Asian Pacific Journal of Tropical Medicine (2010)253-256 253

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Document heading Molecular differentiation of cryptic stage of granulosus and Taenia species from faecal and environmental samples

Diganta Pan, Sumanta De, Asit Kumar Bera, Subhashis Bandyopadhyay, Subrata Kumar Das, Debasis Bhattacharya* Indian Veterinary Research Institute, Eastern Regional Station, 37-Belgachia Road, Kolkata-700 037, West Bengal, India

ARTICLE INFO ABSTRACT

Article history: Objective: Echinococcus granulosus(E. granulosus) Taenia To differentiate cryptic stage of Methods: and Received 29 January 2010 by PCR-RFLP and sequence information of amplicon. DNA were isolated from Received in revised form 20 February 2010 Taenia E. granulosus metacestodes stage of and using DNA isolation kit (Q-BIOgene kit, USA), Accepted 15 March 2010 Available online 20 April 2010 the amplified and purified DNA product was then cloned and sentResults: for sequencing. The generating sequence information was used for amplicons identification. Out of 112 faecal and E.environmental granulosus samples, 16 exhibited positiveTaenia result. The product size of amplicon positive for Keywords: was 310 bp; whereas, for spp. sizes varied from 379 to 388 bp. Restriction Taenia E. granulosus Conclusions: profile of actin II with Csp61 also differed spp. and . The Echinococcus granulosus result of the study indicated that, the primers were useful to differentiate cryptic stage of the two genera which is yet to be reported earlier. Environmental samples PCR- RFLP

