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Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 127-138, March 2021

Toxicological, enzymatic, and immunochemical characterization

of Bothrops asper (Serpentes: Viperidae) reference venom from Panama

Alina Uribe-Arjona

1,3

, Hildaura Acosta de Patiño

2,3*

, Víctor Martínez-Cortés

, David Correa-

Ceballos

3,4

, Abdiel Rodríguez

, Leandra Gómez-Leija

, Natalia Vega

, José María Gutiérrez

& Rafael Otero-Patiño

1. Departamento de Bioquímica y Nutrición, Facultad de Medicina, Universidad de Panamá, Panamá, Ciudad

Universitaria, Estafeta Universitaria, Apartado 3366, Panamá 4, Panamá; allisice@hotmail.com

2. Departamento de Farmacología, Facultad de Medicina, Universidad de Panamá, Panamá, Ciudad Universitaria,

Estafeta Universitaria, Apartado 3366, Panamá 4, Panamá; eleandg@yahoo.com

3. Centro de Investigación e Información de Medicamentos y Tóxicos (CIIMET), Facultad de Medicina, Universidad de

Panamá, Panamá, Ciudad Universitaria, Estafeta Universitaria, Apartado 0824-00167, Panamá, Panamá;

hildaura6@gmail.com, nataliavega@usf.edu

4. Centro para Investigaciones y Respuestas en Ofidiologia (CEREO), Facultad de Ciencias Naturales, Exactas y

Tecnología, Universidad de Panamá, Panamá, Ciudad Universitaria, Estafeta Universitaria, Apartado 3366, Panamá

4, Panamá; pvmartinez@gmail.com, viperidaepanama@gmail.com

5. Centro Regional Universitario de Veraguas, Universidad de Panamá, Veraguas, Panamá, Apartado 3366, Panamá 4,

Panamá; aernesto@hotmail.com

6. Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica; Apartado

11501 San José, Costa Rica; jose.gutierrez@ucr.ac.cr

7. Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia; Calle 7 A Sur No. 35-55, Apto. 505, Medellin,

Colombia; rafaotero@une.net.co

* Correspondence

Received 31-X-2019. Corrected 22-V-2020. Accepted 09-XI-2020.

ABSTRACT. Introduction: It is estimated that 2 000 snakebites occur in Panama every year, 70 % of which are

inflicted by Bothrops asper. Objective: To determine the biochemical and toxicologic effects and to assess the

immunochemical characteristics of a reference pool of B. asper venom representative of Panama. Methods: The

reference venom was prepared as a homogeneous mixture of the venoms obtained from 78 adult snakes collected

in four geographic areas of Panama. Enzymatic and toxicological activities were assessed. The electrophoretic

pattern was studied by SDS-PAGE. Immunoreactivity of various antivenoms was analyzed by Western blot.

Results: B. asper reference venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating,

proteinase and phospholipase A

activities. SDS-PAGE showed the presence of protein bands with molecular

weights ranging from 8 to 70 kDa, with the presence of predominant bands at ≈ 15 kDa and ≈ 30 to 66 kDa,

which likely correspond to phospholipases A

and metalloproteinases,

respectively. Immunoblotting showed a

high degree of recognition by various antivenoms, especially by antivenoms from Colombia and Costa Rica.

Conclusions: Following recommendations by the World Health Organization, this reference venom of B. asper

of Panama will become a useful tool for the preclinical evaluation of antivenoms distributed in this country.

Key words: snakebite; venom; toxicity; immunochemical characterization; antivenom.

Uribe-Arjona, A., Acosta de Patiño, H., Martínez-Cortés, V., Correa-Ceballos, D.,

Rodríguez, A., Gómez-Leija, L., Vega, N., Gutiérrez, J.M., & Otero-Patiño, R.

(2020). Toxicological, enzymatic, and immunochemical characterization of Bothrops

asper (Serpentes: Viperidae) reference venom from Panama. Revista de Biología

Tropical, 69(1), 127-138. DOI 10.15517/rbt.v69i1.39502

ISSN Printed: 0034-7744 ISSN digital: 2215-2075

DOI 10.15517/rbt.v69i1.39502

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Snakebite envenoming is a global public

health problem that, despite its complexity

and magnitude, has not received enough atten-

tion from health authorities, pharmaceutical

companies and research agencies in all parts

of the world (Gutiérrez, Williams, Fan, &

Warrel, 2010). Therefore, the World Health

Organization (WHO) recently included snake

bite envenoming on its list of neglected tropical

diseases (Gutiérrez et al., 2017).

