Maintaining rear-fanged snakes for venom production: an evaluation of mortality and survival rates for Philodryas olfersii and P. patagoniensis in captivity

This study evaluates the mortality and average survival rates of captive female Philodryas olfersii and Philodryas patagoniensis snakes maintained for venom production. Also, two factors likely to reduce captive survival were studied - body condition at admission and seasonality. Mortality peaks occurred during the second month in captivity. More than half the individuals were dead at the end of the third month. This suggests that the first three months in captivity are the most critical in terms of survival and adaptation. Females collected and admitted during spring and summer lived less time than those collected in autumn and winter. As gravidity and egg-laying occur during spring and summer, we suggest that the lower survival rates in these seasons may be due to high costs and stress involved in these reproductive events. Unexpectedly, body mass and body condition were poor predictors of survival in captivity. Our results have important implications in maintaining snakes for venom production. We propose some prophylactic measures to minimize the deleterious impacts of captivity during the adaptation period.(AU)

An alternative method to access in vitro the hemorrhagic activity of snake venoms.

Local and systemic hemorrhages are major problems concerning bites by viper snakes. Therefore, accessing venom hemorrhagic activity is an important feature in order to characterize viper venom major toxicities or to assay antivenom efficacy. The methods currently used to access hemorrhagic activity involve animal experiments and according to the general ethical committees, these procedures should be substituted to in vitro assays in order to minimize animal use in research. In this work, we have developed an immunoassay to detect the content of hemorrhagic metalloproteinases in snake venoms using a neutralizing monoclonal antibody anti-jararhagin (MAJar 3). The correlation between the reactivity of this monoclonal antibody and venom-induced hemorrhage was further revealed by a study comparing the hemorrhagic activity of venom samples collected individually from 88 specimens of Bothrops jararacussu with their reactivity with MAJar 3. As a result, a significant correlation (r=0.942) was achieved between samples hemorrhagic activity and their reactivity with MAJar 3, suggesting that this assay can be used as a substitute of the conventional tests performed in vivo to estimate the hemorrhagic activity.

Sexual dimorphism in venom of Bothrops jararaca(Serpentes: Viperidae).

Bothrops jararaca is an abundant snake in Brazil, and its venom has been studied exhaustively. The species exhibits adult size dimorphism in which female are larger. We registered the growth in Snout-Vent Length and weight of one litter (with 11 females and 12 males). We compared growth curves and venom profile between male and female of B. jararaca in order to establish the relationship of those characters and sex. Their venoms were analyzed when they were 36 months old, concerning SDS PAGE, protein content, proteolytic, hyaluronidasic, phospholipasic, blood-clotting, edematogenic, hemorrhagic, myotoxic activities, and lethality. Differences in the growth curves of the females and the males were significantly different after the 12th month of age, with the females growing faster. Females produced five times more venom than males. The electrophoretic patterns were variable: the venom from males had more protein bands than females. Venom composition varied significantly between males and females. Venom from females is more potent for hyaluronidasic, hemorrhagic, and lethality activities, whereas venom from males is more potent for coagulant, phospholipasic, and myotoxic activities. The variability of proteolytic and edematogenic activities were not significant. The important sexual dimorphism in body size and mass, amount of venom produced, and venom composition in B. jararaca may reflect a divergence in niche partitioning.

Ordered mesoporous silica SBA-15 A new effective adjuvant to induce antibody response

The applicability of SBA-15 mesostructure as an adjuvant and evaluation of its efficiency to induce antibody response, was discussed. It was observed that better encapsulation of biomolecules of variable shape and size can be achieved using a antigen to SBA-15 weight ratio of 1: 2.5. Efficient antibody generation could be achieved because SBA-15 was able to attract antigens effectively due to its high surface area and proper mesopore size. The results show that SBA-15 and related silica mesostructures are promising nanosystems for vaccine delivery.

Biological and immunological properties of the venom of Bothrops alcatraz, an endemic species of pitviper from Brazil.

