In 1960, Charles Shepard published his work on the growth of Mycobacterium leprae (see Chapter 5.1) in the footpads of immunocompetent Carworth Farms white mice . Although M. leprae was first associated with leprosy by Armauer Hansen nearly 90 years earlier, Shepard’s passage of the bacilli in mouse footpads (MFP) was the first successful reproducible propagation of this organism. His was not, however, the first attempt. Since its discovery, numerous investigators have tried to grow M. leprae and reproduce leprosy in a variety of animal models, including common laboratory species such as rodents, rabbits, and monkeys and other mammals, birds, and even cold-blooded animals [reviewed in ]. Most of these attempts were unsuccessful, some because of a natural resistance of the host species to infection, but others likely due to the poor viability of the human biopsy-derived inoculum and a lack of knowledge of the meticulous growth requirements of leprosy bacilli. Shepard and others , ,  went on to confirm the utility of the MFP model for growing M. leprae and, for the first time, the establishment and maintenance of isolates of M. leprae was accomplished. Moreover, drug testing, detection of drug resistance, experimental vaccine evaluation, and immunological studies became feasible.
Mice (Mus musculus) are the experimental animals most commonly used in biomedical research. Due to their small size, they are relatively inexpensive to house and maintain. Mice are also prolific breeders with a relatively short gestation time, allowing for the rapid generation of large numbers of animals. There are hundreds of commercially available well-defined strains, including outbred, inbred, and genetically engineered mutants. Additionally, there is an ever-growing multitude of sophisticated mouse-specific biological reagents available that enable complex and detailed examination of physiological and immunological processes. Mice, in particular the MFP model, have been used extensively in leprosy research.
Brown rats (Rattus norvegicus) were the first mammals specifically domesticated for use in biomedical research in the 19th century . Rats have similar housing and maintenance requirements as mice, and they also have similarly short gestational periods and produce multiple litters in a year. However, while mice have been used across the entire spectrum of biomedical research, rats have been more frequently used to study human and animal physiology due to their larger size. Until the rat genome was sequenced in 2004 , it was more difficult for researchers to create transgenic rats, so their use in genetic studies lagged behind mice . There are now numerous inbred, outbred, and genetic mutant strains of rats commercially available. While useful as biomedical animal models, rats are not frequently used in leprosy research today.
Recently, red squirrels (Sciurus vulgaris) have been identified as natural reservoirs of both M. leprae and M. lepromatosis . Interestingly, only S. vulgaris squirrels from the British Isles seem to be infected with resultant leprosy-like lesions. Schilling et al.  used PCR to test red squirrels from other parts of Europe, along with four other squirrel species from around the world. They found no evidence of either M. leprae or M. lepromatosis in any of the other squirrel species. While red squirrels may represent another potential leprosy zoonosis in addition to armadillos, more research is needed to determine the exact role of red squirrels in leprosy transmission. Currently, squirrels are not being used as a research model for leprosy.
Since the earliest days of leprosy research, investigators have tested the utility of different rodent species including hamsters, gerbils, and mystromys. Syrian golden hamsters were inoculated in footpads, ears, and testicles , , . The lesions that developed in the footpads were smaller than those seen in mice. Chinese hamsters and Mongolian gerbils were also tested with similar results to the Syrian golden hamsters. Binford  reported successful nerve invasion in inoculated African white-tailed rats (Mystromys albacaudatus). However, while these other rodent species have been investigated, the mouse model is still the most commonly used species, and this review will mainly focus on the contributions of the murine model to leprosy research.
In Vivo Infection Models
Shepard chose the footpad as the site of infection because of its cooler temperature, and because other mycobacteria had been successfully cultured at this site. He found that when approximately 104 M. leprae were inoculated into the footpad of immunocompetent animals, the bacilli multiplied with a doubling time of ~13 days. Localized growth in the footpad continued until reaching a peak at ~106 organisms, at which point the bacilli were killed by the host immune system  and slowly cleared. If inoculated with ≥106 M. leprae, there was no bacterial growth as immunization of the mouse occurred at this dose of bacteria. A similar growth plateau of 105 to 106 organisms is also obtained upon inoculation of rat footpads .
