Eläinlääketieteellinen tiedekunta

 

Recent Submissions

  • Venhoranta, Heli (Helsingin yliopisto, 2015)
    Dairy cattle breeding programs rely heavily on artificial insemination (AI). The AI enables intense selection for desired traits, but the use of few elite bulls carries a risk that inherited defects may proliferate in the population. The emergence of inherited defects is a recurrent issue in cattle breeding and rapid identification and management of genetic defects are crucial for preventing economic losses and maintaining good animal welfare. Modern molecular genetics techniques enable efficient detection of causative mutations, information which is needed for DNA testing. In this study, we investigated three inherited congenital bovine defects and located causative mutations for these diseases. 1. A microdeletion in PEG3 domain causes intrauterine growth restriction and stillbirths to almost half of the pregnancies sired by a single Ayrshire AI bull. The deletion, when inherited from the sire, is semi-lethal but the female mutation carriers can breed normally. The deletion truncates the 3 end of the non-coding maternally imprinted MIMT1 transcript and also causes expression changes in other genes of the domain. The expression changes of hundreds of genes in the foetal side of the placenta were also demonstrated. 2. Inherited gonadal hypoplasia with incomplete penetrance in Northern Finncattle and Swedish Mountain breed is associated with homozygosity for the Cs29 allele. This allele is an ectopic segment that is duplicated and translocated from chromosome 6 to 29 and encompasses the KIT gene. Cs29 allele is associated with colour sidedness in various cattle breeds, which coheres with the results that gonadal hypoplasia is connected with white coat colour. The KIT gene is known to regulate the migration of the germ cells and precursors of melanocytes. 3. A new recessive developmental disorder defined as PIRM (ptosis, intellectual disability, retarded growth and mortality) in Ayrshire breed was associated with single base substitution in the UBE3B gene. Mutation causes in-frame exon skipping, resulting in an altered protein lacking 40 amino acids, which likely comprises protein function. In humans, mutations in the UBE3B gene are associated with Kaufman oculocerebrofacial syndrome, with similar pathological effects as for PIRM syndrome. The UBE3B mutation may be connected with the AH1 haplotype, which is associated with reduced fertility and has a carrier frequency of 26.1% in the North American Ayrshire population. The mutations related to these three defects or syndromes can now be easily tested for. The results can be used to avoid risky matings, cull carriers and provide a veterinary diagnostic. The expression analyses proved that the mutations in Ayrshires also affected the RNA expression of the respective mutated gene or even hundreds of other genes. The described genotype-phenotype associations can be used as a basis for approaches to locate quantitative trait loci in cattle or provide new insights into developmental biology and inform translational research across species.
  • 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.
  • 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.
  • Kolmeder, Carolin (Helsingin yliopisto, 2015)
    Human physiological processes are complemented by those of the microbiota, the collection of all microbes living in and on our body. The human intestinal microbiota is one of the most prominent representatives and many associations with a wide spectrum of human diseases have been identified. Analysing faecal material with nucleic acid based approaches revealed the species richness of the intestinal microbiota and its individuality, being unique to each human being. In addition, to date approximately ten million unique genes have been identified from the human intestinal microbiota. These genes add an enormous additional genetic potential to the human genome, but little is known about which of these genes can be expressed into proteins and the conditions under which the protein synthesis occurs. The focus of this thesis work was to increase the knowledge of the biological processes taking place in-vivo, and to establish a baseline of these functions in the intestine of a healthy adult. Faecal material was used to study the metabolic reactions in the lower intestine, thus avoiding invasive sampling like biopsies. The proteins contained in the faecal material, which represent the molecules of most biological reactions, were targeted. At first, a method to access and analyse faecal proteins was developed, a so called metaproteomics approach. Proteins were analysed by mass spectrometry and the vast amount of resulting data was analysed with a wide range of computational methods to get a comprehensive overview of the intestinal functions. Altogether, 81 biological samples collected from 48 adults were analysed. As the main result, it was shown that individuals can be separated by their specific faecal protein profiles. This, in turn, indicates that the collection of intestinal microbial functions taking place in each of us are unique. In addition, the faecal protein profiles from obese individuals were found to be different from those of non-obese individuals. On a phylum level, it appeared that in obese individuals Bacteroidetes were biologically more active than the phylogenetic analysis suggested. This thesis work has identified several core intestinal proteins and helps to understand the functional significance of the intestinal microbiota. Next, we have to address these proteins in well concerted studies and still need to learn more about many of the encoded functions contained in the intestinal microbial genes.
