Bulgarian Journal of Veterinary Medicine, 2019 ONLINE FIRST ISSN 1311-1477; DOI: 10.15547/bjvm.2274

Original article

SEQUENCING AND PHYLOGENETIC ANALYSIS OF MITOCHONDRIAL COX1 AND NAD1 GENES IN AND ISOLATES FROM IRAN

M. VALIZADEH1, F. TAHVILDAR BIDEROUNI1, S. R. SHAHROKHI2, M. GHANIMATDAN3 & A. R. NAGAHI4 1Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran; 2Doctor of Veterinary Medicine, Rasht, Iran; 3Department of Medical Parasitology and Mycology, Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran; 4Department of Medical Parasitology and Mycology, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

Summary Valizadeh, M., F. Tahvildar Biderouni, S. R. Shahrokhi, M. Ghanimatdan & A. R. Nagahi, 2019. Sequencing and phylogenetic analysis of mitochondrial cox1 and nad1 genes in Toxocara canis and Toxascaris leonina isolates from Iran. Bulg. J. Vet. Med. (online first).

Toxocara canis and Toxascaris leonina are the most important ascaridoid of the family Toxocaridae. The present study was aimed to characterisation and analysis of genetic variation within and among T. canis and T. leonina isolates obtained from Iran by sequencing partial mitochondrial cytochrome c oxidase subunit 1 (pcox1) and partial NADH dehydrogenase subunit 1 (pnad1) genes. A total number of 134 adult nematodes belonging to Toxocaridae family were collected from stray dogs in Alborz province, Iran during 2015 and 2016. Polymerase chain reaction (PCR) was per- formed and products were sequenced. Sequences of two mitochondrial cox1 and nad1 genes were compared with other sequences in the GenBank, while multiple sequences alignment analysis was performed using the Bioedit and MEGA6 software and phylogenetic tree was plotted. For all isolates, amplicons of about 450 and 350 base pairs (bp) were successfully produced by PCR for cox1 and nad1, respectively. All sequences of T. canis isolates from present study were 100% homologous across the nad1 gene but not in the cox1 gene. The results indicate that the PCR method based on sequence of cox1 and nad1 genes is a suitable technique for the differentiation of T. canis and T. leonina species and that mtDNA regions could be used as genetic markers for the identification and differentiation of Toxocara species. Key words: cox1, Iran, mtDNA, nad1, Toxocara canis, Toxascaris leonina

INTRODUCTION

Toxocara canis, T. cati and Toxascaris causing accidental infection in humans as leonina are the most important ascaridoid their opportunistic host. However, T. nematodes of the family Toxocaridae, leonina has a low zoonotic potential and Sequencing and phylogenetic analysis of mitochondrial cox1 and nad1 genes in Toxocara canis ... low pathogenicity in humans (Overgaauw The present study was aimed to cha- & van Knapen, 2013). Toxocaridae worms racterisation and analysis of genetic varia- are among the most prevalent endoparasites tion within and among T. canis and T. in dogs and cats (definitive hosts), having a leonina isolates obtained from Iran by worldwide distribution which led to the sequencing partial mitochondrial cyto- high contamination of playgrounds, parks chrome C oxidase subunit 1 (pcox1) and and households with Toxocara eggs partial NADH dehydrogenase subunit 1 (Robertson & Thompson, 2002). (pnad1) genes. Using these sequences, the considered as a typical phylogenetic relationships of these para- neglected disease, is caused by L2 larvae sites compared with isolates from diffe- of Toxocara spp. with a number of clini- rent areas of the world based on the com- cal manifestations such as visceral larva bined sequences of two mt gene frag- migrans (VLMs), ocular larva migrans ments. (OLMs), eosinophilic meningoencepha- litis (EME), covert toxocariasis (CT) and MATERIALS AND METHODS neurotoxocariasis (NT) (Despommier, 2003; Macpherson, 2013). Collecting of nematodes Most metazoan mitochondrial (mt) ge- nomes are circular and small (14–20 kb). A total number of 134 adult nematodes The complete mt genomes of T. canis belonging to Toxocaridae family were (14,332 bp), T. cati (14,029 bp) and T. collected from stray dogs in Alborz leonina (14,310 bp) have been recently province, Iran during 2015 and 2016. determined to provide novel mitochon- Adult worms were taken from apparently drial DNA (mtDNA) markers for taxo- healthy dogs 48 hours after taking nomic and phylogenetic relationship levamisole, washed twice in phosphate- analyses of Toxocara species (Li et al., buffered saline (PBS), stained, and identi- 2008a,b; Liu et al., 2014). The mt ge- fied by using appropriate systematic keys. nomes of the Toxocara species contain 12 DNA extraction, PCR enzymatic amplifi- proteins encoding genes (nad1, atp6, cation and sequencing nad2, cytb, cox3, nad4, cox1, cox2, nad3, nad5, nad6, and nad4L), two ribosomal DNA was extracted using phe- RNA genes (rrnL and rrnS), 22 transfer nol:chloroform:iso-amyl alcohol (25:24:1) RNA (trn) genes, an AT-rich region and followed by washing using chloroform: the non-coding regions (Wickramasinghe iso-amyl alcohol (24:1) and then stored at et al., 2009). -20 ° C. Two mitochondrial genes (cox1 More efforts are spent on development and nad1) were amplified by polymerase of reliable diagnostic methods. Poly- chain reaction (PCR). The forward JB3 merase chain reaction (PCR) and sequen- (5‘TTTTTTGGGCATCCTGAGGTTTAT cing of fragments of mitochondrial cyto- 3‘) and reverse JB4.5 (5‘TAAA chrome C oxidase subunit 1 (cox1) and GAAAGAACATAATGAAAATG‘) pri- NADH dehydrogenase subunit 1 (nad1) mers were used to amplify a 450 bp por- genes, have been used for genetic analyses tion of the mitochondrial cox1 gene and and identification of nematodes in dogs the forward ND1F (5‘TTCTTATGAG and cats (Oguz et al., 2018; Jin et al., ATTGCTTTT 3‘) and reverse ND1R 2019). (5‘TATCATAACGAAAACGAGG‘) pri-

