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Home , Hymenolepis diminuta, Hymenolepis nana, Infestation

Journal of American Science, 2010;6(12) http://www.americanscience.org

Mitochoderial cytochrome c oxidase subunit 1 (cox 1) gene sequence of the species.

Omnia M. Kandil, Mona S. Mahmoud, Nesreen A.T. Allam, Amira H. El Namaky

Corresponding author: Kandil, O.M., Prof. of Parasitology, Parasitology and Diseases Department, Veterinary Division, National Research Center, Dokki, Giza, Egypt, P.O. Box 12622, Fax +20233370931, Mobile +20105414113 [email protected]

Mahmoud, M.S., Prof. of Parasitology, Parasitology and Animal Diseases Department, Veterinary Division, National Research Center, Dokki, Giza, Egypt, P.O. Box 12622, [email protected]

Allam, N. A. T., Researcher of Molecular Biology and Animal Diseases, Parasitology and Animal Diseases Department, Veterinary Division, National Research Center, Dokki, Giza, Egypt, P.O. Box 12622, [email protected]

El Namaky, A.H., Researcher of Parasitology, Parasitology and Animal Diseases Department, Veterinary Division, National Research Center, Dokki, Giza, Egypt, P.O. Box 12622, [email protected]

Abstract: and H. diminuta are the most common cestodes in humans, domestic and wild rodents. Since isolates of H. nana species are morphologically identical, the way they can be reliably distinguished is comparing the parasite in each using molecular techniques. In the current study, Mitochondrial Cytochrome c oxidase gene especially codons within subunit 1 (cox1) of H. diminuta and H. nana Egyptian isolates from different developmental stages (adult and eggs) and hosts origin (human and rat) were amplified, sequenced and aligned. PCR products were approximately 700 bp, 702 bp and 715 bp of H. nana rat isolates, H. diminuta rat isolates, and H. nana human isolates, respectively. Moreover, despite their host susceptibility differences they all gathered in one cluster with three genbank published isolates of H. nana; AB033412.1, AB494472.1 and AY121842.1), forming one clade with 100% similarity, which was non significantly decreased on internal nodes. In addition, clearly far away from H. diminuta published sequence AB033412.1 who's assumed to be genetically closely related to Egyptian H. diminuta than all other H. nana isolates. Both Egyptian murine isolates of Hymenolipidid; H. diminuta and H. nana, were closer to each other than being to H. nana of human origin. The annotated sequences of Egyptian isolates were deposited in GenBank under the following accession numbers; H. diminuta (GU433102), H. nana rat isolate (GU433103), and H. nana human isolate (GU433104). Finally, the development of effective control strategies will only be possible if complete understanding of the epidemiology of is elucidated. [Omnia M. Kandil, Mona S. Mahmoud, Nesreen A.T. Allam, Amira H. El Namaky. Mitochoderial cytochrome c oxidase subunit 1 (cox 1) gene sequence of the Hymenolepis species. Journal of American Science 2010;6(12):1346-1353]. (ISSN: 1545-1003). http://www.americanscience.org.

Keywords: Hymenolepidid, Phylogeny, Cytochrome c oxidase subunit 1 gene (cox1), Sequencing.

Introduction sporadic egg shedding (Thompson et al. 2001). Hymenolepis nana and H. diminuta are the Since isolates of H. nana infecting humans and most common cestodes in humans, domestic and wild rodents are morphologically identical, the only way rats, mice and dogs (Macko and Hanzelova 2008). they can be reliably distinguished is comparing the During various pathological, parasite in each host using molecular techniques immunopathological and physiological alterations are (Macnish et al. 2002a, b). recorded. They similarly stimulate abdominal pain Mitochondrial (mt) genomes are small accompanied by diarrhea and anorexia as well as (usually less than 20000 bp), circular, and maternally increase in the number of mast cells and eosinophiles inherited (Boore 1999). In addition to high copy- in the infected individuals. These uncharacteristic number per cell which has made them attractive and symptoms could not be useful in differential more tractable targets for characterization, population diagnosis clinically or on microscopical examination genetic and phylogenetic studies (Hu et al. 2004; of fecal samples for eggs (Raether and Hänel 2003). McManus et al. 2004). Regions within the It is believed that with Hymenolipis spp., mitochondrial DNA (mtDNA) have been proven in general, may have been under diagnosed due to useful in biology, epidemiology and diagnosis of

