Anaplasma Phagocytophilum in Finnish Dogs

The prevalence of Anaplasma phagocytophilum in Finnish dogs

Suvi Kapiainen, MSc, Bsc Vet Med Licentiate Thesis in Veterinary Medicine Small Animal Internal Medicine Department of Equine and Small Animal Medicine Faculty of Veterinary Medicine University of Helsinki, 2016

Tiedekunta - Fakultet - Faculty Osasto - Avdelning – Department

Faculty of veterinary medicine Department of Equine and Small Animal Medicine Tekijä - Författare - Author

Suvi Kapiainen Työn nimi - Arbetets titel - Title

The prevalence of Anaplasma phagocytophilum in Finnish dogs Oppiaine - Läroämne - Subject

Small Animal Internal Medicine Työn laji - Arbetets art - Level Aika - Datum - Month and year Sivumäärä - Sidoantal - Number of pages

Licentiate thesis March 2016 40 Tiivistelmä - Referat – Abstract

Only little data is available on seroprevalence of tick borne diseases in Finnish dogs. Worldwide these diseases, anaplasmosis being one of them, are relatively widely studied. In some European countries the disease has become more common than earlier and it has spread more towards North just in the last few years. The aim of this study was to investigate the seroprevalence of Anaplasma phagocytophilum –bacterium in Finnish dogs, the PCR-positivity in relation with seropositivity and the risk factors for exposure to a vector borne pathogen. The hypotheseis were that Anaplasma phagocytophilum is underdiagnosed in Finland, there are more Anaplasma phagocytophilum -infections and seropositive dogs in the southern parts of Finland and that hunting dogs are more susceptible for Anaplasma phagocytophilum -seropositivity compared to pet dogs. For material, 390 canine blood samples (serum, EDTA) were collected all around Finland. Of these samples, 50 were from hunting dogs. DNA was extracted from the EDTA-samples and with real-time -PCR the PCR-positive, bacteremic dogs, were spotted. Seropositivity was tested from the serum samples by using commercial SNAP 4Dx-test. The seroprevalence of anaplasmosis in Finnish dogs was 4,6% (18/390) and in hunting dogs 4,0% (2/50). Regionally in Åland there were more seropositive dogs than elsewhere in Finland. In Åland the seropositivity was 45% (9/20). Small dogs were more often seropositive than large dogs. Only two Finnish dogs were PCR-positive (2/390), meaning they had Anaplasma phagocytophilum -infection. These two dogs were seronegative. This study suggests that canine anaplasmosis is an important tick borne disease in Finnish dogs. The results show that dogs are commonly exposed to anaplasmosis especially in Åland islands and small dogs may be more susceptible to infection than large. It is unclear at this point if the prevalence of Anaplasma phagocytophilum -exposure will increase in other parts of the country due to the climate change and the northern spread of Ixodes ticks. The prevalence of actual infection is low. Because of this low number of Anaplasma phagocytophilum -positive dogs, no risk factors for infection could be identified in our study. Therefore, future studies with larger number of infected dogs are necessary to better understand the risk factors associated with Anaplasma phagocytophilum -infections in dogs and in people.

Puutiaisvälitteisten tautien esiintyvyydestä suomalaisissa koirissa on vain vähän tutkimustietoa. Maailmanlaajuisesti nämä taudit, anaplasmoosi yhtenä niistä, ovat suhteellisen laajalti tutkittuja. Joissain Euroopan maissa tauti on yleistynyt ja se on myös levinnyt ylemmäs pohjoiseen Eurooppaan. Tutkimuksen tarkoituksena oli selvittää Anaplasma phagocytophilum -bakteerin esiintyvyyttä suomalaisissa koirissa, bakteremian suhdetta vasta-aineisiin ja arvioida altistumisen riskitekijöitä. Hypoteesejä olivat, että bakteeri on alidiagnosoitu Suomessa, Etelä-Suomessa on enemmän infektioita sekä vasta-ainepositiivisia ja että metsästyskoirat ovat alttiimpia tartunnalle kuin muut koirat. Materiaalina oli 390 koiran verinäytteet (kokoveri ja seerumi), joista 50 oli metsästyskoirien. Näytteitä kerättiin koko Suomesta. Kokoverinäytteistä eristettiin DNA, joka tutkittiin reaaliaikaisen PCR:n avulla bakteremian todentamiseksi.Vasta-ainepositiivisuus tutkittiin veren seeruminäytteistä kaupallisella SNAP 4Dx-testillä. Anaplasma phagocytophilumin vasta-aine-esiintyvyys suomalaisissa koirissa oli 4,6% (18/390) ja metsästyskoirissa 4,0% (2/50). Alueellisesti Ahvenanmaalla oli eniten positiivisia koiria, 45% (9/20). Pienet koirat olivat useammin vasta-ainepositiivisia kuin suuret. Vain kaksi koiraa oli PCR-positiivisia eli vain kahdella oli infektio (2/390). Molemmat näistä koirista olivat vasta-ainetestissä negatiivisia. Tämän tutkimuksen mukaan koiran anaplasmoosi on tärkeä puutiaisvälitteinen tauti suomalaisissa koirissa. Tulosten mukaan koirat altistuvat tartunnalle erityisesti Ahvenanmaalla ja pienet koirat voivat olla alttiimpia tartunnalle. Muualla maassa esiintyvyys oli pienempi, mutta on mahdollista, että se kasvaa tulevaisuudessa ilmaston lämpenemisen vuoksi ja puutiaisten levitessä pohjoisempaan. Varsinaisen infektion esiintyvyys oli pieni. Tästä syystä PCR-positiivisuuden ja riskitekijöiden välisiä suhteita ei voitu analysoida. Aihetta tulee tutkia tarkemmin ja tulevissa tutkimuksissa tulee ottaa laajemmasta joukosta koiria näytteitä, jotta saadaan enemmän tietoa infektioiden ja vasta-ainepositiivisuuksien suhteista sekä parempi ymmärrys Anaplasma phagocytophilum-infektioihin liittyvistä riskitekijöistä koiria ja myös ihmisiä ajatellen. Avainsanat - Nyckelord - Keywords

Anaplasma phagocytophilum, anaplasmosis, dog, seroprevalence, anaplasmoosi, koira, esiintyvyys Säilytyspaikka - Förvaringställe - Where deposited

Eläinlääke- ja elintarviketieteiden talon (EE-talo) Oppimiskeskus Työn johtaja (tiedekunnan professori tai dosentti) ja ohjaaja(t) - Instruktör och ledare - Director and Supervisor(s)

Director Thomas Spillmann Supervisor Cristina Perez Vera

CONTENTS

1 INTRODUCTION…...... 1

2 LITERATURE REVIEW ...... 3

2.1 Anaplasma phagocytophilum (Ap).……………………………………….…. 3

2.2 Geographic distribution and seroprevalence ………………….………....… 3

2.3 Vector, reservoirs …………………………………………….…….....………. 6

2.4 Infection…………………………………………………………………………. 6

2.5 Clinical signs and imaging findings of Ap-infection ….………………..…… 7

2.6 Laboratory findings, diagnostics, persistence…………….……...…...……. 8

2.7 Treatment………………..……………………………………………………. 10

2.8 Ixodes ricinus, the vector ………………………………………………....… 11

3 MATERIAL AND METHODS …………………………………….………………12

3.1 Dogs ……………………………………………………………………………12

3.2 Ap: serology, DNA extraction, real-time -PCR …………………………….14

3.3 Statistical analysis …………………………………………….………………15

4 RESULTS ……………………………………………….………….……...………16

4.1 Dogs ……………………………….…………………..………….……………16

4.2 Ap-seroprevalence ……………….…………………………………....…..…18

4.3 Ap-infection …………………………………………….…….….……….……20

4.4 Statistical analysis ……………………………………..………....…………..20

5 DISCUSSION ………………………………………………………..…………….20

6 CONCLUSIONS AND CLINICAL RELEVANCE ………………….….………..25

7 ACKNOWLEDGEMENTS ……………………………….…………….…………26

8 REFERENCES ……………………………………………………..……………..26

Attachments ..………………………………………………………………………..37 1 INTRODUCTION

The incidence of Anaplasma phagocytophilum (Ap) –infections in humans and dogs has increased during the last decades. Ap is a bacterium that causes the tick borne disease anaplasmosis. The bacterium is zoonotic (Chomel 2011) and transmitted to animals by Ixodes ticks being active during the vegetation period (daily mean temperature being over 5°C) (Jaenson & Lindgren 2011).

Many bacteria have been renamed during this century as molecular methods have progressed. Due to advanced molecular techniques, Ehrlichia phagocytophila, Ehrlichia equi and human granulocytic ehrlichiosis (HGE) agent were also renamed phylogenetically as one species, Ap (Dumler et al. 2001).

The northern position of Finland and the Baltic Sea makes this country in a way isolated. This isolation causes a geographic impact that holds up or inhibits the spreading of many infectious diseases from southern Europe. But as migratory birds may transmit many diseases, human and pet travelling has increased, and climate is changing (Reed et al. 2003, Laakkonen et al. 2009), Ap is regarded as an emerging vector borne pathogen (Dumler et al. 2007). Finnish humans and animals are then susceptible to acquiring infectious diseases, even though Finnish climate and Baltic Sea that surrounds the country in South and West are thought to be main inhibitory factors of the spread of diseases (Reed et al. 2003, Laakkonen et al. 2009).

