This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nonno, R.
Right arrow Articles by Agrimi, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nonno, R.
Right arrow Articles by Agrimi, U.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, September 2003, p. 4127-4133, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4127-4133.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of Cases of Italian Sheep Scrapie and Comparison with Cases of Bovine Spongiform Encephalopathy (BSE) and Experimental BSE in Sheep

Romolo Nonno,1* Elena Esposito,1 Gabriele Vaccari,1 Michela Conte,1 Stefano Marcon,1 Michele Di Bari,1 Ciriaco Ligios,2 Giovanni Di Guardo,3,{dagger} and Umberto Agrimi1

Laboratory of Veterinary Medicine, Istituto Superiore di Sanità,1 Istituto Zooprofilattico Sperimentale del Lazio e della Toscana, Rome,3 Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy2

Received 7 March 2003/ Returned for modification 11 May 2003/ Accepted 22 June 2003


arrow
ABSTRACT
 
Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrPSc from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrPSc. Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrPSc from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrPSc. Scrapie cases displayed homogeneous PrPSc features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrPSc. While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.


arrow
INTRODUCTION
 
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a group of fatal neurodegenerative diseases, including sheep and goat scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease (CJD) in humans. They are characterized by the accumulation of an abnormal protein, named PrPSc (26, 31), which is formed posttranslationally from the normal isoform (PrPC). The two isoforms share the same covalent structure but show different biochemical properties: PrPC (33 to 35 kDa) is soluble and sensitive to protease treatment, while PrPSc is insoluble and partially resistant to treatment with proteinase K, which leaves an unhydrolyzed core fragment of 27 to 30 kDa (26). To date, the agent causing TSEs is still incompletely characterized, although PrPSc is believed to be its major if not unique constituent (32).

Our incomplete understanding of the nature of TSE agents prevents typing with conventional microbiological methods. The existence of different scrapie strains has been nevertheless inferred from transmission studies in inbred mice (8, 17). Scrapie strain discrimination is currently based upon biological typing in a panel of inbred mice, using incubation times and brain pathology scoring as criteria (9).

Recently, molecular strain typing methods have been used in human disease and rodent scrapie models. These methods are based on the electrophoretic features of the protease-resistant core of PrPSc (4, 12, 24, 27, 35), on the relative proteinase K resistance of PrPSc (23), or on the physicochemical behavior of PrPSc during denaturation (29, 34). Glycoform analysis, i.e., the relative amounts of di-, mono-, and nonglycosylated fractions of the protease-resistant core of PrPSc after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, is able to differentiate several PrPSc types which are associated with distinct phenotypes of CJD and various murine TSE strains (12, 24, 27, 35). Additional differences can be shown by the apparent molecular mass of the nonglycosylated protease-resistant core of PrPSc, probably reflecting different cleavage sites of proteinase K (28).

Different lines of evidence suggest that BSE has been accidentally transmitted to other species, including humans (5, 10, 18, 20). Concerns have been raised about the possibility that the BSE agent could have also infected small ruminant populations via contaminated meat and bone meal and that sheep- and goat-derived products might therefore represent a risk for consumers. Sheep experimentally infected with the BSE agent by the oral route develop clinical signs indistinguishable from conventional scrapie (16). Therefore, large-scale scrapie strain characterization is urgently needed in order to recognize the possible presence of BSE infection in sheep populations.

The BSE strain seems to maintain constant biological and molecular properties even after experimental or accidental passages into different species, such as mice, humans, primates, and sheep (7, 10, 15, 25). At the same time, PrPSc from BSE-infected animals and humans displays a fairly typical glycoprofile, characterized by a very high proportion of the diglycosylated fraction, and a low apparent molecular mass of the protease-resistant core (12). These features first suggested a possible epidemiological link between variant CJD and BSE, which is also supported by biological strain typing (10).

Sheep scrapie, by contrast, displays some strain variation, as suggested by conventional biological strain typing of United Kingdom scrapie cases (6). Similar conclusions can be drawn from two independent molecular surveys of United Kingdom scrapie cases (n = 9 and n = 12) (19, 21). Both authors reported evidence of molecular variations among contemporary and archival scrapie cases by using PrPSc glycoform profile and fragment size analysis following protease digestion. By contrast, molecular analysis of French (n = 42) and Irish (n = 16) contemporary natural sheep scrapie cases showed a remarkable homogeneity (2, 38). Little is known about scrapie strain diversity in other European countries, and studies with conventional strain typing in inbred mice are still lacking outside the United Kingdom.

The potential of molecular methods in distinguishing between sheep BSE and natural scrapie is a matter of debate. The molecular mass of the nonglycosylated form of PrPSc has been reported to be lower in sheep BSE than in natural scrapie (3, 19, 37), but scrapie cases with a molecular mass even lower than that with sheep BSE have also been described (21). Furthermore, the experimental scrapie strain CH1641 appears identical to sheep BSE in terms of the molecular mass of PrPSc (3, 21, 37). Similarly, the glycoform ratio of sheep BSE PrPSc was found to be higher than in natural scrapie by Stack et al. (37) but not by other authors (3, 21).

Such differing results might indicate that molecular strain typing is not straightforward and can be very sensitive to methodological variables (38). On the other hand, the potential variability of scrapie strains among different countries makes the interpretation of these results more difficult. This issue might be further complicated by PrP allelic variants in sheep, eventually affecting PrPSc conformation and glycosylation. Three PrP polymorphisms at amino acid residues 136, 154, and 171 (resulting in five different alleles) are known to affect sheep susceptibility to natural scrapie as well as to experimental scrapie and BSE (22). Attempts to elucidate the genotype influence on glycoform analysis were done on a limited number of cases and did not show any evident effect (19, 37, 38).

