The Armadillo as a Model for Leprosy Nerve Function Impairment: Preventative and Therapeutic Interventions.

Mycobacterium leprae infection of peripheral nerves and the subsequent nerve function impairment (NFI), especially in response to reactional episodes, are hallmarks of leprosy. Improved treatments for M. leprae-induced nerve injury are needed, as most if not all of the disability and stigma associated with leprosy arises from the direct or indirect effects of NFI. Nine-banded armadillos (Dasypus novemcinctus), like humans, exhibit the full clinical spectrum of leprosy and extensive involvement of the peripheral nerves. In this study, state-of-the-art technology was used to compare nerve function between uninfected and M. leprae-infected armadillos. Motor nerve conduction velocity (MNCV) and compound muscle action potential (cMAP), which measure changes in the rate of impulse conduction velocity and amplitude, revealed a progression of impairment that was directly correlated with the duration of M. leprae infection and enabled development of an objective nerve impairment scoring system. Ultrasonography accompanied by color Doppler imaging detected enlargement of the M. leprae-infected nerves and increased vascularity, possibly due to inflammation. Assessment of epidermal nerve fiber density (ENFD), which shows a length-dependent innervation in armadillos that is similar to humans, identified small fiber degeneration early after M. leprae infection. Staining for neuromuscular junction (NMJ) integrity, which is an indicator of signal transduction efficiency into skeletal muscle, discerned a markedly lower number and structural integrity of NMJ in M. leprae-infected armadillo footpads. These tools for assessing nerve injury were used to monitor the effects of intervention therapy. Two potential neuro-protective drugs, ethoxyquin (EQ) and 4-aminopyridine (4-AP), were tested for their ability to ameliorate peripheral nerve injury in M. leprae-infected armadillos. 4-AP treatment improved MNCV, cMAP, and EFND compared to untreated animals, while EQ had less effect. These results support the armadillo as a model for M. leprae-induced peripheral nerve injury that can provide insights toward the understanding of NFI progression and contribute to the preclinical investigation of the safety and efficacy of neuro-preventive and neuro-therapeutic interventions for leprosy.

Mycobacterium leprae induces Schwann cell proliferation and migration in a denervated milieu following intracutaneous excision axotomy in nine-banded armadillos.

Nine-banded armadillos develop peripheral neuropathy after experimental Mycobacterium leprae infection that recapitulates human disease. We used an intracutaneous excision axotomy model to assess the effect of infection duration by M. leprae on axonal sprouting and Schwan cell density. 34 armadillos (17 naïve and 17 M. leprae-infected) underwent 3 mm skin biopsies to create an intracutaneous excision axotomy followed by a concentric 4-mm overlapping biopsy 3 and 12-months post M. leprae inoculation. A traditional distal leg biopsy was obtained at 15mo for intraepidermal nerve fiber (IENF) density. Serial skin sections were immunostained against a axons (PGP9.5, GAP43), and Schwann cells (p75, s100) to visualize regenerating nerves. Regenerative axons and proliferation of Schwann cells was measured and the rate of growth at each time point was assessed. Increasing anti-PGL antibody titers and intraneural M. leprae confirmed infection. 15mo following infection, there was evidence of axon loss with reduced distal leg IENF versus naïve armadillos, p < 0.05. This was associated with an increase in Schwann cell density (11,062 ± 2905 vs. 7561 ± 2715 cells/mm3, p < 0.01). Following excisional biopsy epidermal reinnervation increased monotonically at 30, 60 and 90 days; the regeneration rate was highest at 30 days, and decreased at 60 and 90 days. The reinnervation rate was highest among animals infected for 3mo vs those infected for 12mo or naïve animals (mean ± SD, 27.8 ± 7.2 vs.16.2 ± 5.8vs. 15.3 ± 6.5 mm/mm3, p < 0.05). The infected armadillos displayed a sustained Schwann cell proliferation across axotomy time points and duration of infection (3mo:182 ± 26, 12mo: 256 ± 126, naive: 139 ± 49 cells/day, p < 0.05). M. leprae infection is associated with sustained Schwann cell proliferation and distal limb nerve fiber loss. Rates of epidermal reinnervation were highest 3mo after infection and normalized by 12 mo of infection. We postulate that excess Schwann cell proliferation is the main pathogenic process and is deleterious to sensory axons. There is a compensatory initial increase in regeneration rates that may be an attempt to compensate for the injury, but it is not sustained and eventually followed by axon loss. Aberrant Schwann cell proliferation may be a novel therapeutic target to interrupt the pathogenic cascade of M. leprae.

