DIFFERENTIATION OF BRUGIA MA-LAYIAND BRUGIA PAHANGIBYPCR-RFLP OF ITSl AND ITS2

S Nuchprayoon', S Sangprakarnl, AJunpeel, S Nithiuthai2. S Chungpivat2 and Y Poovorawan-'

(Departmentof Parasirology. 3Department of Pediatrics. Chula Medical Research Center. Faculty of Medicine.and 2parasitologyUnit, Department of Pathology. Faculty of Veterinary Science. Chulalongkorn.University,

Bangkok. Thailand

Abstract. Lymphatic filariasis has been targclCdby the World Health Organization for elimination by the year2020. Malayan filariasis. caused by Brl/gia lIIa/ayi,is endemic in southern Thailand where domestic cats serveas a major reservoir host. However, in nature, domestic cats also carry B. pahal1gi infection. In addition tochel11othempyand vector control. control in reservoir hosts is necessary to achieve the elimination of the disease.Thereforc. differentiation between B. ma/ayi and B. pahal1gi in the cat reservoir will help the lymphatic controlprogram (0 monitor and evaluate the real disease situation. It is difficult to differentiate these t\VOBrugia speciesby microscopic examination. The technique is also time-consuming and reyuires expertise. We employed the'polymerase chain reaction-linked restriction fragment length polymorphism (PCR-RFLP) technique of internaltranscribed spacer regions, ITS I and 11'52.ofribosol11alDNA (rDNA) to differentiate B. lIIa/ayifrom B.pahallgispecies. Amongthe restrictionenzymes tested.only the PCR product onTS I digested with Ase I could differentiateB. ma/ayi from B. pa!Jallgi. This PCR-RFLP technique will be useful for lymphatic filariasis control programsfor monitoring and evaluating animal reservoirs.

INTRODUCTION

Lymphaticfilariasis, mainly caused by the filarialnematodes Wuchereria bal1crofti and Brugia malayi,isa debilitatingand tlisfiguring disease. It is estimatedthat I. ] billion people, 20% of the world's population,in mOffthan 80 countries, are at risk of acquiring theinfection. \..bile over' 120 million have already beeninfected(WEG, 2000). Although Bancroftian filariasishasbeen controlled to a Iow level, 0.99 cases/IOO,OOOpopulatioD (Filariasis Division. 1999), Malayanfilariasis is still endemic in southern Thailand.

Lymphaticfilariasis. caused by B. malayi, nocturnalsubperiodic type, is prevalent mainly in NarathiwatProvince. This is due to the many suitable mosquitobreeding sites, large swamp areas. and the existenceof animal reservoir-hosts. In Thailand, Mansonia spare the main mosquito vectors of B. malayi (FilariasisDivision,1998). Domestic cats are important reservoirhosts for B. malayi (Palmieri et al. 1985; Phantana etai, 1987:Kanjanopas et ai, 2001; Chansiri et ai, 2002).Therefore. besides chemotherapy and vector control,theSuccessfulelimination of lymphatic filariasisshouldinclude the control of reservoir hosts. Not only B.llzalayicaninfect domestic cats, but also another filaria!

Correspondence:Dr Surang Nuchprayoon, Departmentof Parasitology. Faculty of Medicine, ChulalongkornUniversity,Bangkok 10330, Thailand.Tel:66 (0) 2256-4387; Fax: 66 (0) 2252-4963E-mail: fmedstt@md2.md.chula.ac.th

Vol34 (SuppI2) 2003

species, such as B. pahangi -(Nithiuthai andChungpivat, 1992; Chungpivat and Sucharit, 1993).Co-infections of both Brugia species in domestic catsis not uncommon in Thailand. This raises the

possibility of misdiagnosis due to the difficulty indifferentiating both species by the convent:,1nalmicroscopic method. Misdiagnosis would jeopardizethe lymphatic filariasis control program.

