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The contemporary relevance of the mouse foot pad model for

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									                  Lepr Rev (2009) 80, 120– 123

                  ED IT ORI A L

                  The contemporary relevance of the mouse foot pad
                  model for cultivating M. leprae

                  V. M. KATOCH
                  Department of Health Research (Govt of India) & Indian Council
                  of Medical Research, Ansari Nagar, New Delhi-110029, India

                  Accepted for publication 28 May 2009

The fight against leprosy has been one of the major success stories of modern medicine. Even
though M. leprae, the causative organism of leprosy, could not be grown in any acceptable
in-vitro medium system, the microbiological need was fulfilled by the development of
various animal models.1 This successful journey began with the report of limited
multiplication of M. leprae in the footpads of ‘CFW’ mice.2 The findings of Shepard were
confirmed by Rees.3 During the last nearly 50 years, the mouse foot pad (MFP) technique has
been extensively evaluated and used. The inbred strains of BALB/C and CBA mice have been
observed to be more susceptible to infection with M. leprae than the C57 BL and C3H mouse
strains. The MFP technique has been used for viability determination, drug screening,4 testing
immunoprophylactic agents, undertaking studies on determination of minimum inhibitory
concentration (MIC) and minimum effective dose of various anti-leprotic compounds, and
also for identification of M. leprae. The pattern of applications has changed from decade to
decade; in the 1960s and 1970s it was used for viability determination, drug screening and
experimental chemotherapy. The information derived from the mouse foot pad about the
effect of single versus continuous administration in animals, and later in human beings, led to
the designing of the WHO recommended multidrug treatment (MDT) for leprosy, comprising
rifampicin, clofazimine and dapsone. These MDT regimen have been key in reducing the
global leprosy problem. During the 1970s and 1980s, the focus of interest shifted to using this
model for testing mycobacteria which could be used as immunotherapeutic and/or
immunoprophylactic agents against leprosy. Identification of BCG, heat killed M. leprae,
ICRC, M. habana, and M. w. are success stories which began with identification of the
protective effect of these mycobacterial strains against leprosy in the mouse foot pad model.
The MFP enabled the studies on the effectiveness of pulsed administration of rifampicin
ofloxacin and minocycline against M. leprae. The growth characteristics and the involvement
of nerve twigs in the mouse foot pad were used as a marker of identification of M. leprae from
the 1960s to the 1990s. During the 1980s the greatest number of publications on different

   Correspondence to:;

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                                      The mouse foot pad model for cultivating M. leprae        121

applications of the MFP were seen with a subsequent decline in interest with the major
successes in leprosy reduction achieved with MDT.
    Major changes have occurred in the disease burden and clinical profile, and there has been
a paradigm shift in the approach resulting from advances in molecular biology and genomics.
Currently, the number of leprosy cases has declined in many countries, and highly bacillated
cases (. 3 þ BI) are contributing a much smaller proportion (, 10%) of the case load.5
There is intense debate going on all over the world as to whether countries and organizations
should continue to support mouse foot pad laboratories which are currently being maintained
by various governments or non governmental organizations. In this context, it is important to
revisit the original application areas of the MFP model:

