Eläinlääketieteellinen tiedekunta


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  • Murros, Anna (Helsingin yliopisto, 2017)
    Yersinia genus includes currently 18 species: Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica, Yersinia frederiksenii, Yersinia intermedia, Yersinia kristensenii, Yersinia bercovieri, Yersinia mollaretii, Yersinia rohdei, Yersinia ruckeri, Yersinia aldovae, Yersinia aleksiciae, Yersinia massiliensis, Yersinia similis, Yersinia entomophaga, Yersinia nurmii, Yersinia pekkanenii and Yersinia wautersii. The history of the genus Yersinia can be dated back to 1883. In 1965, the genus consisted of only three species: Y. enterocolitica, Y. pseudotuberculosis and Y. pestis. Since then the taxonomy of the genus has been under tremendous change over the years, and especially the taxonomy of Y. enterocolitica, of which many new species have been separated from. Still Y. enterocolitica is a group of very heterogeneous bacteria, which can be divided into 6 biotypes and about 30 serotypes and into pathogenic and non-pathogenic strains. This variability makes the identification of Y. enterocolitica very challenging. In the thesis, two Yersinia species; Y. nurmii and Y. pekkanenii are described. These species were characterized by polyphasic taxonomic methods, including of 16S rRNA gene analysis, multilocus sequence analysis (MLSA) of housekeeping genes glnA, gyrB, recA and HSP60, DNA-DNA hybridization studies, 16S and 23S rRNA gene restriction fragment length polymorphism (RFLP), and phenotyping. Y. nurmii was isolated from broiler meat packaged under modified atmosphere, and Y. pekkanenii from water, soil and lettuce samples. In the 16S rRNA gene analysis and the 16S and 23S rRNA gene RFLP analysis, these two species grouped with other Yersinia, but in separate clusters. In the phylogenetic analysis of separate or concatenated housekeeping genes, the species formed unique monophyletic groups in all phylogenetic trees constructed. Y. nurmii had a phenotypic profile most similar to that of Y. ruckeri. Y. pekkanenii could not be differentiated from Y. pseudotuberculosis using phenotypic tests. Methods of polyphasic taxonomy were also used to estimate the taxonomic position of European Y. enterocolitica strains of non-pathogenic biotype 1A. Y. enterocolitica has been divided into two subspecies; Y. enterocolitica subsp. enterocolitica and Y. enterocolitica subsp. palearctica. Both subspecies consist of pathogenic and non-pathogenic biotypes. In this thesis, 212 Y. enterocolitica strains were characterized by numerical analysis of HindIII ribopatterns (16S and 23S rRNA gene RFLP). These strains consisted of 162 strains of biotype 1A and 50 strains of biotypes 2 to 4 isolated from different sources in Europe during years 1997-2013. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). The biotype 1A strains studied were found to form a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and Y. enterocolitica subsp. palearctica, with suggestion of the existence of a subspecies formed by non-pathogenic Y. enterocolitica biotype 1A strains. Finally, while studying Enterobacteriaceae in cold-stored (6 °C), modified atmosphere-packaged (MAP) pig cheek (musculus masseter) and hind leg meat (musculus semimembranosus), it was noticed that the pathogenic Y. enterocolitica subsp. palearctica bioserotype 4/O:3 multiplied into high numbers from a non-detectable level in (MAP) pig cheek meat. The Enterobacteriaceae isolated in this study were identified by 16S and 23S rRNA gene RFLP using the HindIII enzyme. Y. enterocolitica bioserotype 4/O:3 is the most common pathogenic bioserotype word-wide, and it can be transmitted to humans through raw or undercooked pork. Usually the growth of Enterobacteriaceae is inhibited by a modified atmosphere with 20% or more CO2 at refrigerated temperatures. However, in this study, high numbers of Y. enterocolitica bioserotype 4/O:3 was observed in MAP cold-stored pig cheek meat, with a concentration of 30% CO2 and 70% O2 in the packages.
  • Björkman, Stefan (Helsingin yliopisto, 2017)
    We hypothesized that prolonged farrowing decreases subsequent fertility, that is, pregnancy rate. Furthermore, we hypothesized that prolonged farrowing causes retention of placentae and metritis, delays uterine involution and perturbs follicular growth after weaning. We also hypothesized that sows that undergo prolonged farrowing release less oxytocin at subsequent estrus in response to boar stimulation. We determined the farrowing duration (time between expulsion of first and last piglet) of sows and explored whether there is a negative effect on subsequent post-weaning pregnancy rate (n = 148). We explored the relationship between farrowing duration and placenta expulsion (n = 142), and postpartum uterine size and intrauterine fluid (n = 99). For that, placenta expulsion was observed until 24 h after birth of the last piglet and ultrasonography was used during the first week postpartum to examine the uteri of the sows. Uterine size and intrauterine fluid were used as indicators for initial uterine involution and puerperal metritis. Furthermore, we determined the farrowing duration of sows (n = 30) and monitored the subsequent follicular development using transrectal ultrasound twice a day between the third day after weaning and ovulation. At estrus, blood samples were collected in the presence of a boar in order to determine the endogenous oxytocin concentrations and release patterns. The results show that sows with a prolonged parturition (> 300 min) were 3.4 (P = 0.027) more likely not to be pregnant. Sows that experienced total (no expulsion of placental parts; 3%) and partially retained placentae (no expulsion of placental parts after birth of the last piglet; 3%) had longer farrowing durations (1009 ± 275 and 734 ± 136 min) than sows without retained placentae (369 ± 202 min; P = 0.021 and P = 0.004). Farrowing duration conformed to a quadratic relationship with the number of expelled placental parts (P = 0.001), placental expulsion duration (time between expulsion of first and last placental part; P = 0.002) and time between expulsion of last piglet and last placental part (P = 0.024). Use of oxytocin increased number of expelled placental parts (3.8 ± 0.2 vs. 2.9 ± 0.3, P = 0.035), decreased the placental expulsion duration (172 ± 44 vs. 328 ± 26 min, P = 0.011) and time between expulsion of last piglet and last placental part (148 ± 48 vs. 300 ± 24 min, P = 0.025). Furthermore, prolonged farrowing (β ± SE, Wald χ2, Odds; 2.0 ± 0.5, 13.1, 7.6; P = 0.001), obstetrical intervention (1.5 ± 0.7, 4.4, 4.3; P = 0.036) and two or more stillborn piglets (1.4 ± 0.7, 3.8, 3.9; P = 0.052) increased the risk of having enlarged uterine size whereas oxytocin administration (­ 1.5 ± 0.7, 4.7, 0.2, P = 0.040) decreased the risk. Two or more stillborn piglets (2.6 ± 0.9, 8.7, 13.7; P = 0.003), obstetrical intervention (1.8 ± 0.8, 5.0, 6.0; P = 0.025), prolonged farrowing (1.7 ± 0.8, 4.3, 5.7; P = 0.039) and impaired placenta expulsion (3.3 ± 1.7, 4.0, 26.9; P = 0.044) were associated with intrauterine fluid. After weaning, OT concentrations were higher in sows with prolonged farrowing than in sows with shortened farrowing (28.0 ± 7.7 vs. 20.6 ± 7.7, P < 0.01). OT concentrations correlated with diameters of the follicles measured 5 d after weaning and when the follicles reached their maximum size after weaning (r = 0.61, P < 0.01 and r = 0.57, P < 0.01, respectively).