In Finland the first reported Ap infection was in bovine (Tuomi 1966). World widely the first verified human granulocytic ehrlichiosis (anaplasmosis) infections were reported in the USA in 1994 (Bakken et al. 1994) and in Europe (Slovenia) few years later (Petrovec et al. 1997). The first Ap-infection in any Finnish companion animal was reported only few years ago in a cat (Heikkilä et al. 2010). Since then Ap-infection has been reported in Finland in a horse (Valkjärvi et al. 2010) and two dogs (Mäkitaipale 2011). Given the limited data available relating to this pathogen in Finland, it is important to get a better general understanding of the exposure and prevalence of Ap- infection in dogs. With this knowledge it is possible to assess the risk of infection in a

1 given dog and its clinical manifestations. The prevalence of Ap in pet dogs could also act as a good indicator for the corresponding risks to people since dogs live mainly with their owners and owners spend time in the same habitat with their dogs e.g. at home, summer cottages, woods and nature making them susceptible for tick bites and Ap-infection. The American College of Veterinary Internal Medicine (ACVIM) published a consensus statement on ehrlichial infections in small animals (Neer et al. 2002). But since then new studies have been published e.g. on infections, epidemiology, laboratory methods and findings, sings and treatment, and Ap seems to infect dogs more often than before. On the other hand diagnostic methods have been developed which may have increased the positive results on diagnosing Ap infection suspected dogs leading probably to a rather relative than absolute increase.

The objectives of this study were to investigate 1) The seroprevalence of Ap in Finnish dogs; 2) The PCR-positivity in relation with seropositivity and 3) The risk factors for exposure to a vector borne pathogen.

The hypothesis were the following 1) Ap is underdiagnosed in Finland, since Ap is an emerging zoonotic pathogen, its diagnostic tools are not yet that commonly used and its infections can be subclinical and latent without clear sings; 2) There are more Ap-infections and seropositive dogs in the southern parts of Finland, especially in Åland, since Åland is known for its large ticks population and high incidence of tick-borne diseases and 3) Hunting dogs are more susceptible for Ap-seropositivity compared to pet dogs, as they spend more time in the nature and at tick habitats.

2 LITERATURE REVIEW

2.1 Anaplasma phagocytophilum (Ap)

Ap is in the order Rickettsiales, and in the family Ehrlichiaceae that includes members of the genera Ehrlichia, Anaplasma, Cowdria, Wolbachia and Neorickettsia. Ap is a member of the genus Anaplasma. At the same time of naming Ap, Ehrlichia platys was renamed as Anaplasma platys, since it was found to be more closely related to Anaplasma species rather than Ehrlichia (Dumler et al. 2001).

Canine granulocytic anaplasmosis is a zoonotic bacterial arthropod borne disease caused by an obligate intracellular rickettsial organism Ap (Dumler et al. 2001, Carrade et al. 2009). Ap is a gram-negative cocci bacteria with size less than 1,0 μm. It does not have lipopolysaccharide on its surface (Lin et al. 2003) as many other gram-negative bacteria.

2.2 Geographic distribution and seroprevalence

To date two Anaplasma-species have been identified in dogs in Europe: A. platys and Ap. A. platys has been identified in Portugal and Italy (Santos et al. 2009, Ramos et al. 2014) whereas Ap has been identified more widely infecting dogs in Europe e.g. in Italy (Solano-Gallego et al. 2006a), Spain (Solano-Gallego et al. 2006b), Austria (Kirtz et al. 2007), Germany (Barutzki et al. 2006) and the United Kingdom (Volgina et al. 2013). In Northern Europe, Ap has been identified in dogs in Finland’s western neighbor Sweden (Egenvall et al. 2000a) and in Finland (Mäkitaipale 2011). Ap has been also identified in dogs in Russia (Volgina et al. 2013) which is Finland’s eastern neighbor with a long land border. In ticks Ap has been identified in Northern Europe in e.g. Estonia (Mäkinen et al. 2003), European Russia (Alekseev et al. 2001), Sweden (von Stedingk et al. 1997) and Norway (Jenkins et al. 2001). Ap is thought to be endemic throughout Europe (Greene 2012). In Figure 1 is shown the geographic distribution of Ap in Europe.

Figure 1. Geographic distribution of Ap in Europe.

Ap has been diagnosed in many European countries and some studies on its prevalence have been made. Ap prevalence and its distribution have increased in the last few years in many countries as can be seen in Table 1. This increase is a result of a mixture of many factors such as climate and habitat management change, increased tick populations and distribution, and prevailing social and economic conditions (Godfrey & Randolph 2011). Over years Ap infections have become more common and have spread to new niches in Europe possibly due to globalization, urbanization, global warming and increased travelling (Rizzoli et al. 2014). In table 1 is shown prevalence of Ap infections in different European countries.

Table 1. Prevalence of Ap infections in European countries in dogs. IFAT and SNAP 4Dx ELISA are serological methods.

Country Number of Prevalence Method Reference dogs (n) (%) Albania 30 40,0 IFAT Hamel et al. 2009 Austria 1470 56,5 IFAT Kirtz et al. 2007 France 919 2,7 SNAP 4Dx Pantchev et al. 2009 Germany 1124 50,1 IFAT Barutzki et al. 2006 111 43,2 IFAT Jensen et al. 2007 6,3 PCR 5881 21,5 SNAP 4Dx Krupka et al. 2007 522 43,0 IFAT Kohn et al. 2011 5,7 PCR 448 19,4 SNAP 4Dx Barth et al. 2012 Hungary 1305 7,9 SNAP 4Dx Farkas et al. 2014 Italy 460 0,0 PCR Solano-Galleno et al. 2006a 5634 32,8 IFAT Torina & Caracappa 2006 249 38,0 IFAT Pennisi et al. 2012 215 15,0 IFAT Ebani et al. 2013 0,02 PCR Latvia 470 11,4 SNAP 4Dx Berzina & Matise 2013 Poland 192 1,0 PCR Skotarczak et al 2004 Portugal 55 55,0 IFAT Santos et al. 2009 0,0 PCR Romania 1146 5,5 SNAP 4Dx Mircean et al. 2012 Russia 442 1,1 SNAP 4Dx Volgina et al. 2013 82 34,1 SNAP 4Dx Spain 466 11,5 IFAT Solano-Gallego et al. 2006b 1100 3,1 SNAP 4Dx Miro et al. 2013 Switzerland 996 7,5 IFAT Pusterla et al. 1998 Sweden 611 17,7 IFAT Egenvall et al. 2000a 248 20,7 IFAT Jäderlund et al. 2007 UK 120 0,8 PCR Shaw et al. 2005

In conclusion, anaplasmosis is an emerging disease worldwide. However, its prevalence has not been widely studied in Finland. At the time of starting this project, there were no epidemiological studies on Ap-prevalence in Finland. That is why the presented study was initiated and its results became part of a recently published paper addressing this topic (Pérez Vera et al. 2014). An epidemiologic study about the exposure in dogs will provide the basis for future studies that may help understand the public health impact of this disease in Finland.

2.3 Vector, reservoirs

In Europe Ap is transmitted by Ixodes ticks (Ixodes ricinus, I. persulcatus and I. hexagonus) (Baumgarten et al. 1999, Ravyn et al. 1999, Strle 2004) main vector being I. ricinus (Strle 2004). This bacterium infects many mammals e.g. dogs and ruminants (Dumler et al. 2001), bovine species (Stuen 2007), horses (Bjöersdorff et al. 1990), cats (Bjöersdorff et al. 1999), small mammals such as rodents (Liz et al. 2000), European hares (Hulinska et al. 2004) and humans (Petrovec et al. 1997). Ap does not transmit transovarially but transstadially (Dumler et al. 2001) and it stays alive in the environment through its reservoir hosts, including small mammals (Carrade et al. 2009), cervids and other wild ruminants (Stefanidesova et al. 2008). The first study examining the presence of Ap in wildlife reservoir host species in Finland was recently published by Kallio et al. (2014). The authors presented the first evidence of Ap being present in wild rodents (bank voles) in Finland and that 22% of bank voles were infected by the bacterium and acted as reservoir hosts. Also birds are possible hosts and may facilitate the spreading of the bacterium from one area to another (Bjöersdorff et al. 2001).

2.4 Infection

There seems to be no predisposition on which and what kind of dogs the bacterium infects (sex, age, breed, healthy, sick) (Egenvall et al. 1997), but elderly dogs have been

6 more often seropositive (Egenvall et al. 2000a, Kohn et al. 2011), which could be explained by a higher exposure to ticks and Ap throughout their life time.

After tick infestation, the pathogen enters through tick’s saliva to peripheral circulation of the mammal host and targets vertebrate host myeloid and non-myeloid cells, mainly neutrophils and rarely eosinophils (Dumler et al. 2001). With its surface protein the bacterium binds to leucocytes surface and through endocytosis it enters the cell (Webster et al. 1998). The bacterium is present within intracytoplasmic vacuoles (endosomes) as morulae (Dumler et al. 2001). Inside the morulae it multiplies until cell lysis or apoptosis occurs and bacteria are released, or through exocytosis enabling it to infect other granulocytes (Webster et al. 1998). By inhibiting endothelial cell function, transmigration, neutrophil phagocytosis and binding of the neutrophil to the endothelial wall (Choi et al. 2003) Ap slows down leucocytes apoptosis for approximately 24 hours and enables itself to multiply without cell lysis by evading host cell defenses (Ge et al. 2005).

