BACKGROUND
transmissible spongiform encephalopathies  or prion diseases are fatal diseases of the central nervous system, characterized by long incubation periods before neurological symptoms appear.
tses were initially described in sheep as a transmissible disease named scrapie, and in man, as creutzfeldt-jakob disease , a sporadic disease with no detectable link between cases.
the diseases can be diagnosed by vacuolation of neurons in the brain and appearance of a structurally altered form of the host encoded prion protein , termed prpsc.
tses represent a unique class of infectious diseases in which prpsc is considered to be the transmissible agent  <cit> .
tses have attracted much attention since bovine spongiform encephalopthy  was first described in the 1980s  <cit> .
the more or less accepted explanation for the appearance of this condition was the capacity of the scrapie-like agent to spread to cattle fed with insufficiently heated remains of ruminants  <cit> .
in the 1990s a new form of cjd was observed affecting mainly young people  <cit> .
this so-called variant cjd is considered to be caused by the bse agent  <cit>  and to have spread by the consumption of infected food containing residual bovine nervous tissues, although other means of transmission, such as the use of ruminant-derived substances in medical products, can not be ruled out.
furthermore, man-to-man transmissions might have occurred subsequently by blood transfusions.
bse is now largely under control, but two recent reports describing goats with bse have strengthened the fear that the bse agent could again become widespread in some food animals before being diagnosed  <cit> .
in order to prevent this from happening, the tses from livestock must be eradicated.
for this purpose a number of programmes are underway.
for eradication of ruminant tses in europe, where bse cases have occurred in nearly all member states, control programmes have been implemented , e.g., the removal of risk materials, active monitoring of slaughtered cattle, sheep and goats, stamping out of scrapie, and genetic breeding of sheep towards scrapie and bse resistant prpgenotypes.
active monitoring is possible using brain tissue taken late in the incubation phase.
early diagnosis of tses can assist in monitoring programs and has potential in sheep and goats where lymphoreticular tissues generally accumulate infectivity and aberrant prp  <cit> .
indeed, until now, tonsil and other lymphoid tissues have been targets for preclinical scrapie diagnosis in sheep, making reliable diagnosis possible in biopsies early during incubation  <cit> .
however, for the diagnosis to be as early as possible, i.e.
in the preclinical stage, diagnostic methods more rapid than the time consuming microscopic and bioassay techniques currently employed are required.
such a diagnosis should also preferably allow discrimination between tse strains like scrapie and bse, as is described for western blotting  methods applied to brain  <cit> .
a very reliable indicator of disease is the presence of abnormal forms  of prion protein.
it is recognized by immunohistochemistry  as accumulations of prp, or as a partially protease-resistant protein .
by wb of prpres using prp-specific antibodies, a typical triplet banding pattern appears, representative of diglycosylated, monoglycosylated and aglycosyl prpres moieties, migrating with apparent molecular masses between  <dig> and  <dig> kda.
using staining with two antibodies specific for different prp-sites, wb can even discriminate between scrapie and experimental bse infection in sheep brain due to differential cleavage of prpres by proteinase k   <cit> .
in this study we investigated the applicability of prpres wb to retropharyngeal lymph nodes  since these organs are easily accessible at slaughter and are one of the best targets for diagnosis in the lymphoid system  <cit> .
this was performed on three groups of sheep: firstly, sheep from an early and late scrapie pathogenesis study with mainly genotype vrq/vrq ; secondly, arq/arq sheep with orally induced bse; and thirdly, sheep diagnosed during active and passive surveillance in the years 2002– <dig>  the latter group represented six different prp genotypes.
RESULTS
antibody choice and fine epitope specificity
in lymphoid tissues prpres concentrations have been reported to be  <dig> times lower than in brain  <cit> .
therefore, monoclonal antibodies  with superior affinity for ovine prp in western blot  were sought.
ten mabs  with specificity for ovine prp were compared on wb strips by varying two parameters: mab concentration and amounts of ovine scrapie brain digest .
as a result, antibodies 12b <dig>  p <dig>  9a <dig>  and l <dig> were selected as the most reactive.
the epitope specificities of these four mabs were determined by pepscan analysis on solid phase synthetic ovine prp peptides .
synthetic peptides carrying these epitope sequences were also used in solution to investigate by elisa their capacity to block binding to recombinant ovine prp.
indeed, as can be expected for antibodies that are elicited with peptides, all four of these could interfere effectively with antibody binding to prp, confirming the significance of the mapped specificities .
tissue treatment and choice of retro-pharyngeal lymph node
a practical means of homogenisation was chosen for tonsillar and medial retropharyngeal lymph node  tissue by using disposible homogenisers available for mass screening purposes.
prypcon homogenisers were preferred since these consistently yielded satisfactory suspensions while other probes caused formation of gel in the homogenate.
homogenates of tonsils and rln from scrapie-infected sheep were examined after enzymatic hydrolysis with proteinase k  and/or deglycosylation with pngasef .
digestion with pk resulted in the typical triplet of diglycosylated, monoglycosylated and aglycosyl prpres.
subsequent removal of carbohydrate moieties by pngasef led further to a prominent single deglycosylated prpres band.
there were no major differences in prp immunostaining between tonsils and rlns.
in further studies, rln tissue was used as the preferred tissue for evaluating lymphoid tissues, since in contrast to tonsillar tissue, it lead to reproducible scrapie diagnosis and organ size was large enough for repetitive analysis.