- 1346 - Journal of American Science, 2010;6(12) http://www.americanscience.org several parasitic infestations of human and veterinary health perspective, a better understanding of the importance (Ngarmamonpirat et al. 2005; Ando et transmission dynamics of a parasite species al. 2006). Methods used to obtain data from previously believed to be infective only to rodents mt genomes have included DNA sequencing, will be required to answer questions about the restriction fragment length polymorphism (RFLP) potential for transfer of this parasite to humans and/or analysis and single-strand conformation . polymorphism (PCR-SSCP) (Boore and Brown 1998; Avise, 2000). Intraspecific sequence variation Since control of is in coding portions (genes) of the mt genomes seems dependent on the rapid and accurate detection of to range from small to moderate, especially when causative agents this necessitated traditional compared with interspecific variation that have techniques being complemented by molecular tools demonstrated the deep separations among strains of that provide predictive data on genetic variation in same species (Littlewood et al. 2008). and among parasites (Thompson et al. 2004). Thus the present work aims is to characterize, for the first Complete or near-complete mtDNA time, partial sequences of cox1 genes of H. diminuta sequences are available for 12 species of parasitic and H. nana Egyptian isolates to promote basic ; six cestodes including Taenia crassiceps knowledge on their mtDNA composition, to assess (Le et al. 2000), Echinococcus multilocularis the sequence variation level within local (Nakao et al. 2000) and (von Hymenolepidid from different sources, different Nickisch-Rosenegk et al. 2001). Cytochrome c developmental stages (adult worms and eggs) and oxidase (COX) is a 13-subunit protein complex hosts origin (human and rat), and to discuss the located on the inner mitochondrial membrane that potential benefits of such molecular information as catalyzes electron transfer, proton translocation record sheets for ecological, epidemiological, processes, production of up to 95% of the energy of transmission and host-parasite interaction and as eukaryotic living cells (Saraste 1999; Johnston diagnostic approach of infestation in Egypt. 2006), thus directly influence metabolic performance. mt cox sub unit 1 is the most highly conserved among Materials and Methods 3 genes coding for cytochrome oxidase, therefore has Parasites Samples: been employed in several phylogenetic studies H nana eggs were obtained from infected (Traversa et al. 2007). humans in Endemic Diseases institute. Approximately 2000 H. nana eggs were inoculated DNA sequencing of informative regions into 5-week-old male white mice (Movsesyan et al. within the gene encoding for the COX1 protein have 2008). Adult worms were dissected from the small emphasized specific comparative aspects without yet intestine approximately 14 days post-inoculation. H. making a detailed genome description but revealed diminuta worms were obtained from naturally data for basic and applied potential differential infected norvegicus rat from Abu Rawash, Giza, studies on Hymenolopis spp. determining host Egypt. Rats were killed by cervical dislocation and specificity and transmission patterns (Macnish et al. entire was removed from gut. The 2003). Therefore, allow more appropriate approach worms and eggs washed repeatedly in phosphate for control of endemic infestations in Egypt, buffered saline (PBS) and stored at -80 C until used particular where rodent's population is above control for DNA extraction. limits and hygienic measures are not strictly applied. Isolation of DNA from Adult Worms and Eggs Furthermore, for diagnostic purposes since using Templates DNA were purified from H. nana techniques able to overcome inherent limits of the and H. diminuta using QIAmp tissue purification kit classical approaches (Constantine 2003; Thompson (Qiagen, Hilden, Germany) according to et al. 2004). Epidemiologically, despite this manufacturer’s instructions (Macnish et al. 2002a). is a hand-to-mouth rote that in general not very DNA was eluted in 200 µl Tris-EDTA (TE) buffer pathogenic, however it is extremely difficult to be and 1 µl of the extract was added to the polymerase controlled (Littlewood et al. 2008). Till now chain reaction mix. Single adult and/or eggs education in hygiene is probably the only practical for each isolate were used for DNA extraction. way to reduce the incidence in addition to rodent's Oligonucleotide Primers Design eradication (Behera et al. 2008). The genotyping of Entire mt genomes of the following species Hymenolepis isolates in different hosts will help in were aligned Hymenolepis diminuta (accession determine host specificity and transmission patterns number AB033412.1), Taenia crassiceps (accession and thus allow more appropriate approach to control number NC_002547), T. solium (accession number in endemic communities. From a public NC_004022), T. asiatica (accession number

