Preliminary investigation reveals novel pathological consequences of bluetongue virus-1 infection in the endocrine glands of pregnant Indian sheep

Figures & data

Figure 1. Fetal age determination by using (C3-A) real-time ultrasound equipment (Aeroscan-CD-5 Konica Minolta, India) having a convex transducer (C3-A).

Figure 2. Bluetongue virus serotype-1 (BTV-1), 6 ml (5.5 log10 TCID₅₀/ml) by I/D and IV routes, pregnant sheep at 60^th gestation day. Pituitary sections: (A) at 7^th DPI, showing normal capillary plexus (arrow) at C-7 DPI in the infected group, ×100; (B) at 7^th DPI, showing dilated venous capillary (asterisk) plexus with blood, ×100; (C) mild infiltration of the lymphocyte (circle) in the pars nervosa of the pituitary observed at 14^th DPI, H&E, ×400; (D) increased number of the pituicytes (arrowhead) observed in the pars nervosa of the pituitary, 14^th DPI, H&E, ×400; (E) at 30^th DPI, showing an increased number of pituicytes (curved arrow) with an enlarged nucleus in the pars nervosa, H&E, ×200 and (F) at 60^th DPI, infiltration of the eosinophils (circle) between the glandular epithelium of anterior pituitary, H&E, ×400.

Figure 3. BTV-1, 6 ml (5.5 log10 TCID₅₀/ml) inoculated by I/D and IV routes in sheep at 60^th gestation day. H&E section, thyroid sections: (A) at 7^th DPI, hyperplasia of the parafollicular cells (C-cells) with few undergoing apoptosis (asterisk), ×400; (B) at 30^th DPI, enlarged follicles with flattened epithelium (arrowhead) and with dark to lightly stained collide devoid of reabsorption vacuoles, ×200 and (C) at 30^th DPI, showing elongated follicle filled (curved arrow) with the colloid with flattened follicular epithelial cells (arrowhead) on 45^th DPI, H&E, ×200.

Figure 4. BTV-1, 6 ml (5.5 log10 TCID₅₀/ml) inoculated by I/D and IV routes in sheep at 60^th gestation day. H&E section, adrenal sections: (A) at 15^th DPI, distorted histoarchitecture with dilated sinusoids containing sparse neutrophils (asterisk) and a few degenerated cells in the zona fasciculate (arrow), ×200; (B) at 15^th DPI, few cells in the glomerulosa have a condensed nucleus, hyper-eosinophilic cytoplasm and distinct boundaries (arrow), ×200; (C) at 30^th DPI, clumps of the ruptured cells vacuolated cells with eccentrical nuclei in zona glomerulosa (circle), ×200; (D) a higher magnification of (C), ×400; (E) at 45^th DPI, hypertrophy and hyperplasia, cell of zona glomerulosa, H&E, ×400 and (F) at 60^th DPI, section showing a degenerated cell (arrowhead) in the zona fasciculata H&E, ×400.

Figure 5. (A) Control section of the adrenal gland, ×200. (B) Control section of the adrenal gland, ×400. (C) Control section of thyroid, ×100. (D) Control section of thyroid, ×200. (E) Control section of the pituitary gland showing normal pituicytes, ×200.

Figure 6. BTV-1, 6 ml (5.5 log10 TCID₅₀/ml) inoculated by I/D and IV routes in sheep at 60^th gestation day. H&E section, pancreases sections: (A) at 15^th DPI, engorged capillaries (asterisk) in the parenchyma, ×200 and (B) at 30^th DPI, disorganized atrophic pancreatic lobules characterized degenerated exocrine acini (arrow) having pycnotic nuclei and darkly stained cytoplasm, ×200.

Figure 7. Viral load (Viral copy number of BTV) in the pituitary, adrenal and thyroid glands of infected animals at different time points.