this better reproducibility is likely due to a more homogeneous distribution of prpsc in rln compared to tonsils.
also, at slaughter, rlns are more easily accessible in the decapitated head than are tonsillar tissues.
diagnosis of scrapie in sheep during the incubation phase
we wished to know first, whether wb could detect scrapie in the preclinical phase.
as reported previously, vrq/vrq sheep from our scrapie-infected flock are ihc positive in tonsils and rln at two months of age  <cit> .
rln samples of  <dig> such sheep, as well as of  <dig> scrapie negative sheep , were examined by wb.
from the age of three months, wb yielded results identical to ihc .
in 2-month old cases, wb was negative, whereas ihc could detect prp only in fewer than 25% of the follicles.
from the age of four months onwards, all vrq/vrq sheep were positive in both ihc and wb.
a 20-fold concentration step allowing application of  <dig> mg tissue equivalents  per lane instead of  <dig> mg yielded a proportionally stronger signal in the positive cases, but did not change the diagnostic outcome .
the rln samples from the  <dig> scrapie negative sheep remained wb negative.
when tested by wb, brain tissue from the vrq/vrq animals scored positive from  <dig> mo of age onwards, which was some  <dig> mo later than by ihc.
compared to unconcentrated samples, application of  <dig> mg te after a preceding concentration did not lead to earlier detection of prpres.
by wb, scrapie diagnosis using rln from these sheep was already possible at  <dig> mo of age, while a positive diagnosis with brain was obtained only from  <dig> mo of age, thus illustrating that wb on lymphoid tissue can lead to a scrapie diagnosis at a very early stage of infection.
diagnosis in animals in preclinical and clinical phase of experimental bse
rlns from sheep with experimental bse, in a previous  <cit>  and ongoing study, were examined by wb.
all  <dig> infected animals of this study were positive by wb, but only after concentration to 10– <dig> mg te/lane.
.
if no concentration treatment was included and mab l <dig>  the best antibody for bse, was used,  <dig> out of  <dig> animals in the preclinical stage, and  <dig> out  <dig> in the clinical stage, could be diagnosed.
thus, in the case of bse, concentration improves the diagnostic sensitivity.
surprisingly while all animals were positive by wb, one with the shortest duration of infection  was negative by ihc.
antibodies showed different affinities for prpres in bse- and scrapie-infected sheep .
prp-core specific antibodies l <dig> and 9a <dig> recognized prpres in bse samples, with the former being the best antibody.
pk-cleavage site specific antibodies 12b <dig>  hardly bound to prpres from bseinfected samples.
remarkably, 9a <dig> bound less strongly to prpres in bse samples than l <dig>  contrary to findings with scrapie-infected sheep.
it seems, thus, that the 9a <dig> epitope in bse samples could be more prone to pk cleavage than that in scrapie.
variable cleavage of prpsc is a recognized phenomenon, but the extent or pattern of these cleavages could well depend on agent-specific prp-conformational properties, which in situ might result in differences in antibody binding between potentially different tse strains  <cit> .
these results also emphasize that for discrimination between scrapie and bse in dual antibody staining, the use of core antibody l <dig> is preferable to 9a <dig>  especially in weakly positive samples.
diagnosis of scrapie in field cases from surveillance and with mostly unknown ages
rlns from sheep with various prp genotypes and from different sources, were examined by wb and ihc.
in general, again, there was a good correlation between the two methods, since divergent results occurred only for some weak ihc-positive cases .
thus,  <dig> out of  <dig> brain-positive sheep were diagnosed to be scrapie positive with rln by both methods and two others were only positive by ihc.
within the different genotypes, wb of rln scored  <dig> out of  <dig> positive in the arq/arq group, , and  <dig> out  <dig>  in the arr/vrq group.
for the other genotypes , the two tests corresponded fully with each other.
in brain-negative sheep,  <dig> out  <dig> appeared positive with both wb and ihc.
this one lymphoreticular system  positive case was an arq/vrq animal,  <dig> mo of age, derived from our own flock with minimal scrapie-incidence.
further concentration up to  <dig> mgte/lane yielded higher signals proportional to the 10× concentration factor, but the diagnostic result in this set of samples was the same as found for the unconcentrated samples.
molecular weight and glycoprofiles of prpres bands
apparent molecular weights  of the three prpres bands in wb of rln digests were compared for a correlation by type of infection , stage of incubation or genotype.
a bse-specific difference in the mwr existed and was especially distinct for the aglycosyl moiety, which was about 1kda larger in scrapie samples than in bse samples .
this appeared to be the case for both antibodies 9a <dig> and l <dig>  antibody 12b <dig>  only bound well to the scrapie-infected samples  indicating that, as in brain stem tissue  <cit> , a discrimination between scrapie and bse by dual wb staining is possible using lymphoid tissues, i.e.
comparing the immunostaining of a sample for prp-core specific antibody  and prpres n-terminus specific antibodies .
however, no marked differences in mwr for the prpres bands  were observed between preclinical and clinical stages of incubation or between scrapie sheep with different genotypes.
glycoprofiles found in individual rln samples are presented on a triangular graph which allows visualisation of the relative concentration of all three prpres bands, i.e., the di-, mono-, and aglycosyl moieties .
the apparent variations in glycoprofile were further analyzed for correlation with tse-type, tse-incubation stage, and genotype.
firstly, for the tse-types, the diglycosyl fraction of prpres in scrapie samples of the surveillance group and preclinical study were around 54% , while in bse-infected sheep in the preclinical and clinical phases, respectively, it constituted 64%  and 74% .