Mycobacterium leprae Infection in Ticks and Tick-Derived Cells.

Leprosy is a zoonosis in the southern United States involving humans and wild armadillos. The majority of patients presenting with zoonotic strains of Mycobacterium leprae note extensive outdoor activity but only rarely report any history of direct contact with wild armadillos. Whether M. leprae is transmitted to new vertebrate hosts through the environment independently or with the aid of other organisms, e.g., arthropod vectors, is a fundamental question in leprosy transmission. The objectives of this study were to assess the potential for ticks to transmit M. leprae and to test if viable M. leprae can be maintained in tick-derived cells. To evaluate tick transmission, nymphal Amblyomma maculatum ticks were injected with isolated M. leprae. Infection and transmission were assessed by qPCR. Ticks infected as nymphs harbored M. leprae through vertical transmission events (nymph to adult and adult to progeny); and, horizontal transmission of M. leprae to a vertebrate host was observed. Mycobacterium leprae DNA was detected in multiple tick life cycle stages. Likewise, freshly isolated M. leprae (Thai-53) was used to infect a tick-derived cell line, and enumeration and bacterial viability were assessed at individual time points for up to 49 days. Evaluations of the viability of long-term cultured M. leprae (Thai-53 and Br4923) were also assessed in a mouse model. Tick-derived cells were able to maintain viable M. leprae over the 49-day course of infection and M. leprae remained infectious within tick cells for at least 300 days. The results of this study suggest that ticks themselves might serve as a vector for the transmission of M. leprae and that tick cells are suitable for maintenance of viable M. leprae for an extended period of time.

Molecular epidemiology of leprosy: An update.

Molecular epidemiology investigations are notoriously challenging in the leprosy field mainly because the inherent characteristics of the disease as well as its yet uncultivated causative agents, Mycobacterium leprae and M. lepromatosis. Despite significant developments in understanding the biology of leprosy bacilli through genomic approaches, the exact mechanisms of transmission is still unclear and the factors underlying pathological variation of the disease in different patients remain as major gaps in our knowledge about leprosy. Despite these difficulties, the last two decades have seen the development of genotyping procedures based on PCR-sequencing of target loci as well as by the genome-wide analysis of an increasing number of geographically diverse isolates of leprosy bacilli. This has provided a foundation for molecular epidemiology studies that are bringing a better understanding of strain evolution associated with ancient human migrations, and phylogeographical insights about the spread of disease globally. This review discusses the advantages and drawbacks of the main tools available for molecular epidemiological investigations of leprosy and summarizes various methods ranging from PCR-based genotyping to genome-typing techniques. We also describe their main applications in analyzing the short-range and long-range transmission of the disease. Finally, we summarise the current gaps and challenges that remain in the field of molecular epidemiology of leprosy.

Development of LepReact, a defined skin test for paucibacillary leprosy and low-level M. leprae infection.

The persistence of new leprosy cases in endemic areas such as India, Brazil, Bangladesh, and the Philippines has encouraged studies of chemoprophylaxis among contacts of patients. Epidemiological screening tools to enable early detection of infected individuals in endemic populations would be critical to target individuals most in need of intervention. Despite decades of attempts, however, there still are no tests available for the early detection of low-level infection with Mycobacterium leprae. In this report, we describe the development of a leprosy skin test using M. leprae-specific antigens. We selected the chimeric LID-1 fusion protein, formulated to achieve maximum performance at a minimal dose, as a skin test candidate based on its ability to elicit delayed-type hypersensitivity (DTH) reactions in M. leprae immune guinea pigs in a sensitive and specific manner, i.e., with no cross-reactivity observed with other mycobacterial species. Importantly, evaluations in armadillos indicated that intradermal inoculation of formulated LID-1 could distinguish uninfected from M. leprae-infected animals manifesting with symptoms distinctly similar to the PB presentation of patients. Together, our data provide strong proof-of-concept for developing an antigen-specific skin test to detect low-level M. leprae infection. Such a test could, when applied with appropriate use of chemo- and/or immunoprophylaxis, be instrumental in altering the evolution of clinical disease and M. leprae transmission, thus furthering the objective of zero leprosy.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Erratum: Author Correction: LepVax, a defined subunit vaccine that provides effective pre-exposure and post-exposure prophylaxis of M. leprae infection.