  • Rahkila, Riitta (Helsingin yliopisto, 2015)
    Ks. RahkilaAbstract.docx
  • Tuppurainen, Eeva (Helsingin yliopisto, 2015)
    Lumpy skin disease (LSD) is an economically important pox disease of cattle and Asian water buffalo caused by a lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. The disease occurs in Africa and the Near East, causing substantial economic losses for the whole cattle industry in affected countries. The disease is characterized by skin nodules, high fever, lymphadenopathy and loss of production of infected animals. Transmission of LSDV is known to occur mechanically by a variety of blood-feeding insects and to a lesser extent through contaminated feed and water, semen or via direct contact. The disease is classified as notifiable by the World Animal Health Organization (OIE). Currently, Finland is free of LSD. The general aim of the study was to investigate the vector capacity of common sub-Saharan tick species, Rhipicephalus appendiculatus, Amblyomma hebraeum and Rhipicephalus decoloratus for LSDV in cattle via mechanical, intra/transstadial or vertical routes. The specific aim was to investigate if mechanical transmission occurs by R. appendiculatus males and transovarial by R. decoloratus females. As many of the infected animals become viraemic without showing skin lesions, it was investigated if feeding on healthy looking skin of viraemic animals was sufficient for successful mechanical transmission. The final objective was to investigate if the virus was able to grow in vitro in Rhipicephalus spp. tick cell lines. In addition, the presence of the virus or viral DNA in ticks collected from naturally infected animals was investigated. Two animal experiments, using naïve cattle and laboratory-reared ticks were conducted at the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa and samples were tested at the Pirbright Institute, United Kingdom. For the first time, transmission of LSDV (or any pox virus) by hard ticks was demonstrated to occur mechanically/intrastadially by R. appendiculatus males and vertically by R. decoloratus females. Feeding directly on skin lesions was not necessary for transmission of the virus between infected and naïve cattle. No evidence of viral replication in Rhipicephalus tick cell lines was obtained. The presence of the viral DNA was detected in Rhipicaphalus, Amblyomma and Hyalomma ticks collected during natural LSDV outbreaks in South Africa and Egypt. In 2013 - 2015 LSDV is spreading in the Near East at a scale never seen before, posing a threat to the European Union, Caucasus region, Afghanistan and Pakistan. In order to control and eradicate the disease, it is fundamental to understand the role of different arthropod vectors and their importance in the field. The presence of infected tick eggs or different instars in the environment underlines the importance of effective prophylactic tools and sufficient vaccination coverage. In addition, this study contributes to the recommendations set for the international trade of live cattle from affected countries.
