Synapse loss associated with abnormal PrP precedes neuronal degeneration in the scrapie-infected murine hippocampus.

Numbers of neurones, synapses and axon terminals were quantified in a murine scrapie model with severe hippocampal pyramidal cell loss, in which definite clinical scrapie is evident from 226 days post-infection (dpi) and death occurs around 250 dpi. Disease-specific PrP accumulations were first seen at 70 dpi (28% of the incubation period (IP)) in thalamus and as sparse foci within the stratum pyramidale of CA1. By 98 dpi (39% IP), PrP was seen in the stratum radiatum and was found at later stages throughout all levels of the hippocampus. At the ultrastructural level in the stratum radiatum of CA1, a decrease in the numbers of simple synapses from 84 dpi (34% IP) and in perforated synapses from 98 dpi (42% IP) was found using an unbiased stereological method, the disector analysis. Degeneration of axon terminals was found from 98 dpi (39% IP) onwards. Neuronal loss was detected in CA1 from 180 dpi (72% IP). The results suggest that the fundamental lesion in the hippocampus of ME7-infected mice is associated with PrP release from CA1 pyramidal neurones, which perturbs synaptic function and leads to degeneration of preterminal axons, and that subsequent pathological changes including neurone loss are sequelae to this initial insult.

Early unsuspected neuron and axon terminal loss in scrapie-infected mice revealed by morphometry and immunocytochemistry.

Neuronal loss is often quoted as an element of the pathology of the transmissible spongiform encephalopathies, but few data are published. To determine whether neuronal loss is a salient feature of murine scrapie, and whether there is a relationship with the other hallmark lesions of scrapie we compared the numbers of neurons, severity of vacuolation, axonal bouton density and distribution of prion protein (PrP) in the dorsal lateral geniculate nucleus (dLGN) following intraocular infection of C57BL/FaBtDk mice with ME7 scrapie. This route of infection limits the initial spread of infection to the retinal efferents, thus directing infectivity and subsequent pathological changes to the dLGN which is a major projection of the optic nerve. Morphometric assessment of neuron number in the dLGN was made on semi-serial sections from five infected and five normal brain injected controls at four 50-day intervals during the incubation period, and on terminally affected mice. The number of neurons decreased from around 20,000 at 50 days to under 1000 in the terminal group. Significant loss was identified in individual mice at 150 days post-infection, coincident with the onset of vacuolation: neuron number was found to have an inverse relationship to the severity of vacuolation. Axonal boutons in the dLGN (demonstrated by synaptophysin immunolabelling) were reduced at 200 days, and virtually absent in terminal mice. The intensity of PrP immunostaining progressively increased from 150 days, and in a separate experiment PrP was detected from 175 days by polyacrylamide gel electrophoresis of brain extracts.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlative light and electron microscopy studies of PrP localisation in 87V scrapie.

The transmissible neurodegenerative diseases, of which scrapie is the archetype, are caused by unconventional infectious agents. Prion protein (PrP), a widespread host coded, cell surface sialoglycoprotein, is thought to be an essential or, controversially, sole component of these agents. During infection, disease specific accumulations of PrP may be observed in immunostained brain sections of mice infected with the 87V scrapie strain as amyloid plaques or as diffuse or granular foci within the neuropil. Using serial light and electron microscopical preparations we determined immunocytochemically that infection specific PrP is present in amyloid fibrils, and accumulates on the plasmalemma of neurites at the periphery of plaques and in the neuropil, irrespective of the morphological form of PrP accumulation when viewed by light microscopy. In some brain areas with dense granular PrP expression complete disruption of neuropil with loss of neurites was associated with fibrils lying free in expanded extracellular space. These results suggest that normal PrP may be converted to its pathological form at the neuronal plasmalemma or in the extracellular space and, furthermore, that amyloid fibrils are formed following the accumulation and aggregation of subunit proteins at these sites.

Unsuspected early neuronal loss in scrapie-infected mice revealed by morphometric analysis.

