Evaluation of rHyC based dot-ELISA
The dot ELISA was performed with rHyC under optimized conditions to determine the assay specificity and cross reactivity using known positive sera against different important parasitic and bacterial diseases of goats. The diagnostic specificity (DSp) and diagnostic sensitivity (DSn) of the dot ELISA were determined by, using a two-sided contingency table (Table 1.) . Known positive serum samples with brown spots compared to nil in negative serum were reported as positive. The sensitivity and specificity were calculated by using the following formula: Sensitivity = (TP/TP+FN) × 100; Specificity = (TN/TN+FP) × 100; where, TP-true positive; TN-true negative; FP-false positive and FN-false negative. The positive predictive value (PPV) and negative predictive value (NPV) were also calculated based on the optimized assay as shown in Table 1 .
Also, the inter-rater reliability of dot-ELISA was evaluated with Cohen’s Kappa test, taking the rHyC based microtitre plate indirect-ELISA as reference standard.18, 29 A set of 149 sera samples (69 positive and 80 negative) from ICAR-NF laboratory were tested for evaluating the kappa index between both assays for the diagnosis of GWFI.
In a separate experiment, the optimized dot ELISA was tested with neat samples (blood, plasma and serum) and lower dilutions at lower incubation temperature (18 °C) and time (30 min) to check the suitability of the test at field conditions under sub-tropical and temperate climates of GWFI prevalent regions. Samples of a known GWFI positive goat were used for blood and plasma whereas a GWFI negative goat was used to derive negative control samples of blood, plasma and serum. The positive samples of GWFI (blood, plasma and weak positive serum) were tested as neat, 1:2 and 1:10 dilutions and control negative samples for neat blood, plasma and serum. A weak positive serum was taken as positive control to ensure reactivity of weak titre at lower time and temperature of incubation.
Serum samples (n = 274) from different regions of Union territory of Jammu and Kashmir that were available in the ICAR-NF laboratory were tested with the optimized dot-ELISA.