Eläinlääketieteellinen tiedekunta

 

Recent Submissions

  • Aalto-Araneda, Mariella (Helsingin yliopisto, 2020)
    The severe foodborne disease listeriosis is caused by the bacterium Listeria monocytogenes, known as a problematic contaminant of the food chain. This facultative anerobe tolerates many conditions used for controlling harmful bacteria, including high salinity and temperature. Some L. monocytogenes strains tolerate external stressors better than others, which may complicate the control of the bacterium in food-related environments. Strains exposed to one stress condition may also develop tolerance towards another; such cross-adaptation occurs, for instance, between osmotic and heat stress. In food production, L. monocytogenes may encounter these stresses via salting and heat treatments or hot water used in sanitation. Vacuum-packaged ready-to-eat fish products frequently contain L. monocytogenes and have caused several listeriosis outbreaks. They often do not undergo listericidal processes before consumption, and thus, their processing requires stringent preventive measures. The aims of this dissertation were to investigate the strain variability and determinants of L. monocytogenes stress tolerance and to examine the framework of fish-processing plants and their official food control for managing L. monocytogenes contamination. Using optical density measurements of microbial growth, differences in growth ability under osmotic (NaCl) stress were determined for 388 wild-type L. monocytogenes strains. Notable strain variability as well as serotype- and lineage-dependent patterns of L. monocytogenes salt stress tolerance were discovered. Lineage-I-affiliated L. monocytogenes serotype 1/2b and 4b strains grew significantly better at NaCl 9.0% than lineage-II-affiliated serotypes 1/2a, 1/2c, and 3a. By enabling this comprehensive identification of NaCl-tolerant strains, our data assembly and analysis protocol elucidated the biologically relevant intra-species variability of L. monocytogenes salt stress tolerance phenotypes. A comparative whole-genome sequencing approach was implemented to identify underlying determinants of L. monocytogenes stress tolerance phenotypes. Accessory genetic mechanisms of stress resistance were investigated by comparison of heat survival phenotypes and whole-genome sequences of a heat-resistant and a heat-sensitive L. monocytogenes strain. The comparison identified a novel plasmid, pLM58, including an open reading frame annotated as an adenosine triphosphate (ATP) -dependent ClpL-protease-encoding gene, which was present in the heat-resistant strain but absent in the heat-sensitive strain. The curing of pLM58 resulted in a reduction of heat resistance. The conjugation of clpL increased the heat survival of a natively heat-sensitive L. monocytogenes strain. This study described, for the first time, plasmid-borne heat resistance of L. monocytogenes and identified the protease ClpL as a novel mechanism of L. monocytogenes heat resistance. To examine the framework for managing L. monocytogenes contamination in the fish industry, operational practices and efficacy of official control were studied in 21 Finnish fish-processing plants producing vacuum-packaged gravad (cold-salted) and cold-smoked fish products. Product samples were investigated for the presence and quantity of L. monocytogenes in 2014–2015. Additionally, the results of official food control sampling of products and facilities were assessed to retrospectively gain information on L. monocytogenes contamination in the participating fish-processing plant facilities in 2011–2013. The production and hygiene practices of the processing plants were surveyed with an in-depth inspection questionnaire, and the occurrence, control measures, and correction of non-compliances were drawn from their official inspection records. Associations of L. monocytogenes occurrence with fish-processing plant operational practices, compliance, and aspects of official control during the respective years were investigated with statistical modeling. L. monocytogenes product contamination was associated with number of processing machines, deficiencies in the processing environment and machinery sanitation, and staff movement from areas of low hygiene to high hygiene. Performing frequent periodic thorough sanitation was associated with a decreased risk of product contamination. The increased occurrence of L. monocytogenes in the facilities and products of the fish-processing plants was associated with hygiene deficiencies in processing machinery, a lack of demanding control measures for non-compliances, and recurrence of non-compliances. These results identified areas for improvement in the preventive measures of fish-processing plants and official food control, providing ways to reduce L. monocytogenes contamination in the fish industry.
