Critical factors influencing prion inactivation by sodium hydroxide.

BACKGROUND AND OBJECTIVES: Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases caused by aberrantly folded cellular proteins (PrP(Sc); prions) that are generally resistant to conventional pathogen-inactivation techniques. To ensure effective decontamination and inactivation of prions that could be present in source material, we investigated critical factors that influence prion inactivation by NaOH. MATERIALS AND METHODS: A decrease in prion infectivity correlates with the disappearance of the protease-resistant core of PrPSc (PrPRES) observed in biochemical assays. To model prion inactivation, hamster scrapie (strain 263K) brain homogenate (SBH) was incubated for specific periods of time in 0.1 m NaOH at 4 or 18 degrees C, with or without detergent. Neutralized samples were subjected to limited digestion with proteinase K (PK) and then analysed using an endpoint dilution western blot assay and antibody 3F4. Structural changes in prions exposed to NaOH were examined using differential immunoprecipitation. RESULTS: Treatment of SBH with 0.1 m NaOH for 15 min, in the absence of detergent, at 4 and 18 degrees C caused a reduction in the PrP(RES) signal of 3.5 and 4.0 log10 units, respectively, with some residual signal remaining. The presence of the detergent sarkosyl during a 60-min incubation in NaOH further enhanced PrPRES reduction to > or = 4.5 log10 units (i.e. below the limit of detection). NaOH treatment induced conformational changes in PrP that resulted in the exposure of a hidden epitope and enabled prion immunoprecipitation by antibody 3F4. CONCLUSIONS: The use of NaOH can effectively reduce prion levels in an in vitro inactivation assay. After pretreatment of SBH with detergent, NaOH completely eliminates the PrPRES signal. Detergent may liberate lipid membrane-protected PrPSc to improve access to NaOH, which can then inactivate PrPSc by altering its structure. In cases of unidentified exposure to PrPSc during manufacturing, sanitizing procedures combining the use of detergent and NaOH may help to ensure minimal levels of contamination carryover in products.

Measures of bone mineral content in mature dairy cows.

The objectives of this investigation were to assess the relationship between chemical measures and imaging estimates (radiographic photometry and dual-energy x-ray absorptiometry) of bone mineral content in dairy cows and to evaluate the effects of parity, stage of lactation, and site of measurement (fused third and fourth metacarpal bone vs. caudal vertebrae 14 and 15) on bone mineral content. In a preliminary study, the caudal vertebrae were excised from 33 cows following slaughter. Samples were analyzed by radiographic photometry and then analyzed for mineral content chemically. In a second experiment, the caudal vertebrae and right front metacarpal (sample pairs) were excised from 107 Holstein cull cows following slaughter. Parity and days in milk (DIM) of the donor animals were obtained for 43 pairs of samples. Samples were grouped by parity (1, 2, 3, and >or=4) stage of lactation (Stage 1: <90 DIM, Stage 2: 90 to 150 DIM, Stage 3: 151 to 250 DIM, and Stage 4: >250 DIM). Samples were analyzed by radiographic photometry and dual-energy x-ray absorptiometry and then analyzed for mineral content chemically. In both experiments, the relationship between mineral content estimated via the imaging techniques and mineral content measured chemically was poor, likely because of the relative maturity of animals in the sample set and lack of variation in mineral content. Ash content was higher in the metacarpal than in the caudal vertebrae, as were concentrations of Mg (expressed as a proportion of bone ash). No effects of stage of lactation were observed on bone mineral in the caudal vertebrae, but in the metacarpal, P content (proportion of total mineral) was highest in second lactation cows. Total bone mineral content (ash) was not affected by parity in the metacarpal or caudal vertebra, but Ca and P content of the metacarpal increased with parity. Noninvasive imaging techniques are not sufficiently sensitive to detect changes in mineral content or composition of mature cows, and only modest changes in bone mineral were observed with stage of lactation and parity.

Growth of thoroughbreds fed a low-protein supplement fortified with lysine and threonine.

