alone, was determined in bulk tank milk, in-line milk filters of the milking machine, cattle feces, and the farm environment on four dairy farms (STEC and C. jejuni in Studies I and II) or one dairy farm (C. jejuni in Study III). STEC and C. jejuni isolates from the dairy farms were further subjected to phenotypic characterization and whole-genome sequencing, followed by comparative genomic analyses to explore gene contents and phylogenetic relationships between the isolates. Furthermore, questionnaire data were collected to trace back the outbreak source (Study I) and to determine on-farm risk factors associated with milk contamination using a logistic regression model (Study II). Ultimately, the results contributed to the revision of the Finnish legislation that restrict the sales of raw milk in 2017 (Study II). Study I elucidated the reservoirs and transmission routes of atypical, sorbitol-fermenting (SF) STEC O157, which have largely been unknown. The study presents microbiological and epidemiologic evidence that an outbreak of SF STEC O157 with 11 cases originated from a recreational farm housing dairy cattle and was transmitted via the consumption of raw milk. Thus, these results strongly support bovine origin of SF STEC O157. In longitudinal monitoring (Study II), one clone of STEC O157:H7, which represented a bovine-associated lineage, was simultaneously isolated on each of the three dairy farms. STEC O157:H7 persisted in two herds for up to 12 months, and a similar but distinct clone was reintroduced in one herd 2.5 years after the previous detection. These results support evidence that few STEC O157:H7 clones persist on-farm simultaneously. Unlike STEC, both persistent and numerous sporadic C. jejuni strains appeared simultaneously on dairy farms (Studies II and III). Persistence for 11 months or longer was associated with a few C. jejuni genotypes, especially the host generalist sequence type (ST) ST-883. C. jejuni of ST-883 outperformed other STs in environmental fitness, representing the only ST that could be isolated from bulk tank milk and milk filters and the dominant ST found from environmental samples. Therefore, ST-883 imposes a higher contamination pressure on milk than other STs among the farm isolates and represents a candidate for on-farm risk-based monitoring. In the longitudinal monitoring, STEC was rarely isolated from bulk tank milk and milk filters and only simultaneously with fecal isolation. Higher detection rates were obtained from milk filters than milk by both culture methods and real-time PCR. Therefore, milk filters are more reliable sampling targets for monitoring of STEC than milk. Isolation of C. jejuni from milk and milk filters was associated with C. jejuni clone rather than sample material, but the isolation rates of C. jejuni appeared generally poor. To enhance the isolation rates for monitoring purposes, the sampling regime also warrants further consideration. Reduced milk contamination by STEC was associated with on-farm practices: pasturing and culling of dairy cows and rigorous cleansing in the barn. Higher outdoor temperatures were associated with increased milk contamination. In Study III, C. jejuni of ST-883 persistently contaminated bulk tank milk of a dairy farm for seven months or longer after having caused a milkborne outbreak. Although ST-883 survived in refrigerated raw milk longer than other STs from the same farm, the persistence of ST-883 in bulk tank milk was likely affected by other phenotypic traits such as biofilm formation. Outbreak strain of ST-883 reversibly adapted to survival in bulk tank milk, showing biofilm formation in an on/off manner among replicate cultures and cellular heterogeneity by phase variation in genes related to capsule and oxidative stress response. Furthermore, the outbreak strain harbored a pTet-like genomic element, which may have contributed to higher biofilm quantities. This study identified candidate phenotypic and genotypic mechanisms affecting survival and persistence of C. jejuni in milk. Taken together, STEC and C. jejuni can persist on dairy farms for months or longer and contaminate bulk tank milk despite stringent on-farm hygiene measures. Although these measures cannot totally prevent milk contamination, they likely reduce the contamination pressure on milk. Therefore, cost-effective hygiene measures should be applied on all farms that sell raw drinking milk to consumers. Detection of pathogens from milk may be challenging, and milk may also be contaminated by highly virulent STEC and C. jejuni strains that show atypical phenotype, increasing their environmental endurance or hampering their detection. Therefore, only heat treatment of raw milk before consumption can adequately assure its food safety.
