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  • Tuominen, Pirkko (Helsingin yliopisto, 2009)
    The drive for risk-based food safety management, systems and control has spread world-wide in recent decades. Since the term is still internationally undefined, its use and implementation vary, producing different realizations. In this Ph.D. thesis, microbiological risk assessment (MRA) was investigated as a basis for risk-based food safety management, which was defined as ‘food safety management based on risk assessment in order to achieve an appropriate level of protection (ALOP)’. Governments are responsible for commissioning MRAs and also for setting food safety targets up to a certain point, but the practical management measures that need to be in place in order to achieve the targets are to be addressed by the operators. On the plant level, food safety is usually managed through regulation, quality assurance systems and a hazard analysis and critical control point (HACCP) programme with its prerequisites. In Finland, food safety management on the food plant level is implemented through an HACCP-like regulated system termed an own-checking (OC) programme. A quantitative microbiological risk assessment (QMRA) was conducted on salmonella in the beef production chain according to the official standards of the Codex Alimentarius Commission (Codex Alimentarius), and utilized in determining the food safety metrics for beef production. The Finnish Salmonella Control Programme (FSCP) and the main official interventions due to it were examined in the light of risk-based food safety management. The targets set for beef processing plants by the government were converted into quantitative limits, and the results of salmonella monitoring included in the FSCP were examined by the QMRA. The goal of the FSCP was declared in 1994 to ‘maintain the present salmonella situation’, which was considered to refer to the salmonella incidence in humans at that time, and also the de facto ALOP. The requirement for a maximum salmonella prevalence of 1% at defined stages of the beef production chain was embodied in the FSCP. This statement was considered to convey performance objectives (PO) for the aforementioned stages. According to the QMRA, the de facto ALOP was achieved in the referred year 1999, and even the true prevalence levels in the FSCP were estimated to be clearly under the set PO limits with 95% credibility. However, the PO limits were set too high for the de facto ALOP to be maintained in practice. If the salmonella prevalence reached the PO limit of 1% or values near it, the public health risk would increase and overrun the de facto ALOP. The QMRA produced in this work has for the first time provided the possibility to quantitatively asses the relationships between targets set in the FSCP and their impact on public health. At present, imports of beef and beef-derived foods may impose on Finnish consumers a significantly greater exposure than domestic products. If their salmonella prevalence or their share of the foods consumed in Finland increase, the number of human cases could rapidly rise. The models for the QMRA were mainly Bayesian hierarchical models using Markov chain Monte Carlo (MCMC) techniques, which was found to be a flexible and appropriate method for this type of complex modelling. The resulting distributions were also regarded as an advantage compared to the results from models developed with the deterministic approach, because the presentation of results included the extent of the uncertainty, and also in this manner better illustrated the actual operational environment. Based on an inquiry, the personnel in food processing plants had a positive attitude towards food safety management systems, but the knowledge, training and involvement of those employees directly operating on the site with these systems were discovered to be deficient. Therefore, a generic semi-quantitative hygiene risk assessment model, Hygram®, was developed for small and medium-sized food enterprises to offer assistance in understanding, training, and, first of all, detecting the critical steps of the processes, and thereby to contribute to the development of their own-checking systems towards risk-based food safety management. Hygram® was not considered a risk-based tool as such, but whenever the critical limits of the process have been defined as equal to a risk assessment, Hygram® can be used as a risk-based management tool. It can also serve as a tool for systematic hazard analysis and CCP detection when establishing a food safety management system. To conclude, the development of risk-based food safety management is a process in which risk assessment is an essential tool. Scientific, technical, psychological and resource-bound barriers need to be overcome in order to put risk-based management systems into practice. This study showed that QMRA can be valuable in national risk management decision making, although few QMRAs are currently available. Appropriate tools for practical risk management decision making on the industrial level, such as Hygram®, need to be further developed.
