The OROCHI (Observational Research of Clinical Course After mamusHI) Study: A Prospective, Observational, Multicenter Study on the Efficacy of Mamushi (Gloydius blomhoffii) Antivenom Serum.

BACKGROUND: Evidence on the efficacy of mamushi antivenom serum is limited. OBJECTIVE: To investigate the effectiveness of mamushi (Gloydius blomhoffii) antivenom serum. METHODS: The Observational Research Of the Clinical course after mamusHI bite (OROCHI) study was a prospective multicenter study conducted at 24 hospitals in Japan. Patients hospitalized due to mamushi bite were registered. The primary endpoint was the length of hospital stay. Secondary endpoints were adverse effects, pain (numerical rating scale), and grade of swelling. We performed a cohort analysis to compare outcomes between patients treated with mamushi antivenom serum (antivenom group) and those who were not treated with the serum (no-antivenom group). RESULTS: Overall, 106 patients were registered across 18 hospitals between April 22, 2020, and October 31, 2022. Of these, 92 were eligible for the analyses, with 53 and 39 in the antivenom and no-antivenom groups. The median (interquartile) length of hospital stay was not significantly different between the antivenom and no-antivenom groups (5 (3-6) days vs. 3 (1-8) days, P = 0.369). In multivariable analysis, the adjusted odds ratio for a hospital stay of >4 days was 1.331 in patients treated with mamushi antivenom serum (95% confidence interval (CI) = 0.744‒2.015, P = 0.574) and 6.154 in patients treated with cepharanthine (95% CI = 1.442-26.258, P = 0.014). Pain and the grade of swelling were worse in the antivenom group than in the no-antivenom group up to 24 h after arrival, but there were no differences in these outcomes after 48 h. CONCLUSION: Although the effectiveness of mamushi antivenom serum in reducing the length of hospitalization was not demonstrated, beneficial effects on pain and swelling were observed.

Snakebites and Antisnake Venom Utilization in Ghana's Oti Region: A 6-Year Retrospective Study.

Background: Snakebite is a global environmental and occupational hazard and a significant public health threat. In rural areas, snakebite cases often go unreported and undocumented due to the lack of access to well-structured healthcare facilities/infrastructure. In some cases, the need for antisnake venom (ASV) far outstrips supply, negatively affecting treatment outcomes. This study, therefore, assessed the epidemiological characteristics of snakebite cases, their management, and how antivenoms are utilised at the selected hospital in the Jasikan District Hospital. Methods: A 6-year retrospective study using secondary data from antivenom return forms (pharmacy records), clinical records (patient folders), the District Health Information Management System-2 (DHIMS-2) database, and consulting room registers was carried out in selected hospitals in the Jasikan District, Oti, Ghana. Results: The predominant symptom of snakebite was localised pain (71.4%). The snakebite commonly occurred at home (19%) and on farms (18%). Of the 98 snakebite cases, ASV was administered to 73 (74.5%) cases. Supportive treatment applied included prophylactic antitetanus immunoglobulin (ATS) (80.6%), prophylactic antibiotics (63%), corticosteroids (80.6%), and analgesics (63%). 95% (n = 94) of complete recoveries were recorded; three were discharged against medical advice, and one was mortality. The supply and use of antivenom were erratic throughout the months of high incidence, partly due to inconsistent availability at the Regional Medical Stores. The average ASV vials and hospital stay duration were 1.23 ± 0.86 vials and 2.67 ± 1.97 days, respectively. Although the peak of snakebites occurs in April, May, and June, the demand for antivenom in April and May exceeded supply. Conclusion: The outcome of most snakebite case management was appropriate, irrespective of inadequate ASV supply in certain months. The erratic antivenom supply should be aligned with seasonal and facility-use patterns to enhance regional snakebite management.

Bridges between two medical realities: Perspectives of Indigenous medical and nursing students on snakebite care in the Brazilian Amazon.

