Abstracts from Venom Week 2020

Editors: Steven A. Seifert, MD, Michael Schaer, DVM, Stephen P. Mackessy, PhD

This issue of Toxicon contains the abstracts of scientific presentations at Venom Week

2020, held in Gainesville, Florida, March 4 – 7, 2020. Venom Week meetings are periodic,

scientific symposiums on all things venomous. Venom Week Symposiums include

clinicians, veterinarians, venom and antivenom researchers, drug discovery and

development scientists, basic scientists, animal scientists, zoo and collection managers,

regulatory and translational scientists, and others with an interest in venomous animals and

their venoms.

The Chairs and organizers of the meeting were Alfred Aleguas, PharmD and Michael

Schaer, DVM, as well as Erin Dinkel, Brianna Blassneck and other support staff in the

University of Florida CME Office.

The abstract review committee included Alfred Aleguas, PharmD, Daniel Keyler, PharmD,

Stephen Mackessy, PhD, Elda Sanchez, PhD, Michael Schaer, DVM, Steven Seifert, MD,

and Carl-Wilhelm Vogel, MD, PhD.

Venom Week 2020 was supported by its attendees, as well as:

Organizing Sponsor: The North American Society of Toxinology

Diamond Sponsor: Rare Disease Therapeutics

Gold Sponsor: BTG Specialty Pharmaceuticals

Silver Sponsor: bioVeteria

Vendors: Boehringer Ingelheim

Toxins (MDPI)

Venom Vet

Phirex

The Rattlesnake Conservancy

Save the Snakes.

ABSTRACTS AA - ANTIVENOMS AND ANTIDOTES AA1 - INTRASPECIFIC VARIATION OF CROTALUS BASILISCUS VENOM AND NEUTRALIZATION BY BIRMEX ANTIVENOM Colis-Torres, A1, Neri-Castro, E1, Olvera-Rodríguez, A1, Strickland, JL2, Jones, JM3, Alagón, A 1 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México, 2 Department of Biological Sciences, Clemson University, 190 Collings St., Clemson, SC, 29631, USA. 3Herp.mx A.C., Villa del Álvarez, Colima, México. E-mail address: andrea.colis@mail.ibt.unam.mx Background: In Mexico, 4,000 snakebites are reported each year from which approximately 98% are caused by pitvipers, including Crotalus rattlesnakes. The genus Crotalus is vastly represented in Mexico with 44 species. Crotalus basiliscus is the largest rattlesnake in Mexico and it is found along the Pacific coast. The World Health Organization (WHO) lists this species as medically important and its venom is used as an immunogen for the production of “Faboterápico Polivalente Antiviperino” manufactured by Birmex. Despite being important for antivenom production, its venom has been poorly studied. A study that has reported on venom composition in C. basiliscus did not include the location of the individuals used in the study. Venoms from species that are used as immunogens should be well characterized in order to: 1) ensure a homogenous immunogen

during the production of each antivenom batch 2) and to take advantage of intraspecific variation (if it exists) to broaden neutralization against other species. To address the lack of information, the aim of our study is to determine the composition of C. basiliscus venom throughout its range of distribution and asses antivenom efficacy. Methods: We collected samples from as many locations as possible and targeted individuals from different ages. The biochemical and biological characterization was performed to determine hemorrhagic activity, minimum coagulant dose (MCD), lethality measured as Medium Lethal Dose (LD50), fibrinogenolytic activity, phopholipase activity and absence/presence of the two major components (crotoxin and crotamine). Venoms that exert variation were tested against different batches of BIRMEX antivenom. We will employ the antivenomics strategy to determine distinct recognition between components by antivenom. Results: Currently, 23 venom samples have been studied, including 7 juveniles, from 5 different Mexico states. SDS-PAGE and RP-HPLC profiles show significant geographic and ontogenic variation. Juvenile venoms contain crotamine and crotoxin as primary components (except one that lacks crotoxin) while adult venoms vary in serine proteases, metalloproteases and crotoxin proportion. As for lethality, LD50 values suggest juvenile venoms are more potent (0.1 to 0.5 µg/g), whereas adults ranged from 0.5 to 15 µg/g. Adults had higher hemorrhagic values than juveniles who lacked it. Juveniles were capable of excising α chain of fibrinogen while adults cut α and ß chains. Phospholipase activity did not have a specific pattern given that both adults and juveniles have differences between them. Finally, antivenoms were not equally effective recognizing and neutralizing venoms. Conclusion: Crotalus basiliscus venoms exhibit intraspecific and ontogenetic variation. Intraspecific venom variation in C. basiliscus should be taken advantage of by antivenom producers to make mixtures capable of recognizing and neutralizing components against a wider variety of Mexican pit viper species. Ackowledgment: Fordecyt 303045 Key Words: C. basilicus; venom; ontogentics; neurtalization AA2 - CONTINUOUS INFUSION AS AN ALTERNATIVE ADMINISTRATION STRATEGY OF OVINE FAB ANTIVENOM: INITIAL EXPERIENCE IN 41 CASES Shirazi FM1, Delva-Clark H2, Reynolds KM2, Lavonas EJ2,3, Ryall KA2, Dart RC2,3 1Arizona Poison and Drug Information Centers. The University of Arizona College of Pharmacy, Tuscon Arizona 2Rocky Mountain Poison and Drug Safety, Denver Health, Denver, Colorado, USA; 3Department of Emergency Medicine, University of Colorado School of Medicine, Aurora, Colorado, USA E-mail address: heather.delva@rmpds.org

Background: Continuous infusion (CI) of ovine Fab antivenom (CroFab®, BTG Pharmaceuticals; hereafter, FabAV) has been proposed as an alternative administration strategy to bolus dosing. Previous reports are limited to case reports and small case series. Methods: This is a retrospective observational study of patients managed with the advice of a single poison center in Arizona from 01 June 2012 to 01 September 2018. The study population consisted of patients with a rattlesnake envenomation treated with FabAV, did not receive other antivenom, and intentionally received at least one dose of FabAV as a CI (infused over longer than 2 hours) at various time periods during treatment. Data were extracted from poison center records by a trained researcher and verified by a second researcher. Descriptive statistics were used to report demographics, antivenom treatment details, and safety and efficacy outcomes. Results: Forty-one patient encounters were identified. The median age was 55.0 (range: 2.0, 83.0) years. Thirty-five (85.4%) patients were male and 10 (24.4%) were children under age 18 years. CIs consisted of a median of 4 (range: 2, 6) vials of FabAV infused over more than 2 hours (range: 3, 7). A CI was administered to achieve initial control following at least one standard FabAV dose (4 to 6 vials administered over one hour) in 26 cases; 18 out of the 26 (69.2%) patients achieved initial control within 4 hours of completing the CI. Seven patients (26.9%) treated with a CI to achieve initial control were subsequently readmitted, and 2 (7.7%) were retreated with FabAV. Eighteen patients received a CI to treat recurrence of venom effects; 4 out of the 18 (22.2%) achieved control of recurrent venom effects within 4 hours. Two patients received a CI as an alternative dosing method due to an acute hypersensitivity reaction to a standard FabAV infusion; neither patient experienced a reaction when FabAV was administered as a CI. Among all patients receiving a CI, the median total number of FabAV vials administered was 22.0 (range: 12.0, 50.0) vials. Apart from readmission due to recurrent coagulopathy or thrombocytopenia, no serious adverse events were identified. Discussion/Conclusion: CI of FabAV over more than two hours is safe and practical, and potentially reduces the number of acute hypersensitivity reactions. Controlled studies are necessary to evaluate the potential benefit of a CI alternative administration strategy. Key Words: Snake Bites; Crotalus; Rattlesnake; Antivenins; Continuous Infusion AA3 - RECURRENT VENOM EFFECTS FOLLOWING NORTH AMERICAN CROTALIDAE ENVENOMATIONS: A SYSTEMATIC REVIEW Lavonas, EJ1,2, Delva-Clark, H1, Le L1

1 Rocky Mountain Poison and Drug Safety, Denver Health, Denver, Colorado, USA; 2 Department of Emergency Medicine, University of Colorado School of Medicine, Aurora, Colorado, USA E-Mail address: eric.lavonas@dhha.org

Background: Recurrence of local tissue venom effects and hematologic venom effects (HVE) following antivenom treatment have been described in case reports, cohort studies, and clinical trials. No previous attempt has been made to systematically study this literature. Methods: Using controlled vocabulary thesaurus and Emtree terms, PubMed, Ovid Medline, and EMBASE were searched to identify articles published through 01 April 2019 containing patient-level data about human envenomation by North American Crotalidae. Articles describing recurrent, late-onset, or persistent local tissue venom effects or HVE were abstracted. Clinical trials and cohort studies were characterized as analytic studies, while single case reports and convenience sample case series were characterized as case studies. Results: Sixty-six articles reported endpoints of interest, including 24 analytic studies (470 patients) and 42 case studies (58 patients). Most patients received ovine Fab antivenom (CroFab; analytic:306 patients (65.1%); case studies: 46 patients (9.8%)), followed by equine whole-IgG (Wyeth ACP; analytic: 43 patients (9.1%); case studies: 5patients (8.6%)) and equine F(ab’)2 (Anavip; analytic:6 patients (1.3%); case studies: 2patients (3.4%)).The type of antivenom was not reported in 115 (24.5%) patients in analytic studies and 6 (10.3%) patients in case studies. Time from envenomation to first antivenom administration and initial antivenom dose were rarely reported. In the analytic studies, the most commonly reported endpoint of interest was late-onset HVE (220 patients (46.8%)), followed by recurrent HVE (150 patients (31.9%)) and persistent HVE (72 patients (19.5%)).In case studies, the most commonly reported endpoint of interest was recurrent HVE (42 patients (72.4%)), followed by persistent HVE (15 patients (25.9%)) and late-onset HVE (13 patients (22.4%)). Late bleeding was reported in 18 (3.8%) analytic study patients; at least 17 of these involved rattlesnake envenomations from Arizona. Four analytic study patients (0.9%) were considered to experience major bleeding by the study author. Seven case study patients (12.1%) experienced late bleeding, with major bleeding in 5 cases (8.6%, including 1 fatality).The majority of patients in analytic studies were reported from two centers in Arizona and Southern California, in which the proportion of patients with HVE is reported to be high. Discussion/Conclusion: This systematic review finds that late-onset, recurrent, and/or persistent HVE have been reported in patients treated with Fab, F(ab’)2, and IgG-based antivenoms and can lead to hospital readmission and retreatment. Even among patients with late-onset HVE, late bleeding is uncommon, and medically significant late bleeding is rare. Published data do not support an accurate estimate of the proportion of patients suffering these outcomes nationwide. Further studies involving similar populations of Fab and F(ab’)2-treated patients would be valuable to understand comparative risks under real-world conditions. Key Words: Snake Bites, Antivenins, Immunoglobulin Fab fragments, Thrombocytopenia, Afibrinogenemia, Recurrence

AA4 - VARESPLADIB (LY315920) EFFECTIVELY REVERSES EXPERIMENTAL NEURO-MYOTOXIC EFFECTS IN A PIG MODEL OF LETHAL ENVENOMING BY TAIPAN (O. SCUTELLATUS) AND MOJAVE RATTLESNAKE (C. SCUTULATUS)

1Gilliam LL 1Gilliam JN, 2Carter RW, 2Samuel SP, 4Gutierrez JM, 5Williams DJ, 6Surovsteva YV, 2Bulfone T, 2,3Lewin* MR 1Oklahoma State University; 2 Ophirex, Inc.; 3 California Academy of Sciences, 4Instituto Clodomiro Picado, 5University of Melbourne, 6Yale University E-mail address: matt@ophirex.com or mlewin@calacademy.org Background: Two objectives of modernizing treatment for snakebite envenoming are to improve the speed of access to effective interventions and to improve the performance of serum based therapeutics. Small molecule, toxin specific inhibitors of key venom components could begin to address these inadequately addressed needs and examination of how they fit into the bigger picture of multicomponent treatment strategies is necessary above and beyond the task of simply identifying specific inhibitors. Methods: >20 venoms representing different clades and geographical distributions of snakes were screened for sPLA2 activity and then sPLA2 activity was compared in the presence of LY315920 or antivenoms from North America, India and Australia. Mouse studies were conducted to screen for systemic toxicity and the efficacy of LY315920 and/or its orally bioavailable prodrug, LY333013. Three venoms, taipan (New Guinea and Australian) and Mojave rattlesnake were selected from the mouse studies for direct comparison of LY315920/LY333013 and CSL Taipan Specific antivenom, CroFab® or Anavip®. Results: In vitro, IC50 for LY315920 inhibition of snake venom sPLA2swith appropriately matched venom samples was, on average, 6.7E-12M (N=28 venoms) CroFab® 3.8E-07M (N=10) Anavip® 9.9E-7M (N=10) Premium India 1.5E-6M (N=6) Bharat-Indian Polyvalent 2.4E-6M (N=6) Premium Africa 4.9E-6M (N=9) CSL Taipan Specific 9.7E-08 (N=3). LY315920 and different antivenoms (CroFab®, Anavip® and CSL Taipan Specific) were then assessed in a pig model of lethal envenoming comparing different treatment regimens with LY315920 alone, antivenom alone or with LY315920 preceding or following the antivenom at a time point at which antivenom was no longer able to reverse weakness, save the animals and/or there was also evidence of systemic envenoming (e.g. abnormal TEG). LY315920 and/or LY333013were able to prevent lethality or reverse venom-induced weakness at all-time points following taipan (O. scutellatus, Australian and Papuan) and Mojave rattlesnake (C. scutulatus). LY315920 more rapidly reduced markers of muscle breakdown such as AST and CK than comparator antivenoms and compared to controls. 8 of 13 (62%) animals receiving antivenom with manufacturer’s dosing instructions had anaphylactic reactions successfully treated with epinephrine and diphenhydramine. No LY315920 treated animals had evidence of allergic reaction in any of the studies (N=0/18 (0%).17/18 (94%)). LY315920 pigs survived to the end of the studies (minimum 100 hours) with

one animal dying from septicemia following successful resuscitation from taipan envenoming and unsuccessful antivenom therapy the day before. In all studies, control animals died from envenoming within 5.5 hours 10/10 (100%). In 4/4 animals (100%2 Papuan taipan, 2 Mojave) in which antivenom delayed, but failed to prevent weakness LY315920 was able to rapidly reverse the weakness with a single rescue dose of drug (1mg/kg). Discussion/Conclusion: Small molecule, toxin specific inhibitors capable of reversing toxicity of extra-circulatory venom components have good potential to improve outcomes while the longer half-lives of antivenoms and broader spectrum of toxin-neutralizing antibodies make small molecule therapeutics and high quality antivenoms appealing partners for a multi-pronged approach to the treatment of snakebite envenoming when prevention of snakebite has not been achieved. Key Words: Vaespladib, LY315920, Neurotoxicity, Myotoxicity, O. scutellatus, C. scutulatus AA5 - STOCKING ANTIVENOMS IN URGENT CARE CLINICS AND FREE-STANDING EMERGENCY DEPARTMENTS Lund, J1, Peredy, T2,3, Aleguas, A3 1Department of Pharmacy, Sarasota Memorial Healthcare System, Sarasota, FL., USA; 2 Global Physician’s Network, Sarasota, FL, USA; 3 Florida Poison Information Center – Tampa, Tampa, FL., USA E-mail address: Jeremy-Lund@smh.com Background: The US has seen an expansion of immediate care settings in response to patient demands coupled with the barriers to rapidly access primary care and the stigmata of visiting hospital Emergency Departments. Free-standing Emergency Departments as opposed to walk-in clinics accept all levels of acuity including incoming EMS traffic excluding pre-determined specialty care (stroke, MI, burns, major trauma, etc.). In 2018, the Institute for Safe Medication Practices published Medication Safety Best Practices for Hospitals. Best Practice #9 states that hospitals should “ensure all appropriate antidotes, reversal agents, and rescue medications are readily available.” An expert consensus guideline for the stocking of antidotes for hospitals that provide emergency care was first published in 2009 and subsequently updated in 2018. However, these recommendations do not consider the spectrum of facilities that provide immediate care. Anavip® [Crotalidae Immune F(ab’)2 (Equine)] was approved for use in the United Sates in October 2018. This agent provides several purported advantages to Crofab® [Crotalidae Polyvalent Immune Fab (ovine)] resulting in many hospitals adding this antivenin to their formularies. Methods: Collaborative efforts were made between the three authors; a medical

toxicologist and an emergency medicine pharmacist with affiliation at community hospital system and the Clinical Director of their regional Poison Control Center. Historical records of number and species of snake envenomations reported to the PCC were reviewed. Considerations were made for: number of patients, type of snake and average transport time between satellite facilities. Discussions were held with the hospital’s pharmaceutical wholesaler regarding the speed with which antivenin stock could be replenished. The available published studies assessing the average number of vials utilized per patient of F(ab’)2 were analyzed. Lastly, the number of vials of Crotalidae polyvalent immune fab (ovine) recommended for stocking by the expert consensus guidelines was considered. Discussion/Conclusion: With the addition of a new antivenin, many hospitals are considering modifications to formulary choices and stocking levels. Poison Control Centers have not adopted generally accepted guidelines for stocking choices and levels outside of large tertiary referral institutions. Based upon the threshold of at least one bite in the last calendar year, in a geographical area containing Agkistrodon species, we recommend the addition of at least 2x initial treatment recommendations of Anavip at all hospitals greater than one-hour transport time from the main campus or primary hospital within the system. Although individual factors such as pharmacy support, density of surrounding facilities and geographic location will introduce variability, we believe these commonsense factors will lead to more uniform stocking guidelines with the introduction of future antivenins. Key Words: Crotalidae antivenin, Poison Control Center, antivenin stocking recommendation AA6 - A SYNTHETIC METALLOPROTEINASE INHIBITOR DERIVATIVE FROM NATURAL NAPHTHOQUINONES Melo, PA Lab Farmacologia das Toxinas, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro RJ, Brazil E-mail address: melo.pa@gmail.com Background: Local and systemic tissue damage induced by snakebites are associated with permanent incapacitating sequels that could be prevent by the specific antivenoms. Although the antivenoms are good for the systemic toxic effects, some tissue damage such as, skin hemorrhage, dermonecrosis and myonecrosis are not completely neutralized. It is relevant to study and develop new agents with antivenom abilities, either natural or synthetic, that could help and improve the snakebite treatment. Most of the venom compounds are a great variety of enzymes, that induce edema, hemorrhage, coagulopathy and myonecrosis, which are interconnect events. Viperid snake venoms contain a relevant number of metalloproteinases, phospholipase A2, hyaluronidase, which are active enzymes that can be neutralized by the specific antivenins and some

natural and some synthetic compounds. Methods: We combined assays with different approaches for neutralizing snake venom effects, using planed synthetic compounds in biochemical and pharmacological test and select the active new agents. The snake venom enzymes activity used in these models allowed us to create prototypes and new planed synthetic compounds that can specifically neutralize snake metalloproteinase, devoid of other enzymatic inhibitory effect. Many of these metalloproteinase inhibitors and collagenase inhibitors protect the basal lamina in the capillary vessels, prevent hemorrhage, but do not interfere in the coagulation venom effect. Discussion/Conclusion: Our data in this presentation described the effect of planed synthetic quinones, related to lapachol, which revels a selective inhibitory effect on snake venom metalloproteinase activities that’s works very well against Bothrops snake venoms effects in vitro and in vivo. Key Words: Snake venom metalloproteinases, natural and synthetic quinones, Lapachol, Bothrops atrox venom AA7 - SPECIES-SPECIFIC AND GEOGRAPHICAL VARIATION IN VENOME COMPOSTION OF INDIAN COBRA: IMPACT ON POLYANTIVENOM THERAPY Mukherjee, AK*, Chanda, A, Patra, A Department of Molecular Biology and Biotechnology, School of Sciences, Tezpur University, Tezpur 784 028, Assam, India *E-mail address: akm@tezu.ernet.in Background: Approximately 3,000 species of snakes inhabit most parts of the world, except Antarctica and the polar regions. However, only 600 species of snakes are reported to be venomous. World Health Organization (WHO) has classified venomous snakes into three distinct categories-(i) snakes responsible for frequent bites that result in death and disability; (ii) frequency of bites occasional, but envenomation by this class of snakes may lead to death; and (iii) bites by this class of snakes may be common, but they produce the lowest toxicity in humans. Snakebite is a severe problem for several countries of the world; however, this problem is most severe in South Asia, Southeast Asia, and East Sub-Saharan Africa. India has witnessed the highest incidence of snakebite in the world, and approximately 81,000 envenoming take place annually. Of the approximately 300 species of snakes found in India, only 60 species are venomous. Snakebite is an occupational health hazard for Indian subcontinent. The Indian Spectacled Cobra (Naja naja), belonging to the family Elapidae, is one of the “Big Four” venomous snakes of the country and is considered a class I medically important snake. Another species of cobra, N. kaouthia, is prevalent in eastern and northeastern parts of India, but the only treatment against snakebite is immediate administration of equine polyantivenom (PAV) raised against venoms of the “Big Four” snakes of India (N. naja,

Daboia russelli, Echis carinatus, and Bungurus caeruleus). Notably, species-specific (N. najavs. N. kaouthia) and geographical differences in venom composition of N. naja may limit effectiveness of antivenom treatment; therefore, exploring the venom proteome of N. naja and N. kaouthia from different geographical locales and the effect of this variation on antivenom efficacy are warranted. Methods: Indian Spectacled Cobra (N. naja) and Indian Monocled Cobra (N.kaouthia) venoms were obtained from different parts of India and their venom composition was analyzed by biochemical assays and tandem mass spectrometry analysis. The relative abundance of each class of toxins was determined by label-free quantitative analysis. The immunological cross-reactivity between PAV and cobra venom samples was determined by ELISA, Western blot analysis, and immuno-affinity chromatography followed by mass spectrometry analysis of PAV-unbound toxins. Discussion/Conclusion: Toxinological analyses have demonstrated that although some of the toxins are common between N. naja and N. kaouthia venoms from eastern India, species-specific and geographic differences in proteomes of Indian Spectacled Cobra venoms were evident, as revealed by differences in their venom toxicity (LD50) values. Further, PAV raised against the venom of N. naja from a particular geographical location showed partial neutralization of enzyme activity and immunological cross-reactivity against cobra venom samples from other parts of India. Our study suggests that addition of polyclonal antibodies against N. kaouthia venom to PAV and preparation/administration of region-specific PAVswill provide better treatment of cobra bites in India. Key Words: Cobra venom, Naja naja; Naja kaouthia; geographical variation in venom composition, mass spectrometry analysis of venom; antivenom therapy AA8 - SPHINGOMYELINASE D-CROTAMINE RECOMBINANT FUSION PROTEIN AS IMMUNOGEN FOR THE PRODUCTION OF ANTIBODIES AGAINST CROTAMINE Ponce-López, R1,*,Olvera-Rodríguez, A1, Borja-Jiménez, M2, Neri-Castro, E1, Olvera-Rodríguez, L1,Alagón, A1 1Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México;2 Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango, Gómez Palacio, Durango, México E-mail address: rockbert@ibt.unam.mx Background: Crotoxin (MW: ~24 kDa) and crotamine (MW: ~5 kDa) are two of the most studied neurotoxic components found in several rattlesnake venoms (Crotalus spp.). The former compound is well neutralized by antivenoms, whereas the latter is well known for its spastic paralysis symptom provoked in mice with no evidence of

neutralization. Recently, it was reported that crotamine is the major toxin found in some neonate and juvenile Mexican rattlesnake venoms such as Crotalus molossus nigrescens (~50%). Currently, the available Mexican pit viper antivenoms neither recognize nor neutralize crotamine from C. m. nigrescens; possible explained by its low immunogenicity due to its low molecular weight. On the other hand, sphingomyelinase D (SMD) is a highly immunogenic enzyme (MW: ~30 kDa) found in Loxosceles spp. spider venom. To increase immunogenicity of a low molecular weight toxin, we hypothesize that our novel recombinant fusion protein, made of crotamine from C. m. nigrescens and sphingomyelinase D from L. reclusa used as carrier, will contribute significantly to the generation of neutralizing antibodies against crotamine, preventing its spastic paralysis and lethality. Methods: Crotamine cDNA was synthetized from venom gland mRNA of a C. m. nigrescens individual from Mexico, while one plasmid containing L. reclusa SMD was available in the lab. By standard genetic engineering procedures, we cloned in tandem the sphingomyelinase D and one crotamine isoform into the expression vector pQE30. This fusion protein was expressed in Origami Escherichia coli. Using the recombinant protein, we performed immunization protocols in rabbits and, during the scheme, we measured ELISA antibody titers to purified crotamine from C. m. nigrescens venom. Results: The fusion protein was obtained at 3 mg/L of bacterial culture with the expected 37.5 kDa molecular mass as analyzed by SDS-PAGE and western-blot analysis. The fusion protein was lethal in mice (LD50 = 2.8 μg/g weight of mice) showing no effects of spastic paralysis. Based on ELISA and western-blot, serum samples of immunized rabbits reacted to crotamine. Discussion/Conclusion: The SMD-Crotamine fusion protein used as immunogen generates antibodies that recognize crotamine. We are using pre-incubation experiments to analyze the neutralization ability of antibodies to prevent spastic paralysis and lethality from crotamine in mice. Acknowledgment: Fordecyt 303045. Key Words: Crotamine, Sphingomyelinase-D, molecular-carrier, recombinant-protein. AA9 - CROTALUS SIMUS VENOM AS THE IMMUNOGEN TO GENERATE NEUTRALIZING ANTIBODIES AGAINST OTHER NEUROTOXIC AND NON-NEUROTOXIC RATTLESNAKE VENOMS Ponce-López, R1,*, Neri-Castro, E1, Borja-Jiménez, M2, Alagón, A1 1Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México; 2 Facultad de Ciencias Biológicas, Universidad Juárez del Estado de Durango, Gómez Palacio, Durango, México

E-mail address: rockbert@ibt.unam.mx Background: Mexico has a remarkable pit viper diversity, which means that antivenoms produced in this country must be capable of neutralizing the venom of each of the currently reported 44 rattlesnake and the other 28 pit viper species. Crotalus simus venom from Mexico was described as being mainly composed of metalloproteinases at 27.4%, serine proteinases at 30.4% and A2 phospholipases at 22.2% including crotoxin (14.3%). Although this venom is one of the two immunogens employed to produce the antivenom (Antivipmyn®) most used in Mexico, its immunoreactivity knowledge against other Crotalus venoms is limited. The aim of the present work was to generate an experimental antivenom anti-C. simus venom and evaluate its cross reactivity and neutralization to some venoms, containing and non-containing crotoxin, of clinical importance in Mexico. Methods: Three rabbits were hyperimmunized with a C. simus pool sample venom collected in the State of Veracruz, Mexico. During the immunization scheme, serum samples were analyzed by ELISA in order to measure antibody titers against the whole C. simus venom as well as to the three major protein families (metalloproteinases, serine proteinases and crotoxin) found in this venom. Later, serum samples were pooled and used for further analysis by ELISA, western-blot and neutralization (using mice) to five neurotoxic venoms (C. d. terrificus, C. simus, C. basiliscus, C. scutulatus salvini and Ophryacus sphenophrys) and two non-neurotoxic ones (C. tzabcan and C. atrox). Also, rescue experiments were performed with Antivipmyn® and the experimental rabbit serum in mice against C. simus venom. Results: C. simus venom was able to generate high antibody titers in rabbits, both to the whole homologous venom and to each of its three major protein families. Anti-C. simus rabbit serum showed cross reactivity (ELISA and western-blot) to the heterologous venoms. Nevertheless, we observed neutralization only against those venoms containing crotoxin or crotoxin-like molecules. Besides, the spastic paralysis provoked by two venoms containing crotamine (C. basiliscus and C. d. terrificus) was not neutralized even with high doses of serum. Neutralizing potency of Antivipmyn® and the experimental serum decreased, compared to pre-incubation experiments, in rescue experiments. Discussion/Conclusion: Crotalus simus venom from Mexico seems to be an important immunogen for the generation of neutralizing antibodies against highly neurotoxic pit viper venoms, especially for those containing crotoxin or crotoxin-like molecules. Nevertheless, we did not observe neutralization against two non-neurotoxic venoms whose lethality is not dependent on crotoxin, which probably indicates the importance of adding Bothrops asper venom as immunogen. Crotamine is an important evasive component in many Crotalus venoms that was neither neutralized by commercial antivenom nor by the experimental serum. There is much to investigate for improving antivenom efficacy. Acknowledgment: Fordecyt 303045. Key Words: Crotalus simus, crotoxin, crotamine, neutralizing-potency.

AA10 - PARASPECIFIC NEUTRALIZATION OF SNAKE VENOMS: WHAT SNAKE KEEPERS SHOULD KNOW ABOUT THEIR ANTIVENOMS Sanchez, EE1, 2, Suntravat, M1, 2, Salazar Castillo, E1 1 National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA; 2 Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 78363, USA E-mail address: elda.sanchez@tamuk.edu Background: Antivenom availability is a present crisis, particularly in third world countries. Many venomous snakebites occur in areas where medical care is not accessible, and the availability of antivenoms is not feasible due to their high price. Even when antivenom is available, there is no guarantee of their efficacy on specific snake envenomings. The uncertainty of venom neutralization by antivenoms is a concern for facilities that maintain venom snakes, particularly venomous snakes from outside the geographical range from which the antivenoms were produced. These facilities must obtain antivenoms “specific” for the snakes they maintain, and these antivenoms come at a substantial price and an expiration date. Furthermore, although the antivenom may indicate that it covers a particular species, there is still no guarantee that neutralization will be achieved. On the other hand, neutralization of snake venoms by antivenoms not specific for certain snakes has been known to occur. This work aimed to determine the paraspecific neutralization of venom from snakes kept by zoos and other facilities using an effective dose 50 (ED50) assay. Methods: In vivo assays were used to determine the ED50 of several antivenoms on snake venoms to determine their paraspecific neutralizing abilities. A total of three antivenoms (Antivipmyn, SAIMR Polyvalent, and SAIMR Echis carinatus) where used in this study. Antivipmyn was tested on Crotalus vegrandis, Bothrops colombiensis, and B. moojeni. SAIMR Polyvalent was tested on Atheris squamigera, Naja naja karachiensis, N. philippineansis, and N. samarensis venoms. SAIMR Echis carinatus was tested on A. squamigera. The lethal dose 50 (LD50) was determined for each venom on 18-20 g BALB/c mice. Five doses of antivenom were prepared for each antivenom brand or lot. A stock solution containing 30LD50 was prepared for each venom. Each dose of antivenom was diluted in half with the venom stock solution and allowed to incubate for 30 min at 37˚C. A total of 0.2 mL of the antivenom/venom mixture was injected into the tail vein of each mouse, resulting in the injection of 3LD50. The ED50 was determined using the Spearmen-Karber method. Discussion/Conclusion: The data showed that Antivipmyn antivenom is capable of neutralizing 3LD50 of B. moojeni, C. vegrandis, and B. colombienis venoms with ED50s of 10, 12.4, and 85.2 mg/kg, respectively. SAIMR Polyvalent antivenom neutralized N. n. karachiensis, A. squamigera, N. philippinensis, and N. samarensis venoms with ED50s of

68, 105, 313, and 625, respectively. SAIMR Echis carinatus antivenom resulted in an ED50 of 142 mg/kg on A. squamigera venom. This preliminary data suggest paraspecific neutralization of snake venoms, which may verify to be useful to those facilities carrying snakes for which no specific antivenom is assigned. In the case of Antivipmyn, the ability to neutralize venomous snakes of Mexico and South America alleviates the economic burden of having to purchase additional antivenoms since Antivipmyn, Antivip in the U.S., has been FDA approved since 2018. It is the goal of this study to generate an extensive database of paraspecific neutralization of snake venoms by various antivenoms that will provide snake facilities the information they need when acquiring new snakes or when obtaining an unfortunate snakebite.

