1. Structure
• Recent Research Projects:
– UV or cis-Urocanic Acid Pre-exposure Increases the
Severity of Mycobacterium ulcerans Infection in a Hairless
Guinea Pig Model of Buruli Ulcer Disease;
– An Equine Model of Neurolathyrism and Safety
Assessment of Low Toxin Grass Pea (Lathyrus sativus);
– Some Clinical Toxicology Fun (if there is time);
• Other Research Interests.
1

2. UV or cis-Urocanic Acid Pre-exposure Increases The
Severity Of Mycobacterium ulcerans Infection In A
Hairless Guinea Pig Model Of Buruli Ulcer Disease
Cope R.B.1, P. Small2
1Departmentof Environmental and Molecular Toxicology
Oregon State University
2Department of Microbiology, University of Tennessee
Funding Acknowledgement:
US National Institute of Allergy and Infectious Disease
(NIH)
2

3. Features of Buruli Ulcer Disease
(Bairnsdale Ulcer, Daintree Ulcer)
• Non-HIV associated, emergent, chronic, high morbidity, skin
disease caused by Mycobacterium ulcerans infection;
• A single Buruli ulcer can cover up to 15% of the body surface;
• 92% of ulcers are on the limbs;
• Antibiotic treatment in human cases is often ineffective except
in the early nodular stages of the disease (rifampin, rifapentine
and clarithromycin);
• Mainstay of treatment is surgical removal at the nodular stage.

4. Features of Buruli Ulcer Disease
• Transmission:
– The disease is associated with aquatic and swampy
environments with the mycobacterium occurring in
biofilms, soil, aquatic insects, fish and wildlife;
– Acanthamoeba sp. appear to be the host in the
environment;
– M. ulcerans has been detected in mosquitoes and water
bugs;
– Epidemiology in West Africa favors insect-mediated
transmission;

5. Features of Buruli Ulcer Disease
• Transmission:
HOWEVER:
– Case studies have demonstrated Buruli ulcers developing
after wood splinter  gunshot/land-mine wounds and after
exposure of minor skin wounds to swamp water/biofilms;
– Key factor appears to be any form of skin break that allows
penetration of Acanthamoeba sp. into the dermis.

8. Features of Buruli Ulcer Disease
• Optimal treatment is surgical excision and skin grafting but
results are often poor
– The rate of limb amputation, is about 36% of cases
• 100% amputation rate if osteomyelitis is present
– With optimal surgical treatment, approximately 60% of
patients will have significant permanent reduction in
motion of one or more joints
• 63-76% reduction of motion if knees, elbows or wrists
are involved
• Marked restriction is the most common long-term
outcome when ulceration of the hand is present

9. Features of Buruli Ulcer Disease
• Surgical outcomes:
– Significant cosmetic deformity is the usual outcome;
notably worse when the lesion is located on the head
– Recurrence following surgical excision and multiple new
ulcers possibly due to surgery-induced spread are common
– Average number of operations per patient is 2.4 with a
range of 2-5
– Average period of hospitalization is > 6 months

10. Ulcerative form of M. ulcerans infection

11. Sequelae

12. Pathogenesis
Infection
Non-ulcerative Forms Nodule
Necrotic Phase
(Ulceration, Local Immunosuppression, Impaired T cell Homing)
Plaque/edematous
forms Several months to years?
Ulcer? Reactive Phase (+ve DTH Response)
Months to years?
Ulcer That Does Not Heal Spontaneous Healing?
Stellate Scar

13. KM. George et al (1999), Science, 238: 584

14. Specific Aims
• To test the hypothesis that UV-B pre-exposure
enhances M. ulcerans infection in a
Crl:IAF(HA)-hrBR hairless guinea pig model
of human Buruli ulcer disease
• To test the hypothesis that topical cis-urocanic
acid pre-exposure enhances M. ulcerans
infection in a Crl:IAF(HA)-hrBR hairless
guinea pig model of human Buruli ulcer
disease

15. General Aspects of Experiments
• Used Mycobacterium ulcerans MacCallum et
al (ATCC 35840)
– Virulent mycolactone producing wild-type strain
– Prepared from log-phase growth broth cultures
– Grown at 32OC

16. Experiment 1
Group UV-B Treatment
0 kJ Sham irradiated
3 kJ 1 kJ/m2/day for 3 days
30 kJ 10 kJ/m2/day for 3 days

17. 3.5
3
Log Relative Intensity Count
2.5
2
1.5
1
UVR Source
0.5
Solar Light
0
290 300 310 320 330 340 350 360 370 380 390 400
Wavelength (nm)
Relative Irradiance of the Source Versus Solar Radiation
(Normalized to 1 at 300 nm).