In the Brazilian Amazon, snakebite envenomations (SBEs) disproportionately affect Indigenous populations, and have a significantly higher incidence and lethality than in non-Indigenous populations. This qualitative study describes the Indigenous and biomedical healthcare domains for SBE care from the perspective of the Indigenous medical and nursing students in Manaus, Western Brazilian Amazon. In-depth interviews were conducted with five Indigenous students from the Amazonas State University, between January and December 2021. The interviews were analyzed using inductive content analysis. We organized an explanatory model with five themes: (1) participants' identities; (2) causality levels in Indigenous and biomedical systems; (3) therapeutic itineraries in Indigenous and biomedical systems; (4) ideological implications of adding biomedical devices to Indigenous healing systems; and (5) therapeutic failure in and efficacy of Indigenous and biomedical systems. From a noncolonial perspective and seeking to increase the quality and acceptability of health care for the Indigenous populations of the Brazilian Amazon, the training of Indigenous health professionals presents itself as a promising strategy. For this goal, universities should serve as empowering settings for Indigenous health students that support them in their growth and development, raise their awareness of injustice, and catalyze change toward a culturally adapted and effective service for the users.

Paraspecific neutralization capacity of polyvalent snake antivenom against Montivipera wagneri venom.

PURPOSE: This study aims to measure the paraspecific neutralization capacity of nationally produced HSGM polyvalent snake antivenom (HSGM-PSAV), produced using Macrovipera lebetina obtusa, Montivipera xanthina, and Vipera ammodytes montandoni venom, against the lethal effect of the venom of Montivipera wagneri, which is endemic to the Eastern Black Sea and Eastern Anatolia regions of Turkey. METHODS: The neutralization capacity of HSGM-PSAV against the lethal effect of M. wagneri venom was studied using the potency determination testing method specified in the Turkish and European Pharmacopoeia. Lethal dose 50 (LD50) values of the venoms used in immunization, M. wagneri venom in mice, and effective dose 50 (ED50) values of HSGM-PSAV against four types of venoms were calculated using two-fold dilutions. RESULTS: HSGM-PSAV neutralized the lethal effect of M. wagneri venom in mice. The ED50 of the HSGM-PSAV against M. wagneri venom was calculated as 304.42 LD50/mL. CONCLUSION: As a result of this in-vivo study, it was determined that HSGM-PSAV neutralized M.wagneri venom above the antivenom neutralization capacity threshold values (≥50 LD50/mL) specified in the Turkish and European Pharmacopoeia. This result is important preclinical data regarding the usability of HSGM-PSAV in the treatment of poisoning due to M. wagneri bites.

The geographical distribution of scorpions, implication of venom toxins, envenomation, and potential therapeutics in Southern and Northern Africa.

Scorpions are predatory arachnids whose venomous sting primarily affects people in tropical and subtropical regions. Most scorpion stings can only cause localized pain without severe envenomation. Less than one-third of the stings cause systemic envenoming and possibly lead to death. About 350,000 scorpion stings in Northern Africa are recorded yearly, resulting in about 810 deaths. In Eastern/Southern Africa, there are about 79,000 stings recorded yearly, resulting in 245 deaths. Farmers and those living in poverty-stricken areas are among the most vulnerable to getting stung by scorpions. However, compared to adults, children are at greater risk of severe envenomation. Scorpion venom is made up of complex mixtures dominated by peptides and proteins that confer its potency and toxicity. These venom toxins have intra- and interspecies variations associated with the scorpion's habitat, sex, diet, and age. These variations alter the activity of antivenoms used to treat scorpion sting envenomation. Thus, the study of the proteome composition of medically important scorpion venoms needs to be scaled up along their geographical distribution and contributions to envenomation in Southern and Northern Africa. This will help the production of safer, more effective, and broad-spectrum antivenoms within these regions. Here, we review the clinical implications of scorpion sting envenomation in Southern and Northern Africa. We further highlight the compositions of scorpion venoms and tools used in scorpion venomics. We discuss current antivenoms used against scorpion sting envenomation and suggestions for future production of better antivenoms or alternatives. Finally, we discuss the therapeutic properties of scorpion venom.

