Enumeration of Mycobacterium leprae using real-time PCR.

Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories.

Palsy of the rear limbs in Mycobacterium lepraemurium-infected mice results from bone damage and not from nerve involvement.

A small but relatively constant proportion (3-5%) of mice chronically infected with Mycobacterium lepraemurium (MLM) develops bilateral paralysis of the rear limbs. The aim of the study was to investigate whether or not the bilateral leg palsy results from nerve involvement. Direct bacterial nerve infection or acute/delayed inflammation might possibly affect the nerves. Therefore, palsied animals were investigated for the presence of: (a) histopathological changes in the leg tissues including nerves, bones and annexes, and (b) serum antibodies to M. lepraemurium and M. leprae lipids, including phenolic glycolipid I from M. leprae. Histopathological study of the palsied legs revealed that the paralysis was not the result of direct involvement of the limb nerves, as neither bacilli nor inflammatory cells were observed in the nerve branches studied. Antibodies to brain lipids and cardiolipin were not detected in the serum of the palsied animals, thus ruling out an immune response to self-lipids as the basis for the paralysis. Although high levels of antibodies to MLM lipids were detected in the serum of palsied animals they were not related to limb paralysis, as the nerves of the palsied legs showed no evidence of inflammatory damage. In fact, nerves showed no evidence of damage. Paralysis resulted from severe damage of the leg bones. Within the bones the bone marrow became replaced by extended bacilli-laden granulomas that frequently eroded the bone wall, altering the normal architecture of the bone and its annexes, namely muscle, tendons and connective tissue. Although this study rules out definitively the infectious or inflammatory damage of nerves in murine leprosy, it opens a new avenue of research into the factors that participate in the involvement or the sparing of nerves in human and murine leprosy, respectively.

[In vitro and in vivo activities of newly synthesized fluoroquinolones WQ-3345 and WQ-3402 against Mycobacterium leprae].

Activities of newly synthesized fluoroquinolnes WQ-3345 and WQ-3402 against M. leprae were measured by using the Buddemeyer method. The % inhibition of the examined drugs for M. leprae was in the order of RFP > WQ-3402 > SPFX > GFLX > WQ-3345 > LVFX. The anti-M. leprae activity of WQ-3402 was found to be strongest in these five fluoroquinolones when examined by this method, and the activity of WQ-3345 was weaker than that of GFLX. The anti-M. leprae activities of WQ-3345 and WQ-3402 were measured by a mouse footpad method using nude mice. The inhibitory effects on the growth of M. leprae inoculated into the footpads were found to be incomplete after orally administered with WQ-3345 or WQ-3402 respectively at dosages of 10 and 20 mg/kg, and the incomplete inhibition was again was again found even at a dosage of 30, 40, or 50 mg/kg im the latter.

Bactericidal action of oral ampicillin/sulbactam against Mycobacterium leprae.

We reported previously that an injectable form of ampicillin/sulbactam, Unasyn, was bactericidal to Mycobacterium leprae multiplying in mouse foot pads. In this study, we examined the effect of an orally active form of ampicillin/sulbactam, Sultamicillin, on the growth of M. leprae in mice. Three concentrations of the drug, mixed with the feed, were administered from the start until the mice were killed at 6 months; 0.01% of the drug inhibited bacterial growth by 54%, 0.10% by 74% and 0.20% by 93%. To test whether oral ampicillin/sulbactam was bactericidal, 0.50% of the drug, mixed with the feed, was administered to experimentally infected mice for 3 months during the logarithmic phase of bacterial growth, and then discontinued; multiplication of the bacilli was monitored monthly for the next 8 months. The results showed that orally active ampicillin/sulbactam is bactericidal to M. leprae.

Hansen type I disk disease at T1-2 in a dachshund.

A 7-year-old Dachshund was presented with chronic left thoracic limb lameness and acute neurological deficits to the hind limbs following trauma. A lesion was suspected between C7 and T2 on the basis of neurological examinations. Radiography and myelography identified a calcified intervertebral disk at C7-T1 and an extradural unilateral compressive lesion at T1-2. Computed tomography scans of the cranial thoracic spine revealed extrusion of disk material from the T1-2 intervertebral space resulting in marked spinal cord compression. Intervertebral disk disease is rarely reported at this location. The neurological condition deteriorated after a second myelogram, which was done to examine the thoracolumbar spine. A modified dorsal decompression of T1-2 was performed. The dog was euthanased due to further neurological deterioration 8 days after surgery.

