Correlation between the results of a histopathological examination and the detection of abnormal brain fibrils in the diagnosis of bovine spongiform encephalopathy

A statistical comparison was made between the results of the statutory neurohistopathological method for the post mortem diagnosis of bovine spongiform encephalopathy (BSE) and the detection of abnormal brain fibrils (SAF). A total of 773 suspect cases was examined by both methods; it comprised 531 animals born before the feeding of ruminant-derived protein to ruminant species was prohibited and 242 born after the ban. The relative sensitivities and specificities of the methods were calculated for the diagnosis of clinically suspected BSE. The agreement between the results of the methods was excellent for 331 of the cases born before the ban and for all the cases born after it. In these cases the samples were not autolysed. For the 200 cases in which autolysis was recorded there was poor agreement between the diagnostic methods and this was attributed to an apparently reduced specificity of the histopathological diagnosis in the autolysed material. Despite the potentially greater specificity of fibril detection in the diagnosis of scrapie-like disease, this study indicates that a reliance on fibril detection alone may result in some false negative diagnoses, probably owing to the inadequate sampling of the tissues.     

Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain

Bovine spongiform encephalopathy (BSE) is a new disease of cattle which has considerable homology with scrapie, the archetype of the transmissible spongiform encephalopathies. Abnormal brain fibrils, called scrapie associated fibrils (SAF), are specific ultrastructural markers for these diseases. Fibril detection was compared with histopathological diagnosis in the brains of 167 cattle; 157 clinically suspect BSE and 10 clinically normal. Fibrils were detected in samples of pooled brain regions of 67/144 in which vacuolar changes of BSE were confirmed, but absent in the remaining 23 brains, in which no vacuolation was found, including those from the clinically normal cattle and 13 with alternative neuropathological diagnoses. When eight defined anatomic regions from the brains of another 22 affected cows were examined, the sensitivity of fibril detection was greater than 90% for the brain stem areas. Fibril prevalence in these areas approximated to severity of vacuolar changes. When the same defined regions from four of the affected cows were assayed for fibril protein (PrP) by western blotting, the density of immuno-labelling generally correlated with the fibril prevalence. This study confirms the specificity of fibril detection for BSE, shows that the ease of fibril detection depends on anatomic region sampled and suggests an association between PrP accumulation and vacuolar changes in certain neuroanatomic areas.     

Evaluation of the effects of controlled autolysis on the immunodetection of PrP(Sc) by immunoblotting and immunohistochemistry from natural cases of scrapie and BSE

Seventeen clinically suspect scrapie sheep, and twelve suspected BSE-affected cattle were confirmed using routine histopathological examination by the detection of characteristic spongiform change in the medulla brain region taken at the level of the obex. Three sheep and four cows acquired as controls showed no spongiform change. Five aliquots of brain tissue from each of four brain regions were taken (cerebellum, medulla, frontal cerebral cortex and occipital cerebral cortex) from each of the 36 animals. One aliquot was frozen at -70 degrees C, the others were subjected to one of four autolysis regimes at 3 or 7 days at 25 degrees C or 37 degrees C. All samples were tested by Western immunoblotting for detection of PrP(Sc) using the Prionics - Check test (Prionics AG, Zurich, Switzerland). Further samples of medulla from 15 suspect scrapie cases, 10 healthy sheep, 13 suspect BSE cows and 5 healthy cows, were taken adjacent to the obex, and subjected to autolysis at 37 degrees C for 6, 12, 24 and 48 hours before being fixed in 10 per cent formal saline and subsequently examined by a routine immunohistochemical technique for detection of PrP(Sc) protein. The abnormal protein could not be detected in any of the control animals by either technique. PrP(Sc) could be detected by Western immunoblotting in at least one brain area from all the positive animals after autolysis for 7 days at 37 degrees C. The protein could be detected by immunohistochemistry in all cases which were positive by histopathological examination using all autolysis conditions. From the results of this study it is concluded that autolysis does not significantly compromise the diagnosis of scrapie or BSE by either of these diagnostic methods.     

Diagnosis of bovine spongiform encephalopathy: A review

Summary

Cows affected with bovine spongiform encephalopathy (BSE) display chronic neurological signs consisting of behavioural changes, abnormalities of posture and movement, and/or hyperaesthesia. At present, there are no laboratory test available to diagnose BSE in the live animal. In this article, we describe the post‐mortem diagnostic examination of brains from BSE‐suspected cattle as currently performed at ID‐Lelystad. The routine laboratory diagnosis of BSE consists of histopathological examination of the brain and detection of the modified prion protein, PrP^BSE, in brain tissue. These tests, however, have the disadvantage of being laborious and time consuming, so that results are available only after several days.