  • Honkavaara, Juhana (Helsingin yliopisto, 2012)
    The effects of MK-467, a peripherally acting α2-adrenoceptor antagonist, on the cardiopulmonary changes induced by dexmedetomidine, a specific and selective α2- adrenoceptor agonist, were investigated in dogs. Plasma concentrations of both drugs were also quantified, along with influence of MK-467 on the quality of clinical sedation achieved with dexmedetomidine. The main focus of this study was on preventing or attenuating the cardiovascular effects of dexmedetomidine. The effects of three different doses of MK-467, administered simultaneously with the agonist, on the main hemodynamic parameters were evaluated and compared with a single dose of both dexmedetomidine and MK- 467 administered alone. Respiratory effects were evaluated with arterial blood gases, and indices such as oxygen delivery were calculated. The pharmacokinetic interaction between the two drugs was evaluated in vivo and general information on the disposition of intravenously administered MK-467 in dogs was produced. The degree of clinical sedation was subjectively assessed during the studies. The effect of MK-467 on the quality of reversal of dexmedetomidine-induced sedation with atipamezole was also determined. M K-467 dose-dependently reduced or prevented all relevant cardiovascular changes induced by dexmedetomidine. The heart rate, arterial and central venous blood pressure, cardiac index, systemic vascular resistance and oxygen delivery remained within acceptable physiological limits throughout the observational period with all doses of MK-467 administered with the agonist. Moderate hypotension was seen with the highest dose of MK-467. No significant differences in respiratory function were observed between treatments. MK-467, when administered alone, induced sinus tachycardia along with increases in the cardiac index and reductions in systemic vascular resistance. Arterial blood pressures remained unchanged. The degree of clinical sedation was reduced by MK-467, most likely by increasing the disposition of dexmedetomidine, which led to lower plasma concentrations of the agonist. However, the differences in clinical sedation were minor. MK-467 did not interfere with the ability of atipamezole to reverse dexmedetomidine-induced sedation.
  • Niskanen, Taina (Helsingin yliopisto, 2010)
    Yersinia pseudotuberculosis is an animal pathogen widespread in nature. The bacterium has caused infections in humans occasionally. Most of the reported Y. pseudotuberculosis -infections are sporadic, and the sources and vehicles of Y. pseudotuberculosis remain obscure. Y. pseudotuberculosis has emerged as an outbreak -associated pathogen in Finland since 1997. Indirect evidence has indicated that Y. pseudotuberculosis is a potential food borne pathogen, but no specific food has been previously confirmed as a source of the human infections. The aim of this thesis was to determine the prevalence of Y. pseudotuberculosis in different animal species and to evaluate them as a possible source of Y. pseudotuberculosis. Clinically healthy pigs were found to carry Y. pseudotuberculosis. The prevalence of Y. pseudotuberculosis was 4 % and 14 % in fattening pigs at slaughterhouse and at farm level, respectively. All Y. pseudotuberculosis isolates from pigs were pathogenic. Migratory birds representing 57 different bird species were studied for pathogenic Y. pseudotuberculosis during spring and autumn migration. Pathogenic Y. pseudotuberculosis was recovered from two species of birds during spring migration. Y. pseudotuberculosis was first time confirmed to be the cause of a food borne outbreak through domestically produced iceberg lettuce in epidemiological study. Contamination of iceberg lettuce was investigated on farm level in a two-year survey. Several factors predisposing to Y. pseudotuberculosis contamination of lettuce were found on the farms, including a large population of deers and free access of wild animals to unfenced lettuce fields and to irrigation water sources. Pulsed-field gel electrophoresis (PFGE) analysis was optimized for efficient molecular typing method for Y. pseudotuberculosis isolates. PFGE was used to compare Y. pseudotuberculosis isolates from different origin. Y. pseudotuberculosis isolates of different sources showed high genetic diversity, while Y. pseudotuberculosis isolates found in same animal species representing the same serotype showed limited genetic diversity. Identification of environmental Y. pseudotuberculosis isolates was found to be incorrect based solely on biochemical tests. Commercial test kits misidentified some environmental Yersinia spp. isolates as Y. pseudotuberculosis. However, no specific virulence genes of Y. pseudotuberculosis were found in these isolates by polymerase chain reaction (PCR) targeting these gene areas.