In our study, we analyzed the PrPSc glycoprofile and fragment size of 38 Italian sheep scrapie cases in comparison with those in sheep experimentally infected with BSE and BSE in cattle. Our aim was to investigate the potential of molecular strain typing techniques in detecting BSE in sheep and in revealing the possible presence of BSE in the Italian sheep population.


arrow
MATERIALS AND METHODS
 
Animals and tissues. Scrapie was first described in Italy in 1977 (13). Since then, sporadic cases were reported until 1997, when a sudden increase in goat and sheep scrapie outbreaks occurred, probably related to the use of an accidentally contaminated vaccine against Mycoplasma agalactiae (1, 11). With the aim of sampling possibly different scrapie strains, 38 Italian natural scrapie cases were selected among those archived at the Istituto Superiore di Sanità between 1992 and 2000. Scrapie-affected, pathologically confirmed sheep were chosen according to breed, geographic origin, culling year, and presence of the above vaccine as a risk factor for scrapie (Table 1 shows details about sheep breed and geographic distribution of flocks).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Sheep scrapie cases studied

Frozen brain tissue from the medulla oblongata was obtained from all cases. Brain tissues from a Suffolk sheep scrapie case from the United Kingdom (with amino acids ARQ/ARQ at codons 136, 154, and 171, respectively, on both alleles) and a United Kingdom BSE case were provided by the Veterinary Laboratory Agency of Weybridge. Brain tissue from a Cheviot sheep (AHQ/AHQ) experimentally infected with BSE was obtained from the Neuropathogenesis Unit, TSE Resource Centre, Institute of Animal Health, Edinburgh. Two samples of Italian BSE cases were derived from routine active surveillance and provided by M. Caramelli, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin.

PrP genotype determination. Genotype analysis was performed by DNA sequencing using the ABI Prism 310 DNA sequencer (Applied Biosystems). Briefly, DNA was extracted from frozen brain tissue by standard procedures, followed by amplification of two fragments representing the entire PrP coding sequence, as previously described (39).

Amplified fragments were purified with Microcon 100 centrifugal filter devices (Millipore Billerica), and then sequencing reactions were carried out with a BigDye primer sequencing kit (Applied Biosystems).

Tissue preparation. Each brain tissue sample was weighed and homogenized (10% wt/vol) in phosphate-buffered saline containing 10% Sarcosyl (Sigma). The homogenate was incubated for 20 min at room temperature and then centrifuged at 22,000 x g for 20 min (TLA 100.3 rotor; Beckman). Supernatant (1 ml) was added with 50 µl of a stock solution of proteinase K (Sigma) to give a final concentration of 50 µg/ml and then incubated for 1 h at 37°C with gentle shaking. Protease treatment was stopped with phenylmethylsulfonyl fluoride (PMSF) (Sigma) (30 µl of a stock solution, to give a final concentration of 3 mM). The treated homogenate was then ultracentrifuged at 210,000 x g for 40 min (TLA 100.3 rotor; Beckman), and the pellet was resuspended in 100 µl of distilled water and desiccated in speed vacuum (Speed Vac Sc 110; Savant) overnight. The final pellet was stored at -20°C.

Deglycosylation. Pellets were dissolved (0.5 mg of brain equivalent/µl) in denaturing buffer (125 mM Tris-HCl, 10% glycerol, 2% lithium dodecyl sulfate, 0.5 mM EDTA) and heated at 95°C for 10 min. Three microliters of this solution was diluted to 30 µl in 50 mM sodium phosphate buffer (pH 7.4) containing 0.8% Nonidet P40 (Roche) and 40 U of N-glycosidase F/ml (Roche) and incubated for 2 h at 37°C with gentle shaking. Appropriate aliquots were then diluted in NU-PAGE sample buffer (Invitrogen), heated at 95°C for 10 min, and analyzed by Western blotting.

In order to subject only the glycosylated fragments to deglycosylation treatment, samples were first electrophoresed in four consecutive lanes (1 mg of tissue equivalent per lane). The portions of the gel containing the glycosylated protein, determined according to prestained molecular standards (Bio-Rad) and our previous experience, were cut in small pieces and incubated in 0.1% sodium dodecyl sulfate for 24 h to elute proteins from gel. Samples were then centrifuged in a microcentrifuge at 8,000 x g for 10 min, and the supernatant was subjected to methanol precipitation and finally deglycosylated as described above.

Western blotting. Pellets were dissolved in NU-PAGE sample buffer (Invitrogen), heated at 95°C for 10 min, and centrifuged in a microcentrifuge at 12,000 rpm for 5 min. Ten microliters of the supernatants (either undiluted or appropriately diluted in sample buffer) was loaded onto 12% bis-Tris polyacrylamide gels (Invitrogen). Electrophoresis was carried out at 200 V for 40 min, and Western blotting was performed on polyvinylidene difluoride membranes (Millipore) at 100 mA per gel for 40 min in a semidry blotting apparatus (Trans-Blot SD; Bio-Rad). The blots were blocked in phosphate-buffered saline containing 0.1% Tween 20 and 5% nonfat milk powder for 1 h. PrPSc was detected with the monoclonal antibody (MAb) F99/97.6.1 (immunoglobulin G1 [IgG1], 4 µg/ml, provided by K. O'Rourke, USDA, Pullman, Wash.), which binds to the epitope QYQRES from amino acids 220 to 225 of the sheep PrP sequence, or with the polyclonal antibody P7-7 (30) at 1:5,000; both antibodies were used for 1 h at room temperature. The antibody P7-7 was raised against purified, proteinase K-treated and denatured hamster PrPSc, and thus it recognizes several epitopes spanning PrP sequence from amino acid 90 to the C terminus. Horseradish peroxidase-conjugated goat anti-mouse IgG (1:5,000 for 1 h; Sigma) and goat anti-rabbit IgG (1:3,000 for 1 h; Bio-Rad) were used as secondary antibodies. The membranes were developed with the enhanced chemiluminescence method (ECL; Amersham).

