Blood and serum samples
A total of 269 dogs of either sex and all ages suspected for T. evansi infection were selected from different localities in Rayalaseema region of Andhra Pradesh. Dogs showing the signs of lymph adenopathy, chronic emaciation, fever, corneal opacity and hepatitis were included in the present study. Collected blood was stored in separate vials with and without addition of 10 % ethylenediaminetetraacetic acid (EDTA). The blood with anti-coagulant was used to detect T. evansi by wet blood film examination (WBF) (Sivajothi et al. 2013b). The type of organism was diagnosed based on the morphological studies of the stained blood smears (Fig. 1). The blood without anti-coagulant was used for the serum collection. The serum was separated and transferred into sterilized vials after adding one drop of 1:10,000 sodium azide solution and stored at −20 °C till used.
T. evansi parasites were collected from the infected dogs and multiplied in the Wister rats for antigen preparation. T. evansi parasites were separated from the blood of experimentally infected Wister rats at high parasitaemia by DEAE (diethyl amino-ethyl cellulose) anion-exchange column chromatography method. Separated trypanosomes were washed twice in PSG (PBS, pH 8.0 with glucose at 1:1) by repeated centrifugation. The purified parasites were then sonicated at 150 W for 3–4 cycles of 30 s each by ultrasonic disintegrator. The sonicated material was centrifuged at 2400 × g for 20 min at 4 °C. The collected supernatant was designated as purified whole cell lysate T. evansi antigen (WCL Ag), and it was partially purified after precipitating with 50 % saturated ammonium sulphate followed by extensive dialysis against PBS, pH 7.4. The protein content of WCL Ag was estimated and was adjusted to 1.0 mg/ml in PBS, pH 8.0, and stored at −20 °C in 1.0-ml aliquots (Sivajothi et al. 2014c).
Raising of hyper immune sera
The hyper immune serum (HIS) was raised in two healthy New Zealand white rabbits. Pre-immunized serum of these experimental rabbits was also stored at −20 °C till use as negative control serum for standardization of colloidal dye immunobinding assay (CDIA) in the present investigation (Sivajothi et al. 2014c).
Development of CDIA
The test was performed in a flow-through module, in which the whole cell lysate antigen of T. evansi-coated nitrocellulose membranes was pressed tightly to a water-absorbing pad.
In the assay, antibodies in the serum sample are captured by the antigen of T. evansi spotted on to nitrocellulose membrane mounted on a flow-through test device that serve as the assay capture matrix. The bound antibodies are visualized by the addition of protein A colloidal gold conjugate, which served as antigen-antibody-detecting reagent imparting pink colour to the membrane as a dot. Schematic diagram of colloidal dye immunobinding assay for detection of T. evansi antibodies was mentioned in the Fig. 2.
Detection of T. evansi antibodies by CDIA
The nitrocellulose membrane (M/s mdi, Ambala Cantt, India) was placed above the absorbent pads in a flow-through module. One microlitre (1 g/dl) of whole cell lysate antigen was placed at one end of the module (T side), and 1 μl of known negative serum was placed at the other end (C side), which acted as reagent control. The membrane was dried in an incubator at 37 °C for 1 h or overnight at room temperature, to which 100 μl of wash buffer (25 mM PBS pH 7.0 containing 1 % BSA and 0.05 % Tween 20) was added and allowed to be absorbed through the membrane. Then, 100 μl of test serum diluted in wash buffer (1 in 10) was added and allowed to be absorbed through the membrane. Following this, the membrane was washed with wash buffer. Thereafter, 100 μl of wash buffer diluted (1 in 2) protein A colloidal gold conjugate was added. The appearance of two red colour dots in the ‘T’ and ‘C’ sides of the test window indicated a positive reaction (Fig. 2), and the absence of a red colour dot in the T side of the test window indicated a negative reaction. The test was considered invalid if there were no red dots in the window.
Screening of blood and serum samples
A total of 269 blood and serum samples were tested simultaneously with CDIA and wet blood film examination. The assay (CDIA) has been validated with wet blood film examination.