Posteritiivistelmät

ELINTARVIKE- JA YMPÄRISTÖHYGIENIA

1. Eläinperäiset elintarvikkeet ihmisten salmonellatartuntojen lähteinä

Sara Nevalainen, Antti Mikkelä, Markus Linjamäki, Suvi Joutsen ja Pirkko Tuominen
Ruokavirasto, Riskinarvioinnin yksikkö

Eläinperäisiä elintarvikkeita pidetään ihmisten salmonellatartuntojen pääasiallisena lähteenä Euroopassa. Suomessa kansallisen salmonellavalvontaohjelman (Finnish Salmonella Control Programme, FSCP) tulosten perusteella salmonellaesiintyvyys on pysynyt tavoitteessa, mikä on korkeintaan 1 % tuotantoeläimissä ja korkeintaan 0,5 % elintarvikkeissa. FSCP:n ohella Suomen salmonellatilanteeseen vaikuttaa EU:n Suomelle myöntämät erityistakuut, jotka edellyttävät, että tuoreena myyntiin tulevissa lihaerissä salmonella on tutkittu eikä sitä ole todettu ennen saapumista Suomeen. Suurin osa raportoiduista salmonellatartunnoista on peräisin ulkomaan matkailusta, ja kotimaassa saatujen tartuntojen määrä vaihtelee 200–400 tapauksen välillä vuosittain.

Ruokavirastossa arvioitiin kuluttajan riskiä saada salmonellatartunta FSCP:iin kuuluvista broilerin-, kalkkunan-, naudan- ja sianlihan tuotantoketjuista ja vastaavista tuontielintarvikkeista. Päämääränä oli tuottaa tietoa salmonellalähteiden merkityksestä salmonellaseurannan kehittämiseksi ja elintarviketurvallisuustason ylläpitämiseksi sekä kehittää riskinarviointimenetelmiä.

Riskinarviointi tehtiin aiemmin kehitetyillä tuotantoketjuja arvioivilla tilastollisilla malleilla, joiden perusaineistona käytettiin vuosien 2016–2020 FSCP- ja omavalvontatuloksia, EFSA:n, Luke:n ja Tullin tietokantatietoja sekä THL:n tartuntatautitietoja. Malleja yksinkertaistettiin siten, että kotimaiset tuotantoketjut voidaan arvioida myös ilman alkutuotannon osuutta, teurastamovaiheesta elintarvikkeeseen. Tämä mahdollistaa jatkossa kuluttajariskien entistä nopeammat ja tiheämmät arvioinnit. Altisteen määrä, eli salmonellalla kontaminoituneen elintarvikkeen vuosittainen kilomäärä, saatiin arvioimalla salmonellan esiintyvyyttä ja kulutusta kullakin elintarvikkeella. Lähdeattribuutiota varten jokaiselle elintarvikkeelle mallinnettiin vuosittainen serotyyppijakauma, jonka avulla voitiin verrata elintarvikkeissa esiintyvien salmonellojen serotyyppejä ihmisistä löydettyihin serotyyppeihin, ja pystyttiin arvioimaan eri elintarvikkeiden osuuksia ihmisten salmonellatartunnoista. Toisaalta aineiston perusteella voitiin myös arvioida eri serotyyppien ja elintarvikkeiden kykyä aiheuttaa ihmisillä infektioita. Koska Suomessa salmonelloja todetaan vuosittain vähän, lähdeattribuutiomallissa hyödynnettiin aineistoa 13 vuoden seurantajakson ajalta, vuosilta 2008–2020.

Arvion perusteella ihmisten salmonellatartunnoista suurin osa (21 %) on mahdollisesti peräisin kotimaisesta naudanlihasta, ja ulkomaista alkuperää oleva broilerinliha (16 %) aiheuttaa seuraavaksi eniten tartuntoja. Kotimainen siipikarjanliha aiheuttaa arvion perusteella vähiten tartuntoja (broileri 2 %, kalkkuna 4 %). Kaikkiaan kotimaisten elintarvikkeiden osuus ihmisten tartunnoista Suomessa on 38 %, tuontielintarvikkeiden osuus 46 % ja tuntemattomien lähteiden osuus on 16 %. Ihmisille tautitapauksia aiheuttaneista serotyypeistä 84 % oli todettu myös elintarvikkeista 2008–2020. Loppujen tautitapauksien katsottiin olevan peräisin muista lähteistä kuten kasviksista, pastöroimattomasta maidosta, kananmunatuotteista, suorista eläinkontakteista sekä ympäristöstä.

Arvioinnissa käytetty lähdeattribuutiomalli soveltuu nimenomaan Suomen kaltaisille maille, joissa salmonellaa esiintyy hyvin vähän. Tulosten perusteella kotimaisen siipikarjanlihan arvioitiin aiheuttavan hyvin vähän tartuntoja. Siipikarjasta onkin todettu koko 13 vuoden seuranta-ajalta vain muutamia yksittäisiä löydöksiä sekä alkutuotannosta että elintarvikkeista. Kotimainen naudanliha taas aiheuttaisi mallin mukaan eniten tartuntoja. Suomeen tuodun naudan-, sian- ja siipikarjanlihan arvioitiin kuitenkin aiheuttavan suunnilleen saman verran tartuntoja kuin kotimaisen lihan, vaikka tuontilihan kulutus on vain n. viidennes kotimaisen lihan kulutuksesta. Tämä tulos on samansuuntainen kuin aiemminkin on saatu. Tulosta tukee myös se, että lähes kaikki omavalvonnassa todetut elintarvikelöydökset ovat peräisin tuontilihasta. Vaikka kaikkia kontaminoituneita lihaeriä ei todeta ennen kuin ne päätyvät Suomen markkinoille, olisi riski saada salmonella ilman erityistakuita moninkertainen.

2. Elintarviketurvallisuuskulttuuri elintarvikepetosten ehkäisemisen näkökulmasta suomalaisissa elintarviketeollisuuden yrityksissä

Joenperä Jasmin1, Vainio Annukka2 ja Lundén Janne1
1Elintarvikehygienian ja ympäristöterveyden osasto, Eläinlääketieteellinen tiedekunta, Helsingin yliopisto
2Metsätieteiden osasto, Maatalous-metsätieteellinen tiedekunta, Helsingin yliopisto

Elintarviketurvallisuuskulttuurilla tarkoitetaan arvoja, uskomuksia ja normeja, jotka vaikuttavat henkilöstön elintarviketurvallisuutta koskeviin asenteisiin ja käytäntöihin.  Euroopan unionin elintarvikelainsäädäntö edellyttää, että elintarvikealan toimijoiden on luotava asianmukainen elintarviketurvallisuuskulttuuri, ylläpidettävä sitä sekä toimitettava siitä näyttöä. Tutkimme suomalaisten elintarviketeollisuusalan yritysten elintarviketurvallisuuskulttuuria elintarvikepetosten ehkäisemisen näkökulmasta. Tutkimuksen tavoitteena oli pyrkiä havaitsemaan yritysten kulttuurissa mahdollisia heikkouksia, jotka saattavat altistaa elintarvikepetoksille. Elintarvike-turvallisuuskulttuuria ei tiettävästi ole aiemmin tutkittu tästä näkökulmasta.

Toteutimme kyselytutkimuksen kymmenessä suomalaisessa elintarviketeollisuusalan yrityksessä. Kysely sisälsi Likert-asteikollisia väitteitä sekä mittarin, jolla tutkittiin vastaajien näkemyksiä erilaisista vilpillisistä toiminnoista. Kysely kohdennettiin henkilöstölle, joka käsittelee työssään elintarvikkeita tai on muuten tekemisissä niiden turvallisuuden tai laadun kanssa. Vastauksia saatiin 691 kaikkiaan 21 toimipaikasta. Vastaajista 48,9 % oli johtajia, esihenkilöitä, asiantuntijoita tai toimihenkilöitä, 47,2 % työntekijöitä ja 3,9 % muita tai tarkemmin määrittelemättömiä henkilöstön jäseniä. Vastaajista 54,9 %:lla oli jokin elintarvikealaan liittyvä tutkinto (N=681). Aineisto analysointiin SPSS-ohjelmalla. Vastaajaryhmien välisiä eroja tarkasteltiin tilastollisesti Khiin neliö-, Mann-Whitney- sekä Kruskal-Wallis-testeillä.