Key Words: Antivenoms, Effective Dose 50, Paraspecific neutralization AA11 - COMPARISON OF F(AB')2 AND FAB ANTIVENOMS IN RATTLESNAKE ENVENOMATION: FIRST YEARS’ POST-MARKETING EXPERIENCE WITH F(AB’)2 IN NEW MEXICO Mascarenas D1, Fullerton L2, Smolinske SC3, Seifert SA 2,3 1 Division of Pediatric Emergency Medicine, Department of Emergency Medicine, University of New Mexico Health Sciences Center, 2 Department of Emergency Medicine, University of New Mexico Health Sciences Center, 3New Mexico Poison Center, University of New Mexico Health Sciences Center, Albuquerque, NM, USA E-mail address: dnmascar@salad.unm.edu Background: Two antivenoms (AV) are currently available to treat rattlesnake envenomations in the US, a Fab (CroFab®, BTG, UK), and a F(ab’)2 (Anavip®, Bioclon, Mexico) AV, the latter released for use in October, 2018. The Phase 3, comparative clinical trial demonstrated similar hematologic toxicity efficacy and similar Type 1 and Type 3 hypersensitivity reactions. That study also showed a lack of need for maintenance dosing and a lower rate of recurrent hematologic effects for F(ab’)2AV. We compared F(ab’)2AV to FabAV in the first years’ experience of F(ab’)2AV use in New Mexico. Methods: Two trained abstractors reviewed all New Mexico Poison Center case records concerning rattlesnake-bitten patients presenting between May 1 and October 10, 2019. The Poison Center routinely collects data for all exposed patients and enters them into a database (Toxicall v. 4.7.40). At 21 days post-exposure, poison specialists conducted a phone interview regarding symptoms and signs of Type 3 reactions. All cases, including dry bites, were included in analyses of patient demographics, Analytic statistics compared the bivariate predictor variable (F(ab’)2AV vs. FabAV) and outcome variables measuring antivenom control and rescue dosing, hematologic effects, and hypersensitivity reactions. We used Fisher’s exact test, odds ratios, and a 0.05 cutoff for significance.

Results: The 53 patients in the study had an average age of 44 years (range 1 – 77; median 51); 40 (75%) were male, and 13 (25%) were female; 26 (49%) bites involved the upper extremity, 25 (47%) involved the lower extremity, and 2 (4%) involved the trunk. Sixteen patients were not included in bivariate analyses: nine (17%) had a dry bite, one presented > 48 hours post-envenomation and was not treated, and six were transferred out-of-state and lost to follow-up. Of the 37 remaining patients, all received a single type of antivenom during treatment. Eleven (30%) received F(ab’)2AV and 26 (70%) received FabAV. Standard dosing protocols were followed for both antivenoms. There were no significant differences between F(ab’)2 and Fab AV-treated patients by age, sex, bite site, the number of control doses, or the rate of initial hematological abnormalities. No patient in either group had a Type 1 hypersensitivity reaction. No rescue doses were required with F(ab’)2AV for reactivation of local effects. The overall rate of acute, venom-related hematological abnormalities was 59%. The rate of recurrent, persistent or late-, new-onset of hematologic effects was 0% with F(ab’)2AV and 30% (41% of patients with an initial abnormality) with FabAV. No patient was readmitted and none had bleeding complications. The rates of symptoms consistent with a Type 3 reaction with F(ab’)2 and Fab were 36% and 44%, respectively, all mild. Discussion/Conclusions: The effectiveness of F(ab’)2AV and FabAV was similar for local and hematological effects. F(ab’)2AV did not require maintenance doses. There were no recurrent, persistent, or late-, new-onset hematologic effects with F(ab’)2AV. There were no Type 1 hypersensitivity reactions and F(ab’)2AV and FabAV had similar rates of Type 3 hypersensitivity reactions. The higher rates of Type 3 reactions in this study most likely reflect a 21-day personal interview methodology. Key Words: envenomation, rattlesnake, antivenom, hematologic, hypersensitivity AA12 - ANAVIPTM INTERACTION WITH WESTERN PYGMY RATTLESNAKE VENOM: INVITRO ASSESMENT OF REACTIVITY USING SE-HPLC Tanner, DA*, Shults, CA, Sanny, C Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, Tulsa, OK USA E-mail address: dtanner@okstate.edu Background: Every year there are a large number of venomous snake bites occur around the world and especially in tropical areas. This is a problem that is faced worldwide with the World Health Organization classifying venomous snake bites as one of their highest priority neglected tropical diseases. One of the reason for this classification is that there is a short supply of antivenin compared to the number of snake envenomations that occurs each year. The treatment for snake envenomation is antivenom (i.e. antivenin). Most antivenoms are produced from a mixture of similar species in-order to produce a antivenom that would treat envenomation from more than one species of snakes. In some

cases cross-reactivity is seen in antivenoms that provide a protective effect from venom that is not a part of the antivenom. Knowing the cross reactivities of antivenoms could help with the shortage of antivenom as well as provide valuable data of the binding of an antivenom against different venoms. Until recently there has only been one antivenom available for treatment of North American Crotaline envenomation. With the introduction of a Fab2 antivenom into the United States we look at the cross-reactivity of the western pygmy rattlesnake, Sistrurus miliarius streckeri, against Anavip (Fab2). Methods: SE-HPLC was used to assess cross-reactivity. SE-HPLC is a viable method to analyze antivenom-venom reactivity based on separation of higher molecular weight complexes that form vs unreacted components. Estimates of venom-antivenom reactivity was measured in reaction mixtures base on the increase in the elution profile area where higher molecular weight complexes are observed (region 1) and on the decrease in the elution profile area where reactants are observed (region 2). Reaction mixtures contained Anavip (1.0 mg/ml) and S. miliarius venom (0.125, 0.25, 0.5, or 1.0 mg/ml). Controls were Anavip and S. miliarius (1.0mg/ml). Mixtures were incubated at 37*C for 30 minutes then stored at 4*C prior to SE-HPLC. Results: Cross-reactivity was seen between Anavip (Fab2) and S. miliarius venom based on changes in elution profile areas. A decrease in region 2 (reactants) and increase in region 1 (immune complexes) was observed at all venom-antivenom concentrations. The maximum venom-antivemon binding was calculated based on changes in elution profile area to be 67% relative to the antivenom total profile area. Discussion/Conclusion: Apparent saturation of reactive antivenom components was observed at all venom concentrations. Estimates of Anavip reactivity with S. miliarius venom are seen in the changes of elution profile areas showing the formation of larger molecular weight complexes and decrease in reactants. This shows that Anavip could provide protective effects against S. miliarius envenomation. Further studies to determine the composition of unreacted components. Results suggest that binding of Anavip to S.miliarius venom does occur, which is consistent with protective effects of that are observed clinically. Key Words: Anavip, Crotaline, antivenin, SE-HPLC AA13 - RECOMBINANT EXPRESSION OF SCFV 6009F IN PICHIA PASTORIS AND ITS EVALUATION TO RECOGNIZE NEUROTOXINS FROM CENTRUROIDES SP. Adame, M*1, Vázquez, H2, Riaño, L2, Corzo, G2, Villegas, E1 1Centro de investigación en Biotecnología, Universidad Autónoma del Estado de Morelos; 2 Instituto de Biotecnología, Universidad Nacional Autónoma de México. E-mail address: adame.mary@outlook.es

Background: Scorpion sting causes approximately 310,000 envenomations per year in México. The best scorpion envenomation treatment, according to WHO, is through antibodies (IgGs) produced by hyperimmunization of horses with scorpion venoms. An alternative to produce neutralizing IgGs against scorpion envenomations, is the single chain variable fragments (scFvs). Riaño et al. (2005; 2019) reported a scFv from human origin, named 6009F, capable to neutralize 2LD50 of Cn2; the major neurotoxin from the venom of the scorpion Centruroides noxius. scFv 6009F also presented cross reactivity against Cn3, Css2 and Css3, which are neurotoxins from the venoms of C. noxius and C. suffusus suffusus, respectively. The recombinant scFv 6009F was expressed in E. coli TG1 obtaining a yield of 1.1 mg/L. In order to improve such expression yield, in this work scFv 6009F was expressed in two strains of P. pastoris using the plasmid pPIC9, this recombinant scFvs was evaluated for its capacity to recognize and neutralize Cn2. Methods: We cloned scFv 6009F in two strains of P. pastoris, GS115 (Mut+) and KM71 (MutS), using the plasmid pPIC9. The plasmid pSyn was used as template (Riaño-Umbarilla et al., 2005). Recombinant P. pastoris GS115/pPIC9/scFv 6009F or KM71/pPIC9/scFv 6009F were grown in BMMG medium using glycerol as the initial carbon source until they reach an OD595nm of 2. At this point, the carbon source was shifted adding 1% v/v of methanol every 24 h during 96 h. After the recombinant scFv 6009F was purified from the culture supernatant by Ni2+-NTA (Quiagen, Hilden, Germany), SDS-PAGE and Western-blot analyses were performed to confirm the expression and the physical integrity of the scFv 6009F. Circular dichroism and ELISA tests were performed to confirm secondary structure and the ability of scFv 6009F to recognize Cn2. Results: The titer of the recombinant fusion protein express in P. pastoris GS115 and KM71 were 50 and 55 mg/L, respectively. It was possible to observe a band of a28 KDa using SDS-PAGE gels and Western-Blot analysis. The yeast recombinant scFv 6009F recognize Cn2 toxin in an ELISA test reveled with anti-cmyc. Using mice as animal models, the scFv 6009F neutralization trials against Cn2 will be performed. Discussion/Conclusion: scFv 6009F was successfully cloned and expressed in two strains of P. pastoris. After purification, the expression yields of scFv 6009F were higher than that obtained in bacteria (> 50mg/L). Although the expression yields in yeast were higher than those obtained in E. coli TG1 (1.1 mg/mL), culture conditions have to be optimized. The recombinant antibody from P. pastoris could recognize C. noxius toxin Cn2. Key Words: Antivenom, Centruroides, Pichia pastoris, recombinant protein, single chain. BS - BASIC SCIENCE BS1 - CHARACTERIZATION AND NEUTRALIZATION OF LOCAL DAMAGE CAUSED BY THE COMPLETE VENOM OF ATROPOIDES NUMMIFER AND ITS MAIN MYOTOXIN IN A MURINE MODEL

García, B.1,2., Neri, E. 1, Bénard, M.1, Zamundio, F.1, Ocampo, L.2, Morales, E. 2, Alagón, A. 1 1Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México-UNAM, Cuernavaca, Morelos, 61500, México, 2Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México-UNAM, Av. Universidad #3000, Colonia, C.U., Coyoacán, 04510 Ciudad de México E-mail address: biolbelemgo@gmail.com Background: Atropoides nummifer is an endemic snake species to Mexico with a wide distribution within the state of Veracruz. Its venom has low lethal potency and causes principally hemorrhage, inflammation and muscle necrosis in murine models. Phospholipases A2 of the subtype Lys49 are some of the most relevant components responsible for local necrosis generated by viper venoms. Although they lack enzymatic activity, they act specifically on skeletal muscle a few minutes after being inoculated. Some researchers have reported poor neutralization of myotoxic activity by antivenoms of other Atropoides species. Methods: Venom from 29 A. nummifer specimens from the state of Veracruz, Mexico, was pooled. From it, we purified a myotoxic PLA2 using size exclusion followed by cation exchange chromatography. The biochemical and biological activities of the purified PLA2 were studied. Then, we determined the minimum myotoxic dose (MMD) for both venom and the PLA2, measuring release of creatine kinase (CK) to blood serum in groups of 5 mice at 10 min and 3, 6 and 12 h. For histopathologic evaluation, 3 MMDs of whole venom or 2 MMDs of purified PLA2 were injected into the gastrocnemious muscle of mice. At the mentioned times, the animals were euthanized with CO2 inhalation, and blood and tissue samples were obtained. Finally, neutralization of myotoxic activity was tested preincubating 800 and 1800 μg of antivenom (Birmex and Bioclon) with complete venom and purified PLA2, respectively. The antivenoms failed to neutralize the myotoxicity of venom or purified PLA2. Results/Conclusion: A PLA2 with a molecular weight of 13,743.9 Da was purified from the venom of A. nummifer. This protein lacks enzymatic activity and has an MMD of 34.5 μg, higher than that of the whole venom (19.6 μg). Analysis of myotoxicity over time showed that the highest CK levels occurred at 3 and 6 h for both venom and PLA2. Histopathology showed necrosis, hemorrhage and inflammation, all of which increased over time. Mexican antivenom producers should consider the inclusion of A. nummifer venom as an immunogen, to improve the coverage of their products. Key Words: snake, A. nummifer, myotoxic PLA2, creatine kinase, neutralization BS2 - THE ROLE OF CHROMATIN ACCESSIBILITY ON VENOM REGULATION IN CROTALUS ADAMANTEUS

Colston, TJ1*, Bartlett, D1, Broe, M2, Gibbs, HL2, Hogan, M1, Wray, KP1, Margres, MM3, Gilbert, D1 & Rokyta, DR1

1Department of Biological Science, Florida State University, Tallahassee, FL; 2Department of Evolution, Ecology and Organismal Biology, Ohio State University, Columbus, OH; 3Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA. Email address: tim@maddreptiles.com Background: Recent advances in venomics (transcriptomics, proteomics, genomics) have led to a multitude of studies which characterize the composition and activity of snake venoms. In spite of these advances the precise underlying molecular mechanisms regulating venom gene expression have received less attention due to the fragmentary nature of existing genome assemblies and the challenge of adapting existing molecular biology techniques aimed at mapping nucleosome positioning and transcription factor occupancy to nonmodal systems. The latter methods are critical to understanding the influence of cis-regulatory mechanisms involved in venom evolution and diversification, and cannot be adequately analyzed without a well-annotated and contiguous genome assembly. Methods: Here we leverage the fully annotated genome of Crotalus adamanteus and a laboratory assay (ATAC-seq) that targets regions of open chromatin, thus allowing us to map regions of open chromatin, nucleosome positioning and transcription factor occupancy and test the role of chromatin accessibility on venom gene expression. We conduct known and de novo motif analyses to search for motifs associated with venom genes and interrogate venom gland transcriptomes to test for signatures in expression associated with chromatin accessibility. Discussion/Conclusion: We identify several de novo motifs associated with venom genes and significantly differentially expressed known transcription factors. We discuss these findings in light of what is known regarding the role of cis-regulatory mechanisms on venom gene expression. Key Words: Venom, cis-regulation, Crotalus, genome, ATAC-seq BS3 - BIOACTIVE PROPERTIES OF CRUDE VENOM ISOLATED FROM HYPANUS SABINUS Doupnik, CA1,*, Walsh, CJ2, Luer, CA3 1Department of Molecular Pharmacology & Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL 33612; 2Marine Immunology Program, 3Marine Biomedical Research Program, Mote Marine Laboratory, Sarasota, FL 34236 E-mail address: cdoupnik@usf.edu

Background: The Atlantic stingray Hypanus sabinus inhabits Florida’s coastal waters and contributes to the integrity and dynamics of this marine ecosystem. A prominent evolutionary adaptation of stingrays is the presence of a venom-containing barb located on their tail, which they use for defense against predatory species such as sharks. Human encounters occur primarily on beaches where stepping on a stingray in shallow water triggers its whip-like tail reflex, causing skin laceration via the serrated barb coupled with envenomation from the venom-secreting gland cells located on the barb. The typical stingray ‘sting’ is non-lethal and characterized by acute intense pain, followed by tissue necrosis occurring over several days. The pathophysiology of a H. sabinus sting is similar to jellyfish (Chironex fleckeri), however numerous jellyfish tentacle stings can be more life-threatening. To better understand the biological response to stingray venom (SRV), we developed methods for harvesting crude SRV from the barbs of captive H. sabinus for subsequent analysis. Initial efforts have characterized both biochemical and bioactive properties of crude SRV on human cell lines. Identifying the bioactive components in SRV and their mechanism of action may lead to future biomedical applications and treatments. Methods: Crude H. sabinus SRV was obtained by scraping the venom-secreting tissue located along the ventral grooves of a clipped barb. The harvested tissue is then gently homogenized to release venom into 50 mM NH4HCO3. After removal of tissue debris by centrifugation, the SRV sample is lyophilized and stored at -80oC for later analysis. SDS-PAGE analysis of the crude SRV collected from separate barbs, demonstrate protocol reproducibility based on equivalent protein banding patterns and total protein yields. The bioactive effects of the crude SRV were examined on cultured human-derived cell lines monitored in real-time by an impedance-based biosensor system (xCELLigence Real-Time Cell Analyzer). This system enables detection of both acute and long-term changes in cell viability, migration, and growth. Results: The effects of crude SRV on normal human dermal fibroblasts (NHDF’s) and human neuroblastoma cells (SH-SY5Y) were time- and concentration-dependent. At the highest SRV concentration tested (100 Pg /ml), there was a rapid (<15 min) decrease in cell impedance that is consistent with acute cytotoxicity. This response was absent in heat-treated samples (95-100oC for 10 min). For NHDF’s, the acute response to SRV had an EC50 of ~10 Pg /ml. At lower concentrations (3 Pg /ml) SRV slowed the 24-48 hour cell growth curve, consistent with an inhibitory action on cell proliferation. Depleting cell cholesterol with methyl- -cyclodextrin (MECD) pre-treatment was recently shown to inhibit the cytotoxic effects of crude jellyfish venom (JFV) (Lau et al., 2019). Pretreatment of SH-SY5Y cells with 4 mM MECD for 15 min had no effect on either acute or chronic effects of SRV. Discussion/Conclusion: Crude SRV isolated from H. sabinus has potent bioactivity that is effectively inactivated by heat. However unlike JFV, the effects of SRV are insensitive to cellular cholesterol depletion. These results highlight distinct mechanisms for SRV bioactivity, where deploying ‘omic’ methods will help identify SRV components involved in mediating pain and tissue necrosis.

Key Words: Hypanus Sabinus, stingray venom, toxins, cytotoxicity BS4 - THE EVOLUTIONARY ORIGINS AND FUNCTIONAL DIVERSIFICATION OF RATTLESNAKE VENOM TOXINS Dowell, NL1, Giorgianni, MW1, Sanchez, EE2, Carroll, SB1

1Howard Hughes Medical Institute and Department of Biology, University of Maryland – College Park, 4066 Campus Drive, College Park, Maryland 20742, USA; 2 National Natural Toxins Research Center and Department of Chemistry, Texas A&M University – Kingsville, MSC 224, Kingsville, TX 78363, USA E-mail address: ndowell@umd.edu Background: The genetic origin of novel traits is a central, but challenging puzzle in evolutionary biology. The genetic mechanisms and evolutionary forces yielding polymorphisms that encode novel traits are of special interest. Moreover, once invented, diverse variants of a novel trait can be present in closely related species or even within discrete populations of the same species. Animal venoms offer outstanding examples of biochemical novelties where protein components and toxic effects are known and thus are a promising system for identifying the genetic basis of novel trait. Among rattlesnakes, several instances are known of the presence of biochemically distinct neurotoxic or hemorrhagic venom types both within the same species (C. scutulatus and C. helleri) and between closely related species (C. scutulatus and C. atrox) but the genetic basis of these differences were not understood. In one scenario, all genes encoding distinct venom types are present in all individuals and the set of genes expressed determines the observed venom type. Whereas, in an alternative scenario, discrete gene sets encode a specific venom type and the expression of that suite of genes yields the particular venom within an individual. The scarcity of quality genomic data has often obscured the identification of orthologous genes and the contributions of gene content and regulation to the genesis of venom. Therefore, in order to distinguish between the scenarios presented above, we sequenced, assembled and annotated the genomic regions of key toxins associated with the neurotoxic and hemorrhagic venom types in rattlesnakes. Methods: Using a comparative genomics approach we determined the number and organization of genes encoding two key venom components with roles in subduing prey: snake venom metalloproteinases (SVMPs) and phospholipase A2 (PLA2) toxins. We traced the deep, single gene origins of both of these gene families and showed gene duplications lead to gene family expansions. Subsequent genomic rearrangements in the form of whole gene or intragenic exon deletions yielded distinct gene complexes and novel genes, respectively. For species with individuals capable of producing neurotoxic or hemorrhagic venom we found that the associated PLA2 and SVMP gene complexes contain dramatic differences in gene content that encode alternative venom types. Discussion: The PLA2 and SVMP gene family expansions occurred in the lineages leading

to Viperids and not on other branches of the snake tree suggesting that selection initially acted to retain newly duplicated genes and may have facilitated adaptation to new lifestyles. After expansion in the PLA2 family, genetic modification via substitutions yielded distinct classes of proteins (acidic or basic types) with novel molecular interaction modes (monomer versus dimer) thus leading to the evolution of both hemo-/myotoxins (PLA2-gK) and neurotoxins (PLA2-gA2/PLA2-gB2; Mojave toxin A/B). Whereas, in the SVMP family, intra-genetic step-wise exon deletions removed specific domains (disintegrin and cysteine-rich) thus resulting in the three classes of metalloproteinase proteins. Within some species, genomic rearrangements (whole gene deletions) at these gene complexes created modified gene sets that are capable of producing toxins associated with a specific venom type (hemorrhagic or neurotoxic). However, at least one species has horizontally acquired the gene complexes necessary for neurotoxic venom thus highlighting one pathway for the evolution of novelty. Key Words: genetic basis of venom diversity, gene family evolution, gene duplication, gene loss, origins of biological novelty BS5 - INVESTIGATING THE COMPLEXITY OF VENOM RESISTANCE USING INVERTEBRATE VENOMS AND EXTREME QUANTITATIVE TRAIT LOCUS MAPPING IN DROSOPHILA Ellsworth, SA and Rokyta DR Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA E-mail address: sellsworth@bio.fsu.edu Background: Venom resistance has occurred numerous times throughout the animal kingdom. However, most approaches to studying the evolution of venom resistance have focused primarily on studying the evolution of venom complexity and toxicity and the mechanisms and traits that lead to venom resistance have not been studied as in-depth. Categorizing venom complexity and identifying novel compounds has been at the forefront of venom research, while venom resistance, also a complex and diverse trait, has yet to be investigated from a broad molecular standpoint. The few studies looking into the molecular basis of venom resistance have principally focused on the evolution of resistance using mammals and snakes. These studies have identified mechanisms of venom resistance on a single toxin or toxin family, such as, a change in a single ion channel receptor blocking a specific venom protein in grasshopper mice and scorpions. Other studies have looked at more inclusive mechanisms of venom resistance such as circulating blood factors that can bind and inhibit a broad range of venom proteinases in ground squirrels and rattlesnakes. While these studies are important in highlighting some mechanism of venom resistance, this view needs to be expanded to encompass venom resistance as a complex and integrated phenotype. Investigating the complexity of venom resistance has been difficult due to studies using wild populations where the environment is not controlled, or due to studies focusing on one specific molecular mechanism. When working with these wild populations increasing the sample size of the study often will

cause sampling from multiple populations decreasing the amount of experimental control. I plan to overcome these barriers using an experimental evolution approach to categorize and analyze the complexity of venom resistance using fruit flies (Drosophila melanogaster). Using whole venom in D. melanogaster I will be able to control environmental variables, maintain a constant population structure, and use enough individuals in a high throughput experiment to allow for a repeatable and comparative view of different venoms. Drosophila melanogaster have been shown to have variation in venom sensitivity allowing us to isolate and identity alleles associated with venom resistance. Methods: Extreme quantitative trait locus mapping in Drosophila has been used to capture fine scale changes in allele frequencies between two groups of phenotypically different flies from a large variable population. I will inject various invertebrate venoms into a single variable advanced intercrossed population of D. melanogaster and collect the flies that are most resistant to each of the respective venoms. Whole genome sequencing will then be performed on the resistant flies and compared against the base population to identify genomic regions responsible for venom resistance. These genomic regions can then be compared across different venom types to identify similar and distinct mechanisms of venom resistance. Discussion/Conclusion: This dataset will allow for a comparison of the diversity and complexity seen in venom resistance in a model organism. This will provide insight into other potential mechanisms of venom resistance that could be present in natural coevolutionary relationships. Key Words: Venom resistance, venom complexity, invertebrate venom, Drosophila melanogaster BS6 - COMPLEX INTERACTIONS OF BIOTIC AND ABIOTIC FACTORS SHAPE VENOM PHENOTYPES IN THE WESTERN RATTLESNAKES Mackessy, SP1, Smith, CF1, Saviola, AJ2, Schield, D3, Perry, BW3, Parker, J4, Castoe, TA3 1 School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, CO 80639 USA; 2Department of Molecular Medicine and Neurobiology, Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037 USA;3Department of Biology, University of Texas at Arlington, 501 S. Nedderman Drive, Arlington, TX 76019 USA; 4Department of Biology, Fresno City College, Fresno, CA 93704 USA E-mail address: stephen.mackessy@unco.edu Background: The western United States is home to a plethora of rattlesnake species, and one clade, including Crotalus viridis, C. oreganus and C. scutulatus (hereafter CVOS), is

particularly wide-spread and diverse, occurring from southern Canada to northern Mexico and from the Pacific coast to the middle plains states. Various members of this clade produce venoms with radically different phenotypes, ranging from highly hemorrhagic/necrotic type I venoms to potently toxic type II venoms, characterized by the presence of Mojave toxin and homologs. The evolutionary history of the group has been poorly defined and taxonomy has been contentious, and diversification of the CVOS group has also been greatly affected by abiotic factors, including glaciation, desertification and orogeny, further complicating taxonomic status of many populations. A recent study has indicated extensive gene flow where distinct lineages come into contact, indicating that present-day lineages have resulted from a complex interplay of genetic drift, isolation, secondary contact and incomplete reproductive isolation since the early Pliocene (~6 MYA). In addition, predator-prey dynamics have resulted in locally adapted venom phenotypes in at least some populations, and this phenomenon is likely much more broadly distributed. To evaluate gene flow and its effects on venom phenotype, we have collected samples from several regions of contact between species/subspecies where hybridization and admixture are suspected or known, as well as in the center of subspecies distributions (“pure” phenotypes). Methods: Venoms were field-collected in various parts of western Colorado, southern Arizona and New Mexico, southern Idaho and eastern Washington, lyophilized and stored frozen until use. Venoms were assayed for six common enzymes, fractionated via RP-HPLC, analyzed via SDS-PAGE and subjected to shotgun proteomic mass spectrometry analysis. Discussion/Conclusion: In general, the type I-type II compositional dichotomy in venom proteome described earlier was observed for CVOS subspecies/species analyzed. Northwestern subspecies (C. o. oreganus and C. o. lutosus) sampled showed typical type I venom phenotypes, with PI-PIII metalloproteinases dominating the proteome, while type II venoms were noted only in C. o. concolor and C. scutulatus populations. However, several regions of introgression were noted in northwestern Colorado and in southwestern New Mexico. In northwestern Colorado, C. o. concolor and C. v. viridis occur in close proximity, and gene flow between these populations is evident. The effects on venom phenotype are variable; both “pure” phenotypes are observed (C. v. viridis – type I; C. o. concolor – type II), but two additional phenotypes are also seen at lower frequency, one possessing both concolor toxin and moderate metalloproteinase activity (type III), and one lacking both (type IV). Similarly, in New Mexico, hybrids between C. v. viridis and C. scutulatus have long been known,with venom phenotypes showing similar patterns of hybridization. However, evidence of extensive penetration of atypical venom phenotype expression in either population or species pairs was not observed, indicating that co-expression of high levels of neurotoxin and metalloproteinase activity may be maladaptive. Because venom phenotype largely dictates envenomation sequelae, medical personnel should be aware that very different symptoms may be presented depending on specific location of offending snakes. Key Words: admixture, Crotalus, evolution, hybridization, metalloproteinase, toxin

