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  • Salla, Kati (Helsingin yliopisto, 2017)
    Medetomidine (MED), an 2-adrenergic agonist, is widely used in veterinary practice as a sedative and premedication agent, as it provides reliable sedation. However, the marked haemodynamic changes limit its use to only healthy patients. MK-467, a peripheral 2 -adrenoceptor antagonist, has been demonstrated to attenuate the early haemodynamic disturbances induced by MED with intravenous co-administration in dogs. The main aim of this series of investigations was to evaluate the haemodynamic effects of MK-467 when it is combined with MED and selected sedative or anaesthetic agents that are commonly used in veterinary practice in dogs. More specifically, the concomitant administration of MK-467, MED and butorphanol via intravenous or intramuscular routes was studied and compared with sedation induced with MED-butorphanol. The haemodynamic effects after the various doses of MK-467 in relation to the standard dose of MED were evaluated prior to and during isoflurane anaesthesia. In addition, the haemodynamic effect of the administration of MK-467 as a constant rate infusion was assessed during MED-alfaxalone total intravenous anaesthesia. As a comparison, the haemodynamic effects of the co-administration of MK-467 and MED as a premedication regimen in relation to acepromazine-butorphanol or glycopyrrolate-medetomidine prior to and during isoflurane anaesthesia were assessed. Finally, the influence of MK-467 on plasma dexmedetomidine and alfaxalone concentrations was determined. After intravenous administration of MK-467 and MED with or without butorphanol heart rate, cardiac output and oxygen delivery were well maintained, and the increase in systemic vascular resistance and blood pressures was prevented. The profitable effects of MK-467 appeared more slowly after intramuscular than after intravenous administration. The initial MED-induced haemodynamic effects were dose-dependently attenuated by MK-467 during the premedication period but not during isoflurane anaesthesia with these studied doses of MK-467 in relation to a standard dose of MED as a premedication regimen. The addition of MK-467 to MED and alfaxalone produced a stable haemodynamic outcome when they were administered as constant rate infusions. Cardiac output and oxygen delivery were well maintained during both inhalation and total intravenous anaesthetic regimens when MK-467 was involved. The haemodynamic effects after the co-administration of MED-MK-467 or acepromazine-butorphanol mimicked each other prior to and during isoflurane anaesthesia. Pre-treatment with subcutaneous glycopyrrolate failed to improve the MED-induced reduction in cardiac output by means of increasing heart rate prior to or during isoflurane anaesthesia. Peripheral 2- adrenergic receptor blockade by MK-467 resulted in a reduction in plasma dexmedetomidine and alfaxalone concentrations, presumably by altering the haemodynamic function. In conclusion, the addition of MK-467 in a MED-contained sedation or anaesthetic regimen attenuates the rather dramatic MED-induced haemodynamic changes by blocking peripheral 2-adrenergic receptors and by decreasing the plasma concentrations of dexmedetomidine. The overall haemodynamic stability is well maintained during either an isoflurane anaesthesia or an alfaxalone constant rate infusion. The combination of MK-467 with MED may offer an alternative premedication regimen to the combination of acepromazine and butorphanol in healthy dogs.
  • Dyggve, Hanna (Helsingin yliopisto, 2017)
    Doberman hepatitis (DH) is a rare and chronic inflammatory condition of the liver. The early diagnosis of DH is difficult, and Dobermans may be affected for months or years before the diagnosis. There is no curative treatment and the prognosis is poor if the condition is associated with clinical signs of liver failure. Unraveling the disease etiologyand pathogenesis would enhance the diagnostic approaches, and novel therapies could improve the prognosis for Dobermans. Our studies aimed to identify supporting evidence for the suspected autoimmune background of DH by satisfying more descriptive characteristic risk factors that are described in human autoimmune diseases. We focused on major histocompatibility complex (MHC) class II genes, MHC class II regulatory regions, circulating serum anti-nuclear antibodies (ANA), and possible liver-related autoantigens associated with DH. We identified a homozygous dog leukocyte antigen (DLA) risk haplotype and allele for DH. DLA-DRB1*00601/DQA1*00401/DQB1*01303 haplotype elevated the risk of DH susceptibility close to 15-fold. A homozygous risk DLA-DRB1*00601 allele was found in all DH dogs, while this allele was present in 56.8% of the healthy controls. DLA-DQA1*00901/DQB1*00101 and allele DLA-DRB1*01501 appear to protect against DH development. Overall, the DLA region showed very low variation among DH patients, with only two different haplotype