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Fatal lancehead pit viper (Bothrops spp.) envenomation in horses
Toxicon 170 (2019) 41–50
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Fatal lancehead pit viper (Bothrops spp.) envenomation in horses a
T
a
Mizael Machado (DVM) , Tais Meziara Wilson (DVM Msc) , Davi Emanuel Ribeiro de Sousa (DVM)a, Antônio Carlos Lopes Câmara (DVM PhD)b, Fernando Henrique Furlan (DVM PhD)c, Juliana Targino Silva Almeida e Macêdo (DVM PhD)a, Rayane Chitolina Pupin (DVM PhD)d, Ricardo Antônio Amaral de Lemos (DVM, PhD)d, Anibal G. Armién (DVM PhD)e, Severo Sales Barros (DVM PhD)f, Franklin Riet-Correa (DVM PhD)g, Márcio Botelho de Castro (DVM PhD)a,∗ a Veterinary Pathology Laboratory, Veterinary Teaching Hospital, Via L4 Norte, sn/, Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, 70636-200, Brasília, Distrito Federal, Brazil b Large Animal Veterinary Teaching Hospital, Universidade de Brasília (UnB), SIT PqEAT, Granja do Torto, 70297-400, Brasília, Distrito Federal, Brazil c Veterinary Pathology Laboratory, Veterinary Teaching Hospital Universidade Federal de Mato Grosso (UFMT), Av. Fernando Corrêa da Costa 2367, Boa Esperança, 78060-900, Cuiabá, Mato Grosso, Brazil d College of Veterinary Medicine and Animal Science, Universidade Federal de Mato Grosso do Sul (UFMS), Av. Senador Filinto Müller 2443, 79074-460, Campo Grande, Mato Grosso do Sul, Brazil e Ultrastructural Pathology Unit, Veterinary Diagnostic Laboratory, University of Minnesota, 1333 Gortner Ave, MN 55108, St. Paul, MN, USA f Department of Pathology, Veterinary Faculty, Federal University of Pelotas, Campus Capão do Leão, 96010-900, Pelotas, Rio Grande do Sul, Brazil g Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA La Estanzuela, Ruta 50 Km 11, Colonia del Sacramento, Colonia, Uruguay
A R T I C LE I N FO
A B S T R A C T
Keywords: Snakebite Snake Bothrops Coagulopathy Acute kidney injury Endothelial injury Horses
Snake bite envenomations in farm animals are generally overestimated as the cause of mortality in rural areas in Latin America. However, most cases are based only on anecdotal information and assumptions, and lack diagnostic evidence. There are few proven reports of envenomation and death in horses caused by snakebites from members of the Bothrops genus (lancehead pit vipers). This study presents epidemiological and clinical-pathological findings of fatal bothropic envenomation in horses from Central Western Brazil in order to contribute to the correct diagnosis of this condition. A survey of the records of equine necropsies from the Veterinary Pathology Laboratories of the University of Brasilia, Federal University of Mato Grosso and Federal University of Mato Grosso do Sul, from January 2010 to February 2018, was performed. Five fatal cases of bothropic snakebite were identified in 755 necropsies of horses, corresponding to 0.66% of these cases, ranging annually from 0.33% to 0.89%. The main necropsy findings were marked swelling and diffuse subcutaneous hemorrhage, and identification of the fang marks in 2 out of five horses. Hemorrhage in most organs and tissues was the pathological hallmark of systemic envenomation. Myonecrosis, dermonecrosis, and moderate to severe kidney degeneration and necrosis were also observed. Fatal Bothrops snakebites in horses have a low occurrence in Central Western Brazil and most cases occur in the rainy season. The diagnosis of this condition may be substantiated by clinical signs and pathological findings. Local hemorrhage and necrosis, systemic hemorrhagic disorders and injuries related to acute kidney injury are the predominant clinical signs. These findings should be considered in the diagnosis and therapeutic management of these envenomations.
1. Introduction Snakebite envenomation is an important cause of morbidity and mortality in humans and animals worldwide (Cheng and Currie, 2004; Gutiérrez et al., 2009, 2017; Fielding et al., 2011; Machado et al., 2019). Bothrops, Crolatus, Lachesis (Viperidae family) and Micrurus
∗
genus (Elapidae family) are the most dangerous venomous snakes in Latin America (Franco et al., 2001; Malaque and Gutiérrez, 2015). Bothrops account for more than 80% of venomous snakebites in Brazil, and Crotalus sp. causes about 10% of envenomings of humans (Franco et al., 2001). Lancehead pit vipers (Bothrops spp.) also account for most of the venomous snakebites in domestic animals, and a minority of
Corresponding author. E-mail address: [email protected] (M. Botelho de Castro).
https://doi.org/10.1016/j.toxicon.2019.09.002 Received 9 May 2019; Received in revised form 16 July 2019; Accepted 1 September 2019 Available online 06 September 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved.
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incidents occur with rattlesnakes (Crolatus spp.). Lachesis and Micrurus snakebites have never been reported in domestic animals in Brazil (Ferreira Júnior and Barravieira, 2004; Tokarnia and Peixoto, 2006; Chiacchio et al., 2011). In Brazil, snakebite envenomation in farm animals is generally overestimated as the cause of sudden death and mortality in rural areas. However, most cases are based only on anecdotal evidence and assumptions, and there is lack of robust diagnostic evidence (Tokarnia and Peixoto, 2006). In Brazil, there have been reports of fatal snakebites affecting cattle (Grunert, 1967; Grunert and Grunert, 1969) and sheep (Mendez and Riet-Correa, 1995; Tokarnia et al., 2008; Leal et al., 2013). There have also been a few reports of snake envenomation by the genus Bothrops in horses (Raposo et al., 2001; Chiacchio et al., 2011; Silva et al., 2011). Experimental snake envenomations have been carried out in horses (Sousa et al., 2011; Lopes et al., 2012), cattle (Caldas et al., 2008; Graça et al., 2008), sheep (Aragão et al., 2010) and buffaloes (Barbosa et al., 2011) to characterize the relevant clinical-pathological alterations and facilitate the diagnosis in these species. Data on natural cases of Bothrops snakebite envenomation in horses are scarce. Thus, the present study aimed to establish epidemiological, clinico-pathological and ultrastructural findings of fatal Bothrops envenomation in five horses in Central Western Brazil.
Fig. 1. Survey regions of fatal cases of Bothrops envenomation in horses in Brazil.
software (Advanced Microscopy Techniques Corp. Woburn, MA, USA). Image analysis was carried out using ImageJ (NIHR public domain).
2. Materials and methods
3. Results
A survey in the records of equine necropsies from the Veterinary Pathology Laboratories (VPL) of the University of Brasilia (UnB), Federal University of Mato Grosso (UFMT) and Federal University of Mato Grosso do Sul (UFMS) from January 2010 to February 2018 was performed. Bothrops envenomation cases were selected based on the clinical history of snakebite in addition with local signs (hemorrhage, swelling and dermatonecrosis at snakebite site), systemic changes (coagulopathy, circulatory shock and acute kidney injury) and laboratory abnormalities (thrombocytopenia, high CK activities and high serum levels of urea and creatinine) (Malaque and Gutiérrez, 2015). All horses with diagnosis of Bothrops envenomation were retrieved for epidemiological, clinical, laboratory, and pathological data analysis. Four horses (4/5) were submitted to clinical evaluation and all animals were necropsied. The morphological alterations and the location and severity of the hemorrhages in the muscular and subcutaneous tissues, organs and serous cavities were evaluated during necropsies. Fragments of organs and tissues were collected, fixed in 10% buffered formalin (pH 7.0), embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) for histologic evaluation. Additional kidney sections were stained with chromotropic aniline blue to detect hyaline droplets in tubular epithelial cells (De Rijk et al., 2003). Microscopic changes in the kidneys were evaluated, identifying their locations, distribution and intensity. The map with the distribution of the cases and location of the VPL (Fig. 1) was carried out with the Quantum Gis 2.14® software. Electron microscopy (EM) was performed on Horse 5. Kidney was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer followed by a post-fixation in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA, USA). The sample was dehydrated using a 25%–100% ethyl alcohol gradient. It was then infiltrated and embedded with Embed 812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Embedded tissue was sectioned on a Leica UC6 ultramicrotome (Leica Microsystems, Vienna, Austria). One micron sections were stained with Toluidine blue and examine in a light microscope to select the area of interest. Thin sections (60–70 nm) were obtained and collected onto a 200-mesh nickel grid (Electron Microscopy Sciences, Hatfield, PA, USA). Thin section on the grids were contrasted with 5% uranyl acetate and Santos’ lead citrate. These preparations were visualized using a JEOL 1400 transmission electron microscope (JEOL LTD, Tokyo, Japan). Images were obtained using an AMT Capture Engine Version 7.00 camera and
The occurrence of Bothrops snakebite envenomation in necropsy cases was 0.89% (3/337 cases), 0.33% (1/303 cases), and 0.87% (1/ 115 cases) in the VPL of the UnB, UFMS and UFMT respectively, achieving an occurrence of 0.66% (5/755 necropsies) during the 8-year period of this study. Characteristics on fatal Bothrops envenomation in five horses are presented in Table 1. Horses 2, 3, 4, and 5 were clinically evaluated and presented oral and ocular mucous membrane congestion, apathy, tachypnea (30–60 breaths per minute; normal range: 8–16 breaths per minute), tachycardia (50–110 beats per minute; normal range: 28–40 beats per minute), and continuous hemoptysis and epistaxis. All venipunctures and muscular injection sites bled continuously. Three animals (Horses 2, 4 and 5) presented swelling of head, nose and/or lips, causing respiratory distress; whilst Horses 1 and 3 presented marked swelling on the right front limb. Horses 2 and 3 also presented brownish urine (hemoglobinuria/myoglobinuria) and Horse 5 showed severe anuria and jaundice. All horses were clinically treated with intravenous fluid and corticoids but progressed to death in 12–48 h. Laboratory data are summarized in Table 2. The most relevant alterations were: hypoproteinemia (Horses 2 and 4), thrombocytopenia (Horses 2, 3 and 5), elevated creatine kinase (CK) activity (Horses 2 and 4), and elevated serum urea (Horses 2, 4 and 5) and creatinine levels (Horses 2, 3, 4 and 5). Horses 4 and 5 presented severe hemolysis making it impossible to determine the hematological parameters. Horses 2 and 3 presented leukocytosis, and the former also showed normocytic normochromic anemia. The main necropsy findings in all horses were marked swelling and diffuse hemorrhage in the subcutaneous tissues surrounding snakebite sites on the front limbs (Fig. 2A and B) and head (Fig. 3 and Fig. 4). Fang marks (Fig. 3B) were detected in two horses (40%), characterized by parallel circular perforations in the skin, spaced 1.8–2.0 cm apart (Horses 4 and 5). Petechial hemorrhages, ecchymosis and suffusions were detected in subcutaneous and muscular tissues and in thoracic and abdominal cavities and organs (Table 3). Horse 5 also presented severe acute kidney injury characterized by randomly scattered hemorrhages and pale streaks radiating through the renal cortex (Fig. 5). Microscopically, most organs presented mild to moderate hemorrhage. Degeneration and hemorrhagic dissecting myonecrosis (Fig. 6A) characterized by multifocal hyalinization and loss of cytoplasmic 42
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Table 1 Survey data and clinical evolution of fatal cases of bothropic envenomation in horses. Horse
Year
Month
Location
Breed
Sex
Age
Weight (kg)
CC
SS
1 2 3 4 5
2010 2013 2017 2017 2018
October May February November February
Sinop/MT Brasília/DF Sidrolândia/MS Planaltina/DF Brasília/DF
Crioula FBGH QH MB BSH
F F F M F
5y 2½ y 1½ y 8y 4y
274 368 300 320 500
36 h 12 h 24 h 24 h 48 h
RF HN RF HN HN
Breed: MB - Mixed breed; QH - Quarter horse, FBGH - Four beat gait horse; BSH - Brazilian saddle horse; Age: y - years; CC - clinical course since the observation of swelling at snakebite site and clinical signs of envenomation (h - hours); Snakebite site (SS): RF - right forelimb; HN - head/nasal region.
