Detection of PrP(Sc) in rectal biopsy and necropsy samples from sheep with experimental scrapie.

Scrapie diagnosis is based on the demonstration of disease-associated prion protein (PrP(Sc)) in brain or, in the live animal, in readily accessible peripheral lymphoid tissue. Lymphatic tissues present at the rectoanal line were readily obtained from sheep without the need for anaesthesia. The presence of PrP(Sc) in such tissue was investigated in sheep infected orally with scrapie-infected brain material. The methods used consisted of immunohistochemistry and histoblotting on biopsy and post-mortem material. PrP(Sc) was detected in animals with PrP genotypes associated with high susceptibility to scrapie from 10 months after infection, i.e., from about the time of appearance of early clinical signs. In the rectal mucosa, PrP(Sc) was found in lymphoid follicles and in cells scattered in the lamina propria, often near and sometimes in the crypt epithelium. By Western blotting, PrP(Sc) was detected in rectal biopsy samples of sheep with the PrP genotype VRQ/VRQ, after electrophoresis of material equivalent to 8 mg of tissue. This study indicated that rectal biopsy samples should prove useful for the diagnosis of scrapie in sheep.

Mapping PrPSc propagation in experimental and natural scrapie in sheep with different PrP genotypes.

Twenty-one orally inoculated and seven naturally infected sheep with scrapie were examined for PrP(Sc) in peripheral tissues and in the central nervous system (CNS), using immunohistochemistry. In the inoculated group, VRQ (valine at codon 136, arginine at codon 154 and glutamine at codon 171)/VRQ sheep generally had a greater accumulation of the pathologic form of prion protein (PrP(Sc)) in peripheral tissues, as compared with VRQ/ARQ (alanine at codon 136, arginine at codon 154, and glutamine at codon 171) animals at corresponding time points after inoculation. PrP(Sc) was not detected in the ileal Peyer's patch, the spleen, the superficial cervical lymph node, and peripheral nervous tissues of several inoculated VRQ/ARQ animals. All inoculated VRQ/VRQ sheep, but only one of eight inoculated VRQ/ARQ animals, were PrP(Sc)-positive in the CNS. Thus, the propagation of PrP(Sc) seemed slower and more limited in VRQ/ARQ animals. Tissue and cellular localization of PrP(Sc) suggested that PrP(Sc) was disseminated through three different routes. PrP(Sc)-positive cells in lymph node sinuses and in lymphatics indicated spreading by lymph. The sequential appearance of PrP(Sc) in the peripheral nervous system and the CNS, with satellite cells as early targets, suggested the periaxonal transportation of PrP(Sc) through supportive cells. Focal areas of vascular amyloid-like PrP(Sc) in the brain of five sheep, suggested the hematogenous dissemination of PrP(Sc). There was a poor correlation between the amount of PrP(Sc) in the CNS and clinical signs. One subclinically affected sheep showed widespread PrP(Sc) accumulation in the CNS, whereas three sheep had early clinical signs without detectable PrP(Sc) in the CNS. A VV(136) (homozygous for valine at codon 136) sheep inoculated with ARQ/ARR (alanine at codon 136, arginine at codon 154, and arginine at codon 171) tissue succumbed to disease, demonstrating successful heterologous transmission. Less susceptible sheep receiving VRQ/VRQ or ARQ/ARR material were PrP(Sc)-negative by immunohistochemistry, enzyme-linked immunosorbent assay, and western blot.

Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie.

