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  • Rimhanen-Finne, Ruska (Helsingin yliopisto, 2006)
  • Alila-Johansson, Aino (Helsingin yliopisto, 2008)
    This thesis examined the effect of daily and annual lighting conditions on the blood levels of the hormones melatonin, cortisol and leptin and the lipid metabolism of free fatty acids (FFAs) and glycerol in female goats. The blood levels of these substances were also measured in long-term total darkness (endogenous rhythm). The duration of melatonin secretion in goats closely follows the length of the dark phase of the day, except in winter, when the duration is significantly shorter. In total darkness, the goats display two types of endogenous melatonin patterns: a "winter pattern" in winter, early spring, early fall and late fall, and a "summer pattern" in late spring and summer. The results showed that in addition to the light-adjusted overt melatonin rhythm, the endogenous melatonin secretion can be modulated by circannual clock mechanisms and/or long-term photoperiodic history. Serum cortisol levels had no significant daily rhythm at any time of the year, nor are there any differences in the profiles between annual lighting conditions and total darkness. In winter the overall concentrations were higher than in any other season. The results suggest that any/the circadian variation of cortisol secretion is masked by external factors, probably by the feeding schedule, and that the seasonal variation in the overall cortisol levels is likely to be related to the changes in photoperiod. The lipid metabolism of goats seems to be regulated by light; no significant contribution of leptin levels could be shown. Concentrations of plasma FFAs and glycerol exhibit significant daily and seasonal variations. The photoperiod was found to trigger a nocturnal fall and morning rise of FFAs levels, while low levels of glycerol were associated with feeding. In total darkness, FFA and glycerol rhythms were unstable. The close relationships between the daily variations of melatonin and FFAs levels result from both variables being controlled by light-dependent mechanisms. The results do not rule out a possible effect of melatonin on the daily FFAs profiles, but they show that melatonin secretion alone could not completely explain the daily FFAs profiles.
  • Kinnunen, Paula M (Helsingin yliopisto, 2011)
    Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics.
  • Hyytiäinen, Heli (Helsingin yliopisto, 2015)
    Stifle dysfunction is one of the most common reasons for canine hindlimb lameness and an indication for dogs referral to physiotherapy. Until now, there has been a lack of testing batteries in animal physiotherapy, although these are an important part of the evaluation process in various patient groups in human physiotherapy. Using 64 dogs, 43 with stifle dysfunction and 21 healthy dogs, congruity between fourteen physiotherapeutic evaluation methods, commonly used in dogs with stifle dysfunction, and six evaluation methods used by a veterinarian was evaluated. The eight best methods were chosen as items constituting a testing battery, the Finnish Canine Stifle Index (FCSI). The numerical scale of the testing battery was 0-263. Cronbach s alpha for the internal reliability of the total FCSI score was good (0.727). Two cut-offs for the total score were set: 60 and 120, separating adequate, compromised and severely compromised performance level, based on their high sensitivities and specificities. Another 57 dogs, 29 with some type of stifle dysfunction, 17 with some musculoskeletal disease other than stifle dysfunction and 11 healthy dogs, were used to further study the psychometric properties of the testing battery. The dogs with stifle dysfunction showed a significant (P < 0.001) decrease in FCSI total score (93.3 ± 62) compared with the two other groups (29.5 ± 39.6 and 11.7 ± 21.0), demonstrating good responsiveness of the FCSI. Also the inter-tester reliability was excellent (ICC 0.784), with no significant differences between three physiotherapists performing the FCSI. In conclusion, the overall functionality and outcome of rehabilitation in dogs with stifle dysfunction can be reliably evaluated with the new testing battery, the FCSI, developed here.