Growth and protein status were examined in Thoroughbred foals and yearlings offered pasture supplements with different crude protein contents and amino acid compositions. Both supplements contained 3.0 Mcal/kg DM, 10% corn oil, 1.4% calcium, and three sources of fiber. The control supplement contained 14% CP and 22% soybean meal, whereas the experimental supplement contained 9% CP and 3% soybean meal and was fortified with 0.6% lysine and 0.4% threonine. Mares and foals were fed twice daily (0700 and 1400) and kept on 12.14-hectare pastures (mixed grass and ladino clover) until weaning (6 mo). Weanlings continued on specified supplements and pastures for seven additional months. Physical measurements and blood samples were taken monthly for a period of 14 mo. Measurements included BW, ADG, body condition (BC), wither height, hip height, body length, girth, forearm length, front and hind cannon length, and carpus and fetlock circumference. Blood plasma analysis included total protein, albumin, creatinine, and urea nitrogen. Effect of diet and time were evaluated by analysis of variance with repeated measures. No differences (P > 0.05) were found in physical measurements between the control and Lys/Thr groups (ADG 0.7 +/- 0.02 kg/d, BC 4.9 +/- 0.05) for the observational period. Blood data also showed no difference (P > or = 0.05) for the period (albumin 2.9 +/- 0.03 g/dL, total protein 5.7 +/- 0.10 g/dL, creatinine 1.1 +/- 0.02 mg/dL). Differences in ADG between the control and experimental groups were observed in November (0.73 +/- 0.06 vs 0.91 +/- 0.04 kg/d, P = 0.01) and December (0.56 +/- 0.04 vs 0.67 +/- 0.06 kg/d, P = 0.07) and again in April (0.65 +/- 0.12 vs 0.86 +/- 0.06 kg/d, P = 0.06). These results suggest that the foals offered the Lys/Thr grew at the same or greater rates than foals on the control supplement. When a low-protein diet was fortified with the first two limiting amino acids, the protein was utilized more efficiently for growth and development. This study gives further insight into the role of protein quality during a foal's 1st yr of growth.

Hydrolyzable carbohydrates in pasture, hay, and horse feeds: direct assay and seasonal variation.

Carbohydrates may be hydrolyzed or fermented in the digestive tract, and this distinction is important for the evaluation of the diet of herbivores. Both hydrolyzable and fermentable carbohydrates are included in the nonstructural carbohydrate (NSC) fraction as estimated by difference using proximate analysis. Our objectives were to measure hydrolyzable carbohydrates in forages and concentrates, to compare these values with nonstructural carbohydrate, to test for prediction of hydrolyzable carbohydrate concentration in forages from its near-infrared spectrum, and to examine seasonal variation of carbohydrates in pasture. Samples of forages (107) and concentrates (25) were collected, dried, ground, and analyzed for NSC (calculated as 100 - water - CP - fat - ash - NDF), hydrolyzable carbohydrate (CHO-H, direct analysis), and rapidly fermentable carbohydrate (NSC minus CHO-H). Hydrolyzable carbohydrate accounted for 97% or more of the NSC in the concentrates but only 33% in pasture and hay. A two-term polynomial equation fit all the data: CHO-H = 0.154 x NSC + 0.00136 x NSC2, R2 = 0.98, P < 0.0001, n = 132. In 83 pasture samples, CHO-H concentrations were predicted by near-infrared spectra with a calibration R2 of 0.97, a mean of 48 g/kg, and a SE of calibration of 3.5 g/kg DM. In pasture samples collected between September 1995 and November 1996, the coefficient of variation was 31% for both CHO-H and rapidly fermentable carbohydrate (CHO-FR); the largest increments were 31 g/kg of CHO-H from September to October and 41 g/kg of CHO-FR from February to March. The increased risk of certain diseases, such as laminitis and colic, that have been previously associated with an abrupt overload of NSC may be more precisely attributed to CHO-H in grain concentrates, and to CHO-H as well as CHO-FR in pastures.

Dietary carbohydrates and fat influence radiographic bone mineral content of growing foals.

