Eläinlääketieteellinen tiedekunta

 

Recent Submissions

  • Pöntinen, Anna (Helsingin yliopisto, 2019)
    Listeria monocytogenes is a remarkable bacterium, as it is able to shift from a capable environmental saprophyte into a severe intracellular pathogen. As a strictly foodborne pathogen, L. monocytogenes poses a notable risk, particularly to those consumers among the risk groups for whom invasive listeriosis is potentially fatal. Furthermore, modern consumption habits and increasingly favoured ready-to-eat foods, often consumed without proper heating, increase the risk of acquiring the foodborne disease. The aim of this study was to investigate the genetic mechanisms conferring wide-ranging stress tolerance in L. monocytogenes. Two-component systems, comprising a sensor histidine kinase and a cognate response regulator, aid bacteria in sensing and adapting to changes in both surrounding environmental as well as intracellular conditions. The histidine kinases, in particular, have lacked comprehensive studies on their roles in the stress tolerance of L. monocytogenes. Thus, histidine kinases were studied by expressional analyses under cold conditions and by mutationally disrupting each histidine kinase-encoding gene in a parental model strain, L. monocytogenes EGD-e. The modified strains were individually challenged at high (42.5 degrees C) and low (3.0 degrees C) temperatures, high (9.4) and low (5.6) pH levels, and high salt (6% NaCl), ethanol (3.5 vol%) and hydrogen peroxide (5 mM) concentrations. Expressional studies and growth experiments on genetically modified strains proved lisK and yycG to respectively play central roles in the acclimation and immediate growth of L. monocytogenes at low temperatures. The most substantial increase in gene expression under cold conditions was that of the chemotaxis gene cheY with 236-fold upregulation at 3 degrees C. The disrupted ΔliaS strain displayed impaired growth in response to all the other stresses, particularly at a high temperature and under osmotic stress. These studies demonstrated the prominent importance of the histidine kinase-encoding genes yycG and lisK to cold tolerance and liaS, with roles in the growth of L. monocytogenes under multiple stresses. To shed light on the accessory genetic mechanisms that cause large strain variation in L. monocytogenes in withstanding heat treatments, heat resistance-conferring traits were further investigated by means of whole-genome sequencing. Comparing the complete genomes of heat-resistant L. monocytogenes AT3E and -sensitive AL4E strains revealed the heat-resistant strain to harbour a novel 58-kb plasmid, pLM58, which was absent in the sensitive strain. Furthermore, curing of the plasmid in AT3E produced a marked decrease in heat resistance from virtually no reduction to a 1.1 cfu/ml log10 reduction at 55.0 degrees C. In pLM58, a 2,155-bp open reading frame annotated as an ATP-dependent ClpL protease-encoding gene was identified. Conjugation of the coding sequence and the putative promoter of the clpL gene into a natively heat-sensitive L. monocytogenes 10403S strain, in turn, enhanced the survival of the strain from a 1.2 cfu/ml log10 reduction to a 0.4 cfu/ml log10 reduction in heat challenge at 55.0 degrees C. In this study, we presented the first evidence of plasmid-mediated heat resistance in L. monocytogenes and identified the protease ClpL to be a novel plasmid-borne heat-resistance mediator. The emerging resistance of L. monocytogenes to benzalkonium chloride, a quaternary ammonium compound widely used as a detergent in food-processing facilities, is a significant concern for food safety and public health. The resistance of 392 L. monocytogenes isolates from Finland (n = 197) and Switzerland (n = 195) to benzalkonium chloride was assessed. A minimal inhibitory concentration of 20 µg/ml was defined. Altogether, 11.5% of the strains proved to be resistant to benzalkonium chloride. Serotype 1/2c harboured the highest prevalence, 32.4% (11/34), of benzalkonium chloride-resistant strains, while in total, most of the resistant strains belonged to serotype 1/2a. Altogether, 68.9% of the resistant strains harboured at least one of the efflux pump system-encoding genes, bcrABC, emrE or qacH, known to confer benzalkonium chloride resistance in L. monocytogenes. We found resistant strains with partially or completely efflux pump-dependent benzalkonium chloride resistance, with the exception of the known resistance-mediating efflux pumps, suggesting the existence of other resistance-contributing efflux pump systems. The lacking of known efflux pump system-encoding genes in addition to efflux pump-independent benzalkonium chloride resistance, in turn, indicates the contribution of completely novel benzalkonium chloride resistance mechanisms. The aim of these studies was to shed light on the genes contributing to the versatile stress tolerance abilities and strain variation of the severe foodborne pathogen, L. monocytogenes. Knowledge of such traits may aid in developing targeted strategies and measures to identify and control the contamination and risks caused, in particular, by stress-tolerant L. monocytogenes strains.
  • Lopez Valladares, Gloria (Helsingin yliopisto, 2019)
    Isolates of Listeria monocytogenes (N=932) collected from human cases of invasive listeriosis in Sweden between 1958 and 2010 were serotyped and characterised with pulsed-field gelelectrophoresis (PFGE) and AscI restriction enzyme. The genotype diversity of L. monocytogenes isolates was investigated and related to genotypic results from epidemiological information on human infection, in order to detect possible clustering of L. monocytogenes genotypes over time, season, location, age, or gender (Paper I). From 1972 to 1995, serovar 4b was the predominant serovar; however, in 1996, serovar 1/2a became the major serovar among human listeriosis cases in Sweden. Based on the number and distribution of all bands in the profile, 63 PFGE types belonging to serovars 1/2b, 3b and 4b and 119 PFGE types belonging to serovars 1/2a and 1/2c were identified (Paper I). The PFGE types were further assembled into PFGE groups, based on the number and distribution of small bands below 145.5 kb (Papers II and III). As the genomic region of small bands is genetically more conservative than in large bands, the distribution of small bands establishes relatedness of strains and defines genetic markers for both lineages. Cold-smoked salmon (Salmo salar) and gravad salmon packed under modified atmosphere or vacuum from three manufacturers were purchased in Sweden and Germany in 2005 and the occurrence and levels of L. monocytogenes were analysed (Paper IV): 56 products were analysed and eleven harboured L. monocytogenes. From the positive samples, 56 isolates were analysed with AscI, and 11 isolates were further analysed with ApaI: five AscI PFGE types were identified, four belonging to serovar 1/2a and one to 4b. Forty-three (n=43: 76.8%) isolates shared serovar 1/2a and 13 (23.2%) shared serovar 4b and all AscI types were identified among human clinical strains in Sweden. Moreover, three gravad salmon samples harboured two PFGE types each from different lineages, serovar 1/2a and serovar 4b. Although, in most of the products, the level of L. monocytogenes was less than 100 cfu/g, the highest level was 1500 cfu/g. The occurrence of L. monocytogenes was 12.9% in gravad salmon, encountered in three manufacturers (A, B, C) and 28% in cold-smoked salmon only from manufacturer A. Although the level of L. monocytogenes in RTE fish products is generally low, these products, should be considered possible sources of listeriosis in Sweden. A patient may harbour more than one L. monocytogenes PFGE type that can be determined through PFGE and AscI restriction enzyme. However, to avoid misleading conclusions, several L. monocytogenes colonies should be isolated and characterised from different sites from the same patient or mother-baby pairs (Paper V).