main histologic lesions detected in the kidneys (Table 4) were moderate to severe renal degeneration and tubular necrosis, in addition to hyaline and granular intratubular casts (Fig. 7A), multifocal hemorrhage in urinary spaces (Fig. 7B), thrombosis of glomerular capillaries (Fig. 8A) and intracytoplasmic hyaline droplets of various sizes in the distal convoluted tubule epithelial cells (Fig. 8B). The transmission electron microscopy (TEM) of kidney samples showed prominent and diffuse massive necrosis of the convoluted tubules, hydropic vacuolar degeneration of epithelial tubular cells, lysis of organelles and nuclear chromatin, interstitial edema, hemorrhage and extravasation of fibrin (Fig. 9A). Some convoluted tubular epithelial cells presented numerous swollen mitochondria with disorganization and rupture of cristae and electrondense round bodies (Fig. 9B). In some glomerular capillaries, necrotic endothelial cells (Fig. 10A) were seen frequently associated with fibrinous thrombi (Fig. 10B).
Table 2 Hematological and biochemical findings of fatal bothropic envenomation in four horses at admission. Parameter
Horse 2
Horse 3
Horse 4a
Horse 5a
Reference values
Hematocrit (%) Erythrocytes (106/μL) Hemoglobin (g/ dL) Plasma protein (g/dL) Albumin (g/dL) Globulins (g/dL) Platelets (μL) Leucocytes (μL) CKb (IU/L) Creatinine (mg/ dL) Urea (mg/dL)
19 4.2
31 8.3
ND ND
ND ND
24–44 5.5–9.5
6.3
10.5
ND
ND
8–14
4.1
ND
4.3
ND
5.2–7.9
ND ND 33,000 13,400 3543 2.8
ND ND 114,000 16,900 ND 3.9
1.6 2.7 ND ND 2689 3.5
ND ND 31,000 ND ND 9.2
2.6–3.7 2.6–4.0 200,000–500,000 6000–12,000 119–287 1.2–1.9
123
56
96
192
21.4–51.3
c
4. Discussion
a
Not determined due to intense hemoglobinemia/myoglonemia; ND - Not determined. b Creatine phosphokinase activity. c (Kaneko et al., 2008; Grondin and Dewitt, 2010).
Information on pathological changes associated with Bothrops sp. snakebite envenomation in horses is scarce in the scientific literature. In Brazil, Bothrops snakebite envenomation in farm animals has been reported in the Southern (Mendez and Riet-Correa, 1995; Raposo et al., 2001; Leal et al., 2013), Southeast (Tokarnia et al., 2008; Chiacchio et al., 2011), and Northern regions (Silva et al., 2011). In the current study, five cases of fatal Bothrops snakebites in horses were detected in the areas covered by the VPL of the UnB, UFMT, and UFMS, in Central Western Brazil. Although the precise identification of the snakes responsible for the bites was not done, the clinical and pathological manifestations strongly suggests that they were induced by Bothrops
striations of the myofibers with fibrinoid necrosis of the blood vessels and severe hemorrhagic dermonecrosis (Fig. 6B) were also observed in horses 2, 3, and 5, in the anatomical sites surrounding the area of the bite. The lymph nodes draining the affected regions had diffuse hemorrhage in the subcapsular space and in the lymphatic sinuses. The lungs presented congestion and marked multifocal hemorrhage. The
Fig. 2. Horse 3, right front limb. (A) Snake bite site with marked swelling. (B) Severe hemorrhage in subcutaneous and adjacent musculature. 43
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Fig. 3. Horse 4. (A) Head. Pronounced swelling at the bite site. (B) Lip. Fang marks (arrows) characterized by parallel perforations spaced approximately 1.8 cm apart.
spp. Our findings suggest that fatal snakebite envenomation in horses is unusual in Central Western of Brazil. However, the total number of snakebites in horses might be much higher if we consider the possibilities of envenomated horses that recovered without clinical care and underreporting of fatal cases. Large animals should be less likely to die from snakebites due to their weight and the lower venom dose in terms of venom per animal weight (Tokarnia and Peixoto, 2006). Generally, snakes use venom to immobilize prey in order to obtain food, and are able to modulate and reduce the amounts of injected venom in a defensive bite involving large animals (Melgarejo, 2003). Nevertheless, this study features fatal Bothrops snakebites in adult horses weighing up to 500 kg. Differences in the susceptibility of the animals, toxicity variations influenced by environmental conditions and snake feeding, and amount of the injected venom possibly influenced the number of horses that died in the
region throughout the evaluated period. The majority of these fatal envenomations occurred during the rainy season (4/5), with the exception of Horse 2 (dry season). In Latin America, snakebite envenomation is usually seasonal (Radostits et al., 2007; Sazima, 1998) with a higher incidence during warmer and rainy seasons. Bothrops species exhibit lower activity during drought periods and higher activity in the months of higher humidity, heat, and food supply, when the incidence of snakebites is predictably higher (Sazima, 1998; Cruz et al., 2009). In our study, envenomated horses presented extensive hemorrhages near the bite sites, which extended through the adjacent body regions and tissues (neck and all limbs). All horses also presented mild hemorrhages in subcutaneous and muscular tissues not associated with the snakebite sites and bleeding in most internal organs and serous cavities. A hallmark of lancehead pit viper snakebite is the marked local
Fig. 4. Horse 4. Severe hemorrhage at the head and neck. 44
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++ + ++ ++ ++
+++ ++ + ++ +++
++ ++ ++ ++ +++
+ ++ ++ + +++
++ ++ + ++ ++
+ ++ ++ + +++
++ ++ ++ +++ +++
swelling due to intense edema and tissue hemorrhage, which usually occurs near the head, muzzle and limbs where bites occur (Raposo et al., 2001; Chiacchio et al., 2011; Silva et al., 2011). Horses experimentally injected with venoms of B. jararaca, B. jararacussu, B. moojeni and B. neuwiedi also showed marked local swelling (Sousa et al., 2011). These findings are very similar to those observed in the envenomated horses in our study. Bothrops envenomation results in local (hemorrhage, myonecrosis and dermonecrosis) and systemic manifestations (coaguloapathy, systemic hemorrhage, circulatory shock and acute kidney injury) mainly due to the action of metalloproteinases, phospholipases A2 and serine proteinases, the most abundant components in these venoms (Gutiérrez et al., 2017). Phospholipase A2 is also responsible for the venom's powerful myotoxic action, affecting the sarcolemma integrity of skeletal muscle fibers and causing acute myonecrosis (Gutiérrez et al., 2009). These mechanisms explain the extensive myonecrotic areas observed in all horses, and the high CK activities recorded in two cases (Horses 2 and 4). In humans, envenomation by most viperid snakes causes a marked reaction at the snakebite site characterized by hemorrhage, edema, necrosis and blistering, with the severity, extent of lesions and sequelae in the affected limb related to the time of evolution and to the amount of venom injected (Gutiérrez et al., 2006). The blistering at the snakebite site occurs by the loss of the integrity of the dermal-epidermal interface caused by the action of metalloproteinases (Gutiérrez et al., 2017). We did not observe in our study the characteristic blistering or similar histological changes in horses. It is possible that the intensity of skin lesions in horses were less severe than in humans due to the acute fatal clinical course (12–48 h), not progressing to more marked local injuries. The evaluation of new cases of natural Bothrops envenoming in horses with a more prolonged clinical course may allow future comparisons with these lesions in humans. Identification of parallel perforations spaced 1.8–2.0 cm apart (fang marks) confirmed the snakebite site. Fang marks were previously described in sheep with Bothrops envenomation (Leal et al., 2013), and also aided the diagnosis in two horses (Horse 4 and 5). Snakebite fang marks may not be visualized or occur sporadically, as reported in only 25% of the affected animals in a sheep flock (Tokarnia et al., 2008). Bothrops snakebite envenomation were reported in two horses in Brazil, and these marks were observed in only one animal (Raposo et al., 2001; Silva et al., 2011). Although three of the horses evaluated (Horses 1, 2 and 3) did not present fang marks, background information and local and systemic clinical manifestations allowed the diagnosis of Bothrops envenomation. Acute kidney injury (AKI) stands out among the systemic changes in humans and animals with Bothrops envenomation. AKI seems to be associated with the direct action of the Bothrops venom components on renal tubular epithelial cells (Nascimento et al., 2007) and can also occur from the tubulotoxic effects of hemoglobin and/or myoglobin, or simply from impaired perfusion from hypotension (Gutiérrez et al., 2017). Experimental envenomation of mice by Bothrops alternatus revealed deposition of the venom in the kidney glomeruli and tubules, which was associated with renal tubular cell degeneration, desquamation, and necrosis (Gay et al., 2009; Mello et al., 2010). Renal injury may be aggravated indirectly by the release of endogenous mediators such as cytokines, inflammatory peptides, nitric oxide, and arachidonic acid metabolites (Barbosa et al., 2006). The nephrotoxic action of Bothrops venoms may explain the increased serum levels of urea and creatinine observed in our horses. The renal histological changes observed in horses ranged from moderate to severe and are characteristic of systemic envenomings by viperid snakes, such as lancehead pit viper, that have a higher concentration of toxins in the venom that cause cytotoxicity, hemorrhage, coagulopathy, and myonecrosis (Gutiérrez et al., 2017). The ultrastructural injuries observed in the tubular epithelial cells of the kidney and their organelles in our study are similar to those reported in rats
R: right; L: left; F: front limb; H: hind limb; **: snakebite site; Intensity: + mild, ++ moderate, +++ severe, - no lesion.
