Introduction

India is predominantly an agricultural country with about 70-80% of its population dependent on income from agriculture, animal husbandry is an adjunct to crop agriculture cattle were kept for milk production,. Gangrenous syndrome represents symptoms like lameness, swelling at the coronet at the junction of skin and hooves, oedema, necrosis, gangrene and sloughing off of tissues of extremities of animals. It is otherwise known as Degnala disease and/or Gangrenous Syndrome. This disease was reported for the first time by Shirlaw (1939) in Deg rivulet (Nala) which is presently in Pakistan, since then the disease was named as Degnala disease. After that the disease was reported from various part of India like Punjab ( Kalra, et al., 1973), Uttar Pradesh (Mallick et al., 1990), Bengal (Sikdar et al., 2000), Gujarat (Jadhav et al., 2003) and Bihar (Singh et al., 1983).  In Bihar state, India, the disease was firstly reported from Patna and Nalanda districts in 1983 and outbreak of this disease was reported in Bihar in 1998. The occurrence of disease mostly in reported winter season (Kaushik and Sinha, 1999) cold weather and low temperature has synergistic effect in precipitation of clinical symptoms. Prevalence of this disease is more in rice growing state of the country Viz. Haryana, Punjab, Kerala and Bihar (Prasad et al., 2000). This disease is clinically characterized by lameness, swelling at junction of hooves and limb’s (mostly in hind legs), general malaise, absence of fever but may be present in some cases due to secondary bacterial infection, oedema at extremities like tip of tongue, ear, tail, teats and udder (Kaushik and Sinha, 2004). It causes heavy economic loss due to high degree of morbidity and mortality consequently decreases in milk production and functional power in draught animals. To establish a correlation among factors in association with haemato-biochemical profile for early diagnosis and proper treatment of bovine gangrenous syndromes the present study was undertaken.

Materials and Methods

Experimental Animals

A total one hundred fifty (150) clinically affected cattle were taken for the study of gangrenous syndrome as per reported by Clinical complex of Bihar Veterinary College Patna-14. Out of which 14 cattle & 16 buffalo of different age and sex were selected for haemato – bio-chemical analysis of blood, serum and urine. In addition of these 6 apparently healthy cattle and buffalo were taken for control comparison.

Collection of Biological Samples

A total 15 ml blood samples were collected from Jugular vein using sterile glass syringe and needle. Out of which 3ml. of blood was transferred in a glass stopper vial containing sodium citrate (anticoagulant) for study of TLC, PCV, Hb%, ESR and TEC. While 2ml of blood was transferred to another vial containing sodium fluoride and potassium oxalate (anticoagulant) for estimation of blood glucose only. Rest 10ml of blood samples were transferred to a sterile test tube and kept in slant position for coagulation of blood and collection of serum for the study of AST, ALT, total serum protein (TSP), serum albumin, serum globulin, serum uric acid and serum creatinine. However, slides were prepared on the spot for the study of DLC. All serum and blood samples were kept in refrigerator at 4⁰ c in separate sterile vials till analysis.

Urine samples were collected in 100 ml sterilized vials from both healthy and affected animals directly from mid stream during micturation before falling on ground.

Analysis of Biological Samples

Determination of total leucocytes count (TLC), total erythrocyte count (TEC), erythrocyte sedimentation rate (ESR), haemoglobin (Hb%), packed cell volume (PCV) and blood glucose. Determination of total leucocytes count (TLC) and total erythrocyte count (TEC) were done by using Hemocytometer (Neubaur’s Chamber), while erythrocyte sedimentation rate (ESR), Haemoglobin (Hb%) and Packed Cell Volume (PCV) were done by wintrobe tube method, acid hematin and wintrobe method respectively (Brar et al., 2004). Blood glucose was estimated by Folin-wu-method as described by Murtza (1998) and differential leucocytes count (DLC) was done by blood smear prepared earlier and stained with Leishman’s stain. Collected serum sample was used for the study of aspartrate amino transferase (AST), alanine amino transferase (ALT), total serum protein (TSP), serum albumin, serum creatinine and serum uric acid by standard method as described by Murtza (1998). While serum globulin was calculated by subtracting serum albumin to total serum protein, albumin, globulin Ratio was estimated by using formula serum albumin divided by serum globulin. Collected Urine samples were used for quantitative analysis of sugar and protein by Benedicts method and Esbach’s methods respectively as described by Murtza (1998).