  • Rantala, Mari H. (Helsingin yliopisto, 2015)
    Fertility in dairy cows has decreased worldwide, also in Finland. The most common reason for treatments of dairy cattle is various fertility disorders (18 % of all animals in 2013). In practice, decreased fertility warrants use of hormonal estrus synchronization protocols to control follicular waves and luteal regression to achieve acceptable pregnancy rates, without unwanted side effects such as short estrous cycles. In the earlier studies of our research group, short estrous cycles were noted in some cyclic dairy heifers and cows when estrus and ovulation were induced with agonistic analogues of prostaglandin F2α (PGF2α) and GnRH treatments administered 24 h apart. Possible causes for such induced short estrous cycles were further elucidated in the four experiments described in this thesis. Estrus and ovulation were induced with PGF2α and GnRH given 24 h apart during early (Day 7 after ovulation) or late (Day 14 after ovulation) diestrus. Also the effect of gonadorelin doses of 0.1 mg or 0.5 mg given 24 h after PGF2α on the occurrence of short estrous cycles, and on the preovulatory release of LH during 6 h following the gonadorelin administration was investigated. The effect of the time interval between PGF2α and GnRH (0 vs. 24 h) given during early diestrus on the occurrence of short estrous cycles was investigated in cyclic dairy heifers and cows. The expression of endometrial receptors of oxytocin, estrogen-α and progesterone as well as enzymes 20α-hydroxysteroid-dehydrogenase and cyclo-oxygenase-II, on Days 2 and 5 after ovulation was analyzed with real-time quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry. The occurrence of induced short estrous cycles was significantly increased with simultaneous administration of PG and GnRH, but was neither related to the size of the preovulatory follicle nor to the GnRH-induced preovulatory release of LH. Also the basal postovulatory release of LH on Days 1, 3 and 5 after ovulation was similar for induced short and normal length estrous cycles. Lower basal LH concentration after ovulation coincided with higher progesterone concentration. The size of the preovulatory follicle was significantly larger when PG and GnRH were given 24 h apart during early diestrus in comparison with late diestrus, but the occurrence of short estrous cycles was similar. The size of the preovulatory follicle in cows did not correlate with the preovulatory secretion of estradiol-17β. The endometrial expressions of receptors and enzymes analyzed were similar for short and normal length estrous cycles. The results described should be taken into account in estrus synchronization protocols utilizing sequential treatments with PG and GnRH.
  • Hokkanen, Ann-Helena (Helsingin yliopisto, 2015)
    Disbudding entails destroying calves horn buds, and in dairy farming is most often done with a hot-iron. Disbudding is routinely carried out because hornless cattle are considered to be safer for themselves and for humans. Hot-iron disbudding is very painful and causes severe pain-related distress and behavioural changes in calves. Options for treating disbudding-related pain during the procedure, and for 24 hours subsequently, are well known, but continued pain and its management are not much studied in calves after disbudding. Pain can cause restlessness and thus affect calves lying time. Pain in humans and rats also changes sleeping behaviour. Pain connected with disbudding often remains untreated. Reasons for this are unclear. Therefore, more knowledge and research are needed on the recognition of calves pain after hot-iron disbudding, on the duration of pain and on options to treat it in an effective, safe and practical way. Research is also needed on producer knowledge and attitudes towards pain in calves and their decision-making in connection with pain alleviation. The objectives of the work reported in this thesis were all connected with gaining an improved understanding of producer perceptions about pain caused to young calves by hot-iron disbudding, and with options available to increase the use of pain alleviation for this common and painful procedure. Initially we asked dairy producers for their perceptions towards disbudding pain in calves. Then, in order to be able to study the duration of pain after disbudding in the future, we attempted to develop a new device to measure calves lying and sleeping time: a small, neck-based, wireless accelerometer system. Because new methods and various options for pain alleviation are needed, we investigated if sublingual detomidine provided sufficient sedation in calves to allow administration of local anaesthetics prior to disbudding. Because the use of pain alleviation is often a choice faced by producers, we wanted to study Finnish dairy producers interests and motivation regarding pain alleviation in connection with disbudding. We studied Finnish dairy producers perceptions on disbudding-related pain and the need for pain alleviation, and how such perceptions affect the actual practice of pain alleviation. Finnish dairy producers estimated disbudding pain to be severe and producer estimation of pain severity caused by disbudding was correlated with their sensitivity to pain caused by different cattle diseases in general. We were able to develop an accurate device for measuring calves lying and sleeping time. Detomidine oromucosal gel was an effective sedative for calves before infiltration of local anaesthetics and disbudding. Finnish dairy producers who estimated the disbudding-related pain and need for pain alleviation to be high had a veterinarian medicate calves before disbudding more often than producers who ranked disbudding pain and need for pain alleviation lower. Because more studies on duration and alleviation of disbudding pain are needed, our new device for measuring lying and sleeping time in calves could make these studies easier in the future. A non-invasive and user-friendly oromucosal sedation method for calves could enhance the use of local anaesthetics before disbudding by making sedation easier. Our findings among dairy producers support the idea that persons who have knowledge of pain and who think pain alleviation is beneficial and important are also more prone to administer pain alleviation. Education of producers on disbudding-related pain could increase the use of pain alleviation in the future. It could also increase pain alleviation for other cattle diseases because producer perceptions on disbudding-related pain are likely to be connected with pain in cattle in general.