+ ++ + ++ +++ – ++ – – +++ +++ – – – – – +++** + +++ +++ – – + +++** ++** – – + – + – – + – + – – + + + – +++ + +++ +++ 1 2 3 4 5
+++** + +++** + +
+ + + + +
+ + + + +
+ + + + +
– +++** + +++** ++**
+++** – +++** + +
Intestines/mesentery Spleen Diaphragm Aorta Heart Lungs Abdominal Neck RF
LF
RH
LH
Head
RF
LF
RH
LH
Head
Neck
Thoracic
Hemorrhage (petechiae, ecchymosis, suffusion) in organs Serohemorrhagic content in cavities Muscular tissues Subcutaneous tissues Case
Table 3 Location and intensity of hemorrhages in the subcutaneous, muscular tissues, serous cavities and internal organs of horses with fatal bothropic envenomation.
Kidney
Perirenal fat
M. Machado, et al.
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Fig. 5. Horse 5, kidney. Whitish flecked foci of necrosis and striations that extend slightly to the medullar region. (*) Detail view.
distension, necrosis, and rupture, with the consequent hemorrhage (Gutiérrez et al., 2005). The PIII class of metalloproteinases are the most potent hemorrhagic toxins in these venoms (Escalante et al., 2011; Herrera et al., 2015). Diffuse hemorrhage in organs and tissues was the main pathological finding in the envenomated horses in Midwestern Brazil. Additionally, the three horses that were clinically evaluated showed coagulation disorders (Horse 2, 4 and 5), confirmed by severe thrombocytopenia in two cases (Horse 2 and 5). Endothelial injury detected may also aggravate the coagulopathy and disseminated hemorrhages in these horses. The action of snake venom enzymes, mostly thrombin-like serine proteinases and procoagulant metalloproteinases, on the coagulation cascade is responsible for the consumption coagulopathy characteristic
experimentally envenomed by a species of lancehead pit viper (BoerLima et al., 2002). Thrombosis and necrosis of glomerular capillary endothelial cells observed in horses also occurred in rats experimentally envenomated by Bothrops asper (Moreira et al., 1994). Vascular thrombosis is associated with the hemostatic response to the primary vascular damage commonly observed in these envenomations (Moreira et al., 1994). The histological and ultrastructural findings observed herein reinforce the importance of the nephrotoxic action of the lancehead pit viper venom and its probable involvement in AKI. Metalloproteinases are the main venon components of snakes of the family Viperidae involved in the local and systemic hemorrhages due to their direct hydrolytic action on the capillary basement membrane, causing loss of mechanical and hemodynamic stability, capillary
Fig. 6. Horse 4, snakebite site. (A) Skeletal muscle. Hemorrhagic dissecting myonecrosis surrounding the muscle bundles (H&E, scale bar = 250 μm). (B) Skin. Severe focally extensive hemorrhagic dermonecrosis (H&E, scale bar = 250 μm). 46
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Table 4 Location and intensity of histopathological changes in the kidney of horses with fatal bothropic envenomation. Case
1 2 3 4 5
Glomerular capillaries
Urinary space
Tubules
Interstitium
Thrombosis
Hemorrhage
Necrosis
HD
HDP
HC
GC
Hemorrhage
+ ++ + + +++
+ ++ + ++ +++
++ ++ + + +++
++ +++ ++ ++ +++
+ ++ +++ +++ +++
++ +++ ++ ++ +++
++ ++ + ++ ++
++ + + + +++
HD: hydropic degeneration; HDP: hyaline droplets; HC: hyaline casts; GC: granular casts; Intensity: + mild, ++ moderate, +++ severe.
Fig. 7. Horse 5, kidney. (A) Medulla. Tubular necrosis (*) and intratubular casts (arrow heads) (H&E, scale bar = 50 μm). (B) Cortex. Severe glomerular hemorrhage and epithelial tubular cells necrosis (*) (H&E, scale bar = 100 μm).
Fig. 8. Horse 5, kidney. (A) Cortex. Fibrin thrombi (arrows) in the glomerular capillaries (H&E, scale bar = 25 μm). (B) Medulla. Intracytoplasmic hyaline droplets (*) in distal convoluted tubule epithelial cells (Chromotropic aniline blue, scale bar = 25 μm).
47
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Fig. 9. Horse 5, kidney, cortex. (A) Necrotic tubular cell epithelium (asterisk) with margination of the nuclear chromatin, and distended cytoplasm with lysis of organelles (arrow). (TEM, scale bar = 10 μm). (B) Portion of a tubular epithelial cell with numerous swollen mitochondriae with cristae rupture and large amount of electrodense granules and mineral deposition. (TEM, scale bar = 1 μm).
1999; Melgarejo, 2003). Lachesis venom has marked neurotoxic activities (Malaque and Gutiérrez, 2015) which was not observed in horses. Lachesis genus is distributed in Atlantic and Amazon Rainforests (Fernandes et al., 2004) and does not occur in the savanna areas of our study. The spatial distribution and abundance of B. moojeni and B. neuwiedi in vast areas of savanna in Brazil suggests their involvement in the investigated snakebites in horses (Tokarnia and Peixoto, 2006). However, since none of the snakes were seen or captured, the exact species responsible for our cases of envenoming remain unknown. Identification of the venomous snake species causing the bite is only possible in the minority of cases (Tokarnia and Peixoto, 2006; Malaque and Gutiérrez, 2015), and generally can not be used to make therapeutic decisions. Therefore, the diagnosis has to be based on the
of these envenomations, with alteration of the laboratory coagulation tests (Varanda and Giannini, 1999; White, 2005; Sousa et al., 2013), as observed in animals experimentally envenomed by lancehead pit viper venoms (Sousa et al., 2013). Such alterations in hemostasia synergize with the disruptive action of hemorrhagic metalloproteinases in the vasculature, resulting in extensive local and systemic hemorrhage, a characteristic finding in our study. The ability of lancehead pit vipers to induce lethality is likely the result from cardiovascular alterations associated with systemic bleeding and hypovolemia, and acute kidney injury. The most common species of venomous snakes in Brazil are Bothrops alternatus, B. atrox, B. jararacussu, B. jararaca, B. moojeni, B. neuwiedi, Crotalus durissus and Lachesis muta rhombeata (Barraviera and Pereira,
Fig. 10. Horse 5, kidney. (A) Disruption of the endothelial cells and capillary thrombosis (asterisk) with covers exposing the basement membrane, interstitial hemorrhage (star), and tubular cells with hydropic degeneration (cross). (TEM, scale bar = 6 μm). (B) Glomerular capillary with fibrinous thrombus (asterisk) and blunting of the podocytes processes and cytoplasm clearance with organelles lysis (TEM, scale bar = 1 μm).
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distinct clinical-pathological alterations caused by the main venomous snake genera, and their spatial distribution and epidemiology in Brazil (Sazima, 1998; Barraviera and Pereira, 1999). According to these criteria, the diagnosis of fatal Bothrops envenomation was supported in all five horses. Differential diagnoses of Bothrops envenomation include traumatic lesions, clostridial diseases and other hemorrhagic disorders. In all horses investigated, there was no evidence of these diseases, which were excluded due to epidemiology, background information and clinical signs, identification of bite wounds (Horses 4 and 5), absence of gas production in the limb at the site of swelling (Horses 1, 2 and 3) and pathological findings. In conclusion, five fatal cases of horses suffering Bothrops spp. snakebite envenomation were described. Clinical and pathological findings closely correspond to the characteristic features of envenomations by Bothrops spp. in experimentally envenomed horses, other animal species and humans. Our findings highlight the potential severity of such snakebites in horses, and provide evidence for the diagnosis and therapeutic management of these cases.