  • Hallamaa, Raija (Helsingin yliopisto, 2017)
    Summer eczema is one of the most common diseases that causes discomfort and impairs the quality of life of horses worldwide. The lack of a feasible treatment has made this recurrent, insect hypersensitivity-linked allergic pruritus a challenge for veterinary medicine. The first aim of this study was to examine serum phospholipids and their use in the therapy of summer eczema by using an autologous serum preparation. The second aim was to delineate clinical features of summer eczema among Finnhorses. The efficacy of the therapy was investigated in 28 horses in a placebo-controlled study and was also evaluated according to long-term information collected from the owners of the 343 horses treated with this therapy over 12 years. Serum phospholipids and their changes after autoserum therapy were analysed in 10 horses with summer eczema and 10 matched healthy controls by LC-MS. Content of phospholipids in autoserum preparations of 10 affected and 6 healthy horses were analysed by ESI-MS. Horses in the placebo group showed significant aggravation in their clinical signs compared with horses treated with autoserum therapy at the same time (P=0.0329). According to long-term data, 70% of the horses benefited from this autoserum therapy (95% CI 0.64-0.75, P<0.0001) and 16% did not, and 14% of the owners did not provide a clear opinion. No harmful side effects were observed. Horses with summer eczema displayed significantly lower concentrations of phosphatidylcholine (P<0.0001) and sphingomyelin (P=0.0115) in their sera than healthy horses. After a 4-week autoserum therapy, no significant difference between these horses could be demonstrated. The change in clinical signs correlated significantly with the alterations in sphingomyelin concentrations (P=0.0047). Analysis of autoserum preparations revealed that these preparations contained major serum phospholipids, however, in significantly differing concentrations between eczema and healthy horses. Affected horses showed more abundant concentrations of phosphatidylcholine (P=0.042) and sphingomyelin (P=0.0017). In addition, concentrations of these phospholipids correlated significantly with the clinical status. Finnhorses formed the largest group. Most Finnhorses had become affected before the age of 5 years and showed moderate signs. Severity of the signs was not related to age at onset. No significant correlation existed between duration and severity of the disease. This study showed that an autoserum preparation containing serum phospholipids was a favourable method to treat summer eczema. Affected horses displayed significant differentiations in their serum phospholipid profiles and these alterations seemed to change according to the clinical status. Applications of this autoserum therapy for other allergic manifestations of horses should be explored.
  • Kovanen, Sara (Helsingin yliopisto, 2017)
    Campylobacter jejuni is a leading cause of human bacterial gastroenteritis worldwide. This zoonotic pathogen transmits to humans mainly indirectly, via consumption of contaminated food or drinking water or from various environmental sources. In Finland, ~4500 confirmed campylobacteriosis cases are registered annually and most of the infections occur in the summer. Poultry has been generally identified as a major reservoir and source of human campylobacteriosis. Molecular epidemiology of C. jejuni has been widely studied. Especially multilocus sequence typing (MLST), which assigns isolates to sequence types (STs) and clonal complexes (CCs), has provided valuable information about existing C. jejuni genotypes in different sources worldwide. While MLST has advantages to compare larger data worldwide, it has limitations in further distinguishing isolates within STs for epidemiological purposes. Whole-genome sequencing (WGS) enables the investigation of whole bacterial genomes instead of few selected genes. Aim of this thesis was to study C. jejuni isolates in domestic human infections, poultry and water using MLST and further whole-genome (wg) MLST. The most commonly found STs belonged to ST-45 CC, ST-21 CC, ST-283 CC and ST-677 CC. In this thesis, we further explored C. jejuni isolates within same STs, using highly discriminatory wgMLST and also considering the temporal relationship between the isolates from humans and chickens in summer 2012. We were able to recognize human isolates that were both genetically and epidemiologically related, thus most likely sharing the same infection source. We also identified the association between C. jejuni isolates from chicken slaughter batches and human patients. Although 79% of MLST types of human C. jejuni isolates overlapped with the isolates from chickens, only 24% of domestic human infections were related to chickens using both wgMLST and temporal association. Hence, the origin of more than 70% of the infections remained unidentified suggesting other potential transmission routes such as other domestic animals or environment-associated sources. In addition, we used WGS data to characterize genomic features among C. jejuni ST-677 CC isolates. We recognized several genetic characteristics, which are typical for this lineage that has been frequently detected among Finnish patients and associated also with bacteremia.