these  <dig> values were significantly different from each other , and similarly, the aglycosyl and monoglycosyl fractions were statistically different between  <dig> groups of cases – preclinical scrapie, surveillance, preclinical bse and clinical bse .
secondly, linear regression analyses on both bse and preclinical scrapie samples yielded slightly decreasing trends with stage of incubation for the aglycosyl and monoglycosyl prpres fractions, and a concomittant increase in the diglycosyl fraction.
the slopes for these stage-dependent trends, however, were unlikely to deviate significantly from zero for either the bse cases or the cases in the preclinical scrapie study .
this trend was not due to a technical artifact since no correlation between glycoprofile and concentration of prpres existed.
no significant differences were found for the relative concentration of each of the glycoforms of prpres between genotypes in the scrapie surveillance group .
discussion
this study shows that medial retropharyngeal lymph nodes  are a suitable target for monitoring scrapie and bse in sheep, including young lambs.
the animals examined were sheep with natural scrapie or experimental bse in clinical and preclinical stages of the disease, and sheep from an active surveillance program with ihc positive brain but unknown age or clinical status.
the diagnostic technique used was western blotting with several high affinity prp-specific antibodies.
the best results were obtained with antibodies that bind to both scrapie and bse prpres .
sheep of the vrq/vrq genotype scored positive from the age of three months, at a stage when the brain was still negative.
to intercept as many infected sheep as possible, both brain tissue and lymph nodes have to be examined, since vrq animals with allele combinations other than vrq/arr, and many of the infected arq/arq sheep, will be positive in rln much earlier than in brain, whereas a fraction of the arq/arq, and most arr/vrq sheep, may only be positive in the brain.
inclusion in the procedure of a simple concentration step with high prpres recovery improved the sensitivity of detection of bse infected animals.
there is still too little knowledge available about the time of appearance of disease-associated prp in the lrs of sheep with scrapie.
this stage will vary with infection pressure, age, strain and genotype.
for example, the earliest stage in this study, as in some other studies  <cit> , was 2– <dig> months of age in the most susceptible sheep , in heavily infected flocks.
lrs positivity at preclinical stages of natural scrapie in sheep with other genotypes, such as arq/vrq and arh/vrq was noticed at ages varying between 10– <dig> months, while clinical disease developed later, at more than twice that age  <cit> .
preclinical accumulation of prp in lrs tissues has been found in naturally infected suffolk sheep with arq/arq genotype between 8– <dig> months of age  <cit> .
detection of bse prpres by wb in rln of arq/arq sheep was possible at  <dig> months post-infection , the earliest stage investigated, and in all other animals at later stages.
the diagnostic score by wb in our bse-study on rln was nearly similar as to that obtained by ihc, except for the animal with shortest incubation  which by wb was positive but by ihc was negative.
however, in this same animal tonsil and ileal peyer's patches were weakly positive.
discrepancies in the detection of disease-associated prp in rln between ihc and wb might be due to sensitivity of tests or a difference in level of detectable disease-associated prp between left and right side in the head at early stage of appearance.
signs of preclinical infection have been reported in visceral lrs of arq/arq suffolk sheep from  <dig> months post-infection  <cit> .
it must be borne in mind that the dose of  <dig> g brain tissue for oral bseinfection is unusually high and that only arq/arq animals have been investigated.
it is unlikely that such levels have been reached in the field with bse- contaminated with bse.
however, it is likely, based on ihc studies, that the phenotype of bse prpsc in sheep is not altered by either prp-genotype or route of challenge, while on the other hand, it might be assumed that the accumulation of prpsc varies depending on incoming strain and host genotype  <cit> .
scrapie in sheep is not always accompanied by detectable lymphoid involvement  <cit> .
for instance, arr/vrq sheep are usually lrs negative.
our data obtained from rln agree with these observations, with 84%  of rln samples in scrapie positive sheep from surveillance scoring positive by wb.
others report a rate between 88–93% for lrs diagnosis in brain positive animals with only xxq/xxq genotypes   <cit> .
in general, scrapie sheep of all prp genotypes except arr/vrq tend to become positive in lrs, while the arr/vrqs become solely brain positive, and then in only a limited number of animals, at high ages  <cit> .
there are, however, exceptions to the involvement of the lrs with scrapie, as others have reported, and as was also observed in this study  <cit> .
one out of our  <dig> arq/arq sheep with a scrapie diagnosis in the brain was negative in the lymphoid system.
further, we found all brain positive vrq/vrq, vrq/arh and arq/vrq sheep to be rln positive, but jeffrey  <cit>  reported two  arq/vrq sheep to be negative outside the central nervous system.
the first arr/vrq sheep with involvement of the lymphoid tissue was reported by ersdal  <cit>  who described an  <dig> day old lamb with ihc positive peyer's patches in the ileum.
we found two sheep of this genotype with positive lymphoid tissues.
recently,  <dig> cases of susceptible xxq/xxq genotype were reported with positive diagnosis in brain, while negative in all gut-associated lymphoid tissues studied, and which scored positive in the spleen  <cit> .
as well, in infections with the recently described nor <dig> strain of scrapie, no prpres deposits have been detected so far in lymphoid tissues.
the nor <dig> condition has only been diagnosed by examination of the brain, and then, primarily the cerebellum regions  <cit> .
likewise, no deposits of prpsc have been detected in lrs tissues of sheep intracerebrally infected with ch <dig>  an experimental sheep scrapie strain that exhibits similar migrational properties for prpres as bse in sheep  <cit>  .
