S

Ac

IDa

b

c

ARRA

KBLACP

1

weavwiebp2hmB

oH

0h

Veterinary Parasitology 196 (2013) 515–518

Contents lists available at SciVerse ScienceDirect

Veterinary Parasitology

journa l homepage: www.e lsev ier .com/ locate /vetpar

hort communication

utochthonous canine babesiosis caused by Babesia canisanis in Latvia

nese Berzinaa,∗, Valentina Capliginab, Viesturs Baumanisb, Renate Rankab,ina Cirulec, Ilze Matisea

Latvia University of Agriculture, Faculty of Veterinary Medicine, Jelgava, LatviaLatvian Biomedical Research and Study Center, Riga, LatviaInstitute of Food Safety, Animal Health and Environment BIOR, Riga, Latvia

a r t i c l e i n f o

rticle history:eceived 12 January 2013eceived in revised form 4 March 2013ccepted 13 March 2013

a b s t r a c t

This is the first report of confirmed canine babesiosis in Latvia supporting the observedgeographical expansion of this disease. Between 2009 and 2011 three dogs which havenot traveled outside of Latvia were diagnosed with babesiosis. Hematological analysis andserological tests for granulocytic anaplasmosis, ehrlichiosis and borreliosis were negative(Idexx SNAP 4Dx test). Peripheral blood erythrocytes of the three dogs contained large

eywords:abesia canis canisatviautochthonous

Babesia that were identified as Babesia canis canis by PCR. Sequences of partial 18S rRNAgene were 98–100% similar to the sequences of B. canis canis isolated from dogs in otherEuropean countries. We conclude that these are the first autochthonous canine babesiosiscases reported from Latvia.

anine babesiosisCR

. Introduction

Tick-borne diseases in dogs have been diagnosedith increasing frequency worldwide. This tendency is

xplained by a variety of factors, including climate changesssociated with the expansion of the vectors and reser-oir animals, increasing proportion of traveling dogs, asell as increased awareness about these diseases and

mproved diagnostic techniques (Menn et al., 2010; Øinest al., 2010; Solano-Gallego and Baneth, 2011). Canineabesiosis can be caused by several species of hemato-rotozoal organisms of the genus Babesia (Hunfeld et al.,008; Ayoob et al., 2010). In Europe most of the cases

ave been caused by large Babesia Babesia canis canis trans-itted mainly by Dermacentor reticulatus ticks, although

abesia microti induced disease had been diagnosed in

∗ Corresponding author at: Latvia University of Agriculture, Facultyf Veterinary Medicine, Preclinical Institute, Pathology Department, Kr.elmana Street 8, Jelgava LV-3004, Latvia. Tel.: +371 26324105.

E-mail address: inese.berzina@gmail.com (I. Berzina).

304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.vetpar.2013.03.015

© 2013 Elsevier B.V. All rights reserved.

Spain (Ayoob et al., 2010; Solano-Gallego and Baneth,2011). D. reticulatus is described as established tick speciesin United Kingdom and northern part of Russian Federation(Gray et al., 2009), it has been spotted in Lithuania, coun-try that shares its Northern border with Latvia (Zygutiene,2009). Dermacentor spp. tick has been noted on one occa-sion (A. Kruklite, personal communication) and up todate is not considered to be an established tick speciesin Latvia (Bormane, 2007; Karelis et al., 2012). Caninebabesiosis was considered to be endemic in SouthernEurope but recently B. canis canis infections have beenreported in countries located relatively close to Latvia– Poland, Russia and Norway (Caccio et al., 2002; Raret al., 2005; Øines et al., 2010). B. canis canis infected dogsusually present with fever, lethargy, inappetance, anemiaand thrombocytopenia, all of variable severity (Solano-Gallego and Baneth, 2011). Although suspected, caninebabesiosis has never been confirmed in a dog that has

not traveled outside Latvia. Our aim was to determineif autochthonous canine babesiosis cases occur in Latviaand to perform molecular characterization of the isolatedBabesia.