Morulae can be first found from the blood between 4 to 14 days (median six days) after infection/inoculation and they circulate in the blood for only 4 to 8 days during the first 2 weeks after transmission (Egenvall et al. 1998). After infection and morulae development antibodies start to rise as a response for the infection (Carrade et al. 2011). Antibodies against Ap develop approximately one week after infection and persist for 6 to 8 months. If a dog is treated for anaplasmosis with antibiotics the antibodies can decrease to a non-detectable level (Egenvall et al. 2000a, Kohn et al. 2008, Carrade et al. 2011).

2.5 Clinical signs and imaging findings of Ap-infection

Ap infects several mammalian species and the clinical signs are mainly similar between species (Ravnik et al. 2009). After 1-2 weeks of incubation period the infected dog may show severe clinical signs or it can have a self-limiting fever and illness (Egenvall et al. 1998). This is called the acute stage of anaplasmosis. Most dogs do not get clinically sick (Kohn et al. 2011).

Common clinical signs of infection are acute illness with lethargy and fever (4-14 days) and inappetence (Ravnik et al. 2009). Other main signs or findings associated with Ap- infection in dogs include short bacteremia (4-14 days), lameness, anemia and thrombocytopenia as well as impaired immunity (Carrade et al. 2009, Woldehiwet 2010). Lameness is a common sign in anaplasmosis and it developes because of secondary immune-mediated (neutrophilic) polyarthritis. Other findings are pale mucous membranes, tense abdomen and diarrhea/vomiting (Kohn et al. 2008, Ravnik et al. 2009). The infected dog can also have mildly enlarged lymph nodes, tachypnea and endothelial hemorrhage (petechiae, melena and epistaxis caused by thrombocytopenia). More rare signs are collapse, scleral injection, uveitis, mild cough, limb edema and PD/PU (Ravnik et al. 2009, Eberts et al. 2011). Ap can also enhance some immunopathies to develop such as immune-mediated thrombocytopenia and - anemia (Kohn et al. 2008). On the other hand previous studies have shown that dogs can also have Ap-infection with no clinical signs (Jensen et al. 2007, Carrade et al. 2009, Kohn et al. 2011).

In an ultrasound and x-ray an enlarged spleen can be seen as a very common finding in infected dogs (Kohn et al. 2008).

2.6 Laboratory findings, diagnostics, persistence

The main laboratory findings along with short bacteremia and thrombocytopenia are non-regenerative mild to moderate normocytic normochromic anemia (regenerative anemia is rare), lymphopenia, neutrophilia (sometimes left shift), neutropenia (or normal neutrophil concentration), hyperglobulinemia, hypoalbuminemia, increased alkaline phosphatase, mild hyperbilirubinemia, neutrophilic inflammation in synovial fluid. Sometimes the direct Coombs’ test/platelet-bound antibody test can be positive (Ravnik et al. 2009, Carrade et al. 2009, Woldehiwet 2010, Eberts et al. 2011).

Ap can be detected from a direct blood smear, when morulae are seen in neutrophils. Direct blood smear can be considered a good diagnostic tool as morulae have been

8 found in almost 60 % of clinical cases (Kohn et al. 2008). Morulae can be seen in neutrophils after 4 days of infection and they persist usually 4 to 8 days. Ap may participate in the development of subclinical or chronic disease and has been found from dogs blood up to six months after inoculation (Egenvall et al. 2000b). However, blood samples negative for morulae do not exclude an Ap infection calling for more advanced laboratory procedures for confirmation.

Serological tests for Ap are IFAT (immunofluorescence antibody test) and ELISA (enzyme-linked immunosorbent assay). These tests detect antibodies from blood samples. During the acute phase of the infection these tests are usually negative as antibody titers grow later (Eberts et al. 2011). IFAT gives antibody titers (Jensen et al. 2007) whereas the ELISA test SNAP 4Dx is qualitative (Stillman et al. 2014). The SNAP 4Dx test has a synthetic peptide which is derived from the immunodominant p44 major outer surface protein of Ap and it acts as an specific antigen for antibodies in the serum of infected dog. It is a fast in-house test. Cross-reactions with antibodies of other closely related bacterial species such as A. platys (Beall et al. 2008, Diniz et al. 2010) or E. canis (Greig and Armstrong 2006) are common and cannot be ruled out in this test (Diniz et al. 2010) but since neither A. platys nor E. canis are, according to present knowledge, endemic in Finland they are not likely to cause cross-reaction problems when testing Finnish dogs that have not been abroad.

PCR-detection of Ap DNA is a sensitive and specific method. Samples are usually peripheral blood, but tissue samples, and cerebrospinal fluid can also be taken depending on the case. PCR can detect the bacterial DNA in early stages of infection (Scorpio et al. 2011). However detection by PCR is dependent on the number of circulating organisms, which can fluctuate. Thus some infected animals can have negative PCR results (Beall et al. 2008).

Ap DNA has been diagnosed with PCR from several sites in different species; from sheep and horse blood granulocytes (Franzen et al. 2009, Stuen et al. 2008), form horse’s poorly vascularized connective tissue where penetration of antibodies may be difficult (Chang et al. 1998) and from sheep’s skin (Granquist et al. 2010). Latter sites

9 may be places where Ap might persist. Egenvall et al. (2000b) found that Ap persisted in dogs blood and Franzen et al. (2009) provided evidence for persistence of the bacterium for up to 4 months (at least 129 days) in the blood of horses recovering from acute equine granulocytic anaplasmosis. In the latter study, the horses did not have clinical signs of chronic disease. Ap has been seen to persist in blood, but lately also in dogs skin. Results of a study by Berzina et al. (2014) support that Ap can persist in tissues as the authors took skin biopsies from lesional skin sites. Reasons for the lesions were unknown after pathological and other examinations, but as the dogs were seropositive for Ap, they examined the skin biopsies with serological and PCR tests. They found that Ap can persist in the skin of seropositive dogs that are otherwise clinically normal, but are seropositive for Ap and have chronic skin lesions.

2.7 Treatment

Anaplasmosis can be treated, but it may become latent and re-infect the host later on (Barth et al. 2012) so follow-up studies can be recommended. Ap infection is treated with antibiotics. Doxycycline for 30 days at a dosage of 5-10 mg/kg twice a day has been usually given and is the first choice treatment for anaplasmosis (Greig & Armstrong 2006).

Doxycycline has been known to increase liver enzyme activities when treated for anaplasmosis, and imidocarb may be then an option for treatment with a dosage of 6 mg/kg SC once a day. If a sick dog has some other signs, they should be also treated, e.g. vomiting and regurgitation with antiemetics, H+ -blockers like ranitidine and metoclopramide (Kohn et al. 2008). Immune compromised dogs may benefit of corticosteroid treatment. Kohn et al. (2008) treated in their study Ap infected dogs with suspected immune-mediated thrombocytopenia with prednisolone for 7 or 14 days (dosage of 1 mg/kg PO q12h or q24h) tapering it later down after the dogs were clinically better. In the same study a lame dog, with reactive polyarthritis, who did not respond to a 6 day NSAID and doxycycline treatment, was given prednisolone at a dosage of 1 mg/kg PO q12h and it improved quickly.

2.8 Ixodes ricinus, the vector

I. ricinus is the only known vector that transmits Ap from one animal to another in Europe (Jaenson & Lindgren 2011). The most important life limiting factor in ticks’ life in Norther Europe is the temperature which is required for the normal cycle of tick’s different life stages. The stages are egg, larva, nymph and adult. If the climate is optimal the tick can go through its life cycle in one summer. In Finland ticks life cycle takes usually three years and at cold times it overwinters. To go through every stage of life cycle the tick requires a blood meal. Adult females lay eggs near the place where they have detached from the host, so usually ticks are most commonly localized in their hosts’ habitats (Radolf et al. 2012).

I. ricinus is widely spread in Europe and is found through Europe in almost every country. The tick lives in mixed forests, open pastures and moist areas with high humidity. Ap is transmitted to animals by ticks when the daily mean temperature is ≥ 5°C, hence during the vegetation period when ticks are active. Vegetation period is approximately 170 days long in Scandinavia, from spring to autumn. Climate change elongates the vegetation period making spring and autumn weather more suitable for ticks to reproduce for a longer time. During this century climate change has increased tick populations due to prolonged vegetation period in Finland (Jaenson & Lindgren 2011). Also Dautel et al. (2008) came to the conclusion that climate change affects seasonal tick phenology. Already ticks have been found from Northern parts of Sweden at latitude of 68°N (Jaenson et al. 2012) so in the future they will presumably be found even further north. By the end of this century ticks and their pathogens may encompass most of Scandinavia all the way to 70°N, except the mountain regions where vegetation period remains shorter. Hence the risk of being infected by a tick has increased in Finland and elsewhere in Northern Scandinavia (Jaenson & Lindgren 2011). Ticks transmit Ap, so it is presumable that the bacterium will encompass Finland as climate change encompasses ticks towards Northern parts of Finland. In Figure 2 the geographic distribution of Ixodes ricinus in Europe and Western Russia is shown.

Figure 2. The current known distribution of I. ricinus in Europe and in Western Russia officially recorded by the European Center for Disease Control (ECDC) in January 2016. Red areas in the map mark for the distribution.

3 MATERIAL AND METHODS

3.1 Dogs

Three hundred and ninety Finnish dogs were tested for infection and antibodies against Ap. Group 1 included 340 sick or healthy pet dogs and group 2 included 50 healthy hunting dogs. All samples from both groups were collected between August and December after the tick season during the years 2010-2012.

