BIOLOGIJA. 2018. Vol. 64. No. 3. P. 203–207 © Lietuvos mokslų akademija, 2018 The golden jackal Canis aureus – a new species in the Baltic countries Algimantas Paulauskas1*, In the Baltic countries, the golden jackal (Canis aureus) was first recorded in Estonia on 28 February 2013 and three specimens Irma Ražanskė1, of golden jackal were hunted in Latvia in 2014. The first golden jackal in Lithuania was hunted on 7 February 2015. The species Jana Radzijevskaja1, of the golden jackal was identified using morphological and mi- tochondrial DNA control region (CR1) analysis. In Lithuania, Dovilė Nugaraitė1, hunting of these animals is permitted throughout the year. Few studies in the past revealed the potential role of the golden jackal 2 Vaclovas Gedminas as a carrier of intestinal helminths, parasites, and zoonotic diseas- es. In this study, the presence of tick-borne pathogens and other 1 Vytautas Magnus University, parasites in golden jackal specimen were investigated. No patho- Vileikos St. 8, Kaunas 44404, gens (Anaplasma phagocytophilum, Babesia sp., Bartonella sp.) Lithuania were found in the spleen of the golden jackal. However, the flukes Apophallus donicus, nematodes Uncinaria stenocephala, and uni- 2 Kaunas Tadas Ivanauskas Zoological Museum, dentified individuals of classCestoda were detected. Helminths Laisvės Av. 106, Kaunas 44253, A. donicus and U. stenocephala are not new species for Lithuania Lithuania and neighbouring territories. Keywords: Canis aureus, mtDNA, helminths, Uncinaria steno- cephala, Apophallus donicus, Cestoda INTRODUCTION in the areas and countries they had not been re- corded before. Golden jackals have been recent- The golden jackal Canis( aureus) is a native ly sighted as far west as Switzerland and as far Palearctic species with a historic range extending north as Estonia (Rutkowski et al., 2015; Trouw- from North Africa and South-eastern and Central borst et al., 2015). Europe through to Central and East Asia (Jhala In Estonia and Latvia, golden jackals of un- and Moehlman, 2008). The population and dis- known origin have been recorded since 2011 (Ba- tribution of the golden jackal within Europe has nea, 2013; Toom, 2014; Trouwborst et al., 2015). seen considerable changes over recent decades. The first confirmed case of the golden jackal in Since the mid-twentieth century, the distribution- the Baltic countries was reported in Estonia in al range of golden jackals has expanded signifi- 2013. In February 2013, a golden jackal was killed cantly across Central and Eastern Europe with oc- by hunters in Matsalu National Park, West Estonia casional animals being documented to the north (Männil et al., 2014). In 2014, golden jackals were and the west, far from the established populations recorded in several other widely dispersed locali- ties across Estonia, including the north, north- * Corresponding author. Email: [email protected] east and south of the country. Until May 2015, 204 Algimantas Paulauskas, Irma Ražanskė, Jana Radzijevskaja, Dovilė Nugaraitė, Vaclovas Gedminas a total of nine individuals were killed either Conventional PCR and nested PCR methods by hunters or in road accidents across Estonia were used for the detection of Anaplasma phago- (Stratford, 2015). cytophilum, Babesia spp., Bartonella spp. (Mas- First recorded cases of the golden jackal in sung et al., 1998; Rar et al., 2005; Norman et al., Latvia were three individuals killed by a hunter 1995). in July and December 2013 (Männil et al., 2014). Prior to helminthological investigation the Until 2015, a total of ten golden jackals were shot material was frozen at –80°C for 10 days for safe- or found dead in Latvia (Stratford, 2015). ty reasons (i.e., to avoid possible infection with In Lithuania, the first golden jackal was shot Echinococcus spp.) (WHO/OIE Manual, 2001). in February 2015. The species of the hunted The golden jackal was thawed at room tempera- individual was identified through a morpho- ture and separate organs (entire gastrointestinal logical examination. Since that time, other un- tract, lungs, heart, liver, and kidney) were stud- confirmed reports of the golden jackal followed ied helminthologically according to the method- from the districts of Kaunas, Vilnius, Zarasai, ology of Ivashkin et al. (1971). Examination of Varėna, and Biržai. the content of the intestines and of the stomach In the present study, we used mitochondrial was based on the method of consistent flushing. DNA control region data for the confirmation Helminths were picked out and fixed in 70% eth- of species identification, and as few studies in anol for later examination. For the microscopic the past revealed the potential role of the jack- study, trematodes and nematodes were mounted als as carriers of zoonotic diseases, we investi- in glycerin and examined under a Motic BA400 gated the presence of tick-borne pathogens and Tension microscope. Identification was based on other parasites in the golden jackal specimen the key of Kozlov (1977). The diet of the golden from Lithuania. jackal was studied using analysis of the stomach content. Each portion of