  • Turunen, Heta (Helsingin yliopisto, 2020)
    α₂-Adrenoceptor agonists, such as medetomidine and dexmedetomidine are considered to produce reliable sedation, muscle relaxation, and antinociception when used as sedatives or preanaesthetics. One important advantage is that their effects can be reversed with an α₂-adrenoceptor antagonist, atipamezole. However, notable cardiovascular depression associated with α₂-adrenoceptor agonists limit their use to healthy animals. A peripheral α₂-adrenoceptor antagonist vatinoxan (also known as MK-467 and L 659´066) has been shown to reduce these cardiovascular disturbances, without markedly affecting sedation after concomitant use with dexmedetomidine or medetomidine. Vatinoxan is therefore proposed to improve the clinical utility of α₂-adrenoceptor agonists in dogs. The main aim of this series of experiments was to investigate the reduction of medetomidine-induced cardiovascular changes with vatinoxan, atipamezole and ketamine, not only in laboratory dogs, but also in healthy client-owned dogs of various breeds in a clinical environment. The secondary aim was to assess the quality of sedation, hypnosis and recovery when these drugs interacted together. Two of the studies were performed under laboratory conditions with eight purpose-bred beagle dogs that were instrumented prior to each experiment. Heart rate, mean arterial pressure, central venous pressure, cardiac output and arterial blood gas partial pressures were measured and sedation was assessed. Blood samples were collected for plasma drug concentration analyses. Study Ⅰ aimed to investigate the ability of atipamezole to reverse the cardiovascular and sedative effects of medetomidine in the presence or absence of vatinoxan. Study Ⅱ aimed to explore the influence of vatinoxan on the cardiovascular function and quality of anaesthesia and recovery, when the dogs were premedicated with medetomidine and butorphanol, followed by ketamine, and subsequent reversal with atipamezole. In study Ⅲ the clinical utility of vatinoxan was then evaluated in client-owned dogs, sedated with medetomidine and butorphanol combination for non-invasive diagnostic imaging. The need for atipamezole reversal was also monitored. Plasma concentrations of the studied drugs were determined in each study to detect the influence of vatinoxan. In study Ⅰ, atipamezole failed to permanently increase heart rate or cardiac index without the presence of vatinoxan when administered intramuscularly 30 minutes after medetomidine. Momentary decrease in mean arterial pressure was observed shortly after atipamezole administration with and without vatinoxan. However, no clinically relevant hypotension was detected. Atipamezole reversed the sedative effect of medetomidine more efficiently with than without vatinoxan, probably due to increased plasma clearance of medetomidine. Relapse into sedation after intial arousal was observed during recovery when vatinoxan was not included in the treatment. The medetomidine-induced increase in systemic vascular resistance index was attenuated by vatinoxan, with significantly lower values observed throughout the studies Ⅰ and Ⅱ in its presence. Medetomidine-evoked bradycardia and decrease in cardiac index were similarly mitigated by vatinoxan in all of the present studies. In study Ⅱ, heart rate and cardiac index increased temporarily after ketamine administration and the effect was potentiated by vatinoxan. Ketamine decreased mean arterial pressure and mild hypotension was detected in two out of eight laboratory dogs that were given vatinoxan premedication. Hypotension was abolished by atipamezole administration at 60 minutes following ketamine. Vatinoxan reduced the exposure to ketamine. Therefore, the duration of anaesthesia was shorter in the presence of vatinoxan. The quality of recovery after atipamezole was impaired by nausea and increased need to defaecate with and without vatinoxan. In study Ⅲ, intramuscular medetomidine-butorphanol combination with and without vatinoxan provided reliable sedation for diagnostic imaging procedure. Vatinoxan hastened and increased the peak plasma concentrations of intramuscularly co-administered medetomidine and butorphanol. The clinically observed benefit was a faster onset of deeper sedation. Mean heart rate in the treatment group with vatinoxan during the study was approximately 50% higher than in the group without vatinoxan. Atipamezole was required less frequently to reverse medetomidine-evoked sedation when vatinoxan was included in the treatment because of shorter duration of sedation. To conclude, the two α₂-adrenoceptor antagonists, vatinoxan and atipamezole, interacted favourably in medetomidine-sedated dogs. Together they provided more complete reversal of medetomidine’s effects. While medetomidine-induced cardiovascular changes were transiently attenuated by ketamine and atipamezole, more complete improvement in haemodynamics was observed only in the presence of vatinoxan. Although vatinoxan premedicated dogs were more prone to hypotension during ketamine anaesthesia, heart rate and cardiac index were well maintained. The clinical utility of vatinoxan with medetomidine and butorphanol was demonstrated in a clinical trial. Vatinoxan alleviated the medetomidine-induced bradycardia and the drug combination including vatinoxan provided adequate sedation to complete short, non-invasive procedures. Faster decline in the plasma concentrations of co-administered drugs in the presence of vatinoxan was clinically manifested as shorter duration of sedation and anaesthesia when compared to treatment without vatinoxan.