Quantitative and statistical analysis. In each experiment, four 17- or 12-well gels were used in order to include 13 to 20 samples in the same assay. Samples were loaded in three consecutive twofold dilutions, and at least one BSE sample was included in each experiment for direct comparison with scrapie cases. Membranes were subjected to multiple exposures to ECL Hyperfilm (Amersham), and only dilution-exposure combinations within the linear range of the autoradiographic film were included in the analysis. For densitometric analysis, films were scanned and the density of bands was measured with the NIH Image software. Combined signals were defined as 100%, and the contribution of each band was calculated as a percentage.

The molecular masses were measured based on center positions of nonglycosylated bands and molecular markers.

All values were calculated as means ± standard deviations of at least three independent determinations.

Statistical analysis of glycoform profiles and molecular mass of the nonglycosylated bands was carried out by taking into consideration four different parameters (diglycosylated, monoglycosylated, and nonglycosylated band intensities and the diglycosylated-to-monoglycosylated ratio) for each sample. Scrapie cases were divided according to breed, flock, and use of the vaccine and analyzed by one-way analysis of variance. An unpaired t test was used for calculation of two-tailed P values when comparing scrapie and BSE groups.


arrow
RESULTS
 
Genotype. All scrapie cases were ARQ/ARQ, with the only exception being a Sarda sheep which was ARQ/AHQ. In addition, two Sarda breed sheep showed point mutations, one at codon 141 (L/F) and the other at codon 143 (H/R).

Glycoform analysis. When analyzed with MAb F99/97.6.1, proteinase K-treated PrPSc was characterized by the typical three protein bands, whose rank order of density was diglycosylated > monoglycosylated > nonglycosylated in all samples (Fig. 1A and B).



View larger version (58K):
[in this window]
[in a new window]
  		FIG. 1. Western blot of proteinase K-treated PrPSc from scrapie cases and experimental BSE in sheep using MAb F99.97.6.1. (A) Representative immunoblot showing sheep BSE (lane 2) and scrapie cases (lanes 1, 3, 5, 6, 8, and 12, Sarda sheep; lanes 4, 9, and 11, Comisana sheep; lanes 7 and 10, Massese sheep). The positions of molecular mass markers (kilodaltons) are indicated. (B) Representative blot scans from the experiment seen in panel A. Shown are results for sheep BSE and the scrapie case in lanes 2 and 12, respectively, of panel A. The optical densities were normalized by taking the maximum value in each scan as 100% and the minimum as 0%.

Monoglycosylated PrPSc from scrapie samples was represented by a large band frequently appearing as being composed of two peaks in densitometry, depending on sample dilution and film exposure (Fig. 1B). However, this pattern was not faithfully reproduced among different assays, and the close proximity of these two supposed monoglycosylated bands eluded a reliable quantification with our methods. It is worth mentioning that this pattern was never observed in the ovine experimental BSE, whose monoglycosylated PrPSc, when loaded at the appropriate dilution, appeared always as a single narrow band (Fig. 1B).

The diglycosylated-to-monoglycosylated glycoform ratio obtained from all samples is plotted as a scattergraph in Fig. 2A. Taken together, scrapie samples are characterized by a lower ratio (from 49:35 to 59:24) than ovine experimental BSE (77:16) and cattle BSE samples (from 74:21 to 77:16). The scrapie case from United Kingdom appears similar to all other scrapie cases (57:29).



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 2. Scattergraph of proportions of proteinase K-treated diglycosylated and monoglycosylated PrPSc in sheep scrapie, BSE, and an experimental BSE in sheep. (A) Comparison among Italian scrapie cases (n = 38), United Kingdom scrapie (n = 1), BSE (n = 3), and a sheep experimentally infected with BSE, obtained with MAb F99.97.6.1. Error bars represent standard deviations of at least three independent determinations. Scrapie cases appear as a separate group not overlapping with BSE. (B) Comparison of Italian scrapie cases in different sheep breeds (MAb F99.97.6.1). Error bars are omitted. (C) Comparison of scrapie cases in outbreaks related or not with an accidentally contaminated vaccine (MAb F99.97.6.1). Error bars are omitted. (D) Comparison of Italian scrapie cases (n = 19), BSE from the United Kingdom (n = 1), and experimental sheep BSE (n = 1), obtained with the polyclonal antibody P7-7.

When compared with unpaired t test, scrapie and BSE samples (sheep BSE included) showed differences which were highly significant for all parameters analyzed (P < 0.0001, t = 15.59, and df = 41 for diglycosylated band values; P < 0.0001, t = 7.898, and df = 41 for monoglycosylated band values; P < 0.0001, t = 10.91, and df = 41 for nonglycosylated band values; P < 0.0001, t = 15.70, and df = 41 for the diglycosylated-to-monoglycosylated ratio). Mean values ± standard deviations for diglycosylated/monoglycosylated/nonglycosylated band intensities were 54.9 ± 2.7:28.2 ± 2.5:17 ± 2 for scrapie cases and 76.4 ± 2.1:17.9 ± 2.2:5.6 ± 1 for the BSE group.

In Fig. 2B and C, Italian scrapie cases are plotted according to breed and vaccination with the contaminated vaccine. There was no significant variability for any parameter among scrapie cases when compared according to either flock, breed, or vaccination.

With regard to a possible influence of PrP genotype on glycoform ratio, the genotype variability among our scrapie samples was too low to observe any reliable effect. It is worth noting, however, that the only ARQ/AHQ Sarda sheep was characterized by the lowest diglycosylated-to-monoglycosylated glycoform ratio (49:35). Two Sarda breed sheep with point mutations at codon 141 (L/F) or at codon 143 (H/R) showed a glycoform ratio indistinguishable from all others (55:30 and 54:28, respectively).