other techniques, such as elisa and ihc with brain or lrs, might assist in further classification of dubious cases  <cit> .
in summary, while the involvement of peripheral tissues in all clinical or preclinical classical scrapie is not absolute, nevertheless, it occurs with sufficient regularity to be of use in surveillance systems and potentially provides earlier pre-clinical diagnosis than would be achieved by analysis of cns tissues.
different methods for prpres concentration from homogenates were used, depending on the tissue source: centrifugation for rln without additives, and centrifugation after addition of alcohols, for brain tissue.
by allowing  <dig> mg tissue equivalents to be tested, both methods lead to increased sensitivity of detection of prpres by wb compared to no centrifugation.
for rln, it improved bse detection.
however, four scrapie samples, that were weakly positive by ihc, were not detected even after concentration:  <dig> preclinical rln samples from vrq/vrq sheep with scrapie at two months of age, and  <dig> rln samples from routine diagnosis –  <dig> arq/arq and  <dig> arr/vrq sheep.
ihc of these few non-corresponding samples appeared to be only weakly positive, indicating that, with wb, it is possible, in principle, to detect most cases identified by ihc.
in brain samples from animals between 10– <dig> months of age , wb might have missed positive diagnosis in  <dig> preclinical cases, since these analyses possibly obex parts lacking dorsal motor nucleus of the nervus vagus where the first positivity can be found  <cit> .
in our hands the prpsc precipitation technique described by wadsworth  <cit> , which is based on the presence of phophotungstic acid, gave unsatisfactory recoveries  after pk digestion.
recoveries were much better if precipitation preceded pk digestion, but this approach changed the mobility of the prpres bands, making it difficult to rely on molecular weight determination to distinguish between strains.
when analysed by wb, glycoprofiles and apparent molecular weights of prpres in rln digests were similar to those reported for brain tissue of scrapie or bse infected sheep  <cit> .
this means firstly, that, using rln, both tses can be detected in a single screening with the prp-core specific antibodies l <dig> or 9a <dig>  secondly, differentiation of bse and scrapie can be done subsequently, in a dual antibody test using two blots, one with a prp-core specific antibody, such as l <dig> or 9a <dig>  and the other with a prpres n-terminus specific antibody, such as p <dig> or 12b <dig>  all these antibodies have high affinity for ovine prp.
glycoprofile analyses can further support the differential diagnosis of scrapie and bse, where the relative amount of the diglycosyl moiety of prpres is lower in scrapie than bse.
however, glycoprofiling remains an inconclusive tool in discriminating scrapie from bse cases since there is overlap in glycoprofile between the scrapie and bse cases .
in other studies glycoprofile analyses in sheep have yielded too divergent results in scrapie cases to allow a discriminatory diagnosis between scrapie and bse  <cit> .
nevertheless, the relatively high concentration of the diglycosyl prpres fraction in bse-infected animal species, including humans with a variant form of creutzfeldt-jakob disease, remains noteworthy  <cit> .
a new factor in glycoform analyses of prpres is the possible dependence on stage of incubation.
though this was not statistically significant, it was striking that in both our sheep tse incubation studies, experimental bse in arq/arq sheep and natural scrapie in vrq/vrq animals, a correlation was found, revealing an age dependent increase in the diglycosyl moiety of prpres with a concomittant decrease in the a- and monoglycosyl fractions.
this association was not due to a technical artifact since no correlation between glycoprofile and concentration of prpres.
these time-related variations in protein glycosylation could be due to disease status, but normal age dependent variations in posttranslational processes during protein synthesis should also be considered  <cit> .
finally, these glycoprofile variations further indicate that, as a tool for discriminating scrapie from bse, glycoprofiling in sheep is a rather unreliable parameter when the age of the animal and the strain properties of the isolate are not known.
likewise, in brain, further studies are needed to establish a time-dependent relation of prpres-glycosylation.
this study was performed on samples collected during the years 2002– <dig> and appears to be a rather representative cross-section of the dutch sheep population, with respect to the presence of the different genotypes .
it remains to be seen how consistently the rlns score in different age groups, genotypes and preclinical stages.
nevertheless, the efficacy of detecting scrapie and bse infection in current monitoring programs can be highly improved for animals in a stage where the tse agent has not yet invaded the central nervous system, i.e., at ages younger than  <dig> months.
at older ages, both brain and rln testing is needed to optimally assure absence of tse.
it is expected that lymphoid testing also enhance surveillance efficiency in goats, where the lymphoid system usually is involved  <cit>  .
CONCLUSIONS
this study demonstrates that western blotting can be used for routine screening of classical scrapie and bse in retro-pharyngeal lymph nodes of sheep at slaughter with a sensitivity nearly the same as immunohistochemistry.
a concentration step for prpres is required.
the difference in polypeptide length of prpres between bse and scrapie can be unequivocally confirmed by dual antibody staining using two classes of antibodies: one which binds to the n-terminus of prpres in scrapie only, and one which binds to the core of prpres in both bse and scrapie.

