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  • Volume 69 , Number 1
  • Page: 21–5
ORIGINAL ARTICLE

PCR-restriction fragment length polymorphism analysis (PRA) of Mycobacterium leprae f rom human lepromas and f rom a natural case of an armadillo of corrientes, Argentina

Martin Jose Zumarraga1; Edmundo Hector Resoagli2; Maria Elena Cicuta3; Anibal Ramon Martinez4; Maria Isabel Ortiz de Rott5; Sonnia Gracia de Millan6; Karina Caimi7; Andrea Gioffre8; Alicia Alito9; Fabiana Bigi10; Angel Adrian Cataldi11; Maria Isabel Romano12




ABSTRACT

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that allows identification of the species without the need for specialized equipment. Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in internal organs such as lymph nodes, spleen and liver, as well as in nerves and skin. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans. Mycobacterium leprae were recognized in those naturally infected animals through different techniques. Three samples of extracted DNA of the mycobacteria present in the spleen, liver and popliteal lymph node of a naturally infected animal during the Experimental Program in Armadillo (PEA) and three samples of human lepromas were processed by PRA. The patterns of the six samples analyzed were identical and were characteristic of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of armadillos naturally infected with the Hansen bacillus.



RÉSUMÉ

La réaction de Polymerase en chaîne (PCR) associée à l'analyse du polymorphisme des fragments obtenus après digestion par des enzymes de restriction (PRA), qui s'applique ici à l'amplification du segment de 439 paires de base du gène hsp65 qui est présent chez toutes les myeobaetéries, siuvi de deux digestions distinctes par BstEII et Baelll des produits du PCR. représente une méthode facile et rapide qui permet l'identification des espèces sans la nécessité d'un équipement spécialisé. La lèpre chez le tatou à neuf bandes (Dasypus novemcinctus) est caractérisée par la présence de très nombreux bacilles dans les organes internes comme les nœuds lymphatiques, la rate et le foie, ainsi que les nerfs et la peau. Nous avons pu observer cela dans neuf parmi 32 animaux qui furent capturés à Corrientes en Argentine, une région endémique de lèpre humaine. Mycobacterium leprae fut identifiée par di­férentes techniques. Trois échantillons d'ADN isolés de myeobaetéries présentes dans la rate, le foie et le nœud lymphatique poplité d'un animal naturellement infecté durant le programme expérimental chez le tatou (PHA) et trois échantillons de lépromes humains préparés pour une PRA. La distribution des bandes de restriction était identique et caractéristique de M. leprae dans les 6 échantillons testés. Ces études, menées pour la première fois en Argentine, corroborent la découverte initiale faite par notre groupe de la présence de tatous naturellement inféetés par le bacille de Hansen.



RESUMEN

HI análisis por PCR del polimorfismo en el largo de los fragmentos restriclivos (PRA) dei ADN, que se basa en la amplification de una porción de 439 pb del gen hsp65 presente en Iodas las micobacterias. seguido de dos digestiones diferentes dei produeto de PCR. ofrece una alternativa rápida y fácil que permite la identification de la especie, sin la necesidad dc dificultosas pruebas bioquímicas. La lepra salvaje en el armadillo de nueve bandas (Dasypus noveincinctus. Linné, 1758) cursa con la presencia de abundantes bacilos con localization preferentemente linfo-espleno-hepática, con compromiso nervioso y dérmico. tal cual se pudo observar en animales capturados en la província dc Corrientes, Argentina. zona endémica de la enfermedad en humanos. Habiéndose reconocido Mycobacterium leprae, infectando naturalmente estos armadillos, a través de diferentes técnicas, se procesaron, por medio de la técnica de PRA. très muestras de ADN extraído de las micobacterias presentes en bazo, hígado y linfoglándula patelar de un animal naturalmente infectado obtenido dentro del PEA, y de très muestras de lepromas humanos de pacientes de la misma província. Los seis patrones logrados coinciden con los de esta especie, caracterizándose por la obtención uniforme de fragmentos característicos de M. leprae, Estos estúdios. efectuados por primera vez en Argentina, corroboran los hallazgos initiales en Sudamérica hecho por este grupo de investigation, sobre la detection de armadillos naturalmente infectados con el bacilo de Hansen.