  • Heikinheimo, Annamari (Helsingin yliopisto, 2008)
    The present thesis reveals new information, showing that healthy human is a rich reservoir for Clostridium perfringens enterotoxin (CPE)- producing Clostridium perfringens type A. The results show that healthy foodhandlers are commonly asymptomatic carriers of this pathogen. The findings indicate that persons handling foods should be considered a possible source of contamination for C. perfringens type A food poisoning. This work also found that the genotypes that previously were considered as atypical causative agents in food poisonings, in fact cause a notable proportion of C. perfringens type A food poisonings. CPE- producing C. perfringens type A is considered one of the most common causes of food poisonings in the industrialized world. This pathogen is also involved in antibiotic-associated diarrhea and sporadic diarrhea in humans as well as in animals. Recent findings of the variable loci (plasmid vs. chromosome) and different genetic arrangements adjacent to enterotoxin gene (cpe) have shown that CPE- producing C. perfringens type A isolates form in different subpopulations. These subpopulations are responsible for different CPE-associated diseases, i.e. chromosomal cpe-bearing strains are involved in food poisonings, whereas strains carrying cpe in the plasmid are typical in antibiotic-associated or sporadic diarrhea cases as well as in animal diarrheas. Not much is known about reservoirs or transmission routes of cpe-positive isolates. The present thesis aimed to improve diagnostics of cpe-positive C. perfringens by developing molecular methods for detection and isolation. Other objectives were to improve identification and typing of C. perfringens. With novel assays, molecular epidemiology of cpe-positive C. perfringens type A was further investigated. When investigating the presence of cpe-positive C. perfringens in the feces of healthy food- handlers, the organism was carried by 18% of the individuals. The presence of strains representing all previously identified genotypes (plasmid-borne IS1151-cpe, plasmid-borne IS1470-like-cpe, and chromosomal IS1470-cpe) as well as strains carrying previously unrecognized genetic arrangements attached to cpe shows that various different subpopulations of cpe-positive C. perfringens type A occur in the human gastrointestinal tract and that the healthy human is a rich reservoir for cpe-positive C. perfringens type A. The common carriage of cpe-positive C. perfringens type A of healthy food-handlers and the full capacity of these strains to produce CPE indicate that humans handling food should be considered a possible source of contamination of food. By showing that 25% of investigated C. perfringens type A food poisoning outbreaks were caused by strains carrying plasmid-borne cpe, we reveal that plasmid-borne cpe carrying C. perfringens type A is a common cause of food poisonings. The new data presented in this thesis improve the understanding of reservoirs and movements of enterotoxin producing C. perfringens type A. Therefore, CPE-associated diseases may be better controlled and prevented.
  • Hälli, Outi (Helsingin yliopisto, 2008)
    Seasonal infertility is well-defined phenomenon in modern swine production. The outcomes mostly affected are age at puberty in gilts, weaning-to-oestrus interval (WOI) and farrowing rate in sows. The European wild boar, the ancestor of our domestic pig, is a distinct short-day seasonal breeder. Photoperiod is thought to be the most important regulating factor in seasonal breeders. In addition to photoperiod, many environmental and management factors are known to adjust the manifestation of seasonal infertility. The aim of this work was to obtain information about the effect of photoperiod on reproductive endocrinology and on reproductive performance. In addition, the effect of re-modelling the piggery breeding unit on farrowing rate was studied. In the first clinical trial (I), two light regimes were tested in experimental conditions. One group of ovariectomized gilts (16 domestic pigs and 4 European wild boars) was kept under short daylight conditions (8-h light, 16-h dark) and the other group under long daylight conditions (16-h light, 8-h dark). After the treatment period, LH secretion patterns were determined by repeated blood sampling. In contrast to our expectations, LH pulse frequency remained unchanged in both light regimes. However, in the domestic pig, mean and basal LH concentrations were higher in the long-day group than in the short-day group. To investigate the two light regimes under more practical conditions, two trials (II, III) were conducted in commercial piggeries. The outcomes were farrowing rate and WOI. In trial II, the long-day group was kept under a constant 16-h light, 8-h dark regime. The short daylight regime consisted of a light phase of 8-h and a dark phase of 16-h in farrowing and breeding units. The length of the short-day treatment was 6 weeks before breeding. In the gestation unit, the short-day animals were also kept under a 16L:8D regime. The results of this trial revealed that the farrowing rate was 90% in both groups, and the two treatment groups also shared the same median WOI, i.e. 5 days. In trial III, the long daylight regime consisted again of a constant long-day treatment. However, the photophase was 14L:10D. The short-day treatment was extended to 8 weeks and took place exclusively in the farrowing unit. The light regime was 10L:14D. The short-day group was also kept under a 14L:10D regime in the breeding and gestation units. Despite these modifications, the results were the same as in the previous trial. The farrowing rate was 90% and median WOI five days in both groups. The last part (IV) of this thesis was an observational retrospective cohort study with a historical control. A cohort of farms with individual cages in their breeding unit was chosen. These farms either remodelled their breeding cages or switched to different group housing systems in the breeding unit. The farrowing rate before and one year after the remodelling was then compared. The remodelling itself was shown to affect reproductive performance on these farms. In conclusion, the reproductive performance of sows is difficult to control by a single environmental (photoperiod) or management (housing strategy) factor.
