Crotalus Durissus Research Articles

Myotoxicity of Crotoxin on C2C12 Myoblasts and its Inhibition by Crotalus Neutralizing Factor versus Enhanced Resistance in Myotubes: Exploring Toxicity and Membrane Potential.

Crotalus Neutralizing Factor (CNF) is a γ-type Phospholipase A2 (PLA2) inhibitor present in the blood of Crotalus durissus terrificus snake. Particularly, CNF inhibits the toxic action of Crotoxin (CTX), which is a major neurotoxin found in C. d. terrificus venom. CTX induces also myotoxic action and demonstrates high selectivity for skeletal muscle fibers. Consequently, CTX can diffuse beyond the site of infection, which can potentially evoke rhabdomyolysis. The present study has evaluated the effects of CTX on myoblasts and myotubes of muscle cells C2C12 in vitro and the effect of CNF on CTX-induced damage. Cytotoxicity assays were performed by measuring the mitochondrial enzyme dehydrogenase levels. Furthermore, creatine kinase and lactate dehydrogenase levels were used as indicators of muscle damage. Crotoxin has been found to have cytotoxic effects on C2C12 myoblast cells, while CNF has not shown toxic effects on these cells. Furthermore, the findings have shown CNF (50 μg/mL) to abolish CTX toxicity in myoblasts. The myotubes, differentiated cells, showed no change in mitochondrial respiration when exposed to CNF or CTX, showing greater resistance to the toxic actions of crotoxin. The data have confirmed the potential of CNF as an anti-myotoxic agent to prevent CTX-damaged myoblasts and increase resistance to the toxic effects of crotoxin on differentiated cells.

Hyperglycosylation impairs the inhibitory activity of rCdtPLI2, the first recombinant beta-phospholipase A2 inhibitor

Crotoxin, a phospholipase A2 (PLA2) complex and the major Crotalus venom component, is responsible for the main symptoms described in crotalic snakebite envenomings and a key target for PLA2 inhibitors (PLIs). PLIs comprise the alpha, beta and gamma families, and, due to a lack of reports on beta-PLIs, this study aimed to heterologously express CdtPLI2 from Crotalus durissus terrificus venom gland to improve the knowledge of the neglected beta-PLI family. Thereby, recombinant CdtPLI2 (rCdtPLI2) was produced in the eukaryotic Pichia pastoris system to keep some native post-translational modifications. rCdtPLI2 (~41 kDa) presents both N- and O-linked glycans. Alpha-mannosidase digested-rCdtPLI2 (1 mol) strongly inhibited (73 %) CB-Cdc catalytic activity (5 mol), demonstrating that glycosylations performed by P. pastoris affect rCdtPLI2 action. Digested-rCdtPLI2 also inhibited PLA2s from diverse Brazilian snake venoms. Furthermore, rCdtPLI2 (1 mol) abolished the catalytic activity of Lmr-PLA2 (5 mol) and reduced the CTx-Cdc (5 mol) enzyme activity by 65 %, suppressing basic and acidic snake venom PLA2s. Additionally, crotalic antivenom did not recognize rCdtPLI2, suggesting a lack of neutralization by antivenom antibodies. These findings demonstrate that studying snake venom components may reveal interesting novel molecules to be studied in the snakebite treatment and help to understand these underexplored inhibitors.

Sperm storage in males of the Neotropical rattlesnake Crotalus durissus (Squamata: Viperidae): Structure and seasonal variation of the distal ductus deferens

In species with asynchronous reproductive cycles, where gamete production is not aligned with the mating season, either males or females must store sperm. This reproductive tactic is an obligatory feature of male rattlesnakes’ reproductive cycle due to asynchrony between spermatogenesis and mating. Given that the ductus deferens is the primary site of sperm storage in male snakes, we aimed to investigate the morphological and histochemical changes in the distal ductus deferens of C. durissus throughout its reproductive cycle. In this species, spermatogenesis begins in spring and peaks in summer, while testes regress during autumn and winter. The distal ductus deferens of 28 mature male specimens was evaluated using histomorphometric and histochemical methods. Spermatozoa were consistently observed within the lumen of the ductus deferens in almost all specimens. The principal cells of the distal region of ductus deferens reacted positively for Periodic Acid-Schiff and Bromophenol Blue. Secretions were observed in the apical region of the principal cells' cytoplasm and along the epithelium edge, which may be related to gamete maintenance. Increased secretory activity of the principal cells was observed during periods of testicular activity. A reduction in the lumen of ductus deferens occurs during testicular regression, indicating possible fluid resorption by epithelial cells. Fluid resorption might be one of the mechanisms to ensure stored sperm viability, as it provides an increase in the glycoprotein’s concentration.