  • Telkänranta, Helena (Helsingin yliopisto, 2020)
    Tail biting is one of the most important animal welfare problems in commercial pig farming. Its severity varies from scratched skin to partially or entirely eaten tails. A similar though less studied problem is ear biting. Tail and ear biting are abnormal behaviours with multifactorial origins. One of the main factors is a scarcity of materials to fulfil pigs’ innate need to chew and root, which causes some pigs to redirect these needs at other pigs. Of the materials readily available in large enough quantities for farmers, a substantial layer of straw on the floor is considered best to fulfil these needs and to reduce or prevent tail biting. However, such use of straw is rare in intensive farming because the low consumer price of pork has led commercial farms minimising production costs, and one of the consequences is that most farms use slatted or partly slatted pen floors and manage the manure as slurry, which reduces labour costs but precludes the use of straw bedding. Various objects are used on farms as potential outlets for pigs’ needs for oral-nasal manipulation, but their ability to reduce tail biting and to sustain pigs’ interest and to reduce tail and ear biting vary widely. The main aim of the four studies presented in this thesis was to investigate the efficacy of selected materials and object designs in reducing tail and ear biting damage in intensively farmed pigs kept on slatted floors. The studies were carried out on four commercial farms in Finland, and data were collected from a total of 2064 pigs. In each of the studies, experimental treatments represented materials and object designs selected or developed by the experimenters, and control treatments represented the materials and object designs normally used on the farm in question. In Study I, the main research question was whether pre-weaning exposure to sisal ropes and paper would reduce post-weaning tail biting. In the control treatment, 29 farrowing pens were furnished with a suspended plastic ball, and a small amount of wood shavings were provided twice a day. In the experimental treatment, 30 farrowing pens were furnished with the above and in addition ten pieces of suspended sisal rope, and sheets of non-glossy newspaper were distributed twice a day. After weaning, piglets from both treatments were housed in identical pens with three pieces of suspended rope, a plastic chewing stick and wood shavings twice a day. The main findings were that pre-weaning exposure to the supplementary materials reduced the severity of post-weaning tail biting, and that redirection of oral-nasal behaviours at pen-mates was reduced by current, but not past, exposure to the materials. In Study II, the main research questions were whether providing growing-finishing pigs with objects made of recently harvested wood would reduce tail biting and whether the same effect could be attained with metal and plastic objects with moderately added complexity. In the control treatment, 17 grower-finisher pens were furnished with a straw rack and a metal chain. There were four experimental treatments, each furnished with the above and additionally with the following: 14 pens with horizontal wooden beams (W), 13 pens with horizontal plastic crosses (P), 15 pens with hanging metal chains in a branching form (B) and 14 pens with all the above objects (WPB). The main findings were that only the wooden objects reduced the prevalence of tail and ear biting as compared to the control group, and that wooden and plastic objects sustained the interest of the pigs better than metal objects. In Study III, the main research question was whether a small amount of recently harvested wood (approximately 20cm of horizontal wooden beam per pig) would reduce tail biting in breeder gilts that also were provided with minimal amounts of straw. In the control treatment, 12 breeder gilt pens were furnished with a horizontal piece of commercially sourced dry wood and a metal chain with a moderately complex structure (feeder chain), and long straw was distributed once a day, approximately 20g per pig per day. In the experimental treatment, the above solid objects were replaced with objects of recently harvested wood. The main findings were that there was no significant difference between treatments regarding tail biting; and that the frequency of oral-nasal behaviours targeted at other pigs did not differ between treatments