Name: Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production
Uploaded: 2023-10-06T13:10:00
Description: Watch this Scientific Journal Video about Optimizing Scorpion Venom Extraction for Antivenom Production at JoVE.com

Protocols involving invertebrate animals are exempt from Instituto Butantan&#39;s Committee on the Use and Care of Animals&#39; approval. However, the maintenance of scorpions described here follows ethical parameters, and animal welfare is respected according to the needs of the species.
1. Housing
<ol>
	<li>Keep the scorpions in communal polypropylene containers as enclosures (height 35 cm, width 35.5 cm, length 72 cm) containing a maximum of 350 animals.</li>
	<li>Maintai...

Rearing of Brazilian Yellow Scorpion for Venom Extraction

<ol>
	<li>Polis, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The biology of scorpions. , (1990).</li><li>Lacerda, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpion+envenomation+in+the+state+of+S&#227;o+Paulo,+Brazil:+Spatiotemporal+analysis+of+a+growing+public+health+concern.">Scorpion envenomation in the state of S&#227;o Paulo, Brazil: Spatiotemporal analysis of a growing public health concern.</a> PLoS One. 17 (4), 0266138 (2022).</li><li>Reckziegel, G. C., Pinto, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpionism+in+Brazil+in+the+years+2000+to+2012.">Scorpionism in Brazil in the years 2000 to 2012.</a> Journal of Venomous Animals and Toxins Including Tropical Diseases. 20, 46 (2014).</li><li>Guerra-Duarte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpion+envenomation+in+Brazil:+Current+scenario+and+perspectives+for+containing+an+increasing+health+problem.">Scorpion envenomation in Brazil: Current scenario and perspectives for containing an increasing health problem.</a> PLoS Neglected Tropical Diseases. 17 (2), 0011069 (2023).</li><li>Pimenta, R. J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+to+survive+and+kill:+Tityus+serrulatus,+the+Brazilian+yellow+scorpion.">Selected to survive and kill: Tityus serrulatus, the Brazilian yellow scorpion.</a> PLoS One. 14 (4), e0214075 (2019).</li><li>Lacerda, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+areas+vulnerable+to+scorpionism+and+its+association+with+environmental+factors+in+S&#227;o+Paulo,+Brazil.">Detection of areas vulnerable to scorpionism and its association with environmental factors in S&#227;o Paulo, Brazil.</a> Acta Tropica. 230, 106390 (2022).</li><li>Torrez, P. P. Q., Dourado, F. S., Bertani, R., Cupo, P., Fran&#231;a, F. O. d. e. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpionism+in+Brazil:+exponential+growth+of+accidents+and+deaths+from+scorpion+stings.">Scorpionism in Brazil: exponential growth of accidents and deaths from scorpion stings.</a> Journal of the Brazilian Society of Tropical Medicine. 52, 20180350 (2019).</li><li>Amado, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vulnerable+areas+to+accidents+with+scorpions+in+Brazil.">Vulnerable areas to accidents with scorpions in Brazil.</a> Tropical Medicine &amp; International Health. 26 (5), 13561 (2021).</li><li>Brasil, Minist&#233;rioda da Sa&#250;de, Funda&#231;&#227;o Nacional de Sa&#250;de. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual de diagn&#243;stico e tratamento de acidentes por animais pe&#231;onhentos. , (2001).</li><li>Candido, D. M., Lucas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+scorpions+of+the+genus+Tityus+Koch+(Scorpiones,+Buthidae)+for+venom+obtention+at+Instituto+Butantan,+S&#227;o+Paulo,+Brazil.">Maintenance of scorpions of the genus Tityus Koch (Scorpiones, Buthidae) for venom obtention at Instituto Butantan, S&#227;o Paulo, Brazil.</a> Journal of Venomous Animals and Toxins including Tropical Diseases. 10 (1), 000100007 (2004).</li><li>Lucas, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laborat&#243;rio+de+Artr&#243;podes+do+Instituto+Butantan+e+os+aracn&#237;deos+pe&#231;onhentos.">Laborat&#243;rio de Artr&#243;podes do Instituto Butantan e os aracn&#237;deos pe&#231;onhentos.</a> Hist&#243;ria Ci&#234;ncias Sa&#250;de Manguinhos. 10 (3), 000300011 (2003).</li><li>Braga-Pereira, G. F., Santos, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+in+a+sexual+population+of+the+Brazilian+yellow+scorpion+(Tityus+serrulatus,+Buthidae)+as+evidence+of+facultative+parthenogenesis.">Asexual reproduction in a sexual population of the Brazilian yellow scorpion (Tityus serrulatus, Buthidae) as evidence of facultative parthenogenesis.</a> The Journal of Arachnology. 49 (2), (2021).</li></ol>