BS7 - A STING OPERATION: RISK ASSESSMENT AND VENOM EXPENDITURE BY SCORPIONS IN DEFENSIVE CONTEXTS Marston, LA1, Nisani, Z², Fox, GA1, Trujillo, D1, Marcy, S1, Hung, KY3, Hayes, WK1,* 1 Department of Earth and Biological Sciences, School of Medicine, Loma Linda University, Loma Linda, California, 92350 USA; 2Antelope Valley College, Division of Mathematics, Science & Engineering, Department of Biological and Environmental Sciences, Lancaster, California 93536 USA;3Coachella Valley Mosquito and Vector Control District, Indio, California 92201 USA E-mail address: whayes@llu.edu Background: Venomous animals often possess complex venom delivery systems designed to inject their toxic secretion into the tissues of prey and/or predators, though other functions of venom exist. As a limited commodity and costly investment, venom should be efficacious in function and deployed judiciously. Effectiveness of venom deployment depends largely on two critical factors: venom composition and quantity of venom delivered. Venom composition evolves over many generations, whereas venom expenditure can potentially be modulated behaviorally by the individual with any usage (the venom-metering hypothesis). Insufficient venom expenditure will fail to achieve the desired outcome, and excessive use can result in unnecessary metabolic costs of venom regeneration and ecological costs of venom depletion. Thus, natural selection should favor strategies whereby animals (1) choose whether to use their venom; (2) modulate, or meter, the quantity of venom expended; (3) modulate the composition of venom deployed; and (4) deliberately target venom injection to the most vulnerable region of their target. We describe a series of studies that address the first two tactics in the context of defensive stings by scorpions. Methods: We presented a range of targets to elicit defensive stings from either captive or wild scorpions. Experiments on captive scorpions were conducted in daylight, whereas those on wild scorpions were conducted at night with the aid of ultraviolet light and video capture (30 or 60 fps) via cell phones. Targets presented to scorpions included dead mice (Mus musculus), small membrane-covered receptacles, and human fingers. When interactions were videotaped, we conducted field-by-field reviews to quantify number and duration of individual stings. We determine venom expulsion visually, and measured it when possible using capillary tubes. Results:Our initial studies in a laboratory setting demonstrated that the African species Parabuthus transvaalicus makes risk-associated decisions, using larger quantities of venom when stinging or spraying venom (which this species is capable of) under higher levels of defensiveness. Follow-up field studies of Smeringurus mesaensis and Centruroides sculpturatus revealed remarkable reluctance to use their venom defensively. When contacted momentarily by a simulated predator, the scorpions usually employed brief sting-like actions of the metasoma to harmlessly slap the potential predator and generate momentum to flee in the opposite direction. When physically restrained, however,

scorpions were much more likely to deliver stings of longer duration accompanied by venom expulsion. Behavioral differences between the two species were evident. In another laboratory study, we provoked Hadrurus arizonensis to sting after fewer probes and with a higher likelihood of wet stings when predator odors (from Rattus rattus) were present compared to odor absence. Smaller scorpions were also more reactive than the larger ones. Discussion/Conclusion: Collectively, these findings support the view that scorpions assess potential risks and modulate their stinging behavior and venom expenditure accordingly. Although venom composition is often viewed as the critical factor for efficacious venom use, especially in light of shifting prey bases and coevolutionary counteradaptations of prey and predators, the behavioral capacity of scorpions to meter their venom should afford them opportunities to “experiment” with venom composition. Key Words: Scorpions, Centruroides, Hadrurus, Smeringurus, antipredatory behavior, stinging behavior, venom expenditure, venom metering BS8 - ANTIMICROBIAL PEPTIDE DIVERSITY IN THE GIANT DESERT HAIRY SCORPION (HADRURUS ARIZONENSIS) Nystrom, G.1, Ellsworth, S.1, Ward, M.1, Rokyta, D.1 1Florida State University Department of Biological Science E-mail address:gnystrom@bio.fsu.edu Background: Originating more than 400 million years ago, scorpions represent one of the oldest extant lineages of venomous animals. Although venom characterizations have been completed for a handful of species, the vast majority of the approximately 2400 species of scorpions lack any form of venom characterization.Furthermore, most venom characterizations have been completed on venom from species that are considered medically significant. However, venom characterizations from non-medically significant species have revealed venom toxin profiles that are similar to those from medically significant species, indicating the potential for non-medically significant scorpion venoms as sources of novel therapeutic agents. The giant desert hairy scorpions of the genus Hadrurus are a group of non-medically significant scorpions that have been observed to spray themselves with their own venom. As recent analyses have revealed that the venom of Hadrurus spadix has a high abundance and diversity of antimicrobial peptides, it has been suggested that this spraying behavior may serve as a self-cleaning mechanism, providing a novel functional role for scorpion venoms. To further investigate the role of Hadrurus venom in microbiome regulation, we performed an in-depth venom characterization Hadrurus arizonensis and a subspecies, H. arizonensis arizonensis. Methods: We collected scorpions from southern Arizona, extracted venom via electrostimulation, and dissected the venom-glands. Using RNA-seq and quantitative mass spectrometry, we performed a high-throughput venom-gland transcriptomic and

venom proteomic analysis of two individuals each of the Arizona Giant Desert Hairy scorpion (Hadrurus arizonensis) and the subspecies H. arizonensis pallidus. With mass-spectrometry guided annotation we distinguished venom toxins by their presence in both the venom-gland transcriptome and venom proteome. Discussion/Conclusion: We revealed a rich abundance and diversity of antimicrobial peptides, providing evidence for antimicrobial use as a novel function of scorpion venoms. We also identified a rich diversity of potassium ion-channel toxins (KTxs), peptidases, and venom proteins that could not be assigned a functional classification. As K+-channel toxins and antimicrobial peptides are fairly small toxins ( > 200 amino acids), they were challenging to efficiently detect proteomically. This may be, in part, a result of the significant post-translational or proteolytic process involved in protein maturation. Key Words: scorpion, antimicrobial peptide, Hadrurus BS9 - ATTENUATION OF SURGICALLY-INDUCED BRAIN INJURY THROUGH WHOLE AND FRACTIONATED VENOM PROTEIN PRECONDITIOING Travis, ZD1,2,*, Sherchan, P2,3, Cooper, A1, Kelln W1, Hayes, WK1, Zhang, JH2,3,4

1 Department of Earth and Biological Sciences, 2 Neuroscience Institute, 3 Department of Physiology and Pharmacology, and 4 Department of Anesthesiology, School of Medicine, Loma Lunda University, Loma Linda, California 92350 USA E-mail address: zacharydtravis@gmail.com Background: Surgical brain injury (SBI) affects tens of thousands of neurosurgical patients globally every year, but remains one of the least studied traumatic brain injury (TBI) pathophysiologies. Preconditioning comprises a deliberate strategy in which exposure to small doses of a toxic stimulus prepares the body against future massive insult by activating endogenous protective responses. Known inflammatory signaling pathways that have been implicated in post-operative complications are targeted. Viperid and elapid venoms often contain phospholipase A2, snake venom metalloproteases (SVMPs), and snake venom serine proteases (SVSPs), all of which target different inflammatory and hemostatic cascades. We previously explored the preconditioning efficacy of whole venoms and factions from two viperid species (Crotalus atrox and C. helleri) and an elapid (Naja sputatrix). Results suggested that both SVMPs and PLA2s attenuate bleeding and swelling during and subsequent to brain surgery in a laboratory rat (Rattus rattus) model. The current study examined Pseudechis papuanus venom, which contains an exceptional level of secretory PLA2 (90.2% by dry mass), with the remaining portion composed of 3FTXs, SVMPs, cysteine-rich secretory proteins (CRISPs), and l-amino acid oxidases (LAAOs). Because of the protein composition, this venom causes neurotoxicity, hemolysis, pulmonary inflammation, and edema. In this study, we investigated whether whole and purified P. papuanus venom preconditioning (VPC) reduces SBI-induced neuroinflammation by triggering the PLA2/5LOX cascade and attenuates intraoperative hemorrhage by increasing platelet aggregation via the PLA2/COX1 cascade in a SBI rat

model. Sublethal doses of venom were injected subcutaneously three consecutive days prior to SBI. Purified PLA2 VPC may be a beneficial therapy to reduce post-operative neuorinflammation and intraoperative hemorrhage complications from brain surgeries. Methods: We used reverse-phase high-pressure liquid chromatography (RP-HPLC) to separate venom proteins of interests. We then injected rats subcutaneously with varying sublethal doses of whole venom and purified fractions during three consecutive days prior to experimental SBI. We conducted partial frontal lobe resections of rats to mimic a typical surgical procedure. We then used modified Garcia, beam balance, rotarod, and water maze tests to evaluate behavioral outcomes. We measured brain water content to assess cerebral edema, and used a hemoglobin assay to quantify hemorrhage volume. Results: We observed that VPC at an optimal dose significantly reduced edema and improved neurological function 24 h and 72 h after SBI. Furthermore, our long-term study showed an increase in neurological function 30 days after SBI. The VPC regime also significantly reduced intraoperative bleeding, while the sublethal dose caused no skin inflammation at the injection site and no other identifiable toxic effects. Conclusions: These findings suggest that VPC reduces neuorinflammation and intraoperative hemorrhage, and improves neurological outcomes after SBI. Purified PLA2 VPC may be a beneficial therapy to reduce post-operative neuroinflammation and intraoperative hemorrhage complications from brain surgeries. Key words: Venom preconditioning, surgical brain injury, inflammation, hemorrhage, SVMPs, PLA2, SVSP BS10 - IDENTIFICATION OF AMINO ACID RESIDUES IN COBRA VENOM FACTOR RESPONSIBLE FOR CONVERTASE STABILITY BY FORMING BONDS WITH HUMAN FACTOR B Hew, BE1, Fritzinger, DC1, Vogel, C-W1,2 1University of Hawaii Cancer Center, University of Hawaii at Manoa, Honolulu, HI, USA; 2Department of Pathology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, HI, USA. E-mail address: cvogel@cc.hawaii.edu Background: Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. These bimolecular complexes CVF,Bb and C3b,Bb are enzymes that cleave C3. Whereas both convertases exhibit spontaneous decay-dissociation, the CVF,Bb convertase is physico-chemically very stable (t1/2 at 37°C is 7 hrs). In contrast, the C3b,Bb convertase is very unstable (t1/2 at 37°C is 1.5 min). Our previous work using recombinant chimeric proteins of CVF and human C3 demonstrated

that the C-terminal C345C domain harbors important structures responsible for convertase stability. Methods/Results: Here we report the identification of individual amino acid residues of CVF contributing to the stability of the CVF,Bb enzyme by introducing point mutations based on sequence differences between CVF and C3b. In two cases, we replaced CVF residues with the corresponding residues from human C3 in a chimeric protein able to form a very stable convertase (HC3-1496). The two separate substitutions Q1550S (Q1571S in C3 numbering) and E1637V (E1658V in C3 numbering) resulted in chimeric proteins (called HC3-1496-9 and HC3-1496-25) both of which formed convertases exhibiting significantly reduced stability and C3 complement-depleting activity compared to HC3-1496. In the convertase with HC3-1496-9, the hydrogen bond between glutamine 1550 in CVF and Glycine 392 in Bb was lost; in the convertase formed with HC3-1496-25 the ionic bond between glutamic acid 1637 in CVF and lysine 347 in Bb was lost. In a third case, we replaced a C3 residue (P1539T) with the corresponding residue from CVF (P1518T in CVF numbering) in a chimeric protein (HC3-1550) that formed an unstable convertase. The substitution resulted in a chimeric protein (HC3-1550-4) which formed a convertase with significantly increased stability and complement-depleting activity compared to HC3-1550. A new hydrogen bond was formed between Threonine 1518 in CVF and Histidine 389 in Bb. Discussion/Conclusion: Chimeric proteins between human C3 and CVF represent effective tools to identify functionally important amino acids in CVF. CVF residues glutamine 1550, glutamic acid 1637, and threonine 1518 were identified as important residues contributing to convertase stability. All three residues are involved in hydrogen or ionic bonds with Factor B, not formed by the corresponding residues in C3b. Key Words: Cobra venom factor, CVF, complement, chimeric proteins, C3 convertase BS11 - IDENTIFYING TOXIN-TARGETS THROUGH EXPERIMENTAL EVOLUTION AND GENOME-WIDE ASSOCIATION STUDIES Ward, MJ1*, Ellsworth, SA1, Rokyta, DR1

1Florida State University, Department of Biological Sciences, Tallahassee, FL E-mail address: mward@bio.fsu.edu Background: The therapeutic properties offered by venom-derived toxins have led to the development of many life-saving medications. As this medicinal library continues to grow, so does the need to fully understand these toxins and their potential targets, many of which remain unknown. We have taken an experimental-evolution approach to venom target identification by evolving venom resistance in fruit flies, Drosophila melanogaster, using venom from the giant Florida blue centipede, Scolopendra viridis. The advantages of Drosophila genetics enable the identification of genetic (allele frequency) changes

resulting from evolved resistance in comparison to a control population, narrowing down potential targets to a set of candidate genes that have responded to the strong selective pressure of venom. However, results of experimental evolution studies are often noisy, and it can be difficult to distinguish true positives (i.e. alleles that have changed in frequency as a result of selection) from false positives (i.e. alleles that have changed in frequency as a result of drift or other environmental selection pressures). To assist in validation of true positives, a full genome wide association study (GWAS) was also performed. Methods: A genetically mixed population of Drosophila was separated into experimental (venom-injected) and control (PBS-injected) populations, which underwent selection for 18 months (approximately 35 fly generations). Whole-genome sequencing was performed on the ancestral fly population and each of the experimental and control populations to identify allele frequency changes between venom-injected and control groups. A full GWAS was performed by scoring ~200 inbred fly lines for the trait of venom resistance and performing quantitative trait loci (QTL) mapping to identify genomic regions associated with venom resistance. Discussion/Conclusion: Genomic regions identified in both experimental evolution and GWAS datasets will have a higher probability of being a true positive and therefore genes located within these regions are more likely to be venom targets. Results from this work are expected to elucidate the molecular basis of venom resistance while identifying candidate toxin targets, which can be used in downstream analyses leading to both drug development and in understanding the coevolutionary dynamics of predator and prey.

Key Words: Centipede venom, Drosophila, experimental evolution, QTL mapping, toxin targets BS12 - HUMAN FIBROBLAST RESPONSE TO CROTALUS ATROX VENOM AND MODEL WOUND HEALING Melcher L1,2, Peterson I1,2, Hiner, M1, Wertheimer, AM1,2

1VIPER Institute; 2BIO5 Institute, University of Arizona, Tucson AZ 85721 E-mail address: awerth@email.arizona.edu Background: Envenomation is a globally neglected crisis, particularly in rural areas and developing countries. Currently available anti-venom, a critical and life-saving therapy, does not diminish tissue damage at the site of envenomation, and may lead to chronic complications. We intend to improve Crotalus atrox (C. atrox) antivenom effects to also address tissue damage by characterizing and analyzing tissue damage and wound healing following C. atrox envenomation. Our tissue model uses American Type Culture Collection (ATCC) #CRL-2097 neonatal foreskin fibroblast cells to evaluate non immortalized human cell response to venom and track tissue damage, to enhance antivenom therapy effects at the site. Fibroblast cells, found predominately in the interstitial space, are a sensitive cell type to test, thus fibroblast response has greater sensitivity and is likely a useful measure of

tissue damage. Current antivenom therapy consists of a conglomerate of antibodies from hyperimmunized animals (horses or sheep typically). Examining specific mechanisms of tissue damage allows us to target those mechanisms for neutralization, creating antivenom which directly addresses tissue damage. Methods: Fibroblasts were initially seeded in 24 well plates starting with 100,000 cells per well and serially diluted down each row. Cells were cultured for 48 hours, adding 15ug/ml of C. atrox venom to each well to assess the confluence, and imaged at 30 minutes, 1, 4, 24, and 48 hours, assessing damage over time. Fibroblast monolayers were also assessed for wound healing via scratch assay. After creating an initial scratch (a model wound) through a monolayer of 70% confluent cells, cells were exposed to 7.5ug/mL venom and then imaged at 1 hour, 2 hours, 4 hours, 24 hours and 48 hours. Cell death upon 1hr exposure to 20, 40 and 80ug/mL venom was quantified using a commercial lactate dehydrogenase assay (LDH). Results: Imaging revealed that cell monolayers which are confluent appear to survive venom application more often than higher dilutions of cells. Wells with higher confluence slough off layers of cells in clumps and sheets. Less confluent wells showed damaged cells that detached and clumped in smaller quantities, with some surviving cells still adherent and elongated, though often swollen or enlarged. At 24 hours the scratch assay revealed fibroblasts are able to regenerate and start to heal the scratch, with increased regeneration at 48 hours. Fibroblast response to a scratch was also observed with venom addition, which is a more realistic model of envenomation with the presence of local tissue damage. Cells that were scratched and then exposed to venom detached and clumped up more quickly than with a scratch alone. Fibroblasts healed and regenerated successfully when venom was neutralized with hyperimmune serum or immune-purified antibodies. Between 10-30% cell death occurred within 1hr of exposure to high venom concentrations (20-80ug/mL). Combining low dose venom (20ug/mL) with cell lysis solution doubled the amount of cell death compared to lysis solution alone. Discussion/Conclusion: Snake venoms are complex with great variation, even among species. C. atrox venom is particularly necrotic compared to other venoms that may be more hemotoxic or neurotoxic. We found that physical or chemical injury in combination with exposure to venom increased cellular damage and ultimately cell death. Our goal is to systematically identify the specific antibodies and effectively neutralize the toxins responsible for tissue damage, allowing fibroblasts to regenerate and support wound healing. Key Words: Fibroblast, tissue culture, wound healing, venom, Crotalus atrox, envenomation BS13 - HYPERIMMUNE HORSE SERUM AND IMMUNOPURIFIED ANTIBODIES IMPACT ON HUMAN FIBROBLAST CYTOKINE RESPONSE TO CROTALUS ATROX VENOM Park H1,2 Bosak J,1,3 Melcher L, 1,3 Wertheimer, AM1,2,3

1VIPER Institute, 2Applied Bio Sciences (ABS) Graduate Interdisciplinary Program (GIDP), and the 3BIO5 Institute, University of Arizona, Tucson, Arizona E-mail address: haerinp@email.arizona.edu, jbosak@email.arizona.edu, lmelcher@email.arizona.edu, awerth@email.arizona.edu Background: Snakebite is potentially life-threatening. According to the World Health Organization, there are approximately 4.5 - 5.4 million snakebites per year worldwide. Of those bitten, up to 2.7 million develop clinical symptoms with anywhere between 81,000 and 138,000 dying due to complications from the complex mixture of venom toxins. These toxins are typically classified into three categories: neurotoxins, hemotoxins and cytotoxins. Of those who survive the encounter, individuals may be left with physical disability or lifelong tissue damage due to the cytotoxic components of snake venom. Although not frequently addressed, tissue damage might be mitigated with a more efficacious antivenom, specific to the responsible toxins inherent within a specific venom. Our goal was to investigate the underlying mechanisms of venom-induced tissue damage and then neutralize them. Further research into systemic and local inflammatory mediators and cytokines in human cell models is critical to better understand cellular immune response to venom and development of a more effective antivenom. Methods: Human neonatal foreskin fibroblasts were exposed to 6.8µg/ml, 13.6µg/ml and 27.3µg/ml Crotalus atrox (C. atrox) venom and imaged to document cellular morphologic changes over a time course of 1, 15, 30 minutes, 1hr, 2hr, and 24 hours. Hyperimmunized horse plasma and venom were incubated for 15min at 37C with the venom in an attempt to neutralize the C. atrox venom and prevent cell damage. In addition a “rescue” format was tried where hyperimmune horse plasma was added to the fibroblasts 30 minutes after the introduction of the venom. Images were taken for qualitative analysis, and supernatant was retrieved for a quantitative cytokine analysis (ELISA) for pro-inflammatory cytokines TNFα, Il6, and IL8. To further analyze the neutralizing ability of the antibodies within the hyperimmune horse plasma, a Protein G affinity chromatography column was run to elute immunopurified antibodies from the plasma. The presence of these antibodies was confirmed through a 12% SDS-PAGE gel. Discussion/Conclusion: C. atrox venom at 6.8µg/ml resulted in less dramatic morphological changes in the fibroblast monolayer compared to the higher concentrations. The cells were noted to “ball up” and take on a more circular configuration than their usual elongated shape with minor disassociation of the cells from the surface of the well-plate. At the higher concentrations, all fibroblasts were disassociated from the monolayer and presumably die from their interaction with the venom within 24hours. C. atrox venom even at concentrations of 50µg/ml, 100µg/ml and 200µg/ml did not induce TNFα production as hypothesized. The fibroblasts involved in the “rescue” experiments also showed equivalent damage to cells treated with venom alone. Further, the fibroblasts involved in the pre-incubation experiments were only neutralized marginally better by the purified antibodies than the whole hyperimmune horse plasma.

Key Words: Immunopurification, TNFα, human neonatal foreskin fibroblasts, Crotalus atrox venom, tissue damage CT - CLINICAL TOXICOLOGY CT1 - A BREATH OF FRESH AIR: CAPNOGRAPHY, INNOVATION, ASF APPROACH TO TREATMENT AND TRAINING FOR NEUROTOXIC SNAKEBITES IN AUSTERE ENVIRONMENTS Abo, B1,2, Brandehoff, N1,3, Balde, C1,4, Chippaux, J-P1,5,6, Benjamin, JM1,7

1Asclepius Snakebite Foundation, Seattle, WA, USA; 2Miami-Dade Fire Rescue Venom One Venom Response Unit, Doral, FL, USA; 3UCSF-Fresno Department of Emergency Medicine, Division of Medical Toxicology, Fresno, CA, USA; 4Institut Pasteur de Guinée, Kindia, Guinea; 5CRT, Institut Pasteur, Paris 75015, France; 6MERIT, IRD, Université Paris 5, Sorbonne Paris Cité, Paris, 75006, France; 7Whitman College, Department of Biology, Walla Walla, WA, 99362. Email address: benabo@ufl.edu Background: Snakebite envenomation is a global health crisis and is classified as a Category A neglected tropical disease by the World Health Organization. An overwhelming majority of snakebites worldwide occur in remote areas far from equipped medical facilities. Most clinicians that have the opportunity to treat neurotoxic victims are unprepared for the challenges of managing snakebite patients in field conditions. Delays in antivenom treatment, basic life support, and airway management are strongly associated with increased morbidity and mortality in neurotoxic snakebite patients. In certain areas, often far from advanced medical care, neurotoxic envenomations threaten lives with death frequently occuring before definitive care can be accessed. Currently standard of care in developed systems, capnography in conjunction with basic life support is an easily teachable, high-yield tool in assessment-driven patient care and decision making in even the most austere of conditions. Capnography, both waveform and quantitative numbers, provides real-time, non-invasive, objective data to better guide decisions with a better learning curve than subjective assessment. Initially considered technically difficult to measure as well as costly, various durable and portable monitors exist that provide easily interpretable objective visual clues to guide patient care by experts and non-experts alike. Methods: Physicians and paramedics specializing in wilderness emergency medicine and emergency medical services (EMS) from the Asclepius Snakebite Foundation have developed a model to train and equip out of hospital providers, both in clinic and in the field, with essential medications, equipment, and supplies. A standardized training for objective use of capnography used widely in American EMS was implemented at the ASF - Institut de Recherche en Biologie Appliquée de Guinée (IRBAG) clinic in Kindia, Guinea. The ASF - IRBAG clinic receives approximately 700 envenomations per year. Of these, an estimated 25% of envenomations, and 70% of fatalities, are from neurotoxic snakes with death directly associated with inability to assess and manage airway protection. Utilizing a durable, low-cost device and this standardized training, out of hospital providers are able to

better assess and manage patient airways further buying them time to provide life-saving field-stable antivenom. Discussion/Conclusion: In regions where well-equipped and highly-trained advanced practitioners are hard to reach, capnography offers an innovative solution that allows non-physicians to apply the highest standards of airway management towards the fight against morbidity and mortality from neurotoxic bites in sub-Saharan Africa and other resource-limited settings. Studies assessing knowledge retention need to be done to further evaluate the needs and instruction of this critical skill. Key Words: Capnography, EtCO2, Snakebite, Envenomation, Africa CT2 - SEVERE ENVENOMATION FROM A CANEBRAKE RATTLESNAKE (CROTALUS HORRIDUS) LEADING TO CARDIOVASCULAR COLLAPSE WITH FULL RECOVERY Abo B1,2, Roberts, R2,3

1Miami-Dade Fire Rescue Venom One Venom Response Unit, Doral, FL, USA; 2University of Florida College of Medicine, Gainesville, FL, USA; 3North Florida Medical Center, Gainesville, FL, USA Email address: benabo@ufl.edu Background: Unlike the rest of the world, snakebite envenomations in the United States only claim the lives of a few people each year, however, they remain responsible for a significant amount of permanent pain, disability, and disfigurement for a vast majority of affected patients. Often, morbidity is increased by significant delay or lack of proper care despite the existence of safe and effective antivenom. We present a case of severe envenomation from Crotalus horridus leading to severe hemodynamic instability, hypotension, cardiac failure, hemolysis, and rhabdomyolysis with full recovery and ability to return to manual work eight days later. Methods: A male in his fifties initially presented to an outside hospital in a rural hospital setting following an accidental envenomation from a timber rattlesnake. Shortly after the envenomation, the patient syncopized and developed hemolysis and cardiovascular collapse. Initially, the referring hospital did not realize the patient had been envenomated, due to the lack of swelling and skin changes. However, shortly thereafter, the patient collapsed, and required intubation and three vasopressor agents to obtain a heart rate of 160 bpm and blood pressure of 50/30 mmHg. The outside hospital initially was afraid to use the full treatment of available antivenom and transported the patient to a tertiary care center where the patient was noted to have diffuse ischemic changes on ECG and hemolytic crisis including gross hematuria. Prompt use for severe envenomation 12 vials of FDA approved Crofab was infused with almost immediate improvement in heart rate, blood pressure, hemolysis, gross hematuria and anuria while still not showing local evidence of envenomation. On day two of admission to the intensive care unit, the patient started to

show signs of local swelling and erythema with elevations in creatine kinase (CK) and acute kidney injury. More antivenom, fluids and proper elevation of the limb redistributed edema and cleared local changes and kidney injury. The patient was able to be extubated, undergo physical therapy and occupational therapy, and able to be discharged. The patient made a full recovery and was able to return to work doing manual labor without permanent pain, permanent disability or permanent disfigurement. Discussion/Conclusion: Understanding of domestic crotalid envenomations is too often inconsistent and out of date among practitioners providing medical care throughout the United States. Here we highlight the hemolytic and cardiovascular collapse from a neurotoxic and hemotoxic venom that was fully reversed. Proper and adequate use of FDA approved Crofab can not only prevent mortality but significant morbidity and therefore healthcare costs, disability and loss of workforce. Key Words: Crotalidae, Snakebite, Envenomation, United States CT3 - WHEN THEORY AND REALITY DIVERGE: A MONOCLED COBRA ENVENOMATION IN THE UNITED STATES Benjamin Abo1,2, Jeffrey Fobb1, Nicklaus Brandehoff2,3, Diana Dean4, Andrew King4, Stephen Mackessy5, Cara Smith5

1Miami-Dade Fire Rescue Venom One Venom Response Unit, Doral, FL, USA; 2Asclepius Snakebite Foundation, Seattle, WA, USA; 3UCSF-Fresno Department of Emergency Medicine, Division of Medical Toxicology, Fresno, CA, USA; 4Michigan Poison Center; 6University of Northern Colorado. E-mail address: ben@snakebitefoundation.org Background: The monocled Cobra, Naja kaouthia, is an Asian Cobra responsible for a multitude of devastating injuries to its victims both in Asia and among United States victims that work with them or keep them as pets. Polyvalent antivenom from the Thai Red Cross exists and is effective and available from venom banks such as Miami-Dade Venom One and certain places via the antivenom index. Theoretically, African derived and developed SAIMR polyvalent antivenom can effectively neutralize toxins from Asian species. We present an envenomation of a monocled cobra here in the United States with initial failed treatment with SAIMR that ultimately recovered after procuring Thai Red Cross polyvalent antivenom from Venom One and subsequent lab analysis of the specific monocled cobra venom and SAIMR antivenom. Case/Methods: EMS was called to the home of a 26-year old male whose pet snake envenomated his left ankle. Within 20 minutes, he developed vomiting and respiratory failure requiring intubation. The patient was transferred to a tertiary facility where he was completely paralyzed, developed Acute Respiratory Distress Syndrome (ARDS) and renal failure. He received eight vials of SAIMR polyvalent antivenom six hours post-envenomation with minimal response resulting in axial muscle movement but continued fine

motor, bulbar and respiratory paralysis. 18 hours later, he received species-specific Thai Red Cross Cobra antivenom with quick resultant neurologic recovery. Additional doses of antivenom were administered 52 and 61 hours post envenomation given recurrent paralysis with resolution in symptoms. The Patient’s course was further complicated by progressive renal insufficiency with creatinine (Cr) peaking at 5.03 by day nine requiring dialysis, creatine phosphokinase (CPK) peaking at 844, and worsening ARDS. The patient was transferred to an ECMO capable facility but never required cannulation and was extubated with subsequent discharge to inpatient rehab. Venom analysis including protein concentration determination, gel electrophoresis, high performance liquid chromatography, and western blots were performed to test both anti-kaouthia and SAIMR antivenom to the specific venom from the individual monocled cobra. Results: Venom from the individual Naja kaouthia involved has venom comprised of small molecular weight 3 finger toxins (3FTx) and phospholipase A2 (PLA2) enzymes along with differing binding affinities between the Thai Red Cross polyvalent antivenom and SAIMR antivenom. Discussion/Conclusion: Whether for hobby or for professional keeping, handlers and owners of exotic venomous snakes need to be prepared for medical catastrophes. This case of a 26-year old male by Naja kaouthia in the United States with delayed specific antivenom proves the importance of full information early on, the importance of a network of experience and the ability to get proper antivenom and guidance to use such throughout the United States. This patient survived because of the collaboration and hard work from multiple teams across the United States. Furthermore, the laboratory work helps link theoretical cures and clinical symptoms to specific proteins. Key Words: exotic envenomation, Cobra, monocled Cobra, Naja kaouthia CT4 - CLINICAL PRESENTATION AND MANAGEMENT OF CYTOTOXIC AND HEMOTOXIC ENVENOMATIONS AT AN AUSTERE SNAKEBITE CLINIC IN KINDIA, GUINEA Benjamin, JM1,2, Abo, B1,3, Balde, C1,4, Chippaux, J-P1,5,6 , Brandehoff, N1,7

1Asclepius Snakebite Foundation, Seattle, WA, USA; 2Whitman College, Department of Biology, Walla Walla, WA, 99362; 3Miami-Dade Fire Rescue Venom One Venom Response Unit, Doral, FL, USA; 4Institut de Recherche en Biologie Appliquée de Guinée, Kindia, Guinea; 5CRT, Institut Pasteur, Paris 75015, France; 6MERIT, IRD, Université Paris 5, Sorbonne Paris Cité, Paris, 75006, France; 7UCSF-Fresno Department of Emergency Medicine, Division of Medical Toxicology, Fresno, CA, USA Email address: benabo@ufl.edu Background: Snakebite envenoming (SBE) is an environmental and occupational hazard in Africa and a disease of poverty that is responsible for an estimated 30,000 - 50,000 deaths and 90,000 - 150,000 disabilities and disfigurements every year across the continent.