before the provision of the minimal amount of straw, but was lower in the experimental treatment after pigs in both treatments had consumed the straw. In Study IV, the main research questions were whether a minimal amount of recently harvested wood (approximately 10cm length of wood per pig) would reduce tail biting in growing-finishing pigs that were also provided with a straw rack and whether the same quantity of medium-density polythene objects would have the same effect. In the control treatment, 16 growing-finishing pens were furnished with a straw rack. There were two experimental treatments, each furnished with the above and additionally with the following: 16 pens with horizontal wooden objects (W) and 12 pens with horizontal plastic objects (P). The main findings were that there was no significant difference between treatments in their effects on tail biting; and that the wooden but not plastic objects reduced ear biting and the latency to approach an unfamiliar person in a human approach test, as compared to the control group. The main conclusions were that it is possible to reduce post-weaning tail biting by adding pre-weaning material for manipulation, but further studies are required to determine the necessary minimum quantity and quality of the materials. Horizontally placed lengths of recently harvested wood can reduce tail and ear biting and potentially also stress in growing-finishing pigs, but further studies are needed on optimal object design and the necessary minimum quantity. The presence of recently harvested wood can slightly add to the beneficial effects of minimal amounts of straw. There also was a finding relevant to general research methodology in this field: the frequency of object-directed behaviours is not the sole reliable predictor of a material’s efficacy in reducing tail biting as the plastic objects elicited object-directed behaviours to the same extent as the wooden objects, but plastic objects did not reduce tail biting. .
  • Mikkola, Lea (Helsingin yliopisto, 2020)
    This thesis addresses the genetic background of hip dysplasia, a complex hereditary disorder common in many species, including dogs and humans. The highly polygenic nature of hip dysplasia has become evident after many years of arduous research conducted in numerous studies that have explained only a small proportion of the disease heritability in dogs. The studies included in this thesis revealed multiple new loci, a novel regulatory variant for a gene that is imperative to normal joint development and validated numerous loci that have been associated with the disorder. In Study I, genome-wide association analyses uncovered four loci that associated with the disorder with either protective or risk effects. A subsequent targeted resequencing and variant analysis found disease associated variants for multiple genes. Deletion variants in the putative regulatory region of the gene NOG were found to protect against moderate-to-severe hip dysplasia. These variants were shown to downregulate reporter gene expression in vitro. In Study II, different hip dysplasia and osteoarthritis phenotypes were investigated. The genome-wide association analyses revealed three novel loci associated with hip joint incongruity and osteoarthritis. New candidate genes were identified: NOG and NANOS1 for hip joint incongruity and NOX3 and ARID1B for osteoarthritis. In Study III, a total of 21 loci on 14 different chromosomes were validated in across- and within-breed association analyses in a large cohort of dogs comprised of 10 breeds. One locus was specific to the across-breed data. Neddylation-pathway was found to be significantly enriched in a candidate gene set that included 254 genes from the 21 validated loci. These studies have generated new, long-awaited knowledge about the complex genetic background of hip dysplasia and related osteoarthritis in dogs. They also highlighted that mild hip dysplasia and the more severe disease forms may be induced by partially different genetic factors. Validation of associated loci for hip dysplasia has not been conducted to this extent before these studies, so an important step forward was taken in our latest study. Finally, finding causal variants has been uncommon. The regulatory NOG variants were an exciting finding, although their causality in hip dysplasia is yet to be revealed. Overall, our studies emphasise the complexity of the genetic background of hip dysplasia.
