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Background: Finland repeatedly reports some of the highest incidences of tularaemia worldwide. To determine genetic diversity of the aetiologic agent of tularaemia, Francisella tularensis, a total of 76 samples from humans (n = 15) and animals (n = 61) were analysed. Methods: We used CanSNPs and canINDEL hydrolysis or TaqMan MGB probes for the analyses, either directly from the clinical tissue samples (n = 21) or from bacterial isolates (n = 55). Results: The genotypes of the strains were assigned to three previously described basal subspecies holarctica clades. The majority of strains (n = 67) were assigned to B.12, a clade reported to dominate in Scandinavia and Eastern Europe. A single strain was assigned to clade B.4, previously reported from North America, Europe and China. The remaining strains (n = 8) were members of clade B.6. Importantly, new diversity was discovered in clade B.6. We describe two newly designed TaqMan MGB probe assays for this new B.6 subclade B.70, and its previously identified sister clade B.11, a clade dominantly found in Western Europe. Conclusions: The high genetic diversity of F. tularensis subspecies holarctica present in Finland is consistent with previous findings in Sweden. The results suggest a northern and southern division of the B.6 subclade B.10, where B.11 predominates in Western and Central Europe and B.70 is found in Fennoscandia. Further research is required to define whether the vast diversity of genotypes found is related to different habitats or reservoir species, their different postglacial immigration routes to Fennoscandia, or dynamics of the reservoir species.

Several orthopoxviruses (OPV) and Borna disease virus (BDV) are enveloped, zoonotic viruses with a wide geographical distribution. OPV antibodies cross-react, and former smallpox vaccination has therefore protected human populations from another OPV infection, rodent-borne cowpox virus (CPXV). Cowpox in humans and cats usually manifests as a mild, self-limiting dermatitis and constitutional symptoms, but it can be severe and even life-threatening in the immunocompromised. Classical Borna disease is a progressive meningoencephalomyelitis in horses and sheep known in central Europe for centuries. Nowadays the virus or its close relative infects humans and also several other species in central Europe and elsewhere, but the existence of human Borna disease with its suspected neuropsychiatric symptoms is controversial. The epidemiology of BDV is largely unknown, and the present situation is even more intriguing following the recent detection of several-million-year-old, endogenized BDV genes in primate and various other vertebrate genomes. The aims of this study were to elucidate the importance of CPXV and BDV in Finland and in possible host species, and particularly to 1) establish relevant methods for the detection of CPXV and other OPVs as well as BDV in Finland, 2) determine whether CPXV and BDV exist in Finland, 3) discover how common OPV immunity is in different age groups in Finland, 4) characterize possible disease cases and clarify their epidemiological context, 5) establish the hosts and possible reservoir species of these viruses and their geographical distribution in wild rodents, and 6) elucidate the infection kinetics of BDV in the bank vole. An indirect immunofluorescence assay and avidity measurement were established for the detection, timing and verification of OPV or BDV antibodies in thousands of blood samples from humans, horses, ruminants, lynxes, gallinaceous birds, dogs, cats and rodents. The mostly vaccine-derived OPV seroprevalence was found to decrease gradually according to the year of birth of the sampled human subjects from 100% to 10% in those born after 1977. On the other hand, OPV antibodies indicating natural contact with CPXV or other OPVs were commonly found in domestic and wild animals: the horse, cow, lynx, dog, cat and, with a prevalence occasionally even as high as 92%, in wild