Autochthonous canine babesiosis caused by Babesia canis canis in Latvia

Veterinary Parasitology 196 (2013) 515–518

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Short communication

Autochthonous canine babesiosis caused by Babesia canis canis in Latvia Inese Berzina a,∗ , Valentina Capligina b , Viesturs Baumanis b , Renate Ranka b , Dina Cirule c , Ilze Matise a a b c

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

a r t i c l e

i n f o

Article history: Received 12 January 2013 Received in revised form 4 March 2013 Accepted 13 March 2013

Keywords: Babesia canis canis Latvia Autochthonous Canine babesiosis PCR

a b s t r a c t This is the first report of confirmed canine babesiosis in Latvia supporting the observed geographical expansion of this disease. Between 2009 and 2011 three dogs which have not traveled outside of Latvia were diagnosed with babesiosis. Hematological analysis and serological tests for granulocytic anaplasmosis, ehrlichiosis and borreliosis were negative (Idexx SNAP 4Dx test). Peripheral blood erythrocytes of the three dogs contained large Babesia that were identified as Babesia canis canis by PCR. Sequences of partial 18S rRNA gene were 98–100% similar to the sequences of B. canis canis isolated from dogs in other European countries. We conclude that these are the first autochthonous canine babesiosis cases reported from Latvia. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Tick-borne diseases in dogs have been diagnosed with increasing frequency worldwide. This tendency is explained by a variety of factors, including climate changes associated with the expansion of the vectors and reservoir animals, increasing proportion of traveling dogs, as well as increased awareness about these diseases and improved diagnostic techniques (Menn et al., 2010; Øines et al., 2010; Solano-Gallego and Baneth, 2011). Canine babesiosis can be caused by several species of hematoprotozoal organisms of the genus Babesia (Hunfeld et al., 2008; Ayoob et al., 2010). In Europe most of the cases have been caused by large Babesia Babesia canis canis transmitted mainly by Dermacentor reticulatus ticks, although Babesia microti induced disease had been diagnosed in

∗ Corresponding author at: Latvia University of Agriculture, Faculty of Veterinary Medicine, Preclinical Institute, Pathology Department, Kr. Helmana Street 8, Jelgava LV-3004, Latvia. Tel.: +371 26324105. E-mail address: [email protected] (I. Berzina). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.03.015

Spain (Ayoob et al., 2010; Solano-Gallego and Baneth, 2011). D. reticulatus is described as established tick species in United Kingdom and northern part of Russian Federation (Gray et al., 2009), it has been spotted in Lithuania, country that shares its Northern border with Latvia (Zˇ ygutiene, 2009). Dermacentor spp. tick has been noted on one occasion (A. Kruklite, personal communication) and up to date is not considered to be an established tick species in Latvia (Bormane, 2007; Karelis et al., 2012). Canine babesiosis was considered to be endemic in Southern Europe but recently B. canis canis infections have been reported in countries located relatively close to Latvia – Poland, Russia and Norway (Caccio et al., 2002; Rar et al., 2005; Øines et al., 2010). B. canis canis infected dogs usually present with fever, lethargy, inappetance, anemia and thrombocytopenia, all of variable severity (SolanoGallego and Baneth, 2011). Although suspected, canine babesiosis has never been confirmed in a dog that has not traveled outside Latvia. Our aim was to determine if autochthonous canine babesiosis cases occur in Latvia and to perform molecular characterization of the isolated Babesia.