Antivenom potential of the latex of Jatropha mutabilis baill. (Euphorbiaceae) against Tityus stigmurus venom: Evaluating its ability to neutralize toxins and local effects in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Species of the Jatropha genus (Euphorbiaceae) are used indiscriminately in traditional medicine to treat accidents involving venomous animals. Jatropha mutabilis Baill., popularly known as "pinhão-de-seda," is found in the semi-arid region of Northeastern Brazil. It is widely used as a vermifuge, depurative, laxative, and antivenom. AIM OF THE STUDY: Obtaining the phytochemical profile of the latex of Jatropha mutabilis (JmLa) and evaluate its acute oral toxicity and inhibitory effects against the venom of the scorpion Tityus stigmurus (TstiV). MATERIALS AND METHODS: The latex of J. mutabilis (JmLa) was obtained through in situ incisions in the stem and characterized using HPLC-ESI-QToF-MS. Acute oral toxicity was investigated in mice. The protein profile of T. stigmurus venom was obtained by electrophoresis. The ability of latex to interact with venom components (TstiV) was assessed using SDS-PAGE, UV-Vis scanning spectrum, and the neutralization of fibrinogenolytic and hyaluronidase activities. Additionally, the latex was evaluated in vivo for its ability to inhibit local edematogenic and nociceptive effects induced by the venom. RESULTS: The phytochemical profile of the latex revealed the presence of 75 compounds, including cyclic peptides, glycosides, phenolic compounds, alkaloids, coumarins, and terpenoids, among others. No signs of acute toxicity were observed at a dose of 2000 mg/kg (p.o.). The latex interacted with the protein profile of TstiV, inhibiting the venom's fibrinogenolytic and hyaluronidase activities by 100%. Additionally, the latex was able to mitigate local envenomation effects, reducing nociception by up to 56.5% and edema by up to 50% compared to the negative control group. CONCLUSIONS: The latex of Jatropha mutabilis exhibits a diverse phytochemical composition, containing numerous classes of metabolites. It does not present acute toxic effects in mice and has the ability to inhibit the enzymatic effects of Tityus stigmurus venom in vitro. Additionally, it reduces nociception and edema in vivo. These findings corroborate popular reports regarding the antivenom activity of this plant and indicate that the latex has potential for treating scorpionism.

Development and characterization of nanobody against envenomation by Naja naja oxiana.

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

Importance of the Cysteine-Rich Domain of Snake Venom Prothrombin Activators: Insights Gained from Synthetic Neutralizing Antibodies.

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms.

Successful use of anti-venom cross-neutralization effects in the clinical management of Shore Pit Viper envenomation.

As the landscape becomes more urbanized, snakebites have increasingly become uncommon presentations to the emergency departments in Singapore, while snakebites causing significant envenomation are even rarer. In this case report, we discuss a 55-year-old man who had significant envenomation from a Shore Pit Viper (Trimeresurus Purpureomaculatus) and who was successfully treated with haemato-toxic polyvalent antivenom (HPAV). He initially presented with pain, swelling and bleeding over his wound. Due to a deterioration in his coagulation profile, he was given two doses of HPAV which is typically reserved for viperid snakes instead. Following administration of the anti-venom, the patient's coagulation profile improved, and the local soft tissue effects of the venom resolved. He did not manifest any adverse effects and was discharged uneventfully about 72 h after the snakebite. The cross-neutralization potential of HPAV for Shore Pit Viper (Trimeresurus Purpureomaculatus) venom in this case study suggests that there may be a possible common underlying chemical structure and pathophysiology among the venom proteins of various snake species. Given that Trimeresurus-specific antivenom is unavailable in most countries, this cross-neutralization strategy deserves further consideration and evaluation in similar circumstances.

Novel insights into the application of recombinant mutated phospholipases D as antigens for developing new strategies against Loxoscelism.

