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1 Veterinary Laboratories Agency (VLA), New Haw, Addlestone, Surrey KT15 3NB, UK
2 ADAS Rosemaund, Preston Wynne, Hereford HR1 3PG, UK
3 ADAS Defra Drayton, Stratford-upon-Avon, Warwickshire CV37 9RQ, UK
4 VLA Lasswade, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK
Correspondence
James Hope
j.hope{at}vla.defra.gsi.gov.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Donnelly et al., 1997; Ferguson et al., 1997a, b; Wilesmith & Ryan, 1997). Donnelly et al. (1997) showed an enhanced risk of BSE infection in calves born to infected dams within 24 months of onset of clinical BSE. If transmitted from the dam, such infection might arise in utero, in the immediate post-parturition period or by vertical transmission to the calf (Wilesmith et al., 1992; Winter et al., 1989); hence, suckling milk and/or colostrum are possible vehicles for transmission from cow to calf.
Colostrum is a low-volume, protein-rich fluid taken by calves soon after birth at a time (up to 48 h) when absorption of whole proteins from the gut is enabled (Matte et al., 1982; Quigley & Drewry, 1998). Bovine colostrum is not normally part of the human diet. Calves born in a dairy herd normally receive only colostrum for 24–48 h after birth, and reconstituted milk replacer, but no milk, thereafter, whereas calves in a suckler herd commonly receive both colostrum (for up to 48 h) and the dam's milk for extended periods (up to 6 months). Data from a suckler-herd study in the UK indicated no evidence of BSE in offspring born to BSE-infected cows (Wilesmith & Ryan, 1997). This argues against consumption of milk or colostrum from BSE-infected cows as a risk factor for intraspecies transmission of BSE [but see the paper by Donnelly et al. (1997)]. Similarly, in other transmissible spongiform encephalopathy (TSE) diseases, evidence for infectivity in milk or colostrum is negligible.
There is also no direct evidence from past experimental studies for the presence of infectious TSE agent in milk, although, for logistical reasons, the volumes and number of samples examined have been very limited. These have included the intracerebral (i.c.) (0.02 ml, single inoculation), intraperitoneal (i.p.) (0.1 ml, single inoculation) and oral (300 ml over 40 days) dosing of mice with milk collected at early-, mid- and late-lactation points from BSE-infected cows that subsequently developed disease (Bradley, 1993; Taylor et al., 1995). Milk and udder tissue from four cases of terminal-disease bovine BSE were fed to mice (Middleton & Barlow, 1993) at a level of 14.6 ml and 128.9 g (C57Bl mice) and 20.2 ml and 144 g (CRH mice) over a period of 27 days with no evidence of infection. Reports of transmission of disease to mice by i.c. inoculation of human colostrum from a woman with preclinical sporadic CJD (Tamai et al., 1992) have not been confirmed.
In late 1999, several rapid tests for the confirmatory diagnosis of BSE using bovine brain homogenates became available (Moynagh & Schimmel, 1999). These tests are based on the detection of abnormal prion protein (PrPBSE), a marker for TSE infectivity. One, the Bio-Rad Platelia, was claimed to have an analytical sensitivity for PrPBSE equivalent to that of the mouse bioassay for BSE infectivity (Grassi et al., 2001; Moynagh & Schimmel, 1999). The mouse bioassay, using i.c. and i.p. inoculations, is very time-consuming and can only test for infectivity in a very limited volume of milk; therefore, it was decided to validate this type of rapid screening test for PrPBSE and apply it to milk samples. The results would then provide further data required for a quantitative assessment of risk of BSE transmission via milk.
The validation of a milk prion assay in the absence of a positive control, that is, a naturally ‘infectious' milk specimen, poses several problems relating to the classification of a positive result. In order to determine defined levels of confidence that a ‘positive’ result was a chance false-positive rather than a true-positive result, thousands of true-negative samples would require analysis. Alternatively, a second test based on an independent physicochemical principle could be designed to confirm ‘reactives' as true negative or positives. The latter approach was adopted within this study.