  • Tuominen, Pirkko (Helsingin yliopisto, 2009)
    The drive for risk-based food safety management, systems and control has spread world-wide in recent decades. Since the term is still internationally undefined, its use and implementation vary, producing different realizations. In this Ph.D. thesis, microbiological risk assessment (MRA) was investigated as a basis for risk-based food safety management, which was defined as ‘food safety management based on risk assessment in order to achieve an appropriate level of protection (ALOP)’. Governments are responsible for commissioning MRAs and also for setting food safety targets up to a certain point, but the practical management measures that need to be in place in order to achieve the targets are to be addressed by the operators. On the plant level, food safety is usually managed through regulation, quality assurance systems and a hazard analysis and critical control point (HACCP) programme with its prerequisites. In Finland, food safety management on the food plant level is implemented through an HACCP-like regulated system termed an own-checking (OC) programme. A quantitative microbiological risk assessment (QMRA) was conducted on salmonella in the beef production chain according to the official standards of the Codex Alimentarius Commission (Codex Alimentarius), and utilized in determining the food safety metrics for beef production. The Finnish Salmonella Control Programme (FSCP) and the main official interventions due to it were examined in the light of risk-based food safety management. The targets set for beef processing plants by the government were converted into quantitative limits, and the results of salmonella monitoring included in the FSCP were examined by the QMRA. The goal of the FSCP was declared in 1994 to ‘maintain the present salmonella situation’, which was considered to refer to the salmonella incidence in humans at that time, and also the de facto ALOP. The requirement for a maximum salmonella prevalence of 1% at defined stages of the beef production chain was embodied in the FSCP. This statement was considered to convey performance objectives (PO) for the aforementioned stages. According to the QMRA, the de facto ALOP was achieved in the referred year 1999, and even the true prevalence levels in the FSCP were estimated to be clearly under the set PO limits with 95% credibility. However, the PO limits were set too high for the de facto ALOP to be maintained in practice. If the salmonella prevalence reached the PO limit of 1% or values near it, the public health risk would increase and overrun the de facto ALOP. The QMRA produced in this work has for the first time provided the possibility to quantitatively asses the relationships between targets set in the FSCP and their impact on public health. At present, imports of beef and beef-derived foods may impose on Finnish consumers a significantly greater exposure than domestic products. If their salmonella prevalence or their share of the foods consumed in Finland increase, the number of human cases could rapidly rise. The models for the QMRA were mainly Bayesian hierarchical models using Markov chain Monte Carlo (MCMC) techniques, which was found to be a flexible and appropriate method for this type of complex modelling. The resulting distributions were also regarded as an advantage compared to the results from models developed with the deterministic approach, because the presentation of results included the extent of the uncertainty, and also in this manner better illustrated the actual operational environment. Based on an inquiry, the personnel in food processing plants had a positive attitude towards food safety management systems, but the knowledge, training and involvement of those employees directly operating on the site with these systems were discovered to be deficient. Therefore, a generic semi-quantitative hygiene risk assessment model, Hygram®, was developed for small and medium-sized food enterprises to offer assistance in understanding, training, and, first of all, detecting the critical steps of the processes, and thereby to contribute to the development of their own-checking systems towards risk-based food safety management. Hygram® was not considered a risk-based tool as such, but whenever the critical limits of the process have been defined as equal to a risk assessment, Hygram® can be used as a risk-based management tool. It can also serve as a tool for systematic hazard analysis and CCP detection when establishing a food safety management system. To conclude, the development of risk-based food safety management is a process in which risk assessment is an essential tool. Scientific, technical, psychological and resource-bound barriers need to be overcome in order to put risk-based management systems into practice. This study showed that QMRA can be valuable in national risk management decision making, although few QMRAs are currently available. Appropriate tools for practical risk management decision making on the industrial level, such as Hygram®, need to be further developed.