This environmental and occupational dis-

ease affects mainly agricultural workers in

rural communities in tropical regions, afflicting

almost exclusively the impoverished popula-

tion (Williams et al., 2010). The victims of

envenoming generally live in remote commu-

nities, far from medical facilities, thus lacking

timely medical attention. Furthermore, many

people die on the way to medical centers, and

others mostly rely on popular medicine and,

therefore, are not included in the official hospi-

tal records (Otero-Patiño, 2009).

Between 130 000 and 150 000 snakebite

cases are recorded in the Latin American region

each year, with an estimated 2 300 annual deaths

(Chippaux, 2006). Panama has the highest inci-

dence of snakebites in the region, registering

54-62 cases per 100 000 inhabitants (approxi-

mately 2 000 bites per year). The mortality rate

is estimated at 0.54/100 000 inhabitants (Min-

istry of Health, 2011). Many affected people

develop physical and psychological sequelae

and permanent disability, which is estimated

to exceed the number of deaths (WHO, 2010;

Gutiérrez et al., 2017).

In Latin America, snakes of the genus

Bothrops inflict the greatest number of acci-

dents, and B. asper is the principal species

responsible for envenomings in Southern

Mexico, Central America, and Northern South

America. In the Central American region, it

is estimated that B. asper is responsible for

approximately 50-80 % of snakebites, as well

as most of the deaths due to these envenom-

ings (Otero-Patiño, 2009). Popularly, B. asper

is known as “Terciopelo”, “Barba amarilla”,

“nauyaca” or “equis”. It is widely distrib-

uted in the humid lowlands of Mexico, Central

America, Venezuela, Colombia and Ecuador,

living both in forests and in areas of live-

stock and agricultural use, which consequently

increases the likelihood of contacts between

snakes and workers or their homes (Otero-

Patiño, 2009).

The venom of B. asper induces both

local alterations at the site of venom injection

(edema, hemorrhage, dermonecrosis, flictenas,

myonecrosis), and systemic effects that can be

life threatening (defibrination, bleeding distant

from the bite site, nephrotoxicity and cardio-

vascular shock) (Otero et al., 2002; Gutiérrez

& Lomonte, 2003; Gutiérrez, Escalante, &

Rucavado, 2009; Gutiérrez, Rucavado, Chaves,

Díaz, & Escalante, 2009). Snake venom is a

complex mixture, which in addition to a large

variety of toxins, enzymes and proteins without

enzymatic activity, also contains amino acids,

nucleotides, phosphorylated sugars, lipids, and

metal ions. Research in proteomics has shown

a high number of different proteins in the

venom of B. asper (Alape-Girón et al., 2008),

which predominantly belong to the families of

metalloproteinases (41-44 %), phospholipases

(29-45 %), serine proteinases (4-18 %),

L-amino acid oxidases (5-9 %), disintegrins

(1-2 %) and lectin-like C-type proteins (Angulo

& Lomonte, 2009).

There is significant qualitative and quan-

titative variation in the biochemical compo-

sition of venoms, both between and within

species. These differences have their origin

in ontogenetic, geographic, phylogenetic, and

environmental factors, as well as individual

factors (Gutiérrez, Chaves & Bolaños, 1980;

Saldarriaga et al., 2003; Núñez & Otero, 1999;

Alape-Girón et al., 2008).

Martínez (1983a; 1983b) and Quintero,

González, Suárez, & Arantes (2007) studied

some biochemical and toxicological charac-

teristics of B. asper venoms from Panama. A

previous study described slight variations in the

toxic and enzymatic activities in the venoms of

pools of B. asper venoms from several regions

in Panama, although venoms were in general

similar (Vélez et al., 2017).

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Antivenoms remain the only specific and

effective treatment for envenoming caused

by snakebites (Gutiérrez, Williams, Fan, &

Warrell, 2010). Owing to the described varia-

tion in venom composition, antivenoms should

be subjected to preclinical evaluations (neutral-

ization of lethality and other toxic activities)

against the most relevant snake venoms from a

particular country or region (Otero et al., 1995;

Williams et al., 2010; Gutiérrez et al., 2013).