Bioprospection of rattlesnake venom peptide fractions with anti-adipose and anti-insulin resistance activity in vitro

Animal venoms are natural products that have served as a source of novel molecules that have inspired novel drugs for several diseases, including for metabolic diseases such as type-2 diabetes and obesity. From venoms, toxins such as exendin-4 (Heloderma suspectum) and crotamine (Crotalus durissus terrificus) have demonstrated their potential as treatments for obesity. Moreover, other toxins such as Phospholipases A2 and Disintegrins have shown their potential to modulate insulin secretion in vitro. This suggests an unexplored diversity of venom peptides with a potential anti-obesogenic in Mexican rattlesnake venoms. For that reason, this study explored the in vitro effect of Crotalus venom peptide-rich fractions on models for insulin resistance, adipocyte lipid accumulation, antioxidant activity, and inflammation process through nitric oxide production inhibition. Our results demonstrated that the peptide-rich fractions of C. aquilus, C. ravus, and C. scutulatus scutulatus were capable of reverting insulin resistance, enhancing glucose consumption to normal control; C. culminatus, C. molossus oaxacus, and C. polystictus diminished the lipid accumulation on adipocytes by 20%; C. aquilus, C. ravus, and C. s. salvini had the most significant cellular antioxidant activity, having nearly 80% of ROS inhibition. C. aquilus, C. pyrrhus, and C. s. salvini inhibited nitric oxide production by about 85%. We demonstrated the potential of these peptides from Crotalus venoms to develop novel drugs to treat type-2 diabetes and obesity. Moreover, we described for the first time that Crotalus venom peptide fractions have antioxidant and inflammatory properties in vitro models.

Snakebites by venomous snakes in Brazilian serpentaria and zoos over a 10-year period (2012–2021)

Despite the wide range of institutions that maintain venomous snakes in captivity in Brazil there are no comprehensive data on the occurrence of snakebites and envenomations in these places. We examined the range of native and exotic species of venomous snakes kept by Brazilian zoos and serpentaria (scientific and commercial) and assessed the frequency of snakebites in workers handling these snakes during a 10-year period (2012–2021). Twenty-two (73.3%) of 30 institutions returned a standard questionnaire, including 15 serpentaria and 7 zoos that together kept 10,607 venomous snakes in 2022/2023. Commercial and scientific serpentaria had many more snakes (n = 10,550, consisting of 10,499 native specimens and 51 exotic specimens) than zoos (n = 57 native specimens), with two genera accounting for the majority of native species (Bothrops spp. = 84.5% and Crotalus durissus ssp. = 13.5%). Thirty-seven snakebites were reported and involved primarily the hands (33), seven of which occurred during venom extraction and 30 in other circumstances, most of them while handling/manipulating the cages or snake boxes (10) and restraining (9) or feeding (5) the snake. In addition, there were two cases of venom accidently sprayed on the face, including the eyes. Most bites were caused by Bothrops spp. (31), followed by C. durissus ssp. (4), Lachesis muta (1) and Micrurus corallinus (1). Thirty-three bites (89.2%) were treated with antivenom, with four bites to the fingers by Bothrops spp. resulting in local functional sequelae. There were 366,918 venom extractions with a ratio of 1.9 bites/100,000 extractions; no bites were recorded in the six institutions that sedated the snakes prior to venom extraction, which accounted for 22.7% of all extractions. These findings show that although snakebites are rare in Brazilian zoos and serpentaria, severe envenomation may occur. The occurrence of snakebites could be reduced by measures such as sedation of the snakes before venom extraction.