  • Kareinen, Ilona (Helsingin yliopisto, 2015)
    Atherosclerosis is an inflammatory disease characterized by the accumulation of cholesterol in the arterial intima and consequently the formation of atherosclerotic plaques. Formation of these plaques is initiated by the appearance of macrophage foam cell in the arterial intima. Foam cells are formed as excessive cholesterol accumulates in the cytosol of macrophages and finally the net influx exceeds the efflux of cholesterol. Excessive accumulation of chemically modified cholesterol in foam cells finally leads to apoptosis and contributes to the formation of the lipid core in atherosclerotic plaques. The efflux of cholesterol from foam cells is essential for preventing the progression of atherosclerosis. The only unidirectional transporters ABCA1 and ABCG1, expressed on macrophages, transport intracellular cholesterol actively to distinct subpopulations of HDL. ApoA-I, the most important structural and functional component of nascent preβ-migrating HDL particles, receives cholesterol from ABCA1. Lipid-free HDL and apoA-I are sensitive to proteolytic modification leading to loss of function of these molecules. Functional apoA-I is essential for removal of cellular cholesterol and for cholesterol homeostasis. Cholesterol efflux initiates reverse cholesterol transport (RCT) which is the pathway for removal of cholesterol from the periphery for its final excretion into feces. The tiny fraction of total body-RCT that originates from the cholesterol-loaded macrophage foam cells located in the intima, is considered the only RCT component directly involved in atherosclerosis. Mast cells are bone marrow-derived inflammatory cells that are able to activate and secrete various mast cell mediators. Mast cells infiltrate the inflamed arterial intima where they can be activated through several stimuli present in the atherosclerotic intima. Mast cells release several inflammatory compounds of which histamine is probably the best known for its notorious effects in anaphylaxis. In addition to histamine and other vasoactive compounds, such as serotonin and bradykinin, mast cells release upon activation their unique serine proteases, tryptase and chymase. Chymase involvement in the progression of atherosclerosis has been suggested in a number of studies. Chymase is an enzyme capable of degrading LDL and HDL components leading to increased uptake of the modified LDL by macrophage foam cells or resulting in diminished cholesterol efflux, respectively. The purpose of this study was to evaluate whether mast cell-dependent HDL and apoA-I proteolysis would occur in vivo and whether such modification would alter the cholesterol efflux capacities of these cholesterol acceptors and finally affect the macrophage-specific RCT (mRCT). In the present study it was demonstrated for the first time that mast cell activation in vivo resulted in HDL proteolysis. Systemic mast cell activation led to the degradation of lipoprotein particles present in HDL and the entire preβ-HDL and α-HDL subpopulations were reduced in mouse serum following systemic mast cell activation. Systemic activation of mast cells in mice blunted the ability of serum and intraperitoneal fluid to promote cholesterol efflux from macrophage foam cells in vitro. Rat cardiac mast cell activation ex vivo led to the production of truncated apoA-I. ApoA-I was cleaved at the carboxyl-terminal region at Phe229 and Tyr192 or only at Tyr192 depending on the mast cell stimulus. Local peritoneal mast cell activation led to decreased ability of intraperitoneally injected apoA-I to promote macrophage cholesterol efflux in vitro. Furthermore treatment with intact lipid-free apoA-I but not chymase-treated apoA-I increased the overall mRCT from the peritoneal cavity to the intestinal contents within 3 hours. Importantly such an increase was fully blocked by the mast cell-specific degranulating compound 48/80 in mast cell-competent mice but not in mast cell-deficient mice. Interestingly local mast cell activation in the skin was able to promote mRCT from skin to feces. This was due to increased vascular permeability and influx of plasma HDL particles to skin consequently leading to increased mRCT. This stimulatory effect could be reproduced by the sole administration of the mast cell mediators, histamine, serotonin, and bradykinin. Importantly histamine treatment in apoA-I deficient mice was unable to promote mRCT. In conclusion, mast cell chymase is able to proteolyze HDL and lipid-free apoA-I reducing their abilities to promote cellular cholesterol efflux. Proteolysis of lipid-free apoA-I by rat cardiac mast cell chymase can occur within minutes and results in the formation of carboxyl-terminally truncated apoA-I. ApoA-I proteolysis in vivo results in reduced mRCT. Local mast cell activation in the skin results in increased mRCT due to increased availability of cholesterol acceptors in the vicinity of macrophage foam cells. These two seemingly opposite results underline the pleiotropic role of mast cells in the development of atherosclerosis