Vastaajista 21,7 % oli täysin tai jokseenkin samaa mieltä siitä, että yrityksessä voisi tapahtua elintarvikepetos (N=645). Pienempi osa, 13,4 % vastaajista arvioi, että yrityksessä voitaisiin pyrkiä tekemään voittoa epärehellisin keinoin (N=656). Työntekijäryhmään kuuluvat vastaajat olivat näistä väitteistä useammin täysin tai jokseenkin samaa mieltä kuin johtaja- ja asiantuntijaryhmään kuuluvat vastaajat (p<0,001). Vastaajista vain 23,1 % (N=592) oli täysin samaa mieltä siitä, että tietää, minkälaisia elintarvikepetoksia heidän edustamallaan alalla voisi tapahtua.

Lähes kaikki vastaajat kokivat, että yrityksessä tuotettujen elintarvikkeiden laatu ja turvallisuus ovat yrityksessä tärkeä arvo, ja että tämä myös näkyy yrityksen toiminnassa (97,2 % täysin tai jokseenkin samaa mieltä, N=687). Suurin osa vastaajista koki, että laadukkaiden ja turvallisten elintarvikkeiden tuottaminen on kaikkien yrityksen työntekijöiden yhteinen asia (98,7 % täysin tai jokseenkin samaa mieltä, N=688) ja että he voivat omalla toiminnallaan vaikuttaa siihen, että tuotetut elintarvikkeet ovat turvallisia ja laadukkaita (95,2 % täysin tai jokseenkin samaa mieltä, N=676). Vastaajat uskaltaisivat pääsääntöisesti ilmoittaa esihenkilölle tai yrityksen johdolle, jos havaitsisivat toiminnassa tai työnteossa jotakin huolestuttavaa (97,7 % vastaajista oli väitteestä täysin tai jokseenkin samaa mieltä, N=689). Kuitenkin 17 % vastaajista (N=666) oli ainakin jokseenkin samaa mieltä siitä, että huolenaiheesta ilmoittaminen voisi vaikuttaa heihin työntekijöinä negatiivisesti. Työntekijäryhmään kuuluvat vastaajat arvioivat johtaja- ja asiantuntijaryhmään kuuluvia useammin, että ilmoittaminen voisi olla heille haitaksi (p<0,001).

Tutkimuksessa havaittiin sekä positiivisia seikkoja että kehityskohteita yrityksen sisäisen integriteetin parantamiseksi. Vastaajat vaikuttivat esimerkiksi sitoutuneen turvallisten ja laadukkaiden elintarvikkeiden tuotantoon työssään, mutta toisaalta osa arvioi, että yrityksen toimintaan liittyvästä huolenaiheesta ilmoittamisesta saattaisi koitua negatiivisia seuraamuksia. Koska koko henkilöstön tulee pystyä ilmoittamaan mahdollisista huolenaiheista ilman pelkoa haitallisista seuraamuksista, yritysten tulisi hälventää näitä pelkoja. Yritysten henkilöstön olisi myös hyvä tietää, minkälaisia elintarvikepetoksia voi tapahtua, jotta he osaisivat reagoida, mikäli havaitsisivat toiminnassa jotakin epäilyttävää. Koska kysely koostui monivalintakysymyksistä, kyselystä ei saada tarkempaa tietoa esimerkiksi vastaajien perusteluista. Henkilöstöryhmien välisiä eroja ja vastaajien perusteluja esimerkiksi sille, miksi osa piti elintarvikepetosta mahdollisena yrityksessä, voisikin tutkia jatkossa esimerkiksi haastatteluin.

3. Kaupunkirottien Campylobacter jejuni -kantojen karakterisointi kokogenomisekvensoinnilla

Kivistö R1, Hytönen T1, Aivelo T2
1Elintarvikehygienian ja ympäristöterveyden osasto, eläinlääketieteellinen tiedekunta, Helsingin yliopisto, Helsinki, Suomi
2Organismi- ja evoluutiobiologian tutkimusohjelma, Bio- ja ympäristötieteellinen tiedekunta, Helsingin yliopisto, Helsinki, Suomi

Kampylobakteerit ovat maailmanlaajuisesti yleisin bakteeriperäisen suolistotulehduksen aiheuttaja ja niiden merkitys taudinaiheuttajina on huomattava myös Suomessa. Ihmisten tautitapauksissa tyypillisimmin esiintyviä lajeja ovat termotolerantit Campylobacter jejuni ja Campylobacter coli. Kampylobakteeritartunta ilmenee yleensä kuumeisena ripulitautina, joka voi kestää jopa viikon. Kampylobakteerien tartuntareitti on feko-oraalinen ja tartunta saadaan tyypillisimmin saastuneesta elintarvikkeesta tai juomavedestä. Kampylobakteerit ovat zoonoottisia eli ne voivat tarttua eläimistä ihmisiin ja päinvastoin. Monet eläinlajit kantavat kampylobakteereita oireetta ja täten bakteeri voi päätyä elintarvikeketjuun tartunnan saaneen eläimen välityksellä. Kampylobakteerien esiintymistä onkin tutkittu laajasti tuotantoeläimillä ja lisäksi joillakin luonnonvaraisilla eläimillä, mutta rottien osalta tutkimustieto on edelleen puutteellista. Tämän tutkimuksen tarkoituksena oli tarkastella kampylobakteerien esiintyvyyttä suomalaisilla kaupunkirotilla ja selvittää, minkälaisia kampylobakteerikantoja rotilla esiintyy. Tutkimusaineisto on kerätty Helsingin kaupunkirottaprojektissa, joka on vuodesta 2018 lähtien käynnissä ollut, Tuomas Aivelon johtama monitieteinen tutkimushanke. Aineisto koostuu 200 näytteestä, jotka on kerätty Uudenmaan alueelta pyydetyistä rotista (Rattus norvegicus). Näytteitä on otettu sekä kuolleista että elävistä eläimistä. Elävinä pyydystettyjen rottien näytteet ovat peräsuolesta otettuja pyyhkäisynäytteitä ja kuolleina pyydystettyjen rottien näytteet ovat paksu- tai peräsuolesta otettuja suolistonäytteitä. Näytteet on homogenoitu pumpulipuikolla ja viljelty suoraan kampylobakteerille selektiivisille agar-maljoille (mCCDA). Kampylobakteerille positiivisista näytteistä on eristetty bakteerien DNA:ta käyttäen InVitrogen PureLink Genomic DNA Mini Kit-sarjaa (Thermo Fisher Scientific, USA) valmistajan ohjeiden mukaan. Tunnistus on varmistettu qPCR:llä, käyttämällä Campylobacter spp. spesifisiä alukkeita. Genominen DNA on eristetty puhdasviljelmistä ja sekvensoitu Illuminalla. Tutkittujen isolaattien kokogenomisekvenssejä on analysoitu käyttäen vapaasti saatavilla olevia bioinformatiikan työkaluja: Microbial Genomes Atlas Online (MiGa), Pathosystems Resource Integration Center (PATRIC), PubMLST Campylobacter jejuni/coli, ResFinder ja Comprehensive Antibiotic Resistance Database (CARD). Analyyseihin lukeutuu lajintunnistus, MLST-tyypitys, cgMLST-tyypitys ja mahdollisten mikrobilääkeresistenssien selvitys. Tutkituista näytteistä eristettiin 22 kampylobakteeri-isolaattia, joista 18 tunnistettiin Campylobacter jejuni-lajin edustajiksi. Muita neljää isolaattia ei kyetty tunnistamaan luotettavasti, ja ne todennäköisesti edustavat uusia aiemmin kuvaamattomia Campylobacterales-lahkon lajeja. MLST-tyypityksessä havaittiin, että Campylobacter jejuni-isolaattien joukossa esiintyi sekvenssityyppejä ST-45 (CC ST-45), ST-3904 (CC ST-45), ST-11 (CC ST-45) ja ST-9477. Lisäksi löydettiin uusi sekvenssityyppi, ST-11770.  Näistä erityisesti ST-45 on aikaisemmin ollut yleisin siipikarjalla Suomessa tavattu sekvenssityyppi ja yleinen Suomessa myös ihmisten infektioissa. Mikrobilääkeresistenssiä koskevissa analyyseissa ei havaittu hankittua resistenssiä yhdelläkään isolaatilla. Tulokset osoittavat, että tutkituilla rotilla esiintyy erilaisia kampylobakteereita, joista osalla on potentiaalia tarttua ihmisiin ja tuotantoeläimiin. Rottien merkitystä kampylobakteerin leviämisessä tulisi kuitenkin tutkia vielä lisää ennen kuin voidaan tehdä johtopäätöksiä rottien roolista kampylobakteerin levittäjinä.

