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Toxicon

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Local and hematological alterations induced by Philodryas olfersiisnake venom in mice

Juliana S. Oliveira a, Luciana B. Sant'Anna a, *, Manoel C. Oliveira Junior b,Pamella R.M. Souza b, Adilson S. Andrade Souza b, Wellington Ribeiro c,Rodolfo P. Vieira b, e, Stephen Hyslop d, Jos�e C. Cogo e

a Laboratory of Histology and Regenerative Therapy, Institute of Research and Development (IP&D), Vale do Paraíba University (UNIVAP), Avenida ShishimaHifumi, 2911, Urbanova, 12244-000, S~ao Jos�e dos Campos, SP, Brazilb Laboratory of Pulmonary and Exercise Immunology (LABPEI), Nove de Julho University (UNINOVE) and Brazilian Institute of Teaching and Research inPulmonary and Exercise Immunology (IBEPIPE), 01504-000, S~ao Paulo, SP, Brazilc Laboratory of Pharmacology and Biochemistry, Institute of Research and Development (IP&D), Vale do Paraíba University (UNIVAP), Avenida ShishimaHifumi, 2911, Urbanova, 12244-000, S~ao Jose dos Campos, SP, Brazild Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tess�alia Vieira de Camargo, 126, CidadeUniversit�aria Zeferino Vaz, 13083-887, Campinas, SP, Brazile Department of Bioengineering and Biomedical Engineering, Brazil University, Rua Carolina Fonseca, 584/235 (Campus I and II), Vila Santana, 08230-030,Itaquera, S~ao Paulo, SP, Brazil

a r t i c l e i n f o

Article history:Received 25 November 2016Received in revised form22 March 2017Accepted 23 March 2017Available online 24 March 2017

Keywords:Acute inflammationCytokinesEdemaMyonecrosisInflammatory infiltratePhilodryas olfersii venom

* Corresponding author.E-mail address: [email protected] (L.B. Sant'A

http://dx.doi.org/10.1016/j.toxicon.2017.03.0130041-0101/© 2017 Elsevier Ltd. All rights reserved.

a b s t r a c t

Envenomation by the South American opisthoglyphous snake Philodryas olfersii causes local pain, edema,erythema and ecchymosis; systemic envenomation is rare. In this work, we examined the inflammatoryactivity of P. olfersii venom (10, 30 and 60 mg) in mouse gastrocnemius muscle 6 h after venom injection.Intramuscular injection of venom did not affect hematological parameters such as red cell count, he-moglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscularhemoglobin concentration. The venom caused thrombocytopenia (at all three doses), leukopenia andlymphopenia (both at the two highest doses), as well as neutrophilia (30 mg), monocytosis (30 mg) andbasophilia (10 mg). Of the cytokines that were screened [IL-1b, IL-6, IL-10, IL-13, IL-17, TNF-a, IFN-g, MIP-2and KC] and IGF-1, only IGF-1 showed a significant increase in its circulating concentration, seen with60 mg of venom; there were no significant changes in the cytokines compared to control mice. Histo-logical analysis revealed the presence of edema, an inflammatory infiltrate and progressive myonecrosis.Edema and myonecrosis were greatest with 60 mg of venom, while the inflammatory infiltrate wasgreatest with 10 mg of venom. All venom doses caused the migration of polymorphonuclear andmononuclear leukocytes into muscle, but with no significant dose-dependence in the response. Thesefindings show that, at the doses tested, P. olfersii venom does not cause hematological alterations and haslimited effect on circulating cytokine concentrations. These data also confirm that the principal effects ofthe venom in mice are local edema, inflammatory cell infiltration and myonecrosis.

© 2017 Elsevier Ltd. All rights reserved.

1. Introduction

The back-fanged colubroid snake genus Philodryas (Dipsadidae,Xenodontinae), commonly referred to as racers, consists of ~20species with a widespread distribution throughout South America(Zaher et al., 2008, 2014). Snakes of this genus are the principal

nna).

cause of non-front-fanged colubroid envenomations in this conti-nent (Prado-Franceschi and Hyslop, 2002; Weinstein et al., 2011,2013), with the main species involved in human envenomationsbeing P. chamissonis (Otero et al., 2007), P. olfersii (Ribeiro et al.,1999; Correia et al., 2010) and P. patagoniensis (Medeiros et al.,2010); species less commonly involved include P. aestivus (Fowlerand Salom~ao, 1994), P. baroni (Küch and Jesberger, 1993) andP. viridissima (Means, 2010).

The venoms of P. olfersii (Assakura et al., 1992; Acosta de P�erezet al., 2003; Rodríguez-Acosta et al., 2006) and P. patagoniensis

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(Acosta et al., 2003; Peichoto et al., 2004, 2005, 2006; Lopes, 2008)cause edema, hemorrhage and myonecrosis in experimental ani-mals, while in humans the primary manifestations are local effectssuch as pain, edema, erythema and ecchymosis (Ribeiro et al., 1999;Medeiros et al., 2010). A few components have been isolated fromthese venoms, including a myotoxin (Prado-Franceschi et al., 1998)and five fibrinogenolytic proteases (four metalloproteinases andone serine protease, with two of these enzymes also being hem-orrhagic) (Assakura et al., 1992) from P. olfersii, and a metal-loproteinase (patagonfibrase) (Peichoto et al., 2007, 2010, 2011) andcysteine-rich secretory protein (CRISP; patagonin) (Peichoto et al.,2009) from P. patagoniensis. The identification of these isolatedcomponents agrees with proteomic and transcriptomic analysesindicating the presence of metalloproteinases, serine proteases,CRISPs and other components in these venoms (Ching et al., 2006;Peichoto et al., 2012).

Philodryas olfersii venom degrades fibrinogen in vitro and in vivovia the action of metalloproteinases and serine proteinases(Assakura et al., 1994), but is devoid of thrombin-like activity; thisdegradation delays the clotting of fibrinogen by thrombin(Assakura et al., 1992). The venom also has fibrinolytic activity, butis devoid of platelet-aggregating or inhibitory effects (Assakuraet al., 1992). In contrast to these effects on hemostasis, the effectof P. olfersii venom on general hematological parameters is un-known. In addition, compared to P. patagoniensis (Peichoto et al.,2004; Lopes, 2008), the edematogenic response to P. olfersiivenom (Assakura et al., 1992; Acosta et al., 2003) has not beeninvestigated in detail, particularly with regard to the profile of in-flammatory cells involved and the possible changes in the con-centrations of cytokines in the general circulation.

