DISEÑO DE CONTROLES EFECTIVOS

Histopathology lesion included multifocal perivascular lymphocyte cuff in the cerebrum.

Occasionally within the eye, corneal and iridial multifocal ballooning degeneration, pigmented epithelia necrosis, and sclera edema and moderate heterophil infiltration while the harderian gland often had focal extending epithelia necrosis. There is also lymphoplasmacytic optic neuritis. The airsacs were edematous with mild heterophil infiltration and epithelia hypereosinophilia material (fibrin), and sometimes with subepithelia lymphocyte and plasma cell infiltrates. The Bursa of Fabricius showed multifocal follicular depletion with glandular cystic formation while the spleen and thymus had multifocal periateriolar necrosis with hemosiderin laden macrophages. In the ducks (Muscovy, Mallard and Pekin) there were lesions of cerebral and cerebellar neuronal degeneration and necrosis with multifocal perivascular lymphocytic cuffs, (Plate 4.19) and severe lymphoplasmacytic optic neuritis (Plates 4.20 and 4.21). There was respiratory epithelial necrosis with intraluminal cellular debri in the nasal septal and severe vascular congestion (Plate 4.22) and lymphoplasmacytic airsacculitis (Plate 4.23). There was severe myocarditis and pulmonary edema (Plate 4.24) and severe diffuse myocardial necrosis (Plate 4.25). The following cell types were mildly to strongly immunoreative to H5 intranuclear proteins using the ABC immunohistochemical methods. The feather follicle and keratinocytes within the dermis of the Comb, cerebral and cerebellar neurons and glia cells, in particular the meninges, and purkinje cells of the cerebellum, ependymal cells of the choroids plexus, ependyma cells, neurons and glia cells of the spinal cord, also immunoreativity was detected in the central canal. The smooth muscle cells, spindle cells in uvea and outer conjunctival epithelia, fibrocytes and nerve ganglia epineurium, corneal epithelium, sclera and pigmented cells in uvea, including the harderian gland and pectin glandular epithelium were all immunoreactive. Upper and lower cells of filtration angle, anterior chamber are also immunoreative. The glandular epithelium and smooth muscle cells of the eyelid. Mononuclear cells in thymus, Bursa and spleen, the cardiomyocytes were also immunoreactive. The nasal cavity and palatine epithelium, trachea columnar epithelium, parabronchial epithelium of the lung, the esophageal endothelia cells, myocytes in gizzard, jejunum, rectal endothelia cells and hepatocytes and bile duct epithelium, including renal tubular epithelia cells and skeletal muscle myocytes were all immunoreactive. Pekin ducks examined at 4 dpi, as compared to the only Muscovy duck showed histological lesion of multifocal lymphocytic myocarditis (Plate 4.26). There was multifocal

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pancreatic necrosis with duodenal vascular congestion (Plate 4.27), only evident in Muscovy duck no. 4. Bursa of Fabricius (Plates 4.28 and 4.29) show lymphoid depletion and immunohistochemical staining. All tissues of Muscovy ducks examined at 3 dpi were immunopositive by immunohistochemistry, but no positivity was seen in the entire GIT tissues of Pekin. There were consistent histopathologic lesions in these Muscovy ducks at 3 dpi. The spleen and thymus had multifocal periateriolar necrosis with hemosiderin laden macrophages. Furthermore, histopathology lesions observed in the Pekin ducks included comb keratinocyte necrosis, upper respiratory focal epithelia necrosis and desquamation, oedematous and fibrinous lymphoplasmacytic airsacculitis, pulmonary congestion and edema, splenic hemosiderosis, lymphoid depletion in thymus, spleen and Bursa of Fabricius, adrenal and duodenal intramural lympho-plasma-histiocytic ganglioneuritis, lymphocytic myocytis, severe, diffuse pancreatitis and acinar necrosis. Histopathological lesions included severe diffuse meningo-encephalitis with multifocal lymphocytic perivascular cuffs. Bursa of Fabricius in Pekin duck shows follicular necrosis and infiltration by mononuclear inflammatory cells and the heart had diffuse necrotic myocarditis. Mallard duck show neuronal necrosis with diffuse infiltration of mononuclear cells, mainly lymphocytes and plasma cells; and vascular congestion with optic neuritis. The hallmark histopathological lesions in the Pekin and Mallard at 4 dpi included meningoencephalitis with lymphocytic perivascular cuffs. Additionally, there was adrenal necrosis with lympho-plasma-histiocytic ganglioneuritis and the duodenal intramural lympho-plasma-histiocytic ganglioneuritis which was evident in the 4^th and 5 dpi Pekin ducks.