HEVOSET

4. The effects of vatinoxan on detomidine-induced hyperglycemia in horses after oral sugar administration

I.A.M Hallman1*, M.R. Raekallio1, M.J. Weckman1, N.P. Karikoski1
1Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Koetilankuja 1, Helsinki, Finland

The aim of the study was to investigate the effects of an alpha2-adrenoceptor agonist (detomidine HCl) and antagonist (vatinoxan HCl) on blood glucose (BG) concentrations in horses after oral sugar administration.

In total, sixteen Finnhorses were included in the study and assigned by means of a blinded cross-over design with a minimum of 7-days washout period to one of three following treatments: saline (SAL), detomidine HCl (0.02 mg/kg) (DET) and the combination of detomidine and vatinoxan HCl (0.2 mg/kg) (DET+VAT). The horses were fasted overnight, water was offered freely throughout the study. At the start of the study the fasted horses were administered corn syrup 0.45 ml/kg orally and the treatment (SAL, DET or DET+VAT) was given immediately after as a single IV bolus via jugular catheter. Baseline venous blood samples were taken for BG before the oral sugar test and then again at 30, 60, 90, 120, 180, 240, 300 and 360 minutes after treatment. Blood glucose was analyzed immediately after collection with a glucometer (Alpha-TRAK II).

Peak mean BG, 12.6 ± 2.2 mmol/L, occurred 60 minutes after DET and was significantly higher than the mean BG after SAL (7.8 ± 1.0 mmol/L, p < 0.001) and DET+VAT (7.1 ± 0.7 mmol/L, p < 0.001) detected at the same time point. There was no difference in BG at 60 minutes between SAL and DET+VAT (p = 0.07).

Vatinoxan alleviated the detomidine-induced increase in BG after oral sugar administration. The combination of detomidine and vatinoxan may be safer than detomidine alone for sedation of horses, especially in individuals with disturbed energy metabolism and insulin dysregulation.

The authors would like to thank Finnish Foundation of Veterinary Research and Erkki Rajakoski foundation (Hippos ry) for funding this study and Vetcare Ltd. for providing detomidine and vatinoxan for the experiments.

This study was approved by the National Animal Experimentation Board of Finland.

5. Hevosalan sidosryhmien asenteista ravihevosten suuvaurioihin - pilottitutkimus

Kati Tuomola1*, Nina Mäki-Kihniä2, Anna Valros1, Anna Mykkänen3 and Minna Kujala-Wirth4
1 Eläinten hyvinvoinnin tutkimuskeskus, Kliinisen tuotantoeläinlääketieteen osasto, Helsingin yliopisto
2 Itsenäinen tutkija, Pori
3 Kliinisen hevos- ja pieneläinlääketieteen osasto, Eläinlääketieteellinen tiedekunta, Helsingin yliopisto
4 Kliinisen tuotantoeläinlääketieteen osasto, Eläinlääketieteellinen tiedekunta, Helsingin yliopisto

Ravikilpailujen ohjeistuksen mukaan kilpailueläinlääkäri tai kilpailueläinlääkärin avustaja tarkastavat hevosen suun, jos suusta havaitaan verenvuotoa. Eläinlääkäri voi tarvittaessa poistaa hevosen lähdöstä tai vaatia terveystarkastuksen ennen seuraavaa kilpailuun ilmoittamista.

Tarkoitus oli selvittää hevosalan sidosryhmien (N = 93) asenne-eroja liittyen ravihevosten kuolaimiin liittyviin suuvaurioihin. Vuonna 2018 kyselytutkimukseen osallistui 14 eläinlääkäriä ja 14 kilpailueläinlääkärin avustajaa (VETS), 32 ravihevosten valmentajaa (VALMENTAJAT) ja 33 muuta sidosryhmien edustajaa esimerkiksi hoitajia ja omistajia (MUUT).  Vastaajille esitettiin ennen koulutustilaisuutta 12 autenttista valokuvaa kohtalaisista tai vakavista suuhaavoista (luokiteltu vertaisarvioidun luokittelujärjestelmän mukaisesti) ja kuvitteellinen tilanne kilpailuissa. Haavat sijaitsivat hammaslomassa, suupielen sisä- tai ulko-osassa tai posken etuosassa. Vastaajilta kysyttiin sallisivatko he hevosen kilpailla vai eivät (tilanteet S1–7 ennen kilpailuja) tai vaatisivatko he terveystarkastuksen vai eivät (tilanteet S8–12 kilpailun jälkeen). Sidosryhmien yhteys vastausvaihtoehtoihin tutkittiin Pearsonin khiin neliö – testillä. P-arvoa alle 0,05 pidettiin tilastollisesti merkitsevänä.

Tilastollisesti merkitsevä ero havaittiin 6/12 tilanteessa.  Näissä tilanteissa MUUT poistivat hevosen lähdöstä tai vaativat terveystodistuksen useammin kuin muut ryhmät.  Vastaajien osuus, jotka halusivat poistaa hevosen lähdöstä (S1, S2, S4, S6, S7) tai vaatia terveystarkastuksen (S9); S1 (vakava vaurio) VETS 43 %, VALMENTAJAT 22 %, MUUT 52 %, (N = 93, χ2 6.31, df 2, p = 0.04), S2 (vakava) VETS 14 %, VALMENTAJAT 13 %, MUUT 36 % (N = 93, χ2 6.72, df 2, p = 0.04), S4 (kohtalainen) VETS 11 %, VALMENTAJAT 13 %, MUUT 33 % (N = 93, χ2 6.43, df 2, p = 0.04), S6 (vakava) VETS 14 %, VALMENTAJAT 53 %, MUUT 64 % (N = 93, χ2 16.15, df 2, p < 0.001), S7 (vakava) VETS 25 %, VALMENTAJAT 45 %, MUUT 63 % (N = 91, χ2 8.48, df 2, p = 0.01), S9 (kohtalainen) VETS 4 %, VALMENTAJAT 6 %, MUUT 38 % (N = 92, χ2 16.23, df 2, p < 0.001).

Kyselytutkimus antoi viitteitä, että ravureiden suuvaurioihin suhtautumisessa oli eroja sidosryhmien välillä, mutta myös sidosryhmien sisällä. Eläinlääkärit, avustajat ja valmentajat olivat haluttomampia poistamaan hevosia lähdöstä tai vaatimaan hevoselle terveystarkastusta kuin muu sidosryhmä (esim. hoitajat tai omistajat). Erot eettisissä asenteissa voivat aiheuttaa ristiriitoja. Yhteiskunnan luottamus lajin valvontajärjestelmään voi vaarantua, jos hevosia, joilla on vakavia suun vaurioita, ei poisteta kilpailusta.

6. Genome-wide association study suggests genetic candidate loci of insulin dysregulation in Finnhorses

Weckman Minna1, Karikoski Ninja1, Raekallio Marja1, Box Justin1, Kvist Laura2
1Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Finland
2Department of Ecology and Genetics, University of Oulu, Oulu, Finland

Equine metabolic syndrome (EMS) is characterized by obesity, insulin dysregulation (ID), and laminitis and it affects horses and ponies worldwide. Laminitis is a painful foot disease in horses that can lead to euthanasia due to severe lameness. The pathophysiology and epidemiology of EMS/ID is not completely understood, but it is known that both environmental and genetic factors contribute to the phenotype. The environmental factors such as feeding and exercise can be controlled; however, the genetic factors are less modifiable. The objective of this research was to identify potential novel genetic loci influencing ID indicative of EMS in Finnhorses.

A single breed (Finnhorse) case-control genome-wide association study (GWAS) of ID was carried out with DNA samples (hair roots). A total of 78 Finnhorses were selected for the study based on their oral sugar test (OST) results. The OST was performed after overnight fasting by administering 0.45 mL/kg body weight of corn syrup. Blood insulin concentrations were measured before and after the syrup. Horses were categorized as having ID based on either basal or postprandial hyperinsulinemia (n=38). The control horses (n=40) had normal insulin regulation. Genotyping was performed from DNA samples for 65 157 single nucleotide polymorphisms (SNPs) with the Illumina Equine SNP70 BeadChip. Data were further analysed for association and FST-outliers with genomic tools. Significant SNP locations were utilized to screen for candidate genes in GenBank genome browser and by performing gene enrichment and ontology analysis in DAVID v.6.8. The functions of the genes were also studied using the GeneCards software.