  • Bennett, Rachel (Helsingin yliopisto, 2017)
    Background and rationale Alpha2-adrenoceptor agonists, such as medetomidine, are commonly used in veterinary medicine for the sedation and premedication of animals. Their use is associated with a range of undesirable pharmacodynamic effects most notably vasoconstriction, bradycardia (Pypendop and Verstegen 1998), hyperglycaemia (Hsu and Hummel 1981) and diuresis (Thurmon et al. 1978). MK-467 is a peripheral α2-adrenoceptor antagonist (Clineschmidt et al. 1988), which ameliorates the aforementioned side effects of (dex-)medetomidine whilst maintaining the sedative effects (Enouri et al. 2006; Honkavaara et al. 2008; Restitutti et al. 2012; Rolfe et al. 2012). Therefore MK-467 may offer some important clinical benefits, although some questions remain concerning its pharmacodynamic and pharmacokinetic interaction with other anaesthetic drugs. The main objectives of the following studies were to: determine the protein-binding fraction of MK-467, to assess the possible role of MK-467 as a P-glycoprotein substrate in vitro; and to evaluate the impact of MK-467 on the disposition of medetomidine in vivo. During in vivo studies, the impact of MK-467 on the centrally mediated effects: sedation and antinociception and peripherally mediated cardiovascular effects of medetomidine were assessed simultaneously. The protein-binding characteristics of MK-467 were investigated using the technique of equilibrium dialysis. Drug concentrations were measured using the technique of liquid chromatography and tandem mass spectrometry. Protein-binding fraction of MK-467 was approximately 70% and it was unaltered by the presence of medetomidine. Transcellular drug movement was determined using Madin-Darby Canine Kidney cells - wild type (WT-MDCKII) and cells transfected with the human gene encoding P-glycoprotein (MDR1-MDCKII). In addition to MK-467, acepromazine, a putative P-glycoprotein substrate, and dexmedetomidine were also investigated. Based on measured drug concentrations, apparent permeability of the cells was calculated and used to determine the role of active transport in the transcellular movement of the selected drugs. Passive movement of MK-467 was undetectable. Therefore, efflux ratios for MK-467 were not determined. However, movement in the basolateral to apical direction occurred in both cell lines. The identity of the possible transporter remains unclear. Transport ratios for acepromazine were 1.17:1:1.0 and 1.51:1.0 for WT-MDCKII and MDR1-MDCKII transfected cells, respectively. Currently acepromazine cannot be defined as a P-glycoprotein substrate based on these results. Whilst, dexmedetomidine transport ratios were 0.98:1.0 and 1.15:1.0 for WT-MDCKII and MDR1-MDCKII respectively. It does not appear to be a substrate for an active transport mechanism. In vivo drug concentration data underwent non-compartmental analysis. MK-467 increased the volume of distribution and clearance of dexmedetomidine and levomedetomidine, whilst area under the curve and elimination half-life were significantly decreased when compared with medetomidine alone. Medetomidine significantly decreased the clearance of alfaxalone during co-administration, whilst the additional administration of MK-467 counteracted the effect of medetomidine on alfaxalone clearance. The quality and duration of sedation were assessed using a composite sedation score, whilst hypnosis was evaluated by measurement of bispectral index or analysis of the electroencephalogram. Antinociception was determined by the measurement of limb withdrawal times and head lift times following the application of a nociceptive stimulus applied to the nailbed of a hind limb digit. Measured haemodynamic variables included arterial blood pressure, heart rate, cardiac output and systemic vascular resistance. Ventilatory effects of medetomidine and co-administered MK-467 were also assessed by the analysis of arterial and venous blood gas samples taken during these studies. The co-administration of MK-467 did not alter the initial quality of sedation but reduced the duration of sedation produced by medetomidine. MK-467 significantly diminished the antinociceptive action of medetomidine. MK-467 ameliorated the cardiovascular, haemodynamic and ventilatory effects of medetomidine prior to and during general anaesthesia. In conclusion, MK-467 is moderately protein bound and it is unlikely to be subject to drug-drug interactions in vivo. MK-467 shows little passive movement in MDCKII cells but may undergo active cellular efflux. It is unclear whether MK-467 is suitable for use in animals carrying the P-glycoprotein mutation. The addition of MK-467 alters the disposition of co-administered drugs resulting in lower plasma drug concentrations. The reduction in some pharmacodynamic effects may be attributed to the alteration in pharmacokinetics caused by the peripheral α2-adrenoceptor antagonist.
  • Tirkkonen, Birger Taneli (Helsingin yliopisto, 2017)
    More than 150 species of mycobacteria are described, most being opportunistic pathogens and all representing a risk for human and animal health. Human infections derived from environmental mycobacteria are increasing in both industrialized and developing countries. The most susceptible groups are children, the elderly and those, including animals, with immunocompressive conditions. Drug therapy for mycobacteriosis is difficult and not always successful. Infections caused by drug-resistant mycobacteria can be life threatening also for healthy adults and thus represent a real risk for humans. Environmental mycobacterial infections of pigs are usually without clinical signs and the lesions are mainly detected at slaughter. Mycobacterium-infected pork can pass for human consumption due to the poor sensitivity of visual meat control at slaughterhouses, and mycobacteria in pigs also cause economic losses due to condemnation of carcasses. The main challenge is represented by evaluation of the hygiene risk associated with using mycobacteria-contaminated pork. Most environmental mycobacteria species have been isolated from sources such as water, swimming pools, soil, plants and bedding material. In our study mycobacterial growth in piggeries was identified in all bedding materials, sawdust, straw, peat and wood chips in most cases, and water and food samples in many cases, and only occasionally in dust and on wall surfaces. The maximum number of mycobacteria was almost 1 billion (109) per gram of bedding, which is close to the maximum concentration in any growth media. Mycobacteria can multiply in piggeries and contaminate feed and water. Isolation of mycobacteria from pig faeces can be considered an indicator for risk of human infection. Environmental mycobacteriosis in humans and pigs is mainly caused by M. avium subsp. hominissuis. There is little evidence of direct transmission from animals to humans, but particular strains can be recovered from both humans and pigs. In our studies, identical mycobacteria RFLP and MIRU-VNTR fingerprints of porcine and human origins were evident. Interspecies clusters were more common than intraspecies clusters using both methods. Therefore, we concluded that pigs act as a reservoir for virulent M. avium strains and the vector for transmission of infections in humans to pigs, and vice versa, may have an identical source of infection. Culturing mycobacteria is the gold standard for diagnosis, but detection of environmental mycobacteria based on cultivation and biochemical methods can take several weeks. Culture-independent, rapid and accurate techniques for detecting mycobacteria in food and feed chains are urgently needed. In this work we developed a rapid and accurate real-time quantitative PCR for detecting environmental mycobacteria in bedding materials and pig organs. Conclusion: Mycobacteria can multiply in bedding materials and the consequent heavy contamination can cause simultaneous infections in pigs. Mycobacterial DNA was found in pig organ samples, including those without lesions, and similar strains were found from humans and pig organ samples, which suggests that mycobacteria can be transmitted between humans and pigs.