  • Hang, Ingrid (Helsingin yliopisto, 2015)
    Considerable evidence suggests that dietary macronutrients impact upon activities and conditions in the gastrointestinal tract (GIT) including: functions and processes, digestive enzymes secretion, microbial ecology and bacteria-derived metabolism. Knowledge about the modulation of canine intestinal microbiota, bacteria-derived metabolic products,intestinal inflammatory status and adaptive exocrine pancreatic secretion in response to macronutrients is limited. However, such information is necessary to investigate further the complex interplay between host and intestinal microbiota in response to changes of diet. The reasearch for this PhD thesis focused upon the changes of the intestinal microbiota,bacteria-ferivedmetabolicproducts,anintestinalinflammatorymarker and pancreatic enzyme profiles of five healthy Beagle dogs in response to being fed three different diets: high-carbohydrate starch (HCS), high-protein greaves-meal (HPGM), or a balanced dry commercial (DC) diet. Every diet was crossed-over and fed to each dog for three 21-day periods. The microbial deoxyribonucleic acid (DNA) was profiled according to its percentageoftheguanine-cytosinecontent(%G+C)inordertodetectthefluctuations in intestinal microbiota. Thereafter, 16S ribosomal ribonucleic acid (16S rRNA) gene amplicons were obtained from the most abundant %G+C peaks and analysed by sequence analysis. The DC diet sample was associated with high abundances of representatives of the orders Clostridiales, Lactobacillales, Coriobacteriales and Bacteroidales. Sequence diversity was highest for the DC diet samples and included representatives of the orders Lactobacillales and Bacteroidales, which were not detected in samples obtained for the HPGM and HCS diets. The HPGM and HCS diets also had reduced numbers of representatives of the family Lachnospiraceae; specifically Clostridium cluster XIVa. The HCS diet favoured the proliferation of representatives of the order Erysipelotrichales, specifically the Clostridium cluster XVIII, whereas the HPGM diet favoured representatives of the order Fusobacteriales. Bacterial metabolism and intestinal inflammatory status were assessed by determining dry matter, pH, ammonia, short-chained fatty acids (SCFAs), and faecal canine calprotectin concentrations. Faecal ammonia concentrations decreased with the HCS diet. All dogs fed the HPGM diet developed diarrhoea, which led to differences in faecal consistency scores and increased faecal pH. Moreover, decreases in propionic and acetic acids coupled with increases in branched-chain fatty acids and valeric acid caused changes in faecal total SCFAs. Faecal canine calprotectin concentration was also higher for the HPGM diet than with the other diets and correlated positively with valeric acid concentrations.8 Dietary effects on digestive enzyme composition in the serum, in jejunal fluid, and in the faeces were studied by determining the following factors: amylase activity, the concentrations of canine trypsin-like immunoreactivity (cTLI), canine pancreatic lipase immunoreactivity (cPLI), and canine pancreatic elastase (cE1) concentrations with the two radioimmunoassays (RIAs) for determining cTLI and cPLI concentrationswere specifically validated for jejunalfluid and faecal specimen analysis. Both RIAs were linear, accurate, precise, and reproducible. Dog specific serum enzyme concentrations did not differ between diets. Feeding the HCS diet was associated with decreased amylase activities and cPLI concentrations in the lower jejunum, when compared to the corresponding cPLI activities of the HPGM and the DC diet. The HPGM diet decreased the concentrations of cPLI and cE1 in faecal samples, but not in the jejunal fluid. In conclusion, all bacterial clusters discovered in this research represent the normal GIT microbiota of canines. The HPGM diet favoured Fusobacterium and this Gram-negative bacterial genus may be associated with the observed elevated inflammation status. The latter was deduced from the observed diarrhoea and elevated levels of canine faecal calprotectin in all dogs fed the HPGM diet. It seems likely that these research results could be associated with the quality and increased or decreased amounts of dietary protein or carbohydrate being available for fermentation by the intestinal microbiota. The limited capacity of pancreatic enzymes to adapt adequately by a change inprofile inresponse to changes indietary components seems to be an essential factor, which influences the nutrient levels available for the intestinal microbiota.