   i) Viability determination: The MFP was extensively used for assessing and monitoring
      the viability of leprosy bacilli for developing chemotherapy trials and also for studying
      the viability of M. leprae outside the human body.6 While this method was important for
      this research work, the MFP model now is of limited use. Firstly, most patients now
      have a low bacterial load and probably 90% do not have sufficient organisms i.e. about
      5– 10 000/MFP as AFB could be detected in 94% of slit smears in leprosy in a
      population survey from Ghatampur, North India (Kiran Katoch, unpublished data,
      ICMR Taskforce study report 2008). While some investigators have been able to grow
      M. leprae even from paucibacillary smear negative specimens,7 so far these are isolated
      reports. Secondly, there are now validated rapid alternatives for assessing M. leprae
      viability, methods such as ATP bioluminescence,8 PCR,9 RT-PCR10 and real time PCR
      targeting RNA.11 While techniques like ATP bioluminescence, which are based on
      estimation of bacterial ATP and require . 100 live bacilli per specimen, molecular
      methods targeting M. leprae specific DNA,9 or RNA10,11 are able to detect less than
      10 organisms. Such molecular methods are also useful to detect live M. leprae in the
  ii) Screening for Drug resistance: The MFP model was used extensively to determine
      primary and secondary resistance to anti leprosy drugs. While there are some reports of
      drug resistance to one or more drugs,13 the widespread use of MDT has significantly
      reduced the prevalence of drug resistance. Further, because of the requirement of a
      minimum size inoculum (5 –10 000 organisms) for a normal MFP experiment with 5 –6
      mice/specimen, this technique per se has limited usefulness. While MFP has a role in
      defining the ‘viable unit’ for alternate in vitro assays14 – 16 for the purpose of detection of
      drug resistance, various molecular methods detecting DNA mutations have immense
      superiority in terms of feasibility, easiness of application and very high potential
      sensitivity.9,17,18 These methods are good for detecting rifampin resistance genes but
      limited in detecting resistance genes for other anti-leprosy drugs.
 iii) Testing for immunoprophylactic agents: The MFP model has been useful in the
      identification of immunotherapeutic/immunoprophylactic agents against M. leprae.
      While this model is still being used for testing new vaccine candidates,19 it is
      questionable whether we are interested in identifying more agents at present. There is
      now only limited interest in this area.
 iv) Studying Immunopathogenesis: During the last decade, many studies using MFP
      have been looking at the mechanisms of pathogenesis and protection in leprosy
      by dissecting the cells and pathways of the immune response,20 and also studying host-
      parasite interactions using genomic approaches.21 For investigators interested in
122          V. M. Katoch

         investigating the biology of M. leprae and host pathogen interactions, the MFP will
         continue to be useful.
      v) Experimental chemotherapy: As the interest in identifying newer anti leprotic
         compounds has considerably decreased, this application will only be of interest
         to specialised research laboratories who continue to work on identifying newer
         antimycobacterial drugs that could be useful against a variety of infections including
     vi) Identification of M. leprae: After the development of several probes and gene
         amplification methods for M. leprae,9 the need to grow M. leprae in MFP for
         identification purposes is merely of academic interest and no longer important.

To conclude, the usefulness and spectrum of application of the mouse foot pad has drastically
changed over the years. While this model continues to be relevant for selective research
purposes such as understanding the host parasite interaction, confirmation of activity of newer
compounds and also experimental chemotherapy, other earlier applications, such as viability
determination and identification of M. leprae for diagnostic and epidemiological purposes,
can be more effectively done by alternate methods. For growing M. leprae in sufficient
quantities, one needs immuno compromised animals and other animals like armadillos. All
these issues have been extensively debated over the years.1,8,9,17,22 Leprosy is down but not
yet gone. It is true that fewer researchers are interested in the applications of MFP but the
need is still there. It would be important to maintain a few MFP laboratories in some selected
countries/institutions to continue working on currently relevant aspects. At least one MFP
laboratory in countries like India, Brazil, Nepal, Ethiopia, UK, The Netherlands and the USA
should be supported with the support of national, international or non-governmental agencies.

