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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short Communication
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Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine夽
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Alice Rossi a , Álvaro Peix a , Tamara Pavlikovskaya b , Olga Sagach b , Svetlana Nikolaenko b , Nina Chizh b , Vladimir Kartashev c , Fernando Simón d , Mar Siles-Lucas a,∗ a
Instituto de Recursos Naturales y Agrobiología de Salamanca, Agencia Estatal Consejo Superior de Investigaciones Científicas, Salamanca, Spain Central Sanitary and Epidemiological Station of the Ukrainian Ministry of Health, Kiev, Ukraine c Department of Infectious Diseases, Rostov State Medical University, Rostov-na-Donu, Russia d Laboratory of Parasitology, Faculty of Pharmacy & IBSAL, University of Salamanca, Salamanca, Spain b
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Article history: Received 1 August 2014 Received in revised form 9 October 2014 Accepted 27 October 2014 Available online xxx
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Keywords: Dirofilaria repens Dirofilaria immitis Ukraine 12S RNA Subcutaneous Ocular
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1. Introduction
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This short communication describes the phylogenetic analysis of 48 Dirofilaria worms isolated from human patients in Ukraine. 102 cases were both of subcutaneous (47; 46.1%) and ocular (54; 52.9%) locations. Worms from 44 patients (15 subcutaneous and 29 ocular) were subjected to DNA extraction and amplification of a specific fragment of the 12S rRNA subunit, and sequences were used for phylogenetic analysis. Results showed that 13.8% of the ocular cases analyzed at molecular level were caused by Dirofilaria immitis. Very few cases of ocular human dirofilariosis due to D. immitis have been described in the literature to date, majority of them attributed to Dirofilaria repens. Our results show that ocular dirofilariosis cannot be excluded in areas of low endemicity for D. repens were D. immitis is also present. © 2014 Published by Elsevier B.V.
Dirofilariases are vector borne transmitted zoonoses caused by different species of the genus Dirofilaria of which the main reservoirs are domestic and wild canids. Cases of human dirofilariosis are described in areas where infected animals are found and where the culicid mosquitoes feed on animals and humans. Dirofilaria immitis is the causal agent of human pulmonary dirofilariosis worldwide. Human subcutaneous and ocular dirofilariosis is usually caused by Dirofilaria repens in the Old World and by Dirofilaria tenuis, Dirofilaria conjunctivae and Dirofilaria ursi in North America. Both D. immitis and D. repens have been reported in anatomic sites other than the above-mentioned far less often (Simón et al., 2012). Human subcutaneous and ocular dirofilariosis caused by D. repens has been detected with increasing frequency in the Eurasian continent in the last few years, and it has been categorized as emerging disease in Europe (Genchi et al., 2011a). This spreading is attributed to the global warming and the lack of an adequate
夽 Sequences described in this work have been deposited in GenBank under accession numbers KM205372 to KM205415. Q2 ∗ Corresponding author. Tel.: +34 923219606; fax: +34 923219609. E-mail address: [email protected] (M. Siles-Lucas).
treatment of pets in some countries (Kartashev et al., 2014). Around 4000 human cases have been reported worldwide, 3500 of which have been diagnosed in Europe, most of them in Ukraine and the Russian Federation during the last 10 years (Kartashev et al., 2014; Sałamatin et al., 2013). Ocular cases are the most frequently reported, making up 35% of the human cases described worldwide (Genchi et al., 2011b) and more than 40% of the cases found in Ukraine and the Russian Federation (Avdiukhina et al., 1996; Iliasov et al., 2013). Conversely, only 380 cases of human pulmonary dirofilariosis caused by D. immitis have been reported in the literature, most of them in the Americas and Japan, 33 of which have been diagnosed in European countries (Simón et al., 2012). In the present work, 48 worms surgically removed from patients resident in Ukraine with subcutaneous nodules or ocular affection were characterized by molecular techniques. These techniques allowed us to identify an unusually high prevalence of ocular cases caused by D. immitis not reported previously in any other endemic area.