Antibody effect on glycoform analysis. A possible influence of the antibody on glycoform profile determination has been recently reported (38). Therefore, we reanalyzed a subset of samples (19 Italian scrapie cases, the United Kingdom BSE case, and the ovine experimental BSE) with the polyclonal antibody P7-7. The diglycosylated-to-monoglycosylated glycoform ratio obtained from these samples is plotted in Fig. 2D. The glycoform patterns were rather different from those obtained with the MAb F99. The scrapie cases showed a diglycosylated-to-monoglycosylated glycoform ratio close to unity (between 45:36 and 38:39). BSE samples were clearly distinguishable from scrapie cases on the basis of the diglycosylated-to-monoglycosylated glycoform ratio (58:34 and 61:33 for BSE and sheep BSE, respectively).

Molecular mass analysis. Due to the expected interassay variability in the measurement of protein fragment size with gel electrophoresis, we included cattle or ovine BSE in each assay to compare them directly with scrapie samples. In all assays, the nonglycosylated band of PrPSc from the BSE samples, either bovine or ovine, migrated at a higher relative distance than that from scrapie samples, while only minor differences were evident among scrapie samples (see Fig. 1A).

The mean apparent molecular masses of the nonglycosylated band of all samples analyzed are plotted in Fig. 3. The ovine and bovine BSE samples showed consistent lower values than scrapie. The ovine experimental BSE was the lowest of the whole panel of samples.



View larger version (12K):
[in this window]
[in a new window]
  		FIG. 3. Mean molecular masses of proteinase K-treated, nonglycosylated PrPSc bands in Italian scrapie cases (n = 38), United Kingdom scrapie (n = 1), BSE (n = 3), and experimental sheep BSE (n = 1). Error bars represent standard deviations of at least three independent determinations.

An unpaired t test between the grouped scrapie and the BSE samples (including ovine BSE) showed highly significant differences (P < 0.0001, t = 10.80, df = 41), with mean molecular mass values (± standard deviations) of 20.7 ± 0.24 for scrapie and 19.4 ± 0.15 for BSE. Scrapie cases were not different when analyzed by one-way analysis of variance grouped by either flock, breed, or vaccination.

Deglycosylation studies. Deglycosylation of PrPSc and subsequent Western blotting usually results in a single PrPSc band composed of nonglycosylated and deglycosylated protein fragments. For this reason, deglycosylation is frequently used as a further means of molecular mass analysis among PrPSc from different sources.

A subset of scrapie samples was then subjected to enzymatic deglycosylation along with cattle and ovine BSE. After deglycosylation, scrapie and ovine BSE were characterized by a single narrow PrPSc band (Fig. 4). The relative mobility of this band was indistinguishable from that of the nonglycosylated band. In contrast, deglycosylated PrPSc from BSE samples appeared as a larger band (Fig. 4A), and when the sample was loaded at the appropriate dilution, a shift in relative mobility compared to the nonglycosylated band was evident (Fig. 4B). As a result, the fragment size analysis after deglycosylation did not allow differentiation between BSE in cattle and scrapie, while the ovine BSE sample gave a fragment size lower than those of the BSE and scrapie samples. A possible explanation for this finding is that glycosylated and nonglycosylated PrPSc from BSE samples might have slightly different protease sensitivities, resulting in distinct N-terminal truncation sites. In this case, the large protein band obtained in BSE samples after deglycosylation would reflect the presence of nonglycosylated and deglycosylated fragments of different sizes.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 4. Western blots of proteinase K-treated PrPSc using MAb F99/97.6.1. (A) Comparison of a United Kingdom BSE case (lanes 1 and 2), sheep BSE (lanes 3 and 4), and an Italian scrapie case (lanes 4 and 5), either deglycosylated (lanes 2, 4, and 6) or not (lanes 1, 3, and 5). (B) Comparison of the sheep BSE (lanes 1 and 4), an Italian scrapie case (lanes 2 and 5), and an Italian BSE case (lanes 3 and 6), either deglycosylated (lanes 4, 5, and 6) or not (lanes 1, 2, and 3). (C) Immunoblot of PrPSc from sheep BSE (lanes 1 to 4) and United Kingdom BSE (lanes 5 to 8). Proteinase K-treated diglycosylated and monoglycosylated PrPSc fragments were eluted from gel, deglycosylated as described in Materials and Methods, and then loaded at three consecutive dilutions (lanes 2 to 4, sheep BSE; lanes 6 to 8, United Kingdom BSE) for direct comparison with nonglycosylated PrPSc bands in untreated samples (lanes 1 and 5, sheep BSE and United Kingdom BSE, respectively). Deglycosylated PrPSc obtained from the United Kingdom BSE is higher than its respective nonglycosylated fragment.

To obtain a reliable comparison of nonglycosylated and deglycosylated protein fragments, di- and monoglycosylated proteinase K-treated protein fragments from BSE and ovine BSE were eluted from gel and deglycosylated in the absence of any nonglycosylated protein. The deglycosylated fragment obtained was then compared with untreated PrPSc containing the nonglycosylated fragment. Deglycosylated BSE PrPSc migrated at a lower distance than nonglycosylated BSE PrPSc, while the two fragments were indistinguishable in the sheep BSE sample (Fig. 4C).


arrow
DISCUSSION
 
In this study, we characterized 38 Italian sheep scrapie cases, one United Kingdom scrapie case, a sheep experimentally infected with BSE, and three BSE cases for their PrPSc glycosylation profiles and fragment sizes. Overall, scrapie cases gave remarkably homogeneous results and were clearly distinguishable from BSE cases, including the experimental BSE in sheep. Scrapie and BSE were different in terms of both glycoform ratio and molecular mass of the nonglycosylated band. PrPSc from BSE was characterized by a high proportion of diglycosylated protein and by a lower molecular mass of the nonglycosylated fraction compared to all scrapie cases, probably reflecting different N-truncation sites during proteolysis. Notably, the Cheviot sheep experimentally infected with BSE showed the highest diglycosylated-to-monoglycosylated band ratio and the lowest molecular mass in the whole panel of samples.

