leishmaniasis is a group of infectious diseases that affect people and domestic and wild animals worldwide and are caused by species of the genus leishmania , transmitted by sandflies . about 30 species of leishmania infect humans as well as domestic and wild animals in 88 countries . dogs act as a reservoir host of the disease for human infections ; they are considered to be the reservoir for leishmania infantum infection in areas including portugal , the mediterranean basin , central and south america , the middle east , and china . human leishmaniasis is classified into cutaneous ( cl ) , mucocutaneous ( mcl ) , and visceral leishmaniasis ( vl ) according to their clinical manifestations , but dogs usually have both visceral and cutaneous involvement . as the first detailed human case report in the republic of korea ( = korea ) , heu   described 3 cases of kala - azar ( = visceral leishmaniasis ) among returnees from northern china . thereafter , more than 25 human cases of cutaneous and visceral forms of leishmaniasis have been reported in korea [ 3 - 5 ] . only 1 case was autochthonous cutaneous leishmaniasis ( cl ) in a 70-year - old man in kangwon province who has never been abroad . neither autochthonous nor introduced canine leishmaniasis ( canl ) has been reported previously in korea to the best of the authors ' knowledge . here a 12-year - old neutered female mixed - bred dog weighing 5.9 kg presented to the veterinary medical teaching hospital , college of veterinary medicine , seoul national university on july 24 , 2006 after an episode of epistaxis and decreased appetite of 2 days ' duration . the dog was born and raised in a residential area at daeband - dong , dongjak - gu , seoul near where was yongma mountain and was living with its littermate and dam , none of which had been taken to a canl endemic country . physical examinations revealed pale mucous membranes ( gingival and buccal ) and a single diffuse firm round dermal nodule , 1010 cm in diameter , extending from the lateral flank to the lateral abdomen . the skin nodule persisted for a year , and was tentatively diagnosed as panniculitis based on biopsy and histological examinations . numerous intracellular and extracellular organisms with basophilic nuclei and bar - shaped kinetoplasts were observed ( fig . 1 ) , consistent with characteristic features of leishmania sp . based on cytological findings , samples were first incubated in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen , carlsbad , california , usa ) supplemented with 20% ( v / v ) heat - inactivated fetal calf serum ( fcs ) in an atmosphere of 5% co2 and 95% air at 37 for 1 week , after which the medium was replaced with schneider 's drosophila medium ( gibco schneider 's drosophila medium 1x , liquid ) ( invitrogen ) with 20% heat - inactivated fcs , and incubated at 24 . , dna was extracted from the specimen using a commercial dna extraction kit ( accuprep dna extraction kit , bioneer , daejeon , korea ) with minor modifications . in order to diagnose and obtain genomic data , pcr assays were performed with following primer pairs ; r174 ( 5'-ggttcctttcctgatttacg-3 ' ) and r798 ( 5'-ggccggtaaaggccgaatag-3 ' ) which were specific to the partial small subunit ribosomal rna ( ssu rrna ) gene ( 600 bp ) of leishmania , and n13a ( 5'-aacttttctggtcctccggg-3 ' ) and n13b ( 5'-cccccagtttcccgccc-3 ' ) which were specific to a 120 bp fragment of the l. infantum kinetoplast dna minicircle . the pcr products from each gene fragment were purified using a commercial gel extraction kit ( qiaex ii gel extraction kit , qiagen inc . , dna sequencing was carried out using an automated dna sequencer ( abi system 3700 , applied biosystems , inc . , foster city , california , usa ) . using the dna basic module ( dnasis max , miraibio , alameda , california , usa ) , gene sequences of the korean canine leishmania sp . ( leishmania infantum isolated from a dog in this study ) were compared with those of the other known leishmania species . the nucleotide sequences of the 600 bp fragment of the ssu rrna gene and 120 bp fragment of the kinetoplast dna minicircle gene from the leishmania - infected dog described in this study were deposited in genbank under accession numbers eu825208 and eu825207 , respectively . pcr analysis of the aspirated samples showed both the 600 bp band specific for leishmania and 120 bp l. infantum - specific kinetoplast dna minicircle ( fig . 