2. Materials and methods A total of 102 human patients were diagnosed of dirofilariosis in several hospitals from different territories of Ukraine in 2010.
http://dx.doi.org/10.1016/j.actatropica.2014.10.021 0001-706X/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Rossi, A., et al., Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.021
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Dirofilaria spp. worms were isolated either from subcutaneous locations (47 samples; 46.1% of cases), from ocular and periocular locations (conjunctiva or eyelids; 54 samples; 52.9% of cases), or from both locations in the same patient (1 case). Patients were subjected to X-ray examination and none of them showed lung lesions. Worms collected from 44 patients in subcutaneous (15 samples) or ocular and periocular locations (conjunctiva or eyelids; 29 samples) were preserved in ethanol. Preserved parasites were washed twice with cold PBS and subjected to DNA extraction with the Nucleospin® Tissue Kit (Macherey-Nagel), following the manufaturer’s instructions. Extracted DNA was amplified by PCR using primers specific for the 12S rDNA region, similar to those described and used for Dirofilaria species identification by Casiraghi et al. (2004) and Gioia et al. (2010), annealing in a stretch of the 12S sequence that has shown to be highly conserved in nematodes. Primers were designed after alignment of the 12S rDNA partial sequences of D. immitis (GenBank accession number EU169125) and D. repens (GenBank accession number GQ292761), in regions conserved in both species. Alignment was done with the Multalin facility at http://multalin.toulouse.inra.fr/multalin/multalin.html. The designed primers were 12SF (5 -GTTCCAGAATAATCGGCTATAC) and 12SR (5 -ATTGACGGATGGTTTGTACC), comprising a sequence of 514 base pairs (bp) for D. immitis and of 509 bp for D. repens. The PCR reaction was performed in a final volume of 25 l containing 2.5 l of 10× PCR buffer with 15 mM MgCl2 , 2 l of MgCl2 25 mM, 0.5 l dNTPs 10 mM, 25 nM of each primer, 0.1 l High Fidelity DNA polymerase (Fermentas), 18.7 l sterile distilled water and 1 l of purified DNA. PCR conditions were as follows: 30 cycles of 95 ◦ C for 30 s, 46 ◦ C for 30 s and 72 ◦ C for 1 min, with a final extension step of 72 ◦ C for 5 min. PCR products were loaded in 1% agarose gels with ethidium bromide and bands at the expected molecular weight were excised from the gel. Bands were purified with the Gene Jet Gel Extraction Kit (Thermo Scientific), following the manufacturers’ instructions. Purified bands were cloned in the pSC-A vector with the Strataclone PCR cloning kit (Agilent Technologies) as indicated by the manufacturers. Ligation reactions were used to transform Escherichia coli XL1-B cells. Cells were grown in LB-agar plates with 50 g/ml ampicillin overnight at 37 ◦ C. Five colonies for each PCR product were grown overnight in liquid LB plus ampicillin at 37 ◦ C with shacking. Cells were pelleted by centrifugation and the plasmids were extracted with the Nucleospin Plasmid kit (Macherey-Nagel). Extracted plasmids were sequenced with the universal primers T3 and T7. Obtained sequences were subjected to similarity analysis with sequences deposited in GenBank (nrNCBI) using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic relationships among our isolates and those Dirofilaria isolates described before, were evaluated comparing the obtained sequences with the following 12S rRNA sequences available in GenBank: AM779778 (D. repens, subcutaneous human isolate, Italy), AM779777 (D. repens, cat isolate, Italy), KC953031 (D. repens, dog isolate, Turkey), AM779776 (D. repens, dog isolate, Italy), AM779773 (D. repens, cat isolate, Italy); AM779774 (D. repens, subcutaneous human isolate, Italy), GQ292761 (D. repens, subcutaneous human isolate, India), AM779775 (D. repens, dog isolate, Italy), HQ540423 (D. immitis, ocular human isolate, Brazil), EU169125 (D. immitis dog isolate, China), AM779771 (D. immitis, cat isolate, Italy), EU182328 and EU182327 (D. immitis, red panda isolates, China), AM779770 and FN391554 (D. immitis, dog isolates, Italy), AM779769 (D. immitis, cat isolate, Italy), and JN801161 (Ascaris lumbricoides; outgroup). For phylogenetic analysis sequences were aligned using the Clustal X software (Thompson et al., 1997). The distances were calculated according to Kimura’s two-parameter model (Kimura, 1980). Phylogenetic trees of 12S rRNA gene were inferred using the neighbour-joining
analysis (Saitou and Nei, 1987), and maximum likelihood (Rogers and Swofford, 1998). MEGA5 software (Tamura et al., 2011) was used for all analyses.
