• Volume 70 , Number 4
  • Page: 245–9

Dosage and site of entry influence growth and dissemination of Mycobacterium leprae in T900r mice

Gigi J. Ebenezer1; Shantha Arumugam2; Charles K. Job3




ABSTRACT

In the present study, we examined profiles of the interaction of Mycobacterium leprae and Mycobacterium avium complex (MAC) with murine peritoneal macrophages (MΦS) in terms of up-regulation of MΦ expression of proinflammatory and im-munosuppressing cytolines (CKs) after infection. First, the reverse transcription polymerase chain reaction (RT-PCR) assay revealed that both MAC and M. leprae infections up-regulated MΦ mRNA expression IL-12. TNF-α, IL-10, and transforming growth factor-β (TGF-β). except that M. leprae-infected MΦs showed no increase in the IL-12 mRNA expression. Second, the RT-PCR assay also showed some differences between M. leprae- and MAC-infected MΦs with respect to the modes of IL-10 and inducible nitric oxide synthase (iNOS) mRNA expression. That is MAC, but not M. leprae, infection caused a prolonged increase in the expression of IL-10 and iNOS mRNAs. Third, a ribonuclease protection assay revealed that MΦs co-infected with MAC and M. leprae showed the IL-12. TNF-α and IL-10 mRNA expression in an intermediate mode of those of MΦs infected with either M. leprae or MAC alone. This implies that the CK expression of M. leprae-infected MΦs may be modified by co-infection with MAC. These findings may suggest differential interactions of M. leprae and MAC organisms with murine peritoneal MΦs in terms of the activation of signal transduction pathways for expression of some kinds of im-munoregulatory cytokines and immuno-protective enzymes.



RÉSUMÉ

Le rôle, pour l'obtention de lésions lépreuses, de la dose d'inoculum ansi que de I'environnement au site d'inoculation de Mycobacterium leprae, lut examiné en utilisant des souris hautement susceptibles, im-niunologiqnement supprimées de souche T900r. Des doses de 107, 106, 105 et 104 furent inoculées, soit dans les flancs, soit dans la plante des pieds de deux différents groupes de souris. Des biopsies des sites d'inoculation furent réalisées pour évaluer les lésions histologiques et pour compter les bactéries après 6, 8 et 12 mois. M. leprae se sont multipliées localement et se sont disséminées à d'autres parties du corps à toutes les concentrations chez les souris infectées dans la plante des pieds, site à une température de 31 C. Chez les souris inoculées aux flancs à une température de 37C, une multiplication ne fut enregistrée que lorsque les doses de M. leprae étaient élevées et aucune évidence de dissémination ne lut détectée dans ce groupe. La température au site d'entrée et la dose de M. leprae infectieuse pourraient jouer un rôle important dans le développement de la lèpre chez les individus sensibles exposés à M. leprae.



RESUMEN

Se investigó el efecto de la dosis de Mycobacterium leprae y el papel del microambiente en el sitio de inoculación, en la producción de lesiones de la lepra en los ratones T900r inmunológicamente suprimidos y altamente susceptibles. Se inocularon varias dosis de M. leprae ( 107, 106, 105 y 104) tanto en los flancos como en las almohadillas plantares de dos grupos diferentes de ratones. Los sitios de inoculación fueron muestreados para su examen histopatológico y para la cuenta de bacilos al final de 6, 8 y 12 meses de infección. Los bacilos inoculados en la almohadilla plantar (con una temperatura de 30C) se multiplicaron a todas las dosis probadas en el sitio de inoculación y se diseminaron a otras partes del cuerpo. En contraste, en los animales inoculados en los flancos (con una temperatura de 37C), la multiplicación ocurrió solo con las dosis altas de bacilos y no hubo diseminación de los mismos a ninguna concentración probada. Se concluye que la temperatura en el sitio de entrada y la dosis infectante de M. leprae, pueden jugar un papel importante en el desarrollo de la lepra en los individuos susceptibles, expuestos al bacilo.





Many studies have reasonably documented that a large number of bacilli are shed and dispersed from the nose, mouth and ulcerating skin lesions of untreated lep-romatous patients (2,4,5,6,10), but the route of entry of M. leprae, evolution of the type of disease, dissemination of the disease, and its transmission in the community is still not very clear. Several carefully controlled experimental studies on animal models have been done and these experiments in nude mice have shown that M. leprae can be transmitted through broken nasal mucosa(3,9) and abraded skin (8). Our preliminary study to infect intact skin on the flanks of T900r mice, an area with a temperature relatively higher than that of feet, was not successful (5). To further substantiate this finding and to determine whether there was a difference in the growth and dissemination of organisms when M. leprae were inoculated into two different sites (footpad and Hank), a comprehensive study was done and the outcomes are presented.