Parasit

T)

516 I. Berzina et al. / Veterinary

2. Materials and methods

2.1. Sample collection and laboratory analysis

Between November 2009 and July 2011 small ani-mal veterinarians in Latvia were solicited to inform theresearcher (IB) about the dogs with presumptive diagno-sis of babesiosis. Dogs with recent travel history (less thanone year) were excluded.

Veterinarians provided clinical history, laboratory testresults, information on the treatment and outcome andEDTA blood samples (2–5 ml) for each of the dogs. Periph-eral blood was used for blood smear preparation and formolecular characterization of Babesia. Blood smears wereair dried, stained with Wrights Giemsa modified stain(Rapid Differential Stain Kit, VetOne, USA) and evaluatedmicroscopically, the level of parasitemia was expressed aspercentage of erythrocytes with Babesia per 200 eryth-rocytes counted on the blood smear under high power(Bakken et al., 1996).

Hematological and serum biochemical tests wereordered by practicing veterinarians and were performed indifferent laboratories using differing methodologies, there-fore absolute values of these tests could not be compared.To report these changes, we specified whether the specificparameter was normal, increased or decreased denotingrelative severity (mild, moderate, severe) of the change.

The presence of antibodies against Borrelia burgdorferisensu lato, Anaplasma phagocytophilum/Anaplasma platysand Ehrlichia canis, and presence of Dirofilaria immitisantigen were assessed using SNAP 4Dx test (IDEXX Lab-oratories, Westbrook, Maine, USA).

2.2. PCR and sequencing

DNA isolation was performed as described (Berzinaet al., 2012). Babesia spp. specific DNA was detected bynested amplification of the 18S rRNA gene fragment aspublished by Birkenheuer et al. (2003), with modifica-tions. The sequences of the oligonucleotide primers usedin this study were outer forward (5–22F), outer reverse(1661R), inner forward (455–479F) and inner reverse(793–772R) (Birkenheuer et al., 2003). Primers were syn-thetized by Metabion International AG, Germany, and allPCR reagents were obtained from Fermentas Life Sciences,Lithuania. PCR reactions were performed (MastercyclerepgradientS, Eppendorf, Germany) in 25 �l of the reactionmixture containing 1× Taq Buffer with (NH4)2SO4, 2.5 mMMgCl2, 200 �M of each dNTP (deoxynucleoside triphos-phate), 0.5 �M concentration of each primer, 1.5 U of TaqDNA polymerase (recombinant), and 2 �l of DNA templatefor primary reactions or 2 �l of the primary PCR prod-ucts for nested reactions. Cycling conditions were initialdenaturation at 95 ◦C for 5 min, followed by 40 amplifi-cation cycles (95 ◦C for 1 min, 55 ◦C for 1 min, and 72 ◦Cfor 1 min), and a final extension step at 72 ◦C for 5 min.Nested PCR had 30 amplification cycles. Positive and neg-

ative controls were included with each run. Amplifiedproducts were maintained at −20 ◦C until analyzed byagarose gel electrophoresis. PCR products were visualizedby electrophoresis in a 2% agarose gel containing 0.2 �g

ology 196 (2013) 515–518

of ethidium bromide/ml by transillumination with an UVlight. All commonly used quality control measures and pre-cautions were followed.

Sequencing was performed by a set of inner primers(455–479F and 793–772R) in 25 cycles under the followingconditions: 94 ◦C for 30 s, 55 ◦C for 15 s, and 60 ◦C for 4 min(Mastercycler epgradient S, Eppendorf, Germany). Thesequenced material was analyzed by standard techniqueusing an ABI Prism 3100 Genetic Analyzer (Perkin-Elmer,USA).

The BLAST program (http://www.ncbi.nlm.nih.gov/BLASwas used for comparison of sequences obtained in thisstudy versus those previously deposited in GenBank.