The application of the methods described allows us to keep a large number of T. serrulatus individuals and gives us consistent predictability of the number of scorpions needed for annual venom production. This way, we are able to provide enough batches of venom in advance to supply the antivenom manufacturing process. At the same time, the development of pre-established schedules for maintenance, feeding, and venom extraction is an essential part of the activities and assists in complying with the described prot...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Carboard sheet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg tray</td>
			<td>NA</td>
			<td>NA</td>
			<td>cardboard 36 places egg trays</td>
		</tr>
		<tr>
			<td>Electro stimulator device</td>
			<td>Instituto Butantan</td>
			<td>ART-FV-01</td>
			<td>Internally designed for the venom extraction&#160;</td>
		</tr>
		<tr>
			<td>Kraft paper</td>
			<td>NA</td>
			<td>NA</td>
			<td>regular kraft paper</td>
		</tr>
		<tr>
			<td>Making tape</td>
			<td>NA</td>
			<td>NA</td>
			<td>White masking tape (24 mm x 50 m)</td>
		</tr>
		<tr>
			<td>Polyproplene container</td>
			<td>NA</td>
			<td>NA</td>
			<td>70 L Polypropylene box (H &#8211; 35 cm, W &#8211; 35.5 cm, L - 72 cm)</td>
		</tr>
		<tr>
			<td>Polyproplene tray</td>
			<td>NA</td>
			<td>NA</td>
			<td>Polypropylene tray (H &#8211; 3.0 cm, W &#8211; 18.5 cm, L - 30 cm)</td>
		</tr>
		<tr>
			<td>Self-adhesive plastic</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.

Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

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

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.

Thirty standard operational procedures (SOPs) were developed for each procedure, ensuring the repeatability of the procedures among technicians and compliance with quality parameters. The average mortality rate after the extraction procedure is around 12%, which may be considered low, considering the electrostimulation routine and the captivity environment, where stress factors are successfully reduced by applying the procedures.
To keep the animals healthy, a tray covered with cotton and wate...

Watch this Scientific Journal Video about Optimizing Scorpion Venom Extraction for Antivenom Production at JoVE.com

Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

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

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Research

JoVE Journal

Biology

Housing, Captive Maintenance, and Feeding of Brazilian Yellow Scorpions for Venom Extraction

Begin by preparing the enclosures for housing the scorpions. To smoothen the walls of the polypropylene container acting as an enclosure, place 10 centimeters of self adhesive plastic tape over its internal surface, as well as on the upper corners of the container. Then, line the floor of the polypropylene container with craft paper fixed with masking tape on the edges on all sides.Arrange four cardboard trays as vertical substrates on one side of the container with a rigid cardboard sheet between each tray. Place a shallow polypropylene tray containing soaked cotton as a water source on the opposite side of the enclosure. Place the scorpions in the prepared enclosures and house the containers in the room maintained at 24 degrees Celsius.For complete maintenance, first remove the scorpions from the water tray. Then, using long forceps remove the water tray from the enclosure. Place the scorpions in the clean enclosure.Remove all the scorpions left on the enclosure's floor and place them in the clean enclosure. Then remove the remaining live prey from the enclosure. Remove all the disposable substrates from the enclosure and properly discard them before sanitizing the reusable items such as water trays and containers with 0.5%hypochlorite solution.To begin, place the scorpions in communal polypropylene containers as enclosures. Calculate the amount of feed according to the energy intake of the scorpions. Then, count the number of cockroaches as prey items.Then, apply calcium carbonate with alcohol around the top of the enclosure to prevent cockroaches from escaping the enclosure. Place the cockroaches inside the enclosure for two days.

Venom Extraction from Brazilian Yellow Scorpions

On the day of venom extraction, count the live scorpions in the enclosure and using long forceps, transfer them to a glass container. Next, arrange all necessary equipment required for venom extraction on the surface of the airflow cabinet. Plug the electro stimulator power cord into the outlet.Then turn on the airflow cabinet, and electro stimulator. Set the displayed parameters on the electro stimulator machine for venom extraction. Using forceps, hold the metasoma of the scorpion, then immobilize the telson with curved forceps before restraining it with a hand.After restraint, place the telson in direct contact with an electro stimulator conductor and collect the venom drops into a plastic micro tube. Transfer the telson from the hand to the curved forceps and gently place the scorpion on the bottom of the glass container. After all the scorpions are milked, place them back into the enclosure.Store the venom at minus 20 degrees Celsius until lyophilization. The venom production of Tityus serrulatus surpasses 80 grams of liquid venom annually equating to over 13 grams of freeze dried venom. Production indicators such as average venom milked per animal, venom extracted per month, and scorpions extracted per month have shown a positive trend line indicating robust production.