In endemic areas. the routine method for

identifying microfilaria species is microscopicexamination. based on the delineation of particularmorphological features using Giemsa stain, andgeographic location. that is, where the specimens havecome from. Nevertheless, using this technique, it isdifficult to discriminate clearly between closely relatedspecies such as B. l11alayi and B. pahal1gi.Histochemical staining, to detect acid phosphataseactivity, could overcome the problem (Yen and Mak.1978: Chungpivat er ai, 1990: Nithiuthai andChungpivat, 1992: Chungpivat and Sucharit. 1993).However. this technique needs fresh samples to yieldthe best results. Furthermore, both staining methodsrequire expertise to identify and confirm the species.Molecular analysis has been introduced as a new toolto distinguish parasite species. Ribosomal DNA(rDNA) has been a valuable tool to discriminate closelyrelated species among several eukaryotic organisms,including nematodes. The application of internaltranscribed spacers (ITS) to identify the organism hasreceived the most attention by nematologists duringthe past decade (Gasser et al. 1994; 1996; Zhu et al,

67

SOlITHEAST ASIAN .J TROt' MED PUBLIC HEALTH

1998;Almeyda-A11igaset aI,2000; Conole et aI,200 I).Studies of peR-linked restriction fragment lengthpolymorphism (PCR-RFLP) profiles of the nematodes'ITS regions have provided data on nematode diversity,as well as ,the critical taxonomic character useful for

species colnparison and identification (Gasser et al.1994. 1996). We report here the results of PCR-RFLPof ITS I. and ITS2 to differentiate B. lIIalayi from B.pahmzgi.

MATERIALS AND METHODS

Specimen collectionIn co-operation with the Filariasis Division,

Department of Communicable Diseases Control.Ministry of Public Health, Thailand. collection ofhuman blood specimens was performed as previouslydescribed (Triteeraprapab and Songtrus, 1999;Triteeraprapab et aI, 2000; 200 I; Nuchprayoon et aI,200 I). Two milliliters of venous blood from domesticcats infected with B. Illalayi or B. pahangi wereobtained from experimental cats at the ParasitologyUnit, Department of Pathology. Faculty of VeterinaryScience, Chulalongkorn University. Dirofilariaill/mitismicrofilariae were obtained from infected straydogs' blood. Blood from healthy volunteers, non-infected domestic cats and dogs were used as thenegative control. This study was approved by theEthics Committee of the Faculty of Medicine.Chulalongkorn University. Bangkok. Thailand.

All filarial parasites were identified and the speciesconfirmed by Giemsa staining. and special staining foracid phosphatase activity (Yen and Mak, 1978;Chungpivat et aI, 1990; Nithiuthai and Chungpivat,1992; Chungpivat and Sucharit, 1993).

DNA extractionThe extraction method was modified from a

previously reported protocol (William et aI, 1996).Two hundred and fifty microliters of each blood samplewere added to a 1.5 ml microfuge tube and mixed with750 III ofTE buffer (l0 mM Tris-HCL pH 8.0, 1 mMEDTA, pH 8.0). The mixture was centrifuged and thesupematant was discarded. The pellet was washed with750 IIIofTE buffer, pH 8.0 and resuspended in 500 IIIof red cell lysis buffer (RCLB; 1 M sucrose, 10 mMTris-HCI, pH 7.5. 5 mM MgCI2, ]% Triton X-lOO)twice. After centrifugation, the supernatant wasdiscarded, 400 IIIofDSP buffer (20 mM Tris-HCl, pH8.0, 50 mM KCI. 2.5 mM MgCI2, O.Wc Tween 20.150 Ilg/ml Proteinase K) were added and incubated at65"C for 3 hours. Enzyme activity was inactivated byincubation at 90QCfor 10 minutes.

68

Primers designThe forward primer for the ITS 1 (FU-F) \Vas

clesigned from the conserved sequence of filarialparasites reported in the Genbank database (Fig 1). Thereverse primer for the ITS 1 (Di5.8S 660-R) and theforward primer for the ITS2 (Di5.8S 558-F) weredesigned from the rep0l1ed 5.8S rDNA sequence of D.illlmitis(AF217800). The reverse Plimer for ITS2 (FU-R) was designed from the 28S rDNA sequence of D.i11l11litis(AF217800). All oligonucleotide primers werepurchased fromthe Bioservice Unit. NSTDA. Bangkok

Polymerase chain reactionsIn order to obtain sufficient DNA for RFLP study.

semi-nested PCR for-ITS 1and ITS2 were performed.

First PCR ITSl-5.8S-ITS2 region. The entireITS 1-5.8S-ITS2 region (Fig 1) was amplified by PCRusing FL I-F and FL2-R oligonucleotide primers. Thehot-start PCR reaction was performed in a 25 IIIreaction containing PCR buffer (10 mM Tris-HCI, pH9.0, 1.5 mM MgC12. 50 mM KCI) (AmershamPharmacia, Freiburg, Gelll1any);200 ~tMeach of dATP.dCTP. dGTP and dTTP (Proll1ega,Wisconsin, USA):0.625 units Taq DNA polymerase (AmershamPharmacia); 5 pmol of each primer (FLl-F and FU-Ri; and I IIIDNA template. After incubation at 94cCfor 5 minutes. amplification was carried out for 5 cycleswith the following temperature cycling parameters:94"C for 30 seconds of denaturation; 58'C for 30seconds of annealing; and noc for 90 seconds ofextension; followed by 30 cycles of temperaturecycling parameters at 94 QC for 30 seconds ofdenaturation; 55'C for 30 seconds of annealing, and72"C for 90 seconds of extension. The final

amplification cycle included an additional 10 minutes"extension at n'c.