In the monoglycosylated PrPSc from scrapie samples, we could observe a doublet (Fig. 1B), while a single monoglycosylated band characterized the ovine BSE. This could reflect the larger proportion of fully glycosylated PrPSc proteins in BSE as opposed to a less abundant monoglycosylated PrPSc, which might contain a limited number of N-glycosylated variants. A doublet in the monoglycosylated band was also reported in mouse-adapted TSE strains; the relative abundance of the upper and lower monoglycosylated bands varied according to the strain, the brain region, and the PrP genotype of mouse (36).

A sudden increase in scrapie outbreaks was reported in Italy after 1997 (1), with 2 sheep scrapie outbreaks reported in the biennium 1995 to 1996 and 50 between 1997 and 2000 (G. Ru, personal communication). Our series of natural scrapie cases, which includes sheep from 13 different outbreaks in different sheep breeds, is representative of the evolution of scrapie in Italy in that 2 outbreaks occurred before 1997 and 11 in the period from 1997 to 2000. In this context, it is important to emphasize that none of the scrapie cases studied showed PrPSc characteristics compatible with BSE.

Five of our scrapie cases are currently being studied with conventional strain typing methods in inbred mice in parallel with BSE. Preliminary results, based on attack rate, incubation times, lesion profiling, and molecular strain typing after a single passage in mice, indicate differences between scrapie and BSE field isolates (Agrimi et al., unpublished data), thus supporting the present results.

Nine out of the 13 outbreaks studied are linked to the use of a suspected contaminated vaccine and therefore may derive from a common scrapie source. This could partially explain the homogeneous PrPSc characteristics observed among scrapie cases, although the 15 scrapie cases from four outbreaks not related to vaccination and the scrapie case from the United Kingdom were indistinguishable from vaccinated cases, as well. These data could indicate a low scrapie strain variability among cases investigated here. Alternatively, they could simply reflect a low scrapie strain discrimination ability of the molecular typing method used.

The PrP genotype of all sheep with scrapie that we studied was ARQ/ARQ, with the only exception being one animal which was heterozygous R/H at codon 154; this prevented us from studying the impact of different genotypes on the molecular characteristics of PrPSc. These findings are not surprising, given that all Italian scrapie-affected sheep reported to date are ARQ/ARQ or ARQ/AHQ (11, 39).

In terms of discrimination of BSE from scrapie, our findings are consistent with previously reported data showing a low molecular mass of the nonglycosylated PrPSc fraction in sheep with BSE (3, 19, 37), as also reported for BSE transmission to other species (12). Compared with scrapie samples, bovine samples were also characterized by a lower molecular mass of the nonglycosylated PrPSc, although this was slightly higher than that observed in the sheep BSE. Similar findings were reported by Baron and colleagues (3) who compared a BSE-infected sheep inoculated with indigenous BSE with French natural scrapie and BSE cases. Stack and colleagues (37) also analyzed British natural cases of scrapie and BSE, along with two sheep experimentally infected with British BSE cases. Both studies reported a higher molecular mass of the nonglycosylated form in BSE cases than in BSE-infected sheep. Furthermore, in our study, we have demonstrated that the glycosylated and nonglycosylated forms of PrPSc display different proteinase K sensitivities in BSE samples, but not in scrapie and ovine experimental BSE. Taken together, these findings may suggest the possibility that the molecular characteristics of BSE-derived PrPSc undergo some subtle changes in conformation or proteinase sensitivity during adaptation in the ovine species. It is worth remarking that all of the brain samples from sheep experimentally infected with BSE used to date are derived from primary passages of BSE into a new species and that additional passages in the ovine species may well lead to further changes in PrPSc conformation. This point is of primary importance when considering the possible use of molecular strain typing methods for TSE surveillance in sheep populations and could be properly addressed only in secondary and tertiary passages of sheep BSE.

The analysis of the glycoform ratio reported in the present study is in agreement with previous findings showing that the BSE strain is characterized by a high diglycosylated-to-monoglycosylated ratio (12, 24). We observed discrimination of BSE from scrapie cases with both the MAb F99/97.6.1 and the polyclonal antibody P7-7, even though the absolute values obtained with the two antibodies were different. In previous studies aimed at comparing scrapie and BSE, the glycoform analysis did not always lead to a clear-cut differentiation. For example, Baron and colleagues (2) reported similar glycoform profiles for French natural scrapie and BSE cases, while Sweeney and colleagues (38) were able to differentiate Irish scrapie cases from BSE depending on the antibody used. More recently, Stack and colleagues (37) reported some overlapping between scrapie and BSE cases, while the ovine experimental BSE displayed the highest diglycosylated-to-monoglycosylated ratio. These discrepancies could be explained by the different experimental procedures used. These include tissue processing, the antibody used, and the method for quantification of band intensities. The impact of these variables is currently under investigation in our laboratory. It should be considered that host factors also control PrP glycosylation (36), and this could affect the comparison of glycoform profiles obtained in different species, such as sheep and cattle. However, the difference in glycoform profile between scrapie and BSE is also supported by the recent finding that the glycoform ratio in BSE-affected cattle is higher than in cattle experimentally infected with scrapie (33).