  • Felin, Elina (Helsingin yliopisto, 2019)
    Salmonella spp., Yersinia enterocolitica, Toxoplasma gondii and Trichinella spp. are the most relevant biological hazards in the context of meat inspection of pigs in the European Union (EU). These zoonotic pathogens show no clinical symptoms or gross pathological lesions in pigs, and thus are not detectable with current meat inspection procedures, except Trichinella spp. by laboratory analysis. In this study, we analysed the serological prevalence of these pathogens in Finnish fattening pigs and evaluated serological monitoring as a control method. In total, we studied 1353 meat juice samples and 1793 serum samples of fattening pigs using commercial ELISA kits. The seroprevalence of pathogenic Yersinia spp. was the highest of the studied pathogens, and Yersinia antibodies were detected in 57% and 66% of the meat juice and serum samples at the end of the fattening period, respectively. The seroprevalences of Salmonella spp. and T. gondii were low. Salmonella antibodies were detected in 3% of the meat juice samples and in 18% of the serum samples at the end of fattening. T. gondii antibodies were detected in 3% of meat juice samples and 1% of serum samples. Trichinella spp. antibodies were not detected. The seroprevalences at the end of the fattening or at slaughter were not associated with post-mortem findings of the current batch, which was expected. This indicates that we need new tools to control these public health hazards in pork. Meat juice serology at slaughter was feasible and easy to perform. We observed huge differences between farms considering Salmonella spp., Yersinia spp. and T. gondii seroprevalences. This shows that farm-level serological data could be used as part of the food chain information (FCI) for risk-based decisions to improve food safety. Risk-based decisions include slaughtering arrangements, additional carcass processing, targeted sampling at the slaughterhouse and improved biosecurity measures at the farm. However, risk mitigation targets and procedures must be carefully adjusted for each pathogen. With targeted serological monitoring of T. gondii we could effectively target control measures and diminish the pathogen in pork. Serological monitoring of pathogenic Yersinia spp. could be the first step in the huge challenge of Y. enterocolitica in pigs, allowing for the possibility of slaughtering pigs from high-risk farms at the end of the day. Serological monitoring of Salmonella spp. would be beneficial, but would only have a limited positive impact on food safety, because the current situation is already excellent. Serosurveillance of Trichinella spp. would become meaningful, if current testing is to be diminished. In addition to food safety issues, FCI could be useful for visual-only inspection, which should be the most common inspection method. We analysed authentic FCIs and meat inspection findings of 85 slaughter batches of fattening pigs. In addition, we analysed on-farm health status indicators, assessed by a veterinarian, and the meat inspection findings of another 57 slaughter batches. The partial carcass condemnation rate of the current batch was best predicted by the partial carcass condemnation rate of the pigs from the same farm within one year. Constant coughing and tail biting at a farm were associated with partial carcass condemnations. On-farm health indicators (such as the healed tail biting rate at the end of fattening and constant coughing during fattening) together with previous meat inspection results could be used as part of the FCI to make decisions regarding the meat inspection procedure: visual-only or additional inspections. However, farmers must be properly advised to carefully report this information.
  • Pakkanen, Soile (Helsingin yliopisto, 2019)
    Alpha2-adrenoceptor agonists detomidine and romifidine are used in equine medicine for sedation and analgesia. Vatinoxan is an α2-adrenoceptor antagonist that has poor ability to cross the blood-brain barrier, limiting its actions to peripheral α2-adrenoceptors. The main objective of these studies was to evaluate the effect of vatinoxan in preventing the adverse effects, such as cardiovascular compromise, decreased intestinal motility and changes in plasma glucose concentrations, of detomidine and romifidine, while sparing the sedative action in horses. Plasma drug concentrations were investigated to elucidate the pharmacokinetic agonist-antagonist interactions. To assess cardiovascular effects of the drugs, heart rate and blood pressures were measured in horses treated with α2-adrenoceptor agonists and vatinoxan. In horses under general anaesthesia, premedicated with either detomidine alone or with vatinoxan, cardiac index and systemic vascular resistance and tissue oxygen delivery were investigated. Sedation and intestinal borborygmi were scored after treatment with detomidine, romifidine and vatinoxan. Glucose, insulin, ACTH, lactate, FFA, cortisol, triglyceride, and potassium and sodium were analysed from blood samples. Vatinoxan attenuated the bradycardia, hypertension and intestinal hypomotility induced by detomidine and romifidine, while the effect on sedation was minor. Vatinoxan alone increased heart rate, but had no effect on mean arterial blood pressure. Premedication with vatinoxan and detomidine resulted in severe hypotension in anaesthetized horses, and high doses of dobutamine were required for blood pressure support. Combined premedication also resulted in decreased systemic vascular resistance and increased cardiac index and tissue oxygen delivery. The latter two might, at least partly, be related to high doses of dobutamine. Vatinoxan increased volume of distribution and decreased plasma concentration of α2-adrenoceptor agonists. Romifidine induced hyperglycaemia in horses, which vatinoxan alleviated. Vatinoxan alone had no effect on plasma glucose concentration. Serum FFA was significantly higher after vatinoxan alone than after romifidine alone or combined with vatinoxan. In conclusion, vatinoxan alleviated the cardiovascular and intestinal effects of detomidine and romifidine in horses, while no clinically relevant effect occurred on sedation. When vatinoxan was administered before general anaesthesia, marked hypotension followed. Romifidine-induced hyperglycaemia was attenuated by vatinoxan. Vatinoxan increased the volume of distribution and decreased plasma concentrations of detomidine and romifidine. Romifidine had no significant impact on plasma concentration of vatinoxan.
  • Luukkanen, Jenni (Helsingin yliopisto, 2019)
    Official control in slaughterhouses, consisting of meat inspection and food safety inspection, has an important role in ensuring meat safety, animal health and welfare, and prevention of transmissible animal diseases. Meat inspection in the European Union (EU) includes the inspection of food chain information, live animals (ante-mortem inspection), and carcasses and offal (post-mortem inspection). Food safety inspections are performed to verify slaughterhouses’ compliance with food safety legislation and are of the utmost importance, especially if slaughterhouses’ self-checking systems (SCSs) fail. The aim of this study was to investigate the prerequisites for official control such as the functionality of the task distribution in meat inspection and certain meat inspection personnel-related factors. In addition, needs for improvement in slaughterhouses’ SCSs, meat inspection, and food safety inspections, including control measures used by the official veterinarians (OVs) and their efficacy, were examined. In the EU, competent authorities must ensure the quality of official control in slaughterhouses through internal or external audits, and the functionality of these audits was also studied. Based on our results, meat inspection personnel (OVs and official auxiliaries [OAs]), slaughterhouse representatives, and officials in the central authority were mainly satisfied with the functionality of the present task distribution in meat inspection, although redistributing ante-mortem inspection from the OVs to the OAs was supported by some slaughterhouse representatives due to perceived economic benefit. Ante-mortem inspection was assessed as the most important meat inspection task as a whole for meat safety, animal welfare, and prevention of transmissible animal diseases, and most of the respondents considered it important that the OVs perform ante-mortem inspection and whole-carcass condemnation in red meat slaughterhouses. In a considerable number of slaughterhouses, OA or OV resources were not always sufficient and the lack of meat inspection personnel decreased the time used for food safety inspections according to the OVs, also affecting some of the red meat OAs’ post-mortem inspection tasks. The frequency with which OVs observed post-mortem inspection performed by the OAs varied markedly in red meat slaughterhouses. In addition, roughly one-third of the red meat OAs did not consider the guidance and support from the OVs to be adequate in post-mortem inspection. In various parts of this study, OVs’ demand for increased guidance and support from their superiors in control actions of a difficult nature was emphasized. According to our results, the most common non-compliance in slaughterhouses concerned hygiene such as cleanliness of premises and equipment, hygienic working methods, and maintenance of surfaces and equipment. Chief OVs in a few smaller slaughterhouses reported more frequent and severe non-compliances than other slaughterhouses, and in these slaughterhouses the usage of written time limits and enforcement measures by the OVs was more infrequent than in other slaughterhouses. Deficiencies in documentation of food safety inspections and in systematic follow-up of corrections of slaughterhouses’ non-compliance had been observed in a considerable number of slaughterhouses. In meat inspection, deficiencies in inspection of the gastrointestinal tract and adjacent lymph nodes were most common and observed in numerous red meat slaughterhouses. Internal audits performed to evaluate the official control in slaughterhouses were considered necessary, and they induced correction of observed non-conformities. However, a majority of the interviewed OVs considered that the meat inspection should be more thoroughly audited, including differences in the rejections and their reasons between OAs. Auditors, for their part, raised a need for improved follow-up of the audits. Our results do not give any strong incentive to redistribute meat inspection tasks between OVs, OAs, and slaughterhouse employees, although especially from the red meat slaughterhouse representatives’ point of view the cost-efficiency ought to be improved. Sufficient meat inspection resources should be safeguarded in all slaughterhouses, and meat inspection personnel’s guidance and support must be emphasized when developing official control in slaughterhouses. OVs ought to focus on performing follow-up inspections of correction of slaughterhouses’ non-compliance systematically, and also the documentation of the food safety inspections should be developed. Hygiene in slaughterhouses should receive more attention; especially in slaughterhouses with frequent and severe non-compliance, OVs should re-evaluate and intensify their enforcement. The results attest to the importance of internal audits in slaughterhouses, but they could be developed by including auditing of the rejections and their underlying reasons and uniformity in meat inspection.