Recently, at ID‐Lelystad a new post‐mortem test has been developed that enables screening of larger volumes of brain samples for PrP^BSE within 1 day. This BSE test is especially suited for slaughterline monitoring. A preliminary validation study has shown that both sensitivity and specificity are 100% compared to the gold diagnostic standard of histopathology.     

A collaborative Canadian-United Kingdom evaluation of an immunohistochemistry protocol to diagnose bovine spongiform encephalopathy

Collaboration was established in 2001 to evaluate a commercially available immunohistochemistry assay kit for the detection of bovine spongiform encephalopathy (BSE) disease-associated prion protein in formic acid-treated formalin-fixed samples of bovine brain. The kit protocol was evaluated at the National Centre for Foreign Animal Diseases (Winnipeg, Canada) and the Veterinary Laboratories Agency (Weybridge, U.K.). The U.K. laboratory provided paraffin-embedded blocks of brainstem (medulla oblongata at the level of the obex) from 100 positive cases defined by clinical signs and histopathology, and 100 clinically suspect but BSE-negative samples defined by histopathology and immunohistochemistry with anti-PrP monoclonal antibody R145. The Canadian laboratory provided 400 blocks from surveillance cases defined as clinically suspect but negative by histopathology and immunohistochemistry with anti-PrP antibody 6H4. Consecutive sections from each block were cut and coded. Each set of 600 slides was immunolabeled and read in each laboratory. Evaluation parameters included estimates of diagnostic sensitivity and specificity and reproducibility of the results. The kit performed with 100% sensitivity, specificity, and reproducibility in spite of minor differences between the laboratories in brain sample areas, fixation and processing, and in the immunolabeling protocol. Although enzyme linked immunosorbent assays are widely used in high throughput surveillance programs, standardized protocols and reagents for manual immunohistochemistry provide a useful adjunct to surveillance efforts, particularly in laboratories testing small numbers of samples or using immunohistochemistry for confirmation and characterization of BSE cases.     

Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep

Extracts from the cervical spinal cord and from the medulla, thalamus, cerebellum and cerebral cortex of the brains of 10 sheep, histopathologically confirmed as cases of scrapie, were examined by electron microscopy for the presence of scrapie-associated fibrils. Characteristic fibrils were observed in all the extracts except for that from the thalamus of one sheep. No fibrils were found in any extracts from three control sheep. A comparison of these results with a similar study of 22 cases of bovine spongiform encephalopathy (BSE) suggests that in cases of scrapie the area of the brain chosen for the detection of fibrils is less critical than in cases of BSE, in which fibrils are more readily extracted from areas of the brain stem.     

Model calculation to explain the BSE-incidence in Germany

The future development of BSE-incidence in Germany is investigated using a simple epidemiological model calculation. Starting point is the development of the incidence of confirmed suspect BSE-cases in Great Britain since 1988, the hitherto known mechanisms of transmission and the measures taken to decrease the risk of transmission as well as the development of the BSE-incidence in Germany obtained from active post mortem laboratory testing of all cattle older then 24 months. The risk of transmission is characterized by the reproduction ratio of the disease. There is a shift in time between the risk of BSE transmission and the BSE incidence caused by the incubation time of more than 4 years. The observed decrease of the incidence in Germany from 2001 to 2003 is not a consequence of the measures taken at the end of 2000 to contain the disease. It can rather be explained by an import of BSE contaminated products from countries with a high BSE incidence in the years 1995/96 being used in calf feeding in Germany. From the future course of the BSE-incidence in Germany after 2003 a quantification of the recycling rate of BSE-infected material within Germany before the end of 2000 will be possible by use of the proposed model if the active surveillance is continued.     

Lesion profiles and gliosis in the brainstem of 135 Swiss cows with bovine spongiform encephalopathy (BSE)

Lesion profiles are considered to be an important tool for the comparison of the various animal and human spongiform encephalopathies and to obtain information upon prion strain variations. Histological and immunohistochemical reactions (PrPsc, GFAP) in 13 brain areas at 4 levels in the brainstem from 135 BSE-positive and 45 BSE-negative cases were retrospectively evaluated. In this retrospective study a lesion profile based on histological features was worked out on the basis of BSE cases originating from Switzerland over a period of ten years. They were confirmed post mortem by histology and immunohistology. Our findings were reviewed in comparison with lesion profiles published in England. No striking differences comparing type and quality of lesions in the relevant areas between the Swiss and the English cases were evident. Moreover, the lesion profiles and the character of the lesions did not differ between animals born before or after the offal feeding ban, which supports the hypothesis that the Swiss epidemic is sustained by the same single, stable strain of the BSE agent, which is probably the same as in the English epidemic.There was a good correlation between PrPsc accumulation and spongiform changes, in particular in those areas which were morphologically most affected. Astrocytosis in BSE was quantified. A significant rise in GFAP-positive cells could be shown comparing the brain stem nuclei of BSE affected with BSE-unaffected cattle, despite considerable variation between the cases and between the nuclei. The observed astrocytosis did correlate with vacuolation of the neuropil and of perikarya as well as with PrPsc accumulation.     