To this end, the World Health Organization

(WHO) has recommended the preparation of

reference national venom pools of the medi-

cally-relevant snake species and to characterize

such reference venoms, in order to use them in

the preclinical evaluation of antivenoms being

used in the countries.

The venom of B. asper from various coun-

tries has been extensively studied in terms of its

biochemical and toxicological characteristics

(Angulo & Lomonte, 2009; Gutiérrez, Escalan-

te, & Rucavado, 2009; Gutiérrez, Rucavado,

Chaves, et al., 2009). However, no national

reference venom of B. asper has been prepared

and characterized in any country. The present

study was carried out with the aim of character-

izing the toxicological and enzymatic effects,

as well as the immunochemical reactivity, of

a reference venom of B. asper from Panama,

prepared by generating a pool of venoms from

four geographic regions in this country. This

venom mixture can be used in the preclinical

evaluations to test the efficacy of antivenoms

distributed in Panama.

MATERIALS AND METHODS

Animals and venom: The venom was

obtained via manual milking of 78 adult speci-

mens of B. asper collected in the following four

geographical regions of mainland of Panama:

Zone 1: Bocas del Toro, Veraguas’ Caribbean

region, Colon and Guna Yala; Zone 2: Chiriquí

and Veraguas’ Pacific region; Zone 3: Los San-

tos, Herrera and Coclé; and Zone 4: Panama

Oeste, Panama and Darien (Fig. 1). The snakes

were kept in captivity at the Serpentarium

located in the School of Biology at the Uni-

versity of Panama. Once collected, the crude

Fig. 1. Geographical regions where Bothrops asper snakes were collected. Zone 1: Bocas del Toro, Veraguas’ Caribbean

region, Colon and Guna Yala; Zone 2: Chiriquí and Veraguas’ Pacific region; Zone 3: Los Santos, Herrera and Cocle; and

Zone 4: West Panama, Panama and Darien.

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venom was centrifuged for 15 min at 3 000

rpm. The supernatant was frozen at -20 °C,

lyophilized and stored at -20 °C until use. To

ensure geographical representation, a homo-

geneous mixture was prepared using 100 mg

of lyophilized venom from each geographical

zone in order to prepare the reference venom.

Snakes used for the preparation of this refer-

ence venom were adults; 52 % were females

and 48 % were males. Approximately half of

the specimens were collected during the dry

season (December to April) and the other half

during the rainy season (May to November).

For the in vivo tests, mice of the strain

CD-1, with a body weight of 18-20 g, were

used following the recommendations of the

Guide for the Care, Ethics and Use of Labora-

tory Animals of the National Research Council

of the National Academies (2011). The mice

were provided by the Bioterium at the Univer-

sity of Panama. When these studies were car-

ried out, the Ethics Committee in Animal Use

had not been established in our country.

Venom toxicological and enzymatic effects

Hemorrhage: Groups of four mice per

dose received intradermal (i.d.) injections, in

the ventral abdominal area, of a range of doses

of venom (1.0-16.0 μg) in a volume of 100 μl

of phosphate buffered saline solution (PBS)

at pH 7.2. The control group received an

equivalent volume of PBS. The animals were

sacrificed 2 h later by inhalation of anesthetic

(Sevorane®) and the area of hemorrhage in the

inner side of the skin was measured. The Mini-

mum Hemorrhagic Dose (DHm) was the dose

of venom that induced an area of hemorrhage

of 10 mm in diameter, following the method of

Kondo, Kondo, Ikesawa, Murata, & Ohsaka,

(1960), modified by Gutiérrez, Gené, Rojas, &

Cerdas (1985).

Edema-forming: Groups of four mice per

dose received subcutaneous (s.c.) injections, in

the right paw (subplantar), of a range of doses

of venom (0.031-0.5 μg), diluted in a volume

of 5 μl of PBS. As a control, 5 μl of PBS were

injected into the left paw of each mouse. After 3

h, the relative volume (edema) of the right paw

versus the left paw (control) was measured in

each animal, using an LE 7500 digital plethys-

mometer from PanLab Harvard Apparatus. The

Minimum Edema-forming Dose (DEm) was

the dose of venom that caused 30 % increase

in paw volume at 3 h, following the method of

Yamakawa, Nozaki, & Hokama, (1976), modi-

fied by Chaves, Barboza, & Gutiérrez (1995).