combinations compared with seven in the control group. We defined the level of variation of the regulatory promoters in DH patients and controls with the homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 haplotype. Also, the potential polymorphism in the DLA-DRAp promoter and DLA-DRA exon 2 was examined. The promoter DLA-DRAp and exon 2 were identical to the dog reference sequences in both groups. The promoters DRBp, DQAp, and DQBp were monomorphic, with no explaining variation when comparing DH patients with healthy control Dobermans. DLA-DRB1*00601 allele was associated with DRBp*1, DLADQB1*01303 allele with DQBp*6, and DLA-DQA1*00401 allele with DQAp*2. Our result suggests that promoter variants are not associated as risk modifiers for DH in Dobermans with the risk haplotype, but the whole DLA block is associated with DH. The study revealed ANA in DH patients. ANA were further characterized as antihistone autoantibodies (AHA) that were significantly elevated in DH. Thus, immune tolerance has failed in DH. Our results suggest that elevated AHA in conjunction with high alanine aminotransferase (ALT) in a Doberman with or without clinical signs of hepatitis support the DH diagnosis. A negative serum AHA result does not rule out DH, and AHA is not a suitable biomarker for disease progression. We described two novel liver-related target autoantigens in DH. These could be identified as dehydrogenase enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and alcohol dehydrogenase (ADH). DH dogs had significantly elevated serum IgG immunoreactivity towards GAPDH and ADH antigens compared with controls, and the disease was associated with both autoantibodies. This provides evidence for an antigen-driven autoimmune process and elucidates the previously unknown DH pathogenesis. This thesis provides new insights into the suspected autoimmune etiology and pathogenesis of DH. We conclude that the study contributes to the development of useful biomarkers that could be used to improve the currently difficult early diagnosis of DH in Dobermans with elevated ALT levels. Our findings support the theory that DH has an autoimmune origin.
  • Nokireki, Tiina (Helsingin yliopisto, 2017)
    European bat lyssavirus type 2 (EBLV-2) was first isolated in Finland from a Daubenton’s bat (Myotis daubentonii) in 2009. Rabies in bats was already suspected in 1985, when a Swiss biologist died in Finland of lyssavirus infection, later identified as EBLV-2 infection. However, the origin of the infection could not be confirmed at that time. In 2010–2011, during active surveillance study, samples from 774 bats were analyzed for EBLV viral RNA. In addition, sera from 423 bats were analyzed for the presence of lyssavirus antibodies. Antibodies were detected in 2010 and 2011 from two locations and from one location, respectively. All seropositive bats were Daubenton’s bats. No EBLV viral RNA was detected in any of these bats. In 2016, EBLV-2 was detected from a diseased Daubenton’s bat for the second time. These data provide proof that EBLV-2 is endemic in the Finnish Daubenton’s bat population. In phylogenetic analysis, the Finnish EBLV-2 strains formed a monophyletic group separate from other bat-type lyssaviruses with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujand virus (KHUV). EBLV-2 showed limited diversity compared to rabies virus (RABV) and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years. The Finnish EBLV-2 strains and a Swiss strain (1993) were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the Finnish strains (1985 and 2009) appear to have evolved from a common ancestor during the last 200 years. Rabies vaccine is used to protect against rabies virus before or after potential exposure. Since all the currently available vaccines are based on RABV, the vaccines are also used to protect against other lyssaviruses, and additionally against EBLV-2 infection based on cross-protection. We assessed the level of protection afforded by two commercial rabies vaccines, one for humans and one for animals, against intracerebral challenge in mice with EBLV-2 isolated from a bat in 2009. We compared this with protection using the same mouse model against challenge with RABV isolated from a Finnish raccoon dog in 1989. When challenged with RABV, all the vaccinated mice survived. When challenged with EBLV-2, 75% to 80% of the vaccinated mice survived. All vaccinated mice developed sufficient to high virus-neutralizing antibody (VNA) titers against RABV, ranging from 0.5 to 128 IU/ml. RABV-based vaccines also appear to offer good cross-protection against EBLV-2 circulating in the Finnish bat population. To investigate the factors influencing the response to rabies vaccination, we assessed the success of vaccination measured from the antibody response in dogs (n = 10 071) and cats (n = 722) sampled during 2009–2013. We examined the factors influencing the response to vaccination when animals failed to reach a rabies antibody titer of ≥0.5 IU/ml.