  • Õkva, Kai (Helsingin yliopisto, 2012)
    Discussions on laboratory animal welfare issues often refer to the Three Rs replacement, reduction and refinement. Replacement means substituting living animals with non-sentient systems; reduction refers to using fewer animals and refinement causing less pain, suffering and distress to the animals or improving their welfare. This work is focusing on two R-s: reduction and refinement in mice. If one considers reduction as meaning obtaining trustworthy information from using fewer animals, then this can be achieved by improved research strategies, better experimental design and more sophisticated statistical analyses. One can reduce variation within the group by using isogenic inbred animals or by finding ways to reduce variation in outbred animals. One approach to achieve reduction in outbred animals is to include litter and individual features of the animals, e.g weight dynamics, in the statistical analyses. Since the elevated plus-maze (EPM) test is one of the most common tests to evaluate anxiety-like behaviour, it was used to assess the possible effects of litter and weight on the behaviour of outbred mice and the effects of environmental enrichment (EE) on the behaviour of inbred mice. As a research tool, the effects of acute or chronic administration of ethanol or acute therapy with the nitric oxide synthase (NOS) inhibitor L-NOARG were examined in outbred NIH/S mice. The administration of L-NOARG had no effect on the behaviour of mice after acute or chronic ethanol administration but attenuated the anxiogenic effect of ethanol withdrawal. The litter from which the mice had originated had a significant effect on their behaviour in the EPM test. The behavioural indices of mice, originating from different litters, tended to be above or below the mean of the corresponding drug-treatment group, irrespective of the drug treatment. Litter had a significant effect on the initial weight and also on the weight changes occurring during the adaptation period and ethanol inhalation. An approach to refinement, EE, has been introduced to create more natural species-specific living conditions for laboratory animals. At the same time, it has been claimed that EE can affect the results of behavioural studies and also increase variation. The effects of different types of EE and different time periods were studied in inbred C57BL/6J and BALB/c mice. The exposure of male C57BL/6J mice to the different types of EE objects in the form of cage furniture (CF) -nest box, corner and stairs- induced an anxiolytic-like effect in the EPM test and tended to increase the locomotor activity of mice. This apparent anxiolytic-like effect was most pronounced in the third week. The CF, in the form of modified Tapvei OY mouse stairs, produced an anxiolytic-like effect and increased the locomotor activity in female C57BL/6J mice, but not in BALB/c female mice. In conclusion, the NOS inhibitors may have effects on the behavioural changes caused by ethanol withdrawal. Information about the litter of outbred mice could and should be used in statistical analysis in order to reduce variation and the number of mice needed. In the EPM test, different CF items induced an anxiolytic-like effect in male and female C57BL/6J mice, but not in BALB/c female mice. This effect depended on the type of objects and was influenced by time. This anxiolytic like effect can be interpreted as refinement of the housing by improving animal welfare. The effects of CF should be considered in planning enrichment programs for housing institutions, in designing behavioural experiments and in analyzing the results obtained. Therefore the evaluation of CF could provide the valuable information and it is recommended that CF manufacturers collate and distribute the refinement results on the specific CF items they produce.
  • Lindeberg, Heli (Helsingin yliopisto, 2003)
  • Oivanen, Leena (Helsingin yliopisto, 2005)
  • Tast, Anssi (Helsingin yliopisto, 2002)
  • Laukkanen, Riikka (Helsingin yliopisto, 2010)
    Enteropathogenic Yersinia, that is pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis, are zoonotic pathogens causing yersiniosis, the third most frequently reported zoonosis in the EU. Enteropathogenic Yersinia are frequently isolated from the tonsils and intestinal contents of pigs. Similar Y. enterocolitica genotypes have been identified both in pig and human strains and human yersiniosis has been statistically associated with the consumption of pork products in case-control studies, indicating pigs and pork products as an important source of human Y. enterocolitica infections. The link between pathogenic Y. pseudotuberculosis and pork is less clear; however, Y. pseudotuberculosis has also been isolated from carcasses and pork, indicating a possible route from pigs to humans. This work aimed at clarifing the transmission of enteropathogenic Yersinia from farm to slaughterhouse, determining factors affecting the prevalence of enteropathogenic Yersinia on pig farms, and test bagging as an intervention at the slaughterhouse. In addition, methods for the isolation of enteropathogenic Yersinia were evaluated. Isolation of enteropathogenic Yersinia from samples of animal origin is difficult and time-consuming. However, in many cases such as in outbreak investigations, isolates are needed for further typing. Of the isolation methods used, cold enrichment was efficient at isolating enteropathogenic Yersinia, whereas the sensitivity of other methods, such as direct isolation and selective irgasan-ticarcillin-potassium chlorate enrichment, for the isolation of enteropathogenic Yersinia was low. However, none of the isolation methods tested detected all the enteropathogenic Yersinia-positive samples and new isolation methods need to be developed. The transmission of enteropathogenic Yersinia from pigs to carcasses and pluck sets was investigated by collecting samples from individual ear-tagged pigs on the farm and at the slaughterhouse and by analyzing the isolated strains using pulsed-field gel electrophoresis (PFGE). Since the same PFGE types can be isolated from pigs and their subsequent pluck sets and carcasses, the main contamination source of pluck sets and carcasses at the slaughterhouse appears to be pigs that carry enteropathogenic Yersinia