Boer-Lima, P.A., Gontijo, J.A., Cruz-Höfling, M.A., 2002. Bothrops moojeni snake venominduced renal glomeruli changes in rat. Am. J. Trop. Med. Hyg. 67 (2), 217–222. Caldas, S.A., Tokarnia, C.H., França, T.N., Brito, M.F., Graça, F.A., Coelho, C.D., Peixoto, P.V., 2008. Clinic and pathological and laboratory aspects of experimental poisoning by Bothrops alternatus venom in cattle. Pesqui. Vet. Bras. 28, 303–312. https://doi. org/10.1590/S0100-736X2008000600008. Cheng, A.C., Currie, B.J., 2004. Venomous snakebites worldwide with a focus on the Australia-Pacific region: current management and controversies. J. Intensive Care Med. 19, 59–69. https://doi.org/10.1177/0885066604265799. Chiacchio, S.B., Martins, G.T.B., Amorim, R.M., Gonçalves, R.C., Barraviera, B., Ferreira Junior, R.S., 2011. Triple bothropic envenomation in horses caused by a single snake. J. Venom. Anim. Toxins Incl. Trop. Dis. 17, 111–117. https://doi.org/10.1590/ S1678-91992011000100016. Cruz, L.S., Vargas, R., Lopes, A.A., 2009. Snakebite envenomation and death in the developing world. Ethn. Dis. 19, 42–46. De Rijk, E.P.C.T., Ravesloot, W.T.M., Wijnands, Y., Esch, E.V., 2003. A fast histochemical staining method to identify hyaline droplets in the rat kidney. Toxicol. Pathol. 31, 462–464. https://doi.org/10.1080/01926230390213775. Escalante, T., Rucavado, A., Fox, J.W., Gutiérrez, J.M., 2011. Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases. J. Proteomics. 74 (9), 1781–1794. https://doi.org/10.1016/j.jprot.2011.03.026. Fernandes, D.S., Franco, F.L., Fernandes, R., 2004. Systematic revision of the genus Lachesis daudin, 1803 (Serpentes, Viperidae). Herpetologica 60 (2), 245–260. Ferreira Júnior, R.S., Barravieira, B., 2004. Management of venomous snakebites in dogs and cats in Brazil. J. Venom. Anim. Toxins Incl. Trop. Dis. 10, 112–132. https://doi. org/10.1590/S1678-91992004000200002. Fielding, C., Pusterla, N., Magdesian, G., Higgins, J.C., Meier, C., 2011. Rattlesnake envenomation in horses: 58 cases (1992-2009). J. Am. Vet. Med. Assoc. 238, 631–635. https://doi.org/10.2460/javma.238.5.631. Franco, R.L., Rocha, C.C., Jorge, M.T., Ribeiro, L.A., 2001. Snakebites in southern minas gerais state, Brazil. J. Venom. Anim. Toxins Incl. Trop. Dis. 7, 56–68. https://doi.org/ 10.1590/S0104-79302001000100005. Gay, C.C., Maruñak, S.L., Teibler, P., Ruiz, R., Pérez, A.O., Leiva, L.C., 2009. Systemic alterations induced by a Bothrops alternatus hemorrhagic metalloproteinase (baltergin) in mice. Toxicon 53, 53–59. https://doi.org/10.1016/j.toxicon.2008.10.010. Graça, F.A.S., Peixoto, P.V., Coelho, C.D., Caldas, A.S., Tokarnia, C.H., 2008. Clinical and pathological aspects of experimental Crotalus poisoning in cattle. Pesqui. Vet. Bras. 28, 261–270. https://doi.org/10.1590/S0100-736X2008000600001. Grondin, T.E., Dewitt, S.F., 2010. Normal hematology of the horse and donkey. In: Weiss, D.J., Wardrop, K.J. (Eds.), Schalm's Veterinary Hematology. 6. Wiley-Blackwell, Iowa, pp. 821–828. Grunert, E., 1967. Beobachtungen über Schlangenbissverletzungen bei grossen Haustieren in Süd-Brasilien. Dtsch. Tierärztl. Wochenschr. 20, 531–532. Grunert, E., Grunert, D., 1969. Beobachtungen von Bothrops-Schlangenbissverletzungen bei Rind und Pferd in Rio Grande do Sul, Brasilien. Vet.-Med. Nachrichten 3, 217–232. Gutiérrez, J.M., Calvete, J.J., Habib, A.G., Harrison, R.A., Williams, D.J., Warrell, D.A., 2017. Snakebite envenoming. Nat. Rev. Dis. Primers 3, 17063. https://doi.org/10. 1038/nrdp.2017.63. Gutiérrez, J.M., Rucavado, A., Chave, F., Díaz, C., Escalante, T., 2009. Experimental pathology of local tissue damage induced by Bothrops asper snake venom. Toxicon 54, 958–975. https://doi.org/10.1016/j.toxicon.2009.01.038. Gutiérrez, J.M., Rucavado, A., Escalante, T., Díaz, C., 2005. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 45, 97–1011. https://doi.org/10.1016/j.toxicon. 2005.02.029Get. Gutiérrez, J.M., Theakston, R.D.G., Warrell, D.A., 2006. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med. 3 (6), 1–5. https://doi.org/10.1371/journal.pmed.0030150. Herrera, C., Escalante, T., Voisin, M.B., Rucavado, A., Morazán, D., Macêdo, J.K.A., Fox, J.W., 2015. Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage. PLoS Neglected Trop. Dis. 9 (4), 0003731. https://doi.org/10.1371/ journal.pntd.0003731. Kaneko, J.J., Harvey, J.W., Bruss, M.L., 2008. Clinical Biochemistry of Domestic Animals, sixth ed. Academic Press, San Diego. Leal, M.L.R., Aires, A.M.C., Fillapi, A., Trost, M.E., 2013. Clinical and pathological observations associated with snake envenomation in two sheep. Acta Sci. Vet. 41, 1–5. Lopes, C.T.A., Tokarnia, C.H., Brito, M.F., Sousa, M.G., Oliveira, C.M.C., Silva, N.S., Lima, D.H.S., Barbosa, J.D., 2012. Clinical and pathological aspects of experimental crotalic envenoming in horses. Pesqui. Vet. Bras. 32, 843–849. https://doi.org/10.1590/ S0100-736X2012000900005. Machado, M., Wilson, T.M., Sousa, D.E.R., Martins, C.S., Castro, M.B., 2019. Pit viper envenomation in a barn cat. J. Small Anim. Pract. 60 (2), 131. https://doi.org/10. 1111/jsap.12922. Malaque, C.M.S., Gutiérrez, J.M., 2015. Snakebite envenomation in central and South America. Crit Care Toxicol. 1. https://doi.org/10.1007/978-3-319-20790-2_146-1. Melgarejo, A.F., 2003. Venomous snake from Brazil. In: Cardoso, J.L.C., França, F.O.S., Wen, F.H., Malaque, C.M.S., Haddad, J.R.V. (Eds.), Venomous Snake from Brazil: Biology, Clinical and Therapeutics of Snake Envenomation. Sarvier Editora, São Paulo, pp. 33–61 2003. Mello, S.M., Linardi, A., Renno, A.L., Tarsitano, C.A., Pereira, E.M., Hyslop, S., 2010. Renal kinetics of Bothrops alternatus (Urutu) snake venom in rats. Toxicon 55, 470–480. https://doi.org/10.1016/j.toxicon.2009.09.018. Mendez, M.C., Riet-Correa, F., 1995. Snakebite in sheep. Vet. Hum. Toxicol. 37, 62–63. Moreira, L., Borkow, G., Ovadia, M., Gutiérrez, J.M., 1994. Pathological changes induced
5. Conclusions Venomous snakebites in horses represent a diagnostic challenge for practitioners, since rarely the moment of snakebite is witnessed or the snake is identified, thus, the association of clinical, pathological and some epidemiological features (snake habitats and warmer and rainy seasons) is essential for the correct diagnosis. In this study, the diagnosis of fatal lancehead pit viper snakebites in five horses was established by the association of clinical, laboratory and pathological findings. Marked local swelling, disseminated hemorrhages, blood incoagulability, pigmenturia (myoglobinuria/hemoglobinuria) and acute kidney injury are the main changes observed in the acute Bothrops envenoming, and support practioners to suspect of lancehead pit viper snakebites in horses. Ethical statement Not applicable. All information and samples were obtained from records of the Veterinary Pathology Laboratories of horses that had natural death by venomous snakebite. Acknowledgements Special thanks to Dr. Claudio Severo Lombardo de Barros (Federal University of Mato Grosso do Sul) for the technical assistance in the pathological diagnosis and to Anderson Saravia (National Institute of Agricultural Research of Uruguay - Inia, La Estanzuela) for the aid in the making of the map. Thanks are due to Dr. José María Gutiérrez for the critical reading of the manuscript and suggestions. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. References Aragão, A.P., Tokarnia, C.H., Graça, F.A.S., França, T.N., Coelho, C.D., Caldas, A.S., Peixoto, P.V., 2010. Experimental poisoning by Bothropoides jararaca and Bothrops jararacussu in sheep: clinic-pathological and laboratory aspects. Pesqui. Vet. Bras. 30, 717–728. https://doi.org/10.1590/S0100-736X2010000900003. Barbosa, J.D., Sousa, M.G.S., Tokarnia, C.H., Brito, M.F., Reis, A.S.B., Bomjardim, H.A., Lopes, C.T.A., Oliveira, C.M.C., 2011. Comparison between the clinical and pathological picture in the experimental poisoning by crotalic venom. Pesqui. Vet. Bras. 31, 967–973. https://doi.org/10.1590/S0100-736X2011001100005. Barbosa, P.S.F., Martins, A.M.C., Alves, R.S., Amora, D.N., Martins, R.D., Toyama, M.H., Fonteles, M.C., 2006. The role of indomethacin and tezosentan on renal effects induced by Bothrops moojeni Lys 49 myotoxin I. Toxicon 47, 831–837. https://doi.org/ 10.1016/j.toxicon.2006.01.012. Barraviera, B., Pereira, P.C.M., 1999. Snake bites by Bothrops genus. In: Barraviera, B. (Ed.), Clinical and Therapeutic Aspects of Venomous Animals Accidents. EPUC, Rio de Janeiro, pp. 261–280.
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Sousa, L.F., Nicolau, C.A., Peixoto, P.S., Bernardoni, J.L., Oliveira, S.S., Portes-Junior, J.A., Mourão, R.H., Valente, R.H., Silva, A.M.M., 2013. Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of Bothrops complex. PLoS Neglected Trop. Dis. 7 (9), 1–17. https://doi.org/10.1371/journal. pntd.0002442. Sousa, M.G., Tokarnia, C.H., Brito, M.F., Reis, A.B., Oliveira, C.M., Freitas, N.F., Oliveira, C.H., Barbosa, J.D., 2011. Clinical and pathological aspects of the experimental poisoning by Bothrops snakes in horses. Pesqui. Vet. Bras. 31, 773–780. https://doi. org/10.1590/S0100-736X2011000900009. Tokarnia, C.H., Brito, M.F., Malafaia, P., Peixoto, P.V., 2008. Snake bite accident in sheep by Bothrops jararaca. Pesqui. Vet. Bras. 12, 643–648. https://doi.org/10.1590/ S0100-736X2008001200014. Tokarnia, C.H., Peixoto, P.V., 2006. The importance of snake bites as cause of cattle death in Brazil. Pesqui. Vet. Bras. 26, 55–68. Varanda, E.A., Giannini, M.J.S.M., 1999. Biochemistry of snake venoms. In: Barraviera, B. (Ed.), Clinical and Therapeutic Aspects of Venomous Animals Accidents. EPUC, Rio de Janeiro, pp. 205–223. White, J., 2005. Snake venoms and coagulopathy. Toxicon 45 (8), 951–967. https://doi. org/10.1016/j.toxicon.2005.02.030.
by BaH1, a hemorrhagic proteinase isolated from Bothrops asper (Terciopelo) snake venom, on mouse capillary blood vessels. Toxicon 32 (8), 976–987. Nascimento, J.M., Franchi, G.C., Nowill, A.E., Collares-Buzato, C.B., Hyslop, S., 2007. Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin–Darby canine kidney cells. Biochem. Cell Biol. 85, 591–605. https://doi.org/10.1139/O07-067. Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D., 2007. Disease associated with toxins, plants, fungi, cyanobacteria, plant-associated bacteria, and venoms in ticks and vertebrate animals. In: Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D. (Eds.), Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. Saunders Elsevier, Philadelphia, pp. 1851–1915. Raposo, J.B., Méndez, M.C., Bailardi, C.E.G., Raffi, M.B., 2001. A case of snakebite in a horse in southern Brazil - case report. Rev. FZVA 8, 51–57. Sazima, I., 1998. Um estudo de biologia comportamental da jararaca, Bothrops jararaca com uso de marcas naturais. Mem. Inst. Butantan (Sao Paulo) 3, 83–89. Silva, N.S., Silveira, J.A.S., Albernaz, T.T., Campos, K.F., Oliveira, C.M.C., Freitas, N.F.Q.R., Bomjardim, H.A., Barbosa, J.D., 2011. Fatal bothropic snakebite in a horse: a case report. J. Venom. Anim. Toxins Incl. Trop. Dis. 17, 496–500. https://doi.org/ 10.1590/S1678-91992011000400018.