 transmissible spongiform encephalopathies or prion diseases are fatal diseases of the central nervous system, characterized by long incubation periods before neurological symptoms appear. tses were initially described in sheep as a transmissible disease named scrapie, and in man, as creutzfeldt-jakob disease, a sporadic disease with no detectable link between cases. the diseases can be diagnosed by vacuolation of neurons in the brain and appearance of a structurally altered form of the host encoded prion protein, termed prpsc. tses represent a unique class of infectious diseases in which prpsc is considered to be the transmissible agent. tses have attracted much attention since bovine spongiform encephalopthy was first described in the 1980s. the more or less accepted explanation for the appearance of this condition was the capacity of the scrapie-like agent to spread to cattle fed with insufficiently heated remains of ruminants. in the 1990s a new form of cjd was observed affecting mainly young people. this so-called variant cjd is considered to be caused by the bse agent and to have spread by the consumption of infected food containing residual bovine nervous tissues, although other means of transmission, such as the use of ruminant-derived substances in medical products, can not be ruled out. furthermore, man-to-man transmissions might have occurred subsequently by blood transfusions. bse is now largely under control, but two recent reports describing goats with bse have strengthened the fear that the bse agent could again become widespread in some food animals before being diagnosed. in order to prevent this from happening, the tses from livestock must be eradicated. for this purpose a number of programmes are underway. for eradication of ruminant tses in europe, where bse cases have occurred in nearly all member states, control programmes have been implemented, e.g., the removal of risk materials, active monitoring of slaughtered cattle, sheep and goats, stamping out of scrapie, and genetic breeding of sheep towards scrapie and bse resistant prpgenotypes. active monitoring is possible using brain tissue taken late in the incubation phase. early diagnosis of tses can assist in monitoring programs and has potential in sheep and goats where lymphoreticular tissues generally accumulate infectivity and aberrant prp. indeed, until now, tonsil and other lymphoid tissues have been targets for preclinical scrapie diagnosis in sheep, making reliable diagnosis possible in biopsies early during incubation. however, for the diagnosis to be as early as possible, i.e. in the preclinical stage, diagnostic methods more rapid than the time consuming microscopic and bioassay techniques currently employed are required. such a diagnosis should also preferably allow discrimination between tse strains like scrapie and bse, as is described for western blotting methods applied to brain. a very reliable indicator of disease is the presence of abnormal forms of prion protein. it is recognized by immunohistochemistry as accumulations of prp, or as a partially protease-resistant protein. by wb of prpres using prp-specific antibodies, a typical triplet banding pattern appears, representative of diglycosylated, monoglycosylated and aglycosyl prpres moieties, migrating with apparent molecular masses between and kda. using staining with two antibodies specific for different prp-sites, wb can even discriminate between scrapie and experimental bse infection in sheep brain due to differential cleavage of prpres by proteinase k. in this study we investigated the applicability of prpres wb to retropharyngeal lymph nodes since these organs are easily accessible at slaughter and are one of the best targets for diagnosis in the lymphoid system. this was performed on three groups of sheep: firstly, sheep from an early and late scrapie pathogenesis study with mainly genotype vrq/vrq; secondly, arq/arq sheep with orally induced bse; and thirdly, sheep diagnosed during active and passive surveillance in the years 2002– the latter group represented six different prp genotypes.  antibody choice and fine epitope specificity in lymphoid tissues prpres concentrations have been reported to be times lower than in brain. therefore, monoclonal antibodies with superior affinity for ovine prp in western blot were sought. ten mabs with specificity for ovine prp were compared on wb strips by varying two parameters: mab concentration and amounts of ovine scrapie brain digest. as a result, antibodies 12b p 9a and l were selected as the most reactive. the epitope specificities of these four mabs were determined by pepscan analysis on solid phase synthetic ovine prp peptides. synthetic peptides carrying these epitope sequences were also used in solution to investigate by elisa their capacity to block binding to recombinant ovine prp. indeed, as can be expected for antibodies that are elicited with peptides, all four of these could interfere effectively with antibody binding to prp, confirming the significance of the mapped specificities. tissue treatment and choice of retro-pharyngeal lymph node a practical means of homogenisation was chosen for tonsillar and medial retropharyngeal lymph node tissue by using disposible homogenisers available for mass screening purposes. prypcon homogenisers were preferred since these consistently yielded satisfactory suspensions while other probes caused formation of gel in the homogenate. homogenates of tonsils and rln from scrapie-infected sheep were examined after enzymatic hydrolysis with proteinase k and/or deglycosylation with pngasef. digestion with pk resulted in the typical triplet of diglycosylated, monoglycosylated and aglycosyl prpres. subsequent removal of carbohydrate moieties by pngasef led further to a prominent single deglycosylated prpres band. there were no major differences in prp immunostaining between tonsils and rlns. in further studies, rln tissue was used as the preferred tissue for evaluating lymphoid tissues, since in contrast to tonsillar tissue, it lead to reproducible scrapie diagnosis and organ size was large enough for repetitive analysis. this better reproducibility is likely due to a more homogeneous distribution of prpsc in rln compared to tonsils. also, at slaughter, rlns are more easily accessible in the decapitated head than are tonsillar tissues. diagnosis of scrapie in sheep during the incubation phase we wished to know first, whether wb could detect scrapie in the preclinical phase. as reported previously, vrq/vrq sheep from our scrapie-infected flock are ihc positive in tonsils and rln at two months of age. rln samples of such sheep, as well as of scrapie negative sheep, were examined by wb. from the age of three months, wb yielded results identical to ihc. in 2-month old cases, wb