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Rectoanal mucosa-associated lymphoid tissue (RAMALT) is a part of the lymphoid system that can be sampled easily in live animals, especially ruminants. RAMALT biopsy is useful for the diagnosis of transmissible spongiform encephalopathies, including scrapie in sheep and goats and chronic wasting disease (CWD) in cervids. Diagnosis is reliant on detection of abnormal prion protein (PrP(d)), which is associated with lymphoid follicles. For enzyme linked immunosorbent assays (ELISAs) detecting PrP(d) it is necessary to ensure that lymphoid follicles are present in biopsy samples to avoid false-negative results. Monoclonal antibodies known to recognize specific immune cell subsets present in lymphoid tissues of sheep were tested for cross-reactivity with cervine RAMALT and mesenteric lymph nodes (MLNs) preserved in zinc salts fixative. The distribution of cells expressing CD3, CD4, CD79, CD21 and class II molecules of the major histocompatibility complex was determined in these tissues. Cells of each immunophenotype had similar distributions in RAMALT and MLNs and these distributions were similar to those reported previously for sheep and cattle. The identification and validation of cervine lymphoid follicle cell markers (CD79 and CD21) may allow reduction in false-negative results during diagnosis of CWD by ELISA.

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In order to investigate the relationship between the immune response to scrapie infection and genetic susceptibility to the disease in sheep, immune cell subsets and prion protein (PrP) expression were determined in susceptible and resistant Suffolk sheep in the preclinical phase of infection. At 6 months of age, 12 ARQ/ARQ (susceptible) and nine ARR/ARR (resistant) scrapie-free Suffolk lambs were challenged subcutaneously with scrapie inoculum. Prefemoral lymphadenectomies were carried out at 14 and 180 days post-inoculation (p.i.) and serial bleeds were collected at monthly intervals for up to 1 year p.i. An indirect double-labelling procedure was carried out on peripheral blood mononuclear cells (PBMCs) and lymph node cell preparations and analysed using flow cytometry. Prior to scrapie challenge, significantly more PrP(+) cells were detected in PBMCs from the susceptible sheep. Furthermore, following challenge, significantly more CD8(+) and gammadelta(+) T cells were detected in the PBMCs of the resistant sheep. However, at both 14 and 180 days p.i, CD21(+) cell expression was significantly higher in the lymph node preparations of the susceptible sheep. In contrast, more CD4(+) cells were detected in the lymph nodes of the resistant sheep at both time points. It was concluded that significant differences in immune cell subsets and PrP expression occur between ARQ/ARQ and ARR/ARR Suffolk sheep in the preclinical phase of infection.

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Dicalcium phosphate was prepared from industrial crushed bone artificially contaminated with transmissible spongiform encephalopathy agents in two experiments carried out in an accurately scaled-down laboratory model of the industrial manufacturing process. In one experiment, 10 g of mouse brain infected with the 301V strain of mouse-passaged bovine spongiform encephalopathy agent was added to the crushed bone; in the other experiment, 10 g of hamster brain infected with the 263K strain of hamster-passaged scrapie agent was added. Samples of the infectious brain and dried dicalcium phosphate were assayed for the amount of 301V or 263K infectivity present. The titre of infectivity of the 301V-infected brain was 10(7.7) intracerebral ID50/g; that of the 263K-infected brain was 10(8.0) intracerebral ID50/g. The titres of the dried samples of dicalcium phosphate were 10(2.5) ID50/g in the experiment spiked with 301V and 10(2.7) ID50/g in the experiment spiked with 263K. The calculated clearance factors were 10(3.9) for the experiment with 301V and 10(3.8) for the experiment with 263K.

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Dietary exposure to the bovine spongiform encephalopathy (BSE) agent is the probable cause of variant Creutzfeldt-Jakob disease in people. The industrial manufacturing process for the production of gelatine and colloidal protein by the heat and pressure process was downscaled accurately and its capacity to remove or inactivate bse infectivity was investigated. Gelatine was made from bones experimentally contaminated with mouse brain infected with the 301V strain of mouse-passaged bse agent in which the infective titre was 10(8.7) ID50/g. No infectivity was detected in the extracted protein (> or =10(0.45) ID50/g), and the calculated clearance factor was 10(6.5) ID50 or more.