Second PCR for ITSl region. The ITS 1 regionwas amplified by using I III of PCR product froll1thefirst PCR as a DNA template. The PCR reaction wa"performed in a 50 1-11reaction containing PCR buffer.as described above, with 5 pmol of each primer ofFLl-F and Di5.8S 660-R. Amplification was canied outfor 5 cycles with the following temperature cyclingparameters: 94'C for 30 seconds of denaturation; 58'(for 30 seconds of annealing; and noc for 45 secondsof extension. followed by 30 cycles of temperaturecycling parameters: 94 QC for 30 seconds ofdenaturation. 55°C for 30 seconds of annealing; andn"c for 45 seconds of extension. The final

amplification cycle included an additional 10 minutesextension at n'c.

Second PCR for ITS2 region. The ITS2 region

Vol34 (Supp12) 2003

PCR-RFLP OF ITS 1AND ITS2

wasamplified by using 1111of PCR product from thefirstPCR as a DNA template. The PCR reaction was

performed in a 50 111reaction containing PCR bufferwith5 pmol oreach primer, Di5.8S 558-F and FL2-R.After incubation at 94°C for 5 minutes, amplificationwas carried out for 35 cycles with the followingtemperature cycling parameters: 94cC for 30 secondsof denaturation; 55°C for 30 seconds of annealing; andnoc for 45 seconds of extension. The finalamplificationcycle included an additional 10minutes'extension at 72"C.

Restriction fragment length polymorphism (RFLP)After precipitation of each PCR product and

I FLl -F I

resuspension in 10 III sterile distilled water, I 111ofPCR product was digested with 5 units of eachrestriction endonuclease, according to themanufacturer's protocols (New England Biolabs.Massachusetts. USA). The digestion was incubated at37"C for 3 hours. The following enzymes wereevaluated; Ase I. Ace I, HinfI and Rsa I. Analysis ofDNA fragments was performed by 2-2.5% submarineagarose gel electrophoresis.

RESULTS

Oligonucleotide primers were designed based onthe reported conserved sequences of 18S,5.8S and 28S

FLI-F--+

}

DiS.SS 55S-F--+

DiS.SS 660-R~

FL2-R~

188

ITS 1

667 bp

5.8S 28S

ITS!

570 bp

Fig 1- Forward and reverse primers for ITS 1and ITS2. The FLI-F primer is conserved in filarial parasites. All other primers weredesigned from the D. immitis 5.8S and 28S rDNA sequence (AF2 I7800). FLI-F and Di5.8S 660-R are primers for theamplification of ITS 1;.Di 5.8S 558-F and FL2-R primers are for ITS2.

Vol34 (SuppI2) 2003 69

...5' 1 gtgaacctg cggaaggatc attatcgagc ttcaacaaac aacaaacaca tcatcatcat

61 catcattatt actacttcta ctactactac tactactact aatactaata ctaatactac

121 ta.ttaacatc attatgctgc ttattttcat tctttttcaa ataatgcaag aatgaaatag181 aatgaatgaa tgatgtttaa tagattaata gatgaatagt tagtagtagt tagtagttag241 ttgatagat agataaatga aatgatgatg aaatattttt attcgatcaa t.tg.a.atataa301 acggtgatat tcgttggtgt ctatatttta tctaagttat cgcctaaccg tcgatagcga361

tgaagataaa atgatagctt a" . ]tgctcaa ttaagtagac ttaataagca

421

481ttttagctag tatgctacca a DiS.8S558-F cacaca<:;atacatacaa taatatgatataattttat ttataataat t tagcgg gggatcacct ggctcgtgga

541 tcgatgaaga acgcagctag ctgcgataaa tagt cgaat tgcagacgca ttgagcacaa601 agatttcgaa tgcacattgc

accatcgggt tgattcccga tggiacgtct ggttgagggt661 caatatcgat cagattatca aataatatac caaaattgat aca atl . tg721 atatttt tggatatatt atttgttaaa attaattcat caggtg Di5.8S 66J-R ta

