Immuno-pathologic effects of oral administration of chlorpyrifos in broiler chicks

Figures & data

Table 1. Mortality of broiler chicks administered orally various doses of chlorpyrifos.

	Mortality
Groups	Number died	%
CPF-5	3	10.34
CPF-10	6	20.68
CPF-20	10	34.48
Xylene	2	6.89
Control	1	3.44

Each group was comprised of 29 broiler chicks. Groups received chlorpyrifos (CPF diluted in xylene) at 5, 10, and 20 mg/kg BW (CPF-5, CPF-10, CPF-20, respectively) per os daily for 15 days. Xylene and control group received xylene (1 ml/kg BW) and distilled water (1 ml/kg BW), respectively, each day. Total duration of the experiment (treatment and post-treatment periods) was 45 days. From this data, the LD₅₀ value for CPF was calculated to be ≈ 20 mg/kg.

Table 2. Relative weights (% of BW) of organs from chicks given daily oral doses of CPF.

Experimental group	Spleen	Bursa of fabricius	Thymus
Treatment Day 15
CPF-5	0.13 ± 0.02	0.13 ± 0.04*	0.44 ± 0.14
CPF-10	0.11 ± 0.03	0.11 ± 0.03*	0.40 ± 0.16
CPF-20	0.13 ± 0.02	0.25 ± 0.06*	0.45 ± 0.08
Xylene	0.13 ± 0.02	0.35 ± 0.08	0.35 ± 0.10
Control	0.10 ± 0.03	0.37 ± 0.06	0.37 ± 0.09
Post-treatment Day 15
CPF-5	0.13 ± 0.05*	0.05 ± 0.02*	0.38 ± 0.14
CPF-10	0.14 ± 0.02*	0.06 ± 0.02*	0.47 ± 0.04
CPF-20	0.14 ± 0.03*	0.05 ± 0.01*	0.49 ± 0.06
Xylene	0.08 ± 0.01	0.08 ± 0.01	0.37 ± 0.08
Control	0.09 ± 0.01	0.09 ± 0.02	0.42 ± 0.05
Post-treatment Day 30
CPF-5	0.08 ± 0.02	0.06 ± 0.01	0.35 ± 0.03
CPF-10	0.12 ± 0.02	0.08 ± 0.02	0.36 ± 0.06
CPF-20	0.13 ± 0.05	0.06 ± 0.03	0.49 ± 0.07
Xylene	0.17 ± 0.09	0.07 ± 0.02	0.45 ± 0.05
Control	0.10 ± 0.04	0.05 ± 0.03	0.44 ± 0.08

*Value (mean ± SD) significantly different from control at specific time-point (p ≤ 0.05).

Table 3. Anti-SRBC titers in chicks given daily oral doses of CPF.

	Post-primary injection (days)		Post-booster injection (days)
Groups	7	14	7	14
CPF-5	4.8 ± 0.4	1.4 ± 0.5*	3.2 ± 1.30*	2.8 ± 1.1*
CPF-10	2.6 ± 1.5*	1.2 ± 0.4*	2.4 ± 0.9*	2.2 ± 0.8*
CPF-20	1.4 ± 1.1*	0.6 ± 0.5*	3.4 ± 1.1*	2.2 ± 0.8*
Xylene	4.6 ± 0.8	3.0 ± 0.9	4.8 ± 0.8	3.4 ± 0.5
Control	5.8 ± 0.4	3.4 ± 0.5	5.4 ± 0.5	4.4 ± 0.5

*Value (mean ± SD) significantly different from control at indicated time-point (p ≤ 0.05).

Table 4. Anti-Newcastle disease vaccine titres in chicks given daily oral doses of CPF.

Group	Treatment Day 0	Treatment Day 15	Post-treatment Day 15	Post-treatment Day 30
CPF-5	4.8 ± 0.4	5.8 ± 1.6*	5.8 ± 0.8	7.6 ± 1.7
CPF-10	5.0 ± 0.7	5.0 ± 0.7*	5.4 ± 1.1	6.8 ± 0.8
CPF-20	4.8 ± 0.4	3.8 ± 0.8*	4.8 ± 0.8*	6.0 ± 1.0*
Xylene	5.2 ± 0.4	6.8 ± 0.8	5.6 ± 0.9	7.6 ± 1.5
Control	5.0 ± 0.7	7.2 ± 0.8	6.2 ± 0.8	7.8 ± 1.3

*Value (mean ± SD) significantly different from control at specific time-point (p ≤ 0.05).

Table 5. Lymphoproliferative response (mm) in chicks given daily oral doses of CPF.

	Period (h)
Experimental group	24	48	72
Treatment Day 12
CPF-5	0.14 ± 0.03*	0.08 ± 0.05*	0.20 ± 0.11
CPF-10	0.11 ± 0.04*	0.07 ± 0.08*	0.23 ± 0.17
CPF-20	0.12 ± 0.04*	0.14 ± 0.06*	0.13 ± 0.07
Xylene	0.27 ± 0.06	0.25 ± 0.10	0.23 ± 0.15
Control	0.32 ± 0.13	0.30 ± 0.11	0.16 ± 0.12
Post-treatment Day 12
CPF-5	0.39 ± 0.18	0.25 ± 0.09	0.21 ± 0.19
CPF-10	0.27 ± 0.20	0.19 ± 0.10	0.18 ± 0.14
CPF-20	0.23 ± 0.07	0.17 ± 0.10	0.28 ± 0.16
Xylene	0.29 ± 0.16	0.25 ± 0.22	0.10 ± 0.06
Control	0.32 ± 0.10	0.20 ± 0.11	0.18 ± 0.07

*Value (mean ± SD) significantly different from control at specific time-point (p ≤ 0.05).