Statistical Analysis

Descriptive statistical parameters as mean, standard error and regression coefficient were calculated as per described method (Snedecor and Cochran, 1987).

Results

The disease was found more prevalent in most of the district of Bihar like Madhepura, East-Champaran, West-Champaran, Bhagalpur, Saharsa, Samastipur, Darbhanga, Purnea, Muzaffarpur, Patna, Gaya, Mungar and almost each district of Bihar.

Symptomatology of Gangrenous Syndrome

The animals suffering from gangrenous syndrome exhibited variant symptoms. Like swelling of extremities dull and depress appearance, suppressed appetite, weakness, unaltered body temperature but in some cases hyperthermia was found due to secondary bacterial infection in wounds and decubitus. Important lesions noted in the affected animals were inflammation of extremities, disinclination to move, and necrotic changes on extremities like tip of tail, tip of pinna and in some cases on the teats. Beside these sloughing of hooves, muzzle and other distal part of body was also observed. In some case detachment on tongue was also observed due to which affected animal become anorectic and emaciated. The hematological and biochemical parameters were analyzed in cattle and buffalo affected from gangrenous syndrome depicted in Table 1.

Table1: Haemato-biochemical parameters observed in cattle and buffalo suffering from Bovine Gangrenous Syndrome

Discussion

In the present study clinical signs and symptoms of gangrenous syndrome were appeared in both cattle and buffaloes of all age groups. The pathognomic changes of disease comprises of degenerative and gangrenous change in distal extremities of animals like hooves, tail, tip of pinna, muzzles etc. Gangrenous area become slough resulting in ulceration and wounds of varying size, few develops anorexia, and become weak and emaciated (Rashid et al. 2004). The root causes of the disease are various and a great deviation is there regarding etiology. It is considered to be a result of chronic selenium toxicity (Sahay, 2002) or mycotoxicosis (Prasad, 2000). However feeding of rice straw containing high content of oxalate and fungi belonging to genera Aspergillus, Ascidia, Fusarium and Penicillium have been found associated with the diseases (Kalra et al., 1972). The disease has been experimentally produced in buffalo calves by feeding mouldy and moist rice straw collected from the fields (Behra, 1990) (Bhatia, 1978).

In present study the total leukocyte count (TLC) (x10³/µL) was observed as 7.78±0.164; 8.62±0.008; 6.75±0.004 and 8.62±0.08 (x10³/µL) in control and affected group of cattle and buffaloes respectively. Chakrabarty (2002) has reported the normal value of total W.B.C. count as 7.03 and 6.70 (thousand/cc) in normal healthy cow and buffaloes respectively which is in close resemblance with the control group of present study. Sharma et al. (1985) observed the normal physiological range of total leukocyte count as 6.89±0.30 (x10³/µL) in female buffaloes while TLC observed by Prasad (1997) was 6.7 in normal buffaloes. Kaushik et al. (2004) reported the significant increase in total leukocyte count in both cattle and buffaloes suffering from gangrenous syndrome.

The mean haemoglobin (Hb %) (gm %) was found as 12.60±0.21, 9.22±0.32, 9.01±0.187 and 7.37±0.13 in control and affected group of cattle and buffaloes respectively . Chakrabarty (2002) also reported the average normal level of haemoglobin as 11.3 and 12.90 (gm/100ml) in cattle and buffaloes respectively. A significant (p<0.05) decrease of 3.38 and 1.64 gm% was observed in present study in affected group than control group. A decrease in Hb% was observed by Kumar et al. (2003) in foot affected cross bred cows; where as Kaushik and Sinha (2004) reported a non-significant decrease in Hb% in clinical cases of gangrenous syndrome. The decrease in Hb% in present study is also in agreement with Basak et al. (1994) who observed the average Hb% as 6.65 gm/dl in buffaloes suffering from Degnala like disease. The average total erythrocyte count (TEC) (x10⁶/µL) was noted as 5.38 ± 0.14, 4.009±0.081, 5.74±0.135 and 4.12±0.10 in control and affected group of cattle and buffaloes respectively. A significant (p<0.05) low TEC was observed as 4.009 and 4.12 (x10⁶/µL) in cattle and buffaloes respectively in present study. This decrease in TEC was in agreement with Basak et al. (1994) who observed the total erythrocyte count as 3.32 million/c.mm.

