On-line version ISSN 1678-4936
J. Venom. Anim. Toxins vol. 3 n. 1 Botucatu 1997
Bothrops alternatus ENVENOMING IN YOUNG DOGS
1 School of Veterinary Medicine, Universidad Nacional del Nordeste, Corrientes, Argentina, 2 Laboratory of Histopathology of the School of Veterinary Medicine, Universidad Nacional del Nordeste, Corrientes, Argentina
ABSTRACT. Bothrops alternatus venom was intramuscularly inoculated (3 mg/kg) into 12 dogs, 30 to 65 days old. Spontaneous bleeding commenced twenty minutes later. Blood samples obtained 3 and 20 minutes after venom inoculation presented spontaneous clotting formation. Plasmatic fibrinogen decreased within 3 minutes. Partial thromboplastin time (PTT) and one-stage prothrombin time (PT) were found. Plasma did not coagulate 40 minutes after inoculation. Platelet counts did not vary but their function was altered.
Histopathology pointed out severe muscular necrosis and massive hemorrhage in the inoculation area. Regional ganglia showed intense hemorrhage. The 45 and 65-day-old animals showed alveolar thickening of the septum and generalized congestion, but the 30-day-old animals showed thrombosis of small arteries and arterioles. Renal lesions were different with the age. Cortical tubular necrosis was present in puppies, and intense cortical tubular hydropic degeneration was present in adult dogs. Thymus hemorrhage and necrosis were present.
KEY WORDS: dogs, Bothrops alternatus, coagulation, histopathology.
Bothrops alternatus envenoming is one of the most frequent snake accident in Argentina. However, we could not obtain information about this particular type of envenoming. The study of this type of accident started by finding the DL50 of B. alternatus in our laboratory animals. The DL50 was 55.86 µg/20g mouse.
A total of 12 dogs, 30 and 65 days old, weighing around 1.900 kg were inoculated with 3 mg/kg of B. alternatus venom.
Blood samples were collected before inoculation and 3, 20 and 40 minutes after inoculation in 3.8% citrate solution.
We evaluated platelet count in a Newbauer hemacytometer and its functionality by bleeding time (Dukes' method). The intrinsic system of coagulation was controlled by the determination of activated partial thromboplastin time (APTT) with kaolin(2). The extrinsic system was controlled by one-stage prothrombin time (PT) or Quick Time(2). Plasmatic fibrinogen was determined by Clauss' methods.
Each animal was clinically checked and the observation was completed with necropsy data because the dogs died between 12 and 24 hours after venom inoculation. Different tissue samples were obtained in 10% formol for histopathology examination.
Around 2 minutes after envenoming, all the animals showed intense pain. They were moaning and howling with flexion of the affected member. The dogs also defecated and urinated. They stopped walking and laid down. They also showed tachycardia and mydriasis. Within a short time, edema was present in the area of inoculation. Spontaneous bleeding appeared at the site of inoculation and at the incision made to measure the bleeding time. Only one animal showed hemorrhage on the tongue 7 hours after inoculation.
The inoculation area showed intense hemorrhage and muscular necrosis characterized by cytoplasmic eosinophilia, loss of grooves, nuclear pyknosis, abundant histiocytes with hemosiderin and red cells in various stages of autolysis. Hemorrhagic areas were also seen in the thymus.
Regional ganglia showed hemorrhage in cortical and medular area.
Renal tissue lesions were different according to the age of the animals. The 65-day-old animals only showed congestion and degenerative phenomena, while the 30-day olds showed more important lesions with cortical hydropic degeneration.
Alveolar thickening of the septum and generalized congestion with thrombosis in the small arteries and arterioles were observed in the lungs.
Histopathological findings in dogs' livers and kidneys were similar to those with purified phospholipase A2 of B. alternatus in mice(6), while muscular necrosis was similar to those obtained in laboratory animals inoculated with B. jararaca venom(7).