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NC_004826), (accession to the PCR amplification primers were excluded prior number NC_008075), and E. multilocularis to multiple sequence alignment and phylogenetic (accession number NC_000928); and annotated analysis. The confirmed sequences were then sequence of Hymenolepis nana (accession number deposited in the EMBL/GenBank Data Libraries of AF314223.1) (Nakoo et al. 2000, 2002; von the NCBI. In order to improve the homology Nickisch-Rosenegk et al. 2001). It was not deemed statements out group included necessary to include all the available sequences from (AB465239.1), T. solium (AY211880.1), T. multiceps Taenia or Echinococcus as conservation of alignable (GQ228818.1), Echinococcus granulosus positions between genera and being > 30% GC was (AF314223.1), E. multilocularis (AF314223.1) and more important for PCR primer design. PCR primers Spirometra erinaceieuropaei (AB374543.1), as well pair designed cox1-F 5`- as all annotated sequences of Hymenolepis diminuta ACTTCATTGCTTTTGCTTTTTAGA-3` and cox1- (AB033412.1) and Hymenolepis nana (AF314223.1, R 5`-TGCTGTCATAAATGAACCAACAGT-3` AY121842.1, AB494471.1, AB494472.1, were synthesized by Metabion International AG AB033412.1, AF314223.1) by Basic Local (Martinsried/Deutschland). Alignment Search Tool (nBLAST) (www.ncbi.nih.gov/BLAST/) in the NCBI database PCR Amplification Protocol (National Center for Biotechnology Information, Fragments of the mitochondrial cytochrome NIH, Bethesda, Maryland, USA) (Tatusova and c oxidase subunit 1 gene were amplified using Madden 1999). The alignment gaps were treated as designed primers and each PCR mix was prepared in missing data. Phylogeny of Egyptian Hymenolipis 50 µl total volume with 1 µl of template (50 ng), 10 nana and H. diminuta human and rat isolates based pMoles of each primer, 45 µl of Ready TaqMix on coxI gene partial sequences and multiple Complete (Mater Mix, AllianceBio, USA), and alignment analysis were performed with CLUSTAL nuclease free water (Qiagen, Germany) to complete W computer program (Thompson e al. 1994). the total volume of the reactions. PCRs were performed in a PTC-100™ Thermal Cycler (MJ Phylogeny Construction Research Inc., USA) using the following cycling The bootstrap consensus tree inferred from protocol: initial denaturation at 950C for 3 min and 500 replicates is taken to represent the evolutionary then 40 cycles of 940C for 1 min 50 sec, 580C for 1 history of the taxa analyzed (Felsenstein 1985). min 30 sec, and 720C for 1 min. Final extension was Branches corresponding to partitions reproduced in carried out at 720C for 7 min. A reagent blank was less than 50% bootstrap replicates are collapsed. The run as control in every PCR procedure. Positive percentage of replicate trees and node reliability in results by PCR were retested on two further which the associated taxa clustered together in the occasions several days later to examine the bootstrap test is shown next to the branches reproducibility of PCR. Amplified products from the (Felsenstein 1985). The tree is drawn to scale, with PCRs were electrophorised on 1.5% agarose gels branch lengths in the same units as those of the (Bioshop Canada, Burlington, Ontario, Canada) evolutionary distances used to infer the phylogenetic stained with ethidium bromide (0.5 µg/ml) (Bioshop tree. The evolutionary distances were computed using Canada) (Sambrook et al. 1989). A 100 bp ladder the Poisson correction method (Zuckerkand and (Jena Bioscince, GmbH, Germany) was loaded in Pauling 1965) and are in the units of the number of each gel then photographed under UV light with gel amino acid substitutions per site. All positions documentation system. containing gaps and missing data were eliminated Sequencing of cox1 Gene Products from the dataset (Complete deletion option). There PCR-product of each isolates were purified were a total of 99 positions in the final dataset. with QIAquick-spin PCR purification kit (Qiagen, Phylogenetic analyses were conducted in MEGA4 Germany) then directly sequenced from both (Tamura et al. 2007). Neighbor-Joining and directions using ABI Prism™ Dye Terminator Cycle UPGMA methods were used to calculate the Sequencing Ready Reaction Kit (Applied evolutionary relationship of the Egyptian isolates Biosystem, FosterCity, California) according to with genbank references strains (Saitou and Nei manufacturer’s instructions on a 3130XL Genetic 1987). Analyzer (Applied Biosystems). At least two independent PCR products were used for sequencing Results per isolate. PCR Products of mt cox1 Gene Sequences Analysis PCR products were amplified from mt The resulting aligned output was manually genomes using synthesized primers set. Across the adjusted (Lee et al. 2007). Sequences corresponding alignment of mt genomes few regions were suitably

