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The feeding of meat and bone meal which was contaminated with bovine spongiform encephalopathy (BSE) infectivity led to a huge epidemic in the British cattle population. After the emergence of a new variant form of Creutzfeldt-Jakob diseases (nvCJD) in 1996, European consumers lost confidence in beef meat. Given the coincidence by time and place and disease and agent specific characteristics, it must be assumed that nvCJD is caused by the transmission of BSE agent to man. This article gives an overview over the characteristics and diagnosis of transmissible spongiform encephalopathies, namely BSE and nvCJD, in animals and man.

Prion diseases are associated with the accumulation of an abnormal isoform of host-encoded prion protein (PrP(Sc)). A number of prion strains can be distinguished by "glycotyping" analysis of the respective deposited PrP(Sc) compound. In this study, the long-term proteinase K resistance, the molecular mass, and the localization of PrP(Sc) deposits derived from conventional and transgenic mice inoculated with 11 different BSE and scrapie strains or isolates were examined. Differences were found in the long-term proteinase K resistance (50 microg/ml at 37 degrees C) of PrP(Sc). For example, scrapie strain Chandler or PrP(Sc) derived from field BSE isolates were destroyed after 6 hr of exposure, whereas PrP(Sc) of strains 87V and ME7 and of the Hessen1 isolate were extremely resistant to proteolytic cleavage. Nonglycosylated, proteinase K-treated PrP(Sc) of BSE isolates and of scrapie strain 87V exhibited a 1-2 kD lower molecular mass than PrP(Sc) derived from all other scrapie strains and isolates. With the exception of strain 87V, PrP(Sc) was generally deposited in the cerebrum, cerebellum, and brain stem of different mouse lines at comparable levels. Long-term proteinase resistance, molecular mass, and the analysis of PrP(Sc) deposition therefore provide useful criteria in discriminating prion strains and isolates (e.g., BSE and 87V) that are otherwise indistinguishable by the PrP(Sc) "glycotyping" technique.

Prion diseases are closely linked to the conversion of host-encoded cellular prion protein (PrPC) into its pathological isoform (PrPSc). PrP conversion experiments in scrapie infected tissue culture cells, transgenic mice, and cell-free systems usually require unique epitopes and corresponding monoclonal antibodies (MAbs) for the immunological discrimination of exogenously introduced and endogenous PrP compounds (e.g., MAb 3F4, which is directed to an epitope on hamster and human but not on murine PrP). In the current work, we characterize a novel MAb designated L42 that reacts to PrP of a variety of species, including cattle, sheep, goat, dog, human, cat, mink, rabbit, and guinea pig, but does not bind to mouse, hamster, and rat PrP. Therefore, MAb L42 may allow future in vitro conversion and transgenic studies on PrPs of the former species. The MAb L42 epitope on PrPC includes a tyrosine residue at position 144, whereas mouse, rat, and hamster PrPs incorporate tryptophane at this site. To verify this observation, we generated PrP expression vectors coding for authentic or mutated murine PrPCs (i.e., codon 144 encoding tyrosine instead of tryptophan). After transfection into neuroblastoma cells, MAb L42 did not react with immunoblotted wild-type murine PrPC, whereas L42 epitope-tagged murine PrPC was strongly recognized. Immunoblot and fluorescence-activated cell sorting data revealed that tagged PrPC was correctly posttranslationally processed and translocated to the cell surface., (Copyright 1999 Academic Press.)

Prion diseases are characterized by the intraneuronal accumulation of a pathological isoform (PrP(Sc)) of host-encoded prion protein (PrP(C)). While PrP(Sc) displays a partial resistance, PrP(C) is easily degraded by this enzyme. As it turned out in our experiments, PrP(C) of six species is initially degraded to an intermediate fragment of 25-28 kDa prior to complete proteolysis which was solely detected by antibodies binding to epitopes carboxy-terminally of amino acid 144 of PrP(C). The intermediate fragment thus lacked the aminoterminus of PrP(C). These findings are well in line with the putative structure of PrP(C): the amino-terminus consists of a highly flexible and thus more proteinase K sensitive tail while the carboxy-terminus is folded into possibly more resistant alpha-helices and beta-sheets. We observed significant differences in the PK sensitivities of PrP(C) from six different species and from three ovine PrP alleles, while no remarkable variation was seen in PrP(C) from six regions of an ovine brain. This indicates that variations in the sequence of PrP may alter its three-dimensional structure and consequently change its sensitivity towards proteolytic enzymes.