Myotoxicity: Groups of four mice per

dose were received intramuscular (i.m.) injec-

tions, in the right gastrocnemius, of a range of

doses (25 to 50 μg) of B. asper venom in 100

μl of PBS. The control group received the same

volume of PBS. After 3 h, mice were anesthe-

tized and blood samples were collected from

the orbital venous sinus, using heparinized cap-

illaries, to quantify the activity of the enzyme

creatine kinase (CK) in the plasma, with a com-

mercial kit (CK NAC liquiUV, Human). The

Minimum Myotoxic Dose (DMm) was the dose

of venom that induced a four-fold increase in

plasma CK activity, compared with the control

group (Gutiérrez, Arroyo, & Bolaños, 1980).

Myotoxic activity was also expressed as the

CK activity of plasma from mice injected with

50 μg of venom (Segura et al., 2010).

Lethality: Groups of five mice per dose

received intraperitoneal (i.p.) injections of a

range of doses of venom (1 to 7 mg/kg), diluted

in a volume of 500 μl of PBS. The number of

mice dead at 48 h was recorded. Lethal Dose

(LD

) was obtained by the Spearman-Karber

method (WHO, 1981), using TOXICALC soft-

ware provided by the Instituto Clodomiro

Picado (University of Costa Rica).

Coagulant effect: Test tubes with 200 μl

of citrated platelet-free human plasma were

incubated at 37 °C with a volume of 100 μl of

PBS containing different amounts of venom

(0.16-1.8 μg). The clotting time was recorded

in seconds. The Minimum Coagulant Dosage

(DCm) was the dose of venom that induced the

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coagulation of the plasma in 60 s (Gené, Roy,

Rojas, Gutiérrez & Cerdas, 1989).

Defibrinating effect: Groups of four mice

received intravenous (i.v.) injections, into the

caudal vein, with different doses of venom

(0.625-20.0 μg) in a volume of 200 μl of PBS.

After 1 h the mice were anesthetized by inhala-

tion of Sevorane® and cardiac puncture was

realized to obtain whole blood samples, which

were placed in glass tubes and maintained at

room temperature for 1 h. The Minimum Defi-

brinating Dose (DDm) was the minimum dose

of venom that caused anticoagulation in all

injected mice (Theakston & Reid, 1983; Gené

et al., 1989).

Proteolysis: Proteolytic activity was test-

ed by incubating various amounts of venom

in 2 ml of casein (2 % in PBS, pH 8.0) for 30

min. The reaction was stopped by adding 4.0

ml of 5 % trichloroacetic acid. After 5 min, the

tubes were centrifuged at 3 000 rpm for 10 min

and the absorbances of the supernatant were

recorded at 280 nm against the blank reagent.

The Minimum Proteolytic Dose (DPm) was

determined as the amount of venom that pro-

duced an absorbance change of 0.5 at 280 nm

(Lomonte & Gutiérrez, 1983).

Indirect Hemolysis (Phospholipase A

Activity): The activity of phospholipase A

was determined using agarose plates (0.8 %

in PBS, pH 7.2) prepared with human erythro-

cytes, CaCl

(0.01 M) and a suspension of egg

yolk (1:4 in PBS). Volumes of 15 μl of solu-

tion with increasing concentrations of venom

(1.2-30.0 μg) were applied in 3 mm diameter

wells. Then, the plates were incubated for 20

h at 37 °C and the diameter of hemolytic halos

was measured. The Minimum Hemolytic Dose

(DHLm) was the dose of venom that induced

a hemolysis halo of 20 mm diameter in 20 h

(Gutiérrez, Avila, Rojas & Cerdas, 1988).

Immunochemical characterization

of the venom

Quantification of proteins: The pro-

tein content of the venom was estimated by

the Bradford method, as modified by Spec-

tor (1978), using bovine serum albumin as

the standard.

Electrophoresis-SDS PAGE: Electropho-

resis was performed in 15 % polyacrylamide

vertical minigels in the presence of sodium

dodecylsulfate (SDS-PAGE) (Laemmli, 1970).