The armadillo is a mammal of the order Edentata whose origin can be traced to the end of the Eocene epoch, some 40 million years ago, of which today there remain only three living families: Bradypodidae (sloths), Myrmecophagidae (anteaters) and Dasypodidae(armadillos). The last family includes six subfamilies. The sub­family Dasypodidae genus Dasypus com­prises D. novemcinctus, D. hybridus, D. sabanicola and D. kappleri. There are 20 species of armadillos distributed for the most part in the southeastern United States, Central and South America. It is of interest to note that armadillos occur exclusively in the Western Hemisphere (4). It is a valuable animal model for the study of leprosy because of the facility to reproduce the illness and many of the animals, captured in the U.S.A., Mexico and Argentina have been found to be naturally infected with Mycobacterium leprae(1,10,11,14-16).

Wild leprosy in the nine-banded armadillo (Dasypus novemcinctus) is characterized by the presence of multiple bacilli in the internal organs such as the lymph glands, spleen and liver with nerve and skin involvement. We could observe this in 9 out of 132 animals captured in Corrientes, Argentina, an area endemic for leprosy in humans with a prevalence of 6 per 100,000 (14,l5). M. leprae was recognized by 1982 in those naturally infected armadillos through specific stains (Ziehl-Neelsen, Fite-Faraco and King-Young), loss of the acid-fastness with pyridine extraction, positive D-dopa oxidase reaction, noncultivable on Lowenstein-Jensen and Stonebrink media, replication in the mouse foot pad (Shepard technique) (8) and reaction to the armadillo bacterin (AB), similar to the normalizedbacillary lepromin (NBL-40) in diseased persons with different forms of the illness (6).

Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA), which relies on the amplification of a 439-bp portion of the hsp65 gene present in all mycobacteria, followed by two distinct digestions (with BstEII and HaeIII) of the PCR product, offers a rapid and easy alternative that permits the rapid identification of mycobacteria without the need for specialized equipment (13,17). In the present investigation we attempted to use the same methodology to investigate if it was possible to conclusively identify M. lepraefrom two human lepromas and different tissues of the naturally infected nine-banded armadillo.

 

MATERIALS AND METHODS

DNA of M. leprae was extracted and purified from the liver, popliteal and axillary lymph nodes of a naturally infected armadillo [captured during the Experimental Program in Armadillo (PEA)|, and three samples from two human lepromas of the same region. Briefly, 0.5 g of each infected tissue was manually ground with micropestles. Once macerated it was suspended in 300 µl of buffer Tris-EDTA (TE) 1x, with 10 ul of proteinase K (PK; 10 mg/ml) and incubated overnight at 37°C. After adding 10 mg/ml lysozyme solution and incubating for 2 hr at 37°C. 100 µl of SDS 10% and 10 µl of PK were added and incubated 15 min at 65°C. Two extractions of the same volume with phenol-chloroform-isoamyl alcohol (24-24-1) were carried out and one with chloroform-isoamyl alcohol (24-1). Then, 100 pi of NaCl 5 M was added and precipitated overnight with absolute ethanol at -20°C. It was centrifuged at 12,000 g x 15 min and the supernatant was discarded. After two washes with ethanol 70%, it was resuspended in 40 jaI of buffer TE lx. It was used as 1 µl of pure DNA for PCR. A fragment of 439 bp was amplified with the primers Tbl 1 and Tbl2 by the procedure described by Telenti, et al. (I7), and the amplification product was subjected to BstEII and Hae\II enzyme digestions. The fragments obtained were separated by electrofocusing on 3% agarose plus ethidium bromide and visualized under UV. A 50-bp DNA ladder (Gibco BRL, Gaithersburg, Maryland, U.S.A.) was used as a molecular weight marker. DNA fragments were also amplified from M. scrofulaceum, M. szul- gai, M. avium and M. bovis.