The sensitivity of Western blot detection of PrPres using two different extraction procedures on brain material of 30 scrapie-affected sheep was compared. Whereas PrPres could be detected in all sheep after extraction with the first method, 30% did not give any signal after extraction with the second method. However, the second method, when positive, permitted the detection of PrPres from smaller amounts of infected brain tissue. When used with the ruminant specific monoclonal antibody p4, the second method gave positive signals corresponding to less than 12.5 microg of scrapie-infected brain, that, up to now, is the highest sensitivity described for PrPres detection from naturally infected ruminant brains. The overall results showed highly variable levels of PrPres between sheep and are presented in relation to breed, survival time and animal genotype data. Further progress can thus be expected for PrPres detection in prion diseases, if more efficient extraction procedures and more sensitive immunological reagents are used. Such technical improvements could contribute to more accurate diagnosis in animals affected naturally.

Little is known about the pathogenetic basis of characteristic symptoms in transmissible spongiform encephalopathies (TSEs) such as myoclonus and characteristic EEG hyperactivity. We investigated the GABAergic system and its subpopulations in mice inoculated with experimental scrapie (ME7, RML, 22A strains) and Creutzfeldt-Jakob disease (CJD; Fujisaki strain), to study damage to inhibitory neurons. Since recent studies have shown electrophysiological changes in prion protein (PrP) knockout mice, we also studied mice lacking or overexpressing the PrP gene. Antibodies against glutamic acid decarboxylase (GAD), parvalbumin (PV), calbindin (CB), and calretinin (CR) were used to stain GABAergic neurons, and isolectin-B4 to stain perineuronal nets around PV+ neurons. In scrapie infected mice, cortical PV+ neurons were severely reduced while CB+ and CR+ neurons were well preserved. In CJD inoculated mice, loss of PV+ neurons was severe and occurred very early after inoculation. PrP-/- and tg20 mice showed normal appearance of PV, CB, CR, GAD+ neurons and their neuropil, and of isolectin-B4+ perineuronal nets. The early, severe and selective loss of cortical PV+ neurons in experimental scrapie and CJD suggest selective loss of PV+ GABAergic neurons as important event during disease development, possibly as one basis of excitatory symptoms in TSEs.

Five mouse scrapie strains, a mouse-passaged scrapie isolate derived from a field case in sheep in Germany, and 2 mouse-passaged bovine spongiform encephalopathy (BSE) isolates were analyzed by immunoblot in regards to banding patterns of proteinase K-digested pathologic prion proteins (PrPres). To obtain reliable results, the photo-imager technique was used for measurement of staining band intensities. Distinct and reproducible profiles were observed for the different strains or isolates. A British and a German BSE isolate were similar, suggesting the same source of infection. The German scrapie isolate resembled scrapie strain ME7, which has frequently been isolated from sheep scrapie in the past. In selected strains or isolates, no influence of the mouse lines used was observed on PrPres profiles, nor were brain region-specific differences apparent. This investigation suggests that PrPres glycotyping can be an invaluable tool for the in vitro differentiation of BSE and scrapie isolates.

To elucidate the role played by the prion protein in scrapie pathogenesis, we performed experiments with PrP27-30 isolated from scrapie-infected hamster brains in cell culture and studied in vivo the temporal and spatial correlation between deposition of the disease-associated isoform of the prion protein (PrPSc), microglial activation and neuronal cell death in mice infected with scrapie strains 79A, ME7 and RML. The results presented here show that cellular expression of PrPc and the presence of microglia are necessary for the neurotoxicity of PrPSc in vitro. In vivo, accumulation of protease-resistant prion protein was detected early in the incubation period using the histoblot technique. Microglial activation was also detected early in the incubation period of all models studied. Both the time course and the spatial distribution of microglial activation closely resembled the pattern of PrPSc deposition. Microglial activation clearly preceded the detection of apoptotic neuronal cell death which was assessed using the in situ end-labeling technique (ISEL). Taken together, our results indicate that microglial activation is involved in the neurotoxicity of PrPSc both in vitro and in vivo.