In blood samples obtained 3 and 20 minutes after venom inoculation, there were spontaneous APTT and PT clotting formation. Forty minutes after venom inoculation both APTT and PT times were prolonged for more than a minute, as follows: activated partial thromboplastin (normal time = 22 ± 5 seconds) (n = 12), and prothrombin (normal time = 10 ± 2 seconds) (n = 12). Plasmatic fibrinogen showed a remarkable reduction in plasmatic concentration .
Figure 1 shows plasmatic fibrinogen in dog envenoming by B. alternatus . The dose was 3mg/kg i.m. into gastrocnemius muscle in a volume of 0.1 ml phosphate buffered saline solution.
FIGURE 1. Plasmatic fibrinogen in dog envenoming by B. alternatus .
Twenty minutes after venom inoculation, spontaneous bleeding was present with a bleeding time fivefold prolonged. No change was evaluated in platelet counts. Basal values were between 150.000 and 400.000/mm. Mean value was 210.000/mm after inoculation.
Proteinases affecting one or several physiological thrombin substrates are common components of Crotalidae venoms. These enzymes which cause the in vitro coagulation of fibrinogen have already been isolated(9). This fact will induce prolongation of PT in bothropic envenomation with prothrombin and fibrinogen consumption(3). In dogs, blood did not coagulate after 40 minutes with a decrease in plasmatic fibrinogen levels.
The animals presented hemorrhagic necrosis and a considerable bleeding at the envenoming site. This could be the result of the action of the metalloproteinases(4,5) and the activation of other coagulation factors(8) which would induce a severe alteration in hemostasis.
We also found that general coagulation tests, such as prothrombin and partial thromboplastin tests showed a good correlation with the severity of the envenomation(1). The lethality of this envenoming was a consequense of circulatory disturbances with decrease of the arterial blood pressure, bradycardia and dyspnea(6).
This work was possible through grants from the General Secretary of Scientific and Technical Office of UNNE (National University of the Northeast). Thanks are due to Mr. Eduardo Parada from the Ofidiology Center where Bothrops alternatus venom was obtained.
01 BARRANTES A., SOLIS V., BOLANOS R. Alterations in the coagulation mechanisms of patients bitten by Bothrops asper (Terciopelo). Toxicon, 1985, 23, 399-407. [ Links ]
02 CAEN J., LARRIEU M.J., SAMANA M. L'Hémostase, méthodes déxploration et diagnostic pratique. Paris: Expansion Scientifique, 1975. [ Links ]
03 HAAD J.S. Accidentes humanos por las serpientes de los generos Bothrops y Lachesis. Mem. Inst. Butantan, 1981, 45, 403-23. [ Links ]
04 MANDELBAUM F.R., REICHL A.P., ASSAKURA M.T. Hemorrhagic factors from venoms of two species of Bothrops snake. Mem. Inst. Butantan, 1989, 51, 244. [ Links ]
05 MANDELBAUM F.R., ASSAKURA M.T., REICHL A.P. Characterization of two hemorrhagic isolated from the venom of Bothrops neuwiedi (Jararaca pintada). Toxicon, 1984, 22, 193-206. [ Links ]
06 NISENBOM H.E., PERAZZO J.C., MONSERRAT A.J., VIDAL J.C. Contribution of phospholipase A2 to the lethal potency of Bothrops alternatus (víbora de la cruz) venom. Toxicon, 1986, 24, 807-15. [ Links ]
07 SALIBA F. Estudo anatomo-patológico da evolução da necrose produzida experimentalmente por veneno de Bothrops jararaca. Influenza de Sustancia Organo-heparinoide. Mem. Inst. Butantan, 1964, 31, 191-200. [ Links ]
08 SANO MARTINS I.S. Hematological disturbances induced by Bothrops venom. Mem. Inst. Butantan, 1990, 52, 39-40. [ Links ]
09 STOCKER K., FISCHER H., MEIER J. Trombin-like snake proteinases. Toxicon, 1982, 20, 265-73.
[ Links ]
Received 19 March 1996.
Accepted 20 April 1996.