The strongest associations were found in PLINK in SNPs in chromosome’s 3, 10, 17 and 18. SNP BIEC2_383954 (p=8.06×10−6) and BIEC2_387659 (p=9.29×10−6) were both located in chromosome 17. In chromosomes 3, 10 and 18 associations with SNPs BIEC2_782916 (p=8.55×10−5), BIEC2_114160 (p=1.71×10−5) and BIEC2_430810 (p=8.22×10−5) were found. Hierarchical and Bayesian FST-outlier tests also identified these SNPs.

Genes associated to carbohydrate metabolism; Arginine And Glutamate Rich 1 (ARGLU1), Ephrin-B2 (EFNB2), Cullin (CUL4A) and Lysosomal Associated Membrane Protein (LAMP1) were detected close to two SNPs with the smallest p-values in the equine chromosome 17. Both ARGLU1 and EFNB2 have been found to be related to gluconeogenesis and glucose homeostasis. CUL4A is part of the protein complex which interacts with liver receptor homologue-1 (LHR-1). LHR-1-receptor functions include hepatic glucose and lipid metabolism. Additionally, LAMP1 has been shown to play a role in human type I diabetes. Further studies including sequencing of the candidate genes are warranted to find out a more precise significance of these genes in insulin regulation in Finnhorses. Identification of possible mutations in candidate loci associated with the ID may clarify the pathophysiology of EMS in the future.

7. Whole genome sequencing in extended-spectrum beta-lactamase (ESBL) producing Enterobacterales outbreak detection in an equine hospital

Katarina Eskola1, Annamari Heikinheimo2,3, Anna Mykkänen1, Thomas Grönthal1,4
1Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Finland
2Department of Food Hygiene and Environmental Health, Faculty of Veterinary Medicine, University of Helsinki, Finland
3Food and Feed Microbiology Unit, Finnish Food Authority, Finland
4Animal Health Diagnostics Unit, Finnish Food Authority, Finland

Extended-spectrum beta-lactamase producing Enterobacterales (ESBL-E) are an important cause of infections in humans and animals. In addition to beta-lactams, these bacteria are often resistant to many other antimicrobial classes. Recently, the number of reports of ESBL-E infections in equine patients has increased, which is worrisome, as the number of antimicrobial options registered for horses is limited. Equine hospitals can serve as a hub for potential spread of ESBL-E, but tools like next-generation sequencing are rarely used in veterinary epidemiological surveillance. These tools allow for rapid investigation of genetic traits in a single bacterial isolate, such as antimicrobial resistance genes and plasmids, but they can also be used for detailed comparison of multiple isolates. We present a case where whole genome sequencing (WGS) analysis led to the discovery of a large outbreak in an equine hospital.

Three multidrug-resistant Klebsiella pneumoniae isolates with identical phenotypic resistance patterns were initially detected in rectal swab screening specimens of three horses re-admitted to the Equine Teaching Hospital of the University of Helsinki during an ESBL-E prevalence study (October 2020 – March 2021). Selective bacterial culturing, MALDI-TOF identification and disk diffusion susceptibility testing were used to detect ESBL-E isolates. Patient record investigation had revealed recent simultaneous hospital stay of the three horses with ESBL K. pneumoniae. The DNA of the isolates was then extracted and sent to Novogene (UK) for Illumina® WGS generating paired-end 150 bp reads. The raw read data was assembled using Ridom SeqSphere+ software (version 8.3, Ridom Gmbh, Germany) with SKESA assembler, and Center for Genomic Epidemiology tools (ResFinder 4.1, MLST 2.0, PlasmidFinder 2.1) were used to interpret the quality checked data. An epidemiological investigation was initiated, and screening of patients at discharge was performed during February – May 2021 and two weeks in September 2021.

WGS of the three K. pneumoniae isolates, which represented the same sequence type (new type, closest to ST307), suggested clonal spread in the hospital due to near-identical plasmid replicon and genotypic resistance profiles. All of the isolates shared beta-lactamase genes CTX-M-15, TEM-206 and SHV-28. Genes encoding resistance against folate pathway antagonists, amphenicols, quinolones, aminoglycosides and tetracyclines were also detected. The prevalence of ESBL-E in equine patients on admission and at discharge was 3% (5/161) and 20% (49/230), respectively. One clinical ESBL-E infection was diagnosed in summer 2021. The specimens obtained at discharge revealed several ESBL-E species, such as Escherichia coli and Enterobacter cloacae complex. Further genomic investigation of the isolates is in progress.

WGS is a powerful tool to investigate multidrug-resistant bacteria in equine hospitals, and may reveal outbreaks at their early stages. The discordant prevalence of ESBL-E between admitted and discharged horses highlight the potential of equine hospitals as a hub for nosocomial transmission. Furthermore, the heterogenous population of temporally associated ESBL-E isolations during the epidemiological investigation emphasize the possibility that even mobile genetic elements, such as plasmids, can be shared between bacterial species in the same environment. Further research is needed to identify mechanisms and routes through which ESBL-E may spread and transmit in the equine hospitals.

PIENELÄIMET

8. Molecular detection of pathogens from ticks collected from dogs and cats at veterinary clinics in Finland

Fathiah Zakham1,2,3, Essi M Korhonen1,2, Petteri T Puonti2, Robert S Castrén1, Ruut Uusitalo1,4, Olli Vapalahti1,2,5, Tarja Sironen1,2, Paula M Kinnunen6
1Department of Virology, Faculty of Medicine, University of Helsinki, Helsinki, Finland
2Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland
3Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.
4Department of Geosciences and Geography, University of Helsinki, Helsinki, Finland.
5HUSLAB, Hospital district of Helsinki and Uusimaa, Helsinki, Finland
6Companion Animal Business Unit, Nordic Cluster, MSD Animal Health, Espoo, Finland

Ticks carry microbes, which can be pathogenic for humans and animals. To assess this One Health problem, 342 ticks were collected from pet dogs and cats at ten veterinary clinics in Finland as part of the European project “Protect Our Future Too”. The tick species were analyzed and the ticks were screened for tick-borne pathogens with molecular methods.

Most ticks were identified as Ixodes ricinus. In the pathogen screening, Rickettsia spp. were most prevalent, followed in the order of prevalence by Borrelia burgdorferi sensu lato, Anaplasma phagocytophila, B. miyamotoi, Babesia spp., “Candidatus Neoehrlichia mikurensis”, and tick-borne encephalitis virus (TBEV). Ehrlichia canis was not detected. The coinfection rate reached  8% with a predominance of B. burgdorferi sensu lato and Rickettsia spp. In total, 52% % of the ticks carried at least one pathogen.

These results emphasize the importance of awareness of ticks and tick-borne diseases and protection from them, and show that veterinary surveillance can help the early detection of tick-borne pathogens. Multiple pathogens and coinfections should also be considered both in symptomatic humans and animals after tick bites.

9. Comparison of anxiolytic effect of pregabalin versus non-medicinal products during transportation in Finnish client-owned cats

Terttu Lamminen1, John Aspegrén1, Mira Korpivaara1
1 R&D, Orion Corporation Orion Pharma, Espoo, Finland

Anxiolytic effect of pregabalin 5 mg/kg dose in cats was evaluated in two clinical field studies in Finland. The results were compared to those of previously used non-medicinal products to reduce anxiety and fear associated with travel in the same cats.

Both clinical studies, one with a crossover design (n=11) and the other with two parallel groups (n=23), were randomised, blinded, placebo-controlled and included client-owned cats with history of anxiety and fear associated with travelling. Both studies had authority approval. A single dose of pregabalin or placebo was given as oral solution 90 minutes before starting transportation. Anxiolytic effect was evaluated by owners using 5-point rating scale (excellent, good, fair, poor, very poor) for pregabalin and with 4-point rating scale (improvement, mild improvement, no effect, worsening) for previously used non-medicinals. Dichotomised results (first two categories versus other) were evaluated descriptively.

Of 34 cats, 23 received pregabalin and 22 placebo. In total 17 cats (50%) had been given non-medicinals previously, of which 13 cats received pregabalin and 10 placebo. The most commonly used non-medicinals were pheromones (71%), valerian (24%), tryptophan (18%) and alpha-casozepine (12%). Anxiolytic effect was shown in 54% (7/13) of cats given pregabalin, 20% (2/10) given placebo and 0% (0/17) with non-medicinals. Clinical safety of pregabalin was good, with mild transient incoordination and tiredness as most commonly reported adverse events.