the stomach content MATERIALS AND METHODS was mixed with water and individual compo- nents separated into groups. Genomic DNA from the specimen spleen was ex- tracted using the Genomic DNA Purification Kit RESULTS AND DISCUSSION (Thermo Fisher Scientific, Lithuania) according to manufacturer’s instructions. DNA was stored Comparison of the mtDNA sequence identified at –20°C until subsequent handlings. A 460 bp of in this study with those deposited in GenBank hypervariable left domain of the mitochondrial confirmed that the animal hunted in Lithuania DNA control region was amplified using PCR in February 2015 was a golden jackal (accession and sequencing primers WDLOOPL 5’-TCC- number KT123040). The golden jackal haplotype CTGACACCCCTACATTC-3’, H519 5’-CGTT- identified in the present study corresponds with GCGGTCATAGGTGAG-3’ (designed on wolf the haplotype previously identified in Italy, Cro- mtDNA CR (Caniglia et al., 2013)) as described atia, Bulgaria, Serbia, and the Caucasus (Fig. 1). in Fabbri et al., 2014. The products were separat- The golden jackal is a new species in the Baltic ed on 1.5% agarose gel and visualized by etidium region. The confirmed cases of the golden jack- bromide. PCR amplification product of mtDNR al in Latvia and Estonia were recorded in 2013 control region was purified using GeneJET Gel (Männil et al., 2014; Stratford, 2015), two years Extraction Kit (Thermo Fisher Scientific, Lithu- earlier than in Lithuania. ania) and sequenced. The resulting 460-bp-long The Eurasian golden jackal has been reported sequence of the CR mtDNA was revised manu- as a host of pathogens of zoonotic and veterinary ally and aligned with golden jackal sequences importance, including a range of vector-borne from GenBank in MEGA 6. The sequence of CR pathogens such as Ehrlichia canis, Anaplasma mtDNA was submitted to GenBank and assigned phagocytophilum, and others (Waner et al., 1999), the following accession number: KT123040. and only one Babesia species (B. canis) was The golden jackal Canis aureus – a new species in the Baltic countries 205 Fig. 1. Phylogenetic tree of mtDNA sequences created using the Neighbor-Joining method and a bootstrap analysis of 1000 replicates. The sample from the present study is marked ●. Nyctereutes procyonoides are used as an out-group. Abbreva- tions: BG – Bulgaria, HR – Croatia, IT – Italy, RS – Serbia confirmed by molecular methods in golden jack- Ancylostoma caninum and Uncinaria stenoceph- als in Europe, in Romania (Mitkova et al., 2017). ala commonly reported across the entire geo- However, reports of tick-borne pathogens in graphical range of these hosts (Gherman, Mihal- C. aureus are scarce. In this study, different PCR ca, 2017). In the small intestine of the Lithuanian methods were used for the detection of tick- golden jackal, 49 flukes of species Apophallus borne pathogens: Anaplasma phagocytophilum, donicus, 11 nematodes of species Uncinaria Babesia spp., Bartonella spp. The results of PCR stenocephala (Fig. 2), and 29 unidentified indi- analysis were negative for all tested pathogens. viduals of Cestoda were found. Several hookworm (Ancylostomatidae) spe- The diet of golden jackal was studied through cies have been reported in golden jackals, with the stomach content. It was found to consist of Fig. 2. Uncinaria stenocephala, microscopic examination 206 Algimantas Paulauskas, Irma Ražanskė, Jana Radzijevskaja, Dovilė Nugaraitė, Vaclovas Gedminas plants, insects (larvae), birds (feathers), rodents tion range (Croatia and eastern Italian Alps). (bones, limbs), and ungulates (hair). In Croatia, Conserv Genet. 2014; 15(1): 187–99. both animal and plant components were found 4. Gherman CM, Mihalca AD. A synoptic over- in the scat of the golden jackal: the major com- view of golden jackal parasites reveals high ponent was mammals (50.3%), followed by fruit diversity of species. Parasit Vectors. 2017; 10: seeds and vegetables (34.1%), insects (29.5%), 419. birds (including eggs; 24.8%), artificial materi- als (24%), and branches, leaves, and grass (24%) 5. Ivashkin VM, Sobolev AA, Khromova L. Ca- (Radovic, Kovačic, 2010). mallanata of animals and man and diseases caused by them.) In: Skryabin KI, editor. Es- CONCLUSIONS sentials of nematodology. 1971; 22: 1–388. Russian. Result of analysis of a sample of unknown ani- 6. Jhala Y, Moehlman PD. Canis aureus. The IUCN mal from Lithuania revealed that it was a gold- Red List of Threatened Species. http://www.iuc- en jackal. Similar genetic patterns based on nredlist.org/details/3744/0 (2008) [Cited 2018 mtDNA control region sequences were iden- Jan 3]. tified in animals from Bulgaria, Croatia, Italy, Serbia, and the Caucasus. No tick-borne patho- 7. Kozlov MA. A new genus and species of parasi- gens were found in the golden jackal, however, toid of the family Platygastridae (Hymenoptera, the flukesApophallus donicus, nematodes Unci- Proctotrupoidea) from Armenia. Trudy Zoo- naria stenocephala, and unidentified individu- logicheskogo Instituta Akademii Nauk SSSR. als of class Cestoda were detected. 1977; 64: 79–81. 8. Männil P, Jogisalu I, Ozolins J, Maran T.