3. Results and discussion A fragment of the 12S rRNA subunit was amplified out of 44 Dirofilaria worms isolated from subcutaneous (n = 15; 34%) and ocular (n = 29; 66%) locations in Ukrainian patients. Corresponding sequences were compared with those deposited in GenBank, allowing the identification of each isolate at species level. 40 of the isolates (91%) with ocular (n = 25) and subcutaneous (n = 15) locations showed 100–97% identities in BLAST with the first eight matches, corresponding to 12S rRNA sequences of D. repens isolates and 91% identity with the ninth match belonging to D. immitis, and thus were classified as D. repens. This group of 40 sequences only differed in one base: 8 sequences were 509 bp in length (isolates 15, 21, 35, 50, 56, 74, 77 and 82) and 32 sequences 510 bp in length showing one additional base at position 88. The proportion of ocular worms vs. subcutaneous worms was higher for worms displaying the sequence of 509 bp (6 out of 8; 75%) than for worms with the sequence of 510 bp (19 out of 32; 59%), as shown in Fig. 1. Whether those genetic differences influence the anatomical location of D. repens worms should be further analyzed with an extended number of isolates. The remaining four sequences (isolates 23, 25, 48 and 84) showed identities ranging from 100% to 95% with 12S rRNA sequences of D. immitis isolates, and ≤91% identity with the same type of sequence from D. repens, and thus they were classified as D. immitis. The four isolates corresponded with worms found in ocular locations, and showed identical 514 bp in length sequences among them, apart from bases at positions 241 and 437 that were different in isolate number 84 when compared to isolates 23, 25 and 48. Phylogenetic analysis showed the analyzed sequences separated in two main branches, corresponding to the two above-mentioned species (Fig. 1). The neighbour-joining analysis and maximum likelihood analysis resulted in trees with similar topology (data not shown). D. repens and D. immitis are maintained in the same animal reservoirs in the Old World. The epidemiological situation in animals was assessed in the Rostov region of Russia close to Ukraine, showing that around 20% of dogs tested positive in the Knott test for Dirofilaria (Kartashev et al., 2011). Species identification in the infected dogs showed that D. repens and D. immitis had prevalence rates of 44.7% and 30.3%, respectively, and the remaining dogs had mixed infections (Kartashev et al., 2011). Nevertheless, reported cases of human infection due to D. repens widely predominate even in areas with high endemicity of D. immitis in animal reservoirs (rev. in Simón et al., 2012). Epidemiological data on human dirofilariosis in Ukraine and Southwestern Russia also confirm the predominance of D. repens over D. immitis in human infections. In this area, the vast majority of registered cases of human dirofilariosis were subcutaneous or ocular (attributed to D. repens), with only 2 cases of human pulmonary dirofilariosis (attributed to D. immitis) reported until now (Iliasov et al., 2013; Kartashev et al., 2011; Sałamatin et al., 2013; Simón et al., 2012). Here, we only analyzed subcutaneous and ocular affected patients. The proportion of patients was slightly higher for the ocular location in accordance with previous analysis in the region (Avdiukhina et al., 1996; Iliasov et al., 2013; Sałamatin et al., 2013). Ocular dirofilariosis caused by D. immitis has been reported only occasionally, both in animal and human infections. In the New World, ocular infection with D. immitis in the animal reservoir (dog) has been more frequently reported than in the Old World
Please cite this article in press as: Rossi, A., et al., Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.021
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A. Rossi et al. / Acta Tropica xxx (2014) xxx–xxx KM205411 82 UA HU SC KM205410 77 UA HU SC KM205408 56 UA HU OC KM205406 35 UA HU OC KM205405 21 UA HU OC KM205404 15 UA HU SC KM205403 14 UA HU SC 61
KM205402 101 UA HU SC KM205387 52 UA HU SC KM205386 47 UA HU OC KM205385 43 UA HU SC KM205382 34 UA HU OC AM779778 Dirofilaria repens HU IT SC KM205380 29 UA HU OC KM205377 19 UA HU OC AM779777 Dirofilaria repens CAT IT KC953031 Dirofilaria repens DOG TR AM779776 Dirofilaria repens DOG IT
52 KM205372 1 UA HU SC KM205373 2 UA HU OC KM205376 9 UA HU OC KM205381 33 UA HU OC KM205383 37 UA HU OC KM205384 39 UA HU OC KM205393 79 UA HU OC KM205394 86 UA HU OC KM205396 88 UA HU OC KM205400 97 UA HU SC KM205407 50 UA HU OC AM779773 Dirofilaria repens CAT IT KM205374 5 UA HU SC 95
KM205375 8 UA HU SC KM205378 27 UA HU OC KM205379 28 UA HU SC KM205388 57 UA HU OC KM205389 65 UA HU OC KM205390 72 UA HU SC KM205391 73 UA HU OC
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KM205392 76 UA HU OC KM205395 87 UA HU OC KM205397 90 UA HU OC