Leprosy in hypertensive nude rats (SHR/NCrj-rnu).

Since more than a decade ago, we have attempted to develop spontaneously hypertensive rats carrying the nude gene that permits high multiplication of Mycobacterium leprae. A congenic strain carrying nude (rnu) and hypertensive genes was produced using SHR/NCrj females and F344/NJcl-rnu males. Cross-intercross was carried out 12 times to establish the hypertensive nude rat congenic strain. As a result of the genetic monitoring test with NE12F2 generation rats, the genetic profile of the SHR/NCrj-rnu rats was the same as that of the SHR/NCrj rats except for the rnu gene. We have successfully developed a hypertensive congenic nude rat strain (SHR.F344Hfh11; SHR/NCrj-rnu). An increase in the blood pressure in nude rats was found to begin at a slightly delayed age when compared with their hairy litter mates. Both female and male rats showed the highest blood pressure at approximately 20 weeks of age--166 +/- 1.4 and 197 +/- 11 mm Hg in nude rats and 175 +/- 11 and 193 +/- 3.2 mm Hg in their hairy litter mates in female and male rats, respectively. In the present study, comparisons were made on the susceptibility to M. leprae in hypertensive SHR/NCrj-rnu and normotensive F344/NJcl-rnu rats. We have reconfirmed that hypertensive SHR/NCrj-rnu rats of the NE12F3 generation were highly susceptible to M. leprae. In the SHR/NCrj-rnu rats of both sexes excellent massive swelling due to multiplication of M. leprae was observed and, also, nodular lesions were produced in uninoculated fore feet and lips while those sites in the F344/NJcl-rnu rats showed only a slight swelling of the inoculated feet with mild nodular lesions. Although mild lymphocyte proliferation was seen only in the M. leprae-inoculated site with numerous bacilli and partial necrosis in the SHR/NCrj-rnu rats, at noninoculated sites, multiplication of M. leprae was only observed in the cells of the mononuclear phagocyte system. However, in F344/NJcl-rnu rats, lymphocyte proliferation with a few neutrophils was seen at the site of inoculated hind foot pads and everywhere at the site of multiplication of M. leprae. There was a wide difference in the susceptibility to M. leprae between the SHR/NCrj-rnu and the F344/NJcl-rnu rats.

Perfused rat hindlimb is suitable for skeletal muscle glucose transport measurements.

It has been postulated that the perfused rat hindlimb is unsuitable for measurements of muscle glucose transport [P. Hansen, E. Gulve, J. Gao, J. Schluter, M. Mueckler, and J. Holloszy. Am. J. Physiol. 268 (Cell Physiol. 37): C30-C35, 1995]. The aim of the present study was therefore to critically evaluate the suitability of this preparation for glucose transport measurements using the extracellular marker mannitol and the glucose analogs 3-O-methyl-D-glucose or 2-deoxy-D-glucose. In all three muscle fiber types studied, the rate of 2-deoxy-D-glucose uptake during perfusion was linear from 1 to 40 min during maximal insulin stimulation and from 1 to 15 min during maximal electrical stimulation. Uptake of 2-deoxy-D-glucose was not increased by an increase in perfusate flow. Combined stimulation with a maximal insulin concentration and electrical stimulation elicited additive effects on 2-deoxy-D-glucose uptake in slow- and fast-twitch oxidative but not in fast-twitch glycolytic muscle fibers. Furthermore, in muscles having high glucose transport capacities 3-O-methyl-D-glucose is less suitable than 2-deoxy-D-glucose because of rapidly developing nonlinearity of accumulation. Our findings clearly demonstrate that the perfused hindlimb is suitable for measurements of muscle glucose transport and that the most feasible glucose analog for this purpose is 2-deoxy-D-glucose.

Bactericidal activities of single or multiple doses of various combinations of new antileprosy drugs and/or rifampin against M. leprae in mice.