781 atcattgaaa aatgacai1ai1 atcccctgat taagtataat aaaaat ag841 gcaaattttt acttacaaaa tattacatat tggaataaat aatttaatct ttcatatata901 taactgtgat acatcataaa t.atatatata gcaaaagaaa gatataaatt attatcattt961 tttttgatca ttattgctat aattattatc ctgcttgttt aactataata atagcaatga

1021 atgaatcaaa atttaaatga tacgttaaaa tccaaccaat atgttataaa tt.ttctttca1081 tttttgacct caactcagtc gtgattacc gctgaattta o:.gcata taac t.a

--.,,;)

I FL2-R I

SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

rDNAof filarialworms(B. l1Ialayi, W. bal1cJ{~fiiandD. illllllitis). Nevertheless, not all rDNA sequences ofthe selected filarial worms were completely reported.The available filarial rDNA sequences from the onlinedatabase (Genbank) were as follows: 18S rDNA from

B. lllalayi (AF036588), W. bancrofti (AF227134). andD. illlmitis (AF217800): ITS!, 5.8S and ITS2 sequencefrom D. illllllitis (AF217800); partial 28S rDNAsequence from D. illllllitis (AF2 I7800) and B. 111alayi

(AF499 130). The FLI-F primcr was designed fromthe conserved region at the 3-end of B. l7Ialayi. W.

bancrofti and D. illll7litis18SrDNA (Fig I). The FL2-R primer was selected from the beginning 5' end of D.immitis 28S rDNA, whichwasalso conservedamongthe reported 28S rDNA from other nematodes. Afterthe first round of PCR, very faint DNA bands of ITS 1-S.8S-ITS2 were observed. Subsequently. semi-nestedPCR was performed to obtain a high yield of PCRproducts. Two internal primers, Di5.8S 660-R andDi5.8S558-F.weredesignedfromtheD. illll1litis 5.8SrDNA region (AF217800) in order to amplify the ITS 1and ITS2 regions, respectively.

PCR-RFLP patterns onTS! and ITS2The 667 bp band of D. illll1litisITS 1was amplified

as expected(Fig 2A). However.both B. lIlalayi andB. pahangi ITS1had smallerITSI sizesof about590bp. In contrast,the ITS2fromD. il1llllitis was570bp,and for B. lllalayiand B. pahangi ITS2 about 650 bp(Fig2B). BothB. malayi from humansand domesticcats showed the same size of~CR products and similardigestedpatternsforITSI andITS2(Fig2A, B). Therewere 2 bands of about 290 bp and 300 bp from the AseI digestedB. pahangi ITS1(Fig2A),whileabout 140bp, 160 bp and 290 bp bands were from the B. lIlalayidigested ITS I. PCR-RFLP of ITS 1 with Acc I andwith Hinf I did not differentiate B. lIlafayi from B.pahangi. Similary, the PCR-RFLP of ITS2 with Rsa Iand with Ase I did not discriminate either Brugiaspecies.

PCR-RFLPof D. il1ll11itisITSI by Ase I showedthe 455 bp and 212 bp predicted products. All otherdigested ITS I and ITS2 showed the predicted PCR-RFLPpatterns. We demonstrated that the semi-nestedPCR-RFLP of ITS 1 with Ase I could differentiate B.

l11alayi, B. pahal/gi and D. il11l11itis.

DISCUSSION

The conventional method used for the detection

and species differentiation of lymphatic filarialparasites is the identification of microfilariae byGiemsa stain. Although this method is not expensive,

70

it requires skill and experience to differentiate amongclosely-related species. Histochemical stain of acidphosphatase activity is another useful technique forspecies identification. It clearly discriminates differentfilarial nematode species (Yen and Mak. 1978;Chungpivat et af. 1990: Nithiuthai and Chungpivat,1992:Chungpi\'at and Sucharit, 1993). However. theenzymatic activity is rather sensitive to light andrequires fresh specimens. As a result, PCR-RFLP ofthe ITS regions may be an alternative method todifferentiate closely-related species. which areindistinguishableby morphology(Gasseretal. 1998).Furthermore. PCR-RFLP could be used to identifyparasites in developmental stages (Gasser and Chilton.1995: Newton et al. 1998: Almeyda-Artigas er (//,2000) in different geographic distributions (Gasser andChilton, 1995; Ramachandran et aI, 1997).