2 BJVM, ××, No × M. Valizadeh, F. Tahvildar Biderouni, S. R. Shahrokhi, M. Ghanimatdan & A. R. Nagahi mers  to amplify a 350 bp fragment of study, multiple sequences alignment the mitochondrial nad1 gene. PCR reac- (MSA) was performed using BioEdit® tion was performed with a final volume of software and phylogenetic tree was plot- 20 μL, containing 2 µL of template ge- ted using MEGA6® software with maxi- nomic DNA, 25 pmol of each primer and mum likelihood method based on Ki- 10 µL of PCR premix (AmpliTaq Gold mura2-parameter model using bootstrap 360 Master Mix, cat. No. 4398876), value with 1000 replication and a scale of which included 1.25 U Taq DNA poly- 0.001. merase, 200 µM of each dNTPs and 1.5 mM MgCl in a thermocycler under fol- 2 RESULTS lowing conditions: one cycle of 95 °C for 6 min (initial denaturation), followed by For all isolates, amplicons of about 450 35 cycles of 94 °C for 45 s (denaturation), and 350 base pairs (bp) were successfully 60 °C for 1 min (annealing), and 72 °C for produced by PCR for cox1 and nad1, re- 1 min (extension), and a final extension of spectively (Fig. 1). Sequences of the Ira- 72 °C for 6 min. Double-distilled water nian T. canis and T. leonina isolates cox1 (DDW) instead of template DNA was and nad1 genes obtained in this study included in each set of PCR reaction as were submitted to GenBank and the ac- negative control. PCR amplicons were cession numbers of these sequences are detected by 1.5% agarose gel electropho- given in Table 1. resis and ethidium bromide gel staining. PCR products were sequenced by Bioneer Cox1 gene company, South Korea. Five μL of PCR According to the multiple sequences products were digested directly with 2 μL alignment analysis of cox1 gene, two restriction endonuclease Rsa-1 (New Eng- samples of T. canis isolates were 100% land Biolabs (NEB), R0167S) for 6 hours homologous, while in the 369th position of at 37 °C. Restriction fragments of ampli- third isolate sequence was adenine instead cons were detected using a 1.5% (w/v) of Guanine (Fig. 2). The results indicate agarose gel electrophoresis at 70 V for 45 that the sequences of cox1 gene of T. min, ethidium bromide staining and visu- canis1, T. canis2 and T. canis3 isolates alized on a UV trans illuminator. from this study were completely similar to Sequences variability and phylogenetic Iran isolate (Accession no. KC293914) analysis but different from another isolates from Iran (Accession no. KC293906 and Three amplicons of T. canis and two am- KC293901) as well as isolates from China plicons of T. leonina cox1 and nad1 genes (Accession no. AJ920054 and AJ920056) were sequenced in Bioneer company, in several nucleotides. Cox1 gene phy- South Korea (https://www.bioneer.com). logenetic tree results indicate that the se- The sequences were compared with other quences of the T. leonina1 and T. sequences found in GenBank (https:// leonina2 isolates from present study were www.ncbi.nlm.nih.gov/genbank/) using similar to isolates from Iran (Accession the BLAST (https://blast.ncbi.nlm.nih. no. KC293927, KC2 93930, KC293934 ® gov/) and edited with Chromas software. and KC293935) and China (Accession no. To illustrate the evolutionary relationship AJ920063 and JF780946) but different between the samples obtained in this from isolates from Poland (Accession no.

