Armadillo: An emerging animal model for leprosy

Rahul Sharma U.S. Public Health Service National Hansen’s Disease Program LSU School of Veterinary Medicine Skip Bertman Drive Baton Rouge, LA 70803

EXPANDED RANGE OF ARMADILLO IN USA

150 yrs

•Develops fully

disseminated Disease

with extensive nerve

involvement

Nine banded armadillos

(Dasypus novemcinctus)

The only natural hosts

(other then Human) of M.

leprae.

HUMAN INTERECTION

The End

M. leprae Typing Scheme

3-I-2-v1 64%

3-I-2-v1 84%

Human

Armadillo

3-I-2 100%

ARMADILLOS: SOURCE OF ZOONOTIC TRANSMITION OF LEPROSY TO HUMANS

April 27, 2011

88% of Armadillos and 64% of US Human cases With endemic exposure Share the Sylvan strain

VN

TR

N Engl J Med 2011; 364:1626-1633

SNP

M. leprae: The causative agent of leprosy

Only known bacteria capable to invade nerves and induce neuro-

degeneration.

Still not cultivable in-vitro.

Available anti leprosy treatment (MDT) is very efficient bactericidal,

DOESN’T HELP IN NERVE DAMAGE.

Human nerve biopsy: un-ethical, not suitable for Molecular studies.

No other animal model develops leprosy and nerve damage.

ARMADILLO: The only host other than human.

The only immunologically intact host of M. leprae also recapitulates

leprosy as seen in human.

Host of choice to propagate M. leprae for research purpose.

The only animal model to study nerve damage in leprosy.

Genetically Identical quadruplets

Genome sequence available

LEPROSY AND ARMADILLO

Clinical symptoms

Nerve Physiology : MNCV and CMAP

Disseminated Infection

Morphometry

Nerve Fiber density

Comparative Histopathology

Gene expression profile

Global: Cross Species Microarray

Selected Marker: Inflammation, Nerve damage and Growth Factors

CHARACTERIZING THE NEUROPATHY IN ARMADILLOS

CLINICAL LEPROSY IN

HUMAN AND ARMADILLOS

Similar Ulceration in Human and Armadillo feet

Armadillo feet in late stage of laboratory infection

Human patient feet

NERVE FUNCTION ABNORMALITY

Motor Nerve Conduction Velocity (MNCV) and Compound Motor Action Potential (CMAP)

Standard technique identifies MNCV of <49m/sec as abnormal. Results of our sampling similarly suggest armadillo MNCV <40 m/s and CMAP <0.9mV are out of normal ranges.

Naïve Early infection

(6-9months)

Late infection (>2years)

MNCV CMAP MNCV CMAP MNCV CMAP

1

1-3 cm distal half of post tibial nerve

4-6 cm proximal half of post tibial nerve

Orientation of Posterior Tibial Nerve

Endoneurium Epineurioum

Ave

rag

e N

um

be

r o

f L

eu

ko

cyte

s

0

1

2

3

4

5

INFECTED

NAIVE

Degree of Inflammation: leukocyte counts in the posterior tibial nerve

Bacilli count in armadillo nerves by Q-PCR

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06T1

LtP

1-3

T1 L

tP 4

-6

T1 R

tP 1

-3

T1 R

tP 4

-6

T2 L

tP 1

-3

T2 L

tP 4

-6

T2 R

tP1-

3

T2 R

tP4-

6

T3 L

tP1

-3

T3 L

tP4

-6

T3 R

tP1-

3

T3 R

tP4-

6

T4 L

tP1

-3

T4 L

tP4

-6

T4 R

tP1-

3

T4 R

tP4-

6

T5 L

tP1

-3

T5 L

tP4

-6

T5 L

tP1

-3

T5 L

tP4

-6

I6 L

tP1

-3

I6 L

tP4

-6

I6 R

tP1

-3

I6 R

tP4

-6

I7 L

tP1

-3

I7 L

tP4

-6

I7 R

tP1

-3

I7 R

tP4

-6

I8 L

tP1

-3

I8 L

tP4

-6

I8 R

tP1

-3

I8 R

tP4

-6

I9 L

tP1

-3

I9 L

tP4

-6

I9 R

tP1

-3

I9 R

tP4

-6

I10

LtP

1-3

I10

LtP

4-6

I10

RtP

1-3

I10

tR

P4-

6

16S rRNA qRT-PCR

Treated with Rifampicin after developing the disease

Not treated after developing the disease

RLEP qPCR

Total number of myelinated fibers: Infected < naïve

Distribution of fibers: Infected: bimodal, Naïve: Single peak

De-myelination in infected Armadillo nerves

Elementary Morphometry To

tal n

um

be

r o

f fi

ber

s

Diameter of myelin around the axon (micron)

Lower* ENDF among infected animals

Epidermal Nerve Fiber Density (ENFD)

immunostained with PGP9.5, a neuronal marker

*not statistically significant

Cross species microarray hybridization

GENE EXPRESSION PROFILING OF INFECTED ARMADILLO NERVE

Infected Naïve

Human microarray (44K)

Functional classification of differentially regulated genes (>4fold)

Nerve damage in leprosy is a complex process involving various pathways

Neuregulin Signaling

MAPK Signaling

Chemokine Signaling

Ingenuity Pathway Analysis

Genomic Target Function Differential expressed in Condition Reference:

Beta III tubulin stability of the dimer in the microtubule. Response to NGF, colchicines, and mechanical injury J Cell Biol:112:303-312

Brain Res (2006)

1117:80-91

(GFAP) Glial

Fibrillary Acidic

Protein

Cell structure, movement,

communication, mitosis, and blood brain

barrier.