  • Yun, Jinhyeon (Helsingin yliopisto, 2015)
    It is well-known that prepartum sows have a strong instinct to build a nest before parturition. Under commercial conditions, however, the farrowing crate, widely used in modern pig husbandry, restricts this innate behaviour due to the lack of space, materials or both. Restriction of nest-building (NB) behaviour could generate an increase in physiological stress, resulting in a decrease in endogenous hormones, especially oxytocin (OT), which is recognized for its effect on reproductive and behavioural characteristics in mammals. The role of OT in modulation of maternal behaviour, parturition, and lactation has been demonstrated in a wide range of species, including pigs. It is also known that OT is related to the stress reaction, that it reduces anxiety and that it plays a part in emotional reactions in social situations. This study evaluated the effects of provision of space and abundant nesting materials on actual NB behaviour and circulating OT concentrations in prepartum sows. In addition, it also investigated whether facilitating prepartum NB behaviour could improve postpartum maternal characteristics during early lactation, possibly because of elevated OT concentrations in sows. All sows included in the experiment, approximately seven days before the expected parturition date, were housed in: 1) CRATE: the farrowing crate closed (210 × 80 cm), with provision of a bucketful of sawdust, 2) PEN: the farrowing crate opened, with provision of a bucketful of sawdust, and 3) NEST: the farrowing crate opened, with provision of abundant nesting materials. All sows were confined to the farrowing crates, without additional supply of nesting materials, after the first piglet was delivered until seven days post-parturition. Sow blood samples were collected for hormonal assays via indwelling ear vein catheters on days -3, -2, -1, +1, +2, +4 and +7 from parturition, twice a day. Pigs were video-recorded to observe prepartum NB, postpartum nursing and carefulness behaviour. Blood samples from piglets were collected to determine immunoglobulin concentrations. The longest duration of NB behaviour was observed in NEST sows, followed by PEN and CRATE sows respectively (III), and this duration tended to be correlated with prepartum OT (III). Both prepartum OT and prolactin (PRL) concentrations were greater in NEST than in CRATE and PEN sows (III). An interaction was recorded between prepartum OT and PRL concentrations (III). During the periods from days -3 to +7, NEST also brought about an increase in OT concentrations compared with CRATE and PEN (I), and PRL concentrations in NEST sows were also greater than for CRATE sows during those periods (I). From days one to seven of lactation, PRL concentrations were positively correlated with OT (I). Sows in NEST tended to have higher serum NEFA concentrations from days -3 to +7 (II). Piglet growth during early lactation was slower in CRATE than in PEN (II). Post-natal mortality, including piglet deaths resulting from crushing or other factors, indicated no differences between the three treatments (II). During early lactation, piglet serum IgG and IgM concentrations in NEST tended to be greater than in the other treatments (II). The incidence of carefulness behaviour for NEST sows was greater than for the other treatment sows in early lactation (I), and was correlated with OT during the seven days after parturition (I) and with total duration of prepartum NB behaviour (III). The average duration of successful nursing bouts in early lactation was longer for CRATE than for the other sows (I), and negatively correlated with total duration of prepartum NB behaviour (III). In conclusion, it appears that NB behaviour in prepartum sows could be enhanced by the provision of nesting materials. This, coupled with elevated OT and PRL concentrations, could result in improved sow metabolic status, successful colostrum intake measured via neonatal piglet serum IgG and IgM concentrations, and maternal carefulness behaviour during early lactation. This may have potential to increase piglet survival and growth performance during lactation.