3. Results

During the study period seven dogs were diagnosedwith babesiosis and their blood samples and complete clin-ical history were sent to the researcher (IB). Four of thesedogs had traveled out of Latvia (2 to Germany and 2 oth-ers to Ukraine and Russia) and were not evaluated further.None of three remaining dogs had antibodies against A.phagocytophilum/A. platys, B. burgdorferi, E. canis nor hadD. immitis antigen detected. Detailed information on thesignalment, list of clinical and laboratory abnormalities,geographical distribution of babesiosis cases diagnosed inLatvia are provided in the Table 1. Dogs 1 and 2 had ticksattached approximately 1–2 months prior to the onset ofthe clinical signs, no such information was available for dog3.

In dog 1 babesiosis was diagnosed 11 days after theinitial examination when repeated hematological analysiswas sent to the laboratory and blood smear evaluation wasrequested by the veterinarian (all hematological analysisfor this dog were performed at the human hospital). Dog 1also had ultrasonography of abdominal organs performedwith no significant findings. Despite the blood transfusionat the time of diagnosis anemia had worsened and dog diedbefore antibabesial treatment was initiated.

3.1. Genetic characterization of the isolated Babesiaspecies

In all three cases babesial 18S rRNA encoding genewas amplified and the sequencing showed isolates to be98–100% similar to B. canis canis isolates deposited in theGenBank. Isolate from dog 3 had nucleotide (AG → GA)inversion in the position 150 compared to the sequencesfrom dog 1 and 2. Two longest of the three sequenceswere submitted to the GenBank with the following acces-sion numbers JX227980 (dog 2) and JX227981 (dog 3), thesequence of the dog 1 was similar to that of dog 2, butshorter.

4. Discussion

Babesiosis cases reported here were diagnosed based onclinical signs, hematology and molecular analysis and arethe first reported autochthonous canine babesiosis cases inLatvia and the Baltic states.

I. Berzina et al. / Veterinary Parasitology 196 (2013) 515–518 517

Table 1Detailed information on the dogs with autochthonous babesiosis diagnosed in Latvia (signalment, geographical location, onset of and list of clinical signs,laboratory abnormalities, treatment, clinical outcome).

Dog 1 Dog 2 Dog 3

Breed, age (years), sex Darthar, 7, M Golden Retriever, 4, F Mongrel, 3, MLocation/tick species Liepaja/I. ricinus Riga/I. ricinus Riga/I. ricinus

Clinical signsOnset of signs May May MarchFever × × ×Lethargy × × –Anorexia × – ×Laboratory abnormalitiesAnemia Moderate Mild SevereThrombocytopenia Severe Severe SevereHemolysis × × ×% of parasitemiaa 4% (8/200)b 2% (4/200) 0.5% (1/200)AST, ALT Mild ↑ Mild ↑ Moderate ↑Bilirubin (direct) Severe ↑ Mild ↑ Severe ↑Amylase – – Moderate ↑Additional informationTreatment Blood transfusion, symptomatic Imidocarb diproprionate, symptomatic SymptomaticComplications Possible hemoglobin nephrotoxicity No NoOutcome Died Recovered Recovered

×, symptom present; ↓↑, decrease or increase of the analyte; –, sign not observed, analysis not performed. AST, aspartate aminotransferase; ALT, alaninea

ytes per

ittartettwtscruadtnwt2lIdrvyaNinit

minotransferase.a Percentage of erythrocytes with babesia/number of affected erythrocb Babesia were diagnosed 11 days after the initial presentation.