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Toxicon journal homepage: www.elsevier.com/locate/toxicon
Fatal lancehead pit viper (Bothrops spp.) envenomation in horses a
T
a
Mizael Machado (DVM) , Tais Meziara Wilson (DVM Msc) , Davi Emanuel Ribeiro de Sousa (DVM)a, Antônio Carlos Lopes Câmara (DVM PhD)b, Fernando Henrique Furlan (DVM PhD)c, Juliana Targino Silva Almeida e Macêdo (DVM PhD)a, Rayane Chitolina Pupin (DVM PhD)d, Ricardo Antônio Amaral de Lemos (DVM, PhD)d, Anibal G. Armién (DVM PhD)e, Severo Sales Barros (DVM PhD)f, Franklin Riet-Correa (DVM PhD)g, Márcio Botelho de Castro (DVM PhD)a,∗ a Veterinary Pathology Laboratory, Veterinary Teaching Hospital, Via L4 Norte, sn/, Universidade de Brasília (UnB), Campus Universitário Darcy Ribeiro, 70636-200, Brasília, Distrito Federal, Brazil b Large Animal Veterinary Teaching Hospital, Universidade de Brasília (UnB), SIT PqEAT, Granja do Torto, 70297-400, Brasília, Distrito Federal, Brazil c Veterinary Pathology Laboratory, Veterinary Teaching Hospital Universidade Federal de Mato Grosso (UFMT), Av. Fernando Corrêa da Costa 2367, Boa Esperança, 78060-900, Cuiabá, Mato Grosso, Brazil d College of Veterinary Medicine and Animal Science, Universidade Federal de Mato Grosso do Sul (UFMS), Av. Senador Filinto Müller 2443, 79074-460, Campo Grande, Mato Grosso do Sul, Brazil e Ultrastructural Pathology Unit, Veterinary Diagnostic Laboratory, University of Minnesota, 1333 Gortner Ave, MN 55108, St. Paul, MN, USA f Department of Pathology, Veterinary Faculty, Federal University of Pelotas, Campus Capão do Leão, 96010-900, Pelotas, Rio Grande do Sul, Brazil g Instituto Nacional de Investigación Agropecuaria (INIA), Estación Experimental INIA La Estanzuela, Ruta 50 Km 11, Colonia del Sacramento, Colonia, Uruguay
A R T I C LE I N FO
A B S T R A C T
Keywords: Snakebite Snake Bothrops Coagulopathy Acute kidney injury Endothelial injury Horses
Snake bite envenomations in farm animals are generally overestimated as the cause of mortality in rural areas in Latin America. However, most cases are based only on anecdotal information and assumptions, and lack diagnostic evidence. There are few proven reports of envenomation and death in horses caused by snakebites from members of the Bothrops genus (lancehead pit vipers). This study presents epidemiological and clinical-pathological findings of fatal bothropic envenomation in horses from Central Western Brazil in order to contribute to the correct diagnosis of this condition. A survey of the records of equine necropsies from the Veterinary Pathology Laboratories of the University of Brasilia, Federal University of Mato Grosso and Federal University of Mato Grosso do Sul, from January 2010 to February 2018, was performed. Five fatal cases of bothropic snakebite were identified in 755 necropsies of horses, corresponding to 0.66% of these cases, ranging annually from 0.33% to 0.89%. The main necropsy findings were marked swelling and diffuse subcutaneous hemorrhage, and identification of the fang marks in 2 out of five horses. Hemorrhage in most organs and tissues was the pathological hallmark of systemic envenomation. Myonecrosis, dermonecrosis, and moderate to severe kidney degeneration and necrosis were also observed. Fatal Bothrops snakebites in horses have a low occurrence in Central Western Brazil and most cases occur in the rainy season. The diagnosis of this condition may be substantiated by clinical signs and pathological findings. Local hemorrhage and necrosis, systemic hemorrhagic disorders and injuries related to acute kidney injury are the predominant clinical signs. These findings should be considered in the diagnosis and therapeutic management of these envenomations.
1. Introduction Snakebite envenomation is an important cause of morbidity and mortality in humans and animals worldwide (Cheng and Currie, 2004; Gutiérrez et al., 2009, 2017; Fielding et al., 2011; Machado et al., 2019). Bothrops, Crolatus, Lachesis (Viperidae family) and Micrurus
∗
genus (Elapidae family) are the most dangerous venomous snakes in Latin America (Franco et al., 2001; Malaque and Gutiérrez, 2015). Bothrops account for more than 80% of venomous snakebites in Brazil, and Crotalus sp. causes about 10% of envenomings of humans (Franco et al., 2001). Lancehead pit vipers (Bothrops spp.) also account for most of the venomous snakebites in domestic animals, and a minority of
Corresponding author. E-mail address: [email protected] (M. Botelho de Castro).
https://doi.org/10.1016/j.toxicon.2019.09.002 Received 9 May 2019; Received in revised form 16 July 2019; Accepted 1 September 2019 Available online 06 September 2019 0041-0101/ © 2019 Elsevier Ltd. All rights reserved.
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incidents occur with rattlesnakes (Crolatus spp.). Lachesis and Micrurus snakebites have never been reported in domestic animals in Brazil (Ferreira Júnior and Barravieira, 2004; Tokarnia and Peixoto, 2006; Chiacchio et al., 2011). In Brazil, snakebite envenomation in farm animals is generally overestimated as the cause of sudden death and mortality in rural areas. However, most cases are based only on anecdotal evidence and assumptions, and there is lack of robust diagnostic evidence (Tokarnia and Peixoto, 2006). In Brazil, there have been reports of fatal snakebites affecting cattle (Grunert, 1967; Grunert and Grunert, 1969) and sheep (Mendez and Riet-Correa, 1995; Tokarnia et al., 2008; Leal et al., 2013). There have also been a few reports of snake envenomation by the genus Bothrops in horses (Raposo et al., 2001; Chiacchio et al., 2011; Silva et al., 2011). Experimental snake envenomations have been carried out in horses (Sousa et al., 2011; Lopes et al., 2012), cattle (Caldas et al., 2008; Graça et al., 2008), sheep (Aragão et al., 2010) and buffaloes (Barbosa et al., 2011) to characterize the relevant clinical-pathological alterations and facilitate the diagnosis in these species. Data on natural cases of Bothrops snakebite envenomation in horses are scarce. Thus, the present study aimed to establish epidemiological, clinico-pathological and ultrastructural findings of fatal Bothrops envenomation in five horses in Central Western Brazil.
Fig. 1. Survey regions of fatal cases of Bothrops envenomation in horses in Brazil.
software (Advanced Microscopy Techniques Corp. Woburn, MA, USA). Image analysis was carried out using ImageJ (NIHR public domain).
2. Materials and methods
3. Results
A survey in the records of equine necropsies from the Veterinary Pathology Laboratories (VPL) of the University of Brasilia (UnB), Federal University of Mato Grosso (UFMT) and Federal University of Mato Grosso do Sul (UFMS) from January 2010 to February 2018 was performed. Bothrops envenomation cases were selected based on the clinical history of snakebite in addition with local signs (hemorrhage, swelling and dermatonecrosis at snakebite site), systemic changes (coagulopathy, circulatory shock and acute kidney injury) and laboratory abnormalities (thrombocytopenia, high CK activities and high serum levels of urea and creatinine) (Malaque and Gutiérrez, 2015). All horses with diagnosis of Bothrops envenomation were retrieved for epidemiological, clinical, laboratory, and pathological data analysis. Four horses (4/5) were submitted to clinical evaluation and all animals were necropsied. The morphological alterations and the location and severity of the hemorrhages in the muscular and subcutaneous tissues, organs and serous cavities were evaluated during necropsies. Fragments of organs and tissues were collected, fixed in 10% buffered formalin (pH 7.0), embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E) for histologic evaluation. Additional kidney sections were stained with chromotropic aniline blue to detect hyaline droplets in tubular epithelial cells (De Rijk et al., 2003). Microscopic changes in the kidneys were evaluated, identifying their locations, distribution and intensity. The map with the distribution of the cases and location of the VPL (Fig. 1) was carried out with the Quantum Gis 2.14® software. Electron microscopy (EM) was performed on Horse 5. Kidney was fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer followed by a post-fixation in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (Electron Microscopy Sciences, Hatfield, PA, USA). The sample was dehydrated using a 25%–100% ethyl alcohol gradient. It was then infiltrated and embedded with Embed 812 resin (Electron Microscopy Sciences, Hatfield, PA, USA). Embedded tissue was sectioned on a Leica UC6 ultramicrotome (Leica Microsystems, Vienna, Austria). One micron sections were stained with Toluidine blue and examine in a light microscope to select the area of interest. Thin sections (60–70 nm) were obtained and collected onto a 200-mesh nickel grid (Electron Microscopy Sciences, Hatfield, PA, USA). Thin section on the grids were contrasted with 5% uranyl acetate and Santos’ lead citrate. These preparations were visualized using a JEOL 1400 transmission electron microscope (JEOL LTD, Tokyo, Japan). Images were obtained using an AMT Capture Engine Version 7.00 camera and
The occurrence of Bothrops snakebite envenomation in necropsy cases was 0.89% (3/337 cases), 0.33% (1/303 cases), and 0.87% (1/ 115 cases) in the VPL of the UnB, UFMS and UFMT respectively, achieving an occurrence of 0.66% (5/755 necropsies) during the 8-year period of this study. Characteristics on fatal Bothrops envenomation in five horses are presented in Table 1. Horses 2, 3, 4, and 5 were clinically evaluated and presented oral and ocular mucous membrane congestion, apathy, tachypnea (30–60 breaths per minute; normal range: 8–16 breaths per minute), tachycardia (50–110 beats per minute; normal range: 28–40 beats per minute), and continuous hemoptysis and epistaxis. All venipunctures and muscular injection sites bled continuously. Three animals (Horses 2, 4 and 5) presented swelling of head, nose and/or lips, causing respiratory distress; whilst Horses 1 and 3 presented marked swelling on the right front limb. Horses 2 and 3 also presented brownish urine (hemoglobinuria/myoglobinuria) and Horse 5 showed severe anuria and jaundice. All horses were clinically treated with intravenous fluid and corticoids but progressed to death in 12–48 h. Laboratory data are summarized in Table 2. The most relevant alterations were: hypoproteinemia (Horses 2 and 4), thrombocytopenia (Horses 2, 3 and 5), elevated creatine kinase (CK) activity (Horses 2 and 4), and elevated serum urea (Horses 2, 4 and 5) and creatinine levels (Horses 2, 3, 4 and 5). Horses 4 and 5 presented severe hemolysis making it impossible to determine the hematological parameters. Horses 2 and 3 presented leukocytosis, and the former also showed normocytic normochromic anemia. The main necropsy findings in all horses were marked swelling and diffuse hemorrhage in the subcutaneous tissues surrounding snakebite sites on the front limbs (Fig. 2A and B) and head (Fig. 3 and Fig. 4). Fang marks (Fig. 3B) were detected in two horses (40%), characterized by parallel circular perforations in the skin, spaced 1.8–2.0 cm apart (Horses 4 and 5). Petechial hemorrhages, ecchymosis and suffusions were detected in subcutaneous and muscular tissues and in thoracic and abdominal cavities and organs (Table 3). Horse 5 also presented severe acute kidney injury characterized by randomly scattered hemorrhages and pale streaks radiating through the renal cortex (Fig. 5). Microscopically, most organs presented mild to moderate hemorrhage. Degeneration and hemorrhagic dissecting myonecrosis (Fig. 6A) characterized by multifocal hyalinization and loss of cytoplasmic 42
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Table 1 Survey data and clinical evolution of fatal cases of bothropic envenomation in horses. Horse
Year
Month
Location
Breed
Sex
Age
Weight (kg)
CC
SS
1 2 3 4 5
2010 2013 2017 2017 2018
October May February November February
Sinop/MT Brasília/DF Sidrolândia/MS Planaltina/DF Brasília/DF
Crioula FBGH QH MB BSH
F F F M F
5y 2½ y 1½ y 8y 4y
274 368 300 320 500
36 h 12 h 24 h 24 h 48 h
RF HN RF HN HN
Breed: MB - Mixed breed; QH - Quarter horse, FBGH - Four beat gait horse; BSH - Brazilian saddle horse; Age: y - years; CC - clinical course since the observation of swelling at snakebite site and clinical signs of envenomation (h - hours); Snakebite site (SS): RF - right forelimb; HN - head/nasal region.