  • Sihvo, Hanna-Kaisa (Helsingin yliopisto, 2019)
    Abnormally hard breast fillet consistency began to emerge in commercial broiler chickens approximately around 2010. Due to the remarkable muscle hardness, the condition acquired a vernacular name ‘wooden breast myopathy’ (WB). This thesis includes studies of WB morphology and pathogenesis on field samples obtained from slaughterhouses in 2012 and experimental rearing studies of broilers, conducted in 2012 and 2014. The first substudy of this thesis describes the chronic morphology of WB and resulted in the first peer-reviewed publication on WB. The second and third substudies include further analyses on the morphology and pathogenesis of WB. This work characterizes WB as an abnormally firm muscle consistency that is restricted to the pectoralis major muscle in broiler chickens. Additional macroscopic features include pale color, outbulging appearance, occasional hemorrhage and a layer of clear fluid on the muscle surface. WB starts to develop after two weeks of age at the earliest and typically proceeds into a chronic myodegeneration in three to four weeks of age. The lesion begins focally and typically develops into a diffuse lesion that involves the major pectoral muscle completely. Microscopically, WB manifests as a polyphasic myodegenerative disease. This rules out single injuries to the muscle tissue as etiologies for wooden breast myopathy. Ongoing myodegeneration is accompanied by regeneration, and at chronic stages prominent secondary changes such as fibrosis develop. Lymphocytic phlebitis is strongly associated with the myodegeneration, but its role in the pathogenesis of wooden breast is currently disputable. The restricted location of the wooden breast lesion in the pectoralis major muscle distinguishes it from several other myodegenerative diseases that widely affect the skeletal muscle system and often the cardiac and smooth muscle systems too. Other skeletal muscles, such as thigh or dorsal muscles, may occasionally exhibit wooden breast-like lesions, but the syndrome primarily affects the major pectoralis muscle. Relatively reduced microvessel density contributes to the development of wooden breast myopathy. Either the affected birds initially exhibit lower capillary densities, compared to the birds that never succumb to wooden breast, or a relative reduction in the vascular supply,which occurs before the initiation of the degenerative lesion. Decreased dietary selenium does not affect the prevalence of wooden breast myopathy. The morphology of polyphasic myodegeneration renders selenium deficiency as one possible causative factor for wooden breast myopathy, but decreased selenium content in the diet had no effect on the prevalence of wooden breast myopathy in our experimental rearing studies
  • Hewetson, Michael (Helsingin yliopisto, 2018)
    Gastric ulcers can develop in foals and horses of all breeds and uses, and the term equine gastric ulcer syndrome (EGUS) has been coined to describe this disease because of its multifactorial and complicated nature. Currently, detection of EGUS by gastroscopy is the only reliable ante mortem method for definitive diagnosis in horses, and is considered the gold standard against which all other diagnostic tests are compared. Disadvantages of gastroscopy are that it requires the horse to be sedated, it is not readily available to most veterinarians, it is an inefficient expenditure of time, and requires a minimum level of expertise to perform and interpret. A urine sucrose test has been reported to be a reliable method of detecting gastric ulcers in horses; however, technical difficulties associated with urine collection have limited the practical value of the test. It was hypothesized that blood sucrose concentration following nasogastric administration of sucrose can be used as a simple, economical alternative to reliably and practically detect gastric ulcers in horses; and a series of studies were subsequently conducted to develop and validate the test, including determination of the feasibility of the method; sucrose assay development and standardization; and field validation through determination of the performance characteristics of the test in selected populations of horses. The feasibility of the method was determined in 12 adult horses with naturally occurring gastric ulceration. Horses with moderate to severe gastric ulceration demonstrated a significant increase in serum sucrose concentrations at 30, 45, 60 and 90 minutes following nasogastric administration of sucrose. Peak sucrose concentrations occurred at 45 minutes and were correlated with ulcer severity. It was concluded that the determination of sucrose concentration in blood is a feasible alternative to urine when performing sucrose permeability testing in the horse, and may represent a useful screening test for identifying horses with endoscopically visible gastric ulceration. An accurate, yet practical and cost-effective method for quantifying sucrose in equine serum that can be applied to sucrose permeability testing in the horse was subsequently developed and validated using gas chromatography with flame ionization detection (GC-FID). The assay provided an acceptable degree of linearity, accuracy and precision at concentrations of sucrose as low as 2.34 µmol/L and as high as 20.45 µmol/L. Percentage recovery of sucrose from serum ranged from 89 – 102%; and repeatability and intermediate precision (RSD %) ranged from 3.6 to 6.7 % and 4.1 to 9.3 % respectively. The limit of detection was 0.73 µmol/L. It was concluded that the method is valid; and can be applied to the assessment of gastric permeability in the horse. The performance characteristics of the test were subsequently assessed in a large group of adult horses and foals with naturally occurring gastric ulceration by comparing it to gastroscopy as the gold standard. The diagnostic accuracy of blood sucrose for diagnosis of gastric lesions (GL); glandular lesions (GDL); squamous lesions (SQL); and clinically significant lesions (CSL) at 45 and 90 minutes after administration of 1 g/kg of sucrose via nasogastric intubation was assessed using receiver operator characteristics (ROC) curves and calculating the area under the curve (AUC). For each lesion type, sucrose concentration in blood was compared to gastroscopy as the gold standard; and sensitivities (Se) and specificities (Sp) were calculated across a range of sucrose concentrations. Ulcer grading was performed blindly by one observer; and the results were validated by comparing them with that of two other observers, and calculating the level of agreement. Cut-off values were selected manually to optimize Se. Because of concerns over the validity of the gold standard, additional Se, Sp, and lesion prevalence data were estimated and compared using Bayesian latent class analysis. Using the traditional gold standard approach, the prevalence of GL; GDL; SQL and CSL for adult horses was 83%; 70%; 53% and 58% respectively. For foals, the prevalence of GL; GDL; SQL and CSL before weaning was 21%; 9%; 7% and 8% respectively; and increased to 98%; 59%; 97% and 82% respectively after weaning. At the selected cut-offs, Se ranged from 51% to 79% for adult horses; and 84% to 95% for foals, depending upon the lesion type and time of sampling. Sp was poor, ranging from 43% to 72%; and 47% to 71% in adult horses and foals respectively. Estimates of Se and Sp were consistently higher in foals when using a Bayesian approach, however there was little difference between the methods when compared in adult horses. It was concluded that blood sucrose is neither a sensitive or specific test for detecting EGUS in adult horses and is therefore unsuitable as a screening test this study population. In contrast, blood sucrose appears to be a sensitive test for detecting EGUS in foals. Due to its poor specificity, it is not expected that the sucrose blood test will replace gastroscopy, however it may represent a clinically useful screening test to identify foals that may benefit from gastroscopy. Bayesian latent class analysis may represent an alternative method to evaluate the diagnostic accuracy of gastric permeability tests in an attempt to avoid bias associated with the assumption that gastroscopy is a perfect test.
