INTEKNATIONAI. JOURNAL 01' LEPROSY^

\^69. Number IPrimed in the USA.

(ISSN n145-916X)

PCR-Restriction Fragment Length PolymorphismAnalysis (PRA) of Mycobacterium leprae from

Human Lepromas and from a Natural Case of anArmadillo of Corrientes, Argentina'

Martin Jose Zumarraga, Edmundo Hector Resoagli, Maria Elena Cicuta,Anibal Ramon Martinez, Maria Isabel Ortiz de Rott, Sonnia Gracia de Millan,

Karina Caimi, Andrea Gioffre, Alicia Alito, Fabiana Bigi,Angel Adrian Cataldi, and Maria Isabel Romano2

The armadillo is a mammal of. the ordei-Edenuila whose origin can be traced tothe end of the Eocene epoch, some 40 mi I-lion years ago, of which toda), there re-main only three living families: Bratlypodi-doe (sloths). Myrinecophagidae (anteaters)and Dasvpodidae (armadillos). The lastfamily includes six subfaindies. The sub-family Dasypodidue gentis Dasypus com-prises D. novenicincats-, D. kvbridus, D. sa-bonicola and D. kappleri. There are 20species of ai-manos distributed for themost part in the southeastern United Sanes,Central and South America. II is of interestto note that armadillos occur exclusively inthe Western Hemisphere (-1)• It is a valuableanimal mode] for the study of leprosy be-cause of the facility to reproduce the illnessand many of the animais, captured in theU.S.A.. Mexieo and Argentina have beenfound to be naturally infected with MVCO-bacte ri um leprae (1- "-14-'6).

Wild ieprosy in the nine-banded arma-dillo (Da.v.vpits norenicinctus) is character-ized by the presence of multiple bacilli in

' Received for publication on 27 November 2000.Accepted for publication in revise(' forni ou IS Febru-ary 2001.

M. J. Zumarraott. K. Caiini. A. Giofre, A. Alito, F.Bigi. A. Cataldi. M. 1. Romano, Instituto de Biotec-nolotlia (CICV-INTA). Castelar. 13tienos Aires. Ar-gentina. E. H. Resoagli, M. E. Cicuta. A. R. Martinez.M. 1. O. de Rott. S. G. de Millan, Programa Experi-mental eu Armadillo (PEA). Fite ti tad cljCiencias Vet-erinarias (FCV/UNNE), Sgto. Cabral 2.139-3400, Cor-rientes, Argentina.

Reprint requests to Prof. Maria Eleita Cicuta ai theabove address or FAX 54-3783-425753: e-mail: ci-cuta@vet.unne.edu.ar

Lhe internai organs such LIS the lymphglands. spleen and li ver with nerve and skininvolvement. We could observe this in 9 outof 132 animais captured in Corrientes. Ar-gentina. an arca enclemic for leprosy in hu-maus with a prevalence of 6 per 100,000(".15). lvi. leprae was recog.nized by 1982 inthose naturallv infected armadilios throughspecific stains (Ziehl-Neelsen. Fite-Faracoand King-Young), loss of the acid-fastnesswith pyrichne extraction. positive 1)-dopaoxidase reaction, noncultivable on Lõwen-stein-Jensen and Stonebrink media, replica-tion in the mouse foot pari (Sheparti tech-nique) (') and reaction to the armadillo bac-terin (AB). similar to the normalized-bacillary lepmnin ( NBL-40) in diseasedpersons with clifferent forms of the illness (").

Poiymerase chain reaction (PCR)-restric-tion fragment length polymorphism analysis(PRA), which relies ou the amplilication ofa 439-bp portion of the hsp65 gene presentin ali mycobacteria, followed by two dis-tinct di 2.estions ( with BstEII and Haell1) ofthe PC1-Z product. offers a rapid and easy al-ternative that permits the rapid identilicationoí mycobacteria without the need for spe-cialized equipment (''.17). In the present in-vestigation we attempted to use the samemethodoiogy to investicate if it was possibleto conclusively identify M. leprae from twohurnan lepromas and different tissues o fluenaturally infected nine-banded armadillo.

MATERIAIS AND METHODSDNA of. M. leprae was extracted and pu-

rified from the 1 ver, popliteal and axilar)'lymph nodes of a naturally infected ar-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

800 b

xlm~.