From group 1, two hundred and nineteen blood samples were collected from sick or healthy dogs that came to Helsinki University’s Veterinary Teaching Hospital and from veterinary clinics around Finland, including the Åland islands. Approximately 50

12 veterinarians/clinics were contacted via e-mail, letter or phone. The veterinarians were selected randomly from different randomly selected cities around the country. Contact information was obtained from the internet (www.google.fi and www.fonecta.fi, key words: eläinlääkäri, suomi, kunnaneläinlääkäri, yhteystiedot). Twenty nine veterinarians or veterinary clinics of the contacted clinics were willing to participate to the study. They received prepaid envelopes that included the following: information of the study, instructions on taking the samples, needed tubes (ethylenediaminetetra- acetic acid (EDTA) and serum) and questionnaires for the owners, where this information was requested: dog’s name, breed, age, sex status, health status, size, date of collection, address/zip code and travel history outside Finland. Veterinarians were asked to collect the samples during one to two weeks after receiving the instructions, in order to avoid any patient selection, and between August and December, so all the samples would be collected during the same season. The country was divided in to 6 areas (South, East, West, Oulu, Lapland, Åland islands) as seen in Figure 3. In Attachment 1 is the letter and questionnaire for the clinics and owners.

In addition, 121 samples for group 1 were collected in autumn 2010 and 2011 between August and December. They were gathered by the research group “Canine and feline models of human inherited disorders” led by Hannes Lohi, PhD, Professor of Veterinary Molecular Genetics.

For group 2, fifty hunting dog blood samples were collected between August and December 2012. Fourteen of the hunting dog blood samples were collected at a dog show in August 2012. The rest of the samples were collected later, during the autumn and early winter at hunting competitions. The following information was requested from each hunting dog: name, breed, age, sex status, date of collection and home city.

Samples were taken by cephalic or jugular venipuncture in sterile EDTA and sera tubes. Serum was separated in to a sterile tube and all samples (EDTA and sera) were stored at -20 °C until analysis. Approximately 100 µl from each whole blood sample were used to extract the DNA using a commercial kit.

Inclusion criteria for hunting dogs were that they had no clinical signs of illness and had not been abroad.

The study protocol was approved by the Laboratory Animal Board of the Southern Finland Regional State Administrative Agency.

3.2 Ap: serology, DNA extraction, real-time -PCR

Diagnosis of seropositivity was performed through antibody detection in serum using a commercially available SNAP 4Dx® (ELISA) (IDEXX, Laboratories, Inc., USA) that detects antibodies against Ap and Ehrlichia canis, as well as C6 antibodies against Borrelia burgdorferi (Bb) and antigens of Dirofilaria immitis. SNAP 4Dx® was used according to manufacturer’s instructions. In brief, serum samples were first equilibrated at room temperature (18–25°C) for 30 minutes, before 3 drops of sample were pipetted into a new sample tube and then 4 drops of conjugate were added to the sample tube. The sample tube was mixed and then the entire content of the sample tube was added to the sample well. After color appeared in the activation circle, the activator was pushed down. Results were read at 8 minutes. SNAP 4Dx tests were performed by University Lecturer, PhD Sami Junnikkala from Helsinki University.

Diagnosis of Ap-infection was performed by me under the supervision of my supervisor, DVM, PhD, Dipl. ACVIM & ECVIM Cristina Perez Vera, and Research Assistant Kirsi Aaltonen from Haartman Institute using real-time -PCR. At first, DNA extraction of 100 µl of EDTA blood was performed using DNeasy® Blood and Tissue Kit according to the manufacturer’s instructions (Non-nucleated blood protocol, QIAGEN, Germany) and stored at -20 °C until further analysis. Genus-specific primers were used to amplify DNA from the samples. For real-time-PCR the primer sets msp2con33f (5’- GAAGATGAWGCTGATACAGTA-3’) (Oligomer Oy, Helsinki, Finland) and msp2con151r (5’-CAACHGCCTTAGCAAACT-3’) (Oligomer Oy, Helsinki, Finland) were used to amplify a fragment of Ap DNA. In each sample 5 µl of the DNA extracted from each sample was added to 15 µl of reaction mixture. The final reaction mixture had a volume 20 µl which comprised of 5 µl of the sample DNA, 1,5 µM of each primer, 0,5 µM of probe

14 msp2 con86p (6’-FAM-TTATCAGTCTGTCCAGTAACA-TAMRA) (Oligomer Oy, Helsinki, Finland), 10 µl of MasterMix (qPCR ToughMixTM UNG, Quanta BioSciences IncTM) and 3,8 µl of purified water.

Real-time -PCRs were ran under the following thermal cycle conditions: initial activation for 1 min at 95°C, followed by 50 cycles of denaturation step for 10 s at 94°C, an annealing step for 10 s at 53°C and extension-measurement step for 8 s at

72°C. Samples passing the threshold before Ct 40 were considered positive.

False-positive results were avoided by using a positive control; purified PCR-clean water was used as a negative control to indicate any contamination of reagents and enzymes. The positive control was provided by Professor Olli Vapalahti’s zoonotic disease research group (Haartman Institute, Helsinki, Finland). Positive and negative controls were included in each reaction group. Filter tips were used in all stages of DNA extraction and real-time –PCR.

3.3 Statistical analysis

Summary statistics were provided for all of the study variables by status yes/no. The summary statistics included frequencies and percentages. The effects of predetermined factors (sex age-group, size-group, region, travel history and health status) on the prevalence of anaplasmosis were assessed with logistic regression models. Each factor was first analyzed separately with univariate logistic regression models. Each model included only one factor at hand as fixed effect. The factors shown to be meaningful in the univariate models (p-value <0,1) were put into a multivariable logistic regression model. Significant variables at the univariate analysis were then individually entered into a multivariable logistic regression analysis and significance was set at p ≤0,05. The multivariable model included the main effects of all of the meaningful factors from the univariate models. With the multivariable model the possible correlations of the factors could be taken into account.

In the modeling the differences between groups were quantified with odd ratios (OR) and their 95 % confidence intervals (CI). All the models were constructed to model the risk of having anaplasmosis. All of the ORs were also constructed for that probability. All statistical analyses were done at 4Pharma Ltd using SAS® System for Windows, version 9.3 (SAS Institute Inc., Cary, NC, USA).

4 RESULTS

4.1 Dogs

Group 1: Each dog was placed into one of the 6 areas in Finland (South, East, West, Oulu, Lapland, Åland islands) depending on the zip code of its address. No zip code data was available on one dog; that sample was from Helsinki University’s Veterinary Teaching Hospital (group 1). Blood samples included 162 samples from the South (from 10 clinics), East 25 (6 clinics), West 92 (6 clinics), Oulu 28 (2 clinics), Lapland 12 (3 clinics) and Åland 20 (1 clinic). Travel history was available from 193 dogs, of which 27 had travelled abroad (Sweden, Norway, Denmark, Russia, Baltic countries, Germany, Italy, Belgium, Spain, England, France, Poland, Slovakia, Slovenia and USA) and these dogs were included in the study. Group 1 included dogs of 98 different breeds. Other demographic information collected from the questionnaires and their statistical analyses are available in Tables 2 and 3.

Table 2. Frequency table of each factor (region, age, sex, size, travelled abroad and health status) with being seropositive to Ap (seropositivity yes/no) of group 1. Data is presented as number of dogs and percentage (%). Univariate analysis. Statistically significant results are highlighted in bold. Analogous data is available in Perez et al. 2014 article. Factor Parameter Seropositive (n=18) Seronegative (n=322) Univariate n (%) n (%) p-value Region <0,0001 South 2 (1,2) 160 (98,8) East 1 (4,0) 24 (96,0) West 6 (6,5) 86 (93,5) Oulu 0 28 (100,0) Lapland 0 12 (100,0) Åland 9 (45,0) 11 (55,0) Missing data 1

Age <2 years 5 (4,7) 102 (95,3) 0,5001 2-8 years 9 (4,8) 179 (95,2) >8 years 4 (9,1) 40 (90,9) Missing data 1 Sex Male Intact 4 (2,9) 133 (97,1) 0,2488 Male castrated 2 (12,5) 14 (87,5) Female intact 6 (4,0) 145 (96,0) Female spayed 2 (9,5) 19 (90,5) Missing data 4 (26,7) 11 (73,3) Size <10 kg 8 (12,1) 58 (87,9) 0,0196 10-25 kg 5 (6,0) 78 (94,0) >25 kg 4 (2,3) 171 (97,7) Missing data 1 (6,2) 15 (93,8) Travelled 0,4890 Yes 3 (14.8) 24 (85,2) abroad No 15 (9,0) 151 (91,0) Missing data 147 Health status Healthy 9 (3,2) 268 (96,8) - Sick 6 (10,0) 54 (90,0)

Missing data 3

Table 3. Pairwise comparisons, multivariable logistic regression analysis results. Statistically significant results are highlighted in bold. Results for group 1. Estimate Standard Comparison 95% CI p-value (OR) error Lower Upper <10 kg vs 10-25 kg 1,943 0,713 0,478 7,902 0,352 <10 kg vs >25 kg 6,190 0,732 1,468 26,107 0,013 10-25 kg vs >25 kg 3,185 0,762 0,711 14,278 0,130 <10 kg vs atleast 10 kg 3,167 0,587 0,997 10,060 0,051 East vs South 3,926 1,259 0,330 46,731 0,278 West vs South 6,826 0,840 1,308 35,624 0,023 Åland vs South 92,149 0,916 15,191 558,991 <0,001 West vs East 1,739 1,123 0,191 15,855 0,623 Åland vs East 23,472 1,178 2,311 238,428 0,008 Åland vs West 13,500 0,718 3,286 55,456 <0,001 Åland vs rest of Finland 34,267 0,637 9,791 119,935 <0,001

Group 2: 50 hunting dogs off which 44 dogs were from South and six from the West. Group 2 had nine different breeds. Most of the hunting dogs were Finnish hounds (34 of 50), other breeds were Jämtland spitz, Finnish spitz, Labrador retriever, English springer spaniel, German shorthaired pointer, German hunting terrier, West Siberian Laika and working Jack Russel. From group 2 no statistical analysis could be done due to insufficient demographic information.