from farms to the slaughterhouse. However, since non related genotypes could also be isolated from carcasses and pluck sets, the slaughterhouse environment and tools can also contaminate carcasses and pluck sets. The high prevalence of enteropathogenic Yersinia in pigs results in high contamination rates of pluck sets and carcasses. Therefore, interventions at the farm level can decrease the transmission of Yersinia from pigs to pluck sets and carcasses. Farm factors such as production capacity and type may affect the prevalence of enteropathogenic Yersinia on farms. Since the prevalence of pathogenic Y. enterocolitica and Y. pseudotuberculosis varies among farms, within-farm factors can affect how enteropathogenic Yersinia spreads in pigs on farms. In statistical studies, factors affecting Y. pseudotuberculosis included organic production, contacts with pest animals, and the outside environment, whereas the high prevalence of pathogenic Y. enterocolitica was associated with factors such as high production capacity and conventional production. The epidemiology of pathogenic Y. enterocolitica and Y. pseudotuberculosis appears to be different on pig farms and this difference needs to be addressed if interventions on pig farms are considered. However, further information on the factors affecting the prevalence of enteropathogenic Yersinia on pig farms is needed before interventions at the farm level can be used. The effect of bagging of the rectum was studied by sampling tonsils, intestinal content, and carcasses with and without bagging of the rectum and constructing a Bayesian hidden variable model. According to the model, bagging of the rectum reduced significantly the contamination of carcasses at the slaughterhouse. However, since after bagging the prevalence of pathogenic Y. enterocolitica in different parts of the carcass was relatively high, 4 14%, other interventions are also needed. Most of the positive carcass samples were head and chest swabs, indicating that tonsils may be the contamination source.
  • Fossi, Marja (Helsingin yliopisto, 2006)
    Brachyspira pilosicoli causes porcine intestinal spirochaetosis, which is manifested by a mild, persistent diarrhoea among weaned pigs at the age of 7-14 weeks. The growth of diseased pigs is retarded and their feed conversion is lowered, resulting in diminished production. These studies were designed to investigate the molecular epidemiology of B. pilosicoli in Finnish sow herds and to improve laboratory diagnostics for B. pilosicoli. Infectivity of the rare hippurate-negative biotype of B. pilosicoli was examined by an infection trial and eradication of B. pilosicoli from a sow herd was demonstrated. A high genetic diversity was observed among 131 B. pilosicoli strains obtained from 49 sow herds located in the two major pork production areas of Finland. A high discriminatory power of pulsed-field gel electrophoresis (PFGE) was established with either SmaI or MluI used as a restriction enzyme. Common genotypes between the herds were rare, and no clustering of the genotypes according to the two geographical areas was observed. A single genotype could persist in a herd for several years; however, genetic recombination among B. pilosicoli strains might occurr. The epidemic nature of B. pilosicoli infection in Finnish pig farms was shown, and the role of migrating vectors or fomites for horizontal transmission was assessed as minor. The high discriminatory power of PFGE was further exploited to investigate hippurate-negative phenotypes of B. pilosicoli. No relationship between hippurate-negativity and genotypes of B. pilosicoli was detected. This finding was substantiated by comparative analyses of 16S rDNA nucleotide sequences of hippurate-negative and -positive isolates; different nucleotide positions of the strains were not predictive of the hippurate hydrolysis reaction. B. pilosicoli isolates from the same herd could possess an identical genome despite having a different capacity for hydrolysing hippurate. The congruent phylogeny of hippurate-negative and -positive B. pilosicoli was further ascertained by an ultrastructural study; the two biotypes shared all of the features unique to the species B. pilosicoli. In conclusion, the expression of hippurate hydrolysis can vary within a single B. pilosicoli clone, and thus, the hippurate test can not be used alone for differentiation of porcine B. pilosicoli. An occasional hippurate-negativity can disturb the phenotype-based differential diagnostics of B. pilosicoli. A B. pilosicoli-specific D-ribose test was therefore established to strengthen the diagnostics protocol. Sixty unrelated B. pilosicoli strains and 35 strains of other porcine Brachyspira species were studied for D-ribose utilization by an indirect method based on recording the pH reduction of a broth culture in the presence of D-ribose. All B. pilosicoli strains, regardless of the hippurate reaction, could utilise D-ribose, whereas the strains of the other Brachyspira species were D-ribose-negative. These results enabled the construction of an amended classification scheme for phenotypic differentiation of porcine B. pilosicoli. Experimental pigs were inoculated either with a hippurate-negative B. pilosicoli strain or with a B. pilosicoli type strain. Somewhat unexpectedly, the pigs in both trial groups remained healthy. Only the hippurate-negative B. pilosicoli strain could be reisolated from two of eight infected pigs. An explanation for the silent infection or lack of infection may lie in the challenge procedure, strain attenuation, environmental conditions, absence of concomitant enteropathogens or the feeding regimen. The pathogenicity of hippurate-negative B. pilosicoli should be further studied in modified conditions for trial pigs. Eradication of a chronic B. pilosicoli infection without total depopulation was attempted in a 60-sow farrowing herd. The principles for eradication of B. hyodysenteriae, the cause of swine dysentery, were applied. Special attention was paid to sanitary measures, relocation of animals according to age groups to nearby shelters, husbandry principles and adequate medication. The eradication was successful; the diarrhoea of the young growers disappeared, and B. pilosicoli was not detected in any sample during the 4.5-year follow-up.