  • Aalto-Araneda, Mariella (Helsingin yliopisto, 2020)
    The severe foodborne disease listeriosis is caused by the bacterium Listeria monocytogenes, known as a problematic contaminant of the food chain. This facultative anerobe tolerates many conditions used for controlling harmful bacteria, including high salinity and temperature. Some L. monocytogenes strains tolerate external stressors better than others, which may complicate the control of the bacterium in food-related environments. Strains exposed to one stress condition may also develop tolerance towards another; such cross-adaptation occurs, for instance, between osmotic and heat stress. In food production, L. monocytogenes may encounter these stresses via salting and heat treatments or hot water used in sanitation. Vacuum-packaged ready-to-eat fish products frequently contain L. monocytogenes and have caused several listeriosis outbreaks. They often do not undergo listericidal processes before consumption, and thus, their processing requires stringent preventive measures. The aims of this dissertation were to investigate the strain variability and determinants of L. monocytogenes stress tolerance and to examine the framework of fish-processing plants and their official food control for managing L. monocytogenes contamination. Using optical density measurements of microbial growth, differences in growth ability under osmotic (NaCl) stress were determined for 388 wild-type L. monocytogenes strains. Notable strain variability as well as serotype- and lineage-dependent patterns of L. monocytogenes salt stress tolerance were discovered. Lineage-I-affiliated L. monocytogenes serotype 1/2b and 4b strains grew significantly better at NaCl 9.0% than lineage-II-affiliated serotypes 1/2a, 1/2c, and 3a. By enabling this comprehensive identification of NaCl-tolerant strains, our data assembly and analysis protocol elucidated the biologically relevant intra-species variability of L. monocytogenes salt stress tolerance phenotypes. A comparative whole-genome sequencing approach was implemented to identify underlying determinants of L. monocytogenes stress tolerance phenotypes. Accessory genetic mechanisms of stress resistance were investigated by comparison of heat survival phenotypes and whole-genome sequences of a heat-resistant and a heat-sensitive L. monocytogenes strain. The comparison identified a novel plasmid, pLM58, including an open reading frame annotated as an adenosine triphosphate (ATP) -dependent ClpL-protease-encoding gene, which was present in the heat-resistant strain but absent in the heat-sensitive strain. The curing of pLM58 resulted in a reduction of heat resistance. The conjugation of clpL increased the heat survival of a natively heat-sensitive L. monocytogenes strain. This study described, for the first time, plasmid-borne heat resistance of L. monocytogenes and identified the protease ClpL as a novel mechanism of L. monocytogenes heat resistance. To examine the framework for managing L. monocytogenes contamination in the fish industry, operational practices and efficacy of official control were studied in 21 Finnish fish-processing plants producing vacuum-packaged gravad (cold-salted) and cold-smoked fish products. Product samples were investigated for the presence and quantity of L. monocytogenes in 2014–2015. Additionally, the results of official food control sampling of products and facilities were assessed to retrospectively gain information on L. monocytogenes contamination in the participating fish-processing plant facilities in 2011–2013. The production and hygiene practices of the processing plants were surveyed with an in-depth inspection questionnaire, and the occurrence, control measures, and correction of non-compliances were drawn from their official inspection records. Associations of L. monocytogenes occurrence with fish-processing plant operational practices, compliance, and aspects of official control during the respective years were investigated with statistical modeling. L. monocytogenes product contamination was associated with number of processing machines, deficiencies in the processing environment and machinery sanitation, and staff movement from areas of low hygiene to high hygiene. Performing frequent periodic thorough sanitation was associated with a decreased risk of product contamination. The increased occurrence of L. monocytogenes in the facilities and products of the fish-processing plants was associated with hygiene deficiencies in processing machinery, a lack of demanding control measures for non-compliances, and recurrence of non-compliances. These results identified areas for improvement in the preventive measures of fish-processing plants and official food control, providing ways to reduce L. monocytogenes contamination in the fish industry.
