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Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims’ death or permanent disability. To better characterize the inflammatory process induced by this snake’s venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1β in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and—at least bitiscetin-1—induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein—bitiscetin-3—were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit—which differs from that of both bitiscetin-3 subunits—the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.

Historically, snakes have been considered to have a weak functional response to changing prey abundance due to their ectothermic physiology and slow digestion. I measured aspects of food consumption in a captive colony of puff adders ( Bitis arietans ) to evaluate their functional response. I introduce a new metric for this evaluation, the ‘factorial scope of ingestion’, defined as the maximum food intake divided by maintenance consumption. Rates of weight loss during fasting were also quantified to assess the potential for puff adders to persist through periods of low prey abundance. To maintain a constant body mass, puff adders must consume 63% of their body mass in rodents annually. However, when provided with unlimited food, they increased their food intake by an average factor of 12 times above maintenance levels for extended periods, which resulted in dramatic increases in body mass. Regression analysis between annualized food intake and changes in body mass, and direct measures of weight loss, independently estimated an annualized weight loss of ~ 23% body mass for fasting puff adders while metabolising fat. This suggests that a puff adder with a high initial body condition index could fast for more than 2 years. The extreme flexibility of puff adder feeding biology suggests that this species has a significant functional response to high prey abundance, and this response is likely to be much more profound than the functional response of mammalian predators. These findings highlight the importance of snakes as potential ecosystem stabilizers and for the control of agricultural rodent pests.

Variation in snake venoms is well documented, both between and within species, with intraspecific venom variation often correlated with geographically distinct populations. The puff adder, Bitis arietans, is widely distributed across sub-Saharan Africa and into the Arabian Peninsula where it is considered a leading cause of the ~310,000 annual snakebites across the region, with its venom capable of causing substantial morbidity and mortality. Despite its medical importance and wide geographic distribution, there is little known about venom variation between different B. arietans populations and the potential implications of this variation on antivenom efficacy. We applied a range of analyses, including venom gland transcriptomics, in vitro enzymatic assays and reverse phase chromatography to comparatively analyse B. arietans venoms originating from Nigeria, Tanzania, and South Africa. Immunological assays and in vitro enzymatic neutralisation assays were then applied to investigate the impact of venom variation on the potential efficacy of three antivenom products; SAIMR Polyvalent, EchiTAb-Plus and Fav-Afrique. Through the first comparison of venom gland transcriptomes of B. arietans from three geographically distinct regions (Nigeria, Tanzania, and South Africa), we identified substantial variation in toxin expression. Findings of venom variation were further supported by chromatographic venom profiling, and the application of enzymatic assays to quantify the activity of three pathologically relevant toxin families. However, the use of western blotting, ELISA, and in vitro enzymatic inhibition assays revealed that variation within B. arietans venom does not appear to substantially impact upon the efficacy of three African polyvalent antivenoms. The large distribution and medical importance of B. arietans makes this species ideal for understanding venom variation and the impact this has on therapeutic efficacy. The findings in this study highlight the likelihood for considerable venom toxin variation across the range of B. arietans, but that this may not dramatically impact upon the utility of treatment available in the region.