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(e.g., Eberhard et al., 1977; Guterbock et al., 1981; Lavers et al., 1969; Mallett et al., 1971). In Europe, the first documented case of ocular D. immitis in a dog was reported in 2009 in the southeast of Italy (Dantas-Torres et al., 2009). In human patients, very few cases of ocular D. immitis have been reported both in the New World (Moorhouse, 1978 in Australia; Otranto et al., 2011 in northern Brazil) and in the Old World (Avellis et al., 2011 in northeast Italy). Thus, the most striking finding during our analysis was the identification of D. immitis in four of the 44 isolates – 9% over the total – all them found in ocular locations and thus accounting for the 13.8% of the ocular isolates analyzed here. Only two of the above-mentioned parasite isolated from ocular cases due to D. immitis were subjected to 12S rRNA specific PCR and sequencing: the Italian isolate from a dog (Dantas-Torres et al., 2009; GenBank accession number FN391554) and the Brazilian isolate from a human patient (Otranto et al., 2011; GenBank accession number HQ540423). The BLAST identities and the phylogenetic analysis done here, show that those two previously analyzed isolates group with D. immitis sequences (Fig. 1). Nevertheless, the sequence derived from the human ocular isolate of Brazilian origin was slightly different than the rest of D. immitis sequences. Similarly, Otranto et al. (2011) pointed out that this human isolate was distinct from the D. immitis reference sequences, including those of D. immitis infesting dogs in the same area, and concluded that this isolate could be a different zoonotic Dirofilaria species, e.g., an as yet unknown species infesting wild mammals in Brazil (Otranto et al., 2011). Similarly, two isolates of D. repens (GQ292761 and AM779775) lied apart from the rest of D. repens isolates used for the phylogenetic analysis (Fig. 1). The resolution power of the 12S rRNA fragment analyzed here at species level for nematodes, together with the identity close to 100% of the majority of isolates in each species, could indicate that those divergent isolates described before represent other genetic lineages, but no 12S sequences from species of the genus Dirofilaria different from D. immitis or D. repens are currently available. A common genetic marker for nematodes should be investigated in the available isolates of Dirofilaria spp. to better characterize the zoonotic potential of the different Dirofilaria species maintained in domestic and wild animal reservoirs.
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KM205398 91 UA HU SC 93
KM205399 92 UA HU OC KM205401 99 UA HU SC KM205409 74 UA HU OC AM779774 Dirofilaria repens HU IT SC GQ292761 Dirofilaria repens HU IN SC AM779775 Dirofilaria repens DOG IT
HQ540423 Dirofilaria immitis HU BZ OC EU169125 Dirofilaria immitis DOG CH KM205412 23 UA HU OC KM205413 25 UA HU OC
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KM205414 48 UA HU OC KM205415 84 UA HU OC 99
AM779771 Dirofilaria immitis CAT IT EU182328 Dirofilaria immitis PANDA CH EU182327 Dirofilaria immitis PANDA CH AM779770 Dirofilaria immitis DOG IT FN391554 Dirofilaria immitis DOG IT OC AM779769 Dirofilaria immitis CAT IT JN801161 Ascaris lumbricoides
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Fig. 1. Maximum likelihood phylogenetic rooted tree based on 12S rDNA gene sequences showing the taxonomic affiliation of the isolates. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. For each isolate, GenBank accession number, isolate number (for Ukrainian isolates), species, host, country and anatomical location are detailed. HU, human; UA, Ukraine; IT, Italy; TR, Turkey; IN, India; BZ, Brazil; CH, China; SC, subcutaneous; OC, ocular.
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from 1981 to 2011 and Projection by 2030. Biomed. Res. Int., http://dx.doi.org/ 10.1155/2014/858936. Kartashev, V., Batashova, I., Kartashov, S., Ermakov, A., Mironova, A., Kuleshova, Y., Ilyasov, B., Kolodiy, I., Klyuchnikov, A., Ryabikina, E., Babicheva, M., Levchenko, Y., Pavlova, R., Pantchev, N., Morchón, R., Simón, F., 2011. Canine and human dirofilariosis in the Rostov region (southern Russia). Vet. Med. Int. 2, 685713. Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Lavers, D.W., Spratt, D.M., Thomas, C., 1969. Dirofilaria immitis from the eye of a dog. Aust. Vet. J. 45 (6), 284–286. Mallett, G., Spratt, D.M., Derrington, P.C., Thomas, C., 1971. Male Dirofilaria immitis from the eye of a dog. Aust. Vet. J. 47 (3), 124. Moorhouse, D.E., 1978. Dirofilaria immitis: a cause of human intra-ocular infection. Infection 6 (4), 192–193. Otranto, D., Diniz, D.G., Dantas-Torres, F., Casiraghi, M., de Almeida, I.N., de Almeida, L.N., dos Santos, J.N., Furtado, A.P., de Almeida Sobrinho, E.F., Bain, O., 2011. Human intraocular filariasis caused by Dirofilaria sp. nematode, Brazil. Emerg. Infect. Dis. 17 (5), 863–866.
