Introduction
Envenomation resulting from snakebites is an important public health problem in many tropical and subtropical countries including Nigeria, affecting mostly poor people involved in agricultural activities [1,2]. Snakebites cause considerable morbidity and mortality worldwide but are frequently neglected public health issues in tropical and subtropical countries [3,4]. Bitis arietans is a species of viper in the family Viperidae. It is by far the most dangerous snake in Africa, due to its wide distribution [5], its relative abundance in anthropized áreas, and powerful venom and it is responsible for more snake accidents than any other snake on the continent. Currently, two subspecies are recognized: B. arietans arietans and B. arietans somalica, it is accountable for many snakebite fatalities in Africa [6]. Bites from this species can deliver serious local and systemic signs [7]. Based on the degree and type of local effect, bites can be divided into two: those with practically no surface extravasation, and those with hemorrhages evident as ecchymosis and swelling [7]. In both cases, severe pain and tenderness occur, but in the latter, widespread superficial or deep necrosis and compartment syndrome are seen. Serious bites cause limbs to become immovably flexed as a result of significant hemorrhage or coagulation in the affected muscles [7]. Human bite victims may also have severe swelling of the surrounding lymph nodes, shock, blood seeping from puncture wounds, subcutaneous bruises, and edema that can become widespread [8]. Chicken ranks as one of the most common animals in Nigeria that are domesticated with a population of about 140 million [9]. Immunoglobulin Y (IgY) is the main antibody present in birds, reptiles, and lungfish blood with high concentrations in chicken yolk [10,11]. In recent years, chickens have become a source of antibodies for research [12]. This research sought to experiment with a cheaper alternative to snake antivenom production using hyperimmunized chicken eggs.
Materials and Methods
Ethical approval
All procedures performed in the study were under the Declaration of Helsinki. Ethical approval for the study was obtained from the Ahmadu Bello University Committee on Animal Use and Care with approval number ABUCAUC/2019/12.
Experimental animals
The study was conducted in November 2019 and all animal welfare protocols for the snakes, birds, and mice were by current regulations for experimental animals. Twenty egg-laying, brown hens (Gallus gallus domesticus) of the Lohmann brown classic breed approximately 17-week-old were purchased and were kept under an intensive deep litter system. They were fed with layer mash feed and water ad libitum. Twenty Albino mice and nine Wistar rats were purchased from the National Institute for Trypanosomosis Research, Nigeria. Both sexes of B. arietans were captured from the environs of Kaduna and Katsina State, Nigeria. They were identified taxonomically in the Department of Veterinary Pharmacology and Toxicology, Ahmadu Bello University Zaria, Nigeria, and were kept in metal cages.
Collection and crystallization of crude venom
Crude venom of B. arietans was milked from snakes of the same species of different ages and sexes captured from different locations. A modification of Macfarlane [13] was used by introducing cellophane to the top of the beaker [14]. The beaker containing the venom collected was placed in a desiccator with activated silica gel and allowed to crystallize. The crystallized venom was then pooled and transferred into Eppendorf tubes. The tubes were stored in a refrigerator at + 4°C.
Hyper-immunization of chickens
The hyperimmunization protocol utilized is as described by Aguilar et al. [15]. 1/5 of the lethal dose was used. Seventeen-week-old layer chickens, weighing ~1 kg were immunized with the venom. The first and second doses of venom were mixed with an equal volume of Montanide™ ISA 201 w/w (Seppic, France) adjuvant. The third venom dose was mixed with normal saline.
Isolation of IgY from immunized chicken eggs
The method used was a modification of the method as described by Hussain [16] using Poly ethylene glycol 6,000.
Determination of IgY concentration in egg yolk
The IgY content (mg/ml) of the samples was measured photometrically at 280 nm (1:40 dilution with phosphate buffer saline) (Eppendorf BioPhotometer Plus) and calculated according to the Lamber-Beer law with an extinction coefficient of 1.33 for IgY [16].
Venom lethal activity (LD50)
The lethal dose (LD50) of B. arietans venom was determined according to the method developed by Bruce [17].
In vitro test for the presence of B. arietans-specific antibodies in hyperimmunized chickens
The method as described by Ferreira et al. [18] was used to detect the presence of specific antibodies to the venom of B. arietans using agar gel immunodiffusion assay.