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2. Materials and methods 2.1. Sample collection and laboratory analysis Between November 2009 and July 2011 small animal veterinarians in Latvia were solicited to inform the researcher (IB) about the dogs with presumptive diagnosis of babesiosis. Dogs with recent travel history (less than one year) were excluded. Veterinarians provided clinical history, laboratory test results, information on the treatment and outcome and EDTA blood samples (2–5 ml) for each of the dogs. Peripheral blood was used for blood smear preparation and for molecular characterization of Babesia. Blood smears were air dried, stained with Wrights Giemsa modified stain (Rapid Differential Stain Kit, VetOne, USA) and evaluated microscopically, the level of parasitemia was expressed as percentage of erythrocytes with Babesia per 200 erythrocytes counted on the blood smear under high power (Bakken et al., 1996). Hematological and serum biochemical tests were ordered by practicing veterinarians and were performed in different laboratories using differing methodologies, therefore absolute values of these tests could not be compared. To report these changes, we specified whether the specific parameter was normal, increased or decreased denoting relative severity (mild, moderate, severe) of the change. The presence of antibodies against Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum/Anaplasma platys and Ehrlichia canis, and presence of Dirofilaria immitis antigen were assessed using SNAP 4Dx test (IDEXX Laboratories, Westbrook, Maine, USA). 2.2. PCR and sequencing DNA isolation was performed as described (Berzina et al., 2012). Babesia spp. specific DNA was detected by nested amplification of the 18S rRNA gene fragment as published by Birkenheuer et al. (2003), with modifications. The sequences of the oligonucleotide primers used in 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 synthetized by Metabion International AG, Germany, and all PCR reagents were obtained from Fermentas Life Sciences, Lithuania. PCR reactions were performed (Mastercycler epgradientS, Eppendorf, Germany) in 25 ␮l of the reaction mixture containing 1× Taq Buffer with (NH4 )2 SO4 , 2.5 mM MgCl2 , 200 ␮M of each dNTP (deoxynucleoside triphosphate), 0.5 ␮M concentration of each primer, 1.5 U of Taq DNA polymerase (recombinant), and 2 ␮l of DNA template for primary reactions or 2 ␮l of the primary PCR products for nested reactions. Cycling conditions were initial denaturation at 95 ◦ C for 5 min, followed by 40 amplification cycles (95 ◦ C for 1 min, 55 ◦ C for 1 min, and 72 ◦ C for 1 min), and a final extension step at 72 ◦ C for 5 min. Nested PCR had 30 amplification cycles. Positive and negative controls were included with each run. Amplified products were maintained at −20 ◦ C until analyzed by agarose gel electrophoresis. PCR products were visualized by electrophoresis in a 2% agarose gel containing 0.2 ␮g

of ethidium bromide/ml by transillumination with an UV light. All commonly used quality control measures and precautions were followed. Sequencing was performed by a set of inner primers (455–479F and 793–772R) in 25 cycles under the following conditions: 94 ◦ C for 30 s, 55 ◦ C for 15 s, and 60 ◦ C for 4 min (Mastercycler epgradient S, Eppendorf, Germany). The sequenced material was analyzed by standard technique using an ABI Prism 3100 Genetic Analyzer (Perkin-Elmer, USA). The BLAST program (http://www.ncbi.nlm.nih.gov/BLAST) was used for comparison of sequences obtained in this study versus those previously deposited in GenBank. 3. Results During the study period seven dogs were diagnosed with babesiosis and their blood samples and complete clinical history were sent to the researcher (IB). Four of these dogs had traveled out of Latvia (2 to Germany and 2 others 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 had D. immitis antigen detected. Detailed information on the signalment, list of clinical and laboratory abnormalities, geographical distribution of babesiosis cases diagnosed in Latvia are provided in the Table 1. Dogs 1 and 2 had ticks attached approximately 1–2 months prior to the onset of the clinical signs, no such information was available for dog 3. In dog 1 babesiosis was diagnosed 11 days after the initial examination when repeated hematological analysis was sent to the laboratory and blood smear evaluation was requested by the veterinarian (all hematological analysis for this dog were performed at the human hospital). Dog 1 also had ultrasonography of abdominal organs performed with no significant findings. Despite the blood transfusion at the time of diagnosis anemia had worsened and dog died before antibabesial treatment was initiated. 3.1. Genetic characterization of the isolated Babesia species In all three cases babesial 18S rRNA encoding gene was amplified and the sequencing showed isolates to be 98–100% similar to B. canis canis isolates deposited in the GenBank. Isolate from dog 3 had nucleotide (AG → GA) inversion in the position 150 compared to the sequences from dog 1 and 2. Two longest of the three sequences were submitted to the GenBank with the following accession numbers JX227980 (dog 2) and JX227981 (dog 3), the sequence of the dog 1 was similar to that of dog 2, but shorter. 4. Discussion Babesiosis cases reported here were diagnosed based on clinical signs, hematology and molecular analysis and are the first reported autochthonous canine babesiosis cases in Latvia and the Baltic states.