- 1348 - Journal of American Science, 2010;6(12) http://www.americanscience.org conserved to allow primer design. The PCR products (mtDNA), very commonly used in studies of were approximately 700 bp, 702 bp and 715 bp for mt molecular phylogenetics (Avise 2000). Flatworm cox1 gene of H. nana rat isolates, H. diminuta rat mitochondrial genomes have a number of distinct isolates, and H. nana human isolates, respectively, features including all genes are coded on the same (Figure 1). strand (von Nickisch-Rosenegk et al. 2001), utilize a unique mitochondrial genetic code (Boore 1999) and Sequences Analysis truncated stop codons have also been found among a Variation occurred in terms of sequence number of genes (Nakao et al. 2000, 2003). length and nucleotide differences and gaps (nucleotide insertions, deletions, and substitutions), In the current study the earliest genbank but not G+C percentage where the overall numbers records of cox1 gene of Egyptian Hymenolepis spp. did not differ between amplified fragments; A (23%), are declared. So far, only a few is known about the C (10%), G (22%) and T (45%). Where Nucleotide relative divergence rates of mitochondrial DNA in alterations were found to be variable and several hymenolipis species especially Egyptian isolates nucleotide insertions, deletions and substitutions (Vilas et al. 2005), hence only one species belonging were detected with gaps in the same or different to H. diminuta is completely sequenced and positions (Figure 2). published in genbank (Littlewood et al. 2008). However, PCR technology and DNA sequencing Phylogeny Construction techniques permit the identification of species, Similar topologies were observed in the strains, and populations from any stage in their life Egyptian isolates with genbank references strains. history to distinguish among morphologically similar Optimal phylogenetic tree with the sum of branch parasites (Boore 1999). length = 1.91459848 is shown (Figure 3). Egyptian species were genetically distinct from other species Egyptian species were genetically distinct used in this study that are phylogenetically relating to from other species used in this study that are Hymenolipidid. In addition, despite their host phylogenetically relating to Hymenolipidid. Both susceptibility differences they all gathered in one Egyptian murine isolates of Hymenolipidid; H. cluster with three genbank published isolates of H. diminuta and H. nana, were closer to each other than nana; AB033412.1 (gi|6045204), AB494472.1 being to H. nana of human origin. WHO annual (gi|2262378), and AY121842.1 (gi|2221354), reports maintained the traditional host specificity of forming one clade with 100% similarity, which was hymenolepidid till few years ago (Lee et al. 2007). non significantly decreased on internal nodes. Unfortunately, the unexpected discovery of a mixed Moreover, obviously far away from H. diminuta infection with specie which is known to infect rodent published sequence AB033412.1 (gi|1399136) who's as definitive host in surveyed individuals (Thompson assumed to be quite genetically closely related to et al. 2001; Macnish et al. 2003) as well as in dogs Egyptian H. diminuta than all other H. nana isolates. living in the same locality as their infected owners Both Egyptian murine isolates of Hymenolipidid; H. declared the public health impact of new infestations, diminuta and H. nana, were closer to each other than and meditating urgent thorough understanding of the being to H. nana of human origin. epidemiology of these parasites (Jenkins and Andrew 1993; Thompson et al. 1993; Macnish et GenBank accession numbers of Egyptian al. 2003). Since such deviation in patency of amplicons infestation was not previously recorded thus The annotated sequences of Egyptian highlights the growing importance of using molecular isolates were then deposited in the GenBank of NCBI techniques in both the detection and characterization under the following accession numbers; H. diminuta of parasite species in human and animals' populations (GU433102), H. nana rat isolate (GU433103), and H. especially between morphologically similar species nana human isolate (GU433104). (Okamoto et al. 1997; Nakao et al. 2000; von Nickisch-Rosenegk et al. 2001; Macnish et al. Discussion 2003). Mitochonderia play a central role in In a comparison of genetic makeup, our metabolism, apoptosis, disease, and aging (Le et al. result suggests that cox1 gene is generally conserved 2002). They are the site of oxidative phosphorylation, by each isolate nucleotide sequence analysis. Despite essential for the production of ATP, as well as a of variation occurred in terms of sequence length and variety of other biochemical functions. Within these nucleotide differences and gaps (nucleotide subcellular organelles is a genome, separate from the insertions, deletions, and substitutions), but not G+C nuclear chromatin, referred to as mitochondrial DNA percentage where the overall numbers did not differ