Mean packed cell volume (PCV) % in present study was observed as 31.62±0.39, 21.60±0.10, 30.22±0.14 and 17.77±0.08 in control and affected group of cattle and buffaloes respectively. The PCV remain high at birth (42%) but reduces (35%) after 24 month as the calf begins to nurse Benjamin (2007). In this study PCV observed was 31.62±0.39 and 30.22±0.14 in healthy control of cattle and buffaloes. Sharma et al. (1985) has reported the normal PCV of buffaloes 40.2±6.8 (%) while Prasad (1997) has observed 44.30% and 33.70% PCV in buffaloes and cattle respectively.  Significant decrease in PCV was also reported by Kaushik et al. (2004) in both cattle and buffaloes suffering from gangrenous syndrome. Kumar and Behra (2003) observed significantly decreased PVC% in foot affected crossbred cows. Basak et al. (1994) also observed a low PCV (%) in buffaloes suffering from spontaneous degnala like disease. However, Maqbool et al. (1997) reported normal PCV in affected buffaloes. ESR value observed as ‘0’ and 27.78 in cattle and buffaloes in the study respectively. Although increased ESR was observed in affected cattle but varied within the normal level, where as a significant (p<0.05) increase of 18.12 (mm/hr) was noted in ESR of affected group of buffalo in comparison to control group. This increase in ESR was in agreement with the findings of Basak et al. (1994) and Kaushik and Sinha (2004).

The mean alanine spartrate transferase (AST) (IU/ L) value noted as 57.12 ± 0.53, 101.28 ± 0.31, 62.8 ± 0.58 and 108.64 ± 0.23 in control and effected group of cattle and buffalo respectively.  In present study the normal value observed for AST is 57.12 and 62.8 for cattle and buffalo respectively. Significant (p<0.05) increase is AST value was recorded in affected group of both cattle and buffalo. Yeruham et al. (1993) also observed increased limit of AST in cattle due to muscle damage as a result of the prolonged recumbence of sick animal. However, Jadhav et al. (2001) reported no significant change in AST value of buffaloes suffering from Degnala like disease. Alanine amino transferase (ALT) value observed as 12.98 ±0.39, 26.96 ± 0.17, 18.42 ± 0.22 and 39.30 ± 0.15 in control and affected group of cattle and buffalo respectively.  Affected group of both cattle and buffaloes showed an elevated value of ALT supported with Yeruham et al. (1993) and Jadhav et al. (2001). The mean serum creatinine (mg/dl) values were observed as 1.27 ± 0.04; 1.94 ± 0.03; 1.05 ± 0.07 and 1.58 ± 0.04 in control and affected group of cattle and buffaloes respectively. Creatinine is an endogenous substance that arises mostly form phosphocreatinine in muscle under physiological condition.. In present study a significant (p<0.05) increase in serum creatinine value (mg/dl) was observed in affected group of both cattle and buffaloes as compared to healthy control group. This study might be indicative of reduced GFR (glomerular filtration rate) due to degenerative change in kidney. This can further be supported by the finding of Dhillon et al. (1990) and Ranjan et al. (1977) who observed a degenerative change in kidney, where as Gill et al. (2003) observed the atrophy and necrotic changes in kidney of buffalo calves. The average value of serum uric acid (mg/dl) in present study were noted as 1.15 ± 0.02, 1.54 ± 0.05, 1.095 ± 0.03 and 1.44 ± 0.08 in control and affected group of cattle and buffaloes respectively. A significant (p<0.05) increase was noted in affected group of both cattle and buffaloes. Uric acid is an end product of purines. In most of mammals except primates, birds and Dalmatian breed of dog, it get metabolize completely into allantoin by the action of enzyme uricase. The principle site for conversion of uric acid is liver. Uric acid conversion is reduced if any degenerative change in liver occurs (Benjamin, 2007). The study revealed increase in serum uric acid in affected group of both cattle and buffaloes might be due to degeneration in liver which were supported with Gill et al. (2003) and Dhillon et al. (1990) of buffalo calves suffering from chronic selenosis.