The specificity of the Bio-Rad Platelia for PrPBSE rather than PrPC relies on the relative resistance of the abnormal protein to proteinase K (PK) hydrolysis under mild denaturing conditions. The confirmatory test developed hence excluded proteinases and instead achieved this specificity by physical adsorption of aggregates of PrPBSE to a polyanionic ligand, the Seprion reagent (Microsens Biotechnologies), followed by elution and detection by a PrP immunoblot system. This has the advantage that ligands selective for abnormal PrP, such as the Seprion polymer or the 15B3 mAb (Korth et al.,1997), may also bind proteinase-sensitive, infection-related forms of PrP (Nazor et al., 2005).
The quantitative assessment of risk of BSE transmission to humans via milk requires that the limit of detection of the screening and confirmatory tests for abnormal BSE prion protein needs to be calibrated against an infectivity titre. In the absence of an ‘infectious' milk control, the VLA ‘pathogenesis' study provides the most appropriate data for calibration and is accrued from experiments where cattle were fed dilutions of infected cattle-brain homogenate (Wells et al., 1994, 1996, 1998, 1999). Similar (but not identical) homogenates from clinical, confirmed cases of BSE were used to produce dilution curves from which limits of detection in mg equivalents of cattle brain were derived and converted into cattle oral ID50 (CoID50) units.
Cows' milk is mostly water (87.4 %), a complex colloidal dispersion of fat (3.7 %), caseins (2.7 %), whey proteins (0.7 %), lactose (4.8 %) and minerals (0.7 %) and a variable amount of somatic cells, cell fragments and bacteria (Jensen, 1995). Abnormal PrP and infectivity co-partition with cells and membrane sheets during the fractionation of brain tissue in the absence of detergents so, assuming that any abnormal PrP in milk would be similarly associated with cells, we adapted the commercially available rapid BSE test to detect PrPBSE brain homogenates or detergent extracts spiked into a milk-cell concentrate and applied this screening test to similar milk fractions sourced from BSE-challenged cattle and control animals.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 1996, 1998, 1999). Infection was subsequently confirmed in the high dose-challenged milk donors by observation of clinical signs (in all but one animal that was culled because of mastitis) and final histopathological examination of brain tissue at necropsy (Wells et al., 1998). No BSE has been observed at any stage in the surviving control (n=8) or low dose-challenged animals (n=8), nor at necropsy of intercurrent kills (n=2 for both the control and low-dose groups). The cows first calved at an age of between 2 and 3 years and each of the three groups were housed and managed separately with no contact between groups, which had completely separate husbandry regimes. Cows in the high-dose group survived to provide milk from only one or two pregnancies before showing the clinical signs of BSE, whilst the low-dose and control animals have survived longer and have been inseminated, calved and milked for up to four lactations. For each lactation, up to 10 l milk was collected at each of five time points post-calving: 0–7 days and at 10, 20, 30 and, when possible, 40 weeks.
During this experiment, there were bouts of mastitis in 20 cows from within all three groups and all four lactation periods; these observations were recorded here because of the possible confounding effect of concurrent inflammatory disease on the tissue distribution of infectivity in TSE-infected animals (Heikenwalder et al., 2005; Seeger et al., 2005).
Milk was stored at 4 °C and subsequently centrifuged at 500 g for 2 h at 4 °C. The cream and liquid milk were removed, ensuring that all of the cell sediment was retained. Each 1 l equivalent of this cellular fraction was resuspended in PBS (12 ml, 0.05 M, pH 7.4) and stored at –20 °C. These cell concentrates were thawed at 4 °C for 16 h prior to use. The mean dry-weight analysis of this resuspended sediment was 0.038 mg ml–1 (range, 0.0014–0.3580 mg ml–1).
Prion protein (PrP) ELISA.