There is little data on the clinical presentation and management of snakebites in sub-Saharan Africa, and most studies to data have focused on the West African Carpet Viper (Echis ocellatus). The ASF Guinea snakebite clinic in Kindia, Guinea is located in an area where savanna, forest, and grassland ecosystems come together and is therefore home to a great diversity of snake species that are rarely found together in Africa. This results in a high incidence of envenomations by species that are rarely studied in a clinical context. We present a case series discussing the characteristics and clinical management of 25 snakebite patients treated at the ASF Guinea over 25 days between July and August of 2019. Case Series: 25 patients were evaluated for snake envenomations at the ASF Guinea clinic in Kindia during the study period. 16 of these patients were diagnosed with snake envenomations and 9 patients presented with dry bites that resembled snake envenomations due to complications of injury or infection resulting from inappropriate first aid measures or traditional treatments. Three of the 16 patients with envenomations were treated symptomatically for venom ophthalmia (n = 1) or presumed envenomations by Causus or Atractaspis species. The remaining 13 patients were treated with Inoserp Pan-Africa (Inoserp-P, 500 LD50 potency) for presumed envenomations by large African adders (Bitis spp, n = 12) or Naja nigricollis (n = 1). All patients survived and recovered without major disability despite significant treatment delays and a number of severe envenomations, and there were no dangerous reactions to the antivenom. Most patients (n = 9) recovered after a single vial of Inoserp-P; however, 4 patients with severe envenomations required either two (n = 2) or four (n = 2) vials to achieve permanent control. All 15 patients with symptomatic snakebites reported pain and edema. Patients with Bitis or Naja envenomations who could be treated with Inoserp-P (n = 13) were easily distinguished from Causus and Atractaspis envenomations by the presence of persistent local bleeding (n = 12/13), coagulopathy by either WBCT20 or WBCT30 (13/13), and local blistering or necrosis (13/13). Notably, all patients with envenomations by Bitis rhinoceros, B. arietans, or B. nasicornis demonstrated a delayed coagulation characterized by incomplete clotting at 20 minutes (WBCT20) that slowly stabilized to a normal clot by 30 minutes (WBCT30). Discussion/Conclusions: This case series provides valuable information regarding the presentation and treatment of patients with Bitis, Causus, Atractaspis, and Naja envenomations in West Africa. The delayed clotting documented in Bitis envenomations in these patients has also been published in a case series from Benin, and sharply contrasts with the coagulopathy of Echis envenomations where clots become less stable over time due to progressive consumption of clotting factors. Finally, these cases demonstrate the safety and efficacy of Inoserp-P for the treatment of envenomations by Bitis rhinoceros, B. arietans, B. nasicornis, and N. nigricollis in a small series of patients treated in a resource-limited environment. Further research is planned to evaluate these findings in a larger patient population. Key Words: Snakebite, Envenomation, Africa, Antivenom, WBCT CT5 - KETAMINE FOR PAIN CONTROL OF SNAKE ENVENOMATION IN GUINEA: A CASE SERIES

Brandehoff, N1,2, Abo, B1,3, Balde, C1,4, Chippaux, J-P1,5,6 , Benjamin, JM1,7

1Asclepius Snakebite Foundation, Seattle, WA, USA; 2UCSF-Fresno Department of Emergency Medicine, Division of Medical Toxicology, Fresno, CA, USA; 3Miami-Dade Fire Rescue Venom One Venom Response Unit, Doral, FL, USA; 4Institut Pasteur de Guinée, Kindia, Guinea; 5CRT, Institut Pasteur, Paris 75015, France; 6MERIT, IRD, Université Paris 5, Sorbonne Paris Cité, Paris, 75006, France; 7Whitman College, Department of Biology, Walla Walla, WA, 99362. Email address: nick.brandehoff@me.com Background: Snakebite envenomation is a global health crisis and is classified as a Category A neglected tropical disease by the World Health Organization. The economic toll of death and disability in these regions can leave both victims and their families unable to provide food or money, which amplifies the burden placed on the community and further exacerbates the public health crisis. Recently, publications have also illustrated the potential for snake envenomations causing post-traumatic stress disorder (PTSD) on a significant number of patients who are envenomated. Ketamine is a NMDA receptor antagonist used for a variety of pharmacologic applications including anesthesia and acute pain management. Acute pain treatment with ketamine uses much lower doses (0.1 - 0.3 milligram/kilogram (mg/kg) intravenously (IV)) than typical doses used for procedural sedation or agitated delirium (1 - 2 mg/kg IV). Ketamine is easily available in Guinea with an average cost of $3 US for a 500 mg vial. Methods: A standard protocol for ketamine dosing used widely in United States hospitals was implemented at the ASF - Institut de Recherche en Biologie Appliquée de Guinée (IRBAG) clinic in Kindia, Guinea. The ASF - IRBAG clinic is the only dedicated snakebite center in the country and receives approximately 700 envenomations per year. Case Series: Over the course of 25 days, 12 patients who presented with severe pain secondary to Bitis, Causus, and Atractaspis envenomations were treated with low-dose intravenous ketamine. The patients included 7 males and 5 females with a median age of 37.5 (range 14 to 64) and a median presentation time of 6.6 hours (range 5 mins to 96 hours) after the initial bite occurred. Ten envenomations were presumed B. rhinoceros or B. arietans resulting in extensive swelling and blistering of the affected extremities. One envenomation was presumed C. maculatus is causing local swelling and pain. One envenomation was a presumed Atractaspis species causing significant local pain with minimal swelling. All patients expressed having significant pain but could not express the degree of their pain using an analogue pain scale. An initial median dose of 5 mg of ketamine (range 2.5 to 15 mg) was administered IV for pain control. All Bitis envenomations also received initial dose of Inoserp Pan-Africa antivenom. Every patient expressed decreased pain and felt more comfortable within 1 minute after administration of ketamine, confirmed by vital signs including a decrease in heart rate and blood pressure. Nine of the twelve patients only required a single dose. There were no adverse side effects. Discussion/Conclusions: Ketamine appears to be an effective means of pain control for those suffering from painful envenomations. With minimal risk of significant side effects

at acute pain management doses and the average cost per effective dose averaging between $0.03 - 0.06, this may provide a cheap, safe, and effective solution for Sub-Saharan Africa and other resource-limited settings. Controlled studies need to be done to critically assess our observations. Key Words: Ketamine, Snakebite, Envenomation CT6 - SNAKEBITES IN THE SOUTH OF VIETNAM Doan, UV Cho Ray Hospital, Vietnam Email: vanessa@ym.edu.tw Background: Snakebite envenomation is a neglected tropical disease and a significant medical problem in Vietnam. Our goal is to review cases of snakebite in the South of Vietnam to assess the clinical manifestations and their outcome after antivenom use. Methods: This is a retrospective, descriptive study from January 2016 – June 2018 reviewing the clinical findings and outcome of snakebite envenomation at the largest general hospital in Vietnam. Data collected included age, gender, snake species, clinical effects and medical outcome. Results: There were 2405 cases, of whom 1563 (65%) were male. The mean age was 45 (+/-15) years, with 2.5% teenagers. Three species of venomous snakebites were considered necessary to be seen in the clinic: pit viper bites from Trimeresurus albolabris 1276 cases (52%) and Calloselasma rhodostoma 487 (20.2%), and especially from the Colubrid snake Rhabdophis subminiatus 12 cases (0.5%). The predominant signs from Calloselasma rhodostoma bites were mild to severe coagulation disorders with spontaneous systemic hemorrhage often with multiple bullae. These commonly became infected. Notably, red-necked keelback bites (Rhabdophis subminiatus) bites could be mild without developing a coagulation disorder. However, severe Rhadophis subminiatus bites often developed a fatal coagulation disorder. Deadly bites from these non-front fanged snakes that held on for longer than a couple of seconds caused delayed signs and symptoms of severe hemorrhage. Neurotoxic bites results from the Elapidae: Naja siamensis 181 cases (7.5%), Naja kaouthia 45 cases (1.9%), Krait Bungarus candidus 30 cases (1.2%), and King Cobra, Ophiophagus Hannah, 22 cases (0.9%) may cause paralysis, respiratory failure, and eventually, death. Notably, Naja siamensis did not cause any signs and symptoms of neurotoxicity, but severe local necrosis that radiated outward. There are only two monovalent antivenins made in Vietnam, antivenin for Trimeresurus albolabris and Naja kaouthia. Polyvalent antivenin and Malyasia Pit Viper antivenin were imported from Thailand and has been used for neurotoxic venomous bite cases and Calloselasma rhodostoma, respectively. Early antivenin use improved the signs and symptoms of hemotoxic and neurotoxic bites.

Discussion: The lack of antivenin in Vietnam is a huge problem. Many victims with snakebites have suffered severe morbidity or complications such as severe wound infection or amputation for necrotic tissue. The antivenin of Naja Kaouthia has not been used for Naja siamensis bites, we do not know whether it would be effective or not. Patients with Rhabdophis subminiatus bites were fatal in several recent cases because of development of severe coagulation disorders unresponsive to blood transfusions. When patients were admitted to hospital without a clear identification of the snake, a flowchart was created to help the physician make an intelligent guess of the most likely snake involved. Conclusion: An algorithm of snakebites should be established to help clinicians recognize the likely species of snake based on clinical signs and symptoms. This would enable early decision making for antivenin use decreasing mortality, enhancing earlier recovery, and minimizing complications Key words: South Vietnam, snakebites, Rhabdophis subminiatus, Naja siamensis CT7 - SNAKE ENVENOMATION CLINICAL OUTCOME MEASURES VARY WIDELY AND ARE NOT PATIENT-CENTERED: A SYSTEMATIC REVIEW OF CLINICAL STUDIES Campos M1, Rêgo G1, Staton CA2,3, Vissoci J, RN2,3, Gerardo CJ2,3

1Medicine Department, Federal University of Maranhão, São Luis, Maranhão, Brazil; 2 Division of Emergency Medicine, Department of Surgery, Duke University School of Medicine, Durham, NC, US; 3 Duke Global Health Institute, Duke University, Durham, NC, US E-mail address: charles.gerardo@duke.edu Background: With the emergence of potential therapeutic agents for snake envenomation, extensive clinical research is required to establish effectiveness. Currently, there is no internationally agreed upon standard outcome measure for use in snake envenomation clinical studies. Prior clinical trials have primarily used diagnosis-oriented endpoints, such as surrogate markers of coagulopathy, tissue injury or systemic toxicity. We performed a systematic review of prospective clinical studies on snake envenomation globally and identified existing gaps in evidence of outcomes assessments. Methods: In accordance with PRISMA guidelines, we searched the electronic databases MEDLINE, EMBASE, Scopus, Web of Science, and Global Health Library. Article eligibility criterion were snake envenomation, human clinical studies, and prospective design. Basic and translational science studies were excluded. A reference and citation analysis was conducted as well as a data quality assessment. We used a qualitative meta-summary approach to summarize outcomes assessments. No language or year restrictions were applied.

Results: The initial literature search revealed 3,696 articles. Of those, 199 articles fit the eligibility criteria and included data from 31 different countries and 32,342 patients. Most studies (81%) came from tropical countries such as India (15%), Sri Lanka (15%), Brazil (10%) Thailand (9%), and Australia (7%). A minority came from North America. Randomized Controlled Trials (21%) were exclusively conducted in either Sri Lanka, Brazil or the United States. Outcomes assessments were grouped into Venom Level Measurement, Laboratory Studies/Biomarkers, Clinical Severity, Treatment Response, Patient-Centered outcomes, and Adverse Reactions. Laboratory Studies/Biomarkers and Treatment Response were the most common outcome measures. Sixty-four (32%) articles used Clinical Severity, however, most of the scores were not standardized. Few studies (2%) used Patient-Centered outcomes, mostly coming from the clinical studies in the US. Conclusion. Laboratory studies and response to treatment were the most common outcomes assessed and important patient-centered data is scarce. Future studies should develop and use patient-centered measures, and provide their association with existing laboratory studies and treatment responses. Additionally, studies should be performed in low and middle-income countries with patient-centered outcomes in order to guide treatment in the areas with the highest burden of disease. Keywords: Snakebite, Patient-centered outcome, Global Health CT8 - ASSOCIATION OF A CYTOKINE RESPONSE NETWORK WITH FUNCTIONAL RECOVERY FROM SNAKEBITE ENVENOMING Gerardo, CJ1,2, Silvius, E3, Schobel, S4, Eppensteiner, J1, McGowan, L1, Limkakeng, AT1, Kirk, AD5, Elster, EA6 1Division of Emergency Medicine, Department of Surgery, Duke University School of Medicine, Durham, NC, US; 2 Duke Global Health Institute, Duke University, Durham, NC, US; 3 DecisionQ, Arlington, VA, US; 4 The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, US; 5 Department of Surgery, Duke University School of Medicine, Durham, NC, US; 6 Department of Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, US Email address: charles.gerardo@duke.edu Background: Inflammatory pathways activated during snake envenomation are poorly described, and their association with recovery is unclear. An improved understanding of the inflammatory pathways in snake envenomation will help develop diagnostic, prognostic, and therapeutic targets. The purpose of this study was to identify patterns of pre- and post-envenomation cytokines that are associated with recovery from snake envenoming. Methods: Design: Prospective cohort study. Setting: Tertiary care academic medical center emergency department.

Participants: We enrolled consecutive patients with crotaline snake envenomation and obtained serum samples based on previously described criteria for the Surgical Critical Care Initiative (SC2i) Tissue and Data Acquisition Protocol (TDAP) (ClinicalTrials.gov Identifier: NCT02182180). No immunosuppressed patients were enrolled. Data: We measured levels of 35 unique cytokines using Luminex Cytokine 35-Plex Human Panel (Thermo Fisher Scientific, Waltham, MA) pre- and post-antivenom administration. Outcomes: The Patient-Specific Functional Scale, a well-validated patient-reported outcome of functional recovery, was assessed at 0, 7, 14, 21 and 28 days. Individual patient recovery curves over time were assessed and the area under the patient curve (AUPC) determined. Analysis: To account for high dimensionality, we performed Bayesian Belief Network (BBN) modelling, a machine learning technique, to represent relationships with a diagram composed of nodes and arcs. Each node represents a cytokine, and each arc represents a joint-probability distribution (JPD). The JPDs were determined in a step-wise fashion over multiple iterations. We report associations of this cytokine model with the patient’s recovery (AUPC). Results: Twenty-eight snake envenomation patients were enrolled. Preliminary results from 9 patients with pre-antivenom and 11 patients with post-antivenom cytokine data are presented. The group was mostly female (82%) with a mean age of 35 (SD ± 9.8) years. Ten patients were white and one was Asian. The pre-and post-antivenom modeling networks are presented in Figures 1 and 2, respectively. Monocyte Chemoattractant Protein-1 (MCP-1) and Interleukin 1a (IL1a) are most closely related with AUPC in the pre-antivenom network, while the most influential cytokines were IL-13, IL1ra, endothelial growth factor (EGF), and vascular endothelial growth factor (VEGF) Regulated On Activation Normal T Cell Expressed And Secreted (RANTES), IL-1 and interferon gamma (IFNg) were most closely related with AUPC in the post-antivenom network. Macrophage inflammatory protein 1b (MIP1b), ECF, hepatocyte growth factor (HGF), and eosinophil chemoattractant cytokine (EOTAXIN) were most influential Figure 1: Pre-antivenom Network

Figure 2: Post-antivenom Network

Conclusions: Pre- and post-envenomation cytokine networks were associated with functional recovery measured by the AUPC of the PSFS over time. These networks included both innate and adaptive immune system responses. With additional patient data, we can identify cytokine-based predictive models of recovery from snake envenoming. Key Words: cytokine, snakebite, envenoming, functional recovery, immune response CT9 - THINKING OUTSIDE “THE COMPARTMENT BOX”: REVERSING TRENDS IN FREQUENCY OF SNAKEBITE FASCIOTOMY

Chollet, H1, Elliott, C1, Gross, G1, Hanley, BP2, Muenchow, L1, Jin, L1, Gross, O1, Longoria, R2, Betancourt, A1, Merrill, D1, Padilla, D1, Lawrence, A1, Falkoff, M1 1Valley Baptist Medical Center, Harlingen TX, USA; 2 Valley Baptist Medical Center, Brownsville TX, USA E-mail address: venommd@gmail.com Background: Snakebites present a significant problem in Texas. Snakebite envenomation can produce both systemic and local signs and symptoms. One of the most concerning local signs is edema that can produce increased pressure within muscle compartments. These compartments are bounded by strong, rigid fascia that will not stretch or accommodate the swelling. This causes increased pressure on the capillaries and nerves, compromising the neurovascular bundle of the affected area and ultimately leading to a limb-threatening medical emergency known as Acute Compartment Syndrome (ACS). The most commonly affected sites are the leg and the forearm. Fasciotomy is the definitive treatment for ACS with compartment pressure > 30 mmHg; however, routine fasciotomy following a crotaline envenomation is not recommended and there is currently no standardized treatment for envenomation-ACS-like syndrome. The American College of Medical Toxicologists and The American Academy of Clinical Toxicology recommend against fasciotomy in patients with snakebite envenomation. Case Report. We present three cases of crotaline envenomation that occurred in anatomic locations of high risk of developing compartment syndrome: the palm of the hand, forearm and the calf. Each of these patients were managed with adequate doses of Crotalinae polyvalent immune Fab ovine (CroFab®) rather than fasciotomies and all had favorable outcomes. Case #1 A 26-year-old Hispanic male was bitten by Crotalus atrox, patient used his belt as an arterial tourniquet on his right arm. When patient arrived, the tourniquet was removed and had swelling all the way up to his upper arm. He was treated with total of 22 vials of CroFab® until progression was stopped. Patient made full recovery. Case #2 A 43-year-old man presented to the emergency department after being bitten on his left hand by a Crotalus atrox. He received a total of 15 vials of CroFab®. He remained stable throughout his hospital stay and was discharged home with local wound care instructions. No complications. Case #3 A 12-year-old male child was brought to the ED, via EMS after being bitten on left leg, above the ankle by Crotalus atrox. Significant edema soon developed, which was stopped by administration of CroFab®. No complications.

Discussion: Despite lack of the established guidelines for treatment of envenomation-ACS-like syndrome, our experience shows that primary treatment of an envenomation syndrome should be with appropriate antivenom, with dose increased until reversal of pain and edema. This is the not what the CroFab® package insert says to do. Antivenom in sufficient dose decreases intra-compartmental pressures and is first-line therapy while fasciotomy should be reserved for those patients who fail medical therapy and undergo compartment pressure check prior to fasciotomy. Typical envenomation-ACS-like syndrome compartment pressures are 20-22 mmHg or less. Conclusion: With the widespread availability of safe anti-venom, urgent fasciotomy is rarely required in the acute management of crotaline snake envenomation. Key Words: Crotalus envenomation, Fasciotomy, Compartment syndrome CT10 - WHAT ACTIVITIES DO SNAKEBITE ENVENOMATION PATIENTS CHOOSE TO ASSESS DYSFUNCTION? Vissoci, J R N1,2, Tupetz, A1, Phillips, A J1, Kelly, P E,1 Lavonas, EJ3, Gerardo, CJ1, 2

1Division of Emergency Medicine, Department of Surgery, Duke University School of Medicine, Durham, NC, US; 2 Duke Global Health Institute, Duke University, Durham, NC, US; 3 Department of Emergency Medicine, Denver Health Authority, Denver, CO, US. Email address: patrick.kelly@duke.edu Background: The Patient-Specific Functional Scale (PSFS) is a widely used patient-reported outcome measure that identifies patient-chosen activities that are affected by the targeted disease. The PSFS quantitatively measures functional limitations and recovery. However, it also provides qualitative content regarding what functional deficits are important to patients. Therefore, analyzing the content of the activities chosen by snake envenomation patients enhances our understanding of which daily activities are most important for this population. Methods: We performed a post-hoc analysis of two multi-center, prospective studies, conducted at 22 clinical sites across the southeastern United States. All patients in both studies were enrolled in the emergency department with known copperhead envenomation, were followed through their initial hospital encounter, and had outpatient assessments at 3, 7, 14, 21, and 28 days after envenomation. The PSFS is a verbally administered three-item instrument. The patient is asked to identify “three important activities that you are unable to do or are having difficulty with as a result of your snakebite.” The patient rates each item on an 11-point ordinal scale ranging from 0 (“unable to perform activity”) to 10 (“able to perform activity at the same level as before the injury or problem”). We employed a combination of qualitative content analysis and natural language processing to derive categories from the activities chosen by the patients when responding to the PSFS.

Results: Our sample was composed of 86 patients. The average age was 43.0 (SD 17.6) years, and the participants were mostly white (87%). Slightly more male patients (52%) participated in the study. Most patients had lower extremity injuries (62%). A total of 133 unique activities were chosen by the snake envenomation patients when completing the PSFS. The two most commonly cited domains were daily activities (36%, e.g., carrying items, climbing stairs, providing self-care) and sports/exercise (30%, e.g., running, swimming, collective sports). The body functionality domain comprised 14% of the activities chosen by patients (e.g., flexing the affected hand, moving a finger). Although work-related activities represented 5% of all activities, these had a higher average score (Mean 5.1, SD 4.0), followed by "dress/clothing" (Mean 4.5, SD 3.2). Discussion: Our results showed that the main concerns of snake envenomation patients are related to the ability to perform regular daily activities and to engage in sports and exercise. The variety of activities chosen highlight the multi-faceted impact of snake envenomation. Key Words: Copperhead, envenomation, Patient-Specific Functional Scale CT11 - A CASE OF SEVERE SYSTEMIC COPPERHEAD ENVENOMATION WITH DELAYED DIAGNOSIS Kelly, PE1, Gerardo, CJ1,2 1Division of Emergency Medicine, Duke University School of Medicine, Durham, NC, US; 2 Duke Global Health Institute, Duke University, Durham, NC, US E-mail address: patrick.kelly@duke.edu Background: Copperhead snake envenomation does not commonly present with systemic venom effects. This case highlights a severe copperhead envenomation that was initially missed due to the focus on the envenomating species and the lack of initial tissue symptoms, resulting in delayed treatment. Case Report: A 72-year-old man with chronic hypertension presented to the Emergency Department (ED) within 30 minutes of verified copperhead snakebite to his right ankle. On initial evaluation there were only puncture wounds without any signs of local tissue injury, and it was felt to be a dry bite. The patient also reported chest tightness associated with shortness of breath and abdominal discomfort. During his initial observation, the patient had nausea with multiple episodes of vomiting and diarrhea. Approximately 3 ½ hours after the bite, he reported light-headedness and subsequently had a syncopal episode. He was noted to have sinus bradycardia (heart rate = 42 bpm), hypotension (BP = 74/52mmHg), and tachypnea (respiratory rate = 29 bpm). One liter of crystalloid fluid bolus and atropine 0.5mg were administered. Vital signs improved with treatment, but he remained relatively hypotensive (SBP = 90-110/DBP = 60-75 mmHg) compared to his normal blood pressure (SBP = 145-170/DBP = 70-85 mmHg). An EKG showed <1 mm ST segment elevation in anterior and precordial leads. Serial cardiac biomarkers were normal. Initial laboratory evaluation revealed normal platelets, fibrinogen, and coagulation studies. His creatinine

was 1.4 mg/dL with a baseline of 1.2 mg/dL. After 9 hours, he was noted to have progressive local tissue edema, ecchymosis, pain and tenderness. His envenomation syndrome was recognized and crotaline polyvalent immune fab antivenom treatment initiated. The initial 6 vials of antivenom halted progression of his tissue injury and resolved his systemic symptoms. He developed worsening renal function despite resolution of hypotension and his highest creatinine was 2.0 mg/dL. He received a total of 15 vials of antivenom. His edema and tenderness extended to just below his knee at its worst. His worst Snakebite Severity Score was 11. After stabilization and observation, he was discharged with improving edema and a down-trending creatinine. His new baseline appears to be 1.3-1.5 mg/dL with stage 3 chronic kidney disease. Discussion: Snakebite from a copperhead is associated with a lower likelihood of a severe systemic envenomation compared to rattlesnake bites. However, severe systemic symptoms occasionally occur, and clinicians should be aware of the signs of symptoms. Additionally, venom effects are heterogenous and can involve tissue, hemotoxic, or systemic domains. Severity does not strongly correlate between domains, so a patient may have severe systemic envenomation in the absence of severe local tissue injury or hemotoxic venom effects. In this case, the lack of tissue injury and hemotoxic effects did not preclude the possibility of systemic venom effects. Finally, delays in treatment are associated with worse outcomes. Untreated systemic symptoms in this case likely contributed to this patient’s clinical course. Conclusion: In this case of copperhead envenomation, the lack of recognition of severe systemic venom effects resulted in delayed treatment. Key Words: snakebite, severe, copperhead, Agkistrodon CT12 - FORENSIC TOXINOLOGY AND SNAKEBITE DEATH Keyler, DE1 1Department of Experimental & Clinical Pharmacology, University of Minnesota, Minneapolis, Minnesota E-mail address: keyle001@umn.edu Background: Consultation regarding venomous snakes and snakebite can challenge toxinologists in unanticipated ways. Typically, venomous snakebite case consultation involves communication with emergency medical personnel. However, toxinology consultation may be sought for medico-legal reasons. Forensic investigation of snakebite fatalities requires an extension in the scope of toxicological knowledge, available laboratory technologies, and related limitations with respect to interpretation as to the cause of death. Based on these factors a suspected snakebite fatality may be questionable. Case: The author was called by the Chief Investigator, Parish Coroner’s Office, Louisiana regarding 36 year old male who was reported missing during a hot humid day. The victim

left home on his bike with his rifle to go turkey hunting in the bayou. He was found dead approximately 36 hrs later lying supine in the brush 50 feet from his bike. He appeared to have multiple injuries to the face, neck, and upper torso, and there was early maggot colonization. Sherriff’s deputies found two rattlesnakes within 100 feet of the body, which were killed. The Medical Examiner wanted to determine if the cause of death was due to snake envenomationand if excised tissue from what was believed to be the bite site could be assayed for the presence of snake venom to confirm the cause of death, and could one of the killed rattlesnakes be identified asresponsible for envenomation? Results: Postmortem external examination showed the victim’s left neck had a large bruised and ecchymotic area up through the left ear, and blood covered the neck, forehead, and scalp. The face, back of the neck and both arms had multiple-directional linear lacerations, and countless maggots were evident. The killed rattlesnakes were identified as Timber/Canebrake Rattlesnakes (Crotalus horridus) approximately 1 m in length. Discussion: The complexities as to cause of death in this case revolve around the facts that the victim had been dead in a hot humid environment for over 24 hrs, there were multiple puncture wounds and scratches on the body, and lack of analytical confirmation of snake venom presence. Conclusion: Following careful evaluation of the puncture wounds that were believed to be the bite site, and multiple other punctures, excessive blood on the head and neck regions, and appearance of general trauma, the death could not be confirmed to have resulted from a venomous bite by Crotalus horridus, or be linked with either of the snakes found and killed at the scene. Additionally, the puncture wounds in the excised tissue appeared to be irregular and large, and there was no ecchymotic appearance to the tissue observed, which would have been expected with a Crotalus horridus bite wound. The inability to have assayed the excised tissue for the presence of venom components was a limitation. Key Words: death, snakebite, forensic, toxinology. CT13 - COMPARTMENT PRESSURES REQUIRING FASCIOTOMY Lund, J1, Peredy, T2,3, Aleguas, A3 1Department of Pharmacy, Sarasota Memorial Healthcare System, Sarasota, FL., USA; 2 Global Physician’s Network, Sarasota, FL, USA;3 Florida Poison Information Center – Tampa, Tampa, FL., USA E-mail address: aaleguas@tgh.org Background: Approximately 4,000 crotaline envenomations were reported to the American Association of Poison Control Centers (AAPCC) in 2018(1). Roughly one quarter of these envenomations are attributed to rattlesnakes, with about 100 patients suffering a ‘major’ outcome. Rattlesnake envenomations rarely require surgical

intervention. Surgical expert consensus recommendations recently published recommend treating rattlesnake envenomation with appropriate doses of antivenin, and to consider surgical intervention only after assessment of compartment pressures (2). Case Report: A 39-year-old male was bitten on the anterolateral aspect of the left lower extremity (LLE) by an unwitnessed snake. The patient reported immediate debilitating pain. Unable to bear weight, he crawled out of the brushy area where the bite occurred. He was driven to the nearest hospital and received 12 vials (entire stock) of Crofab® [Crotalidae Polyvalent Immune Fab (ovine)]. The patient was transferred to a tertiary referral facility approximately 10 hours after the bite. On arrival, a single puncture wound was evident, with significant swelling. A Crotalus adamanteus envenomation was suspected given the size of the puncture and the local epidemiology of Crotalidae. Initial assessment of the patient’s vital signs were within normal limits. Lab studies included normal coagulation values except for an INR of 1.14 and a fibrinogen of 190 mg/dL. The patient had a Snakebite Severity Score of 3: local wound (2), central nervous system (1). The patient was provided additional doses of CroFab in consultation with the regional Poison Control Center. The following day, a surgical consultation was obtained for on-going swelling and pain unrelieved by antivenin. In the surgical suite, four compartment pressures of the LLE were measured at 40–63 psi. A four-compartment fasciotomy was performed. Two days postoperatively, the LLE was reexamined in the surgical suite and found to have myonecrosis of the tibialis anterior, peroneus longus and brevis muscles. The patient was ultimately discharged home 21 days after envenomation. Discussion: We report a case of a patient requiring fasciotomy for elevated compartment pressures of the LLE following rattlesnake envenomation despite receiving antivenin doses in concordance with PCC recommendations. The patient was likely envenomated directly into the subfascial compartment by an adult C. adamanteus, resulting in pressure myonecrosis extending to three muscle groups. The delay in administration of additional antivenin after initial therapy may have contributed to increased tissue damage and worse outcome. Conclusion: Patients experiencing rattlesnake envenomation rarely require surgical intervention despite elevated compartment pressures. Adequate and timely administration of antivenin and/or expedited transfer to facilities with sufficient stock may obviate the need for fasciotomy. Rare occurrences of intra-fascial envenomation do occur, and it is critical that compartment pressures be measured as a guide to surgical intervention. References: 1. Gummin DD, Mowry JB, Spyker DA, et al. 2018 Annual Report of the American

Association of Poison Control Centers’ National Poison Data System (NPDS): 36th Annual Report. Clin Toxicol 2018:57: 1220-1413.

2. Toschlog EA, Bauer CR, Hall EL, et al. Surgical Considerations in the management of pit viper envenomation. J Am Coll Surg. 2013 Oct;217(4):726-35.

Key Words: Crotalus adamanteus, Fasciotomy

CT14 - UNPREDICTED LATE-, NEW-ONSET THROMBOCYTOPENIA AND HYPOFIBRINOGENEMIA IN FAB ANTIVENOM-TREATED RATTLESNAKE ENVENOMATION Seifert SA1,2, Mascarenas D3, Fullerton L3, Smolinske SC2

1 University of New Mexico Health Sciences Center, Department of Emergency Medicine, 2

New Mexico Poison Center, University of New Mexico Health Sciences Center, 3 Division of Pediatric Emergency Medicine, Department of Emergency Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, USA E-mail address: sseifert@salud.unm.edu Background: At least one case of a Fab-treated rattlesnake envenomation has been reported in which the patient had no predictors of late-, new-onset hematologic effects but who developed late-, new-onset thrombocytopenia of 50 x 109/L. We report two additional cases of late-, new-onset thrombocytopenia, and one case of late-, new-onset hypofibrinogenemia, in Fab antivenom (AV) treated rattlesnake envenomated patients that were unpredicted by current screening methods. Case Reports: Case 1. A 59 year-old male was bitten in the finger by a rattlesnake and treated with ten vials of Fab AV for initial control (six vials, then four vials) and standard maintenance doses (two vials every six hours for three additional doses). Good local control was achieved. No acute hematological abnormalities were noted. There was a 9% increase in platelet count from the pre- to the post-antivenom period. Platelets remained normal throughout hospitalization. The platelet count was noted to be mildly decreased on follow up on Day 6. There were no bleeding complications (see Table 1). Case 2. A 14 year-old male was bitten on the calf by a rattlesnake and treated with six vials of Fab AV for initial control and standard maintenance doses. Good local control was achieved. No acute hematological abnormalities were noted. There was no increase in platelet count from the pre- to the post-antivenom period and platelets remained normal throughout hospitalization. He returned on day 7 with a platelet count of 71,000. Platelets increased to greater than 100 x 109/L on day 10. There were no bleeding complications (see table 1). Case 3. A 35 year-old female was bitten on the finger by a rattlesnake and treated with at least six vials of Fab AV as a loading dose, with no further information on antivenom dosing available. All labs, including a 5.5-hour D-dimer, were normal throughout hospitalization. The patient returned on Day 7 with a fibrinogen of < 100 mg/dL and an INR of 1.4. The abnormalities spontaneously resolved by day 10. There were no bleeding complications (see Table 2).

