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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.

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.

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.

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.

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.

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.

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.

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.