The ITS I and ITS2 regions of rDNA are usefulfor investigatingsome variations among closely-relatedspecies(Gasseret aI, 1996;Ramachandranetal. 1997).The ITS regions are flanked by conserved rDNA genes,and the sequences can be use? 10 design primers toamplify the intervening regions by PCR. Moreover,the presence of multiple copies provides a large numberof target sequences for PCR in most organisms (Longand Dawid, 1980). As a result. the ITS regions offilarial nematodes are suitable targets for amplificationand detection, even in mildly infected individuals.Although the first round PCR was inadequate toprovide a detectable amount of PCR products. asubsequent semi-nested PCR has been utilized toincrease the sensitivity for the detection of the ITSIand ITS2 regions. Since the filarial DNA was extractedfrom whole blood specimens of infected individuals.host genomic DNA might possibly interfere with thePCR reaction. However. the bases at the 3 - end of

both internal primers designed for semi-nested PCRof ITS 1 and ITS2 were not complementary to humanrDNA (U 13369). Therefore, no ITS 1or ITS2 productswere amplified from the semi-nested PCR of the non-infected human blood samples (data not shown). Therewas no reported sequence of ITS 1 and ITS2 fromdomestic cats and dogs. Nevertheless, no PCRproducts of ITS I and ITS2 were amplified from thenon-infected blood samples of domestic cats and dogs(data not shown).

The PCR-RFLP profiles of domestic cat B. /l/alayiand human B. /l/afayi ITS I and ITS2 were similar forall restriction endonucleases used in our study. Our

results supP011edthe suggestion that domestic cats playan important role as animal reservoirs of B. malayi. asreported previously by studies of microfilarialmorphology, periodicity, PCR-RFLP of Hha I repeat

Vol34 (SuppI2) 2003

A..ITS 1

PCR-RFLP OF ITS I-\ND ITS2

Di:6(;7bp

L,.:~

Bm, Bp: 590 bp

B. IT S2

l\'1 UndigestedhEm cEm Bp Di

Bm, Bp: 050bp

L.r

Di:570bp

Rsa I

,455bp 'l

/212 bp J DiI' "I'///'

I/- 300 bp 1.

" 290bp IfBp/" loObp r" Bm

140 bp .-

L4s'e I

rill DigestedhEm rBm Bp Di

M DigestedhEm cEm Bp Di

Fig 2- PCR-RFLP of B. malayi and B. pahal1gi ITS I (A) and ITS2 IB). M: 100 bp ladder molecular weight marker; hBm: human

B. malU\'i; cB m: cat B. malm'i; Bp: B. pahal1gi; Di: D. immitis; Asf' I. Ace!. liil1fl and Rsa I are restriction enzymes used for

the digestion of ITS 1 and ITS 2 PCR products.

genes and PCR ofTrans-spliced Leader Exon I (SLX)(Chansiri et al. 2002).

In summary. we demonstrated that the PCR-RFLPof ITS 1, digested with A.I'e1. could differentiate E,

l11alayi,B. pahangi and D. il11l11itis.Recently. it has

Vo!34 (SuppI2) 2003

been shown that domestic cats in Thailand can carrynot only E, jJahangi and E. 17Il/layi,but also D. inuuitis(Chansiri et 01, 2002). Further study, for the fieldapplication of the PCR-RFLP method to monitor andevaluate infections in the reservoir hosts, would helpthe on-going lymphatic filariasIs control programs.

71

Hinf I .Ace I As'e I- --

r...1 Undigested 1....1 Digested i\'I Digested 1\'1 DigestedhEm rBm Bp Di hBm cBm Bp Di hBm rBm Bp Di hBm cBm Bp Di

SOUTHEAST ASIAN J TROP MED PCI3L1C HEALTH

ACKNOWLEDGEMENTS

This study was supported by the grants for graduatestudents in public universities, the Graduate Schooland Molecular Biology Funds, Research Affairs.Faculty of Medicine. Chulalongkorn University. YPoovofawan was a Senior Research Scholar of theThailand Research Fund. We would like to thank Dr

Saravudh Suvannadabba, Dr Suvith Thampaolo. MsKobkarn Kanjanopas, Ms Sumas Loymak, officers atthe Filariasis Division, and regional officers. for theirsupport during specimen collection. We are th:mkfulto 1\1sWoraporn Sirivichayakul and all staff in theParasitology Unit, Department of Pathology. Facultyof Veterinary Science and staff in the Departmcnt ofParasitology, Faculty of Medicinc, ChulalongkornUniversity for their technical help. We also appreciateDr Benjawan Sichnasai and staff from the RabiesControl Section. Bangkok Metropolitan Area for theirkind assistance.

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