SciELO - Scientific Electronic Library Online

 
vol.52Seroprevalence of Trypanosoma cruzi infection among blood donors in the state of Bahia, BrazilAssociation of vitamin D3, VDR gene polymorphisms, and LL-37 with a clinical form of Chagas Disease índice de autoresíndice de assuntospesquisa de artigos
Home Pagelista alfabética de periódicos  

Serviços Personalizados

Journal

Artigo

Indicadores

Links relacionados

Compartilhar


Revista da Sociedade Brasileira de Medicina Tropical

versão impressa ISSN 0037-8682versão On-line ISSN 1678-9849

Rev. Soc. Bras. Med. Trop. vol.52  Uberaba  2019  Epub 05-Set-2019

http://dx.doi.org/10.1590/0037-8682-0526-2018 

Major Article

Local and systemic effects caused by Crotalus durissus terrificus, Crotalus durissus collilineatus, and Crotalus durissus cascavella snake venoms in swiss mice

Letícia Helena de Carvalho1 

Leda Fabiélen Teixeira1 

Kayena Delaix Zaqueo1  2 

Jéssica Felix Bastos1 

Neriane Monteiro Nery1 

Sulamita Silva Setúbal1 

Adriana Silva Pontes1 

Diana Butzke3  4 

Walter Cavalcante5 

Marcia Gallacci6 

Carla Freire Celedônio Fernandes1 

Rodrigo Guerino Stabeli1  3 

Andreimar Martins Soares3  4 

Juliana Pavan Zuliani1  3 
http://orcid.org/0000-0001-9221-7749

1Fundação Oswaldo Cruz, Laboratório de Imunologia Celular Aplicada à Saúde, Porto Velho, RO, Brasil.

2Instituto Federal de Educação, Ciência e Tecnologia de Mato Grosso, Campus São Vicente, Centro de Referência de Jaciara, Jaciara, MT, Brasil.

3Universidade Federal de Rondônia, Departamento de Medicina, Centro de Estudos de Biomoléculas Aplicadas à Saúde, Porto Velho, RO, Brasil.

4Centro Universitário São Lucas, Porto Velho, RO, Brasil.

5Universidade Estadual de São Paulo, Instituto de Biociências, Departamento de Física e Biofísica, Botucatu, SP, Brasil.

6Universidade Estadual de São Paulo, Instituto de Biociências, Departamento de Farmacologia, Botucatu, SP, Brasil.


Abstract

INTRODUCTION:

Crotalus envenomations cause serious complications and can be fatal without appropriate treatment. Venom isoforms present and inter/intraspecific variations in the venom composition can result in different symptoms presented by bites by snakes from the same species but from different geographical regions. We comparatively evaluated the local and systemic effects caused by Crotalus durissus terrificus (Cdt), C.d. collilineatus (Cdcolli), and C.d. cascavella (Cdcasc) envenomation.

METHODS:

Venom chromatography was performed. Proteolytic, phospholipase, and LAAO activities were analyzed. Edema, myotoxicity, hepatotoxicity, nephrotoxicity, and coagulation alterations were evaluated.

RESULTS:

The venom SDS-PAGE analyses found the presence of convulxin, gyroxin, crotoxin, and crotamine in Cdt and Cdcolli venoms. Crotamine was not present in the Cdcasc venom. Cdt, Cdcollli, and Cdcasc venoms had no proteolytic activity. Only Cdcasc and Cdt venoms had phospholipase activity. LAAO activity was observed in Cdcolli and Cdcasc venoms. Cdcolli and Cdcasc venoms caused 36.7% and 13.3% edema increases, respectively. Cdt venom caused a 10% edema induction compared to those by other venoms. All venoms increased TOTAL-CK, MB-CK, and LDH levels (indicating muscle injury) and ALT, AST, GGT, and ALP levels (markers of liver damage) and were able to induce a neuromuscular blockade. Urea and creatinine levels were also altered in both plasma and urine, indicating kidney damage. Only Cdcolli and Cdcasc venoms increased TAPP and TAP.

CONCLUSIONS:

Together, these results allow us to draw a distinction between local and systemic effects caused by Crotalus subspecies, highlighting the clinical and biochemical effects produced by their respective venoms.

Keywords: Crotalus durissus; Myotoxicity; Nephrotoxicity; Hepatotoxicity; Neurotoxicity; Edema

INTRODUCTION

Crotalus snakebites cause serious problems and can be fatal unless adequately treated. The high lethality occurs due to the frequency with which Crotalus envenomation causes acute renal failure, one of the major causes of death due to snake bites1. In addition to neurotoxicity, myotoxic action and clotting disorders may cause micro-bleeding, which characterizes the systemic effect of envenomation2-3. The local effects that can be observed are mild pain and local or regional paresthesia that may persist for different periods of time, slight edema, and erythema at the bite site4.

Protein isoforms present in C. durissus venom are also of great clinical importance, due to the fact that the venom pool used for antivenom production may be inadequate. This leads to reduced immunogenicity in animals and results in a product that cannot adequately neutralize the clinical manifestations of patients bitten by these snakes5-6.

This study aims to compare the toxic effects (local and systemic) induced by the venom of Crotalus subspecies, C. durissus terrificus, C. durissus cascavella, and C. durissus collilineatus, in individuals bitten by Crotalus snakes. Moreover, this study demonstrates that the variations found can be related to differences in neutralization rates by antivenom actions against the venoms from different subspecies, which is consistent with the results published by Boldrini-França et al.6 and Oliveira et al.7.

METHODS

Venoms and Animals

Cdt, Cdcolli, and Cdcasc venoms were acquired from Instituto Butantan, São Paulo-SP and were maintained at -20ºC in the Amazon Venom Bank at CEBio-UNIR-FIOCRUZ-RO (licenses: CGEN/CNPq 010627/2011-1 and IBAMA 27131-2). All Crotalus sp. venoms used were from a pool of venoms from adults male and female snakes from different parts of Brazil (carried by IBAMA, ONGs, firemen, or physical people).

Male Swiss mice (18-20 g) were housed in temperature-controlled rooms and received water and food ad libitum until used (approved protocol number 2012/09 from the FIOCRUZ-RO Animals’ Ethics Committee).