Inoculation of M. leprae into the footpads of conventional, immunocompetent mice induces a cellular infiltration that is composed mainly of macrophages and epithelioid cells. As the infection progresses, vacuolation increases, bacterial replication ceases, and macrophage activation occurs . Numerous lymphocytes are present, but neutrophils are rare. Tissue necrosis does not occur. At this stage of infection, the nerves are not infected. However, neural damage has been reported very late (>2 years) in the course of the infection . Bacilli are primarily intracellular, often in clumps, and become progressively more beaded as the infection proceeds past the peak of bacterial growth (see Chapter 5.3).
While Shepard’s original model used footpads from immunocompetent mice, other researchers adapted these techniques to immunosuppressed strains to mimic human lepromatous leprosy (LL). The infection of thymectomized and irradiated mice , , congenitally athymic mice , , SCID mice , , , neonatally thymectomized rats , and athymic rats  highlighted the importance of T cells in host defense against leprosy (see Chapter 6.2). As infection progresses over several months, there is a large influx of macrophages into the footpad providing ample cellular substrate for the multiplying bacilli. Eventually, a huge “leproma” consisting of heavily infected foamy macrophages forms and replaces most of the normal footpad tissue (see Chapter 10.2) . These macrophages contain hundreds of bacilli with little to no apparent pathological effect on the host, emphasizing the non-toxic nature of this organism . Bacterial dissemination is rather slow, but bacilli are eventually found in striated muscle cells, perineural cells, Schwann cells, and fibroblasts. A year after infection, bacteria can be found in virtually all organs except for the central nervous system. Compared to immunocompetent rodents, multiplication of M. leprae in immunosuppressed strains often reaches 1010 or more bacilli, and such animals have proven useful for the detection of a low proportion of viable M. leprae in biopsies from patients following drug treatment , . Athymic nude mice are now routinely used for cultures of large numbers of viable M. leprae for experimental use .
While extremely useful, these models did not provide representatives for the investigation of the borderline forms of leprosy (see Chapter 2.4), which are of great clinical importance. The use of gene transfer technology has led to the generation of hundreds of different commercially available genetically engineered mouse strains, such as targeted gene knockouts (KO), conditional KO, tissue-specific KO, and knock-in mutations. Other recent approaches include collaborative cross, diversity outbred, and humanized mice , , . The development of these mouse strains has provided new models for investigating infectious diseases.
Leprosy outcomes manifest differently due to the quality of the host immune response (see Chapter 6.1; Chapter 6.2); therefore, mice with defined defects, especially in host defense pathways such as cytokines, chemokines, cytokine and chemokine receptors, immune modulators, and cell surface markers, have been particularly helpful in the study of leprosy pathogenesis. The host cell-mediated immune response to M. leprae infection has been evaluated in vivo and in vitro in multiple KO strains of mice , , , , , . Interestingly, no single KO of a cytokine, T-cell type, or antimicrobial mechanism transformed any of these strains into an immunosuppressed model comparable to the athymic nude mouse. However, based on their unique characteristic profiles, most KO strains could be placed along the leprosy spectrum (see Chapter 2.4). These findings may help to elucidate mechanisms that may be occurring in borderline leprosy.
M. leprae and M. lepromatosis are the only bacteria that naturally infect human nerves, having a tropism for Schwann cells. However, M. leprae do not readily infect murine Schwann cells in vivo, presumably due to a lack of appropriate receptors. Shetty et al. , , ,  inoculated 1–2x107 viable M. leprae directly into the sciatic nerves of immunocompetent and immunosuppressed mice. Although M. leprae were not observed in the Schwann cells of either strain, a tuberculoid-type granulomatous response (see Chapter 2.4) was elicited in the immunocompetent mice, whereas immunosuppressed mice developed a macrophage response. Interestingly, both bacterial growth and bacterial viability were decreased after intra-neural inoculation, possibly due to the warm temperature of the sciatic nerve. Rambukkana et al.  reported substantial demyelination in both immunocompetent and Rag1-/- mice, which lack T and B cells, after intra-neural inoculation of live M. leprae, dead M. leprae, or whole cell wall fraction. Thus, while murine intra-neural inoculation may not be representative of a natural infection with M. leprae, these studies show that this route of administration is an effective method for investigating both immunologically and non-immunologically mediated nerve damage in leprosy.