18. Experiment 1
Days 1-3: irradiate
72 hours
S/C infect with 3 x 104 CFU in 0.1 ml PBS in left dorsal (irradiated) flank
S/C inject with 0.1 ml PBS in right dorsal (irradiated) flank
Measure lesions at 7, 10, 14 and 21 days post-infection
Day 21:I/D challenge with 0.02 μg burulin S in 0.1 ml PBS in left flank
Sham challenge with I/D 0.1 ml PBS in right flank
24 hours
Read DTH responses; kill and collect samples for histopathology

19. Results
• 30 kJ/m2 UV-B dose induced erythema/edema of the ear tips,
but not the dorsal mid-line;
• M. ulcerans contaminated injection sites showed delayed
healing. No grossly detectable skin lesions were present at any
of the PBS injected sites at 24 hours post-injection;
• M. ulcerans infected sites developed distinct, clearly-
demarcated, subcutaneously situated skin nodules by day 7
post-infection.

20. Effect of UV-B Pre-Exposure on the Healing of
M. ulcerans Contaminated Injection Wounds
1
Probability of Wound Survival
0.8
0.6
p = 0.0097
0.4
p = 0.009
0 kJ
0.2 3 kJ
30 kJ
0
0 5 10 15 20 25
Days Post Injection

21. Effect of UV-B Pre-Exposure on the Size of
M. ulcerans Induced Skin Nodules
16
0 kJ p < 0.004
14 3 kJ UV
Diameter, mm +/- SEM, n = 5
p < 0.009
Mean Maximum Nodule
30 kJ UV p < 0.026
12 p < 0.004
p < 0.021
p < 0.010
10
p < 0.079
p < 0.195
8
6
4
2
0
Day 7 Day 10 Day 14 Day 21
Days Post-Infection

22. Effect of UV-B Pre-Exposure on DTH Responses to
Burulin-S at 21 Days Post-Infection
12
p < 0.001 0 kJ
10 p = 0.002 3 kJ
Mean Diameter of Induration,
30 kJ
mm +/- SEM, n = 5
8
p = 0.035
6
4
2
0

23. Experiment 2
Group UV-B Treatment
0 kJ Sham irradiated
3 kJ 1 kJ/m2/day for 3 days
24 kJ 8 kJ/m2/day for 3 days

24. Experiment 2
Days 1-3: irradiate
72 hours
I/D infect with 2 x 106 CFU in 0.1 ml PBS in left dorsal (irradiated) flank
I/D inject with 0.1 ml PBS in right dorsal (irradiated) flank
Measure lesions at 4, 7, 10, 18, 23, 30 days post-infection
Day 21:I/D challenge with 0.02 μg Burulin S in 0.1 ml PBS in left flank
Sham challenge with I/D 0.1 ml PBS in right flank
24 hours
Day 22: Read DTH responses
Day 30: Kill and collect histology samples

25. Results
• 24 kJ/m2 UV-B dose was sub-inflammatory;
• M. ulcerans inoculated sites developed distinct, clearly-
demarcated skin nodules by day 4 post-infection;
• Injection sites did not heal;
• All animals able to mount a DTH response to burulin-S @ 21
days post-infection. UV-B pre-exposure had no significant (p
> 0.1) effect on DTH responses.