  • Grönthal, Thomas (Helsingin yliopisto, 2017)
    Staphylococcus pseudintermedius (SP) is part of the normal microbiota of dogs and cats. Since the mid-1980s, an ever-increasing number of methicillin-resistant SP (MRSP) isolates have been reported. In the mid-2000s, two predominant MRSP clones, ST71 (sequence type 71) and ST68, spread through Europe and North America, respectively. MRSP isolates are commonly multidrug resistant (MDR), and are thus capable of causing infections that do not respond to routinely used antimicrobials. MRSP appeared in the small animal population of Finland in the late 2000s, also causing numerous infections at the Veterinary Teaching Hospital (VTH) of the University of Helsinki. No data on the epidemiology of MRSP in Finland have been published. This thesis study aimed to explore the epidemiology of MRSP in the Finnish small animal population. This was done by investigating and describing the MRSP outbreak at the VTH, and investigating risk factors for patients being colonized or infected by MRSP in the hospital during the outbreak. The prevalence of MRSP and the risk factors for MRSP carriage were investigated in a canine subpopulation at the Guide Dog School for the Visually Impaired. The susceptibility of SP isolates to antimicrobials in 2011–2015 was also investigated using data from the Clinical Microbiology Laboratory (CML) of the Faculty of Veterinary Medicine, University of Helsinki. Risk factors for an SP isolate being MRSP, as well as for a screening specimen revealing MRPS, were also investigated. Furthermore, the molecular epidemiology of all MRSP isolates stored in 2010–2014 was investigated using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing. Antimicrobial therapy, whether previous or ongoing during sampling, was a risk factor for MRSP in all studies. Furthermore, a prolonged hospital stay and veterinary visits were risk factors among guide dogs. An SP isolate originating from a private clinic (versus the VTH) was a significant risk factor for MRSP among clinical specimens. The same could be seen among screening specimens from patients with risk factors for MRSP. In addition, it was noted that a sizeable proportion (~20–60%) of animals in the studies had been or were being treated with antimicrobials. During the outbreak at the VTH, rigorous hygiene and barrier measures were necessary to achieve control. ST71, the MRSP clone that caused the outbreak, was the predominant clone in 2010–2011, accounting for over 50% of MRSP isolates, even among non-outbreak-related isolates. By 2014 the situation had changed, as ST71 represented only ~10% of MRSP isolates. MRSP clones belonging to CC45 (clonal complex 45) and CC258, and other unrelated STs, dominated the MRSP population by that time. SCCmec type IV was detected in a majority of different STs, indicating the horizontal spread of resistance genes. The prevalence of MRSP was only 3% among guide dogs. The proportion of clinical specimens from small animals that revealed MRSP was similar, 2.5%. However, 9% of screening specimens from high-risk patients revealed MRSP. Overall, 14% of SP isolates were MRSP. Roughly 30–40% of isolates were not susceptible to alternative antimicrobials, such as lincosamides, macrolides, or tetracyclines. MRSP in feline specimens was rare (<1% in all specimens after 2011). Our results give further credence to the hypothesis that antimicrobial therapy and contact with the veterinary environment are risk factors for MRSP in small animals. However, cats do not appear to be a significant source of MRSP. Our data suggest that the epidemiology of MRSP has changed from a predominantly clonal spread to a mix of clonal spread and the spread of genetic elements. The resistance rates among SP are at an alarming level. Decisive action, including the use of non-antimicrobial treatments whenever feasible and more prudent use of antimicrobials, is required to improve the situation.
  • Mikkola, Marja (Helsingin yliopisto, 2017)
    Multiple ovulation and embryo transfer (MOET) has been established in cattle breeding since the 1970s. It is an efficient means to increase the number of offspring from genetically superior females. Despite nearly 50 years of development, the average number of transferable embryos recovered in a single embryo collection has remained nearly constant at approximately six embryos per donor. Several animal-related, environmental and management factors contribute to the outcome of superovulation and embryo recovery. The most prominent factor affecting the success of superovulation is an animal-related attribute: the ovarian follicular population responsive to exogenous gonadotropin stimulation. Environmental factors, such as heat stress or other external factors causing stress, can compromise the embryo yield after superovulation. However, such factors represent a management challenge. The superovulatory outcome is additionally affected by several factors that can conflict with management decisions, including nutritional management of the donor, gonadotropin treatment protocol and semen and technical performance of donor inseminations. The purpose of the work presented in this thesis was to investigate management factors that affect the efficacy of MOET. First, the effect of nutritional protein in the form of rapeseed meal on superovulation of dairy heifers was studied. One-year old heifers were fed diets formulated to meet energy requirements for 800 g daily gain and crude protein either at 14% or 18%, which was higher than the feeding recommendations and the common practice on farms. There was no effect of the higher protein level on the ovulation rate, total number of embryos recovered or the number of transferable embryos. Feeding an energy-adequate diet containing moderate or high protein with respect to feeding recommendations resulted in comparable embryo yields. The efficacy of two commercial FSH products was compared in a retrospective study on superovulations of heifers and cows on Finnish dairy farms and an embryo collection station. A highly purified porcine FSH with a low LH:FSH ratio, Folltropin, was used for 2592 superovulations, and Pluset, containing equal amounts of LH and FSH, was used for 1398 superovulations. Pluset-treated donors had a higher ovulation rate, yielding 1.1 recovered structures (embryos and ova) more than those receiving Folltropin. However, the difference was characterized by more unfertilized ova (UFO). For transferable embryos, the number, quality and developmental stage were similar for both preparations. Therefore, it can be concluded that the efficacy of the preparations is comparable. The effect of sex-sorted semen on efficacy of MOET was investigated from a dataset of commercial embryo collections and transfers. A total of 443 embryo collections produced with sex-sorted semen and 1528 produced with conventional semen were analyzed. Sex-sorted semen decreased the number of transferable embryos and increased proportions of UFO and degenerated embryos, compared with non-sorted semen. The decrease was more evident in cows than in heifers. The proportion of poor quality embryos was higher and there was a slight delay in the embryo developmental kinetics for sexed embryos. The risk of recovering no transferable embryos was increased when sex-sorted semen was used. Pregnancy rates after transfer of embryos produced with sex-sorted semen were 12% lower than for embryos originating from conventional semen. It can be concluded from these studies on sexed semen that the use of sex-sorted semen is profitable because more female calves can be produced from a donor heifer, wasting less recipient resources. For superovulated cows, equal numbers of female calves can be produced per embryo collection, but the need for only half the number of recipients compared with using conventional semen favors the use of sexed semen when female progeny are desired.