1. Introduction community acquired active epilepsy; 26.3% to 53.8% active epilepsy cases in the developing world including Taenia solium (T. solium) Echinococcus granulosus [7] (E. granulosus) and India and Latin America are due to NCC . Metacestode are wellT. knownsolium parasites of medical and stage can be easily differentiated on theCysticercus basis of theirT. economic importance. is severely pathogenic to soliumgross morphologicalT. hydatigena characters either as T. taeniaeformis (for man when the larval stage develops in the central nervous and Echinococcus), strobilocercus ( E. granulosus) system. The disease is not only important in developing and hydatid ( ). But the eggs of countries but also in areas of non endemicity affecting are morphologically indistinguishable to those of other [1-3] T. solium Taenia immigrants, tourists E.. Likegranulosus , tapeworms of the genus . Therefore, the most hydatidosis caused by is responsible for immediate priority is to distinguish egg of the parasite in formation of cysts, mainly in the liver and lungs,Taenia cause faecal and environmental samples. Like other parasitic severehydatigena pathologicalT. hydatigena) effects in intermediate hosts. infection, intra vitam diagnosis of taeniid tapeworms can ( is harbouredRattus by rattus sheep and not be achieved by microscopical detection of worm eggs goats. Semi domesticated blackTaenia rats ( teaniaeformis) serve (T. in faecal samples by routine serological methods. For teaniaeformis)as the intermediate T. solium of E. granulosus that reason copro-DNA test may be used as alternative . For and man approach because parasite DNA are excreted with eggs, and proglottids or parasite cells can be detected from faeces and dogs serve as the’ definitive host of the parasite, [8] respectively. During 80 s abdominal hydatidosis in man has after amplification by PCR . Six different actin isoforms been reported from southern part of India[4], operated cases have been identified by aminoacid sequences. Moreover, of pulmonary hydatid cysts has been documented from actin is the most abundant protein in eukaryotic cells and eastern India[5] and records of consecutive cases of human actins are highly conserved during evolution[9,10]. Two hydatidosis has been reported from northern India[6]. It has actin related sequences (EgactI and Egact II) separated been documented in the literature that, neurocysticercosis by a 4 kb region[11]. The Egact I contained no intron but (NCC) is identified as the single most common cause of an intron of 591 bp was observed in the Egact II. Intron sequences are specifically known because intron is closely *Corresponding author: Dr. Debasis Bhattacharya, Senior Scientist, Indian Veterinary related to the evolution of eukaryotic genomes which has Research Institute, Eastern Regional Station, 37-Belgachia Road, Kolkata-700 037, been established as intron-early and late theories[12,13]. India. E. Tel: 033-25565725 granulosusTherefore, to characterize cryptic interspecific variants of Fax: +913325565725 , analysis of intron of actin II has been taken into [14,15] [16,17] E-mail: [email protected], [email protected] consideration from abroad and India . Considering Diganta Pan et al./Asian Pacific Journal of Tropical Medicine (2010)253-256 254 established theories mentioned above, this communicationE. cattle and buffalo slaughter house (35) and public park granulosusreports for the firstTaenia time differentiation of cryptic stage of (13). Approximately 20 g of faecal and soil samples were and available in India which was further concentrated by centrifugation and floatation was done by supported by PCR-RFLP and sequence information of zinc chloride solution[18]. The same primer described earlier amplicon. was used for screening of faecal and environmental samples. 2.5. Confirmation of amplicon 2. Material and methods 2.1. Collection of metacestodes The amplicons were confirmed by generating sequence information as described earlier. T. solium Cysticercus cellulosae 2.6. Differentiation of E. granulosus and Taenia by PCR- Metacestode stage of ( T. hydatigena) was RFLP collected from skeletal muscle of pigs. Cysts of were collected from naturally infected sheep. Strobilocercus form of metacestode wasRattus collected rattus from liver of semiE. Location of restriction cuts of amplified product on the domesticatedgranulosus black rats ( ). Protoscoleces of basis of sequence information were identified for Csp61 was collected from the lungs of naturally infected using Primer 5.0 (Premier Biosoft International, USA). In buffalo. The metacestodes were identified and stored at -20 曟 till brief, a total volume of 100 毺L of PCR product was purified further use. from gel using QIAquick PCR amplification kit (QIAGEN, 2.2. Isolation of DNA from metacestode stage USA). The elute volume was restricted to 30 毺L. Restriction digestion was conducted in 10 毺L volume which comprised of gel purified PCR product (6 毺L) , 10 伊 Tango buffer (毺L) DNATaenia samplesE. were granulosus isolated from metacestodes stage (Fermentas, USA), and nuclease free distilled water (2 毺L) of and using DNA isolation kit (Genei, Bangalore). The mixture was incubated at 37 曟 for (Q-BIOgene kit, USA). DNA’ isolation was carried out 6 h and digested product was separated using 2.5% agarose according to manufacturer s protocol. In brief, tissue gel. suspension was homogenized in 200 毺L cell suspension solution. The homogenate was further digested by 3. Results proteinase K after addition of RNAse. After digestion, salt out mixture was added and supernatant was collected E. after centrifugation. DNA was precipitated after addition granulosus, Amplicons T. exhibited taeniaeformis, differences T. solium in productT. hydatigenasize for of absolute alcohol (v/v). Isolated DNA was dissolved in and nuclease free distilled water (Genei, Bangalore). DNA (Figure 1). The products after sequencing were shown samples were stored at -20 曟 till further use. to have the span between 310 to 388 bp. The sequence 2.3. Amplification of DNA sample, purification and cloning information was further registered at the Gen Bank of PCR product database under accession numbersE. granulosus EF179177,T. EF187446,solium T. taeniaeformisEF187447, EF192916T. hydatigena for , , and T. solium, respectively. There was no Genomic DNA was used for amplification of DNA samples sequence variation among (379 bp) (EF187447), [14] T. taeniaeformis T. hydatigena using the primers’ described earlier . The design of primer’ (380 bp) (EF 187446) and was: Forward’ 5 GTC TTC CCC TCT ATC GTG GG3 and’ (388 bp) (EF 192916). However, deletion mutation was Reverse 5 CTA ATG AAA TTA GTG CTT ®GTG CGC 3 . foundE. at granulosus21 and 360 positions. Further comparison of data In brief, PCR was performed in Gene Amp PCR system T.with solium T. taeniaeformis of 310 bp fragmentT. hydatigena (EF 179177) with 9700 (Applied Biosystems, USA). A final volume of 25 毺L , and revealed containing DNA (not quantified) , 20毺M of each dNTP dissimilarity between 31.05 to 31.31 per cent. The details (Genei, Bangalore), 10伊PCR buffer (Genei, Bangalore), of nucleotide sequence variation and gap due to probable 2.5 mM MgCl2 (Genei, Bangalore) , 10 pmol of each deletion mutation has beenE. granulosus depicted in FigureTaenia 2. Restriction gene specific primers (IDT, USA) and 1 unit of Taq DNA profile of amplicons of and exhibited polymerase (Fermentas, USA) were used in reaction. The restriction fragments of differentE. granulosus sizes. BecauseTaenia restriction PCR reactions were started with an initial denaturation step sites were different for and which followed by 35 cycles, 95 曟 for 45 sec, 53 曟 for 45 sec could be deduced on the basis of different restriction sites followed by 72 曟 for 90 sec with a final extension of 72 曟 of Csp61. The details of fragment sizes along with their for 10 min. Amplified product was purified using QIAquick restriction sites have been explained in Figure 3. Out of Gel extraction kit (QIAGEN, Germany).TM Further purified 31 canine samples collected from public parks, 3 were product was cloned usingE. InsT/A coli clone product cloning kit positive for taeniidT. eggs, hydatigena and 2 of the 3 positive samples (Fermentas, USA) using DH 5毩 as primary cloning were confirmed as based on size of amplicon, host. Confirmed clone was sent for sequencing to DNA restriction profile (Figure 1&2) and sequence information sequencing facility UDSC (DBT supported, Department of (EF192196). Further investigation on the basis of 22 faecal Biochemistry, University of Delhi, South Campus). DNA samples collected from in and around cattle and buffalo sequencing was carried out in cloned DNA by universal slaughter house, 12 samples were positive for teaniid like primers (M13F and M13R) using cycle sequencing (3730 eggs by microscopy. Out of twelve samples, amplified system, Applied Biosystems, USA). productE. of granulosus 4 samples was indistinguishable from the product 2.4. Evaluation of field sample size of (Figure 1). Out of the total of 59 collected soil samples, 11 were collected from in and around pig slaughter house, and 2 of the A total of 31 canine faecal samples were collected from 11 were positive for taeniid like eggs, whose ampliconsT. solium were public parks and 21 samples were collected from in and further comparable with sequence information of around slaughter house. Soil samples were collected (EF187447). Out of 35 faecal samples collected from in and from in and around pig slaughter house (11), viscinity of around cattle and buffalo slaughter house, 6 were positive on Diganta Pan et al./Asian Pacific Journal of Tropical Medicine (2010)253-256 255 the basis of their product size. Three of the 6 positive samplesT. hydatigenawere confirmed to have homologous sequence information of E. (EF192916) granulosus and the rest 3 positive samples were similar to (EF179177). Out of 13 soil samples collectedTaenia from public parks 2 were amplicon indistinguishable from . AfterT. hydatigensequencinga the sequence information was comparable with (EF192196).