Bothrops alcatraz is a new pitviper species derived from the Bothrops jararaca group, whose natural habitat is situated in Alcatrazes Archipelago, a group of marine islands near São Paulo State coast in Brazil. Herein, the biological and biochemical properties of venoms of four adult specimens of B. alcatraz were examined comparatively to a reference pool of Bothrops jararaca venom. Both venoms showed similar activities and electrophoretic patterns, but B. alcatraz venom showed three protein bands of molecular masses of 97, 80 and 38 kDa that were not present in B. jararaca reference venom. The i.p. median lethal dose of B. alcatraz venom ranged from 5.1 to 6.6 mg/kg, while it was 1.5 mg/kg for B. jararaca venom. The minimum hemorrhagic dose of B. jararaca venom was 0.63, whereas 2.28 mug/mouse for B. alcatraz venom. In contrast, B. alcatraz venom was more potent in regard to procoagulant and proteolytic activities. These differences were supported by western blotting and neutralization tests, employing commercial bothropic antivenom, which showed that hemorrhagic and lethal activities of B. alcatraz venom were less effectively inhibited than B. jararaca venom. Such results evidence that B. alcatraz shows quantitative and qualitative differences in venom composition in comparison with its B. jararaca relatives, which might represent an optimization of venom towards a specialized diet.

Biological and immunochemical characterization of Micrurus altirostris venom and serum neutralization of its toxic activities.

Micrurus altirostris venom from Rio Grande do Sul State, Brazil, was characterized by its biological activities, immunochemical properties and electrophoretic pattern. The results showed a high edematogenic activity, whose peak was observed after 30min of venom injection, as well as a high indirect hemolytic activity. This venom was myotoxic, as shown by a peak of CK release at 6h after injection, and also by the appearance of muscular lesions characterized by necrosis, loss of striated muscle fibers, and the presence of vacuolization, edema and inflammatory infiltrate. This venom showed minimum proteolytic activity and no hemorrhagic, dermonecrotic or coagulant activities. Nonetheless, M. altirostris venom presented high lethal activity. Electrophoretic patterns of Micrurus frontalis and M. altirostris venoms showed different protein bands. Anti-elapidic serum could recognize M. frontalis (homologous) and M. altirostris (heterologous) venoms by Western blotting, and both venoms presented similar titers when assayed by ELISA. The results observed on neutralization tests showed that the anti-elapidic serum produced by Instituto Butantan neutralized myotoxic and hemolytic activities. However, this antivenom could not neutralize the lethal activity of M. altirostris venom. Thus, these data suggest that M. altirostris venom presents different biological, enzymatic and immunological characteristics from other Micrurus venoms, and some activities are not neutralized by the commercial anti-elapidic serum produced in Brazil.

Age-related biological activity of south american rattlesnake (Crotalus durissus terrificus) venom

An in vitro and in vivo comparative study was performed on the effects of Crotalus durissus terrificus venoms from a mother and its 15 newborns. The venoms were tested for protein content, lethality, proteolytic, myotoxic, hemorrhagic, and phospholipase A2 activity. The minimum coagulant dose in plasma and human fibrinogen, protrhombin, and Factor II activations were analyzed. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE). This showed that despite similar total protein content, the biological effects of the venoms were different. Venom from young snakes exhibited higher enzymatic and coagulant activities and higher myotoxicity compared to the mother's. In addition, the PLA2 content paralleled myotoxicity. However, no difference could be detected in their toxicity (LD50 0.08 mg/Kg). High incidence of blood coagulation disorders and elevated circulating myoglobin may characterize systemic envenoming by young C. d. terrificus.

Cloning and characterization of a repetitive 1.9 Kb HindIII DNA fragment from Crotalus durissus terrificus genome

Eukaryote genomes are endowed with varying quantities of repeated DNA families. These families show different patterns of conservation among species, copy numbers, chromosomal distribution, and transcription. Characterization of repeated DNA sequences could help to understand the genome anatomy and organization or be used in molecular systematics and molecular evolution studies. We describe here a repetitive DNA sequence of the HindIII family present in the genome of the rattlesnake Crotalus durissus terrificus. In Brazil, the family Crotalus is comprised only by one species durissus, which include several subspecies. The number and distribution of these subspecies are controversial. In the present study, the genomic DNA of a female rattlesnake was digested with HindIII resulting in a strong 1.9 Kb band. A partial genomic library was constructed from the 1.9 Kb DNAs rescued from the agarose gel after HindIII digestion and ligated to the vector pGEM3Zf(+) (Promega). Analysis of 69 clones, 44 hybridized with the 1.9 Kb probe isolated from one of the clones-clone 76, indicating that the DNA isolated from this clone should represent the 1.9 Kb HindIII fragment. This 1.9 Kb HindIII DNA was completely characterized by sequencing.