rodents, including some previously undetected species and new regions. Antibodies to BDV were detected in humans, horses, a dog, cats, and for the first time in wild rodents, such as bank voles (Myodes glareolus). Because of the controversy within the human Borna disease field, extra verification methods were established for BDV antibody findings: recombinant nucleocapsid and phosphoproteins were produced in Escherichia coli and in a baculovirus system, and peptide arrays were additionally applied. With these verification assays, Finnish human, equine, feline and rodent BDV infections were confirmed. Taken together, wide host spectra were evident for both OPV and BDV infections based on the antibody findings, and OPV infections were found to be geographically broadly distributed. PCR amplification methods were utilised for hundreds of blood and tissue samples. The methods included conventional, nested and real-time PCRs with or without the reverse transcription step and detecting four or two genes of OPVs and BDV, respectively. OPV DNA could be amplified from two human patients and three bank voles, whereas no BDV RNA was detected in naturally infected individuals. Based on the phylogenetic analyses, the Finnish OPV sequences were closely related although not identical to a Russian CPXV isolate, and clearly different from other CPXV strains. Moreover, the Finnish sequences only equalled each other, but the short amplicons obtained from German rodents were identical to monkeypox virus, in addition to German CPXV variants. This reflects the close relationship of all OPVs. In summary, RNA of the Finnish BDV variant could not be detected with the available PCR methods, but OPV DNA infrequently could. The OPV species infecting the patients of this study was proven to be CPXV, which is most probably also responsible for the rodent infections. Multiple cell lines and some newborn rodents were utilised in the isolation of CPXV and BDV from patient and wildlife samples. CPXV could be isolated from a child with severe, generalised cowpox. BDV isolation attempts from rodents were unsuccessful in this study. However, in parallel studies, a transient BDV infection of cells inoculated with equine brain material was detected, and BDV antigens discovered in archival animal brains using established immunohistology. Thus, based on several independent methods, both CPXV and BDV (or a closely related agent) were shown to be present in Finland. Bank voles could be productively infected with BDV. This experimental infection did not result in notable pathological findings or symptoms, despite the intense spread of the virus in the central and peripheral nervous system. Infected voles commonly excreted the virus in urine and faeces, which emphasises their possible role as a BDV reservoir. Moreover, BDV RNA was regularly reverse transcribed into DNA in bank voles, which was detected by amplifying DNA by PCR without reverse transcription, and verified with nuclease treatments. This finding indicates that BDV genes could be endogenized during an acute infection. Although further transmission studies are needed, this experimental infection demonstrated that the bank vole can function as a potential BDV reservoir. In summary, multiple methods were established and applied in large panels to detect two zoonoses novel to Finland: cowpox virus and Borna disease virus. Moreover, new information was obtained on their geographical distribution, host spectrum, epidemiology and infection kinetics. Lehmärokkovirus (Cowpox virus, CPXV), joka kuuluu ortopoxviruksiin (OPV), ja Borna-virus (Borna disease virus, BDV) ovat zoonoottisia, laajalle levinneitä viruksia. Aiemmat isorokkorokotukset ovat ristireaktion avulla suojanneet ihmisväestöä jyrsijävälitteiseltä CPXV:ltä. CPXV:n aiheuttama lehmärokko ilmenee ihmisellä ja kissalla yleisoireisena, mutta hyvänlaatuisena ihotulehduksena, joskin se voi immuunipuutteisille yksilöille olla hengenvaarallinen. Klassinen Bornan tauti taas on aivojen, aivokalvojen ja selkäytimen tulehdus, joka on tunnettu hevosissa ja lampaissa Keski-Euroopassa vuosisatojen ajan. Nykyään BDV tai sen lähisukulainen infektoi muitakin lajeja, esimerkiksi ihmisiä, Keski-Euroopan ulkopuolellakin, mutta varsinaisen ihmisen Bornan taudin olemassaolo sen oletettuine hermostollis-mielenterveydellisine oireineen on kiistanalainen. BDV:n kaltaisten geenien on vastikään havaittu asettuneen pysyvästi muun muassa kädellisten perimään. Tämän tutkimuksen tavoitteina oli selventää CPXV:n ja BDV:n merkitystä Suomessa sekä tunnistaa niiden mahdollisia isäntälajeja. OPV- ja BDV-vasta-aineiden osoittamiseksi tuhansista ihmisten ja eläinten verinäytteistä pystytettiin immunofluoresenssimenetelmä vasta-aineiden sitoutumislujuuden määrityksineen. Lähinnä isorokkorokotteista aiheutuneiden OPV-vasta-aineiden esiintyvyyden havaittiin alenevan syntymävuoden mukaan 100 %:sta 10 %:in. Luonnolliseen CPXV-tartuntaan viittaavat OPV-vasta-aineet olivat tavallisia koti- ja villieläimissä: hevosessa, naudassa, ilveksessä, koirassa, kissassa ja villijyrsijöissä. Jyrsijöissä tartuntoja havaittiin myös aiemmin tuntemattomissa OPV-isäntälajeissa sekä uusilla esiintymisalueilla. BDV-vasta-aineitakin todettiin useassa lajissa. Bornan taudin kiistanalaisuuksien vuoksi sovellettiin immunofluoresenssin lisäksi yhdistelmä-DNA-teknologia- ja peptidipohjaisia varmistusmenetelmiä. Niillä vahvistettiin ihmisen, hevosen, kissan, ja ensimmäistä kertaa villijyrsijöiden (muun muassa metsämyyrän) BDV-infektiot Suomessa. Satojen veri- ja kudosnäytteiden tutkimisessa hyödynnettiin monipuolisia geenimonistusmenetelmiä, joilla osoitettiin OPV- ja BDV-geenejä. OPV-DNA:ta monistui ihmispotilaista ja metsämyyristä, mutta BDV-RNA:ta ei löydetty luonnollisesti infektoituneista yksilöistä. Suomalaiset OPV-DNA-löydökset olivat emäsjärjestykseltään samanlaisia keskenään ja läheistä sukua Venäjältä eristetyn CPXV:n kanssa eroten muista CPXV-kannoista. Saksalaisista myyristä monistuneet DNA-pätkät taas olivat samanlaisia saksalaisten CPXV-varianttien lisäksi apinarokkoviruksen kanssa, mikä kuvastaa OPV:ten lähisukulaisuutta. Potilaita infektoiva OPV varmistui CPXV:ksi, joka todennäköisimmin on aiheuttanut myös havaitut jyrsijäinfektiot. CPXV:tä ja BDV:tä yritettiin eristää potilaiden ja villieläinten näytteistä solulinjojen ja jyrsijöiden avulla. CPXV saatiin eristettyä lapsesta, joka kärsi yleistyneestä lehmärokosta. BDV-eristysyritykset villijyrsijöistä olivat tässä tutkimuksessa tuloksettomia. Kuitenkin rinnakkaisissa tutkimuksissa havaittiin sekä ohimenevä BDV-infektio hevospotilaan aivokudoksella tartutetuissa soluissa että BDV-proteiinia eläinpotilasarkiston aivonäytteistä immunovärjäysen avulla. Useaan riippumattomaan menetelmään pohjautuen voitiin siis osoittaa sekä CPXV:n että BDV:n (tai lähisukulaisen) esiintyvän Suomessa. Kokeellisessa infektiossa BDV levisi laajalle metsämyyrän hermostossa aiheuttamatta merkittävä patologisia muutoksia tai oireita. Moni myyrä eritti virusta virtsassaan ja/tai ulosteissaan. Tämän lisäksi BDV-RNA käänteiskopioitui usein DNA:ksi metsämyyrissä, mikä viittaa siihen, että BDV-geenit voisivat asettua genomiin akuutin infektion aikana. Vaikkakin tarttumistutkimukset ovat vielä tarpeen, koe kokonaisuudessaan osoitti, että metsämyyrät voivat toimia mahdollisena BDV-säilymönä.

ObjectivesThis study sought to evaluate the negative predictive value (NPV) of preoperative dobutamine stress echocardiography (DSE) in patients who fail to achieve target heart rate (HR) and assess the influence of resting wall motion abnormalities (WMAs) without demonstrable ischemia on perioperative events.BackgroundThe prognostic value of a negative-submaximal DSE study before noncardiac surgery is unknown.MethodsConsecutive patients (n = 429) who underwent surgery over a three-year period, preceded by DSE, were included. We compared perioperative event rates among those without inducible ischemia according to whether or not 85% age-adjusted maximum HR was achieved, and whether WMAs were present at rest.ResultsOf 397 negative DSEs, peak HR was