For each run, 15 μg of venom equivalent to 11

μg of protein were used under reducing condi-

tions with β-mercaptoethanol, and 150 V were

applied for 70 min in a Mini-Protean II cham-

ber from Bio-Rad (Richmond, CA, USA). The

reference venom (pool of the four regions) and

venoms from each region were run in parallel

with a molecular mass marker (Bio-Rad-cata-

log No. 161-0304). After separation, gels were

stained with Coomassie Brilliant Blue. Densi-

tometry was performed using myImageAnaly-

sis™ Software from Thermo Fisher Scientific.

Preparation of anti-B. asper serum: Two

New Zealand female rabbits (2-3 kg) were

immunized using variable doses of the refer-

ence venom from Panama, ranging between

250 and 1 000 μg, which were applied s.c.

(the first two doses) and i.m. (the last dose), at

monthly intervals, using appropriate adjuvants

(complete and incomplete Freund’s adjuvants).

After 60 days, blood was collected, and the

serum was separated by centrifugation and

passed through protein A columns to obtain

IgG (specific and non-specific against the

venom). The fractions collected were dialyzed

against distilled water, then lyophilized and

frozen until use (Lomonte, 2002).

Immunological reactivity (Western blot-

ting): The reference venom from Panama

was separated by SDS-PAGE (as previously

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described) and transferred for 2 h to nitrocellu-

lose membranes in a Mini-Transblot® chamber

from Bio-Rad.

As a primary antibody, polyvalent anti-

venoms from different regions of Latin Amer-

ica were used: Argentina (Instituto Biológico

Argentino Saic-lot 1604), Brazil (Institu-

to Vital Brazil SA, lot 095106E), Colom-

bia (Laboratorio Probiol SA, lot Ap310ix10),

Costa Rica (Instituto Clodomiro Picado, lot

4691110POLQ) and Panama (anti-B asper

serum produced in rabbits).

The membranes were incubated at room

temperature with the corresponding antiven-

oms in a dilution of 1:1 000 (in PBS Tween

20) for 3 h. They were then incubated for 2

h at room temperature with a dilution 1:5 000

of anti-horse IgG or anti-rabbit IgG (whole

molecule)-Peroxidase antibody produced in

rabbit, as a secondary antibody. Subsequently,

the reaction was revealed using a solution of

hydrogen peroxide in the presence of 4-Cl-

1-naphthol and diaminobenzidine. The reaction

was stopped by adding distilled water at the

time the bands appeared.

Presentation of the results: All toxico-

logical and enzymatic tests were performed

in triplicate. The results are expressed as the

mean ± S.E.M (standard error of the mean),

except for lethality, in which case variability is

expressed with 95 % confidence intervals.

RESULTS

The reference venom of B. asper from

Panama induced characteristic local and sys-

temic effects of venoms of the Viperidae fam-

ily. This venom possesses lethal, hemorrhagic,

myotoxic, edema-forming, defibrinating and

in vitro coagulant activities; it also shows

proteolytic and phospholipase A

enzymatic

activities (Table 1). In addition to estimat-

ing the Minimum Myotoxic Dose, creatine

kinase (CK) activity 3 h after the intramuscular

injection of 50 μg of venom corresponded to

10 196 ± 1 817 U/l.

When venom protein components were

separated by SDS-PAGE (Fig. 2), fractions

with molecular weights between 8 and 70 kDa

were observed, with the presence of predomi-

nant bands at 70 kDa, 42-48 kDa, 24-37 kDa,

15 kDa and 11 kDa. The percent composition

of the main protein fractions in Panamanian

reference venom can be seen in Table 2.

Fig. 3A shows the SDS-PAGE profile of

the proteins of B. asper reference venom from

Panama. Western blot analysis of the immu-

nological reactivity of several Latin American

TABLE 1

Toxicological and enzymatic activities of the reference venom of Panamanian Bothrops asper

Test Results ± S.E.M.