The Bio-Rad Platelia ‘purification kit’ and ‘detection kit’ provided the reagents for the extraction and PK hydrolysis of abnormal prion protein (PrPBSE) and the subsequent detection of residual PrP, PrPres, by a sandwich ELISA. This set of reagents allows the solubilization of tissue or cell extracts in a homogenization buffer (A), the controlled digestion of proteins, including normal cellular prion protein (PrPC), by low concentrations of PK (<10 µg ml–1) in buffer A, alcohol (buffer B) precipitation of residual proteins including PrPres, denaturation (in buffer C1) and solubilization of the precipitate and dilution (in buffer R6) for assay using a two-mAb (one for capture and one for detection) microtitre plate-based colorimetric immunoassay. By changing the volumes specified by the manufacturer, this protocol was adapted to detect total PrP (no PK-digestion step included) or PrPres (PK added) in BSE-affected brain homogenate-spiked milk-cell samples, BSE-spiked normal bovine-brain homogenates or unaffected brain-homogenate-spiked milk-cell samples.
Briefly, resuspended milk-sediment sample (0.875 ml) was added to an equal volume of Bio-Rad homogenization buffer (A) and agitated by using a vibration homogenizer (Ribolyser; Hybaid). Equal volumes (0.5 ml) of this milk extract (ME) and Bio-Rad buffer A, with or without PK, were mixed and incubated at 37 °C for 10 min before alcohol precipitation with buffer B (0.5 ml) and centrifugation at 15 000 g for 7 min at 20 °C. After centrifugation, the supernatant was removed, buffer C1 (0.2 ml) was added to the pellet and incubated at 100 °C for 5 min, followed by dilution with buffer R6 (0.5 ml). Milk-cell controls, negative and positive (spiked) milk controls and all milk test samples were then assayed by using the microtitre-well system (0.1 ml final sample extract per well). This represents the milk solids from the equivalent of approximately 3 ml whole milk per well.
Bovine-brain pools.
BSE-positive bovine brain, a mixture of spinal cord (C1–C2), medulla and cerebellum, was liquidized on ice and its volume was adjusted with deionized water to prepare a standard pool of 20 % (w/v). This positive-control pool and a similar negative standard prepared from BSE-negative brain tissue were aliquotted and stored at –80 °C prior to use. These pools are referred to as positive (+) and negative (–) 20 % (w/v) bovine-brain standards, respectively.
ELISA standards, analytical sensitivity and the effects of milk.
Serial dilutions of the 20 % (w/v) BSE-positive bovine-brain standard were prepared in 20 % (w/v) non-BSE-infected bovine-brain homogenate. A 0.5 ml aliquot of each dilution was then mixed with an equal volume of Bio-Rad buffer A, with or without PK, and processed for assay as described above. To assess the effect of milk components on this dose–response curve, an ELISA positive-spiked control milk standard curve was prepared by combining each brain-homogenate dilution (0.1 ml) with 0.4 ml ME, mixing with an equal volume of Bio-Rad buffer A, with or without PK, and processing these samples similarly for assay.
Analysis of the milk-cell fractions.
Individual milk-residue samples were analysed initially by using the adapted Bio-Rad Platelia assay. Those found to be >3SD from the mean of the negative controls were deemed ‘reactive’ and were retested by using the same procedure, but surrounded by different control samples on the plate.
Determination of final test-reactive results employed a calculation of the mean absolute deviation (MAD) (Miller & Miller, 2000). The MAD was derived by first calculating the median of all of the data, then calculating the absolute difference of each observation in the whole dataset from this median: this is the difference without regard as to whether it is positive or negative, above or below the median. Finally, the median of this set of differences was calculated to give an estimate SD of the data, with the cut-off set as 3SD of this value above the new median. The final set of reactive samples was determined by using a cut-off calculated from the whole dataset or a cut-off calculated separately for each lactation.
Seprion-PAGE/Western blot analysis of milk cellular fractions.