Venom biochemical characterization

Cdt, Cdcasc, and Cdcolli venoms’ chromatographic profile was produced by molecular exclusion chromatography (Sephadex G75) in an FPLC (Akta Purifier System/GE Healthcare®). The method was performed according to Bercovici et al.8 in which about 35 mg of venom was suspended in 1 mL of 0.05 M ammonium formate buffer, pH 3.5. The peaks obtained were analyzed by SDS-PAGE on 12.5% ​​and/or 15% polyacrylamide gels. The proteins’ concentrations were measured using the Bio-Rad DC Protein Assay method (BIO-RAD).

Proteolytic activity

150 µL of 2% azocasein solution (substrate) was added to 7 µL of each venom (20 µg of Cdt, Cdcolli, or Cdcasc) in a 96-well plate (substrate) and incubated for 1 h at 37ºC. Subsequently, the reaction (Azocasein+venom) was stopped by adding 150 µL of 20% trichloroacetic acid (TCA). After 30 min, the samples were centrifuged at 180 xg for 10 min. Next, 100 µL of the supernatant was added to a 96-well plate, and 100 µL of 500 mM NaOH were added. The absorbance at 440 nm was monitored using spectrophotometer (Biotek)9. Bothrops' venom (from Amazon Venom Bank at CEBio-UNIR-FIOCRUZ-RO) was used as a positive control.

Phospholipase activity

Phospholipase activity was determined using the method described by Holzer and Mackessy10 adapted to 96-well plates. Buffer containing the chromogenic substrate 4-nitrophenyl (3-octanoyloxy) benzoic acid (4N3OAB) was added to 10 µL of each venom (20 µg Cdt, Cdcolli, or Cdcasc) or water (negative control). The solution was analyzed in a spectrophotometer at 425 nm over 30 min for 30 s intervals10.

LAAO Activity

For LAAO activity, the method described by Torii11 adapted to a 96-well plate was followed according to Pontes et al.12. In brief, each venom (10 μg) was added separately to the reaction mixture containing horseradish peroxidase (50 μg/mL), 100 μM l-leucine, and 10 μM 3’3’diaminobenzidine in 100 mM Tris-HCl buffer (pH 7.8). The reaction was incubated at 37°C for 30 min. The reaction was stopped using 10% citric acid and the absorbance was measured on a spectrophotometer at 490 nm.

Paw edema assay

Groups of 5 mice were inoculated with a subplantar injection of 30 µL of 100 μg/kg of Cdt, Cdcolli, or Cdcasc diluted in 150 mM sterile physiological saline in the right hind paw. As a control, sterile physiological saline (30 μL) was injected into the contralateral paw. Paw volume was measured immediately before sample injection (basal time 0 h) and at different time intervals thereafter (0.5, 1, 3, 6, 9, 12, and 24 h). Paw volume was measured using a hydroplethysmometer (Ugo Basile). Results were expressed as percentage paw volume increase in relation to the control paw13.

Myotoxic, Hepatotoxic, and Nephrotoxic activities

Groups of 5 mice received an intramuscular (i.m.) injection of 30 µL of Cdt, Cdcolli, or Cdcasc venom (100 µg/kg of venom diluted in 150 mM sterile physiological saline) in the gastrocnemius muscle. Control group animals received 30 µL (150 mM sterile physiological saline) in the same conditions. After 3, 6, 9, 12, and 24 h, mouse blood was withdrawn from their orbital plexus, dispensed into heparinized tubes, and centrifuged at 2205 xg for 5 min according to Teixeira et al. (2018)13.

Myotoxicity activity was evaluated by measuring creatine kinase (CK), creatine kinase MB isoenzyme (CK-MB), and lactate dehydrogenase (LDH). Hepatotoxic activity was evaluated by measuring alanine transaminase (ALT), aspartate transaminase (AST), gamma-glutamyl transferase (GGT), and alkaline phosphatase (AP) activity. Kidney function was evaluated by measuring plasma creatinine and urea biochemical parameters and total urine proteins and calcium. All determinations were conducted using commercial diagnostic kits purchased from Labtest Diagnostica SA (Brazil).

Coagulation tests in vivo

Groups of 5 mice received in the gastrocnemius muscle an i.m. injection of 30 µL of Cdt, Cdcolli, and Cdcasc (100 µg/kg of venom diluted in 150 mM sterile physiological saline). Control group animals received 30 µL (150 mM sterile physiological saline) in the same conditions. After 3 h, mouse blood was drawn from the inferior vena cava, dispensed into citrated tubes, and centrifuged at 2205 xg for 5 min in order to obtain platelet-poor plasma. Prothrombin time (TAP) and activated partial thromboplastin time (TAPP) were determined using Hemostasis (Labtest, Brazil).

Myographic Study

Cdt, Cdcolli, and Cdcasc venoms neuromuscular activities were assessed in mice phrenic-diaphragm preparations in accordance with the method previously described by Gallacci and Cavalcante14. The amplitudes of indirect and direct twitches were measured at 150 and 420 min.

Statistical analysis

Results were analyzed by ANOVA complemented by the Tukey Kramer test (GraphPad Prism 5.0). Values were expressed as mean ± S.E.M with P<0.05 considered significant.

RESULTS

Biochemical Characterization

Venoms were fractioned by molecular exclusion chromatography using the Sephadex G-75 (GE healthcare®, 10 x 60 cm). The C. durissus venoms chromatographic profile analysis showed three peaks, Cdt-I, Cdt-II, and Cdt-III, for Cdt (Figure 1A); three peaks, Cdcolli-I, Cdcolli-II, and Cdcolli-III, for Cdcolli (Figure 1B); and two peaks, Cdcasc-I and Cdcasc-II, for Cdcasc (Figure 1C).

FIGURE 1: (A) Chromatographic profile of Cdt venom by a size exclusion column. Electrophoresis profile of the fractionation of Cdt venom. A1) A 12.5% and A2) 15% SDS-PAGE in denaturing conditions. PM corresponds to the molecular weight standard; Crotalus durissus terrificus venom (VCdt); and Cdt-I, Cdt-II, and Cdt-III to PI, PII, and PIII, respectively. (B) Chromatographic profile of Cdcolli venom by size exclusion column. Electrophoresis profile of the fractionation of Cdcolli venom. Peaks I, II, and III correspond to DI convulxin (Cvx)/gyroxin (Grx), DII crotoxin (Ctx), and DIII crotamine (Ctm), respectively. B1) 15% SDS-PAGE in denaturing conditions. PM corresponds to molecular weight standard; Crotalus durissus collilineatus venom (VCdcolli); and Cdcolli-I, Cdcolli-II, and Cdcolli-III to PI, PII and PIII, respectively. (C) Chromatographic profile of Cdcasc venom by a size exclusion column. Electrophoresis profile of the fractionation of Cdcasc venom. Peaks I and II correspond to BI convulxin (Cvx)/gyroxin (Grx) and BII crotoxin (Ctx), respectively. C1) 15% SDS-PAGE in denaturing conditions. PM corresponds to the molecular weight standard; Crotalus durissus cascavella venom (VCcasc); and Cdcasc-I and Cdcasc-II to PI and PII, respectively. 