Hemorrhagic activity (DHm) 4.93 ± 0.29 μg

Myotoxic activity (DMm) 10.2 ± 1.68 μg

Edema-forming activity (DEm) 0.32 ± 0.04 μg

Coagulant activity (DCm) 0.53 ± 0.04 μg

Defibrinating activity (DDm) 1.25 ± 0.0 μg

Proteolytic activity (DPm) 1.39 ± 0.1 mg

Phospholipase A

activity (DHIm) 2.93 ± 0.3 μg

Lethal activity (DL

) 4.84 mg/kg (3.98-5.88)

DHm: Minimum Hemorrhagic Dose; DMm: Minimum Myotoxic Dose; DEm: Minimum Edema-forming Dose; DCm:

Minimum Coagulant Dose; DDm: Minimum Defibrinating Dose; DPm: Minimum Proteolytic Dose; DHlm: Minimum

Indirect Hemolytic Dose (Phospholipase Activity A2); DL

: Medium Lethal Dose. The 95 % confidence intervals are

shown in parentheses. S.E.M.: Standard Error of the Mean. The definitions of each dose are detailed in the Materials and

Methods section.

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antivenoms against the reference venom of B.

asper from Panama is shown in Fig 3B. All

antivenoms reacted against most of the bands,

but a higher reactivity was observed for the

antivenoms of Costa Rica and Colombia.

All antivenoms showed immunorecogni-

tion for protein bands of 15-16 kDa and 25-30

kDa, the former bands containing PLA

, such

as myotoxins, and the latter bands contain-

ing metalloproteinases PI, serine proteinases

and PLA

. Fractions of proteins with masses

between 42-48 kDa and 55-70 kDa were highly

recognized by all antivenoms. All antivenoms,

but particularly those from Costa Rica and

Colombia, presented immunoreactivity against

the 11 kDa fraction.

DISCUSSION

This study follows the recommendation

of the WHO Guidelines for the Production,

Control and Regulation of Snake Antivenom

Immunoglobulins (WHO, 2010) in the sense

that it is relevant to prepare national refer-

ence snake venoms of the species of highest

medical impact. Few countries, such as Brazil,

have prepared reference venoms (Araújo et al.,

2017). In our study, a reference venom of B.

asper from Panama has been prepared, includ-

ing representative venom samples from adult

specimens collected in the various regions of

this country. This venom has been character-

ized in terms of its toxicological and enzymatic

activities, as well as its electrophoretic pattern

in SDS-PAGE. This national reference venom

can be used in the preclinical assessment of the

neutralizing efficacy of antivenoms distributed

in this Central American country. The reference

venom from Panamanian B. asper was found to

induce lethal, edema-forming, coagulant, hem-

orrhagic and myotoxic activities. Therefore,

this toxicological profile is similar to the one

previously described for B. asper venoms from

other Central American countries and from

Mexico (Gutiérrez, Chaves & Bolaños, 1980;

Fig. 2. Electrophoretic patterns obtained by SDS PAGE (15 % acrylamide) run under reducing conditions using 15μg of

Panamanian B. asper reference venom, Costa Rican venom and venom from each Panamanian zone. Lane 1: Low Range

Molecular Weight Marker (Bio-Rad); Lane 2: Reference B. asper venom from Panama; Lane 3: B. asper venom from Costa

Rica; Lane 4: B. asper venom from Panama Zone 1 (Atlantic); Lane 5: B. asper venom from Panama Zone 2 (Chiriquí and

Pacific Veraguas); Lane 6: B. asper venom from Panama Zone 3 (Los Santos, Herrera and Cocle); Lane 7: B. asper venom

from Panama Zone 4 (Panama and Darién). Gels were stained with Coomassie Brilliant Blue.

TABLE 2

Percent composition of the protein fractions

of Bothrops asper venom from Panama

Molecular Weight (kDa) Content (Average percent)

55-70 7.1

42-48 8.3

24-37 37.4

15 40.0

11 7.2

Determined by densitometry using My Image Analysis™

software (Thermo Scientific).

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Saravia et al., 2001; Quintero et al., 2007;

Segura et al., 2010).

The most important parameter of toxicity

of a venom is the Median Lethal Dose (LD

The Panamanian B. asper reference venom had

an LD

of 91.9 μg (95 confidence limits: 75.6-

111.7 μg), which corresponds to a dose of 4.84

mg/kg (95 % confidence limits: 3.98-5.88 mg).