In this work, we therefore examined the profile of the cellularinfiltrate associated with the inflammatory response after theintramuscular injection of P. olfersii venom in mice. We alsoquantified a variety of cytokines (IL-1b, IL-6, IL-10, IL-13, IL-17,TNFa, IFNg, MIP-2 and KC) and the growth factor IGF-1 known to beinvolved in the development and modulation of inflammation andexamined the occurrence of myonecrosis.

2. Material and methods

2.1. Venom

Venomwas obtained bymanual extraction from an adult femaleP. olfersii maintained at the Serpentarium of the Center for NatureStudies at UNIVAP (Environmental license SMA 15.380/2012). Thevenom was collected using glass capillary tubes, essentially asdescribed by Ferlan et al. (1983), and was lyophilized and stored at2e6 �C until use. For the experiments, venomwas dissolved in 0.9%NaCl immediately before use and injected into the left gastrocne-mius muscle of mice. Control mice were injected with an equalvolume (50 ml) of saline. Six hours later, the mice were killed andblood and tissue samples were collected for analysis.

2.2. Animals and experimental groups

Male C57BL/6 mice (18e22 g) were housed (5/cage) in poly-propylene cages with a wood shaving substrate at 22 ± 2 �C on a12 h light/dark cycle with lights on at 6 a.m. and free access to food(Purina® rodent chow) and water. The experiments were approvedby the Committee for Ethics in Animal Use of Vale do ParaíbaUniversity (CEUA/UNIVAP, protocol no. A13/CEUA/2015) and weredone in accordance with the ethical guidelines for animal experi-mentation established by the Brazilian Society for Laboratory Ani-mal Science (SBCAL).

For the experiments, 40 mice were randomly allocated to four

groups (n ¼ 10/group): Group 1 e mice injected with phosphate-buffered 150 mM saline (PBS) solution (control group) in the leftgastrocnemius muscle and Groups 2e4 emice injected with 10 mg,30 mg and 60 mg of P. olfersii venom, respectively, in a volume of 50ml/gastrocnemius muscle. Six hours after saline or venom injection,the mice were anesthetized with a mixture of xylazine hydro-chloride (Xilazin™ 2% injectable solution; 10 mg/kg, i.p.) plus ke-tamine hydrochloride (Cetamin™ 10% injectable solution; 100 mg/kg, i.p.). Once satisfactory anesthesia had been reached, blood wascollected via the inferior vena cava in a 1 ml syringe containing0.1 ml of EDTA. Ten microliters of blood were used for a completeblood count and the remainder was centrifuged (900 g, 10 min,4 �C) and the plasma then collected and stored at �80 �C for sub-sequent quantification of inflammatory mediators. After bloodcollection, the left gastrocnemius muscle was removed from theexsanguinated mice and three samples of each muscle were placedin separate polypropylene microtubes and stored at �80 �C.

2.3. Hematological analysis

Hematological analyses were done in an automated hemato-logical analyzer (Sysmex 800i, Roche, Germany) using blood sam-ples collected from the inferior vena cava. The parametersmeasured included red blood cell count (RBC), hemoglobin, he-matocrit, mean corpuscular volume (MCV), mean corpuscular he-moglobin (HCM), mean corpuscular hemoglobin concentration(CHMC), white blood cell (WBC) count, neutrophils, lymphocytes,monocytes and platelets.

2.4. Cytokine quantification

Plasma cytokine levels were quantified by ELISA using com-mercial kits obtained from Biolegend (San Diego, CA, USA) and R&DSystems (Minneapolis, MN, USA). The cytokines investigated wereIL-1b, IL-6, IL-10, IL-13, IL-17,MIP-2,KC, TNF-a and IFN-g and thegrowth factor IGF-1. All of the assays were done according to eachmanufacturer's recommended protocol.

2.5. Histological and quantitative analysis of polymorphonuclearand mononuclear cells

After removal, the gastrocnemius muscle was sectioned intothree parts, frozen in liquid nitrogen, fixed in cardboard chips andembedded in tissue freezingmediummixedwith powderedmilk toincrease the viscosity and support the tissue during cryotomy.Sections 10-mm thick were cut with a Leica DM1250 cryostat andthen stained with hematoxylin-eosin (HE) for semi-quantitativeevaluation of edema, inflammatory infiltrate and muscle degener-ation (myonecrosis), as well as quantitative evaluation of poly-morphonuclear and mononuclear cells in the muscle tissue.

For HE staining, the slides were immersed in acetone for 7 min,washed three times with deionized water and then incubated withhematoxylin for 1 min followed by three washes under runningwater. The sections were then submerged in a differentiating so-lution (9.90 ml of 70% ethanol þ 10 ml of HCl) for 1 s, immersed inwater for 3 min and in 80% ethanol for ~30 s. After this processing,the slides were stained with eosin for 1 min and submerged in 95%ethanol for ~30 s and then washed in absolute ethanol. The slideswere subsequently dried and mounted with Entelan®.

For the semi-quantitative evaluation of edema, inflammatoryinfiltrate and muscle degeneration, the slides were examined witha Nikon Eclipse E200 optical microscope at 400�magnification andthe alterations or extent of damage was scored using the followingarbitrary scale: 0 e no alteration or damage, 1 e mild alteration ordamage, 2 e moderate alteration or damage, 3 e intense alteration

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or damage, 4 e very intense alteration or damage. The individualresponsible for this analysis was blinded or unaware of the treat-ments from which the different sections had been obtained. Themean scores of five microscopic fields, one from each of fiverandomly chosen histological sections from each mouse, were usedto create a single score for each mouse in each experimental group.The mean of these mouse scores provided the overall mean for thecorresponding group.