  • Honkavaara, Juhana (Helsingin yliopisto, 2012)
    The effects of MK-467, a peripherally acting α2-adrenoceptor antagonist, on the cardiopulmonary changes induced by dexmedetomidine, a specific and selective α2- adrenoceptor agonist, were investigated in dogs. Plasma concentrations of both drugs were also quantified, along with influence of MK-467 on the quality of clinical sedation achieved with dexmedetomidine. The main focus of this study was on preventing or attenuating the cardiovascular effects of dexmedetomidine. The effects of three different doses of MK-467, administered simultaneously with the agonist, on the main hemodynamic parameters were evaluated and compared with a single dose of both dexmedetomidine and MK- 467 administered alone. Respiratory effects were evaluated with arterial blood gases, and indices such as oxygen delivery were calculated. The pharmacokinetic interaction between the two drugs was evaluated in vivo and general information on the disposition of intravenously administered MK-467 in dogs was produced. The degree of clinical sedation was subjectively assessed during the studies. The effect of MK-467 on the quality of reversal of dexmedetomidine-induced sedation with atipamezole was also determined. M K-467 dose-dependently reduced or prevented all relevant cardiovascular changes induced by dexmedetomidine. The heart rate, arterial and central venous blood pressure, cardiac index, systemic vascular resistance and oxygen delivery remained within acceptable physiological limits throughout the observational period with all doses of MK-467 administered with the agonist. Moderate hypotension was seen with the highest dose of MK-467. No significant differences in respiratory function were observed between treatments. MK-467, when administered alone, induced sinus tachycardia along with increases in the cardiac index and reductions in systemic vascular resistance. Arterial blood pressures remained unchanged. The degree of clinical sedation was reduced by MK-467, most likely by increasing the disposition of dexmedetomidine, which led to lower plasma concentrations of the agonist. However, the differences in clinical sedation were minor. MK-467 did not interfere with the ability of atipamezole to reverse dexmedetomidine-induced sedation.
  • Knuuttila, Anna (Helsingin yliopisto, 2015)
    Aleutian mink disease virus (AMDV) is a widespread parvovirus mainly affecting American mink (Neovison vison). It can cause a progressive and persistent immune complex-mediated disease (Aleutian disease, AD) in adult mink and an acute and fatal pneumonia in mink kits. The virus has a wide geographical distribution both in farmed mink and in the wild. Aleutian mink disease virus poses a major economic threat to mink farmers and it may affect the conservation and management of indigenous mustelids and other species. Infected farms are difficult to sanitize as the virus is resistant to physical and chemical treatments, it can be transmitted through several vectors and routes, and no effective medications or vaccines currently exist. Since the 1970s, diagnosis on AMDV in farmed mink has been based on the identification of specific antibodies with a counter-current immunoelectrophoresis (CIEP) test. In 2005, the Finnish Fur Breeders Association implemented an eradication program that required the development of a new AMDV-detection protocol to screen ca. 600 000 samples per year. Although AMDV can infect and may cause disease in other mustelids and carnivores, little is known about the epidemiology and evolutionary relationships of AMDV strains in the wild in Finland and elsewhere. Thus, this study aimed to develop a modern automated test for the large-scale serodiagnosis of AMDV in mink and to elucidate the epidemiology and phylogeny of this virus in farmed mink and free-ranging mustelids in Finland. A new antigen for the serological test was developed with a recombinant DNA technique. The major capsid protein (VP2) gene of a Finnish AMDV strain obtained from a farmed mink was amplified, cloned into a baculovirus transfer vector with subsequent recombination to baculovirus genome, and expressed in insect cells. The antigen formed virus-like particles and was confirmed to be antigenic with several serological methods. Subsequently, an enzyme-linked immunosorbent assay (ELISA) was designed for the antigen and automated. Because the small glass capillaries used to collect blood samples in CIEP could not