4.2 Ap-seroprevalence

Group 1: The results and seroprevalence of Ap-infection are shown in Tables 2 and 3. For group 1 the seroprevalence was 5,3% (18 of 340 dogs). If Åland dogs would be excluded from this group, the seroprevalence would be 2,2% (7/320). If travelled Ap- seropositive Åland dogs (n=3) would be excluded from Åland dogs (n=20), the seroprevalence for Åland dogs would be 35,3% (6/17).

Group 2: In group 2 there were 2 Ap-seropositive dogs and seroprevalence for the hunting dogs was 4,0% (2/50).

Groups 1 & 2: In the whole country there were 20 Ap-seropositive dogs: 18 dogs from group 1 and 2 dogs from group 2. Seroprevalence for the whole country was 5,1% (20/390). If Åland dogs would be excluded from the whole country result, the seroprevalence would be 3,0% (11/370). Of these 11 dogs none had been abroad and 7 were clinically healthy so 1,9% (7/370) of Finnish dogs were seropositive for Ap and clinically healthy without ever been abroad. In Figure 3 is a map showing Ap-infection and seropositive areas/dogs.

Figure 3. Finland divided in to six regions. Anaplasma-seropositives are marked as green circles. Anaplasma-PCR-positives are marked as yellow triangles. Groups 1 and 2.

4.3 Ap-infection

There were two Ap real-time –PCR-positive dogs in this study, one from each group. Both of the PCR-positive dogs were seronegative for Ap, clinically healthy and had not been abroad. The other PCR-positive dog from group 1 was from Åland and the PCR- positive hunting dog was from Southern Finland (Figure 3). Prevalence for Group 1 is 0,3% (1/340) and Group 2 it is 2% (1/50). For the whole country the prevalence is 0,5% (2/390).

4.4 Statistical analysis

Results for statistical analysis of group 1 are shown in Tables 2 and 3. Region and size were the factors associated with Ap-exposure. Given that the number of infected dogs was so low, no statistical analyses are available for the risk factors of infection, only exposure. Group 2, the hunting dogs, was not included in the statistical analysis as not all the demographic information was available for them.

5 DISCUSSION

This is the first study investigating purely the prevalence of Ap in Finnish dogs. The results of this study suggest that the presence of Ap-antibodies is not uncommon in Finnish dogs and that the bacterium is present not only in Åland and its dogs, but also in other regions of Finland. The overall seroprevalence of anaplasmosis in Finland was 5,1% (Groups 1 and 2, 20/390). For group 1 the seroprevalence was 5,3% (18/340). On the contrary to the study’s hypothesis, the prevalence 4,0% for hunting dogs (Group 2, 2/50) was less than group 1’s seroprevalence 5,3%. Possible explanations might be group size difference (Group 1 n=340, Group 2 n=50) and that most hunting dogs were from South Finland but none from Åland. Also cross reactions to A. platys, E. canis, E. ewingii and E. chaffeensis are theoretically possible when using the SNAP 4Dx (Greig & Armstrong 2006) and they can affect the results. Cross reactions are also possible in real-time -PCR (Kohn et al. 2011). However these mentioned bacteria are not endemic

20 in Finland, so the possibility of cross reaction in SNAP 4Dx and real- time -PCR in this study is rather unlikely. The seropositive dogs from group 1 were from East, South, West Finland and Åland, and seropositives from group 2 were from South (Fig. 3). As all hunting dog samples were from South and West and only 50 samples were collected, the amount of the samples and the area where they were collected from, was rather small. The results may have been different with a larger group size and larger collecting area. On the other hand, Åland dogs in group 1 raised the prevalence of group 1 and if Åland dogs would have been excluded the seroprevalence would have been 2,8% for group 1 (9/320), versus seroprevalence 4,0% of group 2. This would have indicated that hunting dogs indeed may be more susceptible for Ap infections.

Ap-seroprevalence for dogs from Åland was 45%, which is remarkably higher than elsewhere in Finland (group 1, 2,8%, Åland excluded). But in Åland the travel history/being abroad of the dogs may have affected the prevalence, since the proportion of seropositive dogs was 35,3% when dogs with a travel history were excluded from calculations. In continental Finland travel history had no effect and the Ap-seroprevalence remained to be 2,8% since none of the seropositive dogs in group 1 had travelled (according to information given by the owners). These results suggest that Ap is endemic in Åland and the bacterium is fairly common in the islands’ dogs and perhaps also in people. Åland is also known to be an island that has large population of I. ricinus ticks that transmit zoonoses (Mäkinen et al. 2003). This may be compromising for dog owners also when assessing the risks of getting a tick borne disease like anaplasmosis. So more studies should be made, possibly one study of Åland dogs alone and another of continental Finland’s dogs (pet dogs vs. hunting dogs); this might give more information on the situation.

In this study the total seroprevalence was 5,1% (groups 1 + 2). In other European countries Ap-seroprevalence has grown in the 21st century as Table 1 shows. In Germany, SNAP 4Dx-seroprevalence for Ap was 21,5% a few years ago (Krupka et al. 2007). A more recent study by Kohn et al. (2011) revealed an IFA-seroprevalence of 43%. On the other hand Barth et al. (2012) tested the SNAP 4Dx-seroprevalence to be

19,4% in Germany. The difference of the results may be partly because of the use of different diagnostic methods or because of the recruitment of sera from different regions in Germany. Also in Sweden Ap-seroprevalence has increased in the last decade. Egenvall et al. (2000a) reported a seroprevalence of 17,7% and Jäderlund et al. (2007) of 20,7%. Same kind of changes might be expected in Finland as climate is changing, people travel more with their dogs and spend lots of time in nature with their dogs (Godfrey & Randolph 2011). The current study can serve as the base for future studies when assessing a similar rise of Ap infections in Finnish pet and hunting dogs.

In addition to tick bites, Ap may also be transmitted via blood transfusion (Kohn et al. 2011). In Finland blood donor dogs are tested with SNAP 4Dx when they donate their blood at Helsinki University’s Veterinary Teaching Hospital. This has been the protocol since the year 2011 (oral information from Helsinki University Veterinary Hospital). In our study, no information was collected regarding possible previous blood transfusions in any of our dogs included. But also, given the regular tick borne disease testing, it is unlikely that a dog acquires anaplasmosis via blood transfusion.

Blood samples for this study were collected during August to December in years 2010- 2012. Part of the samples was collected during vegetation period and the rest of them after it in order to get samples from infected dogs after tick infestation. The results of this study might have been different if the samples had been collected at a different time of the year. Ap has been found to cause persistent infection in dogs (Egenvall et al. 2000b, Franzen et al. 2009, Berzina et al. 2014). For this reason collecting the blood samples again the following winter time might have given us a better picture on the possibility of the persistence of Ap-infections in dogs in Finland. This possibility that some dogs would have become seronegative, and that some seronegative would have become seropositive in the winter, cannot be ruled out in our study, since no follow-up could be done. Also, no information was collected from the owners whether their dogs had been bitten by ticks or whether they received ectoparasite prevention. This may have an effect to the possibility of becoming infected by tick borne disease.

For this study it was not possible to collect follow-up information of those 2 real-time - PCR-positive dogs. Both PCR-positive dogs were clinically healthy and had not been abroad, but due to lack of follow-up, it is not known if they developed clinical signs or antibodies, and how long they produced antibodies if they did. A tick infects a dog within hours after infestation and anchoring (Scorpio et al. 2011), and a dog produces antibodies 8-30 days after infection (Egenvall et al. 1998, Scorpio et al. 2011). Serologic studies show that dogs that were experimentally infected by Ap produced antibodies up to 4 months after infection (Egenvall et al. 2000b) and dogs that were naturally infected produced antibodies up to 12 months after infection (Egenvall et al. 1997). Seronegative results were diagnosed 22-36 months after Ap-infection (Eberts et al. 2011) so follow-up studies (SNAP 4Dx and real-time -PCR) would have given a better picture of the infection and its progress in this study.

Previously it has been suggested that subclinical diseases and silent elimination may be common, since Ap-seropositive healthy dogs have been reported (Carrade et al. 2009, Kohn et al. 2011). In this study the two PCR-positive dogs were Ap-seronegative. Previously Kohn et al. (2011) found out that 26,7% of PCR-positive dogs were seronegative (IFA). These dogs may have had an acute infection or the infection was subclinical. In the future studies follow-up samples of dogs and their clinical status should be made in order to get knowledge on how much the infection persists and what kind of diagnostic tests vets should do and how often to take them in the field to catch tick borne diseases.