  • Mascher, Gerald (Helsingin yliopisto, 2018)
    Clostridium botulinum is a dangerous foodborne pathogen that forms highly resistant endospores and the extremely potent botulinum neurotoxin. Whereas endospores enable the survival and transmission of the organism in many harsh environments, the botulinum neurotoxin blocks neurotransmission and causes the severe and potentially lethal disease botulism in humans and animals. Both traits play an important role in the life of this pathogen and temporally overlap in C. botulinum batch cultures, suggesting common regulation. However, the co-regulation of sporulation and neurotoxin synthesis and the significance of both traits during stress conditions have not been examined in detail. This study focused on the role of the master regulator of sporulation Spo0A in neurotoxin synthesis in Group II C. botulinum type E strains, which lack the well-known neurotoxin gene regulator BotR. Furthermore, the role of the two traits during heat stress in Group I C. botulinum ATCC 3502 was investigated. Group II C. botulinum strains represent the main hazard in minimally-processed anaerobically-packaged foods relying on cold storage, as Group II strains are able to grow and produce toxin at temperatures as low as 3 °C. Cold tolerance mechanisms are scarcely known in psychrotrophic Group II C. botulinum. Studying the mechanisms required for adaption and growth at low temperatures is crucial to counter the safety hazards caused by this dangerous pathogen. The role of a two-component signal transduction system in the cold tolerance of Group II C. botulinum type E was investigated. A better understanding of neurotoxin gene regulation and mechanisms contributing to cold tolerance might enable the development of measures to reduce the risk of botulism outbreaks. The sporulation transcription factor Spo0A was shown to control the initiation of sporulation and neurotoxin synthesis in C. botulinum type E Beluga. The non-sporulating spo0A mutants produced drastically less neurotoxin than the wild-type strain, and in vitro binding assays showed that Spo0A binds to a putative Spo0A-binding box (CTTCGAA) upstream of the neurotoxin gene operon, suggesting the direct activation of neurotoxin synthesis by Spo0A. The sequence and location of the putative Spo0A-binding box is conserved among C. botulinum type E strains, and analysis of spo0A mutations in two more type E strains (K3 and 11/1-1) affirmed the important role of Spo0A in neurotoxin type E synthesis. Spo0A is the first neurotoxin regulator reported in C. botulinum type E strains that lack the neurotoxin gene activating alternative sigma factor BotR. However, co-regulation of sporulation and neurotoxin synthesis is probably not limited to type E strains. Analysis of heat shocked continuously growing C. botulinum type A ATCC 3502 cultures revealed simultaneous downregulation of both traits in response to heat stress, which was affirmed by decreased toxin synthesis and abolished sporulation in batch cultures incubated at 45 °C compared to cultures incubated at 39 °C. This suggests that both traits might be co-regulated in C. botulinum type A, possibly also via Spo0A, which was significantly downregulated after heat shock, whereas the expression of genes encoding the known neurotoxin gene transcription activators BotR and CodY was unaffected or even upregulated during the heat shock response. While heat stress had a negative effect on sporulation and neurotoxin synthesis, the expression of genes related to motility was induced after heat shock. This suggests that motility is the preferred choice when facing elevated temperatures, probably to search for environments with less harmful temperatures. In order to grow and produce neurotoxin at cold temperatures, bacteria have to sense low temperatures and adjust their metabolism and structure for efficient growth in cold environments. We identified the first two-component signal transduction system (TCS) induced during the cold-shock response and needed for efficient growth at low temperatures in psychrotrophic C. botulinum type E. Expression of the TCS genes clo3403 (encoding a histidine kinase for sensing) and clo3404 (encoding a DNA regulator for responding) was increased after cold shock and prolonged compared to the expression pattern observed at the optimal growth temperature, suggesting that the TCS CLO3403/CLO3404 is needed for cold adaptation. Furthermore, inactivation of the TCS genes clo3403 and clo3404 resulted in impaired growth with significantly reduced maximum growth rates at low temperatures but not at the optimum temperature compared to wild-type growth. The important role of the TCS CLO3403/CLO3404 for cold tolerance in C. botulinum type E was confirmed by successful complementation of the mutations. In summary, this study demonstrated that sporulation and neurotoxin synthesis are co-regulated via the master regulator of sporulation Spo0A in C. botulinum type E and that heat stress has a negative effect on both traits in C. botulinum ATCC 3502, which also suggests common regulation in type A strains. Sporulation-dependent neurotoxin synthesis might play a central role in the life of this dangerous pathogen and represents a key intervention point for control. Finally, we identified a TCS (CLO3403/CLO3404) important for cold adaptation in psychrotrophic C. botulinum type E, which represents a major hazard in anaerobically-packaged chilled foods.
  • Boström, Anna (Helsingin yliopisto, 2018)
    Epaxial muscle atrophy, decreased cross-sectional area (CSA) and increased fat infiltration has been found in dogs with spinal pathology. However, methods to quantify muscle structure have not been validated, and breed differences in the epaxial muscle architecture of dogs have not been considered in relation to spinal pathology. We aimed to validate a method to quantify epaxial muscle size and composition in dogs using magnetic resonance imaging (MRI) and computed tomography (CT) and to estimate functional roles of the epaxial muscles in Dachshunds and Border terriers. Further aims were to investigate the influence of intervertebral disc disease (IVDD) on epaxial muscle size, composition and internal architecture in dogs. Two blinded observers evaluated the CSA, fat content and muscle attenuation of the Mm. multifidi, M. spinalis et semispinalis and M. longissimus from low field MR and CT images of 10 healthy Dachshunds. The measurement method was reliable, showing substantial to almost perfect intra- and inter-rater reliability for all variables. We used the muscular architecture parameters muscle mass, muscle belly length, fascicle length and physiological cross-sectional area (PCSA) to estimate the force and power production capacity of the epaxial muscles in Dachshunds and Border terriers and in Dachshunds with IVDD. We found significantly higher PCSA and shorter fascicles in M. semispinalis complexus and M. iliocostalis lumborum in Dachshunds compared to Border terriers. The IVDD affected Dachshunds had significantly longer muscle fascicles than non-affected Dachshunds in M. longissimus cervicis and thoracis et lumborum. We also investigated the CSA and fat infiltration of the Mm. multifidi and M. longissimus from high-field MR images in Dachshunds with IVDD and in dogs with fibrocartilaginous embolism (FCE). The FCE dogs had significantly greater CSA and less fat infiltration in the Mm. multifidi and M. longissimus than Dachshunds. Our findings showed that the epaxial muscle CSA and fat content can be reliably measured both on MRI and CT. The Dachshund epaxial muscles have greater potential for force production than Border terriers, suggesting a greater requirement for spinal stability in the Dachshund. The longer fascicles in the M. longissimus of affected Dachshunds may suggest compensation due altered position of the spine. The decreased CSA and increased fat infiltration may be related to compressive IVDD in Dachshunds. We conclude that breed differences must be considered when evaluating muscle structure. Further, our results supports previous evidence that the epaxial muscle structure may be compromised in Dachshunds with IVDD.