The development of rapid TSE strain-typing methods would greatly reduce the risk that the BSE agent could circulate unrecognized in the sheep population. As a possible strategy, the European Commission suggested the use of at least two different and properly validated molecular-typing approaches (among which are methods based on the proteinase K cleavage site and on the ratio of glycoforms of PrPSc fragments) for the identification of suspect BSE cases in sheep. These should then be further analyzed by conventional biological typing methods (14). Our results could represent a starting point in developing such methods, because two different molecular parameters that differentiate BSE and scrapie samples can be analyzed in a single test. Nevertheless, our method may be difficult to apply under routine large-scale testing, particularly due to the use of a purification step in the procedure. Furthermore, it relies upon electrophoresis and Western blotting techniques, which are inherently inadequate for a precise quantification of molecular masses. Our results were obtained by analyzing large numbers of gel runs, and each sample was loaded at different dilutions. The variability of some parameters, such as the molecular mass of the nonglycosylated bands (see Fig. 3), could undermine the reliability of such a method when used in a single gel run and without appropriate BSE and scrapie positive controls. Recently, a more qualitative test has been reported which is based on the use of a MAb that binds to an epitope in the N-terminal end of the proteinase-treated PrPSc fragment and displays a higher affinity for scrapie PrPSc than for BSE PrPSc, probably due to the different N-terminal ends of proteinase-cleaved PrPSc (37).

In summary, the data presented here show that scrapie infection results in brain accumulation of PrPSc displaying molecular features different from those observed in BSE passaged in sheep and in natural BSE cases. The present work represents one of the most extensive surveys of scrapie cases reported to date and provides encouraging information about the feasibility of molecular strain-typing methods for the selection of suspect BSE cases, which then are to be further analyzed by conventional biological strain typing, in Italian sheep. However, more-extensive surveys including scrapie cases from different countries are needed in order to substantiate these findings. In fact, based on our limited knowledge on PrPSc characteristics of natural scrapie, we cannot rule out the occurrence of cases displaying some features similar to BSE PrPSc, regardless of their link to BSE. This was observed in the case of the sheep-passaged CH1641 scrapie isolate (3, 21, 37). Due to the limited availability of tissues from experimental BSE in sheep, we used a single sample of sheep-passaged BSE, though this could be not fully representative of sheep of different breeds and PrP genotypes. Finally, studies on BSE passaged more than once in sheep of different genotypes and breeds are an absolute requirement in order to assess adequately the suitability of PrPSc molecular analysis for BSE surveillance in sheep.


arrow
ACKNOWLEDGMENTS
 
We thank Maurizio Pocchiari, Istituto Superiore di Sanità, for providing us with the P7/7 polyclonal antibody and Kathrine O'Rourke, USDA, Pullman, Washington, for providing us with the F99/97.6.1 MAb. We also thank Moira Bruce, Neuropathogenesis Unit, TSE resource centre, IAH, for generously supplying tissue from sheep BSE and the Veterinary Laboratory Agency (VLA) of Weybridge and M. Caramelli, Centro per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e valle d'Aosta, Turin, for the BSE tissues.

This study has been supported by two grants from the Italian Ministry of Health (Scrapie: applicazione e valutazione di nuovi test diagnostici e di caratterizzazione dei ceppi, ricerca corrente 2000, and Fattori genetici, patogenetici e biochimici responsabili della suscettibilità/resistenza alle EST, fondi 1%, ricerca finalizzata 2001).


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Laboratory of Veterinary Medicine, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Phone: 39-06-49902854. Fax: 39-06-49387077. E-mail: romolo.nonno{at}iss.it. Back

{dagger} Present address: University of Teramo, Faculty of Veterinary Medicine, Teramo, Italy. Back