Insights into the mechanism of crotamine and potential targets involved in obesity-related metabolic pathways

Crotamine (Ctm) is a peptide isolated from Crotalus durissus terrificus venom. This molecule has been demonstrated to diminish body weight gain and enhance browning in adipose tissue, glucose tolerance, and insulin sensitivity; hence, it has been postulated as an anti-obesogenic peptide. However, the mechanism to elicit the anti-obesogenic effects has yet to be elucidated. Thus, we investigated the possible interaction of Ctm with receptors involved in obesity-related metabolic pathways through protein-protein docking and molecular dynamics refinement. To test the anti-obesogenic mechanism of Ctm, we selected and retrieved 18 targets involved in obesity-related drug discovery from Protein Data Bank. Then, we performed protein-protein dockings. The best three Ctm-target models were selected and refined by molecular dynamics simulations. Molecular docking demonstrated that Ctm was able to interact with 13 of the 18 targets tested. Having a better docking score with glucagon-like peptide-1 receptor (GLP-1R) (−1430.2 kcal/mol), DPP-IV (dipeptidyl peptidase-IV) (−1781.7 kcal/mol) and α-glucosidase (−1232.3 kcal/mol). These three models were refined by molecular dynamics. Ctm demonstrated a higher affinity for GLP-1R (ΔG: −41.886 ± 2.289 kcal/mol). However, Ctm interaction was more stable with DPP-IV (RMSD: 0.360 ± 0.015 nm, Radius of gyration: 2.781 ± 0.009 nm). Moreover, the number of interactions and the molecular mechanics energies of Ctm residues suggest that the interaction of Ctm with these receptors is mainly mediated by basic-hydrophobic dyads Y1-K2, W31-R32, and W33-R34. Together, all these results allow elucidating a possible molecular mechanism behind the previously described anti-obesogenic effects.

Crotamine derived from Crotalus durissus terrificus venom combined with drugs increases in vitro antibacterial and antifungal activities.

This study investigated crotamine (CTA), a peptide derived from the venom of the South American rattlesnake Crotalus durissus terrificus, known for its exceptional cell penetration potential. The objective was to explore the antibacterial and antifungal activity of CTA, its ability to inhibit efflux pumps and evaluate the effectiveness of its pharmacological combination with antibiotics and antifungals. In microbiological assays, CTA in combination with antibiotics was tested against strains of S. aureus and the inhibition of NorA, Tet(K) and MepA efflux pumps was also evaluated. CTA alone did not present clinically relevant direct antibacterial action, presenting MIC > 209.7µM against strains S. aureus 1199B, IS-58, K2068. The standard efflux pump inhibitor CCCP showed significant effects in all negative relationships to assay reproducibility. Against the S. aureus 1199B strain, CTA (20.5µM) associated with norfloxacin diluted 10 × (320.67µM) showed a potentiating effect, in relation to the control. Against the S. aureus IS-58 strain, the CTA associated with tetracycline did not show a significant combinatorial effect, either with 2304 or 230.4µM tetracycline. CTA at a concentration of 2.05µM associated with ciprofloxacin at a concentration of 309.4µM showed a significant potentiating effect. In association with EtBr, CTA at concentrations of 2.05 and 20.5µM potentiated the effect in all strains tested, reducing the prevention of NorA, Tet(K) and MepA efflux pumps. In the C. albicans strain, a potentiating effect of fluconazole (334.3µM) was observed when combined with CTA (2.05µM). Against the C. tropicalis strain, a significant effect was also observed in the association of fluconazole 334.3µM, where CTA 2.05µM considerably reduced fungal growth and decreased the potentiation of fluconazole. Against the C. krusei strain, no significant potentiating effect of fluconazole was obtained by CTA. Our results indicate that CTA in pharmacological combination potentiates the effects of antibiotics and antifungal. This represents a new and promising antimicrobial strategy for treating a wide variety of infections.