[This corrects the article DOI: 10.1038/s41541-018-0050-z.].

Differential growth of Mycobacterium leprae strains (SNP genotypes) in armadillos.

Leprosy (Hansen's Disease) has occurred throughout human history, and persists today at a low prevalence in most populations. Caused by Mycobacterium leprae, the infection primarily involves the skin, mucosa and peripheral nerves. The susceptible host range for Mycobacterium leprae is quite narrow. Besides humans, nine banded armadillos (Dasypus novemcinctus) and red squirrels (Sciurus vulgaris) are the only other natural hosts for M. leprae, but only armadillos recapitulate the disease as seen in humans. Armadillos across the Southern United States harbor a single predominant genotypic strain (SNP Type-3I) of M. leprae, which is also implicated in the zoonotic transmission of leprosy. We investigated, whether the zoonotic strain (3I) has any notable growth advantages in armadillos over another genetically distant strain-type (SNP Type-4P) of M. leprae, and if M. leprae strains manifest any notably different pathology among armadillos. We co-infected armadillos (n = 6) with 2 × 109 highly viable M. leprae of both strains and assessed the relative growth and dissemination of each strain in the animals. We also analyzed 12 additional armadillos, 6 each individually infected with the same quantity of either strain. The infections were allowed to fulminate and the clinical manifestations of the disease were noted. Animals were humanely sacrificed at the terminal stage of infection and the number of bacilli per gram of liver, spleen and lymph node tissue were enumerated by Q-PCR assay. The growth of M. leprae strain 4P was significantly higher (P < 0.05) than 3I when each strain was propagated individually in armadillos. Significantly (P < 0.0001) higher growth of the 4P strain also was confirmed among animals co-infected with both 3I and 4P strain types using whole genome sequencing. Interestingly, the zoonotic strain does not exhibit any growth advantage in these non-human hosts, but the varied proliferation of the two M. leprae strains within armadillos suggest there are notable pathological variations between M. leprae strain-types.

LepVax, a defined subunit vaccine that provides effective pre-exposure and post-exposure prophylaxis of M. leprae infection.

Sustained elimination of leprosy as a global health concern likely requires a vaccine. The current standard, BCG, confers only partial protection and precipitates paucibacillary (PB) disease in some instances. When injected into mice with the T helper 1 (Th1)-biasing adjuvant formulation Glucopyranosyl Lipid Adjuvant in stable emulsion (GLA-SE), a cocktail of three prioritized antigens (ML2055, ML2380 and ML2028) reduced M. leprae infection levels. Recognition and protective efficacy of a single chimeric fusion protein incorporating these antigens, LEP-F1, was confirmed in similar experiments. The impact of post-exposure immunization was then assessed in nine-banded armadillos that demonstrate a functional recapitulation of leprosy. Armadillos were infected with M. leprae 1 month before the initiation of post-exposure prophylaxis. While BCG precipitated motor nerve conduction abnormalities more rapidly and severely than observed for control infected armadillos, motor nerve injury in armadillos treated three times, at monthly intervals with LepVax was appreciably delayed. Biopsy of cutaneous nerves indicated that epidermal nerve fiber density was not significantly altered in M. leprae-infected animals although Remak Schwann cells of the cutaneous nerves in the distal leg were denser in the infected armadillos. Importantly, LepVax immunization did not exacerbate cutaneous nerve involvement due to M. leprae infection, indicating its safe use. There was no intraneural inflammation but a reduction of intra axonal edema suggested that LepVax treatment might restore some early sensory axonal function. These data indicate that post-exposure prophylaxis with LepVax not only appears safe but, unlike BCG, alleviates and delays the neurologic disruptions caused by M. leprae infection.

The Armadillo (Dasypus novemcinctus): A Witness but Not a Functional Example for the Emergence of the Butyrophilin 3/Vγ9Vδ2 System in Placental Mammals.