After electrophoresis, convulxin (Cvx) and gyroxin (Grx) were visualized in the lanes associated with Cdt-I, Cdcolli-I, and Cdcasc-I with approximate molecular weights (MW) of ~72 kDa for Cvx and ~30 kDa for Grx. Moreover, crotoxin (Ctx) was found in the Cdt-II, Cdcolli-II, and Cdcasc-II lanes at ~24 kDa. However, crotamine (Ctm) alone was visualized in the Cdt-III and Cdcolli-III lanes at ~4.8 kDa, but was not observed in Cdcasc venom (Ctm-negative) (Figures A2, B1, and C1). It was observed that the estimated protein content for Cdt-I, Cdcolli-I, and Cdcasc-I peaks were 2%, 6%, and 18%, respectively. For Cdt-II, Cdcolli-II, and Cdcasc-II the protein contents were 81%, 67%, and 82%, respectively. Additionally, the estimated protein contents for Cdt-III and Cdcolli-III were 16% and 27%, respectively. The results above apply only to Cdt and Cdcolli because Cdcasc was found to be devoid of crotamine.

Enzymatic Characterization

Results suggested that Cdt, Cdcolli, and Cdcasc venoms do not exert a proteolytic effect on azocasein compared to the negative control (Figure 2A).

Cdcolli and Cdcasc venoms (20 µg) had significant LAAO activity compared to that of the control. This result was determined by the peroxidase reaction, wherein H2O2 was produced. However, Cdt venom showed no LAAO activity (Figure 2B).

Cdcolli venom (20 µg) demonstrated low phospholipase activity on the substrate 4N3OAB at the concentration used. However, Cdcasc and Cdt venoms, using the same substrate and the same concentration, displayed significant catalytic activity compared to control (Figure 2C).

FIGURE 2: Biochemical characterization (A, B, C) and local effect (D) of Cdt, Cdcolli, and Cdcasc venoms. A) Proteolytic activity of Cdt, Cdcolli, and Cdcasc venoms on azocasein. B) Determination of L-amino acid oxidase activity of Cdt, Cdcolli, and Cdcasc venoms. C) Determination of phospholipase activity of Cdt, Cdcolli, and Cdcasc venoms. Results represent the mean ± SEM (n = 5). ***Values significantly different from control (p<0,001) (ANOVA). 

Local and Systemic Effect Characterization

Cdcolli and Cdcasc venoms induced paw edema increases of 76.7% and 40%, respectively, after 1 h. Additionally, Cdt venom induced a paw edema increase of 33.3% after 3 h (Figure 3).

FIGURE 3: Edematogenic effect induced by Cdt, Cdcolli, and Cdcasc venoms in mice. The graph shows a time course of paw edema induced by C. durissus subspecies venoms (100 µg/kg) or sterile physiological saline. Results are expressed as means ± SEM (*** p <0.001 and * p <0.05) of percentage increase of paw volume compared to control (n=5) (ANOVA). 

Mice blood CK levels are depicted in Figure 4A. Cdcolli and Cdcasc induced a significant increase in total-CK liberation compared to control animals 3 h after inoculation. After 6, 9, and 12 h, all venoms induced a significant total-CK increase compared to that of the control (182.3 U/L). Total-CK liberation induced by Cdcolli was statistically different from Cdt from 3 to 12 h after inoculation. However, total-CK liberation induced by Cdcasc was statistically different from Cdt just after 12 h of inoculation. In Figure 4B, animals inoculated with venom had significantly higher MB-CK levels compared to control animals after 3 h of inoculation. At this time, animals inoculated with Cdcolli differed statistically from those inoculated with Cdcasc. This was not observed in mice envenomated with Cdt venom after 6 h of inoculation. After 9 h, all envenomated animals had basal MB-CK levels. However, after 12 h, Cdcolli and Cdt venoms injected in mice induced a significant increase in MB-CK levels compared to that in control animals. Additionally, 24 h after inoculation, all venoms induced a significant MB-CK increase compared to the controls.

Figure 4C shows that all venoms studied increased LDH levels 3 and 6 h after inoculation compared to control animals. However, the animals inoculated with Cdt alone differed statistically from those inoculated with Cdcasc. Moreover, Cdcolli and Cdt venoms increased LDH levels after 12 h compared to controls.

As shown in Figure 4D, none of the venoms changed ALT levels after 3 h compared to control animals. However, Cdcasc and Cdcolli venoms increased ALT levels compared to the controls 6 and 9 h after inoculation, respectively. After 9 h, animals envenomated with Cdcolli differed statistically from those with Cdt and Cdcasc. After 12 h past Cdcolli and Cdt inoculation, ALT levels significantly increased compared to Cdcasc and the control group. Additionally, 24 h after Cdcolli, Cdcasc, or Cdt injection, ALT levels significantly increased compared to controls.

In Figure 4E, significantly higher levels of AST can be seen in animals injected with Cdcasc, Cdcolli, or Cdt compared to those in control animals after 3, 6, 9, and 24 h. However, only animals injected with Cdcolli venom did not display an increase in AST levels 12 h after inoculation. Animals inoculated with Cdcasc differed statistically from those inoculated with Cdcolli and Cdt 3 and 12 h after inoculation. At 9 h after Cdcasc inoculation, Cdcasc action differed from animals inoculated with Cdcasc and Cdt. However, animals inoculated with Cdt and Cdcasc differed from those inoculated with Cdcolli 24 h after inoculation.

The animals injected with Cdt venom showed an increase in ALP levels after 6 h (Figure 4F). Animals injected with Cdcolli venom induced higher ALP levels compared to control animals 9, 12, and 24 h after inoculation. ALP levels in animals inoculated with Cdcolli differed statistically from those inoculated with Cdt and Cdcasc 12 and 24 h after injections.

Figure 4G shows that 3 h after inoculation, only animals injected with Cdcolli venom induced a GGT increase compared to the control and Cdt animals. Moreover, after 6 h, all animals injected with the Crotalus subspecies venoms presented a significant increase in GGT levels. At this time, the GGT levels of animals inoculated with Cdt differs from those animals inoculated with Cdcolli and Cdcasc. After 9 h, mice injected with Cdcolli and Cdcasc venoms had a reduced GGT levels compared to control and Cdt mice.

Figure 4H shows that 3 h after Cdcasc and Cdcolli mice envenomation there was a significant increase in urea levels compared to control animals. Only Cdcolli venom induced a significant increase in urea levels 6 h after envenomation. Additionally, 9 h after Cdt and Cdcolli injection there was a significant increase in urea levels compared to control animals.

For urine urea levels at 3, 6, 12, and 24 h, all Crotalus venoms did not induce urea liberation in mouse urine. However, Cdcolli induced a significant increase in urine urea levels at 9 h after envenomation (Figure 4I). Cdcolli and Cdt induced a significant increase in serum creatinine compared to Cdcasc animals 9 h after envenomation (Figure 4J).

Results showed that 3 h after Crotalus venom injection there was no increase in urine creatinine levels (Figure 4K). In contrast, after 6 h, mice injected with Cdcasc venom had significant increases in urine creatinine levels. At 9 h post injection, increased urine creatinine levels were significant in mice injected with Cdcolli or Cdt venom. Additionally, a significant increase in urine protein levels was observed 3, 6, 9, and 12 h after venom injection (Figure 4L).

Cdcolli venom induced an increase in prothrombin time of 120 sec, which is significantly different from the control group’s prothrombin time of 28 sec. However, Cdt and Cdcasc venom did not increase coagulation time. With respect to activated partial thromboplastin time, Cdcolli and Cdcasc venom induced an increase of 240 sec, statistically different from the control group (92.5 sec, Figure 4N).

FIGURE 4: Myotoxic (A, B, C), hepatotoxic (D, E, F, G), nephrotoxic (H, I, J, K, L), and coagulation (M, N) effects of Cdt, Cdcolli, and Cdcasc venoms in Swiss mice. A) Total creatine kinase (CK) (Total CK); B) MB fraction CK (MB-CK); C) lactate dehydrogenase (LDH); D) alanine aminotransferase (ALT); E) aspartate aminotransferase (AST); F) alkaline phosphatase (ALP); G) gamma-glutamyl transferase (GGT); H) Serum Urea; I) Urinary Urea; J) Serum Creatinine; K) Urinary Creatinine; and L) Proteinuria. M) Prothrombin time (TAP) and N) activated partial thromboplastin time (TAPP). The results are expressed as the means ± SEM of 5 mice in each group. *p<0.05, **p<0.01, ***p<0.001 compared to the corresponding control group for each interval (ANOVA). 

Cdt, Cdcolli, and Cdcasc venoms induced a time- and concentration-dependent blockade of indirectly evoked twitches (Figures 5A and 5B), and at the higher concentration studied (5 μg/mL), they also paralyzed the directly evoked twitches (Figure 5C). As shown in Table 1, the times required for 50% blockade (t50) of indirect twitches were significantly lower than those necessary to blockade the direct contractions. While the blockade of indirect contraction is an unequivocal indicator of a neurotoxic activity, the blockade of direct contractions frequently denotes a myotoxic activity affecting muscle contractility.

TABLE 1: The time (min) required for 50% blockade (t50) of indirectly and directly evoked twitches. 

Experimental Group Indirect Direct
1 μg/mL 5 μg/mL 5 μg/mL
C.d. terrificus Venom 84.36 ± 8.48 (n=4) 42.70 ± 2.3 (n=5) # 56.80 ± 6.43 (n=4) *
C.d. collilineatus Venom 71.20 ± 6.35 (n=6) 42.24 ± 0.88 (n=4) # 64.26 ± 4.01 (n=4) *
C.d. cascavella Venom 100 ± 7.79 (n=4) 57.96 ± 4.43 (n=6) # 249.6 ± 11.7 (n=4) *

#Indicates differences in t50 of indirectly evoked preparations exposed to the same venom at different concentrations (1 μg/mL vs 5 μg/mL). *Indicates differences in t50 between directly and indirectly evoked preparations exposed to the same venom and concentration (5 μg/mL).

FIGURE 5: Effect of Cdt, Cdcolli, and Cdcasc venoms, at concentrations of 1 µg/mL (A) and 5 µg/mL (B) on indirectly and (C) directly evoked twitches in mice phrenic-diaphragm preparations. The ordinate represents the % amplitude of twitches relative to the initial amplitude. 

DISCUSSION

Molecular exclusion chromatography of Crotalus durissus venom identified four major toxins: Cvx15, Gvx16, Ctx17, and Ctm18. Therefore, C. durissus venom has a variable composition, and Ctm may or may not be present in it19. Venom composition is directly associated to age, sex, captivity, and the individual glands of a snake20-22. Ontogenetic and seasonal variations also contribute to molecular diversity and venom complexity7,23.

Oliveira et al.7used proteomic and functional analyses of 22 Cdcolli individuals’ venoms to explore their qualitative and quantitative variations. Moreover, these authors found that different Cdcolli venoms caused envenomings with different changes in biochemical and immunological parameters.

Lourenço et al.24 found Ctm venom heterogeneity but did not observe a statistical difference in C. durissus venom proteolytic activity. The findings of this study are in accordance with our data.

Phospholipase A2 (PLA2) can be responsible for edema induction, since it is directly related to envenoming pathophysiology that accounts for various local and systemic disorders25-27. Furthermore, Cdt, Cdcolli, and Cdcasc venoms were uniform in relation to phospholipase activity with the substrate 4N3OBA. The capacity of some PLA2s to recognize and act on specific targets can explain these differences. The same results were obtained by Santoro et al.28 with Cdcasc and Cdcolli.

Unlike other viperid venoms, Crotalus venoms do not induce significant inflammatory reactions at the bite site in animals or humans29-30. However, there was a report of an edematogenic response induced by Cdt venom that was not dose-dependent and had a fast and transient course41. Some studies have reported that PLA2 induced edema, an effect that in some cases are dependent on PLA2s binding to specific membrane proteins31.