This value is similar to the results obtained

for adult B. asper venoms from other regions

in Latin America, as published by Gutiérrez,

Chaves & Bolaños (1980), Otero et al. (1995),

Saravia et al. (2001), Gutiérrez, Escalante, &

Rucavado (2009) and Segura et al. (2010).

This reference venom from Panama showed

high edematigenous and myotoxic activities.

Hemorrhagic effect was also observed with

this Panamanian reference venom, with a DHm

value of 4.93 ± 0.29 μg. The venoms from spe-

cific regions of Panama, studied by Martínez

(1983b) and Quintero et al. (2007), presented

values of 2.5 μg and 6.3 ± 3.6 μg, respectively.

It should be taken into account that the ref-

erence venom contains snake venoms from

all regions of Panama (including the regions

studied by these authors), which could explain

the intermediate value of the DHm found

in this study.

With regards to in vitro coagulant activ-

ity, the Panama reference venom had a DCm

of 0.536 ± 0.040 μg, which is slightly higher

than the reported value for Costa Rican venom

(DCm = 0.32 ± 0.02 μg) (Segura et al., 2010)

and lower than for Guatemalan venom (3.9 ±

0.08 μg; Saravia et al., 2001). The defibrinat-

ing action in vivo of the Panama reference

venom was evaluated through the DDm, which

was 1.25 μg/mouse. This value is similar to

the Colombian venom (DDm = 1.1 ± 0.3 μg)

(Otero et al., 1995). However, it appears to be

more active than its Costa Rican counterpart

(DDm = 3.0 ± 0.5 μg) (Segura et al., 2010).

The proteolytic activity of viperid venoms

is associated with coagulopathies, edema, local

tissue damage and hemorrhage. The value of

the DPm for the Panamanian reference venom

was 1.39 ± 0.1 mg. This is similar to those pre-

viously reported for the venoms of B. asper of

the Atlantic and Pacific regions of Costa Rica,

which have values of DPm = 1.2 and 1.4 mg,

respectively (Gutiérrez at al., 1985) and slight-

ly higher than that of the B. asper venom from

Honduras (DPm = 2.1 mg) (Rojas et al, 1987).

The reference Panama venom showed

indirect hemolytic activity (induced by phos-

pholipases A

), with a DHlm of 2.93 ± 0.3 μg.

Fig. 3. Electrophoresis and Western blotting assays. A. SDS PAGE electrophoresis of B. asper reference venom from

Panama. MPM: Low Range molecular marker (Bio-Rad); 1. Panamanian B. asper reference venom (15 μg of lyophilized

venom) separated by SDS PAGE (15 %) under reducing conditions; B. Immunological reactivity against B. asper reference

venom from Panama determined by Western blotting using antivenoms produced in Latin America. 1. Antivenom anti B.

asper from Panama (produced in rabbits); 2. Antivenom from Venezuela; 3. Antivenom from Costa Rica; 4. Antivenom from

Colombia; 5. Antivenom from Brazil; 6. Antivenom from Argentina.

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This value is similar to that obtained with B.

asper venoms from Antioquia-Chocó (DHlm =

2.0 μg) in Colombia (Otero et al., 1995).

Electrophoretic analysis by SDS-PAGE of

the reference venom and of venoms from the

different regions of Panama showed qualita-

tively similar patterns, although quantitative

differences were observed, through densito-

metric analysis, in the percentages of each one

of the electrophoretic bands. Previous work

analyzed the variation of B. asper venoms from

these populations in Panama and showed that,

both in their toxic and electrophoretic profiles,

these venoms were similar (Velez et al., 2017).

However, the small variations found in elec-

trophoretic patterns among the populations

highlights the need for including representative

samples of snakes from the various regions in

Panama, in order to have a truly representative

national reference venom preparation.

As for Western blot, the similar patterns

of immunorecognition observed between the

different antivenoms could be attributed to two

aspects: the cross-reactivity and the similarities

that exist between the proteins of the venoms

of the same genus and species, even if they are

from different countries. The high reactivity of

the antivenoms of Costa Rica and Colombia

may be related to the close similarity in the

composition of the B. asper venoms of these

countries and their immunological similari-

ties. Both antivenoms include B. asper venom

in their antigenic mixture, which explains the

high degree of recognition. The experimen-

tal monospecific serum obtained from rabbits

immunized with Panamanian B. asper venom

did not show such strong immunorecognition.