1-5% of human blood T cells are Vγ9Vδ2 T cells whose T cell receptor (TCR) contain a TRGV9/TRGJP rearrangement and a TRDV2 comprising Vδ2-chain. They respond to phosphoantigens (PAgs) like isopentenyl pyrophosphate or (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMBPP) in a butyrophilin 3 (BTN3)-dependent manner and may contribute to the control of mycobacterial infections. These cells were thought to be restricted to primates, but we demonstrated by analysis of genomic databases that TRGV9, TRDV2, and BTN3 genes coevolved and emerged together with placental mammals. Furthermore, we identified alpaca (Vicugna pacos) as species with typical Vγ9Vδ2 TCR rearrangements and currently aim to directly identify Vγ9Vδ2 T cells and BTN3. Other candidates to study this coevolution are the bottlenose dolphin (Tursiops truncatus) and the nine-banded armadillo (Dasypus novemcinctus) with genomic sequences encoding open reading frames for TRGV9, TRDV2, and the extracellular part of BTN3. Dolphins have been shown to express Vγ9- and Vδ2-like TCR chains and possess a predicted BTN3-like gene homologous to human BTN3A3. The other candidate, the armadillo, is of medical interest since it serves as a natural reservoir for Mycobacterium leprae. In this study, we analyzed the armadillo genome and found evidence for multiple non-functional BTN3 genes including genomic context which closely resembles the organization of the human, alpaca, and dolphin BTN3A3 loci. However, no BTN3 transcript could be detected in armadillo cDNA. Additionally, attempts to identify a functional TRGV9/TRGJP rearrangement via PCR failed. In contrast, complete TRDV2 gene segments preferentially rearranged with a TRDJ4 homolog were cloned and co-expressed with a human Vγ9-chain in murine hybridoma cells. These cells could be stimulated by immobilized anti-mouse CD3 antibody but not with human RAJI-RT1Bl cells and HMBPP. So far, the lack of expression of TRGV9 rearrangements and BTN3 renders the armadillo an unlikely candidate species for PAg-reactive Vγ9Vδ2 T cells. This is in line with the postulated coevolution of the three genes, where occurrence of Vγ9Vδ2 TCRs coincides with a functional BTN3 molecule.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients.

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

qPCR-High resolution melt analysis for drug susceptibility testing of Mycobacterium leprae directly from clinical specimens of leprosy patients

BACKGROUND: Real-Time PCR-High Resolution Melting (qPCR-HRM) analysis has been recently described for rapid drug susceptibility testing (DST) of Mycobacterium leprae. The purpose of the current study was to further evaluate the validity, reliability, and accuracy of this assay for M. leprae DST in clinical specimens. METHODOLOGY/PRINCIPAL FINDINGS: The specificity and sensitivity for determining the presence and susceptibility of M. leprae to dapsone based on the folP1 drug resistance determining region (DRDR), rifampin (rpoB DRDR) and ofloxacin (gyrA DRDR) was evaluated using 211 clinical specimens from leprosy patients, including 156 multibacillary (MB) and 55 paucibacillary (PB) cases. When comparing the results of qPCR-HRM DST and PCR/direct DNA sequencing, 100% concordance was obtained. The effects of in-house phenol/chloroform extraction versus column-based DNA purification protocols, and that of storage and fixation protocols of specimens for qPCR-HRM DST, were also evaluated. qPCR-HRM results for all DRDR gene assays (folP1, rpoB, and gyrA) were obtained from both MB (154/156; 98.7%) and PB (35/55; 63.3%) patients. All PCR negative specimens were from patients with low numbers of bacilli enumerated by an M. leprae-specific qPCR. We observed that frozen and formalin-fixed paraffin embedded (FFPE) tissues or archival Fite's stained slides were suitable for HRM analysis. Among 20 mycobacterial and other skin bacterial species tested, only M. lepromatosis, highly related to M. leprae, generated amplicons in the qPCR-HRM DST assay for folP1 and rpoB DRDR targets. Both DNA purification protocols tested were efficient in recovering DNA suitable for HRM analysis. However, 3% of clinical specimens purified using the phenol/chloroform DNA purification protocol gave false drug resistant data. DNA obtained from freshly frozen (n = 172), formalin-fixed paraffin embedded (FFPE) tissues (n = 36) or archival Fite's stained slides (n = 3) were suitable for qPCR-HRM DST analysis. The HRM-based assay was also able to identify mixed infections of susceptible and resistant M. leprae. However, to avoid false positives we recommend that clinical specimens be tested for the presence of the M. leprae using the qPCR-RLEP assay prior to being tested in the qPCR-HRM DST and that all specimens demonstrating drug resistant profiles in this assay be subjected to DNA sequencing. CONCLUSION/SIGNIFICANCE: Taken together these results further demonstrate the utility of qPCR-HRM DST as an inexpensive screening tool for large-scale drug resistance surveillance in leprosy.