arrow
REFERENCES
 
    1
  1. Agrimi, U. G. Ru, F. Cardone, M. Pocchiari, and M. Caramelli. 1999. Epidemic of transmissible spongiform encephalopathy in sheep and goats in Italy. Lancet 353:560-561.
    2. 2
  2. Baron, T. G. M., J.-Y. Madec, and D. Calavas. 1999. Similar signature of the prion protein in natural sheep scrapie and bovine spongiform encephalopathy-linked diseases. J. Clin. Microbiol. 37:3701-3704.[Abstract/Free Full Text]
    3. 3
  3. Baron, T. G. M., J.-Y. Madec, D. Calavas, Y. Richard, and F. Barillet. 2000. Comparison of French natural scrapie isolates with bovine spongiform encephalopathy and experimental scrapie infected sheep. Neurosci. Lett. 284:175-178.[CrossRef][Medline]
    4. 4
  4. Bessen, R. A., and R. S. Marsh. 1994. Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J. Virol. 68:7859-7868.[Abstract/Free Full Text]
    5. 5
  5. Bons, N., N. Mestre-Frances, P. Belli, F. Cathala, D. C. Gajdusek, and P. Brown. 1999. Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents. Proc. Natl. Acad. Sci. USA 96:4046-4051.[Abstract/Free Full Text]
    6. 6
  6. Bruce, M., A. Boyle, S. Cousens, I. McConnell, J. Foster, W. Goldmann, and H. Fraser. 2002. Strain characterization of natural sheep scrapie and comparison with BSE. J. Gen. Virol. 83:695-704.[Abstract/Free Full Text]
    7. 7
  7. Bruce, M., A. Chree, I. McConnell, J. Foster, G. Pearson, and H. Fraser. 1994. Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Philos. Trans. R. Soc. Lond. Ser. B 343:405-411.[Medline]
    8. 8
  8. Bruce, M., and A. G. Dickinson. 1987. Biological evidence that scrapie agent has an independent genome. J. Gen. Virol. 68:79-89.[Abstract/Free Full Text]
    9. 9
  9. Bruce, M., I. McConnell, H. Fraser, and A. G. Dickinson. 1991. The disease characteristics of different strains of scrapie in Sinc congenic mice lines: implications for the nature of the agent and host control of pathogenesis. J. Virol. 72:595-603.[CrossRef]
    10. 10
  10. Bruce, M., R. G. Will, J. W. Ironside, I. McConnell, D. Drummond, A. Suttie, L. McCardle, A. Chree, J. Hope, C. Birkett, S. Cousens, H. Fraser, and J. C. Bostock. 1997. Transmissions to mice indicate that ‘new variant’ CJD is caused by the BSE agent. Nature 389:498-501.[CrossRef][Medline]
    11. 11
  11. Caramelli, M., G. Ru, C. Canalone, E. Bozzetta, P. L. Acutis, A. Calella, and G. Forloni. 2001. Evidence for the transmission of scrapie to sheep and goats from a vaccine against Mycoplasma agalactiae. Vet. Rec. 148:531-536.[Abstract/Free Full Text]
    12. 12
  12. Collinge, J., K. C. L. Sidle, J. Meads, J. Ironside, and A. F. Hill. 1996. Molecular analysis of prion strain variation and the etiology of ‘new variant’ CJD. Nature 383:685-690.[CrossRef][Medline]
    13. 13
  13. Cravero, G. C., F. Guarda, U. Dotta, and R. Guglielmino. 1977. La scrapie in pecore di razza biellese. Prima segnalazione in Italia. Clinica Veterinaria 100:1-5.
    14. 14
  14. European Commission. 2002. Scientific Steering Commission, European Commission. Strategy to investigate the possible presence of BSE in sheep. Opinion adopted by the Scientific Steering Committee at its meeting of 4-5 April 2002.
    15. 15
  15. Foster, J. D., M. Bruce, I. McConnell, A. Chree, and H. Fraser. 1996. Detection of BSE infectivity in brain and spleen of experimentally infected sheep. Vet. Rec. 138:546-548.[Free Full Text]
    16. 16
  16. Foster, J. D., J. Hope, and H. Fraser. 1993. Transmission of bovine spongiform encephalopathy to sheep and goats. Vet. Rec. 133:339-341.[Abstract]
    17. 17
  17. Fraser, H., and A. G. Dickinson. 1973. Scrapie in mice. Agent strain differences in the distribution and intensity of grey matter vacuolation. J. Comp. Pathol. 83:29-40.[CrossRef][Medline]
    18. 18
  18. Fraser, H., G. R. Pearson, I. McConnell, M. E. Bruce, J. M. Wyatt, and T. J. Gruffydd-Jones. 1994. Transmission of feline spongiform encephalopathy to mice. Vet. Rec. 134:449.[Medline]
    19. 19
  19. Hill, A. F., K. C. L. Sidle, S. Joiner, P. Keyes, T. C. Martin, M. Dawson, and J. Collinge. 1998. Molecular screening of sheep for bovine spongiform encephalopathy. Neurosci. Lett. 255:159-162.[CrossRef][Medline]
    20. 20
  20. Hill, A. F., M. Desbruslais, S. Joiner, K. C. L. Sidle, I. Gowland, J. Collinge, L. J. Doey, and P. Lantos. 1997. The same prion strain causes vCJD and BSE. Nature 389:448-450.[CrossRef][Medline]
    21. 21
  21. Hope, J., S. C. E. R. Wood, C. R. Birkett, A. Chong, M. E. Bruce, D. Cairns, W. Goldmann, N. Hunter, and C. J. Bostock. 1999. Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J. Gen. Virol. 80:1-4.[Abstract]
    22. 22
  22. Hunter, N. 1997. PrP genetics in sheep and the implications for scrapie and BSE. Trends Microbiol. 5:331-334.[CrossRef][Medline]
    23. 23
  23. Kuczius, T., and M. H. Groschup. 1999. Differences in proteinase K resistance and neuronal deposition of neuronal prion proteins characterise bovine spongiform encephalopathy (BSE) and scrapie strains. Mol. Med. 5:406-418.[Medline]
    24. 24
  24. Kuczius, T., I. Haist, and M. H. Groschup. 1998. Molecular analysis of bovine spongiform encephalopathy and scrapie strain variation. J. Infect. Dis. 178:693-699.[Medline]
    25. 25
  25. Lasmezàs, C. I., J.-G. Fournier, V. Nouvel, H. Boe, D. Marcé, F. Lamoury, N. Kopp, J.-J. Hauw, J. Ironside, M. Bruce, D. Dormont, and J.-P. Deslys. 2001. Adaptation of the bovine spongiform encephalopathy agent to primates and comparison with Creutzfeldt-Jakob disease: implications for human health. Proc. Natl. Acad. Sci. USA 98:4142-4147.[Abstract/Free Full Text]
    26. 26
  26. Oesch, B., D. Westaway, M. Walchli, M. P. McKinley, S. B. Kent, R. Aebersold, R. A. Berry, P. Tempst, D. B. Teplow, L. E. Hood, S. B. Prusiner, and C. Weissmann. 1985. A cellular gene encodes PrP 27-30 protein. Cell 40:735-746.[CrossRef][Medline]
    27. 27
  27. Parchi, P., R. Castellani, S. Capellari, B. Ghetti, K. Young, S. G. Chen, M. Farlow, D. W. Dickson, A. A. F. Sims, J. Q. Trojanowski, R. B. Petersen, and P. Gambetti. 1996. Molecular basis of phenotypic variability in sporadic Creutzfeldt-Jacob disease. Ann. Neurol. 39:669-680.
    28. 28
  28. Parchi, P., W. Zou, W. Wang, P. Brown, S. Capellari, B. Ghetti, N. Kopp, W. J. Schultz-Schaeffer, P. Gambetti, and S. G. Chen. 2000. Genetic influence on the structural variations of the abnormal prion protein. Proc. Natl. Acad. Sci. USA 97:10168-10172.[Abstract/Free Full Text]
    29. 29
  29. Peretz, D., M. R. Scott, D. Groth, R. A. Williamson, D. R. Burton, F. E. Cohen, and S. B. Prusiner. 2001. Strain-specified relative conformational stability of the scrapie prion protein. Protein Sci. 10:854-863.[CrossRef][Medline]
    30. 30
  30. Pocchiari, M., Y. G. Xi, L. Ingrosso, A. Ladogana, F. Cardone, C. Masullo, Z. Righetto, E. Bigon, A. Di Martino, and L. Callegaro. 1994. Immunodiagnosis of bovine spongiform encephalopathy. Livest. Prod. Sci. 38:41-46.[CrossRef]
    31. 31
  31. Prusiner, S. B., and S. J. De Armond. 1994. Prion diseases and neurodegeneration. Annu. Rev. Neurosci. 17:311-339.[CrossRef][Medline]
    32. 32
  32. Prusiner, S. B. 1989. Scrapie prions. Annu. Rev. Microbiol. 43:345-374.[CrossRef][Medline]
    33. 33
  33. Race, R. E., A. Raines, T. J. Baron, M. W. Miller, A. Jenny, and E. S. Williams. 2002. Comparison of abnormal prion protein glycoform patterns from transmissible spongiform encephalopathy agent-infected deer, elk, sheep, and cattle. J. Virol. 76:12365-12368.[Abstract/Free Full Text]
    34. 34
  34. Safar, J., H. Wille, V. Itri, D. Groth, H. Serban, M. Torchia, F. E. Cohen, and S. B. Prusiner. 1998. Eight prion strains have PrP(Sc) molecules with different conformations. Nat. Med. 4:1157-1165.[CrossRef][Medline]
    35. 35
  35. Somerville, R. A., A. Chong, O. U. Mulqueen, C. R. Birkett, S. C. Wood, and J. Hope. 1997. Biochemical typing of scrapie strains. Nature 386:564.[Medline]
    36. 36
  36. Somerville, R. A. 1999. Host and transmissible spongiform encephalopathy agent strain control glycosylation of PrP. J. Gen. Virol. 80:1865-1872.[Abstract]
    37. 37
  37. Stack, M., M. Chaplin, and J. Clarke. 2002. Differentiation of prion protein glycoforms from naturally occurring sheep scrapie, sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies. Acta Neuropathol. 104:279-286.[Medline]
    38. 38
  38. Sweeney, T., T. Kuczius, M. McElroy, M. Gomerez Parada, and M. Groschup. 2000. Molecular analysis of Irish sheep scrapie cases. J. Gen. Virol. 81:1621-1627.[Abstract/Free Full Text]
    39. 39
  39. Vaccari, G., R. Petraroli, U. Agrimi, C. Eleni, M. G. Perfetti, M. A. Di Bari, L. Morelli, C. Ligios, L. Butani, R. Nonno, and G. Di Guardo. 2001. PrP genotype in sarda breed sheep and its relevance to scrapie. Arch. Virol. 146:2029-2037.[CrossRef][Medline]