Milk concentrate (ME) samples (0.41 ml), unspiked or spiked with serial dilutions of the 20 % (w/v) BSE-positive bovine-brain standard prepared in 20 % (w/v) non-BSE-infected bovine-brain homogenate, were added to Bio-Rad Platelia ‘purification kit’ homogenization buffer (2.70 ml) and homogenized by vibration. This diluted milk sample (2.35 ml) was mixed with 600 µl Seprion capture buffer (Microsens Biotechnologies), Seprion-coated magnetic beads (0.05 ml) were added and the mixture was rotated at 60 °C for 16 h. The sample tubes were then placed in magnetic racks (Dynal Biotech) to fix the bound PrPBSE–magnetic-bead complex and washed four times with Bio-Rad Platelia wash buffer. After a final rinse in deionized water, the complex was dissociated by heating at 100 °C for 10 min in SDS-PAGE sample buffer (0.045 ml) (Prionics-Check Western Blot kit) and the denatured proteins in the extract (0.015 ml) were analysed by SDS-PAGE/Western blot using the 6H4 mAb as detector according to the Prionics-Check Western Blot kit protocol. The equivalent of 8.6 ml whole milk was analysed per track.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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and indicate that the levels of milk fat and milk proteins used in the screening part of this evaluation do not affect the analytical sensitivity of this adapted assay significantly, compared with its sensitivity applied to bovine-brain homogenate.
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). A working estimate of 0.25 mg brain was taken as a reasonable composite of the two disparate assay formats in order to prevent the necessity of two separate calculations. In each well of the ELISA, there is the equivalent of 3 ml whole milk and so, assuming that the recovery of sample is 100 %, the limit of detection of BSE infectivity in 1 l whole milk would be 0.14 (C.I., 0.0013–0.21) CoLD50 l–1. In each track of that Seprion/Western blot, there is the equivalent of milk solids from 8.6 ml whole milk and so, again assuming that the recovery of sample is 100 %, the limit of detection of BSE infectivity in 1 l whole milk is 0.05 (C.I., 0.0005–0.0725) CoLD50 l–1.
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, segregated according to lactation cycle and PK treatment. The effective limit of detection for the assay process, and the subsequent identification of outliers as test-‘reactive’ samples, was determined by a statistical analysis of these milk/colostrum screening data. The critical value of deviation of a suspect value from the sample population mean is usually accepted as 3SD for datasets of 40 or more and was essentially the process employed to determine which individual milk sample would be retested by the Bio-Rad Platelia method. This process requires that the data arise from a fundamentally Gaussian distribution and relies on the value of the mean and median being identical and on the SD being determined as a multiple (>1.5-fold) of the MAD of the data. Neither the median nor the MAD is affected much by a reasonable number of outlying observations, allowing calculation of an appropriate cut-off value relative to the assumed underlying Gaussian distribution and, hence, definition of a population of test ‘reactives’.
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Identification of ‘reactive’ milk samples: lactation-specific cut-offs.
Twenty-two of the 541 cattle-milk samples were classed as ‘reactive’ following MAD analysis of their assay data from the adapted Bio-Rad Platelia procedure with or without the inclusion of PK (Table 1). However, differences existed between the median absorbance values of different lactation cycles and also of the samples taken 1 week post-calving. Therefore, the data were reanalysed by MAD analysis applied to each lactation period separately, giving different cut-off points for each period and for the week 1 post-calving values (Fig. 3
). By this procedure, six new samples were recorded as ‘reactive’ in one or both of the duplicate wells. These samples are highlighted in Table 1. As would be expected for random variation, in this reanalysis, about half of the assays were made more sensitive and half less sensitive relative to a cut-off derived from the whole population of results.