All sheep older than 1 year of age from a flock of the Rygja breed in which clinical scrapie was detected for the first time in two animals (4%) were examined for accumulation of pathogenic prion protein (PrPSc) by immunohistochemistry in the obex, the cerebellum, and the medial retrophayngeal lymph node. In addition, six lambs, 2-3 months old, all offspring of PrPSc-positive dams, were examined for PrPSc in the ileal Peyers' patch (IPP), the distal jejunal lymph node, the spleen, and the medial retropharyngeal lymph node (RPLN). In this flock, 35% (17/48) of the adult sheep showed accumulation of PrPSc, an eightfold increase compared with clinical disease. All positives carried susceptible PrP genotypes. Three sheep had deposits of PrPSc in the RPLN and not in the brain, suggesting that this organ, easily accessible at slaughter, is suitable for screening purposes. Two 7-year-old clinically healthy homozygous V136Q171 ewes showed sparse immunostaining in the central nervous system and may have been infected as adults. Further, two littermates, 86-days-old, showed PrPSc in the IPP. Interestingly, one of these lambs had the intermediate susceptible PrP genotype, VA136QR171. In addition to early immunolabeling in the dorsal motor nucleus of the vagal nerve, a few of the sheep had early involvement of the cerebellum. In fact, a 2-year-old sheep had sparse deposits of PrPSc in the cerebellum only. Because experimental bovine spongiform encephalopathy (BSE) in sheep seems to behave in a similar manner as natural scrapie, these results, particularly regarding spread of infectivity, may have implications for the handling of BSE should it be diagnosed in sheep.

Detection of PrPSc in lymphoid tissues of lambs experimentally exposed to the scrapie agent.

Histoblotting and immunohistochemistry were used to detect disease-associated prion protein (PrP(Sc)) in lymphoid tissues of lambs of known PrP genotype infected with the scrapie agent by stomach tube at the age of 2 months. The ileal and jejunal Peyer's patches and retropharyngeal and distal jejunal lymph nodes were studied 1 week, 5 weeks, 5 months and 11 months after inoculation. Other lymphoid tissues examined included superficial cervical lymph node, tonsil and spleen. PrP(Sc) was not detected in any tissue of any lamb at 1 week post-inoculation. At 5 weeks, PrP(Sc) was detected in tissues of lambs of susceptible PrP genotypes (AV(136)QQ(171) and VV(136)QQ(171)), but not lambs of other PrP genotypes (AA(136)QQ(171), AA(136)QR(171) and AV(136)QR(171)). PrP(Sc) was present in the germinal centres of tonsils, distal jejunal and retropharyngeal lymph nodes, and spleen. In the nodules of ileal and jejunal Peyer's patches, only occasional solitary cells showed the presence of PrP(Sc). At 5 months post-inoculation, increased accumulations of PrP(Sc) were detected in ileal and jejunal Peyer's patches, as well as in the retropharyngeal and distal jejunal lymph nodes of a single lamb inoculated with the agent from a sheep of the same susceptible PrP genotype. Eleven months after exposure to the scrapie agent, PrP(Sc) was detected in all lymphoid tissues examined from sheep of susceptible PrP genotypes. These studies show that PrP(Sc) was detectable in lymphoid tissues 5 weeks after exposure to the scrapie agent by stomach tube in lambs as young as 3 months of age and indicate that the PrP genotype is a significant factor for the rapid uptake and spread of the agent through lymphoid tissues.

A case-control study on scrapie in Norwegian sheep flocks.

Scrapie first was detected in indigenous sheep in Norway in 1981, and from 1995 to 1997 an increase in the number of flocks with scrapie cases was recorded. These flocks were mainly in one geographical region. A study to identify risk factors for scrapie was conducted. The study had three frequency-matched controls selected for every case within the same Veterinary District. A questionnaire was submitted to 176 sheep flocks (42 had been scrapie flocks). The data obtained by the questionnaire were linked to data collected from governmental and industry registers. After imputing missing data using single random imputation, the statistical analysis was performed using multivariable conditional logistic regression. Purchase of female sheep from scrapie flocks, sharing of rams, or sharing of pastures between different flocks were the risk factors associated with the occurrence of scrapie. Of factors potentially sustaining and promoting the infection in the flock, number of winter-fed sheep, number of buildings for housing sheep, rams and ewes shared room during mating period and increase in the flock size were associated with scrapie. We interpret these findings to show that factors involving transfer of sheep between flocks or direct contact between sheep of different flocks are important for the spread of scrapie. Management factors are important for the development of scrapie. However, it was not possible to discriminate between the different management factors in this study at the flock level. Also, factors indicating awareness and interest of the farmer (as well as willingness to contact a veterinarian for diseased sheep) were related to the detection of scrapie in the flock.