  • Rossi, Heini (Helsingin yliopisto, 2020)
    Non-infectious inflammation of the lower airways, the main theme of this thesis, is commonly observed regardless of the breed, age, or use of the horse. Due to similarities with human asthma regarding clinical presentation and several pathophysiological aspects, non- infectious chronic lower airway inflammation in horses is currently named equine asthma (previously inflammatory airway disease or recurrent airway obstruction, depending on the severity). Long-term exposure to organic dust and moulds in stable air is thought to be the most important predisposing factor for the disease. Equine asthma is characterized by airway hyperresponsiveness and mucus accumulation, severity-related airway remodelling, impaired lung gas exchange, and increased quantity of airway inflammatory cells. These changes lead to clinical signs, including chronic cough and nasal discharge, poor performance, and in a severe form of equine asthma, increased respiratory effort at rest corresponding to an “asthma attack” in humans. Despite active research over the years, the aetiology and pathophysiology of equine asthma remain incompletely defined. In this thesis, the role of a transmembrane protein extracellular matrix metalloproteinase inducer (EMMPRIN), a potential new biomarker in equine asthma, and its relationship to matrix metalloproteinases (MMPs) were investigated. In Study I, the respiratory secretions of 29 horses with asthma and 15 asymptomatic controls were examined. Expression of EMMPRIN protein was identified in all horses in bronchoalveolar lavage fluid (BALF) cells, and the expression was highest for the most substantially affected horses with severe asthma and was correlated with MMP-2 and -9 protein expression, MMP-9 gelatinolytic activity, and airway neutrophilia. This is the first study to describe the relationship of EMMPRIN and MMPs in patients of any species clinically affected with asthma, and the results suggest that EMMPRIN is involved in the pathophysiologic processes of asthma in horses. EMMPRIN as a marker of inflammation provides interesting possibilities for further research of horses and other species. The second part of this thesis concentrated on two common diagnostic tools used to assess lower airway diseases in horses. The diagnosis of lower airway inflammation, including asthma, is often aided by the analysis of cytological samples obtained from the trachea (tracheal wash; TW) or from the lungs at alveolar level (bronchoalveolar lavage; BAL). There is considerable variance in the diagnostic methods of equine lower airway diseases between facilities and clinicians, and a true gold standard for diagnosing airway inflammation is lacking. In most previous studies, the cytology of TW and BAL samples has been considered to correlate poorly and BAL has been deemed a superior diagnostic tool in most cases of equine lower airway disease, but sensitivity and specificity have not been evaluated. Moreover, the conventionally used cut-off values for cytology have been largely based on observational studies and expert opinion, and recently, higher values have been proposed. The first aim of Study II of this thesis was to compare the results of these two airway sampling methods in a population of 121 horses with signs related to respiratory disease and in 33 horses with no clinical respiratory signs. A secondary aim was to evaluate the cut-off values of TW and BALF cytology for neutrophils in order to justify their use in clinical practice. TW and BAL showed substantial agreement regarding neutrophils, and only 17.5% of the horses were classified differently (healthy vs. diseased), in contrast to some previous studies with higher disagreement. The neutrophil percentage was found to correlate between TW and BAL results. The sensitivity and specificity of TW were generally higher for TW than for BAL when estimated with conventional as well as with Bayesian statistical methods. The results of this study encourage the use of the current neutrophil cut-off values in clinical diagnosis of airway inflammation instead of elevating the cut-offs. In Study III, equine lower airway inflammation was investigated with regard to general anaesthesia. The complication rate in equine general anaesthesia is high relative to anaesthesia in several other species, postanaesthetic pneumonia being one of the potential complications. However, there is limited knowledge about the effects of general anaesthesia on inflammatory responses in the lungs and on systemic inflammatory markers such as serum amyloid A (SAA) in horses. For example, the level and duration of lower-airway neutrophilia that can be expected after anaesthesia in dorsal recumbency are unknown. Thus, our primary aim was to determine when lung inflammation reaches its maximum and how rapidly BALF cytology returns to baseline after anaesthesia in dorsal recumbency. A secondary aim was to investigate the possible effect of vatinoxan, a novel a2-adrenoceptor antagonist drug, on the BALF cytology results. Anaesthesia in dorsal recumbency resulted in no clinically relevant changes in airway cytology that could be differentiated from the effect of repeated BAL sampling. A marked increase in serum amyloid A was detected in some animals, while others showed no changes from baseline. Vatinoxan as premedication did not consistently affect lung cytology or blood inflammatory markers after anaesthesia. These combined studies on equine lower airway inflammation increase our understanding of mechanisms and factors related to pathophysiology and diagnostics of the condition in the horse. Lower airway inflammation is an important cause of impaired welfare and performance of horses. Broader understanding of the aspects related to the condition will aid in developing new improved clinical and diagnostic procedures and management strategies for equine inflammatory respiratory diseases.