  • Kaukonen, Eija (Helsingin yliopisto, 2017)
    Contact dermatitis in broilers is a multifactorial condition that is most commonly caused by poor litter quality or otherwise unsuitable material affecting the footpad or hock skin. Footpad health is mainly maintained by keeping litter in a dry and friable condition. Hence, footpad lesions reflect litter quality that, more widely, describes the housing conditions and bird health. The evaluation of the prevalence of contact dermatitis denotes a commonly accepted approach to assess the welfare of broiler flocks. However, there is lack of knowledge about footpad lesions in broiler breeders. Although numerous studies on the effect of litter materials on footpad condition have been conducted, experiments with peat are scarce. Also, knowledge of the influence of peat on hock burns and litter quality is lacking. Modern fast-growing broilers spend excessive time resting and this inactivity has been suggested to increase the incidence of impaired gait and leg disorders. Tibial dyschondroplasia (TD) is one of the most common leg pathologies in broilers. Perches or elevated platforms add complexity to the broilers’ environment and may stimulate locomotion. However, research on the use of elevated structures under commercial rearing conditions and possible benefits for broiler leg health is limited. This thesis provides descriptive information about contact dermatitis and breast blisters in broiler breeders throughout the production period with respect to litter condition. Secondly, the study compared the influence of peat bedding with wood shavings and ground straw (fine crushed straw) on contact dermatitis and plumage cleanliness in fast-growing broilers and litter condition in commercial broiler houses. Furthermore, the study examined the use of perches and elevated platforms by broilers, and the impact of the additional equipment on contact dermatitis, plumage cleanliness, walking ability, the occurrence of TD and litter conditions under intensive rearing circumstances. Litter condition in broiler and breeder houses was evaluated according to the Welfare Quality® (WQ) protocol for broiler chicken. Additionally, litter height was measured, and litter quality determined according to moisture, pH and ammonia content. Footpad condition was visually inspected with the WQ-scoring method (broilers), the official Finnish system (broilers) or employing a method modified from the official system (breeders). Hock skin lesions and plumage cleanliness were assessed according to the WQ-protocol. Broiler gait was scored before slaughter following the WQ-protocol. The severity of TD was determined. The use of perches and platforms was monitored by video recording. Additionally, farmers estimated the platform and perch usage twice a week throughout the growing period. The condition of breeder footpads deteriorated towards the end of the production period, with the occurrence of severe lesions reaching a maximum of 64% on average at slaughter. However, hock burns and breast blisters were rarely recorded. The litter layer became drier over time. Although dry and friable litter in breeder houses was associated with healthier footpads, other factors were of greater importance, as footpad lesions, particularly severe lesions, appeared more often towards slaughter age. Broiler footpads were generally in good condition at slaughter age, 80% of the birds having healthy footpads. In broilers, hock burns were more frequently detected than footpad lesions. Inferior footpad and hock skin health was scored on wood shavings rather than on peat, without differences in litter condition and moisture. Moreover, the lack of difference in moisture between ground straw and peat still resulted in poorer litter, footpad and hock skin condition on ground straw. Farms differed for footpad and hock burn condition, and litter quality. In risk analysis, the impact of farmer on contact dermatitis severity exceeded the effect of litter quality. The platforms were used frequently while only single birds used perches. The study indicated no effects of platform treatment on footpad and hock skin health, and litter condition. The birds with access to platforms, however, had enhanced leg health: mean gait score, the percentage of birds scored 3, and TD percentage and severity were lower for birds in platform-equipped houses. Access to platforms most likely enables more versatile movement, such as walking forward, up and down, grasping by feet, and jumping, which may promote leg health and gait. This was the first study to follow footpad health in broiler breeders through the whole production period. The results indicate the need for further investigation because good litter condition alone appears insufficient to keep breeder footpads healthy for their entire life. Further, this thesis provides new knowledge about the applicability of peat as broiler bedding. According to our results, regarding footpad health, peat seems to be the optimal litter material for Finnish conditions. Furthermore, the study underlines the importance of farmer ability to manage litter conditions, regardless of the chosen litter material. Hock burn monitoring could represent a more sensitive indicator of litter condition and possibly also signal leg health status, therefore monitoring hock burns at slaughter should be considered. The advantages of traditional perches for broilers should be re-evaluated as they remained largely unused. However, the extensive use of platforms suggests that broilers are motivated to perch on elevated structures. Hence, platform availability could enhance their emotional wellbeing. Elevated platforms offering additional possibilities for locomotion seem promising because they show apparent potential to enhance leg health without compromising litter condition or footpad health. Based on all these findings, elevated platforms with ramps can be recommended as a way forward to enhance broiler welfare in commercial environments.