Discussion/Conclusions: FabAV-treated, rattlesnake-envenomated patients may develop late-, new-onset hematological abnormalities. To identify at-risk patients, predictive tools were developed, including normal values throughout the early phase of envenomation, and a post-FabAV increase in platelet counts > 20% and/or an elevated D-dimer as predictors of late-, new-onset thrombocytopenia and hypofibrinogenemia, respectively. These cases demonstrate that current predictors have a less than 100% sensitivity and that the absence of indicators has less than a 100% NPV. All Fab-treated, Crotalinae-envenomated patients should be followed with lab for at least one week. Table 1. Platelet Counts

Patient age sex Pre-Fab Post-Fab

Hospital Discharge

Day 4

Day 5

Day 6

Day 7

Day 8

Day 9

Day 10

59 M 220 239 179 200 183 132 138 14 M 303 226 191 71 77 157

Platelet range: 160 – 370 x 109/L Table 2. Fibrinogen (Fib), D-dimer, INR

Fibrinogen (Fib) range: 187 – 446 mg/dL; D-Dimer < 0.5 Key Words: envenomation, rattlesnake, Fab, antivenom, recurrence, platelets, fibrinogen CT15 - SNAKE, SPIDER, SCORPION, INSECT, AND MARINE EXPOSURES AND ENVENOMATIONS IN PREGNANCY IN THE NATIONAL POISON DATA SYSTEM, 2009 - 2018 Ramirez MP1, Smolinske SC2, Warrick BJ2,3, Seifert, SA2,3 1 University of New Mexico School of Medicine, 2 New Mexico Poison Center, University of New Mexico Health Sciences Center, 3 Department of Emergency Medicine, University of New Mexico Health Sciences Center, Albuquerque, NM, USA Email Address: MPRamir39@salud.unm.edu Background: Envenomations during pregnancy have unique considerations regarding management and both maternal and fetal outcomes. We reviewed venomous animal exposures during pregnancy, reported to U.S. poison centers. Methods: The National Poison Data System (NPDS) of the American Association of Poison Control Centers (AAPCC) was searched for cases of envenomation during pregnancy between January 1, 2009 and December 31, 2018 and compared with non-

Patient age sex

Initial Fib

5.5-hour D-dimer

Hospital Discharge Fib

Day 3 Fib / INR

Day 7 Fib / INR

Day 10 Fib / INR

35 F 295 0.27 276 369 / 0.9 < 100 / 1.4 147/1.0

pregnant patient exposures. Odds ratios and descriptive statistics are used where appropriate. Results: There were a total of 3,555 venomous animal exposures in pregnant women (356 per year). Cases by animal type are in Table 1. Antivenom use in selected species is in Table 2. Among treatments, antihistamines were less likely to be used in pregnant patients with bee, wasp, or hornet (12% v. 28%; OR 0.3612; 95%CI (0.2602-0.5012); P<.0001) and scorpion (1.8% v. 4.5%; OR 0.3896; 95%CI 0.282-0.5367; P<.0001) envenomations. Maternal outcomes for all venomous animal exposures in pregnancy are in Table 3. There were three fetal deaths, all in snakebite. There were no maternal deaths and no significant differences in maternal outcomes by gestational age. Discussion/Conclusions: Exposures to and envenomation from venomous animals in pregnancy occur hundreds of times per year in the U.S., most commonly with scorpions, but the most serious outcomes occur in snake envenomation. Pregnant patients have differences in managements and outcomes. Database limitations include the lack of data regarding ultimate pregnancy outcomes in most cases. Table 1. Venomous animal exposures in pregnancy by animal type. Type of Envenomation N

per year HYMENOPTERA Ant or Fire Ant Bites 4 Bee, Wasp, or Hornet Stings 30 TOTAL Hymenoptera cases 33 SPIDERS Black Widow Spider 15 Brown Recluse Spider 9 TOTAL Spider cases 66 SNAKES Copperhead 7 Coral snake 0.3 Cottonmouth 1 Exotic Snakes: Venomous 0.1 Rattlesnake 2 Unknown Crotalinae 3 TOTAL Snake cases 19 SCORPION TOTAL Scorpion cases 210

CATERPILLARS TOTAL caterpillar cases 10 CENTIPEDES/MILLIPEDES TOTAL Centipede or Millipede cases 13 MARINE Fish Stings 2 Jellyfish and Other Coelenterate Stings 3 TOTAL Marine cases 5 TOTAL cases 356

Table 2. Antivenom treatment by selected animal type, pregnant v. non-pregnant patients.

Pregnant Non-pregnant OR

(95%CI) N (%) N (%) Rattlesnake Total 20 9911 + AV 17 (85) 6160 (62) 3.4506

(1.0105-11.7825) - AV 3 (15) 3751 (38) Copperhead Total 69 17431 + AV 20 (29) 7010 (40) NS

- AV 49 (71) 10421 (60) Black Widow Spider Total 145 17179

+ AV 7 (5) 378 (2) 2.2546

(1.0479-4.8508) - AV 138 (95) 16801 (98) + AV = Antivenom given; - AV = No antivenom given NS = Non-significant difference Table 3. Maternal outcomes, all envenomations, pregnant v. non-pregnant patients.

Pregnant N (%)

Non-Pregnant N (%)

OR (95%CI)

Outcomes 3555 454549

No effect 20 (0.6) 4318 (1) 0.5899 (0.3797-0.9165)

Minor effect 1772 (49.9) 176892 (38.9) 1.56 (1.4603-1.6664)

Moderate effect 220 (6.2) 53408 (11.8) 0.4955 (0.4322-0.5681)

Major effect 5 (0.1) 2879 (0.6) 0.221 (0.0918-0.5316)

Key Words: envenomation, pregnancy, antivenom, fetal outcomes CT16 - VINEGAROON EXPOSURES REPORTED TO A U.S. POISON CENTER Smolinske, SC, Warrick, BJ, Seifert, SA New Mexico Poison & Drug Information Center, Albuquerque, New Mexico E-mail address: ssmolinske@salud.unm.edu Background: Vinegaroons (Mastigoproctus giganteus), also known as whip scorpions, are arachnids indigenous to the southwestern United States, parts of Mexico, and southern Florida. They do not bite, but have pedipalps that can pinch. They are best known for having pygidial gland secretions containing 83% acetic acid, which are sprayed upon potential predators. There are no published descriptions of injury to human related to vinegaroon exposures. Our primary aim was to characterize types of exposures and clinical effects reported to a U.S. poison center that serves an area indigenous to this animal. Methods: The database from the New Mexico Poison Center was searched for all cases from 1998 to 2019 regarding human exposures to the vinegaroon. Data captured included age, sex, exposure route, type and duration of symptoms, and part of the body affected. Results: There were 47 exposures documented, with age range from 5 months to 54 years. Females represented 30 cases, males 16, and one unknown. Bites were more commonly described (33 cases), with 11 cases reported dermal exposure to secretions, 3 ocular exposures, and 2 ingestions. Location of injury was upper extremities in 16 cases, lower in 11 cases, and torso in 3 cases. Symptoms were present in 41 cases and included pain in 16, dermal erythema in 11, numbness or tingling in 8, itching in 5, and swelling in 4 cases. Ocular exposure caused pain in all 3 cases, with blurred vision in one case; effects lasted 1 hour, 17 hours, and more than 5 days. Eight non-ocular exposures were followed to outcome, with duration of effects ranging from less than one hour to more than 2 days. Discussion/Conclusion: In a large series of vinegaroon exposures, females predominated, with most exposures occurring from skin contact with secretions. The most common symptoms were pain, erythema, numbness, itching, and swelling, which resolved in less than two days. Ocular exposures were associated with more symptoms

and longer duration of effects. Key Words: vinegaroon, whip scorpion CT17 - THE VALIDITY, RELIABILITY AND MINIMAL CLINICALLY IMPORTANT DIFFERENCE OF THE SF-36 PHYSICAL FUNCTION DIMENSION AND THE PROMIS-10 GLOBAL HEALTH ASSESSMENT IN SNAKE ENVENOMATION Fernandes, NC1, Vissoci, JRN2,3, Lavonas EJ4, Gerardo, CJ2, 3

1 Departamento de Educação Física, Universidade Estadual de Maringá, Maringá, Paraná, Brazil; 2 Division of Emergency Medicine, Department of Surgery, Duke University School of Medicine, Durham, NC, US.; 3 Duke Global Health Institute, Duke University, Durham, NC, US; 4 Department of Emergency Medicine, Denver Health Authority, Denver, Colorado, US. E-mail address: jnv4@duke.edu Background: Valid, reliable, and clinically relevant outcome measures are necessary in snake envenomation clinical studies. The aim of this study was to evaluate the psychometric (validity and reliability) properties of two questionnaires targeting overall health assessment [Short Form 36 Health Survey: Physical Functioning domain (SF-36: PF) and the Patient Reported Outcomes Measurement Information Systems: Physical Functioning 10 questions (PROMIS-10)] in a snake envenomation patient population. Methods: We performed a secondary analysis of data originating from two existing prospective snakebite clinical studies that measured quality of life and functional assessments using the SF-36: PF and the PROMIS-10. Data were collected at 3, 7, 14, and 21 days. We assessed reliability by determining the intraclass correlation coefficient (ICC) for temporal stability at 14 and 21 days. Validity was assessed by the association with correlated constructs (concurrent and criterion validity) for both questionnaires. Concurrent validity was measured by correlation between the patient-specific functional scale and the global impression of change. Criterion validity was assessed by determining the minimal clinically important difference (MCID). We determined the MCID using two methods: (1) the distribution of stable patients according to both standard error of measurement (SEM) and responsiveness techniques, and (2) anchor-based methods to compare between individuals and also to detect discriminant ability of a positive change with a receiver operator characteristic (ROC) curve and optimal cutoff point. Results: A total of 86 patients were evaluated in this study. The mean SF-36: PF scores were 5.4 (SD 3.2), 8.0 (SD 2.2), and 9.1 (SD 1.4) at 3, 7, and 10 days, respectively. The mean PROMIS-10 score was 43.3 (SD 9.7), 49.9 (SD 9.7), and 54.4 (SD 8.7) at 3, 7, and 10 days, respectively. The SF-36: PF had negligible floor effect (maximum of 2.4% at 7 days); however, a ceiling effect was observed at 21 days (43.9%). For the PROMIS-10, also had negligible floor effect (maximum of 1.4% at 7 days); with, a ceiling effect at 21 days

(50%). Both instruments showed good reliability with an ICC of SF-36: PF = 0.63 (95% CI 0.40, 0.78) and PROMIS 10 = 0.68 (95% CI 0.54; 0.79). Both scales showed a strong positive correlation with measures of quality of life and functionality in patients (PSFS), (R = 0.68; 0.59 with SF-36 and R = 0.79; 0.73 with PROMIS-10) at 7 and 14 days respectively. The MCID for the SF-36: PF was 6.0 and for the PROMIS-10 was considered 4.0. Conclusions: The SF-36 PF and the PROMIS 10 are valid and reliable tools to assess quality of life in patients with snake envenomation. Further analysis on their factor structure and item parameters is needed to confirm its psychometric evidence in a snake envenomation population. Keywords: Validation; Psychometric Properties; Quality of life; Snakebite; Venoms CT18 - BITES FROM NON-FRONT-FANGED SNAKES: MEDICAL SIGNIFICANCE, TROUBLESOME TERMINOLOGY AND TAXING TAXONOMY Weinstein, SA Toxinology Department, Women’s and Children’s Hospital, North Adelaide, South Australia 5006 E-mail address: herptoxmed@msn.com Background: Non-front-fanged snakes (NFFC; formerly and artificially taxonomically assembled as "colubrids") comprise about 70% of extant snake species and include several taxa now known to cause lethal or life-threatening envenoming in humans. Although the medical risks of bites by only a handful of species have been well-documented, a growing number of NFFC are implicated in medically significant bites. The majority of these snakes have venoms or other oral products with unknown biomedical properties and their potential for causing harm in humans is unknown. Increasingly, multiple NFFC species are entering the commercial snake trade posing an uncertain risk. In addition, the adjective “venomous” is often incorrectly applied to medically significant bites from some species because “venom” is a biologically, not clinically, based term. Recent debates about what defines ‘venom’ and ‘venomousness’, as well as the turbulent fluidity of taxonomic reassignments in some taxa add further dimensions to analysis of bites by non-front-fanged snakes. Methods: Published case reports describing NFFC bites were assessed for evidence-based value, clinical detail and verified species identification. These data were subjected to meta-analysis and a hazard index was generated for select taxa. Patients personally treated or consulted cases were included and subjected to the same assessment criteria. Cases involving approximately 130 species met the selection criteria, and a small subset designated Hazard Level 1 (most hazardous), contained 6 species with lethal potential. Discussion/Conclusion: Recommended management of these cases includes antivenom for 4 species, Dispholidus typus, Rhabdophis tiginis, R. subminiatus, R. (Balanophis)

ceylonensis; others in this subset without commercially available antivenoms (Thelotornis spp.) are treated with plasma/erythrocyte replacement therapy and supportive care; use of these interventions is decided case-by-case. Heparin, antifibrinolytics and/or plasmapheresis/exchange transfusion have been used in the management of some Hazard Level 1 envenomings, but evidence- based analysis positively contraindicates their use. Hazard Level 2/3 species were involved in cases containing mixed quality data that implicated these taxa (e.g. Boiga irregularis, Philodryas olfersii, Malpolon monspessulanus) with bites that caused rare systemic effects. Recommended management may include use of acetylcholinesterase inhibitors (e.g. neostigmine), airway protection and wound care on a case-by-case basis. Hazard level 3 species comprised a larger group capable of producing significant local effects only, often associated with a protracted bite (eg Heterodon nasicus, Borikenophis (Alsophis) portoricensis, Leptodeira frenata, Hydrodynastes gigas). Management is restricted to wound care. Bites by Hazard level 4 species comprised the majority of surveyed taxa and these showed only minor effects of no clinical importance. This ongoing study has produced a comprehensive evidence-based listing of NFFC snakes tabulated against medical significance of bites, together with best-practice management recommendations. This analysis assumes increasing importance, as there is growing exposure to lesser-known NFFC snakes, particularly in captive collections that may uncover further species of significance in the future. Careful and accurate documentation of bites by verified species of NFFC snakes is required to increase the evidence base and establish the best medical management approach for each species. Key Words: Non-front-fanged snakes, medical significance, low-pressure, high-pressure, antivenom DRUG DISCOVERY & DEVELOPMENT DDD1 - ANTIMICROBIAL ACTIVITY OF CATIONIC PEPTIDE VC15 OBTAINED FROM THE PROTEOLYSIS OF PIN2 González, Z1, Adame, M1, Cuevas, E*1, Corzo, G2, Villegas, E1 1Centro de investigación en Biotecnología, Universidad Autónoma del Estado de Morelos; 2 Instituto de Biotecnología, Universidad Nacional Autónoma de México. E-mail address: elbav@uaem.mx Background: Bacterial resistant against conventional antibiotics has been increasing in the last two decades, this problem led to the research of new antimicrobial molecules with antibiotic potential (WHO, 2018). The venoms from many scorpions have been studied object in the last years due to their importance to public health and the presence of different bioactive molecules like different toxins and antimicrobial molecules. It’s been showed that antimicrobial peptides are potential molecules to their clinical use against bacterial strains with drug resistance. Pin2 it’s an antimicrobial peptide with broad-

spectrum isolated from the Pandinus imperator venom (Corzo et al., 2001) however, Pin2 showed 55% of hemolytic activity against human erythrocytes and susceptibility to bacterial proteases was also observed, which limits their therapeutic potential (Carmona et al., 2013). In this work, we test one short fragment of 15 amino acid (VC15) the sequence was obtained from the proteolysis of Pin2 by human elastase to see if this fragment was able to preserve the activity of the parental peptide. Methods: The antimicrobial peptide VC15 was obtained using a solid phase synthesis using the Fmoc strategy (Fluorenyl-9-methoxycarbonyl) manually in the Department of Molecular Medicine and Bioprocesses of the Institute of Biotechnology-UNAM. The purification of the peptide was carried out using a reverse-phase high-performance liquid chromatography (RP-HPLC) and tested against Escherichia coli (ATCC 25922) and Streptococcus agalactiae (ATCC 25465) using the lineaments of the Clinical Laboratory Standards Institute (CLSI) in microdilution tests and agar diffusion. Results: VC15 showed activity against the two strains, with a minimum inhibition concentration (MIC) of 6.25 µM. The best activity was observed against S. agalactiae in which any concentration tested showed a bactericide effect. A bacteriostatic effect was observed against E. coli during 6 hours with 6.25 and 12.5 µM concentrations, however, the bacteriostatic effect was prolonged to 10 hours with 25 and 50 µM which indicates that the antimicrobial peptide VC15 has a concentration depended effect. Discussion/Conclusion: The fragment VC15 preserved the activity of the parental peptide Pin2 being more toxic against S. agalactiae and less toxic against E. coli with a MIC of 6.25 µM. Hemolytic activity and proteolytic susceptibility tests are necessary to be able to define the antibiotic potential of peptide VC15. Key Words: Antimicrobials peptides, bacterial resistance, Pin2. DDD2 - A SPIDER-VENOM PEPTIDE FOR PREVENTING ISCHEMIC INJURIES OF THJE HEART: APPLICATION TO HEART TRANSPLANT AND MYOCARDIAL INFARCTION Redd, MA1, Scheuer, SE2,3, Saez, NJ1, Macdonald, PS2,3, Palpant, NJ1, King, GF1 1 Institute for Molecular Bioscience, The University of Queensland, Brisbane QLD, Australia; 2 Victor Chang Cardiac Research Institute, Sydney NSW, Australia; 3

Cardiopulmonary Transplant Unit, St. Vincent’s Hospital, Sydney NSW, Australia E-mail address: glenn.king@imb.uq.edu.au Background: Conditions caused by obstruction of blood flow to the heart, such as myocardial infarction (MI), are the most common emergency manifestation of cardiovascular disease. Although acute reperfusion therapies have improved patient outcomes, mortality remains high and MI is one of the largest attributable risks for heart

failure (HF). Globally, 1 in 5 people develop HF, with annual healthcare costs of $108B. Heart transplantation remains the most effective treatment option for HF, but 75% of potential donor hearts are discarded, many due to sensitivity of the donor heart to ischemic injury. Myocardial sensitivity to ischemia-reperfusion injury (IRI) therefore remains a primary point of vulnerability underlying cardiovascular disease, which is the leading cause of morbidity and mortality worldwide. Despite decades of preclinical therapeutic development, there are currently no drugs that block the acute injury response to cardiac ischemia. Methods: During cardiac ischemia, the build-up of acidic metabolites results in decreased intracellular and extracellular pH that can reach as low as 6.0–6.5. Severe acidosis exacerbates the ischemic injury and significantly impacts cardiac function. The proton-gated acid-sensing ion channel 1a (ASIC1a) is known to mediate injury responses during cerebral ischemia, but little is known about its role during cardiac ischemia-reperfusion injury (IRI). We show that genetic ablation of ASIC1a leads to improved functional recovery following global myocardial IRI in ex vivo mouse hearts, and that this effect can be recapitulated by therapeutic blockade of ASIC1a using Hi1a, a potent and specific inhibitors of Hi1a derived from venom of the Australian funnel-web spider. Hi1a yields improved post-IRI cardiac viability and function in multiple whole organ injury models including rodent ex vivo Langendorff IRI and donor heart procurement and storage models. Using human pluripotent stem cells, we show that Hi1a improves cardiomyocyte viability under conditions of acidosis or ischemia in vitro. Consistent with a key role for ASIC1a in human cardiac ischemia, we used summary data from GWAS to show that polymorphisms in the ASIC1 genetic locus are strongly associated with myocardial infarction. Discussion/Conclusion: Collectively, our data provide compelling evidence that ASIC1a is a key target for cardioprotective drugs to reduce the burden of disease associated with myocardial ischemia, and that Hi1a is an exciting lead compound for treatment of cardiac IRI. Key Words: venom peptide, ASIC1a, cardiac ischemia, myocardial infarction, heart transplant DDD3 - RATTLESNAKE VENOM COMPONENTS IN THE CONTROL OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS: IMMUNOMODULATORY PROPERTIES POTENTIATED BY SILICA SBA-15 Sant’Anna, MB1, Giardini, AC1; Lopes, FS1, Kimura, LF1, Chacur, M2; Pagano, RL3; Zambelli, VO1; Sant’Anna, OA4, Picolo, G1*. 1Laboratory of Pain and Signaling, Butantan Institute, São Paulo, Brazil, 2Laboratory of Functional Neuroanatomy of Pain, Institute of Biomedical Sciences , University of São Paulo, São Paulo, Brazil; 3Neuromodulation Laboratory and Experimental Pain, Sírio-Libanês Hospital, São Paulo, Brazil, 4Laboratory of Immunochemistry, Butantan Institute, São Paulo, Brazil.

*E-mail address: gisele.picolo@butantan.gov.br Background: Multiple sclerosis (MS) is an inflammatory, autoimmune and demyelinating disease of the CNS, which causes sensory and motor impairments. MS has no cure, and the available therapies are only able to delay the progression of the disease. Animal venoms and toxins have being considered a rich source for the search of therapeutic drug candidates, since these components in general can exhibit selectivity and affinity for a wide variety of targets in mammalian systems. In this meaning, studies using compounds isolated from crotalic venoms, particularly crotoxin and crotalphine have indicated their analgesic, anti-inflammatory, immunomodulatory and anti-tumoral properties. Then, the effect of both components was investigated in the experimental autoimmune encephalomyelitis (EAE) model, an animal model of multiple sclerosis. Methods: EAE was induced by immunization of female C57BL/6 mice with MOG35–55 peptide (200μg) and M. tuberculosis (400μg) in incomplete Freund’s adjuvant, followed by pertussis toxin injection (300ng, on days 0 and 2).Pain threshold was determined using an electronic pressure-meter test. Clinical signs were assessed according to scores from 0 to 5.Results showed that, CTX, in a single dose, was effective in controlling pain symptoms but does not interfere with clinical signs. However, in 5 consecutive doses, CTX modulates both mechanical hypernociception and clinical signs (motor impairment) in animals with EAE. Furthermore, treatment with CTX reduced production/release of central pro-inflammatory mediators. These effects were potentiated when CTX was adsorbed in silica SBA-15, an inert nanoestruturated mesoporous silica. In relation to crotalphine (CRO), our results showed that CRO caused partial reversion of EAE-induced hyperalgesia and decreased the severity of the clinical signs of EAE when compared to saline-treated animals.CRO reduced the immunoreactivity of the Egr-1, microglia/macrophages and astrocytes markers induced by EAE. In addition, it decreased CD11b expression and increased IL-6 in spinal cord. Discussion/Conclusion: These results reinforce the immunomodulatory effect of crotoxin and crotalphine, confirming this effect in a model of neurodegenerative disease, where both compounds could act attenuating the neuroinflammation induced by EAE. These results confer to both CTX and CRO characteristics to be used as promising candidates for the control of multiple sclerosis. Financial support: FAPESP (2013/07467-1, 2017/17844-8), CNPQ (467211/2014-0) and CAPES (Finance Code 001). Keywords: Experimental Autoimmune Encephalomyelitis, Crotoxin, Crotalphine, Neuroinflammation, SBA-15 silica. DDD4 - FROM CANDIDATE MOLECULES TO CLINICAL TRIALS: A JOURNEY THROUGH TOXINOLOGY Rui Seabra Ferreira Jr1

1Center for the Study of Venoms and Venomous Animals (CEVAP), São Paulo State University (UNESP), Botucatu, SP, Brazil E-mail address: rui.seabra@unesp.br Background: Venomous animals have always been feared for their he capability to kill, but in recent decades, researchers and pharma companies have begun to be interested in their toxins. These toxins act quickly and effectively on the victim’s body, due to massive repertoire of molecules able to bind to specific targets. However, to bridge the gap between basic and applied sciences, to push forward disease research and therapeutics is fundamental. What categories of drug leads are truly “druggable”, sit in “patented bioproduct space” and can be pushed towards clinical trials? Although animal toxins present excellent candidate molecules because they have high specificity for a cellular receptor without side effects, few drugs are approved for human use. We present two successful translational cases of bioproducts from laboratory bench to the bedside. Case 1: Fibrin sealant derived from snake venom. Considering that infectious diseases could be transmitted via human blood, a new heterologous fibrin sealant (HFS) was proposed, whose components are a serine protease (a thrombin-like enzyme) extracted from South American rattlesnake (Crotalus durissus terrificus) venom and a fibrinogen-rich cryoprecipitate extracted from the blood of Bubalus bubalis buffaloes. This new bioproduct has been used as a coagulant, sealant, adhesive, drug delivery, and recently as a scaffold candidate for bone, nerve and cartilage repair associated with mesenchymal stem cells. Thus we show its pre-clinical applications aiming at accelerating the wound healing process and at repairing nervous system trauma, cartilage and bone regeneration. We have also finished an innovative safety trial phase I/II to treat chronic venous ulcers in 40 humans concluding that the product is safe and clinically promising for this purpose due to its preliminary effectiveness. We have observed a significant improvement in 72.1% of the cases studied in only three months of treatment. Case 2: Africanized honeybee antivenom Bees are economically beneficial insects to humans and have provided several products, such as honey, royal jelly, propolis, beeswax, and pollen. However, they represent a public health problem in the Americas with large colonies of Africanized honey bees causing thousands of accidents per year with dozens of deaths. Due to this problem, we have developed a new antivenom to treat multiple stings. One of the major challenges was the standardization of the product, because of the antigenic characteristics of the venom. The apilic antivenom (AA) was developed based on horse hyper-immunization using only main toxic compounds of the venom. Each milliliter of Fab2-AA neutralizes 1.25 mg of venom and 10 mL of antivenom can treat about 100 stings. A multicenter, non-randomized, open-label phase I/II clinical trial with 20 participants to assess safety, neutralizing ability and confirm the lowest effective dose together a specific drug protocol was designed. Its delivery route is intravenous and the administration follows a strict clinical protocol that aims to rapidly neutralize and eliminate the venom toxic effects. The antivenom was considered safe for clinical application and a phase III clinical study will be proposed to evaluate its effectiveness and to promote its registration in regulatory agencies. Conclusion: Toxinologists have the expertise to develop new drugs through translational research, applying modern biotechnology tools, and trying to solve health problems with bioproducts.

Key words: Bioproducts, venoms, toxins, trials, research and development. DDD5 - PNPP-19, A SYNTHETIC PEPTIDE DESIGNED FROM PHONEUTRIA NIGRIVENTER SPIDER TOXIN IS EFFECTIVE FOR GLAUCOMA TREATMENT IN RATS Silva, CN1, Dourado, LFD1, de Lima, ME2, Silva Cunha, A1

1 Faculdade de Farmácia, Universidade Federal de Minas Gerais, Campus Pampulha, Av. Antônio Carlos, 6627, Belo Horizonte-MG, CEP 31270-901, Brasil; 2 Santa Casa de Belo Horizonte: Ensino e Pesquisa, Belo Horizonte- MG, CEP 30150-221, Brasil. E-mail address: carolinnanunes@yahoo.com.br Background: PnPP-19 is a non-naturally and non-toxic 19-amino-acid synthetic peptide designed from Phoneutria nigriventer spider toxin PnTx2-6. This peptide was described as a novel drug for the treatment of erectile dysfunction by activating the nitrergic system. Nitric oxide (NO) has gained great attention recently, as a potential new target for the treatment of glaucoma. This disease results in visual field loss and irreversible blindness and has a multi-factorial etiology. The most studied risk factor for glaucoma is increased intraocular pressure (IOP). Despite the availability of several anti-glaucoma medications, there is still a medical need in this field. Considering the important role of NO in the control of IOP, this study evaluated the safety and efficacy of PnPP-19 in reducing IOP in healthy and glaucomatous rats. Methods: Glaucoma was induced through injection of hyaluronic acid into the anterior chamber of Wistar rats’ eyes. The efficacy of PnPP-19 in reducing IOP was evaluated using a tonometer. Electroretinograms were recorded before and after administration of different doses of PnPP-19 on the eyes. Nitric oxide levels were determined indirectly by measuring nitrite concentration using Griess test. Histological sections of corneas and retinas were prepared. Results/Discussion: These results suggest PnPP-19 as a NO inducer able to reduce the intraocular pressure in healthy and glaucomatous eyes of rats, protecting them against optic nerve damage, after eye drops topical administration. In addition, PnPP-19 showed no toxicity, was safe for ocular application and has a short polypeptide chain, which eases its synthesis that strongly indicates its viability for use. Conclusion: PnPP-19 may emerge as a promising new drug for the treatment of glaucoma. Key words: PnPP-19; glaucoma treatment; nitric oxide; Phoneutria nigriventer.