  • Heikkilä, Helka (Helsingin yliopisto, 2017)
    Osteoarthritis (OA) is considered to be the leading cause of chronic pain in dogs. Oral medication is the mainstay of pain treatment in canine OA, but it can provide insufficient pain relief and intolerable adverse events. Therefore, new treatment modalities are needed for dogs suffering from osteoarthritic pain. Intra-articularly injected botulinum toxin A (IA BoNT A) has shown efficacy in treatment of joint pain in arthritic human patients. The mechanism of the antinociceptive action of IA BoNT A has not been studied in vivo and its adverse effects have not been thoroughly investigated. Our aim was to study the antinociceptive efficacy of IA BoNT A in osteoarthritic dogs and to investigate the effect it has on their pain mediators. Also, we wanted to compare the pain mediators between osteoarthritic and non-osteoarthritic joints and reveal any associations with osteoarthritic pain. Our aim was also to investigate the adverse effects of IA BoNT A and to study whether the toxin spreads from the joint. We investigated the antinociceptive efficacy of IA BoNT A in a randomized, double-blinded, placebo-controlled, 12-week clinical trial in 35 client-owned osteoarthritic dogs after an injection of either IA BoNT A or placebo into the stifle, hip, or elbow joint. We detected significant improvement in the ground reaction forces and Helsinki Chronic Pain Index in the IA BoNT A group. There was also a significant difference in the improvement of the ground reaction forces between the IA BoNT A and placebo groups. We measured substance P (SP), prostaglandin E2 (PGE2), and tumour necrosis factor-alpha (TNF-α) from the synovial fluid (SF) and serum of the 35 osteoarthritic dogs and also analysed SF SP and PGE2 from 13 non-osteoarthritic control joints. IA BoNT A did not affect SF or serum SP or PGE2. TNF-α was not detectable in our samples. SF PGE2 correlated with osteoarthritic joint pain and was significantly higher in osteoarthritic than in non-osteoarthritic joints. We investigated the adverse effects of IA BoNT A in a randomized, placebo-controlled, blinded trial in six healthy laboratory beagle dogs after IA BoNT A and placebo injections into the stifle joints. No significant clinical, cytological, or histopathological adverse effects were detected 12 weeks after the injections, but changes in the electrophysiological recordings in two dogs suggested possible spread of the toxin. Our results indicate that IA BoNT A has efficacy in the treatment of osteoarthritic joint pain in dogs. The antinociceptive effect of IA BoNT A inside the joint seems not to be related to the inhibition in the release of SP or PGE2. SF PGE2 could be a marker of chronic OA and pain in dogs. IA BoNT A does not cause clinical, cytological, or histopathological adverse effects in healthy dogs, although the toxin may spread from the joint.
  • Nordgren, Heli (Helsingin yliopisto, 2017)
    In 2007 Finnish fur farmers detected new kind of signs in farmed mink, foxes and raccoon dogs (Finnraccoon). Mink had severe pyoderma in the head around eyes, mouth or ears or in the feet around the nailbeds. Finnraccoons developed painful abscesses in the paws between the toes. Foxes had severe keratoconjunctivitis that occasionally spread to the eyelids and to facial dermatitis. The disease caused severe symptoms and even increased mortality on the farms. The spread of the disease was typical to an infectious disease. Similar signs have been detected in fur animals in other pelt producing countries as well. Similar signs in mink have been reported for the first time in U.S.A at 1970´s and in Canada 1996. North-American farmers and later researchers linked the onset of the symptoms to the start of feeding mink with feed containing seal byproducts. The University of Helsinki (UH), the Finnish Fur Breeders´ Association (FFBA), and the Finnish Food Safety Authority Evira initiated a collaborative project in 2009 to describe the pathology of the disease, identify the causative organism(s), describe the occurrence of the disease in Finland and to find out possible sources and risk factors for the disease. This thesis includes these studies. Ninety nine fur animals underwent necropsies with complete microbiological examinations, including both diseased and healthy control animals. Due to the common lesions, in diseased animals of all fur animal species, the disease was named Fur animal epidemic necrotic pyoderma (FENP). The bacterium Arcanobacterium phocae was isolated from all diseased animals, but not from healthy controls. The finding of A. phocae is particularly interesting, because this bacterium causes skin inflammations and abscesses in marine mammals such as seals, and there had been found a temporally connection between the onset of the symptoms in mink and the use of seals as feed source in North America. However, the disease occurs also in the countries where seal meat is not used as a feed raw material. Thus, it is probable that there has been a species shift of the bacteria A. phocae from seals to fur animals via feed, and the disease is currently spreading by diseased/carrier animals. The results indicate that the presence of the bacteria A. phocae alone is not enough to cause the disease, as predisposing factors, such as skin or mucosal trauma is needed to evoke the disease. In addition, new Streptococcal species, closely related to Streptococcal species of seals, was detected in these investigations. Furthermore, the infection trial proved that A. phocae caused FENP-like symptoms when mink were experimentally infected. The mail survey study showed that FENP had spread to all areas where fur farming is practiced in Finland and signs of FENP was detected on 40% of responding farms. The results indicated that the disease was introduced to Finland by imported carrier animals and it spread further in the country via domestic purchases. The disease seems to spread on the farms by bird and other wild life contacts. The study revealed also other possible risk factors, such as the farm type; FENP was seen more on larger size farms and on mixed farms (farms farming more than one fur animal species). Also some other predisposing factors were relieved. The results provide a basis for developing preventive methods and treatment of FENP diseased animals to improve animal welfare.