 

MATERIALS AND METHODS

A group of sixty-eight CBA mice were thymectomized at six to seven weeks of age. Three weeks later they were irradiated with 900 rad and a syngeneic bone-marrow cell transfusion as administered intraperi-toneally (11). These immunodeficient mice (T9()()r mice) were now ready for inoculation with M. leprae. M. leprae suspensions were obtained from the lepromatous nodules that developed on the footpads of T9()()r mouse previously inoculated with M. leprae. The mice were randomized, 36 were inoculated intradermal! v in both the flanks and the other 32 in both the footpads. In each ol these two groups four different concentrations of M. leprae inoculations were used. Inocula of M. Leprae containing l() . 10". 10s and \(Y in 0.1 ml were used In the flank group of animals. In the footpad group of animals, die different concentrations were diluted in 0.03 ml for inoculation. The animals were fed with standard food pellets and unlimited water.

One mouse from each M. leprae concentration group was sacrificed at the 6th. 8th and 12th month. All die remaining animals were harvested at the 15th month. The skin along with the subcutaneous tissue and the muscle at the injected area on both sides were dissected. Tissues from the left Hanks were harvested and the AFB were counted. Tissues from the right flanks were immediately fixed in 10% buffered formalin and sent for histopathological studies. Sections of 4 um thickness were made and stained with hematoxylin and eosin (H&E) and modified Fite Faraco stain (7). Autopsies were done on mice sacrificed at the end of the 15th month and clippings from the ear lobes and internal organs, such as the liver, lung, kidney, heart, and spleen, were subjected to histopathological examination. Similar techniques were followed for animals inoculated in the footpad. It was not possible to do an autopsy on one animal in the 104 mouse footpad group because the sacrificed animal was inadvertently discarded.

 

RESULTS

The M. leprae smear counts comparing both groups at different periods of time is given in Table 1. The harvested count showed that 104 in 0.1 ml inoculum did not promote growth in the mice injected in the flank, but growth was seen in all of the mice that were inoculated in the footpad with this concentration of M. leprae. In all other M. leprae concentration groups, there was growth of M. leprae, but it was quantitatively more in the footpad-inoculated animals than in the flank-inoculated animals. In the footpad group of animals, the multiplication was almost double that of the flank group of animals when the inoculation count was 105 and 106, but with the highest concentration of 107, this was not so.

 

 

Table 2 shows organs where Mycobacterium leprae had disseminated. A histopathological examination of the skin from the  flank-inoculated group of animals showed focal granulomas occupying the dermis and subcutaneous tissues. The granulomas consisted of foamy macrophages, some with granular cytoplasm, and few lymphocytes (Fig. 1 ). Acid-fast stain showed macrophages containing bacilli that were mostly fragmented and granular. The ears, liver, spleen, heart, kidney showed no significant lesions, except small nonspecific mononuclear cellular inflammatory infiltration of the liver, lungs and kidneys. Dissemination of bacilli was not seen in this group of animals.

 

 

Fig. 1 . Photomicrograph to show skin from the flanks with a subcutaneous granuloma composed of macrophages (H&E x80).

 

A histopathological examination of footpad-inoculated mice showed granulomas in the footpads in till the animals regardless of the M. leprae concentration (Fig. 2). Granulomas consisting of macrophages filled with bacilli were consistently seen at the 6th, 8th, 12th and 15th month. Autopsied tissue revealed extensive dissemination of acid-fast bacilli (AFB) forming microgran-ulomas in ears (Fig. 3). The bacillary index of the granuloma (BIG) was 4+ (Fig. 4). The lung and liver showed scattered AFB inside the macrophages.

 

Fig. 2. Photomicrograph showing a large subepilhelial granuloma in the footpads composed almost entirely of macrophage (H&E x160).

 

Fig. 3. Photomicrograph of cross-section of ear showing small collections of macrophages forming microgranulomas situated above the cartilage (H&E x80).

 

Fig. 4. Photomicrograph showing clumps of acid-fast bacilli (AFB) in macrophages shown in Fig. 3 (modified Fite Faraco stain x800).

 

DISCUSSION

When M. leprae, in differing concentrations, were injected inlradermally into the Hank skin of the mice, they were taken up by macrophages and aggregated at the site of inoculation (Fig. 1). However, these bacilli were found to be granulated and fragmented signifying degenerated bacteria. The smear count of bacilli was either less than that inoculated or zero when the concentration of M. leprae in the inoculum was the lowest. These features demonstrate that there was no multiplication of bacilli at the flank site. This could be due to the relative increased warmth of the flank (13) compared to the footpad (12). Injection of the M. leprae inoculum into the mouse footpad showed more than a doubling of the organism, and the organisms themselves did not display the fragmentation that was seen at the flank site. This clearly demonstrates the multiplication of M. leprae in the mouse footpad. However the multiplication was not uniform in all of the concentrations. The lowest (104) and the highest (107) inoculum did not demonstrate a high growth rate like that found with the 104 and 105 inoculum. This again clearly demonstrates that optimal levels of inoculum exist that promote growth of the organism in the mouse footpad and inoculating more bacilli than this optimal load does not yield better results.