This study was undertaken to determine to what extent neuronal loss is a feature of scrapie pathology, using an experimental model in which infectivity and subsequent vacuolar lesions are well characterized but in which neuronal loss has not been previously identified. Intraocular infection with ME7 scrapie directs infection through the major projections of the optic nerve, which include the dorsal lateral geniculate nucleus (dLGN) on the contralateral side to the infected eye. Infectivity can be detected in the dLGN at 77 days post-infection and vacuolar lesions are first seen around halfway through the incubation period of 240 days. Morphometric assessment of neuron number in the dLGN was made on gallocyanin stained semi-serial sections from 5 infected and 5 normal brain-injected controls at 4 fifty-day intervals during the incubation period, and on clinically terminal mice. The number of neurons in the dLGN of the infected mice decreased steadily from around 20,000 at 50 days post-infection to under 2,000 in the terminal group. The loss was delayed in the ipsilateral dLGN, although terminal counts were the same for both sides. The onset of neuronal loss was coincident with initial vacuolar changes, and neuronal numbers were inversely proportional to the severity of vacuolation. It is concluded that scrapie infection causes a progressive neuronal loss that can be identified some 30-80 days after infectivity can be detected in the dLGN, long before the onset of clinical disease.

Numbers of neurons in vacuolated and non-vacuolated neuroanatomical nuclei in bovine spongiform encephalopathy-affected brains.

Morphometric studies of the brains of seven Holstein-Friesian cows affected with bovine spongiform encephalopathy (BSE) and five control cows are reported. The numbers of neurons were counted in five selected neuroanatomical nuclei, namely the vestibular complex and red nucleus, both of which consistently show a high degree of neuronal perikaryonal vacuolation, the dorsal vagal nucleus, which shows inconsistent perikaryonal vacuolation, and the caudate and hypoglossal nuclei, which do not usually show vacuolation. The vestibular complex of BSE-affected cattle contained about half the number of neurons present in the controls. No statistically significant neuronal loss was demonstrated at other sites. These results confirm previous findings, showing that neuronal loss is a significant feature of BSE and may therefore contribute to the development of the clinical disease. No association was found between the duration of clinical signs and the number of remaining neurons or the extent of the vacuolation. However, the absence of such association should be viewed cautiously as there was substantial variation in neuronal populations between individuals.

Infection specific prion protein (PrP) accumulates on neuronal plasmalemma in scrapie infected mice.

Prion protein (PrP) is an abundant membrane-associated host protein which accumulates in abnormal, relatively protease-resistant forms in the brains of animals with scrapie and related diseases. Using correlative light and electron microscopy we determined the sites of subcellular localisation of PrP in mice infected with the 87V strain of scrapie. Disease specific accumulation of PrP was observed at light microscopy as amyloid plaques or as diffuse or granular staining within the neuropil, often clearly associated with individual neurons. Serial electron microscopical preparations were immunostained for PrP by the immunogold method. Gold particles were located on amyloid fibrils and on the plasmalemma of neurites at the periphery of plaques and in the neuropil, irrespective of the morphological form of PrP accumulation when viewed by light microscopy. This suggests that amyloid fibrils are formed following the accumulation and aggregation of sub-unit proteins at the plasmalemma and, furthermore, that normal PrP may be converted to its pathological form at this site.

A morphometric and immunohistochemical study of the vestibular nuclear complex in bovine spongiform encephalopathy.

A morphometric and immunohistochemical study of the vestibular nuclear complex was performed on five bovine spongiform encephalopathy (BSE) and five control cow brains. Neurons of the lateral and superior vestibular nuclei were counted at 500-microns intervals in 10-microns-thick sections, using an image analysis system comprising a projection microscope and digitising pad linked to a computer. A bimodal distribution of neuron diameters was recognised in the brains of normal cattle. One population of neurons had a mean diameter of 30 microns and the other had a mean diameter of 60 microns. The vestibular nuclei from BSE cattle had an approximately 50% reduction in total numbers of neurons when compared with controls (P < 0.01). Cattle which were clinically diseased longer had the fewest number of neurons preserved. Diminished numbers of neurons were detected throughout the area studied and affected neurons of all diameters. Immunohistochemical staining for synaptophysin, a protein present in synapses throughout the CNS, showed no significant reduction in axon terminals synapsing with vestibular neurons, including vacuolated neurons of BSE brains, when controls and BSE brains were compared. This suggests that de-afferentation of neurons is not the cause of neuronal loss. Prion protein was detected in the neuropil of the vestibular nuclear complex of BSE brains but not control brains. These studies show that previously unsuspected neuronal loss is a significant feature of BSE.

Investigations of transfer factor activity in resistance to Trichostrongylus colubriformis infections in guinea-pigs.

Resistance to challenge infection with Trichostrongylus colubriformis was studied in an outbred strain of guinea-pigs. Resistance was conferred by previous infections of 50 or 100 infective larvae and by intraperitoneal, intramuscular and subcutaneous administration of dialysed transfer factor prepared from guniea-pigs infected with T. colubriformis, by intraperitoneal administration of transfer factor prepared from the blood of sheep infected with T. colubriformis and on one of three occasions using transfer factor prepared from guinea-pigs which had not been infected with T. colubriformis. No significant resistance was transferred by administration of antigen prepared from adult T. colubriformis, or by non-dialysed transfer factor, or by dialysed transfer factor prepared from T. colubriformis-infected guinea-pig tissue which had been stored for several months at - 20 degrees C.