This study compared the yield, percentage solids, electrophoretic profile, gelatinolytic activity, and brine shrimp lethality of Bitis arietans venom prepared using freeze-drying and desiccator drying. Bitis arietans venom was collected from snakes at Bioken snake farm, Kenya, whereafter it was pooled and divided into two parts. Part 1 was desiccator dried venom (DDV) while part 2 was freeze-dried venom (FDV). The yield and percentage solids in DDV and FDV were compared using Welch’s Student’s t-test and the dried venoms were subsequently subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), 2D electrophoresis, gelatin in-gel zymography, and brine shrimp lethality assays. Mean venom yield and percentage solids did not differ between DDV and FDV (p = 0.5647 and p = 0.4676, respectively). SDS-PAGE and two-dimensional (2D) electrophoresis revealed similar protein profiles for DDV and FDV, showing bands and spot clusters within molecular weight ranges of ~16 kDa to >150 kDa and pH ranging from 3.5 to 9.5. Enzyme zymography revealed comparable gelatinolytic activity between DDV and FDV. However, the brine shrimp lethality assay indicated significantly higher toxicity in DDV (LC50: 86.57 μg/mL) compared to FDV (LC50: 460.37 μg/mL). DDV also showed greater lethality than FDV at 100 μg/mL (p = 0.0416) and 1000 μg/mL (p = 0.0008) but not at 10 μg/mL (p = 0.2465). These findings suggest that DDV exhibits higher toxicity in brine shrimp larvae than FDV, although both drying methods result in similar yields, percentage solids, venom profile, and gelatinolytic activity. Further research is necessary to investigate the mechanism behind this difference and its implications for antivenom production and long-term stability of venom.

The puff adder (Bitis arietans) is a medically important snake species found across much of Africa, yet there is limited literature on the clinical features and pathophysiology of envenoming after a puff adder bite. We conducted a case-series study to describe the clinical features of patients with puff adder bites who were treated in two primary healthcare facilities in Kenya and complemented our case-series with a scoping review of all published cases of puff adder envenoming that contained sufficient clinical details to highlight the major features. Between December 2020 and September 2021, 15 patients were admitted with a suspected puff adder bite (based on the patient's description of the biting snake or confirmed in patients who brought the dead snake or a picture of the biting snake for identification) at the Chemolingot and Mwingi sub-county hospitals in Baringo and Kitui counties, central Kenya. Common local and systemic features on admission included pain (n=15, 100%), swelling (n=14, 93%), and haemorrhage (n=9, 60%). Coagulopathy (n=2, 13%), blistering (n=1, 8%) and shock (n=1, 8%) were less common. In addition, we conducted a literature review and identified 23 studies with detailed descriptions of the clinical features of puff adder envenoming from 37 patients. Local features were common and consistent across cases-swelling (100%, n=37) and pain (95%, n=35). Systemic features were less consistent, with 10 (27%) patients exhibiting hypotension on admission, 10 (27%) patients reporting a fever, and 13 (35%) developing anaemia. Some complications were more common in patients with bites by captive snakes (amputations), compared to patients with bites by wild snakes (hypotension). Snake identification was easier and more accurate after bites by captive snakes, but more challenging for patients bitten in community settings. We combined clinical cases and a literature review to describe the common and less common clinical features of puff adder envenoming. Further clinical research incorporating serial laboratory assays of patients with definitively identified puff adder bites is crucial to better understand the pathophysiology of envenoming by this medically important snake species.

Bitis arietans is a snake of medical importance, as it is responsible for more accidents in humans and domestic animals than all other African snakes put together. The accidents are characterized by local and systemic alterations, such as inflammation, cardiovascular and hemostatic disturbances, which can lead victims to death or permanent disability. However, little is known about the envenomation mechanism, especially regarding the inflammatory response, which is related to severe clinical conditions triggered by the venom. Therefore, the aim of the present study was to evaluate the inflammatory response related to the B. arietans envenomation using a peritonitis mice model. By pharmacological interventions and use of mice genetically deficient of the 5-lipoxygenase enzyme (5-LO−/−) or platelet-activating factor (PAF) receptor (PAFR−/− the participation of eicosanoids and PAF in this response was also investigated. The obtained results demonstrated that the venom induces an in vivo inflammatory response, characterized by an early increased vascular permeability, followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity, accompanied by the production of the eicosanoids LTB4, LTC4, TXB2 and PGE2, as well as the local and systemic production of IL-6 and MCP-1. These inflammatory events were attenuated by the pre-treatment with anti-inflammatory drugs that interfere in lipid mediators’ functions. However, 5-LO−/− mice did not show a reduction of inflammatory response induced by the venom, while PAFR−/− mice showed a reduction in both the PMN leukocytes number and the local and systemic production of IL-6 and MCP-1. This study demonstrated that the Bitis arietans venom contains toxins that trigger an inflammatory process, which is partially dependent on lipid mediators, and may contribute to the envenomation pathology.

