Научная статья на тему 'Characteristic non-synonymous SNP in coxl mtDNA of Russian isolates of Trichinella spiralis'

Characteristic non-synonymous SNP in coxl mtDNA of Russian isolates of Trichinella spiralis Текст научной статьи по специальности «Биологические науки»

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Russian Journal of Nematology
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East Asia / genetic variability / wild and domesticated hosts / Trichinella / trichimellosis

Аннотация научной статьи по биологическим наукам, автор научной работы — Sergei E. Spiridonov, Lydia A. Bukina, Ivan V. Seryodkin, Irina M. Odoyevskaya

Non-synonymous nucleotide restitution in the coxI mitochondrial gene of Russian isolates of Trichinella spiralis can serve as a genetic marker to distinguish these isolates from West European and North American ones.

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Характерный не-синонимичный однонуклеотидный полиморфизм последовательности сoxI митохондриальной ДНК российских изолятов Trichinella spiralis.

Обнаруженный не-синонимичный однонуклеотидный полиморфизм в последовательности сoxI митохондриальной ДНК четырех российских изолятов Trichinella spiralis из Южной Сибири и Дальнего Востока Российской Федерации может быть использован как генетический диагностический маркер. С его помощью возможно отличать изоляты Trichinella spiralis, характерные для Западной Европы и Северной Америки, от изолятов из восточной части Российской Федерации.

Текст научной работы на тему «Characteristic non-synonymous SNP in coxl mtDNA of Russian isolates of Trichinella spiralis»

Russian Journal of Nematology, 2016, 24 (2), 127 - 129

Characteristic non-synonymous SNP in cox I mtDNA of Russian isolates of Trichinella spiralis

Sergei E. Spiridonov1, Lydia A. Bukina2, Ivan V. Seryodkin3 and Irina M. Odoyevskaya4

'Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 119071 Moscow, Russia e-mail: s_e_spiridonov@rambler.ru 2 Federal State Educational Institution of Higher Education Vyatka State Agricultural Academy, October Avenue, 133, 610017, Kirov, Russia 3Paciflc Geographical Institute, Far Eastern Branch of the Russian Academy of Sciences Radio st., 7,

690041, Vladivistok, Russia

4K.I. Skrjabin All-Russian Institute for Fundamental and Applied Parasitology of Animals and Plants, Bolshaya

Cheremushkinskaya Street 28, 117218 Moscow, Russia

Accepted for publication 12 October 2016

Summary. Non-synonymous nucleotide restitution in the coxI mitochondrial gene of Russian isolates of Trichinella spiralis can serve as a genetic marker to distinguish these isolates from West European and North American ones.

Key words: East Asia, genetic variability, wild and domesticated hosts, Trichinella, trichinellosis.

Trichinella spiralis (Owen, 1985) is a dangerous parasitic nematode of humans and animals. This parasite is capable of successfully spreading in anthropogenic habitats (Rosenthal,

2008). Typically, pigs and rats are the hosts in such sites. Infestation of wild animals with this nematode throughout Eurasia was also reported (Pozio et al,

2009). Every year, outbreaks of trichinellosis caused by T. spiralis are reported from different Eurasian countries, thus demanding a deeper understanding of T. spiralis epidemiology. To have a molecular tool to distinguish between separate intraspecific groups of this cosmopolitan parasite would be useful; however, the very low genetic variability reported for T. spiralis (Rosenthal et al., 2008) is the obvious obstacle for the design of such a tool. Nearly complete genetic homogeneity, expressed as a high level of fixation in microsatellite and mitochondrial markers, was demonstrated for the T. spiralis strains isolated in Western Europe, North Africa and the Americas (Rosenthal et al., 2008).

from East Asia (China, Thailand) display a more pronounced genetic variability (Rosenthal et al., 2008). The territory of the Russian Federation is situated just between East Asian and West European parts of T. spiralis distribution area with different levels of genetic variability. The question of which T. spiralis haplotypes inhabit this vast territory is of importance from both scientific and applied points of view. Below we have proposed a marker to distinguish between West European and Russian isolates of this species.

