Presenter: Owen Paiva1

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The New Guinea Small-eyed Snake - Micropechis
ikaheka A Case Study of Basic Research and
Antivenom Suitability

• Presenter Owen Paiva1
• Mr. Owen Paiva1, Assoc. Prof. Teatulohi
  Matainaho1, Mr. David Williams2, Prof. Martin
  Lavin3, Dr. Geoff Birrell3, Dr. Liam St. Pierre3,
  Dr. Stephen Earl3
• UPNG School of Medicine Health Sciences1,
  University of Melbourne Australian Venom Research
  Unit2, Queensland Institute of Medical Research3

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Outline

• Papua New Guinea
• Venomous snakes of PNG
• New Guinea Small-eyed snake
• Associated problems with New Guinea small-eyed
  snakebite
• Aim, Objectives
• Methods 2D-PAGE Immunoassays
• Results of both experiments
• Summary
• Conclusion
• Acknowledgements

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• Papua New Guinea (PNG) is a country diverse in
  people, culture, language and geography
• The terrain is covered mostly by rugged
  mountains, tropical rainforests, coastal
  lowlands, swamps, and rolling foothills (with
  very little road network and poor transportation
  means in many areas)

PNG Business Directory, http//www.pngbd.com
Encarta 2006
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PNG - Fast Facts

• Population 5,887,000 people in 2005 majority
  (85) in rural areas
• Capital Port Moresby
• Area 462,840 km2 (178,000 miles2)
• Languages English, Motu, Pidgin but more than
  800 indigenous languages

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• Papua New Guineas rich flora fauna is host to
  some of the most venomous snakes found in the
  Australasian region (Currie, Emerg. Med., 2000)

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Papuan Taipan
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Death Adder spp.
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Brown snake
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Papuan Black Snake
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• Among these venomous snakes is the unique and
  endemic New Guinea Small-eyed snake Micropechis
  ikaheka
• Also known colloquially by locals as the White
  snake because of its colour

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New Guinea Small-eyed Snake
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New Guinea Small-eyed Snake Distribution
Courtesy of David Williams
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• The New Guinea small-eyed snake has been reported
  to be the cause of several snakebite cases in
  both West Papua PNG (Blasco Hornabrook, PNG
  Med J, 1972 Hudson Pomat, Trans Roy Soc Trop
  Med Hyg, 1988 Hudson, PNG Med J, 1988 Warrell
  et al, Q J Med, 1996)
• Signs symptoms neurotoxicity, dark-coloured
  urine, muscle pain, painful lymph nodes,
  incoagulable blood, hypotension, dizziness,
  nausea, and vomiting (Warrell et al, Q J Med,
  1996 Hudson, PNG Med J, 1988)

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Cases of Suspected Micropechis Envenoming

• Patient Sex/Age Time Where Bite site Signs
  Pre- Antivenom Clinical
• of bite bitten symptoms hospital administered
  outcome
• 
  ..
• 1 M/29 7pm Garden Left foot Vomiting, Razor
  cut Polyvalent given Discharged
• abdominal to bite site 19hrs post-bite
• pain, chewed
• weakness bark
• M/32 7pm Right foot Dark- None Polyvalent given
  Discharged coloured 2 hrs
  post-bite urine 2 days D/C home,
  post-bite, re-admitted 2
  days dizziness, later weakness,
  nausea, vomiting, fitting
  
• M/31 11am Garden Left hand Dark- Blackstone Polyva
  lent not given Discharged coloured b
  ecause no signs of urine, envenomation
  nausea, vomiting, bleeding
  from bite site

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• Although small-eyed snakebite cases that have
  been treated with CSL Australo-Papuan polyvalent
  antivenom have had positive outcomes, (Warrell et
  al, Q J Med, 1996 Hudson, PNG Med J, 1988)
• It is not known which component(s) of the
  Australo-Papuan polyvalent antivenom was
  neutralizing the venom toxins

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• Challenges associated with snakebite
• Lack of snakebite prevention first aid
  knowledge in communities where this snake is the
  common cause of snakebite
• Poor access to primary health care
  transportation
• Lack of antivenom
• Lack of proper health infrastructure
• More importantly, there is NO SPECIFIC ANTIVENOM
  for treating victims bitten by the New Guinea
  Small-eyed snake