- 1349 - Journal of American Science, 2010;6(12) http://www.americanscience.org between amplified fragments mostly triggered by a isolates is built on genetic background (Okamoto et variety of hosts' biological conditions (Macnish et al. al. 1997). However, there results are conflicting with 2002a, b). Such data showed consistent patterns with both the characteristic cryptic species of H. nana other researcher groups in this regard. They reported (Macnish et al. 2002a, b), and Schmidt classification that mitochondrial DNA sequences of the where H. nana should be closer to H. microstoma Platyhelminthes accumulate nucleotide substitutions than H. diminuta (Schmidt 1986). These at a much higher rate than sequences in comparisons observations that were revealed from the present of genetic distances (Littlewood and Bray 2001; study which should not be applied unambiguously to Vilas et al. 2005). Base substitutions and additions host-parasite associations since it does not take into are characterized by high T content which can, at consideration other factors related to the ecology of times, represent poly-T structures. In addition, this the hosts and the dynamics of the host-parasite may be a consequence of frame-shift mutations or assemblages (Johnston 2006). However, it should premature stop codons, however, protein-coding highlight the danger of triggering changes in genetic genes of the mtDNA are error-checked by translating interspecificity subsequently definitive host the nucleotide sequences (Benasson et al. 2001). susceptibility. Since, mt cox1 resultant phylogenetic Specific substitution rates include metabolic rates and tree did not support the current hypotheses on the body mass, generation time, differential fixation of basis of morphological evidence for the separation of slightly deleterious mutations, DNA repair species (Littlewood et al. 2008). mechanisms, and nucleotide composition (Vilas et al. 2005). In conclusion, molecular protocol According to the inferred topology of amino developed in this study will provide the tools for acids phylogeny of Egyptian hymenolipidid, hosts achieving supplementary comprehensive effect on the evolutionary relationship between epidemiological portrait of infestation in Egypt. isolates was clear despite their intra species Consequently, should be applied on much broader differences which agree with Johnston (2006). This scale in screening for Hymenolipis spp. infestations. could explain the closer relation of Hymenolipis spp. Sufficient clarification of evolutionary relationship of (H. nana and H. diminuta) collected from rat to be Hymenolipis spp. by other ribosomal DNA content, arranged in one cluster despite the disparities in host and complete mt genome sequencing and its genes species and morphology which is in continence with arrangement are essentials. These data will ultimately Littlewood et al. (2008). These results agree with aid investigations on dynamics of morphological and previous reports supported variant biological features developmental evolution, as well as the biology of of H. diminuta that are not always identical between .

M A B C

1000 700 500

100

Fig. 1 PCR products of mt cox1 gene amplified by specified primers pair from Egyptian isolates of (A) Hymenolipis diminuta 703 bp fragment, (B) H. nana rat isolate 699 bp fragment, (C) H. nana human isolate 715 bp fragment, and (M) 100 bp DNA Ladder.

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Fig. 2 Nucleotides multiple alignment of partial mt cox1 gene sequences of Egyptian H. diminuta, H. nana human isolate, H. nana rat isolate, and reference gi|13991366: H. diminuta, gi|226237884: H. nana isolat: HnanaMon, gi|22213549:H. nana, gi|6045204: H. nana, gi|14009612: Echinococcus granulosus genotype 1, gi|15042575: Echinococcus equinus , gi|193884329: Spirometra erinaceieuropaei, gi|239997751: Taenia multiceps, gi|28856111: , and gi|260162222: Taenia saginata, isolate: TsagT017KANTH. Black columns represents homology between sequences.

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Fig. 3 Rooted phylogenetic tree based on amino acids sequences of in silico translated partial mt cox1 gene sequences of Egyptian H. diminuta, H. nana human isolate, H. nana rat isolate and reference gi|13991366: H. diminuta, gi|226237884: H. nana isolate: HnanaMon, gi|22213549: H. nana, gi|6045204: H. nana, gi|14009612: Echinococcus granulosus genotype 1, gi|15042575: Echinococcus equinus, gi|193884329: Spirometra erinaceieuropaei, gi|239997751: Taenia multiceps, gi|28856111: Taenia solium, and gi|260162222: Taenia saginata, isolate: TsagT017KANTH. Similar topologies were developed when both Neighbor-Joining and UPGMA methods were applied (MEGA4 software).

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