Enzymatic Activity And Brine Shrimp Lethality Of Venom From The Large Brown Spitting Cobra (Naja Ashei) And Its Neutralization By Antivenom ...
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Enzymatic Activity And Brine Shrimp Lethality Of
Venom From The Large Brown Spitting Cobra (Naja
Ashei) And Its Neutralization By Antivenom
CURRENT STATUS: UNDER REVIEW
Mitchel Otieno Okumu
University of Nairobi
mytchan88@gmail.comCorresponding Author
ORCiD: https://orcid.org/0000-0002-9316-990X
James Mucunu Mbaria
University of Nairobi College of Agriculture and Veterinary Sciences
Joseph Kangangi Gikunju
Jomo Kenyatta University of Agriculture and Technology
Paul Gichohi Mbuthia
University of Nairobi College of Agriculture and Veterinary Sciences
Vincent Odongo Madadi
University of Nairobi College of Biological and Physical Sciences
Francis Okumu Ochola
Moi University
DOI:
10.21203/rs.3.rs-20006/v1
SUBJECT AREAS
Toxicology
KEYWORDS
Snake venom phospholipase A2, brine shrimp lethality assay, snake venom toxicity,
Naja ashei
1Abstract
Objective There has been little focus on the enzymatic and lethal activities of Naja ashei venom and
their neutralization by antivenom. This study aimed to determine the snake venom phospholipase
A2/svPLA2 activity and brine shrimp lethality of N. ashei venom and their neutralization by two
antivenoms (I and II). The venom of other snakes in East Africa including the puff adder (Bitis
arietans), green mamba (Dendroaspis angusticeps), black mamba (Dendroaspis polylepis), Egyptian
cobra (Naja haje), red spitting cobra (Naja pallida), and the Eastern forest cobra (Naja subfulva) were
used for comparison.
Results: N. subfulva venom had the highest svPLA2 activity while D. angusticeps venom had the least
activity. N. subfulva venom was the most toxic in the 24-hour brine shrimp lethality assay (BSLA),
while N. ashei venom was the most toxic in the 48 and 72-hour assays. N. haje venom was the least
toxic in all assays. One ml of antivenom I neutralized 0.075 µg of svPLA2 in N. ashei venom compared
to 0.051 µg by antivenom II. Antivenom I was ineffective in neutralizing N .ashei venom-induced
lethality but 1 ml of antivenom II neutralized 0.21 mg of N. ashei venom.
Introduction
More than 5 million snakebites occur every year disproportionately affecting the poorest nations
globally [1]. Up to 138, 000 people die from snakebite and 400, 000 are disabled [1].
Cobras are venomous snakes that may be spitting or non-spitting in nature [2]. They are often
cultural icons and objects of reverence [3, 4]. African spitting cobras are associated with morbidity
and mortality in sub-Saharan Africa [3–11]. Toxicological/pathological effects include blistering,
edema, necrosis, respiratory paralysis and ophthalmia [8–13]. Naja ashei is one of the African spitting
cobras and is a category 1 snake in Kenya, Ethiopia, Somalia, Uganda, and a category 2 snake in
Tanzania [14]. Figure 1. Category 1 snakes are highly venomous, cause many bites, and are
associated with high levels of morbidity, disability or mortality [14]. Category 2 snakes are highly
venomous, may cause morbidity, mortality, disability or death but lack epidemiological/clinical data to
implicate them in snakebite [14].
The skull structure, mitochondrial DNA, venom composition, antiproliferative, and antibacterial
2properties of N. ashei have previously been reported [3, 7, 8, 15–17]. However, there has been little
focus on the enzymatic, and lethal effects of this venom and the capacity of antivenom to neutralize
them. This study aimed to evaluate the svPLA2 activity and lethal effects of N. ashei venom and the
capacity of commercially available antivenom to neutralize these effects.
Materials And Methods
Snake venom
Venom was extracted from 21 specimens of wild-caught puff adders (B. arietans), green mambas (D.
angusticeps), black mambas (D. polylepis), large brown spitting cobras (N. ashei), Egyptian cobras (N.
haje), red spitting cobras (N. pallida), and Eastern forest cobras (N. subfulva) maintained at the
Bioken Snake Farm (Kenya). Table S1. Venoms were snap frozen, lyophilized, and stored as powder at
-20 °C. They were reconstituted in phosphate-buffered saline (PBS) at the time of use.
Animals (brine shrimp)
One hundred grams of brine shrimp eggs were commercially sourced from yourfishstuff in the
Borough of Lebanon, New Jersey, USA (Batch number; X001M8M5IZ). The eggs were hatched at the
Department of Public Health, Pharmacology, and Toxicology, University of Nairobi and brine shrimp
larvae were used for experiments on venom lethality and its neutralization.
