Friday, April 20, 2012

Ultrastructural findings in pigs experimentally infected with bovine spongiform encephalopathy agent

Ultrastructural findings in pigs experimentally infected with bovine spongiform encephalopathy agent

Pawel P. Liberski1, Beata Sikorska1, Gerald A.H. Wells2, Steve A.C. Hawkins3, Michael Dawson3, Marion M. Simmons2 1Department of Molecular Pathology and Neuropathology, Medical University of Lodz, Poland, 2TSE Department, and 3Specialist Scientific Services Department, Animal Health and Veterinary Laboratories Agency, Weybridge, Addlestone, Surrey, United Kingdom Folia Neuropathol 2012; 50 (1): 89-98

A b s t r a c t

We report here an electron microscopic study of selected nervous system tissues from pigs infected experimentally with the agent of bovine spongiform encephalopathy (BSE). Generally, the ultrastructural neuropathology of BSE-affected pig brain resembled that of BSE-affected cattle brain. Spongiform change, in the form of membrane-bound vacuoles separated by septae into secondary chambers, dominated the pathology. Numerous astrocytic processes were visible in close conjunction with elongated microglial cells. Neuronal degeneration presented as either dystrophic neurites or by the formation of autophagic vacuoles. Altered subcellular organelles: mitochondria, electron-dense bodies, autophagic vacuoles, neurofilaments and “branching-cisterns” accumulated in abnormal neurites. Autophagic vacuoles appeared as neuronal cytoplasm of increased electron-density sequestrated by intracytoplasmic membranes. Tubulovesicular structures were numerous, particularly in the cerebellum. Unusual crystalloids were observed in the white matter. In conclusion, experimental BSE in pigs demonstrated ultrastructural pathology in keeping with that observed in other spongiform encephalopathies.

Key words: prions, BSE, pigs, ultrastructure.


Material and methods

Experiment design and inoculation procedure

The pigs were infected at 1-2 weeks of age by multiple- route parenteral inoculation with a homogenate of bovine brain from natural BSE cases, as described in full previously [76,86,87]. All challenges were carried out in accordance with the Animals (Scientific Procedures) Act, 1986, under licence from the UK Home Office. Animals were sedated with azoperone (Stresnil; Janssen Animal Health) and killed by the intravenous injection of pentobarbitone sodium followed by exsanguination. When clinical disease developed, animals were killed and samples collected immediately postmortem.


Electron microscopy

Multiple samples, comprised 2-3 mm3, of cerebral cortex, brain stem at the level of vestibular nuclei, ventral horns of the spinal cord, cerebellum and dorsal root ganglia, selected on the basis of the previously determined prevalence of light-microscopy changes [76,87], were fixed immediately after dissection in 2.5% glutaraldehyde, freshly prepared in phosphate buffer (pH 7.4), then postfixed in 1% osmium tetroxide and processed for routine electron microscopy. Comparable areas of brain from uninoculated pigs served as controls.



In general, the ultrastructural features of BSE-affected pig brain were similar to those of BSE-affected cattle [46,66,67] and humans with TSEs [60,61]. Spongiform change in the form of membrane-bound vacuoles (Fig. 1) separated by membranes curled into secondary chambers dominated the pathology. A dense astrocytic reaction was accompanied by abundant elongated microglial cells. Of particular note was the finding of numerous astrocytic processes in close conjunction with microglial cells. Neuronal degeneration presented as either neuroaxonal dystrophy, as evidenced by dystro - phic neurites, or autophagic vacuoles. Dystrophic neurites accumulated altered subcellular organelles: mitochondria (Fig. 1B), electron-dense bodies, neurofilaments and “branching-cisterns” (Fig. 2). Autopha - gic vacuoles appeared as a part or parts of the neuronal cytoplasm sequestrated by intracytoplasmic membranes (Fig. 3). Sequestrated cytoplasm was of higher electron density than the remaining cytosol. Discontinuity of plasma membranes was occasionally seen (Fig. 3B, arrow). Tubulovesicular structures (TVS) were numerous with the highest number of affected processes in the cerebellum (Fig. 4). Many large multivesicular bodies were seen (Fig. 5).



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