Exploring the antibacterial potential of venoms from Argentinian animals.

The resistance to antimicrobials developed by several bacterial species has become one of the main health problems in recent decades. It has been widely reported that natural products are important sources of antimicrobial compounds. Considering that animal venoms are under-explored in this line of research, in this study, we screened the antibacterial activity of venoms of eight snake and five lepidopteran species from northeastern Argentina. Twofold serial dilutions of venoms were tested by the agar well-diffusion method and the minimum inhibitory concentration (MIC) determination against seven bacterial strains. We studied the comparative protein profile of the venoms showing antibacterial activity. Only the viperid and elapid venoms showed remarkable dose-dependent antibacterial activity towards most of the strains tested. Bothrops diporus venom showed the lowest MIC values against all the strains, and S. aureus ATCC 25923 was the most sensitive strain for all the active venoms. Micrurus baliocoryphus venom was unable to inhibit the growth of Enterococcus faecalis. Neither colubrid snake nor lepidopteran venoms exhibited activity on any bacterial strain tested. The snake venoms exhibiting antibacterial activity showed distinctive protein profiles by SDS-PAGE, highlighting that we could reveal for the first time the main protein families which may be thought to contribute to the antibacterial activity of M. baliocoryphus venom. This study paves the way to search for new antibacterial agents from Argentinian snake venoms, which may be a further opportunity to give an added value to the local biodiversity.

Unraveling the distinctive venomous features of the saturniid Hylesia sp.: An integrative approach of a public health concern in Argentina.

The saturniid genus Hylesia is well known for the cutaneous lepidopterism induced by airborne setae on contact with the skin. Although several cases of such dermatitis have been reported in Argentina, no information about their venoms and toxicological implications on human health is available yet. Thus, we conducted a morphological analysis of the setae/spines and a toxinological characterization (through biological assays and proteomic techniques) of the bristle extract from caterpillars and moths of Hylesia sp. from Misiones, Argentina. By scanning electron microscopy, we revealed the various and distinctive types of urticating structures: harpoon-shaped or spiny setae in caterpillars, and setae with barb-like structures in female moths. Their venom electrophoretic profiles were substantially different, presenting proteins related to toxicity, such as serpins and serine peptidases. The female moth venom exhibited higher caseinolytic activity than the caterpillar venom, and coincidentally only the former noticeably hydrolyzed fibrinogen and gelatin. In addition, the female venom displayed a dose-dependent procoagulant effect. The injection of this venom into mouse skin led to the rapid detection of an increased number of intact and degranulated mast cells in the dermis; a few areas of focal subcutaneous hemorrhage were also observed after 5 h of injection. Altogether, this study provides relevant information about the pathophysiological mechanisms whereby Hylesia sp. from northeastern Argentina can induce toxicity on human beings, and paves the way for treatment strategies of accidents caused by this saturniid lepidopteran.

First insights into the biochemical and toxicological characterization of venom from the Banded Cat-eyed Snake Leptodeira annulata pulchriceps.

With the aim to widen the current knowledge of toxinological implications of bites from rear-fanged snakes and biological roles of their venoms, this study focuses on the biochemical composition and toxic effects of the venom of Leptodeira annulata pulchriceps from Argentina. We analyzed the protein composition by electrophoresis and mass spectrometry, and enzymatic properties by quantitative assays on different substrates. Additionally, we evaluated local and systemic toxicity in mice, and tested its cross-reactivity with elapid and viperid antivenoms used in Argentina. This venom showed features reminiscent of venoms from snakes of Bothrops genus, containing components ranging from ~17 to 75 kDa, which are mainly tissue-damaging toxins such as proteinases. Although showing low lethality to mice (LD50 = 20 µg/g body weight), prominent hemorrhage developed locally in mice intramuscularly and intradermally injected with the venom, and the minimum hemorrhagic dose was found to be 12.7 µg/mouse. This study is the first comprehensive investigation of the venom of L. a. pulchriceps, and sheds new light on differences between this and those of the other two subspecies of L. annulata. Additionally, the study provides new insights into the venom components of "colubrid" snakes, advocating for considering bites from this rich diversity of snakes as a public health problem that needs to be addressed worldwide.

