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45Russian Journal of Nematology, 2018, 26 (2), 123 - 128
The diversity of Trichinella in natural habitats of
the Russian Far East
1 2 3
Irina M. Odoyevskaya , Ivan V. Seryodkin ' and Sergei E. Spiridonov4
'All-Russian Scientific Research Institute of Fundamental and Applied Parasitology of Animals and Plants named after K.I. Skryabin, Bolshaya Cheremushkinskaya street 28, 117218 Moscow, Russia
E-mail: odoevskayaim@rambler.ru 2Pacific Geographical Institute, Far Eastern Branch of the Russian Academy of Sciences, Radio st., 7,
690041, Vladivistok, Russia 3Far Eastern Federal University, Sukhanova st., 8, 690091, Vladivistok, Russia 4Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect 33, 119071 Moscow
Accepted for publication 26 November 2018
Summary. The juveniles of Trichinella nematodes were collected from the muscular tissue of different predaceous mammals in the natural habitats of the Russian Far East (Primorsky Region of the Russian Federation). Identification of Trichinella nematodes up to the species level was based on the analysis of nucleotide sequences of mitochondrial DNA. The amplified sequence contained CoxI gene and adjacent tRNA sequences. Three species of trichinellids were detected in the studied material: T. spiralis, T. pseudospiralis and T. nativa. The latter species demonstrated the strongest genetical diversity as five haplotypes of T. nativa were discovered in the studied material. New hosts were reported for Trichinella nematodes in the Russian Far East including a leopard cat (Prionailurus bengalensis). Other mammal hosts included an Asian badger (Meles leucurus), a brown bear (Ursus arctos), a lynx (Lynx lynx), a red fox (Vulpes vulpes) and a sable (Martes zibellina).
Key words: CoxI mtDNA, predaceous mammal hosts, Trichinella, Trichinella spiralis, Trichinella pseudospiralis, Trichinella nativa.
The nematodes of the genus Trichinella pose a significant threat to human health, as in most acute cases the consumption of meat containing the juveniles of these nematodes can be fatal. The ability of Trichinella nematodes to complete their life cycle with the use of different vertebrate animals and ability to survive in the organism of invertebrates makes the task of the veterinary control very complicated. Usually, the cases of human infection with Trichinella are related to the consumption of pork as a product of animal husbandry or the meat of wild game from natural habitats. In Russian Federation, the outbreaks of trichinosis are often related to the consumption of a bear's meat or meat of other predaceous mammals, either wild or kept on fur farms (Odoyevskaya et al., 2013; Uspensky et al., 2018). Although the circulation of Trichinella larvae in anthropogenic ecosystems has been studied quite intensively, the circumstances of these parasitic nematodes
circulation in the natural habitats remain little studied. The last two decades have been a time of significant progress in understanding the taxonomic structure of the genus Trichinella (Pozio & Murrell, 2006; Pozio & Zarlenga, 2013). According to modern concepts, this genus includes 12 species, which are genetically characterised and demonstrate a certain level of uniformity throughout the areal of distribution (Pozio et al., 2009). Nine of these 12 species correspond to the described species, while three species yet have to be characterised morphologically and named. Two widely distributed species of Trichinella (T. nativa Britov & Boev, 1972 and T. pseudospiralis Garkavi, 1972) have been described based on material collected in the Russian Federation (Britov & Boev, 1972; Garkavi, 1972). The findings of T. nativa are usually associated with wild sylvatic carnivores; the second species is associated with sylvatic mammals and birds, but was also reported from domestic pigs
© Russian Society of Nematologists, 2018; doi:10.24411/0869-6918-2018-100012
(Gottstein et al, 2009). In recent years, intensive collections of samples of the muscle tissue of dead animals were carried out in the Primorsky Region by one of the authors (I.V.S.). It made possible returning once more to the problem of the taxonomic diversity of Trichinella in natural ecosystems of the Russian Far East.