Transmissible spongiform encephalopathies are closely linked to the accumulation of a pathological isoform of a host-encoded prion protein (PrP(C)), designated PrP(Sc). In an attempt to generate mono- and polyclonal antibodies to ruminant PrP, 32 mice were vaccinated with peptide vaccines which were synthesized according to the amino acid sequence of ovine PrP. By this approach five PrP-reactive polyclonal antisera directed against four different domains of the protein were stimulated. Splenocytes of mice which had developed PrP-reactive antibodies were used for the generation of monoclonal antibodies (MAbs). Obtained PrP-specific MAbs were directed to three different domains of ruminant PrP which differed from the three previously described major MAb binding sites in rodent PrP. MAbs exhibited reactivity with non-denatured ruminant PrP(C) in ELISA and immunoprecipitation and with denatured ovine and bovine PrP(Sc) in immunoblot. Cross-reactivity was observed with PrP(C) of nine other mammalian species and with pathological PrP preferably of ruminants and weakly with that of hamster and mouse. The generated MAbs will be useful tools for the development of diagnostic tests for BSE and scrapie as well as for pathogenesis studies of these diseases.

The prion protein (PrP) is a cell surface glycoprotein normally associated with neurones. Expression of the prion protein in cultured mouse myoblasts and myotubes suggests that the prion protein may play a physiological role in skeletal muscle. When myotubes differentiate from myoblasts prion protein expression is upregulated. Accompanying this increase is an upregulation of Cu/Zn superoxide dismutase (SOD-1) in myotubes. Muscle cells derived from mice deficient in cellular PrP (PrPc) show little increase in SOD-1 after differentiation from myoblasts to myotubes. Myoblasts and myotubes are resistant to the toxicity of a neurotoxic prion protein peptide (PrP106-126). However, in the presence of murine microglia, PrP106-126 causes a reduction in cell number. This effect is greater on myotubes than myoblasts. Even in the presence of microglia PrP106-126 is not toxic to muscle cells derived from PrP-deficient mice. Our results suggest that PrPc expression is associated with regulation of cellular resistance to oxidative stress in skeletal muscle.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.

Pathological prion protein (PrPSc) which is a conformational isoform of a host-encoded protein designated (PrPC) serves as a specific marker protein for the immunochemical diagnosis of transmissible spongiform encephalopathies (TSE). The generation of suitable antibodies to PrPSc therefore underlies the specificity and sensitivity of diagnostic assays. However, most antibodies reported to date are directed to a limited number of epitopes only. PrPC is a highly conserved cell membrane protein in all mammalian species studied to date. In an attempt to generate antibodies to further regions of PrP we raised antisera in rabbits and chicken against sixteen synthetic peptides which represent the complete aminoacid sequence of ovine PrP. By this approach immunotolerance was overcome and immunoblot-reactive antibodies were stimulated to epitopes at almost any site of ovine PrPC and PrPSc. A large number of different antibodies cross-reacted also with affinity-purified PrPCs from other mammalian species including cow, goat, pig, man, dog, cat, mink, mouse, hamster and guinea pig. No epitope, however, was recognized exclusively on the pathological or cellular isoform of PrP indicating that both isoforms occur in highly denatured conformations on the immunoblots. Antibodies to the amino-terminus are suitable for immunoprecipitation of PrP. The availability of rabbit and chicken anti-peptide antibodies to PrP will greatly improve immunochemical diagnosis and pathogenetic studies on these diseases.

Ten racing pigeons were infected experimentally with the paramyxovirus (PMV) type 1 of the pigeon. Within twelve weeks of observation, they were euthanized at different times. Their brains were examined for proteinase K resistant fibrils and histopathologically for spongiform lesions. No proteinase K resistant fibrils and no spongiform lesions could be detected in any case. Therefore, it is estimated that PMV type 1 of the pigeon is not likely to induce pathogenic mechanisms assumed for transmissible spongiform encephalopathies.

Background: Prion diseases belong to a group of neurodegenerative disorders affecting humans and animals. The human diseases include kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome (GSS), and fatal familial insomnia (FFI). The pathogenic mechanisms of the prion diseases are not yet understood. Monoclonal antibodies provide valuable tools in the diagnosis, as well as in the basic research, of several diseases; however, monospecific antisera or monoclonal antibodies (mAbs) against human prion proteins were, until now, not available., Materials and Methods: We have developed an immunization protocol based on nucleic acid injection into nontolerant PrP0/0 mice. DNA or RNA coding for different human prion proteins including the mutated sequences associated with CJD, GSS, and FFI were injected into muscle tissue. Mice were primarily inoculated with DNA plasmids encoding the prion protein (PRNP) gene and boosted either with DNA, RNA, or recombinant Semliki Forest Virus particles expressing PRNP. Hybridomas were then prepared., Results: Different mAbs against human prion proteins were obtained, and their binding behavior was analyzed by peptide enzyme-linked immunosorbent assay, Western blot, immunofluorescence, and immunoprecipitation. Their cross-reactivity with prion protein from other species was also determined. Our mAbs are directed against four different linear epitopes and may also recognize discontinuous regions of the native prion protein., Conclusions: These antibodies should allow us to address questions concerning the nature of the prion protein as well as the initiation and progression of prion diseases. Moreover, these mAbs can now be used for the diagnosis of prion diseases of humans and animals.