Unveiling toxicological aspects of venom from the Aesculapian False Coral Snake Erythrolamprus aesculapii.

Most colubrid snake venoms have been poorly studied, despite the fact that they represent a great resource for biological, ecological, toxinological and pharmacological research. Herein, we explore the venom delivery system of the Aesculapian False Coral Snake Erythrolamprus aesculapii as well as some biochemical and toxicological properties of its venom. Its Duvernoy's venom gland is composed of serous secretory cells arranged in densely packed secretory tubules, and the most striking feature of its fang is their double-curved shape, exhibiting a beveled bladelike appearance near the tips. Although E. aesculapii resembles elapid snakes of the genus Micrurus in color pattern, this species produces a venom reminiscent of viperid venoms, containing mainly tissue-damaging toxins such as proteinases. Prominent hemorrhage developed both locally and systemically in mice injected with the venom, and the minimum hemorrhagic dose was found to be 18.8 µg/mouse; the lethal dose, determined in mice, was 9.5 ±â€¯3.7 µg/g body weight. This work has toxicological implications that bites to humans by E. aesculapii could result in moderately severe local (and perhaps systemic) hemorrhage and gives insight into future directions for research on the venom of this species.

Understanding toxicological implications of accidents with caterpillars Megalopyge lanata and Podalia orsilochus (Lepidoptera: Megalopygidae).

Megalopygids Megalopyge lanata and Podalia orsilochus are common causative agents of accidents in agricultural workers. These accidents are provoked by dermal contact at their larval stage and are characterized by cutaneous reactions, such as burning pain, edema and erythema, typically mild and self-limited. There is very little information about their venoms and their toxicological implications on human health. Thus, we employed proteomic techniques and biological assays to characterize venoms (bristle extracts) from caterpillars of both species collected from Misiones, Argentina. The electrophoretic profiles of both venoms were substantially different, and they presented proteins related to toxicity, such as serinepeptidases, serpins and lectins. P. orsilochus venom exhibited higher caseinolytic activity than M. lanata venom, agreeing with the fact that only P. orsilochus venom hydrolyzed human fibrin(ogen). In addition, the latter shortened the clotting time triggered by calcium. While the venom of M. lanata induced a mild inflammatory lesion in mouse skin, P. orsilochus venom caused prominent necrosis, inflammatory infiltration and hemorrhage at the site of venom injection. On the other hand, P. orsilochus venom was better recognized by Lonomia obliqua antivenom, although many of its proteins could not be cross-reacted, what may explain the difference in the clinical manifestations between accidents by Podalia and those by Lonomia. Altogether, this study provides relevant information about the pathophysiological mechanisms whereby both caterpillars can induce toxicity on human beings, and paves the way for novel discovery of naturally occurring bioactive compounds.

Assessment of the potential toxicological hazard of the Green Parrot Snake (Leptophis ahaetulla marginatus): Characterization of its venom and venom-delivery system.

Snakes are the major group of venomous vertebrates, and the rear-fanged snakes represent the vast majority of species and occur worldwide; however, relatively few studies have characterized their venoms and evaluated their potential hazards for humans. Herein we explore the protein composition and properties of the venom of the rear-fanged Green Parrot Snake, Leptophis ahaetulla marginatus, the most common snake found in the Iguazu National Park (Argentina), as well as the main features of its venom delivery system. This species has venom reminiscent of elapid venoms, composed mainly of components such as 3FTxs, CRiSPs and AChE, but it shows low toxicity toward mammals (LD50 > 20 µg/g mouse). The histology of its Duvernoy's venom gland is similar to that of other colubrids, with serous secretory cells arranged in densely packed secretory tubules. The posterior end of its maxilla exhibits 1-3 blade-shaped and slightly recurved fangs but without grooves. This study provides an initial analysis of the biological role of venom in Leptophis, with implications for potential symptoms that might be anticipated from bites by this species.

First report of parasitism by Hexametra boddaertii (Nematoda: Ascaridae) in Oxyrhopus guibei (Serpentes: Colubridae).