Histamine in lambs with abomasal bloat, haemorrhage and ulcers.

The median concentration of histamine in abomasal fluid of lambs with abomasal haemorrhage and/or ulcers (group 2) was significantly (P < 0.05) higher than the concentrations in lambs presenting abomasal bloat (group 1) and in the healthy and the diseased controls. In group 2, there was also a strong correlation (R2 = 0.81) between the histamine concentrations in abomasal tissue and abomasal fluid, although the median value of histamine in the abomasal tissue was not statistically higher in this group than in the others. The urine of lambs in group 2 also had numerically higher median concentration of histamine than the other groups. Five out of eight tested strains of Lactobacillus spp. and one out of two strains of Clostridium sordellii, isolated from abomasal contents of lambs with abomasal disease, were strong producers of histamine. Bacterial production is one possible source for the increased histamine concentrations in lambs suffering from abomasal haemorrhage and/or ulcers.

Abomasal bloat, haemorrhage and ulcers in young Norwegian lambs.

An abomasal syndrome affecting mainly three- to four-week-old lambs was studied by using a case-control design involving 88 cases and 85 controls. The principal clinical signs were tympany and colic. The cases were divided into three groups according to the main gross pathological findings in 82 of them. The 28 lambs in group 1 had abomasal tympany, the 29 lambs in group 2 had severe damage to the abomasal mucosa, and the 25 lambs in group 3 consisted mainly of lambs with various other diseases in combination with abomasal changes. The lambs in group 1 had a significantly (P<0.05) lower mean (se) abomasal pH (2.7 [0.19]) than those in group 2 (4.1 [0.32]), group 3 (3.7 [0.39]) or the controls (3.3 [0.13]). The ruminal pH values ranged from 3.5 to 7.4 but there were no significant differences between the groups. Lambs with ulcers had a significantly (P<0.05) higher frequency of trichophytobezoars, than the cases without ulcers or the controls. The tympanic lambs in group 1 had a significantly higher mean packed cell volume, and higher mean red and white blood cell counts, and a significantly lower mean cell volume and mean cell haemoglobin concentration than the healthy control lambs.

Prion protein gene polymorphisms in sheep with natural scrapie and healthy controls in Norway.

Two-hundred and forty healthy sheep and 32 cases of natural scrapie in Norway were analysed for disease-linked polymorphisms in the prion protein (PrP) gene. Scrapie was strongly associated with the presence of a valine polymorphism at codon 136 (V136), as 68.8% of the cases were homozygous (VV136) and 15.6% were valine/alanine heterozygous (VA136). All cases were homozygous arginine/arginine at codon 154 (RR154), except two which were homozygous histidine/histidine (HH154). All cases except two were homozygous glutamine/glutamine at codon 171 (QQ171), the two exceptions being heterozygous glutamine/arginine (QR171). More than 80% of all scrapie cases in Norway have occurred in a Cheviot-related crossbred type of sheep called Rygja. This type of sheep, which is largely restricted to the south-western coast, carries the V136 allele at a higher frequency than do other breeds of Norwegian sheep. Polymorphisms at codons 138 and 151 are also described.

Fungi on Narthecium ossifragum leaves and their possible involvement in alveld disease of Norwegian lambs.