Burn injury BMC Infect Dis. 2008

Jun 24;8(1):84

MAG (Myelin-

Associated

Glycoprotein)

M yelination during nerve regeneration. Apotransferrin (aTf) Stimulation J Neurosci :2006;

26:7532-40

MBP

(Myelin Basic Protein) myelination of nerves CNS myelinogenesis

Physiol Behav. 2007 Sep

10;92(1-2):46-53

NCAM (Neural Cell

Adhesion Molecule)

neurite outgrowth during optic nerve regeneration Exp Neurol 2003, 182

(1):180-5

N-Cadherin At certain CNS synapses, presynaptic to

postsynaptic adhesion Regenerating visual structures

Exp.

Neurol.2002:177: Page

: 396-406

Neurofilament

major determinant of axonal caliber Accumulation of neurofilaments in neurons

Brain Res. (2000)

2;866(1-2):326-32.

Neuragulin development of the nervous system. Response to Schwann cell-derived neurotrophic factors J. Neurosci 2004:24:

6218-27

PGP9.5 (UCHL1)

normal synaptic and cognitive function Human prostate cancer cells express

Prostate. 2007 Dec

1;67(16):1761-9

PMP 22 (Peripheral

Myelin Protein) Expressed mainly in Schwann cells during de-myelination

J of Anatomy (2002)

200: 377 - 390

NGF β survival and maintenance of sympathetic

and sensory neurons. Traumatic Brain Injury J Trauma. 2008 Jun 19

DLK-1 Required for activation of MAPK Nerve Re generation Cell 2009;138:1005-

1018.

Neuronal markers selected for quantitative RT-PCR:

Development of quantitative RT-PCR

Human gene sequence was used as template to Retrieved Armadillo gene sequence

was used to design the Taq Man probe and primers.

Relative quantification by using ΔΔCt method

GAP3DH was used as normalizer

-2

-1.5

-1

-0.5

0

0.5

1

1.5

Log

RQ

Left distal half Left proximal half Right distal half Right proximal half

Differential expression of selected nerve marker during the active infection with M. leprae compared to naïve Armadillo nerve (n=5)

Infected vs Naive

• Inflammation,

• Upregulation of neuronal component ( Active damage / Repair effort)

• No activation of MAPK (no Regeneration)

armadillos treated with Rifampicin for one year compared to naïve armadillo

-2.5

-2

-1.5

-1

-0.5

0

0.5

1

PGP9.5 PMP 22 B Tubulin Neurofilament NCAM NGF B MAG DLK1 IFN TNF

Log

RQ

Left distal half Left proximal half Right distal half Right proximal half

After treatment

• Consistent Inflammation even after treatment

• No activation of MAPK (no Regeneration)

Intra-neural Inoculation of M. leprae in Nine-banded

Armadillos (Dasypus novemcinctus).

Surgical removal, Orientation and gene expression

at various time points after infection

AFB and histopathology

Molecular enumeration

Q-RT-PCR

• In deep anaesthesia, post tibial nerve

was exposed

• Total 107 M. leprae were injected

directly into the nerves of 14 armadillos.

• Nerves were surgically removed in pairs

at 3, 7, 14, 21, 30, 60 and 90 days post

inoculation.

A

Fig 2

A

Enumeration of M. leprae at different time points after intraneural innoculation

3 Day

s

17 D

ays

21 D

ays

30 D

ays

60 D

ays

90 D

ays

Baculli /

cm

of

nerv

e

1e+1

1e+2

1e+3

1e+4

1e+5

1e+6

1e+7

Distal 3 cm

Poteriar 3 cm

Greater involvement of distal nerve

No Acid Fast Bacilli (AFB) in microscopy.

Total number of bacteria retain in the nerve (~ 9.18+3 M. leprae ) was significantly lower

than natural or intra-venous Infection (9.3E+04 bacilli/mm of nerve).

IFN-

Nai

ve

3 Day

s

17 D

ays

21 D

ays

30 D

ays

60 D

ays

90 D

ays

Lo

g R

Q

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

TNF-

Nai

ve

3 Day

s

17 D

ays

21 D

ays

30 D

ays

60 D

ays

90 D

ays

Lo

g R

Q

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

2.0

DLK-1

Nai

ve

3 Day

s

17 D

ays

21 D

ays

30 D

ays

60 D

ays

90 D

ays

Lo

g R

Q

-2.5

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

NGF

X Data

Naive

3 Days

17 Days

21 Days

30 Days

60 Days

90 Days

Lo

g R

Q

-4

-3

-2

-1

0

1

A B

C D

Higher cytokine production

Down regulation of Regeneration and Growth factors

CONCLUSIONS: Nerve damage in leprosy is the result of :

Inflammation,

Demyelination / degeneration

Failure of regeneration efforts

Armadillo is an animal model for:

Study molecular events associated with neuropathy.

reproducing the observation seen in-vitro and other

in-vivo intervention studies.

Test any neuro-regenerative or protective Agents.

Acknowledgments:

Dr. R. Truman, PhD Dr. D Williams, PhD Dr. T Gillis, PhD Dr. R Lahiri, PhD

Dr. D Scollard, PhD

Maria Penna Tana Pittman Kile Andrew Ale All others

Armadillo Contract and NHDP for grants