For the quantitative analysis of polymorphonuclear and mono-nuclear inflammatory cells in gastrocnemius muscle, histo-morphometry was used in conjunction with image analysis, in anadaptation of similar analyses reported elsewhere (Vieira et al.,2008; Ramos et al., 2010; Gonçalves et al., 2012; Vieira et al.,2012). A total of 20 slides (five from each experimental group)were analyzed. From each slide, five microscopic fields wererandomly selected for analysis. Photomicrographs were capturedwith a digital video camera (Leica DF425) coupled to an opticalmicroscope (Leica DM2500) and scanned at 1024 � 768 pixels, 24bits/pixel resolution at a global magnification of 400�. Image-ProPlus 4.0 software was then used to calculate the total area of thephotomicrograph and clear area (area without tissue section). Thedifference in area obtained by subtracting the clear area from thetotal area of the photomicrograph yielded the tissue area.

Polymorphonuclear (PMN) and mononuclear (MN) cells werecounted manually and the number of polymorphonuclear cells/mm2 of tissue was calculated using the formula (number of PMN x1000 cells) ÷ tissue area ¼ PMN/mm2. Likewise, the number ofpolymorphonuclear cells/mm2 of tissue was calculated using theformula (number of MN x 1000 cells) ÷ tissue area ¼ MN/mm2. In

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Fig. 1. Changes in the number of circulating total leukocytes (A), platelets (B), neutrophilsP. olfersii venom. The mice were injected in the left gastrocnemius muscle with venom (10, 3they were anesthetized for blood sampling from the inferior vena cava (blood collected intautomated hematological analyzer. The columns represent the mean ± SD (n ¼ 10/group). *followed by the Newman-Keuls multiple comparisons test). The ends of each horizontal ba

both cases, tissue area was calculated as described in the precedingparagraph.

2.6. Statistical analysis

Quantitative (numerical) data were expressed as the mean ± SDwhenever appropriate. Prior to statistical analysis, the Shapiro-Wilk normality test was applied to examine the distribution ofthe data. Data with a normal distribution were analyzed by one-way analysis of variance (ANOVA) followed by the Newman-Keulstest for multiple comparisons among groups. For data without anormal distribution, the non-parametric Kruskal-Wallis test wasused followed by the Dunn test for multiple comparisons. In allcases, the level of significance was set at 5% (p < 0.05). All dataanalyses and statistical comparisons were done using Prism 5.0software (GraphPad Inc., La Jolla, CA, USA).

3. Results

3.1. Hematological parameters

The intramuscular injection of P. olfersii venom (10, 30 and60 mg) produced no significant alterations in the following he-matological parameters: red cell count, hemoglobin, hematocrit,mean corpuscular volume, mean corpuscular hemoglobin andmean corpuscular hemoglobin concentration (see Supplemen-tary table).

In contrast, as shown in Fig. 1, therewas a significant decrease inthe number of leukocytes (leukopenia), particularly with 30 mg and

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(C), monocytes (D), lymphocytes (E) and basophils (F) in BALB/c mice injected with0 or 60 mg in 50 ml of PBS) or the same volume of PBS alone (control mice) and 6 h latero EDTA), followed by exsanguination. The cells in plasma samples were counted in anp < 0.05, **p < 0.01 and ***p < 0.005 for the comparisons indicated (one-way ANOVAr indicate the two columns being compared in each case.

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60 mg of venom. There was also a significant reduction (~30%) inplatelet count, but no significant difference in the response to thethree doses of venom. There was a significant increase in thenumber of neutrophils and monocytes with 30 mg of venomcompared to the control group (PBS solution). For neutrophils,venom doses of 10 mg and 60 mg also produced an increase, but thiswas considerably less marked than with 30 mg of venom. Formonocytes, the dose of 10 mg caused an increase in cell numberswhereas with 60 mg the number of monocytes was not significantlydifferent from that of the PBS control. The changes in the number oflymphocytes mirrored those for total leukocytes but were rathermore pronounced, with 30 mg and 60 mg producing the greatestreductions (lymphopenia). There was a significant increase in thenumber of basophils (basophilia) with 10 mg of venom, but not withthe other doses.

3.2. Cytokine quantification

Fig. 2 shows the plasma concentrations of IGF-1 and variouscytokines measured by ELISA in PBS- and venom-treated mice 6 hafter venom injection. Therewas a very large increase (p < 0.001) inserum IGF-1 levels with 60 mg of venom compared to the control(PBS) group and the other two doses of venom. There was no sig-nificant increase in KC levels compared to the PBS group. However,the concentration with 30 mg of venom was significantly greaterthan that seen with 10 mg and 60 mg, primarily because of areduction associated with the latter two doses. There were nosignificant changes in the concentrations of various other pro-inflammatory cytokines (MIP 2, IFN-g TNF-a, IL-1b, IL-6 and IL-17) or the anti-inflammatory interleukins IL-10 and IL-13 for thethree venom doses.

3.3. Histological analysis

Gastrocnemius muscle inoculated with PBS alone showed thecharacteristics of normal skeletal striated muscle, namely, multi-nucleated cylindrical cells, peripheral nuclei and transverse stria-tions visible in light microscopy, with the presence of mild edemaand a discrete inflammatory infiltrate. In muscle inoculated with10 mg of P. olfersii venom, there was edema, an intense inflamma-tory infiltrate characterized by a predominance of poly-morphonuclear cells, and mild degeneration of the muscle fibersseen as membrane rupture and fibrillar disorganization. In muscleinoculated with 30 mg of venom, there was moderate edema and aninflammatory infiltrate characterized by a predominance of poly-morphonuclear cells accompanied by foci of muscle degenerationwith fiber destruction (myonecrosis). In muscle injected with 60 mgof venom there was intense edema, inflammatory infiltrate with apredominance of polymorphonuclear cells, and muscle degenera-tion, the latter involving membrane rupture, fibrillar disorganiza-tion and myonecrosis (Fig. 3).

Semi-quantitative analysis (Fig. 4) showed significant edemaand muscle degeneration with 60 mg of venom compared to thecontrol group (PBS alone) after 6 h. In contrast, the inflammatoryinfiltrate was greatest with 10 mg of venom compared to the controlgroup. Muscle degeneration, but not edema or inflammatory infil-trate, showed dose-dependence. The intramuscular injection ofP. olfersii venom caused a significant increase in poly-morphonuclear leukocyte density/mm2 with 30 mg and 60 mg ofvenom compared to the control group (PBS alone), but therewas nodifference in the responses among venom doses. Although all threevenom doses increased the number of mononuclear leucocytes/mm2, this increase was not significantly different from the control(PBS) group, nor was there any difference in the responses amongvenom doses (Fig. 5).

