Alligators: A Menace or A Savior

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Antiviral Activity of Serum from the American
Alligator (Alligator mississippiensis)
M. Merchant, M. Pallansch, R. Paulman, J. Wells
Alligators A Menace or A Savior
By Timothy J. Gill
Amoebacidal Effects of Serum from the American
Alligator (Alligator Mississippiensis)
M. Merchant, D. Thibodeaux, K. Loubser, R. Elsey
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Introduction
Alligators are extremely aggressive animals.
Throughout the year, they will have numerous
disputes, especially over territory and females.
Even in captivity, territorial disputes will
still break out.
Despite often losing limbs and receiving
dangerous cuts, Alligators rarely show infection.
This is extremely rare considering the living
environments of these creatures. Studies have
shown antibacterial activity in Alligator serum.
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Untapped Potential
Alligator Serum specifically, and other lower
vertebrate animals, are an unused source of
possible benefits to medicine. All three papers
by M. Merchant were the first reports of activity
in crocodilian species, with the antiviral
activity, the first of the lower vertebrate
animals. Alligators ability to avoid infection
in everyday life indicates a possibility for a
vast improvement in human medicine.
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Main Focus

• Testing Alligator Serum for antiviral activity
  against three enveloped viruses.
• West Nile Virus (WNV) from the 1999 epidemic.
• Herpes Simplex Virus -1 (HSV-1).
• Human Immunodeficiency Virus-1 (HIV-1).
• Alligator Serum was also tested for amoebacidal
  activity against three strains of Naegleria
  species and four strains of Acanthamoeba species.
• Concentration dependent effects
• Saturable nature of amoebacidal effects
• Kinetic properties
• Temperature dependence
• Pretreatment with heat, proteases, and EDTA.

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How to get access to Alligator Blood

• Wild Alligators were captured at night through
  use of spotlight and a snare.
• Blood was collected from the spinal vein by use
  of an 18 gauge 60 mL syringe.
• The spinal vein was used as the target for taking
  blood because of its size and location. This
  vein is a result of an enlarged occipital sinus
  which covers both the dorsal and ventral areas of
  the brain.
• The connections of the spinal vein indicate that
  it plays a major role in the blood transportation
  through the Alligators body.

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Amoebacidal Activity of Alligator Serum

• Alligator serum was tested for concentration
  effects. The Percent survival of amoebas
  decreased from 73.7 survival at 25 serum to
  16.7 survival at 100 serum.

Different concentrations of amoeba cultures were
used to determine if the serum can be saturated.
A density of 3.4 x 104 cells/ml resulted in the
smallest percentage of survival. Above densities
of 7 x 104 cells/ml, percent survival did not
significantly differ.
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Amoebacidal Activity of Alligator Serum

• The increase in activity against Amoeba as serum
  concentration increases is significant and is
  almost linear. Although 100 of amoeba are not
  killed by pure Alligator serum, the drop to 16
  shows an extremely effective activity against
  amoeba.

• Testing the serum against different
  concentrations showed that the capacity to kill
  can be saturated at high densities. Above
  densities of 12 x 104 the complement system
  became overwhelmed. It was determined that the
  total number of amoeba killed by the serum
  remained the same, despite density of cultures.

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Amoebacidal Activity of Alligator Serum

• Samples of N. gruberi were exposed to alligator
  serum at temperatures ranging from 5 degrees to
  40 degrees. Maximal activity was observed
  between temperatures of 15 and 30 degrees C.
  Above 30 degrees, the activity dropped to 75 at
  35 degrees, and 67 at 40 degrees. Below 15
  degrees, the activity expresses a much greater
  decrease to 54 at 10 degrees and 22 at 5
  degrees.

• To test the kinetic properties of alligator
  serum, cell densities of 8 x 104 cells/ml were
  set up and incubated with serum for varying
  lengths of time. After only 5 minutes, survival
  decreased to 61 and after 60 minutes dropped all
  the way to 6.

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Amoebacidal Activity of Alligator Serum

• The temperature curve of amoebacidal activity is
  similar to that of the antibacterial activity.
  Optimal activity occurred at 30 degrees. Other
  studies have suggested that alligators are
  biochemically and physiologically optimized at
  30-31 C. Even though the activity drops above 30
  degrees, it is most likely not significant, since
  the alligators almost always maintain their body
  below 35 degrees. The drastic decrease below 15
  degrees suggests that alligators may be
  immunocompromised during the winter months.

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Amoebacidal Activity of Alligator Serum

• The Kinetic results are similar to the
  antibacterial effects discussed in the 2003
  paper. The effects are rapid, resulting in amoeba
  lysis occurring within the first five minutes of
  exposure. Although significant activity can be
  seen within the first few minutes, longer
  exposure results in almost complete elimination
  of amoeba cultures.

