CZECH POLAR REPORTS 4 (1): 40-46, 2014 Molecular taxonomic study of Trichinella spp. from mammals of Russian Arctic and subarctic areas Irina M. Odoevskaya1*, Sergei E. Spiridonov2 1GNU K. I. Skryabin All-Russian Institute of Helminthology, Bolshaya Cheremushins- kaja, 28, Moscow, 117259, Russia 2Centre for Parasitology, AN Severtsov´s Institute of Ecology and Evolution. Russian Academy of Science, 33 Leninsky prospect, Moscow, 117071, Russia Abstract Analysis of taxonomic affiliation of Trichinella species circulating in the Chukotka Autonomous Region and some subarctic areas of the Russian Federation showed that the representatives of T. spiralis and the Arctic trichinellas - T. nativa (genotype T2) and Trichinella sp. (genotype T6) can be found there. The partial sequences of Coxb (704 bp) of these Arctic Trichinella spp. from Russia differ from Coxb sequences of those genotypes (T2 and T6) deposited in NCBI GenBank (1-3 bp). The cultivated larvae of Trichinella sp., which were established from muscular tissue sample of stray cat (shot on the fur farm in Chukotka peninslula) differ at molecular level (Coxb) even more significantly; 21-24 bp difference between Trichinella sp. and T. nativa and 46-47 bp difference between the same isolate and T. spiralis were recorded. Key words: mammals, parasitic nematodes, phylogenetic tree, taxonomy, Trichinella sp.T6 DOI: 10.5817/CPR2014-1-4 Introduction The presence of parasitic nematodes of bes 2010, Seymour 2012). The connection the genus Trichinella Railliet 1895 in Artic of human infection to the consumption of ecosystems and their importance as epizo- the meat of several terrestrial and marine otic and epidemiological factor in these mammals (walruses, bears, bearded seals, areas were reported from different regions, ringed seals) was demonstrated (Rausch e.g. Chukotka peninsula in Russia, North- 1970, Serhir et al. 2001, Leclair et al. ern Canada and Alaska, USA (Lukashenko 2004, Pozio 2007, Seymour 2012). Nowa- et al. 1971, Forbes 2000, Gajadhar et For- days, molecular taxonomical methods used ——— Received April 29, 2014, accepted August 1, 2014. *Corresponding author: Ирина Одоевская <[email protected]> Acknowledgements: This study was supported by the grant № 14-04-01064 from the Russian Foundation for Basic Researches. 40 I. M. ODOEVSKAYA et S. E. SPIRIDONOV for the identification of parasitic organ- and T6 have been found in a number of isms, including also nematodes are more marine mammals: walruses (Odobenus and more common, but up to now no study rosmarus rosmarus, Odobenus rosmarus was particularly focused on the molecular divergens), ringed seals (Phoca hispida), determination of Trichinella species from spotted seals (Phoca largha), bearded Russian Arctic region. seals (Erignathus barbatus), harp seals It is known that two genotypes of (Phoca groenlandica), Steller sea lions Trichinella, Trichinella nativa (T2) and (Eumetopias jubatus), as well as represent- not-yet-described as an independent spe- atives of the order Cetacea (Cetacea), the cies Trichinella sp. (T6) are found at the suborder of toothed whales (Odontoceiti), Arctic coasts, e.g. of Greenland, Canada beluga (Delphinapterus leucas) (Dunams- and Alaska (Dunams-Morel et al. 2012). Morel et al. 2012, Forbes 2000, Gajadhar Trichinella of these two genotypes were et Forbes 2010, Leclair et al. 2004, Møller found in animals common for Arctic eco- et al. 2005, Murrell et al. 2000, Pozio et systems, such as wild white arctic foxes Zarlenga 2005, Serhir et al. 2001). The (Alopex lagopus), red foxes (Vulpes vul- results of molecular taxonomic study of pes), wolves (Canis lupus), brown bears Trichinella spp. collected in the Russian (Ursus arctos), polar bears (Ursus mari- arctic and subarctic territories are pre- timus) and wolverines (Gulo gulo). Simi- sented below. larly, Trichinella genotypes T. nativa (T2) Material and Methods In our study, we have carried out strains and frozen samples at the K. I. molecular genetic studies of 5 isolates of Skryabin All-Russian Institute of Helmin- Trichinella spp. isolated from the muscles thology. of terrestrial and marine mammals of DNA was extracted from a suspension Chukotka: sledge dog (Canis familiaris), of Trichinella larvae L1, which were iso- stray cat (Felis familiaris), a wild fox lated from experimentally infected ani- (Alopex lagopus), a seal (Phoca hispida), mals. The laboratory animals (mice or and a brown bear (Ursus arctos). Several Syrian hamsters), were per-orally fed with samples of Trichinella collected from the suspension of larvae obtained through mammals outside of Chukotka were also digestion of the muscle tissue of hunted studied, inlcuding samples from polar animals with artificial gastric juice. Labo- bears (U. maritimus) and wolverine (G. ratory animals were euthanized with ether gulo) from Sakha-Jakutia republic, Strays on 45-60 day post infection to obtain cats (F. familiaris) and dogs (C. familiaris) samples of muscle tissue for digestion. from Kirov and Voronezh regions, brown DNA was extracted from