MATERIAL AND METHODS

Isolates studied. Five isolates of T. spiralis were available, collected in the Czech Republic and different regions of Russia from different hosts, including sable, hunted in Lazo district of Primorskii krai (Table 1). The samples of muscular tissue positive for Trichinella juveniles were transported to the laboratories in Moscow (I.M. Odoyevskaya) or Kirov (L. A. Bukina), where the

Unlike these similar T. spiralis isolates, the isolates

Table 1. Origin of Trichinella spiralis isolates used for DNA extraction and sequencing.

Host Geographic origin Laboratory label Deposition N

1 Domestic pig (Sus scrofa) Czech Republic Tri74 KU321693

2 Wolf (Canis lupus) South-east part of Siberia Tri84 KU321694

3 Dog (Canis lupus familiaris) South-east part of Siberia Tri40 KU321695

4 Fox (Vulpes vulpes) Primorskii Region Tri109 KU321696

5 Sable (Martes zibellina) Primorskii Region Tri117 -

Spiridonov S.E. et al.

Table 2. Nucleotide differences between Trichinella spiralis isolates in the partial cox1 mtDNA sequence.

Species, isolate designation, NCBI accession number, Locality of origin 1 2 3 4 5 6 7 8

1 T. spiralis AF293969 - France --

2 T. spiralis ISS31 GU386314 - USA 0 --

3T. spiralis ISS3 KM357422 - Poland 0 0 --

4 T. spiralis T74 - Czech Republic 0 0 0 --

5T. spiralis T40 - South-East of Siberia 2 2 2 2 --

6 T.spiralis T84 - South -East of Siberia 2 2 2 2 --

7 T spiralis T109 - Primorskii region 4 4 4 4 2 2 --

8 T spiralis T117 - Primorskii region 4 4 4 4 2 2 0 --

detected Trichinella juveniles were used to establish the laboratory cultures in mice and hamsters. Trichinella juveniles were obtained from the laboratory inoculated animals through the digestion of muscular tissues with artificial gastric juice (8-10 g hydrochloric acid of 1,175 density and 3% pepsin l-1). Trichinella juveniles were filtered from the digest, concentrated on the bottom of the tube and used for DNA extraction with Wizard® SV Genomic DNA Purification System columns (Promega Corp., USA).

PCR protocol, sequencing and analysis. A pair of primers of original design were used to obtain a partial sequence of mitochondrial gene of cytochrome oxidase I (coxI). The composition of primers was based on primers 37F and 42R (Hu et al., 2002) with some nucleotides replaced to meet the restitution characteristics for trichinellid coxI mtDNA: 37F_Tri GCA GTA AAT TTA GAA TTT AAA C and 42R_Tri CCT AAT ATT CAT GGT GTT CAT A. DNA was extracted from juvenile suspensions with Promega® Wizard. The size of amplicon obtained with these primers was about 1300 bp. PCR products were purified through gel electrophoresis and precipitation and sequenced with Genotech, Moscow. Four sequences were obtained for each amplicon (twice with both forward and reverse primers). The sequences of other species and genotypes of Trichinella were downloaded from GenBank and used to compare with the obtained ones. The sequences were aligned using Clustal X with default values for gap opening and gap extension penalties. Different methods of alignment analysis (maximum parsimony, neighbour joining and maximum likelihood) were performed with PAUP 4.0b.10 (Swofford, 1998) and MEGA5 (Tamura et al., 2011).

RESULTS

Approximately 1300 bp long amplicon was obtained with designed primers for 4 Trichinella spiralis isolates from Russia and one from Czech Republic. Nucleotide base calling of approximately

1310-1320 bp for each isolate was obtained and resulted in approximately 1320 bp long alignment. Pairwise nucleotide differences between the studied isolates were calculated with PAUP 4.0b.10 and are presented in Table 2. All four studied Russian isolates of T. spiralis (Tri40, Tri84, Tri109 and Tri117) differed from the European isolates by 2-4 bp. Thus, three CoxI mtDNA haplotypes of this species were detected. Analysis of the amino acid sequences for this gene obtained after translation of nucleotide sequences revealed the presence of one non-synonymous nucleotide substitution in T. spiralis: alanine in the Russian isolates instead of threonine in the European ones (Fig.1.). This nucleotide substitution was present in all studied Russian isolates of T. spiralis.