  • Toresson, Linda (Helsingin yliopisto, 2018)
    Cobalamin (cbl) deficiency is a common sequela to chronic enteropathies (CE) in dogs. Numerous metabolic and clinical consequences have been reported in association with cbl deficiency, as has a poorer prognosis of the underlying disease. The recommended treatment for dogs is multiple parenteral cbl injections. However, to the best of our knowledge, no studies have been performed on dogs with CE and low cbl concentrations to evaluate the effect of oral cbl supplementation, nor has the currently recommended parenteral cbl protocol been systematically validated. This is in contrast to human medicine, where comparative studies have shown equal efficacy of parenteral and high-dose oral cbl supplementation. Oral supplementation is a cost-effective and very simple treatment in human patients since it can be performed at home, as opposed to parenteral treatment. This thesis aimed to evaluate the effects of oral cbl supplementation on serum cbl concentrations in dogs with CE and low serum cbl concentrations. Further, a comparison of oral and parenteral supplementation was performed and intracellular markers of cbl deficiency, such as homocysteine (HCY) and methylmalonic acid (MMA), were analyzed and compared during the treatment period. First, a retrospective study was performed on dogs with CE and low serum cbl concentrations that had received oral cbl supplementation in clinical practice by the author. A significant difference was found when comparing serum cbl concentrations before and after supplementation. All dogs had serum concentrations within or above the cbl reference interval after supplementation. The increase in serum cbl concentrations was not correlated to Canine Inflammatory Bowel Disease Activity Index at inclusion, degree of cobalamin deficiency or if a change of diet or addition of immunosuppressive treatment was started during supplementation. Second, a prospective block-randomized study was performed, comparing serum cbl concentrations in dogs treated with peroral (PO) versus parenteral (PE) cbl supplementation. All dogs of both groups were within the upper half of the reference interval or above the reference interval limit after 28 days of cbl supplementation. The PO group had significantly lower serum cbl concentrations at this time-point than the the PE group. Ninety days after starting supplementation, the PO group had a significantly higher serum cbl concentration than the PE group. Methylmalonic acid (MMA) decreased significantly in both groups between baseline and 28 days of treatment, with no further reduction after 90 days compared to 28 days in either group. No significant differences in MMA concentrations emerged between the groups at any time-point. Serum HCY concentrations did not differ between baseline and 28 days after initiation of cbl supplementation in any group. Ninety days after cbl supplementation was started, a small increase in serum HCY concentration compared to 28 days was noted in both groups. In parallel with the MMA results, there were no significant differences in HCY concentrations between the groups at any time point. The studies on intracellular markers of cbl deficiency suggest that both treatment protocols are equally effective on a cellular level. In conclusion, this thesis provides evidence-based data that oral cbl supplementation can be used as an alternative treatment to the traditional parenteral protocol. Our results suggest that, similar to humans, an alternative intestinal absorptive pathway of cbl may exist in dogs.
  • de Vries, Annemarie (Helsingin yliopisto, 2018)
    This thesis includes studies investigating several options to minimize the undesirable cardiovascular side effects commonly seen with some of the routinely used sedative and anaesthetic agents in horses, under both clinical and research conditions. In the first study, the effect of dobutamine on arterial blood pressure and cardiac output was investigated in horses anaesthetised with isoflurane for routine clinical procedures. In these horses, dobutamine was infused when mean arterial blood pressure was below 70 mmHg. An improvement in arterial blood pressure and cardiac output, measured by the lithium dilution technique, was observed after the start of dobutamine infusion, although an increase in sympathetic tone induced by surgical stimulus likely also contributed. In two studies involving ponies anaesthetised for minor surgical procedures, total intravenous anaesthetic techniques were evaluated. In one, the combined infusion of propofol with ketamine was examined; in the other, involving colts undergoing castration, a continuous intravenous infusion of alfaxalone was compared to intermittent intravenous injections of the same agent. In both studies, good quality of anaesthesia and surgical conditions were achieved with no apparent cardiovascular depression. Two studies investigated the influence of vatinoxan on alpha-2 adrenoceptor-induced cardiovascular side effects. The effects of the intravenous co-administration of vatinoxan with romifidine on the cardiopulmonary system were investigated in one study. The peripheral antagonist successfully attenuated the effects of romifidine on the cardiovascular system, without affecting the level of sedation. In a further research study, vatinoxan was combined with detomidine for pre-anaesthetic medication in horses anaesthetised with isoflurane for arthroscopy, and resulted in improved cardiac function and oxygen delivery compared to the same horses anaesthetised with an identical protocol but without vatinoxan. However, arterial blood pressure was unacceptably low during the initial anaesthetic phase in horses receiving vatinoxan. The optimal dose ratio of vatinoxan and the respective alpha-2 adrenoceptor agonists romifidine and detomidine, needs to be further investigated, especially when these drugs are part of volatile agent-based anaesthetic protocols, in order to optimize peripheral perfusion without unacceptable decreases in arterial blood pressure.
  • Seppä-Lassila, Leena (Helsingin yliopisto, 2018)
    The acute phase response (APR) is a component of the innate immune system, a general and non-specific response that limits the spread of infection and removes factors potentially harmful to the body. Infection, neoplasm or tissue trauma initiates an acute phase response, resulting in visible systemic signs of illness, fever, reduction of appetite and depression. APR also induces a variety of less discernible changes. One measurable sign of APR is the increased synthesis of acute phase proteins (APP) by the liver, mediated by cytokines. In bovine medicine, increased concentrations of the APPs haptoglobin (Hp), serum amyloid A (SAA) and fibrinogen (Fb) have been associated with several diseases, including respiratory tract infections in calves, metritis, mastitis, and hoof diseases. The aim of this thesis was to explore further the associations between APPs and common diseases in calves and assess the ability of APPs to differentiate between severities of disease during different stages. The studies also addressed the possibility of using the variation in APP concentrations in apparently healthy animals to predict the growth. The thesis is based on four individual studies, performed as field studies on commercial Finnish farms. APP measurements and collection of data for explanatory variables were got from dairy calves on dairy farms, beef calves in suckler herds and calves on a calf-rearing farm. The reference values for Hp, SAA and Fb were established from the healthy population of dairy calves (Study I). The association between the diseases and APPs were assessed in different calf populations, and increased concentrations of Hp, SAA and Fb were recorded in calves with respiratory tract infection and umbilical inflammation (Studies I, II, III). Fb, Hp and SAA differed between surgically confirmed diagnoses of local umbilical inflammation and severe umbilical inflammation reaching the abdominal cavity (Study I). Decreased haptoglobin concentrations were associated with weak signs of eimeriosis (diarrhoea or low oocyst count or presence of non-pathogenic Eimeria spp.; Study II). Increased albumin concentrations at around two weeks of age were associated with better growth rate over the first 30 days of life (Study III) in a population of beef calves. However, a longer-term effect was recorded for SAA concentrations around two to three weeks of age (Studies III and IV). A negative association between increased SAA and growth rate was evident for beef calves over the period 30–200 days of age in (III). A similar association between SAA and growth was apparent in rearing dairy calves over two months, seven months and even at slaughter, at 15–18 months (Study IV). The results support the concept that APPs are suitable markers for inflammation and several calf diseases. The novel finding of an association between SAA and growth rate represents opportunities for purposes other than aiding clinical work. The increase in non-specific markers of inflammation can indicate general disturbance in animal health.