26. Day 15

27. The Effects of UV-B Pre-Exposure on M. ulcerans Ulcer
Development Time
20
p = 0.024
18 0 kJ
16 3 kJ
Time, Days +/- SEM n = 6
Mean Ulcer Development
24 kJ
14
12
10
8
6
4
2
0

28. Effect of UV-B Pre-Exposure on the Size of M. ulcerans
Induced Skin Lesions
25
p ≤ 0.035 p ≤ 0.035
0 kJ p < 0.035 p < 0.035 p ≤ 0.05
3 kJ
Mean Lesion (Induration + Ulcer)
20 p = 0.028 p = 0.031
24 kJ
Diameter +/- SEM, n = 6
15
p = 0.023
p = 0.338 p = 0.094
10 p = 0.154
5
0
Day 4 Day 7 Day 10 Day 14 Day 18 Day 23 Day 30
Days Post-Infection

29. Protein
Catabolism
(particularly filaggrin: histidine-rich
protein that is a cementing substance
for the tonofibrils in the keratin
complex of the stratum corneum)
L-Histidine
NH2
UV

30. Experiment 3
Group Topical Treatment
1 No treatment
2 Vehicle only
3 0.1 mg trans-urocanic acid
4 0.5 mg trans-urocanic acid
5 1 mg trans-urocanic acid
6 0.1 mg cis-urocanic acid
7 0.5 mg cis-urocanic acid
8 1 mg cis-urocanic acid

31. Experiment 3
Days 1-3: topical treatments
72 hours
I/D infect with 1.5 x 107 CFU in 0.1 ml PBS in left dorsal flank
I/D inject with 0.1 ml PBS in right dorsal flank
Measure lesions at 5, 10, 15, and 21 days post-infection
Day 21:I/D challenge with 2 μg M. ulcerans cell wall fragments in 0.1 ml PBS in left
flank. Sham challenge with I/D 0.1 ml PBS in right flank.
24 hours
Day 22: Read DTH responses
Kill and collect histology samples

32. 2
Product of Lesion Perpendicular Diameters mm
Mean +/- SEM; n = 6
50
100
150
200
250
300
0
Negative Control
Vehicle Only
0.1 mg Trans
P > 0.6
0.5 mg Trans
1 mg Trans
Day 5
P > 0.001
0.1 mg Cis
0.5 mg Cis
P > 0.4
1 mg Cis
Negative Control
Vehicle Only
0.1 mg Trans
P > 0.8
0.5 mg Trans
1 mg Trans
Day 10
0.1 mg Cis
P > 0.001
0.5 mg Cis
P > 0.4
1 mg Cis
Negative Control
Vehicle Only
0.1 mg Trans
P > 0.8
0.5 mg Trans
1 mg Trans
Day 15
0.1 mg Cis
P > 0.001
0.5 mg Cis
P > 0.4
1 mg Cis
Negative Control
Vehicle Only
0.1 mg Trans
P > 0.8
0.5 mg Trans
1 mg Trans
P > 0.001
Day 21
0.1 mg Cis
0.5 mg Cis
P > 0.4
1 mg Cis

33. Maximum Diameter of Induration (mm)
Mean +/- SEM; n = 6
10
12
14
16
0
2
4
6
8
Negative Control
Vehicle Only
0.1 mg Trans
P > 0.9
0.5 mg Trans
1 mg Trans
P < 0.001
0.1 mg Cis
24 Hours Post-Challenge
0.5 mg Cis
P > 0.9
1 mg Cis
Negative Control
Vehicle Only
0.1 mg Trans
P > 0.9
0.5 mg Trans
1 mg Trans
P < 0.001
0.1 mg Cis
48 Hours Post-Challenge
0.5 mg Cis
P > 0.6
Effect of Topical Treatments on DTH Responses to M.
1 mg Cis
ulcerans Cell Wall Fragments at 21 Days Post-Infection

34. Summary
Variable Nodular Disease Ulcerative Disease
↑ Nodule size
↑ Lesion size
Delayed healing of
UV
injection sites
No effect on DTH
↓ DTH
↑ Overall lesion size
Cis-Urocanic Acid --
↓ DTH

35. Proposed Mode of Action
UV-cUCA- Neuropeptide-Histamine-Prostanoid Photoimmunosuppression Pathway.
Dermal non-myelinated sensory
Trans-UCA cis-UCA c-type nerve fibers Calcitonin gene related peptide
UV Keratinocytes Nerve growth factor Dermal mast cell degranulation
Tumor necrosis
factor alpha
Histamine
Green = fast pathway ( minutes to hours)
Blue = slow pathway (8-12 hours) Cyclooxygenase/Prostanoid dependent pathway
Photoimmunosuppression
35

