Standardized guidelines for Africanized honeybee venom production needed for development of new apilic antivenom.

Africanized bees have spread across the Americas since 1956 and consequently resulted in human and animal deaths attributed to massive attacks related to exposure from Argentina to the USA. In Brazil, more than 100,000 accidents were registered in the last 5 years with a total of 303 deaths. To treat such massive attacks, Brazilian researchers developed the first specific antivenom against Africanized honey bee sting exposure. This unique product, the first of its kind in the world, has been safely tested in 20 patients during a Phase 2 clinical trial. To develop the antivenom, a standardized process was undertaken to extract primary venom antigens from the Africanized bees for immunization of serum-producing horses. This process involved extracting, purifying, fractionating, characterizing, and identifying the venom (apitoxin) employing mass spectrometry to generate standardized antigen for hyperimmunization of horses using the major toxins (melittin and its isoforms and phospholipase A2). The current guide describes standardization of the entire production chain of venom antigens in compliance with good manufacturing practices (GMP) required by regulatory agencies. Emphasis is placed upon the welfare of bees and horses during this process, as well as the development of a new biopharmaceutical to ultimately save lives.

Preliminary Insights of Brazilian Snake Venom Metalloproteomics.

Snakebite envenoming is one of the most significantly neglected tropical diseases in the world. The lack of diagnosis/prognosis methods for snakebite is one of our motivations to develop innovative technological solutions for Brazilian health. The objective of this work was to evaluate the protein and metallic ion composition of Crotalus durissus terrificus, Bothrops jararaca, B. alternatus, B. jararacussu, B. moojeni, B. pauloensis, and Lachesis muta muta snake venoms. Brazilian snake venoms were subjected to the shotgun proteomic approach using mass spectrometry, and metal ion analysis was performed by atomic spectrometry. Shotgun proteomics has shown three abundant toxin classes (PLA2, serine proteases, and metalloproteinases) in all snake venoms, and metallic ions analysis has evidenced that the Cu2+ ion is present exclusively in the L. m. muta venom; Ca2+ and Mg2+ ions have shown a statistical difference between the species of Bothrops and Crotalus genus, whereas the Zn2+ ion presented a statistical difference among all species studied in this work. In addition, Mg2+ ions have shown 42 times more in the C. d. terrificus venom when compared to the average concentration in the other genera. Though metal ions are a minor fraction of snake venoms, several venom toxins depend on them. We believe that these non-protein fractions are capable of assisting in the development of unprecedented diagnostic devices for Brazilian snakebites.

Experimental Bothropsatrox Envenomation: Blood Plasma Proteome Effects after Local Tissue Damage and Perspectives on Thromboinflammation.

The clinical manifestations of Bothrops atrox envenoming involve local and systemic changes, among which edema requires substantial attention due to its ability to progress to compartmental syndromes and sometimes cause tissue loss and amputations. However, the impact of edema on the poisoned body's system has not been explored. Thus, the present study aimed to explore the systemic pathological and inflammatory events that are altered by intraplantar injection of B. atrox venom in a mouse model through hematologic, lipidic, and shotgun proteomics analysis. Plasma samples collected showed a greater abundance of proteins related to complement, coagulation, lipid system, platelet and neutrophil degranulation, and pathways related to cell death and ischemic tolerance. Interestingly, some proteins, in particular, Prdx2 (peroxiredoxin 2), Hba (hemoglobin subunit alpha), and F9 (Factor IX), increased according to the amount of venom injected. Our findings support that B. atrox venom activates multiple blood systems that are involved in thromboinflammation, an observation that may have implications for the pathophysiological progression of envenomations. Furthermore, we report for the first time a potential role of Prdx2, Hba, and F9 as potential markers of the severity of edema/inflammation in mice caused by B. atrox.

A fingerprint of plasma proteome alteration after local tissue damage induced by Bothrops leucurus snake venom in mice.