  • Kirk, David (Helsingin yliopisto, 2015)
    Clostridium botulinum presents a risk to food safety through the production of endospores. These spores are highly heat-resistant and may withstand temperatures used in food processing. Despite this, the process of spore formation is poorly understood in C. botulinum. This study aimed to analyse in Group I C. botulinum ATCC 3502 the role of sigma (σ) factors σF, σE, σG, and σK. The role of these σ factors is well known in other spore formers, activating in an ordered cascade to regulate gene transcription during sporulation. To study gene expression during sporulation in C. botulinum ATCC 3502, we identified a suitable normalisation reference gene for reverse-transcription real-time PCR (RT-qPCR). Mutants of sigF, sigE, sigG, and sigK were examined on the transcriptional level during sporulation, and each strain was characterised for growth and spore formation. Furthermore, the role of σK in stress tolerance was investigated under cold, NaCl, and pH stresses. Transcriptional analysis, from exponential to stationary phases of growth, of eight candidate reference genes was performed. The candidate genes were 16S ribosomal RNA (rrn), the ATP metabolism enzymes adenosine kinase (adK) and glutamate dehydrogenase (gluD), the DNA-binding protein gyrase (gyrA), and ribosome-related proteins alanyl-tRNA synthetase (alaS), GTP-binding Era (era), RNA polymerase β subunit (rpoC) and 30S ribosomal protein S10 (rpsJ). Of these candidates, only 16S rrn was stable during the study period. 16S rrn was used as the normalisation reference gene for RT-qPCR analysis of spo0A, sigF, sigE, sigG, and sigK expression during the same growth period. Expression of spo0A was highest during exponential growth, suggesting a role in early sporulation. Induction of sigF, sigE, and sigG expression occurred on entry into stationary growth, indicating a role in sporulation. Expression of sigK appeared biphasic, being expressed in both exponential and stationary phases, suggesting σK may play a dual role in sporulation. The genes of σF, σE, σG, and σK were mutated using the ClosTron tool. RT-qPCR analysis of the sigF and sigE sense mutants suggested that the sporulation pathway was disrupted in the early stages. This was confirmed by electron microscopy, which showed that all sigF and sigE mutants were unable to form spores. They halted sporulation after asymmetric cell division, stage II of the seven-stage sporulation cycle. The sigG sense mutant showed delayed transcription of the sporulation pathway and both sigG mutants possessed a thin spore coat but no cortex. This indicated that σG may be responsible for cortex, but not coat, formation in C. botulinum. The sigK sense mutant did not express the early-sporulation genes spo0A and sigF. Both sigK mutants appeared to halt sporulation early. Sporulation was restored by complementing the sigK mutation in trans. These results suggested that σK plays an essential role in early sporulation of C. botulinum ATCC 3502, and adds further weight to the possibility of a dual role in sporulation overall in this strain. Expression of sigK was assessed in C. botulinum ATCC 3502 after cold, osmotic (NaCl), and acidic shock. After cold and osmotic shock, expression of sigK was induced. Both sense and antisense sigK mutants were then grown under stress conditions of low temperature, high NaCl, and low pH. Under low temperature and high NaCl conditions, but not in low pH, growth of the mutant strains was negatively affected compared to parent strain growth, suggesting that σK may play a role in tolerance to low temperature and high salinity stress conditions.