Dissemination of M. leprae to the internal organs as seen in the footpad-inoculated animals was completely absent in the flank-inoculated mice. Multiplication at the site of entry and dissemination to the internal organs, when the inoculum is injected into the mouse footpad and when no growth or dissemination occurs when injected into the flank, demonstrates that local environmental changes, such as relatively cooler temperatures and an optimum dose of M. leprae at the site of entry, are conducive factors for the development of the disease.

In a recent study, it was found that a majority of the leprosy mono-lesions were located in the uncovered parts of the body with a special predilection for the posterior aspects of the lower extremities (1). In children, these sites are most vulnerable to trauma. They are also relatively cooler, and our animal study demonstrates that M. leprae multiply better in cooler parts, such as the footpad, and disseminate more to the ear lobes. This may explain why the first and early lesions occur in the extremities and face where the temperature is low. In mice, the entry of M. leprae through a relatively cooler entry point (i.e.. the footpad) allows better growth of M. leprae locally, needs smaller doses of inoculum to promote growth, and allows dissemination of the organism to the internal organs. The site of entry and the dose of M. leprae may have a role in determining whether the person will be infected or not.

 

REFERENCES

1. Abraham, S., Manimozhi, N., Joseph, G. A.. Kurian, N.. Rao. P. S. S. S. and Job, C. K. Epidemiological significance of first skin lesion in leprosy. Int. J. Lepr. 66 (1998) 131-138.

2. Barton. R. P. E. A clinical study of the nose in lepromatous leprosy. Lepr. Rev. 45 (1974) 135-144.

3. Chehl, S.. Job, C. K. and Hastings, R. C. Transmission of leprosy in nude mice. Am. J. Trop. Med. Hyg. 34 (1985) 1161-1166.

4. Davey. T. F. and Rees, R. J. W. The nasal discharge in leprosy: clinical and bacteriological aspects. Lepr. Rev. 45 (1964) 121-134.

5. Ebenezer. G. J., Akumucam, S. and Job, C. K. Infection by M. leprae is governed by the temperature at the entry point: a preliminary note. Int. J. Lepr. 67(1999) 162-164.

6. Hubsctier, S., Gridhar. B. K. and Desikan, K. V. Discharge of Mycobacterium leprae from the mouth in lepromatous leprosy patients. Lepr. Rev. 50(1979)45-50.

7. Job, C. K. and Chacko, C. J. G. A modified Fite's stain for demonstration of M. leprae in tissue sections. Indian J. Lepr. 58 (1986) 17-18.

8. Job, C. K., Chehl, S. and Hastings, R. C. New finding on the mode entry of M. leprae in nude mice. hit. J. Lepr. 58 (1990) 726-729.

9. McDermott-Lancaster. R. D. and McDougall. A. C. Mode of transmission of M. leprae in nude mice. Int. J. Exp. Path. 71 (1990) 689-700.

10. McDougall, A. C. and Rees. R. J. W. Ulcerating lepromatous leprosy in a patient with dapsone resistant Mycobacterium leprae. Lepr. Rev. 44 (1973)59-64.

11. Shantha, A., Job. C. K. and Ebenezer. G. J. Preparation of thymectomized and irradiated T900r mice: a modification. Indian J. Lepr. 69 (1997) 200-201.

12. Shepard. C. C. Temperature optimum of Mycobacterium leprae in mice. J. Baeteriol. 90 (1965) 1271-1275.

13. Short, D. J. and Woodnott. D. P. Measurement of temperature and humidity. In: The IAT Manual of Laboratory Animal Practice and Techniques. 2nd edn. London: Crosby Lockwood & Son Ltd. 1969, pp. 70-71.

 

 

 

 

 

 

 

 

 

 

1. M.D.. Head. Department of Hislopathology and Experimental Pathology.
2. B.Sc. Senior Laboratory Technician. Grade II. Department of Experimental Pathology
3. M.D.. FRC Path, EAMS, Emeritus Scientist. Schieffelin Leprosy Research and Training Center, Kurigiri, Vellore, District, Tamilnadu, India 632106.

Reprint requests to:
Dr. Gigi Ebenezer
Head. Department of Hislopalhology and Experimental Pathology, Schieffelin Leprosy Research & Training Center
Kurigi, Vellore, District
Tamilnadu, India 632106

Received for publication on 3 July 2002.
Accepted for publication on 7 October 2002.

2020 © International Journal of Leprosy and other Mycobacterial Diseases all right reaserved GN1