Morphological alterations caused by manual venom extraction on the main venom gland of Bothrops asper and Crotalus simus snakes (Serpentes: Viperidae): Long-term implications for antivenom production.

The only scientifically validated treatment for snakebite envenomation is the administration of antivenoms. For their production, small quantities of snake venom are injected in animals to elicit a specific antibody response. Snakes are kept in captivity, and their venom is regularly extracted to assure antivenom access. It has already been reported that the pressure exerted upon the venom gland during this extraction can cause tissue damage and fibrosis, leading to a decrease in the venom yield. We described the histopathology of venom glands for B. asper and C. simus snakes used for antivenom production. Based on these reported tissue abnormalities, we quantify the tissue injury by a generated damage-SCORE and fibrosis. A variety of histopathological damages were found such as fibrosis, edema, necrosis, hemorrhage, and formation of anomalous structures, especially in C. simus, which is more prone to suffer severe damage. The level and severity of the damage depend on the frequency and the number of venom extractions. Furthermore, we design an experimental intensive venom extraction scheme with which we could confirm the causality of these effects. In addition to the histopathological damages, the LD50 and biochemical venom composition were also affected giving experimental evidence that the venom extraction not only causes tissue damage but also affects the composition stability and toxicity of the venom. In order to produce quality and effective antivenoms, an improvement of the management of snake collections could be established, such as rotation groups to assure the quality of the venom yielded.

Cross-reactivity and cross-immunomodulation between venoms of the snakes Bothrops asper, Crotalus simus and Lachesis stenophrys, and its effect in the production of polyspecific antivenom for Central America.

A mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys is used as immunogen to produce the polyspecific Central American antivenom (PoliVal-ICP). In this work, we studied the ability of each of these venoms to modulate the antibody response induced by the other two venoms included in the immunization mixture. For that, equine monospecific, bispecific and polyspecific antivenoms were prepared and compared regarding their ability to neutralize the phospholipase A2, coagulant and lethal activities of each venom, and their anti-venom antibodies concentration. Results indicate that there is low cross-reactivity and cross-neutralization between venoms of B. asper, C. simus and L. stenophrys, hence justifying the use of all of them as immunogens for the production of the Central American antivenom. It was also found that the venom of B. asper reduces the anti-crotalic response while the venom of C. simus does not affect the anti-bothropic response. On the other hand, the venoms of B. asper and C. simus increase the anti-lachesic response, and L. stenoprhys venom reduced both the anti-bothropic and anti-crotalic responses. On the basis of these results, the immunization strategy can be adjusted by preventing or taking advantage of cross-immunomodulation between venoms, in order to maximize the antibody response towards all venoms. Immune responses can be improved by injecting horses with several immunogen mixtures, composed by one or two of the three venoms, and administering them at different times during the immunization, eventually generating a high titer against the three venoms. Our results suggest that addressing the issue of immunomodulation by venoms might improve antivenom manufacture worldwide.

Lachesis stenophrys venom reduces the equine antibody response towards Bothrops asper venom used as co-immunogen in the production of polyspecific snake antivenom.

The anti-bothropic activity of an antivenom prepared from the plasma of horses immunized with Bothrops asper venom (anti-B antivenom) was compared with a similar formulation produced from the plasma of horses immunized with a mixture of B. asper and Lachesis stenophrys venoms (anti-BL antivenom). Likewise, a comparison between the anti-lachesic activity of the anti-BL antivenom and a similar formulation prepared from horses immunized only with L. stenophrys venom (anti-L antivenom) was performed. The anti-BL antivenom had lower concentration of anti-bothropic antibodies than the anti-B antivenom. This difference was associated to a lower response towards all components of B. asper venom, but particularly towards some D49-phospholipases A2 (PLA2s) and PIII-metalloproteinases. Consequently, the anti-BL antivenom was less effective neutralizing lethal, coagulant, defibrinogenating, PLA2, and myotoxic activities of B. asper venom. On the other hand, anti-BL and anti-L antivenoms showed similar concentration of anti-lachesic antibodies, and similar capacity to recognize the HPLC fractions of L. stenophrys venom and to neutralize lethal, coagulant, proteolytic, hemorrhagic, PLA2 and myotoxic activities induced by this venom. It is concluded that, when used as co-immunogens, the venom of L. stenophrys reduces the antibody response towards B. asper venom, whereas the latter does not affect the anti-lachesic response.

Epidemiology of rapes in Costa Rica: characterization of victims, perpetrators and circumstances surrounding forced intercourse.

Since the year 2000, the number of rapes in Costa Rica has increased at a rate of 42 cases per year. In 2011, 1786 rape cases were reported to the prosecution offices throughout the country, but only 1081 reports continued through the investigation process by the Judicial Investigation Agency. A randomly collected sample of 272 reports received by Judicial Investigation Agency, between July 2012 and June 2013, were prospectively studied. The analysis was limited to cases reported within 30 days following the rape. Results indicate that most of the provinces in the country show an incidence of about 38 cases/100,000 inhabitants. Ninety-six percent of the victims were women, 50% of which were between 10 and 19 years old. More than 99.5% of violators were men. The rape was perpetrated by a single aggressor in 85% of the cases. It was found that 48% of the victims were within the first 11 days of their menstrual cycle at the time of the attack. Twenty-nine percent of rapes occurred in "high rape-risk" circumstances--e.g., victims attacked by strangers in public outdoors or indoors. Twenty-five percent of rapes occur in "moderate rape-risk" circumstances--e.g., victims attacked indoors at public locations or at the home other than the victim's by relatives, sentimental partners or acquaintances. Fifteen percent of rapes occurred in "low rape-risk" circumstances--e.g., victims attacked in their homes by relatives or sentimental partners. In 67% of the cases the perpetrator was an acquaintance of the victim. Eleven percent of the cases corresponded to rapes in which the perpetrator was a partner or ex-partner of the victim. Fourteen percent and 25% of rapes could be classified as "proactive drug-facilitated rapes" or "opportunistic drug-facilitated rapes", respectively. Semen in the vaginal fluid of victims and the genetic profile of the alleged perpetrator were detected in 55% and 33% of the cases, respectively.

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: implications for the production of polyspecific snake antivenoms.

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.