A method to identify BSE suspects in emergency slaughtered cattle during ante mortem examination

A method is presented by which a maximal number of BSE-infected cattle that had escaped the filter of clinical examination are identified during ante mortem examination. This approach might prove to be an efficient and cost-effective method for veterinary and non-veterinary meat inspectors to remove infected animals prior to slaughter. In a case-control study, the clinical signs of 224 randomly selected sick slaughtered animals were compared with the clinical signs of 26 sick slaughtered animals in which BSE infection was diagnosed using a rapid test post mortem. In addition, the clinical signs of the sick slaughtered BSE-positive animals were compared with the clinical signs of a group of BSE suspects identified during the same time period and in which BSE infection could be confirmed. As a result of this study a mathematical model was developed to identify BSE suspects. This model contains a total of 7 variables (clinical signs) that proved to be of importance. These signs were aggressive behaviour, grinding of teeth, protruding eyeballs, reduced rumination, inability to stand, overexcitability, and difficulty in standing up. The presented model has a sensitivity of 61.5% (16 out of 26 BSE-positive animals slaughtered while sick were identified) and a specificity of 99.6% when compared to a rapid test conducted post mortem.     

Pathogenesis of experimental bovine spongiform encephalopathy (BSE): estimation of tissue infectivity according to incubation period

This paper reports the results of tissue infectivity assays of bovine spongiform encephalopathy (BSE) agent in orally exposed cattle at stages during the incubation period. Estimations of the titre of infectivity in central nervous system (CNS), certain peripheral nerve ganglia and distal ileum tissue were made according to time post exposure from the relationship between incubation period and dose for RIII mice and C57bl mice using data from titrations of brain material from cases of BSE. The rate of increase of infectivity in the bovine CNS was then estimated, taking into account these tissue infectivity titres, the variability of the brain titre of clinical field cases of BSE, and the probability density of the expected number of months before clinical onset of each infected bovine. The doubling time for CNS was shown to equal 1.2 months. The titre in the thoracic dorsal root ganglia (DRG) was, on average, approximately 1 log units less than CNS, and cervical DRG approximately 0.5 log less than thoracic DRG. The pattern of increase of infectivity in the distal ileum is that of an initial increase up to 14-18 months post exposure, followed by a decrease, which is likely to be highly variable between animals. These results will be informative for future risk assessments of BSE, especially in relation to reviewing current control measures.     

Structural weakening of intramuscular connective tissue during postmortem aging of pork

We studied structural changes in the endomysium and perimysium during postmortem aging of pork using the cell‐maceration/scanning electron microscope method. Immediately post mortem, endomysia sheaths that house individual muscle fibers displayed a honeycomb‐like structure. The sheaths of the endomysium consisted of tightly arranged collagen fibrils in a random network. The perimysium comprised several layers of wavy sheets made up of tightly bundled collagen fibers. While the structure of the intramuscular connective tissues remained almost unchanged up to five days post mortem, the endomysium had resolved into individual collagen fibrils, and the thick sheets of the perimysium had separated into collagen fibers and fibrils at 8 days post mortem. These results provide direct evidence for structural weakening of the endomysium and perimysium during postmortem aging of pork. The shear‐force value of raw pork decreased rapidly within six days post mortem and then decreased slowly until 14 days post mortem. Since the rapid increase in tenderness is mainly due to structural weakening of myofibrils, we conclude that the disintegration of the endomysium and perimysium contributes to tenderization of pork during extended postmortem aging.     

Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method

Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.     

Studies of embryo transfer from cattle clinically affected by bovine spongiform encephalopathy (BSE)

Semen from 13 bulls, eight with clinical bovine spongiform encephalopathy (BSE), was used to artificially inseminate (AI) 167 cows with clinical BSE, and their resultant embryos were collected non-surgically seven days after AI. The viable and non-viable embryos with intact zonae pellucidae were washed 10 times (as recommended by the International Embryo Transfer Society) then frozen. Later, 587 of the viable embryos were transferred singly into 347 recipient heifers imported from New Zealand, and 266 live offspring were born of which 54.1 per cent had a BSE-positive sire and a BSE-positive dam. The recipients were monitored for clinical signs of BSE for seven years after the transfer, and the offspring were monitored for seven years after birth. Twenty-seven of the recipients and 20 offspring died while being monitored but none showed signs of BSE. Their brains, and the brains of the recipients and offspring killed after seven years, were examined for BSE by histopathology, PrP immunohistochemistry, and by electron microscopy for scrapie-associated fibrils. They were all negative. In addition, 1020 non-viable embryos were sonicated and injected intracerebrally into susceptible mice (20 embryos per mouse) which were monitored for up to 700 days, after which their brains were examined for spongiform lesions. They were all negative. It is concluded that embryos are unlikely to carry BSE infectivity even if they have been collected at the end-stage of the disease, when the risk of maternal transmission is believed to be highest.     