  • Rintakoski, Simo (Helsingin yliopisto, 2014)
    In Denmark, Finland, Norway and Sweden the National Dairy Disease Registers (NDDRs) collect and store disease information at the individual cow level. Because these registers are monitored nationally they offer access to data that cover most of the dairy population in each country. Data from these registers are used, for example, for herd health and animal welfare assessments, production management, genetic evaluations and epidemiologic research. Since the register data are not usually designed for research purposes their quality is unknown. Understanding the magnitude of data loss during data transfer and human influence in disease recording process is important. The knowledge will enhance the ability to improve register quality, which in turn will improve reliability of frequency measure calculations from the register data. This thesis investigated the quality (measured as completeness and correctness) of the Finnish NDDR and compared register qualities among the four countries. In Finland the quality of recorded information was excellent, but approximately 17% of disease information was lost during the data transfer steps. A large proportion of the data loss was due to artificial insemination (AI) technicians not transferring events. The majority of those events occurred close to culling of the cow, suggesting early removal of the cow health cards so that AI technician did not have access to those cards. Additionally, fewer diagnostic events were transferred from purchased cows compared to those born on farm suggesting problems with animal identification. An efficient way to improve completeness in the Finnish NDDR is to have veterinarians electronically transfer diagnostic information during farm visits. The Finnish system has already been modified accordingly. This thesis also showed how register quality for four reproductive disorders (metritis, retained placenta, assisted calving and oestrous disturbances) varied among the four countries. Metritis and oestrous disturbance events were well represented in the NDDRs. Farmer-observed completeness (the proportion of all farmer observations that were recorded in the NDDR) was around 0.80 and did not differ significantly among the countries. Assisted calving and retained placenta events showed more among-country variation. Farmer-observed completeness was highest in Denmark and lowest in Finland, ranging between 0.31 and 0.89. The comparison of completeness-adjusted incidences to incidences calculated from the registers showed that incidences were underestimated for assisted calving and retained placenta. Underestimation was highest in Finland. This thesis also demonstrated how both farmer and veterinary intentions toward starting veterinary treatment of mild clinical mastitis could explain the reasons for different mastitis incidence rates among the countries. The results suggest that when intentions towards veterinary treatment are greater, mild cases receive veterinary treatment more often than when intentions are reduced. Therefore, greater farmer and veterinarian intentions can increase the disease incidence calculated from the NDDR data. According to the study, intentions were greater in Denmark than in other countries.