In vivo anti-lethality assay of the venom of B. arietans
The anti-lethal potential of the candidate antivenom was determined against the crude venom of B. arietans. Various venom doses comprising 10 mg/kg, 5 mg/kg, 2.5 mg/kg, 1.25 mg/kg, and 0.625 mg/kg using (standard antivenom, candidate antivenom, and plain chicken antibodies) as diluents for groups A, B, and C, respectively, were prepared and incubated at 37°C for 30 minutes and then injected intraperitoneally into the mice. The mice were observed for 24 hours.
Neutralization of hemolytic activity
The method described by Gomes and De [19] using caprine erythrocytes was used for the anti-hemolytic activity.
Neutralization of hemorrhagic activity
Data analysis
Values obtained were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance was used followed by Tukey’s post-hoc test for multiple comparisons of groups. GraphPad prism version 9.0.2 for windows (GraphPad software San Diego, California, USA) was used. Values of p < 0.05 were considered significant.
Results
Median lethal dose of B. arietans venom in mice
The LD50 of the venom of B. arietans administered intraperitoneally to mice was determined to be 0.9 mg/kg.
In vivo anti-lethality assay of the venom of B. arietans
The group treated with the standard drug recorded survival at all the venom doses (100% survival) while the test group recorded survival at 1.25 mg/kg and 0.625 mg/kg only (40% survival). The group that was treated with the plain chicken antibodies recorded mortality at all the venom doses used (0 % survival) (Table 1).
Table 1.
In vivo anti-lethality assay of the venom of Bitis arietans.
| Venom dose | Standard antivenom | Candidate antivenom | Plain chicken antibodies |
|---|---|---|---|
| 10 mg/kg | +a | -b | -b |
| 5 mg/kg | +a | -b | -b |
| 2.5 mg/kg | +a | -b | -b |
| 1.25 mg/kg | +a | +a | -b |
| 0.625 mg/kg | +a | +a | -b |
a,bMeans with different superscript alphabets across rows are significantly (p < 0.05) different. +=Survival, -=Mortality.
Neutralization of hemorrhagic activity
The venom of B. arietans showed marked hemorrhage at 10 mg/ml, 5 mg/ml, and 2.5 mg/ml, the values obtained were 12.11 ± 3.83 (Fig. 1), 7.01 ± 1.83 (Fig. 2), and 3.14 ± 0.00 cm2 (Fig. 3), respectively. The anti-hemorrhagic activity of the candidate antivenom at 10 mg/ml; 5mg/ml and 2.5mg/ml venom concentrations were 4.12 ± 0.79 (Fig. 1), 2.65 ± 0.77 (Fig. 2), and 4.16 ± 0.77 cm2 (Fig. 3), respectively. The candidate antivenom showed higher inhibitory effect (p < 0.05) against the hemorrhagic activity of B. arietans venom (2.5 ± 0.83 cm2 lesion) than the standard antivenom (EchiTab plus-ICP®) (5.1 ± 1.18 cm2 lesion) at 5 mg/ml venom concentration (Fig. 2). The standard antivenom (EchiTab plus-ICP®) recorded anti-hemorrhagic activity at 10 mg/ml, 5 mg/ml, and 2.5 mg/ml venom concentrations. The values obtained were 4.33 ± 1.53 (Fig. 1), 5.26 ± 1.51 (Fig. 2), and 2.61 ± 0.61 cm2 (Fig. 3), respectively. The values obtained from the plain chicken anti-body group for 10 mg/ml, 5 mg/ml, and 2.5 mg/ml venom concentrations were 4.07 ± 0.51 (Fig. 1), 3.33 ± 0.80 (Fig. 2), and 1.70 ± 0.30 cm2 (Fig. 3), respectively.
Figure 1.
Anti-hemorrhagic activity of the candidate antivenom, the standard antivenom, and the plain antibodies against Bitis arietans venom and the hemorrhagic activity of the crude venom of Bitis arietans at 10 mg/ml. Values are represented as Mean ± SEM. a,b,cMeans with different superscript letters are significantly different (p < 0.05).
Figure 2.