Table 6. In vivo phagocytic response (OD increase %; carbon clearance assay) in chicks given daily oral doses of CPF.

	Period (min)
Experimental group	3	15
Treatment Day 12
CPF-5	63.2 ± 7.8	23.5 ± 5.5*
CPF-10	72.4 ± 7.8	21.4 ± 2.3*
CPF-20	81.1 ± 2.9	35.3 ± 3.4*
Xylene	72.3 ± 14.9	18.7 ± 5.6
Control	68.5 ± 9.9	12.8 ± 3.2
Post-treatment Day 12
CPF-5	75.7 ± 8.7	25.7 ± 4.9*
CPF-10	75.8 ± 12.6	22.9 ± 6.0*
CPF-20	76.9 ± 4.8	25.6 ± 6.9*
Xylene	79.1 ± 11.1	17.7 ± 1.0
Control	73.1 ± 1.8	11.8 ± 4.3

*Value (mean ± SD) significantly different from control at specific time-point (p ≤ 0.05).

Table 7. Histologic changes in bursa of Fabricius, spleen, and thymus due to oral CPF dosing.

Organ and type	Treatment group
of lesion	CPF-5	CPF-10	CPF-20	Xylene	Control
Bursa of Fabricius
Vesicular cytoplasm	+	++	+++	+	−
Nuclear fragmentation	+	++	+++	−	−
Congestion	+	+	++	+	−
Cellular depletion	+	++	+++	−	−
Inflammation	+	+	++	−	−
Inter-follicular CT proliferation	−	+	+++	−	−
Spleen
Vesicular cytoplasm	++	++	+++	−	−
Nuclear fragmentation	+	++	+++	−	−
Congestion	+++	+++	+++	+	−
Cellular depletion	+	++	++	−	−
Reticular cell hyperplasia	++	++	+++	−	−
Thymus
Vesicular cytoplasm	+	+	++	−	−
Nuclear fragmentation	+	+	+++	−	−
Congestion	+	++	+++	+	−
Myoid cell hyperplasia	+	+	+	−	−

Group A (5 mg CPF/kg BW), Group B (10 mg CPF/kg BW), Group C (20 mg CPF/kg BW), Group D (xylene, 1 ml/kg BW), E (Distilled water, 1 mL/kg BW).

Pathology scoring: Normal (−), Mild (+), Moderate (++), Severe (+++).

Figure 1. Bursa of Fabricius of broiler chick administered chlorpyrifos (20 mg/kg BW) at post-treatment Day 15. Representative photomicrograph shows vacuolar degeneration (arrow heads), proliferation of fibrous connective tissue between lymphoid follicles (arrows), and follicles depleted from lymphoid tissue (D). H&E stain. Magnification = 200×.

Figure 2. Bursa of Fabricius of broiler chick administered chlorpyrifos (20 mg/kg BW) at post-treatment Day 15. Representative photomicrograph shows necrosis of follicle in the center, edema in the follicle (arrow heads), and proliferation of fibrous connective tissue around the lymphoid follicles (arrows). H&E stain. Magnification = 400×.

Figure 3. Bursa of Fabricius of broiler chick administered chlorpyrifos (20 mg/kg BW) at post-treatment Day 15. Representative photomicrograph shows fragmentation of nuclei (apoptotic changes) in lymphoid follicle (arrows). H&E stain. Magnification = 400×.

Figure 4. Spleen of broiler chick administered chlorpyrifos (10 mg/kg BW) at day 15 of treatment. Representative photomicrograph shows congestion and degenerative changes. H&E stain. Magnification = 40×.

Figure 5. Spleen of broiler chick administered chlorpyrifos (20 mg/kg BW) at post-treatment Day 15. Representative photomicrograph shows congestion and fragmentation of nuclei (apoptotic changes) (arrows). H&E stain. Magnification = 400×.

Figure 6. Thymus of broiler chick administered chlorpyrifos (10 mg/kg BW) at day 15 of treatment. Representative photomicrograph shows congestion and degenerative changes. H&E stain. Magnification = 40×.

Figure 7. Thymus of broiler chick administered chlorpyrifos (20 mg/kg BW) at post-treatment Day 15. Representative photomicrograph shows hyper-cellularity along with immature monocytes in the medullary region, and necrosis of myoid cells (arrows). H&E stain. Magnification = 100×.

Figure 8. Thymus of broiler chick administered chlorpyrifos (20 mg/kg BW) at post-treatment Day 15. Representative photomicrograph shows condensed nuclei, vacuolar degeneration (arrow heads), myoid cells necrosis, and perforation of myoid cells (arrows). H&E stain. Magnification = 400×.