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BACKGROUND AND OBJECTIVES: The risk of haemophiliacs contracting variant Creutzfeldt-Jakob disease (vCJD) via treatment with factor VIII concentrates is not known. Therefore, in order to determine the extent to which the vCJD agent might be removed during the preparation of factor VIII concentrate, the partitioning of a bovine spongiform encephalopathy (BSE)-derived agent was measured over the main purification step used to prepare the Scottish National Blood Transfusion Service high-purity factor VIII concentrate (Liberate). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to 'spike' a solution of factor VIII of intermediate purity. The 'spiked' starting material was subjected to solvent-detergent treatment and then to anion-exchange chromatography with Toyopearl DEAE-650M. All fractions were tested for 301V infectivity using a murine bioassay, including the procedures used to clean the ion-exchange media after use. RESULTS: BSE 301V infectivity was reduced by 2.9 log(10) in the fibrinogen fraction and by 2.7 log(10) in the factor VIII fraction. Over 99% of the added 301V infectivity remained bound to the ion-exchange column after elution of factor VIII. A large quantity of infectivity was subsequently removed by washing the ion-exchange media with 2 m NaCl. No further BSE 301V infectivity was detected in column eluates after treatment with 0.1 m NaOH or a second wash with 2 m NaCl. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity would be substantially removed by the ion-exchange process used in the preparation of fibrinogen and factor VIII concentrate. Although 301V infectivity remained bound to the ion-exchange matrix following elution of factor VIII, this appeared to be eliminated by the procedure used for cleaning the ion-exchange media after each use.

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BACKGROUND AND OBJECTIVES: There is still uncertainty over how the agent of variant Creutzfeld-Jakob disease (vCJD) would partition during the manufacture of plasma derivatives. In this study, a BSE-derived agent was used as a vCJD model to determine the extent to which infectivity could be removed by selected steps used in the manufacture of intravenous immunoglobulin (IVIG). MATERIALS AND METHODS: Murine-passaged BSE (strain 301V), in the form of a microsomal fraction prepared from infected brain, was used to "spike" the starting material in three experiments. The partitioning of BSE infectivity was measured over Fraction I+III precipitation, borosilicate microfibre depth filtration and Seitz depth filtration, with these steps being examined individually and in series. RESULTS: Most 301V infectivity partitioned into Fraction I+III (log reduction 2.1). Infectivity remaining in Supernatant I+III was reduced by AP20 glass-fibre depth filtration (log reduction 0.6) and subsequently removed to below the limit of detection by Seitz KS80 depth filtration, giving an overall log reduction of > or = 2.9 for the three steps in series. By contrast, glass-fibre depth filtration gave a log reduction of 2.4 when challenged directly with "spiked" feedstock. Seitz KS80 depth filtration gave a log reduction of > or = 3.1 when challenged directly with 'spiked' feedstock and also removed residual infectivity to below the limit of detection when applied as the final step in series. CONCLUSIONS: Results using a BSE-derived agent suggest that vCJD infectivity should be substantially removed from immunoglobulin G (IgG) solutions by Fraction I+III precipitation and Seitz KS80 depth filtration. The three different process steps examined acted in a complementary manner to one another when operated in series. However, the data demonstrated that it would be inappropriate to add together the reduction factors that had been derived for each step in isolation.

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Fifty mg aliquots of macerated mouse-brain infected with the 22A strain of scrapie agent were treated by exposing them without mechanical mixing to (a) distilled water for 2 h, (b) 5% sodium dodecyl sulphate (SDS) for 2 h, (c) autoclaving at 121 degrees C for 15 min in distilled water, (d) autoclaving at 121 degrees C for 15 min in 5% SDS, or (e) boiling in 5% SDS for 15 min. Prior to injection into mice, all samples were washed by a procedure that is described and was shown not to reduce infectivity titres. Although the infectivity titre of the sample that was autoclaved in SDS was reduced considerably, infectivity was present in all of the samples exposed to cold or hot SDS.