4. Discussion

Philodryas olfersii and P. patagoniensis are the two species mostcommonly involved in envenomation by Philodryas snakes in Brazil(Ribeiro et al., 1999; Medeiros et al., 2010) and their venoms are thebest studied among South American non-front-fanged colubroidsnakes. However, compared to P. patagoniensis (Acosta et al., 2003;Peichoto et al., 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012),relatively few studies have examined the biological activities ofP. olfersii venom. Assakura et al. (1992, 1994) showed that P. olfersiivenom was hemorrhagic, fibrinogenolytic and edematogenic, andsubsequent studies confirmed the edematogenic (Acosta et al.,2003) and hemorrhagic (Rocha et al., 2006; Rodríguez-Acostaet al., 2006) activities and the ability of this venom to cause myo-necrosis in vitro (Prado-Franceschi et al., 1996, 1998; Collaço et al.,2012) and in vivo (Acosta et al., 2003). In this work, we examinedthe edema and myonecrosis caused by P. olfersii venom in mousegastrocnemiusmuscle, and also assessed the ability of the venom toalter hematologic parameters and increase the circulating cytokineconcentrations. The doses of venom used here were chosen basedon Rocha and Furtado (2007), who reported a minimum hemor-rhagic dose of 24 mg/mouse, a minimum necrotizing dose of 79,1mg/mouse and a lethality (LD50) of 62.43 mg/mouse for this venom.An interval of 6 h post-venom was studied because literature re-ports of envenoming by P. olfersii indicate that this interval showstypical changes associated with an acute inflammatory reaction.

The lack of significant changes in the hematological parameters6 h after the intramuscular injection of P.olfersii venom indicatedthat there were no systemic alterations in these parameters asso-ciated with the venom doses, route of administration and timeinterval examined here. The unaltered hemoglobin and hematocritalso indicated that there was no intravascular hemolysis, perhapsreflecting the absence of PLA2 activity in this venom (Assakuraet al., 1992; Peichoto et al., 2012).

In contrast to the lack of effect on hematological parameters,significant thrombocytopenia was seen with all doses of venom,although there was no difference in the extent of the responseamong the three doses. Possible changes in circulating plateletnumbers have not previously been examined after injection ofPhilodryas venoms. Metalloproteinases (Assakura et al., 1992, 1994;Ching et al., 2006; Rocha et al., 2006; Peichoto et al., 2012) and C-type lectins (Ching et al., 2006; Peichoto et al., 2012) in P. olfersiivenom could possibly contribute to this decrease in platelet num-ber, whereas PLA2, thrombin-like enzymes and procoagulant en-zymes are unlikely to be involved since the venom is devoid ofthese activities (Assakura et al., 1992; Ching et al., 2006; Peichotoet al., 2012). However, the venoms of P. baroni (S�anchez et al.,2014) and P. patagoniensis (Peichoto, 2007) do not aggregate hu-man washed platelets in vitro, but inhibit collagen- and thrombin-induced aggregation. Similarly, the metalloproteinase patagonfi-brase from P. patagoniensis venom does not aggregate humanwashed platelets but inhibits collagen- and ADP-induced aggrega-tion without affecting that induced by thrombin or ristocetin; theinhibitory activity is independent of the protein's enzymatic ac-tivity (Peichoto et al., 2007). The thrombocytopenia observed herewas similar to that reported by Yamashita (2013) in mice inoculatedwith Bothropsjararaca venom. In contrast, Graça et al. (2008) foundno significant variations in the platelet count in an experimentalstudy of crotalic envenomation in cattle treated with venom of theSouth American rattlesnake Crotalus durissus terrificus.

Philodryas olfersii venom caused leukopenia that was greatestwith 30 mg of venom. Leukopenia is generally defined as a globaldecrease in the number of white blood cells and is most oftencaused by a reduction in the number of neutrophils (the mostcommon type of leukocyte) followed by a reduction in lymphocytes

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Fig. 3. Histological analysis of mouse gastrocnemius muscle injected with P. olfersii venom (10, 30 or 60 mg in 50 ml of PBS) or PBS alone (control). Male Balb/c mice were injectedwith venom or PBS in the left gastrocnemius muscle and 6 h later the animals were killed with an overdose of anesthetic and exsanguinated. The muscle was removed andprocessed for histological analysis as described in section 2.5. Asterisks e edema, triangles e inflammatory infiltrate, and arrows e muscle fiber degeneration. H-E staining. Scalebars: 20 mm in all panels.

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(the second most common type of leukocyte) (Stock and Hoffman,2000). However, as shown here, the significant decrease in leuko-cytes apparently resulted from a decrease in the number of lym-phocytes rather than neutrophils since the latter showed asignificant increase in number with this dose of venom (30 mg).Acosta de P�erez et al. (2003) also reported a neutrophilic infiltratein mouse gastrocnemius muscle injected with 40 mg of P. olfersiivenom from Argentina. Our findings contrast with those for venomof the pitviper B. jararaca that caused an increase in the totalleukocyte count 3 and 6 h after venom administration i.v.(Yamashita, 2013).

There was a significant increase in the number of circulatingbasophils in mice injected with 10 mg of venom, but not with theother doses, the counts for which were similar to basal (control)

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Fig. 4. Local tissue responses in mouse gastrocnemius muscle injected with P. olfersii venomwith venom (10, 30 or 60 mg in 50 ml of PBS) or PBS alone (control) in the left gastrocnemexsanguination. The muscle was removed and processed for histological analysis as describeas follows: 0 e no alteration or damage, 1 e slight alteration or damage, 2 e moderate altedamage. The results are expressed as box-plots showing the median, interquartils and rangeANOVA followed by the Newman-Keuls multiple comparisons test). The ends of each horiz

values. This increase in basophil number appeared to correlatewiththe significant increase in inflammatory infiltrate assessed semi-quantitatively in the gastrocnemius muscle of mice inoculatedwith this same dose of venom. Basophils release mediators capableof enhancing vascular permeability and inducing the migration ofinflammatory cells (neutrophils and macrophages) (Cruvinel et al.,2010). However, the precise relationship between the increase incirculating basophil numbers and the increase in inflammatoryinfiltrate in envenomed muscle remains to be established. Thenumber of circulating neutrophils generally increases 1e6 h afterinjury (Leech, 1997). In agreement with this, 6 h after P. olfersiivenom administration there was a predominance of neutrophils inthe inflammatory infiltrate present in gastrocnemius muscleinjected with different doses of P. olfersii venom, as well as an