The current study summarizes the postmortem examination of a specimen of Oxyrhopus guibei (Serpentes, Colubridae) collected in Iguazu National Park (Argentina), and found deceased a week following arrival to the serpentarium of the National Institute of Tropical Medicine (Argentina). Although the snake appeared to be in good health, a necropsy performed following its death identified the presence of a large number of roundworms in the coelomic cavity, with indications of peritonitis and serosal adherence. Additional observations from the necropsy revealed small calcifications in the mesothelium of the coelomic cavity; solid and expressive content in the gallbladder; massive gastrointestinal obstruction due to nematodes; and lung edema and congestion. Histopathological analyses of lung sections also showed proliferative heterophilic and histiocytic pneumonia. Parasites isolated from both the intestine and coelomic cavity were identified as Hexametra boddaertii by a combination of light and scanning electron microscopic examination. Results from this necropsy identify O. guibei as a new host for H. boddaertii, and is the first report of a natural infection by Hexametra in Argentina. Since Hexametra parasites may contribute to several pathological conditions in humans, and with the recent availability of O. guibei specimens through the illegal pet trade, it is necessary to consider the possibility of zoonotic helminth transmission of Hexametra from snake to human.

[Accidents with caterpillar Lonomia obliqua (Walker, 1855). An emerging problem]. / Accidentes causados por la oruga Lonomia obliqua (Walker,1855). Un problema emergente.

Lonomia obliqua (Walker, 1855) is a moth from the family Saturniidae, widely distributed in tropical rainforests of South America. In its larval stage (caterpillar) it is characterized by bristles that cover the animal's body. These structures are hard and branched spiny evaginations of the cuticle, underneath which a complex mixture of toxic molecules is stored. When spicules are brought into contact with the skin of people, toxins enter passively through the injury, causing not only local but also systemic poisoning (primarily hemorrhagic manifestations). When the whole animal is accidentally crushed, the insect's chitinous bristles are broken and the venomous secretions penetrate the human skin, reaching the blood circulation. Due to the numerous registered cases of erucism in Southern Brazil, the Butantan Institute has produced an antivenom able to neutralize the deleterious effects produced by contact with L. obliqua caterpillar bristles. In Argentina, these kinds of accidents are rare and restricted to the province of Misiones. Taking into account that to date there is no report in this country about clinical cases submitted to a specific treatment (antivenom), our aim is to communicate here six cases of Lonomia caterpillar-induced bleeding syndrome that were treated in the Hospital SAMIC of Puerto Iguazú (Misiones, Argentina) during 2014 with the antilonomic serum produced in Brazil. It is worthy to note that all patients evolved favorably within the first few hours, and for this reason, the use of this antivenom is recommended to treat the cases of Lonomia erucism in Argentina.

Accidentes causados por la oruga Lonomia obliqua (Walker, 1855): Un problema emergente / Accidents with caterpillar Lonomia obliqua (Walker, 1855). An emerging problem

Lonomia obliqua (Walker, 1855) es una mariposa nocturna de la familia Saturniidae, ampliamente distribuida en selvas tropicales de Sudamérica. Su larva (oruga) se caracteriza por poseer espículas ramificadas puntiagudas a lo largo de su cuerpo, que contienen una mezcla compleja de moléculas tóxicas en su interior. Cuando las espículas contactan con la piel de las personas, las toxinas ingresan pasivamente a través de la lesión, generando un envenenamiento caracterizado por manifestaciones no solo locales sino también sistémicas (fundamentalmente manifestaciones hemorrágicas). Debido al elevado número de casos que se produjeron en Brasil en las últimas décadas, el Instituto Butantan ha producido un antiveneno capaz de neutralizar los efectos deletéreos de los accidentes por contacto con L. obliqua. En Argentina, los accidentes por Lonomia son poco frecuentes y se limitan a la provincia de Misiones. Teniendo en cuenta que a la fecha no hay en la literatura descripciones de casos clínicos ocurridos en el país con tratamiento específico (antiveneno), el propósito del presente trabajo es comunicar seis casos de accidentes por contacto con orugas Lonomia que fueron atendidos en el Hospital SAMIC de Puerto Iguazú (Misiones, Argentina) durante el año 2014, y que fueron tratados con el suero antilonómico producido en Brasil. Se destaca la evolución rápida y favorable de todos los pacientes, por lo que se recomienda el uso de este antiveneno para tratar los casos de erucismo por Lonomia en la Argentina.