Biotechnological potential of Brazilian snake venom in the production of biologically active drugs: Literature review

Despite being feared, Brazilian venomous snakes can be reservoirs of important resources of medicinal interest, since the venoms of these animals have an invaluable potential for biologically active substances found in nature. Pharmacological and biochemical studies carried out in recent decades have shown the diversity of proteins with enzymatic activity, toxins, peptides, bioactive amines, among other compounds in snake venoms. The aim of this literature review was to describe the pharmaceutical biotechnological potential of Brazilian snake venom and to demonstrate the importance of these toxins and their pharmacological derivatives for public health. Since 1894, when they were studied by Vital Brazil, molecules derived from Brazilian snake venom have shown significant biological activity. These molecules have been proven to be efficient antimicrobial, antifungal and antiprotozoal agents, mainly due to their activity as serine proteases, hyaluronidases, L-amino acid oxidases (LAAO) and acetylcholinesterases, among other well-known substances. Brazilian snake venoms have thus been the target of a series of studies, which have borne excellent fruit, for example in the development of various drugs, such as Captopril®, which is derived from a peptide isolated from the venom of Bothrops jararaca, and fibrin sealant, derived from molecules in the venom of Crotalus durissus terrificus associated with cryoprecipitate from the blood plasma of buffaloes. With the analysis of biotechnologies applied to various molecules with the capacity for pharmacological, biologically active action, it is clear that this field is vast for new research and due to the great diversity of compounds in snake venoms. Certainly, as research progresses, new biologically active drugs derived from snake venom should become available for use and benefit of the population. Thus, studies related to the chemical composition of snake venoms should be encouraged, as well as those related to their pharmaceutical biotechnological potential.

The relevance of vascular adjustments to hemodynamic control in the face of temperature change in Crotalus durissus.

The presence of cardiac shunts in ectothermic tetrapods is thought to be consistent with active vascular modulations for proper hemodynamic support. Local control of blood flow modulates tissue perfusion and thus systemic conductance (Gsys) is assumed to increase with body temperature (Tb) to accommodate higher aerobic demand. However, the general increase of Gsys presses for a higher right-to-left (R-L) shunt, which reduces arterial oxygen concentration. In contrast, Tb reduction leads to a Gsys decrease and a left-to-right shunt, which purportedly increases pulmonary perfusion and plasma filtration in the respiratory area. This investigation addressed the role of compensatory vascular adjustments in the face of the metabolic alterations caused by Tb change in the South American rattlesnake (Crotalus durissus). Cardiovascular recordings were performed in decerebrated rattlesnake preparations at 10, 20 and 30°C. The rise in Tb increased metabolic demand, and correlated with an augmentation in heart rate. Although cardiac output increased, systemic stroke volume reduced while pulmonary stroke volume remained stable. Although that resulted in a proportionally higher increase in pulmonary blood flow, the R-L shunt was maintained. While the systemic compliance of large arteries was the most relevant factor in regulating arterial systemic blood pressure, peripheral conductance of pulmonary circulation was the major factor influencing the final cardiac shunt. Such dynamic adjustment of systemic compliance and pulmonary resistance for shunt modulation has not been demonstrated before and contrasts with previous knowledge on shunt control.

Arsenic and metal levels in snake tissues from Lagoa Santa Karst, Brazil.

Concentrations of one metalloid (As) and eight metals (Cd, Cr, Cu, Hg, Mn, Ni, Pb, and Zn) were determined in tissues (muscle, liver, and kidney) of eight snake species (Bothrops neuwiedi, Crotalus durissus, Dipsas mikanii, Epicrates crassus, Helicops modestus, Micrurus carvalhoi, Oxyrhopus guibei, and Oxyrhopus trigeminus) from Lagoa Santa Karst. Except for Cu and Zn, all other analyzed elements were detected in concentrations within the ranges previously reported for snakes inhabiting polluted areas, emphasizing Hg (specific Hg mean concentrations varied from 0.87 to 9.76μgg-1 d.w). The highest mean concentrations of all elements except Zn were found in muscle samples of the false corals O. guibei (means ranged from 2.01 [Pb] to 9.76 [Hg]). The highest Zn mean concentration (13.77μgg-1 d.w) was detected in the kidney of the water snake H. modestus. No significant correlation was found between element concentrations and body size for all species. Significant interorgan differences were observed for As, Cr, Cu, Hg, Mn, Pb, and Zn concentrations in the three tissues in H. modestus. Significant interspecific differences were found in at least one organ for all elements. Significant pairwise differences were found between diet specialist species and between these species and broader diet species, while no significant difference was found between the broader diet species. The bioaccumulation of As and metals in snakes from Lagoa Santa Karst could be associated with natural rock dissolution and erosion processes but also with the wide-scale mining in the region and the increased agriculture and urbanization.