This might be due to the fact that commercial

antivenoms produced in horses subjected to

cycles of repeated immunizations show greater

neutralization than experimental antivenoms

produced in a single round of immunization

(Gutiérrez et al., 2010).

These immunochemical results suggest

that the antivenoms tested in this study could

be effective in neutralizing the main effects

inflicted by the toxins of the venom. However,

the preclinical efficacy of antivenoms should

be assessed by studying the neutralization of

toxic and enzymatic activities of this reference

venom, since immunoreactivity by ELISA or

Western blot does not necessarily imply neu-

tralization of toxicity (Gutiérrez et al., 2010).

For this reason, immunochemical tests, such as

Western blotting, should not be used as a basis

to recommend the therapeutic use of a specific

antivenom (Otero-Patiño, 2009), although they

are useful to determine immunological simi-

larities between venoms.

In conclusion, this study described the

preparation of a national reference venom of

B. asper from Panama, prepared by pooling

venom samples from specimens collected in

various regions of the country. It is therefore

a representative venom from this country. The

toxicological, enzymatic, and electrophoretic

characterization of this reference venom under-

scores strong similarities with venoms from

this species collected in other countries. In

the light of the recommendations of the World

Health Organization (WHO), this national ref-

erence venom will be useful for assessing the

preclinical efficacy of antivenoms distributed

in this Central American country.

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

and requirements. All financial sources are

fully and clearly stated in the acknowledge-

ments section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

This study received financial support from

the School of Medicine, School of Veteri-

nary Medicine, Regional University Center of

Veraguas and the Vice-rectorate of Research

and Postgraduate Studies of the University

of Panama, SENACYT (COL06-017; EST09-

087 B; INF10-051), and Institute Clodomiro

Picado, University of Costa Rica. We want

to thank to Department of Biochemistry and

136

Rev. Biol. Trop. (Int. J. Trop. Biol.) • Vol. 69(1): 127-138, March 2021

Nutrition and the Department of Pharmacology

of the School of Medicine of the University of

Panama. To Jackeline Morán, Hermes Fuentes,

Marcos Salazar and Adolfo Castillo for the sup-

port provided. And our gratitude to Tomás Diez

and Juan Miguel Pascale for their scientific and

technical advice.

RESUMEN

Caracterización toxicológica, enzimática e inmu-

noquímica del veneno de referencia de Bothrops asper

(Serpentes: Viperidae) de Panamá. Introducción: Se

estima que 2 000 mordeduras de serpiente ocurren en

Panamá cada año, el 70 % de las cuales son infligidas

por Bothrops asper. Objetivo: Determinar los efectos

bioquímicos y toxicológicos y evaluar las características

inmunoquímicas del veneno de referencia de B. asper

representativo de Panamá. Métodos: El veneno de referen-

cia se preparó como una mezcla homogénea de los venenos

obtenidos de 78 serpientes adultas recolectadas en cuatro

áreas geográficas de Panamá. Se evaluaron las actividades

enzimáticas y toxicológicas. El patrón electroforético se

estudió mediante SDS-PAGE. La inmunoreactividad de

varios antivenenos se analizó mediante transferencia de

Western. Resultados: El veneno de referencia de B. asper

tiene actividades letales, hemorrágicas, miotóxicas, forma-

doras de edema, coagulantes, desfibrinante, proteolítica

y de fosfolipasa A

. El análisis de SDS-PAGE mostró la

presencia de bandas de proteínas con pesos moleculares

que varían de 8 a 70 kDa, con la presencia de bandas

predominantes a ≈ 15 kDa y ≈ 30 a 66 kDa, que probable-

mente corresponden a fosfolipasas A2 y metaloproteinasas,

respectivamente. La inmunotransferencia mostró un alto

grado de reconocimiento por varios antivenenos, espe-

cialmente por antivenenos de Colombia y de Costa Rica.

Conclusiones: Siguiendo las recomendaciones de la Orga-

nización Mundial de la Salud, este veneno de referencia de

B. asper de Panamá se convertirá en una herramienta útil

para la evaluación preclínica de antivenenos distribuidos

en este país.

Palabras clave: mordedura de serpiente; veneno; toxici-

dad; caracterización inmunoquímica; antiveneno.

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