36. Questions?
36

37. Current Research Project:
An Equine Model of Neurolathyrism
Safety Assessment of Low Toxin Grass Pea (Lathyrus sativus)
Collaborators: Prof. Peter Spencer and Dr Valerie Palmer, OHSU
Funding Acknowledgement: Third World Medical Research Foundation
37

38. Neurolathyrism
 Neurolathyrism is a neurodegenerative spastic paralysis caused by
consumption of grass pea (L. sativus)
 Toxin is β-N-oxalyl-α,β-diaminopropionic acid (β-ODAP)
[Synonym β-oxalylaminoalanine (BOAA)]
 Rumors about other undefined neurotoxic nitriles
38

39. CH2 CH COOH CH2 CH COOH
NH NH2 CH2 NH2
CO COOH
COOH
β-ODAP L-Glutamate
39

40. Glutamatergic
input BOAA, SCN-
3-nitropropionic acid
domoic
acid
Soma NMDA AMPA KA
Axon
Dendrite
Mito
Excito toxcity
Soma
Axon
Na+ H2O Ca2+
Cellular target is the Ca2+ alpha-amino-3-hydroxy-5-methyl-4-isoxazole
propionic acid receptor (AMPA receptor)
Methionine and cysteine are protective
40

41. History
• Ancient disease: oldest definitively described neuro-toxic
disease
– The disease was known to ancient Hindus, to Hippocrates
(460–377 BC), Pliny the Elder (23–79 AD), Pedanius
Dioskurides (50 AD) and Galen (130–210 AD)
– Outbreaks of neurolathyrism have been systematically
reported since the 1700’s in:
•Bangladesh •Romania
•India, •Spain
•Pakistan, •Ukraine
•France •Afghanistan
•Italy •China
•Germany •Algeria
•Romania •Ethiopia
•Spain •Eritrea
•South Sudan Gracias a la almorta.
Francisco Goya 41

42. Known Recent epidemics
• 1931-1932: Ukraine (The Holodomor)
• 1941-1946: Spain
• 1973: China
• 1976: Bangladesh
• 1976: Ethiopia
• 1998: Nepal (non published)
• 1998: Afghanistan (non published)
• 1997-99: Ethiopia/Eritrea
• Present: Afghanistan
• Present: Somalia (Darfur) and South Sudan
42

43. Afghanistan 2005 43

44. Bangladesh 2006 44

45. Current Prevalence
• Ethiopia 6 per 1000
• India 5.3 per 1000
• Bangladesh 1.4 per 1000
45

46. Why Grass Pea?
• Often the last source of food available during a
drought
• Often the first food available following the end of a
drought
• Starvation food source
46

47. Why Grass Pea?
• Particularly useful food crop if it can be detoxified
• High protein: source of protein and essential amino acids in areas
where protein malnutrition is comon and there are few protein sources
• Resist both water shortage and water logging i.e. survives both
droughts and floods
• Easy to grow
– Requires no fertilizers
– Requires no insecticides
– Requires no machinery
– Improves soil fertility (nitrogen fixer)
– Compatible with rice production
47

48. Why Grass Pea?
• High protein/energy supplement for ruminants: aid to
goat and cattle production in arid/semi-arid areas
• Detoxified grain may be suitable for poultry, hogs and
fish: major source of protein in many areas in Africa
and Asia
48

49. Risk factors
• Heavy physical activity
• Male gender
• Young age (15-25 years)
49

50. Risk factors
• Low cysteine and methionine intake
• Consumption of 400 g of grass pea for more than 2
weeks (acute disease can occur with higher
consumption for shorter period)
• Notably, grass pea eaters appear relatively well-
nourished when compared with the general
population (starving)
50

51. Clinical features
• Permanent “non-progressive”* upper motor neuron disorder
with symmetrical spastic paralysis
– (*Evidence of sensorimotor neuropathy and signs of muscle denervation almost
30 years after the onset of the disease in 7% of Romanian Jews affected by
neurolathyrism)
• Largely pyramidal distribution
– Leg weakness
– Exaggerated thigh, adductor, patellar and ankle reflexes
– Bilateral presence of ankle clonus and extensor plantar
reflexes/Babinski responses
– Scissor/cross-legged sitting posture and gait
51

