Mierob Ecol (1989) 17:105-110

MICROBIAL ECOLOGY 9 Springer-Verlag New York Inc. 1989

Earthworms Near Leprosy Patients' Homes Are Negative for Acid-Fast Bacilli by Fite Stain, Providing No Link Between Leprous Armadillos (Dasypus novemcinctus) and Human Leprosy Leslie A. Blake, t Burton C. West, ~ C y n t h i a H. Lary,* Marjorie E. R. Fowler, 2 a n d J o h n R. T o d d t XSecfionof Infectious Diseases, Department of Medicine and the 2Department of Pathology, Louisiana State University School of Medicine, Shreveport, Louisiana 71130, USA

Abstract.

Enzootic leprosy has been recognized in a r m a d i l l o s in L o u i s i a n a since 1975. C o n t a c t with a r m a d i l l o s is being assessed as a risk factor for leprosy in three white w o m e n , lifelong residents o f separate rural areas in northern Louisiana, which is a region w i t h o u t e n d e m i c leprosy. N o n e has had any k n o w n exposure to h u m a n leprosy. Each was aware o f a r m a d i l l o s (Dasypus novemcinctus) near or u n d e r her h o m e for decades. In considering possible e n v i r o n m e n t a l sources for Mycobacterium leprae, we o b s e r v e d that all three had e a r t h w o r m growth areas for fishing bait where soil was kept m o i s t n e a r their homes. T h e w o r m s attracted armadillos. Since armadillos subsist on w o r m s , grubs, a n d insects and because o f the c o m m o n feature o f a " ' w o r m f a r m " near each h o m e , we reasoned that e a r t h w o r m s might contain M. leprae and be part o f a cycle i n v o l v i n g the a r m a d i l l o a n d h u m a n beings. W o r m s f r o m each h o m e w o r m f a r m were studied. One site was s a m p l e d twice at patient l ' s h o m e , five sites were s a m p l e d once at patient 2's h o m e , a n d three sites were s a m p l e d once at patient 3's h o m e . A sample consisted o f 3 - 4 w o r m s , which were washed, purged, fixed live in 10% formalin, e m b e d d e d in paraffin, sectioned, and stained with the Fire stain. Each was sagittally sectioned a n d e x a m i n e d by three i n d e p e n d e n t observers. N o acid-fast bacilli or other acid-fast structures were identified. We conclude that it is unlikely that e a r t h w o r m s are an e n v i r o n m e n t a l SOurce or reservoir o f M. leprae.

Introduction EnZootic leprosy was described in L o u i s i a n a armadillos in 1975 [17]. It has been found in 4.6% o f a r m a d i l l o s on the T e x a s gulf coast and up to 29.6% in northern Louisiana, as it has been m o r e fully characterized in these two states [6, 13, 16]. A leprous Mexican a r m a d i l l o has recently been d i s c o v e r e d [ 1]. Like

Present address: Division of Infectious Diseases and Immunology, Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72202.

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Fig. 1. This map of the of Louisiana illustrates the location of the homes of the three women with autochthonous leprosy. All are in the northern, nonendemic part of the state. State

so m a n y aspects o f leprosy, these fascinating observations are not fully understood because Mycobacterium leprae has never been cultivated in vitro. Practical, but c u m b e r s o m e nonculture methods, especially histochernical ones, are required for its investigation, making leprosy unique a m o n g h u m a n infections. Contact, broadly defined and no longer thought to require skin contact, has been considered the m e t h o d o f transmission o f leprosy from an infected person to a susceptible one. Other sources o f leprosy have not been proven, but are now being considered, Seven armadillo handlers have gotten leprosy [5, 9, 10], H u m a n beings are thought to be infected by leprous armadillos through physical contact, e.g., wrestling, involving breaks in the skin or inhalation o f infectious droplets. A risk for acquiring leprosy from armadillo exposure or an intermediate or c o m m o n reservoir is gradually being established [2, 14, 18]. Transmission via fomites, dust, water, soil, plants, or insects is a n o t h e r possibility