Antivenom
Antivenoms used in this study are described in Table S2 and were acquired from hospitals in Kenya.
Protein content determination of the venoms and antivenoms
Lowry’s method was used [18]. Serial dilutions (0.05-2.0 mg/ml) of bovine serum albumin were
prepared in triplicate, 0.2 ml of each dilution was pipetted into 10 ml glass tubes, 2 ml of alkaline
copper sulphate was added to each tube and incubated at room temperature for 10 minutes. 0.2 ml
of Folin-Ciocalteau reagent was added and tubes incubated for 30 minutes. Absorbance was read at
660 nm. Protein content was inferred from the calibration curve [18]. Table S3.
SvPLA 2 activity of venoms and neutralization of N. ashei venom by antivenom
The svPLA2 activity of the venoms was determined by the methods of Haberman and Hardt 1972 and
Felix Silva et al [19, 20] with modifications. 10 ml of a 1:3 v/v suspension of egg yolk in phosphate-
buffered saline (PBS) was added to 90 ml PBS and 300 ml of a 1% w/v agarose solution. 125 µl of
0.1 mM Calcium chloride (CaCl2) was added to the mixture, which was poured into sterile Petri dishes
3and 6 mm wells were made on the solidified media. Figure S1. 10 µl of venoms (0.5–22.5 µg/ml) were
pre-incubated at 37 °C for 60 minutes and pipetted into the wells. These were incubated at 50 °C for
24 hours. A 1:10, v/v solution of Carbol Fuchsin (CF) was used to visualize the halos and Vernier
calipers were used for measurement. PBS was used as a negative control. The minimum
phospholipase concentration (MPC50) was calculated using regression analysis. All experiments were
performed in triplicate.
Neutralization of svPLA2 activity
The method of Iwanaga and Suzuki 1979 was used with modifications. [21]. 10 µl of a 2 × MPC50 dose
of N. ashei venom was mixed with 20 µl of antivenom (25–400 µg/ml) in 96-well ELISA plates,
incubated at 37 °C for 20 minutes and 200 µl of substrate (1.1% egg yolk suspension in 0.1M PBS +
125 µl 0.2 mM CaCl2) was added to each well, incubated at room temperature and the change in
absorbance of the substrate was measured spectrophotometrically from 0 to 30 minutes at 620 nm
[21]. All experiments were performed in triplicate (Figure S2) and the % PLA2 activity was calculated
by taking the absorbance of the wells with venom alone as 100%. Table S4.
Venom induced brine shrimp lethality
The method of Meyer et al 1982 was used [22]. Ten, 48-hour old brine shrimp larvae were transferred
from a hatching trough to 5 ml sample vials. Figure S3. Aliquots (5, 50 and 500 µl) of 5 mg/ml stock
solutions of the venoms were pipetted into the sample vials and made up to the mark with marine salt
solution (38.5% w/v) to a final concentration of 10, 100, and 1000 µg/ml respectively. PBS was used
as control. Surviving larvae were counted after 24, 48, and 72 hours. All experiments were performed
in quintuples. Probit analysis was used to determine the median lethal concentration (LC50)[23–25]
and the toxicity of the venoms was classified using toxicity indices [26, 27].
Neutralization of brine shrimp induced lethality by antivenom
The World Health Organization (WHO) protocol was used with modifications [14]. Various doses of
antivenoms (25–400 µl of 100 mg/ml stock solutions) were mixed with a 2LC50 dose of N. ashei
venom, incubated at 37 °C for 30 minutes, and added to the vials. Figure S3. Surviving larvae were
counted after 24 hours. The least amount of antivenom required to prevent death in 50% of brine
4shrimps was calculated using probit analysis.
Results
svPLA2 activities of the venoms was dose-dependent. Table 1. N. subfulva venom had the highest
activity while D. angusticeps venom had the least activity. Wells containing venom alone recorded the
largest decrease in absorbance. Table S4. Regression analysis established that one ml of antivenom I
neutralized 0.075 µg of N. ashei venom (Figure S3) while one ml of antivenom II neutralized 0.051 µg
of N. ashei venom. (Figure S4).
N. subfulva venom was the most toxic in the 24-hour lethality assay, N. ashei venom was the most
toxic in the 48 and 72-hour assays, and N. haje venom was the least toxic at all the time intervals.