Studies of cutaneous leishmaniasis utilized a murine ear pinnae model and discovered interesting differences in disease development and anti-Leishmania immune responses in the ear pinnae infection versus the footpad infection . Since murine ear pinnae are also cooler areas similar to footpads, intradermal inoculation of viable M. leprae into ear pinnae was attempted to study the growth kinetics and development of anti-M. leprae immune responses . By 30 weeks post inoculation, there was limited growth of M. leprae in the ears, and the draining lymph nodes showed markedly more cellular infiltration, especially with regard to IFNγ-secreting CD4+ T cells, compared to that of footpad-inoculated mice. The cellular infiltration in draining lymph nodes as well as at the inoculation site was significantly reduced following rifampin treatment of mice shortly (1–3 weeks) after inoculation (see Chapter 5.2). This modulatory inflammatory response seen in ear pinnae was proposed as a potential model for testing new anti-leprosy drugs.
In Vitro Infection Models
In 1965, Chang and Neikirk presented one of the earliest reports of the use of murine macrophages for leprosy research at the joint conference of The Leonard Wood Memorial and The Armed Forces Institute of Pathology . Mouse peritoneal macrophages were infected in vitro with M. leprae obtained from 11 different patient biopsies from varied geographical regions. The infected cultures were maintained over 10 weeks, and the authors reported elongation of the intracellular AFB. A much later study, using nude MFP-derived M. leprae inoculated into mouse peritoneal macrophages, also showed bacterial elongation but no growth . Moreover, this latter study established that IL-10 is needed to maintain intracellular bacterial metabolic activity, as measured by palmitic acid oxidation rate (see Chapter 5.3). Without IL-10, metabolic activity of intracellular bacteria maintained within macrophages in vitro significantly declined within 3–4 weeks.
Krahenbuhl and his group were instrumental in establishing the use of in vitro murine macrophage cultures as a valuable model for understanding basic leprosy immunology. Using this macrophage model, his group established that infected mouse peritoneal macrophages become refractory to IFN-γ induced macrophage activation both in vivo and in vitro , . Further research found the fate of live versus dead M. leprae in macrophages to be quite different. Live M. leprae prevented phagolysosomal fusion whereas phagolysosomal fusion occurred in macrophages when infected with gamma-irradiated (dead) M. leprae . This finding was one of the first indications that live and dead bacilli are perceived differently by the host cell.
Using fluorescent microscopy, Alves et al.  examined the colocalization of viable or heat-killed M. leprae bacilli within the acidified lysosomal compartments in RAW (mouse) macrophages and observed differences in endosomal trafficking of live versus dead M. leprae in the cell line. The viability of intracellular bacilli rapidly decreases after macrophage activation, and this intracellular killing is dependent on the L-arginine-dependent production of reactive nitrogen intermediates , . Together, these data suggest that following infection, viable M. leprae creates a niche for itself by resisting lysosomal digestion and inhibiting IFN-γ induced activation, as macrophages become potent killers of intracellular bacilli following activation.
The MFP model also allowed for the immunological investigation of the M. leprae-induced granuloma (see Chapter 2.4). After infection with M. leprae, T cells and macrophages are the primary cells that respond to the infection. However, because of leprosy’s extremely slow progression and its capacity to remain asymptomatic long term, it is difficult to define the early cellular interactions of this immune response to infection. Therefore, neither the dynamics of the T-cell and macrophage interactions within the microenvironment of the granulomatous lesion nor their role in disease development and progression are fully understood.