be utilized in the ELISA test, a wicking technique using a filter paper blood comb was developed. The performance of this test was compared to CIEP (an imperfect gold standard) by testing blood/serum samples from farmed mink. The results were analyzed with Bayesian modelling allowing for conditional dependence. The new automated ELISA test was found to be accurate with a diagnostic sensitivity of 96.2% (95% probability interval [PI], 91.5 99.0) and specificity of 98.4% (95% PI, 95.3 99.8), and was therefore determined suitable for the serodiagnosis of AMDV. The epidemiology and phylogenetics of AMDV were inferred from organ and/or blood samples from farmed mink and free-ranging mustelids. The samples were screened with the newly-developed ELISA (described above) or CIEP test for anti-AMDV antibodies and with previously described or newly-developed PCR assays for AMDV DNA. Test results were studied with statistical, phylogenetic, and sequence analysis methods. Aleutian mink disease virus was found to be prevalent in the wild in Finland. A new host species, the European badger (Meles meles), with a prevalence of 27% (7/26; 95% confidence interval [CI], 13 46), was identified. In addition to the badger, infection markers were found in 54% (31/57; 95% CI, 42 67) of feral American mink and in one European polecat (Mustela putorius) (1/14; 95% CI, 1 29). No infection was found in Eurasian otters (Lutra lutra) (24; 95% CI, 0 10), European pine martens (Martes martes) (183; 95% CI 0 1), least weasels (Mustela nivalis) (2; 95% CI, 0 67), stoat (Mustela erminea) (1; 95% CI, 0 85), or wolverine (Gulo gulo) (1; 95% CI, 0 85). Positive animals were distributed throughout western, southern, and eastern Finland (10/17 of sampled regions). American mink (odds ratio [OR], 335) and badger (OR, 74) had higher odds of infection compared to other species. Also, animals sampled during the first sampling period (2006 2009; OR, 5) had higher odds of infection compared to the second period (2010 2014). No significant association was detected between infection and age, sex, or region. Furthermore, mink farms were not associated with higher odds of AMDV infection nor appeared to serve as a major source of infection for free-ranging mustelids at the municipal or regional level. Based on these results, it appears that domestic and sylvatic transmission pathways are largely decoupled, but it seems probable that infections occasionally move between the farmed and wild populations (e.g., via infected escapees/intruders). A phylogenetic analysis, including Finnish, Estonian, and global strains, indicated that AMDV strains form at least five main clusters. It also inferred that the virus has been introduced to Finnish farms on at least three occasions. Unfortunately, it could not be discerned whether the occurrence of AMDV in Finland is natural or a consequence of the global mink trade. In addition to its main hosts (farmed and wild mink), similar strains of AMDV were found in pine martens, polecats, and badgers. Interestingly, Estonian badgers carried a divergent strain, possibly representing a new amdoparvovirus. Other than the strain found in Estonian badgers, and the tendency of strains from Finnish farmed and feral mink to diverge into separate clusters, AMDV strains did not cluster according to location, year, species, or pathogenicity. The nucleotide differences between Finnish AMDV sequences, based on partial non-structural protein 1 gene, ranged from 0% to 14% and similar levels of variability were observed in farmed and natural populations. As a result of these studies, an automated ELISA test for the serodiagnosis of AMDV was developed and validated with high diagnostic sensitivity and specificity. The test offers a low cost, easy sampling, rapid throughput of large sample numbers, reduced processing time, and automated data management. The new test can be utilized for the monitoring, control and eradication of the virus, calculating the seroprevalence, and confirming the infection status of farms or individual mink. In addition, new information on AMDV epidemiology and genetic variation in Finnish farmed mink and free-ranging mustelids were established with potential impact on the biosecurity of farms, outbreak investigations, and the conservation of threatened mustelid species. Moreover, the new diagnostic tools and additional sequence data generated in this study can be utilized in the future research on the epidemiology of AMDV.