Figure 3. Restriction fragment length polymorphism analysis of Acin II gene amplified product. M= Molecular weight marker (arrows from the bottomE. granulosus 100, 200, 300, 400, 500 bp), Lane( 1-3)= Taenia sp., Lane 4= ; Lane1-3: 171 bp (lower band), 176 bp (upper band) , Lane 4: 81 bp (lower band), 184 bp (upper band). Figure 1. E. granulosus Amplified products of Actin II gene of and Taenia 4. Discussion sps. M= Molecular weight marker (arrows from the bottomE. granulosus100, 200, 300, 400, 500 bp), This is not possible to identify taeniid cestodes on the basis of Lane 1: Amplicon of (310 bp), T. teaniaeformis traditional methods particularly after microscopic examination Lane 2: Amplicon of (380 bp), of eggs. In order to overcome limitations molecular approaches T. solium Lane 3: Amplicon of (379 bp), has been explored like PCR-RFLP, single stranded T. hydatigena Lane 4: Amplicon of ( 388 bp) conformation polymorphism (SSCP), amplification of DNA of parasite and base excision sequence scanning thymine- base reader analysis with mitochondrial genes[19-22]. Although immunological methods are available based on primary binding assay to measure antigen or antibody but preparation of worms recovered from experimental is impractical [20] forcoxI regular use . In the recent past, cytochrome c oxidase ( ) and cytochrome b (cytb) genes were used because their nucleotide sequences have been determinedT. saginatacompletely. TheseT. solium genes have been used to discriminate and [20]. Although SSCP may be useful approach without DNA sequencing but the methodology is time consuming[22]. On the contrary, duringTaenia the presentE. granulosus investigation we could easily discriminate and on the basis E.of granulosusproduct size using primer for amplification of actin II of . Further, products have been confirmed on the basis of their sequence informationEchinococcus and RFLP becauseTaenia on the basis of Csp61 recognition sites and could be differentiated. The different restriction sites have been described through figuresin silico along with their predicted product sizes indeduced silico by primer premier 5.0. Therefore, our analysis matched with the bench top observation. It can be summarized that, PCR based RFLP is not only useful for identificationTaenia of taeniidEchinococcus specimens but also for the speculation of and prevalence in a particularT. soliumarea and forEchinococcus screening environmental samples. Because and eggs are infective to man and cause infection. During the present study panel of parasites is less because we could not include other parasites [23] [24] described earlier .Taenia Because in earlier study this has been indicated that, egg discriminated by molecular Figure 2 T. solium T. hydatigena T. multiceps T. . Alignment of sequence information of Actin II of , tool may be speculated as , or T. taeniaeformis T. hydatigena E. granulosus taeniaeformis Taenia , and . Echinococcus. Therefore, present investigation on and (.) indicates similarity of sequence information. (-) indicates gap. is useful and novel approach which is yet to be Diganta Pan et al./Asian Pacific Journal of Tropical Medicine (2010)253-256 256 Table 1 Showing results of copro-DNA and parasitological tests of faecal and environmental samples. Number of Number positive Type of samples Number positive by PCR samples(112) by microscopy(16) Taenia2 Canine faecal sample ( from (confirmed as based on product Faecal samples(53) 31 3 T. hydatigena public park) size and on the basis of sequence information.) “ 4 Canine faecal sample ( from in Echinococcus ” 22 12 (confirmed as based on and around slaughter house ) product size and sequence information.) “ Taenia2 From in and around slaughter (confirmed as based on product Soil samples (59) ” 11 2 T. solium house size and on the basis of sequence information.) Taenia3 From in and around cattle and (confirmed as based on product 35 6 T. solium buffalo slaughter house size and on the basis of sequence information.) Taenia2 (Confirmed as based on product Collected from public parks 13 2 T. hydatigena size and on the basis of sequence information.)

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