In conclusion, pregabalin oral solution dose 5 mg/kg was found to be clearly more effective than non-medicinals for alleviation of acute anxiety and fear associated with transportation in cats.

10. Concurrent use of tasipimidine and clomipramine - An Interaction study

J. Lindstedt1, M. Kirjavainen1, M. Korpivaara1
1Research and Development, Orion Pharma, Espoo, Finland

The objective of the study was to evaluate pharmacokinetics of tasipimidine alone and in combination with clomipramine, and to study clinical effects of the combination on functional alertness and cardiovascular parameters (blood pressure (BP), heart rate (HR) and ECG) in six dogs equipped with telemetry devices.

The study was performed in 3 Phases. Pharmacokinetics and functional alertness were studied after a single dose (Phase 1) and after the four days repeated twice daily dosing of tasipimidine 30 µg/kg and clomipramine at approximately 1 mg/kg, alone and in combination (Phase 2). In Phase 3, the combination was studied with a reduced dose of tasipimidine (20 µg/kg) as single and repeated dosing.

The dogs were dosed orally by a treatment administrator twice  daily (Phases 2 and 3) and the clinical assessments were made by a person blinded to the treatment. The wash-out period between the phases was 7 to 10 days.

Dogs’ alertness was slightly reduced by combination of tasipimidine at the dose of 30 µg/kg with clomipramine. Decreasing tasipimidine dose to 20 µg/kg in the combination caused less reduction in alertness.

Tasipimidine alone and in combination with clomipramine was well tolerated and safe in respect to cardiovascular effects (BP, HR and ECG). The exposure levels of tasipimidine increased dose-proportionally and were similar alone and in combination with clomipramine. Slightly slower absorption of clomipramine was observed when dosed concomitantly with tasipimidine.

The effect on functional alertness supports the use of lower dose of tasipimidine (20 µg/kg) when combined with clomipramine 1.0 mg/kg.

11. A novel detection method for canine sleep-disordered breathing

Iida Niinikoski a, Sari-Leena Himanen b, Mirja Tenhunen c, d, Liisa Lilja-Maula a, Minna M. Rajamäki a
a Department of Equine and Small Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, PO Box 57, 00140 Helsinki, Finland,
b Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland,
c Department of Clinical Neurophysiology, Medical Imaging Centre, Pirkanmaa Hospital District, Tampere, Finland,
d Department of Medical Physics, Tampere University Hospital, Medical Imaging Centre, Pirkanmaa Hospital District, Tampere, Finland

Sleep-disordered breathing (SDB), defined as any difficulty breathing during sleep, has previously been described in brachycephalic dogs (BDs). At-home devices for detection of SDB have been developed for humans, but in dogs the evaluation of SDB events has required laboratory assessment. A commonly used diagnostic value for SDB is the Apnea-Hypopnea Index/Respiratory-Event Index (AHI/REI), which represents the number of apnea and hypopnea events per hour.

The objective of this observational cross-sectional study was to evaluate the usability of a portable sleep apnea collar (Nukute Ltd, Finland) for the detection of SDB in dogs. We hypothesized that the collar is well-tolerated, and that brachycephaly predisposes to SDB. A total of 24 pet dogs, 12 BDs (nine French Bulldogs, one Cavalier King Charles Spaniel, one Bullmastiff, one English Bulldog) and 12 normocephalic control dogs, were included. All dogs wore the sleep apnea collar for one night at home. AHI/REI and snore percentage were recorded.

Brachycephalic dogs have a significantly higher AHI/REI value (Hodges-Lehmann estimator for the median difference = 3.5, 95 % CI 2.2 – 6.8; P < 0.0001) and snore percentage (Hodges-Lehmann estimator = 34.15, 95 % CI 13.6 – 60.8; P < 0.0001) compared with controls. A strong positive correlation between AHI/REI and snore percentage was detected (rs= 0.79, P < 0.0001). The sleep apnea collar was easy to use and caused only minor discomfort.

Brachycephaly is associated with SDB. The sleep apnea collar is a feasible way of determining SDB in dogs.

12. High serum creatinine concentration is associated with metabolic perturbations in dogs

Claudia Ottka1,2,3, Katariina Vapalahti1,2, Ann-Marie Määttä4, Nanna Huuskonen4, Sinikka Sarpanen5, Liisa Jalkanen6, Hannes Lohi1,2,3.
1 PetBiomics Ltd., Helsinki, Finland
2 Department of Veterinary Biosciences and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland
3 Folkhälsan Research Center, Helsinki, Finland
4 Movet Ltd., Kuopio, Finland
5 Kuopion Eläinlääkärikeskus Ltd., Kuopio, Finland
6 Pieneläinvastaanotto Punaturkki Ltd., Kuopio, Finland

Renal diseases, such as chronic kidney disease (CKD) are common in dogs. While the kidneys have multiple important metabolic functions, the occurrence of metabolic disturbances in canine renal diseases has not been studied extensively. The objective of this study is to identify metabolic changes in blood samples exhibiting elevated blood creatinine, indicating reduced renal filtration.

Samples consisted of clinical blood samples analysed by a proton nuclear magnetic resonance (1H NMR)-based metabolomics platform. The case group included 23 samples with creatinine > 125 μmol/l, and the control group 873 samples with creatinine within the reference interval. Biomarker association with elevated creatinine concentration was evaluated utilizing three statistical approaches: Wilcoxon rank-sum test and logistic regression analysis (FDR-corrected p-values), and classification using random forest. Medians of the biomarkers were compared to reference intervals. A heatmap and box plots were used to represent the differences.

All 3 statistical approaches identified similar analytes associated with increased serum creatinine concentrations. The levels of citrate, tyrosine, branched-chain amino acids, valine, leucine, albumin, linoleic acid percentage and the ratio of phenylalanine to tyrosine differed significantly using all statistical approaches, acetate differed using the Wilcoxon test and random forest, docosapentaenoic acid percentage only using logistic regression (p < .05), and alanine only using random forest.

This study identified multiple metabolic changes associated with elevated blood creatinine, including prospective diagnostic markers and therapeutic targets. Further research is needed to verify the association of these changes to the canine patient’s clinical state. The 1H NMR metabolomics platform is a promising tool for improving diagnostics and management of canine renal diseases.

13. A novel nuclear magnetic resonance spectroscopy-based metabolomics platform for dogs: validation and sample handling

Claudia Ottka1,2,3, Katariina Vapalahti1,2,3, Jenni Puurunen1,2,3, Laura Vahtera2,3, Hannes Lohi1,2,3.
1 PetBiomics Ltd., Helsinki, Finland
2 Department of Veterinary Biosciences and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland
3 Folkhälsan Research Center, Helsinki, Finland

Metabolomics has been proven to be an invaluable research tool by providing comprehensive insight to systemic metabolism. However, the lack of scalable and quantitative methods with known reference intervals (RIs) and documented reproducibility has prevented the use of metabolomics in the clinical setting. The objective of this study was to validate the developed quantitative nuclear magnetic resonance (NMR) spectroscopy-based metabolomics platform for canine serum and plasma samples and determine optimal sample handling conditions for its use.

Altogether, 8247 canine samples were analyzed using a Bruker’s 500 MHz NMR spectrometer. Using statistical approaches derived from international guidelines, we studied method precision, measurand stability in various long- and short-term storage conditions, as well as the effect of prolonged contact with red blood cells (RBC), and differences among blood collection tubes. We also screened interferences with lipemia, hemolysis, and bilirubinemia. The results were compared against routine clinical chemistry methods, and RIs were defined for all measurands.

We determined RIs for 123 measurands, most of which were previously unpublished. The reproducibility of the results of the NMR platform appeared generally outstanding, and the integrity of the results can be ensured by following standard blood drawing and processing guidelines.

Owing to the advantages of quantitative results, high reproducibility, and scalability, this canine metabolomics platform holds great potential for numerous clinical and research applications to improve canine health and well-being.

14. Age, breed, sex and diet influence serum metabolite profiles of 2000 pet dogs

Jenni Puurunen1,2,3, Claudia Ottka1,2,3†, Milla Salonen2,3†, Julia E. Niskanen2,3, Hannes Lohi1,2,3.
1 PetBiomics Ltd., Helsinki, Finland
2 Department of Veterinary Biosciences and Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland
3 Folkhälsan Research Center, Helsinki, Finland

Metabolomics is the comprehensive investigation of metabolism in a biological system. Since an individual’s metabolism reflects health and disease states well, metabolomics holds vast potential in understanding physiology and multifactorial diseases. However, it is vital to understand the metabolic effects of basic physiological characteristics, such as age or breed, to be able to separate them from metabolic alterations caused by pathological processes. This study aimed to investigate how physiological and diet-related factors influence canine metabolism.