Hydrolyzable carbohydrate intake in horse diets may become excessive when rapidly growing pastures are supplemented with grain-based concentrates. The substitution of fat and fiber for hydrolyzable carbohydrate in concentrates has been explored in exercising horses but not in young, growing horses. Our objective was to compare bone development in foals that were fed pasture and concentrates rich in sugar and starch (corn, molasses) or fat and fiber (corn oil, beet pulp, soybean hulls, oat straw). Forty foals were examined, 20 each in 1994 and 1995. In each year, 10 mares and their foals were fed a corn and molasses supplement (SS) and 10 others were fed a corn oil and fiber supplement (FF). The concentrates were formulated to be isocaloric and isonitrogenous, and mineral content was balanced to complement the pastures and meet or exceed NRC requirements. Dorsopalmar radiographs were taken of the left third metacarpal monthly from birth to weaning and then every other month until 1 yr of age. Bone density was estimated using imaging software and an aluminum stepwedge. Radiographic examination indicated differences in medial, lateral, and central bone mineral content of the metacarpal III. Bone mineral content increased with age, and a plateau was observed during winter. Bone mineral content was lower in weanlings and yearlings fed the FF supplement than in those fed SS. Subjective clinical leg evaluations indicated differences in physitis, joint effusion, and angular and flexural limb deformities in response to age, and possibly to season. Regression analysis indicated positive relationships between bone mineral content and body weight, age, and body measurements. Nutrient and chemical interactions, such as the binding of calcium by fat and fiber, may alter the availability of elements necessary for bone development.

Deconditioning reduces mineral content of the third metacarpal bone in horses.

Diet and exercise are two management factors that affect bone density and strength. We proposed that bone density and calcium status would be affected by deconditioning for 12 wk and by dietary Ca concentration. Eleven highly conditioned Arabian horses were taken out of training and placed in stalls for 12 wk. Horses were walked on a mechanical walker in two 30-min sessions, 7 d/wk. Diets were designated CC (.36% Ca) and HC (.62% Ca). Data were collected every 21 d. Serum or plasma were analyzed for total and ionized Ca, parathyroid hormone, osteocalcin, hydroxyproline, electrolytes, and blood gases. Bone mineral content (BMC) of the left third metacarpal bone was estimated by radiographic photometry using an aluminum step wedge, which was exposed in each radiograph, as a reference standard for an image analysis system. During deconditioning, BMC decreased by approximately 1.1 g/2 cm, or .45% per week. This decrease was unaffected by dietary Ca. Serum Ca concentration increased with deconditioning. The results suggest that dietary Ca at twice the currently recommended level did not prevent the loss of BMC in response to deconditioning. Loss of BMC during 12 wk of stall confinement may weaken bones, increasing the risk of skeletal injuries when training is resumed.

Designing an incentive compensation program that works.

To encourage innovation and improve departmental and organizational bottom lines, Winchester Hospital, Winchester, Massachusetts, developed and implemented a middle management and staff incentive compensation plan. Middle managers and staff who participate in projects that provide savings to the hospital are paid a share of the savings. The plan allows staff the option of designing "horizontal" projects, in which a project leader carries out a project individually or with peers, or "vertical" projects, in which a project leader directs a team of key individuals. Increasing numbers of vertical projects have been proposed since the plan's inception in 1994. In addition to increasing the hospital's operating income, the program provides cost-effective, variable compensation, which especially appeals to motivated, high-performing employees; motivates employees to overcome roadblocks; facilitates a team approach that bridges traditional departmental boundaries; and encourages a beneficial and collegial competitiveness among managers.

The mechanical properties of equine third metacarpals as affected by age.