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; Fig. 3). The number of different animals sampled to provide specimens varied with each group for operational reasons. In total, 80 specimens were sampled from unchallenged animals, 90 specimens from low dose-challenged animals and 49 specimens from the high dose-challenged animals. Six of these specimens proved ‘reactive’ by using both the PK and non-PK ELISA protocols; significantly, these specimens were all collected from different cows in the first week post-calving: three from the unchallenged group in their first, second and third lactations, respectively, and three from the low dose-challenged group in their third (n=2) or fourth (n=1) lactation (Table 1). One specimen from animal No. 276 from the low-dose group proved ‘reactive’ only in the PK ELISA. Searching through the data for other associations with ‘reactivity’ was less fruitful. From the unchallenged group, five of the 10 cows provided the eight milk specimens that were reactive in the Bio-Rad Platelia screening test, with one cow (No. 1) providing four specimens. Five samples were obtained within the first week post-calving from lactation periods 1–4, two from the 20 week post-calving period in lactations 3 and 4 and one from the 40 week collection from lactation 1. The low-dose group provided seven cows with 14 reactive specimens, which included nine samples from the first week post-calving from lactations 1–4 and one each from the 40 weeks lactation 1, 30 weeks lactation 2 and 10, 20 and 30 weeks periods from lactation 4. The high dose-challenged group saw fewer ‘reactives' overall, with four individual cows producing six ‘reactive’ milk samples, five of which were first-week collections from lactation periods 1–3 and one at 20 weeks in the second lactation cycle. PK is used routinely in TSE screening tests to discriminate between the normal and abnormal isoforms of the prion protein, but several samples from both the unchallenged and low-dose groups and none from the high-dose group gave ‘reactives' by using the PK ELISA protocol and, so, we conclude that there was no correlation between BSE challenge/disease status and test ‘reactivity’.
No association between ‘reactivity’ and mastitis
Mastitis is a common disease in dairy herds. Macrophage and polymorphonuclear leukocytes or neutrophils predominate in both milk and colostrum, whilst lymphocytes constitute up to 15 % of the total leukocyte population. Recently, the presence of chronic lymphocytic inflammation has been shown to facilitate ectopic PrPSc accumulation, raising the possibility that the udder may be a site of prion propagation in inflammatory conditions such as mastitis (Heikenwalder et al., 2005; Seeger et al., 2005). Indeed, Ligios et al. (2005) have now observed PrPSc in the mammary gland of sheep suffering from mastitis.
In this study, we recorded many cases of mastitis in all three groups of milk donors. However, we were unable to find any evidence that mastitis stimulates replication of abnormal prions in the udder under conditions thought to mimic the natural oral BSE infection. Only one animal (of 10) (No. 133) was reactive (at lactation 2) and had a recorded bout of mastitis (during its first lactation) in the high-dose group, although all of the cattle (5/10) in the unchallenged group displaying reactive results suffered from periodic bouts of mastitis and six of the seven reactive (of 10) cows in the low dose-challenged group also had at least one incidence of mastitis. These data and their statistical analysis are summarized in Table 3.
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Development of a confirmatory test: Seprion-PAGE/Western blot analysis
The method that we developed uses a charged polymer (the Seprion ligand) to extract abnormal prion protein from solution, followed by denaturation of the bound PrPBSE and SDS-PAGE/immunoblot analysis. The Seprion technology uses synthetic polymers that bind PrPSc specifically in the presence of PrPC (Lane et al., 2003). Capture is via polyionic interaction between the chemical ligand and PrPSc and requires an ionic surfactant to competitively inhibit any weak binding of PrPC. The selectivity of the Seprion-ligand capture technology eliminates the need for PK digestion to remove PrPC and hence distinguishes this protocol from those PrPBSE-diagnostic methods that rely on PK hydrolysis of PrPC for their specificity. The specificity and sensitivity of the Seprion technology are demonstrated in Fig. 2, where the sensitivity of the Western blot and the ELISA is compared. Abnormal PrP was extracted from samples by using the Seprion polymer, denatured, separated by SDS-PAGE and visualized by Western blot using mAb 6H4. In the initial characterization studies, no banding was observed in 12 individual ME samples from unchallenged cattle and from pooled MEs derived from milk collected at weeks 10, 20 and 30 from unchallenged cattle by this Seprion/Western immunoblot technique. In addition, all milk samples spiked with BSE-negative brain homogenate remained negative for immunoreactive staining at 30 kDa, whilst there was a dose–response effect for the milk samples spiked with increasing amounts of BSE-infected brain standards.