  • Kantala, Tuija (Helsingin yliopisto, 2017)
    Hepatitis E virus (HEV) infections are common in both humans and animals. HEV genotypes 1 and 2 (HEV-1 and HEV-2) only infect humans and are endemic to Asia, Africa, and Central America, where they cause large, usually waterborne hepatitis epidemics. HEV-3 and HEV-4 are zoonotic and in addition to humans, they also infect animals. Especially HEV-3 is common in swine globally. The porcine infection is usually asymptomatic. In humans, HEV-3 and HEV-4 infections are often asymptomatic or only cause mild symptoms, but they can also cause chronic hepatitis that can lead to liver fibrosis and cirrhosis and even death in immunocompromised patients. The aims of this work were to investigate occurrence of HEV and antibodies against HEV in human patients with unexplained acute hepatitis, in veterinarians, and in production pigs in Finland. Antibodies against HEV were present in 27.6% of human patients diagnosed with acute non-A C hepatitis and as a marker for acute hepatitis E infection, anti-HEV IgM antibodies in 11.3% of the patients. All HEV isolates obtained from the patients belonged to HEV-1. Most of the patients with acute infections had recently visited HEV-1 endemic areas in Asia, Africa, or Mexico, indicating that their infections were obtained during travels. However, the possibility of infections acquired in Finland could not be excluded, since no traveling data were available for several HEV-positive patients. Of all production pigs of different ages investigated, 15.5% were positive for HEV RNA and 86.3% for antibodies against HEV. Longitudinal follow-up studies on pigs revealed that the pigs were infected with HEV at the age of 2 3 months, when the prevalence of HEV RNA-positive pigs was at its peak, 34.6%. Thereafter, the prevalence of HEV RNA-positive pigs declined to 21.1% at 3 4 months of age and to 2.9% in slaughter-aged pigs. High HEV antibody seroprevalences of over 80% were detected in all age groups tested, from weaner-aged pigs to sows. All HEVs from pigs were of HEV-3, subtype e. Genetically separate clusters of HEV isolates were obtained from different swine farms, suggesting that genetic variations in viruses from different locations occur. In addition, two different isolates were obtained from the same farm, and also HEV-negative swine farms were seen. The pigs were commonly shedding HEV at the time they were transferred from farrowing farms to fattening farms, creating a possible risk of zoonotic infection for pig handlers. When pigs from HEV-negative and HEV-positive farms arrive at the same fattening farm, infection at a later age during the fattening stage must also be considered possible, which constitutes a risk for HEV entering the food chain in pork at the time of slaughter. With apparent anti-HEV antibody prevalence of 10.2%, Finnish veterinarians commonly have antibodies against HEV. HEV seropositivity was unexpectedly associated with working as a small animal practitioner and negatively associated with having contacts with swine. However, contradictory to swine contacts, the seroprevalence appeared to be higher in those who had had needle stick by a needle that had previously been injected into a pig than in those who had not, suggesting that contact with blood or tissue fluid from swine might be a risk factor for HEV infection in veterinarians. In addition, those small animal practitioners who had traveled outside Europe during the previous five years appeared to be more often seropositive than those who had not, suggesting that some infections might have been travel-related. Although pigs seem to play a role in the hepatitis E infections of veterinarians, there are possibly multiple factors involved, including also other reservoirs of HEV than pigs. Hepatitis E virus must be considered a possible cause of acute hepatitis in humans in Finland, especially in patients who have returned from areas endemic to HEV-1 and HEV-2. Although no cases of possibly zoonotic HEV-3 infections acquired in Finland were detected in humans in this study, their possibility should not be overlooked since HEV is widespread in production pigs in Finland and routes for zoonotic infection exist.
  • Pohjola, Leena (Helsingin yliopisto, 2017)
    There is an increasing interest in keeping backyard poultry in many countries, including Finland. However, several studies in Western Europe and North America have identified the involvement of backyard poultry flocks in avian influenza virus outbreaks occurring in commercial poultry. In addition, commonly without any signs of illness, poultry can be carriers of enteric bacterial agents that are human pathogens. Farm management and biosecurity practices among 178 backyard poultry flocks were investigated using a questionnaire. Furthermore, the main causes of mortality of backyard chickens were studied through a retrospective study of necropsy data from the Finnish Food Safety Authority Evira from 2000 to 2011. In addition, voluntary backyard poultry farms were visited during October 2012 and January 2013, and blood samples, individual cloacal samples as well as environmental boot sock samples were collected from 51 farms and 457 chickens. The results of the questionnaire study revealed that the backyard poultry farms in Finland were mainly small (91 % ≤ 50 birds) and most flocks (98 %) had access to outdoors. Biosecurity practices, such as hand washing and changing shoes after bird contact were rare, 35 % and 13 % respectively. The farms were mainly located distantly (94 % > 3 km) from commercial poultry farms. The subjectively reported flock health was good (96 %). The most common postmortem diagnosis were Marek s disease (27 %) and colibacillosis (17 %). Of the zoonotic bacterial pathogens, Campylobacter jejuni and Listeria monocytogenes were frequently detected on the farms, 45 % and 33 %, respectively. Yersinia enterocolitica was also often isolated on the farms (31 %); however, all isolates were yadA negative, i.e. non-pathogenic. C. coli, Y. pseudotuberculosis and Salmonella enterica were rarely detected (2 %). All enteric bacteria were highly susceptible to most of the antimicrobials studied and only few AmpC- and no ESBL-producing E. coli were found. Avian encephalomyelitis virus, chicken infectious anemia virus and infectious bronchitis virus (IBV) antibodies were commonly found from the studied flocks, 86 %, 86 % and 47 %, respectively. The IBV detected from backyard poultry flocks were QX-type IBV strains differing from the strains found from commercial farms, suggesting different routes of infection for commercial and backyard poultry. The results indicated that among backyard poultry flocks pathogens circulate posing a risk to transmit infection to commercial poultry in Finland, but because of the distant locations and small flock sizes, the risk is relatively small. Notifiable avian diseases that also are of zoonotic potential (AIV and NDV) are very rare. Backyard chickens are a reservoir of C. jejuni strains and thus a potential source of C. jejuni infection for humans. Because of the lack of good hygiene after bird contact, the risk of transmission of the pathogen from birds to humans exists
  • Selby, Katja (Helsingin yliopisto, 2017)