Univariate analysis of frequencies on each factor (region, age, sex, size, travelled abroad and health status) is shown in Table 2. Multivariable logistic regression analysis is shown in Table 3. Of the information that was asked from the owners and gathered from the dogs, two factors, region and size, were statistically significant in this analysis (Group 1). In group 1 anaplasmosis was significantly more common in Åland than in East, South, West and rest of Finland. In West there were significantly more seropositive dogs than in South. Small dogs (less than 10 kg) were more often seropositive than large dogs (over 25 kg). Statistical analysis on group 2 could not be done, so groups are incomparable to each other at this stage.

There was no statistical significance on the travel history, health status or sex, but for region and size. The results of the regional differences suggest that pet owners should carefully consider the increased risk of contact with infected ticks when living or travelling in Åland should use ectoparasitic treatments for exposed dogs during the vegetative season. To the authors knowledge no body size predisposition has been shown in previous studies. In some studies it has been suggested that older dogs have been more often seropositive than younger (Egenvall et al. 2000a, Kohn et al. 2011) but size has not been earlier acknowledged to have an effect of becoming infected. Small dogs may be susceptible to tick infestation as they may be closer to ticks habitat when going out with their owners as ticks live in forests, open pastures and grass areas (Jaenson & Lindgren 2011).

As seen in this study a dog can be Ap-seropositive or real-time –PCR-positive (have an acute infection) without any clinical signs and without being abroad. This statistically insignificant finding in this study seems still to be in line with other studies that suggest/say that a dog can have Ap infection without any clinical signs (Jensen et al. 2007, Carrade et al. 2009, Kohn et al. 2011). However, some studies have reported that Ap infection can persist and cause chronic health problems (Egenvall et al. 2000b, Berzina et al. 2014). If the infection persists in dogs, signs may fluctuate causing some difficulties for the clinicians to establish a diagnosis. This leads to the demand for performing follow-up examinations if dogs are presented in endemic areas with fluctuating clinical signs.

As a result of this study it is advisable that future studies collect information from the owners about prophylactic ectoparasite treatments and about the burden of tick infestation noticed during the vegetation period. This way it will be possible to evaluate how heavily the dogs really have been exposed to ticks and which dogs are at higher risk of getting anaplasmosis. On the other hand, it has been previously studied that dogs that were treated against tick infestations were as seropositive as untreated dogs, regardless of ectoparasitic treatments. Also in the same study the researchers

24 noticed that dogs having been infested by ticks were as seropositive as dogs which according to their owners did not have ticks (Kohn et al. 2011).

Ap is an emerging pathogen and it should be noticed when travelling to tick rich areas or treating a patient with anaplasmosis-type clinical signs. Finnish people like to walk in the woods and nature and go to their summer cottages. Thousands of Finnish citizen hunt (approximately 300 000 have a license to hunt) and own hunting dogs. This leads to a situation where dogs and their owners have an increased risk of getting bitten by ticks and acquiring tick borne infections. Since dogs live in close proximity of people they can also transmit ticks to humans. Therefore it is important to intensify studies about zoonotic tick borne diseases in Finnish dogs.

6 CONCLUSIONS AND CLINICAL RELEVANCE

This study suggests that canine anaplasmosis has the potential to become an important tick borne disease in Finnish dogs. The results show that dogs are commonly exposed to this tick borne disease especially in Åland islands, and this suggests that people in Åland are also at an increased risk to Ap infection. It is unclear at this point if the prevalence of Ap-exposure will increase in other parts of the country. However, given the climatic change and the northern spread of Ixodes ticks it must be considered to be likely or even inevitable.

Even though Finnish dogs are exposed to Ap, this study showed that the prevalence of the actual infection seems to be rather low. Because of this low number of Ap-positive dogs in real-time -PCR, no risk factors for infection could be identified in our study. Therefore, future studies with larger number of infected dogs and bigger groups are necessary to better understand the risk factors associated with Ap-infections in dogs and in people.

7 ACKNOWLEDGEMENTS

I would like to thank my supervisor Cristina Pérez Vera, DVM, PhD, Diplomate of ACVIM & ECVIM, for her advice, directions and patience, and also for being a great teacher. I would also like to thank the director of my thesis, Professor Thomas Spillmann, and my teacher, PhD Sami Junnikkala for their help and advice, and my gratitude goes also to the staff of Veterinary Teaching Hospital and Haartman Institute, especially Kirsi Aaltonen from the Haartman Institute. Also I am very grateful for the dog owners and veterinarians who participated to this study. I would also like to thank 4Pharma! and Jouni Junnila for SNAP 4 Dx tests.

8 REFERENCES

Alekseev A, Dubinina H, Van De Pol I, Schouls LM. Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J Clin Microbiol 2001, 39: 2237-2242.

Bakken J, Dumler J, Chen SM, Eckman M, Van Etta L, Walker D. Human Granulocytic Ehrlichiosis in the Upper Midwest United States - A New Species Emerging? JAMA 1994, 272: 212-218.

Baumgarten B, Röllinghoff M, Bogdan C. Prevalence of Borrelia burgdorferi and granulocytic and monocytic ehrlichiae in Ixodes ricinus ticks from southern Germany. J Clin Microbiol 1999, 37: 3448-3451.

Barth C, Straubinger R, Sauter-Louis C, Hartmann K. Prevalence of antibodies against Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum and their clinical relevance in dogs in Munich, Germany. Berl Munch Tierarztl Wochenschr 2012, 125: 337-344.

Barutzki D, De Nicola A, Zeziola M. Reule M. Seroprevalence of Anaplasma phagocytophilum infection in dogs in Germany. Berl Munch Tierarztl Wochenschr 2006, 119: 342-347.

Beall MJ, Chandrashekar R, Eberts MD, Cyr KE, Diniz PP, Mainville C, Hegarty BC, Crawford JM, Breitschwerdt EB. Serological and molecular prevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia species in dogs from Minnesota. Vector Borne Zoonotic Dis 2008, 8: 455-464.

Berzina I, Matise I. Seroprevalence against Borrelia burgdorferi sensu lato and occurence of antibody co-expression with Anaplasma phagocytophilum in dogs in Latvia. Ir Vet J 2013, 66: 9.

Berzina I, Krudewig C, Silaghi C, Matise I, Ranka R, Müller N, Welle M. Anaplasma phagocytophilum DNA amplified from lesional skin of seropositive dogs. Ticks Tick Borne Dis 2014, 5: 329-335.

Bjöersdorff A, Svendenius L, Owens J, Massung R. Feline granulocytic ehrlichiosis - a report of a new clinical entity and characterisation of the infectious agent. J Small Anim Pract 1999, 40: 20-24.

Bjöersdorff A, Bergström S, Massung RF, Haemig D, Olsen B. Ehrlichia-infected ticks on migrating birds. Emerg Infect Dis 2001, 7: 877-879.

Carrade D, Foley J, Börjesson D, Sykes J. Canine granulosytic anaplasmosis: a review. J Vet Intern Med 2009, 23: 1129-1141.

Carrade D, Foley J, Sullivan M, Foley CW, Sykes JE. Spatial distribution of seroprevalence for Anaplasma phagocytophilum, Borrelia burgdorferi, Ehrlichia canis, and Dirofilaria immitis in dogs in Washington, Oregon, and California. Vet Clin Pathol 2011, 40: 293-302.

Chang Y, Novosel E, Dubovi S, Wong F, Chu C, Chang F, Del Piero S, Shin S, Lein D. Experimental infection of the human granulocytic ehrlichiosis agent in horses. Vet Parasitol 1998, 78: 137-145.

Choi K, Garyu J, Park J, Dumler J. Diminished adhesion of Anaplasma phagocytophilum–infected neutrophils to endothelial cells is associated with reduced leukocyte surface selectin expression. Infect Immun 2003, 71: 4586–4594.

Chomel B. Tick-borne infections in dogs -an emerging infectious threat. Vet Parasitol 2011, 179: 294-301.

Dautel H, Dippel C, Kämmer D, Werkhausen A, Kahl O. Winter activity of Ixodes ricinus in a Berlin forest. Int J Med Microbiol 2008, 298: 50-54.

Diniz P, Beall M, Omark K, Chandrashekar R, Daniluk DA, Cyr KE, Koterski JF, Robbins RG, Lalo PG, Hegarty BC, Breitschwerdt EB. High prevalence of tick-borne pathogens in dogs from an Indian reservation in northeastern Arizona. Vector Borne Zoonotic Dis 2010, 10: 117–123.

Dumler J, Barbet A, Bekker C, Dasch G, Palmer G, Ray S, Rikihisa Y, Rurangirwa F. Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila. Int J Syst Evol Microbiol 2001, 51: 2145-2165.

Dumler JS, Madigan JE, Pusterla N, Bakken JS. Ehrlichiosis in humans: epidemiology, clinical presentation, diagnosis, and treatment. Clin Infect Dis 2007, 45: 45-51.

Ebani VV, Bertelloni F, Turchi B, Cerri D. Serological and molecular survey of Anaplasma phagocytophilum in Italian hunting dogs. Ann Agric Environ Med 2013, 20: 289-292.

Eberts M, Diniz P, Beall M, Stillman B, Chandrashekar R, Breitschwerdt E. Typical an atypical manifestations of Anaplasma phagocytophilum infections in dogs. J Am Anim Hosp Assoc 2011, 47: 86-94.