Partial autolysis of μ/m‐calpain during post mortem aging of chicken muscle

The objective of this study was to investigate changes occurring in μ/m‐calpain in post mortem chicken muscles and to determine the origin of the unknown bands found in calpain casein zymography. The unknown bands were reported with slightly greater mobility compared to conventional μ/m‐calpain bands in casein zymography. Identification of these bands was accomplished using native polyacrylamide gel electrophoresis, liquid chromatography tandem mass spectrometry and with protein phosphatase treatment. Results showed that the unknown bands were corresponding to μ/m‐calpain, and dephosphorylation by protein phosphatase did not change their appearance. The calpain samples were then incubated with various concentrations of Ca²⁺ to determine the relationship between changes in μ/m‐calpain and the appearance of the unknown bands. The products of μ/m‐calpain partial autolysis were found to be consistent with the appearance of the unknown bands. Therefore, the appearance of these bands did not result from phosphorylation of μ/m‐calpain as previously hypothesized, but from partial autolysis of μ/m‐calpain. Also their presence suggests that μ/m‐calpain undergoes partial autolysis during aging which may play certain roles in meat quality improvement.     

Clinical sign profile likelihood ratios for bovine spongiform encephalopathy suspects

This study has described a method for generating the probability of a bovine spongiform encephalopathy (BSE) suspect being a true positive BSE case. A weighting equivalent to the clinical sign frequencies recorded in histopathologically confirmed BSE cases was assigned to 14 clinical observations and a clinical profile score for the case was generated by the summation of the weightings. This method was applied to 50 histopathologically confirmed (true positive cases) BSE suspects and 50 histopathologically unconfirmed (false positive cases) BSE suspects. The profile scores for the true positive BSE suspect cases were statistically significantly higher than the profile scores of the false positive BSE suspect cases (P = 0.0014) using a Mann-Whitney U non-parametric test. The mean profile score for the true positive cases was 944 and for the false positive scores was 879. Likelihood ratios for BSE suspects with different clinical profile scores were computed using different clinical profile score cut off point and ranges. A BSE suspect with profile score of 727 or above (the lowest cut off point) and 1037 or above (the highest cut off point) had likelihood ratios for being BSE of 1. 09 and 4.00 respectively. The likelihood ratios for a BSE suspect being a true positive BSE case in the profile score ranges 627-866, 867-966, 967-1036 and 1037-1067 were 0.35, 1.06, 1.30 and 4.0 respectively. Further investigations or a revisit may be justified in animals with a low probability of being BSE.     

Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4°C and −20°C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at −20°C, but not in any of the extracts stored at 4°C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4°C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.     

Control of the collagen fibril diameter in the equine superficial digital flexor tendon in horses by decorin

The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.     

Cerebellar hypoplasia in three sibling cats after intrauterine or early postnatal parvovirus infection

The present report describes the case of an intrauterine or early postnatal parvovirus infection with subsequent cerebellar hypoplasia in three kittens from the same litter. Clinical examination of affected cats revealed neurologic signs indicative of cerebellar ataxia. Due to poor prognosis, animals were euthanised and submitted for necropsy. Post mortem examination demonstrated variable degrees of cerebellar hypoplasia. Histologically, brain lesions were characterised by segmental loss of the external and internal granular layer and decreased numbers of Purkinje cells. Reactive proliferation of astrocytes in the central nervous system was verified by the detection of GFAP-expressing glial cells in affected areas using immunohistochemistry. Furthermore, parvovirus antigen was detected immunohistochemically in neuronal cells of the cerebellum, but not in other parts of the brain and spinal cord or non-neuronal tissues. The present report demonstrates the usefulness of post mortem examination and detection of viral antigen by immunohistochemistry for the discrimination of neurologic disorders in feline species. Neurologic deficiencies due to cerebellar hypoplasia caused by in utero or perinatal feline parvovirus infection should be taken into consideration as differential diagnoses for ataxia in neonatal and juvenile cats.     

Abnormalities of collagen fibrils in a rabbit with a connective tissue defect similar to Ehlers-Danlos syndrome

A morphometric ultrastructural study was performed to confirm the presence of an abnormality of the collagen fibrils in a rabbit with a connective tissue defect similar to Ehlers-Danlos syndrome. Median fibril diameter and perimeter were not altered but their ranges were significantly increased. As indicated by the median fibril ‘form factor’, fibrils were significantly more irregular in shape; the range of irregularity in shape was also increased. Fibril periodicity was unchanged. The results are discussed in relation to collagen fibril structure and fibril abnormalities in similar diseases in man and other animals.