suspension of bears from Kirov and Primorskii (Vladi- larvae by using of columns system of vostok area) regions. The Trichinella Wizard ® SV Genomic DNA Purification isolates from a rat (Ossetia) were used as a System according to the protocol of control group. Laboratory culture of Tri- Promega company. chinella nelsoni (obtained from Central Partial sequence of mitochondrial Coxb Helminthological Laboratory in Sophia, gene was obtained with primers Tricob F1 Bulgaria) was used as outgroup for phy- (forward) CAA TCC ATT AGG TAC logenetic analysis. All these samples were ACA CTC AC and Tricob R3 (reverse) kept in the collection of living Trichinella TAA GTA AGA TTT CAA TGG CG 41 TRICHINELLA IN CHUKOTKA (Rosenthal et al. 2008). The following spectrophotometer Nanodrop 2000. Direct protocol was used for polymerase chain sequencing was performed with the same reaction (PCR): primary denaturation primers as used for the primary PCR. (94°C) – 4 min, then 35 cycles of Obtained chromatograms were read with denaturation at 94°C – 10 sec, annealing at Chromas 1.45., exported to FASTA- 55°C – 30 sec, elongation at 72°C – 30 format and used to obtain alignments with sec. Upon completion of 35 cycles, a final Clustal X 1.81. The programme Genedoc elongation was performed at 72°C – 3 min. 2.5. was used to remove flanking parts to The separation of PCR amplicons was obtain rectangular alignment and export performed in 1% agarose gel and the the data into Nexus-format. Such align- profiles were visualized on Gel Imager. ments were analysed with PAUP 4.0b10 The PCR amplicons were cuted from the and MEGA 5.0 (Swofford 1998, Tamura agarose gels and the isolation of DNA et al. 2011). Phylogenetic analysis was from the gel cubes was carried out by conducted by the combination of several using a kit of Promega company - Wizard methods including maximum parsimony, ® SV Gel and PCR Clean-Up System. maximum likelihood and Bayesian infer- Eluted DNA was precipitated with 96% ence. All these methods demonstrated ethanol with 5M ammonium acetate and similar topologies of resulting phyloge- re-diluted in 30-50 µl of water. The netic trees. Thus, only maximum parsi- concentration of DNA was estimated with mony tree is presented below. Results and Discussion The alignment of the Cob1 mtDNA polar bear and wolverine are demon- sequences obtained for the Trichinella strating the close relationships with samples from Chukotka and other Arctic Trichinella spiralis. Only one sequence – and subarctic areas of the Russian sample 51 from stray cat (Chukotka) was Federation resulted in the partial sequence out of obvious relationship with known of length 704 bp. The phylogenetic tree Trichinella sequences (Fig. 1.), clustering obtained with the method of maximum with basal node for T. nativa. parsimony obtained after the analysis of More detailed overview of revealed this alignment was generated and it is nucleotide differences is presented in the presented on Fig. 1. According to the Table 1. In accordance to the phylogenetic phylogenetic tree the studied Trichinella tree the studied trichinellids are divided samples are divided into two main groups. into two main groups. One group, con- Samples from the seal, husky, polar fox sisting of three Chukotka Trichinella sam- (sample 54) from Chukotka as also from ples from: seal, husky dog and polar fox polar bear from Jakutia and from stray cat from fur farm (sample 54), together with and dog of Kirov and Voronezh regions several samples from other areas of Russia are clustering together with the sequences (brown bear, Kirov region; stray cat, of the species Trichinella nativa and Voronezh region; stray dog, Kirov region). Trichinella sp. ‘T6” deposited in NCBI There is demonstrated low number of GenBank (Dunams-Morel et al. 2012), differences in nucleotide sequence to those whereas samples from polar bear and polar of Trichinella nativa (JQ430661) and fox (sample 24) from Chukotka, as also Trichinella sp., genotype T6 (JQ430674) from brown bear from Vladivostok area, observed in database. Obviously, all ana- 42 I. M. ODOEVSKAYA et S. E. SPIRIDONOV lyzed isolates belong to the complex of GU339148) is 50 bp – approximately 8%. species “nativa-T6”. There are no signifi- The sample of Trichinella sp. from stray cant differences in the studied Cob1 cat, hunted at the fur-farm, demonstrated mtDNA sequence according to which is 21-24 bp difference (about 3%) with all possible to discriminate between T. nativa T. nativa sequences, and 46-47 bp dif- and T6. Remaining sequences of studied ference (7%) with T. spiralis (Table 1). Trichinella samples are clustering with the Trichinella sample from stray dog (Kirov sequence of T. spiralis (GU339148). The region) demonstrated the difference in sequential difference of T. spiralis and 3 bp (less than 1%) to other T. nativa T. nativa Cob1 genes (JQ430661 and samples. Fig. 1. Phylogenetic tree of partial Coxb gene sequences of studied Trichinella spp. samples from the Russian Arctic and those of other Trichinella species - Trichinella sp. T6, T.