Sabūnas et al. Parasites Vectors (2019) 12:177 https://doi.org/10.1186/s13071-019-3406-y Parasites & Vectors RESEARCH Open Access Diroflaria repens in dogs and humans in Lithuania Vytautas Sabūnas1,2, Jana Radzijevskaja1, Povilas Sakalauskas1, Saulius Petkevičius3, Birutė Karvelienė3, Jolanta Žiliukienė4, Indrė Lipatova1 and Algimantas Paulauskas1* Abstract Background: In Lithuania, the frst case of canine subcutaneous diroflariosis was recorded in 2010. Since then, an increasing number of cases of canine diroflariosis have been documented in diferent veterinary clinics throughout the country. Human diroflariosis was diagnosed in Lithuania for the frst time in September 2011. However, to the authors’ knowledge, there are no published data on the presence and prevalence of autochthonous diroflariosis in dogs and humans in the country. The present study provides information about the predominant species and preva- lence of Diroflaria in dogs and describes the cases of human diroflariosis in Lithuania. It also outlines PCR detection of the bacterial endosymbiont Wolbachia that contributes to the infammatory features of flarioid infection. Results: A total of 2280 blood samples and six adult worms from pet and shelter dogs were collected in the central and eastern regions of Lithuania in 2013–2015. Based on their morphological appearance, morphometric measure- ments and molecular analysis, all the adult nematodes were identifed as Diroflaria repens. The diagnosis of micro- flariae in blood samples was based on blood smear analysis and Knott’s test. The PCR and sequence analysis of the ribosomal DNA ITS2 region and cox1 gene confrmed the presence of D. repens. Overall, 61 (2.7%) of the 2280 blood samples were found to be positive for the presence of D. repens. The infection rate of D. repens was signifcantly higher in shelter dogs (19.0%; 19/100) than in pet dogs (1.9%; 42/2180) (χ2 100.039, df 1, P < 0.0001). Forty-nine DNA sam- ples of D. repens-infected dogs were tested for the presence of the bacterial= endosymbiont= Wolbachia and, of these, 40 samples (81.6%) were found to be positive. Three ocular and six subcutaneous cases of human diroflariosis were diagnosed in Lithuania in the period 2011–2018. Conclusions: To the authors’ knowledge, this is the frst report of autochthonous D. repens infection in dogs and humans in Lithuania. The present data demonstrate that D. repens is the main etiological agent of diroflariosis in Lithuania. The DNA of the flarioid endosymbiotic bacterium Wolbachia was detected in the vast majority of dogs infected with D. repens. Keywords: Diroflariosis, Autochthonous, Diroflaria repens, Dogs, Humans, Wolbachia, Lithuania Background (s.l.) and Aedes vexans [1–3]. Carnivores are the defni- Diroflariosis is an emerging vector-borne parasitic tive hosts. Te majority of cases in humans and animals zoonotic infection caused by nematodes of the genus are caused by two Diroflaria species, Diroflaria repens Diroflaria. Te parasites are transmitted by a large vari- and Diroflaria immitis, both of which can infect numer- ety of mosquito species belonging to the Culicidae fam- ous mammalian species [4]. ily, including some species of the Lithuanian mosquito Among mammalian hosts, domesticated dogs (Canis fauna, i.e. Culex pipiens (s.l.), Anopheles maculipennis familiaris) function as reservoirs and are the most important source for human transmission of both spe- *Correspondence: [email protected] cies [4]. Diroflariosis caused by D. repens and D. immi- 1 Department of Biology, Faculty of Natural Sciences, Vytautas Magnus tis has been also reported in wild carnivores, e.g. wolves University, Vileikos str. 8, LT-44404 Kaunas, Lithuania Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sabūnas et al. Parasites Vectors (2019) 12:177 Page 2 of 10 (Canis lupus), red foxes (Vulpes vulpes) and golden jack- Methods als (Canis aureus) [5–7]. Sample collection Te life-cycle of D. repens comprises fve larval stages. Samples collected from dogs Adult D. repens worms in dogs produce the frst-stage In co-operation with veterinary clinics and animal shel- larvae (microflariae) and release them into the blood- ters in Kaunas (central Lithuania) and Vilnius (eastern stream. Only mature helminths of the genus Diroflaria Lithuania), a total of 2280 dogs (2180 pet dogs and 100 can produce microflariae [4]. shelter dogs) were investigated for the presence of micro- Canine subcutaneous diroflariosis (D. repens) is often flariae in their blood in 2013–2015. None of the dogs considered asymptomatic, although in some cases the examined had been imported from endemic countries parasites cause subcutaneous nodules, while circulating or had ever travelled outside Lithuania. Dogs younger microflariae cause dermatological