DDD6 - INHIBITION OF CANCER CELL MIGRATION BY DISINTEGRINS FROM THE VENOM OF CROTALUS POLYSTICTUS SNAKE López-Márquez, P1, Olvera-Rodríguez, F2, Neri-Castro, E2, Bach, H3, Alagón, A2, Vergara, I1,*

1Department of Chemical and Biological Sciences, Sciences School, Universidad de las Américas Puebla, Cholula, México. 72810; 2 Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México; 3Department of Medicine, Division of Infectious Diseases, University of British Columbia, Vancouver, BC V6H3Z6, Canada. E-mail address: irene.vergara@udlap.mx Background: There are several examples of proteins from animal venoms that have potential therapeutic leads for cancer treatment. Disintegrins can target specific integrins, which are transmembrane receptors that participate in cell adhesion to the extra cellular matrix, cell migration and proliferation within the tissues. Different research groups have studied and demonstrated that integrins play a fundamental role in the survival, proliferation and metastatic process in many types of cancer. Cancer term describes a large group of diseases that can affect any part of the body, produced by the transformation of normal cells into tumor cells in a multi-stage process. Currently, cancer is an upsetting health issue. In 2018, there were registered more than 9.5 million deaths worldwide. In general, treatments for cancer include chemotherapy and radiotherapy; these treatments have saved many lives but they have important side effects and, in some cases, low efficacy. These facts inspire researchers for continue investigating new developments in cancer treatments. The aims of the present study were to isolate, to characterize and test the anti-migration cell activity in-vitro of disintegrins from Crotalus polystictus venom, in order to find possible candidates for the development of anti-metastatic drugs with potential use as cancer treatment, in combination with other therapies. Methods: A venom pool of C. polystictus (200 mg) was fractionated by gel filtration chromatography using Sephacryl S-300 resin. Six fractions were obtained and loaded in a SDS-PAGE. The fractions with a close molecular weight to desintegrins were re-fractionated by RP-HPLC in a C-18 column, and the molecular mass of each component was measured by mass spectrometry. The fractions identified as desintegrins were tested in three different concentrations on THP-1 monocytes to determine their cytotoxicity. The anti-migration cell activity of disintegrins was measured in-vitro using the wound healing assay on a confluent monolayer of cells of kidney (caki-1) and lung (A549) cancer cell line. In order to monitoring the cell migration, we captured images using an inverter microscope at the beginning and at different intervals during 48 h. Discussion/Conclusion: The gel chromatography of the C. polystictus venom yielded six main fractions. Fraction 3 was re-fractionated by RP-HPLC and 11 peptides showed a molecular weight in the range of disintegrins (6-7 kDa) previously reported. The C. polystictus venom showed a high percentage of disintegrins (4%) compared with other snake venoms of the same family. The cytotoxicity of the most abundant disintegrins in the

venom showed that none of them has relevant cytotoxic effects. Two disintegrins showed promising activity as anti-metastatic molecules since they stopped cell migration of kidney and lung cancer cells in vitro.

Key Words: C. polystictus venom, disintegrins, cancer, anti-metastatic DDD7 - BACTERIAL EXPRESSION OF A SNAKE VENOM METALLOPROTEINASE INHIBITOR (SVMPI) FOUND IN THE NORTH AMERICAN OPOSSUM (DIDELPHIS VIRGINIANA): SOLUBILITY ENHANCEMENT USING A MALTOSE BINDING PROTEIN (MBP) AFFINITY TAG Werner, RM Grand Valley State University, Department of Chemistry, 312 Padnos Hall of Science, Allendale, MI 49401 E-mail: wernerro@gvsu.edu Background: Several mammalian species, most notably members of the Didelphidae family (opossums) produce endogenous inhibitors to a variety of snake venom components including metalloproteinases and phospholipases. While the genes of several of these inhibitors have been sequenced, to this point samples of homogenous protein can only be isolated directly from animal serum. Due to the scarcity of pure samples, investigations of the 3D-structure and detailed mechanism of inhibition utilized by these proteins has been hampered.

Methods: We present our efforts to purify, characterize, and crystalize a bacterially expressed snake venom metalloproteinase inhibitor (SVMPI) produced by the N. American opossum (D. virginiana). This protein is homologous to previously isolated SVMPIs found in the serum of D. virginiana (oprin) and several South American opossum species (DM43).

Discussion/Conclusion: When expressed as a fusion with maltose binding protein (MBP), this non-glycosylated protein has good solubility but its inhibitory properties are reduced relative to a C-terminal 6His-oprin. Our progress towards the crystallization and structural characterization of this protein will be presented.

Key Words: Didelphis virginiana, snake venom metalloproteinase inhibitor, SVMPI, maltose binding protein, MBP. EP- EPIDEMIOLOGY EP1 - PUFFERFISH EXPOSURE AND TETRODOTOXICITY IN DOGS IN PERTH, WESTERN AUSTRALIA

Hanson, J1, Sharp, CR1 1School of Veterinary Medicine, College of Science, Health, Engineering, and Education, Murdoch University, Murdoch, Western Australia. E-mail address: c.sharp@murdoch.edu Background: Pufferfish ingestion is anecdotally a common reason for dogs to present to veterinary emergency rooms (ERs) in Western Australia (WA). Despite this, epidemiologic data is sparse. The objective of this study was to describe the epidemiology of pufferfish exposure and tetrodotoxicity in dogs in Perth, WA. We hypothesised that while pufferfish exposure is a common reason for dogs to present the ER, pufferfish poisoning (tetrodotoxicity) is rare, but has a good prognosis with aggressive supportive care. Methods: This was a retrospective case series. Electronic medical records (EMR) at The Animal Hospital at Murdoch University (TAHMU) were searched using search terms including ‘puffer fish’, ‘blow fish’, and ‘tetrodotoxin’. Cases were included if pufferfish ingestion was confirmed. Patient information was recorded using a standardised data collection sheet and subsequently entered into REDCap (Research Electronic Data Capture); a secure, customisable, online database. In order to determine the prevalence of pufferfish ingestion, the total number of all cases seen on an emergency basis at TAHMU was determined. A fee code search was used to identify emergency cases over the same time period as included pufferfish cases. Descriptive statistics were performed. Case Series: 325 dogs had confirmed pufferfish ingestion. The time period over which these cases were seen was from January 1st 2007 (the first date on which the current hospital EMR was in use) to October 2018. Over the same time period, a total of 57,687 emergency cases were seen, such that the prevalence of pufferfish exposure resulting in ER presentation was 0.56%. Median age of affected dogs was 24 months. Common breeds included the Labrador retriever (84/325, 24.8%), mixed breeds (51/325, 15.7%), and the Staffordshire Bull Terrier (30/325, 9.2%). The majority (211/325, 64.9%) remained asymptomatic. Clinical signs were predominately gastrointestinal (63.6% of symptomatic dogs), mental inappropriateness (17.6%), neuromuscular (14.2%), and respiratory (3.4%). Induction of emesis was the most common intervention. 38/325 were hospitalised, while the remainder were treated as outpatients. Seven dogs required mechanical ventilation; all survived to discharge. Overall, 324/325 survived to discharge, with one case being euthanised. Conclusions: This study confirmed that while pufferfish exposure is common, tetrodotoxicity is less common. Nonetheless, the prognosis for recovery from tetrodotoxicity is excellent with supportive care. Key Words: veterinary, tetrodotoxin, epidemiology, hypoventilation

EP2 - GEOGRAPHICAL DISTRIBUTION AND HEALTH CARE DISPARITIES OF SCORPION STINGS IN BRAZIL Wen, FH1, Monteiro, WM2, Scheidt, JF3, Andrade L3,4, Ye, J5, Staton, CA5,6, Gerardo, CJ5,6, Vissoci, JRN.5,6

1 Butantan Institute, São Paulo, São Paulo, Brazil; 2 Tropical Medicine Foundation Doutor Heitor Vieira Dourado, Manaus, Amazonas, Brazil. 3 Medicine Department, State University of Maringá, Maringá, Paraná, Brazil; 4 Postgraduate Program in Health Sciences, Health Sciences Center, State University of Maringá, Maringá, Paraná, Brazil; 5 Division of Emergency Medicine, Department of Surgery, Duke University School of Medicine, Durham, NC, US; 6 Duke Global Health Institute, Duke University, Durham, NC, US. Email address: jnv4@duke.edu Background: Scorpion stings have become an emerging public health concern in Brazil, with a tripling of cases from 2013 to 2017. Deaths also increased by 50% during the same period, mostly impacting the elderly and children. Preventing the morbidity and mortality related to these stings depends on timely access to appropriate care and administration of antivenom. Although Brazil’s national health system provides universal healthcare at no direct cost to patients, socioeconomic inequities still greatly impact outcomes. Our study objectives were 1) to determine the prevalence of venomous scorpion stings in Brazil using a geographic information system (GIS); and 2) to evaluate geospatial associations between mortality, clinical complications, and socioeconomic development. Methods: Using data from 2010 to 2016 in Brazil’s national healthcare databases, we identified areas of high scorpion sting prevalence, mortality and incidence of clinical complications through GIS. Complications were defined as localized to the envenomation site or systemic sequelae of envenomation. These outcomes were spatially associated with geographic location and socioeconomic development as measured by the Human Development Index (HDI). Areas with a higher density of stings and outcomes associated with HDI were identified with Geographically Weighted Regression. Results: The Southern and Northern regions had the highest prevalence of scorpion stings (Moran’s I = 0.375). However, mortality and morbidity were concentrated in the Southeast and the state of Amazonas. Lower access to scorpion antivenom was observed in the North and Northeast regions, as well as the coastal area of the Southeast region. The highest volume of cases with treatment delays (> 3 hours to reach the hospital) were concentrated in the Northern region, mostly in the Amazon forest area, as well as in the interior area of the Northeast region. Delayed access to care was mostly associated with mortality and morbidity in the Northern and Northeast regions. In the Southeast region, lower literacy levels and lower income were associated with mortality and morbidity.

Figure 1. Geographically weighted regression model estimates for the association between accessibility, time and sociodemographic indicators with negative outcomes for scorpion stings in Brazil from 2010 to 2016. Discussion/Conclusion: Despite nationalized healthcare and antivenom production in Brazil, the system faces additional barriers to equitably prevent morbidity and mortality from scorpion stings. In regions with lower socioeconomic development, such as the Northern, delays in timely care are associated with worse outcomes. In highly developed regions, such as the Southeast, social vulnerability is associated with worse outcomes. Developing strategies to raise awareness and access to timely antivenom administration are necessary next steps to improve outcomes from scorpion stings in Brazil. Keywords: Snake envenomation, snake bite, Brazil, geographic information systems HISTORY OF TOXINOLOGY HT1 - ANTIVENOM USE IN VETERINARY MEDICINE A THIRTY-FIVE YEAR PERSONAL JOURNEY Peterson, M Gem Veterinary Clinic, 703 S Washington Ave., Emmett, Idaho, U.S.A. 83617 Email address: Petersonkate@netscape.net Discussion: After graduating from veterinary school in 1983 I moved to Tucson, Arizona in 1985 was employed in an emergency and critical care 24-hour referral practice. The average annual pit viper envenomation case load in this veterinary emergency practice was 55-80 dogs and cats. Standard treatment protocol for rattlesnake envenomation initially was intravenous Fort Dodge (Wyeth) antivenom crotalidae polyvalent® (ACP) as a 10ml injection, intravenous corticosteroids, antibiotics, and intravenous crystalloid infusion. Over

the next year this protocol transitioned to same treatment however corticosteroid use was discontinued and antivenom was administered in the i.v. fluid infusion. In 1987 Fort Dodge provided ACP for a clinical trial in 32 dogs (unpublished). In the late 1980’s and early 1990’s Fort Dodge intermittently placed their antivenom on backorder and for several years the product was not available. This was an untenantable situation for practicing veterinarians experiencing heavy envenomation caseloads and they sought alternative antivenoms. Protherics (now BTG) supplied Crofab® (ovine Fab1 polyvalent antivenom) for a prospective clinical trial in 115 dogs1. Due to the high cost of Crofab® and the limited availability of Fort Dodge’s ACP veterinarians practicing along the southern U.S. border began crossing into Mexico and bringing Instituto Bioclon crotalid antivenom back into the United States for use. Bioclon began developing an interest in marketing their antivenom for veterinary use in the United States and restricted the access of this antivenom from local Mexican border town pharmacies, closing off this route of procurement. Veterinarians then began importing Bioclon’s Anavip® (equine Fab2) antivenom under compassionate use U.S.D.A. import permits. Woods et al published an important canine prospective safety study with this antivenom2. Some veterinarians imported the Costa Rician PolyVet® crotalid antivenom (liquid equine IgG) but found some limitations which generally precluded its use. A crotalid antivenom manufactured in Argentina (VenomVet®) (equine origin IgG) has been licensed for veterinary use which is now commonly used. Mg Biologics has a veterinary licensed modified equine plasma crotalid antivenom product Rattler®. Other equine plasma products have been manufactured but have had issues with reactions and antibody concentration and have not been widely used. Currently Licensed veterinary products include Boehringer-Ingelheim (purchased Fort Dodge) antivenom crotalidae® equine whole IgG antibody, MT Venom Venom Vet® Fab2 equine origin, and Mg Biologics Rattler® Antivenin modified equine plasma (whole IgG). Products also used in veterinary medicine but not licensed for veterinary market include BTG’s Crofab® ovine origin Fab1, and Bioclon Anavip® equine origin Fab2. References: 1. Peterson M, Matz M, Seibold K, Plunket S, Johnson S, Fitzgerald K. A randomized

multicenter trial of crotalidae polyvalent immune fab antivenom for the treatment of rattlesnake envenomation in dogs. J Vet Emerg Crit Care 2011; 21(4):335-345.

2. Woods C, Young D. Clinical safety evaluation of F(ab’)2 antivenom (Crotalus durissus – Bothrops asper) administration in dogs. J Vet Emerg Crit Care 2011;21(5):565-569.

Key Words: antivenom, veterinary, personal LM - LABORATORY METHODS

LM1 - SERUM CHOLESTEROL CONCENTRATION IN DOGS FOLLOWING DABOIA (VIPERA) PALAESTINAE ENVEBOMATION AS A POTENTIAL MARKER OF THE SEVERITY OF THE ENVENOMATION Klainbart S1*, Kelmer E1, Segev G2 Beery-Cohen I, 1 Aroch I2 Departments of Small Animals Emergency and Critical Care,1 and Internal Medicine.2 The Veterinary Teaching Hospital, Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment - Hebrew University of Jerusalem. Israel. E-mail address: Klainbart@gmail.com

Background: Daboia (Vipera) palaesinae is the commonest venomous snake in Israel and neighboring countries, responsible for most envenomations in humans and dogs, with mortality rates of 0.5%-2% and 3.7%-15%, respectively. In humans envenomed by D. palaestinae, and in rabbits injected with its venom, serum cholesterol concentration (sChol) is inversely correlated with the severity of envenomation. This study examined sChol in dogs envenomed by D. palaestinae, and its utility as a marker of the severity and outcome of envenomation. Methods: Data were retrospectively collected from the medical records of dogs proven with D. palaestinae envenomation (years 1989- 2017), whereas sChol was measured at presentation. Results: The study included 193 dogs. The overall mortality rate was 8.8%. sChol was lower (P=0.046) in the non-survivors (n=17; median, 87 mg/dL; range: 78-196) compared to the survivors (n=176; median, 175 mg/dL; range: 109-210). Mean serum albumin concentration was significantly (P=0.004) lower in the non-survivors (16 dogs; 2.7±0.8 g/dL) compared to the survivors (159 dogs; 3.2±0.7 g/dL). sChol was positively correlated with serum albumin concentration (r=0.67; P<0.001). sChol was higher (P=0.011) in dogs with normal mentation at presentation compared with those presenting mentation abnormalities. sChol was lower (P<0.05) in dogs with leukocytosis, hypoproteinaemia or thrombocytopenia compared to those in which these analytes were within reference interval. Discussion/Conclusion: In dogs envenomed by D. palaestinae, sChol at presentation may serve as a marker of the severity and outcome of the envenomation, warranting future studies to investigate sChol concentration changes over time throughout hospitalization and their association with the outcome. Key Words: Albumin, Antivenom, Fresh frozen plasma, Canine, Viperid. LM2 - ECHIS COLORATUS ENVENOMATION IN A DOG, CLINICAL, HEMOSTATIC AND THROMBOELATOMETRIC FINDINGS AND TREATMENT Klainbart S1*, Kelmer E1, Aroch I2, Atamna R3

Departments of Small Animals Emergency and Critical Care,1 Internal Medicine,2 and Surgery.3 The Veterinary Teaching Hospital, Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agriculture, Food and Environment - Hebrew University of Jerusalem. Israel. E-mail address: klainbart@gmail.com Background Echis coloratus is a Viperid, endemic to the Middle East. Clinical reports describing E. coloratus envenomation in humans are scarce, while natural envenomations in animals were not reported. The envenomation can culminate to systemic coagulopathy. This report describes a confirmed E. coloratus envenomation in a dog, with assessment of the global hemostasis by thromboelastometry. Case Report: A 6-year Belgian Shepherd was presented in shock, with oral mucosal bleeding and swelling due to snakebite. Laboratory tests showed prolonged prothrombin-time (PT) and activated partial thromboplastin-time (aPTT). Because Daboia palaestinae is the most common venomous snake in Israel, immunoglobulin-G monovalent D. palaestinae antivenom was administered, with supportive care. The dog improved clinically, was discharged, and was readmitted with active bleeding from the bite site, and the dead snake was identified as E. coloratus. PT, aPTT and fibrinogen concentration were unmeasurable. Thromboelastometry showed severe hypocoagulability.The dog was treated with a polyvalent antivenom against venoms of several Middle Eastern snakes, including E. leucogaster, (3 units; IV), fresh-frozen plasma (FFP; 3 units), and packed red blood cells (1 unit). Bleeding completely ceased, and thromboelastometry results improved. The dog was discharged. Three days later, all hemostatic test results, including thromboelastometry were normal. Discussion/Conclusion: This is the first report of naturally occurring E. coloratus envenomation in dogs, and the first one of thromboelastometry in any such envenomation. Thromboelastometry was useful for monitoring and managing E. coloratus envenomation venom induced coagulopathy, and guided FFP and polyvalent antivenom treatment, along with medical assessment, when the biting snake species is unknown. Key Words: Coagulation, INTEM, EXTEM, polyvalent antivenom LM3 - CROSS-REACTIVITY OF VENOMS AND ANTIVENOMS DETERMINED IN VITRO USING SIZE-EXCLUSION CHROMATOGRAPHY Sanny, C*, Shults, CA Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, Tulsa, OK USA *E-mail address: charles.sanny@okstate.edu Background: Size-exclusion chromatography (SEC) can be used to characterize venom-

antivenom immune complex formation. Complex formation would be expected between antivenom and venom used in the antivenom production process. Binding of antivenom with venom not used in the production process might be predicted due to similarities of venom composition, but would typically be confirmed by standard assays, such as inhibition of venom lethality or neutralization of venom toxicity. Evaluation of complex formation is relevant since venom-antivenom binding is required for neutralization of venom lethality or toxicity, though binding does not guarantee protection. SEC may be a useful tool in comparing the binding of different antivenoms to a particular venom, as well as binding of different venoms to a particular antivenom. The data presented in this study demonstrates the use of SEC to evaluate the cross-reactivity of two distinctly different antivenoms with venoms used in antivenom production (Table 1).

Table 1. Antivenoms and venoms used in cross-reactivity study Antivenom Venom

Ovine Fab (FabAV); Crotalidae Polyvalent Immune Fab (Ovine)

Crotalus atrox (North America); used in FabAV production

Equine F(ab′)2 [F(ab′)2AV]; Bothropic antivenom, Butantan Institute, Brazil

Bothrops jararaca (Brazil); used in F(ab′)2AV production

Methods: Venom, antivenom, and venom-antivenom mixtures were prepared at 4oC in 50 mM sodium phosphate, pH 7.0, containing 0.15 M NaCl (column elution buffer) and incubated for 30 minutes at 37oC. (Samples were stored at 4oC prior to SEC.) Samples (20 PL) were injected into the SEC column (TSKgel G3000SWxl 7.8 mm ID x 30 cm, 5 Pm, TOSOH Bioscience) at a constant flow rate of 1 mL/min. Elution profiles were monitored using a photodiode array detector (Waters). Three regions within the elution profiles were chosen for integration based on comparison of control and venom-antivenin mixture profiles. Venom-antivenin binding was estimated from differences between control and venom-antivenin mixture region areas (i.e. Area). Concentration-dependent changes in Area were fit to a hyperbolic dose-response functions (Eq.1) to estimate 'Areamax (maximum binding) and C50 [effective concentration of reactants (C) at one-half 'Areamax)].

'Area = 'Areamax [C/(C50+C)] Eq. 1 Venom and antivenom reactants and venom-antivenom mixtures were comprised of: (1) C. atrox venom, FabAV; (2) C. atrox venom, F(ab’)2AV; (3) B. jararaca venom, F(ab’)2AV; and (4) B. jararaca venom, FabAV.

Results: Complex formation was apparent in all four combinations of venoms and antivenoms. C. atrox venom binding to FabAV was greater than to F(ab’)2AV. B. jararaca venom, however, bound to F(ab’)2AV greater than to FabAV. Venoms used in antivenom production tended to bind preferentially to the respective antivenom product. Concentrations of venoms at one-half 'Areamax (C50) were similar for all four combinations of venom and antivenom. Conclusion: The data presented in this study demonstrate the use of SEC to evaluate the binding of different antivenoms to a particular venom, as well as binding of different venoms to a particular antivenom. SEC may have application in evaluating cross-reactivity of different types of antivenoms and venoms.

Key Words: venoms, antivenoms, venom-antivenom binding, size-exclusion chromatography

PH – PHARMACOLOGY PH1 - VENOM ABSORPTION AFTER SNAKEBITE PaniaguaDa, VergaraI.b, Roman Rc, Romero Cd, Benard-Valle Mc, Calderon Ac, Jimenez Lc, Bernas M Je,f,Witte M Hf, Boyer L Vg and Alagon Ac

aFacultadde Ingeniería, Arquitectura y Diseño, Universidad Autónoma de Baja California, Ensenada, Baja California, Mexico;bDepartment of Chemical and Biological Sciences, Sciences School, Universidad de las Americas Puebla, Cholula, Mexico; cDepartamento de Biologıa Molecular y Bioprocesos, Instituto de Biotecnologıa Universidad Nacional Autonoma de Mexico, Cuernavaca,Mexico; dCentro Universitario UAEM Amecameca, Universidad Autonoma del Estado de Mexico, Amecameca de Juarez, Mexico; eDepartment of Medical Education, TCU and UNTHSC Schoo of Medicine, Fort Worth, TX, USA; fDepartment of Surgery, University of Arizona, Tucson, AZ, USA;gVenom Immunochemistry, Pharmacology, and Emergency Response (VIPER) Institute, University of Arizona, Tucson, AZ, USA E-mail address: dayanira.paniagua@uabc.edu.mx Background: The knowledge of venom pharmacokinetics is essential to improve the understanding of envenomation pathophysiology, while the understanding of the pharmacokinetics of antivenoms is essential to determine the timing and optimal dose of antivenom required to bind and thus neutralize all venom in snakebite patients. When pharmacokinetics of Micurus fulvius venom was analyzed after an experimental subcutaneous injection, it was demonstrated that lymphatic absorption plays a major role in pharmacokinetics of coral snake venom, suggesting that venom bioavailability is limited by the venom absorption from subcutaneous tissue. This evidence supports the notion that the bite site is a venom depot. However, the effect of AV on systemic bioavailability and neutralization of M. fulvius venom and its mayor components was unclear. Since venom and antivenom are inoculated by different routes during clinical envenomation, a mismatch between pharmacokinetics is expected. In clinical envenomation, venom is inoculated by an extravascular route, and an absorption process take place through the interstitial space and extracellular matrix until the entry into the blood capillaries and early lymphatics before it reaches the bloodstream. Meanwhile, antivenom is injected intravenously as soon as the patient reaches the hospital. Methods: We analyzed the antivenom effect on lymphatic absorption and systemic bioavailability of coral snake venom using sheep as large animal model in a rescue type experiment. Venom was injected subcutaneously, while F(ab’)2 antivenom was injected intravenously two hours after envenomation and both, venom and antivenom levels were

followed by ELISA assays in serum and lymph samples for 6 hours, and in tissue harvested post-mortem.The results were compared with the pharmacokinetics of Micrurus fulvius venom previously reported. Discussion/Conclusion: After rescue with F(ab’)2 antivenom, the venom in the bloodstream was immediately neutralized. The antivenom extravasated from bloodstream, and it was able to neutralize the venom absorbed by lymph due to subcutaneous administration, although this neutralization seems to be slow and incomplete. The antivenom was unable to reach the residual venom depot at the site of injection, such results showed that venom may remain active with slow delivery to the bloodstream for ongoing systemic distribution. After the antivenom treatment, serum venom levels showed a subtle secondary rise. To discern the nature of the secondary rise, the proportion of the main components of the venom in the bloodstream were estimated before and after antivenom injection. A change in the proportion of the main components that depends of antivenom administration was observed. This finding showed that the secondary rise was caused by specific venom proteins that are not well recognized by the antivenom and illustrated that a secondary rise in venom detection may reflect venom components of varied clinical importance, unrepresentative of whole venom; thus, reliance on overall levels of circulating AV as a marker of clinical protection may be an oversimplification. Key Words: Equine F(ab’)2 antivenom; venom depot; coral snake; lymphatic absorption; neutralization; pharmacokinetics; thoracic duct. TOX – TOXINS TOX1 - HOW THE BROWN RECLUSE GOT ITS BITE: WHAT AN INSECTICIDAL TOXIN WITH NASTY MAMMALIAN CONSEQUENCES CAN TELL US ABOUT THE EVOLUTION OF VENOM PHENOTYPES Zobel-Thropp, PA¹; Santana-Propper, E¹; Delgado. K¹; Cordes, MHJ²; Binford, G.J.1* ¹Department of Biology, Lewis & Clark College; Portland, Oregon, USA ² Department of Chemistry & Biochemistry, University of Arizona, Tucson, Arizona, USA E-mail address: binford@lclark.edu Background: The spider family Sicariidae includes brown recluse (Loxosceles) and 6-eyed sand spiders (Sicarius and Hexopthalma) and is among the few spider lineages with an intrinsic toxin of medical consequence. They are famous for bites that cause mammalian dermonecrosis resulting from toxic phospholipase Ds (PLDs). Venoms of individual spiders include multiple related proteins in a PLD gene family (SicTox) that vary in substrate specificity. These PLDs are potent insecticidal neurotoxins, and given that sicariids are predators of arthropods, this is likely their primary biological role. The SicTox gene family is broadly present in chelicerates and homologs have deep evolutionary histories that are consistent with these enzymes serving important unknown non-venomous functions, and independent recruitment as venom toxins or pathogens in multiple disparate lineages. The recruitment of SicTox as a venom toxin in sicariids occurred before the most

recent common ancestor of the family over 100 Mya on Western Gondwana before the separation of Africa and South America. Understanding the distribution and diversity of this toxin is not only clinically relevant, the lineage is also a riveting evolutionary example of the origin of a novel toxin. As such it provides an opportunity to focus closely on the consequences of the addition of a novel toxin to the evolutionary dynamics of the overall molecular phenotypic complex of venom. We have comparatively analyzed the venom proteomes of representative spiders in the higher-level spider clade Synspermiata with sampling that includes diverse sicariids, pholcids, and spitting spiders with the goal of inferring the interrelated evolutionary histories of SicTox PLD toxins and other toxins evolving as components in these complex phenotypes. Methods: Using transcriptomic and proteomic analyses we have identified and quantified the venom components of 11 species of Synspermiata. These taxa were selected to represent a broad phylogenetic spread to help confirm the limits of the distribution of venom expressed SicTox PLD toxins within this clade. We compare the phylogenetic distributions of toxin lineages focusing on transitions that are evolutionarily coincident with the recruitment of SicTox as a functionally important toxin. Discussion/Conclusion: With the exception of a single SicTox homolog detected in Scytodes spitting spiders, these proteins were unique to sicariids where they make up nearly 50% of the composition of venoms. Diverse metalloproteinases, and small venom peptides were widespread and abundant contributors to venoms of all taxa. Contrary to predictions we did not detect a reduction of other toxic components with the recruitment of SicTox as a neurotoxin, but rather sicariid venoms have an expansion of component complexity relative to other synspermiata. Key Words: brown recluse venom, venom phenotype, phylogenetic distribution, evolution, SicTox PLD toxins TOX2 - SUBSTRATE SPECIFICITY OF RECLUSE SPIDER PHOSPHOLIPASE D TOXINS Cordes, M.H.J.1,*, Copple, J.S.1, Hauri, G.N.2, Pham, C.H.2, Binford, G.J.2 1 Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona, 85721-0088; 2Department of Biology, Lewis and Clark College, Portland, Oregon, 97219 E-mail address: cordes@email.arizona.edu Background: Recluse spider venoms contain phospholipase D toxins that cause dermonecrotic lesions in humans and neurotoxic effects in insect prey. These interfacial enzyme toxins cleave choline/ethanolamine head groups from sphingolipids or lysolipids in biological membranes, yielding cyclic phospholipid products. In previous studies, we showed that individual toxins may strongly prefer choline- or ethanolamine-containing substrates. The determinants of this substrate specificity, as well as its significance for

human health or predation, remain poorly understood. Two competing hypotheses for the determinants of specificity involve either a) sequence differences in the head group binding pocket of the active site, or b) sequence differences in the i-face of the protein, which interacts with the surface of membrane bilayers. Methods: Here we present site-directed mutagenesis experiments on Loxosceles and Sicarius venom toxins, coupled with phosphorus NMR and fluorescence-based enzyme activity assays. Results: We find that two substitutions near the active site pocket play at least some role in governing substrate preference, and preliminarily lead to approximately tenfold changes in relative velocity toward choline- and ethanolamine-containing substrates. Discussion/Conclusion: Structural modeling does not suggest a straightforward rationale for the effect of these substitutions but hints that water-mediated contacts may mediate recognition of ethanolamine while steric complementarity may help mediate recognition of choline. Future studies may focus on i-face substitutions as well as the effect of substrate preference on prey and mammalian toxicity. Key Words: Loxosceles venom, substrate specificity, phospholipase D, phospholipid head TOX3 - UNVEILING THE ACTIVITIES OF PNPP-19, A TOXIN-DERIVED SYNTHETIC PEPTIDE Freitas, A.CN2; Pacheco, DF2, Lemos, VS2.; Carmona, AK3; Peigneur, S4; Tytgat, J4; Pimenta, AM.; Hathway G5; Cruz, J2; Duarte IDG2; de Lima, ME1,2 1Santa Casa-Belo Horizonte: Ensino e Pesquisa, Belo Horizonte, MG, Brazil. 2Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. 3Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo, SP, Brazil 4Toxicology and Pharmacology, KU Leuven, 3000 Leuven, Belgium; 5Arthritis Research UK Pain Centre, School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, U.K. Email address: lima.mariaelena@gmail.com; mariaelena@santacasabh.org.br Background: PnTx2-6 (δ-CNTX-Pn1c), a potent neurotoxin (LD50 = 0.7 mg/Kg) from the venom of the spider Phoneutria nigriventer, has 48 aminoacid residues (aa), six disulfide bridges and is a promiscuous toxin, targeting different Navs. It causes priapism and pain. Based on its sequence and searching for a less toxic molecule, able to preserve its potentiator activity in erectile function, a smaller peptide (19 aa) named PnPP-19 was designed and synthesized. As previously demonstrated, PnPP-19 was active in erectile function (1). In the current work this peptide was assayed for a possible activity in pain models and its mechanism was investigated. Methods: Nociceptive thresholds were measured in male Wistar rats, by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective antagonist receptor drugs.