Anti-hemorrhagic activity of the candidate antivenom, the standard antivenom, and the plain antibodies against Bitis arietans venom and the hemorrhagic activity of the crude venom of Bitis arietans at 5 mg/ml. Values are represented as Mean ± SEM. a,b,cMeans with different superscript letters are significantly different (p < 0.05).
Figure 3.
Anti-hemorrhagic activity of the candidate antivenom, the standard antivenom, and the plain antibodies against Bitis arietans venom and the hemorrhagic activity of the crude venom of Bitis arietans at 2.5 mg/ml. Values are represented as Mean ± SEM. a,b,cMeans with different superscript letters are significantly different (p < 0.05)
Neutralization of hemolytic activity
The hemolytic activity of the venom of B. arietans at 10 mg/ml, 5 mg/ml, and 2.5 mg/ml venom concentrations was observed to be 0.15 ± 0.02 (Fig. 4), 0.09 ± 0.03 (Fig. 5), and 0.13 ± 0.02 nm (Fig. 6), respectively. The anti-hemolytic activity of the candidate antivenom at venom concentrations of 10 mg/ml, 5 mg/ml, and 2.5 mg/ml were observed to be 0.08 ± 0.00 (Fig. 4), 0.10 ± 0.00 (Fig. 5), and 0.10 ± 0.01 nm (Fig. 6), respectively. The candidate antivenom exhibited significantly higher (p < 0.05) anti-hemolytic activity against the hemolytic activity of B. arietans venom (0.08 ± 0.00 nm) than (EchiTab plus-ICP®) standard antivenom (0.13 ± 0.02 nm) at 10 mg/ml venom concentration (Fig. 4). The anti-hemolytic activity exhibited by the standard antivenom at 10 mg/ml, 5 mg/ml, and 2.5 mg/ml were observed to be 0.13 ± 0.02 (Fig. 4), 0.10 ± 0.03 (Figure 4), and 0.12 ± 0.02 nm (Fig. 4), respectively. The values obtained from the plain chicken anti-body group for 10 mg/ml, 5 mg/ml, and 2.5 mg/ml venom concentrations were 0.15 ± 0.02 (Fig. 4), 0.09 ± 0.03 (Fig. 5), and 0.13 ± 0.02 nm (Fig. 6), respectively.
Figure 4.
Anti-hemolytic activity of the candidate antivenom, the standard antivenom, and the plain antibodies against Bitis arietans venom and the hemolytic activity of the crude venom of Bitis arietans at 10 mg/ml. Values are represented as Mean ± SEM. a,b,cMeans with different superscript letters are significantly different (p < 0.05).
Figure 5.
Anti-hemolytic activity of the candidate antivenom, the standard antivenom, and the plain antibodies against Bitis arietans venom and the hemolytc activity of the crude venom of Bitis arietans at 5 mg/ml. Values are represented as Mean ± SEM. a,b,cMeans with different superscript letters are significantly different (p < 0.05).
Figure 6.
Anti-hemolytic activity of the candidate antivenom, the standard antivenom, and the plain antibodies against Bitis arietans venom and the hemolytc activity of the crude venom of Bitis arietans at 2.5 mg/ml. Values are represented as Mean ± SEM. a,b,cMeans with different superscript letters are significantly different (p < 0.05).
Agar gel immunodiffusion assay
The Agar gel immunodiffusion assay carried out showed lines of precipitation for the wells inoculated with the standard antivenom and the well inoculated with the candidate antivenom. The wells inoculated with the plain chicken antibodies showed no line of precipitation. The central well was inoculated with the crude venom of B. arietans. This shows that specific antibodies against the venom of B. arietans were present in the candidate antivenom and the standard antivenom (Fig. 7).
Figure 7.
Agar gel immunodiffusion assay. Test antivenom (black arrows), standard drug (blue arrow), and plain chicken antibodies (green arrows).
Determination of Igy concentration in egg yolk
The IgY content (mg/ml) of the sample was measured photometrically at 280 nm and calculated according to the Lamber-Beer law to be 0.77 mg/ml.