  • Tapio, Heidi (Helsingin yliopisto, 2020)
    Detomidine and medetomidine are α2-adrenoceptor agonists that are used in equine medicine for sedation and analgesia. These drugs, however, also influence the cardiopulmonary and gastrointestinal system, mainly by increasing systemic vascular resistance and decreasing cardiac performance and intestinal motility. Vatinoxan, in turn, is primarily a peripherally acting α2-adrenoceptor antagonist that has been demonstrated to attenuate some of the undesired effects of α2-adrenoceptor agonists with no major influence on the level of sedation in many species. One aim of the present studies was to evaluate the effects of vatinoxan with a constant rate infusion (CRI) of medetomidine in standing and anesthetized healthy horses. Another aim was to evaluate the effects of vatinoxan when it is administered after detomidine in standing healthy horses. We hypothesized that vatinoxan would alleviate the cardiopulmonary and gastrointestinal effects of these α2-adrenoceptor agonists, while preserving their sedative effects under these experimental conditions. The horses received, in a cross-over design in three separate studies, an α2-adrenoceptor agonist either alone or with vatinoxan for sedation or for premedication before general anesthesia. To evaluate the cardiopulmonary function, the heart rate, respiratory rate, cardiac output, arterial blood pressures, central venous pressure, and pulmonary arterial pressures (this last one in two of the studies) were recorded. Additionally, arterial and venous blood gases were analyzed. Systemic vascular resistance, oxygen delivery, and other selected cardiopulmonary parameters were calculated afterwards. The effects of the drugs on intestinal motility were evaluated by auscultation of the borborygmi or by determining the amount of fecal output. The level of sedation was scored, and the recovery times were recorded in anesthetized horses. Finally, the drug concentrations in plasma were determined. In standing horses receiving a CRI of medetomidine, vatinoxan attenuated the early, although minor, cardiopulmonary changes induced by medetomidine. Furthermore, the intestinal motility was markedly improved by vatinoxan. In anesthetized horses receiving an adjunctive CRI of medetomidine, premedication with vatinoxan induced significant hypotension. Despite this hypotension, which was successfully treated with dobutamine, the cardiac performance and tissue oxygen delivery were better maintained in these horses than in those receiving the adjunctive medetomidine CRI without vatinoxan. The fecal output decreased after general anesthesia, and this decrease was not influenced by vatinoxan. Finally, vatinoxan, administered after detomidine, partially reversed the cardiopulmonary effects and intestinal hypomotility induced by detomidine. Vatinoxan decreased the level of sedation at the beginning of the CRI of medetomidine in standing horses, but not when administered with a single dose of medetomidine before general anesthesia or after detomidine in standing horses. The recovery times with or without vatinoxan also did not differ from each other. The plasma concentration of both enantiomers of medetomidine mostly correlated with the level of sedation, being lower in the presence of vatinoxan when the level of sedation was reduced. To conclude, vatinoxan attenuated the cardiopulmonary changes and intestinal hypomotility induced by detomidine or a CRI of medetomidine in standing horses. The effects of vatinoxan on the level of sedation could be considered relatively minor. With the dosing used in this study, vatinoxan induced marked hypotension in anesthetized horses and did not influence the intestinal hypomotility associated with general anesthesia. Thus, vatinoxan represents a potential drug for alleviating or reversing the undesired effects of medetomidine and detomidine in standing horses. Its hypotensive effects, however, warrant more research before it could be usable in anesthetized horse.