ECDC. European Centre of Disease Prevention and Control. Tick maps. http://ecdc.europa.eu/en/healthtopics/vectors/vector-maps/Pages/VBORNET-maps- tick-species.aspx , haettu 15.2.2016.

Egenvall A, Hedhammar A, Bjöersdorff A. Clinical features and serology of 14 dogs affected by granulocytic ehrlichiosis in Sweden. Vet Rec 1997, 140: 222-226.

Egenvall A, Bjöersdorff A, Lilliehöök I, Olsson Engvall E, Karlstam E, Artursson K, Hedhammar A, Gunnarsson A. Early manifestations of granulocytic ehrlichiosis in dogs inoculated experimentally with a Swedish Ehrlichia species isolate. Vet Rec 1998, 143: 412-417.

Egenvall A, Bonnett B, Gunnarsson A, Hedhammar A, Shoukri M, Bornstein S, Artursson K. Sero-prevalence of granulocytic Ehrlichia spp. and Borrelia burgdorferi sensu lato in Swedish dogs 1991-94. Scand J Infect Dis 2000a, 32: 19-25.

Egenvall A, Lilliehöök I, Bjöersdorff A, Engvall E, Karlstam E, Artursson K, Heldtander M, Gunnarsson A. Detection of granulocytic Ehrlichia species DNA by PCR in persistently infected dogs. Vet Rec 2000b 146: 186-190.

Farkas R, Gyurkovszky M, Lukacs Z, Aladics B, Solymosi N. Seroprevalence of some vector-borne infections of dogs in Hungary. Vector Borne Zoonotic Dis 2014, 14: 256- 260.

Franzén P, Aspan A, Egenvall A, Gunnarsson A, Karlstam E, Pringle J. Molecular evidence for persistence of Anaplasma phagocytophilum in the absence of clinical

29 abnormalities in horses after recovery from acute experimental infection. J Vet Intern Med 2009, 23: 636-642.

Ge Y, Yoshiie K, Kuribayashi F, Lin M, Rikihisa Y. Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation. Cell Microbiol 2005, 7: 29–38.

Godfrey E, Randolph S. Economic downturn results in tick-borne disease upsurge. Parasit Vectors 2011, 4: 35.

Granquist EG, Aleksandersen M, Bergström K, Dumler SJ, Torsteinbø WO, Stuen S. A morphological and molecular study of Anaplasma phagocytophilum transmission events at the time of Ixodes ricinus tick bite. Acta Vet Scand 2010, 52: 43.

Greene CE, editor: Infectious diseases of the dog and cat. 4th ed. Saunders Elsevier, St. Louis 2012.

Greig B, Armstrong P. Canine granulocytotropic anaplasmosis (A. phagocytophilum infection). In Greene CE (ed). Infectious diseases of the dog and cat. 3 rd ed. Saunders Elsevier, St. Louis 2006: 219-224.

Hamel D, Silaghi C, Knaus M, Visser M, Kusi I, Rapti D, Rehbein S, Pfister K. Detection of Babesia canis subspecies and other arthropod-borne diseases in dogs from Tirana, Albania. Wien Klin Wochenschr 2009, 121: 42-45.

Heikkilä H, Bondarenko A, Mihalkov A, Pfister K, Spillmann T. Anaplasma phagocytophilum infection in a domestic cat in Finland: Case report. Acta Vet Scand 2010, 52: 62.

Hulinska D, Langrova K, Pejcoch M, Pavlasek I. Detection of Anaplasma phagocytophilum in animals by real-time polymerase chain reaction. APMIS 2004, 112: 239-247.

Jaenson TG, Lindgren E. The range of Ixodes ricinus and the risk of contracting Lyme borreliosis will increase northwards when the vegetation period becomes longer. Ticks Tick Borne Dis 2011, 2: 44-49.

Jaenson TG, Jaenson DG, Eisen L, Petersson E, Lindgren E. Changes in the geographical distribution and abundance of the tick Ixodes ricinus during the past 30 years in Sweden. Parasit Vectors 2012, 5: 8.

Jenkins A, Kristiansen B, Allum A, Aakre R, Strand L, Kleveland E, van de Pol I, Schouls L. Borrelia burgdorferi sensu lato and Ehrlichia spp. in Ixodes ticks from southern Norway. J Clin Microbiol 2001, 39: 3666-3671.

Jensen J, Simon D, Murua Escobar H, Soller J, Bullerdiek J, Beelitz P, Pfister K, Nolte I. Anaplasma phagocytophilum in dogs in Germany. Zoon & Publ Health 2007, 54: 94- 101.

Jäderlund K, Egenvall A, Bergström K, Hedhammar A. Seroprevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in dogs with neurological signs. Vet Rec 2007, 160: 825-831.

Kallio E, Begon M, Birtles R, Bown K, Koskela E, Mappes T, Watts PC. First Report of Anaplasma phagocytophilum and Babesia microti in Rodents in Finland. Vector Borne Zoonotic Dis 2014, 14: 389-393.

Kirtz B, Czettel B, Thum D, Leidinger E. Anaplasma phagocytophilum in einer österreichischen Hundepopulation: eine Prävalenz-studie (2001–2006). Kleintierpraxis 2007, 52: 562-568.

Kohn B, Galke D, Beelitz P, Pfister K. Clinical features of canine granulocytic anaplasmosis in 18 naturally infected dogs. J Vet Intern Med 2008, 22: 1289-1295.

Kohn B, Silaghi C, Galke D, Arndt G, Pfister K. Infections with Anaplasma phagocytophilum in dogs in Germany. Res Vet Sci 2011, 91: 71-76.

Krupka I, Pantchev N, Lorentzen L, Weise M, Straubinger RK. Tick-transmitted, bacterial infections in dogs: seroprevalences of Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis in Germany. Prakt Tierarzt 2007, 88: 776- 788.

Laakkonen J, Terhivuo J, Huhtamo E, Vapalahti O, Uzcátegui N. First report of Ixodes frontalis (Acari: Ixodidae) in Finland, an example of foreign tick species transported by a migratory bird. Mem Soc Fauna Flora Fenn 2009, 85: 16–19.

Lin M, Rikihisa Y. Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Infect Imm 2003, 71: 5324–5331.

Liz JS, Anderes L, Sumner JW, Massung RF, Gern L, Rutti B, Brossard M. PCR detection of granulocytic ehrlichiae in Ixodes ricinus ticks and wild small mammals in western Switzerland. J Clin Microbiol 2000, 38: 1002-1007.

Mircean V, Dumitrache MO, Györke A, Pantchev N, Jodies R, Mihalca AD, Cozma V. Seroprevalence and geographic distribution of Dirofilaria immitis and tick-borne infections (Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, and Ehrlichia canis) in dogs from Romania. Vector Borne Zoonotic Dis 2012, 12: 595-604.

Miro G, Montoya A, Roura X, Galvez R, Sainz A. Seropositivity rates for agents of canine vector-borne diseases in Spain: a multicentre study. Parasit Vectors 2013, 6: 117.

Mäkinen J, Vuorinen I, Oksi J, Peltomaa M, He Q, Marjamäki M, Viljanen MK. Prevalence of granulocytic Ehrlichia and Borrelia burgdorferi sensu lato in Ixodes ricinus ticks collected from Southwestern Finland and from Vormsi Island in Estonia. APMIS 2003, 111: 355-362.

Mäkitaipale J. Anaplasma phagocytophilum -tartunta koiralla: kaksi tapausselostusta. Suomen Eläinlääkärilehti 2011, 117: 4.

Neer TM, Breitschwerdt EB, Greene RT, Lappin MR. Consensus statement on ehrlichial disease of small animals from the infectious disease study group of the ACVIM. American College of Veterinary Internal Medicine. J Vet Intern Med 2002, 16: 309-315.

Pantchev N, Schaper R, Limousin S, Norden N, Weise M, Lorentzen L. Occurrence of Dirofilaria immitis and tick-borne infections caused by Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and Ehrlichia canis in domestic dogs in France: results of a countrywide serologic survey. Parasitol Res 2009, 105: 101-114.

Pennisi MG, Capri A, Solano-Gallego L, Lombardo G, Torina A, Masucci M. Prevalence of antibodies against Rickettsia conorii, Babesia canis, Ehrlichia canis, and Anaplasma phagocytophilum antigens in dogs from the Stretto di Messina area (Italy). Ticks Tick Borne Dis 2012, 3: 315-318.

Pérez Vera C, Kapiainen S, Junnikkala S, Aaltonen K, Spillmann T, Vapalahti O. Survey of selected tick-borne diseases in dogs in Finland. Parasit Vectors 2014, 23: 285.

Petrovec M, Lotric Furlan S, Zupanc TA, Strle F, Brouqui P, Roux V, Dumler JS: Human disease in Europe caused by a granulocytic Ehrlichia species. J Clin Microbiol. 1997, 35: 1556-1559.

Pusterla N, Pusterla JB, Deplazes P, Wolfensberger C, Müller W, Hörauf A, Reusch C, Lutz H. Seroprevalence of Ehrlichia canis and of canine granulocytic Ehrlichia infection in dogs in Switzerland. J Clin Microbiol 1998, 36: 3460–3462.

Radolf JD, Caimano MJ, Stevenson B, Hu LT. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes. Nat Rev Microbiol 2012, 10: 87-99.