Other than the association with edema induction, other studies have emphasized the participation of PLA2s in myotoxicity caused by crotalic envenoming. A large part of this action at least is due to two components: Ctm and Ctx32-34. The toxicity induced by Ctx is generated by the CB subunit, which is a PLA235.

Several biological activities including cytotoxicity; mild myonecrosis; apoptosis induction; platelet aggregation, induction, and/or inhibition; as well as hemorrhagic, hemolytic, edematogenic, antibacterial, antiproliferative, antiparasitic, and anti-HIV activities36have been attributed to LAAO. In 201537, a very small amount (1.8% of the total venom) of the first LAAO from Cdt yellow venom, Bordenein-L, was isolated. Few studies have been conducted to determine the mechanisms of action of LAAOs in the induction of edema compared to other classes of snake toxins. Studies investigating different toxins revealed that the action of these proteins is related to the release of inflammatory mediators such as histamine, prostaglandin, kinins, and serotonin38. However, the edematogenic activity of LAAOs does not seem to be mediated by the same mechanisms described for other toxins, since these enzymes do not lose their edematogenic activity in the presence of antihistamines.

There are essentially two clinical models for myotoxicity: local and systemic. For instance, rhabdomyolysis constitutes a generalized muscle breakdown and causes myoglobin and CK increases in circulation that may lead to renal dysfunction, which is one of the envenoming characteristics of crotalics2,28,39.

Results showed that all Crotalus subspecies can induce alterations in CK (Total and MB) levels in different ways. The variance between the extravasation of Total-CK induced by crotalic venoms can be explained by the venom’s PLA240 and LAAO activity. Cdcolli had the highest activity in the muscular system, which differs from results found by Santoro et al.28that showed that Cdt had its highest activity in muscles. This could be the result of the two toxins involved in myotoxicity, Ctm and Ctx, working synergistically. Both are found in high concentration in Cdcolli venom.

The significant increase in MB-CK circulation caused by Cdt and Cdcolli venoms can be explained by PLA2 action on the myocardium muscle, the occurrence of early lesions, and the release of mediators that contribute to delayed injury. However, Cupo et al.,41observed an absence of clinical signs of myocardiotoxicity and the presence of normal serial ECG and echocardiography. Siqueira et al.42 reported myocardium damage in a victim bitten by a C.d. terrific snake. C. durissus venom's toxicity in heart muscle is controversial and poorly understood. It was observed by electrocardiogram changes43 and acute myocardial infarction41 in Cdt bitten patients. TOTAL-CK and MB-CK results in the present study are in agreement with the works mentioned above in which CK increases were verified in the first 2 to 3 h after venom inoculation. Additionally, the myotoxic activity results of our work are in line with the data of Saraiva et al.44. They measured mice plasma CK levels 3 h after Cdt venom intramuscular injection in the right gastrocnemius muscle. According to Barraviera et al.45 in their retrospective study of Crotalus bitten victims, 100% of the analyzed patients presented increased CK levels.

Cupo et al.41,43 showed that the LDH (heart fraction) concentrations in Cdt bitten patients were higher than those of other LDH isoforms. Rowlands et al.46 performed autopsies on Pseudechis australis envenoming victims and observed rhabdomyolysis with necrosis foci in the myocardium. De Siqueira et al.42 reported damage to the myocardium after Cdt envenoming. Autopsies showed diffuse edema myocytolysis and rare micro-infarcts foci.

Accordingly, it was suggested that the Cdcasc venom induced liver injury 6 h after inoculation, whereas Cdcolli and Cdt venoms induced the same reaction after 9 h and 12 h, respectively. AST levels also changed after individual inoculation of these three venoms. These differences in activities triggered by the venoms can be attributed to Ctx actions and its PLA2 subunit and/or LAAO action on specific sites in the hepatocytes membranes47.

Elevation of ALT and AST may be related to side effects caused by biological factors released by tissue injury. ALP and GGT alterations are shown here reinforcing the hepatotoxicity induced by Crotalus envenoming. Barraviera et al.48 showed a positive correlation between bromsulphalein retention and ALT serum levels as well as AST and ALT serum increase in Cdt bitten patients. The authors proposed that these alterations were associated with liver dysfunction. In other research, Barraviera et al.45 performed an anatomopathological exam on a patient who died and were diagnosed with extensive hepatic necrosis. However, França et al.47 measured rat serum levels of ALT, AST, ALP, and GGT after Cdt venom inoculation and showed acute hepatotoxicity. Snake venom from other families, such as Elapidae Nana naja venom, also induces liver changes49. However, the mechanisms involved in hepatic injury are still not understood, which makes them an important question in the field.

Given the creatinine excretion reduction caused by envenoming, it can be inferred that it is related to bloodstream retention. However, the presence of albumin in urine indicates kidney injury because this protein has a high molecular weight and is not able to cross the glomerular membrane in physiological conditions. Reduction or loss of kidney function, known as acute renal failure, is the main complication of crotalic envenoming. This condition can be attributed to systemic manifestations, dehydration, and especially myotoxicity. Furthermore, it can develop into a severe renal ischemia, leading to kidney function loss2,39,50. However, Amora et al.51 used isolated rat kidney to find that Ctx and PLA2 were involved in this process, since the renal effects observed would be due to the venom components’ synergistic action.

Another systemic manifestation of crotalic envenoming is the alteration of the blood coagulation system52. These characteristics may be attributed to the enzymes present (serineproteases and metalloproteinases) that primarily affect the hemostatic system53. The difference in these enzymes expression patterns in these three subspecies might explain the different results shown in coagulation.

De Oliveira et al.54 reported on the activity of fibrinolytic enzymes, mainly in the fibrinogen Bβ and Aα subunits in the Cdt and Cdcolli venoms. This is considered a characteristic of the serineproteases present in these venoms. Accordingly, the blood incoagulability observed in severe cases of C. durissus bites are derived from fibrinogen consumption52.

The studied venoms were able to induce a contraction blockade. The blockade of indirect contraction is an unequivocal indicator of a neurotoxic activity, the blockade of direct contractions usually denotes myotoxic activity affecting muscle contractility55.