At this point, it is unclear how these dogs acquired thenfection, since B. canis canis has not been isolated fromicks in Latvia (Bormane, 2007). Import of the infectedicks, expansion of the vector tick D. reticulatus habitatnd an adaptation of Babesia to transmission by Ixodesicinus ticks are the suggested routes of the infection inerritories previously free of canine babesiosis (Dautelt al., 2006; Cieniuch et al., 2009; Øines et al., 2010). Athe time of writing this article (autumn 2012) informa-ion on additional five canine babesiosis cases in dogsithout travel history outside Latvia was received (not

ested by PCR). This fact raises our suspicion that babesio-is is more common in Latvia and possibly neighboringountries, suggesting more convenient and establishedoutes of infection than imported tick. Our ongoing molec-lar analysis of ticks collected from dogs in Latvia mightdd useful information on the species of ticks attackingogs and on the pathogens they carry. It is notewor-hy that the seroprevalence of another tick-borne disease,amely, canine granulocytic anaplasmosis in dogs in Latviaas higher in areas inhabited by I. ricinus compared

o those inhabited by Ixodes persulcatus (Berzina et al.,012). Up to date three Babesia species have been iso-

ated from ticks in Latvia – B. microti (from I. ricinus and. persulcatus), Babesia bovis (from I. ricinus) and Babesiaivergens (isolated in regions where both tick specieseside) (Bormane, 2007). Latvian Food and Veterinary Ser-ice has no information on the babesiosis cases duringear 2011–2012 in any of the farm animal species (equinend bovine babesiosis are notifiable diseases in Latvia).o human babesiosis cases were diagnosed in last year

n Latvia (Latvia Center of Infectology). Personal commu-ications with small animal veterinarians from Lithuania

ndicate that canine babesiosis is frequently diagnosedhere, but no further investigations have been carried

200 erytrocytes.

out as to the species of Babesia involved, geographicaldistribution of the babesiosis cases or routes of infec-tion.

Presentation in spring with rather mild to moderateclinical signs and changes in the laboratory test resultsin cases reported here are similar to B. canis canis infec-tions diagnosed in dogs elsewhere (Ayoob et al., 2010;Øines et al., 2010; Adaszek et al., 2011; Solano-Gallegoand Baneth, 2011). B. canis canis isolates from Latviandogs were similar to those from dogs in various Euro-pean countries, including the recent isolate from dog inNorway (Øines et al., 2010). A study in Poland showed vari-ations in the nucleotides in B. canis canis 18S rRNA partialgene sequences and we found similar nucleotide inver-sions (Adaszek and Winiarczyk, 2008). It is unclear whetherthese differences are important in terms of pathogenicityof the B. canis canis (Adaszek and Winiarczyk, 2008).

Microscopical detection of Babesia in the blood smear isan established diagnostic tool that should be undertakenin all suspected dogs (Adaszek and Winiarczyk, 2008). Itis unclear why the marked thrombocytopenia in dog 1 didnot trigger microscopical examination of the blood smear.Although no necropsy was performed, we suspect that anacute renal failure due to hemoglobin toxicity, hypoxia andworsening anemia might be contributing to the lethal out-come in the dog 1 (Ayoob et al., 2010). None of the dogswas tested for Babesia after the resolution of clinical signsand no information on the relapse of the disease has beenreceived up to the time of writing this article.

Acknowledgments

This study was funded by European Union and Euro-pean Social Funds (Agreement No. 2009/0180/1DP/1.1.2.1.2/09/IPIA/VIAA/017, “Support for Doctoral

Parasit

518 I. Berzina et al. / Veterinary

Studies Program of Latvia University of Agriculture”,04.4–08/EF2.D1.20’). SNAP 4Dx tests were kindly donatedby IDEXX Laboratories.

References

Adaszek, L., Winiarczyk, S., 2008. Molecular characterization of Babesiacanis canis isolates from naturally infected dogs in Poland. Vet. Para-sitol. 152, 235–241.

Adaszek, L., Carbonero Martinez, A., Winiarczyk, S., 2011. The factorsaffecting the distribution of babesiosis in dogs in Poland. Vet. Parasitol.181, 160–165.

Ayoob, A.L., Hackner, S.G., Prittie, J., 2010. Clinical management of caninebabesiosis. J. Vet. Emerg. Crit. Care (San Antonio) 20, 77–89.