BJVM, ××, No × 3 Sequencing and phylogenetic analysis of mitochondrial cox1 and nad1 genes in Toxocara canis ...

1 2 3 4 M N 1 2 3 4 M N

500 bp 500 bp

450 bp 350 bp

A B

Fig. 1. PCR bands of cox1 (~450 bp) (A) and nad1 (~350 bp) gene (B) of Toxocara on 1.5% agarose gel. M: 100 bp DNA marker, N: negative control.

Table 1. T. canis and T. leonina isolates and their accession numbers of cox1 and nad1 genes of the present study

Isolates Accession No. cox1 Accession no. nad1 1 T. canis1 MK913431 MK913428 2 T. canis2 MK913432 MK913429 3 T. canis3 MK913433 MK913430 4 T. leonina1 MK591004 MK616562 5 T. leonina2 MK591005 MK616563

KX963448) and another isolate from well as isolates from China (Accession no. China (Accession no. JF7800950) with AJ920385, AJ920386 and AJ920387). 96% homology (Fig. 3). Also, nad1 gene phylogenetic tree indi- cate that the T. leonina isolates from pre- Nad1 gene sent study were similar to isolate from According to the multiple sequences Iran (Accession no. KC293956) and dif- alignment analysis of nad1 gene all se- ferent with isolates from China (Accession quences of T. canis isolates from present no. AJ893360) and Iran (Accession no. study were 100% homologous (Fig. 4). KC293955) (Fig. 5). Nad1 gene phylogenetic tree results show that the T. canis isolates of the present DISCUSSION study were similar to isolate from Iran (Accession no. KC293915) with a homol- Ascaridoid nematodes have a significant ogy of 100% but there were differences adverse impact on human and with two other isolates from Iran (Acces- health worldwide (Bethony et al., 2006). sion no. KC293920 and KC293922), as

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M. Valizadeh, F. Tahvildar Biderouni, S. R. Shahrokhi, M. Ghanimatdan & A. R. Nagahi

Fig. 2. Similarity degree and multiple sequencing alignment between T. canis cox1 gene isolated from this study (marked with a star) and other isolates.

Fig. 3. Phylogenetic tree of T. canis (A) and T. leonina (B) cox1 gene isolated from this study (marked with a star) with maximum likelihood method using the Kimura-2 parameter model. Ascaris suum was considered as the outside group.

BJVM, ××, No × 5 Sequencing and phylogenetic analysis of mitochondrial cox1 and nad1 genes in Toxocara canis ...

Fig. 4. Similarity degree and multiple sequencing alignment between T. canis nad1 gene isolated from this study (marked with a star) and other isolates.

Fig. 5. Phylogenetic tree of T. canis (A) and T. leonina (B) nad1 gene isolated from this study (marked with a star) with maximum likelihood method using the Kimura-2 parameter model. was considered as the outside group.

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T. canis (dog roundworm) is an impor- tions (Wang et al., 2011; Lin et al., 2012; tant worldwide-distributed helminth Wang et al., 2013; Chang et al., 2015; parasite of dogs and T. leonina is a com- Poon et al., 2017). Toxocara species mon ascaridoid of dogs and could be differentiated from each other cats (Becker et al., 2012). The preva- and from other ascaridoids by direct PCR lence of T. canis in dogs ranged from based on cox1 and nad1 sequences. 5.5% to 64.7% (Chen et al., 2012) while There are some reports on molecular T. leonina is more prevalent in wild and differentiation of T. canis from other domestic carnivores (Okulewicz et al., species based on PCR-linked restriction 2012). fragment length polymorphism (PCR- Zoonotic infections of nematodes are RFLP) assays analyses of rDNA, while common in many developing countries. no data are available from PCR-RFLP Fecal infection with Toxocara egg in analyses of mitochondrial cox1 and nad1 carnivores has been reported in various genes. The data obtained by in silico regions of Iran. In many areas however, analyses revealed that only one enzyme accurate information about the preva- (MboII) is appropriate for PCR-RFLP on lence of Toxocara spp. does not exist. the cox1 gene and there is not any suit- Most studies in Iran have been carried able enzyme for PCR-RFLP on the nad1 out on seroepidemiology of toxocariasis gene (Mikaeili et al., 2017). in apparently healthy people with no A few studies on the cox1 and nad1 clinical signs, especially in <12-year-old sequence analysis of T. canis, T. cati and children (Tahvildar Biderouni et al., T. leonina has been done before (Li et 2019). The high prevalence of human al., 2008a; Mikaeili et al., 2015; He et seropositive rate was reported in Fars and al., 2018). He et al. (2018) reported that Tehran Provinces (Sadjjadi et al., 2001; cox1 gene sequencing is enough to accu- Agin, 2012; Zibaei & Sadjjadi, 2017) rately distinguish and identify T. cati. moreover the high percentage of carni- The intra-specific sequence variations vore’s infestation has been reported from within T. cati were 03.6% for cox1 (He East-Azerbaijan, Razavi Khorasan and et al., 2018) Similarly Li et al. (2008b) Mazandaran Provinces in Iran (Daryani described sequences variation in three et al., 2009; Shemshadi et al., 2014; Ha- mitochondrial DNA regions among and jipour et al., 2016). within T. canis, T. cati, T. malaysiensis, Analysis of genetic sequence variabi- T. vitulorum and T. leonina in order to lity in parasites is widespread and has define genetic markers for their specific important outcomes for the molecular identification and differentiation. the diagnosis, genetic structure, epidemiol- intra-specific sequence variations were ogy, , population biology as 0.23.7% for cox1 and 02.8% for nad1, well as for the effective control of para- the inter-specific sequence differences sites (Stensvold et al., 2011). Mitochon- were 7.912.9% for cox1 and 10.7 drial DNA (mtDNA) has been used as a 21.1% for nad1. Phylogenetic analyses genetic marker to identify nematodes and revealed that T. malaysiensis was more cestodes and study genetic structures and closely related to T. cati than to T. canis phylogenetics. mtDNA is approved as a (Li et al., 2008a). genetic marker for helminthic parasites This study was designed for analysis epidemiological and genetics investiga- of genetic variation among and within