Application of the confirmatory test
Of the 28 individual samples found to be reactive by using the Bio-Rad ELISA (Table 1), 12 gave positive band signals by using the Seprion/Western blot method (Table 2). All of these ‘positives' were colostrum samples collected within 1 week of calving, irrespective of challenge group. Immunoreactive bands in the molecular mass ranges of >80 kDa (HMM band), 25–29 kDa and 20–22 kDa were observed and one or more of these bands was seen in each sample (Fig. 4). Twenty-two bands from eight cases, including all HMM and 25–29 kDa bands, were still observed when the primary mAb (6H4) was omitted from the method protocol. Changing the secondary-antibody detection conjugate failed to improve on this lack of specificity and we conclude that the majority, if not all, of these signals are due to cross-reactions between the secondary antibody and proteins (possibly immunoglobulins) present in the samples taken in the first week post-calving.
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, involving the experimental infection of a transgenic mouse line expressing bovine PrP (boTg) with BSE prions administered i.c. These authors demonstrated that vertical transmission occurred when mating took place close to the clinical stage of the disease. In order to ascertain whether the feeding of milk was a possible route of infection, a volume of delipidated milk, equivalent to 25 % of the offspring's intake during lactancy (the period during which the cow is lactating), was injected i.c. into boTg110 mice. Analysis of survival times failed to show any significant differences as a result of the inoculations and the histology and immunohistochemistry of the brains failed to detect any PrPres. Additionally, colostrum from a BSE-affected cow failed to transmit infection (monitored by subpassage) or disease after i.c. inoculation into another transgenic mouse overexpressing the bovine prion protein gene; although the volume of liquid administered was understandably small, this tgbovXV line has been estimated to be 10-fold more sensitive to BSE exposure than cattle (Buschmann & Groschup, 2005).
Throughout our study, no abnormal prion protein was identified in the cellular fraction of milk from cattle incubating BSE by using these biochemical methods at their limits of detection. Whilst this does not exclude milk as being a potential vehicle for the transmission of prions, it does concur with our current understanding of the pathogenesis of BSE. Infectivity in lymphoid tissue has only been found in the distal ileum (Wells et al., 1998; Terry et al., 2003) and the palatine tonsil of the tongue (Wells et al., 2005), and non-neural involvement in BSE is relatively low, lessening the risk of infectivity in inflamed tissues with ectopic lymphoid follicles and its secretion in milk.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Wells, G. A. H., Hawkins, S. A., Green, R. B., Austin, A. R., Dexter, I., Spencer, Y. I., Chaplin, M. J., Stack, M. J. & Dawson, M. (1998). Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet Rec 142, 103–106.
Wells, G. A. H., Hawkins, S. A., Green, R. B., Spencer, Y. I., Dexter, I. & Dawson, M. (1999). Detection of sternal bone marrow infectivity in the clinical phase of experimental bovine spongiform encephalopathy (BSE). Vet Rec 144, 292–294.
Wells, G. A. H., Spiropoulos, J., Hawkins, S. A. C. & Ryder, S. J. (2005). Pathogenesis of experimental bovine spongiform encephalopathy: preclinical infectivity in tonsil and observations on the distribution of lingual tonsil in slaughtered cattle. Vet Rec 156, 401–407.
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Received 2 September 2005;
accepted 10 March 2006.
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, solid line) and control (healthy bovine) brain homogenate (
, dashed line) on the Bio-Rad Platelia dose–response serial-dilution curves of BSE-affected bovine-brain homogenate.
, dashed line) and the Bio-Rad Platelia ELISA applied with (
, solid line) inclusion of its PK-hydrolysis step. For each curve, two dilution series of (i) BSE-positive- and (ii) BSE-negative-brain-spiked samples of milk-sediment preparations were assayed and each curve point value was calculated as the difference of value (i)–value (ii). Vertical arrows indicate the respective assay cut-off value of a positive result, determined as the mean+3SD of a set of authentic negative tissue samples.
), milk (–PK, 
), colostrum (+PK, 
). The base of the filled triangle to the left of each set of data represents the median value, and the black bar the mean absolute deviation (MAD) cut-off value, for each dataset. Each point represents the mean of two absorbance measurements per sample.