  • Markkula, Annukka (Helsingin yliopisto, 2013)
    Listeria monocytogenes causes potentially fatal illness to susceptible people and is found in various foods. It typically enters processed foods via a contaminated processing environment, in which it may have persisted for years. To study the role of raw material as a potential source of contamination of food processing plants by L. monocytogenes, the prevalence and genetic diversity of this species in tonsils of pigs and in raw fish was examined. A total of 14% and 4% of tonsils of pigs and raw fish, respectively, harboured L. monocytogenes. From 38 pig tonsil isolates and 11 raw fish isolates, 24 and nine different types were recovered using pulsed-field-gel electrophoresis (PFGE) typing. The results indicate that a wide variety of L. monocytogenes strains enters pork slaughterhouses and fish processing plants in the raw materials, which are thus potential sources of direct or indirect contamination of processing plants by this pathogen. Since identical PFGE types were recovered from both raw and processed fish, it is likely that raw fish are an initial source of the L. monocytogenes found in processed fish. Some strains entering a plant along with raw fish may contaminate and persist in the processing environment, causing recurrent contamination of the final products via contact surfaces. Alternatively, L. monocytogenes strains in raw fish may survive non-listericidal processes, resulting in contamination of the final product. To identify novel factors contributing to survival of L. monocytogenes in food processing environment, the roles of specific genes in stress response were investigated, using flhA and motA that encode flagellar factors involved in cold stress tolerance, and lmo0866, lmo1246, lmo1450, and lmo1722 encoding DEAD-box RNA helicases involved in cold, heat acid, alkali, osmotic, ethanol, and oxidative stress tolerance. Increased relative transcription levels of flhA, motA, lmo0866, lmo1450, and lmo1722, restricted growth of the single gene deletion mutant strains EGD-eΔflhA, EGD-eΔmotA, Δlmo0866, Δlmo1450, and Δlmo1722 at 3°C, and increased minimum growth temperatures of Δlmo0866, Δlmo1450, and Δlmo1722 revealed that FlhA, MotA, Lmo0866, Lmo1450, and Lmo1722 had roles in growth of L. monocytogenes EGD-e under cold stress conditions. The restricted growth of Δlmo0866 in 3.5% ethanol, and its increased maximum growth temperature and growth rate at 42.5°C, indicated that Lmo0866 had roles also in ethanol and heat stress tolerance of strain EGD-e. The role of Lmo1450 in the growth of strain EGD-e under heat, alkali, and oxidative stress conditions was shown by the restricted growth rate of Δlmo1450 at 42.5°C, in pH 9.4, and in 5 mM H2O2. The slightly decreased growth rate and maximum optical density of Δlmo1246 at 3°C indicated that the role of Lmo1246 in cold stress tolerance was negligible. Under all the other conditions, the growth of Δlmo1246 and the wild-type EGD-e were identical, suggesting that Lmo1246 had no role in growth of L. monocytogenes EGD-e under heat, pH osmotic, ethanol, or oxidative stress conditions. The deletion of flhA, motA, lmo0866, lmo1450, and lmo1722 impaired the motility of strain EGD-e, whereas the motility of Δlmo1246 did not differ from that of the wild type. This indicates that DEAD-box RNA helicases Lmo0866, Lmo1450, and Lmo1722 have roles in motility of strain EGD-e. Moreover, these results suggest that motility and cold stress tolerance of L. monocytogenes are linked, and that motile flagella may be needed for full cold stress tolerance of strain EGD-e.
  • Virtanen, Sonja (Helsingin yliopisto, 2014)
    Yersinia enterocolitica is a foodborne zoonotic pathogen. Among domestic animals, pigs are considered the major reservoir of Y. enterocolitica bioserotype 4/O:3. The pathogen is found in pig carcasses and pluck sets at slaughterhouses. Carcass contamination at the slaughterhouse originates from pigs that are already infected on farms. Considerable variation exists in the prevalence of Y. enterocolitica between different pig farms. The aim of this study was to determine the factors in farm management that can be used to prevent the presence and spread of this pathogen within and between farms. Multiple-locus variable-number tandem repeat analysis (MLVA) has been developed and used for genotyping of Y. enterocolitica strains of human origin. This genotyping method was used here to investigate its discriminatory ability, advantages and limitations, and use in genotyping Y. enterocolitica strains isolated from pigs. Among Y. enterocolitica 4/O:3 strains that originated from humans, pigs, and pork products from four European countries, the use of MLVA was found to have high discriminatory power. Similar MLVA types were detected among humans and pigs, human clinical isolates from limited geographical locations indicating the presence of past unidentified epidemics and also from pigs that originated from the same farms. MLVA proved to be able to detect farm-specific genotypes, but mild variation was common in strains originating from the same farms. Sampling of the farms revealed the spread of similar MLVA types among farms that had previously transported pigs between each other. Pigs were found to be a major source of transmission of this pathogen between all production types, including farrowing, farrow-to-finish, and fattening farms. Piglets from certain breeding farms served as a major source of infection for fattening pigs. These piglets carried farrowing farm-specific MLVA types of Y. enterocolitica to the fattening farm, and the infection spread throughout the fattening unit. Farm management practices and their association with carriage and shedding of Y. enterocolitica in pigs were studied by a purpose-designed questionnaire for farms whose pigs were previously sampled at slaughterhouses. The use of municipal water, organic production, and purchase of feed from a certain feed company were found to be protective factors against the carriage of Y. enterocolitica. In contrast, snout-to-snout contacts between pens and buying feed from another company were discovered as risk factors for fecal shedding of the pathogen. In total, 30 farms were further visited and sampled for enteropathogenic Yersinia, and the management practices and conditions were recorded during each sampling visit. The use of municipal water, the use of an all-in all-out system in the units of weaned piglets and fattening pigs, buying piglets from no more than one supplier at a time, and generous use of bedding were associated with lower prevalence of Y. enterocolitica on farms.