Twenty-five pairs of equine third metacarpals (McIII) were collected from horses of varying breeds and ranging in age from 1 d to 27 yr. Standard three-point bending tests were performed on the McIII to obtain comparative data on breaking load (BL), breaking strength (BS), and elasticity (E). Broken bones were reassembled and 2-cm sections cut from the bone 1 cm below the nutrient foramen and at the site of the break. Bone mineral content (BMC) and geometrical properties of these sections were determined. Maximum BMC (17.3 g/2 cm) was reached at 6.0 +/- 1.8 yr of age. Horses in this study achieved 76% of maximum BMC by 1 yr of age. Maximum BL (1,888 kg) was reached at 4.6 +/- 1.8 yr of age. The coefficient of determination (r2) between BMC and BL was .92 (P < .001). Breaking strength peaked at 2,272 kg/cm2 at 6.3 +/- 1.2 yr of age. The correlation between BS and BMC was .84 (P < .0001). At 1 yr of age BS was 1,919 kg/cm2, or 85% of maximum BS. Elasticity increased linearly with log 10 age (r2 = .91). The r2 of BMC and E was .79 (P < .001). Area moment of inertia (I) of the cortex of the bone was best approximated by the area of an ellipse (r2 = .91; P < .01). Results suggest that bone strength in horses peaks after most race horses have completed their careers.

Criteria to evaluate bone mineralization in cattle: II. Noninvasive techniques.

An experiment was conducted to evaluate the capabilities of dual photon absorptiometry (PA), radiographic photometry (RP), and ultrasound (U) to estimate bone mineral content (BMC) and bone strength of a group of bovine third metacarpals (McIII). Metacarpals were chosen for evaluating BMC and bone strength because of their accessibility and susceptibility to biomechanical stress. The right and left McIII of 14 Angus heifers (24 to 32 mo of age) were collected at slaughter and all soft tissues (including periosteum) were removed. The BMC was estimated at both the midpoint and 3 cm proximal to the midpoint on the McIII diaphysis. Metacarpals then were tested by three-point bending to determine breaking load (BL) and breaking strength (BS). Bones were reassembled and two 2-cm sections were removed, one at the midpoint and one 1 cm proximal to the midpoint section. Sections then were ashed and ash content was expressed as grams per 2-cm slice and defined as BMC. Correlation coefficients (r) between BMC vs PA, RP, and U were .908 (P less than .0001), .967 (P less than .0001), and .565 (P less than .0001), respectively; r values between BS vs PA, RP, and U were .406 (P less than .05), .429 (P less than .05), and .499 (P less than .01), respectively, and r values between BL vs PA, RP, and U were .870 (P less than .0001), .865 (P less than .0001), and .588 (P less than .001), respectively. These data indicate that noninvasive techniques are useful in predicting BMC and BL in the bovine.

Criteria to evaluate bone mineralization in cattle: I. Effect of dietary phosphorus on chemical, physical, and mechanical properties.

Fourteen Angus heifers (210 +/- 6 kg initial BW) were allotted randomly to either a low P (LP: .12% P, DM basis) or an adequate P (AP: .20% P, DM basis) diet fed for 14 to 16 mo under drylot conditions on concrete floors to determine the influence of dietary P on chemical, physical, and mechanical properties of bone. Three weeks postpartum, after 14 to 16 mo on their diets, heifers were slaughtered and the right and left third metacarpals (McIII) were excised; soft tissue was removed and metacarpals were frozen in .9% saline. Metacarpals were subjected to a three-point flexure test using an Instron Testing Machine with a crossload speed of 50 mm/min to determine mechanical properties. Broken McIII were reassembled and a 2-cm section was removed at point of loading for determination of chemical and physical properties. Breaking load (BL) was greater (P less than .05) for McIII from the AP than for those from LP heifers (1,348 vs 1,179 kg). Breaking strength (BS) was greater (P less than .05) for AP than for LP heifers (202.5 vs 189.2 MPa). Animals receiving AP diets had greater (P less than .01) bone mineral content (12.6 vs 11.2 g/2-cm slice) and percentage of bone ash (68.0 vs 67.2%) than did LP animals. No differences (P greater than .10) were observed between treatment groups in Ca, P, or Mg percentage in bone ash. Circular, elliptical, radiographic, and planimeter area indices all were greater (P less than .05) in AP than in LP animals (1,048, 729, 1,069, and 570 vs 932, 660, 957, and 523 mm2, respectively). These data indicate that mechanical properties of bovine third metacarpals are sensitive to dietary P and reflect P status in the bovine. Mineral content of bone was highly correlated with its mechanical and physical properties.

Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV 3-6 months after immunization.

Formalin-inactivated (FI) RSV, purified F glycoprotein in alum, and RSV infection (intranasal) were compared for their immunogenicity, efficacy, and ability to enhance pulmonary histopathology during RSV infection 3 and 6 months following immunization by the intramuscular route. Purified influenza virus in alum was used as a control immunogen. At 1 month following immunization with one dose of purified F glycoprotein (5 micrograms), cotton rats developed levels of F antibodies (ELISA) higher than the other groups, but these antibodies had the lowest level of neutralizing activity, Little increase in antibody titre was seen following a second dose of FI-RSV or purified F vaccine given at 1 month. Animals that received 5 micrograms F, 0.5 microgram F, or were almost completely resistant to pulmonary RSV infection following challenge at 3 months, but were susceptible by 6 months. Animals immunized with 5 micrograms of purified F glycoprotein developed alveolar and bronchiolar histopathology following RSV challenge at 3 or 6 months which was comparable to that of animals immunized with FI-RSV. These levels significantly exceeded those in animals previously immunized with influenza A virus vaccine which exhibited little histopathology. Animals previously infected with RSV also developed bronchiolar, but not alveolar, histopathology suggesting that the bronchiolar histopathology seen in RSV challenged cotton rats is a normal component of the immune resolution of RSV infection. These results suggest that the immune response of cotton rats to immunoaffinity purified F glycoprotein can result in enhanced bronchiolar and alveolar histopathology following RSV challenge. Thus, caution should be exercised in studies in humans using a purified F glycoprotein subunit vaccine.

Detection of respiratory syncytial virus (RSV) infected cells by in situ hybridization in the lungs of cotton rats immunized with formalin-inactivated virus or purified RSV F and G glycoprotein subunit vaccine and challenged with RSV.

The replication of RSV in unimmunized cotton rats was evaluated by quantitating the amount of infectious virus in the lung and the number of RSV infected cells in a histopathological section of lung by in situ hybridization. RSV infected cells were detected only in alveoli and bronchioles and constituted only a small minority of the cell population. The temporal patterns of rise to the peak number of infected cells (day 4) and the peak titer of infectious virus (day 3) were similar. The clearance of both infected cells and infectious virus was nearly complete by day 7. In animals previously immunized with purified RSV glycoproteins or formalin-inactivated RSV there also was a good correlation between the number of infected cells detected by in situ hybridization and the amount of infectious virus recovered. It was previously demonstrated that cotton rats immunized with formalin-inactivated vaccine developed enhanced pulmonary histopathology following challenge with RSV. In such animals, there was approximately a 90% reduction in the number of infected cells compared to control unimmunized, RSV-challenged animals. Formalin-inactivated RSV vaccine-enhanced lung histopathology developed despite the effective elimination of virus and virus-infected cells suggesting that the enhanced pathology is the result of an exaggeration of normal immune mechanisms involved in clearance of virus infection, an aberrant immune response during infection, or both.

Evaluation in chimpanzees of vaccinia virus recombinants that express the surface glycoproteins of human respiratory syncytial virus.

The immunogenicity and protective efficacy of recombinant vaccinia viruses that express the two major protective antigens of human respiratory syncytial virus (RSV), the F and G glycoproteins, were evaluated in chimpanzees. In previous studies in rodents and monkeys the F and G proteins expressed by the same recombinants were highly immunogenic and induced high levels of resistance to RSV replication following subsequent challenge. In contrast, in chimpanzees, a single intradermal immunization induced only moderate levels of F and G-specific serum antibodies as measured by an enzyme-linked immunosorbent assay, and these antibodies did not efficiently neutralize RSV infectivity in vitro. This poor antibody response in chimpanzees to the F and G glycoproteins occurred despite efficient replication of the vaccinia virus vector as evidenced by lesion size and serum antibody response to vaccinia virus. Upon intranasal RSV challenge, it was observed that prior immunization with the F and G recombinants effected only a marginal reduction in the magnitude and duration of RSV shedding from the nose and trachea and did not reduce illness. However, the RSV challenge induced a strong secondary antibody response, resulting in very high titres (greater than 8000 reciprocal mean titre) of serum neutralizing antibodies. The poor protective efficacy observed here is discussed with regard to the permissiveness of the chimpanzee to RSV replication, the general requirements for effective immunization against RSV, and the limitations of experimental animals for evaluating candidate RSV vaccines.