  • Niskanen, Taina (Helsingin yliopisto, 2010)
    Yersinia pseudotuberculosis is an animal pathogen widespread in nature. The bacterium has caused infections in humans occasionally. Most of the reported Y. pseudotuberculosis -infections are sporadic, and the sources and vehicles of Y. pseudotuberculosis remain obscure. Y. pseudotuberculosis has emerged as an outbreak -associated pathogen in Finland since 1997. Indirect evidence has indicated that Y. pseudotuberculosis is a potential food borne pathogen, but no specific food has been previously confirmed as a source of the human infections. The aim of this thesis was to determine the prevalence of Y. pseudotuberculosis in different animal species and to evaluate them as a possible source of Y. pseudotuberculosis. Clinically healthy pigs were found to carry Y. pseudotuberculosis. The prevalence of Y. pseudotuberculosis was 4 % and 14 % in fattening pigs at slaughterhouse and at farm level, respectively. All Y. pseudotuberculosis isolates from pigs were pathogenic. Migratory birds representing 57 different bird species were studied for pathogenic Y. pseudotuberculosis during spring and autumn migration. Pathogenic Y. pseudotuberculosis was recovered from two species of birds during spring migration. Y. pseudotuberculosis was first time confirmed to be the cause of a food borne outbreak through domestically produced iceberg lettuce in epidemiological study. Contamination of iceberg lettuce was investigated on farm level in a two-year survey. Several factors predisposing to Y. pseudotuberculosis contamination of lettuce were found on the farms, including a large population of deers and free access of wild animals to unfenced lettuce fields and to irrigation water sources. Pulsed-field gel electrophoresis (PFGE) analysis was optimized for efficient molecular typing method for Y. pseudotuberculosis isolates. PFGE was used to compare Y. pseudotuberculosis isolates from different origin. Y. pseudotuberculosis isolates of different sources showed high genetic diversity, while Y. pseudotuberculosis isolates found in same animal species representing the same serotype showed limited genetic diversity. Identification of environmental Y. pseudotuberculosis isolates was found to be incorrect based solely on biochemical tests. Commercial test kits misidentified some environmental Yersinia spp. isolates as Y. pseudotuberculosis. However, no specific virulence genes of Y. pseudotuberculosis were found in these isolates by polymerase chain reaction (PCR) targeting these gene areas.
  • Heikinheimo, Annamari (Helsingin yliopisto, 2008)
    The present thesis reveals new information, showing that healthy human is a rich reservoir for Clostridium perfringens enterotoxin (CPE)- producing Clostridium perfringens type A. The results show that healthy foodhandlers are commonly asymptomatic carriers of this pathogen. The findings indicate that persons handling foods should be considered a possible source of contamination for C. perfringens type A food poisoning. This work also found that the genotypes that previously were considered as atypical causative agents in food poisonings, in fact cause a notable proportion of C. perfringens type A food poisonings. CPE- producing C. perfringens type A is considered one of the most common causes of food poisonings in the industrialized world. This pathogen is also involved in antibiotic-associated diarrhea and sporadic diarrhea in humans as well as in animals. Recent findings of the variable loci (plasmid vs. chromosome) and different genetic arrangements adjacent to enterotoxin gene (cpe) have shown that CPE- producing C. perfringens type A isolates form in different subpopulations. These subpopulations are responsible for different CPE-associated diseases, i.e. chromosomal cpe-bearing strains are involved in food poisonings, whereas strains carrying cpe in the plasmid are typical in antibiotic-associated or sporadic diarrhea cases as well as in animal diarrheas. Not much is known about reservoirs or transmission routes of cpe-positive isolates. The present thesis aimed to improve diagnostics of cpe-positive C. perfringens by developing molecular methods for detection and isolation. Other objectives were to improve identification and typing of C. perfringens. With novel assays, molecular epidemiology of cpe-positive C. perfringens type A was further investigated. When investigating the presence of cpe-positive C. perfringens in the feces of healthy food- handlers, the organism was carried by 18% of the individuals. The presence of strains representing all previously identified genotypes (plasmid-borne IS1151-cpe, plasmid-borne IS1470-like-cpe, and chromosomal IS1470-cpe) as well as strains carrying previously unrecognized genetic arrangements attached to cpe shows that various different subpopulations of cpe-positive C. perfringens type A occur in the human gastrointestinal tract and that the healthy human is a rich reservoir for cpe-positive C. perfringens type A. The common carriage of cpe-positive C. perfringens type A of healthy food-handlers and the full capacity of these strains to produce CPE indicate that humans handling food should be considered a possible source of contamination of food. By showing that 25% of investigated C. perfringens type A food poisoning outbreaks were caused by strains carrying plasmid-borne cpe, we reveal that plasmid-borne cpe carrying C. perfringens type A is a common cause of food poisonings. The new data presented in this thesis improve the understanding of reservoirs and movements of enterotoxin producing C. perfringens type A. Therefore, CPE-associated diseases may be better controlled and prevented.