The hydrolysis of PnPP-19 by neutral endopeptidase (EC 3.4.24.11, NEP) was monitored by HPLC and the cleavage sites evaluated by LC–MS. NEP activity inhibition by PnPP-19 and Leu-enkephalin was determined spectrofluorimetrically (2). Proteins of interest were evaluated by Western blotting technique. Nitric oxide (NO) levels were determined by Griess methodology (3). Calcium currents and calcium images were evaluated in DRG neurons by Patch Clamp technique and incubation with Fura 2-AM, respectively. Whole-cell currents from oocytes were recorded from 1 to 2 days after injection using the two-microelectrode voltage-clamp technique. Xenopus laevis oocytes were isolated and co-injected with GIRK1, GIRK2, and RGS4 cRNA, with the addition of either hMOR, hKOR, hDOR, hMORW318L, or hMORW318Y/H319Y cRNA. Discussion/Conclusion: PnPP-19 induced peripheral antinociception in rats. Specific antagonists of μ/δ opioid receptors and CB1 receptors, partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase or of anandamide uptake enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 μmol·L-1 were determined for PnPP-19 and Leu-enkephalin respectively, as inhibitors of NEP activity. These assays suggested that antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, μ/δ opioid receptors (2). In another approach, we concluded that the peripheral antinociceptive effect of PnPP-19 results from activation of NO-cGMP-KATP pathway (3,4) and that activation of eNOS and nNOS might be required for such effect. PnPP-19 also activated specifically, but with low potency, the μ-opioid receptors transfected in oocytes of Xenopus (4). The peptide also blocked calcium channels in DRG neurons (4). In conclusion, PnPP-19 is the first synthetic peptide, derived from a spider toxin, acting directly on opioid receptors, more specifically, on μ-opioid receptor subtype. Novel ligands of the μ -opioid receptors are of clinical and social importance since the current analgesic drugs, such as morphine, fentanyl and others cause, besides of their beneficial pharmacological effect, undesirable side effects. References (1) Silva et al., J.Urology, 194, 1481-1490, 2016 (2) Freitas et al., Brit. Journal of Pharmacol., 173, 1491–15012016 (3) Freitas et al., Nitric Oxide 64, 31-38, 2017 (4) Freitas et al., Toxins, 10, 43; 2018 Supported by: FAPEMIG and CNPq. Key Words: PnPP-19, antinociceptive activity, opioid receptors, calcium channels. TOX4 - VENOM GLAND TRANSCRIPTOMICS AND VENOM PROTEOMICS OF THE COTTONMOUTH SNAKE (AGKISTRODON PISCIVORUS) REVEALS THAT GEOGRAPHIC ISOLATION CONTRIBUTES LITTLE TO VENOM DIVERSITY Lawrence, KC1, Colston, TJ1, Holding, ML1, Rokyta, DR1

1Florida State University E-mail address: klawrence@bio.fsu.edu Background: It is at the core of evolutionary biology that organisms must adapt to their environment in order to survive. Selection pressures that vary with geography such as predation and resource availability select for traits that convey fitness, which may lead to speciation. One such trait is venom production in North American pit vipers. Venom is a complex mixture of toxin and nontoxin proteins that are used in prey capture and defense. It is under heavy selection pressure due to its direct influence on individual survival, thus differences in venom composition among populations may be associated with geographic variation. The Cottonmouth, Agkistrodon piscivorus, is a venomous snake that is found throughout the Southeastern United States. A semiaquatic pit viper, A. piscivorus generally resides in or around the water and opportunistically feeds on local prey. There are three recognized subspecies (Western, Eastern, and Florida), and two putative species (Continental and Florida), defined by their geographic range. Although morphometric differences have been observed, variation in venom composition has been widely understudied. We hypothesize that variation in venom composition should reflect these proposed species classificationsas A. piscivorus becomes more specialized to their local environment. Methods: Venom gland tissue and whole venom samples were collected from nine individual A. piscivorus from across their geographic range. We used quantitative transcriptomic and proteomic approaches to generate a genotype-phenotype map and determine the effect of geographic isolation on toxin abundance. Results/Discussion: Venom composition was not significantly different among the A. piscivorus analyzed, with consistently high expression of the phospholipase A2 toxin family.Because A. piscivorus is a semiaquatic pit viper, selection pressures may not vary enough among geographic locations to cause significant variation in venom composition. Conclusion: The hypothesis that A. piscivorus venom will vary with geographic isolation concordant with proposed subspecies classifications was not supported. More sampling across the geographic range and further genomic analysis is needed to clarify these classifications. Key Words: Agkistrodon venom, geographic variation, transcriptomics, proteomics TOX5 - BIOLOGICAL ACTIVITIES OF TWO PHOSPHOLIPASES A2 FROM NORTH AMERICAN SNAKE VENOMS Salazar, E1*, Zavala, C1, Cantu, A1, Sanchez, O1, Suntravat, M1,2, Galan, J1,2, Sánchez, EE1,2

1 National Natural Toxins Research Center, Texas A&M University- Kingsville, Kingsville, TX, USA 78363; 2 Chemistry Department, Texas A&M University- Kingsville, Kingsville, TX, USA 78363 *E-mail address: emelyn.salazarcastillo@tamuk.edu Background: Snake venoms are one of the main sources of secreted phospholipases A2 (PLA2s), stable, ubiquitous and versatile toxins that are found in vary amounts in these venoms. Despite sharing a high homology, PLA2s have shown a broad spectrum of biological activities including neurotoxicity, myotoxicity, cardiotoxicity, hemolysis, modulation of the hemostatic system and inflammation. These molecules are pharmacologically active and can exert the effects mentioned through mechanisms that may depend or not of their enzymatic activity and, can be done by their own or with the aid of other components. Acidic and basic PLA2s were purified using reverse phase HPLC fractionation from Crotalus adamanteus and Agkistrodon piscivorus piscivorus crude venoms. Both N-terminal amino sequences revealed Asp at position 49 (D-49), which were homologous with catalytically active PLA2s from Viperidae venoms. These toxins were determined to have indirect hemolytic activity through a turbidimetric method employing red blood cells and egg yolk as phospholipid substrates. In addition, these toxins promoted hemolysis, inflammation and myotoxicity in a mouse model. Methods: In vitro assays were employed to further characterize these viperid PLA2s. The toxins were tested against cell lines to determine cell viability and cell activation, specifically the effects on the release of pro-inflammatory mediators and markers of cell damage. In addition, the effects of PLA2s on the hemostatic system were analyzed using a global assay through a Sonoclot analyzer. Discussion/Conclusion: Viperid PLA2s had no effect on cell viability. However, significant morphological changes were seen on myotubes, including the release of pro-inflammatory mediators and markers relating to cell damage. In addition, it was observed that these PLA2s caused significant changes on the coagulation system and mild changes on platelet function in human whole blood. These findings can be used for further in vivo and in vitro experimentation to characterize enzymes belonging to this family. This is significant for the production of specialized antivenoms that target PLA2s and their biological activities. Key Words: Phospholipases A2, Agkistrodon piscivorus piscivorus, Crotalus adamanteus, myotoxicity, hemostatic system, cell activation TOX6 - SNAKE VENOM CYSTEINE-RICH SECRETORY PROTEINS (SVCRISPS) FROM NORTH AMERICAN VIPERS: MECHANISM OF ACTION ON BLOOD AND LYMPHATIC ENDOTHELIAL CELLS Suntravat, M1,2*, Sanchez, O1, Reyes, A1, Bernal, J1, Salazar, E1, Sanchez, EE1, 2

1 National Natural Toxins Research Center (NNTRC), Texas A&M University-Kingsville, MSC 224, 975 West Avenue B, Kingsville, TX 78363, USA; 2 Department of Chemistry, Texas A&M University-Kingsville, MSC 161, Kingsville, TX 78363, USA E-mail address: montamas.suntravat@tamuk.edu Background: Snakebite is a substantial global health problem, causing serious injury to 2.7 million men, women, and children and claiming an estimated 125,000 deaths annually. Our studies are focused on the vascular biology of snake venom Cysteine-Rich Secretory Proteins (svCRiSPs), a large family of toxins that are important components in the venom of many species of snakes including Viperidae and Elapidae. In spite of the widespread distribution of svCRiSPs in snake venoms, little is known of the contribution that they make to the local pathophysiology of snakebite. The aim of this work was to investigate the role of svCRiSPs from five of the most medically significant species of North American vipers (Crotalus atrox, C. adamanteus, C. scutulatus scutulatus, C. horridus, and Agkistrodon piscivorus) in snakebite, focusing specifically on the cellular and molecular mechanisms underlying vascular biology in snakebite. Methods: We evaluated the biologic activity of svCRiSPs (Css-CRiSP, Catrox-CRiSP, Cada-CRiSP, Chor-CRiSP, and App-CRiSP) by using both in vitro assays on human dermal lymphatic endothelial cells (HDLECs) and human dermal blood endothelial cells (HDBECs) permeability and in in vivo Miles assay of vascular permeability. Css-CRiSP is the most potent cause of directly and acutely increase vascular permeability both in vitro and in vivo compared to other crotaline CRiSPs. To elucidate the main pathway underlying the endothelial permeability induced by Css-CRiSP, we initially screened the changes in protein expression and phosphorylation in HDLECs and HDBECs 30 min after treatment with Css-CRiSP using reverse phase protein arrays (RPPA). The antibodies used in RPPA analysis specifically recognize proteins acting on multiple signaling pathways, including receptor tyrosine kinases, PI3K-AKT signaling, MAPK pathway, AMPK signaling, PKC signaling, mTOR pathway, cell motility, focal adhesion as well as cell stress and immune response, DNA repair, cell cycle, and apoptosis/autophagy regulators. We observed that protease activating receptor (PAR) and several signaling molecules, which are part of the cell mobility, immune response, and MAPK pathway, were elevated in HDBEC and HDLEC cells in varying degrees of protein expression and phosphorylation. Additionally, Css-CRiSP induced nitric oxide release in both HDBECs and HDLECs. Discussion/Conclusion: These preliminary observations suggest that Css-CRiSP induce the increased endothelial monolayer permeability in HDBECs and HDLECs via different mechanisms. Further studies are required to confirm the intracellular mechanisms through which Css-CRiSP reaches to provoke vascular permeability. Knowledge gained from these studies provides insights into the molecular mechanisms that underlie the effects of svCRiSPs on vascular function and contributes to a new level of understanding of the pathophysiology of snakebite and the development of novel therapeutic strategies for the treatment of snakebite and possibly other vascular and lymphatic diseases.

Key Words: Snake venom cysteine-rich secretory proteins (svCRiSPs), vascular permeability, lymphatic cells, signaling mechanisms VAB – VENOMOUS ANIMAL BIOLOGY VAB1 - VENOM RESISTANCE AGAINST TWO GRASSLAND RATTLESNAKES IN AN EASTERN COLORADO RODENT COMMUNITY Balchan, NR, Mackessy, SP School of Biological Sciences, University of Northern Colorado, 501 20th St., CB 92, Greeley, CO 80639 USA E-mail address: neil.balchan@unco.edu Background: The Red Queen hypothesis describes the coevolutionary dynamic between predator and prey where both partners must evolve in tandem to remain competitive. These arms-races play out in an array of interacting pairs, ranging from water fleas and their parasites, to larger animals like garter snakes and newts. In several cases, rodents have demonstrated resistance to the venoms of their snake predators. For example, the California Ground Squirrel (Otospermophilus beecheyi) exhibits high resistance to the venom of the Pacific Rattlesnake (Crotalus oreganus). Conversely, cases exist where a prey species apparently lacks physiological resistance to the venom of its predator - the Cape Ground Squirrel (Xerus inauris) lacks venom resistance to the predatory Puffadder (Bitis arietans) and Snouted Cobra (Naja annulifera). The development and maintenance of venom resistance mechanisms may be dependent on a variety of selective pressures present in an ecosystem as a whole. Furthermore, understanding venom resistance patterns at the community level may allow for better understanding of evolutionary processes as a whole within an ecosystem. My research evaluates patterns of venom resistance in a Colorado grassland ecosystem, where the Desert Massasauga (Sistrurus tergeminus edwardsii) and Prairie Rattlesnake (Crotalus viridis) prey upon a suite of rodent species. Methods: Field sites were located in northern Colorado (single snake predator) and southern Colorado (two snake predators) to understand better the impact of geography and interspecific interactions. Venoms were collected from Prairie Rattlesnakes at both field sites, and Desert Massasaugas at the southern field site. Rodents were live-trapped at both field sites for use in assays. Serum based assays were used to determine resistance to snake venom metalloproteases within various snake-rodent pairings. LD50 tests were run with several rodent-snake pairings to determine whole organism toxicity of crude venoms. Finally, affinity chromatography was used to isolate venom-binding proteins present in rodent sera. Discussion/Conclusion: Resistance to rattlesnake venoms was present in several species of Colorado rodent, including Deer Mice (Peromyscus maniculatus), Ord’s Kangaroo Rats

(Dipodomys ordii), Northern Grasshopper Mice (Onychomys leucogaster), and Eastern Woodrats (Neotoma floridana). Plains Pocket Mouse (Perognathus flavescens) serum from both field sites failed to inhibit Desert Massasauga venom metalloproteases from southern field site snakes, indicating a lack of local adaptation to this predator. Northern Grasshopper Mouse and Eastern Woodrat serum generated strong inhibition against all rattlesnake venom metalloproteases. LD50 data indicate that Deer Mice are least resistant to the venoms of their co-occurring Prairie Rattlesnakes, and that feral House Mice (Mus musculus) are no more resistant to Prairie Rattlesnake venoms than lab mice. Preliminary affinity chromatography results suggest that venom-binding serum proteins may be present in Ord’s Kangaroo Rats, Northern Grasshopper Mice, and Eastern Woodrats. Further study should explore the role of snake diets in the development of venom resistance, as strong selection pressures may necessitate greater levels of venom resistance in rodents. Key Words: local adaptation, evolution, predator-prey dynamics, geographical variation, metalloproteinase, toxin VAB2 - HUMAN DIMENSIONS IN SNAKE CONSERVATION Daly-Crews, A1, Griffin, L1, 1The Rattlesnake Conservancy E-mail address: Director@savethebuzztails.org Background: Translocation, commonly known as relocation, is the act of removing an animal from their home range to another potentially suitable habitat. Snakes are often translocated due to conflict with land use associated with children, pets, and livestock, as well as in situations where the animal appears to be in danger (crossing roads, etc.). Human conflict with wildlife, especially snakes, is increasing in areas where urban development encroaches on natural areas. In response, commercial industry (such as animal control), concerned citizens, conservationists, and others are tasked with relocating “nuisance” snakes to areas well beyond where they may interact with the aggrieved party again. Scientists and conservationists also translocate imperiled snakes in areas at risk for development, during reintroduction efforts, and to bolster genetic viability of isolated populations. However, little is known about the characteristics of people who regularly translocate snakes and how they make decisions about translocation. Methods: To explore this, our team designed a brief questionnaire and distributed it through social media groups associated with conservationists, reptile enthusiasts, commercial industry, and wildlife professionals between the months of February and April 2019. The questionnaire examined who is translocating snakes, frequency, and motivations. We also explored the relocation of venomous snakes in particular Discussion/Conclusion: 74.8 percent of participants (n=140) translocated snakes sometimes or all of the time, and three quarters (74.2%) moved fewer than 20 snakes annually. 40 percent moved less than 5 snakes annually. Only about 5 percent of participants who translocate snakes say they have no experience with snakes. The

remaining participants identified as snake enthusiasts (private herpetoculturalists, fielder herpers (amateur reptile observers or photographers, similar to "birding"), (53%), biologists or scientists (26%), or both (6.7%). The majority (88.5%) of participants translocated snakes found on someone’s property who did not want it there and/or the landowner was threatening to kill the animal. Around half (46.2%) of participants translocated snakes that were on roads Almost a third of participants (29.8%) report moving snakes less than half a mile from the original capture site, and less than 3% said they moved snakes more than 2 miles from capture site. However, nearly 40% of participants separately indicated that they moved snakes to the nearest protected area, regardless of distance, so these findings need to be interpreted cautiously. Participants also reported translocating venomous snakes. Of participants who translocate snakes, 83.7% said they relocate venomous snakes. Despite this, over a quarter (26.1%) reported having no training--formal or informal--in handling venomous snakes. Around a third of participants (34.1%) had informal training on handling venomous snakes, while another third (31.8%) had formal training, such as a venomous course or through their profession. Eight percent of participants reported both formal and informal training. To our knowledge, our results are the first to describe characteristics of individuals engaged in snake translocation and their motivations for doing so. When done improperly, translocation may increase mortality in individual animals and inadvertently spread pathogens. As such, understanding who is translocating snakes and why is critical for researchers and conservationists developing best management practices for translocation and developing conservation strategies for imperiled species. The Rattlesnake Conservancy plans to evaluate the long-term impacts to multiple crotaline species to compliment development of relocation guidelines for the average public, nuisance wildlife trappers, researchers, and conservationists. Key Words: Snake translocation, human dimensions, field VAB3 - A TIME SERIES ANALYSIS OF VENOM PROTEIN REGENERATION IN THE GIANT FLORIDA BLUE CENTIPEDE (SCOLOPENDRA VIRIDIS) AND THE HENTZ STRIPED SCORPION (C. HENTZI)

Fry, L1, Nystrom, GS1, Ellsworth, SA1, Rokyta, DR1,*

1 Department of Biological Sciences, Florida State University, Tallahassee, Florida 32306 Tallahassee, Florida 32306 Corresponding author: drokyta@bio.fsu.edu

Email address: lgf16@my.fsu.edu

Background: Many animals, including snakes, invertebrates, fish and even mammals, have evolved to produce venom with varying levels of toxicity (1). Animal venoms are complex proteinaceous secretions of which composition is dependent on the species producing the venom. Organisms depend on their venom to forage for prey and defend themselves against predators which contributes to their overall fit-ness. The regeneration process of venom and

toxicity, after full depletion, has been widely studied in snakes to understand the best time point to extract venom in the lab. Studies have also been done on scorpions, though to a lesser degree, and found that venom regeneration was an asynchronous process where different components regenerate at different rates (2). Although recent studies have shown that the composition of centipede venom is complex and toxin rich (3), the process of venom regeneration in centipedes has not been studied. In this study, we analyzed the regeneration of venom in S. viridis, a species of centipede found in north Florida along with C. hentzi, a species of scorpion also found in North Florida. To create a venom regeneration timeline, the protein content from five different time points, after an initial venom extraction, were compared using reversed-phase high- performance liquid chromatography (RP-HPLC).

Methods: We collected the centipedes and scorpions from the Apalachicola National Forest in northern Florida. The animals were housed at Florida State University and starved for three weeks prior to an initial venom extraction. Using electrostimulation, venom was successively from both the centipedes and the scorpions at five different time points after an initial extraction. Venom samples were lyophilized and stored at -80 until the venom was quantified. Reverse-phase high performance liquid chromatography was used to create venom profiles for the five different time points for both the centipedes and scorpions.

Discussion: We found a significant difference in the RP-HPLC profiles at different time points in S. viridis. The venom complexity was able to be analyzed with the RP-HPLC, but the composition of the venom (what proteins contribute to which peak) was not studied. Future studies aimed at isolating specific peaks on the venom profile and characterization of the protein could help better understand centipede venom.

References: 1. Casewell, NR, Wuster, W, Vonk, FJ, Harrison, RA, Fry, BG (2013). Trends in ecology & evolution, 28(4), 219-229. 2. Carcamo-Noriega, EN, Possani, LD, Ortiz, E. (2019). Toxicon, 157, 87-92. 3. Undheim, EA, Fry, BG., King, GF (2015). Toxins, 7(3), 679-704. 4. Ward, MJ, Rokyta, DR (2018). Toxicon, 152, 121-136. Key Words: Protein regeneration, Time-series analysis, C. Hentzi VAB4 - CHANGE FOR THE BETTER: VENOM ONTOGENY CORRESPONDS TO DIETARY SHIFTS IN SOUTHWESTERN NORTH AMERICAN RATTLESNAKES Hayes, WK1,*, Gren, ECK2, Kelln, W1, Fox GA1, Cochran, R1, Macomber, FB1, Kim, K1 Cooper, AM1, Lee, KH3, Dugan, EA4, Travis, ZD1 1 Department of Earth and Biological Sciences, School of Medicine, Loma Linda University, Loma Linda, California, 92350 USA; 2 University of Montana, Bitterroot College campus, and Bitterroot Community Science Center, Hamilton, Montana 59840

USA; 3 College of Osteopathic Medicine, Touro University Nevada, Henderson, Nevada 89104 USA; 4 Dugan Biological Services, LLC, Upland, California 91784 USA E-mail address: whayes@llu.edu Background: As important mesopredators in many ecosystems, rattlesnakes (genera Crotalus and Sistrurus) generally consume a diverse range of small prey. Although some species have more specialized diets than others, many species exhibit dietary shifts during ontogeny, often from ectothermic to endothermic prey species. Because rattlesnakes are gape-limited, the size of prey also shifts during ontogeny to include increasingly larger prey. Changes in prey species and prey size are accompanied by varying risks and benefits associated with prey acquisition, energy intake, and digestion. Because these snakes rely on venom to procure their food, shifts in venom composition also occur. We examined ontogenetic changes in the venom composition of several southwestern North American rattlesnake species to test three hypotheses: that (1) change is greatest in species or populations possessing largely proteolytic (type I or type B) rather than largely neurotoxic (type II or type A) venoms; (2) change is greater in species that transition from reptile to mammal prey than those which specialize on mammals throughout their life; and (3) change occurs continuously as an individual grows, even after attaining reproductive size. Methods: We separated the venom components of individual venoms representing six rattlesnake species using reverse-phase high-pressure liquid chromatography (RP-HPLC), and quantified the protein content of individual peaks and/or elution regions of the resulting chromatograms. We identified specific toxins using mass spectrometry. To associate ontogenetic changes in venom with diet, we dissected prey material from the stomachs or guts of preserved rattlesnakes to ascertain ontogenetic variation in diet. Analyses for two species (Crotalus atrox, C. pyrrhus) remain to be completed. Results: Ontogenetic changes in venom composition existed in all species examined. Consistent with Hypothesis 1, venom changes were indeed substantially greater in species or populations possessing largely proteolytic venoms (C. cerastes, C. helleri type I, C. ruber) compared to those possessing largely neurotoxic venoms (C. helleri type II,C. scutulatus type II). Hypothesis 2, however, proved wrong; dietary shifts from primarily lizards (ectotherms) to primarily small mammals (endotherms) were associated with limited (C. scutulatus type II and C. helleri type II) to modest (C. cerastes, C. helleri type I) changes in venom composition, but the most profound changes occurred in C. ruber, which feeds largely on rodents throughout its life. Venoms in C. ruber shifted from primarily myotoxins in neonates (62-74% of protein content) to primarily snake venom metalloproteases (SVMPs) in adults (44–73%), which coincided abruptly with the transition to reproductive maturity (60–70 cm snout-vent length)and a shift from predominantly heteromyid rodent prey to predominantly cricetid rodent and leporid (rabbit) prey. Consistent with Hypothesis 3, venom change persisted as snakes grew, with the highest proportion of SVMPsin C. cerastes and C. ruber occurring in the largest specimens. Discussion/Conclusion: Our findings demonstrate the remarkedly varied patterns of venom ontogeny that exist within a closely related group of vipers, and even within a single

species. They also underscore the likely adaptive importance of venom composition changes coincident with dietary shifts, at least in rattlesnakes that possess proteolytic venoms. Key Words: Rattlesnakes, Crotalus, venom ontogeny, diet ontogeny VAB5 - THE GENETIC BASIS OF SENSORY PERCEPTION IN PITVIPERS Hogan, Michael1,Whittington, A Carl, Rokyta, Darin R* Rokyta Venom Lab, Florida State University E-mail address: mhogan@bio.fsu.edu Background: Venom and other traits involved in subduing and digesting prey are only relevant following the detection of prey, since the act of predation generally initiates when the predator perceives the presence of a potential prey item. Prey-based selective pressures should therefore drive the evolution of genes responsible for prey detection, resulting in signals of adaptive evolution in sensory genes comparable to venom genes. Pitviper sensory systems are among the most biologically impressive and phenotypically unique in the animal kingdom, consisting of four primary senses: trichromatic color vision through low-light sensitive vertical pupils; bi-focal thermal or infrared perception via two specialized facial pits; acute mechanosensation enabling the detection of low-frequency vibrations ventrally through the ground; and bi-directional chemosensation implementing a specialized forked-tongue to manually probe and deliver airborne scent molecules to chemoreceptors located inside the vomeronasal organ. Using a complete multi-sensory genetic framework, sensory repertoires of North American pitviper species were evaluated and tested for predatory character displacement and sensory optimization through sequence analyses across lineages. Methods: Using a systematic homology and RNA-seq guided approach, we annotated all putative sensory genes present in the genome of the eastern diamondback rattlesnake (Crotalus adamanteus), highlighting an extreme diversity of putative chemoreceptor genes (∼800 full-length genes) compared to the other senses. Chemoreceptor gene phylogenies were reconstructed from coding sequences identified from the genome to test for signals of gene expansion and positive selection corresponding with gene expression, identifying biologically relevant chemoreceptor genes for this species. Chemoreceptor gene expression variations relating to sex and age were also compared for Crotalus adamanteus revealing biases relating to development and mate detection. This represents the most thorough genetic characterization of snake olfaction to date and provides a foundation for multispecies gene comparisons via bioinformatic approaches. Discussion/Conclusion: Genome organization of predatory loci into tandem-repeat-arrays likely enabled snakes to rapidly diversify and expand genes involved in predation, such as venom and chemoreceptor genes. Key Words: Crotalus, predatory phenotype evolution, genome organization, sensory

perception VAB6 - DISTRIBUTION AND SEVERITY OF SNAKE FUNGAL DISEASE CAUSED BY OPHIDIOMYCES OPHIODIICOLA AMONG TIMBER RATTLESNAKES (CROTALUS HORRIDUS) IN PENNSYLVANIA Januszkiewicz, E1,2, LaDuke, TC1, Chinnici, N2 1Department of Biology, East Stroudsburg University, East Stroudsburg, Pennsylvania, USA; 2Dr. Jane Huffman Wildlife Genetics Institute, East Stroudsburg, Pennsylvania, USA E-mail address: ericjanuszkiewicz@gmail.com Background: Snake Fungal Disease (SFD) is a recently emerging disease caused by a fungal infection from Ophidiomyces ophiodiicola.It has been found to infect over 30 different species including European species. It is a type of keratinophilic fungus that opportunistically infects snakes and feeds on the epidermis. An immune response results in necrosis of the infected tissue and can cause swelling, lesions, pustules, and crusts on the individual. This pathogen affects different species differently which warrants further investigation. Methods: A total of 243 individual rattlesnakes were captured, swabbed, and tested for the presence of this fungal pathogen using real-time PCR. A DNA extraction method was developed for this fungus. Specific primers and a probe were used in real-time PCR to determine if pathogen DNA was present or not. Select samples were chosen for DNA sequencing to further confirm the presence of the pathogen DNA. Discussion/Conclusion: Of the 243 individuals swabbed, 24% tested positive. This is the first study to assess the presence, prevalence, and severity of SFD among timber rattlesnakes (Crotalus horridus) in Pennsylvania. There were no relationships found between infection rates and color phase, sex, or length. The cause of emergence and spread of this pathogen is largely unknown. However, this study provides evidence to support the notions that SFD severity can vary seasonally and that timber rattlesnakes are able to recover from infections. Long-term monitoring studies would be helpful to further examine the effects this fungal pathogen may have on populations and elucidate the driving forces behind these infections. Key Words: Snake Fungal Disease, pathogen, timber rattlesnakes VAB7 - EXPLORING VENOM VARIATION AND POPULATION DYNAMICS IN THE STRIPED BARK SCORPION (CENTRUROIDES VITTATUS)

Koffinas, L1, Ward, MJ1, Ellsworth, SE1, Nystrom, GS, Martinez, P, Saul, RM, Rokyta, DRR1,*

1Department of Biological Science, Florida State University, Tallahassee, Florida 32306 Tallahassee, Florida 32306 Email address: drokyta@bio.fsu.edu Background: Venom is often referred to as a toxic cocktail of proteins and peptides utilized by animals for both predation and defense. Some scorpion species, however, have been observed using their venom for alternative functions, such as courtship and mating. Intraspecific venom variation is prevalent in many venomous animal taxa and is generally accepted as being due to local adaptation of populations within a given species. Interspecific venom variation within a set population, however, has been more difficult to study, as it requires repeated sampling from the same individuals, in addition to sampling a large number of individuals from the same population. These requirements have made the exploration of intrapopulation venom variation difficult for a number of venomous species, particularly vertebrates, where sampling strategy, animal collection, and safety standards are under strict guidelines. Venomous invertebrates, however,offer an ethical alternative in the study of venom variation. Methods: Using two populations of striped bark scorpions (Centruroides vittatus) found in San Antonio and Kisatchie, I am examining the extent of venom variation both within and among these two populations, and then determining whether or not sex contributes to this variation. Three biological replicate venom samples from each individual were collected.Venom samples are analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC) to determine the extent of compositional venom variation. Additionally, we examined potential functional differences of venom between male and female venoms by performing lethal dose 50 (LD50) assays in crickets, using all-male and all-female pooled venom samples. Currently, we are also performing PCR amplification of the cytochrome oxidase sub unit 1 (COX 1) gene, which is known to be variable due to speciation among Centuroides species. Results: We found a significant difference in variation in the compositional analyzes between the two populations of C. vittatus (P= 0.00009). There was a significant difference between the sexes of both populations (P=0.0195) with a significant interaction of population and sex (P=0.0188) suggesting that sex contributes to the observed between population variation. We performed 3 post hoc tests for variation within the sexes between populations, with a Bonferroni correction (α= 0.0125). We found a significant difference between the males of the two populations (P=0.007) as well as between the females (P=0.00009). Additionally, the males and females within each population were also compared, with no significant difference based on sex in the San Antonio Population (P=0.6) and a stark difference between the sexes of the Kisatche population (P=0.00009). The LD50 assay results are still being analyzed, but preliminary results suggest no significant difference in functionality of the two populations.