Spores of Pithomyces chartarum (Berk. & Curt.) M.B. Ellis were only rarely seen on leaves of Narthecium ossifragum (L.) Hudson collected in summer from five areas in western Norway in which alveld, a photosensitization disease of lambs, is endemic. Cladosporium magnusianum (Jaap) M.B. Ellis was found on all 118 leaf samples collected in the summers of 1990 and 1991. The hypothesis that P. chartarum contributes to the aetiology of alveld could not be supported, but it is possible that C. magnusianum may have a role in the causation of the disease.

Regional enteritis in lambs.

Thirty-two lambs with regional enteritis were examined. Eighteen were received alive and examined clinically, while the remaining were dead on arrival. One of the sick lambs recovered without treatment. Occurrence and epidemiology, clinical signs, clinical pathology, necropsy findings, light microscopy, immunoperoxidase studies, scanning electron microscopy and transmission electron microscopy are reported. The disease was characterised by stretching of the fore- and hindlimbs, ill thrift, intermittent diarrhoea, leucocytosis, thickened ileal wall and hyperplasia of the ileal mucosa. None of the methods applied revealed any causal agents. Possible aetiological factors are discussed.

Ovine white-liver disease (OWLD). Botanical and chemical composition of pasture grass.

The most important grass species on the ovine white-liver disease (OWLD) pastures (S) were Poa spp., Agropyron repens and Lolium perenne, while the control pastures (H), where lambs grew well, consisted of Poa spp., Festuca rubra and Agrostis tenuis. The soil was more acidic on the H pastures as compared with the S pastures. OWLD grass (S grass) contained marginal to deficient amounts of cobalt during the first 2 months of grazing. During 2 years out of 3, the average Co content was slightly lower in the S grass as compared with the content in the H grass. The lowest average grass Co was, however, found during one year in the H grass, in spite of the fact that the H lambs also this year grew well, and were 13 kg heavier than the S lambs after 3 1/2 months on pasture. Results thus indicate that the H lambs some years were subclinically Co deficient, without developing clinical symptoms or OWLD, and that factors other than marginal/deficient grass Co are of importance as to whether OWLD will develop or not. S grass differed from H grass by having significantly lower copper, molybdenum, manganese and zinc content, lower protein N/amid N ratios and higher aluminium and iron contents. According to the results, marginal to deficient grass Co is essential for development of OWLD, but cofactors play a part.

Ovine white-liver disease (OWLD). Vitamin B12 and methyl malonic acid (MMA) estimations in blood.

At pasture outlet, mean plasma vitamin B12 varied between 210 and 1,200 pmol/l in 1 month old lambs, 19% of them had values below 250 pmol/l. In those put on OWLD pastures, mean values dropped after 2-4 weeks, and mostly stayed below 150 pmol/l throughout grazing. Plasma methylmalonic acid (MMA) rose above 5 mumol/l 2-8 weeks after outlet, and above 15 mumol/l 4 weeks later. Reduced growth occurred 3-8 weeks after plasma B12 dropped below 150 pmol/l, and 4-6 weeks after MMA rose above 5 mumol/l. Clinical OWLD was most often associated with plasma B12 less than 150 pmol/l and MMA greater than 15 mumol/l. Cobalt fertilization of pastures induced satisfactory plasma B12/MMA values for 3 succeeding years. Elevated plasma B12 was found 3 weeks after Co pellet dosing. The use of Co lick resulted in large individual variations in plasma B12/MMA. The control lambs, which were healthy and grew well on pastures which some years contained marginal/deficient cobalt, had plasma B12/MMA values which varied considerably. One year values indicated functional Co deficiency, but none developed OWLD, and growth was satisfactory, but less than other years. In these lambs, high MMA was not always associated with low B12, or depressed growth. OWLD occurred in Co/B12 deficient lambs, but Co/B12 deficient lambs on other pastures did not develop OWLD.

Ovine white-liver disease (OWLD). Changes in blood chemistry.