Lonomia obliqua (Walker, 1855) is a moth from the family Saturniidae, widely distributed in tropical rainforests of South America. In its larval stage (caterpillar) it is characterized by bristles that cover the animal’s body. These structures are hard and branched spiny evaginations of the cuticle, underneath which a complex mixture of toxic molecules is stored. When spicules are brought into contact with the skin of people, toxins enter passively through the injury, causing not only local but also systemic poisoning (primarily hemorrhagic manifestations). When the whole animal is accidentally crushed, the insect’s chitinous bristles are broken and the venomous secretions penetrate the human skin, reaching the blood circulation. Due to the numerous registered cases of erucism in Southern Brazil, the Butantan Institute has produced an antivenom able to neutralize the deleterious effects produced by contact with L. obliqua caterpillar bristles. In Argentina, these kinds of accidents are rare and restricted to the province of Misiones. Taking into account that to date there is no report in this country about clinical cases submitted to a specific treatment (antivenom), our aim is to communicate here six cases of Lonomia caterpillar-induced bleeding syndrome that were treated in the Hospital SAMIC of Puerto Iguazú (Misiones, Argentina) during 2014 with the antilonomic serum produced in Brazil. It is worthy to note that all patients evolved favorably within the first few hours, and for this reason, the use of this antivenom is recommended to treat the cases of Lonomia erucism in Argentina.

Venom proteomes of South and North American opisthoglyphous (Colubridae and Dipsadidae) snake species: a preliminary approach to understanding their biological roles.

Opisthoglyphous snake venoms remain under-explored despite being promising sources for ecological, evolutionary and biomedical/biotechnological research. Herein, we compared the protein composition and enzymatic properties of the venoms of Philodryas baroni (PbV), Philodryas olfersii olfersii (PooV) and Philodryas patagoniensis (PpV) from South America, and Hypsiglena torquata texana (HttV) and Trimorphodon biscutatus lambda (TblV) from North America. All venoms degraded azocasein, and this metalloproteinase activity was significantly inhibited by EDTA. PooV exhibited the highest level of catalytic activity towards synthetic substrates for serine proteinases. All venoms hydrolyzed acetylthiocholine at low levels, and only TblV showed phospholipase A(2) activity. 1D and 2D SDS-PAGE profile comparisons demonstrated species-specific components as well as several shared components. Size exclusion chromatograms from the three Philodryas venoms and HttV were similar, but TblV showed a notably different pattern. MALDI-TOF MS of crude venoms revealed as many as 49 distinct protein masses, assigned to six protein families. MALDI-TOF/TOF MS analysis of tryptic peptides confirmed the presence of cysteine-rich secretory proteins in all venoms, as well as a phospholipase A(2) and a three-finger toxin in TblV. Broad patterns of protein composition appear to follow phylogenetic lines, with finer scale variation likely influenced by ecological factors such as diet and habitat.

A comparative study of the effects of venoms from five rear-fanged snake species on the growth of Leishmania major: identification of a protein with inhibitory activity against the parasite.

Leishmania parasites of several species cause cutaneous and visceral disease to millions of people worldwide, and treatment for this vector-borne protozoan parasite typically involves administration of highly toxic antimonial drugs. Snake venoms are one of the most concentrated enzyme sources in nature, displaying a broad range of biological effects, and several drugs now used in humans were derived from venoms. In this study, we compared the effects of the venoms of the South American rear-fanged snakes Philodryas baroni (PbV), Philodryas olfersii olfersii (PooV) and Philodryas patagoniensis (PpV), and the North American rear-fanged snakes Hypsiglena torquata texana (HttV) and Trimorphodon biscutatus lambda (TblV), on the growth of Leishmania major, a causative agent of cutaneous leishmaniasis. Different concentrations of each venom were incubated with the log-phase promastigote stage of L. major. TblV showed significant anti-leishmanial activity (IC50 of 108.6 µg/mL) at its highest concentrations; however, it induced parasite proliferation at intermediate concentrations. PpV was not very active in decreasing the parasitic growth, and a high final concentration (1.7 mg/mL) was necessary to inhibit proliferation by only 51.5% ± 3.6%. PbV, PooV and HttV, at final concentrations of 562, 524 and 438 µg/mL respectively, had no significant effect on L. major growth. The phospholipase A2 of TblV (trimorphin) was isolated and assayed as for crude venom, and it also exhibited dose-dependent biphasic effects on the parasite culture, with potent cytotoxicity at higher concentrations (IC50 of 0.25 µM; 3.6 µg/mL) and stimulation of proliferation at very low concentrations. Anti-leishmanial activity of TblV appears to be solely due to the action of trimorphin. This is the first report of anti-leishmanial activity of rear-fanged snake venoms, and these results suggest novel possibilities for discovering new protein-based drugs that might be used as possible agents against leishmaniasis as well as tools to study the biology of Leishmania parasites.