We analysed serum samples of 2068 healthy pet dogs using a canine-specific nuclear magnetic resonance spectroscopy metabolomics platform, which quantitatively measures 123 metabolite measurands. We examined the effects of age, breed, sex, sterilisation, body size, diet, and fasting duration before blood sampling on the metabolite levels with generalised linear models. In these models, the false discovery rate corrected significance threshold was set at p < 0.05.

Age, breed, sex, sterilisation, diet, and fasting time profoundly affected canine metabolism, although the observed effects were largely metabolite-specific. Breed and age caused considerable variation in the metabolite concentrations, and breeds with very different body conformations systematically differed in their levels of several lipid measurands. However, only a few changes exceeded or undercut the established reference intervals.  

This study provides a solid foundation for understanding how physiological factors affect canine metabolism. The results stress the importance of controlling physiological factors in study designs and statistical analyses when conducting metabolomics studies and aid the interpretation of results.

TUOTANTOELÄIMET

15. Selective Dry Cow Therapy Effect on Milk Yield and Somatic Cell Count

Riitta E. Niemi, Mari Hovinen, and Päivi J. Rajala-Schultz
Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Finland

Antibiotic dry cow therapy (DCT) at dry-off is an effective mastitis control measure. Selective DCT means that only infected or presumably infected cows are treated instead of administering DCT for all cows, and it is the prevailing practice in the Nordic countries. Our objective was to compare milk yield and somatic cell count (SCC) during the first half of lactation between selectively DCT-treated and -untreated cows on commercial dairy farms, taking into account risk factors for reduced milk yield and high SCC.

The study utilized 2015–2017 Dairy Herd Improvement (DHI) data from 172 conventional Finnish dairy farms with 4,720 multiparous cows. Selective DCT was the standard practice on all these farms. The response variables were cow-level milk yield (kg/d) and naturally log-transformed composite SCC × 1,000 cells/mL (lnSCC), which were repeatedly measured on test days during 154 DIM. The main explanatory variable of interest was cow-level DCT at dry-off. The statistical method was a linear mixed-effects model with two-level random intercepts, cows nested within herds, and an AR(1) correlation structure.

The mean herd-level proportion of DCT-treated multiparous cows was 21%, and the median (IQR) herd-level DCT-treatment proportion was 12.5% (from 3.3% to 33.3%), showing that in Finland overall DCT use is low. Based on DHI recordings, geometric means of bulk tank SCC, and clinical mastitis incidence, the quality of milk and udder health nevertheless are good.  SCC on the last test day prior to dry-off had a different effect on milk yield in DCT-treated cows compared with the effect on those untreated, so that a higher SCC led to an increased difference in yield between treated and untreated cows. Considering this interaction, at an SCC of 200,000 cells/mL prior to dry-off, the administration of DCT corresponded to a yield increase of 0.97 kg/d. The persistence of subclinical mastitis over a dry period can lead to a negative economic impact during the subsequent lactation. Our result thus emphasizes the importance of successful selection criteria for cows in need of DCT. DCT reduced SCC during the following lactation. At an SCC of 200,000 cells/mL prior to dry-off, the results correspond to an SCC decrease of 20,000 cells/mL at 45 DIM for DCT-treated cows compared to those untreated. On the other hand, a higher late lactation SCC was associated with higher SCC again during the subsequent lactation.

SCC on the last test day prior to dry-off had a different effect on milk yield in DCT-treated cows, when compared with the effect on those untreated, so that a higher SCC correlated with greater difference in yield between treated and untreated cows. DCT reduced SCC during the following lactation. A higher late-lactation SCC was associated with higher post-calving SCC. Findings indicate that, for a cow likely to have an intramammary infection, a missed DCT treatment undesirably affects subsequent lactation milk yield and SCC. These findings highlight the importance of careful selection of cows to be treated, but in addition, our results demonstrate that selective-DCT practice can maintain good udder health and milk quality.

16. Economic perspectives on blanket and selective dry cow therapy

Anna-Maija Heikkilä1, Riitta E. Niemi2, Päivi J. Rajala-Schultz2
1Natural Resources Institute Finland (Luke), Latokartanonkaari 9, FI-00790 Helsinki, Finland
2Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Paroninkuja 20, 04920 Saarentaus, Finland

Dry cow therapy (DCT) is an efficient and widely used measure to control intramammary infections (IMI) in dairy herds. On a blanket-DCT practice (BDCT), all cows receive antibiotics at dry-off. On a selective-DCT practice (SDCT), treatments are targeted only at infected cows. Our objective was to compare the cost-effectiveness of SDCT and BDCT, considering the risk of IMI and uncertainty related to inputs and outputs of milk production.

Because a DCT practice affects both revenue and costs, a margin calculation is a useful method. We built a stochastic calculation model to show a margin for costs that are equal in both DCT policies. Data for modeling were generated by running 100,000 Monte Carlo simulations. The value of fixed variables, parameters for the distribution of stochastic variables, and probabilities for receiving DCT at dry-off or for acquiring IMI after calving were extracted from the literature and official statistics. In a sensitivity analysis, we tested effects of diverse IMI probabilities.

Results indicated the annual margin per cow in both SDCT and BDCT herds. Within a same herd size and milking system, the distribution of the margin in BDCT herds deviated from that of SDCT herds with the difference in median being €23─€29 per cow (1%) in favor of BDCT. If SDCT herds could reduce their current risk of post-calving IMI from 0.24 to 0.20 and BDCT herds would have their current risk of 0.18, an equal difference would be in favor of SDCT.

Antimicrobial resistance is a serious global concern that emphasizes non-antibiotic IMI prevention. Financial differences between SDCT and BDCT are minimal and, therefore, the focus in decision-making should be on the health status of the herd. SDCT is a competitive management practice, especially in herds succeeding to keep their IMI risk low.

17. Necropsy-based study on Finnish dairy cows – underlying causes of death

K. A. Hagner,¹ H. S. Nordgren,¹ K. Aaltonen,2 K. Sarjokari,3 H. Rautala,4 T. Sironen,1,2 A. Sukura,¹ and P. J. Rajala-Schultz 4
¹Department of Veterinary Biosciences, Faculty of Veterinary Medicine
2Department of virology, Faculty of Medicine, University of Helsinki
4Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki

During the recent decades, many reports have shown increased dairy cow mortality. The problem is widespread in modern production systems, associates with impaired animal health and welfare and causes negative economic and environmental consequences. Secondary register data or questionnaires for producers or veterinarians are often the source of the mortality data, and necropsies or histopathologic analyses are usually not performed. For this reason, true causes of dairy cow mortality may remain unidentified making it challenging to implement effective preventive measures. The aims of our study were to 1) determine causes and risk factors of on-farm mortality of Finnish dairy cows, 2) determine the usefulness of routine histopathologic analysis in bovine necropsies, and 3) assess how reliable producers’ perception about the cause of death is.

Necropsies for 319 dairy cows were performed at an incineration plant (Honkajoki Ltd.). The necropsy data were combined with cow- and herd-level information obtained from on-line questionnaires. Based on anamnestic information and macroscopic and histopathologic findings the final underlying, intermediate, and immediate diagnoses were set. The final underlying causes of death were grouped into eight major categories: accidents, calving disorders, digestive disorders, locomotor disorders, metabolic disorders, udder/teat disorders, other known reasons, and unknown reasons.

The most common underlying cause of death was udder disorders, mostly mastitis (26.6%). Other common underlying causes were digestive disorders (15.4%), calving associated disorders (12.2%), and locomotion disorders (11.9%). The cause of death remained undetermined only for 10 cows (3.1%). Most cows had also an intermediate (69.9%) or immediate (46.1%) cause of death. Most common consequences were acute (23.2%) and chronic (6.9%) abomasal ulcers, arthritis (17.2%), embolic inflammation in kidneys, liver, or lungs (13.8%), and aspiration pneumonia (13.8%).

Large proportion of the study cows (46.7%) died during the first 30 days after calving, especially during the first five days (63.6%) revealing the vulnerability of transitional cows. The underlying causes of death varied during different stages of lactation and with parity. Udder disorders were common in both late (31.0%) and early lactation (21.5%), but calving-related disorders (22.3%) were, as expected, common only in early lactation. The occurrence of mastitis as a cause of death increased markedly after second calving. Digestive and metabolic disorders were more common in older cows (at or over 4 parities).