  • Hälli, Outi (Helsingin yliopisto, 2008)
    Seasonal infertility is well-defined phenomenon in modern swine production. The outcomes mostly affected are age at puberty in gilts, weaning-to-oestrus interval (WOI) and farrowing rate in sows. The European wild boar, the ancestor of our domestic pig, is a distinct short-day seasonal breeder. Photoperiod is thought to be the most important regulating factor in seasonal breeders. In addition to photoperiod, many environmental and management factors are known to adjust the manifestation of seasonal infertility. The aim of this work was to obtain information about the effect of photoperiod on reproductive endocrinology and on reproductive performance. In addition, the effect of re-modelling the piggery breeding unit on farrowing rate was studied. In the first clinical trial (I), two light regimes were tested in experimental conditions. One group of ovariectomized gilts (16 domestic pigs and 4 European wild boars) was kept under short daylight conditions (8-h light, 16-h dark) and the other group under long daylight conditions (16-h light, 8-h dark). After the treatment period, LH secretion patterns were determined by repeated blood sampling. In contrast to our expectations, LH pulse frequency remained unchanged in both light regimes. However, in the domestic pig, mean and basal LH concentrations were higher in the long-day group than in the short-day group. To investigate the two light regimes under more practical conditions, two trials (II, III) were conducted in commercial piggeries. The outcomes were farrowing rate and WOI. In trial II, the long-day group was kept under a constant 16-h light, 8-h dark regime. The short daylight regime consisted of a light phase of 8-h and a dark phase of 16-h in farrowing and breeding units. The length of the short-day treatment was 6 weeks before breeding. In the gestation unit, the short-day animals were also kept under a 16L:8D regime. The results of this trial revealed that the farrowing rate was 90% in both groups, and the two treatment groups also shared the same median WOI, i.e. 5 days. In trial III, the long daylight regime consisted again of a constant long-day treatment. However, the photophase was 14L:10D. The short-day treatment was extended to 8 weeks and took place exclusively in the farrowing unit. The light regime was 10L:14D. The short-day group was also kept under a 14L:10D regime in the breeding and gestation units. Despite these modifications, the results were the same as in the previous trial. The farrowing rate was 90% and median WOI five days in both groups. The last part (IV) of this thesis was an observational retrospective cohort study with a historical control. A cohort of farms with individual cages in their breeding unit was chosen. These farms either remodelled their breeding cages or switched to different group housing systems in the breeding unit. The farrowing rate before and one year after the remodelling was then compared. The remodelling itself was shown to affect reproductive performance on these farms. In conclusion, the reproductive performance of sows is difficult to control by a single environmental (photoperiod) or management (housing strategy) factor.
  • Hang, Ingrid (Helsingin yliopisto, 2015)
    Considerable evidence suggests that dietary macronutrients impact upon activities and conditions in the gastrointestinal tract (GIT) including: functions and processes, digestive enzymes secretion, microbial ecology and bacteria-derived metabolism. Knowledge about the modulation of canine intestinal microbiota, bacteria-derived metabolic products,intestinal inflammatory status and adaptive exocrine pancreatic secretion in response to macronutrients is limited. However, such information is necessary to investigate further the complex interplay between host and intestinal microbiota in response to changes of diet. The reasearch for this PhD thesis focused upon the changes of the intestinal microbiota,bacteria-ferivedmetabolicproducts,anintestinalinflammatorymarker and pancreatic enzyme profiles of five healthy Beagle dogs in response to being fed three different diets: high-carbohydrate starch (HCS), high-protein greaves-meal (HPGM), or a balanced dry commercial (DC) diet. Every diet was crossed-over and fed to each dog for three 21-day periods. The microbial deoxyribonucleic acid (DNA) was profiled according to its percentageoftheguanine-cytosinecontent(%G+C)inordertodetectthefluctuations in intestinal microbiota. Thereafter, 16S ribosomal ribonucleic acid (16S rRNA) gene amplicons were obtained from the most abundant %G+C peaks and analysed by sequence analysis. The DC diet sample was associated