Loxosceles gaucho spider venom and its sphingomyelinase fraction trigger the main functions of human and rabbit platelets.

Loxosceles venoms can promote severe local and systemic damages. We have previously reported that Loxosceles gaucho spider venom causes a severe early thrombocytopenia in rabbits. Herein, we investigated the in vitro effects of this venom and its sphingomyelinase fraction on the main functions of platelets. Whole venom and its fraction induced aggregation of both human and rabbit platelets. Aggregation was dependent of plasma component(s) but independent of venom-induced lysophosphatidic acid generation. There was no increase in the levels of lactate dehydrogenase during platelet aggregation, ruling out the possibility of platelet lysis. The increased expression of ligand-induced binding site 1 (LIBS1) induced by L. gaucho venom and its sphingomyelinase fraction, as well as of P-selectin by the whole venom, evidenced the activation state of both human and rabbit platelets. Adhesion assays showed an irregular response when platelets were exposed to the whole venom, whereas the sphingomyelinase fraction induced a dose-dependent increase in the platelet adhesion to collagen. These findings evidence that L. gaucho venom and its sphingomyelinase fraction trigger adhesion, activation, and aggregation of both human and rabbit platelets. Thus, this work justifies the use of rabbits to investigate Loxosceles venom-induced platelet disturbances, and it also supports research on the role of platelets in the pathogenesis of loxoscelism.

Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom.

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 degrees C, primarily producing a approximately 32.6 kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca(2+), patagonfibrase was only partially autolysed, giving rise mainly to one fragment of approximately 52.2 kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.

Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase fromPhilodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom

Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37 ¡ÆC, primarily producing a ¡­ 32.6 kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca2+, patagonfibrase was only partially autolysed, giving rise mainly to one fragment of ¡­ 52.2 kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.

Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom.

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions that remain unknown. In the present study we describe the purification and characterization of a CRiSP (patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate its biological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whose NH(2)-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purified protein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibition of platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smooth muscle induced by high K(+). However, it caused muscular damage to murine gastrocnemius muscle, an action that has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important for studies of the structure-function and evolutionary relationships of this family of proteins that are widely distributed among snake venoms.

Purification and characterization of a cysteine-rich secretory protein from Philodryaspatagoniensis snake venom

Cysteine-rich secretory proteins (CRiSPs) are widespread in reptile venoms, but most have functions thatremain unknown. In the present study we describe the purification and characterization of a CRiSP(patagonin) from the venom of the rear-fanged snake Philodryas patagoniensis, and demonstrate itsbiological activity. Patagonin is a single-chain protein, exhibiting a molecular mass of 24,858.6 Da, whoseNH2-terminal and MS/MS-derived sequences are nearly identical to other snake venom CRiSPs. The purifiedprotein hydrolyzed neither azocasein nor fibrinogen, and it could induce no edema, hemorrhage or inhibitionof platelet adhesion and aggregation. In addition, patagonin did not inhibit contractions of rat aortic smoothmuscle induced by high K+. However, it caused muscular damage to murine gastrocnemius muscle, an actionthat has not been previously described for any snake venom CRiSPs. Thus, patagonin will be important forstudies of the structure-function and evolutionary relationships of this family of proteins that are widelydistributed among snake venoms.