Journal of Clinical Microbiology, September 2003, p. 4127-4133, Vol. 41, No. 9
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.9.4127-4133.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Stack, M., Gonzalez, L., Jeffrey, M., Martin, S., Macaldowie, C., Chaplin, M., Thorne, J., Sayers, R., Davis, L., Bramwell, J., Grimmer, S., Bellworthy, S. (2009). Three serial passages of bovine spongiform encephalopathy in sheep do not significantly affect discriminatory test results. J. Gen. Virol. 90: 764-768 [Abstract] [Full Text]  
  • Baron, T., Biacabe, A.-G. (2007). Molecular Behaviors of "CH1641-Like" Sheep Scrapie Isolates in Ovine Transgenic Mice (TgOvPrP4). J. Virol. 81: 7230-7237 [Abstract] [Full Text]  
  • Jacobs, J. G., Langeveld, J. P. M., Biacabe, A.-G., Acutis, P.-L., Polak, M. P., Gavier-Widen, D., Buschmann, A., Caramelli, M., Casalone, C., Mazza, M., Groschup, M., Erkens, J. H. F., Davidse, A., van Zijderveld, F. G., Baron, T. (2007). Molecular Discrimination of Atypical Bovine Spongiform Encephalopathy Strains from a Geographical Region Spanning a Wide Area in Europe. J. Clin. Microbiol. 45: 1821-1829 [Abstract] [Full Text]  
  • Stack, M., Jeffrey, M., Gubbins, S., Grimmer, S., Gonzalez, L., Martin, S., Chaplin, M., Webb, P., Simmons, M., Spencer, Y., Bellerby, P., Hope, J., Wilesmith, J., Matthews, D. (2006). Monitoring for bovine spongiform encephalopathy in sheep in Great Britain, 1998-2004. J. Gen. Virol. 87: 2099-2107 [Abstract] [Full Text]  
  • Vaccari, G., Bari, M. A. D., Morelli, L., Nonno, R., Chiappini, B., Antonucci, G., Marcon, S., Esposito, E., Fazzi, P., Palazzini, N., Troiano, P., Petrella, A., Guardo, G. D., Agrimi, U. (2006). Identification of an allelic variant of the goat PrP gene associated with resistance to scrapie.. J. Gen. Virol. 87: 1395-1402 [Abstract] [Full Text]  
  • Moudjou, M., Treguer, E., Rezaei, H., Sabuncu, E., Neuendorf, E., Groschup, M. H., Grosclaude, J., Laude, H. (2004). Glycan-Controlled Epitopes of Prion Protein Include a Major Determinant of Susceptibility to Sheep Scrapie. J. Virol. 78: 9270-9276 [Abstract] [Full Text]  
  • Baron, T., Crozet, C., Biacabe, A.-G., Philippe, S., Verchere, J., Bencsik, A., Madec, J.-Y., Calavas, D., Samarut, J. (2004). Molecular Analysis of the Protease-Resistant Prion Protein in Scrapie and Bovine Spongiform Encephalopathy Transmitted to Ovine Transgenic and Wild-Type Mice. J. Virol. 78: 6243-6251 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nonno, R.
Right arrow Articles by Agrimi, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nonno, R.
Right arrow Articles by Agrimi, U.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»