Discussion
Snakebite envenomation remains a significant public health concern in many regions, particularly where venomous snakes like B. arietans, commonly known as the puff adder, are prevalent (Fig. 8). Recent research focusing on the venom of B. arietans and the development of corresponding antivenoms has shed light on key factors influencing antivenom efficacy and potential strategies for improvement. The scope of the study was to determine the hemorrhagic and hemolytic activities of the venom alongside the activity of the candidate antivenom on the aforementioned assays and also its ability to neutralize the venom. The LD50 of the venom used in this study was 0.9 mg/kg; this implies that the venom was still able to retain its potency to exert its lethal effect as it falls in the same range (0.9–3.7 mg/kg) earlier determined by Brown [21] for intraperitoneal administration. Other authors also reported an LD50 of 1.78 mg/kg and 0.71 mg/kg [8,22]. The median lethal dose neutralization is a required preclinical test for antivenom efficacy determination [23]. Several authors recorded success in neutralization of the lethal doses obtained in their studies [24,25,5,26]. The candidate antivenom developed was able to neutralize the lethal dose of the B. arietans venom in this study. However, the inability to neutralize higher doses of the venom can be attributed to the concentration of the antivenom [25]. The IgY concentration in the hyperimmunized chicken egg yolk was lower than those obtained in other studies. Other factors could be the type of venom used for the hyperimmunization of the chickens and, the duration of hyperimmunization [5,27]. Snake venom-induced hemorrhages are a vital component of the pathophysiology of viperid envenomation [28]. This deleterious property of the venom on the vascular system is carried out chiefly by hemorrhagins present in the venom [28]. The venom of B. arietans from this study has shown that it possesses hemorrhagic properties. This phenomenon has also been reported by Matthew et al. [8]. The candidate antivenom, standard antivenom, and plain chicken antibodies showed anti-hemorrhagic properties at different concentrations of the venom. Other authors have reported similar results in which antibodies produced from chickens have been able to inhibit the hemorrhagic activity of snake venom [29]. The ability of the plain antibodies to have some anti-hemorrhagic properties can be attributed to the fact that they possess some compounds that though not specific, have some inhibitory capabilities against the toxin. This has been shown by previous research that non-specific compounds can inhibit certain activities of snake venom which was evident in the plain antibody group [12–15].
Viperids have been implicated in one of the major clinical signs associated with snakebite envenomation which is hemolysis. Determining the hemolytic properties of the venom used in this study thereby became one of the objectives of this research. The venom of B. arietans demonstrated its ability to cause hemolysis at all concentrations used in this study. This finding agrees with previous studies that showed the ability of the venom of B. arietans to cause hemolysis [8]. The candidate antivenom exhibited its anti-hemolytic properties in this study. The anti-hemolytic property of antibodies extracted from eggs obtained from hyperimmunised birds has also been reported in studies carried out by Meenatchisundaram [30]. The agar gel immunodiffusion test carried out in this study revealed the presence of precipitin lines for the candidate antivenom group, the standard antivenom group but none for the plain chicken antibody group. This indicates that specific antibodies against the venom of B. arietans were present in the test group and specific antibodies against the venom of B. arietans can be produced by hyper-immunizing chickens with the venom of the aforementioned snake [30]. The development and evaluation of antivenoms targeting B. arietans venom have yielded promising results. However, challenges such as variations in IgY concentration in hyperimmunized chicken egg yolks and the duration of hyperimmunization have been identified as potential factors affecting antivenom efficacy. Addressing these challenges through optimized immunization protocols could enhance antibody production and improve antivenom potency.
Conclusion
The venom of B. arietans has lethal, hemorrhagic, and hemolytic properties. Hyperimmunization of chickens with the venom of B. arietans can produce specific antibodies against the venom of B. arietans and specific antibodies (IgY) can be gotten from eggs laid by birds immunized with the venom of B. arietans and these antibodies have inhibitory properties against the lethal, hemorrhagic and hemolytic properties of the venom of B. arietans.
Acknowledgement
The authors thank the National Veterinary Research Institute, Vom, and the staff of the Toxicology Laboratory, Department of Veterinary Pharmacology and Toxicology, A.B.U. Zaria for their technical expertise and assistance in conducting this research.
Conflict of interest
The authors declare no potential conflict of interest.
Authors’ contributions
JSO, MM, JA, and POY designed the study, supervised the research, and edited the manuscript; JSO, KOJ, CCU, MOO, OA, OO, and AU performed the research and analyzed the data; JSO and KOJ drafted the manuscript. All authors have read and agreed to the published version of the manuscript.