  • Derman, Yağmur (Helsingin yliopisto, 2015)
    The capability of Clostridium botulinum to survive food processing and grow in foods is a substantial hazard to human health. Clostridium botulinum can thrive under stress conditions and the wide genetic variation among C. botulinum strains reflects differences in their growth properties. However, the strain variation in growth, molecular mechanisms, and genetic markers behind the stress tolerance of C. botulinum are poorly understood. This study focused on the variation in growth of Group II C. botulinum strains at temperatures out of the optimal range and characterized the role of the genes that are involved in the stress response of Group I ATCC 3502 and Group II Beluga strains. At extreme temperatures, Group II C. botulinum strains showed significant variation. Some strains showed better growth at 37 °C than their generally accepted optimum growth temperature of 30 °C. Although not absolute, the minimum growth temperatures of 24 Group II strains in our experimental setting were higher than expected. The AFLP typing indicated a weak association between genetic background and growth at extreme temperatures. In addition, clustering analysis showed that the type B and F strains are closely related, since they were clustered together, and the type E strains formed a separate cluster. Our data indicated that Group II C. botulinum strains show considerable differences in their growth properties, and this should be taken into account in designing safety measures against this foodborne pathogen. The study implied that csp genes play important roles in NaCl, pH, and ethanol stress and motility of C. botulinum ATCC 3502. The growth of the cspB mutant was impaired in relation to the wild-type strain under all stress conditions tested, suggesting a universal role in stress response. Additionally, functional cspC was required for efficient growth during NaCl, pH, and ethanol stress. The cspA did not take part in NaCl, pH, and ethanol stress responses, and inactivation of this gene enhanced growth, which suggested a possible growth repressor role. In addition, cspB was not involved in motility, but cspA and cspC were crucial for flagellation and motility. The two-component system CBO2306/CBO2307 of Group I C. botulinum ATCC 3502 and CLO3403/CLO3404 of the Group II Beluga strain were induced up to 4.4- and 3.4-fold, respectively, after cold shock, but not at optimum growth temperatures. This indicated that both systems were involved in rapid response to cold shock. This argument was further confirmed by inactivation of the CBO2306/CBO2307 or CLO3403/CLO3404 genes, which resulted in impaired ability of the mutant strains to grow at low temperatures compared with the wild-type strains. In addition, the histidine kinase encoding clo3403 was required for motility. The cbo2802 that encodes the DEAD-box RNA helicase in C. botulinum ATCC 3502 was cold-inducible. The RT-qPCR analysis showed a 7.6-fold rapid increase in the relative expression of cbo2802 after cold shock. The significance of cbo2802 in the growth at suboptimal temperature of C. botulinum ATCC 3502 was further characterized by disruption of this gene. The mutants without functional cbo2802 showed impaired growth and restricted motility at suboptimal temperature. This study concluded that strains of Group II C. botulinum pose considerable variation in growth properties, which may be of critical importance in predictive modeling of growth in foods. The major cold-shock protein-encoding gene cspB seems to play a universal role in stress response and cspC a role in NaCl, pH, ethanol stress, and motility. The cspA gene is not involved in the stress response of C. botulinum ATCC 3502, but is important for flagella formation. The CBO2306/CBO2307 in Group I C. botulinum ATCC 3502 and CLO3403/CLO3404 in the Group II Beluga strain are part of the cold stress response machinery. Moreover, clo3403 is required for motility. We report for the first time in C. botulinum that DEAD-box RNA helicase is crucial for growth at suboptimal temperature and motility.