was negative, whereas ihc could detect prp only in fewer than 25% of the follicles. from the age of four months onwards, all vrq/vrq sheep were positive in both ihc and wb. a 20-fold concentration step allowing application of mg tissue equivalents per lane instead of mg yielded a proportionally stronger signal in the positive cases, but did not change the diagnostic outcome. the rln samples from the scrapie negative sheep remained wb negative. when tested by wb, brain tissue from the vrq/vrq animals scored positive from mo of age onwards, which was some mo later than by ihc. compared to unconcentrated samples, application of mg te after a preceding concentration did not lead to earlier detection of prpres. by wb, scrapie diagnosis using rln from these sheep was already possible at mo of age, while a positive diagnosis with brain was obtained only from mo of age, thus illustrating that wb on lymphoid tissue can lead to a scrapie diagnosis at a very early stage of infection. diagnosis in animals in preclinical and clinical phase of experimental bse rlns from sheep with experimental bse, in a previous and ongoing study, were examined by wb. all infected animals of this study were positive by wb, but only after concentration to 10– mg te/lane.. if no concentration treatment was included and mab l the best antibody for bse, was used, out of animals in the preclinical stage, and out in the clinical stage, could be diagnosed. thus, in the case of bse, concentration improves the diagnostic sensitivity. surprisingly while all animals were positive by wb, one with the shortest duration of infection was negative by ihc. antibodies showed different affinities for prpres in bse- and scrapie-infected sheep. prp-core specific antibodies l and 9a recognized prpres in bse samples, with the former being the best antibody. pk-cleavage site specific antibodies 12b hardly bound to prpres from bseinfected samples. remarkably, 9a bound less strongly to prpres in bse samples than l contrary to findings with scrapie-infected sheep. it seems, thus, that the 9a epitope in bse samples could be more prone to pk cleavage than that in scrapie. variable cleavage of prpsc is a recognized phenomenon, but the extent or pattern of these cleavages could well depend on agent-specific prp-conformational properties, which in situ might result in differences in antibody binding between potentially different tse strains. these results also emphasize that for discrimination between scrapie and bse in dual antibody staining, the use of core antibody l is preferable to 9a especially in weakly positive samples. diagnosis of scrapie in field cases from surveillance and with mostly unknown ages rlns from sheep with various prp genotypes and from different sources, were examined by wb and ihc. in general, again, there was a good correlation between the two methods, since divergent results occurred only for some weak ihc-positive cases. thus, out of brain-positive sheep were diagnosed to be scrapie positive with rln by both methods and two others were only positive by ihc. within the different genotypes, wb of rln scored out of positive in the arq/arq group,, and out in the arr/vrq group. for the other genotypes, the two tests corresponded fully with each other. in brain-negative sheep, out appeared positive with both wb and ihc. this one lymphoreticular system positive case was an arq/vrq animal, mo of age, derived from our own flock with minimal scrapie-incidence. further concentration up to mgte/lane yielded higher signals proportional to the 10× concentration factor, but the diagnostic result in this set of samples was the same as found for the unconcentrated samples. molecular weight and glycoprofiles of prpres bands apparent molecular weights of the three prpres bands in wb of rln digests were compared for a correlation by type of infection, stage of incubation or genotype. a bse-specific difference in the mwr existed and was especially distinct for the aglycosyl moiety, which was about 1kda larger in scrapie samples than in bse samples. this appeared to be the case for both antibodies 9a and l antibody 12b only bound well to the scrapie-infected samples indicating that, as in brain stem tissue, a discrimination between scrapie and bse by dual wb staining is possible using lymphoid tissues, i.e. comparing the immunostaining of a sample for prp-core specific antibody and prpres n-terminus specific antibodies. however, no marked differences in mwr for the prpres bands were observed between preclinical and clinical stages of incubation or between scrapie sheep with different genotypes. glycoprofiles found in individual rln samples are presented on a triangular graph which allows visualisation of the relative concentration of all three prpres bands, i.e., the di-, mono-, and aglycosyl moieties. the apparent variations in glycoprofile were further analyzed for correlation with tse-type, tse-incubation stage, and genotype. firstly, for the tse-types, the diglycosyl fraction of prpres in scrapie samples of the surveillance group and preclinical study were around 54%, while in bse-infected sheep in the preclinical and clinical phases, respectively, it constituted 64% and 74%. these values were significantly different from each other, and similarly, the aglycosyl and monoglycosyl fractions were statistically different between groups of cases – preclinical scrapie, surveillance, preclinical bse and clinical bse. secondly, linear regression analyses on both bse and preclinical scrapie samples yielded slightly decreasing trends with stage of incubation for the aglycosyl and monoglycosyl prpres fractions, and a concomittant increase in the diglycosyl fraction. the slopes for these stage-dependent trends, however, were unlikely to deviate significantly from zero for either the bse cases or the cases in the preclinical scrapie study. this trend was not due to a technical artifact since no correlation between glycoprofile and concentration of prpres existed. no significant differences were found for the relative concentration of each of the glycoforms of prpres between genotypes in the scrapie surveillance group. discussion this study shows that medial retropharyngeal lymph nodes are a suitable target for monitoring scrapie and bse in sheep, including young lambs. the animals examined were sheep with natural scrapie or experimental bse in clinical and preclinical stages of the disease, and sheep from an active surveillance program with ihc positive brain but unknown age or clinical status. the diagnostic technique used was western blotting with several high affinity prp-specific antibodies. the best results were obtained with antibodies that bind to both scrapie and bse prpres. sheep of the vrq/vrq genotype scored positive from the age of three months, at a stage