Zoonotic Leprosy in the Southeastern United States.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and have been implicated in zoonotic transmission of leprosy. Early studies found this disease mainly in Texas and Louisiana, but armadillos in the southeastern United States appeared to be free of infection. We screened 645 armadillos from 8 locations in the southeastern United States not known to harbor enzootic leprosy for M. leprae DNA and antibodies. We found M. leprae-infected armadillos at each location, and 106 (16.4%) animals had serologic/PCR evidence of infection. Using single-nucleotide polymorphism variable number tandem repeat genotyping/genome sequencing, we detected M. leprae genotype 3I-2-v1 among 35 armadillos. Seven armadillos harbored a newly identified genotype (3I-2-v15). In comparison, 52 human patients from the same region were infected with 31 M. leprae types. However, 42.3% (22/52) of patients were infected with 1 of the 2 M. leprae genotype strains associated with armadillos. The geographic range and complexity of zoonotic leprosy is expanding.

Mechanisms of nerve injury in leprosy.

All patients with leprosy have some degree of nerve involvement. Perineural inflammation is the histopathologic hallmark of leprosy, and this localization may reflect a vascular route of entry of Mycobacterium leprae into nerves. Once inside nerves, M. leprae are ingested by Schwann cells, with a wide array of consequences. Axonal atrophy may occur early in this process; ultimately, affected nerves undergo segmental demyelination. Knowledge of the mechanisms of nerve injury in leprosy has been greatly limited by the minimal opportunities to study affected nerves in man. The nine-banded armadillo provides the only animal model of the pathogenesis of M. leprae infection. New tools available for this model enable the study and correlation of events occurring in epidermal nerve fibers, dermal nerves, and nerve trunks, including neurophysiologic parameters, bacterial load, and changes in gene transcription in both neural and inflammatory cells. The armadillo model is likely to enhance understanding of the mechanisms of nerve injury in leprosy and offers a means of testing proposed interventions.

The armadillo as an animal model and reservoir host for Mycobacterium leprae.

Apart from humans, armadillos are the only known natural hosts of Mycobacterium leprae. They are well developed as hosts for in vivo propagation of M leprae and are advancing as models for studying the pathogenesis of leprosy and translational research. Armadillos are immunologically intact. They exhibit the full Ridley-Jopling spectrum of histopathologic responses to M leprae and uniquely manifest extensive neurological involvement that closely recapitulates human leprosy. In addition, free-ranging armadillos in some regions are known to harbor a naturally occurring infection with M leprae, and zoonotic transmission between armadillos and humans has been implicated in a large number of new case presentations. We review the role of the armadillo as a model for leprosy and reservoir for human infection.

Mechanisms of nerve injury in leprosy

All patients with leprosy have some degree of nerve involvement. Perineural inflammation is the histopathologic hallmark of leprosy, and this localization may reflect a vascular route of entry of Mycobacterium leprae into nerves. Once inside nerves, M leprae are ingested by Schwann cells, with a wide array of consequences. Axonal atrophy may occur early in this process; ultimately, affected nerves undergo segmental demyelination. Knowledge of the mechanisms of nerve injury in leprosy has been greatly limited by the minimal opportunities to study affected nerves in man. The nine-banded armadillo provides the only animal model of the pathogenesis of M leprae infection. New tools available for this model enable the study and correlation of events occurring in epidermal nerve fibers, dermal nerves, and nerve trunks, including neurophysiologic parameters, bacterial load, and changes in gene transcription in both neural and inflammatory cells. The armadillo model is likely to enhance understanding of the mechanisms of nerve injury in leprosy and offers a means of testing proposed interventions.

