Incidence
Snakebite is one of the most important acute life-threatening medical emergency conditions affecting both humans and animals, recognized and declared as a neglected tropical disease by World Health Organization
(Ahmad et al., 2016). Ample data on companion animal and equine snakebite is available as compared to ruminants
(Bolon et al., 2019). During the present study 80 cases of viper snakebites were confirmed in buffaloes admitted over a period of 10 years from 2010 to 2019, with an overall incidence of 0.98%.
The month-wise incidence of viper bites in buffaloes revealed the highest occurrence in August (18.75%) and July (16.25 %) months. Season wise highest incidence was reported in monsoon (56.25%), followed by post-monsoon (21.25%), winter (16.25%) and least in summer (6.25%). The present findings agree with higher rates of snakebites and deaths during the same season due to snakebite in humans and zebu cattle
(Mohapatra et al., 2011; Bhikane et al., 2020). Monsoon commences with rainfall, rapid growth of tall, lush and dense pasture in the grasslands, which is an important habitat for snakes. Age-wise highest incidence of viper snakebite was observed in buffaloes aged > 4 years (58.75%), followed by 1-4 years (36.25%) and least in young buffaloes below < 1 year (5%). Higher incidence in > 4 year and 1-4-year age groups might be attributed to complete dependence of adult buffaloes on grazing for meeting nutritional requirements compared to young bubaline population, which are generally kept indoors and stall fed.
Sex-wise incidence of viper bites was almost 96.25% in female buffaloes while 3.75% in male buffaloes. Highest incidence in female buffaloes could be attributed to sole rearing of female animals for milk production which are more prone to come across snakes while grazing. While male buffaloes are generally reared up to the end of lactation or age of 1 year, during which they are mostly stall fed and rarely allowed for grazing reducing their risk of exposure to the snakes. Out of the total affected buffaloes with viper snake envenomation, 91.25% were of the local Marathwadi breed, owing to its native tract while 8.75% were graded Murrah buffaloes. When the data pertaining to the probable time of bite was analyzed, it was found that maximum bites occurred during the morning (41.25%) followed by afternoon (26.25%) and evening (23.75%) hours while least in night hours (8.75%). Almost 91.25% (73) buffaloes were bitten during grazing, while about 8.75% (7) buffaloes were bitten while resting in the stall/shed during night hours which was in agreement with the findings of
Bhikane et al., (2020).
Clinical signs
No significant changes were observed in body temperature (100.31±0.56 vs. 100.41±0.15°F) while highly significant (P<0.01) increase in heart rate (68.00±3.14 vs. 54.14±0.79 bpm) and significant (P<0.05) increase in respiration rate (29.78±3.06 vs. 21.42±0.70/min) was observed in viper bite affected buffaloes compared to healthy control buffaloes. The most characteristic sign of viper envenomation observed in buffaloes was ascending edematous swelling with pain and lameness over limbs (Fig 1a and 1b). In most of the cases of bite over limbs, the swelling was found extending upward towards dewlap and brisket in forelimb bites. Asymmetrical edematous swelling over the face with pain and severe dyspnea was a consistent finding in viper bite over the face region (Fig 2a). In one case of viper bite over the tail region in buffalo, ascending swelling was observed over the tail (Fig 2b). Analysis of data based on the site of snakebite revealed maximum cases of viper bites on forelimbs (42.50%) followed by hind limbs (32.75%), face (23.75%) and tail (1.25%). The findings were in agreement with those reported by
Bhikane et al., (2020) and
Bolon et al., (2019). The highest number of bites observed on limbs (75.00%) might be attributed to their direct and closer contact with ground during either walk or grazing in dense grassland followed by face (23.75%) during grazing time.
The mucous membranes showed variable changes
viz., congestion and petechiae in the initial stage while pallor in the late stage. Signs of local and mucosal bleeding were prominent in most of the affected buffaloes. Variable degree of local bleeding (Fig 2 c.) was observed at the site of bite in 56.25% of cases while mucosal bleeding in the form of hematochezia/ melena (8.75%, Fig 3 a), epistaxis (6.25%, Fig. 3 b), vaginal bleeding (1.25%) and hematuria (1.25%) was also evident. The general signs observed in all envenomed buffaloes were inappetence to anorexia, suspended rumination, dullness, depression and decreased rumen motility.