Rogers, J.S., Swofford, D.L., 1998. A fast method for approximating maximum likelihood’s of phylogenetic trees from nucleotide sequences. Syst. Biol. 47, 77–89. Saitou, N., Nei, M., 1987. A neighbour-joining method: a new method for reconstructing phylogenetics trees. Mol. Biol. Evol. 4, 406–425. Sałamatin, R.V., Pavlikovska, T.M., Sagach, O.S., Nikolayenko, S.M., Kornyushin, V.V., Kharchenko, V.O., Masny, A., Cielecka, D., Konieczna-Sałamatin, J., Conn, D.B., Golab, E., 2013. Human dirofilariosis due to Dirofilaria repens in Ukraine, an emergent zoonosis: epidemiological report of 1465 cases. Acta Parasitol. 58, 592–598. Simón, F., Siles-Lucas, M., Morchón, R., González-Miguel, J., Mellado, I., Carretón, E., Montoya-Alonso, J.A., 2012. Human and animal dirofilariosis: the emergence of a zoonotic mosaic. Clin. Microbiol. Rev. 25 (3), 507–544. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739. Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acid Res. 25, 4876–4882.
Please cite this article in press as: Rossi, A., et al., Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.021
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Contents lists available at ScienceDirect
Acta Tropica journal homepage: www.elsevier.com/locate/actatropica
Short Communication
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Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine夽
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Alice Rossi a , Álvaro Peix a , Tamara Pavlikovskaya b , Olga Sagach b , Svetlana Nikolaenko b , Nina Chizh b , Vladimir Kartashev c , Fernando Simón d , Mar Siles-Lucas a,∗ a
Instituto de Recursos Naturales y Agrobiología de Salamanca, Agencia Estatal Consejo Superior de Investigaciones Científicas, Salamanca, Spain Central Sanitary and Epidemiological Station of the Ukrainian Ministry of Health, Kiev, Ukraine c Department of Infectious Diseases, Rostov State Medical University, Rostov-na-Donu, Russia d Laboratory of Parasitology, Faculty of Pharmacy & IBSAL, University of Salamanca, Salamanca, Spain b
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Article history: Received 1 August 2014 Received in revised form 9 October 2014 Accepted 27 October 2014 Available online xxx
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Keywords: Dirofilaria repens Dirofilaria immitis Ukraine 12S RNA Subcutaneous Ocular
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This short communication describes the phylogenetic analysis of 48 Dirofilaria worms isolated from human patients in Ukraine. 102 cases were both of subcutaneous (47; 46.1%) and ocular (54; 52.9%) locations. Worms from 44 patients (15 subcutaneous and 29 ocular) were subjected to DNA extraction and amplification of a specific fragment of the 12S rRNA subunit, and sequences were used for phylogenetic analysis. Results showed that 13.8% of the ocular cases analyzed at molecular level were caused by Dirofilaria immitis. Very few cases of ocular human dirofilariosis due to D. immitis have been described in the literature to date, majority of them attributed to Dirofilaria repens. Our results show that ocular dirofilariosis cannot be excluded in areas of low endemicity for D. repens were D. immitis is also present. © 2014 Published by Elsevier B.V.
Dirofilariases are vector borne transmitted zoonoses caused by different species of the genus Dirofilaria of which the main reservoirs are domestic and wild canids. Cases of human dirofilariosis are described in areas where infected animals are found and where the culicid mosquitoes feed on animals and humans. Dirofilaria immitis is the causal agent of human pulmonary dirofilariosis worldwide. Human subcutaneous and ocular dirofilariosis is usually caused by Dirofilaria repens in the Old World and by Dirofilaria tenuis, Dirofilaria conjunctivae and Dirofilaria ursi in North America. Both D. immitis and D. repens have been reported in anatomic sites other than the above-mentioned far less often (Simón et al., 2012). Human subcutaneous and ocular dirofilariosis caused by D. repens has been detected with increasing frequency in the Eurasian continent in the last few years, and it has been categorized as emerging disease in Europe (Genchi et al., 2011a). This spreading is attributed to the global warming and the lack of an adequate
夽 Sequences described in this work have been deposited in GenBank under accession numbers KM205372 to KM205415. Q2 ∗ Corresponding author. Tel.: +34 923219606; fax: +34 923219609. E-mail address: [email protected] (M. Siles-Lucas).
treatment of pets in some countries (Kartashev et al., 2014). Around 4000 human cases have been reported worldwide, 3500 of which have been diagnosed in Europe, most of them in Ukraine and the Russian Federation during the last 10 years (Kartashev et al., 2014; Sałamatin et al., 2013). Ocular cases are the most frequently reported, making up 35% of the human cases described worldwide (Genchi et al., 2011b) and more than 40% of the cases found in Ukraine and the Russian Federation (Avdiukhina et al., 1996; Iliasov et al., 2013). Conversely, only 380 cases of human pulmonary dirofilariosis caused by D. immitis have been reported in the literature, most of them in the Americas and Japan, 33 of which have been diagnosed in European countries (Simón et al., 2012). In the present work, 48 worms surgically removed from patients resident in Ukraine with subcutaneous nodules or ocular affection were characterized by molecular techniques. These techniques allowed us to identify an unusually high prevalence of ocular cases caused by D. immitis not reported previously in any other endemic area.