Bothrops spp. is responsible for about 70% of snakebites in Brazil, causing a diverse and complex pathophysiological condition. Bothrops leucurus is the main species of medical relevance found in the Atlantic coast in the Brazilian Northeast region. The pathophysiological effects involved B. leucurus snakebite as well as the organism's reaction in response to this envenoming, it has not been explored yet. Thus, edema was induced in mice paw using 1.2, 2.5, and 5.0 µg of B. leucurus venom, the percentage of edema was measured 30 min after injection and the blood plasma was collected and analyzed by shotgun proteomic strategy. We identified 80 common plasma proteins with differential abundance among the experimental groups and we can understand the early aspects of this snake envenomation, regardless of the suggestive severity of an ophidian accident. The results showed B. leucurus venom triggers a thromboinflammation scenario where family's proteins of the Serpins, Apolipoproteins, Complement factors and Component subunits, Cathepsins, Kinases, Oxidoreductases, Proteases inhibitors, Proteases, Collagens, Growth factors are related to inflammation, complement and coagulation systems, modulators platelets and neutrophils, lipid and retinoid metabolism, oxidative stress and tissue repair. Our findings set precedents for future studies in the area of early diagnosis and/or treatment of snakebites. SIGNIFICANCE: The physiopathological effects that the snake venoms can cause have been investigated through classical and reductionist tools, which allowed, so far, the identification of action mechanisms of individual components associated with specific tissue damage. The currently incomplete limitations of this knowledge must be expanded through new approaches, such as proteomics, which may represent a big leap in understanding the venom-modulated pathological process. The exploration of the complete protein set that suffer modifications by the simultaneous action of multiple toxins, provides a map of the establishment of physiopathological phenotypes, which favors the identification of multiple toxin targets, that may or may not act in synergy, as well as favoring the discovery of biomarkers and therapeutic targets for manifestations that are not neutralized by the antivenom.

Molecular identification and phylogenetic analysis of Bothrops insularis bacterial and fungal microbiota.

Bothrops insularis, known as the golden lancehead snake, has its natural habitat restricted to Queimada Grande Island on the southern coast of Brazil. This culture-dependent study aimed to identify microorganisms obtained from the mouth, eyes, and cloaca of this species. Swabs from 20 snakes were collected for fungal and bacterial isolation. DNA was extracted from all samples, and identification was performed by amplifying the ITS1-5.8S-ITS2 regions and the 16S rDNA gene, respectively. All strains were identified and deposited in the GenBank nucleotide database. MEGA v6.0 software was utilized to construct phylogenetic trees. In total, 100 strains were isolated and characterized, from which 42 fungi were distributed into 23 species and 58 bacteria into 13 species. The genus Fusarium was predominant since 11 strains and probably a new species was isolated from this fungus. Pseudomonas aeruginosa and Enterococcus faecalis were the predominant groups of aerobic bacteria isolated. Phylogenetic analyses between bacterial and fungal sequences suggest a similarity between the microorganisms found on the island and on the continent. These findings may be attributed to anthropic actions resulting from both expeditions to the island and actions of migratory birds, which are the main sources of food for snakes.

Crotalus durissus terrificus crotapotin naturally displays preferred positions for amino acid substitutions.

BACKGROUND: Classically, Crotalus durissus terrificus (Cdt) venom can be described, according to chromatographic criteria, as a simple venom, composed of four major toxins, namely: gyroxin, crotamine, crotoxin and convulxin. Crotoxin is a non-covalent heterodimeric neurotoxin constituted of two subunits: an active phospholipase A2 and a chaperone protein, termed crotapotin. This molecule is composed of three peptide chains connected by seven disulfide bridges. Naturally occurring variants/isoforms of either crotoxin or crotapotin itself have already been reported. METHODS: The crude Cdt venom was separated by using RP-HPLC and the toxins were identified by mass spectrometry (MS). Crotapotin was purified, reduced and alkylated in order to separate the peptide chains that were further analyzed by mass spectrometry and de novo peptide sequencing. RESULTS: The RP-HPLC profile of the isolated crotapotin chains already indicated that the α chain would present isoforms, which was corroborated by the MS and tandem mass spectrometry analyses. CONCLUSION: It was possible to observe that the Cdt crotapotin displays a preferred amino acid substitution pattern present in the α chain, at positions 31 and 40. Moreover, substitutions could also be observed in ß and γ chains (one for each). The combinations of these four different peptides, with the already described chains, would produce ten different crotapotins, which is compatible to our previous observations for the Cdt venom.