[21. Armadillos are exquisitely susceptible to M. leprae and they develop disease with m a n y similarities to h u m a n leprosy. Armadillo inoculation for leprosy research began in 1968, and for a time it was theorized that these armadillos were responsible for the natural infection in armadillos, However, recent work using armadillo serum collected between 1961 and 1964 showed antibodies to the species-specific phenolic glycolipid 1 (PGL-1), a c o m p o n e n t o f the cell wall o f M . leprae, demonstrating that armadillos were infected in Louisiana at least as early as 1961 [ 15]. Although contact with infected h u m a n beings is a possible origin for armadillo infection, an e n v i r o n m e n t a l reservoir has been suspected. In addition, recently (1) an organism indistinguishable from M. lepraeby animal inoculation and microbiologic tests was isolated from sphagnum moss in Norway and water in the i v o r y Coast [7]; (2) in Bombay, mycobacterial organisms isolated from soil by mouse foot pad injection and tested for the M. leprae specific P G L - I were positive [8]; and (3) soil from armadillo burrows in Louisiana was positive for PGL-1 [2]. E n v i r o n m e n t a l sources are compatible with epidemioiogic aspects o f h u m a n leprosy, since it exhibits a rural distribution, a male predominance, and is sometimes thought to be inoculated into broken

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skin o n t h e d i s t a l l o w e r e x t r e m i t i e s t h r o u g h t r a u m a [2]. S u c h p o t e n t i a l e n v i ronmental sources are relevant to armadillo infection, since armadillos subsist on W o r m s a n d g r u b s a n d o b t a i n t h e i r m e a l s b y b u r r o w i n g i n t o t h e g r o u n d . R e c e n t c a s e s o f l e p r o s y in o u r c a r e a r e t h o u g h t t o b e a u t o c h t h o n o u s b e c a u s e leprosy is e s s e n t i a l l y n o t e n d e m i c in n o r t h e r n L o u i s i a n a a n d n o n e o f t h e p a t i e n t s has b e e n e x p o s e d t o l e p r o s y o r o t h e r k n o w n r i s k f a c t o r s e x c e p t a r m a d i l l o s [ 18]. The t h r e e w o m e n r e p o r t e d h e r e all h a d m o i s t p a t c h e s o f s o i l u s e d a s w o r m farms n e a r t h e i r h o m e s . A l t h o u g h t h e y d e n i e d d i r e c t c o n t a c t w i t h a r m a d i l l o s , in e a c h c a s e a r m a d i l l o s h a d f r e q u e n t l y b e e n s e e n b u r r o w i n g a n d r o o t i n g i n t h e Worm f a r m s . E a c h p a t i e n t s p e n t a g r e a t d e a l o f t i m e o u t d o o r s , o f t e n w a l k i n g b a r e f o o t . E a c h r e p o r t e d t h a t a r m a d i l l o s w o u l d g o u n d e r h e r h o m e t o dig. T h u s , the w o r m f a r m s a p p e a r e d t o b e p o t e n t i a l s i t e s o f c o n t a c t a m o n g a r m a d i l l o s , WOrms, a n d h u m a n b e i n g s . W e c o n s i d e r e d t h a t w o r m s , as a r m a d i l l o f o o d s t u f f s , m i g h t b e a n i n t e r m e d i a t e in t h e t r a n s m i s s i o n o f l e p r o s y .

Materials and Methods Patients The three patients were women, living in separate rural homes at least 100 km from the nearest .Other case (Fig. 1). Case 1 was diagnosed in 1983, case 2 in 1985, and case 3 in 1986; all were ~rnproving on the treatment for leprosy.

"Worm Farms" Each home had one or more areas of soil intentionally kept moist to cultivate earthworms, which Were used for fishing bait. Each home was visited and earthworms of the family Lumbricidae were Obtained.

Case 1. Worms were taken from one site on two separate occasions. This soil was shielded by a Piece of plywood which trapped kitchen runoff water, providing continuous moisture and many WOrms. Armadillos were frequently seen burrowing in this area. Case 2. Worms were obtained from five sites. Two were in neighboring overgrown fields. A third Was near the spring house for a shallow spring used as the family's water supply. Several armadillo burrows were located in this immediate area. A fourth site was near the house, and the fifth was next to a large bayou, close to the home, where armadillos drank and burrowed. Armadillos favored digging underneath the house and could be heard as they scraped their carapaces on the undersurface of the floor. Case 3. Worms were sampled from three sites. Two directly adjacent to the house were subject to Water runoff and had large worm beds. Another was a wooded area where family members played. Armadillos had been observed many times rooting and burrowing in these areas. ttistopathology Three to eight worms from each site were transported in soil, washed, placed in normal saline (9 iV~ioter NaCI) for 24 hours, and purged. Three or four worms from each sample were fixed live in v0formalin, dehydrated, embedded in paratfin, and longitudinally sectioned. Samples were split

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and stained either with hematoxylin and eosin or with the Fire stain [4] as modified by rinsing instead of blotting to remove oil, and air drying instead of blotting following counterstaining with methylene blue (unpublished laboratory manual, USPHS Hospital, Carville, LA).