Passive transfer of immunity to trichostrongyle species infections in sheep. Investigations of specificity and limitations of 'transfer factor' activity with Trichostrongylus axei infections.

The ability of transfer factor (TF) preparations to transfer resistance to challenge with Trichostrongylus axei in sheep and to correct partially the immune immaturity of young lambs was confirmed. Non-dialysed TF consistently transferred resistance but dialysed TF frequently failed, perhaps demonstrating a loss of activity during TF preparation procedures. Specificity of transfer was demonstrated when T. axei TF transferred resistance to T. axei challenge but Haemonchus contortus TF failed to do so.

Successful passive transfer of resistance to Fasciola hepatica infection in rats by immune serum and transfer factor.

Resistance to Fasciola hepatica was shown by a 64 per cent reduction in fluke recovery compared with control rats was recorded in rats inoculated with serum from sheep experimentally infected with F hepatica. A comparable reduction (55.6 per cent) was obtained using transfer factor prepared from lysed leucocytes of infected rats. No significant reduction was obtained using transfer factor prepared from sheep or calves.

Investigation of Haemonchus contortus infections in sheep. Comparison of irradiated larvae and transfer factor treatment.

A comparison is made between the resistance conferred to Haemonchus contortus challenge by irradiated larval treatment and transfer factor (TF) treatment in four-month-old lambs and seven-month-old lambs. As in previous investigations the irradiated larval treatment failed to confer resistance to the four-month-old lambs challenged with 10,000 third stage larvae while similar irradiated larval treatment in seven-month-old lambs reduced the worm burdens by 40 per cent compared to controls. The TF treatment produced a 34 per cent reduction in the challenge infection in the four-month-old lambs and a 45 per cent reduction in the seven-month-old lambs compared to the control lambs. It is concluded that TF activity operates independently of immune competence.

Investigations of 'transfer factor' activity in the transfer of immunity to Trichostrongylus axei infections in sheep.

Three investigations are described in which non-dialysed and dialysed leucocyte lysates, 'transfer factor' prepared from the blood of sheep infected with Trichostrongylus axei successfully transferred immunity to challenge infection with that parasite in susceptible lambs. Similar leucocyte lysates from parasite-free lambs failed to transfer a similar resistance to challenge infection. 'Transfer factor' treatment produced a 30--72 per cent reduction in a total worm burden compared to susceptible control lambs. In the first two investigations the donor and recipient lambs were genetically dissimilar and in the third investigation were of different breeds. The resistance transfer was considered to operate independently of immune incompetence.

Haematological and biochemical aspects of an E. coli septicaemia in brown leghorn chickens.

A series of experiments is described in which E. coli septicaemia was produced in 6-week-old Brown Leghorn male chickens by air sac inoculation of a pathogenic strain of E. coli. Groups of birds were slaughtered for examination on days 1 to 5 post infection and haematological and biochemical examinations performed on blood samples collected just prior to slaughter. Changes in erythrocyte values and serum chemistry were observed and the clinical pathology is discussed. Consideration is given to the role of incidental haemodilution in the haematological changes observed.

Haematological and blood chemistry "comparison values" for clinical pathology in poultry.

Twelve blood chemistry values, six haematological values and bodyweight were estimated for six-week-old specific-pathogen-free brown leghorns. Observations were based on 208 birds sampled from 11 groups. Subsequently the same values were estimated for specific pathogen free brown leghorns and broiler hybrids of differing ages. These estimates have been designated "comparison values" and were intended for reference use in clinical pathology. Results were compared with 14 "comparison value" estimated obtained previously for six-week-old commercial broiler hybrids. Haematological values varied with age and type of bird. Differences were observed between bird types in serum protein, serum cholesterol, uric acid, calcium and potassium levels and in weight. The significance of these differences is discussed.

Effect of transportation on blood metabolites in broilers.

1. Groups of 30 broilers from seven flocks were bled and weighed at the farm and a further sample of 10 broilers from each flock was transported from 8 to 16 km to the laboratory prior to bleeding and weighing. 2. One haematological and 11 biochemical analyses were made on each blood sample. 3. Although transportation did lead to significant differences in certain variables, the trends were consistent and sampling following transit is considered acceptable for clinical pathology studies.