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in mice: (A) edema, (B) inflammatory infiltrate and (C) myonecrosis. Mice were injectedius muscle and 6 h later the animals were killed with an overdose of anesthetic andd in section 2.5. Semi-quantitative analysis was done using an arbitrary scoring system,ration or damage, 3 e intense alteration or damage, and 4 e very intense alteration or(n ¼ 5 mice/group). *p < 0.05 and **p < 0.01 for the comparisons indicated (one-wayontal bar indicate the two columns being compared in each case.

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es/m

m2

Venom (μg)

BA

Fig. 5. Analysis of inflammatory cell infiltrate (A e polymorphonuclear cells and B e mononuclear cells) in mouse gastrocnemius muscle injected with P. olfersii venom. Mice wereinjected with venom (10, 30 or 60 mg in 50 ml of PBS) or PBS alone (control) in the left gastrocnemius muscle and 6 h later the animals were killed with an overdose of anesthetic andexsanguination. The muscle was removed and processed for histological analysis as described in section 2.5. The columns represent the mean ± SD (n ¼ 10/group). *p < 0.05 for thecomparisons indicated (one-way ANOVA followed by the Newman-Keuls multiple comparisons test). The ends of each horizontal bar indicate the two columns being compared ineach case.

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increase in the number of circulating neutrophils, with the greatestincrease seen with 30 mg of venom. Graça et al. (2008) reported amoderate increase in the total leukocyte count in nine out of tencattle injected with C. d. terrificus venom; this increase was char-acterized by neutrophilia, relative lymphopenia, eosinopenia andmonocytosis, with their findings for lymphopenia and monocytosisbeing similar to those observed here for P. olfersii.

Cytokines are important mediators in the inflammatoryresponse to snake venoms (Petricevich et al., 2000; Petricevich,2004; Cruz et al., 2008). In this regard, the increase in the num-ber of circulating neutrophils seen with 30 mg of venom waspossibly related to the greater circulating concentration of KC seenwith this dose of venom relative to that in mice treated with 10 mgand 60 mg of venom, although the levels of KC were not significantlydifferent from the control group (injected with PBS). KC belongs tothe CXC class of chemokines and is selective for the recruitment ofpolymorphonuclear leukocytes. Wengner et al. (2008) demon-strated that KC participates in neutrophil chemotaxis by activatingthe CXCR2 receptor expressed in these cells, leading to an increasein the number of this cell type in the peripheral circulation. Thismechanism could explain the increase in circulating neutrophils inmice injected with 30 mg of venom.

Petricevich et al. (2000) observed an increase in the serumlevels of TNF-a, IL-1b, IL-6, IL-10 and IFN-g in mice injected intra-peritoneally with the mean lethal dose (LD50) of Bothropsasper andB. jararaca venoms. Similarly, Cruz et al. (2008) reported an increasein the serum levels of TNF-a, IL-10 and IL-6 15e30 min after theadministration of one LD50 of C. d. terrificus venom, with peakconcentrations occurring ~2 h after envenomation, followed by adecrease thereafter; there were no significant changes in the serumconcentration of IFN-g. Overall, pro-inflammatory cytokines pre-dominated in the early phases of envenomation, whereas anti-inflammatory cytokines predominated in the later stages (Cruzet al., 2008). In contrast to these findings, 6 h after the injectionof all three doses of P. olfersii venom there were no significantchanges in the circulating concentrations of IL-1b, IL-6, IL-10, IL-13,IL -17, IFN-g and TNF-a, although there was a trend towards anincrease in the levels of IL-6, IL-1b, IL-10 and especially TNF-a. Thisfinding indicates that the release of cytokines from the site ofvenom injection into the general circulation is not a major feature

of envenomation by P. olfersii in mice. However, local production ofat least some of these cytokines could be involved in muscledamage since in mouse phrenic nerve-diaphragm and chickbiventer cervicis preparations in vitro P. olfersii venom enhances theexpression of IFN-g and TNF-a, as assessed immunohistochemically(Collaço et al., 2012).

Growth factors such as IGF-1 act as positive regulators in thecontrol and activation of satellite cells located between the basallamina and plasma membrane of the muscle fiber and are indis-pensable for muscle regeneration. Takahashi et al. (2003) demon-strated that IGF-1 promoted muscle regeneration after transfer byelectroporation in experimentally injured mouse muscle. Lopes(2008) observed that from the sixth hour after the inoculation ofP. patagoniensis venom onwards there was regression of the myo-necrotic lesions and the initiation of muscle fiber regeneration,such that 24 h later normal fibers were surrounded by an inflam-matory infiltrate. As shown here, there was a very marked increasein the circulating IGF-1 concentration in mice 6 h after the injectionof 60 mg of P. olfersii venom, perhaps in response to the extensivemuscle degeneration seen with this dose.

Philodryas olfersii venom caused edema, an inflammatory infil-trate and myonecrosis but no hemorrhage after 6 h. The absence ofhemorrhage at 6 h post-venom may reflect a previous finding thatthe hemorrhagic response to P. olfersii venom peaks at 2e4 h afterinjection (Rocha and Furtado, 2007). All venom doses stimulatedthe migration of polymorphonuclear and mononuclear cells intogastrocnemius muscle, but with no clear dose-dependence. Aninflammatory infiltrate has also been observed with P. olfersiivenom from Argentina (Acosta et al., 2003) and for the venoms ofP. baroni (S�anchez et al., 2014) and P. patagoniensis (Acosta et al.,2003; Peichoto et al., 2004), as well as purified proteins such asthe metalloproteinase patagonfibrase (Peichoto et al., 2007, 2011)and the CRISP patagonin (Peichoto et al., 2009).