Results Altogether, 38 w o r m s were sectioned for study and one Fite-stained, complete longitudinal section o f each w o r m was thoroughly e x a m i n e d by three observers. N o n e was found to contain acid-fast bacilli. Positive Fite stain controls were acid-fast bacilli in the ear biopsy specimen o f a naturally infected armadillo found near case l's h o m e and skin biopsy specimens from each o f the three patients.

Discussion This study was undertaken because these cases o f sporadic leprosy, like 5070% o f patients with leprosy, have no evidence o f acquisition from another case. T h a t environmental, n o n h u m a n sources exist as reservoirs, sources of transmission, and vectors o f leprosy is implied by our cases and others [2, 14, 18]. However, our findings are that the worms collected from the vicinity of the three patients' h o m e s did not h a r b o r acid-fast bacilli, and by inference, M. leprae. Our m e t h o d o f detection using sections o f e a r t h w o r m s histochemically stained, and then painstakingly searched for acid-fast bacilli by each o f three observers, is traditional, but relatively insensitive. Culture is usually e m p l o y e d as a far m o r e sensitive m e t h o d o f detection o f microorganisms, but does not apply because M. leprae has never been cultivated in vitro. T h e most sensitive m e t h o d for detection o f M. leprae in e a r t h w o r m s would have been to inject earthworms prepared in some m a n n e r into armadillos, and then follow the injected armadillos for a prolonged period, inspecting them and periodically sacrificing some o f them, searching for the d e v e l o p m e n t o f leprosy. However, this was considered impractical. A n o t h e r alternative to the Fire stain would have been the m e a s u r e m e n t o f PGL-1 in e a r t h w o r m s in a m a n n e r similar to that applied to e n v i r o n m e n t a l soil samples with a m o n o c l o n a l antibody to PGL-1 using an enzyme-linked i m m u n o s o r b e n t assay or high pressure liquid chromatography [2, 8]. Although detection o f M. leprae by m e a s u r e m e n t o f P G L - l is m o r e sensitive than the Fite stain, its superiority in assessing ecological samples is not established. An advantage o f measuring P G L - 1 is in its specificity in distinguishing M. leprae from other e n v i r o n m e n t a l mycobacteria. Finally, concentration o f acid-fast bacilli by homogenization and differential centrifugation o f earthworms was considered, but we d i d not find or develop a m e t h o d that could concentrate acid-fast bacilli from tissue, and thus be superior to histology using the Fite stain. Our single m e t h o d o f approach, namely, the painstaking examination o f stained sections, is intrinsically limited, but we think it is sufficient for the conclusions we draw. A search o f literature concerning earthworms and mycobacteria revealed no information. Armadillo foodstuffs have been found to contain e n v i r o n m e n t a l mycobac-

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teria other than M. leprae. Armadillos themselves are often infected by environmental mycobacteria, and such organisms might serve as cofactors and predispose to superinfection with M. leprae. Arthropods in parts of the world have been shown to harbor M. leprae and have been considered potential vectors of leprosy in man [ t l]. The association of leprosy with mites, lice, and fleas earlier in this century in leprosy-endemic countries was indirectly proven by the disappearance of leprosy after the standards o f living improved and these insects were controlled [2, 3]. In addition, arthropods have been established as potential sources of leprosy, inasmuch as mosquitoes have transferred M. leprae, which subsequently grew, to mouse foot pads [ 12]. Armadillos have harbored M. leprae for at least 28 years. Their disease may be due to exposure to leprous humans or contaminated articles, but it is more likely that in the wild, armadillos could contaminate one another either directly or indirectly, e.g., through spread of infected droplets or via insect transmission. It is also possible that other nonhuman sources o f M . leprae exist, particularly in view of its rural distribution. Yet, though M. leprae has been detected in Soil, presumptively identified in water and sphagnum moss, and observed in a number of insects [2], it has not been reported in earthworms. We conclude that within the limits of the sample size and the histochemical method we Utilized, leprosy in human beings and enzootic leprosy in armadillos are not associated with acid-fast bacilli in earthworms, an armadillo food. A link between armadillo leprosy, environmental sources, and human leprosy remains to be established. Acknowledgments. This study was supported in part by the Ed E. and Gladys Hurley Foundation, and the American Leprosy Missions, Inc. This work was presented in part in New Orleans, Louisiana at the meeting of the Southern Section of the American Federation for Clinical Research, January 28, 1987 (Clin Res 1987;35: 20A). Leslie A. Blake received a Student Award from the Southern Section of the American Federation for Clinical Research for her presentation. We thank Virginia R. McBride for technical assistance and Tommie Lue Maddox for secretarial assistance.