BJVM, ××, No × 7 Sequencing and phylogenetic analysis of mitochondrial cox1 and nad1 genes in Toxocara canis ... isolates obtained from stray dogs of sion no. JF780946). The also report that Karaj, Iran and comparison with data none of the nad1 sequences of T. cati, T. available from other isolates found in canis and T. leonina from Iran were GenBank. Based on multiple sequences 100% homologous with reference se- alignment, there was sequence variation quences in GenBank, except for an iso- within and among the cox1 gene of T. late of T. canis which showed 100% ho- canis1, T. canis2 and T. canis3 isolates mology to an isolate from Australia (ac- from this study and T. canis isolates from cession no. AJ920383). Phylogenetic Iran (Accession no. KC293906 and analysis indicates the differences within KC293901) and China (Accession no. Iranian isolates of T. cati, and a large AJ920054 and AJ920056). These three difference between isolates of T. cati isolates were identical and exhibiting from Iran and three isolates of T. cati 100% homology with an isolate from from China (accession nos. JF780941, Iran (Accession no. KC293914). Se- JF780942, JF780945 for cox1 and quence variations within T. canis isolate JF833957 and JF833959 for pnad1) (Mi- from this study were 00.3% for cox1 kaeili et al., 2015). and 0% for nad1. However, the sequence In conclusion, the results of present variations among T. canis isolate from study indicate that PCR method based on this study and other isolates found in sequence of cox1 and nad1 genes of GenBank were 02.5% for cox1 and mtDNA is a suitable technique for the 0.61.1% for nad1. differentiation of T. canis and T. leonina T. leonina isolates from present study species. Also we demonstrated the se- showed 100% homology with isolates quence variation in mitochondrial genes from Iran (Accession no. KC293927, in T. canis and T. leonina isolates from KC293930, KC293934 and KC293935) Karaj, Iran. Genetic characterisation of and China (Accession no. AJ920063 and these species can be used for future stu- JF780946) across the cox1 gene and dies of genetic variability and specific 100% homology with isolate from Iran identification of T. canis and T. leonina. (Accession no. KC293956) across the The results of the present study and pre- nad1 gene. sequence variations within T. vious studies have indicated that mtDNA leonina isolate from this study were 0% regions may be used as genetic markers for both cox1 and nad1 genes. However, for the identification and differentiation the sequence variations among T. leonina of Toxocara species. Also, genetic vari- isolate from present study and other iso- ability among Toxocara isolates could be lates found in GenBank were 06% for revealed by sequences of mtDNA re- cox1 and 110% for nad1. gions. More attention to molecular and In a study performed in Iran, Mikaeili bioinformatics studies can be helpful in et al. (2015) noted that mitochondrial this regard. DNA regions could be used confidently for the identification of the nematode ACKNOWLEDGEMENTS helminths. They reported that seven iso- lates of T. leonina collected from dogs in This article was extracted in part from the Iran did not differ in cox1 gene from an thesis prepared by Manoochehr Valizadeh to isolate of T. leonina collected from a fulfill the requirements required for earning grey wolf (Canis lupus) in China (acces- the PhD degree of medical parasitology. The authors gratefully acknowledge the financial

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