  • Rossi, Heini (Helsingin yliopisto, 2020)
    Non-infectious inflammation of the lower airways, the main theme of this thesis, is commonly observed regardless of the breed, age, or use of the horse. Due to similarities with human asthma regarding clinical presentation and several pathophysiological aspects, non- infectious chronic lower airway inflammation in horses is currently named equine asthma (previously inflammatory airway disease or recurrent airway obstruction, depending on the severity). Long-term exposure to organic dust and moulds in stable air is thought to be the most important predisposing factor for the disease. Equine asthma is characterized by airway hyperresponsiveness and mucus accumulation, severity-related airway remodelling, impaired lung gas exchange, and increased quantity of airway inflammatory cells. These changes lead to clinical signs, including chronic cough and nasal discharge, poor performance, and in a severe form of equine asthma, increased respiratory effort at rest corresponding to an “asthma attack” in humans. Despite active research over the years, the aetiology and pathophysiology of equine asthma remain incompletely defined. In this thesis, the role of a transmembrane protein extracellular matrix metalloproteinase inducer (EMMPRIN), a potential new biomarker in equine asthma, and its relationship to matrix metalloproteinases (MMPs) were investigated. In Study I, the respiratory secretions of 29 horses with asthma and 15 asymptomatic controls were examined. Expression of EMMPRIN protein was identified in all horses in bronchoalveolar lavage fluid (BALF) cells, and the expression was highest for the most substantially affected horses with severe asthma and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. This is the first study to describe the relationship of EMMPRIN and MMPs in patients of any species clinically affected with asthma, and the results suggest that EMMPRIN is involved in the pathophysiologic processes of asthma in horses. EMMPRIN as a marker of inflammation provides interesting possibilities for further research of horses and other species. The second part of this thesis concentrated on two common diagnostic tools used to assess lower airway diseases in horses. The diagnosis of lower airway inflammation, including asthma, is often aided by the analysis of cytological samples obtained from the trachea (tracheal wash; TW) or from the lungs at alveolar level (bronchoalveolar lavage; BAL). There is considerable variance in the diagnostic methods of equine lower airway diseases between facilities and clinicians, and a true gold standard for diagnosing airway inflammation is lacking. In most previous studies, the cytology of TW and BAL samples has been considered to correlate poorly and BAL has been deemed a superior diagnostic tool in most cases of equine lower airway disease, but sensitivity and specificity have not been evaluated. Moreover, the conventionally used cut-off values for cytology have been largely based on observational studies and expert opinion, and recently, higher values have been proposed. The first aim of Study II of this thesis was to compare the results of these two airway sampling methods in a population of 121 horses with signs related to respiratory disease and in 33 horses with no clinical respiratory signs. A secondary aim was to evaluate the cut-off values of TW and BALF cytology for neutrophils in order to justify their use in clinical practice. TW and BAL showed substantial agreement regarding neutrophils, and only 17.5% of the horses were classified differently (healthy vs. diseased), in contrast to some previous studies with higher disagreement. The neutrophil percentage was found to correlate between TW and BAL results. The sensitivity and specificity of TW were generally higher for TW than for BAL when estimated with conventional as well as with Bayesian statistical methods. The results of this study encourage the use of the current neutrophil cut-off values in clinical diagnosis of airway inflammation instead of elevating the cut-offs. In Study III, equine lower airway inflammation was investigated with regard to general anaesthesia. The complication rate in equine general anaesthesia is high relative to anaesthesia in several other species, postanaesthetic pneumonia being one of the potential complications. However, there is limited knowledge about the effects of general anaesthesia on inflammatory responses in the lungs and on systemic inflammatory markers such as serum amyloid A (SAA) in horses. For example, the level and duration of lower-airway neutrophilia that can be expected after anaesthesia in dorsal recumbency are unknown. Thus, our primary aim was to determine when lung inflammation reaches its maximum and how rapidly BALF cytology returns to baseline after anaesthesia in dorsal recumbency. A secondary aim was to investigate the possible effect of vatinoxan, a novel a2-adrenoceptor antagonist drug, on the BALF cytology results. Anaesthesia in dorsal recumbency resulted in no clinically relevant changes in airway cytology that could be differentiated from the effect of repeated BAL sampling. A marked increase in serum amyloid A was detected in some animals, while others showed no changes from baseline. Vatinoxan as premedication did not consistently affect lung cytology or blood inflammatory markers after anaesthesia. These combined studies on equine lower airway inflammation increase our understanding of mechanisms and factors related to pathophysiology and diagnostics of the condition in the horse. Lower airway inflammation is an important cause of impaired welfare and performance of horses. Broader understanding of the aspects related to the condition will aid in developing new improved clinical and diagnostic procedures and management strategies for equine inflammatory respiratory diseases.