The discrepancy between the apparently greater inflammatoryinfiltrate observed with 10 mg of venom compared to the other twodoses and the lack of significant differences among the doses withregard to the number of polymorphonuclear and mononuclearleukocytes in the inflammatory infiltrate probably reflects differ-ences in the methodological approaches used for the quantitativeand semi-quantitative assessments, with the semi-quantitative

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analysis providing amore comprehensive assessment of themuscletissue. It is also possible that the photomicrographs used for thequantitative analysis of mice injected with 10 mg of venom wereobtained from areas with a lower inflammatory infiltrate that wasnot representative of the true response for this venom dose.

Myonecrosis is a common finding in experimental studies withPhilodryas venoms in vitro (Prado-Franceschi et al., 1996, 1998;Carreiro da Costa et al., 2008; Collaço et al., 2012) and in vivo(Acosta et al., 2003; Acosta de P�erez et al., 2003; Peichoto et al.,2004, 2007; Lopes, 2008; S�anchez et al., 2014). As shown here,myonecrosis was present 6 h after the inoculation of the threedoses of P. olfersii venom and similar activity was reported forP. olfersii venom from Argentina (Acosta et al., 2003). Although wedid not investigate the time-course of myonecrosis, Lopes (2008)noted that myonecrosis started within 30 min after the injectionof 10 mg of P. patagoniensis venom. The venom componentsresponsible for myonecrosis remain poorly studied, although amyotoxin has been identified in P. olfersii venom (Prado-Franceschiet al., 1998). In addition, a CRISP (patagonin) from P. patagoniensisvenom causes myonecrosis but no edema or hemorrhage (Peichotoet al., 2009). Venom metalloproteinases may also contribute to themyonecrosis since Philodryas venoms have consistently beenshown to be very proteolytic when compared to Bothrops (lance-head) snake venoms (Assakura et al., 1992; Acosta et al., 2003;Rocha et al., 2006; Carreiro da Costa et al., 2008; S�anchez et al.,2014). PLA2 is not involved since these venoms are generallydevoid of this enzyme (Assakura et al., 1992; Peichoto et al., 2004,2012; Ching et al., 2006).

In conclusion, the results described here indicate that P. olfersiivenom (10e60 mg) does not affect erythrocyte parameters or theserum levels of pro- and anti-inflammatory interleukins, but in-creases the serum levels of IGF-1. The venom-induced inflamma-tory response was characterized by edema and a cellular responseinvolving leukopenia and lymphopenia, with neutrophilia, mono-cytosis, basophilia and thrombocytopenia. Myonecrosis withouthemorrhage was seen 6 h after venom injection. These various al-terations are probably mediated by metalloproteinases, serineproteinases, CRISPs and C-type lectins present in the venom.

Ethical statement

1) This material has not been published in whole or in partelsewhere.

2) The manuscript is not currently being considered for publi-cation in another journal.

3) All authors have been personally and actively involved insubstantive work leading to the manuscript, and will hold them-selves jointly and individually responsible for its content.

Conflict of interest

The authors have no conflict of interest with the publication ofthis work.

Acknowledgments

JSO was supported by a studentship from Coordenaç~ao deAperfeiçoamento de Pessoal de Nível Superior (CAPES, grant no.33051011009-PO).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2017.03.013.

Transparency document

Transparency document related to this article can be foundonline at http://dx.doi.org/10.1016/j.toxicon.2017.03.013.

References

Acosta, O., Leiva, L.C., Peichoto, M.E., Maru~nak, S., Teibler, P., Rey, L., 2003. Hemor-rhagic activity of the Duvernoy's gland secretion of the xenodontine colubridPhilodryas patagoniensis from the north-east region of Argentina. Toxicon 41,1007e1012.

Acosta de P�erez, O., Leiva de Vila, L., Peichoto, M.E., Maru~nak, S., Ruíz, R., Teibler, P.,Gay, C., Rey, L., 2003. Edematogenic and myotoxic activities of the Duvernoy'sgland secretion of Philodryas olfersii from the north-east region of Argentina.Biocell 27, 363e370.

Assakura, M.T., Salom~ao, M.G., Puorto, G., Mandelbaum, F.R., 1992. Hemorrhagic,fibrinogenolytic and edema-forming activities of the venom of the colubridsnake Philodryas olfersii (green snake). Toxicon 30, 427e438.

Assakura, M.T., Reichl, A.P., Mandelbaum, F.R., 1994. Isolation and characterizationof five fibrin(ogen)olytic enzymes from the venom of Philodryas olfersii (greensnake). Toxicon 32, 819e831.

Carreiro da Costa, R.S., Prudencio, L., Ferrari, E.F., Souza, G.H., de Mello, S.M., PriantiJúnior, A.C., Ribeiro, W., Zamun�er, S.R., Hyslop, S., Cogo, J.C., 2008. Neuromus-cular action of venom from the South American colubrid snake Philodryaspatagoniensis. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 148, 31e38.

Ching, A.T.C., Rocha, M.M.T., Paes Leme, A.F., Pimenta, D.C., Furtado, M.F.D.,Serrano, S.M.T., Ho, P.L., Junqueira-de-Azevedo, I.L.M., 2006. Some aspects of thevenom proteome of the Colubridae snake Philodryas olfersii revealed from aDuvernoy's (venom) gland transcriptome. FEBS Lett. 580, 4417e4422.

Collaço, R.C.O., Cogo, J.C., Rodrigues-Simioni, L., Rocha, T., Oshima-Franco, Y., Ran-dazzo-Moura, P., 2012. Protection by Mikania laevigata (guaco) extract againstthe toxicity of Philodryas olfersii snake venom. Toxicon 60, 614e622.

Correia, J.M., Santana Neto, P.L., Pinho, M.S.S., da Silva, J.A., Amorim, M.L.P.,Escobar, J.A.C., 2010. Poisoning due to Philodryas olfersii (Lichtenstein, 1823)attended at Restauraç~ao Hospital in Recife, state of Pernambuco, Brazil: casereport. Rev. Soc. Bras. Med. Trop. 43, 336e338.

Cruvinel, W.M., Mesquita Júnior, D., Araújo, J.A.P., Catelan, T.T.T., de Souza, A.W.S., daSilva, N.P., Andrade, L.E.C., 2010. Immune system e Part I. Fundamentals ofinnate immunity with emphasis on the molecular and cellular mechanisms ofinflammatory response. Rev. Bras. Reumatol. 50, 434e461.