when the brain was still negative. to intercept as many infected sheep as possible, both brain tissue and lymph nodes have to be examined, since vrq animals with allele combinations other than vrq/arr, and many of the infected arq/arq sheep, will be positive in rln much earlier than in brain, whereas a fraction of the arq/arq, and most arr/vrq sheep, may only be positive in the brain. inclusion in the procedure of a simple concentration step with high prpres recovery improved the sensitivity of detection of bse infected animals. there is still too little knowledge available about the time of appearance of disease-associated prp in the lrs of sheep with scrapie. this stage will vary with infection pressure, age, strain and genotype. for example, the earliest stage in this study, as in some other studies, was 2– months of age in the most susceptible sheep, in heavily infected flocks. lrs positivity at preclinical stages of natural scrapie in sheep with other genotypes, such as arq/vrq and arh/vrq was noticed at ages varying between 10– months, while clinical disease developed later, at more than twice that age. preclinical accumulation of prp in lrs tissues has been found in naturally infected suffolk sheep with arq/arq genotype between 8– months of age. detection of bse prpres by wb in rln of arq/arq sheep was possible at months post-infection, the earliest stage investigated, and in all other animals at later stages. the diagnostic score by wb in our bse-study on rln was nearly similar as to that obtained by ihc, except for the animal with shortest incubation which by wb was positive but by ihc was negative. however, in this same animal tonsil and ileal peyer's patches were weakly positive. discrepancies in the detection of disease-associated prp in rln between ihc and wb might be due to sensitivity of tests or a difference in level of detectable disease-associated prp between left and right side in the head at early stage of appearance. signs of preclinical infection have been reported in visceral lrs of arq/arq suffolk sheep from months post-infection. it must be borne in mind that the dose of g brain tissue for oral bseinfection is unusually high and that only arq/arq animals have been investigated. it is unlikely that such levels have been reached in the field with bse- contaminated with bse. however, it is likely, based on ihc studies, that the phenotype of bse prpsc in sheep is not altered by either prp-genotype or route of challenge, while on the other hand, it might be assumed that the accumulation of prpsc varies depending on incoming strain and host genotype. scrapie in sheep is not always accompanied by detectable lymphoid involvement. for instance, arr/vrq sheep are usually lrs negative. our data obtained from rln agree with these observations, with 84% of rln samples in scrapie positive sheep from surveillance scoring positive by wb. others report a rate between 88–93% for lrs diagnosis in brain positive animals with only xxq/xxq genotypes. in general, scrapie sheep of all prp genotypes except arr/vrq tend to become positive in lrs, while the arr/vrqs become solely brain positive, and then in only a limited number of animals, at high ages. there are, however, exceptions to the involvement of the lrs with scrapie, as others have reported, and as was also observed in this study. one out of our arq/arq sheep with a scrapie diagnosis in the brain was negative in the lymphoid system. further, we found all brain positive vrq/vrq, vrq/arh and arq/vrq sheep to be rln positive, but jeffrey reported two arq/vrq sheep to be negative outside the central nervous system. the first arr/vrq sheep with involvement of the lymphoid tissue was reported by ersdal who described an day old lamb with ihc positive peyer's patches in the ileum. we found two sheep of this genotype with positive lymphoid tissues. recently, cases of susceptible xxq/xxq genotype were reported with positive diagnosis in brain, while negative in all gut-associated lymphoid tissues studied, and which scored positive in the spleen. as well, in infections with the recently described nor strain of scrapie, no prpres deposits have been detected so far in lymphoid tissues. the nor condition has only been diagnosed by examination of the brain, and then, primarily the cerebellum regions. likewise, no deposits of prpsc have been detected in lrs tissues of sheep intracerebrally infected with ch an experimental sheep scrapie strain that exhibits similar migrational properties for prpres as bse in sheep. other techniques, such as elisa and ihc with brain or lrs, might assist in further classification of dubious cases. in summary, while the involvement of peripheral tissues in all clinical or preclinical classical scrapie is not absolute, nevertheless, it occurs with sufficient regularity to be of use in surveillance systems and potentially provides earlier pre-clinical diagnosis than would be achieved by analysis of cns tissues. different methods for prpres concentration from homogenates were used, depending on the tissue source: centrifugation for rln without additives, and centrifugation after addition of alcohols, for brain tissue. by allowing mg tissue equivalents to be tested, both methods lead to increased sensitivity of detection of prpres by wb compared to no centrifugation. for rln, it improved bse detection. however, four scrapie samples, that were weakly positive by ihc, were not detected even after concentration: preclinical rln samples from vrq/vrq sheep with scrapie at two months of age, and rln samples from routine diagnosis – arq/arq and arr/vrq sheep. ihc of these few non-corresponding samples appeared to be only weakly positive, indicating that, with wb, it is possible, in principle, to detect most cases identified by ihc. in brain samples from animals between 10– months of age, wb might have missed positive diagnosis in preclinical cases, since these analyses possibly obex parts lacking dorsal motor nucleus of the nervus vagus where the first positivity can be found. in our hands the prpsc precipitation technique described by wadsworth, which is based on the presence of phophotungstic acid, gave unsatisfactory recoveries after pk digestion. recoveries were much better if precipitation preceded pk digestion, but this approach changed the mobility of the prpres bands, making it difficult to rely on molecular weight determination to distinguish between strains. when analysed by wb, glycoprofiles and apparent molecular weights of prpres in rln digests were similar to those reported for brain tissue of scrapie or bse infected sheep. this means firstly, that, using rln, both tses can be detected in a single screening with the prp-core specific antibodies l or 9a secondly, differentiation of bse and scrapie can be done subsequently, in a