Plasma proteome of buffaloes.

The proteomic approach has aroused the interest of veterinary medicine researchers, especially regarding the production of biopharmaceuticals and diagnosis of diseases in farm animals. Water buffaloes have gained prominence in the world economy due to the quality of their milk, meat, and leather, in addition to being an important donor of blood components. This work aimed to identify and characterize the proteins present in the blood plasma of Murrah buffaloes (Bubalus bubalis) through 2D electrophoresis, in gel protein digestion followed by mass spectrometry technique and for albumin depletion, in solution protein digestion followed by shotgun analysis. Our results showed the identification of 112 protein spots and 35 individual proteins, respectively. The abundant proteins were represented by albumin, fibrinogen-α, fibrinogen-ß, fibrinogen-γ, immunoglobulins in general, α-1-antiproteinase, α-1B-glycoprotein, α-2-HS-glycoprotein, α-macroglobulin, apolipoprotein A1, antithrombin-III, endopin 2B, fetuin-B, retinol-binding protein, serotransferrin, transthyretin and vitamin D-binding protein. Most of these proteins are related to the signaling pathways of the complement system and coagulation cascade. The results allowed a better understanding of the protein composition of these blood components, thus promoting studies on animal health in the search for molecular markers of zoonotic diseases in buffaloes.

Heterologous fibrin sealant derived from snake venom: from bench to bedside - an overview.

Hemostatic and adhesive agents date back to World War II, when homologous fibrin sealant came onto scene. Considering that infectious diseases can be transmitted via human blood, a new heterologous fibrin sealant was standardized in the 1990s. Its components were a serine protease (a thrombin-like enzyme) extracted from the venom of Crotalus durissus terrificus snakes and a fibrinogen-rich cryoprecipitate extracted from the blood of Bubalus bubalis buffaloes. This new bioproduct has been used as a coagulant, sealant, adhesive and recently as a candidate scaffold for mesenchymal stem cells and bone and cartilage repair. This review discusses the composition of a new heterologous fibrin sealant, and cites published articles related to its preclinical applications aiming at repairing nervous system traumas and regenerating bone marrow. Finally, we present an innovative safety trial I/II that found the product to be a safe and clinically promising candidate for treating chronic venous ulcers. A multicenter clinical trial, phase II/III, with a larger number of participants will be performed to prove the efficacy of an innovative biopharmaceutical product derived from animal venom.

Individual venom profiling of Crotalus durissus terrificus specimens from a geographically limited region: crotamine assessment and captivity evaluation on the biological activities.

Crotalus durissus terrificus (Cdt) venom major components comprise crotoxin, crotamine, gyroxin and convulxin. Crotamine exerts a myotoxic action, among others, but its expression varies even amid snakes from the same region. Biochemical, enzymatic and pharmacological variations of venoms may be associated with the geography, climate, gender, age, and diet, as well as captivity time and venom extraction intervals. The present study aimed to characterize the Cdt venom from the Botucatu region, (SP, Brazil), by assessing its biochemical, pharmacological and enzymatic properties. Venoms from newly captured snakes and already-captured animals were characterized comparatively to verify the sexual, environmental (length of captivity) and ontogenetic variations that could influence the venom composition. Protein concentration, SDS-PAGE and RP-HPLC were performed and the coagulant, toxic (LD50) and crotamine activities were assayed. Individual SDS-PAGE analyses (315 samples) were performed and the biological activities of the venom of 60 adults (captive and newly captured males and females) and 18 newborns were compared with the Brazilian Reference Venom. Crotamine was found in 39.7% (125/315) of the samples, as determined by SDS-PAGE and RP-HPLC. Protein concentration differed significantly between adults (75%) and newborns (60%). RP-HPLC and SDS-PAGE analyses showed highly variable protein concentration and copious crotoxin isoforms; however, the LD50 values decreased during the captivity time. Cdt venom biological activities were similar among adult groups, but diminished during the captivity period. The current findings demonstrate that venoms vary significantly in terms activity and protein concentration, despite originating from the same specie and region.