Table 2. N. ashei, N. pallida, and N. subfulva venoms were toxic at all the time intervals while N. haje
venom was non-toxic at all the time intervals. B. arietans venom was non-toxic after 24 hours but
toxic after 48 and 72 hours. Table 2.
Discussion
svPLA2 make up to 30% of viperid and elapid venoms [28, 29], and the local effects of venomous
snakebite in sub-Saharan Africa have been linked to svPLA2’s [12, 30–33]. Our results suggest that
the Eastern forest cobra had the highest svPLA2 activity while mamba venoms had the least activity.
This observation seems to corroborate clinical reports on venomous snakebite in Sub-Saharan Africa
where cobra bites are associated with severe muscle and tissue damage, and painful progressive
swelling which are generally absent in mamba bites [30, 34–39]. Both antivenoms had poor
neutralization capacity against the svPLA2 activity of N. ashei venom. It is no surprise therefore that
research into compounds like varespladib, methylvarespladib, and medicinal plant extracts continue
to be pursued as possible adjuncts in the management of svPLA2-induced effects of viper and elapid
venoms [20, 28, 31, 40, 41].
To the best of our knowledge, this is the first report to establish that the non-spitting Eastern forest
cobra (N. subfulva) is more potent than the red spitting cobra (N. pallida) and the large brown spitting
cobra (N. ashei) in as far as svPLA2 activity is concerned. However, considering that both the Eastern
forest cobra (N. subfulva) and the Egyptian cobra (N. haje) are non-spitting cobras, it is not clear why
5they have varying svPLA2 activity.
The brine shrimp lethality assay has been used to evaluate the toxicity of medicinal plant extracts
[42, 43], heavy metals [44], metal ions [45], cyanobacteria [46], algae [47], dental material [48],
nanoparticles [49], and marine natural products [50, 51]. It is rapid, simple, inexpensive, robust, and
results may be obtained after 24 hours [52]. It has few requirements, cysts are readily available
worldwide, and a large number of test organisms of the same age and physiology can be obtained to
start the test [52]. The results are highly reproducible and there is a good correlation between the
brine shrimp LC50 values and LD50 values obtained from acute oral toxicity testing in rodents [43, 52,
53]. The venom of D. angusticeps had the highest protein content but was generally non-toxic. It is
likely that venom proteins in D. angusticeps may have other pharmacological effects not discernible
by this assay. N. subfulva venom was the most toxic and had the second-highest protein content
suggesting that brine shrimps are sensitive to N. subfulva venom proteins. The venom of B. arietans
was non-toxic after 24 hours but toxic after 48 and 72 hours of exposure implying that this venom has
delayed toxicity in brine shrimps. Antivenom II had a lower protein content than antivenom I but was
more effective in neutralizing the lethal effects of N. ashei venom suggesting that antivenom II is
more sensitive in recognizing and neutralizing proteins responsible for venom-induced lethality than
antivenom I.
Conclusions
There is variation in the svPLA2 and lethal activities of N. ashei venom relative to other cobras,
mambas, and the puff adder. The capacity of antivenoms to neutralize svPLA2 and lethal activities of
N. ashei venom also varies. Further evaluation of the capacity of antivenoms to neutralize other
toxins in N. ashei venom is warranted. The potential of the brine shrimp model as an alternative to
the murine model in venom neutralization assays warrants further research.
Limitations
Naja ashei venom used in this study was from wild-caught snakes in Kenya. It may therefore, not be
possible to extrapolate the observed effects to the larger East African region.
Abbreviations
6svPLA2: Snake venom phospholipase A2; CF: Carbol Fuchsin; BSLA: Brine shrimp lethality assay; LC50:
lethal concentration responsible for 50 % mortality; MPC50: The minimum phospholipase
concentration responsible for 50% response; DNA: Deoxyribonucleic acid; mM: Millimole; v/v: Volume
by volume; µl: Microliter; µg/ml; Microgram per milliliter; mm: Millimeter; ELISA: Enzyme-linked
Immunosorbent assay; PBS: phosphate-buffered saline; CaCL2: Calcium chloride; WHO: World Health
Organization; mg/ml: Milligram per milliliter; R2: Coefficient of determination; ND: Not determined,
LD50: Median lethal dose
Declarations
Ethics approval and consent to participate
This study was approved by the Biosafety, Animal Use, and Ethics Committee of the Faculty of
Veterinary Medicine, University of Nairobi. REF BAUEC/2019/220 issued on 24th April 2019. The
animals (brine shrimps; Artemia salina) used in this study were commercially sourced thus owner
consent was not required.
Consent for publication
Not applicable
Availability of data and material
All data generated or analyzed during this study are included in this published article [and its
supplementary information files].