with high abundances of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for the DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples obtained for the HPGM and HCS diets. The HPGM and HCS diets also had reduced numbers of representatives of the family Lachnospiraceae; specifically Clostridium cluster XIVa. The HCS diet favoured the proliferation of representatives of the order Erysipelotrichales, specifically the Clostridium cluster XVIII, whereas the HPGM diet favoured representatives of the order Fusobacteriales. Bacterial metabolism and intestinal inflammatory status were assessed by determining dry matter, pH, ammonia, short-chained fatty acids (SCFAs), and faecal canine calprotectin concentrations. Faecal ammonia concentrations decreased with the HCS diet. All dogs fed the HPGM diet developed diarrhoea, which led to differences in faecal consistency scores and increased faecal pH. Moreover, decreases in propionic and acetic acids coupled with increases in branched-chain fatty acids and valeric acid caused changes in faecal total SCFAs. Faecal canine calprotectin concentration was also higher for the HPGM diet than with the other diets and correlated positively with valeric acid concentrations.8 Dietary effects on digestive enzyme composition in the serum, in jejunal fluid, and in the faeces were studied by determining the following factors: amylase activity, the concentrations of canine trypsin-like immunoreactivity (cTLI), canine pancreatic lipase immunoreactivity (cPLI), and canine pancreatic elastase (cE1) concentrations with the two radioimmunoassays (RIAs) for determining cTLI and cPLI concentrationswere specifically validated for jejunalfluid and faecal specimen analysis. Both RIAs were linear, accurate, precise, and reproducible. Dog specific serum enzyme concentrations did not differ between diets. Feeding the HCS diet was associated with decreased amylase activities and cPLI concentrations in the lower jejunum, when compared to the corresponding cPLI activities of the HPGM and the DC diet. The HPGM diet decreased the concentrations of cPLI and cE1 in faecal samples, but not in the jejunal fluid. In conclusion, all bacterial clusters discovered in this research represent the normal GIT microbiota of canines. The HPGM diet favoured Fusobacterium and this Gram-negative bacterial genus may be associated with the observed elevated inflammation status. The latter was deduced from the observed diarrhoea and elevated levels of canine faecal calprotectin in all dogs fed the HPGM diet. It seems likely that these research results could be associated with the quality and increased or decreased amounts of dietary protein or carbohydrate being available for fermentation by the intestinal microbiota. The limited capacity of pancreatic enzymes to adapt adequately by a change inprofile inresponse to changes indietary components seems to be an essential factor, which influences the nutrient levels available for the intestinal microbiota.
  • Õkva, Kai (Helsingin yliopisto, 2012)
    Discussions on laboratory animal welfare issues often refer to the Three Rs replacement, reduction and refinement. Replacement means substituting living animals with non-sentient systems; reduction refers to using fewer animals and refinement causing less pain, suffering and distress to the animals or improving their welfare. This work is focusing on two R-s: reduction and refinement in mice. If one considers reduction as meaning obtaining trustworthy information from using fewer animals, then this can be achieved by improved research strategies, better experimental design and more sophisticated statistical analyses. One can reduce variation within the group by using isogenic inbred animals or by finding ways to reduce variation in outbred animals. One approach to achieve reduction in outbred animals is to include litter and individual features of the animals, e.g weight dynamics, in the statistical analyses. Since the elevated plus-maze (EPM) test is one of the most common tests to evaluate anxiety-like behaviour, it was used to assess the possible effects of litter and weight on the behaviour of outbred mice and the effects of environmental enrichment (EE) on the behaviour of inbred mice. As a research tool, the effects of acute or chronic administration of ethanol or acute therapy with the nitric oxide synthase (NOS) inhibitor L-NOARG were examined in outbred NIH/S mice. The administration of L-NOARG had no effect on the behaviour of mice after acute or chronic ethanol administration but attenuated the anxiogenic effect of ethanol withdrawal. The litter from which the mice had originated had a significant effect on their behaviour in