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Pretreatment of Alligator Serum

• Alligator serum was pretreated with heat,
  proteases, and EDTA, in order to determine what
  causes the activity expressed against amoeba
  cultures.
• Serum was preheated at 56 C for 30 min, resulting
  in 85 amoeba survival, a drastic increase from
  the 16 survival expressed by untreated serum.
• Serum was also treated with protease derived from
  Streptomyces griseus. This resulted in 90
  amoeba survival.
• Serum was treated with 30 mM ethlenediaminetetraac
  etic acid (EDTA) resulting in 88 survival.

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Pretreatment of Alligator Serum

• Heat treatment produced a reduction in the
  immunity, which is characteristic of a serum
  complement activity.
• Reduction of activity during pretreatment with
  protease is indicative of activity caused by 1 or
  more proteins.
• Effects of EDTA on activity is another factor,
  indicating a serum complement system plays a role
  in amoebacidal activity.
• Amoeba survival was not entirely eliminated by
  any of these pretreatments which indicates that
  factors other than the complement system are
  responsible for some activity.

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Testing alligator serum against enveloped viruses

• Alligator serum was tested against the adapted
  IIIB strain of HIV-1 using a cell-based assay in
  which the virus was used to infect a human T
  lymphoblastoid cell line.
• Activity against the West Nile Virus was tested
  through a cell-based assay in which Vero African
  green monkey kidney cells were infected with the
  WNV strain NY-99.
• Serum was also used to test against Herpes
  simplex virus-1 using a cell-based assay in which
  Vero cells were infected with HSV-1 strain HF.

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Activity against West Nile Virus strain NY-99.
The NY-99 strain originates from the epidemic
that struck New York City in 1999. The assay of
the WNV strain is based on infection of cultured
cells and monitoring the inhibition of cytopathic
effects. Increased population densities and
accessible transport has caused the danger of WNV
to amplify.
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Activity against West Nile Virus strain NY-99.
Results for anti-WNV activity are representative
of the means of three independent determinations.
Ribavirin was used as the positive control.
Alligator serum shows moderately high activity
against WNV at low concentrations. 16 serum
expressed 77 of maximal activity, but also
showed a 24 reduction in cell viability. Full
potential of anti-WNV properies are difficult to
obtain due to the toxicity of serum toward the
Vero cells. The IC50 was determined to be 4.3
serum and the TC50 was 36.3 serum.
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Activity against Herpes Simplex Virus 1, strain HF
The Antibody-dependent cellular cytotoxicity
(ADCC) is believed to be an important mechanism
Measuring Chromium release has indicated the
importance ADCC in recovery from HSV.
Approximately 90 of adults have developed an
immunity to HSV, so it is imperative to fight the
disease in children. Some medications are able
to alleviate some pain, but the search remains
for more effective results.
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Activity against Herpes Simplex Virus 1, strain HF
The results are representative of three
independent determinations. Alligator serum
showed moderate antiviral activity against HSV-1.
Treatment with 16 and 5 serum provided 76 and
61 protection. These concentrations resulted in
only minor reductions in cell viability(15 and
7). The IC50 value was determined to be 3.4
serum while the TC50 was 39.1 serum.
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Activity against IIIB strain of HIV-1
Human Immunodeficiency Virus Type 1(HIV-1) is a
well-known and well studied disease that has
caused a lot of trouble since the first case in
1984. Many breakthroughs have been made, but no
cost effective treatment exists. Since
individuals are able to carry the disease without
showing signs, transmission is much more
difficult to contain. Monoclonal antibodies have
been determined to activate the Human complement
system. These antibodies have shown potent
activity against primary and T-line strains of
HIV-1.
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Activity against IIIB strain of HIV-1
Results for activity against HIV-1 was determined
through three determinations. Alligator serum
exhibits potent activity against HIV-1. 5 and
1.6 serum concentrations were the most effective
at reducing viral CPE without affecting cell
viability. The 5 sample produced 100 reduction
in viral cytopathic effects while having 0 effect
on viability. The 1.6 sample produced an 89
reduction in CPE while also having 0 effect on
viability. The IC50 was determined to be 0.85
serum while the TC50 was 32.8 serum.
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Activity against IIIB strain of HIV-1
The activity against HIV-1 above serum
concentrations of 5 can not be assessed due to
the toxicity of the serum towards the CEM-SS
cells. Heat treatment of the serum eliminated
all effects, indicating that the HIV-1 activity
is most likely due to the complement system.
Since a lot of knowledge exists about the human
complement system, it is likely that future
studies will be able to isolate the factors of
the alligator system that produce such an
efficient response to the virus.
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Conclusions

• The natural immunity of alligators towards
  viruses that they have not naturally encountered
  in the evolution is interesting and brings about
  questions of other untapped resources.
• Alligator serum was least effective, of the three
  viruses, against the WNV, which is supported by
  reported West Nile infections in alligator
  populations.
• The HIV-1 activity as well as the amoebacidal
  activity indicate the alligator has developed a
  strong serum complement system.
• Activity against HSV-1 and WNV show other
  mechanisms are active in alligator serum.

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Acknowledgements
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