Granuloma cells can be isolated from the footpad and cultured in vitro , . Studies using such cells showed that heavily infected granuloma macrophages are refractory to IFN-γ activation ,  and that this unresponsiveness may be due to elevated production of prostaglandin E2 , . Observations of inflammation in the footpads has been correlated with distinct immune cell phenotypes and cytokine and chemokine expression and production of the granuloma cells .
M. leprae and M. lepromatosis are the only bacteria known to invade peripheral nerves in humans, where they show a unique tropism towards Schwann cells (see Chapter 9.1; Chapter 9.2). Due to the inherent difficulties in obtaining human peripheral nerve tissues, much of our understanding of M. leprae’s interactions with peripheral nerves comes from experiments with mouse or rat primary Schwann cells. These rodent studies have contributed significantly to our understanding of M. leprae pathogenesis in peripheral nerves.
An early study in the 1980s found that Schwann cells (primary rat sciatic nerve derived) lacked an M. leprae-specific uptake system because M. leprae, along with other mycobacterium species and inert particles (latex beads), were all phagocytosed indiscriminately . However, another study showed that lipase pretreatment of mouse Schwann cells decreased M. leprae adherence, while trypsin pretreatment enhanced adhesion. Based on these findings, M. leprae adhesion to Schwann cells appears dependent on surface lipid receptors .
Using primary rat Schwann cells, Rambukkana et al. , , ,  showed that M. leprae specifically binds to the G-domain of the laminin-α2 chain present on Schwann cell basal lamina and utilizes it as a bridge molecule to ultimately interact with laminin receptor b4-integrin for internalization. PGL-1, a molecule specific to the M. leprae cell wall, binds laminin-α2 with its terminal trisaccharide. It appears that entry of M. leprae into Schwann cells is a complex process involving at least two phases, binding to basal lamina of the Schwann cells-axon unit and then invading Schwann cells, involving multiple host and bacterial molecules.
Both myelinating and non-myelinating Schwann cells are able to phagocytose M. leprae with similar efficiencies. In a rat sciatic nerve Schwann cells-neuron co-culture model, infected Schwann cells can myelinate without any apparent difference in myelin architecture compared to uninfected Schwann cells. However, another study using a rat Schwann cells-dorsal root ganglion neuron co-culture showed rapid demyelination of the myelinated Schwann cells within 24 hours of bacterial invasion without any indications of apoptosis and without any inflammatory cells. This bacterial-induced demyelination is independent of bacterial viability because demyelination also occurs with irradiated (dead) bacilli , .
Both adult and neonatal rat Schwann cells are able to produce IL-1 in response to M. leprae antigens, which allows the Schwann cells to function as antigen-presenting cells , , . Ford and Britton conclusively showed that rat Schwann cells could present M. leprae hsp70 to antigen specific T cells. The secondary activation of these T cells was inhibited by anti-MHC-II monoclonal antibodies and not by anti-MHC-I antibodies . The ability of Schwann cells to present immunodominant antigens to MHC class II-restricted antigen-specific T lymphocytes should be further investigated to understand its role in the activation of immunological reactions frequently associated with nerve damage.
Viable M. leprae are able to induce reprogramming of infected murine Schwann cells through the upregulation of numerous genes responsible for embryonic development, transcription, chromatin remodeling, cell signaling, and cell division. Eventually, these M. leprae-infected Schwann cells are reprogrammed into progenitor cells with migratory and immunomodulatory properties conducive to bacterial dissemination. Moreover, the reprogrammed cells can also efficiently transfer bacilli to co-cultured fibroblasts within 24 hours , , . This is a novel phenomenon only observed in in vitro mouse primary Schwann cell
s cultures, but further studies are required to establish whether this process is indeed taking place within the peripheral nerves of leprosy-affected individuals.
The inability to culture M. leprae in an artificial medium not only impedes the understanding of vital molecular and cellular events in the pathogenesis of leprosy, it also prevents researchers from easily and definitively distinguishing between live and dead bacilli (see Chapter 5.3). The capability to determine bacterial viability is essential for testing the efficacy of new drugs and/or new treatment regimens. While there have been advancements in addressing these issues, such as the use of molecular techniques to measure M. leprae viability , the MFP model remains the gold standard for the study of leprosy, particularly for the testing of new drugs. New and powerful assays have shed light on many aspects of drug resistance and the response to drug treatments in clinical trials, but all of them rely on the mouse model to be successful.