  • 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)
    Våra hypoteser var att en utdragen förlossning leder till nedsatt fruktsamhet d.v.s. dräktighetsfrekvens, kvarbliven efterbörd och livmoderinflammation, fördröjd involution av livmodern och störningar i follikel tillväxten efter avvänjningen. En ytterligare hypotes var att suggor efter en utdragen förlossning svarar på galt stimulation med lägre oxytocin utsöndring under den påföljande brunsten. Suggornas förlossningstid (tiden mellan den första och sista grisens födelse) och negativ effekt på den påföljande dräktighetsfrekvensen (n = 148) efter avvänjningen undersöktes. Vi undersökte sambandet mellan grisningens längd och lossnandet av efterbörden (n=142), och postpartum livmoderns storlek och intrauterin vätska (n=99). Vi observerat lossnandet av efterbörden ända till 24 timmar efter sista grisens födelse och ultraljud tillämpades under den första postpartum veckan för att undersöka suggornas livmoder. Livmoderns storlek och mängden intrauterin vätska användes som indikationer på initial livmoder involution och puerperal metrit. Vi uppmätt förlossningstidens längd hos suggor (n = 30) och övervakade den efterföljande utvecklingen hos folliklarna med ultraljudsundersökning två gånger per dag mellan avvänjningens tredje dag och ovulationen. Den endogena oxytocin halten och utsöndringsmönstret fastställdes genom att analysera blodprover insamlade under brunsten i närvaro av en galt. Resultaten visar att suggor efter en utdragen förlossning (>300 min) hade 3.4 (P=0,027) lägre sannolikhet att bli dräktiga. Suggor som uppvisade totalt kvardröjd efterbörd (ingen efterbörd levererades; 3 %) och delvis kvardröjd efterbörd (ingen efterbörd levererad efter den sista grisens födelse; 3 %) hade en mer utdragen förlossning (1009±275 och 734± 136 min) än suggor vars efterbörd inte var kvarbliven (369± 202 min; P= 0,021 och P = 0,004). Bildade förlossningens längd ett kvadratiskt förhållande till antalet utstötta placentadelar (P = 0,001), placentans utstötnings tid (tiden mellan utstötningen av placentans första och sista del; P = 0,002) och tiden mellan utstötningen av den sista grisen och den sistadelen av placentan (P = 0,024). Oxytocin ökade andelen utstötta placentadelar (3.8±0.2 vs 2.9±0.3 P = 0,035), förkortade efterbördens utstötningstid (172±44 vs 328±26 min, P = 0,011) och tiden mellan födseln av den sista grisen och den sista delen av placentan (148± 48 vs 300± 24 min, P=0,025). Dessutom ökade risken för förstorad livmoder genom en utdragen förlossning (β±SE, Wald x2, Odds; 2.0±0.5, 13.1, 7.6;P=0,001), obstetrisk intervention (1.5±0.7, 4.4,4.3;P=0,036) och två eller flera dödfödda grisar (1.4±0.7,3.8,3.9;P=0,052). Behandling med oxytocin (-1.5±0.7,4.7,0.2,P = 0,040) minskade denna risk. Två eller flera dödfödda grisar(2.6 ±0.9,8.7, 13.7; P=0,003), obstetrisk intervention (1.8±0.8,5.0,6.0;P=0,025), utdragen förlossning(1.7±0.8, 4.3, 5.7;P=0,039)och försämrad utstötning av placentan (33±1.7,4.0,26,9;P=0,044) sammanhängde med intrauterin vätskeansamling. Efter avvänjningen var oxytocin koncentrationen högre hos suggor vars förlossning varit utdragen än hos suggor som haft en snabbare förlossning (28.0±7.7 vs.20.6±7.7, P<0.01). OT koncentrationerna korrelerade med folliklarnas diameter både då de uppmätts 5 dagar efter avvänjningen, och då de uppmätts då folliklarna nått sin maximala storlek efter avvänjningen (r=0.61,P=0.01 och r=0.57, P<0.01).
  • 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.