  • Niemelä, Tytti (Helsingin yliopisto, 2019)
    Intra-articular (IA) inflammation resulting in lameness is a common health problem in horses. Exogenous IA hyaluronic acid has been shown to provide an analgesic effect in equine and human osteoarthritis. High molecular weight non-animal stabilized hyaluronic acid (NASHA) has gained popularity in the treatment of human arthritic conditions due to its long-acting pain-relieving effects. In animal models, hyaluronan has been demonstrated to reduce pain by protecting nociceptive nerve endings and blocking pain receptor channels. Inflammatory and degenerative activity inside the joint can be studied in vivo by analysis of synovial fluid (SF) biomarkers. In addition to pro-inflammatory mediators, several anabolic and anti-inflammatory substances are produced during the disease process. The response of SF biomarkers after IA hyaluronan injection, alone or in combination with other substances, has been examined only in a few equine studies. Aim of the studies was to examine the efficacy of IA NASHA in relieving lameness and related signs of IA inflammation in clinical and experimental studies. We also investigated the effect of NASHA on various SF biomarkers. In addition, we explored possible adverse effects of NASHA on clinical signs of IA inflammation and SF. In the clinical study, we compared the response to treatment of lameness localized in the equine metacarpophalangeal joint injected with NASHA or placebo (saline). We also investigated the response of SF prostaglandin E2 (PGE2), substance P, aggrecan chondroitin sulphate 846 epitope (CS846), and carboxypeptide of type II collagen (CPII) concentrations to the NASHA and placebo treatments. After collection of baseline SF samples followed by IA diagnostic anaesthesia, horses in the treatment group received 3 ml of a NASHA product (20 mg/ml) intra-articularly, and those in the placebo group received an equivalent volume of sterile 0.9% saline solution. The horses were re-evaluated and a second SF sample was obtained after a two-week period. In the experimental study, the aim was to measure the SF interleukin-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB), transforming growth factor beta 1 (TGF-β1), and tumour necrosis factor alpha (TNF-α) concentrations before and after surgically induced cartilage defect and sham operation as a control in horses. We also investigated whether the concentrations of selected biomarkers in SF changed following NASHA injection. Results of the clinical study indicate that a single IA NASHA injection is not better than a single saline injection for reducing lameness in horses with synovitis or mild osteoarthritis. However, IA NASHA may have some beneficial effects in modifying mild clinical signs. The decrease in the SF concentration of the cartilage-derived biomarker CS846 only in the NASHA group suggests that less damage and hence less repair to the cartilage has occurred post-injection. Creation of the cartilage defect and sham operation lead to an increase of synovial fluid IL-1ra and TNF-α concentrations in the experimental study, but NASHA failed to produce changes in SF biomarkers.The significant increase in SF white blood cell count after IA NASHA may indicate a mild inflammatory response. However, as no clinical adverse effects were observed, IA NASHA appeared to be well tolerated.
