Citrated plasma samples were sent to the University of Miami Avian and Wildlife Laboratory for clinical determination of total protein, plasma electrophoresis (pre-albumin, albumin, alpha 1 globulins, alpha 2 globulins, beta globulins, gamma globulins) and aspartate aminotransferase and creatine phosphokinase activities.

Range finding trial in which kestrels were fed diets containing varying quantities of brodifacoum and signs of intoxication were monitored.

Range finding trial in which kestrels were fed diets containing varying quantities of brodifacoum and signs of intoxication were monitored.

Trial examining blood clotting function response in kestrels initially fed a diet containing chlorophacinone (CPN) or brodifacoum (BROD), and following a recovery period, kestrels were challenged with a diet containing chlorophacinone. Kestrels received two 25 ± 0.1 g NBP meatballs daily for a 7-day period containing either vehicle, 1.5 µg CPN/g wet wt diet (i.e., 1.5 ppm chlorophacinone) or 0.5 µg brodifacoum/g wet wt (i.e., 0.5 ppm brodifacoum) during an initial exposure phase. Following 7 day recovery period, these kestrels were then fed 0.75 µg CPN/g wet wt diet (i.e., 0.75 ppm chlorophacinone) for a 7 day challenge exposure phase. Hereafter, these groups are designated control-chlorophacinone challenge (CON-CPN), chlorophacinone-chlorophacinone challenge (CPN-CPN), and brodifacoum-chlorophacinone challenge (BROD-CPN).

Trial examining blood clotting function response in kestrels initially fed a diet containing chlorophacinone (CPN) or brodifacoum (BROD), and following a recovery period, kestrels were challenged with a diet containing chlorophacinone. Kestrels received two 25 ± 0.1 g NBP meatballs daily for a 7-day period containing either vehicle, 1.5 µg CPN/g wet wt diet (i.e., 1.5 ppm chlorophacinone) or 0.5 µg brodifacoum/g wet wt (i.e., 0.5 ppm brodifacoum) during an initial exposure phase. Following 7 day recovery period, these kestrels were then fed 0.75 µg CPN/g wet wt diet (i.e., 0.75 ppm chlorophacinone) for a 7 day challenge exposure phase. Hereafter, these groups are designated control-chlorophacinone challenge (CON-CPN), chlorophacinone-chlorophacinone challenge (CPN-CPN), and brodifacoum-chlorophacinone challenge (BROD-CPN).

Trial in which coagulopathy and the time course of recovery of clotting function was determined in kestrels fed a diet containing brodifacoum.

- Observations of test subjects, - Body weight - Estimates of test diet consumption - Hematocrit - Clotting time parameters (prothrombin time, Russell’s viper venom time, fibrinogen concentration) - Residues of brodifacoum and chlorophacinone in tissue

- Observations of test subjects, - Body weight - Estimates of test diet consumption - Hematocrit - Clotting time parameters (prothrombin time, Russell’s viper venom time, fibrinogen concentration) - Residues of brodifacoum and chlorophacinone in tissue

A heterologous thrombin clotting time assay (TCT) was used to measure the time for conversion of fibrinogen to fibrin using commercially available reagents. Human reference material included in the kit was diluted with imidazole buffered saline (IBS; 0.0125M imidazole-0.109 M sodium chloride, pH7.4) to generate a standard curve. Each kestrel plasma sample was thawed at 37ºC and diluted with IBS, and following incubation at 37ºC, the reaction was then initiated by the addition of bovine thrombin reagent supplied with the assay kit, with clotting time measured to 0.1 s. Fibrinogen concentration was determined in a single assay for each of the 3 study trials, with reference samples interspersed among study samples. The intra-assay variation of kestrel study samples (duplicate determinations for trials 1 and 3) and reference samples (multiple determinations of plasma pools in trials 1, 2 and 3) were calculated. For Russell’s viper venom time (RVVT), reconstituted RVV Factor X activator was diluted 1:10 with IBS and maintained at room temperature. Citrated plasma samples were diluted with phosphate buffer (8.3 mM phosphate buffer, pH 7.2) (3:5 dilution), incubated at 37ºC and diluted RVV was added and incubated for 10 s. The clotting reaction was initiated with 25 mM CaCl2, with clotting time measured to 0.1 s. Baseline and/or terminal study samples, and multiple inter-assay (inter-batch) human reference and kestrel plasma sample pools, were measured using this assay. For the prothrombin time (PT) assay, crude chicken hatchling thromboplastin (CHT) was prepared by the method of Quick, as modified and previously described (Rattner et al 2010). Plasma samples were diluted phosphate buffer (4:5 dilution), incubated at 37ºC, and the reaction was initiated by the addition of CHT in 25 mM CaCl2 (1:8 dilution of CHT working solution) with clotting time measured to 0.1 s. Baseline and/or terminal study samples, and multiple inter-assay (inter-batch) kestrel plasma sample pools, were measured using this assay.

A heterologous thrombin clotting time assay (TCT) was used to measure the time for conversion of fibrinogen to fibrin using commercially available reagents. Human reference material included in the kit was diluted with imidazole buffered saline (IBS; 0.0125M imidazole-0.109 M sodium chloride, pH7.4) to generate a standard curve. Each kestrel plasma sample was thawed at 37ºC and diluted with IBS, and following incubation at 37ºC, the reaction was then initiated by the addition of bovine thrombin reagent supplied with the assay kit, with clotting time measured to 0.1 s. Fibrinogen concentration was determined in a single assay for each of the 3 study trials, with reference samples interspersed among study samples. The intra-assay variation of kestrel study samples (duplicate determinations for trials 1 and 3) and reference samples (multiple determinations of plasma pools in trials 1, 2 and 3) were calculated. For Russell’s viper venom time (RVVT), reconstituted RVV Factor X activator was diluted 1:10 with IBS and maintained at room temperature. Citrated plasma samples were diluted with phosphate buffer (8.3 mM phosphate buffer, pH 7.2) (3:5 dilution), incubated at 37ºC and diluted RVV was added and incubated for 10 s. The clotting reaction was initiated with 25 mM CaCl2, with clotting time measured to 0.1 s. Baseline and/or terminal study samples, and multiple inter-assay (inter-batch) human reference and kestrel plasma sample pools, were measured using this assay. For the prothrombin time (PT) assay, crude chicken hatchling thromboplastin (CHT) was prepared by the method of Quick, as modified and previously described (Rattner et al 2010). Plasma samples were diluted phosphate buffer (4:5 dilution), incubated at 37ºC, and the reaction was initiated by the addition of CHT in 25 mM CaCl2 (1:8 dilution of CHT working solution) with clotting time measured to 0.1 s. Baseline and/or terminal study samples, and multiple inter-assay (inter-batch) kestrel plasma sample pools, were measured using this assay.