main histologic lesions detected in the kidneys (Table 4) were moderate to severe renal degeneration and tubular necrosis, in addition to hyaline and granular intratubular casts (Fig. 7A), multifocal hemorrhage in urinary spaces (Fig. 7B), thrombosis of glomerular capillaries (Fig. 8A) and intracytoplasmic hyaline droplets of various sizes in the distal convoluted tubule epithelial cells (Fig. 8B). The transmission electron microscopy (TEM) of kidney samples showed prominent and diffuse massive necrosis of the convoluted tubules, hydropic vacuolar degeneration of epithelial tubular cells, lysis of organelles and nuclear chromatin, interstitial edema, hemorrhage and extravasation of fibrin (Fig. 9A). Some convoluted tubular epithelial cells presented numerous swollen mitochondria with disorganization and rupture of cristae and electrondense round bodies (Fig. 9B). In some glomerular capillaries, necrotic endothelial cells (Fig. 10A) were seen frequently associated with fibrinous thrombi (Fig. 10B).
Table 2 Hematological and biochemical findings of fatal bothropic envenomation in four horses at admission. Parameter
Horse 2
Horse 3
Horse 4a
Horse 5a
Reference values
Hematocrit (%) Erythrocytes (106/μL) Hemoglobin (g/ dL) Plasma protein (g/dL) Albumin (g/dL) Globulins (g/dL) Platelets (μL) Leucocytes (μL) CKb (IU/L) Creatinine (mg/ dL) Urea (mg/dL)
19 4.2
31 8.3
ND ND
ND ND
24–44 5.5–9.5
6.3
10.5
ND
ND
8–14
4.1
ND
4.3
ND
5.2–7.9
ND ND 33,000 13,400 3543 2.8
ND ND 114,000 16,900 ND 3.9
1.6 2.7 ND ND 2689 3.5
ND ND 31,000 ND ND 9.2
2.6–3.7 2.6–4.0 200,000–500,000 6000–12,000 119–287 1.2–1.9
123
56
96
192
21.4–51.3
c
4. Discussion
a
Not determined due to intense hemoglobinemia/myoglonemia; ND - Not determined. b Creatine phosphokinase activity. c (Kaneko et al., 2008; Grondin and Dewitt, 2010).
Information on pathological changes associated with Bothrops sp. snakebite envenomation in horses is scarce in the scientific literature. In Brazil, Bothrops snakebite envenomation in farm animals has been reported in the Southern (Mendez and Riet-Correa, 1995; Raposo et al., 2001; Leal et al., 2013), Southeast (Tokarnia et al., 2008; Chiacchio et al., 2011), and Northern regions (Silva et al., 2011). In the current study, five cases of fatal Bothrops snakebites in horses were detected in the areas covered by the VPL of the UnB, UFMT, and UFMS, in Central Western Brazil. Although the precise identification of the snakes responsible for the bites was not done, the clinical and pathological manifestations strongly suggests that they were induced by Bothrops
striations of the myofibers with fibrinoid necrosis of the blood vessels and severe hemorrhagic dermonecrosis (Fig. 6B) were also observed in horses 2, 3, and 5, in the anatomical sites surrounding the area of the bite. The lymph nodes draining the affected regions had diffuse hemorrhage in the subcapsular space and in the lymphatic sinuses. The lungs presented congestion and marked multifocal hemorrhage. The
Fig. 2. Horse 3, right front limb. (A) Snake bite site with marked swelling. (B) Severe hemorrhage in subcutaneous and adjacent musculature. 43
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Fig. 3. Horse 4. (A) Head. Pronounced swelling at the bite site. (B) Lip. Fang marks (arrows) characterized by parallel perforations spaced approximately 1.8 cm apart.
spp. Our findings suggest that fatal snakebite envenomation in horses is unusual in Central Western of Brazil. However, the total number of snakebites in horses might be much higher if we consider the possibilities of envenomated horses that recovered without clinical care and underreporting of fatal cases. Large animals should be less likely to die from snakebites due to their weight and the lower venom dose in terms of venom per animal weight (Tokarnia and Peixoto, 2006). Generally, snakes use venom to immobilize prey in order to obtain food, and are able to modulate and reduce the amounts of injected venom in a defensive bite involving large animals (Melgarejo, 2003). Nevertheless, this study features fatal Bothrops snakebites in adult horses weighing up to 500 kg. Differences in the susceptibility of the animals, toxicity variations influenced by environmental conditions and snake feeding, and amount of the injected venom possibly influenced the number of horses that died in the
region throughout the evaluated period. The majority of these fatal envenomations occurred during the rainy season (4/5), with the exception of Horse 2 (dry season). In Latin America, snakebite envenomation is usually seasonal (Radostits et al., 2007; Sazima, 1998) with a higher incidence during warmer and rainy seasons. Bothrops species exhibit lower activity during drought periods and higher activity in the months of higher humidity, heat, and food supply, when the incidence of snakebites is predictably higher (Sazima, 1998; Cruz et al., 2009). In our study, envenomated horses presented extensive hemorrhages near the bite sites, which extended through the adjacent body regions and tissues (neck and all limbs). All horses also presented mild hemorrhages in subcutaneous and muscular tissues not associated with the snakebite sites and bleeding in most internal organs and serous cavities. A hallmark of lancehead pit viper snakebite is the marked local
Fig. 4. Horse 4. Severe hemorrhage at the head and neck. 44
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++ + ++ ++ ++
+++ ++ + ++ +++
++ ++ ++ ++ +++
+ ++ ++ + +++
++ ++ + ++ ++
+ ++ ++ + +++
++ ++ ++ +++ +++
swelling due to intense edema and tissue hemorrhage, which usually occurs near the head, muzzle and limbs where bites occur (Raposo et al., 2001; Chiacchio et al., 2011; Silva et al., 2011). Horses experimentally injected with venoms of B. jararaca, B. jararacussu, B. moojeni and B. neuwiedi also showed marked local swelling (Sousa et al., 2011). These findings are very similar to those observed in the envenomated horses in our study. Bothrops envenomation results in local (hemorrhage, myonecrosis and dermonecrosis) and systemic manifestations (coaguloapathy, systemic hemorrhage, circulatory shock and acute kidney injury) mainly due to the action of metalloproteinases, phospholipases A2 and serine proteinases, the most abundant components in these venoms (Gutiérrez et al., 2017). Phospholipase A2 is also responsible for the venom's powerful myotoxic action, affecting the sarcolemma integrity of skeletal muscle fibers and causing acute myonecrosis (Gutiérrez et al., 2009). These mechanisms explain the extensive myonecrotic areas observed in all horses, and the high CK activities recorded in two cases (Horses 2 and 4). In humans, envenomation by most viperid snakes causes a marked reaction at the snakebite site characterized by hemorrhage, edema, necrosis and blistering, with the severity, extent of lesions and sequelae in the affected limb related to the time of evolution and to the amount of venom injected (Gutiérrez et al., 2006). The blistering at the snakebite site occurs by the loss of the integrity of the dermal-epidermal interface caused by the action of metalloproteinases (Gutiérrez et al., 2017). We did not observe in our study the characteristic blistering or similar histological changes in horses. It is possible that the intensity of skin lesions in horses were less severe than in humans due to the acute fatal clinical course (12–48 h), not progressing to more marked local injuries. The evaluation of new cases of natural Bothrops envenoming in horses with a more prolonged clinical course may allow future comparisons with these lesions in humans. Identification of parallel perforations spaced 1.8–2.0 cm apart (fang marks) confirmed the snakebite site. Fang marks were previously described in sheep with Bothrops envenomation (Leal et al., 2013), and also aided the diagnosis in two horses (Horse 4 and 5). Snakebite fang marks may not be visualized or occur sporadically, as reported in only 25% of the affected animals in a sheep flock (Tokarnia et al., 2008). Bothrops snakebite envenomation were reported in two horses in Brazil, and these marks were observed in only one animal (Raposo et al., 2001; Silva et al., 2011). Although three of the horses evaluated (Horses 1, 2 and 3) did not present fang marks, background information and local and systemic clinical manifestations allowed the diagnosis of Bothrops envenomation. Acute kidney injury (AKI) stands out among the systemic changes in humans and animals with Bothrops envenomation. AKI seems to be associated with the direct action of the Bothrops venom components on renal tubular epithelial cells (Nascimento et al., 2007) and can also occur from the tubulotoxic effects of hemoglobin and/or myoglobin, or simply from impaired perfusion from hypotension (Gutiérrez et al., 2017). Experimental envenomation of mice by Bothrops alternatus revealed deposition of the venom in the kidney glomeruli and tubules, which was associated with renal tubular cell degeneration, desquamation, and necrosis (Gay et al., 2009; Mello et al., 2010). Renal injury may be aggravated indirectly by the release of endogenous mediators such as cytokines, inflammatory peptides, nitric oxide, and arachidonic acid metabolites (Barbosa et al., 2006). The nephrotoxic action of Bothrops venoms may explain the increased serum levels of urea and creatinine observed in our horses. The renal histological changes observed in horses ranged from moderate to severe and are characteristic of systemic envenomings by viperid snakes, such as lancehead pit viper, that have a higher concentration of toxins in the venom that cause cytotoxicity, hemorrhage, coagulopathy, and myonecrosis (Gutiérrez et al., 2017). The ultrastructural injuries observed in the tubular epithelial cells of the kidney and their organelles in our study are similar to those reported in rats
R: right; L: left; F: front limb; H: hind limb; **: snakebite site; Intensity: + mild, ++ moderate, +++ severe, - no lesion.