MATERIAL AND METHODS
Fragments of muscular tissue were collected from wild fallen predatory mammals in Primorsky Region of the Russian Federation. The list of studied samples is given in Table 1. The tissue samples were examined by 'the compression method'. Samples positive for Trichinella juveniles were then digested in artificial gastric juice. The juveniles were isolated by sedimentation in a Baermann funnel and frozen. After thawing, 5-12 first-stage juveniles from each sample were collected with a needle and transferred to Eppendorf 0.5 ml tubes with transparent walls in a 2-3 ^l drop of sterile water on the inner surface of a tube. It was possible to check the presence and calculate the number of collected juveniles. Then, 22 ^l of sterile water and 25 ^l of lysis buffer containing proteinase K and mercaptoethanol were added (Holterman et
al., 2006). After incubation for 1.5 hours at 65 ° C, followed by heating at 99° C for 5 minutes, 1-1.6 ^l of the obtained homogenate was used as template for PCR. The PCR protocol corresponded to that described earlier (Odoyevskaya & Spiridonov, 2016). The primer pair 37F_Tri GCA GTA AAT TTA GAA TTT AAA C and 42R_Tri-CCA AAT ATT CAT GGT GTT CATT was used. A 1400 bp long portion of mitochondrial genome containing Ala-tRNA, Cys-tRNA, Tyr-tRNA and CoxI mtDNA gene was amplified. The obtained PCR products were purified by electrophoresis in 0.8% agarose gel, isolated from the gel using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, USA) and finally cleaned by precipitation with 70% ethanol in the presence of ammonium acetate. Amplified DNA fragments were sequenced with the same primers used for PCR. Chromatograms were analysed using the Chromas 1.45 program (www.technelysium.com.au/ chromas.html). Obtained sequences were saved as FASTA format. The closest matches for the obtained sequences were found in NCBI GenBank using the BLAST algorithm (Altshul et al., 1990). The resulting set of sequences containing newly obtained ones and downloaded from GenBank were aligned in Clustal X (Thompson et al., 1997). The flanking parts of different
Table 1. Origin of the studied Trichinella isolates and accession numbers in NCBI GenBank.
Locality Host Isolate designation Species NCBI GenBank accession number
Primorsky Region, Russian Far East Sable Tri113 Trichinella nativa MK189165
Primorsky Region, Russian Far East Leopard cat Tri122 Trichinella nativa MK189166
Primorsky Region, Russian Far East Asian badger Tri126 Trichinella nativa MK189167
Primorsky Region, Russian Far East Sable Tri132 Trichinella nativa MK189168
Primorsky Region, Russian Far East Sable TrN2 Trichinella spiralis MK257742
Primorsky Region, Russian Far East Leopard cat TrN3 Trichinella nativa MK032471
Primorsky Region, Russian Far East Leopard cat TrN12 Trichinella pseudospiralis MK257739
Primorsky Region, Russian Far East Red fox TrN13 Trichinella nativa MK034472
Primorsky Region, Russian Far East Lynx TrN17 Trichinella nativa MK257736
Primorsky Region, Russian Far East Brown bear TrN24 Trichinella nativa MK032473
Primorsky Region, Russian Far East Red fox TrN26 Trichinella nativa MK257738
Kamchatka Peninsula, Russian Far East Domestic pig Tri79 Trichinella pseudospiralis MK257740
length were removed in Genedoc 2.7. (Nicholas et al., 1997) to obtain the rectangular matrix of data. This data matrix was used for phylogenetic analysis in the MEGA7 program (Kumar et al., 2016). Three methods of analysis were used: maximum parsimony (MP), neighbour joining (NJ) with 1000 bootstrap pseudo-repeats and maximum likelihood (ML) under Tamura-Nei model with gamma-distributed sites (TN93+G), Nearest-Neighbor-Interchange as tree inference option and with rapid bootstrapping under 500 replicates. The Trichinella sequences representing separate haplotypes were deposited with NCBI GenBank (accession numbers in Table 1).
RESULTS
The obtained rectangular matrix with mismatching flanking parts removed had a length of 1135 bp. Among these positions of the alignment, 808 positions were constant, 67 positions were non-informative and 260 positions were informative. The topologies of the trees obtained by all the methods (MP, NJ and ML) were similar and values of bootstrap support for clades were presented on Fig. 1. The maximal (100%) level of support was observed for two species (T. spiralis and T. pseudospiralis) and for a clade consisting of T. nativa and sequence of Trichinella sp. T6 genotype. Bootstrap support for isolates of T. nativa was quite low (70-77% depending on the method of analysis).