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom (“traditional approach”) and immunization with selected key toxins isolated from the snake venom (“toxin oriented” approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Sexual selection studies often focus on morphological traits that are important only in the later stages of mate acquisition. Comparatively little is known about traits that lead to mate acquisition, such as mate-searching activities. We experimentally manipulated body condition (i.e., the energy reserves) in male puff adders (Bitis arietans) prior to the mating season using supplemental feeding in the field, and used radio-telemetry and DNA paternity analyses to characterize the relationships between male energy reserves, mating activities, and reproductive success. We found that male mobility is a strongly sexually selected trait because males that travelled further in search of females have higher female encounter rates and reproductive success. However, supplemental feeding did not have a significant effect on mating activities or reproductive success, because control snakes compensated by foraging more often during the mating season. The time invested in foraging by control snakes did not come at the costs of decreased mating activities or opportunity compared to fed snakes, because the latter spent the spare time resting. Our experimental field research directly demonstrates the link between male mobility and reproductive success, identifying the ultimate mechanism leading to the evolution of prolonged male mate-searching activities in snakes, and indicates that male puff adders, presumed capital breeders, adjust their breeding tactics according to resource availability.

Background: The present study investigated the efficacy of Conyza bonariensis, Commiphora africana, Senna obtusifolia, Warburgia ugandensis, Vernonia glabra, and Zanthoxylum usambarense against Bitis arietans venom (BAV), Naja ashei venom (NAV), and Naja subfulva venom (NSV). Methods: 40 extracts and fractions were prepared using n-hexane, dichloromethane, ethyl acetate, and methanol. In vitro efficacy against snake venom phospholipase A 2 (svPLA 2 ) was determined in 96-well microtiter and agarose-egg yolk coagulation assays. in vivo efficacy against venom-induced cytotoxicity was determined using Artemia salina . Two commercial antivenoms were used for comparison. Results: The 96-well microtiter assay revealed poor svPLA 2 inhibition of BAV by antivenom (range: 20.76% ± 13.29% to 51.29% ± 3.26%) but strong inhibition (>90%) by dichloromethane and hexane fractions of C. africana , hexane and ethyl acetate extracts and fraction of W. ugandensis , dichloromethane fraction of V. glabra , and the methanol extract of S. obtusifolia . The methanol extract and fraction of C. africana , and the hexane extract of Z. usambarense strongly inhibited (>90%) svPLA 2 activity in NAV. The hexane and ethyl acetate fractions of V. glabra and the dichloromethane, ethyl acetate, and methanol extracts of C. africana strongly inhibited (>90%) svPLA 2 in NSV. The agarose egg yolk coagulation assay showed significant inhibition of BAV by the dichloromethane fraction of C. africana (EC 50 = 3.51 ± 2.58 μg/mL), significant inhibition of NAV by the methanol fraction of C. africana (EC 50 = 7.35 ± 1.800 μg/mL), and significant inhibition of NSV by the hexane extract of V. glabra (EC 50 = 7.94 ± 1.50 μg/mL). All antivenoms were non-cytotoxic in A. salina but the methanol extract of C. africana and the hexane extracts of V. glabra and Z. usambarense were cytotoxic. The dichloromethane fraction of C. africana significantly neutralized BAV-induced cytotoxicity , the methanol fraction and extract of C. africana neutralized NAV-induced cytotoxicity, while the ethyl acetate extract of V. glabra significantly neutralized NSV-induced cytotoxicity. Glycosides, flavonoids, phenolics, and tannins were identified in the non-cytotoxic extracts/fractions. Conclusion: These findings validate the local use of C. africana and V. glabra in snakebite but not C. bonariensis, S. obtusifolia, W. ugandensis , and Z. usambarense. Further work is needed to isolate pure compounds from the effective plants and identify their mechanisms of action.