Changes in blood chemistry were examined in vitamin B12 deficient lambs which developed ovine white-liver disease (OWLD), and were compared with values of cobalt/B12 supplemented lambs on the same pastures, as well as clinically healthy, but sometimes B12 deficient, lambs on other pastures (H). In the OWLD group, signs of hepatic damage were seen concurrently with reduction in weight gain, or 1-3 weeks before, and comprised elevation of serum glutamate dehydrogenase (GLDH) and decrease of phospholipid and cholesterol. Drop of plasma glucose and elevation of gamma GT also came in the earlier phase of the disease. All other blood changes developed later, and were partly regarded as reflections of the inappetence or hepatic injury. The changes included a drop in packed cell volume (PCV) and mean corpuscular volume (MCV), elevation of serum iron, and reduction of total serum protein and urea. Generally Co/B12 supplementation prevented hepatic damage and normalized blood values. The clinically healthy H lambs also showed signs of hepatic damage, especially one year when they were B12 deficient, indicating that simple B12 deficiency causes a moderate liver damage as well. For diagnostic purposes, clinical pathology is recommended mainly on a flock basis.

Ovine white-liver disease (OWLD). Serum copper and effects of copper and selenium supplementation.

Serum copper was generally higher in lambs affected with ovine white-liver disease (OWLD) than in cobalt/vitamin B12 supplemented lambs grazing the same pastures. Although the copper content of the grass was very low on the OWLD pastures, dosing lambs with Cu alone resulted in worsening of the clinical condition and aggravation of clinical pathology. Dosing with selenium had no effect on OWLD. Dosing with a combination of Co, Se and Cu resulted in normal lamb growth and normal laboratory tests. Lambs growing well on other pastures (H) showed elevated serum Cu when they were subclinically B12 deficient.

Ovine white-liver disease (OWLD). Trace elements in liver.

Trace elements in liver were examined in vitamin B12 deficient lambs which developed ovine white-liver disease (OWLD), in cobalt/vitamin B12 supplemented lambs on the same pastures as well as clinically healthy, but sometimes subclinical B12 deficient lambs on other pastures (H). Liver Co was marginal to deficient in both OWLD lambs (S lambs) and H lambs. Supplementation with B12 or Co elevated liver Co. Liver copper was significantly lower in OWLD lambs than in the H lambs, and Co/B12 supplementation on pasture generally had no significant effect on the contents. Dosing lambs on OWLD pastures with copper oxide needles (SCuO), however, resulted in high/toxic liver Cu. Dosing with Co, Se and Cu glass boluses resulted in adequate liver Cu, except for 1 lamb with toxic amounts indicating dissolution and absorption of the bolus. OWLD lambs had significantly lower liver molybdenum than H lambs, and Co/B12 supplementation elevated values, while CuO treatment depressed them. Liver zinc, manganese and selenium are also reported.

Ovine white-liver disease (OWLD). Pathology.

Microscopic liver changes could earliest be found after 1 month on OWLD pasture, and include extensive fatty change with large spherical vacuoles in hepatocytes, varying size of hepatocytes and nuclei, and formation of Councilman bodies. Later came ceroid deposits, biliary hyperplasia and mesenchymal proliferation. Changes occurred in all lambs which died or were killed due to OWLD, and altogether 83% of the lambs grazing OWLD pastures showed typical or suspect changes. Widespread haemosiderosis of the spleen was common. In severely affected lambs, sclerosis of the Peyer's patches and of the germinative centres of the intestinal lymph nodes were seen, as were neuronal atrophy and patchy microcavitation of areas in the brain stem. Four had polyvasculitis. Cobalt/vitamin B12 supplemented lambs showed no specific changes. Lambs which grew well on other pastures (H lambs), but which were subclinically Co/B12 deficient some years, showed no fulminant hepatic OWLD, but 15% developed some features seen in OWLD. They showed no extensive fatty change. Results indicate that OWLD is a manifestation of B12 deficiency worsened by factors triggering early hepatic fatty change resulting in a more severe liver damage with loss of intracellular homeostasis rendering the hepatocytes vulnerable to other elements, like copper.