References 1. Amezcua E, Escobar-Gutibrrez A, Storrs EE, Dhople AM, Burchfield HP (1984) Wild Mexican armadillo with leprosy-like infection [letter]. Int J Lepr 52:254-255 2. Blake LA, West BC, Lary CH, Todd JR IV 0987) Environmental non-human sources of leDrosy. Rev Infect Dis 9:562-577 3. Dungal N 0960) Is leprosy transmitted by insects? Lepr Rev 31:25-34 4. Fite GL, Cambre PJ, Turner MH (1947) Procedure for demonstrating lepra bacilli in paraffin sections. Arch Pathol 43:624-625 5. Freiberger CF, Fudenberg HH ( 198 I) An appefile for armadillo. Hosp Pract 16: ) 37, 141, 144 6. Job CK, Harris EB, Allen JL, Hastings RC (1986) A random survey of leprosy in wild ninebanded armadillos in Louisiana. Int J Lepr 54:453--457 7. Kazda j (1981) Occurrence of non-cultivable acid-fast bacteria in the environment and their relation to klycobacterium leprae. Lepr Rev 52(suppl 1):85-91 8. Kazda j, Ganapati R, Revankar C, Buchanan TM, Young DB, Irgens LM (1986) Isolation ~ Mycobacterium leprae from soil in Bombay. Lepr Rev 57(suppl 3): 201-208

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9. Lumpkin LR 1II, Cox GF, Wolf JE Jr (1983) Leprosy in five armadillo handlers. J Am Acad Dermatol 9:899-903 10. Lumpkin LR III, Cox GF, Wolf JE Jr (1984) Leprosy in armadillo handlers [letter]. J Am Acad Dermatol 10:1073 I I. Narayanan E, Shankara Mania K, Kirchheimer WF, Balasubrahmanyan M (1972) Occurrence of Mycobacwrium leprae in arthropods. Lepr Rev 43:194-198 12. Narayanan E, Sreevatsa, Kirchheimer WF, Bedi BMS (1977) Transfer of leprosy bacilli from patients to mouse foot pads by Aedes aegyptL Leprosy in India 49:181-189 13, Smith JH, Folse DS, Long EG, Christie JD, Crouse DT, Tewes ME, Gatson AM, Ehrhardt RL, File SK, Kelly MT (1983) Leprosy in wild armadillos (Dasypus novemcinctus) of the Texas Gulf Coast: Epidemiology and mycobacteriology. J Reticuloendothel Soc 34:75-88 14. Thomas DA, Mines JS, Thomas DC, Mack TM, Rea TH (1987) Armadillo exposure among Mexican-born patients with lepromatous leprosy. J Infect Dis 156:990-992 15. Truman RW, Shannon EJ, Hagstad HV, Hugh-Jones ME, Wolff A, Hastings RC (1986) Evaluation of the origin of Mycobacterium leprae infections in the wild armadillo DasypuS novemcinctus. Am J Trop Med Hyg 35:588-59316. Walsh GP, Meyers WM, Binford CH (1986) Naturally acquired leprosy in the nine-banded armadillo: A decade of experience, 1975-1985. J Leukocyte Biol 40:645-656 17. Walsh GP, Storrs EE, Burchfield HP, Cottrell EH, Vidrine MF, Binford CH (1975) Leprosylike disease occurring naturally in armadillos. J Reticuloendothel Soc 18:347-351 18. West BC, Todd JR, Lary CH, Blake LA, Fowler MER, King JW (1988) Leprosy in six isolated residents of northern Louisiana: Time-clustered cases in an essentially non-endemic area. Arch Intern Med 148:1987-1992

Earthworms near leprosy patients' homes are negative for acid-fast bacilli by fite stain, providing no link between leprous armadillos (Dasypus novemcinctus) and human leprosy.

Enzootic leprosy has been recognized in armadillos in Louisiana since 1975. Contact with armadillos is being assessed as a risk factor for leprosy in ...
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