dual antibody test using two blots, one with a prp-core specific antibody, such as l or 9a and the other with a prpres n-terminus specific antibody, such as p or 12b all these antibodies have high affinity for ovine prp. glycoprofile analyses can further support the differential diagnosis of scrapie and bse, where the relative amount of the diglycosyl moiety of prpres is lower in scrapie than bse. however, glycoprofiling remains an inconclusive tool in discriminating scrapie from bse cases since there is overlap in glycoprofile between the scrapie and bse cases. in other studies glycoprofile analyses in sheep have yielded too divergent results in scrapie cases to allow a discriminatory diagnosis between scrapie and bse. nevertheless, the relatively high concentration of the diglycosyl prpres fraction in bse-infected animal species, including humans with a variant form of creutzfeldt-jakob disease, remains noteworthy. a new factor in glycoform analyses of prpres is the possible dependence on stage of incubation. though this was not statistically significant, it was striking that in both our sheep tse incubation studies, experimental bse in arq/arq sheep and natural scrapie in vrq/vrq animals, a correlation was found, revealing an age dependent increase in the diglycosyl moiety of prpres with a concomittant decrease in the a- and monoglycosyl fractions. this association was not due to a technical artifact since no correlation between glycoprofile and concentration of prpres. these time-related variations in protein glycosylation could be due to disease status, but normal age dependent variations in posttranslational processes during protein synthesis should also be considered. finally, these glycoprofile variations further indicate that, as a tool for discriminating scrapie from bse, glycoprofiling in sheep is a rather unreliable parameter when the age of the animal and the strain properties of the isolate are not known. likewise, in brain, further studies are needed to establish a time-dependent relation of prpres-glycosylation. this study was performed on samples collected during the years 2002– and appears to be a rather representative cross-section of the dutch sheep population, with respect to the presence of the different genotypes. it remains to be seen how consistently the rlns score in different age groups, genotypes and preclinical stages. nevertheless, the efficacy of detecting scrapie and bse infection in current monitoring programs can be highly improved for animals in a stage where the tse agent has not yet invaded the central nervous system, i.e., at ages younger than months. at older ages, both brain and rln testing is needed to optimally assure absence of tse. it is expected that lymphoid testing also enhance surveillance efficiency in goats, where the lymphoid system usually is involved.  this study demonstrates that western blotting can be used for routine screening of classical scrapie and bse in retro-pharyngeal lymph nodes of sheep at slaughter with a sensitivity nearly the same as immunohistochemistry. a concentration step for prpres is required. the difference in polypeptide length of prpres between bse and scrapie can be unequivocally confirmed by dual antibody staining using two classes of antibodies: one which binds to the n-terminus of prpres in scrapie only, and one which binds to the core of prpres in both bse and scrapie. 
moreover, bse infection could be discriminated from scrapie infection by faster electrophoretic migration of the prpres bands. using dual antibody staining with selected monoclonal antibodies like 12b and l these differences in migration could be employed for an unequivocal differentiation between bse and scrapie. diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy. with sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes are considered suitable organs for testing. firstly, in a study with preclinical scrapie cases, wb of rln from infected sheep of vrq/vrq genotype – vrq represents, respectively, polymorphic prp amino acids and – allowed a diagnosis mo earlier compared to wb of brain stem. with respect to glycosylation of prpres, bse cases exhibited a greater diglycosylated fraction than scrapie cases. secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002– yielded positive results depending on genotype: all sheep with genotypes arh/vrq, vrq/vrq, and arq/vrq scored positive for prpres, but arq/arq and arr/vrq were not all positive. furthermore, a slight time dependent increase of diglycosylated prpres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and bse study. optimal prpres detection in rln by wb was achieved by proper tissue processing, antibody choice and inclusion of a step for prpresconcentration.
 diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy. with sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes are considered suitable organs for testing. western blotting of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and bse. in this report, wb is developed for rapid diagnosis in rln and to study biochemical characteristics of prpres.  optimal prpres detection in rln by wb was achieved by proper tissue processing, antibody choice and inclusion of a step for prpresconcentration. the analyses were performed on three different sheep sources. firstly, in a study with preclinical scrapie cases, wb of rln from infected sheep of vrq/vrq genotype – vrq represents, respectively, polymorphic prp amino acids and – allowed a diagnosis mo earlier compared to wb of brain stem. secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002– yielded positive results depending on genotype: all sheep with genotypes arh/vrq, vrq/vrq, and arq/vrq scored positive for prpres, but arq/arq and arr/vrq were not all positive. thirdly, in an experimental bse study, detection of prpres in all arq/arq sheep, including preclinical cases, was possible. in all instances, wb and ihc were almost as sensitive. moreover, bse infection could be discriminated from scrapie infection by faster electrophoretic migration of the prpres bands. using dual antibody staining with selected monoclonal antibodies like 12b and l these differences in migration could be employed for an unequivocal differentiation between bse and scrapie. with respect to glycosylation of prpres, bse cases exhibited a greater diglycosylated fraction than scrapie cases. furthermore, a slight time dependent increase of diglycosylated prpres was noted between individual sheep, which was remarkable in that it occurred in both scrapie and bse study.  the present data indicate that, used in conjunction with testing in brain, wb of rln can be a sensitive tool for improving surveillance of scrapie and bse, allowing early detection of bse and scrapie and thereby ensuring safer sheep and goat products. 