Ramos RA, Latrofa MS, Giannelli A, Lacasella V, Campbell BE, Dantas-Torres F, Otranto D. Detection of Anaplasma platys in dogs and Rhipicephalus sanguineus group ticks by a quantitative real-time PCR. Vet Parasitol 2014, 205: 285-288.

Ravnik U, Tozon N, Strasek K, Avsic Zupanc T. Clinical and haematological features in Anaplasma phagocytophilum seropositive dogs. Clin Microbiol Infect 2009, 15: 39-40.

Ravyn M, Korenberg E, Oeding J, Kovalevskii Y, Johnson R. Monocytic Ehrlichia in Ixodes persulcatus ticks from Perm, Russia. Lancet 1999, Feb 353:722-723.

Reed K, Meece J, Henkel J, Shukla S. Birds, Migration and Emerging Zoonoses: West Nile Virus, Lyme Disease, Influenza A and Enteropathogens. Clin Med Res 2003, 1: 5-12.

Rizzoli A, Silaghi C, Obiegala A, Rudolf I, Hubálek Z, Földvári G, Plantard O, Vayssier- Taussat M, Bonnet S, Spitalská E, Kazimírová M. Ixodes ricinus and Its Transmitted Pathogens in Urban and Peri-Urban Areas in Europe: New Hazards and Relevance for Public Health. Front Public Health 2014, 2: 251.

Santos AS, Alexandre N, Sousa R, Nuncio MS, Bacellar F, Dumler JS. Serological and molecular survey of Anaplasma species infection in dogs with suspected tick borne disease in Portugal. Vet Rec 2009, 164: 168-171.

Scorpio D, Dumler J, Barat N, Cook J, Barat C, Stillman B, DeBisceglie K, Beall M, Chandrashekar R. Comparative strain analysis of Anaplasma phagocytophilum infection and clinical outcomes in a canine model of granulocytic anaplasmosis. Vector Borne Zoonotic Dis 2011, 11: 223-229.

Shaw S, Binns S, Birtles R, Day M, Smithson R, Kenny MJ. Molecular evidence of tick- transmitted infections in dogs and cats in the United Kingdom. Vet Rec 2005, 157: 645- 648.

Skotarczak B, Adamska M, Rymaszewska A, Supron M, Sawczuk M. Maciejewska A. Anaplasma phagocytophila and protozoans of Babesia genus in dogs from endemic areas of Lyme disease in north-western Poland. Wiad Parazytol 2004, 50: 555-561.

Solano-Gallego L, Trotta M, Razia L, Furlanello T, Caldin M. Molecular survey of Ehrlichia canis and Anaplasma phagocytophilum from blood of dogs in Italy. Ann N Y Acad Sci 2006a, 1078: 515-518.

Solano-Gallego L, Llull J, Osso M, Hegarty B, Breitschwerdt E. A serological study of exposure to arthropod-borne pathogens in dogs from northeastern Spain. Vet Res 2006b, 37: 231-244.

Stefanidesova K, Kocianova E, Boldis V, Kostanova Z, Kanka P, Nemethova D, Spitalska E. Evidence of Anaplasma phagocytophilum and Rickettsia helvetica infection in free- ranging ungulates in central Slovakia. Euro J Wildl Res 2008, 54: 519-524.

Stillman BA, Monn M, Liu J, Thatcher B, Foster P, Andrews B, Little S, Eberts M, Breitschwerdt EB, Beall MJ, Chandrashekar R. Performance of a commercially available in-clinic ELISA for detection of antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii and Dirofilaria immitis antigen in dogs. J Am Vet Med Assoc 2014, 245: 80-86.

Strle F. Human granulocytic ehrlichiosis in Europe. Int J Med Microbiol 2004, 293: 27- 35.

Stuen S. Anaplasma phagocytophilum-the most widespread tick-borne infection in animals in Europe. Vet Res Commun 2007, 31: 79-84.

Stuen S, M. Bråten M, K. Bergström K, K. Bårdsen K. Cyclic variation in lambs infected with Anaplasma phagocytophilum. Vet Rec 2008, 163: 338–340.

Torina A, Caracappa S. Dog tick-borne diseases in Sicily. Parassitologia 2006, 48: 145- 147.

Tuomi J. Studies in epidemiology of bovine tick-borne fever in Finland and a clinical description of field cases. Ann Med Exp Biol Fenn 1966, 44: 1-62.

Valkjärvi L, Karikoski N, Mykkänen A, Tulamo RM. Tapausselostus: Anaplasma phagocytophilum -tartunta hevosella. Acta Vet Fen 2010, 116:10.

Volgina NS, Romashov BV, Romashova NB, Shtannikov AV. Prevalence of borreliosis, anaplasmosis, ehrlichiosis and Dirofilaria immitis in dogs and vectors in Voronezh Reserve (Russia). Comp Immunol Microbiol Infect Dis 2013, 36: 567-574. von Stedingk L, Gürtelschmid M, Hanson H, Gustafson R, Dotevall L, Engvall E, Granström M. The human granulocytic ehrlichiosis (HGE) agent in Swedish ticks. Clin Microbiol Infect 1997, 3: 573-574.

Webster P, IJdo J, Chicoine L, Fikrig E. The agent of Human Granulocytic Ehrlichiosis resides in an endosomal compartment. J Clin Invest 1998, 101: 1932-1941.

Woldehiwet Z. The natural history of Anaplasma phagocytophilum. Vet Parasitol 2010, 167: 108-22.

Attachment 1

HELSINGIN YLIOPISTO

Tarvitsemme apuasi!!!

Tutkimusaihe: Niveljalkaisvälitteiset taudit suomalaisilla koirilla

Mikä on tutkimuksen tarkoitus?

Tutkimuksen tarkoituksena on selvittää koirien niveljalkaisvälitteisten tautien esiintyvyyttä Suomessa. Keräämme verinäytteitä (EDTA ja seerumi) tällä hetkellä koirilta ympäri Suomen.

Projekti:

Testataan eri puolilta Suomea olevia koiria puutiaisvälitteisten tautien varalta (serologia ja PCR). Koirat voivat olla sairaita tai terveitä ja matkustaneita tai ei- matkustaneita. Näytteiden keruu tapahtuu syys-joulukuussa 2012. Lähetämme tutkimukseen osallistuville klinikoille tarvittavat putket sekä vastauskuoren takaisinlähetystä varten. Tutkimukseen osallistuvista koirista tarvitsemme seuraavat tiedot: a. Signalmentti (ikä, rotu, sukupuoli) b. Sirunumero/tatuointi ja kotikaupunki c. Näytteenottopäivämäärä d. Sairas vai terve e. Matkustanut vai ei-matkustanut

Mitä puutiaisvälitteisiä tauteja tutkimme? Testaamme Snap-testillä A. phagocytophilumin, E. canisin ja B. burgdorferin vasta-aineita. Lisäksi tutkimme PCR:n avulla Bartonellaa ja A. phagocytophilumia sekä puutiaisaivokuumeviruksen (TBEV) serologiaa.

Mitä putkia tarvitsemme?

EDTA- ja seerumiputket. Olkaa hyvä, ja pitäkää näytteet (EDTA, eroteltu seerumi) jääkaapissa (tai pakastimessa), kunnes voitte lähettää ne meille. Lähetyksen tultua pakastamme näytteet ja säilytämme ne tulevia testauksia varten.

OHJEET 1. Kerätkää 1 EDTA- ja 1 seeruminäyte kustakin klinikallanne käyneestä koirasta yhden-kahden viikon aikana syys-joulukuussa. Pyydämme, että lähetätte seeruminäytteen sentrifugoituna ja eroteltuna.

2. Lähetyskuoret ovat valmiiksi maksettuja, joten postikuluja tutkimukseen osallistumisesta ei tule. Jos tarvitsette enemmän kirjekuoria, lähetämme niitä pyynnöstä lisää.

3. Neuloja emme valitettavasti pysty lähettämään. Jos tarvitsette niitä, ottakaa meihin yhteyttä.

4. Ilmoittakaa kunkin putken kanssa koiran nimi ja näytteenottopäivämäärä.

Jokainen näyte on tärkeä meille, ja toivomme saavamme niitä mahdollisimman paljon. Olkaa hyvä ja ottakaa yhteyttä, mikäli teille tulee kysymyksiä mieleen!

** Periaatteessa potilaskohtaisia testien tuloksia ei lähetetä. Jos eläinlääkäri tai omistaja haluaa tulokset, ottakaa meihin yhteyttä, niin lähetämme ne postitse tai sähköpostitse.

Kiitos jo etukäteen yhteistyöstänne!

Osoitteemme postittamista varten:

Koiratutkimus/Perez Kliinisen hevos- ja pieneläinlääketieteen osasto Tunnus 5019843 info H9013 00003 VASTAUSLÄHETYS

Lisätiedot ja yhteydenotot: Dipl. ACVIM Cristina Perez (englanniksi), eläinlääkäri ja sisätautien diplomaatti, [email protected], tai FM, ELK Suvi Kapiainen, eläinlääketieteen opiskelija. [email protected]. puh: 040-7521678.

Täytä tämä lomake kullekin koiralle

1. Nimi, sukupuoli:

2. Rotu :

3. Ikä : tunniste (siru, tatuointi):

4. Sairas tai terve?:

5. Koiran asuinkunta?:

6. Keräyspäivä:

7. Yhteystiedot (eläinlääkäri):

8. Haluatteko saada tulokset?

9. Matkailuhistoria (kesämökki, ulkomaille jne? Minne (kunta, maa)?)