  • Hanifeh, Mohsen (2018)
    Chronic enteropathy (CE) in dogs refers to a group of inflammatory conditions of the intestinal tract with unknown etiology. However, the occurrence of an aberrant immune response to antigens derived from endogenous microbiota is likely to play an important role in the pathogenesis of canine CE. Thus, finding inflammatory markers that reflect disease severity would be clinically useful. Matrix metalloproteinase (MMP) -2 and -9 degrade extracellular matrix under both physiological and pathological conditions. Mucosal MMP-2 and -9 activities have been reported to be upregulated in the intestine of humans with inflammatory bowel disease (IBD) and also in animal models of human IBD. However, their identification in the intestinal mucosa of healthy Beagles and their involvement in the pathogenesis of canine CE are unknown. Elevated intestinal mucosal levels of S100A12 and myeloperoxidase, as markers of gut inflammation, have been reported in human patients with IBD. Also, increased concentrations of S100A12 in feces and serum have been reported in dogs with CE. However, intestinal mucosal S100A12 concentrations and MPO activities have not previously been investigated in dogs with CE and in healthy Beagles. The aims of this project were to validate laboratory methods for the determination of MMP-2 and -9, S100A12, and MPO in the intestinal mucosa samples of healthy Beagles, to measure their mucosal levels in dogs with CE, and to compare these results to healthy Beagles. The project also sought to determine the relationship between the levels of the four markers and the canine clinical IBD activity index (CIBDAI), histopathologic findings, clinical outcome, and serum albumin concentrations in dogs with CE. Intestinal mucosal biopsies were collected from 40 dogs with CE (duodenum [n = 35], ileum [n = 12], colon [n = 15], and cecum [n = 6]). Stored intestinal tissue samples from 18 healthy Beagle dogs served as controls (duodenum, ileum, colon [n = 18, each], and cecum [n = 6]). MMP-2 and -9 activities, S100A12 concentrations, and MPO activities were measured using gelatin zymography, ELISA, and spectrophotometric methods, respectively. The methods for determination of MMP-2 and -9, S100A12, and MPO were successfully validated in the intestinal mucosa samples of healthy Beagles. Compared to healthy Beagles, mucosal pro- and active MMP-2 positive samples were significantly higher in duodenum, ileum, and colon of dogs with CE, while mucosal pro-MMP-9 positive samples were significantly higher in the duodenum and colon. None of the intestinal mucosal samples in healthy Beagles showed gelatinolytic activity corresponding to the control bands of active MMP-2 and -9. In dogs with CE, however, mucosal active MMP-9 activities showed a significant positive association with the severity of neutrophils infiltration in duodenum, eosinophils in the cecum. Ileum activities were positively associated with the CIBDAI score. Compared with healthy controls, both mucosal S100A12 concentrations and MPO activities were increased in the duodenum and colon of dogs with CE, while the mucosal MPO activity was also increased in the ileum and cecum. In dogs with CE, mucosal S100A12 concentrations had an association with the severity of epithelial injury and total histopathological injury in the colon; and with the presence of neutrophils and macrophages in the duodenal mucosa or with hypoalbuminemia. Moreover, mucosal MPO activity had a relationship with the severity of epithelial injury and total histopathological injury in the duodenum of dogs with CE. Overall, the results of this project demonstrate an upregulation of mucosal pro- and active MMP-2 and pro-MMP-9, S100A12, and MPO in the intestine of dogs with CE compared to healthy Beagles and it seems that they are involved in the pathogenesis of canine chronic enteropathies. These results provide supporting evidence to more deeply assess the clinical utility of MMP-2 and -9, S100A12, and MPO as possible diagnostic biomarkers in dogs with CE.
  • Hiitiö, Heidi (Helsingin yliopisto, 2018)
    Mastitis is the most common and economically damaging production disease of dairy cows. In the majority of cases it is induced by intramammary infection with a pathogen. Identification of mastitis-causing pathogens enables treatments to be targeted appropriately, avoiding unnecessary antimicrobial treatments, and enabling design of the best possible herd level management protocols. Diagnosis of bovine IMI has been traditionally based on microbial culture of aseptically taken quarter milk samples. Due to the requirements of modern dairy production and development of molecular technologies, milk samples can be examined for target pathogens with DNA-based methods such as multiplex real-time PCR assays. Despite several years of routine use of PCR assays in some countries, scientific knowledge concerning interpretation of the results is sparse. This dissertation focuses on providing new information for diagnosing bovine mastitis using multiplex real-time PCR. In order to accomplish that, first a commercial multiplex real-time PCR assay was compared with the current reference method, conventional culture (I). It showed that PCR was a reliable diagnostic method for Staphylococcus aureus, but less reliable for diagnosing true non-aureus staphylococci infections. In an experimental challenge study (IV), elimination of Staphylococcus epidermidis and S. simulans was followed with PCR in addition to conventional culture. The results showed that PCR yielded target-positive results for several days after the culture results were negative. Subsequently, the time of sampling related to the onset of inflammation may be of importance when interpreting the PCR results. Secondly, quarters with suspected mastitis were sampled using two experimental techniques, in addition to the conventional aseptic technique (II, III), and examined with multiplex real-time PCR. In the needle technique, the whole teat and teat area were bypassed. With the cannula technique, the teat orifice and the teat canal were bypassed. The results showed that samples taken with alternative techniques were of better hygienic quality. We also reported that over half of the non-aureus staphylococci findings in our PCR results originated from sites other than the mammary gland. A diagnosis of a true IMI or mastitis, with possible outcome of an antimicrobial treatment course for the animal, cannot be based solely on a PCR result, but must be combined with all the available information about a cow`s history, symptoms, changes in milk and indicators of inflammation. Some microbes detected with PCR can be considered to be contaminants, especially when present with low levels of DNA. If the signs of the cow or the degree of udder inflammation do not fit the usual signs caused by the pathogen detected, the possibility of contamination should be considered.
  • Kant, Ravi (Helsingin yliopisto, 2018)
    This thesis details an in silico exploration of the genetic potential of the Gram-positive genus Lactobacillus through the establishment and analysis of a pan-genome dataset. Lactobacilli are an intensively researched and studied group of bacteria, which is in part owed to their exploitation for various man-made food and industrial purposes and their advocated beneficial use as gut probiotics. Bacterial pan-genomics is an outgrowth of the comparative genomics field and by comparing the genomes from many strains of the same species the data obtained serves to catalogue the entire genetic repertoire available to a particular species. In effect, the pan-genome represents the complete assortment of putative genes in a species (or genus) of bacteria. The research presented in this thesis is comprised of three distinct studies (I, II, and III), each of which used the pan-genome approach to give a theoretical account of Lactobacillus genetics. In study I, a pan-genome was assembled at the genus level using 20 fully sequenced genomes from 14 different Lactobacillus species. Here, complete genome sequences were selected for creating a broader framework that would allow more comprehensive genomic comparisons. Varying aspects were apparent among the genome sequences used, including sizes that ranged from ~1.8 to ~3.3 Mbps and a G+C content of between ~33% and ~51%. The assembled pan-genome was sized at 14,000 protein-encoding genes, and out of which a small 383-gene Lactobacillus core genome (LCG) was derived. The genetic content of the LCG was used for reconstructing a molecular phylogeny, which then permitted a taxonomic grouping of the 20 genomes into three main clades. Additional classifications of the LCG involved identifying core group and signature group genes, as well as so-called ORFan genes that were further sorted as either LCG-specific or group-specific. In study II, a pan-genome of the Lactobacillus rhamnosus species was constructed from 13 different genomes. L. rhamnosus is a highly adaptable bacterium that thrives in a variety of hosts and environments. Presumably, there is little doubt that numerous genetic peculiarities are the source of the niche-related phenotypes that enable the inherent ecological adaptability of L. rhamnosus strains. For this, the genetic content of the assembled pan-genome was examined for those geno-phenotypic variations occurring at the cell-surface level and whether these correlate to a particular habitat preference of various L. rhamnosus strains. The L. rhamnosus pan-genome itself had an estimated size of 4,893 protein-encoding genes, which was further partitioned into the 2,095-gene core and 2,798-gene accessory genomes. Pan-genomic comparisons were benchmarked against the gut-adapted L. rhamnosus GG strain and focused primarily on seven functionally characterized surface-exposed proteins. Most notably, the operonic genes for the mucoadhesive SpaCBA pilus were part of the accessory genome and can be regarded as a genomic novelty in L. rhamnosus. Nonetheless, for those L. rhamnosus strains with a functional SpaCBA piliation trait, this would improve niche-specific fitness and presumably prolong transient (allochthonous) colonization of mucosal epithelial surfaces in the gut or elsewhere in the body. In study III, a pan-genomic appraisal was performed on the Lactobacillus ruminis species by compiling the genomes of nine different strains obtained from human, bovine, porcine, and equine digestive tracts. L. ruminis is a piliated and flagellated strict anaerobe and one of the few indigenous (autochthonous) lactobacilli in the gut. The pan-genome was utilized to pinpoint the molecular basis for the intractable colonization behavior of L. ruminis, where the focus was on those geno-phenotypes associated with cellular surface morphology and anaerobic fermentation and respiration. The size of the L. ruminis pan-genome was predicted to contain 4,301 protein-related genes, while the number of genes in the core and accessory genomes was 1,234 and 3,067, respectively. As inferred from the pan-genomic data, the presence of certain surface proteins and a substitute anaerobic energy-yielding metabolism might represent the adaptive phenotypes that help make L. ruminis a gut-autochthonic species.