Discussion: We found significant differences between these two populations of C.vittatus, leading us to believe that populations of this species are variable in venom composition. The San Antonio population of C. vittatus was found to have no significant functional or compositional variationbased on sex, however, we did find significant variation due to sex within the Kisatche population. Conclusion: Previous work has shown the degree of intrapopulation and intersexual variation is variable, and that female venom variation seems to drive population divergence. This also seems to be the case in C. vittatus as previous studies have shown significant differences between male and female venoms in other populations. Intersexual venom variation is thought to be due to differences in ecology and behavior between males and females within a species. Our results suggest that ecological and behavioral differences among populations, eluding inconsistent pat-terns of venom variation across populations Centruroides vittatus. Key Words: Centruroides, venom, venom variation, population dynamics VAB8 - A STING TO DIE FOR: VENOM COMPOSITION ANDCOMPLEXITY IN SOUTHWESTERN NORTH AMERICAN SCORPIONS Marcy, S1, Sarfo-Poku1, C, Fox, GA1, Cooper, AM1, Marston, L1, Kelln, W1, Hung, KY2, Hayes, WK1,* 1 Department of Earth and Biological Sciences, School of Medicine, Loma Linda University, Loma Linda, California, 92350 USA; 2 Coachella Valley Mosquito and Vector Control District, Indio, CA 92201 USA E-mail address: smarcy@llu.edu Background: Scorpion venom comprises a complex blend of inorganic salts, heterocyclic compounds, bioactive peptides, and proteins used primarily for predation and defense. The peptide and protein composition differs regionally within the paired venom glands (i.e., venom heterogeneity) and changes sequentially during expulsion, and can also vary during ontogeny, between the sexes, and among different geographic locations. Venom complexity, which has been largely neglected by researchers, can also vary. Numerous factors potentially influence the number of toxins and their individual representation in venom, including diet breadth, predator diversity, the range of biological roles (e.g., predation, defense, and reproduction), environmental variation, and evolutionary history. Venom complexity is sometimes characterized loosely by the number of toxins present, but we can borrow from ecological theory by using diversity indices that additionally take into account the relative quantity of each toxin within the venom mixture. An alternative approach is to use fractal analysis to characterize contortedness of the chromatogram waveform. To better understand variation in scorpion venom composition and complexity, we characterized ontogenetic, intersexual, and/or geographic variation within three North American scorpion groups: three species of the genus Hadrurus, Smeringurus mesaensis,

and the only medically significant scorpion in the United States, Centruroides sculpturatus. Methods: We used reverse-phase fast protein (RP-FPLC) or high-pressure (RP-HPLC) liquid chromatography to separate venom components of individual venoms. We identified specific toxins using mass spectrometry. We quantified the protein content of individual peaks and/or elution regions of the resulting chromatograms, and compared these using multivariate statistical models. We further subjected chromatogram waveforms of S. mesaensisto fractal analysis, and will eventually compare peaks and their protein content using the Shannon diversity index. Results: Venom composition variation existed at all levels examined among the three taxonomic groups. In H. arizonensis, profound geographic variation far exceeded the mild intersexual variationthat existed. Geographic variation in venom composition among populations of H. arizonensis was comparable to interspecific variation among the three Hadrurus species examined. In S. mesaensis, fractal analyses similarly revealed that differences among populations were much greater than the mild differences detected between the sexes, but pairwise comparisons of fractal similarity indicated that differences among populations were surprisingly independent of the geographical distances separating them. Our studies of C. sculpturatus are nearing completion; however, we expect statistical analyses to confirm mild ontogenetic shifts and mild intersexual differences in the relative abundance of certain toxins, and surprisingly mild geographic variation. We further expect analyses of complexity, yet to be undertaken, to show that C. sculpturatus secretes a less complex venom than the other two genera. Differences in complexity might also exist during ontogeny or at the other levels of variation. Discussion/Conclusion: Collectively, these findings contribute to our understanding of the factors that influence venom composition and complexity in scorpions, and offer insight regarding potential avenues to explore adaptive function. Because C. sculpturatus venom can cause severe scorpionism (envenomation) in humans, we plan on testing the neutralizing capacity of a commercially available antivenom against the natural venom variation we have identified. Key Words: Scorpions, Centruroides, Hadrurus, Smeringurus, venom variation, ontogenetic variation, sexual variation, geographic variation VAB9 - RISK ASSESSMENT AND THE EFFECTS OF HABITAT COMPLEXITY ON THE DEFENSIVE BEHAVIORS OF THE SOUTHERN STRIPPED SCORPION, VAEJOVIS CAROLINIANUS Harty, CN1, David, EM1, Hector, JB1, Corbit, AG1,*, Nelsen, DR1,* 1 Department of Biology and Allied Health, Southern Adventist University, Collegedale TN 37315. * Co-Principle Investigators

E-mail address: dnelsen@southern.edu Background: Scorpions are predators and prey of/to a myriad of other organisms; thus, selection should favor individuals that are able to acquire, process, and act on relevant environmental signals in order to avoid predation. Although relatively few, studies have consistently found that scorpions are able to control their use of venom: both in their choice to release venom or not, and, when released, in the volume expelled during a given sting. These decisions occur in association with many factors including satiety, venom availability, prey size, prey struggle intensity, prey type, and perceived threat (risk assessment). In addition, previous research suggests that some species use their pectens to create mental maps of their environment in order to navigate back home after foraging. To date, no research has investigated if Vaejovis carolinianus is capable of risk assessment, or if any scorpion species uses physical information about their environment to modulate their defensive behaviors. Methods: We tested 18 scorpions (N = 9 females and males respectively) in a repeated-measures design. Scorpions were placed in a circular arena (56 mm diameter) with the base coated in a thin layer of mulch, and supplied with varying numbers (0, 2, or 4) of square (10.38 ± 0.13 cm) hides. Scorpions were placed in the arenas overnight and their movements were recorded using infrared cameras. Defensive behaviors were elicited by prodding scorpions once per second over a two-minute observation time. Prods were directed toward the scorpion’s chelae, prosoma (behind their median eyes), and ventral surface of the two distal most metasoma segments. Each location was prodded 10 times in succession, cycling through each location 4 times in a repeated pattern of chelae, metasoma, and prosoma. Defensive behaviors were recorded and analyzed for stinging, chela use, and movement. Movement data was obtained from the overnight recordings using a frame-subtraction algorithm implemented in Matlab. Defensive behaviors data were analyzed with Jamovi (v.1.0.7) using a linear and generalized linear mixed models. Discussion/Conclusion: We found that V. carolinianus shows clear evidence of risk assessment with stinging being elicited sooner and occurring more frequently when prodded on the prosoma compared to either of the other locations (chelae and metasoma). We also found potential interactions between sex and number of available hides and the use of stinging behavior. Our findings suggest that V. carolinianus are able to acquire and processes environmental information in ways that allows them to alter their defensive strategies. Our findings also suggest that the availability of hides may be a relevant signal that affects their willingness to use stinging behavior. However, more work is needed to determine if and how habitat structure influences stinging and venom use behavior. Key Words: Venom metering, Venom optimization, Learning walks VAB10 - PRELIMINARY INVESTIGATION OF SURVIVORSHIP AND THE EMERGENCE OF SEXUAL DIMORPHISM AND DEFENSIVE BEHAVIORS IN THE EARLY ONTOGENESIS OF VAEJOVIS CAROLINIANUS

Lang, A1, Sewell, E1, Martin, M1, Kuhlman, R1, Pergerson, R1, Corbit, AG1,*, Nelsen, DR1,*

1 Southern Adventist University * Co-Principle Investigator E-mail address: dnelsen@southern.edu Background: Sexual differences are common in the animal kingdom and can encompass both morphology and behavior. Many scorpion species exhibit sexual dimorphism and some evidence suggests behavioral differences as well. One study found that Centruroides vittatus (striped bark scorpion) females sting more often in response to defensive stimuli than do males. We investigated sexual differences in Vaejovis carolinianus (southern devil scorpion) which inhabits forested areas in the southeastern U.S. This species exhibits sexual dimorphism as adult females are not only larger than males but exhibit wider spacing between their pectines. This species may also show behavioral differences due to sex since at least one study indicates that that females are typically found under rocks while males prefer leaf litter. Our study takes an ontogenetic approach, observing the development of V. carolinianus starting from maternal independence. This allows for the study of the emergence of sexual dimorphism along with changes in general body size. It also allows for the testing of defensive behaviors between sexes and throughout ontogenesis. A concern going into our study was survivorship, as previous research investigating the post-birth development of Vaejovis bilineatus noted a high mortality throughout the experiment, resulting in no scorpions surviving into adulthood. As a result, we expanded our research questions to include data on survivorship of V. carolinianus. Our goals in this study were to investigate the following questions: is the spacing between pectines diagnostic of sex in early instars, as it is for adults? How does pectine spacing change, if at all, throughout ontogenesis? How does Vaejovis carolinianus change in size, mass, and defensive behaviors throughout early development? Finally, what factors predict survivorship across early development? Methods: We captured 200 scorpions from the forest around the campus of Southern Adventist University (near Chattanooga TN) between August 27, 2019 and September 24, 2019. We separated females carrying juveniles on their backs, and when the juveniles voluntarily left their mother’s back, we placed each into their own container. Each week, we massed, photographed, and behaviorally tested each scorpion. To test for defensive behavioral changes, we prodded each scorpion in a circular testing arena over a 30 second time interval and video recorded the trials. We did a frame-by-frame analysis of the videos and recorded the number of stings, tail wags, percentage of time spent running, and other large and small movements. We used Image J to measure the space between pectines and the width of the first metasomal segment. Additionally, we recorded the date of death and the corresponding mother for each scorpion to investigate survivorship. For data analysis, mixed models were run using Jamovi (1.0.7) and cox regression was run using SPSS (version 25). Discussion/Conclusion: Preliminary data suggests that as the initial mass of the scorpion increased, the probability of death decreased. However, when mass was held constant, a larger metasoma 1 width indicated a lower probability of survival. Nine weeks after the

scorpions dispersed from their mothers’ backs, we do not observe sexual dimorphism based on pectine spacing. In terms of behavior, as the scorpions got older and increased in mass, they ran less often but showed no detectable differences in the frequency of stinging. Overall, we did not observe sexual differences during the twelve weeks of the study for these juvenile scorpions. Key Words: Vaejovis carolineanus, juvenile scorpions, sexual dimorphism, survivorship, behavioral ontogeny VAB11 - OUT OF SIGHT OUT OF MIND: THE ROLE OF VISION IN RISK ASSESSMENT AND VENOM METERING IN VAEJOVIS CAROLINIANUS Lee, C1, Park, B1, Tsai, J1, Carter, D1, Nelsen, D1,*, Corbit, A1* 1Department of Biology and Allied Health, Hickman Science Center, Southern Adventist University, Collegedale, TN 37315, USA; *Principal Investigator E-mail address: dnelsen@southern.edu Background: The venom metering or venom optimization hypothesis suggests that, because venom is a metabolically costly commodity, organisms must regulate its usage effectively. Evidence suggests that defensive venom metering is reliant on the repetition or persistence of threat, evaluated through the process of risk assessment. Scorpions, like other organisms, must acquire, transmit, process, and respond to signals from their environment. In addition, they must either integrate multiple different sensory modalities (vision, chemical, tactile...) simultaneously, prioritize specific signals above others, or a combination of the two. Scorpions use their sensory organs when assessing prey and potential risk factors to determine the amount of venom required to adequately defend themselves while simultaneously being metabolically conservative. However, there is still uncertainty as to which environmental signals contribute the most to this decision-making process. The purpose of this study is to examine the role vision plays in risk assessment of a standardized threat using Vaejovis carolinianus by assessing the volume of venom released, as well as the latency to and frequency of stings under various lighting and vision ablation conditions. Methods: Vaejovis carolinianus vision was reversibly ablated with layer of dark red wax, added either over their eyes (both median and lateral pairs) or across the width of the carapace just posterior to the median eyes. Additionally, the ablated/nonablated individuals were exposed to alternative chromatic conditions: red or green light. The subjects were then agitated with a parafilm covered prod, and their behavior was recorded over a one-minute duration. After each strike and subsequent release of venom onto the parafilm membrane, a one microliter capillary tube (Drummond cat. #1-000-0010, internal diameter = 0.2 mm) was used to collect the venom. The type (clear/proteinaceous) and volume of venom was calculated. Each scorpion was tested

under every treatment combination. We used a repeated-measures design with each scorpion being exposed to each of the four conditions. All data were analyzed using a both linear and generalized linear mixed model using (Jamovi 1.0.7). Discussion/Conclusion: We did not detect a noticeable effect of the varying light or ablation conditions on venom expenditure. However, we found a statistically significances due to sex, with females more likely to deliver venom when they sting and in larger quantities. These initial results suggest that vision might not be an important sensory modality for modulating defensive behavior in this species. Key Words: vision ablation, scorpion, venom optimization hypothesis VAB12 - THE PROGRESSIVE EFFECTS OF ELECTRICAL EXTRACTION ON VENOM COMPOSITION AND SEXUAL VENOM VARIATION IN THE SOUTHERN DEVIL SCORPION VAEJOVISCAROLINIANUS Czaykowsky, C1, Carritte, M1, Cooper, P1, Mangunsong, J1, Peeke, J1, Corbit, AG1,*, Nelsen, DR1,* 1Southern Adventist University, 4881 Taylor Circle, Collegedale, Tennessee 37315 *Lab Supervisors E-mail address: dnelsen@southern.edu Background: The arachnid order Scorpionesis one of the oldest known venomous, terrestrial groups of animals. This group is represented by approximately 1,750 species whose morphology has largely remained unchanged. Scorpion venom contains many low molecular weight compounds: including enzymes, peptides, phospholipases, mucoproteins, biogenic amines, and other substances capable of producing a pathophysiological effects in their victims. Venom is of particular interest, not only for its medical impact in humans, but also for its therapeutic potential. Hence, there is a strong demand for efficient venom extraction,that not only acquires the venom, but also maintains the health and wellbeing of the scorpions for continued venom production. Several different extraction techniques are described in the literature. These include electrical stimulation, manual extraction, and glandular dissection, with electrical stimulation being the most commonly used technique. However, electrical stimulation is associated with physical trauma and a general decline in venom yield through time. In addition, questions remain as to whether the venom extracted from repeatedly milked individuals maintains its quality and thus accurately reflects the natural composition of wild populations. Further, the literature concerning electrical stimulation does not do a good job of reporting the extraction parameters, with most reporting either only the volts or current used but not both. In this study we used the southern devil scorpion, Vaejovis carolinianus, to test if electrical venom extraction negatively affected the scorpions physically and in terms of venom composition. Additionally, we investigated sexual venom variation.

Methods: Male and female scorpions were collected from the forest surrounding Southern Adventist University (near Chattanooga TN) from late August through early October. Scorpions were housed individually in 8 oz deli containers with a mulch substrate. Each scorpion was watered and fed weekly. Scorpions were anesthetized with CO2, restrained, and venom was electrically extracted by applying a 0.1 amp and 9 volt pulse through feather weight forceps soaked in hypersaline water. Expressed venom was collected in 5 uL capillary tubes and each tube was immediately frozen after venom expulsion ceased. Each scorpion was milked a total of three times, with a two weeks interval between each milking to allow for venom regeneration. One group was allowed a 3 weeks regeneration time to examine the effects of a longer period for regeneration. We recorded body condition (particularly of the metasoma and stinger) and presence/absence of venom expression. Venom composition was analyzed using RP-HPLC. Discussion/Conclusion: Preliminary RP-HPLC results suggest that there are minor but consistent chromatographic differences between the venoms from successive milkings and between sexes. Physically, we noticed a significant reduction in total number of individuals that expressed venom after each milking and a significant increase the proportion of individual with tail damage. Our findings suggest that electrical extraction of venom at 0.1 amp and 9 volts in this species is sufficient to produce long term consequences, both in terms of venom composition and physical wellbeing. Thus, future research needs to investigate if changing the electrical parameters can maintain venom composition, produce a consistent venom yield, and allow scorpions to thrive through this process. Key Words: Vaejovis carolinianus, scorpion venom, venom profile, RP-HPLC, toxicity, electrical venom extraction VAB13 - EVOLUTIONARY DRIVERS OF VENOM VARIATION IN THE PRAIRIE RATTLESNAKE (CROTALUS VIRIDIS VIRIDIS) Smith, CF1*, Schield, D2, Balchan, N1, Perry, B2, Nikolakis, Z2, Orton, R2, Castoe, TA2, Mackessy, SP1

1School of Biological Sciences, 501 20th Street, University of Northern Colorado, Greeley, CO 80639 USA; 2Department of Biology & Amphibian and Reptile Diversity Research Center, 501 S. Nedderman Drive, University of Texas at Arlington, Arlington, TX 76019 USA Background: Snake venoms are complex mixtures comprised primarily of potent bioactive proteins used for prey incapacitation and defense. Venom composition has been shown to vary geographically, taxonomically, ontogenetically, and with dietary preference. One of North America’s most wide-ranging rattlesnakes, Crotalus v. viridis (the Prairie Rattlesnake), is found from northern Mexico through the plains of the western United States to southern Canada. Coupled with its wide species range, C. v. viridis is also a

habitat generalist, and inhabits areas in close proximity to humans, increasing the likelihood of human encounters and bites from this snake. Typically described as a Type 1 venom, Crotalus v. viridis venom is known to induce hemorrhage and muscle degradation due to the presence of large enzymes like snake venom metalloproteases (SVMPs), snake venom serine proteases (SVSPs) and smaller nonenzymatic muscle toxins (myotoxins). Though previous research on C. v. viridis has shown geographic variation in some venom toxins, to date, no analysis has investigated broad-ranging variation in the entire venom proteome of this species. We investigate venom variation in C. v. viridis throughout the United States by determining geographic population-level patterns in the abundance of major toxins. We also identify the environmental, dietary, and phylogenetic drivers of the venom variation observed in this wide-ranging species. Methods: Venom compositional analysis was performed using SDS-PAGE, reverse-phase high performance liquid chromatography (HPLC), mass spectrometry and biochemical assays. The relationships between broad environmental variables (e. g. precipitation, temperature, elevation, habitat) and venom composition were used in preliminary ecological analysis in ArcMap. Gut content analysis of preserved museum specimens was used to determine dietary trends throughout the range of C. v. viridis, and RADseq was used to determine phylogeographic history. Discussion/Conclusion: The geographic distribution of venom toxins throughout the range of C. v. viridis straddles the Type 1-Type 2 venom compositional dichotomy previously characterized in rattlesnakes, as there appears to be a trade-off between dominantly enzymatic and nonenzymatic venom phenotypes. Further, variation in the abundance of major toxins correlates with latitudinal shifts in dietary availability and preference through the range of C. v. viridis. Ultimately, patterns of venom variation in C. v. viridis can provide clues about venomous snake foraging ecology and the major drivers of venom evolution. Key Words: Crotalus venom, venom evolution, snake venom metalloproteases, myotoxins VAB14 - TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF THE VENOM FROM THE WOLF SPIDER, HOGNA LENTA Weedon, EJ, Ellsworth, SA, Rokyta, DR Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA Email Address: ejw17@my.fsu.edu Background: Spiders are a skillful class of venomous invertebrates that utilize their venom to immobilize their prey and as a defense mechanism against threats. Spider venom is composed of peptides that have inhibiting properties called neurotoxins which specializes in blocking ion channels in cells and can have effects ranging from temporary paralysis to severe nervous system damage. Lycosidae is a family of venomous spiders found worldwide from sandy beaches to mountainous terrains. Spiders in Lycosidae are

characterized by their active hunting style unlike other web spinning spiders. Much like their habitats, these spiders range in size from a few millimeters to about three centimeters with males usually being smaller than the females. Their diet consists of mostly insects and smaller spiders. Female wolf spiders provide material care for their young and carry newly hatched offspring on her back until they can venture on their own. While venom produced from these spiders are harmless to people, it contains several neurotoxins responsible for its venomous properties. Other studies have identified that lycotoxins, a type of antimicrobial peptide, are commonly found in the Lycosidae family. However, an in-depth investigation characterizing the complete diversity of lycosid venom has yet to be completed. To better understand Lycosidae venom and its properties, the venom proteome and venom gland transcriptome of Hogna lenta were analyzed to provide an in-depth characterization of the venom. Methods: Two individuals, a male and a female were collected in Apalachicola National Forest in northern Florida and underwent venom extractions through electrostimulation. .Venom samples were analyzed using mass spectrometry and this data was compared against the transcriptomic data to identify potential toxins. Four days following venom extractions, venom glands from both individuals were extracted and mRNA was isolated for sequencing. Raw sequence data was assembled using multiple bioinformatic programs and contigs were annotated using UniProt toxin database and BLASTX. The assembled contigs were then compared to the analyzed proteins and were considered toxins if they were detected in both the transcriptome and proteome. Conclusion: Because so little is known about the chemical makeup of Lycosidae venom, there is potential to further scientific understanding of venom as a complex evolutionary mechanism. An in-depth analysis and characterization of Lycosidae venom has the potential to help identify not only the conserved components of venom but also components unique to each species. Preliminary results have indicated that lycotoxin and other neurotoxins are present in the Hogna lenta venom. This species of wolf spider will be the first of its family to have a complete transcriptome and proteome assembled. Key words: Hogna venom, proteome, transcriptome, lycotoxins VAC - VENOMOUS ANIMAL COLLECTIONS VAC1 - HEAT SEEKING MISSLES IN CAPTIVITY: HUSBANDRY OF BOTHROPS Harrison J. 1* 1Kentucky Reptile Zoo, 200 L and E Railroad Pl., Slade KY 40376, USA E-mail address: kyreptilezoo@kyreptilezoo.org

Background: Snakes in the genus Bothrops are useful in venom research but can be challenging captives. They are also a significant cause of snakebite in Central and South America, necessitating their venom collection to produce antivenoms. These snakes can be large and fast moving, dangerous to care for, and also easily stressed and difficult to acclimate in captivity. Recently animal rights advocates have been pushing for more humane treatment of all animals maintained for scientific or medical use in captivity. It is important to develop and spread methods of successful maintenance and reproduction of these snakes so they can continue to be used for research and antivenom. Methods: Various husbandry techniques will be shared, including methods of acclimation to captivity, breeding and raising of young, restraint techniques, ways to reduce stress, and extraction methods. Methods that are specific to Bothrops will be noted. Discussion/Conclusion: Snakes in the genus Bothrops can be maintained for decades in captivity and reproduce successfully while providing venom. Key Words: Bothrops, captive husbandry, venom production, breeding VAC2 - COME HITHER, KING: ATTEMPTING TO TARGET TRAIN KING COBRAS (OPHIOPHAGUS HANNAH) Wiley, K1,* 1Kentucky Reptile Zoo, 200 L and E Railroad Pl., Slade KY 40376, USA E-mail address: kyreptilezoo@kyreptilezoo.org Background: As the world’s largest venomous snake, king cobras can present some challenges in captivity, simply because of their large size and potential to inject huge quantities of venom in a bite. Operant conditioning is a way to improve safety and animal welfare by shaping desired behaviors and movements without using force or traditional handling methods. Applying training methods to snakes is challenging as they cannot hear the clicker typically used as a bridge. Training reptiles with an aggressive feeding response can also be challenging as they can be very reactive to anything associated with food. Methods: Target training, using traditional operant conditioning with a food reward, was applied to four king cobras, all chosen for being of moderate size. All snakes were adults and already feeding on prekilled food. Discussion/Conclusion: Target training can be a viable option to manage large venomous snakes in captivity, but challenges exist and it is not a substitute for safe handling methods. Used in conjunction with more typical methods it can enhance keeper

safety and potentially serve to enrich the snakes’ lives as well. Key Words: Ophiophagus hannah, king cobra, captive management, husbandry, enrichment, operant conditioning, target training VET – VETERINARY TOXINOLOGY VET1 - RETROSPECTIVE EVALUATION OF MICRURUS FULVIUS (EASTERN CORAL SNAKE) ENVENOMATION AND THE USE OF MECHANICAL VENTILATION IN DOGS & CATS Campos, SK (1), Allen-Durrance, AE (1), Schaer, M (1), Lynch, AM (2) (1) Department of Small Animal Clinical Sciences, University of Florida, Gainesville, Florida, USA (2) Department of Clinical Sciences, North Carolina State University College of Veterinary Medicine, Raleigh, North Carolina, USA Email address: scampos@ufl.edu Background: Coral snake venom contains potent α-neurotoxins that competitively block the postsynaptic nicotinic acetylcholine receptor site of the neuromuscular junction, leading to diffuse lower motor neuron signs ranging from generalized musculoskeletal weakness to paralysis and death by ventilatory failure. In addition to the potent neurotoxin, the venom contains phospholipase A2 which can cause intravascular hemolysis in some dogs and rodents, clinically manifesting as hemoglobinuria, hemoglobinemia, and anemia. Mechanical ventilation (MV) is indicated in envenomated animals when severe respiratory muscle weakness has developed, leading to progressive hypoventilation, hypercarbia, and eventually hypoxemia. The objectives of this study were to describe the use of mechanical ventilation in the management of Eastern coral snake envenomation in dogs and a cat. Methods: Retrospective data was collected and included signalment, physical and laboratory characteristics at presentation, clinical course during hospitalization, management including antivenom administration, mechanical ventilation settings, duration of ventilation, length of hospitalization, cost of care, and survival to discharge. Coral snake antivenom (either a F(ab’)2 or IgG coral snake antivenom) was administered to 7 of the 8 animals. All animals had progressive hypoventilation and received positive pressure ventilation, specifically volume controlled, synchronized intermittent mandatory ventilation with pressure support. Discussion/Conclusion: 7 out of 8 patients survived; the single dog that was not successfully weaned from the ventilator developed AKI with subsequent evidence of multiple organ dysfunction including acute respiratory distress syndrome resulting in death. Of the survivors, the median duration of MV was 58 hours (range 25-84 hours) and the median duration of hospitalization was 8.2 days (range 6-11 days). Ventilator associated

complications occurred in 8 of 8 animals, but overall outcome was excellent with 7 of 8 surviving to discharge. Ventilatory failure secondary to Eastern coral snake envenomation necessitating mechanical ventilation carries an excellent prognosis and better than reported for other causes of lower motor neuron disease. Adverse reactions to antivenom were rare, regardless of the type administered. Keywords: small animal critical care, environmental toxins, neuromuscular disorders, peripheral nervous system disorders, small animal respiratory VET2 - USE OF SONOCLOT® TO ASSESS THE IN VITRO EFFECT OF AGKISTRODON CONTORTRIX LATICINCTUS ON COAGULATION IN CANINE AND EQUINE WHOLE BLOOD Moore, A1, Lyon, S1, Di Girolamo, N1, Gilliam, L1* 1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74078 E-mail address: l.gilliam@okstate.edu Background: Coagulopathy is common following pit viper envenomation. Species susceptibility to snake venom has been recognized for decades; however, peer reviewed studies of coagulation differences among species following venom exposure are lacking. Coagulation disturbances among species differ and the tendency toward hyper or hypocoagulation following envenomation could alter treatment strategies.Venom induced coagulopathy is multi-factorial. Traditional coagulation testing (platelet count, PT, and aPTT) does not provide a comprehensive view of coagulation and while useful for determining hypocoagulation they are not as useful in determining hypercoagulation. In addition, platelet counts do not assess platelet function. A dynamic look at coagulation more completely evaluates the coagulation process. Dynamic viscoelastic coagulometry, Sonoclot®, is one method of dynamic coagulation testing. Sonoclot® utilizes a whole blood sample and rapid, simple patient side technology to assess the coagulation process. The software calculates values for platelet function, activated clotting time (fibrin formation), clot rate (fibrinogen to fibrin) and time to peak (time to reach peak clot strength). This technology could allow for a more comprehensive view of coagulation in envenomation cases. The objective of this study was to investigate the ability of Sonoclot® to detect coagulation disturbances caused by Agkistrodon contortrix laticinctus in both dogs and horses. Methods: The study enrolled 13 healthy equine and 8 healthy canine subjects. Blood was collected from the jugular vein of each subject and separated into two sodium citrate tubes. Lyophilized Agkistrodon contortrix venom was weighed in the amount of 10 milligrams. A total of 1 milliliter of sterile water was added to make a 10mg/ml solution. This stock solution was used to make serial dilutions. Venom was added to blood tubes to a concentration of 25ug/ml, 50ug/ml and 100ug/ml. Samples sat undisturbed for 30 minutes

and then Sonoclot® analysis was performed per manufacturer instructions. Generalized linear mixed models were employed to evaluate the association between species, venom concentration and the 5 outcomes (ACT, clot rate, platelet function, time, max clot), with individual animals included as random effects. Results: There was a significant effect of venom on ACT at concentration of 25, 50 and 75 ug/ml in dogs and 50 ug/ml in horses. There was a significant difference in clot rate depending on the species, and at venom concentrations at 25 and 50 ug/ml. There was a significant difference in platelet function depending on the species, and a significant effect of venom at concentrations of 75 and 100 ug/ml in dogs. There was a significant difference in time depending on the species, but no overt effect of venom concentrations. Regarding max clot there was a significant effect of venom at 25 ug/ml in dogs as compared to no venom, but no overall difference between species. Discussion/Conclusion: Sonoclot was useful in detecting variations in factors related to coagulation in both species’ whole blood samples after the addition of Agkistrodon contortrix venom. Most values evaluated were not linearly affected by increasing concentrations of venom. Sonoclot may be a good addition to traditional coagulation testing when evaluating patients suffering from Agkistrodon contortrix envenomation. Key Words: Agkistrodon venom, viscoelastic testing, coagulation, canine, equine VET3 - ONE HEALTH TOXINOLOGY Woods, C1 1Institute for Healthcare Innovation, Midwestern University, 19555 N. 59th Avenue, Glendale AZ 85308 USA. E-mail address: cwoods@midwestern.edu Background: The One Health Initiative concerns inter-disciplinary collaborations among healthcare professionals to advance scientific knowledge and medical care. In the context of drug development, One Health collaborations propose to de-risk product development, improve clinical trial design, and permit parallel development for human and veterinary medical applications. By some estimates, the cost of developing a new chemical entity exceeds a billion dollars when accounting for failure rates. Excessive failure rates suggest pre-clinical studies are suboptimal for predicting human clinical outcomes. There is a growing interest in accessing veterinary patients to improve translational drug development and the design of human clinical trials. This presentation will cover a One Health approach in the context of toxinology, and address (a) implementing comparative veterinary studies (VCSs) for venom derived drug development, (b) associated costs and regulations of comparative veterinary studies, and (c) how One Health collaborations may lower clinical development costs. Methods: To establish VCSs potential impact on the cost of drug development, the

author selected pain as the therapeutic category. Pain is a therapeutic focus for many venom derivatives, and is considered a naturally occurring veterinary comparative disease to humans. Pain drug development costs were obtained from published literature and commissioned reports to establish a pre-clinical through registration expenditure table. Revised drug development costs were calculated to account for the cost of VCSs and the cost of capital associated with longer development timelines. Then, PII percent failure rates were incrementally adjusted to determine the financial impact of integrating VCSs prior to Phase I human studies. Results: The analysis revealed that VCSs have the potential to dramatically lower the cost to develop pain drugs despite increased development timelines and cost of VCSs. Conclusions: VCSs propose to de-risk drug candidates prior to entering human clinical trials. In addition, VCS may serve to increase asset valuation and provide a revenue stream from veterinary applications, and are feasible from a regulatory, cost and temporal perspective. Key Words: One Health, drug development, comparative medicine, translational medicine