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Table 1 Detailed 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 Location/tick species

Darthar, 7, M Liepaja/I. ricinus

Golden Retriever, 4, F Riga/I. ricinus

Mongrel, 3, M Riga/I. ricinus

Clinical signs Onset of signs Fever Lethargy Anorexia

May × × ×

May × × –

March × – ×

Laboratory abnormalities Anemia Thrombocytopenia Hemolysis % of parasitemiaa AST, ALT Bilirubin (direct) Amylase

Moderate Severe × 4% (8/200)b Mild ↑ Severe ↑ –

Mild Severe × 2% (4/200) Mild ↑ Mild ↑ –

Severe Severe × 0.5% (1/200) Moderate ↑ Severe ↑ Moderate ↑

Additional information Treatment Complications Outcome

Blood transfusion, symptomatic Possible hemoglobin nephrotoxicity Died

Imidocarb diproprionate, symptomatic No Recovered

Symptomatic No Recovered

×, symptom present; ↓↑, decrease or increase of the analyte; –, sign not observed, analysis not performed. AST, aspartate aminotransferase; ALT, alanine aminotransferase. a Percentage of erythrocytes with babesia/number of affected erythrocytes per 200 erytrocytes. b Babesia were diagnosed 11 days after the initial presentation.

At this point, it is unclear how these dogs acquired the infection, since B. canis canis has not been isolated from ticks in Latvia (Bormane, 2007). Import of the infected ticks, expansion of the vector tick D. reticulatus habitat and an adaptation of Babesia to transmission by Ixodes ricinus ticks are the suggested routes of the infection in territories previously free of canine babesiosis (Dautel et al., 2006; Cieniuch et al., 2009; Øines et al., 2010). At the time of writing this article (autumn 2012) information on additional five canine babesiosis cases in dogs without travel history outside Latvia was received (not tested by PCR). This fact raises our suspicion that babesiosis is more common in Latvia and possibly neighboring countries, suggesting more convenient and established routes of infection than imported tick. Our ongoing molecular analysis of ticks collected from dogs in Latvia might add useful information on the species of ticks attacking dogs and on the pathogens they carry. It is noteworthy that the seroprevalence of another tick-borne disease, namely, canine granulocytic anaplasmosis in dogs in Latvia was higher in areas inhabited by I. ricinus compared to those inhabited by Ixodes persulcatus (Berzina et al., 2012). Up to date three Babesia species have been isolated from ticks in Latvia – B. microti (from I. ricinus and I. persulcatus), Babesia bovis (from I. ricinus) and Babesia divergens (isolated in regions where both tick species reside) (Bormane, 2007). Latvian Food and Veterinary Service has no information on the babesiosis cases during year 2011–2012 in any of the farm animal species (equine and bovine babesiosis are notifiable diseases in Latvia). No human babesiosis cases were diagnosed in last year in Latvia (Latvia Center of Infectology). Personal communications with small animal veterinarians from Lithuania indicate that canine babesiosis is frequently diagnosed there, but no further investigations have been carried

out as to the species of Babesia involved, geographical distribution of the babesiosis cases or routes of infection. Presentation in spring with rather mild to moderate clinical signs and changes in the laboratory test results in cases reported here are similar to B. canis canis infections diagnosed in dogs elsewhere (Ayoob et al., 2010; Øines et al., 2010; Adaszek et al., 2011; Solano-Gallego and Baneth, 2011). B. canis canis isolates from Latvian dogs were similar to those from dogs in various European countries, including the recent isolate from dog in Norway (Øines et al., 2010). A study in Poland showed variations in the nucleotides in B. canis canis 18S rRNA partial gene sequences and we found similar nucleotide inversions (Adaszek and Winiarczyk, 2008). It is unclear whether these differences are important in terms of pathogenicity of the B. canis canis (Adaszek and Winiarczyk, 2008). Microscopical detection of Babesia in the blood smear is an established diagnostic tool that should be undertaken in all suspected dogs (Adaszek and Winiarczyk, 2008). It is unclear why the marked thrombocytopenia in dog 1 did not trigger microscopical examination of the blood smear. Although no necropsy was performed, we suspect that an acute renal failure due to hemoglobin toxicity, hypoxia and worsening anemia might be contributing to the lethal outcome in the dog 1 (Ayoob et al., 2010). None of the dogs was tested for Babesia after the resolution of clinical signs and no information on the relapse of the disease has been received up to the time of writing this article. Acknowledgments This study was funded by European Union and European Social Funds (Agreement No. 2009/0180/1DP/ 1.1.2.1.2/09/IPIA/VIAA/017, “Support for Doctoral

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