53. Clinical features
• Sensation, sphincter, cerebellar, cranial nerve and
cognitive/cortical functions are are spared
• Changes are permanent
• Does not appear to affect life-span
• No treatment
53

54. Problems/Research Opportunities
• Little or no human pathology
• Published reports are rare and mostly examined cases 30+
years after the onset of disease (predominantly from the
epidemic in Spain in the 1940’s)
• No systematic neuropathology studies
54

55. Problems/Research Opportunities
• Available neuropathology studies, which have focused on the
spinal cord, show predominantly distal symmetrical
degeneration of lateral and ventral corticospinal tracts, distal
degeneration of spinocerebellar and gracile tracts and Hirano
bodies in anterior horn cells with no reduction of the lower
motor neuron pool
• MRI has never been performed (Amazing!)
• Pathology resembles primary lateral sclerosis
(LMN pool is preserved)
55

56. Research Opportunities
• Rodent models:
– Good models of osteolathyrism/vascular lathyrism
(β-aminopropionitrile, Lathyrus odoratus, sweet pea)
(Hydrazine compounds [semicarbazide], isoniazid)
NOT RELEVENT TO NEUROLATHYRISM
– Repeated injection of β-ODAP results in rats with paraparesis of
the legs, though at a low incidence rate of 0.032. These paralyzed
rats displayed severe atrophy of the ventral root of the lumbar cord
as well as degeneration of lower motor neurons.
PATHOLOGY DOES NOT MATCH THE HUMAN DISEASE
(HUMAN DISEASE IS EXCLUSIVELY UMN) 56

57. Research Opportunities
• Low β-ODAP seeds: NO SAFETY ASSESSMENT –
ARE THESE GRAINS REALLY SAFE?
(the mysterious “other neurotoxic nitriles”)
• CURRENTLY NO GOOD ANIMAL MODELS
57

58. Research Opportunities
• Potential animal models
– Domestic animals: disease occurs in horses, cattle, sheep,
pigs, elephants, ducks, geese, hens, and peacocks
– Sheep are relatively resistant: tolerate a diet of 50% grass
pea with no problems
– Reports of neurolathyrism in species other than the horse
and sheep are anecdotal, incomplete and old
(more mythology than science?)
58

59. Research Opportunities
• Potential animal models: Macaque
• Onset of disease depends on dose: can reproduce disease via
sub-acute high dose or chronic low dose
• Produces pyramidal disease that resembles the human disease
59

60. Research Opportunities
• Unlike humans, NEUROLOGICAL IMPROVEMENT
OCCURRED after cessation of grass pea administration
• Neuropathology studies showed no evidence of neuronal
degeneration in motor cortex or spinal cord
• Electrophysiology in macaques does not match the human
disease
60

61. Problems/Research Opportunities
• Potential animal models: Horse
• Horse is reputed to be the most susceptible species: Stockman
(1929)
• Diet made exclusively of L. sativus precipitated signs
after 10 days.
• Horses fed only 1–2 quarts per day succumbed after 2–3
months, and neurological manifestations appeared a
month or more after the diet was withdrawn
• Neurological changes were permanent
• Natural cases of neurolathyrism occur sporadically in
horses in eastern Oregon
61

63. Current Research Objectives
• Establish an equine model of neurolathyrism
– Reproduce clinical neurolathyrism in miniature
horses using high β-ODAP and derive the Bench
Mark Dose
– Systematic neuropathology examination of the
equine disease
– Systematic electrophysiological examination of the
equine disease
63

64. Other Research Interests
• Chemoprevention of ethanol-induced
hepatotoxicity
• Computational/in silico
toxicology/alternatives to in vivo testing
• In vitro alternatives to OECD toxicology
testing
64

65. Questions?
65

66. Clinical/Diagnostic Toxicology Fun
What is your diagnosis?
(Unlikely to occur in Australia)
66

67. 67

68. 68

69. 69

70. Timber (Canebrake) Rattlesnake
(Crotalus horridus) 70

71. A gentle reminder that in companion animals, 71
many snake bites occur on the head!