Crotoxin induces cytotoxic effects in human malignant melanoma cells in both native and detoxified forms.

Introduction: Melanoma, a highly aggressive skin cancer originating in melanocytes, poses a significant threat due to its metastatic potential. While progress has been made in treating melanoma with targeted therapies and immunotherapies, challenges persist. Crotoxin (CTX), the principal toxin in Crotalus durissus terrificus snake venom, exhibits various biological activities, including anti-tumoral effects across multiple cancers. However, its clinical use is limited by toxicity. Thus, exploring alternatives to mitigate adverse effects is crucial. Methods and Results: This study investigates the antitumoral potential of CTX in its native and in a detoxified form, in melanoma cells. Firstly, we demonstrated that detoxified CTX presented reduced phospholipase activity. Both forms proved to be more cytotoxic to SK-MEL-28 and MeWo melanoma cells than non-tumoral cells. In SK-MEL-28 cells, where cytotoxic effects were more pronounced, native and detoxified CTX induced increased necrosis and apoptosis rates. We also confirmed the apoptosis death demonstrated by the activation of caspase-3 and 7, and the formation of apoptotic bodies. Furthermore, both CTX caused cell cycle arrest at the G2/M phase, interfering with melanoma cell proliferation. Cell migration and invasion were also suppressed by both CTX. These results confirm the antitumoral potential of CTX. Discussion: The maintenance of the antiproliferative effects in the detoxified version, with reduced enzymatic activity often liked to harm effects, supports further studies to identify active parts of the molecule responsible for the interesting effects without causing substantial toxic events, contributing to the future use of CTX-derived drugs with safety and efficacy.

Ascorbyl palmitate (ASC16) as a potential inhibitor of toxicity induced by Crotalus durissus terrificus venom

It is well known that C. d. terrificus venom causes pathophysiological effects such as neuropathies, coagulopathies, and even death. Previous studies have reported that ASC16 can interact with monomeric phospholipases A2 from the venom of various snake species (e.g., Vipera russelli and Echis carinatus). As a result, ASC16 has been proposed as an inhibitor of the toxic effects induced by the heterodimeric complex (crotoxin) and other components of the venom of C. d. terrificus. To investigate this further, in silico studies were designed using the crotoxin (CTX) protein complex as a model, and experimental assays were conducted to evaluate the inhibitory effect of ASC16 on CTX, as well as on other venom enzymes such as thrombin-like enzyme (TLE), phosphodiesterase (PDE) and l-aminoxidase (LAAO). For in vitro assays, specific substrates were used, and lethal activity was measured over 48 h using an in vivo murine experimental model (CF01). In silico studies have indicated that the hydrophilic portion of ASC16 adopts a stable conformation while interacting with the catalytic site of crotoxin. At the highest concentrations, ASC16 significantly inhibited the activities of PLA2 (40.89 ± 0.09 %), TLE (11.03 ± 0.69 %), PDE (51.33 ± 2.83 %), and LAAO (56.79 ± 2.91 %). Furthermore, ASC16 neutralized the 2 LD50 lethality of crotalic venom. These findings lay the groundwork for designing promising adjuvants that can facilitate the incorporation of a larger quantity of proteins in immunization schemes. Consequently, this approach aims to achieve higher antibody titers, reduce the number of required immunizations, and minimize local damage in the producer animal.

A Snake Venom Peptide and Its Derivatives Prevent Aβ42 Aggregation and Eliminate Toxic Aβ42 Aggregates In Vitro.