C. durissus subspecies venoms contain large amounts of Ctx6,28,56-58, a potent β-neurotoxin that induces neuromuscular transmission blockade and progressive muscle paralysis59. In addition, Ctx also induces in vivo and in vitro myotoxic activity, which may be underlying the blockade of direct muscle contractions induced by crotalic venoms60-62. Prior to the establishment of the neuromuscular blockade, Cdt and Cdcolli venoms induce initial facilitation of muscle contractions. This effect can be attributed to the presence of Ctm in both venoms28,58. This myonecrotic toxin binds to voltage-sensitive Na+ channels on the skeletal muscle sarcolemma, leading to a large influx of Na+ ions, which causes depolarization, strong contraction, injury, and myonecrosis63-65. In contrast, the absence of this molecule in Cdcasc venom may explain the t50 for direct evoked twitches, which was six times higher than those observed for the other venoms (Table 1).

Taken together, the data obtained in this study created a new view of the intraspecific variation of local and systemic effects caused by Cdt, Cdcolli, and Cdcasc venoms. Based on this evidence and the changes in the liver, kidney, muscle systems, and coagulation induced by these envenoming processes, besides variations in protein and enzymatic composition, we can evaluate the differences between local and systemic effects caused by subspecies of C. durissus. This highlights the clinical and biochemical effects produced by their respective venoms. The differences in some pharmacological activities observed in our study are in accordance with published data6,66,44.

ACKNOWLEDGMENTS

The authors express their gratitude to Financiadora de Estudos e Projetos (FINEP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Projeto NanoBiotec, and Rede de Biodiversidade e Biotecnologia da Amazônia Legal (BIONORTE/CNPq/MCT).

REFERENCES

1. Barraviera B, Bonjoro Júnior JC, Arkaki D, Domingues MA, Pereira PC, Mendes RP, et al. A retrospective study of 40 victims of crotalus snake bites. Analysis of the hepatic necrosis observed in one patient. Rev Soc Bras Med Trop. 1989;22(1):5-12. [ Links ]

2. Azevedo-Marques MM, Cupo P, Coimbra TM, Hering SE, Rossi MA, Laure CJ. Myonecrosis, myoglobinuria and acute renal failure induced by south american rattlesnake (Crotalus durissus terrificus) envenomation in Brazil. Toxicon. 1985;23(4):631-6. [ Links ]

3. Martins AM, Toyama MH, Havt A, Novello JC, Marangoni S, Fonteles MC, Monteiro HAS. Determination of Crotalus durissus cascavella venom components that induce renal toxicity in isolated rat kidneys. Toxicon. 2002;40(8):1165-71. [ Links ]

4. Cruz LS, Vargas R, Lopes AA. Snakebite envenomation and death in the developing world. Ethn Dis. 2009;19(1 Suppl 1):S1-42-6. [ Links ]

5. Faure G, Bon C. Several isoforms of crotoxin are present in individual venoms from the South American rattlesnake Crotalus durissus terrificus. Toxicon. 1987;25:229-34. [ Links ]

6. Boldrini-França J, Correa-Netto C, Silva MM, Rodrigues RS, De La Torre P, Perez A, et al. Snake venomics and antivenomics of Crotalus durissus subspecies from Brazil: Assessment of geographic variation and its implication on snakebite management. J Proteomics. 2010;73:1758-76. [ Links ]

7. Oliveira IS, Cardoso IA, Bordon KCF, Carone SEI, Boldrini-França J, Pucca MB, et al. Global proteomic and functional analysis of Crotalus durissus collilineatus individual venom variation and its impact on envenoming. J Proteomics . 2018;S1874-3919(18)30066-6. [ Links ]

8. Bercovici D, Chudziniski AM, Dias NO, Esteves MI, Hiraichi E, Oishi NY, et al. Crotalus durissus terrificus venom. Mem Inst Butantan. 1987;49:69-78. [ Links ]

9. Gomes MS, De Queiroz MR, Mamede CC, Mendes MM, Hamaguchi A, Homsi-Brandeburgo MI, et al. Purification and functional characterization of a new metalloproteinase (BleucMP) from Bothrops leucurus snake venom. Comp Biochem Physiol C Toxicol Pharmacol. 2011;153:290-300. [ Links ]

10. Holzer M, Mackessy SP. An aqueous endpoint assay of snake venom phospholipase A2. Toxicon . 1996;34:1149-55. [ Links ]

11. Torii K. A new pharmacological and physiological aspects of L-amino acids Folia. Jpn J Pharmacol. 1997;110:28-32. [ Links ]

12. Pontes AS, Da S Setúbal S, Xavier CV, Lacouth-Silva F, Kayano AM, Pires WL, et al. Effect of l-amino acid oxidase from Calloselasma rhodosthoma snake venom on human neutrophils. Toxicon . 2014;80:27-37. [ Links ]

13. Teixera LF, de Carvalho LH, de Castro OB, Bastos JSF, Néry NM, Oliveira GA, et al. Local and systemic effects of BdipTX-I, a Lys-49 phospholipase A2 isolated fromBothrops diporus snake venom. Toxicon . 2018;141:55-64. [ Links ]

14. Gallacci M, Cavalcante WL. Undersing the in vitro neuromuscular activity of snake venom Lys49 phospholipase A2 homologues. Toxicon . 2010;55:1-11. [ Links ]

15. Prado-Franceschi J, Brazil OV. Convulxin, a new toxin from the venom of the South American rattlesnake Crotalus durissus terrificus. Toxicon . 1981;19:875-87. [ Links ]

16. Barrabin H, Martiarena JL, Vidal JC, Barrio A. Isolation and characterization of gyroxin from Crotalus durissus terrificus venom. Anim Plant Microbial. 1978;113-33. [ Links ]

17. Slotta KH, Fraenkel-Conrat H. Estudos químicos sobre os venenos ofídicos. Purificação e cristalização do veneno da cobra cascavel. Mem Inst Butantan . 1938a12:505-13. [ Links ]

18. Gonçalvez JM, Vieira LG. Estudos sobre venenos de serpentes brasileiras I Análise eletroforética. An Acad Bras Cienc. 1950;22:141-50. [ Links ]

19. Barrio A, Brasil OV. Neuromuscular action of the Crotalus durissus terrificus (Laurenti) poisons. Acta Physiol Latinoam. 1951;1:291-308. [ Links ]

20. Aguilar I, Guerrero B, Maria Salazar A, Girón ME, Pérez JC, Sánchez EE, et al. Individual venom variability in the South American rattlesnake Crotalus durissus cumanensis. Toxicon . 2007;50:214-24. [ Links ]