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The study was designed to determine the effect on bovine spongiform encephalopathy (BSE) and scrapie agents of the solvent extraction processes used in the past by British renderers. The raw material was mouse spleen infected with either the 22A strain of scrapie agent or the 301V strain of BSE agent. Samples were exposed to hexane, heptane, petroleum spirit or perchlorethylene at the relevant temperatures for the appropriate times. Control samples were exposed to the same range of temperatures for the same range of times in saline. Other samples were exposed to the hot solvents, followed by treatment with dry heat at 100 degrees C for 30 minutes and steam at 100 degrees C for 30 minutes. Further samples were exposed only to the dry heat and steam cycles. No single complete process was significantly more effective than any of the others, and they all produced only slight inactivation, less than one log on average for both strains of agent. The average degree of inactivation produced by exposure to hot saline was generally comparable to that produced by exposure to the hot solvents. This was also true for the samples exposed only to dry heat and steam compared with those exposed to hot solvent before treatment with dry heat and steam, and suggests that the slight inactivation was caused by the heat rather than by the solvents. It is concluded that the solvent extraction processes used by renderers in Britain had little capacity to inactivate BSE and scrapie agents.

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When scrapie agent is exposed to partially inactivating autoclave cycles, the fraction of infectivity that survives remains thermostable during relatively long periods of autoclaving. This resistant subpopulation can also be differentiated from the main population by its prolonged incubation periods in assay animals, compared with control material. Stabilisation of this subpopulation may occur through the smearing and drying of infected tissue that can occur prior to autoclaving, in which the disease-specific form of PrP protein (PrP(Sc)) could become rapidly heat-fixed. This may paradoxically be what protects this fraction of PrP(Sc) from further inactivation during autoclaving. Data are presented showing that the thermostability acquired by the resistant subpopulation is a stable characteristic; autoclaving for a second time results in very little further loss of infectivity. These observations suggest that inactivation procedures that do not involve rapid and effective fixation of PrP(Sc) may be better candidates for dealing effectively with scrapie-like agents.

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An in vitro cell culture assay using myeloma cells and hybrid lymphocytes was developed which detected pathogenic Listeria strains in just 6 h. Three separate hybridoma cell lines, murine Ped-2E9 and EM-7G1 and human RI.37 and murine myeloma NS1 cells, proved equally sensitive in responding to virulent Listeria species. Listeria monocytogenes along with other Listeria spp., collected from food and clinical sources, were inoculated at 10(8) cfu/ml into a suspension of Ped-2E9 (10(6)/ml). Pathogenic Listeria spp. killed 80% of hybridoma cells by 4 h, as determined by trypan blue exclusion test. Conversely, none of all nonpathogenic Listeria spp. killed the hybridoma cells. Ped-2E9 cells exposed to three strains of L. monocytogenes strains showed 96-97.5% death in 6 h measured by trypan blue staining and release of 91-97% of lactate dehydrogenase (LDH) enzyme. RI.37 cells showed similar results. A multiplicity of exposure (MOE) of 100 L. monocytogenes to 1 hybridoma cell or of 10:1 killed about 80% of the hybridoma cells in 4 or 6 h respectively. The in vitro virulence assay of L. monocytogenes with hybridoma cells compared favorably with the immunocompromised mouse model, yielding results in 6 h instead of 3 days. Intracellular L. monocytogenes and L. innocua were not recovered from Ped-2E9 hybridoma cells after 2 or 4 h of exposure. However, attachment of both L. monocytogenes and L. innocua cells on Ped-2E9 cell surfaces were observed under epifluorescence microscopy. Direct contact of hemolysin positive L. monocytogenes with hybridoma cells is essential to cause death, since hybridoma cells were not killed when they were separated from the growing bacteria by a 0.45 microns filter.

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Ferric sulfate solution is an accepted soft tissue hemostatic agent for use in dermatology and dentistry. This study was designed to test its effect on osseous healing when used during surgery to control osseous hemorrhage. Standardized osseous defects were created bilaterally in the naturally edentulous zone in rabbit mandibles. The control site was sutured immediately after clot formation in the defect. The contralateral experimental site received ferric sulfate application until complete hemostasis was achieved. The defect was filled with ferric sulfate solution to maximize any effect on healing and then closed with sutures. The experimental and control specimens were examined histologically after 18 and 46 days and scored for healing. Statistical analysis by Wilcoxon signed rank test showed significant adverse effects on osseous healing when ferric sulfate solution was left in situ.

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