A histopathologic analysis changed preliminary gross diagnosis in 18.2% of the cases. Producers’ perception about the cause of death agreed with the necropsy-based underlying cause of death in 42.8% of the cases. It was most consistent for mastitis, calving disorders, leg diseases, and accidents. In cases where producers had no insights about the cause of death the necropsy revealed the final underlying cause in 88.2% of cases. Based on these findings, we suggest that necropsies should be utilized more in dairy industry to obtain reliable information to build control programs for cow mortality. Including routine histopathologic analysis in necropsies, more precise information is obtained. Targeting preventive measures on transitional cows to diminish mortality might be profitable as the number of deaths was highest at this time.

18. Antimicrobial susceptibility of mastitis-causing staphylococci

Suvi Taponen, Riitta E. Niemi, Heli Simojoki, Päivi J. Rajala-Schultz
Department of Production Animal Medicine, Faculty of Veterinary Medicine

Microbiologic diagnosis and knowledge on antimicrobial resistance (AMR) of pathogens form a basis for effective treatment of intramammary infections (IMI). Staphylococci are the most prevalent causes of IMI in Finland, isolated in over 60% of mastitis cases. The commercial PCR assay (PathoProof Complete-16™) tests for the blaZ gene in staphylococci, but no other AMR is routinely monitored. Our objectives were to evaluate antimicrobial susceptibility of mastitis-causing staphylococci, and to compare their phenotypic and genotypic beta-lactamase resistance.

Milk samples positive for Staphylococcus aureus or non-aureus staphylococci (NAS) in the PCR assay were cultured, and the isolates identified to species level using MALDI-ToF MS. In addition to detection of the blaZ, mecA or mecC genes, AMR of the isolates to cefoxitin, ceftiofur, enrofloxacin, gentamycin, oxacillin, penicillin and tetracycline was evaluated using the disc diffusion method. Agreement beyond chance between phenotypic and genotypic beta-lactamase resistance was assessed with kappa-coefficient.

Antimicrobial susceptibility of 504 isolates (260 S. aureus, 244 NAS) from different herds across the country was evaluated. The most common NAS species were S. simulans, S. epidermidis, S. chromogenes and S. haemolyticus (74% of all NAS). Of all the staphylococci, 27% carried the blaZ gene and 19% were phenotypically resistant to penicillin. Considerable differences existed between species, ranging from 70% of S. epidermidis, 9% of S. aureus, and 5% of S. simulans being phenotypically resistant to penicillin. Eight NAS isolates carried the mecA gene (of which 6 S. epidermidis) and one S. aureus carried mecC. Overall agreement between blaZ carriage and phenotypic penicillin resistance was substantial (kappa=0.64).

Resistance to penicillin was most common, resistance to other antimicrobials was rare. A lot of variability existed between staphylococcal species, S. epidermidis exhibiting most resistance. Methicillin resistance in S. aureus isolates from mastitis milk was rare. Phenotypically penicillin-susceptible staphylococci carried the blaZ gene, suggesting that genotypic AMR testing may be more reliable when choosing medication.

19. Salmonella eradication on 57 dairy cattle farms in Finland

Olli Ruoho¹, Leena Seppä-Lassila², Miia Kontturi³
¹ Animal Health ETT, Seinäjoki, Finland
² Finnish Food Authority, Risk Assessment Unit, Helsinki, Finland
³ Finnish Food Authority, Animal Health Diagnostic Unit, Helsinki, Finland

Finland has a salmonella control program that covers cattle, pigs, and most of the poultry species. The aim of the control program is to maintain Salmonella prevalence below 1% in the food chain and thus reduce the risk of human salmonellosis. If Salmonella is detected, it must be eradicated from the farm.  On a dairy farm eradication includes e.g., sampling of animals and the environment, eliminating the possible infection source, and thorough cleaning and disinfection. For the present group insurances have been available for farms to partly cover the costs of eradication.

Our aim was to investigate Salmonella eradications on dairy farms, describe the principles of the eradication process and the most likely cause of the infection. Based on these findings, it would be easier to advice the prevention and guide the eradication of Salmonella on dairy farms.

Our data included 57 dairy farms with Salmonella infection and an eradication that an experienced veterinary advisor (Dipl. ECBHM Olli Ruoho) had planned and guided during 2009–2019. These herds had an average 76 milking cows (range 17-260). The most common serotypes detected were S. Typhimurium (n=30), S. Enteritidis (n=11) and S. Altona (n=4). We calculated the length of the eradication from the start and end date of the official restrictions of the farm. Based on environmental and animal sampling at the beginning of the eradication process, we defined the Salmonella infection of the farm as non-existent, restricted, average, or vast.

The eradication lasted 21-533 days (mean 114, median 90). It lasted 52-533 days (mean 156, median 120) on 31 farms (54%) where the infection was considered as average or vast. Other typical reasons for a prolonged eradication were: if the source of the infection was undetected at the beginning, if salmonella positive animals were not culled in time, if cleaning and disinfection procedures were inadequate or lack of labour leading to exhaustion. On 46 farms (81%) the source of the infection was birds or rodents, and in most of these cases birds. Occasionally, the possible source was Salmonella positive animals, humans, or contaminated feed. However, the management of salmonella risk in commercial feeds is at high level in Finland.

Eradicating Salmonella from a dairy farm is possible. However, proper planning is essential as well as working systematically throughout the eradication process. Furthermore, a competent supervision and guidance are important as well as having enough skilled labour on the farm. Nevertheless, the focus should be on Salmonella prevention by improving the on-farm biosecurity like protecting the barn and especially feed storage from birds. This includes furnishing the barns with anti-bird nets, covering all the feed storage, maintaining the barn and farm area tidy that uneaten feed is taken away regularly, and enhancing the overall pest control. Moreover, the whole sector should discuss the means how to reduce the risk of spreading Salmonella infections from dairy farms to calf rearing units and thereby to beef production chain since all calves can not be tested for Salmonella.

20. Prevalence of digital dermatitis using mirror scoring in Finnish dairy herds

Hertta Pirkkalainen1, Aino Riihimäki1, Heli Simojoki1, Timo Soveri1, Päivi Rajala-Schultz1, Tuomas Hintikka2, Sinikka Pelkonen2, Miia Kontturi3, Minna Kujala-Wirth1
1Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki
2Finnish Food Authority, Animal Health Diagnostic Unit, Kuopio
3Finnish Food Authority, Animal Health Diagnostic Unit, Helsinki

Digital dermatitis (DD) is a severe bacterial claw disease found worldwide. The disease can be classified into 5 different M-stages by clinical examination. The aim of this study was to find the herd level prevalence of acute (M1, M2, M4.1) and chronic (M3, M4) DD lesions in Finnish dairy cattle population and test scoring in milking parlors and pens. Prevalence studies concerning DD have not been conducted on national level earlier. Another aim was to estimate the sensitivity and specificity of mirror scoring on standing cows in a pen or in a milking parlor without washing the feet.

Altogether 81 randomly selected herds were visited by the project veterinarians. During the farm visit, hind feet of lactating cows (n = 7,010) were scored with a mirror without washing the feet, either when the cows were standing in a pen (n = 4,992) or in the milking parlor (n = 2018). In addition, 256 feet were chosen with cross-sectional sampling and scored in a trimming chute.

Animal-level sensitivity for scoring M2 lesions with a mirror was 55% and specificity was 97%; for all active DD lesions (M1, M2, or M4.1), sensitivity was 36% and specificity was 96%. Sensitivity for scoring any DD lesions was 90% and specificity was 82%. The bias-corrected sensitivity and specificity for scoring any DD lesions were 79% and 92%, respectively. The bias-corrected sensitivity and specificity for scoring M2 DD lesions were 10% and 100%.

We found that 33.3 % of the herds had acute lesions (M1, M2 or M4.1) but cow level prevalence was less than 0.7 %. In most farms only 1-2 cows were affected. The within-herd prevalence of M2 lesions (in herds where at least 1 cow had a M2 lesion) was 5.7% and varied between 0.4% and 18.8%. Herds with active DD lesions also had more any DD lesions than herds without active DD lesions.

In our study, the prevalence of acute DD lesions was higher than previously thought, with only one herd without any DD lesions. Scoring in the pen or milking parlor had low sensitivity but could be used to monitor within-herd DD dynamics regularly. The scoring method could also be used to investigate national prevalence of DD in herd level (DD present, DD absent), but a careful approach should be used when interpreting the results. Education concerning DD is needed for all involved in the cattle field. Measures for controlling and preventing DD in Finland must be introduced without delay.