the EPM test. The behavioural indices of mice, originating from different litters, tended to be above or below the mean of the corresponding drug-treatment group, irrespective of the drug treatment. Litter had a significant effect on the initial weight and also on the weight changes occurring during the adaptation period and ethanol inhalation. An approach to refinement, EE, has been introduced to create more natural species-specific living conditions for laboratory animals. At the same time, it has been claimed that EE can affect the results of behavioural studies and also increase variation. The effects of different types of EE and different time periods were studied in inbred C57BL/6J and BALB/c mice. The exposure of male C57BL/6J mice to the different types of EE objects in the form of cage furniture (CF) -nest box, corner and stairs- induced an anxiolytic-like effect in the EPM test and tended to increase the locomotor activity of mice. This apparent anxiolytic-like effect was most pronounced in the third week. The CF, in the form of modified Tapvei OY mouse stairs, produced an anxiolytic-like effect and increased the locomotor activity in female C57BL/6J mice, but not in BALB/c female mice. In conclusion, the NOS inhibitors may have effects on the behavioural changes caused by ethanol withdrawal. Information about the litter of outbred mice could and should be used in statistical analysis in order to reduce variation and the number of mice needed. In the EPM test, different CF items induced an anxiolytic-like effect in male and female C57BL/6J mice, but not in BALB/c female mice. This effect depended on the type of objects and was influenced by time. This anxiolytic like effect can be interpreted as refinement of the housing by improving animal welfare. The effects of CF should be considered in planning enrichment programs for housing institutions, in designing behavioural experiments and in analyzing the results obtained. Therefore the evaluation of CF could provide the valuable information and it is recommended that CF manufacturers collate and distribute the refinement results on the specific CF items they produce.
  • Autio, Karoliina (Helsingin yliopisto, 2015)
    Cancer is one of the most common reasons for death in dogs, cats and humans. New therapeutic modalities are necessary to improve disease outcome. One promising approach is oncolytic virotherapy. Until now, the only oncolytic virus evaluated in a clinical trial in veterinary medicine has been canine oncolytic adenovirus, but a clinical trial has been started with oncolytic vaccinia virus (VV) in pet dogs. In cats, oncolytic viruses have not been evaluated in clinical settings. Tumour treatment in dogs and cats could also serve as a model for human cancer therapy. The purpose of the thesis was to evaluate preclinically whether genetically modified oncolytic VV and Semliki Forest virus (SFV) could offer a new treatment modality for dogs and cats. Oncolytic VV was rendered tumour selective by a dual ablation of vaccinia growth factor and thymidine kinase, making it more cancer-specific than previously used VVs in veterinary research. To further increase the efficacy of the VV, an immunostimulatory gene, CD40L, was added to the virus backbone. Avirulent SFV A7(74) has a natural trophism for cancer cells and was not genetically manipulated. Both viruses infected and killed tested cancer cell lines, and VV also infected most of the primary surgical tumour tissues tested. In the nude mouse xenograft model, double deleted VV (vvdd) significantly reduced tumour growth. Interestingly, when in intact monolayers, SCCF1 cells were not killed by VV, but secreted infectious, morphologically abnormal virions. One dog experienced a possible seizure after VV administration, but no other serious adverse events occurred. Vaccinia DNA declined quickly in the blood after virus administration, but was still detectable one week later by qPCR. Only samples taken directly after the VV infusion contained infectious virus, which was not found in any other blood, saliva, urine or faecal samples. Necropsies did not reveal any pathological changes associated with virus administrations. In conclusion, our results show that oncolytic VV and SFV can infect and kill tested canine cancer cell lines, and that VV has antitumoral activity in the mouse xenograft model and it can infect fresh tumour biopsies. In addition, intravenous administration of the viruses did not induce life-threatening adverse events in healthy dogs. These agents thus warrant further evaluation in veterinary medicine.
  • Lindeberg, Heli (Helsingin yliopisto, 2003)
  • Oivanen, Leena (Helsingin yliopisto, 2005)