    Clostridium botulinum, the causative agent of botulism in humans and animals, is frequently exposed to stressful environments during its growth in food or colonization of a host body. The wide genetic diversity of the strains of this foodborne pathogen has been thoroughly studied using different molecular biological methods; however, it is still largely unknown how this diversity reflects in the ability of different C. botulinum strains to tolerate environmental stresses. In contrast to cold tolerance, which has been the focus of intensive research in recent years, the molecular mechanisms C. botulinum utilizes in response to heat shock and during adaptation to high temperature stress are poorly understood. The aims of this study were to investigate the strain variation of Group I and II C. botulinum with regard to growth at low, high, and optimal temperature; the roles of hrcA, the negative regulator of Class I heat shock genes (HSG) and dnaK, a molecular chaperone coding Class I HSG, in the response of the Group I C. botulinum strain ATCC 3502 to heat and other environmental stresses; and the molecular mechanisms this strain employs in response to acute and prolonged heat stress. The maximum and minimum growth temperatures of 23 Group I and 24 Group II C. botulinum strains were studied. Further, maximum growth rates of the Group I strains at 20, 37, and 42°C and of the Group II strains at 10, 30, 37, and 42°C were determined. Within their groups, the C. botulinum strains showed significant variation in growth-limiting temperatures and their capability to grow at extreme temperature, especially at high temperature. Largest strain variation was found for Group I within type B and for Group II within type E strains, which further showed more mesophilic growth tendencies than the other Group II strains. However, the genetic background of the selected C. botulinum strains reflected only weakly in their growth characteristics. Group I strains showed larger physiological variation despite being genetically more closely related than Group II. A number of strains of both groups showed faster growth at temperatures above than at their commonly assumed optimal growth temperatures of 30°C for Group II and 37°C for Group I strains. In addition, they possessed higher maximum growth temperatures than the average of the studied strains. These strains can be expected to have higher than assumed optimal growth temperatures and pronounced high temperature stress tolerance. Good correlation was detected between maximum growth temperatures and growth rates at high temperature, although not for all strains. Therefore direct prediction from one studied growth trait to the other was impossible. These findings need to be taken into account when estimating the safety of food products with regard to C. botulinum by risk assessment and challenge studies. The role of Class I HSGs in C. botulinum Group I strain ATCC 3502 was studied by quantitative real-time reverse transcription PCR and insertional inactivation of the Class I HSGs hrcA and dnaK. During exponential and transitional growth, Class I HSGs were constantly expressed followed by down-regulation in the stationary phase. Exposure of mid-exponentially growing culture to heat shock led to strong, transient Class I HSG up-regulation. Inactivation of hrcA resulted in over-expression of all Class I HSGs, which confirmed its role as negative regulator of Class I HSGs in C. botulinum. Both inactivation mutants showed impaired high temperature tolerance as indicated by reduced growth rates at 45°C, a reduced maximum growth temperature, and increased log-reduction after exposure to lethal temperature. The growth of the dnaK mutant was more strongly affected than that of the hrcA mutant, emphasizing the importance of the molecular chaperone DnaK for C. botulinum. Reduced growth rates were evident for both mutants under optimal conditions and heat stress, but also under low pH, and high saline concentration. This suggests a probable role for Class I HSG in cross protection of C. botulinum against other environmental stresses. C. botulinum ATCC 3502 was grown in continuous culture and exposed to heat shock followed by prolonged high temperature stress at 45°C. Changes in the global gene expression pattern induced by heat stress were investigated using DNA microarray hybridization. Class I and III HSGs, as well as members of the SOS regulon, were employed in response to acute heat stress. High temperature led to suppression of the botulinum neurotoxin coding botA and the associated non-toxic protein-coding genes. During adaptation and in the heat-adapted culture, motility- and chemotaxis-related genes were found to be up-regulated, whereas sporulation related genes were suppressed. Thus, increase in motility appeared to be the long-term high-temperature stress-response mechanism preferred to sporulation. Prophage genes, including regulatory genes, were activated by high temperature and might therefore contribute to the high temperature tolerance of C. botulinum strain ATCC 3502. Further, remodeling of parts of the protein metabolism and changes in carbohydrate metabolism were observed.
  • Viitanen, Sanna (Helsingin yliopisto, 2017)
    Bacterial pneumonia (BP) is an acquired inflammation of the lower airways and lung parenchyma secondary to bacterial infection. BP is difficult to induce experimentally in healthy dogs; the pathogenesis is therefore considered complex, involving several underlying mechanisms. BP was first described in dogs decades ago, but it is still one of the most common systemic bacterial infections in dogs, with a significant morbidity and mortality. Several aspects of BP, including the applicability of inflammatory biomarkers in its diagnosis and follow-up as well as the role of respiratory viruses in its clinical picture and development, warrant further studies. This thesis aimed to describe clinical findings during the disease and recovery periods in dogs with BP and to evaluate the applicability of acute-phase proteins as diagnostic and follow-up markers in BP. The prevalence and role of viral co-infections in dogs with BP were also investigated. We evaluated the diagnostic applicability of serum C-reactive protein (CRP) and noted that CRP is significantly elevated in BP relative to dogs with other lower respiratory tract diseases, such as chronic bronchitis, bacterial tracheobronchitis, canine idiopathic pulmonary fibrosis, and eosinophilic bronchopneumopathy, as well as in cardiogenic pulmonary edema. Our results indicate that serum CRP concentration may be used as an additional biomarker in the diagnosis of canine BP. Serum CRP, serum amyloid A (SAA), and haptoglobin (Hp) were followed during the disease and recovery periods. The follow-up study showed that serum CRP and SAA reflected well the recovery process and declined rapidly after initiation of successful therapy and could therefore be used as markers of treatment response in dogs with BP. Currently, markedly longer antibiotic courses are recommended in dogs with BP than in humans with pneumonia. Since serum CRP is a sensitive inflammatory biomarker, it was hypothesized that normalization of serum CRP could be used as an indicator for the cessation of antimicrobial therapy. In our study, we treated a group of dogs according to conventional recommendations. In another group, antimicrobial therapy was ended 5-7 days after CRP normalization. When the normalization of CRP was used to guide antimicrobial therapy, treatment length was significantly reduced without increasing the number of relapses. According to these results, normalization of serum CRP may be applied to guide the length of antimicrobial therapy in dogs with BP. Respiratory viruses, primarily canine parainfluenza virus, were found frequently in lower respiratory tract samples in dogs with BP. This indicates that viruses may play an important role in the etiology and pathogenesis of BP. Viral co-infections did not affect disease severity or clinical variables. Our findings add new knowledge about the natural course of BP as well as about the possible applications of acute phase protein measurements in the diagnosis and follow-up of BP. The utilization of acute phase protein measurements may allow a more precise diagnosis of BP, enable the early identification of patients with a poor response to treatment, and diminish the use of antimicrobial drugs.