2 ) [ 7 - 10 ] . nucleotide sequence comparisons of the ssu rrna gene of the organism with that of other parasites available in genbank revealed that the organism shared greater than 99% identity with 10 of 13 strains of leishmania spp . and the sequence comparison of the kinetoplast minicircle dna of the organism with that of other parasites available in genbank revealed that the organism shared 96.5 - 98.3 nucleotide identities with llm-719 , lem-2298 , and ipt1 strains of l. infantum . the organism also showed relatively high nucleotide identities of the kinetoplast minicircle dna with leishmania chagasi and leishmania donovani , sharing 95.6 - 96.5% similarity ( table 2 ) . however , the organism revealed to have very low nucleotide ( 69.7% ) identities with leishmania amazonensis ( table 2 ) . based on these data , the korean leishmania species from the dog described in this study was confirmed to be l. infantum and the dog was therefore diagnosed with canl . following the diagnosis , in this report the diagnosis was made on direct visualization of specific leishmania amastigotes in fine needle aspirate smears of the cutaneous lesion , positive results using pcr assay , and dna sequencing . cytological or histological identification of amastigotes is virtually 100% specific for the definitive diagnosis of leishmaniasis , and pcr demonstration of leishmanial dna is sensitive both in human and animal patients [ 1,5,9 - 12 ] . most cases of human and animal leishmaniasis in non - endemic areas have been associated with travel to endemic regions . however , in this case of canl , the dog had never been taken to an endemic region . in addition , known vector species for leishmania spp . have not been recorded in korea , and autochthonous animal infections have never been previously documented . in a recent case of human autochthonous cutaneous leishmaniasis in korea , the route of infection and the species of leishmania responsible were not identified , and the patient had no history of travel to an endemic country , which were shared features with this canine case . such transmission is rare , and is thought to occur through mothers who contracted severe canl before or during pregnancy . the mother of the infected dog was not taken outside of korea while pregnant and was subsequently proven to be free of leishmania by pcr . another possible source of infection is from a blood transfusion that the dog received 2 years prior to the illness . the donor in this case was born in korea , had never visited an endemic area , and was clinically healthy . none of other dogs that had received blood transfusion from the same donor showed clinical manifestations of leishmaniasis . screening tests using a commercial elisa test kit ( snap leishmania test , idexx laboratories , wetherby , uk ) for detection of leishmania were performed on all donor dogs in the blood bank and no positive results were returned . therefore , the route of infection through blood transfusion can be ruled out . because sandflies , the vectors of leishmania , are intolerant of temperature changes , leishmaniasis acquired through accidental importation of sandflies is unlikely . further , the family was also unlikely to be the source of infection because all family members showed negative results on a serological test for detection of leishmania . it has been suggested that sandflies may exist in korea because of an increase in temperature and climate changes over the past few decades , but this has not been confirmed . although we were unable to identify where or how the dog described in this case study acquired leishmania organism , it is possible that other cases of autochthonous leishmaniasis exist in korea . our findings suggest that more epidemiological investigations are needed , and clinicians in korea should be alert to the possibility of leishmaniasis in animal patients presenting with cutaneous lesions and undiagnosed epistaxis , even if there is no history of travel to known endemic areas .
a 12-year - old spayed female mixed - bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank . 
 no abnormalities were found in coagulation profiles and blood pressure . 
 cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background , consistent with leishmaniasis . in vitro culture was unsuccessful but pcr of the nodular aspirate identified the organisms as leishmania infantum , and the final diagnosis was canine leishmaniasis . 
 no history of travel to endemic countries was noted . 
 because the dog had received a blood transfusion 2 years before the illness , serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial leishmania elisa test kit , and there were no positive results . 
 additional 113 dogs with hyperglobulinemia from seoul were also screened with the same kits but no positive results were obtained . to the best of the author 's knowledge 
 this is the first autochthonous case of canine leishmaniasis in korea .