 

The Figure PRA gel after digestion with BstEII (lanes I to 11) and HaeIII (lanes 12 to 22) of439-bp DNA fragments amplified from M. scrofulaceum (lanes I and 12), M. szulgai (lanes 2 and 13), M. avium (lanes 3 and 14), M. bovis (lanes 4 and 15) and M. leprae from armadillo (lanes 6, 7, 10, 17, 18 and 21 ) and from human lepromas (lanes 5, 8, 9, 16, 19 and 20): molecular weight marker (lanes 11 and 22).

 

RESULTS

The results of the PRA are shown in The Figure. The patterns of the six samples of lepromatous tissues analyzed were identical and coincident with those of M. leprae (9,13) that are characterized by uniformly obtaining two fragments of 315 and 135 bp with the digestion with BstEII and two fragments of 265 and 130 bp with the digestion with HaeIII that agree with the published sequences of the antigen of 65-kDa.

 

DISCUSSION

Since M. leprae are noncultivable mycobacteria, the diagnosis of leprosy is based on the demonstration of at least two of the following: characteristic lesions of the skin, loss of sensibility, nerve involvement or the presence of acid-fast bacilli in smears of skin lesions (13). In this context, the quick diagnosis by molecular biology methods has renovated the interest in scientific groups that have described a variety of techniques, such as amplification of M. leprae-specific repetitive sequences (19), in situ hybridization (2), nested-primer gene amplification of a 347-bp product from a bacterial genomic library (l2), amplification of the gene that encodes an antigen of 36-kDa size (7), PCR based on the selective amplification of a 530-bp fragment of the gene encoding the proline-rich antigen of M. leprae C), a specific 360-bp DNA-probe encoding 80% of the 18-kDa protein gene of M. leprae (5), or PCR using a unique sequence of 16S ribosomal RNA (3).

We conclude that the PRA method is relatively simple and that it was able to compare a variety of mycobacterial species in a single experiment, allowing the conclusive identification of M. leprae. These studies, made for the first time in Argentina, corroborate the initial discoveries in South America made by our investigative group on the detection of naturally infected armadillos with the Hansen's bacillus (8,14,15) The similar reaction of the comparative test made in humans with armadillo bacterin (AB) obtained from this naturally infected animal and bacillary lepromin (LBN-40) confirm that it was always natural or wild leprosy acquired by this animal from the environment (6).

Acknowledgment. We are grateful to M. V. Juan Carlos Sampietro for his technical assistance.

 

REFERENCES

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1. Instituto de Biotecnologia (CICV-INTA). Castelar. Buenos Aires. Argentina.
2. Programa Experimental en Armadillo (PEA). Facultad de Ciências Veterinárias (FCV/UNNE), Sgto. Cabral 2.139-3400, Corrientes, Argentina.
3. Programa Experimental en Armadillo (PEA). Facultad de Ciências Veterinárias (FCV/UNNE), Sgto. Cabral 2.139-3400, Corrientes, Argentina.
4. Programa Experimental en Armadillo (PEA). Facultad de Ciências Veterinárias (FCV/UNNE), Sgto. Cabral 2.139-3400, Corrientes, Argentina.
5. Programa Experimental en Armadillo (PEA). Facultad de Ciências Veterinárias (FCV/UNNE), Sgto. Cabral 2.139-3400, Corrientes, Argentina.
6. Programa Experimental en Armadillo (PEA). Facultad de Ciências Veterinárias (FCV/UNNE), Sgto. Cabral 2.139-3400, Corrientes, Argentina.
7. Instituto de Biotecnologia (CICV-INTA). Castelar. NBuenos Aires. Argentina.
8. Instituto de Biotecnologia (CICV-INTA). Castelar. NBuenos Aires. Argentina.
9. Instituto de Biotecnologia (CICV-INTA). Castelar. NBuenos Aires. Argentina.
10. Instituto de Biotecnologia (CICV-INTA). Castelar. NBuenos Aires. Argentina.
11. Instituto de Biotecnologia (CICV-INTA). Castelar. NBuenos Aires. Argentina.
12. Instituto de Biotecnologia (CICV-INTA). Castelar. NBuenos Aires. Argentina.

Reprint requests to Prof. Maria Eleita Cicuta at the above address or FAX 54-3783-425753; e-mail: cicuta@vet.unne.edu.ar

Received for publication on 27 November 2000.
Accepted for publication in revised form ou 13 February 2001.

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