Enzymatic Activity And Brine Shrimp Lethality Of Venom From The Large Brown Spitting Cobra (Naja Ashei) And Its Neutralization By Antivenom

doi:10.21203/rs.3.rs-20006/v1

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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

https://doi.org/10.21203/rs.3.rs-20006/v1

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published 06 Jul, 2020

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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.

Toxicology

Snake venom phospholipase A2

brine shrimp lethality assay

snake venom toxicity

Naja ashei

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 properties 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.

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.

SvPLA₂ 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 and 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 shrimps was calculated using probit analysis.

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.

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 they 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.

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.

svPLA₂: 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, LD_50:Median lethal dose

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 24^th 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 draft: 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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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*	*N. haje*	*N. pallida*	*N. subfulva*
Concentration (µg/ml)
0.5	4.67±0.52	3.17±1.17	3.33±1.51	9.5±0.55	2.17±0.41	6.00±0.63	14.00±1.30
1.0	4.83±0.75	3.00±0.89	3.33±0.52	10.00±0.63	3.67±0.41	9.00±0.63	15.83±1.37
2.0	8.83±2.23	4.17±0.75	3.50±0.55	11.83±1.60	4.17±0.41	10.33±0.52	17.42±2.42
4.0	8.67±0.82	4.33±0.52	4.33±0.82	12.16±0.98	5.67±0.82	10.67±0.52	20.75±2.73
8.0	12.00±1.41	4.33±1.37	3.33±0.82	12.00±0.89	6.50±0.55	11.67±0.52	21.50±2.12
10.0	10.83±1.17	3.17±0.98	4.00±0.63	13.83±1.60	7.33±0.52	11.67±0.52	20.33±2.42
12.5	12.50±0.95	4.00±1.55	3.33±0.82	14.42±2.42	7.33±0.52	12.33±0.52	22.58±1.11
15.0	14.33±1.17	3.00±0.63	5.00±0.89	17.08±2.11	8.33±0.52	13.17±0.41	26.17±2.64
17.5	12.83±2.02	3.67±0.52	3.33±0.52	18.75±1.54	9.17±0.41	14.17±0.41	24.33±3.22
20	14.25±1.29	3.33±0.52	2.50±0.55	16.67±1.21	9.67±0.52	14.67±1.75	25.25±1.84
22.5	14.58±1.69	3.17±0.41	2.50±1.22	17.67±0.41	10.50±0.55	15.17±0.41	25.50±0.89
Regression equation	y=6.2x+5.9	y=0.02x+3.6	y=-0.12x+3.6	y=5.1x+10.0	y=4.6x+3.2	y=4.6x+8.1	y=7.00x+15.7
R²value	0.9412	0.0004	0.0083	0.8070	0.9442	0.9246	0.9212
MPC₅₀ value	1.689	ND	ND	0.770	2.849	0.774	0.421

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

Mortality per test dose

Toxicity

Snake species

Common name

Family

Duration of exposure

µg/ml

100

µg/ml

1000

µg/ml

LC₅₀ (µg/ml)

(Uncensored)

Meyer’s toxicity

index^[26]

Clarkson’s toxicity

index^[27]

arietans

Puff adder

Viperidae

1,153,200.34

437.21

206.64

Non-toxic

Toxic

Non-toxic

Moderately toxic

D. angusticeps

Green mamba

Elapidae

1808.35

1103.20

559.32

Non-toxic

Toxic

Non-toxic

Low toxic

polylepis

Black mamba

Elapidae

411.37

2043.6

641.47

Toxic

Non-toxic

Toxic

Moderately toxic

Non-toxic

Low toxic

ashei

The large brown spitting cobra

Elapidae

63.02

4.73

0.153

Toxic

Highly toxic

haje

Egyptian cobra

Elapidae

1,668,100.54

2972.03

2142.57

Non-toxic

pallida

Red spitting cobra

Elapidae

59.55

14.26

4.46

Toxic

Highly toxic

subfulva

Forest cobra

Elapidae

47.94

12.46

13.72

Toxic

Highly toxic

Vincristine

(Positive control)

171.83

2.099

All died

Toxic

Highly toxic

LC₅₀: Concentration of snake venom responsible for 50 % mortality of brine shrimp larvae

SupplementarymaterialOkumuetal.docx

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published 06 Jul, 2020

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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

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Snake venom

Animals (brine shrimp)

Antivenom

Protein content determination of the venoms and antivenoms

SvPLA₂ activity of venoms and neutralization of N. ashei venom by antivenom

Neutralization of svPLA₂ activity

Venom induced brine shrimp lethality

Neutralization of brine shrimp induced lethality by antivenom

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1

Enzymatic Activity And Brine Shrimp Lethality Of Venom From The Large Brown Spitting Cobra (Naja Ashei) And Its Neutralization By Antivenom

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials And Methods

Snake venom

Animals (brine shrimp)

Antivenom

Protein content determination of the venoms and antivenoms

SvPLA2 activity of venoms and neutralization of N. ashei venom by antivenom

Neutralization of svPLA2 activity

Venom induced brine shrimp lethality

Neutralization of brine shrimp induced lethality by antivenom

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1

SvPLA₂ activity of venoms and neutralization of N. ashei venom by antivenom

Neutralization of svPLA₂ activity