The obtained phylogram (Fig. 1) also showed that the level of nucleotide variability within the studied species differed significantly. For the species T. pseudospiralis, the differences between distant intra-specific groups were 58 bp.
The sequence of the same DNA locus obtained for T. pseudospiralis isolated from domestic pigs in Kamchatka peninsula was also included in this analysis. This isolate T. pseudospiralis differed from the isolate of this species from Primorsky Region by one nucleotide. Although both of these Russian isolates showed significant proximity to the previously studied isolates 'ISS176', 'ISS13' and 'ISS588', the Kamchatka isolate can be classified as a separate intra-specific haplotype. Within the studied species T. spiralis and T. nativa, the limits of nucleotide variability for this locus are less significant and amount, respectively, to 2-4 bp and 1-6 bp. The level of differences in the studied DNA region between T. nativa and Trichinella sp. 'T6' is 7-8 bp. At least two haplotypes can be distinguished within nematodes of the species T. spiralis from Primorsky Region. The nematodes of laboratory samples Tri117 and TrN2 differ in two nucleotide substitutions from the isolate Tri109. This latter with non-complete labelling was provided by Prof. V.A. Britov and, as seems, also originated from Far East of the Russian Federation. The isolates of T. nativa from Primorsky Region belong to several haplotypes (Table 2).
Table 2. Nucleotide differences between Trichinella nativa isolates in the partial Coxl mtDNA sequence
Trichinella species 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
1 T. nativa Tri122 -
2 T. nativa Tri 100 3 -
3 T. nativa Tri 108 6 3 -
4 T. nativa ISS10 NC025752 5 2 1 -
5 T. nativa Tri 107 5 2 1 0 -
6 T. nativa TrN17 2 1 4 3 3 -
7 T. nativa TrN24 1 2 5 4 4 1 -
8 T. nativa Tri 102 1 2 5 4 4 1 0 -
9 T. nativa TrN23 1 2 5 4 4 1 0 0 -
10 T. nativa Tri 126 1 2 5 4 4 1 0 0 0 -
11 T. nativa TrN13 1 2 5 4 4 1 0 0 0 0 -
12 T. nativa Tri 132 1 2 5 4 4 1 0 0 0 0 0 -
13 T. nativa Tri 113 2 3 6 5 5 2 1 1 1 1 1 1 -
14 T. nativa TrN3 2 3 6 5 5 2 1 1 1 1 1 1 0 -
15 Trichinella sp. T6 8 7 8 7 7 6 7 7 7 7 7 7 8 8 -
77 papuae NC 025754
77 spiralis Tri 109 laboratory culture
100/100/100
91/86/81
i
77 spiralis Trill7 sable 77 spiral/¿'TrN2 sable 77 spiralis AF293969 77 spiralis 'ISS31' GU386314
77 sp iral is AF293969
77 patagoniensis KJVI357412
- 77 nelson/ NC 025753
Trichinella 'T8' KM357419 - 77 murrelli NC 025751 — Trichinella sp. 6T9' KM357420 77 britovi NC 025750 Trichinella sp. T6' KJVL357418 77 naiiva 'ISSIO'NC 025752 77 nativa Tril07 brown bear 77 nativa Tril08 sable 77 nativa TrilOO sable 77 nativa TrN17 lynx 77 nativa Tril32 sable 77 nativa TrN24 brown bear 77 naiiva TrN26 red fox 77 naiiva TrN13 red fox 77 nativa Tril02 sable 77 naiiva Tril22 leopard cat
77 nativa Tril26 badger
77 nativa TrN3 leopard cat 77 naiiva Tri 113 sable
100/100/100
77/75/70
H
100/100/100
100/100/97
10
• 77 pseudospiralis 'ISS470'KJVI357411 77 pseudospiralis Tri79 Kamchatka peninsula 77 pseudospiralis KJM357410 77 pseudospiralis KJVL357409 77 pseudospiralis TrN12 leopard cat 77 pseudospiralis NC 025749 77 pseudospiralis TSS588' KM357409
Fig. 1. The taxonomic position of the Trichinella isolates from Russian Far East. The bootstrap support values are presented in the format MP/NJ/ML.