2. Materials and methods A total of 102 human patients were diagnosed of dirofilariosis in several hospitals from different territories of Ukraine in 2010.
http://dx.doi.org/10.1016/j.actatropica.2014.10.021 0001-706X/© 2014 Published by Elsevier B.V.
Please cite this article in press as: Rossi, A., et al., Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.021
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Dirofilaria spp. worms were isolated either from subcutaneous locations (47 samples; 46.1% of cases), from ocular and periocular locations (conjunctiva or eyelids; 54 samples; 52.9% of cases), or from both locations in the same patient (1 case). Patients were subjected to X-ray examination and none of them showed lung lesions. Worms collected from 44 patients in subcutaneous (15 samples) or ocular and periocular locations (conjunctiva or eyelids; 29 samples) were preserved in ethanol. Preserved parasites were washed twice with cold PBS and subjected to DNA extraction with the Nucleospin® Tissue Kit (Macherey-Nagel), following the manufaturer’s instructions. Extracted DNA was amplified by PCR using primers specific for the 12S rDNA region, similar to those described and used for Dirofilaria species identification by Casiraghi et al. (2004) and Gioia et al. (2010), annealing in a stretch of the 12S sequence that has shown to be highly conserved in nematodes. Primers were designed after alignment of the 12S rDNA partial sequences of D. immitis (GenBank accession number EU169125) and D. repens (GenBank accession number GQ292761), in regions conserved in both species. Alignment was done with the Multalin facility at http://multalin.toulouse.inra.fr/multalin/multalin.html. The designed primers were 12SF (5 -GTTCCAGAATAATCGGCTATAC) and 12SR (5 -ATTGACGGATGGTTTGTACC), comprising a sequence of 514 base pairs (bp) for D. immitis and of 509 bp for D. repens. The PCR reaction was performed in a final volume of 25 l containing 2.5 l of 10× PCR buffer with 15 mM MgCl2 , 2 l of MgCl2 25 mM, 0.5 l dNTPs 10 mM, 25 nM of each primer, 0.1 l High Fidelity DNA polymerase (Fermentas), 18.7 l sterile distilled water and 1 l of purified DNA. PCR conditions were as follows: 30 cycles of 95 ◦ C for 30 s, 46 ◦ C for 30 s and 72 ◦ C for 1 min, with a final extension step of 72 ◦ C for 5 min. PCR products were loaded in 1% agarose gels with ethidium bromide and bands at the expected molecular weight were excised from the gel. Bands were purified with the Gene Jet Gel Extraction Kit (Thermo Scientific), following the manufacturers’ instructions. Purified bands were cloned in the pSC-A vector with the Strataclone PCR cloning kit (Agilent Technologies) as indicated by the manufacturers. Ligation reactions were used to transform Escherichia coli XL1-B cells. Cells were grown in LB-agar plates with 50 g/ml ampicillin overnight at 37 ◦ C. Five colonies for each PCR product were grown overnight in liquid LB plus ampicillin at 37 ◦ C with shacking. Cells were pelleted by centrifugation and the plasmids were extracted with the Nucleospin Plasmid kit (Macherey-Nagel). Extracted plasmids were sequenced with the universal primers T3 and T7. Obtained sequences were subjected to similarity analysis with sequences deposited in GenBank (nrNCBI) using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Phylogenetic relationships among our isolates and those Dirofilaria isolates described before, were evaluated comparing the obtained sequences with the following 12S rRNA sequences available in GenBank: AM779778 (D. repens, subcutaneous human isolate, Italy), AM779777 (D. repens, cat isolate, Italy), KC953031 (D. repens, dog isolate, Turkey), AM779776 (D. repens, dog isolate, Italy), AM779773 (D. repens, cat isolate, Italy); AM779774 (D. repens, subcutaneous human isolate, Italy), GQ292761 (D. repens, subcutaneous human isolate, India), AM779775 (D. repens, dog isolate, Italy), HQ540423 (D. immitis, ocular human isolate, Brazil), EU169125 (D. immitis dog isolate, China), AM779771 (D. immitis, cat isolate, Italy), EU182328 and EU182327 (D. immitis, red panda isolates, China), AM779770 and FN391554 (D. immitis, dog isolates, Italy), AM779769 (D. immitis, cat isolate, Italy), and JN801161 (Ascaris lumbricoides; outgroup). For phylogenetic analysis sequences were aligned using the Clustal X software (Thompson et al., 1997). The distances were calculated according to Kimura’s two-parameter model (Kimura, 1980). Phylogenetic trees of 12S rRNA gene were inferred using the neighbour-joining
analysis (Saitou and Nei, 1987), and maximum likelihood (Rogers and Swofford, 1998). MEGA5 software (Tamura et al., 2011) was used for all analyses.