Mean level of total serum protein (gm /dl) were noted as 7.654±0.05, 8.535±0.159,7.346±0.02 and 8.940±0.05 in control and affected group of cattle and buffaloes. The value has increased significantly (p<0.05) in clinically affected group as compared to control (healthy) group of cattle and buffaloes. This increase in total serum protein of affected group of both cattle and buffaloes in comparison to control group might be due to increase in serum globulin level and proliferation of plasma cell as a result of immune response. This clearly indicates that increase in globulin might be due to protective mechanism.  Ghose et al. (1993) observed low level of total serum protein in clinically affected animals suffering from degnala disease where as Jadhav et al. (2001) observed non-significant change in total serum protein in clinical cases of degnala disease. Serum albumin (gm %) were found as 2.674±0.03, 2.254±0.036, 2.59±0.029 and 2.69±0.023 in control and affected group of cattle and buffaloes respectively. A significant (p<0.05) decrease was noted in affected cattle than control, while a significant (p<0.05) increase was observed in affected group of buffaloes than control group. Maqbool (1997) reported the total albumin value varied from 2.60 to 2.90 g/dl in normal group of buffaloes. Total albumin of affected of group of cattle decreased (p<0.05) significantly while Kaushik and Sinha (2004) reported an insignificant change in albumin in clinical gangrenous syndrome. The increase in total albumin of buffaloes in present study was in agreement with Hokonohara et al. (2003). The mean globulin (gm %) value was noted as 4.98 ± 0.03, 6.27± 0.138, 4.75±0.031 and 6.23±0.055 in control and affected group of cattle and buffaloes respectively. Maqbool (1997) reported the average total globulin varied form 4.60 to 4.70 for healthy buffaloes. A significant (p<0.05) increase was observed in affected group of both cattle and buffaloes as compared  to control group  which  was in  agreement with Kaushik and Sinha (2004) and Hokonohara (2003), where as   Jadhav et  al. (2001) reported a non-significant variation in total globulin concentration in clinical cases of Degnala disease buffaloes.

The geometric mean of Albumin and globulin ratio were found 0.536, 0.363, 0.541 and 0.427 in control and affected group of cattle and buffaloes respectively.. The normal value of A: G ratio in bovine is 0.70 (Kaneko et al., 1997). In cattle relative amounts of albumin and globulin remains nearly equal or globulin tend to be in excess of albumin (Benjamin, 2007). However, in liver disease the ratio usually altered by a decrease in albumin or increase in globulin.  A decrease in A: G was noted in present study which is in agreement with the findings of Kaushik and Sinha (2004). However, Jadhav et al. (2001) observed no significant variation in A: G ratio in clinical cases of degnala disease. A decrease in A: G ratio might be due to hyerglobunemea and chronic liver degeneration (Chakrabarti, 2002). The blood glucose (mg/dl) was observed as 47.16±0.64; 43.64±0.19; 52.38±0.58 and 44.69±0.27 in control and affected group of cattle and buffaloes respectively. Normal glucose level in whole blood of healthy cattle varied as 45-75 mg/dl (Dhanotiya, 2004). The mean blood glucose value of cattle and buffaloes of control group was noted within normal range. However, a significant (p<0.05) decrease of 3.52 and 7.69 (mg/dl) was observed in affected group of both cattle and buffaloes respectively.  This study supported with Ghose et al. (1993) and Gupta et al. (1983) where as Kumar and Behara (2003) observed a non-significant changes in blood glucose level in foot affected crossbred cows.

Urine sugar was significantly increased (p<0.05) in affected group of cattle and buffaloes as compared to control group of cattle and buffaloes respectively. This might be due to decreased re absorption of renal glucose (Radostits et al., 2004) and higher level of glucose in urine is also present in chronic liver disease (Chakrabarti, 2002). It may also occur in any other cause of hyperglycemia or decreased tubular glucose re absorptive capacity supported with Ranjan et al. (1977). The  protein level in urine of affected group were observed as 1.297 (gm%) and 1.206 gm.% of cattle and buffaloes which were significant (p<0.05) increased by 0.968 gm % and 0.88 gm % in cattle and buffaloes than control group respectively supported by  Gill et al. (2003), Rodostits et al. (2004).

Conclusion

Clinical cases of 150 out of which 16 buffaloes and 14 cattle of all age and sex were examined and noted for the gross lesions of the disease. The study showed that   Hematological and biochemical  alterations  in the Clinical gangrenous syndrome represents symptoms like lameness, swelling at the coronet at the junction of skin and hooves, Oedema, necrosis, and sloughing off of tissues of extremities of animals . Hence the study revealed and conclude that hematological and biochemical alterations should be considered in order to achieve early diagnosis and initiation of appropriate therapeutic regimen for a favorable outcome of the disease.

Acknowledgement

The authors are highly grateful to Dean, Bihar Veterinary College, Patna-800014, Bihar, India for giving necessary facilities to conduct this investigation

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