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• Snake venoms are a complex mixture of
  polypeptides and other molecules that adversely
  affect multiple homeostatic systems within their
  prey in a highly specific and targeted manner
  (St. Pierre et al, J Proteome Res, 2007)
• Basic research into the venom composition of the
  New Guinea small-eyed snake would improve our
  understanding of the clinical syndromes of
  envenoming seen in snakebite victims, and
• Provide better insights into antivenom suitability

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Aim

• To isolate identify toxic peptides from the
  venom of the New Guinea Small-eyed snake, and
• To determine the neutralisation of small-eyed
  snake venom toxins by different antivenom through
  immunoassay studies

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Objectives

• To separate venom peptides by gel electrophoresis
• To identify toxic venom peptides by mass
  spectrometry
• To determine venom toxin-binding of various
  antivenom via immunoassay studies

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Methods

• 2-Dimensional Polyacrylamide Gel Electrophoresis
  (2D-PAGE)
• Venom samples were collected and pooled from live
  specimens kept at the UPNG Serpentarium
• Venom samples were freeze-dried and appropriate
  amounts were prepared for a two-phase 2D-PAGE
  analysis

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Venom protein separation by charge

• First dimension Isoelectric Focusing (IEF) of M.
  ikaheka venom was done in a BioRad Protean IEF
  unit at 20C using a three-phase gradient voltage
  program 250V for 15mins, 250-8000V for 3 hrs,
  then 8000V to a total of 40,000V/hrs

IEF Unit
Venom on IPG strips in tray
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Venom protein separation by size

• In the second dimension, the IPG strips
  containing IEF-separated venom proteins were
  applied to 12 Tris-HCl acrylamide gels (BioRad
  criterion, 13x10cm) for electrophoresis at 200V
  for 65 minutes

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• The 2D-PAGE gel was then silver-stained to view
  the protein spots

Isoelectric point (pI)
Protein Markers
Decrease in protein size
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MS of 2D-PAGE Protein Spots

• 72 protein spots were cut out from the gel and
  destained
• The peptides were trypsin-digested, dried,
  spotted on a MALDI plate, and subsequently
  analyzed using a 4700 Proteomics Analyzer
  (Applied Biosystems)
• Mass spectrometry (MS) data were acquired using
  2000 shots of a NdYAG laser at 355nm with a 200
  Hz repetition rate and fixed intensity
• The top 50 peptides detected for each spot in the
  MS mode were automatically selected for tandem MS
  (MS/MS) analysis using 3000 laser shots at a
  fixed greater intensity
• MALDI-TOF/TOF-MS/MS data from the 4700 Proteomic
  Analyzer were analyzed using the GPS Explorer
  software (Version 3.5, build 321, Applied
  Biosystems)

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MS of 2D-PAGE Protein Spots

• For each spot a combined MS and MS/MS analysis
  was done using a Mascot search engine (Version
  1.9) and the Celera Discovery System database
  (containing 1, 335 729 sequences, dated May 5,
  2006)
• Criteria for positive protein identification
• Mascot scores gt95 confidence threshold
• Candidates whose protein mass and pI correlated
  with the 2D-PAGE spot were automatically accepted
• Candidates with Mascot scores lt95 confidence
  threshold but whose identity matched a known
  snake venom protein were also included

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Gel Spots with Protein Identity
Phospholipase A2
Serine protease
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Gel Spots with Protein Identity
Phospholipase A2
Cysteine-rich venom protein
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2D-PAGE Protein Spot Identification
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Results of 2D-PAGE

• The MS results of the 2D-PAGE protein spots
  identified several venom proteins including
  phospholipase A2, serine protease and
  cysteine-rich venom protein
• Phospholipase A2 (PLA2) isoenzymes MiPLA2,
  MiPLA3, an MiPLA4 are known to have
  anticoagulant, myotoxic, and haemoglobinuria-induc
  ing activities (Lok et al, FEBS J, 2005 Gao et
  al, Biochim. Biophys. Acta, 2001)
• Cysteine-rich venom proteins have been reported
  to contribute to envenomation via a number of
  mechanisms including the blockage of cyclic
  nucleotide-gated ion channels and the inhibition
  of smooth muscle contraction through calcium ion
  channel blockage (Brown et al, Proc. Natl. Acad.
  Sci. USA, 1999 Yamazaki et al, Eur. J Biochem.,
  2002)