21. Furtado MFD, Maruyama M, Kamiguti AS, Antonio LC. Comparative study of nine Bothrops snake venoms from adult female snakes their offspring. Toxicon . 1991;29:219-26. [ Links ]

22. Francischetti IM, Gombarovits ME, Valenzuela JG, Carlini CR, Guimarães JA. Intraspecific variation in the venoms of the South American rattlesnake (Crotalus durissus terrificus). Comp Biochem Physiol C Toxicol Pharmacol. 2000;127:23-36. [ Links ]

23. Furtado MFD, Santos MC, Kamiguti AS. Age-related biological activity of South American rattlesnake (Crotalus durissus terrificus) venom. J Venom Anim Toxins Incl Trop Dis. 2003;9:186-201. [ Links ]

24. Lourenço A Jr, Zorzella Creste CF, de Barros LC, Delazari dos Santos L, Pimenta DC, Barraviera B, et al. Individual venom profiling of Crotalus durissus terrificus specimens from a geographically limited region: Crotamine assessment, captivity evaluation on the biological activities. Toxicon . 2013;69:75-81. [ Links ]

25. Ponce-Soto LA, Martins-De-Souza D, Marangoni S. Neurotoxic, myotoxic and cytolytic activities of the new basic PLA2 isoforms BmjeTX-I and BmjeTX-II isolated from the Bothrops marajoensis (marajo lancehead) snake venom. Protein J. 2010;29:103-13. [ Links ]

26. Fatima L, Fatah C. Pathophysiological and pharmacological effects of snake venom components: Molecular Targets. J Clin Toxicol. 2014;4:190. [ Links ]

27. Marangoni FA, Ponce-Soto LA, Marangoni S, Lucci EC. Unmasking snake venom of Bothrops leucurus: Purification and pharmacological and structural characterization of new PLA2 Bleu TX-III. Biomed Res Int. 2013;2013:941467. doi: 10.1155/2013/941467 [ Links ]

28. Santoro ML, Sousa-e-Silva MCC, Goncalves LRC, Almeida-Santos SM, Cardoso DF, Laporta-Ferreira IL, et al. Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake (Crotalus durissus terrificus, C-durissus cascavella , C-durissus collilineatus). Comp Biochem Physiol C-Toxicol Pharmacol. 1999;122:61-73. [ Links ]

29. Rosenfeld G. Symptomatology, pathology, treatment of snake bites in South America. In: Bucherl W, Buckley EE, editors. Venomous Animals and their venoms. New York: Academic Press, 1971;p 345-84. [ Links ]

30. Azevedo-Marques MM, Hering SE, Cupo P. Acidente crotálico. In: Cardoso JLC, França OSF, Wen FH, Málaque CMS, Haddad Jr V (orgs.). Animais peçonhentos no Brasil: biologia, clínica e terapêutica dos acidentes. São Paulo: Sarvier; 2003. p91-8. [ Links ]

31. Iglesias CV, Aparicio R, Rodrigues-Simioni L, Camargo EA, Antunes E, Marangoni S, et al. Effects of morin on snake venom phospholipase A2 (PLA2). Toxicon . 2005;46,751-58. [ Links ]

32. Cameron DL, Tu AT. Chemical and functional homology of myotoxin a from prairie rattlesnake venom, crotamine from south american rattlesnake venom. Biochim Biophys Acta. 1978;532:147-54. [ Links ]

33. Kouyoumdjian JA, Harris JB, Johnson MA. Muscle necrosis caused by the sub-units of crotoxin. Toxicon . 1986;24:575-83. [ Links ]

34. Mebs D, Ownby CL. Myotoxic components of snake venoms: Their biochemical and biological activities. Pharmacol Ther. 1990;48:223-36. [ Links ]

35. Gopalakrishnakone P, Dempster DW, Hawgood BJ, Elder HY. Cellular and mitochondrial changes induced in the structure of murine skeletal muscle by crotoxin, a neurotoxic phospholipase A2 complex. Toxicon . 1984;22:85-98. [ Links ]

36. Paloschi MV, Pontes AS, Soares AM , Zuliani JP. An update on potential molecular mechanisms underlying the actions of snake venom L-amino acid oxidases (LAAOs). Curr Med Chem. 2017;2520-30. [ Links ]

37. Bordon KC, Wiezel GA, Cabral H, Arantes EC. Bordonein-L a new L-amino acid oxidase from Crotalus durissus terrificus snake venom: isolation, preliminary characterization, enzyme stability. J Venom Anim Toxins Incl Trop Dis . 2015; 21:26. [ Links ]

38. SA Ali, S Stoeva, A Abbasi, JM Alam, Rakez Kayed, M Faigle, et al. Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom. Archives of Biochemistry and Biophysics. 2000;384: 216-226. [ Links ]

39. Azevedo-Marques MM, Hering SE, Cupo P. Evidence that Crotalus durissus terrificus (South American rattlesnake) envenomation in humans causes myolysis rather than hemolysis. Toxicon . 1987;25:1163-1168. [ Links ]

40. Gutiérrez JM, Ownby CL. Skeletal muscle degeneration induced by venom phospholipases A2: Insights into the mechanisms of local and systemic myotoxicity. Toxicon . 2003;42:915-31. [ Links ]

41. Cupo P, Azevedo-Marques MM, Hering SE. Acute myocardial infarction-like enzyme profile in human victims of Crotalus durissus terrificus envenoming. Trans R Soc Trop Med Hyg. 1990;84:447-51. [ Links ]

42. Siqueira JE, de L Higuchi ML, Nabut N, Lose A, Souza JK, Nakashima M. Lesão miocárdica em acidente ofídico pela espécie Crotalus durissus terrificus (cascavel): relato de caso. Arq Bras Cardiol. 1990;54:323-25. [ Links ]

43. Cupo P, de Azevedo-Marques MM, Hering SE. Absence of myocardial involvement in children victims of Crotalus durissus terrificus envenoming. Toxicon . 2003;42:741-45. [ Links ]

44. Saraiva P, Rojas E, Arce V, Guevara C, López JC, Chaves E, et al. A Geographic ontogenic variability in the venom of the neotropical rattlesnake Crotalus durissus: Pathophysilogical , therapeutic implications. Rev Biol Trop. 2002;50:337-46. [ Links ]