Over a century has passed since Alois Alzheimer first described Alzheimer's disease (AD), and since then, researchers have made significant strides in understanding its pathology. One key feature of AD is the presence of amyloid-β (Aβ) peptides, which form amyloid plaques, and therefore, it is a primary target for treatment studies. Naturally occurring peptides have garnered attention for their potential pharmacological benefits, particularly in the central nervous system. In this study, nine peptide derivatives of Crotamine, a polypeptide from Crotalus durissus terrificus Rattlesnake venom, as well as one d-enantiomer, were evaluated for their ability to modulate Aβ42 aggregation through various assays such as ThT, QIAD, SPR, and sFIDA. All tested peptides were able to decrease Aβ42 aggregation and eliminate Aβ42 aggregates. Additionally, all of the peptides showed an affinity for Aβ42. This study is the first to describe the potential of crotamine derivative peptides against Aβ42 aggregation and to identify a promising d-peptide that could be used as an effective pharmacological tool against AD in the future.

Defensive plasticity in South American rattlesnakes Crotalus durissus

Reptiles exhibit defensive responses in diverse ways, influenced by situational factors. Snakes, such as the South American rattlesnake Crotalus durissus, display complex defensive responses, adapting tactics based on circumstances when encountering potential predators. Environmental and intrinsic parameters shape snake behaviour during predator encounters. This study employs distinct stimuli to investigate behavioural changes and whether snakes adapt defensive strategies. We compare the behaviours of rattlesnakes before and after an aversive stimulus involving physical contact. Following encounters with higher-risk stimuli, snakes become more defensive.Findings reveal that snakes alter strategies based on threat intensity. This phenomenon might be influenced by the potential cost of defense and prior interactions with predators. The study underscores the intricate nature of snake defensive behaviour. Keywords: behavioural plasticity, antipredator behaviour, aversive stimulus, behavioural cost, active behaviours

Expanding anti-venom strategies: Camelid polyclonal antibodies with high capacity to recognize snake venom

Camelid immunoglobulins represent a unique facet of antibody biology, challenging conventional understandings of antibody diversification. IgG2 and IgG3 in particular are composed solely of heavy chains and exhibit a reduced molecular weight (90 kDa); their elongated complementarity determining region (CDR) loops play a pivotal role in their functioning, delving deep into enzyme active sites with precision. Serum therapy stands as the primary venom-specific treatment for snakebite envenomation, harnessing purified antibodies available in diverse forms such as whole IgG, monovalent fragment antibody (Fab), or divalent fragment antibody F (ab')2. This investigation looks into the intricacies of IgGs derived from camelid serum previously immunized with crotamine and crotoxin, toxins predominantly in Crotalus durissus venom, exploring their recognition capacity, specificity, and cross-reactivity to snake venoms and its toxins. Initially, IgG purification employed affinity chromatography via protein A and G columns to segregate conventional antibodies (IgG1) from heavy chain antibodies (IgG2 and IgG3) of camelid isotypes sourced from Lama glama serum. Subsequent electrophoretic analysis (SDS-PAGE) revealed distinct bands corresponding to molecular weight profiles of IgG's fractions representing isotypes in Lama glama serum. ELISA cross-reactivity assays demonstrated all three IgG isotypes' ability to recognize the tested venoms. Notably, IgG1 exhibited the lowest interactivity in analyses involving bothropic and crotalic venoms. However, IgG2 and IgG3 displayed notable cross-reactivity, particularly with crotalic venoms and toxins, albeit with exceptions such as PLA2-CB, showing reduced reactivity, and C. atrox, where IgGs exhibited insignificant reactivity. In Western blot assays, IgG2 and IgG3 exhibited recognition of proteins within molecular weight (≈15 kDa) of C. d. collilineatus to C. d. terrificus, with some interaction observed even with bothropic proteins despite lower reactivity. These findings underscore the potential of camelid heavy-chain antibodies, suggesting Lama glama IgGs as prospective candidates for a novel class of serum therapies. However, further investigations are imperative to ascertain their suitability for serum therapy applications.