Competing interests
The authors declare no conflict of interest
Funding
This study did not receive any external funding.
Authors’ contributions
Conceptualization: MO, FO, and JG; data curation: all authors; formal analysis: MO; investigation: all
authors; methodology: MO; project administration: MO and FO, resources; all authors; software: MO;
supervision; JM, JG, PM, and VO; validation: FO, JM, JG, PM, and VO; visualization: MO; writing original
7draft: MO: writing review and editing: all authors
Acknowledgments
The authors would like to acknowledge Ms. Claire Taylor and Mr. Boniface Momanyi of the Bioken
Snake Farm (Watamu, Kenya) for providing the venom samples. We would also like to thank Ms.
Vivian of the Department of Animal Physiology, University of Nairobi who helped us in freeze-drying
the venom samples and Dr. Nduhiu, Mr. Mainga, Mr. Maloba, Mr. Nderitu and Mr. Bett of the
Department of Public Health, Pharmacology and Toxicology for their assistance in the phospholipase
and brine shrimp lethality assays. Special gratitude to Dr. Nelson Odhiambo and Edna Opiyo of the
Nyakach Sub County Hospital for providing the antivenom samples that were used in this study. We
also wish to thank Dr. Joshua Onono for his invaluable insight in several aspects of statistical analysis.
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Tables
Table 1: Dose-dependent svPLA2 activity and MPC50 values of N. ashei venom relative to the activity
of B. arietans, D. angusticeps, D. polylepis, N. haje, N. pallida, and N. subfulva snake venoms
Mean svPLA2 response (mm
Venom sample B. arietans D. angusticeps D. polylepis N. ashei
Concentration
(µg/ml)
0.5 4.67±0.52 3.17±1.17 3.33±1.51 9.5±0.55
1.0 4.83±0.75 3.00±0.89 3.33±0.52 10.00±0.63
2.0 8.83±2.23 4.17±0.75 3.50±0.55 11.83±1.60
4.0 8.67±0.82 4.33±0.52 4.33±0.82 12.16±0.98
8.0 12.00±1.41 4.33±1.37 3.33±0.82 12.00±0.89
10.0 10.83±1.17 3.17±0.98 4.00±0.63 13.83±1.60
12.5 12.50±0.95 4.00±1.55 3.33±0.82 14.42±2.42
15.0 14.33±1.17 3.00±0.63 5.00±0.89 17.08±2.11
17.5 12.83±2.02 3.67±0.52 3.33±0.52 18.75±1.54
20 14.25±1.29 3.33±0.52 2.50±0.55 16.67±1.21
22.5 14.58±1.69 3.17±0.41 2.50±1.22 17.67±0.41
Regression equation y=6.2x+5.9 y=0.02x+3.6 y=-0.12x+3.6 y=5.1x+10.0
R2 value 0.9412 0.0004 0.0083 0.8070
MPC50 value 1.689 ND ND 0.770
PLA2: Phospholipase A2 , R2 : Coefficient of determination, MPC50; Minimum phospholipase
concentration, ND; Not determined due to the weak coefficient of determination.
Table 2: Brine shrimp lethality of N. ashei venom relative to the lethality in B. arietans, D.
angusticeps, D. polylepis, N. haje, N. pallida, N. subfulva snake venoms and vincristine (standard)
after 24, 48, and 72 hours of exposure
14Mortality per test dose
Snake species Common Family Duration of 10 100 1000
name exposure µg/ml µg/ml µg/ml
B. Puff adder Viperidae 24 0 0 12
arietans 48 1 5 45
72 9 13 50
D. angusticeps Green mamba Elapidae 24 0 0 29
48 1 1 39
72 7 2 48
D. Black mamba Elapidae 24 1 1 41
polylepis 48 3 2 43
72 7 4 45
N. The large brown Elapidae 24 0 48 50
ashei spitting cobra 48 26 50 50
72 40 50 50
N. Egyptian cobra Elapidae 24 0 0 3
haje 48 0 3 15
72 2 20 22
N. Red spitting Elapidae 24 2 48 50
pallida cobra 48 15 50 50
72 32 50 50
N. Forest cobra Elapidae 24 10 30 50
subfulva 48 17 50 50
72 23 50 50
Vincristine _ _ 24 0 30 46
(Positive control) 48 35 50 50
72 50 50 50
LC50: Concentration of snake venom responsible for 50 % mortality of brine shrimp larvae
Figures
15Figure 1
Distribution of Naja ashei in Africa. Source: The authors.
Supplementary Files
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SupplementarymaterialOkumuetal.docx
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