Snake venoms are responsible for several toxic actions in the body of human and animal
viz., necrotizing, anticoagulant and procoagulant fractions, neurotoxic, cardiotoxic, myotoxic, nephrotoxic, cytotoxic, hemolytic and hemorrhagic fractions producing edema, inflammation, hemorrhages, necrosis and altered blood coagulation
(Goddard et al., 2011; Kumar et al., 2022). The clinical signs observed in the present study are in agreement with the findings of
Rodriguez et al., (2016); Altug and Isler (2019);
Bhikane et al., (2020) and
Sawane et al., (2023). In the present study, fang marks were visible only in 13.75% (11) cases on unpigmented (white) areas upon shaving of the bite site in early hours while fangs were not visible in 86.25% (69) cases due to pigmented dark and thick skin with hair coat.
Hemato-biochemistry
Mean (±S.E.) values of hemato-biochemical parameters revealed a significant (P<0.05) increase in WBC count (9.77±0.82 vs. 7.57±0.37×10
3/ml) and PCV (35.52±1.76 vs. 30.52±1.13%). The highly significant (P<0.01) increase in neutrophil count (51.31±3.03 vs. 32.52±4.90%) and highly significant (P<0.01) decrease in platelet count (37.4±10.87 vs. 203.08±15.04×10
3/ml) was observed in the viper bite affected buffaloes as compared to healthy buffaloes with non-significant changes in RBC count, hemoglobin concentration and lymphocyte count. Highly significant (P<0.01) increase in capillary blood clotting time (26.16±2.30 vs. 7.11±0.11 minutes) was observed in viper bite affected buffaloes compared to healthy counterparts. Hematological findings in the present study are in agreement with the findings of
Rodriguez et al., (2016) and
Bhikane et al., (2020) in cattle,
Armentano and Schaer (2011) in dogs and
Lenchner et al., (2014) in cats. Mucosal or local bleeding in viper bite affected buffaloes might be attributed to abnormal function of coagulation factors, effects of venom on capillary endothelium and platelets leading to severe thrombocytopenia and prolonged capillary blood clotting time
(Goddard et al., 2011). The bleeding syndromes in the form of epistaxis, melena, hemoglobinuria or hematuria could be attributed to venom-induced platelet disorders (
Kamiguti, 2005). Assessment of kidney function revealed a highly significant (P<0.01) increase in BUN (37.08±5.11 vs. 16.63±0.92 mg/dl) and creatinine (2.46±0.36 vs. 0.68±0.05 mg/dl) as compared to healthy buffaloes which was also reported previously by several authors in snakebite affected animals
(Goddard et al., 2011; Bhikane et al., 2020; Sawane et al., 2023).
Viperine envenomation in buffaloes was diagnosed by using circumstantial evidences obtained from animal owner, dead snake presented at the time of admitting the patient by few owners, set of clinical signs supported by bleeding profile (thrombocytopenia, prolonged capillary blood clotting time) and renal function tests (elevated BUN and creatinine). As per the data pertaining to circumstantial evidences regarding snakebite, 47.50% cases of bites were witnessed by farmers during grazing followed by signs of fear and excitement, while 8.75% cases were bitten during the resting period. In the rest of the cases (43.75%), the animal owners reported evidence of jumping, bellowing and sudden restlessness followed by the onset of progressive swelling over limbs or face.
Post-mortem findings
The post-mortem examination of buffaloes died due to viperine snake envenomation revealed excessive ascending swelling of the affected limb as compared to the unaffected limb (Fig 4a and 4b). The external skin surface of the affected limb frequently showed cyanosis or blue discoloration (Fig 4c), exudation of blood-stained serous fluid (Fig 4d.) and cracks due to overstretching. On incision to affected limb, the heavy hemorrhages were evident within underlying subcutaneous tissue and skeletal muscles (Fig 5a, 5b and 5c). The gross lesions observed in other visceral organs include the presence of blood-tinged pericardial fluid (Fig 5d), patchy or ecchymotic hemorrhages in lungs (Fig 6a, 6b and 6c) and moderate hepatomegaly along with mild icteric discoloration (Fig 6d).