Viperidae snakes infected by mammalian-associated trypanosomatids and a free-living kinetoplastid

Trypanosomatids have achieved significant evolutionary success in parasitizing various groups, yet reptiles remain relatively unexplored. The utilization of advanced molecular tools has revealed an increased richness of trypanosomatids in vertebrate hosts. The aim of this study was to identify the trypanosomatid species infecting Bothrops moojeni and Crotalus durissus kept in captivity from 2000 to 2022. Blood samples were obtained from 106 snakes: 73C. durissus and 33 B. moojeni. Whole blood was collected for hemoculture, blood smears and centrifugated to obtain the blood clot that had its DNA extracted and submitted to Nested PCR (18S rDNA gene) to detect Trypanosomatidae. Positive samples were quantified and submitted to both conventional (Sanger) and next generation sequencing (NGS). Cloning of the amplified PCR product was performed for only one individual of C. durissus. To exclude the possibility of local vector transmission, attempts to capture sandflies were conducted using six CDC-LT type light traps. Molecular diagnosis revealed that 34% of the snakes presented trypanosomatid DNA, 47.94% in C. durissus and 3.9% in B. moojeni. The cloning process generated four colonies identified as a new MOTU named Trypanosomatidae sp. CROT. The presence of DNA of five trypanosomatids (Trypanosoma cruzi TcII/VI, Trypanosoma sp. DID, Trypanosoma cascavelli, Trypanosomatidae sp. CROT, Leishmania infantum and Leishmania sp.) and one free-living kinetoplastid (Neobodo sp.) was revealed through NGS and confirmed by phylogenetic analysis. The haplotypic network divided the T. cascavelli sequences into two groups, 1) marsupials and snakes and 2) exclusive to marsupials. Therefore, the diversity of Kinetoplastea is still underestimated. Snakes have the ability to maintain infection with T. cruzi and L. infantum for up to 20 years and the DNA finding of Neobodo sp. in the blood of a C. durissus suggests that this genus can infect vertebrates.

Pre-clinical Efficacy and Immunogenicity of IgY Antibodies Directed Against Crotalus durissus cumanensis Venom

Background:: Crotalus durissus cumanensis (C.d.c.) is the most widely distributed snake in Venezuela, causing the majority of snakebite envenoming Objective:: The purpose of this study was to produce IgY antibodies against a C.d.c. venom pool from different Venezuelan regions and evaluate their neutralization capacity on various venom toxic activ-ities. Methods:: Anti-C.d.c. venom antibodies are purified from chicken egg yolks by precipitation with polyethylene glycol and further analyzed by Multiple Antigen Blot Assay, indirect ELISA, Western blot, and Inhibition assays. In addition, we evaluate the phospholipase, edematogenic, and hemor-rhagic activities. In addition, a new envenoming simulation study using anti-C.d.c. venom IgY in mice is presented. Results:: In this study, we show that anti-C.d.c. venom IgY is capable of neutralizing 4LD50 doses of the Cdc venom (i.e., 1.76 mg of IgY neutralized 14 μg of C.d.c. venom) and effectively neutralizing the phospholipase, edematogenic and hemorrhagic activities. Additionally, the anti C.d.c. venom IgY specifically recognizes polypeptide bands with apparent molecular masses of ~ 54.55, 30.39, 24.1, 14.02, and 9.44 kDa by western blot. The IgY specificity is demonstrated by a dose-dependent inhi-bition, in which antibodies pre-adsorbed with the C.d.c. venom does not recognize the proteins con-tained in the venom. Furthermore, in the simulation study of envenoming, the mice inoculated with IgY showed no response. Conclusion:: Our results support the use of anti-venom IgY as an alternative to traditional equine therapy in animals and, eventually, in human patients bitten by C.d.c snakes. conclusion: Our results support the use of anti-venom IgY as an alternative to traditional equine therapy in animals and, eventually, in human patients bitten by C.d.c snakes.