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• Immunoassay and Antivenom-binding
• Studies
• Lyophilized M. ikaheka venom was reconstituted in
  PBS to a concentration of 10mg/ml prior to being
  applied to 12 Tris-HCl acrylamide gels (BioRad
  criterion, 13x10cm) for electrophoresis at 200V
  for 65 mins

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• Following electrophoresis, the gels containing
  separated venom proteins were blotted onto
  nitrocellulose membranes (to immobilize the
  proteins) at 30V overnight, using the BioRad
  Transblot Cell (BioRad, Electrophoretic Transfer)
  
• 2 gels were each stained separately with either
  Coomassie or silver stain

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Immunoblotting
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Antivenom-binding

• The venom protein blots were then incubated
  separately with different antivenoms (primary
  antibody) and subsequently incubated with
  anti-horse antibody (secondary antibody), and
• Finally chemiluminescent reagents were added, and
  the blots were viewed under ultraviolet (UV)
  light to determine antivenom-toxin binding

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Antivenom-binding
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M. ikaheka on 1D-PAGE
New Guinea Small-eyed snake venom
250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37
kDa 25 kDa 20 kDa 15 kDa 10 kDa
Coomassie Stain
Silver stain
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Antivenom-binding
M. ikaheka venom
250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37
kDa 25 kDa 20 kDa 15 kDa 10 kDa
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Antivenom-binding
M. ikaheka venom
250 kDa 150 kDa 100 kDa 75 kDa 50 kDa 37
kDa 25 kDa 20 kDa 15 kDa 10 kDa
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Results of Antivenom-binding Studies

• CSL tiger snake monovalent antivenom, CSL sea
  snake antivenom, and the CSL Australia New Guinea
  polyvalent antivenom were efficaciously-bound to
  the venom toxins of the New Guinea small-eyed
  snake
• The various antivenom used were all expired stock
  (1987 1994)

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Summary

• Basic scientific research with practical
  implications to improving the management of
  people bitten by the New Guinea small-eyed snake
  and
• Establishing basic capacity building with regard
  to human resource (skills) and infrastructure
  (building equipment) needed for this research
  to be carried out

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Summary

• The positive results are manifold and which
  included the following
• The isolation and identification of toxic venom
  proteins from the New Guinea Small-eyed snake has
  enabled a better understanding of the toxins
  present, their functions effects, and the role
  they may play in envenomation
• Antivenom-binding studies determined that apart
  from polyvalent antivenom, tiger snake and sea
  snake antivenom could provide cost-saving
  alternatives to definitive treatment
• Further in vivo and clinical studies could prove
  useful in determining the value of tiger snake
  and sea snake antivenom in treating bites from
  the M. ikaheka

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Summary

• This work utilized snake venom as a tool in the
  training of young Papua New Guinean scientists in
  acquiring basic scientific research skills and
  techniques which can used in a variety of areas
  such as molecular biology, immunology,
  pharmacology and biochemistry
• The collaboration between the UPNG medical school
  and the AVRU, UniMelb, has assisted in creating
  appropriate capacity-building with regard to the
  establishment of a UPNG Serpentarium and the
  imparting of valuable knowledge in finding
  solutions to the snakebite problem in PNG

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Conclusion

• Model?...especially for other developing
  countries to foster international collaborations
  or similar relationships that could expand local
  capacities to tackle snakebite issues
• Give a man fish, and you feed him for a day
  teach a man to fish and you feed him for a
  lifetime

• Make fire for a man and you warm him for a
  day, set him on fire and you warm him for a
  lifetime

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Acknowledgements

• Mr David Williams Dr Ken Winkle (Australian
  Venom Research Unit, Pharmacology, University of
  Melbourne)
• Assoc. Prof Teatulohi Matainaho (Pharmacology,
  SMHS, UPNG)
• Prof Martin Lavin, Dr. Geoff Birrell, Dr. Liam
  St. Pierre, and Dr. Stephen Earl (Venomics Group,
  Cancer Radiation Biology Lab, Queensland
  Institute of Medical Research)
• Dr. Paul Masci (Princess Alexandria Hospital,
  Brisbane)
• This work funded and supported by the AVRU, PNG
  Office of Higher Education and the Department of
  Environment and Conservation

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• Em tasol (Thats all folks!)
• Tenk u tru
• Questions.comments.for this lab rat??