Loxoscelism is the pathological condition triggered by a brown spider bite. The venom of these spiders is rich in phospholipases D (PLDs), which can induce virtually all local and systemic manifestations. Recombinant mutated PLDs from clinically relevant Loxosceles species in South America have been investigated as potential antigens to develop novel therapeutic strategies for loxoscelism. However, certain gaps need to be addressed before a clinical approach can be implemented. In this study, we examined the potential of these recombinant mutated PLDs as antigens by testing some variations in the immunization scheme. Furthermore, we evaluated the efficacy of the produced antibodies in neutralizing the nephrotoxicity and sphingomyelinase activity of brown spider venoms. Our findings indicate that the number of immunizations has a greater impact on the effectiveness of neutralization compared to the amount of antigen. Specifically, two or three doses were equally effective in reducing dermonecrosis and edema. Additionally, three immunizations proved to be more effective in neutralizing mice lethality than one or two. Moreover, immunizations mitigated the signs of kidney injury, a crucial aspect given that acute renal failure is a serious systemic complication. In vitro inhibition of the sphingomyelinase activity of Loxosceles venoms, a key factor in vivo toxicity, was nearly complete after incubation with antibodies raised against these antigens. These findings underscore the importance of implementing an effective immunization scheme with multiple immunizations, without the need for high antigen doses, and enhances the spectrum of neutralization exhibited by antibodies generated with these antigens. In summary, these results highlight the strong potential of these antigens for the development of new therapeutic strategies against cutaneous and systemic manifestations of loxoscelism.

Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms.

The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 µg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

Taking the sting out of scorpions: Electrophysiological investigation of the relative efficacy of three antivenoms against medically significant Centruroides species.

In this study, we report the innovative application of whole-cell patch-clamp electrophysiology in assessing broad-spectrum neutralisation by three different antivenoms, of venoms from the medically significant scorpion genus Centruroides. Envenomations by as many as 21 species from the Centruroides genus result in up to 300,000 envenomations per year in Mexico, which poses significant and potentially life-threatening pathophysiology. We first evaluated the in vitro manifestation of envenomation against two human voltage-gated sodium (hNaV) channel subtypes: hNaV1.4 and hNaV1.5, which are primarily expressed in skeletal muscles and cardiomyocytes, respectively. The neutralisation of venom activity was then characterised for three different antivenoms using a direct competition model against the more potent target, hNaV1.4. While broad-spectrum neutralisation was identified, variation in neutralisation arose for Centruroides elegans, C. limpidus, C. noxius and C. suffusus venoms, despite the presence of a number of these venoms within the immunising mixture. This raises questions regarding the truly "broad" neutralisation capacity of the antivenoms. This study not only extends previous validation of the in vitro investigation of antivenom efficacy utilising the whole-cell patch-clamp technique but also underscores the potential of this animal-free model in exploring cross-reactivity, experimental scalability, and most importantly, informing clinical management practices regarding the administration of antivenom in Mexico.

Experimental production and efficacy testing of mono-specific antibodies against the venom of carpet viper (Echis ocellatus) from savannah Nigeria.

Echis ocellatus is one of the commonest snakes responsible for envenomation in Nigeria. Antivenom is the only effective treatment, but the country suffers from a limited supply of effective antivenom. This study therefore aimed to explore the feasibility of effective, mono-specific antibodies production through immunization in rabbits using the venom of Echis ocellatus from Nigeria. The World Health Organization guide on antivenom production was employed in the immunization and the resultant antibodies were purified using protein A agarose column chromatography. Antibody titer reached a high plateau by 2-month immunization, and SDS PAGE of the sera suggests the presence of intact immunoglobulins accompanied with the heavy (50 kDa) and light (25 kDa) chains. The venom has an intravenous LD50 of 0.35 mg/kg in mice, and the venom lethality at a challenge dose of 2 LD50 was effectively neutralized by the antibodies with a potency value of 0.83 mg venom per g antibodies. The antibodies also neutralized the procoagulant activity of the venom with an effective dose (ED) of 13 ± 0.66 µl, supporting its use for hemotoxic envenomation. The study establishes the feasibility of developing effective, mono-specific antibodies against the Nigerian Carpet viper.