Bakken, J.S., Krueth, J., Wilson-Norskog, C., Tilden, R.L., Asanovich, K.,Dumler, S.J., 1996. Clinical and laboratory characteristics of humangranulocytic ehrlichiosis. JAMA 275, 199–205.

Berzina, I., Capligina, V., Bormane, A., Pavulina, A., Baumanis, V., Ranka,R., Granta, R., Matise, I., 2012. Association between Anaplasmaphagocytophilum seroprevalence in dogs and distribution of Ixodesricinus and Ixodes persulcatus ticks in Latvia. Ticks Tick Borne Dis.,http://dx.doi.org/10.1016/j.ttbdis.2012.08.003.

Birkenheuer, A.J., Levy, M.G., Breitschwerdt, E.B., 2003. Development andevaluation of a seminested PCR for detection and differentiation ofBabesia gibsoni (Asian Genotype) and B. canis DNA in canine bloodsamples. J. Clin. Microbiol. 41, 4172–4177.

Bormane, A., 2007. Ixodes ricinus L. and Ixodes persulcatus P. Sch. (Acari:Ixodidae) distribution, significance and molecular epidemiology oftransmitted infectious diseases in Latvia. Ph.D. Thesis (in Latvian).

Caccio, S.M., Antunovic, B., Moretti, A., Mangili, V., Marinculic, A., Baric,R.R., Slemenda, S.B., Pieniazek, N.J., 2002. Molecular characterization

ology 196 (2013) 515–518

of Babesia canis canis and Babesia canis vogeli from naturally infectedEuropean dogs. Vet. Parasitol. 106, 285–292.

Cieniuch, S., Stanczak, J., Ruczaj, A., 2009. The first detection of BabesiaEU1 and Babesia canis canis in Ixodes ricinus ticks (Acari, Ixodidae) col-lected in urban and rural areas in northern Poland. Pol. J. Microbiol.58, 231–236.

Dautel, H., Dippel, C., Oehme, R., Hartelt, K., Schettler, E., 2006. Evidencefor an increased geographical distribution of Dermacentor reticulatesin Germany and detection of Rickettsia sp. Int. J. Med. Microbiol. 296,149–156.

Gray, J.S., Dautel, H., Estrada-Pena, A., Kahl, O., Lindgren, E., 2009. Effectsof climate on ticks and tick-borne diseases in Europe. Interdiscip. Per-spect. Infect. Dis. 1–12, http://dx.doi.org/10.1155/2009/593232.

Hunfeld, K.P., Hildebrandt, A., Gray, J.S., 2008. Babesiosis: recent insightsinto an ancient disease. Int. J. Parasitol. 38, 1219–1237.

Karelis, G., Bormane, A., Logina, I., Lucenko, I., Suna, N., Krumina, A.,Donaghy, M., 2012. Tick-borne encephalitis in Latvia 1973–2009:epidemiology, clinical features and sequelae. Eur. J. Neurol. 19, 62–68.

Menn, B., Lorentz, S., Naucke, T.J., 2010. Imported and travelling dogsas carriers of canine vector-borne pathogens in Germany. ParasitesVectors 3, http://dx.doi.org/10.1186/1756-3305-3-34.

Øines, Ø., Storli, K., Brun-Hansen, H., 2010. First case of babesiosis causedby Babesia canis canis in a dog from Norway. Vet. Parasitol. 171,350–353.

Rar, V.A., Maksimova, T.G., Zakharenko, L.P., Bolykhina, S.A., Dobrotvorsky,A.K., Morozova, O.V., 2005. Babesia DNA detection in canine blood andDermacentor reticulatus ticks in southwestern Siberia, Russia. Vector

Borne Zoonotic Dis. 5, 285–287.

Solano-Gallego, L., Baneth, G., 2011. Babesiosis in dogs and cats – expand-ing parasitological and clinical spectra. Vet. Parasitol. 181, 148–160.

Zygutiene, M., 2009. Tick-borne pathogens and spread of Ixodes ricinus inLithuania. EpiNorth 2, 63–71.