     Gupta UD, Katoch VM. Animal models in leprosy. Ind J Med Microbiol, 1995; 13: 57 –64.
     Shepard CC. The experimental disease that follows the injection of human leprosy bacilli into foot pads of mice.
     J Exp Med, 1960; 112: 445.
     Rees RJW. Limited multiplication of acid fast bacilli in the foot pads of mice inoculated with M. leprae. Br J Exp
     Pathol, 1964; 45: 207–218.
     Shepard CC, Chang YT. Effect of several anti-leprosy drugs on multiplication of human leprosy bacilli in foot
     pads of mouse. Proc Soc Exp Biol Med, 1962; 109: 636– 638.
     Arora M, Katoch K, Natrajan M et al. Changing profile of disease in leprosy patients diagnosed in a tertiary care
     centre during 1995– 2000. Ind J lepr, 2008; 80: 257–265.
     Desikan KV. Viability of M. leprae outside the human body. Lepr Rev, 1977; 48: 231–235.
     Wakade AV, Shetty VP. Isolation of Mycobacterium leprae from borderline tuberculoid, mid borderline and
     indeterminate cases using normal mouse foot pad technique – a study of 209 cases. Lepr Rev, 2006; 77: 366– 370.
     Katoch VM, Sharma VD. Recent advances in the microbiology of leprosy. Ind J Lepr, 2000; 72: 363– 379.
     Katoch VM, Lavania M, Chauhan DS et al. Recent advances in molecular biology of leprosy. Ind J Lepr, 2007;
     79: 151 –166.
     Jadhav RS, Kamble RR, Shinde VS et al. Use of reverse transcription polymerase chain reaction for detection of
     Mycobacterium leprae in slit skin smears of leprosy patients. Ind J Lepr, 2005; 77: 116–127.
     Sharma R, Lavania M, Katoch K et al. Development and evaluation of Real-Time RT-PCR assay for quantitative
     estimation of viable Mycobacterium leprae in clinical samples. Ind J Lepr, 2008; 80: 315–321.
     Lavania M, Katoch K, Katoch VM et al. Detection of viable Mycobacterium leprae from environmental soil
     samples: insights into possible sources for transmission of leprosy. Infect Genet Evol, 2008; 8: 627–631.
     Norman G, Joseph G, Ebenzer G et al. Secondary rifampin resistance following multi-drug therapy – a case
     report. Int J Lepr Other Mycobact Dis, 2003; 71: 18–21.
     Agrawal VP, Shetty VP. Comparison of radiorespiromteric Budemeyer assay, ATP assay and mouse foot pad test
     in detecting viable Mycobacterium leprae from clinical specimens. Ind J Med Microbiol, 2007; 25: 358–363.
                                             The mouse foot pad model for cultivating M. leprae             123
     Levy L, Ji B. The mouse foot pad technique for cultivation of Mycobacterium leprae. Lepr Rev, 2006; 77: 3–4.
     Gupta UD, Katoch K, Sharma RK et al. Analysis of quantitative relationship between MFP, ATP bioluminescence
     and gene amplification assay. Int J Lepr Other Mycobact Dis, 2001; 69: 328– 334.
     Williams DL, Gillis TP. Molecular detection of drug resistance in Mycobacterium leprae. Lepr Rev, 2004; 75:
     118 –130.
     Sapkota BR, Ranjit C, Macdonald M. Reverse line probe assay for rapid detection of rifampicin resistance in
     Mycobacterum leprae. NMCJ, 2006; 8: 122–127.
     Roche PW, Neupane KD, Failbus SS et al. Vaccination with DNA of the Mycobacterium tuberculosis 85B antigen
     protects mouse foot pad against infection with M. leprae. Int J Lepr Other Mycobact Dis, 2001; 69: 93–98.
     Hagge DA, Saunders BM, Ebenzer GJ et al. Lymphotoxin alpha and TNF have essential dependence but
     independent roles in the evolution of the granulomatous response in experimental leprosy. Am J Pathol, 2009;
     174: 1379–1389.
     Williams DL, Torrero M, Wheeler PR et al. Biological implications of Mycobacterium leprae gene expression
     during infection. J Mol Microbiol Biotechnol, 2004; 8: 58– 72.
     Also Special volumes/issues of International Journal of Leprosy 1987; Leprosy Review 1986, 57(suppl 3) and
     (Indian Journal of Leprosy 1991).

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