+ ++ + ++ +++ – ++ – – +++ +++ – – – – – +++** + +++ +++ – – + +++** ++** – – + – + – – + – + – – + + + – +++ + +++ +++ 1 2 3 4 5
+++** + +++** + +
+ + + + +
+ + + + +
+ + + + +
– +++** + +++** ++**
+++** – +++** + +
Intestines/mesentery Spleen Diaphragm Aorta Heart Lungs Abdominal Neck RF
LF
RH
LH
Head
RF
LF
RH
LH
Head
Neck
Thoracic
Hemorrhage (petechiae, ecchymosis, suffusion) in organs Serohemorrhagic content in cavities Muscular tissues Subcutaneous tissues Case
Table 3 Location and intensity of hemorrhages in the subcutaneous, muscular tissues, serous cavities and internal organs of horses with fatal bothropic envenomation.
Kidney
Perirenal fat
M. Machado, et al.
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Fig. 5. Horse 5, kidney. Whitish flecked foci of necrosis and striations that extend slightly to the medullar region. (*) Detail view.
distension, necrosis, and rupture, with the consequent hemorrhage (Gutiérrez et al., 2005). The PIII class of metalloproteinases are the most potent hemorrhagic toxins in these venoms (Escalante et al., 2011; Herrera et al., 2015). Diffuse hemorrhage in organs and tissues was the main pathological finding in the envenomated horses in Midwestern Brazil. Additionally, the three horses that were clinically evaluated showed coagulation disorders (Horse 2, 4 and 5), confirmed by severe thrombocytopenia in two cases (Horse 2 and 5). Endothelial injury detected may also aggravate the coagulopathy and disseminated hemorrhages in these horses. The action of snake venom enzymes, mostly thrombin-like serine proteinases and procoagulant metalloproteinases, on the coagulation cascade is responsible for the consumption coagulopathy characteristic
experimentally envenomed by a species of lancehead pit viper (BoerLima et al., 2002). Thrombosis and necrosis of glomerular capillary endothelial cells observed in horses also occurred in rats experimentally envenomated by Bothrops asper (Moreira et al., 1994). Vascular thrombosis is associated with the hemostatic response to the primary vascular damage commonly observed in these envenomations (Moreira et al., 1994). The histological and ultrastructural findings observed herein reinforce the importance of the nephrotoxic action of the lancehead pit viper venom and its probable involvement in AKI. Metalloproteinases are the main venon components of snakes of the family Viperidae involved in the local and systemic hemorrhages due to their direct hydrolytic action on the capillary basement membrane, causing loss of mechanical and hemodynamic stability, capillary
Fig. 6. Horse 4, snakebite site. (A) Skeletal muscle. Hemorrhagic dissecting myonecrosis surrounding the muscle bundles (H&E, scale bar = 250 μm). (B) Skin. Severe focally extensive hemorrhagic dermonecrosis (H&E, scale bar = 250 μm). 46
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Table 4 Location and intensity of histopathological changes in the kidney of horses with fatal bothropic envenomation. Case
1 2 3 4 5
Glomerular capillaries
Urinary space
Tubules
Interstitium
Thrombosis
Hemorrhage
Necrosis
HD
HDP
HC
GC
Hemorrhage
+ ++ + + +++
+ ++ + ++ +++
++ ++ + + +++
++ +++ ++ ++ +++
+ ++ +++ +++ +++
++ +++ ++ ++ +++
++ ++ + ++ ++
++ + + + +++
HD: hydropic degeneration; HDP: hyaline droplets; HC: hyaline casts; GC: granular casts; Intensity: + mild, ++ moderate, +++ severe.
Fig. 7. Horse 5, kidney. (A) Medulla. Tubular necrosis (*) and intratubular casts (arrow heads) (H&E, scale bar = 50 μm). (B) Cortex. Severe glomerular hemorrhage and epithelial tubular cells necrosis (*) (H&E, scale bar = 100 μm).
Fig. 8. Horse 5, kidney. (A) Cortex. Fibrin thrombi (arrows) in the glomerular capillaries (H&E, scale bar = 25 μm). (B) Medulla. Intracytoplasmic hyaline droplets (*) in distal convoluted tubule epithelial cells (Chromotropic aniline blue, scale bar = 25 μm).
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Fig. 9. Horse 5, kidney, cortex. (A) Necrotic tubular cell epithelium (asterisk) with margination of the nuclear chromatin, and distended cytoplasm with lysis of organelles (arrow). (TEM, scale bar = 10 μm). (B) Portion of a tubular epithelial cell with numerous swollen mitochondriae with cristae rupture and large amount of electrodense granules and mineral deposition. (TEM, scale bar = 1 μm).
1999; Melgarejo, 2003). Lachesis venom has marked neurotoxic activities (Malaque and Gutiérrez, 2015) which was not observed in horses. Lachesis genus is distributed in Atlantic and Amazon Rainforests (Fernandes et al., 2004) and does not occur in the savanna areas of our study. The spatial distribution and abundance of B. moojeni and B. neuwiedi in vast areas of savanna in Brazil suggests their involvement in the investigated snakebites in horses (Tokarnia and Peixoto, 2006). However, since none of the snakes were seen or captured, the exact species responsible for our cases of envenoming remain unknown. Identification of the venomous snake species causing the bite is only possible in the minority of cases (Tokarnia and Peixoto, 2006; Malaque and Gutiérrez, 2015), and generally can not be used to make therapeutic decisions. Therefore, the diagnosis has to be based on the
of these envenomations, with alteration of the laboratory coagulation tests (Varanda and Giannini, 1999; White, 2005; Sousa et al., 2013), as observed in animals experimentally envenomed by lancehead pit viper venoms (Sousa et al., 2013). Such alterations in hemostasia synergize with the disruptive action of hemorrhagic metalloproteinases in the vasculature, resulting in extensive local and systemic hemorrhage, a characteristic finding in our study. The ability of lancehead pit vipers to induce lethality is likely the result from cardiovascular alterations associated with systemic bleeding and hypovolemia, and acute kidney injury. The most common species of venomous snakes in Brazil are Bothrops alternatus, B. atrox, B. jararacussu, B. jararaca, B. moojeni, B. neuwiedi, Crotalus durissus and Lachesis muta rhombeata (Barraviera and Pereira,
Fig. 10. Horse 5, kidney. (A) Disruption of the endothelial cells and capillary thrombosis (asterisk) with covers exposing the basement membrane, interstitial hemorrhage (star), and tubular cells with hydropic degeneration (cross). (TEM, scale bar = 6 μm). (B) Glomerular capillary with fibrinous thrombus (asterisk) and blunting of the podocytes processes and cytoplasm clearance with organelles lysis (TEM, scale bar = 1 μm).
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distinct clinical-pathological alterations caused by the main venomous snake genera, and their spatial distribution and epidemiology in Brazil (Sazima, 1998; Barraviera and Pereira, 1999). According to these criteria, the diagnosis of fatal Bothrops envenomation was supported in all five horses. Differential diagnoses of Bothrops envenomation include traumatic lesions, clostridial diseases and other hemorrhagic disorders. In all horses investigated, there was no evidence of these diseases, which were excluded due to epidemiology, background information and clinical signs, identification of bite wounds (Horses 4 and 5), absence of gas production in the limb at the site of swelling (Horses 1, 2 and 3) and pathological findings. In conclusion, five fatal cases of horses suffering Bothrops spp. snakebite envenomation were described. Clinical and pathological findings closely correspond to the characteristic features of envenomations by Bothrops spp. in experimentally envenomed horses, other animal species and humans. Our findings highlight the potential severity of such snakebites in horses, and provide evidence for the diagnosis and therapeutic management of these cases.