DISCUSSION
The nematodes of the genus Trichinella were intensively studied in the last 10 years with all available methods of molecular genetics. Quite recently full mitochondrial genomes of all 12 known species were published together with the phylogeny inferred from these data (Mohandas et al., 2014). In this study the mitochondrial genomes of five intraspecific forms of T. pseudospiralis were also published. Complete nuclear genomes of all species of Trichinella were partially annotated (Korhonen et al., 2016). For each of the species, from 11 to 16 thousand genes were identified, where more than a thousand ones were single-copy genes. The genus phylogeny inferred from the analysis of these single copy genes was proposed. In the main features, the phylogeny inferred from the Trichinella nuclear genome was similar to mitochondrial ones. Analysis of the data obtained on the basis of the molecular clock model allowed to determine the lifetime of the common ancestor of the nematodes of the genus Trichinella and Trichuris suis as 384-204 million years ago. The time of Trichinella division into two evolutionary lines with the species able or unable for incapsulation in the host muscle tissues was defined as 28-15 million years ago. The authors of this comprehensive analysis (Korhonen et al., 2016) concluded from their phylogeographic analysis that the Trichinella nematodes evolutionary origin is in the prehistoric areas that now constitutes Eurasia. It means that the territory of the Russian Federation was an area for expansion of species and intraspecific groups of trichinellids. The haplotypes of e.g. T. pseudospiralis isolated in the Primorsky Region of the Russian Federation were similar to three haplotypes isolated in Eurasia (T4.1-3), and significantly differed from the North American (T4.4.) and Australian (T4.5) haplotypes. As reported before, the haplotype of T. spiralis from Primorsky Region differed from the haplotypes widespread in the Western Europe and North America (Spiridonov et al., 2016). A study of T. nativa isolates from different regions of the Russian Federation showed a complex haplotype structure of this species (Odoyevskaya & Spiridonov, 2016), but the comparable level of haplotype diversity was found between isolates originated from Primorsky Region alone.
The diversity in the nucleotide sequences of the studied part of mitochondrial genome in three Trichinella species found in the Russian Far East demonstrated three different patterns. Trichinella
spiralis genetic uniformity throughout Western Europe, Americas and Australia was reported before (Rosenthal et al., 2008), with only East Asian isolates being different and probably representing the basal form for this species. The isolates found in the Russian Far East also differ from West Eurasian ones and probably represent the same East Asian intraspecific form as Chinese isolates. Trichinella pseudospiralis is quite uniform in Eurasia with only North American and Australian isolates being prominently different. It can be speculated that genetic uniformity of T. pseudospiralis can be explained by their ability to travel over long distances in the muscular tissues of their bird hosts. It is the T. nativa that demonstrates haplotype diversity in the material from Russian Far East and throughout the Russian territory (Odoyevskaya & Spiridonov, 2016). Rosenthal et al. (2008) demonstrated that Trichinella species with wildlife hosts demonstrate the highest level of genetic variability compared with species associated with domestic animals. This genetic variability can be considered as a manifestation of the fragmentation of the trichinellid species inside its area. Such a fragmentation might have been a result of association with only local food chains significantly limiting the gene flow.
ACKNOWLEDGEMENT
This study was supported by the Russian Science Foundation (project no. 14-16-00026).
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И.М. Одоевская, И.В. Серёдкин, С.Э. Спиридонов. Taxonomic diversity of Trichinella nematodes in the natural ecosystems of Primorsky Region of the Russian Federation.
Резюме. Личинок нематод рода Trichinella извлекали из мышечной ткани павших хищных животных, останки которых собирали в природных экосистемах Приморского края на Дальнем Востоке Российской Федерации. Определение полученных личинок было основано на анализе нуклеотидных последовательностей CoxI гена и последовательностей tRNA митохондриального генома. Было выявлено три вида трихинеллид: T. spiralis, T. pseudospiralis и T. nativa. Последний вид продемонстрировал самый высокий уровень генетического разнообразия - было выявлено не менее пяти гаплотипов этого вида, отличающихся на 1-6 п.н. Амурский кот (Prionailurus bengalensis) отмечен как новый хозяин для T. pseudospiralis и T. nativa на россйском Дальнем Востоке. Также трихинеллы выявлены у азиатского барсука (Meles leucurus), бурого медведя (Ursus arctos), рыси (Lynx lynx), лисицы (Vulpes vulpes) и соболя (Martes zibellina).