  • Culebro Escandon y Perez, Alejandra (Helsingin yliopisto, 2018)
    Campylobacter jejuni and Campylobacter coli are the most common cause of bacterial gastroenteritis, affecting approximately 96 million people around the world every year. Campylobacteriosis is characterized by diarrhoea, abrupt abdominal pain, and fever. Generally, the disease is self-limiting and resolves within a few days. Occasionally, neurological symptoms manifest clinically three weeks post-infection, indicating the onset of Guillain-Barré syndrome (GBS). GBS is a polyradiculoneuropathy characterized by an acute progressive and symmetrical motor weakness of the extremities with varying degrees of areflexia. GBS has been established as a post-infectious sequelae, whose principal aetiological agent is C. jejuni. The C. jejuni-GBS link was the first confirmed case of human autoimmune disease caused by ganglioside mimicry. C. jejuni lipooligosaccharides (LOS) have been established as the main structure responsible for ganglioside mimicry. Studies on the genetic basis of ganglioside-like LOS expression in C. jejuni have identified the genes associated with the addition of sialic acid (Neu5Ac) to the LOS chain; neuA, neuB, neuC, and a gene encoding a sialyltransferase from the glycosyltransferase CAZy family 42 (GT-42). These genes are found within the LOS biosynthesis locus of C. jejuni. The existence of neuA, neuB, neuC, and GT-42 genes in C. coli is unknown, despite the fact that this species has also been isolated from GBS patients. The primary aim of this study was to investigate the diversity of the C. coli LOS biosynthesis locus with particular interest in identifying genetic features associated with the synthesis and transfer of Neu5Ac. C. coli was found to possess a more diverse LOS biosynthesis locus than previously thought. A total of 27 different LOS locus classes containing a GT-42 encoding gene were identified. Among these, 16 are potentially able to synthesize sialylated LOS structures. Interestingly, several LOS locus classes resemble those of C. jejuni involved in ganglioside mimicry. Thus, bacterial factors implicated in GBS aetiology can cross species barriers. Also, C. coli has a larger GT-42 enzyme repertoire than C. jejuni. Two of the most common GT-42 encoding genes in C. coli were found to be associated to the presence of nonulosonic acid in LOS structures. Marked differences in diversity in the LOS locus were observed between C. coli clades, suggesting a potential role of this structures in niche adaptation. The importance of LOS to C. coli ecology and host-pathogen interaction remains to be explored.
  • Kettunen, Karoliina (Helsingin yliopisto, 2018)
    Efficacious and consistent enforcement of food safety legislation is a cornerstone in protecting public health and preventing distorted competition between food business operators (FBOs). In case of non-compliance, food control authorities must provide the FBO with advice and requests to ensure that the FBO corrects the violation. If these actions do not induce compliance or the operations cause a health hazard, food control authorities are to employ administrative enforcement measures such as orders or prohibitions. The objective of this thesis was to explore the risk-basis, efficacy and consistency of using administrative enforcement measures in local official food control in Finland. In addition, the aim was to investigate the importance and uniformity of local official food control from the perspective of FBOs. The methods include analysis of administrative enforcement decisions and inspection reports made by local authorities, survey and interview of local food control officials and survey of FBOs in approved dairy, fishery and meat establishments under local official food control. The results indicate that administrative enforcement measures are important control actions undertaken to force FBOs to correct multiple, recurrent or critical food safety violations. The enforcement process was initiated more rapidly and the duration of the process was shorter in critical than in non-critical food safety violations. However, the use of enforcement measures was often preceded with repeated requests to correct non-compliances, and the duration of enforcement processes was often rather long. Food control officials perceived the process as laborious and slow, and the use of enforcement measures varied among and within the local food control units. Some units had a strong routine for using the measures, while others used them rarely or not at all. Unclear alignments and uncertainty in using enforcement measures decreased the efficacy and consistency of enforcement. Many officials perceived the guidelines of the new disclosure system of inspection reports, the Oiva system, as helpful in enhancing the consistency of using enforcement measures. Respondent FBOs in approved dairy, fishery and meat establishments considered official food control to be important for food safety and appeared to be generally satisfied with its quality. However, many FBOs perceived the consistency of official food control as poor, and particularly small-sized FBOs were critical of the relevance of control actions. The Oiva system raised early-stage concerns among FBOs regarding the fairness and consistency of control. A perception of good co-operation with the official by FBOs was associated with their positive views about official food control and its importance. The results of this thesis indicate that local food safety enforcement is generally risk-based and control actions are progressively adjusted. However, the efficacy and consistency of using administrative enforcement measures could be improved and further efforts are needed to establish the Oiva system and unify the interpretations. Provision of more practical training for officials and further development of operating instructions and peer-review systems between and within control units would likely increase the efficacy and consistency of enforcement. Improving the consistency of control actions would also enhance the trust of FBOs in the relevance and justness of control.
  • Mahiout, Selma (Helsingin yliopisto, 2018)
    The aim of this thesis study was to gain more information on the physiological and toxicological functions of the aryl hydrocarbon receptor (AHR). The effects of two novel selective AHR modulators (SAHRMs) were investigated in vitro and in vivo. In addition, the involvement of the AHR in the avoidance of novel food was examined. The AHR is an evolutionarily ancient, apparently over 600-million-year-old protein. It is a ligand-activated transcription factor that modulates the expression of various genes within cells. One of the most studied groups of compounds that activate the AHR are dioxins. They are environmental contaminants primarily formed as by-products of various industrial processes, and many of them are toxic. Dioxins are chemically very persistent and lipid soluble, and thus accumulate in the food chain. Therefore, humans are also exposed to small amounts from food. In Finland, the most common source of dioxins is fatty wild fish from the Baltic Sea. The AHR has been recognised as the mediator of dioxin-induced toxicity for decades. More recently, it has also been shown to be involved in several physiological functions of the body, including the regulation of reproduction, foetal development, the immune system and autoimmunity. However, our understanding of the mechanisms of both the toxicological and physiological functions of the AHR remains incomplete. As a consequence, for instance, human health risk assessment of dioxins is challenging. Furthermore, better understanding of the physiological effects of the AHR could help elucidate the aetiology and pathogenesis of certain diseases, and therefore also benefit the discovery of novel pharmacological therapies. As lead compounds for drug discovery, SAHRMs are particularly interesting. They only elicit subsets of AHR-mediated effects, often without the major toxic outcomes of dioxins. Moreover, they could be valuable tools in elucidating the so far incompetently understood, multifaceted physiological roles of AHR, and the underlying molecular mechanisms. This thesis research had two main objectives. The first was related to studying the in vitro and in vivo toxicity of two novel SAHRMs, which are intended as drug compounds for the treatment of autoimmune diseases. The aim was to determine whether they appear suitable for pharmacological use from the pre-clinical safety perspective. Furthermore, finding out the extent to which their effects resemble or differ from those of the most toxic dioxin, TCDD, was of interest. The second objective was to accumulate more knowledge on a peculiar novel food avoidance behaviour, previously characterised in rats and mice after exposure to TCDD. This behaviour resembles a recognised behaviour model, conditioned taste aversion (CTA), which is also exhibited in humans, for instance in conjunction with nausea related to cancer treatment. The aim here was to verify whether the aforementioned rodent response is a physiological effect of the AHR or, more specifically, a consequence of TCDD exposure. Based on the results, the novel SAHRMs are very effective AHR activators, both in vitro and in vivo, and are in fact comparable to TCDD. However, their toxicity profiles are distinct from that of TCDD, and they appear considerably less toxic in rats. Therefore, the novel SAHRMs appear promising as possible drug compounds, and also highly interesting as tools for AHR research. Despite the differences in toxicity, one of the novel SAHRMs, as well as all of the three other AHR activators tested in this study, induced a strong avoidance response resembling that previously observed to TCDD, but shorter lasting. In addition, the reaction was not inducible in AHR knock-out rats. Thus, this study confirmed that the novel food avoidance behaviour is mediated by the AHR. The effect appears protective against potentially harmful ingested foods, and is therefore another physiological function of the AHR.