In an attempt to determine whether scrapie infectivity can be found in the peripheral nervous system of a scrapie-diseased sheep, mice were inoculated intracerebrally or intraperitoneally with 10-fold dilutions of homogenates of Nervus (N.) axillaris, N. ulnaris, N. medianus, N. ischiadicus, N. tibialis, N. fibularis, and N.saphenus. Mice were observed for clinical signs of scrapie for 700 days and their brains were analyzed for accumulation of pathological prion protein by immunoblot. Substantial amounts of infectivity were found in all peripheral nerves tested except N.saphenus. Infectivity at titers of approximately 10(4.5) mouse infectious units (MIU)/g were detected in N. axillaris and N. ischiadicus, of approximately 10(3.0) MIU/g in N. ulnaris, N. medianus, N. tibialis, and N.fibularis, and of 10(6) MIU/g in the cerebellum. Since muscles are traversed by the nerve tracts tested, mutton of scrapie-diseased animals should not be regarded as being free of scrapie agent.

The so-called prion diseases are probably caused by the conformational conversion of the cellular prion protein (PrPc) into an abnormal, pathological form (PrPsc). PrPc is widely expressed in neuronal tissues, but its function is not known. From electrophysiological measurements in prion-less mice it was proposed that PrPc may contribute to the structural integrity of central synapses containing gamma-aminobutyric acid type A (GABAA) receptors. We tried to substantiate this hypothesis by obtaining evidence for a structural link between the GABAA receptor and PrPc. Preparations of PrPc and GABAA receptors, respectively, from cow brain were analysed for PrPc-GABAA receptor complexes. No evidence for such complexes could be obtained in our experiments, although the protein purification schemes used should favour the preservation of intermolecular linkages. We conclude that further data concerning interactions of PrPc with other proteins are needed to obtain insight into its normal functional role.

Neuronal loss is a salient yet poorly understood feature in the pathology of transmissible spongiform encephalopathies (prion diseases). Cell culture experiments with neurotoxic prion protein fragments suggest that neuronal cell death in these diseases may be due to apoptosis. To test this hypothesis in vivo we used the in situ end-labeling (ISEL) technique and electron microscopy to study cell death in an experimental scrapie system in the mouse. ISEL, which relies on the incorporation of labeled nucleotides in fragmented DNA by terminal transferase, showed labeled nuclei in the brains and retinae of mice infected with the 79A strain of scrapie, whereas no labeling was observed in control animals. In the retina the highest numbers of labeled nuclei were found in the outer nuclear layer 120 days post infection followed by massive cell loss in this layer. In the brain, labeled nuclei were mainly found in the granular layer of the cerebellum of terminally ill mice. This corresponded to the presence of small dark nuclei with condensed and occasionally fragmented chromatin at the light and electron microscopical levels. Our results support the hypothesis that neuronal loss in spongiform encephalopathies is due to apoptosis. This may explain the almost complete absence of inflammatory response in prion diseases in the face of widespread neuronal cell death, and may also have therapeutic implications in the future.

Bovine spongiform encephalopathy (BSE) has been described as an epidemic central nervous disorder in cattle from the United Kingdom. The disease is thought to have emerged by an interspecies transmission of the scrapie agent of sheep to cattle, after feeding scrapie-contaminated meat and bone meal (MBM). The disease has caused substantial economic losses for the British cattle industry. Because of strict veterinary regulations for the import of adult British cattle by the European Union and for MBM by most of the member states the spread of BSE to continental Europe could be efficiently controlled, and only few cases have been described outside the UK. Here we report the first German case of BSE diagnosed in a Scottish Highland cow. The affected cow was imported into Germany before the import ban for cattle from the UK was implemented. BSE was confirmed by histopathology, immunohistochemistry, animal experiments, immunoblotting and by electron microscopic detection of scrapie-associated fibrils (SAFs).