  • Bergman, Paula Susanna (Helsingin yliopisto, 2019)
    In sow farms, animals are actively removed and replaced to maintain target efficiency levels, herd health status and a static herd size. Sow removal has a critical effect on pig farm functionality and profitability. Excessive removal decreases lifetime production and especially during periods of small profit margins, becomes burdensome. It is also recognized as a welfare issue. The overall aim of this thesis was to investigate the current reality of piglet production in the rapidly changing Finnish production conditions with special emphasis on removal. The thesis is based on three individual studies which utilize diverse data from real commercial piglet producing farms. The types of data collected for each are different and the methods used reflect the differences. We showed systematic and temporal differences in removal between individuals, parities, farms and replacement circumstances in Finland. The results also demonstrated the economic value of improved animal health and removal. We benchmarked national culling and mortality rates retrieved from mandatory registrations. Especially, the average on-farm mortality may be considered relatively high although the rates accorded with published literature. However, the large differences between farms and several farms succeeding in obtaining low levels of removal imply that it is conceivable to strive for and reach certain special targets. Unfortunately, we demonstrated that optimal lifetime of a sow is not a fixed number and as such, no generally applicable policy for replacing sows can be determined. Neither can excessive removal be improved by single improvements only, because of limited resource reserves and other shortages within the individual farm. However, a few factors were found to be linked with an increased risk for removal and higher removal levels in these studies: e.g. the smallest litter sizes and the number of stillborn piglets at the sow level, and features indicative of semi-intensive or intensive farming compared with a combination of environmental animal welfare indicators (mortality) and a non-intensified farming style (culling) at the farm level. Traditional production approach of maximizing the net monetary income or quantitative measures in piglet production is likely to be changed in response to consumer’s increasing concerns around animal well-being, environmental sustainability and one health. We introduced an empirical base that could be used to motivate debate on future development of piglet production systems and delivered useful evidence relevant for stakeholders to engage in and promote research into identification, monitoring and management of sow removal and health. It may be more motivating for Finnish piglet producers, herd advisers and the industry to have specified removal levels to work towards. Our study can also be considered valid for emphasizing awareness of multidisciplinary approaches in integrating accurate epidemiological livestock data into larger frameworks as well as identifying current bottlenecks in available data and modelling methodology.
  • Hasan, Shah (Helsingin yliopisto, 2019)
    In modern pig production there has been a steady increase in the litter size during recent decades. Large litters represent a major challenge for the sow, increase the farrowing duration and compromise sow welfare by prolonging this stressful process. Moreover, the continuous increase in litter size is a major cause of pre-weaning piglet mortality by increasing the proportion of low birth weight piglets, which are less vital and have reduced colostrum intake. Approximately 30% of hyper-prolific sows produce insufficient colostrum for their piglets. Colostrum plays an essential role in the health, survival and growth of piglets by providing energy, immunoglobulins, growth factors and many other bioactive components. Both colostrum yield and its composition are highly variable among sows, yet mechanisms and factors that regulate colostrogenesis are not fully understood. The aim of this study was to evaluate sow colostrum IgG concentration on farm using a Brix refractometer, to thus improve the management of neonatal piglets immediately after initiation of farrowing. We established colostrum evaluation criteria, to be used at herd level, by comparing the Brix results with those from ELISA. We aimed to investigate sow physiology around farrowing, acute phase protein (APP) response and their association with colostrum yield (CY), colostrum composition and piglet colostrum intake (CI). The impact of sow CY, colostrum composition and quality on piglet performance were also investigated. We also aimed to determine the effects of yeast hydrolysate (YD) and resin acid-enriched composition (RAC) inclusion in sows’ gestation and lactation diets on CY, colostrum immunoglobulins, nutritional composition and subsequent litter performance. We also sought to determine the influence of these two