The bactericidal activities against Mycobacterium leprae of single or multiple doses of various combinations of new antileprosy drugs [minocycline (MINO), clarithromycin (CLARI), ofloxacin (OFLO), and sparfloxacin (SPFX)] and/or rifampin (RMP) were titrated in immunocompetent mice by the proportional bactericidal method. Drugs were administered by gavage at the following dosages (mg/kg) per dose: RMP 10, MINO 25, CLARI 100, OFLO 150, and SPFX 50. All 15 regimens exerted significant bactericidal activities, at least 96% of viables were killed. The activity of a single dose MINO + CLARI was only slightly inferior to that of RMP, and the activities of a single dose OFLO/SPFX + MINO + CLARI were similar to that of RMP. This suggests that either MINO + CLARI or OFLO/SPFX + MINO + CLARI may be administered once monthly together with RMP 600 mg for the treatment of multibacillary (MB) leprosy, and monthly administration of MINO + CLARI or OFLO/SPFX + MINO + CLARI may also be employed for the treatment of RMP-resistant MB leprosy. Because the killing effects of multiple doses of the combinations were so powerful, comparison of the bactericidal activities of these regimens was beyond the sensitivity of the immunocompetent mouse model, and are being tested in the nude mouse model. Although SPFX is more active against M. leprae than OFLO on a weight-to-weight basis, when both drugs were administered in mice at dosages equivalent to clinically tolerated dosages in humans, SPFX did not show more superiority than OFLO, and its real advantage over OFLO in the treatment of leprosy remains unclear.

Adoptive cell transfer of resistance to Mycobacterium leprae infections in mice.

Cells were transferred from mice intradermally vaccinated with killed Mycobacterium leprae to sublethally irradiated recipients. Unseparated cells from lymph nodes or spleens of M. leprae vaccinated mice were found to cause significant inhibition of the growth of a subsequent M. leprae challenge in mouse footpads for up to 26 weeks after vaccination. Vaccination with live BCG and cells transferred from BCG-vaccinated mice caused no significant inhibition of M. leprae growth in mouse footpads. Cell separation into fractions containing predominantly B and T lymphocytes showed that the inhibition of growth was due to M. leprae-sensitized T lymphocytes. M. leprae vaccinated mice were also skin tested with soluble M. leprae antigen and showed maximum delayed hypersensitivity responses 4 weeks after vaccination.

Dapsone resistance in North India.

An attempt has been made to screen the resistant strains of M. leprae in lepromatous patients from the eight northern States of India. By using the mouse foot pad technique it was found that in a total of 69 clinically suspected patients 33 (47.8%) harboured M. leprae with some degree of dapsone resistance. A detailed epidemiological study in these parts of the country may reveal the prevalence rate.

Experimental lepromatous leprosy in the white-handed gibbon (Hylobatus lar): successful inoculation with leprosy bacilli of human origin.

Leprosy bacilli of human origin were inoculated into a white-handed gibbon by the i.v. and i.p. routes, and also locally into ears, testis and around an ulnar nerve. The animal was observed closely during a period of nearly 15 years and did not exhibit any clinical evidence of cutaneous or neurological disease. At death, a wide range of tissues was taken for bacterial counts and histological examination, and a disseminated and progressive infection was demonstrated. Acid-fast bacilli were found in many sites; their morphological appearance distribution in nerves, and pattern of multiplication in mouse foot-pads, and also the presence of anti-mycobacterial antibody in the serum and the absence of specific lymphocyte transformation were all in keeping with an infection by Mycobacterium leprae, at an early lepromatous stage. This is probably the first fully documented report of experimental lepromatous infection in a primate. The findings are discussed in relation to the long incubation period of le promatous leprosy and the difficulties of diagnosing the disease at an early stage in man.

Experimental murine leprosy: growth of Mycobacterium lepraemurium in C3H and C57/BL mice after footpad inoculation.

Forty-three female C57/BL and C3H mice were inoculated with 2.7 X 10(6) Mycobacterium lepraemurium into each hind footpad. The foot thickness and the number of acid-fast bacilli in the footpad and popliteal and inquinal lymph nodes were recorded. In addition the morphological index and the mean bacillary length were determined in the footpad and in the popliteal lymph node. The bacilli multiplied in both strains during the first 4 weeks after inoculation. After that time no further increase in acid-fast bacilli was observed in the C57/BL strain; the bacilli became elongated and the morphological index decreased. These changes were preceded by a local swelling of the footpad due to the onset of an immune reaction. Thus, under the present conditions, C57/BL mice were able to resist experimental infection with M. lepraemurium by developing an immune response. In C3H mice no indication of an immune reaction was detected, and the bacilli continued to multiply throughout the observation period. The mouse footpad model seems to provide an excellent basis for the use of experimental murine leprosy to study immunity to mycobacterial infections. Certain aspects of the present model are discussed in relation to the mouse footpad model as used in the study of M. leprae infection in mice.