  • Herva, Tuomas (Helsingin yliopisto, 2015)
    Animal welfare (AW) is an issue of growing concern in Finland as well as in other developed countries. A public debate has focused on the potential AW problems resulting from current production systems. Possibilities to find mutual benefit for animals, farmers, industry and society have received less attention. According to the reviewed literature the inconsistency of determination and perception of AW appeared to be a major barrier to enhance AW. Farmers should be confident that their measures to promote AW satisfy public opinion and are ecomically sustainable. The main objective of the study was a thorough understanding of relationships between AW and beef production economics to find barriers and opportunities for enhanced AW. A version of the Animal Needs Index (ANI/TGI 35L), modified for Finnish beef production, called A-index was used for AW assessments. The A-Index was modified and evaluated based on Test Theory. On-field associations between A-index and production parameters were determined on 180 farms and over 12 000 bulls using statistical multilevel models. Economic evaluation of AW was based on comparison between cold and warm housing using the confirmed association between AW and production results. AW was associated with good production results. A-Index and the best subset of items used as welfare score (WFS) were covering different aspects of AW. The association between the used measures and production results, reflecting AW in certain degree, can be considered as a proof of the criterion validity of A-Index and WFS. Cold housing with enhanced welfare and bedding based on own straw at a reasonable price was economically favourable. Profitability of cold housing was sensitive to fluctuation in bedding price. Developing a reasonably priced market for bedding material would be a major way to enhance AW. Rubber covered slats were found to be a profitable way to enhance AW in warm housing. A reform of the subsidy system was suggested to be needed to fulfil the aims of the subsidy regime to support AW.
  • 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.
  • Mölsä, Sari (Helsingin yliopisto, 2014)
    Cranial cruciate ligament (CCL) disease is one of the most common causes of lameness in dogs. Surgical treatment is recommended to stabilize the stifle joint, alleviate pain, and delay the progression of osteoarthritis (OA). A variety of surgical techniques has been introduced and can be broken down into the more traditional intracapsular ligament replacement and extracapsular suture techniques and the newer neutralizing dynamic osteotomy techniques. Although an enormous amount of literature is available concerning this disease, surprisingly few studies have assessed surgical outcome with objective evaluation methods and comparison between groups. Previous studies have demonstrated significant improvement of limb function after CCL surgery. However, OA has been shown to progress in most patients, and this may cause deterioration in the function of the surgically treated limb over time. Long-term follow-up studies that include objective evaluation methods designed for assessing orthopedic outcome are needed. This thesis aimed to evaluate the long-term surgical outcome and signs of chronic pain after CCL surgery. Also, outcomes between the intracapsular, extracapsular and osteotomy techniques were compared. A multimodal approach, including an owner questionnaire and assessment of chronic pain using the validated Helsinki Chronic Pain Index (HCPI), orthopedic and radiographic examinations, force plate analysis, and as a new evaluation method, a physiotherapeutic examination was included. To provide reference values, clinically healthy Rottweilers and Labrador Retrievers were examined. A long-term retrospective follow-up with a mean time interval of at least 2.7 years between the surgery and evaluation was conducted. Of the 206 surgically treated dogs that were evaluated by their owners, 31.1% had a HCPI value ≥ 12, indicating pain. Of the 47 dogs evaluated by a veterinarian 31.9% showed a pain response to flexion/extension of the surgically treated joint. In addition to the unilaterally treated CCL rupture, contralateral stifle joint pathology and other orthopedic problems were frequently diagnosed during the evaluation. When symmetry of weight bearing was evaluated in dogs with no other orthopedic findings (n=21),approximately 30% of dogs had decreased dynamic and static weight bearing in the surgically treated limbs. Also, goniometric angles of the surgically treated limbs remained inferior to those of healthy limbs, and impairment of active range of motion was frequently observed. The owner assessments revealed no significant differences between the surgical techniques in long-term outcome. However, differences were seen in clinical evaluation of dynamic and static weight bearing between intracapsular and osteotomy technique groups. Although the retrospective study design and low sample size have to be acknowledged and may cause bias to the results, osteotomy techniques may offer long-term limb function that is superior to that achieved with the intracapsular technique. The low number of dogs treated with the extracapsular technique did not allow comparison of dynamic or static weight bearing to other technique groups.