signs such as pruritus, than 6 months were excluded due to the long life-cycle of erythema, alopecia, difused dermatitis and itching [8]. Diroflaria. Te sex, age and body size of the shelter dogs Te traditional picture of human diroflariosis caused were recorded. by D. repens is associated with subcutaneous nodules Te shelter and pet dog blood samples were taken and ocular locations. However, there have been some from the cephalic vein, collected in EDTA-containing reports of human diroflariosis in unusual sites. Parasites vacutainers and then stored at 4 °C or -20 °C until DNA have been found in the lungs, scrotum, penis, spermatic isolation. cord, epididymis and female mammary glands [9, 10]. In Te microflariae were detected based on blood most human cases of ocular diroflariosis caused by D. smear microscopy and the modifed Knott’s test. PCR repens, the parasites have been found in nodules or cysts and sequence analysis were applied in order to confrm in the eye or in periocular tissues [10]. Only a few cases diagnosis and molecularly characterise the Diroflaria of humans showing circulating microflariae have been species. reported. Due to incomplete development, humans are In six dogs presented in small animal clinics, the adult not suitable hosts for disease transmission [11–13]. worms were removed from skin nodules using a surgical Many flarioid nematode species harbour intracellular technique. Adult nematodes were identifed by morpho- bacteria of the genus Wolbachia (Rickettsiaceae) [14]. logical and molecular methods. Wolbachia is found in all flarioid life stages and is essen- tial for embryogenesis, normal development, fertility and the long-term survival of the adult worm [15–17]. Wol- Samples collected from humans bachia is released into the blood and interacts with host Data on human diroflariosis cases (patients’ travel his- tissues when adult worms and microflariae die, stimulat- tory and other anamnestic data) were collected from the ing infammation and inducing immune responses [16, National Public Health Surveillance Laboratory (NVSPL), 18]. where each of the nine human diroflariosis cases during In Lithuania, the frst case of canine diroflariosis was the period 2011–2018 had been registered. Adult worms recorded in 2010 in the small animal clinic of the Veteri- were removed from human patients during surgical pro- nary Academy in Kaunas in central Lithuania [19]. Since cedures by ophthalmologists and surgeons in diferent then, a growing number of canine diroflariosis cases have areas of Lithuania. Helminths were subsequently sent to been documented in diferent veterinary clinics through- the NVSPL for further investigation. Species identifca- out the country. Human diroflariosis was diagnosed in tion was undertaken using morphological and morpho- Lithuania for the frst time in September 2011. However, metric analysis. To confrm the diagnosis in the frst case to the authors’ knowledge, no data have been published (Patient no. 1; Table 1), helminths were sent for investiga- about the presence and prevalence of autochthonous D. tion to the Swiss Institute of Parasitology of the Univer- repens infection in dogs and humans in Lithuania. Tere- sity of Zurich (SCUP). fore, the aim of the present study was to determine the predominant species and prevalence rate of diroflariosis Canine blood smear in dogs and to describe the cases of human diroflariosis After blood collection, a thin blood smear was imme- in Lithuania. A further objective of this work was to per- diately prepared. Te slides were air-dried, fxed with form molecular detection of the bacterial endosymbiont methanol and stained with Giemsa stain. Slides were Wolbachia, which plays an important role in D. repens observed for microflaria by light microscopy at 100× biology and contributes to the infammatory pathology of and 500× (oil immersion) magnifcation [20]. the infection. Sabūnas et al. Parasites Vectors (2019) 12:177 Page 3 of 10 Table 1 A summary of information about human infections of D. repens in Lithuania in the period 2011–2018 Patient no. Sex Age Locality Travelling history Year Location in host 1 Female 76 Kaunas None 2011 Ocular 2 Female 55 Vilnius Turkey 2012 Subcutaneous (head) 3 Male 7 Vilnius None 2012 Subcutaneous (abdomen) 4 Female 66 Vilnius None 2013 Subcutaneous (head) 5 Female 66 Ukmergė None 2013 Subcutaneous (chest) 6 Female 76 Kaunas None 2014 Ocular 7 Female 51 Vilnius None 2014 Ocular 8 Male 79 Utena None 2015 Subcutaneous (penis) 9 Male 28 Klaipėda Germany, Poland 2018 Subcutaneous (chest) Modifed Knott’s test Acanthocheilonema reconditum, Acanthocheilonema Briefy, 1 ml of EDTA blood was added to 9 ml of dracunculoides, Brugia pahangi and Brugia malayi) 2% formalin, mixed by inversion and centrifuged at [23].