A case report of mis-snaken identity: When misdiagnosis really bites.

When patients present with an unknown puncture wound, emergency physicians need to consider regional hazards, in addition to standard mechanical injury etiologies. In the Southwestern United States, one such hazard is the rattlesnake. In this report, we present a case in which a rattlesnake envenomation was not considered as a possible cause for a puncture wound of unknown origin, which resulted in an envenomation left untreated for 7 days. A full dry bite observation period of 12 h with serial physical exams and laboratory analysis with guidance from the region poison control center might have led to earlier recognition of an envenomation and antivenom administration. A male patient in his late 70's felt a painon his right ankle while in his backyard in southern Arizona. He did not see the cause and assumed he had sustained an insect bite. He went to the ED that day with minor pain and swelling and was discharged home. One week later, he re-presented severely anemic with edema and ecchymosis to the entire right lower extremity that developed over several days after his first ED visit. He was admitted for antivenom and blood transfusion and discharged on hospital day three. For as long as humans continue to interact with the natural world, venomous creature encounters are going to continue to happen. Rattlesnake envenomation should be included in a physician's differential diagnosis even if one is not witnessed, especially in regions with high rattlesnake activity. In addition to assessing for other potential causes of undifferentiated puncture wounds, serial physical examinations and laboratory testing (with guidance of the regional poison center) are necessary to rule out rattlesnake envenomation.

Infections following rattlesnake envenomation and use of antibiotics.

BACKGROUND: There are 7000-8000 venomous snake bites annually in the USA. Antibiotics are commonly administered to bite victims because infection is difficult to differentiate from local tissue injury following envenomation. METHODS: The Arizona Poison and Drug Information Center (APDIC) in Tucson oversees antivenom administration for 14 Arizona counties. Records (1999-2021) were searched for antibiotic use and confirmed infections after a rattlesnake bite. RESULTS: There were 4160 calls to APDIC regarding rattlesnakes. After excluding bites to animals, 'dry bites', prisoners and records with missing data, 2059 records were evaluated. Systemic antibiotics were administered to 206 patients (10% of bite victims). Twenty patients (0.97%) had confirmed infections, including cellulitis (n=10), fasciitis (n=4), abscess (n=3) and osteomyelitis (n=3). Five of the victims had positive blood cultures. The presence of tissue necrosis, leukocytosis, fever and elevated fibrinogen levels did not discriminate between toxic effects of venom and infection. CONCLUSIONS: Confirmed infections following a rattlesnake bite are uncommon (0.97% of bites). Physicians should refrain from prescribing antibiotics, as they are not justified for most rattlesnake bite victims and the variety of pathogens encountered precludes use of any single effective antibiotic.

Envenomation by the Indian ornamental tarantula (Poecilotheria regalis): A case report on treatment with Latrodectus mactans antivenom.

Poecilotheria spiders are considered theraphosids of underestimated clinical importance, with bites from these species inducing symptoms such as severe pain and intense muscle cramps. However, there is no specific treatment for the envenomation caused by these species, which, while native to India and Sri Lanka, are widely distributed worldwide. The present study reports the case of a 31-year-old man bitten by a Poecilotheria regalis specimen. The patient's clinical presentation was similar to Latrodectus envenomation, and patient was treated with an L. mactans antivenom. Most of patient's symptoms improved (fasciculations, pain, erythema, and local swelling), except muscle cramps. A toxicological study conducted on mice did not show that L. mactans antivenom has a neutralizing effect on the toxicity of P. regalis. The present report discusses the envenoming process of Poecilotheria species and the possible neutralizing effect exerted by L. mactans antivenom.

A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

Comparative functional characterization and in vitro immunological cross-reactivity studies on Daboia russelii and Craspedocephalus malabaricus venom.