3. Results and discussion A fragment of the 12S rRNA subunit was amplified out of 44 Dirofilaria worms isolated from subcutaneous (n = 15; 34%) and ocular (n = 29; 66%) locations in Ukrainian patients. Corresponding sequences were compared with those deposited in GenBank, allowing the identification of each isolate at species level. 40 of the isolates (91%) with ocular (n = 25) and subcutaneous (n = 15) locations showed 100–97% identities in BLAST with the first eight matches, corresponding to 12S rRNA sequences of D. repens isolates and 91% identity with the ninth match belonging to D. immitis, and thus were classified as D. repens. This group of 40 sequences only differed in one base: 8 sequences were 509 bp in length (isolates 15, 21, 35, 50, 56, 74, 77 and 82) and 32 sequences 510 bp in length showing one additional base at position 88. The proportion of ocular worms vs. subcutaneous worms was higher for worms displaying the sequence of 509 bp (6 out of 8; 75%) than for worms with the sequence of 510 bp (19 out of 32; 59%), as shown in Fig. 1. Whether those genetic differences influence the anatomical location of D. repens worms should be further analyzed with an extended number of isolates. The remaining four sequences (isolates 23, 25, 48 and 84) showed identities ranging from 100% to 95% with 12S rRNA sequences of D. immitis isolates, and ≤91% identity with the same type of sequence from D. repens, and thus they were classified as D. immitis. The four isolates corresponded with worms found in ocular locations, and showed identical 514 bp in length sequences among them, apart from bases at positions 241 and 437 that were different in isolate number 84 when compared to isolates 23, 25 and 48. Phylogenetic analysis showed the analyzed sequences separated in two main branches, corresponding to the two above-mentioned species (Fig. 1). The neighbour-joining analysis and maximum likelihood analysis resulted in trees with similar topology (data not shown). D. repens and D. immitis are maintained in the same animal reservoirs in the Old World. The epidemiological situation in animals was assessed in the Rostov region of Russia close to Ukraine, showing that around 20% of dogs tested positive in the Knott test for Dirofilaria (Kartashev et al., 2011). Species identification in the infected dogs showed that D. repens and D. immitis had prevalence rates of 44.7% and 30.3%, respectively, and the remaining dogs had mixed infections (Kartashev et al., 2011). Nevertheless, reported cases of human infection due to D. repens widely predominate even in areas with high endemicity of D. immitis in animal reservoirs (rev. in Simón et al., 2012). Epidemiological data on human dirofilariosis in Ukraine and Southwestern Russia also confirm the predominance of D. repens over D. immitis in human infections. In this area, the vast majority of registered cases of human dirofilariosis were subcutaneous or ocular (attributed to D. repens), with only 2 cases of human pulmonary dirofilariosis (attributed to D. immitis) reported until now (Iliasov et al., 2013; Kartashev et al., 2011; Sałamatin et al., 2013; Simón et al., 2012). Here, we only analyzed subcutaneous and ocular affected patients. The proportion of patients was slightly higher for the ocular location in accordance with previous analysis in the region (Avdiukhina et al., 1996; Iliasov et al., 2013; Sałamatin et al., 2013). Ocular dirofilariosis caused by D. immitis has been reported only occasionally, both in animal and human infections. In the New World, ocular infection with D. immitis in the animal reservoir (dog) has been more frequently reported than in the Old World
Please cite this article in press as: Rossi, A., et al., Genetic diversity of Dirofilaria spp. isolated from subcutaneous and ocular lesions of human patients in Ukraine. Acta Trop. (2014), http://dx.doi.org/10.1016/j.actatropica.2014.10.021
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A. Rossi et al. / Acta Tropica xxx (2014) xxx–xxx KM205411 82 UA HU SC KM205410 77 UA HU SC KM205408 56 UA HU OC KM205406 35 UA HU OC KM205405 21 UA HU OC KM205404 15 UA HU SC KM205403 14 UA HU SC 61
KM205402 101 UA HU SC KM205387 52 UA HU SC KM205386 47 UA HU OC KM205385 43 UA HU SC KM205382 34 UA HU OC AM779778 Dirofilaria repens HU IT SC KM205380 29 UA HU OC KM205377 19 UA HU OC AM779777 Dirofilaria repens CAT IT KC953031 Dirofilaria repens DOG TR AM779776 Dirofilaria repens DOG IT