  • Tapio, Heidi (Helsingin yliopisto, 2020)
    Detomidine and medetomidine are α2-adrenoceptor agonists that are used in equine medicine for sedation and analgesia. These drugs, however, also influence the cardiopulmonary and gastrointestinal system, mainly by increasing systemic vascular resistance and decreasing cardiac performance and intestinal motility. Vatinoxan, in turn, is primarily a peripherally acting α2-adrenoceptor antagonist that has been demonstrated to attenuate some of the undesired effects of α2-adrenoceptor agonists with no major influence on the level of sedation in many species. One aim of the present studies was to evaluate the effects of vatinoxan with a constant rate infusion (CRI) of medetomidine in standing and anesthetized healthy horses. Another aim was to evaluate the effects of vatinoxan when it is administered after detomidine in standing healthy horses. We hypothesized that vatinoxan would alleviate the cardiopulmonary and gastrointestinal effects of these α2-adrenoceptor agonists, while preserving their sedative effects under these experimental conditions. The horses received, in a cross-over design in three separate studies, an α2-adrenoceptor agonist either alone or with vatinoxan for sedation or for premedication before general anesthesia. To evaluate the cardiopulmonary function, the heart rate, respiratory rate, cardiac output, arterial blood pressures, central venous pressure, and pulmonary arterial pressures (this last one in two of the studies) were recorded. Additionally, arterial and venous blood gases were analyzed. Systemic vascular resistance, oxygen delivery, and other selected cardiopulmonary parameters were calculated afterwards. The effects of the drugs on intestinal motility were evaluated by auscultation of the borborygmi or by determining the amount of fecal output. The level of sedation was scored, and the recovery times were recorded in anesthetized horses. Finally, the drug concentrations in plasma were determined. In standing horses receiving a CRI of medetomidine, vatinoxan attenuated the early, although minor, cardiopulmonary changes induced by medetomidine. Furthermore, the intestinal motility was markedly improved by vatinoxan. In anesthetized horses receiving an adjunctive CRI of medetomidine, premedication with vatinoxan induced significant hypotension. Despite this hypotension, which was successfully treated with dobutamine, the cardiac performance and tissue oxygen delivery were better maintained in these horses than in those receiving the adjunctive medetomidine CRI without vatinoxan. The fecal output decreased after general anesthesia, and this decrease was not influenced by vatinoxan. Finally, vatinoxan, administered after detomidine, partially reversed the cardiopulmonary effects and intestinal hypomotility induced by detomidine. Vatinoxan decreased the level of sedation at the beginning of the CRI of medetomidine in standing horses, but not when administered with a single dose of medetomidine before general anesthesia or after detomidine in standing horses. The recovery times with or without vatinoxan also did not differ from each other. The plasma concentration of both enantiomers of medetomidine mostly correlated with the level of sedation, being lower in the presence of vatinoxan when the level of sedation was reduced. To conclude, vatinoxan attenuated the cardiopulmonary changes and intestinal hypomotility induced by detomidine or a CRI of medetomidine in standing horses. The effects of vatinoxan on the level of sedation could be considered relatively minor. With the dosing used in this study, vatinoxan induced marked hypotension in anesthetized horses and did not influence the intestinal hypomotility associated with general anesthesia. Thus, vatinoxan represents a potential drug for alleviating or reversing the undesired effects of medetomidine and detomidine in standing horses. Its hypotensive effects, however, warrant more research before it could be usable in anesthetized horse.

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