400 h350 b300 b250 b200 b150 b

100 13

50 bi

419111*W:4,

ah,̂

BstEll^Ilaelll

/met./múmia11011n1(11 of Lepro.sy^ 2001

"Dir. FiGuRE. PRA gel alter digestion with llstEll (lanes 1 to 11) and //tic111 (lanes 12 to 22) of 439-hp DNAfragments amplilied from M..sym.fuhuicom (lanes 1 and 12), M. sriu/gui (lanes 2 and 13). M. aritem (lanes 3 and14), M. boris (lanes 4 and 15) and M. hiptioe from annadillo (lanes 6, 7, 10, 17. 18 and 21) and from human lep-romas (lanes 5, 8, 9, 1(1, 19 and 20); molecular weight marker (lanes II and 22).

madillo 1captured during the ExperimentalProgram in Armadillo (PEA)1, and threesamples from two human lepromas, of thesame region. 13rielly, 0.5 g of each infectedtissue was manually ground with micropes-tles. Once macerated it was suspended in300 pl of buffer Tris-EDTA (TE) lx, withI() pl of proteinase K (PK; 1 O mg/m1) andincubated overnight ai 37°C. After adding10 mg/mllysozyme solution and incubatingfor 2 hr ai 37°C, 100 pl of SDS 10% and I()pl of PK were aldeei and incubated IS minat 65,C. Two extractions of the same vol-ume with phenol-chloroform-isoamyl alco-hol (24-24-1) were carried out and one withchloroform-isoamyl alcohol (24-1). Then,100 pl of NaCI 5 M Wati added and precipi-tated overnight with absolute ethanol ai—20°C. It was centrifuged at 12,000 g x 15min and the supernatant \vas discarded. AI'-ter two washes with ethanol 70%, it was re-suspended in 40 pl of buffer TE I x. 11 wasused as I pl of pure DNA for PCR. A frag-ment of 439 bp was amplitied with theprimers Tb I I and TbI2 by the proceduredescribed by Telenti, et ai. (17), and the am-

plilication product was subjected to BstElland Haelll enzyme digestions. The frag-ments obtained were separated by electro-focusing on 3% agarose plus ethidium bro-mide and visualized under UV. A 50-bpDNA ladder (Gibco BRL, Gaithersburg,Maryland, U.S.A.) was used as a molecularweight marker. DNA fragments were alsoamplilied from Al. scrojalaceuni, M.

M. aviltai and M. boris.

REsuursThe results ui the PRA are shown in The

Figure. The patterns of the six sampleslepromatous tissues analyzed were identicaland coincident with those ofM. leprae ('.13)that are characterized by uniformly obtain-ing two fragments of 315 and 135 bp withthe digestion with B.slEII and two fragmentsof 26'5- and 130 bp with the digestion withHaelll that agree with the published se-(vences of the antigen of. 65-kDa.

DISCUSSIONSince AI. leprae are noncultivable myco-

bacteria, the diagnosis of leprosy is based

69, 1^Zunuirraga, et al.: Arnuidino Lepmsv in Argentino

on the demonstration of ai least two of thefollowing: characteristic lesions of the sido,loss ol sensibility, nerve involvement or thepresence of acid-fast bacilli in smears ofskin lesions ("). In this context, the quickdiagnosis by molecular biology methodslias renovated the interest in scientificgroups that have described a varietytechniques, such as amplincation ofM. /ep-rae-specific repetitive sequenceshybridization (2), nested-primer gene ampli-lication of a 347-bp product from a bacte-rial genomic library (12), amplitication ofthe gene that encodes ao antigen of 36-k Dasize (7), PCR baseei on the selectivecaibo of a 530-bp fragment of the gene en-coding the proline-rich antigen of M. leprae(5), a specitic 360-bp DNA-probe encoding80% of the 18-kDa protein gene of M. lep-me ( S), or PCR usine a unique sequence of16S ribosomal RNA (

We conclude that the PRA method is rel-atively si ia ^and that it was able to com-pare a variety of mycobacterial species in asingle experiment, allowing the conclusiveidentitication of M. leprae. These studies.mate for the first time in Argentina, corrob-orate the inalai discoveries in SouthAmerica mate by our investigative groupon the detection of naturally infected ar-madillos with the Hansen's bacillus^b).

The similar reaction of the comparative testmade in humans with armadillo bacterin(AB) obtained from this naturally infectedanimal and bacillary lepromin (LBN-40)contirm that it was ;Always natural orleprosy acquired by this animal from theenvironment (6).

SUMNIARYPolymerase chain reaction (PCR)-restric-

tion fragment length polymorphism(PRA) which relies on the amplitication of a439-bp portion of the hsp65 gene present inali inycobacteria, followed by two distinctdigestions (with BstE11 and une! II) of thePCR product, offers a rapid and easv alterna-tive that allows identilication of Lhe specieswithout the need for specialized equipment.