Investigations on extracts from Erysipelothrix (E.) rhusiopathiae carried out within the last few years yielded the identification of a species-specific proteinaceous antigen of 66-64 kDa. The protective properties of this protein presented in particular in crude NaOH and NaOH-EDTA but not in acid and heat extracts could be demonstrated in mice and pigs vaccinated with the electroeluted 66-64 kDa antigen or treated with polyclonal and monoclonal antibodies, respectively. The identification and characterization of the 66-64 kDa protein as a protective antigen of (E.) rhusiopathiae can be regarded as a basis for a possible replacement of the official mouse protection test in vaccine testing by an in vitro assay.

The species specific nature of an antigenic determinant previously discovered in the scrapie form of prion protein (PrPD) from cattle, sheep and mice, was further investigated in normal prion protein (PrPC) from these and other species. This was carried out with eight different anti-peptide sera raised in rabbits against various synthetic peptides representing segments of the amino acid (aa) sequence 101-122 of ovine, bovine, murine and hamster PrP. Antipeptide serum against a peptide representing aa 107-122 of ovine PrP showed almost specific reaction and crossreacted in immunoblot with caprine and human PrP only. Antisera to the corresponding bovine sequence stained bovine and porcine PrP and to a minor extent PrP of goat, man, cat, and mink, while antiserum to the murine aa sequence reacted with rodent and monkey PrP only. In contrast, antiserum to the corresponding hamster sequence displayed a broader reactivity pattern, just like the four other anti-peptide sera to various ovine and bovine sequences. Antisera were also tested for reactivity with the pathogenic isoforms of PrP of sheep, cow, hamster and mouse and showed generally similar reactivity patterns as by using PrPC. In conclusion, the region close to the actual or putative proteinase K cleavage sites of PrP seems to exhibit high structural variability among mammalian species.

Sera from 265 finishing pigs belonging to 70 herds, in which severe respiratory disorders were observed, were examined for antibody prevalence to porcine reproductive and respiratory syndrome virus (PRRSV), influenza virus subtypes H3N2 and H1N1, porcine respiratory corona virus (PRCV) and a recently described porcine paramyxovirus (PPMV). By immunoperoxidase-monolayer assay 69.1% of these sera were positive for PRRSV. Hemagglutination inhibiting activity was found in 55.1% of the sera for influenza virus subtype H1N1 (strain A/swine/Arnsberg/1/81). in 51.3% for influenza virus subtype H3N2 (strain A/Hong Kong/1/68) and in 14.3% for PPMV. PRCV specific antibodies, as determined by differential competitive blocking enzyme-linked immunosorbent assay were demonstrated in 192 of 236 (81.3%) sera. In order to reveal associations interspecific coefficients were calculated for antibody prevalence between PRRSV and the other viruses. Positive associations to PRRSV titres were found to PRCV, PPMV and influenza virus subtype H1N1 titres. chi-square analysis showed the statistical significance of associations regarding PRCV and influenza virus subtype H1N1. Depletion of lung macrophages after PRRSV infection is discussed as a possible mechanism for the promotion of secondary infections.

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders which are linked to abnormal isoforms of the prion protein (PrP), which is expressed in different cells of various mammalian species. Susceptibility to disease and reduced transmission rates upon the first passage to another species are thought to be a result of functional and biochemical differences of the PrP as a consequence of amino acid sequence among species. In 1985 an epidemic of bovine spongiform encephalopathy (BSE) started after accidental transmission of scrapie by feeding infected sheep and goat meat and bone meal products to cattle. In this report we present data demonstrating species-specific epitopes in bovine, ovine and murine PrP that are based on amino acid substitutions at positions 108 and 110. Rabbit antisera to synthetic peptides representing amino acid sequence 108 to 123 of PrP of cattle, sheep and mice reacted strongly with modified PrP of the homologous host but not, or only poorly, with PrP of heterogeneous origin. Cross-reactivity was observed, however, with antisera to bovine and ovine peptide sequences 102 to 117, thus stressing the importance of the location of the amino acid substitution in synthetic peptides used for immunization. Based on these data, BSE PrP and ovine and murine scrapie PrP can be distinguished from each other, and these differences might help elucidate the species barrier effect.