Cruz, A.H., Garcia-Jimenez, S., Mendonça, R.Z., Petricevich, V.L., 2008. Pro- and anti-inflammatory cytokines release in mice injected with Crotalus durissus terrificusvenom. Med. Inflamm. 2008, 874962. http://dx.doi.org/10.1155/2008/874962.

Ferlan, I., Ferlan, A., King, T., Russell, F.E., 1983. Preliminary studies on the venom ofthe colubrid snake Rhabdophis subminatus (red-necked keelback). Toxicon 21,570e574.

Fowler, I.R., Salom~ao, M.G., 1994. Activity patterns in the colubrid snake genusPhilodryas and their relationship to reproduction and snakebite. Bull. Chic.Herp. Soc. 29, 229e232.

Gonçalves, C.T.R., Gonçalves, C.G.R., Almeida, F.M., Lopes, F.D., Dur~ao, A.C.S.,Santos, F.A., Silva, L.F., Marcourakis, T., Castro Faria Neto, H.C., Vieira, R.P.,Dolhnikoff, M., 2012. Protective effects of aerobic exercise on acute lung injuryinduced by LPS in mice. Crit. Care 16, R199.

Graça, F.A.S., Peixoto, P.V., Coelho, C.D., Caldas, S.A., Tokarnia, C.H., 2008. Aspectosclínico-patol�ogicos e laboratoriais do envenenamento crot�alico experimentalem bovinos. Pesq. Vet. Bras. 28, 261e270.

Küch, U., Jesberger, U., 1993. Human envenomation from the bite of the SouthAmerican colubrid snake species Philodryas baroni (Berg, 1895). Snake 25,63e65.

Leech, S.J., 1997. Review of muscle healing. J. Physiol. X, 15e18.Lopes, P.H., 2008. Local Changes Induced by the Toxic Secretion of Philodryas

patagoniensis (Girad, 1857) (Serpentes: Colubridae). PhD thesis (in Portuguese).University of S~ao Paulo, S~ao Paulo, Brazil. Available from: http://www.teses.usp.br/teses/disponiveis/41/41135/tde-06082008-185402/publico/PriscilaHessLopes.pdf.

Means, D.B., 2010. Ophidism by the green palmsnake. Wild. Environ. Med. 21,46e49.

Medeiros, C.R., Hess, P.L., Nicoleti, A.F., Sueiro, L.R., Duarte, M.R., Almeida-Santos, S.M., França, F.O.S., 2010. Bites by the colubrid snake Philodryas pata-goniensis: a clinical and epidemiological study of 297 cases. Toxicon 56,1018e1024.

Neira, P.O., Jofr�e, L.M., Oschilewski, D.L., Subercaseaux, B.S., Mu~noz, N.S., 2007.Mordedura por Philodryas chamissonis. Presentacíon de un caso y revisi�on de laliteratura. Rev. Chil. Infectol. 24, 236e241.

Peichoto, M.E., 2007. Caracterizaci�on y aislameinto de proteínas del veneno dePhilodryas patagoniensis que habita la regi�on nordeste de Argentina. PhD thesis.Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires,Argentina, p. 173.

Peichoto, M.E., Acosta, O., Leiva, L., Teibler, P., Maru~nak, S., Ruíz, R., 2004. Muscle andskin necrotizing and edema-forming activities of Duvernoy's gland secretion ofthe xenodontine colubrid snake Philodryas patagoniensis from the north-east ofArgentina. Toxicon 44, 589e596.

Peichoto, M.E., Leiva, L.C., Guiam�as Moya, L.E., Rey, L., Acosta, O., 2005. Duvernoy's

J.S. Oliveira et al. / Toxicon 132 (2017) 9e17 17

Author's Personal Copy

gland secretion of Philodryas patagoniensis from the northeast of Argentina: itseffects on blood coagulation. Toxicon 45, 527e534.

Peichoto, M.E., Teibler, P., Ruíz, R., Leiva, L., Acosta, O., 2006. Systemic pathologicalalterations caused by Philodryas patagoniensis colubrid snake venom in rats.Toxicon 48, 520e528.

Peichoto, M.E., Teibler, P., Mackessy, S.P., Leiva, L., Acosta, O., Gonçalves, L.R.C.,Tanaka-Azevedo, A.M., Santoro, M.L., 2007. Purification and characterization ofpatagonfibrase, a metalloproteinase showing a-fibronogenolytic and hemor-rhagic activities, from Philodyras patagoniensis snake venom. Biochim. Biophys.Acta 1770, 810e819.

Peichoto, M.E., Mackessy, S.P., Teibler, P., Tavares, F.L., Burckhardt, P.L., Breno, M.C.,Acosta, O., Santoro, M.L., 2009. Purification and characterization of a cysteine-rich secretory protein from Philodryas patagoniensis snake venom. Comp. Bio-chem. Physiol. C Toxicol. Pharmacol. 150, 79e84.

Peichoto, M.E., Paes Leme, A.F., Pauletti, B.A., Batista, I.C., Mackessy, S.P., Acosta, O.,Santoro, M.L., 2010. Autolysis at the disintegrin domain of patagonfibrase, ametalloproteinase from Philodryas patagoniensis (Patagonia green racer; Dip-sadidae) venom. Biochim. Biophys. Acta 1804, 1937e1942.

Peichoto, M.E., Zychar, B.C., Tavares, F.L., Gonçalves, L.R.C., Acosta, O., Santoro, M.L.,2011. Inflammatory effects of patagonfibrase, a metalloproteinase from Philo-dryas patagoniensis (Patagonia green racer; Dipsadidae) venom. Exp. Biol. Med.236, 1166e1172.

Peichoto, M.E., Tavares, F.L., Santoro, M.L., Mackessy, S.P., 2012. Venom proteomes ofSouth and North American opisthoglyphous (Colubridae and Dipsadidae) snakespecies: a preliminary approach to understanding their biological roles. Comp.Biochem. Physiol. D 7, 361e369.

Petricevich, V.L., 2004. Cytokine and nitric oxide production following severe en-venomation. Curr. Drug Targets Inflamm. Allergy 3, 325e332.