479

Y

T.spiralis Poland KM357422 TCATCAATTACAGGAAGAATCAAC T.spiralis Russia KU321696 TCATCAATTGCAGGAAGAATCAAC

T.spiralis Poland KM357422 SSITGSIN T.spiralis Russia KU321696 SSIAGSIN

Fig.1. The position of non-synonymous nucleotide substitutions in the coxI mtDNA nucleotide and amino acid sequences of the Russian isolate of Trichinella spiralis.

Arrowhead is indicating the position of this SNP in the complete sequence of mitochondrial genome of T. spiralis from Poland.

DISCUSSION

Despite intensive molecular studies on Trichinella, only three complete sequences of the coxI mtDNA gene were available in the NCBI GenBank. Several other sequences of T. spiralis deposited are quite short (approx. 400 bp) and correspond to the 3' end of this coxI mtDNA sequence. As primers of our own design amplify a

Characteristic SNP of Trichinella spiralis isolates from Russia

5' portion of this gene and the site with non-synonymous substitution was at the beginning of this sequence, only three deposited sequences of T. spiralis were available for comparison. Our study showed that the sequence obtained for the Czech isolate of T. spiralis was identical with sequences deposited of this species from France, Poland and USA. The similarity of West European and North American strains was demonstrated earlier and considered to be a result of the introduction of this pathogenic nematode to North America by European colonists (Rosenthal et al., 2008). All the Russian isolates have several characteristic nucleotide differences with the West European/American isolates. Among them, one nucleotide substitution was non-synonymous, i.e. resulting in the mutation of a single amino acid in the encoded protein. Genetic isolation of T. spiralis strains of East Asian origin was demonstrated earlier by Rosenthal et al., 2008, but their conclusion was based on the analysis microsatellite repeats and the 3100 bp long sequence of several spanning mtDNA genes (cytb, tRNA-Ser, SSU rDNA, tRNA-Val, LSU rDNA, atp6, and cox3). As the mtDNA fragment studied and those used in the cited paper are not overlapping, we are unable to predict whether East Asian isolates of T. spiralis do contain the described non-synonymous SNP, but we believe that the genetic marker discovered can be used in wider studies of intraspecific groups of T. spiralis throughout Eurasia. The finding of T. spiralis in the sables in Primorskii Region of Russian Far East is indicative of the presence of T. spiralis in non-anthropogenic habitats in East Asia.

ACKNOWLEDGEMENTS

This study was supported by the Russian Foundation for Basic Researches (project no. 14-04-01064a) and by the Russian Science Foundation (project no. 14-16-00026).

REFERENCES

Hu, M., Chilton, N.B., Gasser, R.B. 2002. Long PCR-based amplification of the entire mitochondrial genome from single parasitic nematodes. Molecular and Cellular Probes 16: 261-267. Pozio, E., Rinaldi, L. G., Marucci, V. Musella, Galati, F., Cringoli, G., Boireau, P., G. La Rosa. 2009. Hosts and habitats of Trichinella spiralis and Trichinella britovi in Europe. International Journal for Parasitology 39: 71-79. Rosenthal B. M. 2008. How has agriculture influenced the geography and genetics of animal parasites? Trends in Parasitology 25: 67-69. Rosenthal B. M., La Rosa, G., Zarlenga, D., Dunams D., Yao Chunyu, Liu Mingyuan, E. Pozio. 2008. Human dispersal of Trichinella spiralis in domesticated pigs. Infection, Genetics and Evolution 8: 799-805.

SWOFFORD, D.L. 1998. PAUP*: PhylogeneticAnalysis Using Parsimony, Version 4. Sunderland,, MA, USA, Sinauer Associates. 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. Molecular Biology and Evolution 28: 2731-2739.

Спиридонов С.Э., Букина Л.А., Середкин И.В., Одоевская И.М. Характерный несинонимичный однонуклеотидный полиморфизм последовательности coxl митохондриальной ДНК российских изолятов Trichinella spiralis.

Резюме. Обнаруженный не-синонимичный однонуклеотидный полиморфизм в последовательности coxl митохондриальной ДНК четырех российских изолятов Trichinella spiralis из Южной Сибири и Дальнего Востока Российской Федерации может быть использован как генетический диагностический маркер. С его помощью возможно отличать изоляты Trichinella spiralis, характерные для Западной Европы и Северной Америки, от изолятов из восточной части Российской Федерации.

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