The histopathological examination of skeletal muscles from affected limb revealed the presence of extensive myonecrosis (evident from hypereosinophilic dissociated or fragmented myofibers and loss of striations), widespread hemorrhages (Fig 7a and 7b), mild to moderate mononuclear cell infiltration and edema (Fig 7c). The marked intradermal or hypodermal hemorrhages were also evident in skin sections of the affected limb (Fig 7d). Microscopic lesions observed in lungs were the frequent presence of fibrinous microthrombi within pulmonary vasculature, widespread interstitial and intra-alveolar hemorrhages and edema, as well as presence of proteinaceous fluid within bronchial lumens (Fig 8a, 8b, 8c and 8d). Multifocal to coalescing areas of massive hemorrhages (Fig 9a, 9b and 9c) and coagulative necrosis (Fig 9d) were evident in hepatic parenchyma of viperine envenomated animals causing marked distortion of hepatic architecture. The lesions such as marked degeneration of tubular epithelium, presence of casts or cellular debris within tubular lumens and Bowman’s space and atrophy of glomerular tufts were evident in kidneys of envenomed buffaloes (Fig. 10a, 10b, 10c and 10d).
Post-mortem lesions due to snake envenomation are specific to each snake. Localized swelling at the site of the bite is attributed to exudation of serous fluid and inflammatory reaction to venom components, which is often deeply blood stained
(Constable et al., 2017). Post-mortem examination of a cow presumed to have died from the viper bite would show petechial and ecchymotic to frank hemorrhages in the lung, liver, tracheal lumen, peritoneum, epicardial and sub-endocardial surfaces and also pale linear streaks in the right ventricular myocardium
(Banga et al., 2009). Additionally, reported the histopathological changes like hemorrhages at the site of bite as well as in various organs including lungs along with necrotic changes in skeletal and cardiac muscles. These gross and microscopic changes reported by various earlier research workers were also in consonance with the pathological findings observed in present study. These findings observed in present study were also in consonance with the pathological findings observed by
Bhikane et al., (2020).
Treatment
A total of 80 confirmed cases of viper bite in buffaloes were treated by using therapeutic protocol mentioned above. In one case with severe dyspnea due to extensive swelling over sub-mandibular region, tracheotomy was performed. Mean (± S.E.) values of vital clinical parameters and hemato-biochemical parameters before treatment and on sixth day in viper bite affected buffaloes (n=25) are depicted in Table 1. Appreciable reduction in edematous swelling at the site of bite was observed from second day of treatment with complete resolution by 8-10 days. Significant (P<0.05) reduction in heart rate and respiration rate was observed on 6
th day of treatment compared to pre-treatment values suggestive of restoration of heart rate and respiration rate towards normal physiological range. Hematological parameters showed highly significant (P<0.01) reduction in RBC, hemoglobin and PCV with non-significant alterations in leukogram and differential leukocyte count on 6
th day. Bleeding profile showed highly significant (P<0.01) increase in platelet count while decrease in capillary blood clotting time. Kidney function test showed significant (P<0.05) reduction in the values of BUN and creatinine on 6
th day of treatment as compared to pre-treatment values.
Efficacy of antivenom therapy in snakebite cases depends on antivenom specificity to snake venom, potency of antivenom, dose, severity of envenomation and the time elapsed between bite and antivenom administration
(Otero et al., 2002). In the present study, 20-40 ml of PASV was found effective in the therapeutic management of viper envenomation in buffaloes along with supportive therapy. The dose of PASV found effective in the present study is in agreement with the earlier reports in cattle, horses and new world camelids (
Altug and Isler, 2019;
Bhikane et al., 2020; Fielding et al., 2011; Chiacchio et al., 2011; Dykgraaf et al., 2006). The poor therapeutic response was observed in lately presented cases (> 24 hours) with bleeding diathesis in the present study was in agreement with one report in new world camelids
(Dykgraaf et al., 2006). The aggravation of envenomation and absence of free circulating venom for neutralization might be responsible for poor response to antivenom therapy in lately presented cases (
Bawaskar, 2004). The emergency tracheotomy in one buffalo with bite on face and severe dyspnea due to submandibular swelling assisted in relieving the respiratory distress thereby increasing the survival chance. In the present study, out of 80 buffaloes treated for viperine envenomation with follow up in 76 cases had showed complete clinical recovery in 67 buffaloes with success rate of 88.15%, which signifies early treatment using PASV along with supportive therapy is able to produce complete clinical recovery in buffaloes with viper envenomation.