Although hallmark of histopathological lesions were fewer, the disease was accompanied by lymphoplasmacytic meningoencephalitis and polioencephalitis with lymphocytic perivascular cuffs, which was also seen in the eyelid and multifocal lymphocytic myocarditis. In the Pekin ducks, the following cell types were mildly to strongly immunoreative to H5 intranuclear proteins using the ABC immunohistochemical methods. The feather follicle and keratinocytes within the dermis of the Comb, cerebral and cerebellar neurons and glia cells, in particular the meninges, and purkinje cells of the cerebellum, ependymal cells of the choroids plexus, ependyma cells, neurons and glia cells of the spinal cord, also immunoreativity was detected in the central canal linning cells and, also the corneal epithelium. The glandular epithelium and smooth muscle cells of the eyelid. Mononuclear cells in thymus, spleen and adrenal, also the cardiomyocytes, The nasal cavity medial ophthalmic branch of trigeminal nerve and palatine epithelium, trachea columnar epithelium, parabronchial epithelium of the lung, including renal tubular and testicular seminiferous interstium and skeletal

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muscle myocytes. In the Mallard, the feather follicle and keratinocytes within the dermis of the comb, cerebral and cerebellar neurons and glia cells, in particular the meninges, and purkinje cells of the cerebellum, ependymal cells of the choroids plexus, ependyma cells, neurons and glia cells of the spinal cord, also immunoreativity was detected in the central canal linning cells and, also the corneal epithelium, and the glandular epithelium and smooth muscle cells of the eyelid. Mononuclear cells in thymus, spleen and adrenal, also the cardiomyocytes, The nasal cavity medial ophthalmic branch of trigeminal nerve and palatine epithelium, trachea columnar epithelium, parabronchial epithelium of the lung, ncluding renal tubular and testicular seminiferous interstium and skeletal muscle myocytes.

Generally Immunoreactivity was successful in all ducks but with variation in the GIT tissues. It was seen in the endothelia cells of some GIT tissues and in Bursa of Fabricius in Muscovy ducks examined at 3 dpi, but none was seen in the entire GIT tissues of Pekin. The Pekin only showed immunoreactivity in the nasal cavity medial ophthalmic branch of trigeminal nerve (Plate 4.30) and palatine epithelium. Although the serosal lining cells of the GIT tissues were IHC positive in Mallard at 5 dpi, they remain negative in the Pekin still, and in both at 6 dpi. Immunoreativity was restricted to the cerebral neurons and glia cells and around the vessels only in one Mallard and not detected in the second Mallard 8 dpi.

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Plate 4.18. Muscovy duck 3days p.i, clade 1 HPAI. Torticollis and partial paralysis of right limb.

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Plate 4.19. Cerebrum; Mallard duck 4 days p.i, clade 1 HPAI. Multifocal Perivascular lymphocytic cuffing, neuronal necrosis, with multifocal infiltration of mononuclear cells, mainly lymphocytes;

vascular congestion. HE. Bar=100µm

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Plate 4.20. Optic nerve - Eye; Muscovy duck 3 days p.i, clade 1 HPAI Optic neuritis with diffuse infiltration of mononuclear cells, mainly lymphocytes and plasma cells; and vascular congestion. HE. Bar=100µm

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Plate 4.21. Optic nerve and pectin - Eye; Mallard duck 4 days p.i, clade 1 HPAI Neuronal necrosis with diffuse infiltration of mononuclear cells, mainly lymphocytes and plasma cells; and vascular congestion. HE. Bar=100µm

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Plate 4.22. Muscovy duck 3 days p.i, clade 1 HPAI. Respiratory epithelium. Necrotizing and hemorrhagic rhinitis. Mucous epithelium is hypertrophied, matted and necrotic, infiltrated by mononuclear cells with multifocal desquamation. Vascular congestion and hemorrhage HE.

Bar=100µm

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Plate 4.23. Airsac-Pulmonary; Pekin duck 4 days p.i, clade 1 HPAI. Airsacculitis : the mesothelium is diffusely infiltrated by mononuclear cells, mainly lymphocytes HE. Bar=100µm

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Plate 4.24. Heart: Pekin duck 6 days p.i, clade 1 HPAI. Fibrinous pericarditis and myocardial necrosis, diffuse. Lung: pulmonary congestion and edema.

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Plate 4.25. Heart: Pekin duck 5 days p.i, clade 1 HPAI. Myocarditis, diffuse and coronary vascular congestion.

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Plate 4.26. Heart; Pekin duck 6 days p.i, clade 1 HPAI. Myocarditis, severe diffuse: Cardiomyocyte necrosis and mononuclear cell infiltration, mainly lymphocyte and plasma cells . HE. Bar=100µm.

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Plate 4.27. Pancreas: Muscovy duck 3 days p.i, clade 1 HPAI. Necrotic pancreatitis, multifocal with vascular congestion.

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Plate 4.28. Bursa of Fabricius; Pekin duck 4 days p.i, clade 1 HPAI. Bursitis, follicular necrosis and lymphoid depletion and lymphocytic repopulation. HE. Bar=100µm

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Plate 4.29. Bursa of Fabricius; Muscovy duck 3 days p.i, clade 1 HPAI. Follicular mononuclear cells are focally and strongly immunoreactive to H5 nuclear protein. Bar=100µm

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Plate 4.30. Trigerminal nerve-Nasal septum; Pekin duck 4 days p.i, clade 1 HPAI. Neuronal and axonal necrosis with focally diffuse infiltration of lymphocytes. The neurons are diffusely and strongly immunoreactive to H5 nuclear protein. Bar=100µm

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4.4.1.4 Real-Time Reverse Transcriptase Polymerase Chain Reaction (rRT-PCR)