Superinfection in mice previously infected with Mycobacterium leprae.

Previous studies of the protection of mice by prior infection with Mycobacterium leprae in one hind footpad against challenge with M.leprae in the opposite hind footpad had produced conflicting results; therefore, the problem was restudied. In several experiments, BALB/c mice were inoculated first in the right hind footpad with 5,000 M. leprae and then challenged in the left hind footpad with 5,000 M. leprae of the same strain at intervals after primary infection, at the same time that uninfected mice were inoculated. Multiplication of the M. leprae of the secondary challenge inoculum occurred at the same rate and to the same level as multiplication in uninfected mice when challenges were made soon after primary infection. Multiplication was slowed but proceeded to the same level in previously infected as in uninfected mice when the challenges were administered between 76 and 106 days after primary infection (47 to 17 days before the M. leprae of the primary inoculum had multiplied to the level of 10-6 organisms per footpad). Finally, the M. leprae of a secondary challenge administered at the time that the organisms of the primary inoculum had multiplied to 10-6 per footpad or later not only multiplied more slowly in previously infected than in control animals, but multiplication in the previously infected animals reached a lower maximum. These results are similar to those observed when mice previously infected with M. bovis (BCG), M. marinum, Toxoplasma gondii, or Besnoitia jellisoni were challenged with M. leprae.

Susceptibility of thymectomized and irradiated mice to challenge with several organisms and the effect of dapsone on infection with Mycobacterium leprae.

B6C3F1 mice that had been thymectomized at 8 to 12 weeks of age, subjected to 950 R of whole-body X irradiation, and transfused with syngeneic bone marrow were challenged in a footpad with Mycobacterium leprae or M. marinum, or intravenously or intraperitioneally with Listeria monocytogenes. Also, mice inoculated with M. leprae in a hind footpad were administered dapsone in the mouse chow. The thymectomized-irradiated (T + R) mice did not survive as well as non-thymectomized mice when housed in the vivarium with no special precautions, but survived sufficiently well to permit the completion of some long-term experiments. M. leprae multiplied to a higher "ceiling" and survived longer in the T + R mice than in the non-thymectomized controls. But a ceiling to multiplication of M. leprae was imposed, and finally the organisms were killed. The histopathological appearance of the footpad tissues, studied by electron microscopy, was consistent with the measurements of bacterial numbers and viability. Swelling of the footpad after local inoculation with M. marinum was greater in T + R mice than in non-thymectomized controls. Similarly, the number of L. monocytogenes following intravenous challenge was greater in the spleens of T + R than of non-thymectomized mice, and the survival of the T + R mice was impaired after intraperitoneal challenge with L.monocytogenes, compared to the survival of non-thymectomized mice. None of these differences was striking, suggesting that these T + R mice had retained or regained some immune competence. The effects of dapsone treatment of T + R mice inoculated with M. leprae were much the same as those of treatment of non-thymectomized mice. Because these T + R mice were not greatly immunosuppressed, they would not have provided a model of human lepromatous leprosy suitable for chemotherapeutic studies.

Effect of T-cell depletion on the growth of BCG in the mouse footpad.

The growth of Mycobacterium bovis (BCG Montreal) and M. tuberculosis Erdman was determined in normal and T-cell depleted (THXB) mice when injected subcutaneously into a hind footpad. The bacilli multiplied only to a limited extent within the footpad itself but the infection quickly spread to the draining popliteal lymph node to eventually reach the liver, spleen and lung. The amount of systemic growth seen in the THXB mice was 10-100 times greater than in the normal controls, all of which developed a tuberculin hypersensitivity and an immune response in 14-18 days. T-cell depletion completely inhibited the expression of tuberculin sensitivity by the infected host as well as ablating the antituberculous response against both the vaccinating BCG population and a superinfecting Erdman challenge inoculum. Incorporation studies in the THXB mice indicated a striking reduction in cell division within the draining lymph node but there was an unexpected elevation in the level of incorporation by the lung cells as the BCG infection progressed. The significance of these findings is discussed in relation to the possible use of the BCG footpad model for studies of leprosy immunity.