Wild leprosy in the nine-banded ar-madillo (Das.vpus noreincinents) is chame-terized by the presence of multiple bacilli ininternai organs such as lymph nodes, spleenand tiver, as well as in nerves and skin. Wecould observe this in 9 out of 132 animais

captured in Corrientes, Argentina. an arcaendeinic for leprosy in humans. Mvcobacte-riam lepra(' were recognized in those natu-rallv intected animais through differenttechniques. Three samples of extracted1)NA of the mycobacteria present in thespleen. tiver and popliteal lympli node of anaturallv infectecl animal during the Exper-imental Program in Armadillo (PEA) andthree samples of human leprmnas wereprocessed by PRA.

The patterns of the six samples analyzedwere iclentical and were characteristic of M./epmc. These studies. made for the firsttime in Argenlina. corroborate the inalaidiscoveries in South America made by otn-investigative grou) on the detection of ar-madillos naturally infected with the I lansenbacillus.

IZESUNIENEl análisis por KR (lel polimorfismo en el largo de

los fragmentos restricti \ os (/'RA 1 del ADN. que se

basa en la amplilicación de una porción de 439 pb del

gen 11.11165 presente en todas las inicobacterias, seguido

de dos digestiones diferentes del proibido de PCR.ofrece una alternativa rápida y fácil que permite laidentificación de la especie. sin Ia necesidad de ditielll-

lOSIIS pruebas bioquímicas.La lepra salvaje en el arinadillo de nueve bandas

(1)a.s.v1ies noreincinclids. Lintk', 1758f cursa con la

presencia de abundantes bacilos con locali/ación prel-erentemente linfo-espleno-liepzitica, con compromiso

nervioso y (térmico. tal cual se pudo observar en a111-males capturados en la província de Corrientes. Ar-

2entina. fona endémica de la enfermedad en humanos.

Ilabiéndose reconocido Myroharterium /cprac, in-

fectando naturalmente estos annadillos, a través de

cliferentes técnicas. se procesaron, por mediu de la téc-

nica de PRA. ires muestras de ADN extraído de las mi-

cobacterias presentes eu bato. liíuado y linfoglándula

piela' de mi animal naturalmente infectado obtenidodentro (lei y de Ires inuestras de lepromas hu-

manos de pacientes de la misma provi ncia.Los seis patrones logrados coinciden comi los de esta

especie. caracteri/ándose por la obtención uniforme de

fragmentos característicos de Al. lepnie. Estos estu-

dios. efectuados por primem Vel en Argentina. corrob-

oran los hallzi/gos iniciziles en Sudamérica lieclio poreste grupo de investigación, sobre la detecciOn de ar-

madillos natural inclue infectados comi el bacilo de

Hansen.

RÉSUMÉI,a réaction de Polymérase en cliimte (PCIZ) asso-

cie à l' analyse (lu polymorphisme (les fragments

obtentis après digestion par (les enty fies de restriction

(I)RA qui s'applique ici à l'ampl i ficai ion clu segment

24^ Internaiional .1011/71a1 of. Lepmsy^ 2001

de 439 paires de base du gène Itspó5 qui est présenteliez toutes les inycobactéries, siuvi de deux digestionsdistinctes par BstEll et Mieni des produits du PCR.représente une méthode làcile et rapide qui perinetl'identilication (les espéces sans la necessite d'unéquipement spécialisé.

La lèpre clic/ te tabu à tient' bandes (Das.vpus

novenwin(tus) est caractérisée par ia présence de trèsnombreux bacilles dans les organes internes comine^7.les no:mis lympliatiques. la rate et te foie, ainsi que lesnerfs et la peau. Nous avons pu observer cela dansneuf partiu i 32 animaux qui furem capturés à Corri-entes en Argentine. une région endémique de lèpre hu-^8.maine. Mycohacterium lepme Int identifiée par dif-férentes techniques. Trois échantillons d'ADN isolésde inycobactéries présentes dans la rate, le foie et ten(clid lympliatique poplité d'Un animal naturellementinfecté duram le programme expérimental clic/ te^9.tatou (P1-1A) eI trois échantillons de lépromes humainspréparés pour une PRA. La distribution des bandes derestriction était identique et caractéristique de M. lep-

rim dans les 6 échantillons testés. Ces études, menées^10.pont- la premiére foi'. en Argentine. corroborem la dé-converte initiale falte par noti-e. groupe de ia pré.sencede tabus naturellement inféctés par le bacille deHansen.

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