45. Barraviera B, Bonjorno Júnior JC, Arakaki D, Domingues MA, Pereira PC, Mendes RP, et al. A retrospective study of 40 victims of Crotalus snake bites. Analysis of the hepatic necrosis observed in one patient. Rev Soc Bras Med Trop . 1989;22:5-12. [ Links ]

46. Rowlands JB, Mastaglia FL, Kakulas BA, Hainsworth D. Clinical and pathological aspects of a fatal case of mulga (Pseudechis australis) snakebite. Med J Aust. 1969;1:226-30. [ Links ]

47. França RF, Vieira RP, Ferrari EF, Souza RA, Osorio RAL, Prianti-Jr ACG, et al. Acute hepatotoxicity of Crotalus durissus terrificus (South American rattlesnake) venom in rats. J Venomous Anim Toxins Incl Trop Dis. 2009;15(1):61-78. [ Links ]

48. Barraviera B, Coelho KYR, Curi PR, Meira DA. Liver dysfunction in patients bitten by Crotalus durissus terrificus (Laurenti, 1768) snakes in Botucatu (State of São Paulo - Brazil). Rev Inst Med Trop Sao Paulo. 1995;37:63-69. [ Links ]

49. Al-Quraishy S, Dkhil MA, Abdel Moneim AE. Hepatotoxicity and oxidative stress induced by Naja haje crude venom. J Venom Anim Toxins Incl Trop Dis . 2014;20:42. [ Links ]

50. Pinho FMO, Zanetta DMT, Burdmann EA. Acute renal failure after Crotalus durissus snakebite: a prospective survey on 100 patients. Kidney Int. 2005;67:659-67. [ Links ]

51. Amora DN, Sousa TM, Martins AM, Barbosa PS, Magalhães MR, Toyama MH, et al. Effects of Crotalus durissus collilineatus venom in the isolated rat kidney. Toxicon . 2006;47:260-4. [ Links ]

52. Bucaretchi F, De Capitani EM, Branco MM, Fernandes LC, Hyslop S. Coagulopathy as the main systemic manifestation after envenoming by a juvenile South American rattlesnake (Crotalus durissus terrificus): case report. Clin Toxicol (Phila). 2013;51:505-8. [ Links ]

53. Serrano SM, Maroun RC. Snake venom serine proteinases: Sequence homology vs substrate specificity, a paradox to be solved. Toxicon . 2005;45:1115-32. [ Links ]

54. De Oliveira DG, Murakami MT, Cintra AC, Franco JJ, Sampaio SV, Arni RK. Functional and structural analysis of two fibrinogen-activating enzymes isolated from the venoms of Crotalus durissus terrificus, Crotalus durissus collilineatus. Acta Biochim Biophys Sin (Shanghai). 2009;41:21-29. [ Links ]

55. Cavalcante WL, Hernez-Oliveira S, Galbiatti C, Razzo-Moura P, Rocha T, Ponce-Soto L, et al. Biological characterization of Bothrops marajoensis snake venom. J Venom Res. 2011;2:37-41. [ Links ]

56. Beghini DG, Toyama MH, Hyslop S, Sodek LC, Novello JC, Marangoni S. Enzymatic characterization of a novel phospholipase A2 from Crotalus durissus cascavella rattlesnake (Maracambóia) venom. J Protein Chem. 2000;19:603-07. [ Links ]

57. Lennon BW, Kaiser II. Isolation of a crotoxin-like protein from the venom of a south american rattlesnake (Crotalus durissus collilineatus). Comp Biochem Physiol - Part B Biochem. 1990;97:695-99. [ Links ]

58. Rangel-Santos A, Dos-Santos EC, Lopes-Ferreira M, Lima C, Cardoso DF, Mota I. A comparative study of biological activities of crotoxin and CB fraction of venoms from Crotalus durissus terrificus, Crotalus durissus cascavella and Crotalus durissus collilineatus. Toxicon . 2004;43:801-10. [ Links ]

59. Bon C, Bouchier C, Choumet V, Faure G, Jiang MS, Lambezat MP, et al. Crotoxin, half-century of investigations on a phospholipase A2 neurotoxin. Acta Physiol Pharmacol Latinoam. 1989;39:439-48. [ Links ]

60. Breithaupt H. Neurotoxic and myotoxic effects of Crotalus phospholipase A and its complex with crotapotin. Naunyn Schmiedebergs Arch Pharmacol. 1976;292:271-78. [ Links ]

61. Gopalakrishnakone P, Hawgood BJ. Morphological changes induced by crotoxin in murine nerve and neuromuscular junction. Toxicon . 1984;22:791-804. [ Links ]

62. Gutiérrez JM, Ponce-Soto LA, Marangoni S, Lomonte B. Systemic and local myotoxicity induced by snake venom group II phospholipases A2: Comparison between crotoxin, crotoxin B and a Lys49 PLA2 homologue. Toxicon . 2008;51:80-92. [ Links ]

63. Vilca-Quispe A, Ponce-Soto LA, Winck FV, Marangoni S. Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis. Toxicon . 2010;55:745-53. [ Links ]

64. Ponce-Soto LA, Martins-de-Souza D, Marangoni S. Structural and pharmacological characterization of the crotamine isoforms III-4 (MYX4_CROCu) and III-7 (MYX7_CROCu) isolated from the Crotalus durissus cumanensis venom. Toxicon . 2010;55:1443-52. [ Links ]

65. Cavalcante WL, Ponce-Soto LA, Marangoni S, Gallacci M. Neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis. Toxicon . 2015;96:46-49. [ Links ]

66. Calvete JJ, Sanz L, Cid P, de la Torre P, Flores-Diaz M, Dos Santos MC, et al. Snake venomics of the Central American rattlesnake Crotalus simus, the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America. J Proteome Res. 2010;9:528-44. [ Links ]

Organization Funding: The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support. This study was supported by grants (479316-2013-6) from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Juliana Pavan Zuliani was a recipient of productivity grant 306672/2014-6 from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Leticia Helena de Carvalho was the beneficiary of CAPES Master's fellowship.

Recebido: 18 de Março de 2019; Aceito: 18 de Julho de 2019

Corresponding author: Dra. Juliana Pavan Zuliani. e-mail: juliana.zuliani@fiocruz.br

Conflict of Interest: The authors declare that they have no competing interests.

Creative Commons License This is an open-access article distributed under the terms of the Creative Commons Attribution License