Name: Venom Extraction from Brazilian Yellow Scorpions
Uploaded: 2023-10-06T13:10:00
Description: Watch this Scientific Journal Video about Venom Extraction from Brazilian Yellow Scorpions at JoVE.com

venom extraction from brazilian yellow scorpions

Rearing of Brazilian Yellow Scorpion for Venom Extraction

Scorpions are arthropods belonging to the class Arachnida, order Scorpiones, which comprises approximately 2,621 species1,2. These animals have a wide geographical range and are present on all continents except Antarctica1. Envenomation caused by scorpions results in the morbidity or death of thousands of people every year globally3. In 2019, it was estimated that there were more than 1.2 million accidents and 3,500 annual deaths caused by these animals. In Brazil, the number of cases has been increasing exponentially, reaching more than 100,000 cases per year since 20174,5,6. The uncontrolled urbanization observed in Brazil in the past decades, without adequate sewage treatment and regular collection and disposal of garbage, associated with environmental degradation and climate changes, has provided conditions for the proliferation of invasive scorpions, such as T. serrulatus, increasing the contact with humans and thus resulting in harmful accidents4,7,8. There are about 178 scorpion species in Brazil, but stings of medical importance are caused by the genus Tityus, with four species (T. serrulatus, T. bahiensis, T. stigmurus, and T. obscurus) being of medical concern, with Tityus serrulatus being responsible for the most severe cases and deaths7,9.
Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing approximately 70,000 vials of scorpion antivenom for the Brazilian health system. Briefly, the steps involved in antivenom manufacturing include the inoculation of venom-derived antigens in equines, the collection and purification of rich-antibodies plasma, resulting in 5 mL vials of scorpion antivenom. Each vial is capable of neutralizing, at least, 1 mg of scorpion venom per mL of antivenom. The Arthropods Bioterium is an integral part of the industrial center, responsible for supplying the source material for the scorpion antivenom.
The Arthropods Bioterium at Instituto Butantan was originally named the Laboratory of Arthropods and was established in 1967. However, it was only in 1995 that the Laboratory moved to an exclusive facility dedicated to housing venomous arthropods10,11. At that time, the scorpions were kept in a room of 12 m2 with no controlled temperature and distributed among approximately 13 enclosures, each housing a maximum of 300 animals10. Due to the increasing demand for venom over the years and the consequent need to increase the number of animals, along with improvements in the venom extraction process, a major change occurred in 2016. The Laboratory was integrated into the industrial center of Instituto Butantan and was renamed the Arthropods Bioterium. Additionally, in 2016, the Bioterium moved to a new purpose-built facility. The new scorpion room covers an area of 24 m2 and contains approximately 48 polypropylene enclosures, each housing up to 300 animals, as shown in Figure 1. This results in a total of 10,000 to 20,000 individuals being maintained under controlled captive conditions, with variations throughout the year. The scorpions are carefully managed under strict husbandry protocols to ensure their welfare while meeting the high production demands and adhering to good practices and ethical standards for animal care. For venom extraction, a specific room was designed and constructed, housing two airflow cabinets as shown in Figure 2A. These cabinets serve as safety measures to prevent technicians from inhaling venom particles during the extraction procedure. Personal protective equipment (PPE) is mandatory for the technicians, including synthetic aprons and FFP2 masks, to avoid any contamination and ensure their safety.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65737/65737fig01v2.jpg" /> 
Figure 1: General view of the scorpion&#39;s room of the Arthropods Bioterium. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65737/65737fig02.jpg" /> 
Figure 2: Working area. (A) General view of the venom extraction room of the Arthropods Bioterium. (B) Airflow cabinet work surface. All the materials and equipment are prepared and arranged on the top of the airflow cabinet work surface. The black arrow indicates the electrostimulator device, and the red arrow indicates the forceps. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
About 90% of the T. serrulatus scorpions in the Arthropods Bioterium originate from different sites, collected by municipal health agents as part of a surveillance and control program for these animals. The scorpions are caught in urban areas and then sent to Instituto Butantan as part of a collaborative program. T. serrulatus scorpions are parthenogenetic, meaning that birth occurs as a consequence of the development of offspring from unfertilized eggs, without the presence of an individual of the opposite sex12. Due to this type of reproduction, some animals are born in captivity in our facility and are maintained until they reach the adult phase, following a similar husbandry protocol as described below. Once they reach the adult stage, they are added to the venom extraction routine. Upon arrival, the scorpions undergo initial screening, and those in good health conditions are kept in collective enclosures containing a maximum of 350 individuals, divided by their origin. For each enclosure, a control form is filled out daily, providing data on the date of feeding and the type of prey offered (cricket or cockroach), water supply, cleaning procedures, the number of dead animals, and the number of remaining live ones inside the enclosure.
In a pre-scheduled agenda, animals are subjected to electric stimulation, and the resulting venom of hundreds of individuals is then lyophilized, resulting in batches of freeze-dried venom. The standardized venom obtained from this process is destined for horse immunization as part of the manufacturing process, as well as to provide reference material for quality control of the active substance and the final product.
There are very few facilities in the world capable of keeping such a large number of scorpions and performing the volume of venom extractions in accordance with good manufacturing practices and ethical use of the animals, as done in the Arthropods Bioterium of Instituto Butantan. Therefore, our objective is to describe the scorpion maintenance protocols and venom extraction procedures used in T. serrulatus husbandry, which successfully provide the necessary amount of venom for the production of scorpion antivenom.

Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Fångenskap och giftextraktion av Tityus serrulatus (brasiliansk gul skorpion) för produktion av motgift

This protocol describes the method to maintain scorpions for the extraction of high amounts of venom, which is used in the production of neutralizing equine-delivered immunoglobulins or antivenoms. There is a high demand for scorpion antivenom in tropical countries, which is the keystone for treating human envenomation cases. The maintainancy procedures reproduce optimal natural conditions, keeping the scorpions in captivity, thus with considering animal welfare and good laboratory practice.In parallel, venom extraction is based on electrical stimulation in the scorpion telson, with minimal harmful effects on the animals. This protocol enabled us to increase exponentially the number of animals maintained in captivity, ranging from 4, 000 to 20, 000 individuals in 10 years. At the same time, venom has been extracted sufficiently to immunize horses to obtain plasma with the specific antibodies, which is the starting material for the manufacturing process that results in around 100, 000 vials of anti-venom per year.The venom milking procedures different from others described in the literature in which the telson is removed, thus limiting the number of extraction per animal. The accumulated experience of the technicians of our facility allow us to extract venom from 200 to 300 animals in around 1 to 1 1/2 hours without anesthetizing the animals or making other invasive procedures.

Watch this Scientific Journal Video about Venom Extraction from Brazilian Yellow Scorpions at JoVE.com

Venom Extraction from Brazilian Yellow Scorpions  

Giftutvinning från brasilianska gula skorpioner

På dagen för giftextraktionen räknar du de levande skorpionerna i inhägnaden och överför dem till en glasbehållare med hjälp av en lång pincett. Ordna sedan all nödvändig utrustning som krävs för giftextraktion på ytan av luftflödesskåpet. Anslut elektrostimulatorns nätsladd till uttaget.Slå sedan på luftflödesskåpet och elektrostimulatorn. Ställ in de visade parametrarna på elektrostimulatormaskinen för giftextraktion. Använd en pincett, håll i skorpionens metasoma och immobilisera sedan telsonen med en böjd pincett innan du håller fast den med en hand.Efter fasthållning, placera telsonen i direkt kontakt med en elektrostimulatorledare och samla upp giftdropparna i ett mikrorör av plast. Överför stjärtfenan från handen till den böjda pincetten och placera försiktigt skorpionen på botten av glasbehållaren. När alla skorpioner har mjölkats sätter du tillbaka dem i inhägnaden.Förvara giftet vid minus 20 grader Celsius tills det frystorkas. Giftproduktionen av Tityus serrulatus överstiger 80 gram flytande gift årligen, vilket motsvarar över 13 gram frystorkat gift. Produktionsindikatorer som genomsnittligt mjölkat gift per djur, gift som extraherats per månad och skorpioner som extraherats per månad har visat en positiv trendlinje som indikerar robust produktion.

Research

JoVE Journal

Biology

Venom Extraction from Brazilian Yellow Scorpions

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow ...