Effect of claw disorders on haemato-biochemical parameters and acute phase protein levels in crossbred cattle

Lameness is one of the major problem concerning dairy industry and is ranked 3^rd after mastitis and infertility (Weaver et al., 2005). The economic losses incurred are due to low milk production, weight loss, culling cost, reduced reproductive performance and treatment costs. The condition leads to severe pain and discomfort to the animal and as such raises animal welfare issue. In India, with the improvement of germplasm of dairy cattle, the milk producing ability has increased tremendously but these high yielders are predispose to lameness (Green et al., 2002). Hematology is primarily aimed to monitor the health status and to detect possible diseases. The general condition of lame animal is often affected and hematology, as one of the important methods of paraclinical diagnosis also gets changed (El-Ghoul et al., 2000). In addition, haematology can be used to evaluate animal stress and welfare levels (Brucka-Jastrzêbska et al., 2007). The presence of serum proteins and enzymes like AST and ALT in varying concentration are indicators of pathological differentiation in body tissues (Milinković-Tur et al., 2005). Acute phase proteins are released from the liver after activation of hepatocytes by the pro-inflammatory cytokines (IL-6 and TNF-α) which are secreted by the monocytes in response to bacterial toxins or tissue injury. These biomarkers are now being widely used to improve diagnosis, monitoring treatment and prognosis of many diseases. Haptoglobins (Hp) has diagnostic value in the lame dairy cows (Jawor et al., 2008). Accordingly, study was framed to evaluate haematological and biochemical alterations including acute phase protein levels in dairy cattle suffering from lameness due to claw disorders.

Thirty four HF crossbred dairy cattle from 11 farms (3 organized and 8 unorganized) suffering from lameness were selected from various locations in the Jammu division and to compare the levels of various parameters, 10 healthy animals were also sampled.
 
Hoof Examination
 
Each lame animal was properly restrained and different foot lesions were identified after performing hoof trimming as per standard procedure given by Raven (1989).
 
Categorization of lame animals
 
34 lame animals were categorized under various groups based on: a) Locomotion score-I, II, III and IV as per Wells et al., (1993). Seven animals were placed under LS 1 score, 5 animals in LS 2, 16 animals in LS 3 and 6 animals in LS 4. b) Lesion-wise-Affected animals were placed under two groups based on zone description of hoof: Group I comprised of 17 animals having lesions like white line haemorrhage (WLF), heel erosion (HE) and over grown hoof (OGH) whereas, group II comprised of 17 animals having lesions like sole haemorrhage (SH), sole ulcer (SU), toe ulcer (TU) and toe haemorrhage (TH). c) Severity-According to the severity of the lesions and the duration of disease, lame animals were designed as acute cases (having history of lameness <4 days duration) in which 11 animals were identified and chronic cases (having history of lameness >4 days duration) in which 23 animals were identified.
 
Sampling
 
Blood samples were collected from each cattle affected with lameness. For the estimation of biochemical constituents, a part of blood sample (15 ml) was collected by jugular venipuncture into a 30 ml stoppered mineral free heparinised glass vials (dipped overnight in 2N HcL). For determining Hb, PCV, TLC, TEC, DLC, 2 ml of blood sample was collected from each selected animal in sterile plastic tubes containing dipotassium salt of EDTA as anticoagulant (Hi Media Mumbai, @ 2 mg/ml of blood). To prevent haemolysis of blood samples during transportation ice box was used. Blood samples meant for mineral and biochemical estimations were centrifuged at 3000 rpm for 30 minutes to separate plasma immediately after collection to prevent haemolysis. Plasma samples were stored at -10°C in deep freeze for subsequent analysis.
 
Analysis
 
The haematological parameters were analyzed from collected blood samples as per Jain (1993). Standard kits procured from Erba Mannheim and Agappe Diagnostics Ltd were used for determination of biochemical parameters which included total plasma proteins (Biuret Method), albumin (BCG Dye Method). The liver enzymes were also determined using standard kits which included ALT, AST (Modified IFCC Method) and ALP. Fibrinogen was determined by refractometer method described by Schalm (1980). Plasma was used for the estimation of haptoglobin (Hp) employing ELISA kits supplied by the Bioassay Technology Laboratory. C-reactive protein (CRP) estimation was done with the help of ELISA kits supplied by the Cloud -Clone Corp. Data was statistically analyzed using student’s t-test (Independent samples test) and General linear model:multivariate analysis (Turkey) using SPSS version 16 software.

The average values of Hb, TEC, PCV and TLC among the lame and healthy animals having LS-0 are shown in Table 1. Significant (p<0.05) decline in the levels of Hb, TEC, PCV was observed among lame animals along with significant (p<0.05) increase in TLC. Significant (p<0.05) increase in average values of neutrophil and monocyte counts along with significant (p<0.05) decline in lymphocyte count among lame animals was recorded. Compared with acute lameness cases, animals with chronic lameness showed significant (p<0.05) decline in Hb, TEC and PCV levels. Severity-wise, animals with LS-4 had significantly (p<0.05) declined levels of Hb, TEC and PCV. Conflicting reports on effect of lameness on haematological parameters are available in the literature. Normocytic-normochromic anaemia in lame animals was reported by Pairizi and Khatafizadeh (2006), whereas Al-qudah and Ismail (2012) reported non-significant decline in RBC, Hb and PCV levels in lame cows. The factor responsible for anemia in lameness is unclear however, variety of cytokines involved in the chronic inflammatory process might be contributing to development of anemia (Duncan et al., 1994).
 

        
Significant (p<0.05) increase in TLC along with non-significant (p>0.05) increase in average values of neutrophil, eosinophil and monocyte counts were observed which corroborates with finding of Al-qudah and Ismail (2012). Neutrophil count of the animals with LS 1, 2 and 4 was significantly higher than control group animals. These changes are probably due to reactive leukocytosis and stress. The increase in the number of monocytes in the lame cattle is probably due to chronic inflammatory response (Jain, 1993). It was concluded that hematological parameters change in severe lameness and these changes were probably related to inflammatory events and possibly to malnutrition of affected animals due to pain and anorexia.
        
Significant (p<0.05) rise in the levels of AST, ALT, TPP and globulin along with non significant (p>0.05) increase in average values of ALP, albumin and A-G ratio was recorded (Table 1). Score-wise, animals with LS-3 had significantly elevated level of AST, ALT, TPP and globulin. Sprecher et al., (1997) and Seyrek et al., (2008) reported that cows with lameness score 1 and 2 should be considered healthy and those with scores of 3-5 as lame. The albumin level was higher among animals with LS 3, while it was lowest in cows with LS 4 which could be due to disease severity. This decline in albumin level could be due to the severity of disease. Whitaker et al., (1999) reported that albumin and globulin levels help to evaluate chronic inflammatory diseases. Study concludes that globulin level increases while albumin decreases in illness. Acute phase reaction initiates reduction in albumin synthesis (Gabay and Kushner 1999). Contrary to present study, Yaylak et al., (2009) reported non-significant (p>0.05) alterations in the level of AST, ALT, TPP, TG, cholesterol and albumin level by lameness scores. The variations in the finding could be due to varying etiology of lameness as in present study only claw disorders were considered.
        
Significant (p<0.05) increase in average value of haptoglobin, CRP and fibringogen was observed among lame animals. Overall average values of haptoglobin, CRP and fibrinogen among the lame animals were 924.59± 59.13µg/ml, 65.00± 1.98µg/ml and 8.75± 0.51mg/dl compared with 607.02± 49.13µg/ml, 13.79± 0.80µg/ml and 2.78± 0.17mg/dl among healthy animals, respectively (Table 1). Bagga (2014) reported elevated levels of serum CRP and amyloid A (SAA) levels in lame cows. Increased concentrations of Hb and fibrinogen have been described in lame cows with clinical signs of claw diseases earlier by Jawor et al., (2008) and Smith et al., (2010). Tothova et al., (2014) reported fivefold higher levels of Hp and fibrinogen in heifers with clinical signs of hoof diseases and lameness. Hp primarily functions by binding free Hb released from RBC’s and thereby inhibit oxidative activity (Yang et al., 2003). This binding of Hp with Hb decreases availability of haem residue for growth of bacteria (Murata et al., 2004). Fibrinogen level increases 2-3 fold during inflammatory reaction leading to increased blood viscosity, aggregation of RBC thereby contribute to development of atherosclerotic plaques (Medcalf 2007). Heegaard et al., (2000) concluded that severity of infection and extent of underlying tissue damage influences magnitude and duration of acute phase response.

Study concluded that animals suffering from various claw disorders show significant (p<0.05) decrease in Hb, PCV, TEC and lymphocyte count with significant (p<0.05) increase in neutrophil count. Similar trend was observed in animals showing varying locomotion score, lesions and duration of lameness. Biochemical analysis revealed significant (P<0.05) increase in AST, ALT, TPP and globulin among lame animals. Lameness due to claw disorders is associated with a systemic acute phase response as significant (p<0.05) increase in levels of fibrinogen, Hp and CRP was observed.