A pepsin-sensitive R-like antigen with a molecular mass of 65 kilodaltons was extracted from Streptococcus uberis strain ATCC 19436 by trypsinisation and purified by diethyl-aminoethanol anionic exchange chromatography. The antigen reacted with sera from infected cows and stimulated opsonic antibody in the guinea pig. The amino acid composition of the antigen was generally similar to that previously reported for the R antigen of group C streptococci.

Although vaccination is widely practiced against infection by Erysipelothrix rhusiopathiae in pigs and turkeys, the protective antigen(s) involved have not been fully characterized or purified to homogeneity. Antigens of E. rhusiopathiae strain T28, serotype 2b, and of FRANKFURT XI, serotype N, in culture supernatant and in extracts made with hot acid, 10 mM NaOH, ultrasound or EDTA were compared by SDS-PAGE and immunoblotting and in a mouse protection test. EDTA and 10 mM NaOH yielded highly protective extracts; culture supernatant was less protective and ultrasonic or hot acid extracts stimulated little or no protection in mice. Protective antisera from swine, horses and mice recognized prominent bands of molecular mass (m.m.) of 66-64 and 40-39 kDa in EDTA and 10 mM NaOH extracts. Mice immunized with preparations of the 66-64 kDa band purified by preparative electrophoresis were protected. Both antigens were trypsin sensitive, contained no detectable polysaccharide, and showed a marked tendency to aggregate in the absence of SDS.

DNA- fingerprints (Hind III) of Streptococcus uberis field isolates from New York State and Europe showed substantial homogeneity, but were different to those of the type strain of the newly proposed psychrophilic species S. parauberis. S. uberis strains had major SDS-heat extracted antigens of molecular masses (Mr) less than 14, 40-41, 42-43, 59-61, 80-86 and 118-122 kDa following immunoblotting with rabbit hyperimmune sera. Bovine sera and milk reacted with the 40-41 and 118-122 kDa antigens. Variations in the Mr of particular bands were too unevenly distributed to permit formation of subgroups. Although cross reactive, the sizes of the antigens of S. parauberis strain NCDO 2020 were substantially different to those of S. uberis, the most prominent antigen having a Mr of 50 kDa. The antigenic and genetic data therefore strongly support the introduction of S. parauberis as a distinct species. S. uberis strains reacted with antiserum to Lancefield groups B, E, G and P, their grouping reactions showing no correlation with DNA and immunoblot fingerprints. Lancefield grouping of S. uberis therefore appears to have little value in identification.

A simple custom-made gel holder for preparative SDS-PAGE to separate aggregated bacterial antigens is described. The gel holder fits easily into commercially available gel electrophoresis apparatus and proteins or peptides are collected in a stream of distilled water through a channel in the gel. The performance of the device was illustrated by the successful separation and purification of fragments of aggregated Streptococcus equi M protein and of Erysipelothrix rhusiopathiae protective antigens. Yields of up to 1.2 mg per run were obtained. The purified proteins retained immunological reactivity and were of sufficient purity for amino acid compositional and sequence analysis.

The molecular weights of the proteins produced in different extracts of Streptococcus equi were compared on immunoblots with antisera against acid extracted and mutanolysin extracted M-protein. Acid and alkaline extracts of S. equi contained some peptides of similar molecular weight that reacted with antiserum against an acid extracted 41,000 m.w. fragment suggesting that these fragments contained common epitopes. Comparison of the amino acid compositions of the 35,000 m.w. fragment of the alkaline extract and the 41,000 m.w. fragment of the acid extract suggest that these immunologically reactive fragments were probably derived from the same protein. Little cross-reactivity was observed between antisera against S. equi acid extracted protein and the native 58,000 m.w. M-protein. This suggests that conformational epitopes on the native M molecule are not present after acid treatment.

The production of protective antigen in modified serum-free nutrient broth (H. Feist, K.-D. Flossmann, and W. Erler, Arch. Exp. Veterinaermed. 30:49-57, 1976) and in brain heart infusion broth supplemented with 10% horse serum (BHIS) was evaluated for six strains of Erysipelothrix rhusiopathiae serotypes 1a, 2, 2b, 4, and N. All six strains grew to higher cell densities in modified Feist medium than in BHIS and produced larger amounts of 64,000- to 66,000- and 39,000- to 40,000-molecular-weight antigens involved in immunity to erysipelas. A vaccine produced in Feist medium from E. rhusiopathiae SE-9 (serotype 2) was highly effective in a mouse protection test. We therefore suggest that modified Feist medium is an excellent, if not superior, alternative to BHIS for production of erysipelas vaccine.