BACKGROUND: Snake venom is a complex mixture of organic and inorganic constituents, including proteins and peptides. Several studies showed that antivenom efficacy differs due to intra- and inter-species venom variation. METHODS: In the current study, comparative functional characterization of major enzymatic proteins present in Craspedocephalus malabaricus and Daboia russelii venom was investigated through various in vitro and immunological cross-reactivity assays. RESULTS: The enzymatic assays revealed that hyaluronidase and phospholipase A2 activities were markedly higher in D. russelii. By contrast, fibrinogenolytic, fibrin clotting and L-amino acid oxidase activities were higher in C. malabaricus venom. ELISA results suggested that all the antivenoms had lower binding potential towards C. malabaricus venom. For D. russelii venom, the endpoint titration value was observed at 1:72 900 for all the antivenoms. In the case of C. malabaricus venom, the endpoint titration value was 1:2700, except for Biological E (1:8100). All these results, along with the avidity assays, indicate the strength of venom-antivenom interactions. Similarly, the western blot results suggest that all the antivenoms showed varied efficacies in binding and detecting the venom antigenic epitopes in both species. CONCLUSIONS: The results highlight the need for species-specific antivenom to better manage snakebite victims.

Loxosceles amazonica Brown Spider venom: Insights into enzymatic activities, immunorecognition, and novel phospholipase D isoforms.

The Loxosceles genus represents one of the main arachnid genera of medical importance in Brazil. Despite the gravity of Loxosceles-related accidents, just a handful of species are deemed medically important and only a few have undergone comprehensive venom characterization. Loxosceles amazonica is a notable example of a potentially dangerous yet understudied Loxosceles species. While there have been limited reports of accidents involving L. amazonica to date, accidents related to Loxosceles are increasing in the North and Northeast regions of Brazil, where L. amazonica has been reported. In this work, we provide a complementary biochemical and immunological characterization of L. amazonica venom, considering its most relevant enzymatic activities and its immunorecognition and neutralization by current therapeutic antivenoms. Additionally, a cDNA library enriched with phospholipase D (PLD) sequences from L. amazonica venom glands was built and subsequently sequenced. The results showed that L. amazonica venom is well immunorecognised by all the tested antibodies. Its venom also displayed proteolytic, hyaluronidase, and sphingomyelinase activities. These activities were at least partially inhibited by available antivenoms. With cDNA sequencing of PLDs, seven new putative isoforms were identified in the venom of L. amazonica. These results contribute to a better knowledge of the venom content and activities of a synanthropic, yet understudied, Loxosceles species. In vivo assays are essential to confirm the medical relevance of L. amazonica, as well as to assess its true toxic potential and elucidate its related pathophysiology.

Expression and characterization of scFv-6009FV in Pichia pastoris with improved ability to neutralize the neurotoxin Cn2 from Centruroides noxius.

Small single-chain variable fragments (scFv) are promising biomolecules to inhibit and neutralize toxins and to act as antivenoms. In this work, we aimed to produce a functional scFv-6009FV in the yeast Pichia pastoris, which inhibits the pure Cn2 neurotoxin and the whole venom of Centruroides noxius. We were able to achieve yields of up to 31.6 ± 2 mg/L in flasks. Furthermore, the protein showed a structure of 6.1 % α-helix, 49.1 % ß-sheet, and 44.8 % of random coil by CD. Mass spectrometry confirmed the amino acid sequence and showed no glycosylation profile for this molecule. Purified scFv-6009FV allowed us to develop anti-scFvs in rabbits, which were then used in affinity columns to purify other scFvs. Determination of its half-maximal inhibitory concentration value (IC50) was 40 % better than the scFvs produced by E. coli as a control. Finally, we found that scFv-6009FV was able to inhibit ex vivo the pure Cn2 toxin and the whole venom from C. noxius in murine rescue experiments. These results demonstrated that under the conditions assayed here, P. pastoris is suited to produce scFv-6009FV that, compared to scFvs produced by E. coli, maintains the characteristics of an antibody and neutralizes the Cn2 toxin more effectively.