Seasonality in Crotalus durissus venom

Rattlesnakes belonging to the genus Crotalus are widely distributed throughout the Americas. In Brazil, symptoms commonly associated with envenomation by Crotalus durissus collilineatus include myalgia, rhabdomyolysis, renal failure, neurotoxicity, and progressive paralysis, which are related to the protein composition of this venom. Snake venom composition exhibits compositional variability that may reflect geographic distribution, age, captivity, diet, sex, and even individual genetics. Although seasonality is also considered a possible source of variation, there are few reports of such variability in snake venom. In this work, venoms of the same eight C. durissus collilineatus were extracted every three months for two years, to analyze seasonal changes in composition and activities. To this end, venom composition was analyzed by protein quantification, SDS-PAGE, and HPLC, and the LAAO, PLA2 and coagulant activities were measured. Venoms of these C. d. collilineatus showed minor seasonal differences in venom activities and no composition differences were found. LAAO and coagulant activities displayed a pattern of seasonal change, while PLA2 activity seemed to have no seasonality tendency. Also, there are sexual differences, in which males seem to be more stable than females in regard to some activities. Individual variability occurs even in seasonal variation of activities, highlighting the importance of controlling circumstances of venom extraction before comparing results between groups of snakes.

Effects of crotamine in human prostate cancer cell line

Our study presents the anticancer potential of crotamine from Crotalus durissus terrificus in human prostate cancer cell line DU-145. Crotamine isolation was conducted through RP-FPLC, its molecular mass analyzed by MALDI-TOF was 4881.4 kDa, and N-terminal sequencing confirmed crotamine identity. Crotamine demonstrated no toxicity and did not inhibit migration in HUVEC cells. Although no cell death occurred in DU-145 cells, crotamine inhibited their migration. Thus, crotamine presented potential to be a prototype of anticancer drug.

Effects of Prolonged Fasting on Cloacal Microbiota of Crotalus durissus with Different Corporal Scores

Aims: This study provides a detailed analysis of the composition and diversity of the bacterial community within the cloacal cavity of Crotalus durissus (Cascavel) under the influence of diet and the corporal score of breeding snakes through metagenomics analysis. For this aim, the study also provides a body condition score protocol for the classification of snakes as thin, normal, and obese. Study Design: The experimental groups were established from snake body conditions such as visibility of ribs, the aspect of the side view, the upper and ventral view, and the aspect of the final portion, of the neck and square bone. Place and Duration of Study: The research was conducted at S-Inova Laboratory at Don Bosco Catholic University, between December 2017 and February 2019 (Mato Grosso do Sul, Brazil). Methodology: There are 3 groups of score corporations: thin (n=3), normal (n=3), and obese (n=3). The first sample of each animal was obtained 7 days after feeding and the second sample after 50 days of fasting. The metagenomic DNA was extracted from all 18 swab samples through DNeasy® PowerSoil® Kit (Qiagen). Thereafter the samples were submitted to sequencing, performed using the 2 x 250bp paired-end sequencing on a single run of an Illumina HiSeq, followed by the application of bioinformatics tools. Results: The most representative phyllo for all the samples was Proteobacteria (69,4%), followed by Bacteroidetes (19,2%) and Synergists (11,2%). In the group of obese snakes, the phylum Proteobacteria still being the group with the higher relative abundance in the two moments, with a decrease of 9,6%, as well as the phylum Bacteroidetes which decreased by 5,5% in the second moment (after fasting). Differently, the groups of the normal and thin have a decrease of 37,2% and 29,4% respectively of the phylum Proteobacteria and an increase of the relative abundance of 35% and 27,9% of the phylum Bacteroidetes. Before prolonged fasting the groups obtained 213 shared OTUs which reduced to 206 after the prolonged fasting, suggesting that, in general, the treatment influences microbial communities of the groups, which was possible to observe when analyzed and compared individually. The index of Shannon diversity showed that before the fasting, the groups of obese snakes and normal presented a bigger richness of species, differently from the thin group that showed more diversity after 50 days of fasting. The variance analysis showed that the obese snakes presented a bigger proximity to the samples collected before the prolonged fasting. The samples showed greater proximity in PCA analyses and ANOVA when considering the prolonged fasting time than when compared by body score. Conclusion: With this study, it was possible to conclude that prolonged fasting can influence the cloacal microbiota of the snake Crotalus durissus, except in the individuals considered obese.