Boer-Lima, P.A., Gontijo, J.A., Cruz-Höfling, M.A., 2002. Bothrops moojeni snake venominduced renal glomeruli changes in rat. Am. J. Trop. Med. Hyg. 67 (2), 217–222. Caldas, S.A., Tokarnia, C.H., França, T.N., Brito, M.F., Graça, F.A., Coelho, C.D., Peixoto, P.V., 2008. Clinic and pathological and laboratory aspects of experimental poisoning by Bothrops alternatus venom in cattle. Pesqui. Vet. Bras. 28, 303–312. https://doi. org/10.1590/S0100-736X2008000600008. Cheng, A.C., Currie, B.J., 2004. Venomous snakebites worldwide with a focus on the Australia-Pacific region: current management and controversies. J. Intensive Care Med. 19, 59–69. https://doi.org/10.1177/0885066604265799. Chiacchio, S.B., Martins, G.T.B., Amorim, R.M., Gonçalves, R.C., Barraviera, B., Ferreira Junior, R.S., 2011. Triple bothropic envenomation in horses caused by a single snake. J. Venom. Anim. Toxins Incl. Trop. Dis. 17, 111–117. https://doi.org/10.1590/ S1678-91992011000100016. Cruz, L.S., Vargas, R., Lopes, A.A., 2009. Snakebite envenomation and death in the developing world. Ethn. Dis. 19, 42–46. De Rijk, E.P.C.T., Ravesloot, W.T.M., Wijnands, Y., Esch, E.V., 2003. A fast histochemical staining method to identify hyaline droplets in the rat kidney. Toxicol. Pathol. 31, 462–464. https://doi.org/10.1080/01926230390213775. Escalante, T., Rucavado, A., Fox, J.W., Gutiérrez, J.M., 2011. Key events in microvascular damage induced by snake venom hemorrhagic metalloproteinases. J. Proteomics. 74 (9), 1781–1794. https://doi.org/10.1016/j.jprot.2011.03.026. Fernandes, D.S., Franco, F.L., Fernandes, R., 2004. Systematic revision of the genus Lachesis daudin, 1803 (Serpentes, Viperidae). Herpetologica 60 (2), 245–260. Ferreira Júnior, R.S., Barravieira, B., 2004. Management of venomous snakebites in dogs and cats in Brazil. J. Venom. Anim. Toxins Incl. Trop. Dis. 10, 112–132. https://doi. org/10.1590/S1678-91992004000200002. Fielding, C., Pusterla, N., Magdesian, G., Higgins, J.C., Meier, C., 2011. Rattlesnake envenomation in horses: 58 cases (1992-2009). J. Am. Vet. Med. Assoc. 238, 631–635. https://doi.org/10.2460/javma.238.5.631. Franco, R.L., Rocha, C.C., Jorge, M.T., Ribeiro, L.A., 2001. Snakebites in southern minas gerais state, Brazil. J. Venom. Anim. Toxins Incl. Trop. Dis. 7, 56–68. https://doi.org/ 10.1590/S0104-79302001000100005. Gay, C.C., Maruñak, S.L., Teibler, P., Ruiz, R., Pérez, A.O., Leiva, L.C., 2009. Systemic alterations induced by a Bothrops alternatus hemorrhagic metalloproteinase (baltergin) in mice. Toxicon 53, 53–59. https://doi.org/10.1016/j.toxicon.2008.10.010. Graça, F.A.S., Peixoto, P.V., Coelho, C.D., Caldas, A.S., Tokarnia, C.H., 2008. Clinical and pathological aspects of experimental Crotalus poisoning in cattle. Pesqui. Vet. Bras. 28, 261–270. https://doi.org/10.1590/S0100-736X2008000600001. Grondin, T.E., Dewitt, S.F., 2010. Normal hematology of the horse and donkey. In: Weiss, D.J., Wardrop, K.J. (Eds.), Schalm's Veterinary Hematology. 6. Wiley-Blackwell, Iowa, pp. 821–828. Grunert, E., 1967. Beobachtungen über Schlangenbissverletzungen bei grossen Haustieren in Süd-Brasilien. Dtsch. Tierärztl. Wochenschr. 20, 531–532. Grunert, E., Grunert, D., 1969. Beobachtungen von Bothrops-Schlangenbissverletzungen bei Rind und Pferd in Rio Grande do Sul, Brasilien. Vet.-Med. Nachrichten 3, 217–232. Gutiérrez, J.M., Calvete, J.J., Habib, A.G., Harrison, R.A., Williams, D.J., Warrell, D.A., 2017. Snakebite envenoming. Nat. Rev. Dis. Primers 3, 17063. https://doi.org/10. 1038/nrdp.2017.63. Gutiérrez, J.M., Rucavado, A., Chave, F., Díaz, C., Escalante, T., 2009. Experimental pathology of local tissue damage induced by Bothrops asper snake venom. Toxicon 54, 958–975. https://doi.org/10.1016/j.toxicon.2009.01.038. Gutiérrez, J.M., Rucavado, A., Escalante, T., Díaz, C., 2005. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 45, 97–1011. https://doi.org/10.1016/j.toxicon. 2005.02.029Get. Gutiérrez, J.M., Theakston, R.D.G., Warrell, D.A., 2006. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med. 3 (6), 1–5. https://doi.org/10.1371/journal.pmed.0030150. Herrera, C., Escalante, T., Voisin, M.B., Rucavado, A., Morazán, D., Macêdo, J.K.A., Fox, J.W., 2015. Tissue localization and extracellular matrix degradation by PI, PII and PIII snake venom metalloproteinases: clues on the mechanisms of venom-induced hemorrhage. PLoS Neglected Trop. Dis. 9 (4), 0003731. https://doi.org/10.1371/ journal.pntd.0003731. Kaneko, J.J., Harvey, J.W., Bruss, M.L., 2008. Clinical Biochemistry of Domestic Animals, sixth ed. Academic Press, San Diego. Leal, M.L.R., Aires, A.M.C., Fillapi, A., Trost, M.E., 2013. Clinical and pathological observations associated with snake envenomation in two sheep. Acta Sci. Vet. 41, 1–5. Lopes, C.T.A., Tokarnia, C.H., Brito, M.F., Sousa, M.G., Oliveira, C.M.C., Silva, N.S., Lima, D.H.S., Barbosa, J.D., 2012. Clinical and pathological aspects of experimental crotalic envenoming in horses. Pesqui. Vet. Bras. 32, 843–849. https://doi.org/10.1590/ S0100-736X2012000900005. Machado, M., Wilson, T.M., Sousa, D.E.R., Martins, C.S., Castro, M.B., 2019. Pit viper envenomation in a barn cat. J. Small Anim. Pract. 60 (2), 131. https://doi.org/10. 1111/jsap.12922. Malaque, C.M.S., Gutiérrez, J.M., 2015. Snakebite envenomation in central and South America. Crit Care Toxicol. 1. https://doi.org/10.1007/978-3-319-20790-2_146-1. Melgarejo, A.F., 2003. Venomous snake from Brazil. In: Cardoso, J.L.C., França, F.O.S., Wen, F.H., Malaque, C.M.S., Haddad, J.R.V. (Eds.), Venomous Snake from Brazil: Biology, Clinical and Therapeutics of Snake Envenomation. Sarvier Editora, São Paulo, pp. 33–61 2003. Mello, S.M., Linardi, A., Renno, A.L., Tarsitano, C.A., Pereira, E.M., Hyslop, S., 2010. Renal kinetics of Bothrops alternatus (Urutu) snake venom in rats. Toxicon 55, 470–480. https://doi.org/10.1016/j.toxicon.2009.09.018. Mendez, M.C., Riet-Correa, F., 1995. Snakebite in sheep. Vet. Hum. Toxicol. 37, 62–63. Moreira, L., Borkow, G., Ovadia, M., Gutiérrez, J.M., 1994. Pathological changes induced
5. Conclusions Venomous snakebites in horses represent a diagnostic challenge for practitioners, since rarely the moment of snakebite is witnessed or the snake is identified, thus, the association of clinical, pathological and some epidemiological features (snake habitats and warmer and rainy seasons) is essential for the correct diagnosis. In this study, the diagnosis of fatal lancehead pit viper snakebites in five horses was established by the association of clinical, laboratory and pathological findings. Marked local swelling, disseminated hemorrhages, blood incoagulability, pigmenturia (myoglobinuria/hemoglobinuria) and acute kidney injury are the main changes observed in the acute Bothrops envenoming, and support practioners to suspect of lancehead pit viper snakebites in horses. Ethical statement Not applicable. All information and samples were obtained from records of the Veterinary Pathology Laboratories of horses that had natural death by venomous snakebite. Acknowledgements Special thanks to Dr. Claudio Severo Lombardo de Barros (Federal University of Mato Grosso do Sul) for the technical assistance in the pathological diagnosis and to Anderson Saravia (National Institute of Agricultural Research of Uruguay - Inia, La Estanzuela) for the aid in the making of the map. Thanks are due to Dr. José María Gutiérrez for the critical reading of the manuscript and suggestions. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. References Aragão, A.P., Tokarnia, C.H., Graça, F.A.S., França, T.N., Coelho, C.D., Caldas, A.S., Peixoto, P.V., 2010. Experimental poisoning by Bothropoides jararaca and Bothrops jararacussu in sheep: clinic-pathological and laboratory aspects. Pesqui. Vet. Bras. 30, 717–728. https://doi.org/10.1590/S0100-736X2010000900003. Barbosa, J.D., Sousa, M.G.S., Tokarnia, C.H., Brito, M.F., Reis, A.S.B., Bomjardim, H.A., Lopes, C.T.A., Oliveira, C.M.C., 2011. Comparison between the clinical and pathological picture in the experimental poisoning by crotalic venom. Pesqui. Vet. Bras. 31, 967–973. https://doi.org/10.1590/S0100-736X2011001100005. Barbosa, P.S.F., Martins, A.M.C., Alves, R.S., Amora, D.N., Martins, R.D., Toyama, M.H., Fonteles, M.C., 2006. The role of indomethacin and tezosentan on renal effects induced by Bothrops moojeni Lys 49 myotoxin I. Toxicon 47, 831–837. https://doi.org/ 10.1016/j.toxicon.2006.01.012. Barraviera, B., Pereira, P.C.M., 1999. Snake bites by Bothrops genus. In: Barraviera, B. (Ed.), Clinical and Therapeutic Aspects of Venomous Animals Accidents. EPUC, Rio de Janeiro, pp. 261–280.
49
Toxicon 170 (2019) 41–50
M. Machado, et al.
Sousa, L.F., Nicolau, C.A., Peixoto, P.S., Bernardoni, J.L., Oliveira, S.S., Portes-Junior, J.A., Mourão, R.H., Valente, R.H., Silva, A.M.M., 2013. Comparison of phylogeny, venom composition and neutralization by antivenom in diverse species of Bothrops complex. PLoS Neglected Trop. Dis. 7 (9), 1–17. https://doi.org/10.1371/journal. pntd.0002442. Sousa, M.G., Tokarnia, C.H., Brito, M.F., Reis, A.B., Oliveira, C.M., Freitas, N.F., Oliveira, C.H., Barbosa, J.D., 2011. Clinical and pathological aspects of the experimental poisoning by Bothrops snakes in horses. Pesqui. Vet. Bras. 31, 773–780. https://doi. org/10.1590/S0100-736X2011000900009. Tokarnia, C.H., Brito, M.F., Malafaia, P., Peixoto, P.V., 2008. Snake bite accident in sheep by Bothrops jararaca. Pesqui. Vet. Bras. 12, 643–648. https://doi.org/10.1590/ S0100-736X2008001200014. Tokarnia, C.H., Peixoto, P.V., 2006. The importance of snake bites as cause of cattle death in Brazil. Pesqui. Vet. Bras. 26, 55–68. Varanda, E.A., Giannini, M.J.S.M., 1999. Biochemistry of snake venoms. In: Barraviera, B. (Ed.), Clinical and Therapeutic Aspects of Venomous Animals Accidents. EPUC, Rio de Janeiro, pp. 205–223. White, J., 2005. Snake venoms and coagulopathy. Toxicon 45 (8), 951–967. https://doi. org/10.1016/j.toxicon.2005.02.030.
by BaH1, a hemorrhagic proteinase isolated from Bothrops asper (Terciopelo) snake venom, on mouse capillary blood vessels. Toxicon 32 (8), 976–987. Nascimento, J.M., Franchi, G.C., Nowill, A.E., Collares-Buzato, C.B., Hyslop, S., 2007. Cytoskeletal rearrangement and cell death induced by Bothrops alternatus snake venom in cultured Madin–Darby canine kidney cells. Biochem. Cell Biol. 85, 591–605. https://doi.org/10.1139/O07-067. Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D., 2007. Disease associated with toxins, plants, fungi, cyanobacteria, plant-associated bacteria, and venoms in ticks and vertebrate animals. In: Radostits, O.M., Gay, C.C., Hinchcliff, K.W., Constable, P.D. (Eds.), Veterinary Medicine: A Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats. Saunders Elsevier, Philadelphia, pp. 1851–1915. Raposo, J.B., Méndez, M.C., Bailardi, C.E.G., Raffi, M.B., 2001. A case of snakebite in a horse in southern Brazil - case report. Rev. FZVA 8, 51–57. Sazima, I., 1998. Um estudo de biologia comportamental da jararaca, Bothrops jararaca com uso de marcas naturais. Mem. Inst. Butantan (Sao Paulo) 3, 83–89. Silva, N.S., Silveira, J.A.S., Albernaz, T.T., Campos, K.F., Oliveira, C.M.C., Freitas, N.F.Q.R., Bomjardim, H.A., Barbosa, J.D., 2011. Fatal bothropic snakebite in a horse: a case report. J. Venom. Anim. Toxins Incl. Trop. Dis. 17, 496–500. https://doi.org/ 10.1590/S1678-91992011000400018.
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