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.

Efficient muscle regeneration after highly haemorrhagic Bothrops alternatus venom injection.

Bothrops alternatus snake venom is particularly characterized for inducing a prominent haemorrhage and affecting hemostasis as a consequence of 43.1% of metallo-proteinases and less than 10% of PLA2 (almost all non-myotoxic phospholipases) in its venomics. In addition, myonecrosis is the major local effect in viper envenoming which might lead to permanent sequela. Then, the rebuilding of the microvasculature at the local injured site acquires significance since represents one of the pivotal stages for subsequent skeletal muscle regeneration either at morphological or functional aspects. Due to the significance played by vasculature in this process, it is important to study by histology and immunohistochemical techniques, the muscular damage and the sequence of skeletal muscle reconstruction (degree of damage, reconstitution of muscle fibres and capillaries). In this work, we injected intramuscularly 50 or 100 µg per mouse of B. alternatus venom in gastrocnemius muscles. We provided a complete description and characterization of the different stages of myogenesis after mild (50 µg) and severe (100 µg) local injury induced by B. alternatus venom toxins. The regeneration was evaluated 24 h, 3, 7, 14 and 28 days after receiving venom injection. Finally, both doses induced an extended necrosis at the site of injection where, when critical steps in the regenerative process are taking place, an efficient tissue rebuilding is achieved. B. alternatus venom is characterized by the high percentage of exclusively class P-III metalloproteinases, and by the lack of class P-I metalloproteinases in its venom composition. This could explain the effectiveness of muscle regeneration after venom injection despite the severity of the initial phase of envenoming.

Suspected natural lysosomal storage disease from ingestion of pink morning glory (Ipomoea carnea) in goats in northern Argentina.

This study describes an occurrence of pink morning glory (Ipomoea carnea) intoxication in goats in northern Argentina. The clinical signs displayed by the affected animals were ataxia, lethargy, emaciation, hypertonia of the neck muscles, spastic paresis in the hind legs, abnormal postural reactions and death. The clinico-pathologic examination revealed that the affected animals were anemic and their serum level of aspartate aminotransferase was significantly increased. Cytoplasmic vacuolation in the Purkinje cells and pancreatic acinar cells was observed by histological examination. The neuronal lectin binding pattern showed a strong positive reaction to WGA (Triticum vulgaris), sWGA (succinylated T. vulgaris) and LCA (Lens culinaris). Although I. carnea is common in tropical regions, this is the first report of spontaneous poisoning in goats in Argentina.

Experimental intoxication of guinea pigs with Ipomoea carnea: behavioural and neuropathological alterations.

Ipomoea carnea is a toxic plant that affects goats, with symptoms being characterised by nervous disorders and death. Swainsonine and calystegines are the principal toxic components isolated from I. carnea, which also yields lysergic acid derivatives. The aim of this study was to improve the clinical characterisation of experimental intoxication by I. carnea in guinea pigs through the evaluation of behavioural changes and to perform a thorough histopathological analysis of the affected CNS. Leaves of I. carnea were administered to guinea pigs. Open-field gait analysis and monoamine levels were measured. The poisoned animals exhibited increased vocalisation, lethargy, and a reduction in the locomotion frequency after the fourth week of intoxication, as demonstrated in the open-field test. Significant differences were observed in hind-limb gait width by the last week of intoxication. After 65 days, the guinea pigs were euthanised, necropsied, and examined using light and electron microscopy. At the end of the experiment, plasma serotonin decreased. In contrast, dopamine decreased, and noradrenaline increased in urine. Brain sections were evaluated with conventional histological methods and immunohistochemistry (IHC), as well as by transmission electron microscopy (TEM). Vacuoles were observed throughout the brain, but they were particularly prominent in the brainstem. In addition, there were PAS-negative regions, and the Nissl substance was dispersed or absent, which was confirmed with the Kluver-Barreda stain. Moderate microgliosis was observed by immunohistochemistry. In the medulla oblongata, numerous ubiquitin-positive spheroids together with neuronal degeneration were observed in the nucleus gracilis/cuneatus. Furthermore, vacuoles were observed in astrocytes, oligodendrocytes, and endothelial cells by TEM. Our results showed that the behavioural effects may have been caused by alterations in the brain in conjunction with changes in monoamine levels. This research confirms the utility of this model for studying the pathogenesis of plant-induced lysosomal storage diseases.

Isolation and functional characterization of a new acidic PLA(2) Ba SpII RP4 of the Bothrops alternatus snake venom from Argentina.

An acidic protein with phospholipase A(2) activity was purified to homogeneity from the venom of the Northeast Argentinian viperid Bothrops alternatus by two chromatographic steps: a conventional gel filtration on Sephadex G-75 and reversed phase on C18 HPLC column. A molecular mass of 14185.48 Da was determined by mass spectrometry, displaying a homodimer conformation. The kinetic assay demonstrated a catalytically active phospholipase A(2) in correspondence with Asp49 PLA(2) group. The enzyme designated Ba SpII RP4 contains an amino acid composition of 121 residues and a calculated theoretical pI value of 4.88. Amino acid sequence alignments with other Bothrops PLA(2) revealed a high degree of homology sequence (90-56%). Ba SpII RP4 did not show myotoxic activity upon muscular fibers at doses up to 100 microg i.m. route injection or lethal response when it was i.p. injected at the hightest dose of 200 microg. This toxin generates slight biological activities like paw edema inflammation and a delay in the clotting time, although Ba SpII RP4 exhibited catalytic activity. The primary amino acid sequence, determined a quadruple-time of flight (Q-TOF) hybrid mass spectrometer Q-TOF Ultima from Micromass (Manchester, UK) equipped with a nano Zspray source operating in a positive ion mode and tandem mass spectrum, an ESI/MS mass spectrum (TOF MS mode) "de novo amino acid sequencing", also provides more database about the small group of the non-myotoxic PLA(2)s isolated up to the present.