21. Herd level risk factors for active digital dermatitis lesions in Finnish free stall dairy herds

Aino Riihimäki1, Hertta Pirkkalainen1, Heli Simojoki1, Timo Soveri1, Päivi Rajala-Schultz1, Sinikka Pelkonen2, Miia Kontturi3, Minna Kujala-Wirth1
1Department of Production Animal Medicine, Faculty of Veterinary Medicine, University of Helsinki, Helsinki
2Finnish Food Authority, Animal Health Diagnostic Unit, Kuopio
3Finnish Food Authority, Animal Health Diagnostic Unit, Helsinki

Digital dermatitis (DD) is a severe bacterial hoof disease found worldwide. The disease can be classified into five different stages, of which DD M1, DD M2 and DD M4.1 are considered active lesions. Based on earlier studies, prevalence of active lesions in Finland was low. The aim of this study was to find herd-level risk factors for active DD lesions.

Free stall dairy herds were randomly selected from Finnish dairy herds that had > 45 lactating cows. Study population included 81 herds and 6877 cows. We scored all hind feet of adult cows for DD with a mirror when cows were standing in a pen or in the milking parlor. If at least one active lesion was detected in a herd, the herd was considered as an active DD lesion herd.

Farmers also filled out a questionnaire, which included 67 closed questions. Questions were divided into five categories: overall management, herd and barn characteristics, animal purchasing history, hoof trimming and hoof bath practices, and hoof disease history on the farm.

A multivariable logistic regression model was used for modelling risk factors. Selection of potential risk factors for a full model was based on a causal diagram and univariate screening. The risk factors for the model were chosen with univariate analysis. Herd and area were used as random effects. A predictor variable with p < 0.25 in an univariable analysis was selected for the multivariable model.

Multicollinearity was evaluated between categorial explanatory variables using phi-test and Cramer’s V. Most of the explanatory variables were strongly correlated which led to the selection of the predictor variable to the final model that best described the situation. All biological meaningful two-way interactions were tested in the final model.

The mean herd size was 87 cows (range 49–241). Of all the study cows, 67% were milked in an automatic milking system (AMS) and 33% in a milking parlor. Out of study herds, 33% had active DD lesions. No significant biological interactions were detected.

Larger herd size was a risk factor (OR 1.8 / cow nr loge; p=0.05) for active DD lesions. Compared to AMS, barns with parlor milking were associated with the presence of active DD lesions (OR 3.7; p=0.02). Occurrence of interdigital phlegmon during the previous year was associated with higher odds of active DD lesion (OR 3.6; p=0.04).

In this study, larger herd size, barn with a parlor milking and occurrence of interdigital phlegmon were risk factors for having active DD lesions in the herd. As AMS farms are more common in Finland, further research on management practices and their effect on prevalence of active DD lesions in the herd is warranted.

22. A unique Quality Assurance Programme for Finnish sheep herds

Eero Rautiainen & Johanna Rautiainen
Sheep World Ltd. (Lammasmaailma Oy), Finland

Animal diseases and deficiencies in animal husbandry weaken the productivity and profitability of livestock farming and endanger animal welfare. According to several sources, the most significant health problems in sheep herds include gastrointestinal parasite infections, infectious diseases in lambs such as coccidiosis, perinatal lamb mortality and ewe mastitis, listeriosis and disturbances in calcium and energy metabolism. Subclinical problems may only occur as slow growth and suboptimal reproductive performance.

The aim of the work was to draw up a quality assurance programme to prevent animal diseases and improve treatment practices by influencing the root causes of the problems in typical Finnish family herds engaged in professional sheep farming with 80-400 ewes. The objectives of the quality assurance programme were set to support high level of biosecurity, feeding in accordance with the production stage and professional animal husbandry.

When drawing up the programme, we applied some of the HACCP principles. Initially, we described the events of the ewe production year in chronological order and constructed a unified lamb breeding process. We set a perspective on ewe on the assumption that ewe's health and wellbeing form the basis for the health of lambs. Next, we assessed which support functions typically associated with sheep farming are necessary for the management of the production process and for which good practice guidelines (GMP) can be drawn up. Thirdly, we assessed what kind of indicators a producer would need to monitor the management of the process. In addition, we decided that the programme should integrate the parasite prevention programme we developed earlier.

In the work, we identified seven subsequent stages of production in the ewe production year, which can be treated as sub-processes of lamb husbandry and for which it is possible to establish control points for the management of the process. The production stages were named and their outputs were defined: (1) Selection of ewes (healthy ewes with good body condition); (2) heat synchronisation (short lambing period); (3) Ewe clinic (grouping of ewes into feeding groups); (4) Late pregnancy feeding (good birth weights of lambs); (5) Maintenance of good lactation (good growth of small lambs); (6) Weaning of ram lambs and (7) weaning of ewe lambs (steadily growing lambs with low risk of parasitic diseases, healthy ewes). In addition, we identified the necessary support functions related to the sub-processes. These include, for example, the use of ewe body scoring, pregnancy scanning, quarantine of purchase animals, and feeding plan based on analysed feedingstuffs. Thirdly, we selected and drew up three types of indicators for producers: Indicators describing the state of the process at key control points (e.g. the length of the tupping period); Indicators describing the utilisation of key support measures (e.g. the use of adaptable pen structures); Indicators describing the success of production (e.g. lamb mortality).

The quality assurance programme we have drawn up (Sheep Health 2020) creates good conditions for the comprehensive management of animal disease risks and compliance with good practices in sheep herds. Instead of partial optimisation, it is more profitable to avoid underperformances at any individual production stage.

23. An epidemic of salmonellosis caused by rapeseed meal containing salmonella at a dairy farm – case report

Olli Ruoho
Animal Health ETT, Seinäjoki, Finland

Finland has zero tolerance policy for salmonella. In addition to the official salmonella monitoring program, producers take many self-monitoring samples, e.g. in connection with the animal trade. If salmonella is found on a cattle, swine or poultry farm, the farm will be subject to official restrictions and salmonella eradication must be started. The principles of eradicating salmonella from the animals and the environment are to assure hygienic feed and water and not contaminate the feed with feces. The production facilities are cleaned and disinfected according to a separate plan.

A dairy farm in Western Finland was tested positive for Salmonella Infantis in May 2019. The loose-housing dairy had 210 cows and 150 young livestock. Salmonella eradication was started immediately. Animal movements to the farm an out of the farm were ceased. Milk was delivered to the dairy to be diverted to heat-treated products. All animals were examined for salmonellosis by faecal samples. About 80 environmental swab samples were taken e.g. from feeding tables, water throughs, feeding equipment and feed storages.

The animals were fed TMR (total mixed ration) with a TMR-wagon. TMR-feed and each of its components were examined. The main component was pre-dried grass silage harvested from their own fields. In addition, the feed included rapeseed meal, minerals and ground barley and oats.

At first 25 % of the animals were salmonella positive. Some environmental samples from feeding tables, feeding equipment and water throughs were positive but all feed was negative. The hygienic procedures were concentrated to the contaminated critical points. Feeding tables and drinking throughs were disinfected twice a day. Feed handling and feeding equipment was cleaned and disinfected as well as possible.

Next month, however, 60 % of faecal samples were positive for salmonella. Hence, new environmental and feed samples were taken. Salmonella was detected in an environmental swab sample from the inner surface of the rapeseed meal silo cover. This site had not been sampled before because a crane was needed for access. The rapeseed meal was imported. It was not heat-treated but tested on a sample of 1 bulk sample / 25,000 kg with negative result.

The silo was emptied, washed, and disinfected. Rapeseed meal was excluded from animal nutrition and replaced by industrial heat-treated protein feed. The hygienic measures proceeded.        

The eradication procedure was monitored by faecal and environmental samples every second or fourth week. The salmonella was eradicated in four months, as confirmed by two successive monthly faecal samples from the animals and one environmental sampling. Hence, the restrictions could be lifted.

The use of non-heat-treated rapeseed meal in animal feeding involves a risk of salmonella that cannot be eliminated completely by sampling. Contamination does not necessarily occur in feed samples alone but requires extensive swab and dust sampling of the feed environment throughout the whole feeding system. Feed storages and feeders may contain salmonella inoculants which, when mixed with feed, can cause infection in the herd.