Petricevich, V.L., Teixeira, C.F., Tambourgi, D.V., Guti�errez, J.M., 2000. Increments inserum cytokine and nitric oxide levels in mice injected with Bothrops asper andBothrops jararaca snake venoms. Toxicon 38, 1253e1266.

Prado-Franceschi, J., Hyslop, S., 2002. South American colubrid envenomations.J. Toxicol. e Toxin Rev. 21, 117e158.

Prado-Franceschi, J., Hyslop, S., Cogo, J.C., Andrade, A.L., Assakura, M., Cruz-H€ofling, M.A., Rodrigues-Simioni, L., 1996. The effects of Duvernoy's glandsecretion from the xenodontine colubrid Philodryas olfersii on striated muscleand the neuromuscular junction: partial characterization of a neuromuscularfraction. Toxicon 34, 459e466.

Prado-Franceschi, J., Hyslop, S., Cogo, J.C., Andrade, A.L., Assakura, M.T., Reichl, A.P.,Cruz-H€ofling, M.A., Rodrigues-Simioni, L., 1998. Characterization of a myotoxinfrom the Duvernoy's gland secretion of the xenodontine colubrid Philodryasolfersii (green snake): effects on striated muscle and the neuromuscular junc-tion. Toxicon 36, 1407e1421.

Ramos, D.S., Olivo, C.R., Lopes, F.D.T.Q.S., Toledo, A.C., Martins, M.A., Os�orio, R.A.L.,Dolhnikoff, M., Ribeiro, W., Vieira, R.P., 2010. Low-intensity swimming trainingpartially inhibits lipopolysaccharide-induced acute lung injury. Med. Sci. SportsExer. 42, 113e119.

Ribeiro, L.A., Puorto, G., Jorge, M.T., 1999. Bites by the colubrid snake Philodryasolfersii: a clinical and epidemiological study of 43 cases. Toxicon 37, 943e948.

Rocha, M.M.T., Furtado, M.F.D., 2007. An�alise das atividades biol�ogicas dos venenosde Philodryas olfersii (Lichtenstein) e P. patagoniensis (Girard) (Serpentes,Colubridae). Rev. Bras. Zool. 24, 410e418.

Rocha, M.M.T., Paix~ao-Cavalcante, D., Tambourgi, D.V., Furtado, M.F.D., 2006.Duvernoy's gland secretion of Philodryas olfersii and Philodryas patagoniensis(Colubridae): neutralization of local and systemic effects by commercialbothropic antivenom (Bothrops genus). Toxicon 47, 95e103.

Rodríguez-Acosta, A., Lemoine, K., Navarrete, L., Gir�on, M.E., Aguilar, I., 2006.Experimental ophitoxemia produced by the opisthoglyphous lora snake (Phil-odryas olfersii) venom. Rev. Soc. Bras. Med. Trop. 39, 193e197.

S�anchez, M.N., Timoniuk, A., Maru~nak, S., Teibler, P., Acosta, O., Peichoto, M.E., 2014.Biochemical and biological analysis of Philodryas baroni (Baron's green racer;Dipsadidae) venom: relevance to the findings of human risk assessment. Hum.Exp. Toxicol. 33, 22e31.

Stock, W., Hoffman, R., 2000. White blood cells 1: non-malignant disorders. Lancet355, 1351e1357.

Takahashi, T., Ishida, K., Itoh, K., Konishi, Y., Yagyu, K.I., Tominaga, A., Miyazaki, J.I.,Yamamoto, H., 2003. IGF-1 gene transfer by electroporation promotes regen-eration in a muscle injury model. Gene Ther. 10, 612e620.

Vieira, R.P., Andrade, V.F., Duarte, A.C., dos Santos, A.B., Mauad, T., Martins, M.A.,Dolhnikoff, M., Carvalho, C.R., 2008. Aerobic conditioning and allergic pulmo-nary inflammation in mice. II. Effects on lung vascular and parenchymalinflammation and remodeling. Am. J. Physiol. Lung Cell. Mol. Physiol. 295,670e679.

Vieira, R.P., Toledo, A.C., Silva, L.D., Almeida, F.M., Damaceno-Rodrigues, N.R.,Caldini, E.G., Santos, A.B., Rivero, D.H., Hizume, D.C., Lopes, F.D., Olivo, C.R.,Castro Faria Neto, H.C., Martins, M.A., Saldiva, P.H., Dolhnikoff, M., 2012. Anti-inflammatory effects of aerobic exercise in mice exposed to air pollution. Med.Sci. Sports Exer. 44, 1227e1243.

Weinstein, S.A., Warrell, D.A., White, J., Keyler, D.E., 2011. Venomous Bites fromNon-venomous Snakes: a Critical Analysis of Risk and Management of “Colu-brid” Snake Bites. Elsevier, London, UK.

Weinstein, S.A., White, J., Keyler, D.E., Warrell, D.A., 2013. Non-front-fangedcolubroid snakes: a current evidence-based analysis of medical significance.Toxicon 69, 103e113.

Wengner, A.M., Pitchford, S.C., Furze, R.C., Rankin, S.M., 2008. The coordinated ac-tion of G-CSF and ELR þ CXC chemokines in neutrophil mobilization duringacute inflammation. Blood 111, 42e49.

Yamashita, K.M., 2013. Pathogenesis of Systemic Hemostatic Disturbances Inducedby Venom of the Bothrops jararaca Snake. MSc dissertation (in Portuguese).University of S~ao Paulo, S~ao Paulo. Available at: www.teses.usp.br/teses/disponiveis/5/5167/tde20052013/KarineMikiYamashita.pdf.

Zaher, H., Scrocchi, G., Masiero, R., 2008. Rediscovery and redescription of the typeof Philodryas laticeps Werner, 1900 and the taxonomic status of P. oligolepisGomes, 1921 (Serpentes, Colubridae). Zootaxa 1940, 25e40.

Zaher, H., Arredondo, J.C., Valencia, J.H., Arbel�aez, E., Rodrigues, M.T., Altamirano-Benavides, M., 2014. A new Andean species of Philodryas (Dipsadidae, Xen-odontinae) from Ecuador. Zootaxa 3785, 469e480.