The development of a reliable immune-based laboratory diagnostic test for early or subclinical leprosy is a top priority as early treatment is essential to prevent nerve damage and disability and to block transmission. However, sensitivity is an issue because subclinical leprosy presents minimum immune stimulation. Furthermore, the unknown duration since exposure and the variability in human immune responses confound results. An important advantage of rodent models of infection is that they eliminate many of these issues, especially the problems of unknown length of exposure to M. leprae and of potential cross-reactivity with other common mycobacterial species. Hence, the MFP has been utilized in an effort to develop standardized protocols for immunodiagnosis. One such study involved evaluation of the cell-mediated immune response, gauged in terms of IFN-γ secretion, by splenic lymphocytes from infected mice stimulated in vitro with M. leprae antigens . In an attempt to model subclinical or early leprosy, various doses of M. leprae and durations of infection were titrated in MFP to establish the minimum dose at the earliest time point required for the development of a measurable cell-mediated immune response. For a consistent and significant response three months after inoculation, a minimum dose of 1x105 live M. leprae per footpad was required. Both BALB/c and C57Bl/6 mice responded similarly although the response was more robust in C57Bl/6 mice.
This model was able to pick-up most, though not all, of the recombinant M. leprae antigens recognized by patient sera or peripheral blood mononuclear cells , , . Thus, this ‘early’ disease model may be used to screen M. leprae antigens for immunodiagnostic purposes that may then be further evaluated in the natural hosts of M. leprae. T cells isolated from the site of an M. leprae infection (i.e., M. leprae-induced foot pad granuloma) could also be examined for their antigen responsiveness , .
Drug Trials and Drug Resistance
Since being introduced in 1981 by the World Health Organization, multi-drug therapy (MDT; see Chapter 2.6) has been used successfully to treat leprosy. However, the number of new leprosy cases each year has not shown a significant decrease and the presence of drug resistance remains a concern, indicating a need for new drugs to treat leprosy.
The mouse model has been used extensively for testing new drug therapies for leprosy . The ability to test new antibiotics in the MFP is part of a very useful and successful drug pipeline (Figure 1). The pipeline consists of three stages: (1) determining efficacy against non-dividing M. leprae in axenic cultures, (2) determining efficacy against non-dividing intracellular M. leprae in macrophage cultures, and (3) determining efficacy against multiplying M. leprae in MFP assays. The MFP step is traditionally performed using immunocompetent mouse strains, such as Balb/c or Swiss Webster, to test the proposed drug for bactericidal and bacteriostatic effects against multiplying bacteria , , , . However, because this assay has limited sensitivity, it can be modified to include athymic nude mice for a more robust measure of drug efficacy. This model has been used to test the efficacy of several different compounds, including PA-824, nitazoxanide, and rifapentine , .
FIG 1 NHDP drug screening pipeline.
Test drugs, which have been verified to have a target in M. leprae, are screened for bactericidal activity against non-dividing bacilli in axenic culture and non-dividing intracellular bacilli in macrophage culture. Drugs showing activity are then evaluated for both bactericidal and bacteriostatic effects against multiplying M. leprae in the MFP assay.
While these studies have shown promise for new therapies, potential resistance to current drug regimens remains an issue. For years, the mouse model was the only reliable and accurate method to test for drug resistance in M. leprae. However, the time, costs, and technical expertise associated with using the mouse model, as well as advancements in molecular techniques , , has led to a decline in the usage of the mouse model for detecting drug resistance in most laboratories.