Dietary phosphorus concentrations related to breaking load and chemical bone properties in heifers.

Effects of dietary P on breaking load and chemical properties of bone were evaluated in growing beef heifers. Initially, 14 weaned Angus heifers received ad libitum a low P diet (.10% P dry basis) for 270 d. Heifers were subsequently randomly allotted into two groups (7 heifers/group) and received ad libitum either .12 or .20% P (dry basis). The experimental endpoint was 245 d of the treatment phase for nonpregnant and 3 wk postpartum for pregnant heifers. Results indicate that chemical and physical properties of bovine bones are sensitive to dietary P. Mineralization of rib bones was less at .12% P (dry basis). Rib bone density (g/cm3) and mineral content expressed on a per unit volume basis (mg/cm3) were the most sensitive parameters measured. Breaking load results of 12th ribs also indicated a greater amount of mineral present in heifers receiving .20% P (153.8 vs. 114.3 kg). Vertebral bone density and mineral concentrations on a per unit volume basis were also sensitive to dietary P and clearly ranked the two dietary P amounts, whereas percentage of bone ash or mineral concentration expressed on an ash basis did not.

Compensation strategy motivates executives to achieve goals.

A compensation strategy helps healthcare organizations motivate key executives to achieve business goals. Such a strategy has five elements: base salary, annual incentive plans, long-term incentive plans, perquisites, and benefits. In designing a compensation package, an organization should consider the managerial model of its executives plus the institution's culture and its position in the business life cycle.

Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus.

Posttranslational processing and cell surface expression were examined for three C-terminally truncated mutants of the G protein of respiratory syncytial virus expressed from engineered cDNAs. The truncated mutants, encoded by cDNAs designated G71, G180, and G230, contained the N-terminal 71, 180, and 230 amino acids, respectively, of the 298-amino-acid G protein. To facilitate detection of G71, which reacted inefficiently with G-specific antisera, we constructed a parallel set of cDNAs, designated G71/13, G180/13, and G230/13, to encode the same truncated species with the addition of a C-terminal 13-amino-acid reporter peptide which could be detected efficiently with an antipeptide serum. G71, G180, and G230 were expressed as species of Mr 7,500, 48,000, and 51,000, respectively, compared with 84,000 for parental G protein. The proteins encoded by G180 and G230, like parental G protein, contained both N-linked and O-linked carbohydrate. Also, the protein encoded by G71/13 appeared to be O glycosylated, showing that even this highly truncated form contained the structural information required to target the protein for O glycosylation. As for parental G protein, the estimated Mrs of the proteins encoded by G180 and G230 were approximately twice the calculated molecular weight of the polypeptide chain. Experiments with monensin showed that most of this difference between the calculated and observed Mr was due to posttranslational processing in or beyond the trans-Golgi compartment, presumably owing to the addition of carbohydrate or aggregation into dimers or both. Like parental G protein, all three truncated forms accumulated abundantly at the cell surface, and in each case the C terminus was extracellular. Thus, the N-terminal 71 amino acids of the G protein contained all the structural information required for efficient membrane insertion and cell surface expression, whereas the extracellular domain was dispensable for these activities. Cotton rats were immunized with recombinant vaccinia viruses expressing the G71, G180, G230, or parental G protein to compare their abilities to induce serum antibodies and resistance to challenge virus replication. The G71 and G180 recombinants failed to induce significant levels of G-specific antibodies or resistance to challenge, whereas the immunogenicity of G230 equaled or exceeded that of parental G protein. This suggested that the C-terminal 68 amino acids of the 236-amino-acid ectodomain do not contribute to the major epitope(s) of the G protein that is involved in inducing protective immunity.