feed additives on the taxonomic profile of the hindgut microbiota of sows. Additionally we aimed to determine whether changing the gut microbiota of sows influences the microbial colonization of piglets after birth. There was a correlation between Brix refractometer measurement of colostrum and the corresponding IgG concentration measurements of ELISA (r = 0.63, P < 0.001). A classification of colostrum quality was suggested; low levels of IgG (14.5 ± 1.8 mg/ml) were recorded for colostrum samples with Brix readings below 20%. Borderline colostrum IgG content (43.8 ± 2.3 mg/ml) had Brix readings of 20% to 24%, adequate colostrum IgG content (50.7 ± 2.1 mg/ml) had Brix readings of 25% to 29% and very good IgG colostrum content (78.6 ± 8.4 mg/ml) had Brix readings >30%. Sow CY was positively correlated with plasma haptoglobin (Hp) (P = 0.029), number of live-born piglets (P < 0.01) and negatively correlated with farrowing duration (P = 0.01). Piglet CI was positively associated with piglet weight at birth (P < 0.001) and negatively associated with the number of live-born piglets in the litter (P < 0.001). Both piglet CI and birth weight were positively associated with piglet average daily gain (ADG) (P < 0.001). Risk for piglet death, or a piglet being treated with an antibiotic before weaning, increase with a decrease in sow back fat thickness at farrowing (P = 0.04). Similarly, we found that piglets of litters with low BWB and low CI had a higher risk of death before weaning (P < 0.001). Piglets born from sows having lower levels of colostrum IgA and serum amyloid A (SAA) and high plasma progesterone at the end of farrowing had higher risk of neonatal diarrhea (P = 0.04; P = 0.05; P = 0.04). Piglets born from sows having higher back fat thickness at weaning had higher risk of developing weaning diarrhea (P = 0.02). There was a significant increase in CY in sows fed with YD during gestation (P = 0.04) and a higher level of fat in their colostrum (P = 0.01). In RAC fed sows there was a significant increase in colostrum IgG content. Piglets weaning weights of RAC fed sows were higher in two trial herds. Inclusion of YD and RAC in gestation and lactation diet increased the abundance of beneficial and fermentative fecal bacteria (Roseburia, Paraprevotella, Eubacterium, Romboutsia and Clostridium sensu stricto) significantly (P < 0.01) while, opportunistic pathogens, especially Proteobacteria, were suppressed. In addition, piglets from the sows fed YD had more beneficial microbial populations with higher diversity and fewer opportunistic pathogens at one week of age. However, feeding YD, higher CY, colostrum composition and piglet birth weight were all correlated with specific sow gut microbiota. In conclusion, Brix measurement of a sow’s fresh colostrum is an inexpensive, rapid and sufficiently accurate method of estimating IgG concentration, providing indication of differentiation between good and poor IgG content of colostrum. Although colostrum is vital for piglet survival, both CY and composition are highly variable among sows. Sow body condition and physiological status around farrowing affect CY and colostrum immunoglobulin contents. Piglet survival and risk of death before weaning also depend on CI. Therefore, to support CY and CI, measures should be taken to ease the process of farrowing, increase piglet vitality and improve colostrum availability for piglets. Functional feed ingredients such as YD and RAC may increase colostrum availability, improve colostrum IgG and its energy content for neonate piglets, and also promote beneficial maternal microbial sources for neonates.
  • Summa, Maija (Helsingin yliopisto, 2019)
    Human noroviruses (HuNoVs) are yearly responsible for a large number of acute human gastroenteritis cases globally in all age groups. Typically, the virus transmits via the fecal-oral route from person to person, causing strong symptoms such as nausea, vomiting, and diarrhea, which usually disappear in a few days. However, HuNoVs cause also numerous food-related illnesses in developed countries, including Finland, inducing gastroenteritis outbreaks through contaminated water and foodstuffs. According to the reports of the European Commission, both in Europe and in Finland the most common foods causing HuNoV outbreaks are shellfish, berries (especially frozen raspberries), vegetables, and mixed foods, which most likely became contaminated by a sick food handler. Noroviruses belong to the Caliciviridae family and are classified into seven genogroups. HuNoVs belong to genogroups I (GI), II (GII), and IV (GIV). Other genogroups contain only animal noroviruses. Noroviruses are generally regarded as host-species-specific, but the possibility of zoonotic transmission and infections has been discussed for over a decade for several genotypes. The purpose of this study was to develop a simple and rapid method for detection of HuNoVs in food. The potential zoonotic nature of HuNoVs, particularly whether