Screening Vaccine Candidates
In addition to their usefulness in drug studies, murine models are beneficial for screening potential leprosy vaccine candidates (see Chapter 6.3). The concept of utilizing a vaccine that is cross protective for both tuberculosis and leprosy, two poverty-associated diseases caused by closely related organisms, has been considered for years. The most widely used and studied vaccine is live attenuated M. bovis Bacille Calmette-Guérin (BCG), which currently remains the only global TB vaccine , and numerous studies have shown it has some effectiveness against leprosy as well (see Chapter 6.3). An early study by Shepard et al.  used Swiss Webster mice to measure the immune response elicited by vaccines of either M. bovis BCG, heat-killed M. leprae, or a combination. In terms of sensitization, all three produced similar immune responses except at the lowest dose, where the combination was more effective. In human trials, BCG has variable protection against leprosy, ranging from an average of 26% in experimental studies to an average of 61% in observational studies . A vaccine composed of M. indicus pranii has also shown protection against both diseases  and is being tested as an adjunct to chemotherapy , .
Recombinant protein vaccines are another option. LepVax  is composed of three different antigens (ML2055, ML2380, and ML2028) that have been shown to provoke a robust immune response (see Chapter 6.3). A Th1-stimulating adjuvant, GLA-SE, is added to the vaccine to elicit a stronger cell-mediated immune response. When C57BL/6 mice were immunized with LepVax and challenged with M. leprae, the bacterial load in the immunized mice was 85% less compared to mice immunized with GLA-SE alone. Another study comparing M. tuberculosis-Ag85B-ESAT6/GLA-SE with M. leprae Ag85B-ESAT6/GLA-SE  showed that the M. tuberculosis formulation protected against M. leprae infection significantly better than the M. leprae formulation, providing optimism for a successful cross-protective vaccine.
Relapse versus reinfection
Currently, there is no clinical methodology for distinguishing between relapse and reinfection in leprosy cases. Relapse is a critical indicator of drug therapy effectiveness, while reinfection is more indicative of active transmission. As there is no way of discerning between the two, both researchers and clinicians struggle to ascertain the effectiveness of a given drug therapy. A few studies in recent years have demonstrated the ability to distinguish between relapse and reinfection by using molecular genotyping techniques, including typing specific single nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTR) in M. leprae , , , , . While these approaches have been shown to be effective, they still face limitations in addressing this issue.
A recent study by Stefani et al.  showed that a whole-genome sequencing analysis of M. leprae from recurrent leprosy patients can characterize the recurrences as relapse, reinfection, or drug resistance. Samples from two of the three patients in the study were passaged in BALB/c mice, and then DNA was extracted and sheared to prepare a library for whole genome sequencing. Overall, they were able to show that two of the three cases of recurrence were due to relapse, while the third was due to reinfection. More studies are needed to adequately address if recurrent cases of leprosy are due to relapse or reinfection, and mice continue to serve as a
n valuable resource in this endeavor.
Limitations of Rodent Models
Although rodents are a vital resource for research, investigators must be cognizant of the immunological differences between rodents and humans. Immunocompetent mice are highly resistant to infection with M. leprae, and they are not an ideal model for the study of nerve infection with the bacilli. They also lack a homologue for granulysin, a key anti-mycobacterial protein generated by human NK and CD8+ T cells  (see Chapter 6.2). In addition, NOS2-generated reactive nitrogen intermediates, which are toxic radicals of activated macrophages that have antimycobacterial properties , , are produced much more robustly by murine macrophages compared to human macrophages , . Mouse strains also vary in their ability to recognize certain mycobacterial antigens such as CFP-10 , . Another drawback to the mouse model is its inability to display the full clinical manifestations of the leprosy spectrum that is seen in humans. Fortunately, genetic knockout strains of mice can address this issue in many cases . Finally, there is no known rodent model, or any animal model for that matter, for leprosy reactions, which is a critical shortcoming for the investigation of these serious phenomena.
Despite these drawbacks, rodents, especially mice, are invaluable models for leprosy studies. With the ease of use, availability of numerous biological reagents, availability of numerous genetically defined strains, and readily assessable granulomatous footpad lesions, mice are ideal for studying the basic immunological parameters of infection. For many years they have also proven useful for the evaluation of potential new anti-leprosy drugs and to determine experimental vaccine efficacy, which further confirms their status as the gold standard animal model for leprosy research.
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