The armadillo as an animal model and reservoir host for Mycobacterium leprae

Apart from humans, armadillos are the only known natural hosts of Mycobacterium leprae. They are well developed as hosts for in vivo propagation of M leprae and are advancing as models for studying the pathogenesis of leprosy and translational research. Armadillos are immunologically intact. They exhibit the full Ridley-Jopling spectrum of histopathologic responses to M leprae and uniquely manifest extensive neurological involvement that closely recapitulates human leprosy. In addition, free-ranging armadillos in some regions are known to harbor a naturally occurring infection with M leprae, and zoonotic transmission between armadillos and humans has been implicated in a large number of new case presentations. We review the role of the armadillo as a model for leprosy and reservoir for human infection.

The armadillo as a model for peripheral neuropathy in leprosy.

Leprosy (also known as Hansen's Disease) is a chronic infectious disease caused by Mycobacterium leprae that primarily targets the peripheral nervous system; skin, muscle, and other tissues are also affected. Other than humans, nine-banded armadillos (Dasypus novemcinctus) are the only natural hosts of M. leprae, and they are the only laboratory animals that develop extensive neurological involvement with this bacterium. Infection in the armadillo closely recapitulates many of the structural, physiological, and functional aspects of leprosy seen in humans. Armadillos can be useful models of leprosy for basic scientific investigations into the pathogenesis of leprosy neuropathy and its associated myopathies, as well as for translational research studies in piloting new diagnostic methods or therapeutic interventions. Practical and ethical constraints often limit investigation into human neuropathies, but armadillos are an abundant source of leprotic neurologic fibers. Studies with these animals may provide new insights into the mechanisms involved in leprosy that also might benefit the understanding of other demyelinating neuropathies. Although there is only a limited supply of armadillo-specific reagents, the armadillo whole genomic sequence has been completed, and gene expression studies can be employed. Clinical procedures, such as electrophysiological nerve conduction testing, provide a functional assessment of armadillo nerves. A variety of standard histopathological and immunopathological procedures including Epidermal Nerve Fiber Density (ENFD) analysis, Schwann Cell Density, and analysis for other conserved cellular markers can be used effectively with armadillos and will be briefly reviewed in this text.

The armadillo: a model for the neuropathy of leprosy and potentially other neurodegenerative diseases.

Leprosy (also known as Hansen's disease) is an infectious peripheral neurological disorder caused by Mycobacterium leprae that even today leaves millions of individuals worldwide with life-long disabilities. The specific mechanisms by which this bacterium induces nerve injury remain largely unknown, mainly owing to ethical and practical limitations in obtaining affected human nerve samples. In addition to humans, nine-banded armadillos (Dasypus novemcinctus) are the only other natural host of M. leprae, and they develop a systemically disseminated disease with extensive neurological involvement. M. leprae is an obligate intracellular parasite that cannot be cultivated in vitro. Because of the heavy burdens of bacilli they harbor, nine-banded armadillos have become the organism of choice for propagating large quantities of M. leprae, and they are now advancing as models of leprosy pathogenesis and nerve damage. Although armadillos are exotic laboratory animals, the recently completed whole genome sequence for this animal is enabling researchers to undertake more sophisticated molecular studies and to develop armadillo-specific reagents. These advances will facilitate the use of armadillos in piloting new therapies and diagnostic regimens, and will provide new insights into the oldest known infectious neurodegenerative disorder.

Insights from animal models on the immunogenetics of leprosy: a review

A variety of host immunogenetic factors appear to influence both an individual's susceptibility to infection with Mycobacterium leprae and the pathologic course of the disease. Animal models can contribute to a better understanding of the role of immunogenetics in leprosy through comparative studies helping to confirm the significance of various identified traits and in deciphering the underlying mechanisms that may be involved in expression of different disease related phenotypes. Genetically engineered mice, with specific immune or biochemical pathway defects, are particularly useful for investigating granuloma formation and resistance to infection and are shedding new light on borderline areas of the leprosy spectrum which are clinically unstable and have a tendency toward immunological complications. Though armadillos are less developed in this regard, these animals are the only other natural hosts of M. leprae and they present a unique opportunity for comparative study of genetic markers and mechanisms associable with disease susceptibility or resistance, especially the neurological aspects of leprosy. In this paper, we review the recent contributions of genetically engineered mice and armadillos toward our understanding of the immunogenetics of leprosy.