animals can serve as transmitters for these viruses, was also investigated. In the past two decades, numerous methods for detecting HuNoVs in food have been developed. However, many of these are time-consuming and the sensitivity of the methods has been highly variable. In this work, four published extraction methods for detection of HuNoV in food (lettuce, ham, and frozen berries) were compared. The method based on alkaline elution and polyethylene glycol (PEG) precipitation was found to be the most reliable detection method for all three food matrices tested. The recovery efficiency of the method with frozen raspberries was on average 28%. Two rapid methods for detection of HuNoV in frozen raspberries were also presented. The rapid method based on direct RNA extraction yielded the same recovery levels (32%) as the PEG precipitation method. The method proved to be sensitive because it detected HuNoV also with a virus level of 100 genome copies in a 25 g sample. Moreover, the method detected HuNoV in naturally contaminated berry samples that were linked to outbreaks of disease. A treatment with either a chloroform-butanol mixture or dilution of the food samples for the RT-PCR reaction was efficient in reducing the effect of PCR inhibitors. The same effect was achieved with PEG as a supplement in the food samples. Thirty-nine frozen berry samples purchased from local stores in 2010, 2014, and 2017 were screened. All berries tested negative for HuNoVs GI and GII. HuNoV genome was detected in the feces of 31 birds, two rats, and four pet dogs. The genotypes found in six bird samples and all dog samples were the same as those commonly found in human samples at the time of sampling. HuNoVs can be detected in food samples also in small numbers using the rapid method presented in this study. The use of PEG as a supplement was found to reduce inhibition of the RT-PCR reaction in the two rapid methods, and therefore, the commonly used chloroform-butanol treatment, which easily loses viruses during processing, could be omitted. The results of animal samples strongly indicate that wild birds, pet dogs, and possibly also rats may be involved in the transmission of HuNoVs to food, water, and surfaces
  • Adam, Magdy (Helsingin yliopisto, 2019)
    The impact of the peripherally selective α2-adrenoceptor antagonist, vatinoxan, on selected pharmacodynamic and pharmacokinetic properties of two selective α2-adrenoceptor agonists, medetomidine and dexmedetomidine, were investigated in sheep. Moreover, certain interactions between vatinoxan and atipamezole, a specific α2-adrenoceptor antagonist, were evaluated. The initial objective of this study was to identify a dose of vatinoxan that would best mitigate the undesirable cardiopulmonary changes produced by intramuscular (IM) medetomidine-ketamine in sheep. Specifically, three doses of vatinoxan (150, 300 and 600 µg/kg) or saline were combined in the same syringe with medetomidine (30 µg/kg) and ketamine (1 mg/kg) and given IM. Systemic hemodynamics, arterial blood gas tensions, clinical sedation and plasma drug concentrations were compared, both before and after reversal with IM atipamezole (150 µg/kg). The middle dose of vatinoxan (300 µg/kg), which appeared to be optimal among the other doses, was then added to medetomidine (30 µg/kg) and co-administered IM, followed by atipamezole for reversal. Last, the influence of intravenous pre-treatment with vatinoxan on dexmedetomidine-induced cardiopulmonary alterations was investigated in sevoflurane-anesthetized sheep. Following concomitant IM administration, vatinoxan dose-dependently attenuated some of medetomidine’s cardiopulmonary side effects. Vatinoxan did not significantly affect the level of sedation or the plasma concentrations of drugs when ketamine was included in the same syringe. Conversely, vatinoxan significantly increased the plasma concentrations of medetomidine, and accelerated the onset and intensified the degree of sedation when compared with the agonist alone. Moreover, recoveries after atipamezole-reversal were more complete in the presence of vatinoxan. No deleterious effects were noted between vatinoxan and atipamezole. Pre-treatment with vatinoxan prevented all dexmedetomidine-induced pulmonary alterations in sheep anesthetized with sevoflurane. In conclusion, vatinoxan alleviated or prevented the unwanted cardiopulmonary effects of (dex-) medetomidine by blocking the peripheral α2-adrenoceptors. Presumably, when co-administered IM in the same syringe, vatinoxan accelerated the absorption of medetomidine and increased its concentration in blood, which resulted in a faster and more intense sedation than when the agonist was used alone. Vatinoxan also decreased later exposure to dexmedetomidine, which appeared to improve atipamezole’s efficacy to reverse both the central and peripheral effect of the agonist.
  • 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.

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