  • Cheng, Jing (Helsingin yliopisto, 2016)
    The interactions between a host and his/her microbiota have co-evolved over time and they exert profound effects on each other. Intestinal microbiota has been linked with a number of diseases, such as irritable bowel syndrome; it is considered to be a major etiopathological factor since it can alter intestinal homeostasis. However, the role of intestinal microbiota, especially commensals, is unclear in celiac disease. To date, most efforts for detecting potential microbial changes affected by celiac disease have focused on adult individuals and have examined fecal materials, although it is known that early life is the critical period for the microbiota to colonize and establish their niche in the human intestine. At this time in healthy individuals, there is continuing cross-talk with the host e.g. via the immune system, leading to the establishment of homeostasis in both metabolic and immunological programming. Since the intestinal epithelium is the main interface for host- microbe interactions, the role of mucosa-associated microbiota may be distinct from that of fecal microbiota, but both the normal fluctuations in intestinal microbiota and the composition of duodenal mucosa-associated microbiota are still not fully clarified. The aims of thesis were to characterize the development and stability of intestinal microbiota in healthy young children and to compare the microbial features between children and adults. Furthermore, the aim was to investigate host-microbe interactions in celiac disease by studying duodenal mucosa-associated microbial signatures and mucosal gene expression in healthy children and their counterparts with celiac disease. The microbiota profiles were characterized by using the human intestinal tract chip (HITChip), which is a bacterial phylogenetic microarray. The amounts of Bifidobacterium spp. in children and adults were verified with real time qPCR. The levels of mucosal gene expressions were quantified with reverse transcriptase quantitative PCR. The results showed that intestinal microbiota is not fully matured at the age of five in children. A common core microbiota, including several butyrate-producing bacteria, was identified in children and it was developing towards core microbiota found in adults. The different progression pattern of major bacterial taxa may reflect the physiological development steps in children. Moreover, differences were observed between healthy- and celiac disease- associated microbial signatures. The differences may reflect changes in epithelial integrity associated with the disease. On the other hand, the studies on both microbiota and mucosal gene expression indicated that the persistently enhanced Th1 type immune responsiveness in subjects with celiac disease after treating with gluten-free diet might result from the increased expression of TLR9, which recognizes unmethylated CpG motifs in bacterial DNA via the direct stimulation of immune cells and/or intestinal epithelial cells. The results of this thesis project suggest that specific symbiotic and dysbiotic microbial signatures may provide potential functional diagnostic or therapeutic targets for promoting healthy/natural microbiota development. Long-term studies in a controlled environment with an adequate number of participants will be necessary to decode the disturbed microbial signatures. These trials should be combined with systematic pathological surveillance to reveal how the changes in the microbiota influence the onset of disease.
  • Viljamaa-Dirks, Satu (Helsingin yliopisto, 2016)
    Crayfish plague is a severe disease of European crayfish species and has rendered the indigenous crayfish populations vulnerable, endangered or even extinct in the most of Europe. Crayfish plague is caused by an oomycete Aphanomyces astaci, a fungal-like water mould that lives its vegetative life in the cuticle of crayfish and infects other crayfish by producing zoospores. Zoospores swim around for a few days in search of crayfish, and when they find one they attach onto its surface, encyst and germinate to start a new growth cycle as new growing hyphae penetrate the crayfish tissues. Unrestricted growth of A. astaci leads to the death of the infected animal in just a few weeks. Crayfish plague induced mortalities started in Italy around 1860. Although the disease was known about since 1860 its cause remained unknown for several decades. Little was done to prevent the spread of the disease. A lively crayfish trade probably facilitated the spread of the crayfish plague, which reached Finland in 1893. The crayfish plague has remained the most important disease problem of the Finnish noble crayfish Astacus astacus, since then. The consensus was that the disease killed all infected animals in a short time, and it appeared almost impossible to restore the flourishing crayfish populations to the levels that existed before. Following the example of neighbouring Sweden, a North American crayfish species, the signal crayfish Pacifastacus leniusculus that appeared resistant to crayfish plague was introduced to Finland in 1960s. As expected, the signal crayfish slowly started to replace the lost populations of the noble crayfish to become an important part of the crayfish fisheries. The introduction of the signal crayfish significantly added to the management problems of the noble crayfish stocks left. Signal crayfish appeared to be a chronic carrier of the crayfish plague agent, and spread the disease to the dwindling vulnerable noble crayfish populations. Later research showed that the crayfish plague agent is a parasite of North American crayfish that in normal circumstances does not harm the host animal. Intriguingly, the crayfish plague agent carried by the signal crayfish, genotype Ps1, is different from the pathogen originally introduced into Europe, genotype As. The diagnosis of crayfish plague especially when based on the isolation of the pathogen is challenging and accordingly the genotype difference was mostly unrecognized until recently. In this study we determined the genotype of the causative agent from most of the detected Finnish crayfish plague cases between 1996 -2006. It appeared that most of the epidemics in the immediate vicinity of signal crayfish populations were caused by genotype Ps1, whereas genotype As was more prevalent in the noble crayfish areas. Interestingly, a difference was seen in the outcome of the infection. The Ps1 infection was always associated with acute mortalities, while As infections were also frequently found in existing but weak populations. The persistent nature of an As infection could be verified in noble crayfish from a small lake in southern Finland. This finding explained why many of the efforts to introduce a new noble crayfish population into a water body after a crayfish plague induced mortality were futile. The main conclusion from the field study data of this research was the difference in virulence between the Ps1 and As genotype strains. This was also verified in a challenge trial with noble crayfish. While the Ps1 strains did not show much variation in their growth behaviour or virulence, there was much more variation in the As strains. The As genotype arrived in Finland more than 100 years ago, and since that date it seems to have adapted to the novel host, the noble crayfish, to some extent. In order to gain insight into a possible vector of this genotype, we studied another North American crayfish species present in Europe, the spiny-cheek crayfish Orconectes limosus from a Czech pond. This crayfish species appeared to carry a novel genotype of A. astaci, named Orconectes genotype, designated Or . It seems possible that many of the North American crayfish species carry their own type of crayfish plague agent, with variable features such as virulence. These differences should be further tested in the future. The results of this study alleviate the necessity to study the noble crayfish mortalities for the verification of crayfish plague, including the study for the genotype of the A. astaci strain. Crayfish fisheries and conservation management decisions should not be made without a prior control of the donating population and the receiving water body for the eventual presence of a low-virulent A. astaci.