Figure 8. BTV-1 infected sheep at 65^th DPI, ABC-IHC-DAB, immunohistochemically stained section, polyclonal BTV-1 primary antibody. (A) Thyroid section showing positive immunolabeling in the cytoplasm of the follicular epithelial cell (asterisk) at 14^th DPI, ×200. (B) Adrenal gland section showing positive immunolabelling in the cytoplasm of the cell of zona fasciculata (arrowhead) at 7^th DPI, ×400. (C) Adrenal gland section showing positive immunolabelling in the cytoplasm of the cell of zona reticularis projecting in the medullary region (dotted circle) of the gland at 14^th DPI, ×400. (D) Pituitary gland showing positive immunolabeling in the cytoplasm of the cell of the adenohypophysis (curved arrow) at 14^th DPI, ×200. (E) Antibody control section devoid of any immunolabeling, IHC-DAB, 14^th DPI, ×200.

Figure 9. BTV-1 infected sheep at 60^th DPI, ABC-IHC-DAB, immunohistochemically stained section, polyclonal caspase-3 primary antibody. (A) Thyroid follicles showing the positive immunostaining for anti caspase-3 in the parafollicular cells of the thyroid as well as in the cytoplasm of the endothelial cell lining the blood vessels (circle). (B) Thyroid follicular epithelial showing the positive immunostaining for anti caspase-3 in the follicular epithelial cells (arrowhead), 30^th DPI, DAB-IHC, ×200 (inset: higher magnification, ×400). (C) Positive immunolabeling in the cytoplasm of the infiltrated macrophage (arrowhead), 30^th DPI, ×400. (D) No signals were detected in the control group thyroid sections, 30^th DPI. (E) No immunostaining was observed in the primary antibody control groups, ×400.

Figure 10. BTV-1 infected sheep at 60^th DPI, ABC-IHC-DAB, immunohistochemically stained section, polyclonal caspase-3 primary antibody. (A) Adrenal gland showing the positive immunostaining for anti caspase-3 in the cytoplasm of the cell of zona reticularis cells (circle), ×200. (B) Higher magnification, ×400. (C) Positive immunolabeling in the cytoplasm of zona fasciculata cells (asterisk) that arranged in the linear manner, ×400, 30^th DPI. (D) No signals were detected in the control group adrenal sections, 30^th DPI, DAB-IHC, ×100.

Table 1. Serum hormone concentration (mean ± SE) in the mid-infected group (GrIMP) and control group at different days postinfections.

	DPI
	0^th DPI	3^rd DPI	7^th DPI	14^th DPI	21^th DPI	45^th DPI	60^th DPI
Cortisol
GrIMP	56.19 ± 19.9*	83.19 ± 16.14	78.85 ± 21.87	64.62 ± 23.82	78.39 ± 18.02	74.19 ± 43.08	54.66 ± 54.85
Control	140.57 ± 17.1	87.12 ± 17.13	100.35 ± 10.62	78.58 ± 5.06	77.92 ± 7.69	78.58 ± 3.58	78.58 ± 5.06
T-3
GrIMP	6.9 ± 3.72*	9.34 ± 2.1*	8.43 ± 3.07	10.2 ± 11	13.5 ± 10.67	11.05 ± 2.76	7.37 ± 8.53
Control	23.06 ± 4.18	22.74 ± 4.04	7.31 ± 1.3	9.32 ± 0.16	7.49 ± 0.81	9.13 ± 0.15	9.49 ± 1.32
Thyroxine (T-4)
GrIMP	64.69 ± 32.24	61.04 ± 33.12	50.51 ± 33.55	50.23 ± 33.17	73.29 ± 32.62	107.46 ± 49.81	125.97 ± 50.94
Control	31.37 ± 8.98	24.07 ± 10.62	28.64 ± 6.62	26.11 ± 11.93	27.55 ± 4.68	25.99 ± 5.18	32.39 ± 7.33
Progesterone
GrIMP	27.01 ± 10.41*	28.17 ± 9.25*	37.97 ± 13.14	43.24 ± 10.54	27.33 ± 8.86*	43.65 ± 24.73	43.65 ± 24.73
Control	77.2 ± 3.77	74.73 ± 3.6	39.21 ± 14.68	42.17 ± 18.48	59.12 ± 9.5	50.99 ± 7.4	23.5 ± 2.74

Data are presented in the form of mean ± SEM. Statistical analysis was performed using paired T-test. *P values less than 0.05 were considered statistically significant.