  • Yu, Xia (2018)
    The mammalian gut microbiota is composed of autochthonous species that permanently colonize the host intestine, and of allochthonous species that are only transiently able to occupy the intestinal environment. In this thesis research, Lactobacillus ruminis and Lactobacillus rhamnosus GG were investigated as paradigms for each type of microbe–host interaction, with special emphasis on the in vitro characterization of their adaptation factors in the host GIT. L. rhamnosus GG has two pilus operons: spaCBA encoding the well-studied SpaCBA pili and spaFED putatively encoding SpaFED pili. The expression of SpaFED pili in L. rhamnosus GG under laboratory conditions has not thus far been reported. In this study, a nisin-induced expression system was used for the generation of SpaFED or SpaF-deleted pili in Lactococcus lactis NZ9000. The results revealed that SpaFED pili were essential in mediating lactococcal adhesion to intestinal cell lines, to certain extracellular matrix proteins, and to porcine mucins, the tip pilin SpaF playing a central role as a focal adhesion factor. With regard to immunomodulation, SpaFED pili appeared to dampen the immune responses, which was largely attributed to the SpaF pilin, in HEK 293-blue cells expressing TLR2, and in Caco-2 cells. While encountering human peripheral blood monocyte-derived dendritic cells, neither immune response enhancement nor immune dampening by SpaFED pili was observed. Consistent with genomic analyses, transmission electron microscopy demonstrated that pili in gut autochthonous L. ruminis ATCC 25644 of human origin consisted of the tip (LrpC), basal (LrpB), and backbone (LrpA) pilins. Recombinant L. lactis strains were constructed, producing either LrpCBA or LrpC-lacking pili of L. ruminis. Recombinant LrpCBA pili mediated lactococcal adherence to ECM proteins and intestinal epithelial cells, and also dampened TLR2-dependent NF-κB signaling and IL-8 production in HEK cells, whereas L. ruminis itself induced evidently elevated immune responses. When incubated with Caco-2 cells, L. ruminis and recombinant lactococcal constructs expressing pili with and without LrpC pilin lowered IL-8 production. Subsequently, a novel L. ruminis strain was isolated from porcine feces and demonstrated to be flagellated and piliated. We analyzed the abilities of this new isolate and three other L. ruminis strains to adhere to host cells and extracellular matrix proteins, to inhibit pathogen binding and growth, to maintain epithelial barrier functions, and to modulate immune responses in vitro. The results indicated that the strains shared several characteristics, such as binding to ECM proteins and HT-29 cells, inhibition of pathogen adhesion and growth, maintenance of epithelial barrier functions in epithelial cells, and activation of innate immune responses to various extents. In conclusion, this thesis study demonstrated the adhesiveness of SpaFED and LrpCBA pili, which may respectively promote the gut retention of L. rhamnosus GG and of L. ruminis. Moreover, pilus-mediated dampening of innate immune responses might be a strategy for these two gut bacterial species to induce immune tolerance in the host. Additionally, L. ruminis inhibited enteropathogen adhesion and growth, as well as maintained intestinal barrier function, which could be regarded as beneficial to the host and which may in turn favor the persistent residence of L. ruminis.
  • Salla, Kati (Helsingin yliopisto, 2017)
    Medetomidine (MED), an 2-adrenergic agonist, is widely used in veterinary practice as a sedative and premedication agent, as it provides reliable sedation. However, the marked haemodynamic changes limit its use to only healthy patients. MK-467, a peripheral 2 -adrenoceptor antagonist, has been demonstrated to attenuate the early haemodynamic disturbances induced by MED with intravenous co-administration in dogs. The main aim of this series of investigations was to evaluate the haemodynamic effects of MK-467 when it is combined with MED and selected sedative or anaesthetic agents that are commonly used in veterinary practice in dogs. More specifically, the concomitant administration of MK-467, MED and butorphanol via intravenous or intramuscular routes was studied and compared with sedation induced with MED-butorphanol. The haemodynamic effects after the various doses of MK-467 in relation to the standard dose of MED were evaluated prior to and during isoflurane anaesthesia. In addition, the haemodynamic effect of the administration of MK-467 as a constant rate infusion was assessed during MED-alfaxalone total intravenous anaesthesia. As a comparison, the haemodynamic effects of the co-administration of MK-467 and MED as a premedication regimen in relation to acepromazine-butorphanol or glycopyrrolate-medetomidine prior to and during isoflurane anaesthesia were assessed. Finally, the influence of MK-467 on plasma dexmedetomidine and alfaxalone concentrations was determined. After intravenous administration of MK-467 and MED with or without butorphanol heart rate, cardiac output and oxygen delivery were well maintained, and the increase in systemic vascular resistance and blood pressures was prevented. The profitable effects of MK-467 appeared more slowly after intramuscular than after intravenous administration. The initial MED-induced haemodynamic effects were dose-dependently attenuated by MK-467 during the premedication period but not during isoflurane anaesthesia with these studied doses of MK-467 in relation to a standard dose of MED as a premedication regimen. The addition of MK-467 to MED and alfaxalone produced a stable haemodynamic outcome when they were administered as constant rate infusions. Cardiac output and oxygen delivery were well maintained during both inhalation and total intravenous anaesthetic regimens when MK-467 was involved. The haemodynamic effects after the co-administration of MED-MK-467 or acepromazine-butorphanol mimicked each other prior to and during isoflurane anaesthesia. Pre-treatment with subcutaneous glycopyrrolate failed to improve the MED-induced reduction in cardiac output by means of increasing heart rate prior to or during isoflurane anaesthesia. Peripheral 2- adrenergic receptor blockade by MK-467 resulted in a reduction in plasma dexmedetomidine and alfaxalone concentrations, presumably by altering the haemodynamic function. In conclusion, the addition of MK-467 in a MED-contained sedation or anaesthetic regimen attenuates the rather dramatic MED-induced haemodynamic changes by blocking peripheral 2-adrenergic receptors and by decreasing the plasma concentrations of dexmedetomidine. The overall haemodynamic stability is well maintained during either an isoflurane anaesthesia or an alfaxalone constant rate infusion. The combination of MK-467 with MED may offer an alternative premedication regimen to the combination of acepromazine and butorphanol in healthy dogs.

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