52 KM205372 1 UA HU SC KM205373 2 UA HU OC KM205376 9 UA HU OC KM205381 33 UA HU OC KM205383 37 UA HU OC KM205384 39 UA HU OC KM205393 79 UA HU OC KM205394 86 UA HU OC KM205396 88 UA HU OC KM205400 97 UA HU SC KM205407 50 UA HU OC AM779773 Dirofilaria repens CAT IT KM205374 5 UA HU SC 95
KM205375 8 UA HU SC KM205378 27 UA HU OC KM205379 28 UA HU SC KM205388 57 UA HU OC KM205389 65 UA HU OC KM205390 72 UA HU SC KM205391 73 UA HU OC
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KM205392 76 UA HU OC KM205395 87 UA HU OC KM205397 90 UA HU OC
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(e.g., Eberhard et al., 1977; Guterbock et al., 1981; Lavers et al., 1969; Mallett et al., 1971). In Europe, the first documented case of ocular D. immitis in a dog was reported in 2009 in the southeast of Italy (Dantas-Torres et al., 2009). In human patients, very few cases of ocular D. immitis have been reported both in the New World (Moorhouse, 1978 in Australia; Otranto et al., 2011 in northern Brazil) and in the Old World (Avellis et al., 2011 in northeast Italy). Thus, the most striking finding during our analysis was the identification of D. immitis in four of the 44 isolates – 9% over the total – all them found in ocular locations and thus accounting for the 13.8% of the ocular isolates analyzed here. Only two of the above-mentioned parasite isolated from ocular cases due to D. immitis were subjected to 12S rRNA specific PCR and sequencing: the Italian isolate from a dog (Dantas-Torres et al., 2009; GenBank accession number FN391554) and the Brazilian isolate from a human patient (Otranto et al., 2011; GenBank accession number HQ540423). The BLAST identities and the phylogenetic analysis done here, show that those two previously analyzed isolates group with D. immitis sequences (Fig. 1). Nevertheless, the sequence derived from the human ocular isolate of Brazilian origin was slightly different than the rest of D. immitis sequences. Similarly, Otranto et al. (2011) pointed out that this human isolate was distinct from the D. immitis reference sequences, including those of D. immitis infesting dogs in the same area, and concluded that this isolate could be a different zoonotic Dirofilaria species, e.g., an as yet unknown species infesting wild mammals in Brazil (Otranto et al., 2011). Similarly, two isolates of D. repens (GQ292761 and AM779775) lied apart from the rest of D. repens isolates used for the phylogenetic analysis (Fig. 1). The resolution power of the 12S rRNA fragment analyzed here at species level for nematodes, together with the identity close to 100% of the majority of isolates in each species, could indicate that those divergent isolates described before represent other genetic lineages, but no 12S sequences from species of the genus Dirofilaria different from D. immitis or D. repens are currently available. A common genetic marker for nematodes should be investigated in the available isolates of Dirofilaria spp. to better characterize the zoonotic potential of the different Dirofilaria species maintained in domestic and wild animal reservoirs.
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KM205398 91 UA HU SC 93
KM205399 92 UA HU OC KM205401 99 UA HU SC KM205409 74 UA HU OC AM779774 Dirofilaria repens HU IT SC GQ292761 Dirofilaria repens HU IN SC AM779775 Dirofilaria repens DOG IT
HQ540423 Dirofilaria immitis HU BZ OC EU169125 Dirofilaria immitis DOG CH KM205412 23 UA HU OC KM205413 25 UA HU OC
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KM205414 48 UA HU OC KM205415 84 UA HU OC 99
AM779771 Dirofilaria immitis CAT IT EU182328 Dirofilaria immitis PANDA CH EU182327 Dirofilaria immitis PANDA CH AM779770 Dirofilaria immitis DOG IT FN391554 Dirofilaria immitis DOG IT OC AM779769 Dirofilaria immitis CAT IT JN801161 Ascaris lumbricoides
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Fig. 1. Maximum likelihood phylogenetic rooted tree based on 12S rDNA gene sequences showing the taxonomic affiliation of the isolates. Bootstrap values calculated for 1000 replications are indicated. Bar, 5 nt substitution per 100 nt. For each isolate, GenBank accession number, isolate number (for Ukrainian isolates), species, host, country and anatomical location are detailed. HU, human; UA, Ukraine; IT, Italy; TR, Turkey; IN, India; BZ, Brazil; CH, China; SC, subcutaneous; OC, ocular.
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